Instituto Nacional de Ciência e tecnologia de Biologia ... de resumos V Encontro do... · – MsC....

V Encontro Annual do INBEB – www.inbeb.org.br Página 1

Instituto Nacional de C&T de Biologia Estrutural e Bioimagem - INBEB

O Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB) é um instituto de pesquisa multicêntrico, constituído por uma entidade sede e por uma rede de grupos de pesquisa em Biologia Estrutural e Bioimagem distribuídos em diversos estados brasileiros. A entidade sede do INBEB é o Centro Nacional de Bioimagem (CENABIO), localizado na Universidade Federal do Rio de Janeiro (UFRJ). Em novembro de 2013, o Cenabio conquistou o status de Órgão Suplementar da UFRJ, efetivando o professor Adalberto Ramon Vieyra (IBCCF/UFRJ) como seu 1º diretor.

O INBEB tem a missão de criar e consolidar uma infraestrutura técnico-científica que permita o desenvolvimento de pesquisas nacionais de qualidade, acerca das estruturas de sistemas biológicos, desde o nível macromolecular até o imageamento de organismos inteiros. Para isso, o CENABIO conta com equipamentos modernos e corpo técnico qualificado, oferecendo amplo suporte tecnológico para a comunidade científica. Sua inserção no INBEB permite mobilizar e agregar, de forma articulada, os melhores grupos de pesquisa na área, além de promover mais interações e oportunidades de formação profissional. É neste âmbito que apresentamos no Programa do V Encontro Anual do INBEB a programação do evento, assim como os resumos dos 142 pôsteres inscritos no encontro. Esperamos que todos possam aproveitar a oportunidade para interagir, aperfeiçoar seus conhecimentos e estabelecer novas parcerias.

Apoio:

Prof. Wanderley de Souza Vice-coordenador do INBEB

Prof. Adalberto R. Vieyra Diretor do Cenabio

Prof. Jerson Lima da Silva Coordenador do INBEB

V Encontro Anual do INBEB – www.inbeb.org.br Página 2

Índice Programação ........................................................................................................................................................................... 3

Quarta-feira - 07/05/2014 .................................................................................................................................................. 3

Quinta-feira - 08/05/2014 .................................................................................................................................................. 5

Sexta-feira - 09/05/2014 ..................................................................................................................................................... 7

Sessões de pôsteres ................................................................................................................................................................ 9

Data das apresentações ...................................................................................................................................................... 9

Certificados ......................................................................................................................................................................... 9

Listas de apresentadores .................................................................................................................................................. 10

Graduandos ................................................................................................................................................................... 10

Mestrandos ................................................................................................................................................................... 11

Doutorandos ................................................................................................................................................................. 12

Pós-doutorandos .......................................................................................................................................................... 14

Pesquisadores ............................................................................................................................................................... 14

Resumos ................................................................................................................................................................................ 15

Graduandos....................................................................................................................................................................... 15

Mestrandos ....................................................................................................................................................................... 24

Doutorandos ..................................................................................................................................................................... 35

Pós-doutorandos .............................................................................................................................................................. 53

Pesquisadores ................................................................................................................................................................... 60

http://www.inbeb.org.br/


Programação

Quarta-feira - 07/05/2014 Auditório do bloco N, CCS/UFRJ (Acesso pelo interbloco I/J e pela Rua César Pernetta) 9:30 – 10:00h Abertura. – Diretor do CENABIO, Prof. Adalberto Vieyra (IBCCF/CENABIO/UFRJ). 10:00 – 10:30h “Um breve histórico do Centro Nacional de Ressonância Magnética Nuclear”. – Coordenador do INBEB, Prof. Jerson Lima da Silva (IBqM/CNRMN/CENABIO/UFRJ). 10:30 – 10:50h - Coffee-break. 10:50 – 11:50 h “A Personal View of the History of NMR in Structural Biology and Medical Diagnosis”. – Nobel de Química de 2002 Prof. Kurt Wüthrich (The Scripps Research Institute/Eidgenössische Technische Hochschule Zürich/Prof. Visitante do IBqM/UFRJ/INBEB). 11:50 – 12: 10h Inauguração dos novos magnetos e visitação ao Centro Nacional de Ressonância Magnética Nuclear. 12:10 – 14h – Almoço. 14:00 -14:40h



“As pesquisas desenvolvidas no Centro Nacional de Ressonância Magnética Nuclear”. – Prof. Fabio Almeida (IBqM/CNRMN/CENABIO/UFRJ). 14:40 – 15:20h “Novas capacidades do Centro Nacional de Ressonância Magnética Nuclear”. – Profª. Monica Freitas (IBqM/CNRMN/CENABIO/UFRJ). 15:20 -16h “Determinação de estrutura de proteínas de membrana por Ressonância Magnética Nuclear em estado sólido”. – Prof. Barth-Jan van Rossum (Leibniz Institute für Molekulare Pharmakologie, Berlin, Germany). 16:00 – 18:00h – Sessão de pôster com coffee-break.



Quinta-feira - 08/05/2014 Auditório do bloco N, CCS/UFRJ (Acesso pelo interbloco I/J e pela Rua César Pernetta) 9:30 - 10:30h “Tutorial na determinação da estrutura de proteínas de membrana por RMN em estado sólido: 1ª parte”. – Prof. Barth-Jan van Rossum (Leibniz Institute für Molekulare Pharmakologie, Berlin, Germany). 10:30 - 10:50h - Coffee-break. 10:50 - 12:30h “Tutorial na determinação da estrutura de proteínas de membrana por RMN em estado sólido: 2ª parte”. – Prof. Barth-Jan van Rossum (Leibniz Institute für Molekulare Pharmakologie, Berlin, Germany). 12:30h – 14:00h – Almoço. 14:00h – 15:00h “Traditional and new technologies combined for a dynamic portrait of Toxoplasma gondii” - Profª. Marcia Attias (IBCCF/INBEB/UFRJ). 15:00h – 17:30h Mesa-redonda: “PAPEL DO INBEB NOS GRUPOS DE PESQUISA DE FORA DA SEDE”. 1. “A consolidação do grupo de pesquisa em Biologia Estrutural (LA14) na Região Norte do país com apoio do INBEB”. – Dra. Ana Paula Rodrigues/Pós-doc/UFPA/INBEB; 2. “O Inbeb na Ilha de Santa Catarina”.



– Dr. Hernán Terenzi/Prof. Associado/UFSC/INBEB. 3. “Estudo da Estrutura das Chaperonas Hsp40” – Dra. Regina Adão/Pós-doc do IQ/UNICAMP/INBEB. 4. “Efeitos do Sildenafil no Sistema Nervoso Central: Potencial Aplicação Farmacológica.” – MsC. Ana Karolina Santana Nunes/UFPE/INBEB 5. “O papel do INBEB na estruturação do laboratório de Bioinformática e Modelagem Molecular da UFBA”. – Dr. Marcelo Castilho/Prof. Associado/UFBA/INBEB 17:30h – 19:30h - Sessão de pôster com coffee-break.



Sexta-feira - 09/05/2014 Auditório do bloco N, CCS/UFRJ (Acesso pelo interbloco I/J e pela Rua César Pernetta) 10:00h – 12:00 h Mesa-redonda: “JOVENS PESQUISADORES DO INBEB”. 1. “Estrutura e dinâmica do domínio RRM1 do regulador pós-transcricional HuR: implicações para o reconhecimento específico de RNAm”. - Prof. Anderson Pinheiro (IQ/INBEB/UFRJ). 2. "The three-dimensional structure of the cytostome-cytopharinx complex of Trypanosoma cruzi epimastigotes". – MsC. Carolina de Lima Alcantara (IBCCF/INBEB/UFRJ). 3.“Terapia celular com células mesenquimais de medula óssea em cães com cardiomiopatia chagásica crônica”. – Profª. Adriana Bastos de Carvalho (IBCCF/CENABIO/UFRJ). 4. “Minha experiência no laboratório 17 do INBEB no programa Ciências sem Fronteiras”. – Profª. Karine Verdoorn (UFRJ- Campus Macaé/INBEB). 12:00h -13:30h – Almoço. 13:30h – 16:00h Mesa-redonda: “PARCERIAS PÚBLICO-PRIVADAS”.



- Representante do Instituto D´Or de Pesquisa e Ensino (IDOR), Dr. Jorge Moll; - Representante do Banco Nacional do Desenvolvimento (BNDES), Dr. Pedro L. Palmeira Filho; - Representante da Fundação do Cancer, Dr. Marcos Moraes; - Representante da Bruker Corporation, Dr. Mark Chaykovsky; - Representante do Hospital Sírio e Libanês, Dr. Fabio Gregory. 16:00h -16:20h - Coffee-break. 16:20h -17:20h “Impacto do INBEB medicina translacional”. - Prof. Antônio Carlos Campos de Carvalho (IBCCF/CENABIO/UFRJ/DECIT/MS). 17:20 – 18:00h Encerramento com premiação dos pôsteres. - Diretor do CENABIO, Prof. Adalberto Vieyra e Comitê Gestor do CENABIO/INBEB.



Sessões de pôsteres

Data das apresentações Trabalhos de número ímpar: dia 07/05

Trabalhos de número par: dia 08/05

Certificados Após a arguição dos avaliadores, serão entregues certificados de apresentação apenas em nome dos apresentadores inscritos que forem avaliados. Os demais autores terão este caderno de resumos como certificado de inscrição dos trabalhos no evento.



Listas de apresentadores Os resumos estão separados por titulação e ordenados alfabeticamente pelo primeiro nome do apresentador.

Graduandos

G 1 - Ana Beatriz Padilha de Figueirêdo

G 2 - Antonio Leonardo Freitas Casalinho

G 3 - Bruno Jennings de Almeida

G 4 - Caio Bidueira Denani

G 5 - Carlla Assis de Araujo e Silva

G 6 - Clarice S C Machado

G 7 - Claudia Monteiro da Rocha

G 8 - Dahienne Ferreira de Oliveira

G 9 - Daniel Meira dos Anjos

G 10 - Fenando Limoeiro Lara de Oliveira

G 11 - Hector Franco Barbosa Rhault Loponte

G 12 - Juliana Bosco Santos

G 13 - Juliana Santos Santana

G 14 - Lara Soares Junqueira

G 15 - Leandro Coelho Teixeira Pinheiro

G 16 - Lilian Hernández Alvarez

G 17 - Luciana Domett Siqueira

G 18 - Matheus Campello P. L. P. Baptista

G 19 - Mirian Kelley Alves

G 20 - Nathali Pereira da Costa Campos

G 21 - Raiane França Delvalle dos Santos

G 22 - Ramon Pinheiro Aguiar

G 23 - Raysla Alves Pires

G 24 - Roger Borges dos Santos

G 25 - Stéphanie Casali Rocha

G 26 - Stephanie Fernanda Fulaz

G 27 - Stephanny Miranda Alves de Souza

G 28 - Wesley Júnio Alves da Conceição



Mestrandos

M 1 - Alan Cesar Nunes de Moraes

M 2 - Alessandro Miranda de Souza

M 3 - Aline Araujo Alves

M 4 - Aline Miyoko Sakaguchi Yamashita

M 5 - Ana Carolina Loyola Machado

M 6 - Barbara Barbosa Succar

M 7 - Beatriz Bastos Fonseca

M 8 - Bruno José Martins da Silva

M 9 - Caroline Martins Almeida

M 10 - Cristine Saibert

M 11 - Cristóvão Freitas Iglesias Junior

M 12 - Dany Naranjo Feliciano

M 13 - Diana Pelizzari Raymundo

M 14 - Gabriel Nascimento dos Santos

M 15 - Gabriela Veras de Moraes

M 16 - Glauber Ribeiro de Sousa Araujo

M 17 - Júlia Araújo de Freitas

M 18 - Lienne Silveira de Moraes

M 19 - Lucas Vellasco

M 20 - Luiz Carlos dos Santos Ribeiro

M 21 - Luiza Fernandes

M 22 - Murilo Martins Pedrote

M 23 - Nathália Rocco Machado

M 24 - Paula Cristina Rodrigues Frade

M 25 - Paula Mattos da Silva

M 26 - Pedro Ernesto Lopes Leão

M 27 - Rafael Mesquita Stoque

M 28 - Raquel Gama Gomes Leite

M 29 - Rodrigo Dias Requião

M 30 - Talita Stelling de Araujo

M 31 - Thamires Quadros Froes

M 32 - Tiago Souza Salles

M 33 - Verônica Leite Queiroz

M 34 - Victor da Conceição David



Doutorandos

D 1 - Alane Bernardo Ramos

D 2 - Almir Jordão da Silva Junior

D 3 - Ana Clara Vicente dos Santos

D 4 - Ana Karolina De Santana Nunes

D 5 - André Luiz Oliveira Poleto

D 6 - Anita Leocadio Freitas Mesquita

D 7 - Antonio Real Hohn Neto

D 8 - Aracelys López Castilla

D 9 - Banny Silva Barbosa

D 10 - Beatriz Ferreira de Carvalho Patricio

D 11 - Bruno Macedo da Silva

D 12 - Carlos Henrique Dumard

D 13 - Davi Marcos Souza de Oliveira

D 14 - Edlene Lima Ribeiro

D 15 - Elaine da Conceição Petronilho

D 16 - Elaine Hilário de Souza

D 17 - Erivan Schnaider Ramos Junior

D 18 - Estefania Azevedo

D 19 - Flora Magno de Jesus Oliveira

D 20 - Franco Henrique Andrade Leite

D 21 - Gustavo Monnerat Cahli

D 22 - Isalira Peroba Ramos de Góes Freitas

D 23 - Ivone Rosa de Andrade

D 24 - João Guilherme de Moraes Pontes

D 25 - Josineide Pantoja Da Costa

D 26 - Juliana Cunha Vidal

D 27 - Juliana Dias Alves Pinto

D 28 - Julliana Ferreira Santanna

D 29 - Larissa Nogueira de Almeida

D 30 - Laura Machado de Faria

D 31 - Leonardo Maciel de Oliveira Pinto

D 32 - Leonardo Vazquez

D 33 - Luana P. Borba-Santos

D 34 - Luis Henrique Seabra de Farias

D 35 - Luiza Helena Daltro Cardoso

D 36 - Luiza Villarinho Pereira Mendes

D 37 - Luzia da Silva Sampaio

D 38 - Mara Cristina Pimenta dos Santos



D 39 - Marcelle de Souza Ferreira

D 40 - Maria Micaela Lopez Alarcón

D 41 - MARIANA G. A. T. CUNHA

D 42 - Moema Monteiro Batista

D 43 - NATALIA DO CARMO FERREIRA

D 44 - Nathalia dos Santos Alves

D 45 - Paula Viegas Pereira Signoretti

D 46 - Paulo César Arantes

D 47 - Pedro Henrique Monteiro Torres

D 48 - Rachel Santos de Menezes

D 49 - Raísa da Rocha Reis

D 50 - Reinaldo Souza de Oliveira Júnior

D 51 - Rosemberg de Oliveira Soares

D 52 - Sabrina Ribeiro Gonsalez

D 53 - Samir Pereira da Costa Campos

D 54 - Sura Wanessa Santos Rocha

D 55 - Tácio Vinício Amorim Fernandes

D 56 - Tatiana Christina Paredes

D 57 - Thais Russo Abrahão

D 58 - Tiago Bortolotto



Pós-doutorandos

PD 1 - Aline A. Zuma

PD 2 - Carla Maria de Souza Menezes

PD 3 - Carlos Alberto Marques de Carvalho

PD 4 - Carolina Galvão Sarzedas

PD 5 - Cherley Borba Vieira de Andrade

PD 6 - Clarissa Rodrigues Nascimento

PD 7 - Clelton Aparecido dos Santos

PD 8 - Douglas Ricardo Norberto

PD 9 - Fernanda Magalhães Ferrão

PD 10 - Humberto Muzi Filho

PD 11 - Luana Heimfarth

PD 12 - Lucio Ayres Caldas

PD 13 - Mariangela Dametto

PD 14 - Paulo André da Silva

PD 15 - Pedro Alexandre de A. G. L. Loureiro

PD 16 - Rafael Soares Lindoso

PD 17 - Shana Priscila Coutinho Barroso

PD 18 - Tatiana Kelly Silva Fidalgo

PD 19 - Victor do Valle Pereira Midlej

Pesquisadores

P 1 - André Meyer Alves de Lima

P 2 - Diego Enry Barreto Gomes

P 3 - Manuela Leal da Silva



Resumos Graduandos G1 - ANÁLISE POR RESSONÂNCIA MAGNÉTICA DA PERMANÊNCIA DE CÉLULAS DE MEDULA ÓSSEA NO VÍTREO APÓS LESÃO DO NERVO ÓPTICO

1,2-FIGUEIRÊDO, A.B.P; 1,2-MESENTIER-LOURO,L;1,2-ZAVERUCHA-DO-VALLE,C; 1,2-NASCIMENTO-DOS-SANTOS,G; 2-TEIXEIRA,C;2-TOVAR-MOLL,F;1,2-MENDEZ-OTERO,R;

1,2-SANTIAGO,M.F 1-Instituto de Biofísica Carlos Chagas Filho;2-Intituto Nacional de Ciência e Tecnologia

de Biologia Estrutural e Bioimagem,INBEB Em mamíferos adultos, lesões no nervo óptico provocam degeneração retrógrada das células ganglionares da retina e morte progressiva, especialmente por apoptose. Várias abordagens terapêuticas vem sendo estudadas para aumentar a sobrevida neuronal e regeneração axonal daqueles neurônios em diferentes modelos de lesão, incluindo a terapia celular. Nesse sentido, em trabalhos anteriores demonstramos que tanto as células mononucleares quanto as células mesenquimais derivadas da medula óssea geram neuroproteção e aumentam a extensão axonal após lesão no nervo óptico. No presente trabalho, procuramos, então, analisar o destino das células transplantadas in vivo por imagem através de ressonância magnética e ex vivo através de reações histoquímicas. Para isso, utilizamos ratos adultos da variedade Lister-hooded e as células derivadas da medula óssea foram marcadas com nanopartículas superparamagnéticas (SPIO - Feratrack) e injetadas no vítreo dos animais imediatamente após lesão no nervo óptico. Os animais foram então analisados por ressonância magnética 1 dia e 2, 14 e 18 semanas após o transplante. Demonstramos, então, que por ressonância magnética foi possível observar um sinal hipointenso derivado das células marcadas em todos os períodos analisados e não houve redução evidente de sinal ao longo do experimento. Além disso, foi possível identificar células marcadas com SPIO ex vivo 2 e 18 semanas após a injeção através da reação de azul da Prússia. Dezoito semanas após a injeção, observamos uma concentração de células marcadas no vítreo próximas à região do disco óptico. Isso sugere a permanência a longo prazo das células injetadas nos olhos dos animais hospedeiros, demonstrando a eficiência do transplante e levantando novas questões sobre o possível efeito terapêutico dessas células." CNPQ,FAPERJ,INBEB G2 - STRUCTURAL AND PHYSICOCHEMICAL STUDIES OF THE HEPATITIS C VIRUS CORE PROTEIN Braga, V.L.A.1; Casalinho, A. L. F.1; Albernaz, F. P. 1; Mendes-Silva, A.1; Bianconi, M. L.1,

Rangel, L. P.1; Peabody, D.2; Silva, J.L .1; Gomes, A.M.O.1; Souza, T.L.F.3 & Oliveira, A.C.1 1Programa de Biologia Estrutural/IBqM/UFRJ, Brasil. 2Department of

Molecular Genetics and Microbiology and Cancer Research and Treatment Center/UNMSM/USA. 3Faculdade de Farmácia/UFRJ/Brazil.

"Introduction: Hepatitis C virus (HCV) is the major cause of chronic liver disease. Cell culture systems for virus propagation are few and recent, so the core protein (HCVcp) has been described as a great target of studies. HCVcp is involved in the formation of the nucleocapsid, viral infection, and different cellular processes. Two different sizes of core protein, 179 and 124 amino acids, are involved with HCV assembly and pathogenesis. Small regions of the HCVcp, comprised of peptides 22-39, 50-67 and 85-102, were identified to be important for the nucleocapsid assembly. The structural characterization, and the elucidation of the mechanisms involved in lipid and nucleic acid interactions of HCVcp are new approaches to understand the HCV assembly process. Material and Methods: Our experiments were performed with the protein HCVcp124, obtained in an E. coli expression system, or with the peptides 22-39, 50-67, and 85-102, synthesized by Genscript, in the absence or in the presence of different membrane models (micelles) and non-specific nucleic acids. Structural and physicochemical aspects of the interaction of HCVcp with the RNA or with the viral envelope during HCV assembly were investigated by circular dichroism (CD), fluorescence spectroscopy and isothermal titration calorimetry (ITC). Results and Discussion: Although HCVcp is an intrinsically unstructured protein, denaturation studies by CD and fluorescence spectroscopy demonstrated the presence of few structural elements. Only the peptide 85-102 of HCVcp adopted an alpha-helix structure in n-octylglucopyranoside and SDS micelles with partial internalization of its tryptophan residues. HCVcp124 interacts with different DNAs and is able to form nucleocapsid-like particles (NLPs). The presence of all three peptides does not prevent the formation of these NLPs. ITC measurements showed that the peptide 50-67 was the only one able to interact with DNAs. Conclusions: Our data are relevant to better understand the assembly mechanisms and can contribute to the identification of new targets for inhibitors of HCV viral infection." FAPERJ, CAPES, CNPq, Pronex and INBEB. G3 - THE AXIS NALP3-ASC-CASPASE-1-IL-1BETA IS DECISIVE FOR THE PERITONITIS INDUCED BY BACTEROIDES FRAGILIS AND STERILE CECAL CONTENTS

1-DE ALMEIDA, B.J.; 1-CASTELPOGGI, J.; 2-LOBO, L.; 2-FERREIRA, E.O.; 2-DOMINGUES, R.M.P.; 3-ZAMBONI, D.; 2-BELLIO, M.; 1-SCHARFSTEIN, J.; 1-OLIVEIRA, A.C.

1- Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro; 2- Instituto de Microbiologia Paulo de Góes, UFRJ, Rio de Janeiro; 3 - Faculdade de Medicina de

Ribeirão Preto, USP, São Paulo. The cytokine IL-1β has pleiotropic effects in several cells and tissues and plays a crucial role in amplifying the host inflammatory response during sepsis and abscess development. Its secretion is dependent of inflammasome activation, a multiprotein cytoplasmic complex that activates caspase-1 which converts pro-IL-1β into its mature form. Bacteroides fragilis is a strictly anaerobic Gram-negative bacterium frequently



isolated from clinical infections, such as intra-abdominal abscesses and bacteremia. When intestinal integrity is disrupted, B. fragilis and colonic contents escape into the peritoneum, featuring a virulent bacteria-host interaction. The murine model of abscess induction consists of ip. inoculum of B. fragilis + sterile cecal contents (SCC). Our aim was to evaluate the inflammasome activation and IL-1 secretion induced by B. fragilis and/or SCC. Initially, we observed that SCC alone induces in vitro IL-1β production by BMDC and BMDM in a MyD88-dependent manner, whereas no effect was observed with B. fragilis alone, which was confirmed in in vivo assays. Next we used macrophages deficient in Nalp3, ASC and caspase-1, and a drastic reduction in the IL-1 production was observed. Consistently, in vivo analyses showed that caspase-1, ASC and IL-1R knockout mice were protected from decrease on body weight and exhibit reduced inflammatory abscesses, as compared to WT mice. Besides, WT mice present an intense cellular infiltration into the peritoneal cavity in response to B. fragilis + SCC, which was dramatically reduced in caspase-1-, ASC- and IL-1R-deficient mice. Taken together, our results strongly suggest that the development of abdominal peritonitis is critically dependent on inflammasome/IL-1β pathway by colonic contents. Based on our mice studies, we suggest that the development of such abscesses in susceptible patients might be controlled by procedures that restore the epithelium barrier, preventing the transposition of colonic contents to the peritoneal cavity, and/or by inhibitors of the inflammasome/IL-1β pathway. CNPq, FAPERJ, INBEB, PRONEX G4 - THE ROLE OF BOVINE LACTOFERRIN/HEPARAN SULPHATE INTERACTION ON HUMAN RHINOVIRUS 14 INFECTION

1 - DENANI, C.B.; 1 - SANTOS, R.A.; 2 - CARVALHO, C.A.M.; 2 - SILVA, J.; 2 - OLIVEIRA, A.C.; 2 - GOMES, A.M.O.; 1 - GONÇALVES, R.B.

1 - Departamento de Bioquímica, Universidade Federal do Estado do Rio de Janeiro; 2 - Instituto de Bioquímica Médica Leopoldo DeMeis, Universidade Federal do Rio de

Janeiro. Rhinovirus, the causative agent of the common cold, is responsible for enormous damages to the world economy and health. Lactoferrin (Lf), a glycoprotein present in mucosal secretions of mammals, is widely studied for its antiviral activity. The mechanisms of Lf antiviral activity already described include intracellular biochemical processes, interaction with cell surface and competition for virus receptors. Our aim is to study the interaction of lactoferrin with HeLa H1 cells and how it interferes with the HRV14 infection cycle, investigating the possible interaction of Lf with molecules present at the cell surface such as glycosaminoglycans and ICAM-1 receptors. We used confocal microscopy to investigate the interaction between HRV14 and HeLa-H1 cells. Virus infectivity and Lf ability to bind nonspecifically to other cellular surface molecules, like heparan sulfate, were also investigated by the use of fluorescent labeling of Lf and sulfation inhibition. Time-lapse microscopy experiments showed that bLf causes morphological changes to the cellular surface. Structures similar to transitional blebs are observed immediately after bLf addition. After 30 minutes, bLf is observed bound to the cell membrane, while after 60 minutes a perinuclear distribution of bLf could be observed. Cells treated with sodium chlorate for sulfation inhibition present less bLf binding as compared to control cells, suggesting a role for heparan sulfate during bLf binding to the cell. Our data show that bLf binds to HeLa H1 cells surface and is slowly internalized (60 min) moving to the perinuclear region of the cell. Sulfation inhibition suggests that the initial interaction of bLf is dependent on negative molecules, such as heparan sulfate. The bLf binding to glycosaminoglycans may be the key for an unspecific antiviral mechanism of bLf. The slow internalization of the molecule suggests that it may interfere with intermediate to late stages of the viral infection cycle.

FAPERJ, CNPq, FINEP, CAPES G5 - TRATAMENTO COM CÉLULAS-TRONCO MESENQUIMAIS DA MEDULA ÓSSEA EM UM MODELO IN VITRO DA DOENÇA DE ALZHEIMER 1-GODOY, MA; 2-SARAIVA L; 1-VASCONCELOS-DOS-SANTOS, A; 1-BEIRAL, HJV; 1-SINIS, LC;; 1-SILVA, LRP; 1-BRAGA, CV; 1-LEAL, RB; 1-SILVA, CAA; 1-VIEYRA, A; 2-DE FELICE, FG;

2-FERREIRA, ST; 1-MENDEZ-OTERO, R 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro 2-

Instituto de Bioquímica Medica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil

"A doença de Alzheimer é uma doença neurodegenerativa cuja incidência tende a aumentar com o envelhecimento da população mundial. Até o momento, não existem alternativas terapêuticas eficazes. Os oligômeros abeta são considerados as principais neurotoxinas para o desencadeamento dos efeitos deletérios nas sinapses e estresse oxidativo presentes na doença de Alzheimer. A terapia celular é uma perspectiva para o tratamento desta desordem neurodegenerativa. Na medula óssea existem células-tronco hematopoiéticas, que dão origem à linhagem hematopoiética e células-tronco mesenquimais (MSCs), que originam as células do tecido conectivo da medula óssea. A principal hipótese sobre o mecanismo de ação das células mesenquimais é sua ação parácrina, liberando fatores tróficos que poderiam atuar na estimulação de sobrevivência neuronal, neurogênese e modulação da inflamação. Nosso objetivo é avaliar o potencial neuroprotetor das células-tronco mesenquimais da medula óssea sobre os efeitos deletérios gerados pela exposição aos oligômeros abeta em neurônios hipocampais, assim como a interação dos oligômeros com as células mesenquimais. Para isso, foram estabelecidas culturas de MSCs provenientes de tíbias e fêmures de ratos Wistar adultos e após 3 passagens, foi realizada a cocultura por 24h dessas células com neurônios dissociados hipocampais, obtidos do hipocampo de embriões Wistar com 18 a 20 dias. Após esse período foi realizada a exposição aos oligômeros abeta por 6h a 24h (500 nM) nas coculturas ou somente nas MSCs. Os principais parâmetros avaliados serão: estresse oxidativo, integridade das sinapses, expressão de pTAU nos neurônios, assim como a viabilidade, proliferação e estresse oxidativo nas MSCs expostas aos oligômeros. Nós concluímos até o momento que os oligômeros são capazes de se ligar as mesenquimais, sem alterar sua viabilidade durante todo o período analisado (máximo de 72h de exposição). As células mesenquimais são resistentes ao estresse oxidativo gerado pelos oligômeros após 24h de exposição, diferente do observado nos neurônios. Além disso, uma análise inicial sugere que a cocultura com células tronco mesenquimais protege as culturas hipocampais contra o estresse oxidativo após a exposição por 6h aos oligômeros abeta. Assim, o tratamento com células tronco mesenquimais parece ser uma boa opção como abordagem terapêutica para os sintomas iniciais da doença de Alzheimer." CAPES, CNPq, INBEB, FAPERJ G6 - IN SILICO EVALUATION OF PHARMACODYNAMIC AND PHARMACOKINETIC PARAMETERS AND IN VITRO MUTAGENICITY ANALYSIS OF ANTI-PRION COMPOUNDS 1- MACHADO, C. S. C., 1- NATALIA C. FERREIRA, 1- PATRÍCIA N. FERNANDES, 1- WESLEY A. CONCEIÇÃO, 2- ALESSANDRA MASCARELLO, 2- LOUISE D. CHIARADIA-DELATORRE, 2-

ROSENDO A. YUNES, 2- RICARDO J. NUNES, 1- YRAIMA CORDEIRO 1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 2= Departamento de

Química, Universidade Federal de Santa Catarina.



"Introduction: Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders that affect humans and other animals. TSEs develop after the conversion of PrPC into a pathological isoform, PrPSc that can form aggregates, which are deposited in the central nervous system inducing neurodegeneration. Until now there is no treatment and they are invariably fatal. In an attempt to identify new anti-prion compounds, we realized a screening in neuroblastoma cells infected with scrapie (ScN2a) and we identified ~40 compounds effective in reducing the levels of PrPSc . Material and Methods: We analyzed in silico the possible interaction of PrPC with selected active compounds. We used molecular docking seeking the less energetic molecular complexes, considering electrostatic and geometric aspects of the molecules involved. We used the SWISSDOCK server to evaluate the interactions between proteins and small ligands. CHARMM force field was used to predict parameters like interaction energy and stoichiometry. Since these compounds are potential drug candidates, we performed in silico tests in order to predict the pharmacokinetic properties of these compounds. Moreover, the Salmonella typhimurium reverse mutation assay (Ames test) was done to validate the results from the in silico mutagenicity prediction. Results and Conclusions: The ChemSilico predictor showed three chalcones with potential for future drugs, due to the good results in the calculated pharmacokinetic parameters. It was predicted that these molecules possess good ability to passively cross membranes, and the blood brain barrier, are lipophilic and non-mutagenic. Preliminary results from the AMES test confirmed that they are non-mutagenic, in agreement with in silico predictions. Molecular docking showed that the compounds may bind directly to PrP globular domain (1AG2.pdb), preferentially interacting in the loop between helix 2 and β-sheets of mice PrPC. In summary, these chalcones possess good pharmacokinetic and pharmacodinamycs characteristics that warrant their use as anti-prion compounds." CNPq, FAPERJ, PIBIC G7 - YELLOW FEVER VIRUS-INDUCED MITOCHONDRIAL DYSFUNCTION: CHANGES IN MITOCHONDRIAL ENERGETIC METABOLISM AND APOPTOSIS INDUCTION

Daniel Sanches 1, Samir Pereira da Costa Campos 1, Claudia Monteiro da Rocha 1, Luciane Pinto Gaspar 2, Marcos da Silva Freire 2, Mariana Figueiredo Rodrigues 1, Jerson

Lima da Silva 1, Andre Marco de Oliveira Gomes 1, Andréa Cheble de Oliveira 1 1 Universidade Federal do Rio de Janeiro (UFRJ) (Instituto de Bioquímica Médica), 2

Fundação Oswaldo Cruz - Bio-Manguinhos (Instituto de Tecnologia em Imunobiológicos) "Yellow fever is a hemorrhagic disease of great relevance in Africa and South America, where it occurs as small outbreaks. It is caused by the yellow fever virus (YFV), a flavivirus such as the Dengue Virus, both being transmitted by mosquitos. It has been described that the cytopathological effect observed during YFV infection is related to apoptosis. During this process, the mitochondrial pathway has been reported to play an important role in viruses-induced apoptosis. Once the mitochondrial pathway is triggered, loss of mitochondrial membrane potential (Δym) occurs and caspases can be activated, ending in apoptotic process. In this study, we investigate the role of mitochondrial pathway of apoptosis in YFV infection and its consequence to mitochondrial energetic metabolism. We infected Vero cells with YFV using a MOI=1. We analyzed the cell viability by Live/Dead and LDH assay. Apoptosis was investigated through PhosphatidylSerine (PS) exposure and TUNEL, while the role of mitochondrial pathway was studied by the Δym through fluorescence microscopy. The role of the mitochondrial pathway was examined by using Bongkrekic acid, an adenine nucleotide

translocator (ANT) inhibitor. The mitochondrial energetic metabolism was studied by oxygraphy. Apoptosis was detected 72 hours post infection (h.p.i.) through TUNEL and PS exposure. Its dependence of caspases activation was observed using z-Vad-fmk, a pancaspase inhibitor. Also, we observed loss of Δym 72 h.p.i., indication of the mitochondrial pathway activation. Apoptosis seems to depend on ANT activity. Oxygraphy data show a significant increase of oligomycin-sensitive oxygen consumption at 72 h.p.i., which suggests an increase in oxygen consumption rate related to ATP synthesis. Our data suggest that the mitochondrial pathway is activated, contributing partially for the caspase-dependent cell death induced by YFV. Our data also demonstrate changes on mitochondrial energetic metabolism associated to virus infection. CNPq, CAPES, FAPERJ, FINEP, PRONEX, INBEB G8 - CARDIOPROTEÇÃO INDUZIDA POR FATORES HUMORAIS, LIBERADOS PELO PRECONDICIONAMENTO ISQUÊMICO EM CORAÇÕES ISOLADO DE RATOS.

1 - OLIVEIRA, F. D.; 1, 2 - BATISTA, G.; 1 - MACIEL, L.; 1- NASCIMENTO, J. H. M. 1 - Laboratório de Eletrofisiologia Cardíaca Antonio Paes de Carvalho / Instituto de

Biofísica Carlos Chagas Filho , Universidade Federal do Rio de Janeiro 2 - Faculdade de Farmárcia, Universidade Federal do Rio de Janeiro.

As doenças do sistema cardiovascular são a causa predominante de morte nos países industrializados. O pré-condicionamento isquêmico (PCI) é um mecanismo endógeno de cardioproteção contra lesões de isquemia e reperfusão. Assim, é importante elucidar os mecanismos do PCI para explorar sua possível utilização terapêutica. Objetivos: Avaliar a atividade cardioprotetora dos fatores humorais liberados no precondicionamento isquêmico. Método: Corações de ratos Wistar macho foram perfundidos com solução de Krebs-Henseleit em sistema de coração isolado com fluxo constante de 10 ml/min. Um balão de látex foi inserido no ventrículo esquerdo e conectado a um transdutor de pressão para registro da pressão intraventricular. Os grupos experimentais: Controle (n=5): submetidos a 30 min. de isquemia e 60 min. de reperfusão (I/R); PCI (n=5): submetidos a 3 ciclos de 5 min. de isquemia e 5min. de reperfusão, antes do I/R; RECEP (n=5): perfusão do efluente coletado durante o PCI, por 15 min. antes da I/R; Perfusão do efluente coletado, durante o PCI por 15 min., antes da I/R; Antagonista (DPCPX 20nM, Naloxone 10nM, Gliburide 10µM, 5HD 100µM) (n=5). Ao final da reperfusão, os corações foram seccionados e incubados com TTC (1%), para determinação planimétrica da área de infarto. Resultados: Os resultados mostraram um aumento da PDVE nos grupos PCI e RECEP, comparados ao grupo CTRL. este efeito foi atenuado nos grupos com antagonistas. Em relação a área de infarto, houve cardioproteção nos grupos RECEP e PCI. Nos grupos com DPCPX e Naloxone houve bloqueio da cardioproteção, enquanto nos grupos Gliburide e 5HD este efeito foi apenas atenuado. Conclusão: O efluente coletado do precondicionamento isquêmico é capaz de gerar cardioproteção e os antagonistas dos receptores de opióides, adenosina A1, mKATP e KATP foram capazes de atenuar ou reverter o efeito cardioprotetor do efluente, sugerindo um possível envolvimento dessas vias no preconcionamento isquêmico. FAPERJ, CNPq, CAPES G9 - POSSIBLE INVOLVEMENT OF INNATE IMMUNITY IN PRION PROPAGATION MECHANISM: AGGREGATION TRIGGERED BY NEUTROPHIL EXTRACELLULAR TRAPS (NETS) 1-DOSANJOS, D. M. ; 1-AZEVEDO, E. P. ;1- VIEIRA, T. C. R. G. ; 1-FOGUEL, D. ; 2-SARAIVA,

E. M. ; 3 - MORAES, M. C. ; 1-SANTOS, J. B ; 1-SILVA, J. L.



1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Instituto de Microbiologia, Universidade Federal do Rio de Janeiro; 3 - Instituto de Química,

Universidade Federal Fluminense "Prion diseases are fatal neurodegenerative protein-misfolding diseases related to a protein known as prion protein (PrP). The constitutive cellular isoform of PrP (PrPC), present in the cell surface, can be converted into its abnormal, β-sheet-rich isoform (PrPSc) that undergoes aggregation. PrPSc can be transmissible and infectious. Evidences suggest that adjuvant factors may play a role in the conversion process. Our group previously reported that DNA can convert PrPC into a β-sheet conformation leading to its aggregation. Innate immune system cells have been proven to play critical roles in the incunabular pathogenic events of prion diseases and may be involved in its progression. Following peripheral exposure, the replication of prions within the lymphoid tissue has been shown to be important for the efficient spread of the disease to the brain. Neutrophil extracellular traps (NETs) are large DNA networks decorated with histones and granule proteins that trap and kill pathogens, being extruded by activated neutrophils in response to several stimuli. NETs were reported of being released in lymphoid tissues. Furthermore, NETs were recently found in association with amyloid fibrils in tissues of patients with other protein-misfolding diseases and these fibrils triggered the release of NETs.1 Regarding such observations and considering the large quantity of DNA in NETs we asked if it could induce PrPC aggregation in vitro. Murine recombinant PrP23-231 was added into the supernatants from activated human neutrophils containing NETs. Aggregation was measured by light scattering and electron microscopy. The results show NETs dose-dependently triggering an instantaneous PrPC aggregation. It gradually declined remaining detectable after 1 hour. The aggregates showed amorphous morphology and absence of fibrils. NETs previous treatment with DNase I inhibited PrPC aggregation, pointing out the importance of the DNA integrity for its effect. Our results show that NETs are able to trigger stable PrPC aggregation and that its DNA networks are crucial for this process. Differences in NETs protein composition among different donors seems to be related to distinct aggregation features. PrP-coated magnetic beads were used to “fish out” PrP-ligands from a pooled sample containing NETs from 7 human donators. MALDI-TOF mass spectrum of proteins as potential ligands isolated from NETs shows a mass ion at around 13.5 kDa. Our data suggest that NETs are an intriguing factor that must be appraised in the studies concerning prion propagation mechanisms." INBEB, FAPERJ, CNPq, CAPES G10 - ANOTAÇÃO E MODELAGEM DE ENDOGLUCANASE DE SERRATIA SP ORIUNDA DO METAGENOMA DO CARAMUJO AFRICANO ACHATINA FULICA.

1,2-LARA, FLO ; 2- SOARES, RO ; 1-GOMES, DEB ; 1-DA SILVA, ML 1 - Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de

Metrologia, Qualidade e Tecnologia ; 2- Universidade Federal do Rio de Janeiro "A biomassa lignocelulósica tornou-se um dos principais alvos da produção de combustíveis de segunda geração devido ao seu potencial como matéria-prima para fabricação de bioetanol. Por estar amplamente disponível no Brasil, principalmente através do resíduo industrial da fermentação de etanol, estudar os organismos e / ou enzimas com potencial para degradar biomassa lignocelulósica e extrair fermentáveis de polissacarídeos de cadeia longa torna-se um alvo em potencial para tal fim. Neste trabalho, analisamos com técnicas de bioinformática e modelagem molecular uma das enzimas oriundas do metagenoma do suco gástrico do caramujo Achatina fulica. Utilizamos diferentes programas e bancos de dados para anotação da mesma.

Através da utilização do BlastP (não redundante), KEGG, COG. supFam, CDD, UniProt, MetaCyc e dbCAN identificamos a enzima em questão como uma endo-1,4-D-glucanase, membro da família 8 glicosil hidrolases, mais especificamente a Subunidade Z da Celulose Sintase de Serratia sp. Além disso, utilizamos ferramentas para predição de estrutura secundária (PSIPRED), presença de peptídeo sinal (SignalP), localização celular (PSORT) e diferentes metodologias de alinhamento (PROMALS e ClustalW). Na técnica de Modelagem Comparativa, utilizamos a Subunidade Z de celulose Sintase de E. Coli K-12 (PDBID 3QXQ) como molde, tendo essa enzima 50% de identidade, 66% de similaridade e 100% de cobertura com o alvo . Foram construídos 100 modelos candidatos e para avaliar esses modelos analisamos os Gráficos de Ramachandran (PROCHECK) , valores de RMSD entre molde e modelos (função super do PyMOL), bem como os escores GA341 e DOPEscore (MODELLER). O modelo escolhido para a Celulose Sintase obteve 89.8% aminoácidos em zonas permitidas do gráfico de ramachandran, 0.5% em zonas proibidas, DOPEscore de -22368.76953 e RMSD relativo ao molde de 0.354 A. À partir deste modelo, poderemos realizar estudos de docking e dinâmica molecular, de modo à entender as interações que ocorrem no sítio ativo dessa enzima." CNPq G11 - IN VIVO STUDIES OF THE HYPERGLYCEMIA INFLUENCE ON THE GLYCOPHENOTYPE AND THE PROGRESSION OF COLON CARCINOME CELLS 1 - Loponte, H. F.; 1 - Vasconcelos-dos-Santos, A.; 1 - Mantuano, N. R.; 1 - Oliveira, I.A.;

2 - Moll, F.; 1 - Dias, W.B.; 1 - Todeschini, A.R. 1 - IBCCF, 2 - CENABIO. Rio de Janeiro, Brazil.

Cancer cells depend on altered metabolism and nutrient uptake to generate and keep the malignant phenotype. Recent work suggests that the metabolite availability to the hexosamine and Golgi O-glycosylation pathways exerts control over cell signaling, gene expression and cell migration (Alisson-Silva etal., Plos One, 8: e60471, 2013). Indeed, high glucose concentration increases O-glycosylation of FN (onFN), which generates the onFN, and modulates tumorigenesis. Herein we implicate hyperglycemia in facilitating cancer progression and increasing onFN expression in vivo. Cells from colon carcinoma of C57BL/6 mice (MC-38 cell line) were subcutaneously inoculated in streptozotocin (STZ)-treated or untreated C57BL/6 mice. Subcutaneous tumors from STZ-treated group were larger and more vascularized when compared with vehicle group. Tumors from STZ-treated mice presented high expression of α2-6-Neu5Ac and α1-3-/ α1-6-Fucose (Fuc) containing glycoconjugates as well as increased expression of onFN. These data suggest that metabolite availability modulates cell surface glycosylation contributing to tumor formation and progression. CNPq, FAPERJ, CAPES G 12 - Prion Protein Coated Magnetic Beads: A New Approach For Ligands Screening 1,2 Santos, J.B.;1,3 de Moraes, M.C.;1- dos Anjos, D.M.; 1- da Silva, J.L. 1-Instituto de Bioquímica Médica, IBqM-UFRJ, RJ; 2- Faculdade de Medicina, Unirio, RJ; 3-Departamento de Química Orgânica, UFF, Niterói/RJ Prion diseases are characterized by prion protein (PrP) aggregation and eventual neurodegeneration. The conversion of the constitutive cellular isoform of PrP (PrPC) into an abnormal one - scrapie PrP (PrPSc) - is the most important step leading to the development of these diseases [1]. The mechanism of conversion is still unclear. Therefore, the development of analytical tools for the identification of PrP-ligands, which can act as cofactors or inhibitors in the conversion mechanism of PrP, has become an interesting task. The main purpose of this work is the development of PrP-ligands screening assay based on bioaffinity chromatography, to rapidly identify ligands which may contribute to understand and completely elucidate this conversion mechanism, which probably requires a cellular cofactor. This work describes the PrPC



covalent immobilization onto the surface of silica-based magnetic beads (Bioclone) to isolate ligands from complex mixtures. PrP was labeled with fluorescein isothiocyanate prior to immobilization so as to evaluate the success of the immobilization procedure by fluorescence microscopy of the PrPC coated-beads. To validate the purposed bioaffinity assay, the PrPC-coated magnetic beads were used to “fish out” ligands from an equimolar mixture of thiamine (Syrian hamster PrP-ligand), quinacrine (human PrP-ligand) and caffeine (negative control). An UHPLC-MS/MS method was developed and validated to quantify the amount of isolated ligands by the bioaffinity assay. The selected ratio PrP:beads amount was 1:3 (mg), which yielded around 60% of PrP immobilization. The binding of labeled PrPC was observed by fluorescence microscopy and showed a substantial coating. In the bioaffinity assays, the PrP-coated magnetic beads isolated 40% of the quinacrine from the equimolar (300nM) compounds mixture. The next step is applying this method for screening new compounds, cellular products and extracts. The developed method represents an useful tool to investigate the affinity and interaction of PrP with cellular components and complex matrices. FAPERJ, CNPq G13 - DESENVOLVIMENTO DE ESTRATÉGIAS PARA O ESTUDO DE PROTEÍNAS MAL ENOVELADAS POR RESSONÂNCIA MAGNÉTICA NUCLEAR 1-SANTANA, J.S.; 1-SOUSA, A.R.N.; 1- SANT'ANNA, R.O.; 1- FOGUEL, D.; 1- FREITAS, M.S.

1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro,

Brasil. "Doenças amiloidogênicas, em sua maioria, estão relacionadas com o mal enovelamento de determinadas proteínas. A doença de Alzheimer, doença de Parkinson, doença de Creutzfeldt-Jakob, doença do Neurônio Motor, Polineuropatia Amiloidótica Familiar e a doença de Huntingnton são exemplos de amilóidoses e são de grande impacto na saúde pública. Os estudos com as proteínas envolvidas nas doenças supracitadas tem possibilitado um ganho na compreensão quanto aos mecanismos envolvidos no mal enovelamento proteico. A importância de se estudar a má conformação através da caracterização da estrutura tridimensional, possibilita observar o que acarreta o mal enovelamento, favorecendo o desenvolvimento de farmacos, terapias ou até mesmo estimar possíveis grupos propensos a desenvolver tais doenças. O objetivo do nosso trabalho tem sido monitorar padrões estruturais relacionados a formação de pequenos e grandes oligômeros que são causados pelo enovelamento incorreto, visando estabelecer um protocolo de experimentos In cell onde possamos melhorar o compromisso entre o tempo de aquisição e resolução dos espectros de Ressonância Magnética Nuclear. Com o intuito de estudar algumas das doenças supracitadas, foram usadas como modelos as proteínas: Transtirretina , Transtirretina monomérica, Prion e α-Sinucleína. Todas expressas In cell com o objetivo de propiciar um ambiente celular na tentativa de analisar possíveis mudanças em relação aos experimentos realizados In Vitro com a proteína purificada. Foi utilizada deuteração da amostra com uso de sequências de pulso que minimizam os efeitos inseridos pelo acoplamento dipolar devido a viscosidade do ambiente intracelular que leva a uma redução do T2 que consequentemente, leva ao alargamento de linha. Levando em consideração os aspectos apresentados, esse trabalho, visa estabelecer um protocolo experimental, In cell, que reduzirá o tempo de preparo da amostra, otimizará o tempo de análise dos dados, podendo trazer novas informações sobre a interação da proteína com o micro ambiente intracelular." CNPq, FAPERJ, AvH, CAPES, DAAD

G14 - EFEITOS DE CICLOSPORINA A E CICLOHEXIMIDA SOBRE O CONDIONAMENTO E EXTINÇÃO DE MEMÓRIAS AVERSIVAS

1- JUNQUEIRA, S. L.; 2- MOULIN, T. C.; 3- ALMEIDA-CORRÊA, S. 4- AMARAL, O. B. 1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Instituto

de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 3-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 4-Instituto de Bioquímica

Médica, Universidade Federal do Rio de Janeiro "Memórias aversivas não são estáticas, podendo ser labilizadas ou modificada após sua consolidação a partir de processos como reconsolidação e extinção. Dentre diversas moléculas envolvidas, trabalhos anteriores descrevem a proteína fosfatase calcineurina (CaN) como uma enzima crucial no processo da extinção do condicionamento aversivo contextual, contudo seu exato papel sobre este tipo de memória ainda é contraditório. Neste estudo, investigamos os efeitos do bloqueio de CaN pelo fármaco ciclosporina A (CsA) e os comparamos com a influência da cicloheximida (CHX), um inibidor de síntese proteica, mecanismo esse necessário para a aquisição da memória. Para isso, condicionamos aversivamente camundongos Swiss machos a um contexto e em seguida, os reexpomos ao mesmo local para a extinção desta memória. Em diferentes grupos aplicamos os fármacos CsA e CHX 1h pré-extinção ou pré-treino por injeção intracerebroventricular (ICV) freehand. A medida utilizada para calcular o medo foi a contagem do tempo de congelamento diante do contexto aversivo (freezing) durante as sessões de extinção e teste." FAPERJ, CNPq. G15 - ANÁLISE TRIDIMENSIONAL DO SISTEMA NERVOSO CENTRAL POR MICROSCOPIA ÓTICA APÓS CLARIFICAÇÃO TECIDUAL. 1- TEIXEIRA-PINHEIRO, L.C. 1- NASCIMENTO-DOS-SANTOS, G. ; 1- MENDEZ-OTERO, R.; 1-

SANTIAGO, M.F. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro

"O tecido nervoso possui camadas densas de lipídios que o fazem ser pouco permeável à luz e a anticorpos usados em imunohistoquimica, o que limita a visualização por microscopia ótica. Sendo assim, faz-se necessária a secção mecânica do tecido para a visualização de áreas que de outra forma não seriam acessíveis. Novas técnicas de clarificação tecidual, como CLARITY e 3DISCO, vem sendo desenvolvidas com o objetivo de permitir a visualização de camadas mais internas de tecidos intactos. Neste trabalho propomos a comparação de dois protocolos distintos de clarificação tecidual em diferentes tecidos do SNC com o objetivo de aplica-las e adapta-las aos principais modelos de estudo utilizados por nosso grupo (retina e nervo ótico). Camundongos foram perfundidos com PBS seguido de uma solução de 4% de paraformoldeido, 4% de acrilamida, 0.05% de bis-acrilamida e 0.25% de iniciador VA-044 em PBS (solucao de hidrogel). Ratos Lister foram divididos em 3 grupos, o grupo 1 foi perfundido em solução de 4% de paraformoldeido e pós fixados em solução de hidrogel, o grupo 2 foi perfundido em solução de hidrogel e o grupo 3 perfundido em solução de hidrogel sem bis-acrilamida. Os cérebros foram dissecados e armazenados na solução de hidrogel correspondente a cada grupo e colocados em 37°C por 3 horas para a polimerização da solução. O tecido em solução polimerizada foi cortado em fatias de 1, 2 e 3mm ou mantido intacto. O cérebros e as fatias foram então colocados em uma solução de acido bórico a 200mM com 4% de dodecil sulfato de sódio (solução de clareamento) a 37°C ou temperatura ambiente ate estarem visivelmente transparentes. As amostras foram então lavadas em PBS, marcadas por imunohistoquímica e colocadas em 80% de glicerol para serem visualizadas por microscopia confocal." CAPES, FAPERJ, INBEB, CNPq.



G16 - RATIONAL DRUG DESIGN FOR FASCIOLA HEPATICA CATHEPSIN L3

1,2-HERNANDEZ, L.A. ; 3- VALIENTE, P.A. ; 4- GOMES, D.E.B. ; 2-PASCUTTI, P.G. 1- Centro Nacional de Sanidad Agropecuaria; 2- Universidade Federal do Rio de Janeiro

; 3- Universidad de La Habana ; 4- Instituto Nacional de Metrologia, Qualidade e Tecnologia

"Cathepsin L3 of Fasciola hepatica (FhCL3) is a cystein protease considered as an attractive target for the design of fasciolicidal drugs, but its crystallographic structure is still unknown. In order to search for potential inhibitors of FhCL3, we calculated a homology model of its 3D structure based on the primary sequence of this protease using the 3D structure of F. hepatica cathepsin L1 (PDB:2O6X) as template. Our model suggests that the binding site of FhCL3 has distinctive structural features compared to those of both human and bovine cathepsins L which, in turn, indicates the feasibility of designing selective inhibitors of FhCL3. A docking study was then conducted based on the FhCL3 model using. MYBRIDGE compound database against FhCL3 and both bovin and human cathepsin L, and five compounds selective to FhCL3 and with high energy scores were found. Subsequently, molecular dynamics (MD) simulations were performed to study the stability and structural properties of the free enzyme and the five ligand-enzyme complexes. Besides the five ligand-enzyme complexes, the model of FhCL3 in complex with nitrile, a well-known inhibitor of the cystein protease family, was included as a positive control. MD simulations were used as well to refine the binding modes of the ligands in the enzyme pocket. Hydrogen bonds present in the ligand-enzyme complexes were analyzed, as well as contact residues. Finally, MM-GBSA binding free energy calculations were performed based on the MD trajectories generated for each complex. These calculations suggest that two out of the five compounds identified through virtual screening have favorable binding free energy values comparable to that of nitrile, the positive control." CAPES G17 - ESTUDO DOS MECANISMOS DE DEFICIT COGNITIVO ASS I EN E E I EN : P PE IN SIS I

1- D'AVILA, J.C.; 1-SIQUEIRA, L.D.; 3- AZEVEDO, E.P.; 1- ARAÚJO, J.P.; 3- FOGUEL, D.; 2- BOZZA, F.A.

1- Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz - FIOCRUZ; 2- Instituto de Pesquisa Clínica Evandro Chagas - FIOCRUZ; 3- Instituto de Bioquímica Médica,

Universidade Federal do Rio de Janeiro en elhecimento o ulacional é um fen meno global. en elhecimento é o maior fator de risco ara doen as neurodegenerati as, ue t m como caracter stica comum a resen a de neuroinflama o caracteri ada ela ati a o da microglia, as células imunol gicas residentes do sistema ner oso central. urante o rocesso de en elhecimento, a efic cia das fun es da microglia se deteriora e esta disfun o microglial ode estar relacionada aos danos cogniti os associados ao en elhecimento. s mecanismos de les o res ons eis ela disfun o microglial associada ao en elhecimento ainda n o s o bem com reendidos. ossa hi tese é de ue a disfun o microglial associada ao en elhecimento en ol e déficits em ias de degrada o fagocitose e autofagia e de gera o de es écies reati as de o ig nio P o idases e mitoc ndrias defeituosas ue odem determinar o ac mulo de prote nas neurot icas e estresse o idati o. ste trabalho tem como ob eti o in estigar os mecanismos da neuroinflama o e dano tecidual em um modelo de en elhecimento com re u o cogniti o, com nfase no erfil de ati a o microglial e nos mecanismos de les o tecidual. disfun o microglial foi indu ida com a inflama o sist mica cr nica

de bai a intensidade, rodu ida atra és de in e es intra eritonais semanais de bai as doses de li o olissacarideo. ados reliminares demostram ue h ati a o microglial mais ronunciada em idosos, tanto nai e uanto desafiados com P cronicamente, em com ara o com os gru os o ens. s testes de com ortamento mostraram ue o desafio inflamat rio cr nico de bai a intensidade afeta o desem enho cogniti o dos animais idosos e n o dos animais o ens frente ao desafio com P cr nico, re elando assim, uma maior susce tibilidade dos idosos inflama o sist mica cr nica. s dados obtidos neste estudo oder o contribuir ara o desen ol imento de tera ias mais efica es ara doen as neurodegenerati as associadas ao en elhecimento. FAPERJ, CNPq, CAPES G18 - REGULATION OF GLUTAMATE AND D-SERINE LEVELS IN MICE CEREBRAL CORTEX

1- BAPTISTA, M.; 1- POLETO, A.; 1- MADEIRA, C.; 1- PANIZZUTTI, R. 1- Instituto de Ciências Biomédicas, UFRJ

D-serine is the main co-agonist for the glutamate activation of the N-methyl D-aspartate receptor (NMDAr), an ionotropic receptor important for memory and neuroplasticity. In vitro studies using neuron and astroglial cultures have shown that activation of glutamate receptors can stimulate the release of D-serine. Even though the mechanisms that regulates the release of D-serine in vivo has not been established. Also, it is unknown whether D-serine can regulate the extracellular levels of glutamate in vivo. The goal of this project is to study the effect of glutamate receptors activation on the extracellular levels of D-serine and glutamate in the mice cerebral cortex. To do so, we performed microdialysis in freely moving mice and used high performance liquid chromatography to analyze the extracellular levels of amino acids. Preliminary data show that increasing concentrations of D-serine in the perfusate led to higher concentrations of glutamate in the dialysate. On the other hand, increasing concentrations of glutamate in the perfusate did not affect the concentrations of D-serine in the dialisate. In vivo brain microdialysis provides a proper model to analyze the interaction of these neuroactive amino acids and how they modulate each other’s release. Further studies using specific agonists or antagonists of glutamate receptors are needed to clarify the mechanism involved in such interactions. FAPERJ G19 - USO DE UMA RESINA FUNCIONALIZADA PARA PURIFICAÇÃO DE FIBRAS AMILÓIDES

MIRIAN KELLEY1, DANIELA GAMA1, DEBORA FOGUEL1, FERNANDO L. PALHANO1 [1] Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro, Rio de

Janeiro, Brasil Fibras amilóides são responsáveis por uma série de doenças degenerativas, como Parkinson e Alzheimer, que atingem cada vez mais pessoas devido ao envelhecimento da população mundial. O tratamento dessas doenças, quando existe, é pouco eficaz. Acredita-se que isso se deve, em parte, ao diagnóstico tardio, sendo de grande valia o desenvolvimento de métodos de diagnóstico precoce. Uma técnica com potencial aplicação no diagnóstico precoce de doenças amilóides é a cromatografia de afinidade, sendo interessante a associação da molécula de penta tiofeno à resina de agarose, pois esta molécula tem grande afinidade por fibras amilóides. Objetivamos então produzir uma resina funcionalizada e um protocolo eficaz que sejam capazes de detectar fibras amil ides com alta sensibilidade e es ecificidade. Para tal, usamos fibras de β1-40 e α-sinucleína produzidas in vitro, associadas respectivamente às doenças de Alzheimer e de Parkinson. Como controle usamos albumina solúvel. Para detecção das amostras usamos a técnica de Dot-Blot. bser amos ue as fibras de β1-40 se ligam tão



fortemente à resina com penta tiofeno que apenas ácido fórmico 96% foi capaz de deslocar um pequeno percentual dessa fibra da coluna, ficando a maior parte desta resa na mesma. J a α-sinucleína, demonstrou ter se deslocado da resina com eluição em 8M de uréia pH 2,2+2% SDS. Por outro lado, a albumina eluiu facilmente da resina com solução de lavagem (PBS+1% Triton X-100+0,5M NaCl). Nossas perspectivas são encontrar um sol ente eficiente ra elui o da fibra de β1-40, avaliar se a es ecificidade ara α-sinucleína ocorre apenas com fibras ou também com monômeros e fazer testes usando fibras produzidas in vivo usando como modelo leveduras com ou sem agregados amiloides. FAPERJ, CNPq, CAPES. G20 - DOES RESVERATROL PREVENTS P53 AGGREGATION? 1,2- COSTA, D.C.F.; 1,2- CAMPOS, N.P.C.; 2,3- ZANPHORLIN, L.; 2,3- RAMOS, C.H.I.; 1,2-

SILVA, J.L. 1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de

Janeiro, Brazil; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil; 3- Instituto de Química, Universidade Estadual de

Campinas, São Paulo, Brazil. p53 has an essential role in preventing cancer development by inducing cell cycle arrest and/or apoptosis in response to cellular stress. Mutations in the p53 gene are described in over 50% of all human cancers. Besides the mutations, cellular aggregation of p53 can also inactivate the protein, leading to malignancy. Resveratrol, a natural polyphenol found in grapes and red wine, is able to induce p53-dependent cell death in a variety of cell lines. Although several indirect mechanisms of p53 activation by resveratrol have been proposed, there is no evidence that this bioactive compound can interact with p53. Thus, we investigated a possible interaction between resveratrol and the p53 core domain (p53C). In addition, we tested the potential of resveratrol in preventing wild-type and mutated p53 aggregation in vitro and in tumor cells. Experiments were performed by using fluorescence spectroscopy and fluorescence microscopy techniques. Our data suggest that an interaction between resveratrol and the wild-type p53C does occur. We also found that resveratrol has the ability to inhibit the wild-type p53 core domain as well as the R248Q p53 mutant to undergo in vitro aggregation. Additionally, resveratrol (50 and 100 µM) induces the formation of nuclear p53 aggregates in MDA-MB-231 human breast cancer cells. Our study provides evidence that resveratrol can directly modulate p53 and may pave the way for a better understanding of the mechanisms involved in p53 protein aggregation as a therapeutic strategy for cancer treatment. FAPERJ, CNPq, INCT/INBEB G21 - DISSECTION OF PRION PROTEIN AND LIPID INTERACTION

1,2,3-Santos, R.F.D; 1,2,3-Vieira, 1,2,3-TCRG; Silva, JL. 1-Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, 2-Instituto de

Bioquímica Médica Leopoldo De Meis, 3- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro,

21941-902, Brazil. Introduction: Transmissible spongiform encephalopathies are a group of fatal diseases, which affect mammals, caused by an abnormal isoform of the prion protein (PrP). Conversion of cellular PrP (PrPC) into the pathological conformer, PrPSc, involves contact between both isoforms and probably requires a cellular factor. Recombinant PrP can be converted to an abnormal form via seeded polymerization in vitro techniques in the presence of lipids. Objectives: The importance of lipid molecules for conversion has been revealed, but little is known about the structural features implicit

in this interaction. A detailed understanding of this interaction may provide new insights into toxic mechanisms associated with this disease. Material and Methods: In the present work, we used Rayleigh and Dynamic Light Scattering, and fluorescence measurements in order to provide information on the chemical and physical properties of the murine recombinant PrP (rPrP 23-231) interaction with Phosphatidylethanolamine (PE) and Phosfatidic Acid (PA) vesicles. Results and iscussion: We found that increasing concentrations of hos holi ids’ esicles raised rPrP light scattering, forming large aggregates (>1 μm). The tryptophan fluorescence emission intensity was increased and blue-shifted, showing change of the environment/properties of the protein tryptophan groups. The observed effect of phospholipids on the tryptophan fluorescence could arise from the lipid-induced conformational change of PrP, or membrane protein insertion. Conclusions: Taken together, our results indicate that PE and PA induce PrP oligomerization followed by formation of large aggregates. The conformational changes observed could play a critical role in nucleating the formation of toxic species. CNPq, FAPERJ G22 - UNRAVELING THE FUNCTION OF THIOREDOXIN-INTERACTING PROTEIN (TXNIP) THROUGH STRUCTURAL STUDIES

Aguiar, R. P.; Amorim, G. C.; Valente, A. P.; Almeida, F. C. L. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

"Thioredoxin-interacting protein (TXNIP), also known as vitamin D up-regulated protein, is strongly up-regulated by vitamin D. TXNIP belongs to the α-arrestin protein family and functions regulating oxidative stress response and glucose uptake. TXNIP binds directly to thioredoxin (hTrx) controlling all Trx-dependent regulatory processes, such as apoptosis and response to oxidative stress. It modulates inflammatory response, cardiac function, cell signaling and apoptosis. TXNIP plays a crucial role in several pathological conditions such as cancer, diabetes and cardiovascular diseases.Little structural information is available for this class of proteins. Our goals are to solve the structure of TXNIP domains and understand details of the interaction with hTrx. We cloned and expressed the N-terminal (1-149, D1) and C-terminal domain (163-301, D2) using the native sequence and mutating all cysteines by serine, with the exception of Cys63 for D1 and Cys247 for D2. We used standard methods to isotope label the cysteine-mutated domain (15N and 15N/13C). D1 and D2 have been expressed in E. coli and purified. Nuclear magnetic resonance triple resonance experiments were collected. The software NMRPipe and CCPN-NMR were used for processing and assignment. We successfully expressed native and cys-mutated D1 and cys-mutated D2. D1 showed low-affinity interaction with hTrx. We almost fully assigned the resonances of D1 and map its interaction with hTrx. We constructed a NMR-derived model of the complex D1-Trx1. Contrary to D1, D2 showed strong interaction with hTrx.These data is crucial for understanding the mechanism of interaction with hTrx. We showed that D1 binds to hTrx through Cys37, possibly regulating the interaction, which occurs primarily thought D2, the hTrx-binding domain." CNPq, FAPERJ G23 - ANOTAÇÃO E MODELAGEM ESTRUTURAL DA ENDO-1,4-D-GLUCANASE DE STENOTROPHOMONAS SP. PROVENIENTE DO METAGENOMA DO CARAMUJO ACHATINA FULICA.

1,2-R A PIRES, 2-M L da SILVA 1- Universidade Federal do Rio de Janeiro, 2- Instituto Nacional de Metrologia,

Qualidade e Tecnologia – INMETRO



O Brasil é referência mundial na produção de etanol, porém o etanol produzido a partir de biomassa (celulósico) ainda não é viável economicamente. Devido a suas vantagens, como a redução do uso de petróleo, minimização da poluição ambiental, e a convivência com a cultura de alimentos sem concorrência, pesquisas nesse setor tem crescido consideravelmente, representando desta forma uma solução próspera para os problemas atuais do uso de combustíveis fósseis. Desta forma, é necessário desenvolver o conhecimento acerca da hidrólise enzimática com uso de enzimas para que esta tecnologia sustentável se torne viável e com valores interessantes para a indústria. Este estudo analisou sequências obtidas pelos métodos de pirosequênciamento da microbiota do suco gástrico do caramujo africano Achatina fulica para a triagem de enzimas potencialmente envolvidas na degradação de celulose e sua modelagem estrutural através da técnica de modelagem comparativa. Inicialmente, das enzimas obtidas no metagenoma, optou-se por analisar estruturalmente a endo-1,4-D-glucanase de Stenotrophomonas sp.. Essa proteína teve seu modelo construído com base na estrutura da endoglucanase de Escherichia coli k-12 (PDBCod 3QXQ). A escolha pelo melhor modelo dentre os 100 candidatos construídos pelo MODELLER foi feita utilizando os critérios de RMSD entre molde e modelo e Gráfico de Ramachandran. Obteve-se um modelo tridimensional de ótima qualidade para a enzima endo-1,4-D-glucanase de Stenotrophomonas sp., com RMSD 0,182 Å entre molde e modelo, Gráfico de Ramachandran com 95,8% dos aminoácidos nas regiões favoráveis (Vermelha) 3,6% nas regiões permitidas (Amarela) 0,6% nas regiões generosamente permitidas (Bege) e 0.0% nas regiões proibidas (Branca). Com o modelo gerado em mãos, a próxima etapa do trabalho consistirá em desenvolver estudos de Dinâmica Molecular para melhor elucidar o potencial catalítico na degradação de celulose. CNPq G24 - CALORIMETRIC AND STRUCTURAL ANALYSIS OF THE INTERACTION OF INHIBITOR OF APOPTOSIS PROTEIN XIAP WITH SMAC MIMETIC COMPOUNDS

1- SANTOS, R.B.; 2- OLIVEIRA, A.C.; 1- SOUZA, T.L.F. 1- Faculdade de Farmácia, UFRJ, Rio de Janeiro, Brazil. 2- Programa de Biologia

Estrutural, IBqM, UFRJ, Rio de Janeiro, Brazil. Apoptosis resistance, in cancer, can be generated by high expression of inhibitor of apoptosis proteins (IAPs) family, which is responsible by the inhibition of initiator and effector caspases. Smac/DIABLO is an endogenous inhibitor of XIAP due to its direct interaction on the XIAP-BIR3 domain. Smac mimetics have been considered potential drug candidates, owing to its capacity to bind on XIAP-BIR3 and to sensitize apoptosis in cancer cell. The goal of this work is better understand the effects of the binding of Smac mimetics on the structure and stability of the XIAP-BIR3 and to add useful information to drug design. Smac mimetic compounds with structural similarities, but with different activities, were selected to us investigate the individual thermodynamic contributions of functional groups. We used circular dichroism (CD), fluorescence spectroscopy and isothermal titration calorimetric (ITC) to perform the structural and thermodynamic analyses. Denaturation with guanidine hydrochloride (GdnHCl) was used to estimate the domain stability. Analysis of tryptophan fluorescence spectra showed that this domain has a high stability and that the chemical denaturation occurs with two transitions. The domain underwent a different stabilization in the presence of the different compounds and the denaturation, differently, occurred with a single transition. Additionally, the Smac mimetics promote a significant change in the CD spectra of free XIAP-BIR3 domain, indicating changes in the secondary structure content. Results obtained by ITC revealed that, besides have different affinities, the enthalpy-entropy contributions of each compounds are significantly different. The significant changes in the structure and

stability of XIAP-BIR3 promoted by the interaction with Smac mimetics, and that contribute to the difference in the thermodynamic binding parameters, may be a relevant factor to be considered in affinity optimization of anti-IAP drug candidates. INBEB, FAPERJ, CNPq, CAPES. G25 - ANÁLISE DOS EFEITOS ESTRUTURAIS DE ÍONS COBRE E ÁCIDOS NUCLEICOS NA PROTEÍNA PRÍON

1- CASALI, S.; 1- MACEDO B.; 1- GOMES, M.P.; 1- CORDEIRO, Y. 1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro

Com o aumento da expectativa de vida média em alguns países, crescem também as preocupações quanto às doenças neurodegenerativas. Destacam-se neste grupo as encefalopatias espongiformes transmissíveis (EETs), cujo agente etiológico é a proteína príon (PrP). Sua forma celular (PrPC) sofre uma conversão estrutural, levando à formação da proteína prion scrapie (PrPSc), responsável pela agregação e a propagação de espécies tóxicas no sistema nervoso central, onde é majoritariamente expressa. A PrP é considerada promíscua, graças à descrição de diversos ligantes para ela, dentre os quais encontram-se o íon cobre e os ácidos nucléicos, sendo o Cu2+ o ligante mais descrito na literatura. Esses ligantes poderiam estar envolvidos tanto na patogênese, facilitando o processo de conversão, quanto na função da PrP nos mamíferos. É importante destacar que a interação da PrP com esses dois ligantes poderia acontecer ao mesmo tempo no ambiente celular. Nosso grupo procura entender os efeitos dos íons cobre na agregação e na estrutura da PrP selvagem de camundongo (PrPWT), obtida por expressão heteróloga em E. coli, levando em consideração também o efeito de moléculas de DNA no sistema. Este projeto constitui uma continuação de um conjunto de dados desenvolvido por nosso grupo, o qual inclui também peptídeos da PrP. Observamos anteriormente que cobre e pequenas moléculas de DNA (D67 e D44) apresentam efeito sinérgico quanto à indução da agregação e alteração das estruturas da PrP, sendo este mais pronunciado para os DNAs. Atualmente, estamos refinando esses dados e adicionando outros utilizando não mais domínios da PrP isolados, mas a PrP recombinante inteira, cujos resultados ainda são preliminares, mas incluem dicroísmo circular, espalhamento de luz e fluorescência, além de calorimetria e microscopia eletrônica. Esperamos, assim, trazer mais informações a respeito da interação dual da PrP com Cu2+ e ácidos nucleicos, que podem auxiliar a compreender a patogênese das EETs. INBEB, CNPq, FAPERJ G26 - INNATE SUPRAMOLECULAR INTERACTIONS IN BIOGENIC NANOSILVER

BALLOTTIN, D.1; 1- FULAZ, S.1; TASIC, L. 1 Universidade Estadual de Campinas - Instituto de Química 1 - Laboratório de Química

Biológica. The aim of this work was to characterize the capping proteins around silver nanoparticles produced by biosynthetic method using Fusarium oxysporum fungus. The AgNP were characterized by TEM (spherical morphologies) and DLS (106 nm) and zeta potential (-37 mV). Adhered proteins were quantified using Bradford Test and BSA as standard (1.91 mg mL-1 in the fungal filtrate and 0.14 mg mL-1 in AgNP suspension) and analyzed by electrophoresis (MW from 14.0 to 103.3 kDa). FTIR experiment results exhibited characteristic proteins bands as, for example, in the region 3400-3500 cm-1 (N-H stretching), 2920-2950 cm-1 (C-H stretching vibrations), from 1620 to 1650 cm-1 due to aromatic rings, and at 1380 cm-1 and 1030 cm-1 associated to C-N vibrations. Raman spectroscopy gave information about the involviment of cysteine residues, free amine and amide groups in the AgNP formation and stalilization (changes in the



intensity of Ag-N (240 cm-1) and Ag-S (233 cm-1) bands). Fluorescence spectrum showed maximums at 350 nm, 422 nm and 489 nm that correspond, respectively, to amino acid residues from fungal proteins (350 nm, tryptophan, tyrosine, cystine) and AgNP. Shift in the emission maximum and decreasing in intensity (quenching) indicates the interaction of the protein with AgNP surface. Circular Dichroism analysis allowed to assess not only the structural composition of proteins present in FF and AgNP, as well as conformational changes when these proteins interact with nanoparticles. There was a slight loss of secondary structure, since changes in the percentage contents of helix α and random coil were observed. INBEB, FAPESP G27 - PEPTÍDEO BIOATIVO EM CÂNCER LUNASINA: UMA CARACTERIZAÇÃO FÍSICO-QUÍMICA E ESTRUTURAL

SOUZA, S.M.A.; LIMA, L.M.T.R.; SOUZA, T.L.F. Faculdade de Farmácia da UFRJ

Lunasina é um peptídeo de 43 aminoácidos, o qual foi descoberto em soja por sua atividade antimitótica, mas também é presente em outras sementes e grãos, tais como cevada, trigo, amaranto e centeio. Sua principal ação é alterar a dinâmica de acetilação/desacetilação de histonas de células que sofrem transformação levando-as à morte. Lunasina também é capaz de modular a expressão de genes e tem sido mostrada sua atividade hipocolesterolêmica, anti-inflamatória e antioxidante. Muitos estudos têm mostrado sua atuação em diferentes linhagens de células tumorais em modelos in vitro e in vivo. Visto a sua promissora utilização como um biofármaco, e a escassez de informações estruturais deste peptídeo, o objetivo do nosso trabalho foi analisar sua estrutura em diferentes pHs simulando condições fisiológicas. Para essas análises utilizamos dicroísmo circular (CD), espectroscopia de absorção no infravermelho utilizando transformada de Fourier (FITR), espectroscopia por emissão de fluorescência e cromatografia líquida de alta eficiência (CLAE). Medidas de dicroísmo circular

-hélice. Medidas por CD e FTIR indicam que Lunasina é majoritariamente desestr -hélice, a qual sofre ligeira estabilização em pHs ácidos. Os resultados obtidos nos experimentos de espectroscopia por emissão de fluorescência sugerem que o triptofano se encontra exposto ao solvente e que não sofre alterações nos diferentes pHs testados. Análises por filtração em gel (CLAE) demonstraram uma diminuição do raio hidrodinâmico do peptídeo com a diminuição do pH, sugerindo que em pHs ácidos ocorra uma compactação estrutural. Diante desses resultados, concluímos que a Lunasina possui estrutura majoritariamente desordenada em solução com a presença de um pequeno

conteúdo de alfa-hélice que se torna mais estruturada em pHs ácidos. Esta flexibilidade estrutural pode estar relacionada com as diferentes funções exercidas pela Lunasina. INBEB, FAPERJ, CNPq. G28 - CONFORMATIONAL ENSEMBLES OF PRION PROTEIN: A STRATEGY FOR MAPPING INTERACTIONS WITH PHYSIOLOGICAL AND THERAPEUTIC LIGANDS

1,2-WESLEY ALVES; 1- REINALDO OLIVEIRA; 1- PEDRO TORRES; 2- BRUNO MACEDO; 1- RAFAEL LINDEN; 2- YRAIMA CORDEIRO; 1- PEDRO PASCUTTI

1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro

The prion protein (PrPC) is ubiquitously expressed in mammals, and anchored by GPI on the outer leaflet of the plasmatic membrane. Three alpha helices, one short anti-parallel beta-sheet, a large highly flexible domain, and two glycans compose the structure of PrPC. The functional properties of PrPC are controversial, although a number of its natural ligands have been described. Laminin receptor precursor (LRP), stress inducible protein-1 (STI1) and neural cell adhesion molecule (NCAM) are three such proteins ligands for which cognate interaction domains have been mapped onto each pair of ligands. Our goal is to test whether the main function of PrPC may be to scaffold signaling modules at the cell surface, through allosteric interactions with its protein ligands, thus leading to effects on cell proliferation, differentiation and death. We use techniques of molecular modelling, docking and dynamics to generate conformational ensembles including PrPC with its flexible N-terminus, and to stabilize structural models of interaction of PrPC together with its ligands. In addition, we use circular dichroism (CD), X-ray small angle scattering, fluorescence anisotropy and calorimetry for in vitro assays of binding and conformational effects among recombinant mouse PrPC and synthetic cognate peptides from each ligands. CD and calorimetric experiments showed apparent conformational changes undergone by PrPC in the presence of cognate peptides added in a particular order, consistent with results from docking, and suggesting that the affinity of the globular domain of PrPC for a given peptide is distinct, depending on the previous binding of another ligand peptide. A novel protocol of molecular dynamics was produced, specific for the generation of conformational ensembles based on PrPC, including parameters for glycans. Promissory data are consistent with the hypothesis that PrPC scaffolds cell surface protein ensembles, and additional spectroscopy and simulation will provide further testing of allosteric regulation of PrPC structure. INBEB, FAPERJ, CNPq, CAPES



Mestrandos M1 - ANÁLISE DAS ALTERAÇÕES OVARIANAS INDUZIDAS PELA QUIMIOTERAPIA PARA O TRATAMENTO DO CÂNCER DE MAMA 1-MORAES, ACN; 2-ANDRADE, CBV; 2-RAMOS,IP; 3-SALATA,C; 1,4- NASCIMENTO ALR; 1-

CARVALHO, JJ; 1-STUMBO,AC 1-Departamento de Histologia e Embriologia, Universidade do Estado do Rio de Janeiro; 2 Laboratório de Cardiologia Celular e Molecular do Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 3 - Laboratório de Ciências Radiológicas,

Universidade do Estado do Rio de Janeiro "Introdução: Uma das mais utilizadas estratégias de tratamento para o câncer de mama (CM) é a quimioterapia. O regime TC (docetaxel e ciclofosfamida) é o mais recente, portanto existem poucos estudos relacionados a ele em relação a efeitos tardios, como a osteoporose precoce. Mulheres na pré-menopausa submetidas à quimioterapia para o tratamento do CM apresentam significante perda óssea a partir do primeiro ano após o início do tratamento. A mais provável causa desta perda deve-se ao fato destes quimioterápicos levarem a menopausa precoce. Como regra geral, a quimioterapia para câncer de mama parece acrescentar cerca de 10 anos de idade ao ovário em termos de função reprodutiva. Sabendo-se que alguns quimioterápicos para o tratamento do CM, aumentam o risco de menopausa precoce, espera-se neste estudo, determinar se o recente regime quimioterápico TC pode induzir danos ovarianos. Materiais e Métodos: Ratas Wistar, com 3 meses, foram divididas em 2 grupos, tratados com quimioterapia (TC) e controle tratados com placebo (NaCl 0,9%). Os animais foram eutanasiados 5 meses após o término do tratamento. Foi realizada a coleta de sangue, ovários e dos úteros para posterior análise. As técnicas utilizadas foram: aferição da massa uterina e ovariana e concentração de estradiol sorológico por radioimunoensaio. Resultados/Discussão: Os animais do grupo TC demonstraram redução significativa (p<0.01) da massa do útero em relação ao controle, sugerindo atrofia uterina gerada pela baixa de estrogênio (p<0.05), devido à ação dos quimioterápicos. Esse dado é corroborado pela redução dos níveis de estradiol (p<0,001). Não houve alteração da massa ovariana entre os grupos. Futuramente serão realizadas análises por imunohistoquímicapara caspase3, TGF-beta, Colágeno tipo I e III, além de análise ultraestrutural por microscopia eletrônica." CAPES, FAPERJ M2 - A INFLUÊNCIA DA MANIPULAÇÃO EXTRACELULAR DE NA+ SOBRE OS TRANSPORTADORES RENAIS DE NA+ E CA2+: ESTUDOS IN VITRO E IN VIVO.

1-SOUZA, A.M.; 2,3-FERRÃO, F.M.; 2,3-LOWE, J.; 1-CUNHA, V.M.N.; 1,3-LARA, L.S. 1- Instituto de Ciências Biomédicas, UFRJ, 2- Instituto de Biofísica Carlos Chagas Filho, UFRJ, 3-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem.

O acúmulo de Na+ no organismo não é exatamente proporcional a retenção de água. O aumento localizado da concentração de Na+ pode contribuir para a hipertensão e lesões em órgãos alvos, inclusive nos rins. Pouco se sabe como o aumento da ingestão de sal ativa vias de sinalização que contribuem para o desenvolvimento e progressão da lesão renal. Determinar os mecanismos moleculares ativados pela presença de alta concentração de Na+ que levam a progressão da necrose tubular. Para os ensaios in vitro, células LLC-PK1 foram incubadas por 1 h com NaCl 140 mEq (controle) e 170 mEq (alta [Na+]). Foram medidas as atividades das ATPases transportadoras de Ca2+, Na+. Nos entudos in vivo, ratos Wistar machos foram utilizados no modelo DOCA/Sal (dieta

com 4% de NaCl e acetato de desoxocorticosterona 8mg/Kg 2x/semana). Após 4 semanas os animais foram separados em gaiolas metabólicas e sacrificados e os rins removidos. A região cortical foi dissecada para medidas de atividade enzimática. Aprovado pela CEUA da UFRJ. Nas células LLC-PK1, o aumento da [Na+] aumenta a atividade Ca2+- -1×min-1, n=3, p<0,05), sendo este evento mediado pelo aumento da atividade da SERCA. Nesta condição, a atividade da (Na++K+)ATPase aumenta 3 vezes, sendo a Na+-ATPase insensível. No modelo animal, tanto o grupo DOCA quanto o DOCA/Sal apresentaram um aumento na pressão sistólica arterial e diminuição da função renal, associada ao aumento da proteinúria. A atividade da (Na++K+)ATPase do grupo DOCA/Sal é reduzida

as ATPases renais respondem ao aumento da concentração extracelular de Na+ de formas diferentes, dependendo do tempo de exposição. Os ensaios in vitro sugerem que modulação do Ca2+intracelular pode ser o segundo mensageiro chave da função tubular renal mediada pela alta ingestão de sal. FAPERJ, CNPq M3 - ACTIN ROLE ON TRANSFERRIN ENDOCYTOSIS BY TRYPANOSOMA CRUZI EPIMASTIGOTES

1-ALVES, A. A.; 1-ALCANTARA, C. L.; 1-VIDAL, J. C.; 1-CUNHA-E-SILVA; N. L. 1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.

Trypanosoma cruzi epimastigote forms present high endocytic activity, differing from trypomastigote and amastigote forms. Epimastigotes have two endocytic portals (Soares, Parasitol. Res. 99:321, 2006): the flagellar pocket and the cytostome-cytopharynx complex, described as a funnel-shaped opening, the cytostome that invaginates deeply, forming the cytopharynx (Milder & Deane, J. Protozool. 16:730, 1969). Our group showed recently that specialized microtubules support the cytostome-cytopharynx, playing a crucial role on its ultrastructure. Corrêa and coworkers (Exp. Parasitol. 119:58, 2008) have shown a drastic reduction in transferrin uptake and alterations in cytopharynx structure after treatment with Cytochalasin. Nevertheless, the role of the cytoskeleton on endocytosis by T. cruzi is not clear. In this work we focused on actin filaments, which are poorly characterized in T. cruzi, and its role on endocytosis. We have used transferrin-FITC as tracer in endocytosis assays by epimastigotes pretreated with Cytochalasin D, which destabilizes actin filaments, and Latrunculin B, which binds to G-actin and hinders its polymerization. The results were quantified by fluorimetry and also observed by fluorescence microscopy. We further performed immunoelectron microscopy on ultrathin sections of parasites embedded in LR White using a polyclonal antibody anti-Tc actin produced by Cevallos et al (Exp. Parasitol. 127:249, 2011). Our results showed that Cytochalasin D and Latrunculin B were both able to decrease transferrin endocytosis by about 80 %, without affecting parasite viability. By fluorescence microscopy, we found the endocytic tracer retained at the cytostome. Using transmission electron microscopy, we observed that anti-TcActin antibody recognized thin filaments not yet described at the cytostome opening, among other structures. These data suggest that actin acts on the initial stages of the endocytosis in T. cruzi. PIBIC/CNPq, CAPES, FAPERJ, CNPq M4 - S-NITROSOGLUTATIONA REDUTASE MODULA A PROLIFERAÇÃO E FUSÃO DE MIOBLASTOS

1-YAMASHITA, A.M.S.; 1-FIGUEIREDO-FREITAS, C.; 2-MERMELSTEIN, C.S.; 2-POSSIDONIO, A.C.; 2-SOARES, C.P.; 1-SORENSON, M.M.



1-Instituto de Bioquímica Médica Leopoldo De Meis, Universidade Federal do Rio de Janeiro; 2-Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro.

A miogênese é o processo de formação da fibra muscular. Durante a miogênese do tecido muscular esquelético diversos fatores levam o mioblasto a sair do ciclo celular, se alongar, se alinhar, se aderir e fundir, formando miotubos. O ambiente redox da célula pode atuar regulando a diferenciação do mioblasto. Foi mostrado que o radical livre óxido nítrico (NO) tem um papel importante durante a formação do tecido muscular esquelético. Uma das vias de sinalização por NO envolve a oxidação dos grupos tiol em proteínas, formando SNO-proteína. Para controlar o estado redox intracelular, todas as células sintetizam antioxidantes como a glutationa (GSH). O excesso de NO pode levar a um aumento na produção de S-nitrosoglutationa (GSNO). Uma enzima importante para controlar os níveis de GSNO é a S-nitrosoglutationa redutase (GSNO-R), que reduz GSNO para GSH promovendo a denitrosilação de proteínas. Desta forma a GSNO-R controla os níveis de S-nitrosotióis (SNOs). Diversos tecidos expressam GSNO-R, porém muito pouco se sabe sobre essa enzima no músculo esquelético. O objetivo deste trabalho é analisar o papel da GSNO-R durante a diferenciação muscular esquelética. Foi utilizada cultura primária de músculo esquelético peitoral de embrião de galinha. Foi encontrada atividade e a expressão da GSNO-R no tecido muscular esquelético. A cultura foi crescida por 24 h e tratada por 48 h com inibidor da GSNO-R, o composto C3 (4-{[2-[(2-ciano benzil) tio]-4-oxotieno [3,2-d] pirimidina-3 (4H)-il]metil} ácido benzóico), S-nitrosocisteína (CysNO), ou o inibidor da óxido nítrico sintase 1 (L-NAME). Os tratamentos com o C3 ou com a CysNO levaram a um aumento dos SNOs. Imunofluoresc ncia contra rote nas musculares desmina e α-actinina) e para núcleos (DAPI) permitiram quantificar a proliferação e a fusão dos mioblastos. Os tratamentos com o C3 ou com a CysNO levaram a um aumento do número de núcleos das células mononucleadas (mioblastos) e uma diminuição do índice de fusão. O L-NAME reverteu os efeitos do tratamento com o C3 e levou a um aumento da espessura dos miotubos. Estes dados demonstram a importância do NO e pela primeira vez revelam o papel da GSNO-R modulando a diferenciação dos mioblastos. INBEB, FAPERJ, CNPq, CAPES M5 - ENDOSYMBIOSIS IN TRYPANOSOMATIDS: THE ASSOCIATION BETWEEN THE SYMBIOTIC BACTERIUM AND THE GLYCOSOMES OF STRIGOMONAS CULICIS, AS REVEALED BY MICROSCOPY TECHNIQUES

Machado, A.C.L1, Catta-Preta, C.M.C.1, De Souza, W.1, Motta,C.M.1 1Laboratório de Ultraestrutura Celular Hertha Meyer, IBCCF-UFRJ

Strigomonas culicis is a trypanosomatid protozoan that co-evolves in a mutualist relationship with a symbiotic bacterium and constitutes an excellent model to study the origin of organelles and cell evolution. An interesting aspect of this relationship is the intense metabolic exchange between the eukaryotic host and the prokaruyotic endosymbiont. Based in this fact, the motion of our study was to investigate the association between the symbiont and energetic organelles, as the mitochondrion and glycosomes. Different microscopy approaches were used to observe the ultraestructure of S. culicis, as confocal microscopy, transmission electron microscopy (TEM) and three dimensional (3D) reconstruction using electron tomography. The immunofluorescence assays revealed the great proximity between symbiont and glycosomes, that formed clusters around the bacterium.. This association was confirmed by TEM since the bacterium envelope was seen juxtaposed to the glycosome membrane, however a fusion was not observed between both structures, as revealed by electron tomography,. Such association was not observed between the bacterium and the mitochondrion. Moreover, some mitochondrial branches were sometimes observed parallel to the bacterium outermambrane, but this was a rare event. Preliminary data with the

aposymbiotic strain of S. culicis showed that glycosomes were randomically distributed in the cytoplasm, either in the anterior or in the posterior part of the cell body. Taken together, our results suggest that glycosome distribution is oriented according to the symbiont localization. In accordance to this idea, our previous studies revealead that the bacterium enhances O2 consumption and can benefits from host glycosomes energy production Our next step is to analyse the symbiont association with other host cell structures by the focused ion beam technique INBEB, CAPES, CNPq, FAPERJ M6 - EVALUATION OF ANTIPLATELET PROFILE OF SYNTHETICS PEPTIDES

SUCCAR,B.B.1; SILVA,M.C.C1; GERALDO,R.B.1; JULIANO,M.A.2; WERMELINGER,L.S.3 E ZINGALI,R.B.1

1-Instituto de Bioquímica Médica Leopoldo de Meis, IBqM-UFRJ, RJ; 2-Departamento de Biofísica, Escola Paulista de Medicina-UNIFESP, SP; 3-Faculdade de Farmácia, DACT-

UFRJ, RJ, Brazil. "Introduction: Thrombosis can occur in the arterial or venous circulation, being the most common cause of cardiovascular disease associated death. Nowadays, antiplatelet drugs are effective in reducing arterial and venous thrombosis. However, there are some side effects, which limits their use. Improvement on safety and effectiveness of antiplatelet drugs is required. In this study, we analyzed the antiplatelet profile of five new synthetics peptides (PS1045-C, PS1045-D, PS1045-E, PS1045-F and PS1045-H). Methods: First of all, a theoretical study was performed using Clustal–W, Swiss Model and Swiss-PDB viewer, and docking programs. This study lead to the design of new cyclic peptides. Then, an automated bench-top simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from Shimadzu) was use for synthesis of all the peptides. These peptides were tested in human platelet aggregation assays using thrombin (10nM), ADP (3-4µM) and collagen (2.5µM) as agonists. Discussion and Results: The peptides PS1045-F and PS1045-H (200µM) are the most potent to inhibit platelet aggregation (75%) when induced by ADP and thrombin. Additionally, the peptides PS1045-C and PS1045-E (200µM) were able to inhibit platelet aggregation (50%) induced by the same agonists. None of the cyclic peptides displayed inhibitory activity against collagen-induced human platelet aggregation and the peptide PS1045-D does not inhibit in any agonist. Preliminary results showed that four out of the five synthesized peptides were able to inhibited platelet aggregation in a concentration similar to that observed in the literature. Additional studies used in vivo models are required in other to investigate the antithrombotic profile of these molecules. Conclusion: Our study demonstrated that peptides might be as a tools to development a new drug. Sources of research support: CAPES, FAPERJ, CNPQ, IMBEB M7 - CRIOFIXAÇÃO DE BIOFILMES DE CANDIDA ALBICANS COM PRESERVAÇÃO DA MATRIZ EXTRACELULAR POLIMÉRICA OBSERVADO POR MICROSCOPIA ELETRÔNICA DE VARREDURA

1- FONSECA, B. B.; 1- VILA, T. V. M.; 2 - ISHIDA, K.; 1- DE SOUZA, W.; 1- ROZENTAL, S. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro; 2- Departamento de Microbiologia, Universidade de São Paulo, São Paulo.

Os biofilmes são comunidades microbianas embebidas em matrizes poliméricas (MEC) produzidas por elas próprias. Os biofilmes podem desenvolver-se em qualquer superfície úmida, seja ela biótica ou abiótica e são frequentemente relacionados com infecções invasivas. Fungos da espécie Candida albicans são importantes formadores de



biofilmes, sendo estes relatados por serem mais resistentes a drogas antifúngicas do que as células planctônicas (FEMS Yeast Res. 6:979-986, 2006). Neste estudo, a microscopia eletrônica de varredura (MEV) foi utilizada para estudar a morfologia do biofilme e a preservação da MEC. Em estudos anteriores, com a utilização de fixadores químicos, não conseguimos obter uma boa preservação da MEC, com isto no presente trabalho tivemos como objetivo utilizar a criofixação para melhor visualização da MEC. Realizamos um estudo comparativo de três fixadores químicos diferentes para biofilmes de fungos: rotina, sacarose e vermelho de rutênio onde observamos uma boa preservação de células nos biofilmes, no entanto, a MEC foi extraída do biofilme. Assim propomos uma combinação de congelamento de alta pressão (HPF) com a substituição a frio (FS) para melhor fixação de biofilmes de C. albicans e para avaliar a sua capacidade de preservar a MEC. Os resultados mostraram que o método de criofixação preservou melhor a estrutura geral do biofilme, permitindo a visualização de hifas e blastoconídeos incorporado em uma matrix densa e bem preservada. Nenhum fixador químico nos possibilitou o estudo e preservação do biofilme e da MEC, apesar de serem técnicas bem aceitas para biofilmes bacterianos. O método de criofixação se mostrou o melhor para a preservação da MEC e do biofilme de C. albicans. FAPERJ, CNPq, CAPES, INBEB M8 - PHYSALIS ANGULATA INDUCES AUTOPHAGIC PROCESS DURING DIFFERENTIATION OF MONOCYTES INTO MACROPHAGES 1,2- SILVA, B. J. M, 1,2,3- RODRIGUES, A. P. D, 1,2- FARIAS, L.H.S, 4- NASCIMENTO, J.L.M,

1,2- SILVA, E.O. 1- Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Brazil; 2- Instituto Nacional de Ciência

e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão; Brazil 3- Laboratório de Microscopia, Instituto Evandro Chagas,

secretaria de vigilância em saúde, ministério da saúde, Belém, Pará, Brazil; 4- Laboratório de Neuroquímica, Instituto de Ciências Biológicas, Universidade Federal do

Pará, Belém, Pará, Brazil. "Monocytes and macrophages belong to mononuclerar phagocyte system, and are derived from myeloid precursors in the bone marrow. Monocytes are blood circulation cells that in response to cytokines and inflammation are induced to migrate into tissues and differentiation into macrophages and dendritic cells.The process of differentiation requires activation of autophagy, AKT pathway and caspase-8. After that macrophages can undergo the activation process and protect the organism against pathogens and for establishing an effective immune response. This study aim to evaluate the action of an aqueous extract obtained from Physalis angulata roots (AEPa) in the autophagic process and cell differentiation of monocytes into macrophages. Bone marrow cells were obtained by flushing femurs extract, and maintained in cultures treated with AEPa at a concentration of 100 µg/mL. Ultrasctructural analysis showed that treated cells increased cytoplasmic volume, numerous cytoplasmic projections, apparent increase in the number of endoplasmic reticulum and the presence of elongated mitochondria. In addition, was observed the presence of autophagic vacuoles in the cytoplasm of treated cells with AEPa. Flow cytometry analysis showed increased labeling LC3 in cells treated with AEPa, indicating autophagic process. Nitro blue tetrazolium (NBT) cytochemistry revealed that only cells with small cytoplasm volume showed an increased production of ROS in the group of cells treated with AEPa. Flow cytometric analysis of CD11b and F4/80 protein revealed that AEPa induces the differentiation of monocytes into macrophages.No cytotoxic effect was observed in cells treated with AEPa when compared to the control. These results demonstrate that AEPa is able to induce autophagy in bone marrow cells promoting monocyte-macrophage differentiation.

CAPES, CNPq/UFPa, INBEB, FAPERJ, FAPESPA, MCT/CNPq/FNDCT/PROCAD-NF CAPES. M9 - IMMUNOMODULATORY EFFECTS OF KOJIC ACID ON MONONUCLEAR CELLS OBTAINED OF MURINE BONE MARROW

ALMEIDA, C. M. 1,3; SILVA, B. J. M 1,3; RODRIGUES, A. P. D. 2,3; FARIAS, L.. H. S. 1,3;AZEVEDO, E.P.C.3,4; FOGUEL,D.3,4; SILVA, E. O. 1,3

¹ Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Brasil. 2 Laboratório de Microscopia

Eletrônica, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde do Ministério da Saúde, Belém, Pará, Brazil 3 Instituto Nacional de Ciência e Tecnologia em Biologia

Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brasil 4 Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Ilha do Fundão,

Brasil. Bone marrow is soft and sponge-like material found inside bones that contains hematopoietic cells responsible for development and proliferation of peripheral blood cells. This process occurs through a complex interaction between stromal cells, cytokines and growth factors that compound the bone marrow microenvironment. The monocytes proliferation generated in the bone marrow and the differentiation in macrophages plays a key role in the immune response. This process is important against pathogens through the production of proteolytic enzymes and reactive oxygen species (ROS). In this context, the research for drugs that enhance the innate immune response is needed to restore the homeostasis and the immune response. Kojic acid (KA), a secondary metabolite, synthesized by some species of fungi from Aspergillus genera, has several applications as food additive, cosmetics, antitumor agent, macrophage activator and anti-leishmanial agent. Thus, the aim of this study is to evaluate the immunomodulatory effects of kojic acid (KA) in the bone marrow cells of mice. These cells were obtained by flushing femurs, and maintained in cultures treated with KA at the concentration of 50 and 100 µg/mL for 24-96h of culture. It was observed by optical microscopy that KA promoted increased cell adhesion, spreading ability and high number of cytoplasmatic projections and vacuoles in cytoplasm in the mononuclear cells from bone marrow. To confirm these results, cytometer analysis of surface markers showed that the KA induced cell differentiation in the bone marrow when these cells were treated with KA for 96h. In addition, Akt pathway was analyzed by western blot. KA seems to be able to activate the Akt signaling pathway that has a critical regulatory role in cellular development, homeostasis and differentiation. Furthermore, no cytotoxic effects were observed in cells treated with KA when compared to the untreated bone marrow cells. Thus, KA acts as an immunomodulatory and is able to induce bone marrow monocyte-macrophage differentiation process. Supported by CAPES, CNPq/UFPa, INBEB, FAPERJ, FAPESPA, MCT/CNPq/FNDCT/PROCAD-NF CAPES. M10 - BINUCLEAR COPPER(II) COMPLEXES MODIFIED WITH PYRENE: DNA CLEAVAGE AND BINDING PROPERTIES

1- SAIBERT, C., 2- DA SILVA, M. P., 2- NEVES, A., 1- TERENZI, H. 1- Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina; 2- Departamento de Química, Universidade Federal de Santa

Catarina In last years, binuclear complexes of copper(II) have gained attention because of the large activity as DNA cleavage agents. We report here two new binuclear copper(II) complexes, derived from the Cu2(L-TCl) (1), that present strong DNA cleavage properties: Cu2(L-Tdab) (2) and Cu2(L-TN-Mepydab) (3), where L-TCl is 2-chloro-4,6-bis(di-2-picolylamino)-1,3,5-triazine; L-Tdab is 2-diaminobutane-4,6-bis(di-2-



picolylamino)-1,3,5-triazine and L-TN-Mepydab is 2-N-methylpyrenyldiaminobutane-4,6-bis(di-2-picolylamino)-1,3,5-triazine. In this study, we compare the activity of the complexes 2 and 3 towards 1, searching for structure-activity relationships, especially for the pyrene-derived complex (3) since pyrene is a well know intercalating agent. The analysis on the cleavage of DNA was made following the conversion of supercoiled form of pBSK-II plasmid into its cleaved forms using agarose gel. Similarly, the cleavage of a fluorescent end-labeled hairpin DNA by the complexes was investigated using urea page polyacrylamide gel electrophoresis. Complexes 2 and 3 showed high activity in terms of plasmid DNA cleavage, even at mild conditions (pH 7.0, 37°C), low concentration and incubation time. The substitution of chlorine by butanodiamine and the posterior addition of a pyrene moiety seem to increase the affinity of the complexes for DNA, since KM decreases accordingly. Although, changing on the rate of cleavage was not observed. The reaction mechanism seems to be oxidative and the complexes have preference for the minor groove of DNA. The high resolution gel analysis suggests that the complexes cleave DNA in the vicinity of a hairpin structure, mainly for complex 2 that presents a better cleavage pattern on the urea page polyacrylamide gel. The high activity of 3 in comparison to 1 and 2 by the addition of pyrene motifs represents a good strategy to increase the cleavage activity of metal complexes. However, the greater cleavage pattern showed by the complex 2 upon the fluorescent end-labeled hairpin DNA needs to be more investigated. CNPq, CAPES, MCT, FINEP, FAPESC, INCT - Biologia Estrutural e Bioimagem M11 - OTIMIZAÇÃO DE HISTOGRAMAS 1D E 2D APLICADO A RESULTADOS DE SIMULAÇÃO DE DINÂMICA MOLECULAR.

1- IGLESIAS JR, C. F.; 1- FERNANDES, T. V. A.; 1- OLIVEIRA JR, R.; 1 - PASCUTTI, P. G 1 - Instituto de Biofísica Carlos Chagas Filho

Sumariar resultados de simulações de dinâmica molecular em histogramas fornecem três peças muito importantes de informação sobre as distribuições, como: forma, localização central e spread. Quando se faz um histograma, se precisa escolher o ótimo tamanho do bin que será utilizado para evitar os seguintes problemas: escassez de amostras em cada bin fazendo com que a altura da barra em cada bin sofra uma flutuação estatistica significativa e a perda de resolução impedindo que o histograma represente a forma real da distribuição. Ter histogramas otimizados é crucial para construirmos superficies de energia livre que representam um mapa com um amplo espectro de conformações, que macromoléculas biológicas normalmente pode acessar através molecular simulações de dinâmica.Com isso nosso objetivo foi criar um software que disponibiliza aplicação de métodos e regras para se obter o tamanho ideal de um bin que resolve estes dois problemas descritos acima.Os métodos Scotts rule e Freedman-Diaconis rule estão disponíveis para otimização de histogramas em uma dimensão e Método Shimazaki para otimização de histogramas em duas dimensões. O software possui a licensa open-source e foi construido utilizando a linguagem de programação Python. Para ilustrar o potencial do software, nos o utilizamos para analisar os resultados de simulações de dinâmica molecular da proteina BID e suas formas ativas tBID e tBID-myr no qual desempenha um papel chave na apoptose e estão relacionadas a algumas doenças degenerativas. Gerando histogramas otimizandos para valores de RMSD e Raio de Giro nos podemos criar superfícies de energia livre e identificar conformações das proteinas BID, tBID e tBID-myr que estão em regiões de menor energia livre e nas conformações das proteinas tBID e tBID-myr encontramos cavidades que podem ser exploradas como alvo farmacologico. Ja que tBID e tBID-myr somente atuam na apoptose e as cavidas encontradas não estão na superficie da proteina BID. INBEB, CNPq

M12 - SIMULAÇÕES DE DINÂMICA MOLECULAR DE COMPLEXOS DE CATEPSINA B DE FASCIOLA HEPATICA COM CA074, UM INIBIDOR DIPEPTIDIL NITRILA E POTENCIAIS INIBIDORES SELETIVOS

1,2- FELICIANO, D.N.; 3-VALIENTE, P.A.; 4-GOMES, D.E.B.; 2- PASCUTTI, P.G. 1- Centro Nacional de Sanidad Agropecuaria; 2- Universidade Federal do Rio de Janeiro;

3- Universidad de la Habana; 4- Instituto Nacional de Metrologia, Qualidade e Tecnologia

A Catepsina B ( fCatB ), a principal protease secretada a partir da fase juvenil de Fasciola hepatica, é um alvo relevante para o tratamento quimioterapico da fasciolíase[1] e também um alvo em potencial para o desenvolvimento de drogas para tratar diversar enfermidades humanas [2]. No entanto, a estrutura tridimensional (3D ) da fCatB ainda não foi resolvida experimentalmente. Isto constitui uma desvantagem importante para o design baseado em estrutura de novos inibidores usando abordagens de triagem virtual. Inibidores da fCatB revogam o ciclo de vida do parasita e apresentam uma baixa seletividade para humanos ( hCatB ) e bovinos ( bCatB ), suas proteases homólogas em seus hospedeiros definitivos.Prevemos inibidores seletivos putativos de fCatB combinando modelagem comparativa (multiple structures complex template), Molecular Docking (CA074, um inibidor da dipeptidil nitrila e compostos da bases de dados Hitfinder-Myabridge) e simulações de dinâmica molecular . Uma previsão de cavidades, interacções de ligante de ensaio e simulações de dinâmica molecular foram feitas. Além disso, 13 resíduos potencialmente determinantes na especificidade de substrato foram identificados podendo sugerir que árvore deles poderia melhorar o design de inibidores seletivos da catepsina B de Fasciola hepatica em relação às Catepsinas de mamíferos. INBEB, CAPES, FAPERJ, CNPq M13 - BEX3, UMA PROTEÍNA NEGLIGENCIADA RELACIONADA AO CÂNCER E DOENÇAS NEURODEGENERATIVAS, FORMA OLIGÔMEROS QUE PROTEGEM CONTRA AGREGAÇÃO E PROTEÓLISE

RAYMUNDO, D. P. 1, CABRAL, K. M. S. 1, HILL, L. F.2, SILVA, V. S. 1, CORDEIRO, Y. 3, CIRAUQUI, N.3 E ALMEIDA, M. S.1.

"1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 2- Instituto de Biofísica Carlos Chagas Filho,

Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 3- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil."

"A proteína BEX3 (Brain expressed X-linked 3) é específica de mamíferos placentários e é codificada por um gene que está em uma região do cromossomo X que contém genes relacionados com a evolução do neurocórtex. BEX3 atua nas vias extrínsecas e intrínsecas da apoptose através da interação com p75NTRDD e Smac, respectivamente. Além destes, outros parceiros de interação com a BEX3 já foram identificados como DRG-1, 14-3-3e e Hamartina. Embora a BEX3 esteja envolvida com apoptose e provavelmente com câncer, além de ser conservada em diversas espécies de mamíferos, muito pouco foi estudado a respeito da estrutura desta proteína. Ao longo do presente estudo, realizamos a caracterização estrutural da BEX3. Ensaios de Ressonância Magnética Nuclear (RMN), Fluorescência Intrínseca do Triptofano (FIT), Proteinase K (PK) e Dicroísmo Circular (CD) mostraram que esta proteína possui estrutura secund ria em hélice α e regi es desordenadas no - e C - terminal, caracterizando esta proteína como parcialmente enovelada. A BEX3 parece formar um oligômero atra és de uma or o em hélice α. lém disso, e erimentos de modelagem molecular ajudaram a descrever os resultados experimentais obtidos e ainda



levantaram a hipótese de que BEX3 se apresenta na forma de duas subunidades antiparalelas (coiled-coil). Dados de Microscopia de Força Atômica (AFM), crosslinking, Espalhamento de Raios-X a baixo ângulo (SAXS), Tioflavina T (ThT) e Gel Filtração (GF) mostram que BEX3 forma um oligômero solúvel com a formação de um núcleo hidrofóbico (FIT, Bis-ANS e PK). Além disso, resultados de ensaios de fluorescência por ThT e eletroforese em gel de poliacrilamida em condições desnaturantes (SDS-PAGE) sugerem que esta proteína forma agregados que podem ter implicações fisiológicas." CNPq, INBEB, FAPERJ M14 - CELL AND GENE THERAPY: COMBINING TWO APPROACHS TO PROMOTE NEUROPROTECTION AND NEUROREGENERATION AFTER A MODEL OF CNS INJURY.

1- NASCIMENTO-DOS-SANTOS, G.; 1- MESENTIER-LOURO, L.A.; 1- TEIXEIRA-PINHEIRO, L.C.; 1- SILVA-JUNIOR, A.J.; 1- FIGUEIRÊDO, A.B.P.; 2- TEIXEIRA, C.; 2- TOVAR-MOLL, F.; 1-

MENDEZ-OTERO, R.; 1- PETRS-SILVA, H.; 1- SANTIAGO, M.F. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-

Instituto Nacional de Tecnologia de Biologia Estrutural e Bioimagem. After injury, the axons in the central nervous system (CNS) are not able to regenerate great distances and permanently lose their connections in the brain. Two promising approaches to reverse this condition are the cell and gene therapies. In previous work, we demonstrated that the intravitreous transplantation of bone marrow mononuclear cells (BMMC) were capable of promoting an increase in retinal ganglion cells (RGCs) survival and axonal outgrowth 14 days after optic nerve crush. In the present work, we evaluated the therapeutic potential of mesenchymal stem cell transplantation (MSC) and pigment epithelium derived factor (PEDF) gene therapy using adeno-associated viral vectors (AAV) in a model of rat optic nerve crush. In MSC transplantation adult (3-5 months old) Lister rats underwent unilateral optic nerve crush followed by MSC or vehicle injection into the vitreous body. Sixteen and 28 days after injury, RGC survival was evaluated assessing the number of Brn3a-positive cells in flat-mounted retinas, and nerve regeneration was investigated after anterograde labeling of the optic nerve axons. For gene therapy, adult (2-3 months old) Lister rats underwent unilateral optic nerve crush 30 days after AAV-PEDF, AAV-GFP or vehicle injection into the vitreous body. Twenty eight days after injury, RGC survival was also evaluated assessing the number of Brn3a-positive cells in flat-mounted retinas. Cell therapy with MSC increased the number of Brn3a positive cells in the retina and the number of axons distal to the crush site both at 16 and 28 days after optic nerve crush when compared to control. The PEDF gene therapy using adeno-associated viral vectors also increased the number of Brn3a positive cells in the retina. Considering the results, our propose is to combine these two therapies in order to enhance the effects we have obtained so far by transplantation of the MSC previously transducted with AAV-PEDF. CAPES, FAPERJ, INBEB, CNPq. M15 - WHAT IS THE BEST WAY TO PROCESS CAT INTESTINE TO VISUALIZE TOXOPLASMA GONDII?

VERAS, GM1; DUBEY, JP3; DE SOUZA, W1, 2; ATTIAS, M1 1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil. 2Diretoria de Metrologia Aplicada às Ciências da Vida do INMETRO 3United

States Department of Agriculture, Animal Parasitic Diseases Laboratory, EUA Toxoplasmosis is a worldwide disease caused by the intracellular protozoan Toxoplasma gondii. This parasite can infect any nucleated cell of any warm-blooded animal. The transmission can be from the mother to the foetus by tachyzoites, by eating raw or undercooked meat containing tissue cysts or by ingestion of contaminated food and

water with sporulated oocysts. The sexual cycle occurs only into the felids small intestine where the oocysts are formed and released with the feces to the environment. The aim of this work was to visualize the enteroepithelial stages of Toxoplasma gondii in cat intestinal villus by scanning electron microscopy after cold fracture. For this, cats were contaminated with tissue cysts, intestine segments or intestinal villi that were gently scrapped from the ileum were fixed with 2.5% glutaraldehyde and 1% formaldehyde, post-fixed with 1% of osmium tetroxide (OsO4) and 1.25% of potassium ferrocyanide in 0.1 M sodium cacodylate buffer, pH 7.2, for 40 minutes. Then, the intestinal villi were embedded in 10% gelatin with or without 2% of chitosan, re-fixed with 0.5% glutaraldehyde and 0.5% formaldehyde in 0.1 M sodium cacodylate buffer for 1 hour at 4oC. After this, the samples were cut into small pieces and infiltrated in 25-50% glycerol per 1 hour, then frozen by immersion in liquid nitrogen and fractured with a razor blade. Fracture was followed by thawing and glycerol deinfiltration in water. Some of the specimens were immediately dehydrated in crescent series of ethanol, critical point dried or went to maceration protocol, where the samples were placed in 0.1% OsO4 solution in cacodylate buffer for 10 days before dehydration and critical point drying. Segments of the ileum were post-fixed and dehydrated in crescent series of ethanol. Then, the samples were frozen by immersion in liquid nitrogen, fractured with a razor blade, thawed in 100% ethanol and critical point dried. After gold sputtering, the samples were visualized in a Quanta 250 or in a Quanta 450 FEG scanning electron microscopes. Cleaved planes showed different, and feline exclusive, stages of the parasite at the upper portion of intestinal villi. Merozoites, schizonts, gamonts and oocysts were visualized. Observation of the intestinal villus revealed moreinformation than the intestine fragments because of the parasite localization, usually between the nucleus and the microvilli. Either method (cleavage in frozen ethanol or cleavage of cryoprotected samples, followed by long term maceration) proved to be adequate to the observation of intraepithelial forms of Toxoplasma gondii. However, observations made on a FEG-SEM had, as expected, a much higher resolution. In conclusion, we can recommend fracture of samples frozen in 100% ethanol as a time saving and reliable method of exposing the inside organization of tissues and cells. INBEB, FAPERJ, CNPq M16 - CHARACTERIZATION OF CRYPTOCOCCUS NEOFORMANS POLYSSACHARIDE CAPSULE BY HIGH-RESOLUTION SCANNING ELECTRON MICROSCOPY

Glauber R de S. Araújo1,2, Daniel G. I. Vieira1, Kildare Miranda1,2, Thais C. Souto-Padrón3, Danielle Cavalcanti2 , Daniela Leão2, Wanderley de Souza1,2 and Susana

Frases1,2* 1Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos

Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 2Diretoria de Metrologia Aplicada a Ciências da Vida, Instituto Nacional de Metrologia, Qualidade

e Tecnologia, Rio de Janeiro, Brazil. 3Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

The encapsulated fungus Cryptococcus neoformans is a common cause of life-threatening disease in immunocompromised individuals. Its major virulence determinant is the polysaccharide (PS) capsule. The native form of polysaccharides appears to involve association with other molecules and create highly variable ultrastructural configurations. Thus, the expression of ultrastructural polysaccharide molecules can determine the progress and effectiveness of the innate immune response against pathogens. In this scenario, there is a clear need for studies focused on a better understanding the ultrastructure characterization of C. neoformans capsule. An unsolved problem in cryptococcal biology is whether the PSs composing the capsule are



linear or complex branched polymers, as well as the implications of this structural composition in pathogenesis. We analyzed the dependence of capsular PS molecular mass and the radius of gyration and we noticed a strong evidence against a simple linear PS configuration. Shape factors calculated from dynamic and static light scattering measurements in solution and viscosity measurements revealed values consistent with polymer branching. All this physical data showed evidences of ultrastructure modification during the pathogenic process. For that reason, we decided to use High Resolution Electron Microscopy to visualize this PS complexity. Our results showed PS spherical-like structures similar to other branched that was confirmed analyzing the PS capsule by high resolution scanning electron microscopy and different fiber sizes forming the PS capsule by atomic force microscopy (AFM). FAPERJ, CNPq M17 - INVOLVEMENT OF THE TRANSCRIPTION FACTOR NFAT IN PARKINSON'S DISEASE

1- DE FREITAS, J.A; 1- FOGUEL, D. 2- ROBSS, B.K. 1- Instituto de Biquímica Médica 2- Faculdade de Odontologia de Nova Friburgo,

Universidade Federal Fluminense Parkinson's disease (PD) is a neurodegenerative disorder that is caused by the death of midbrain dopaminergic neurons and affects nearly 5 million individuals worldwide. There are a large number of studies suggesting that the mechanism which leads to neuronal loss in PD consists by an abnormal accumulation of a protein known as α-synuclein and subsequent formation of intracellular protein aggregates called Lewy bodies. Studies have shown that α-synuclein aggregates alter membrane fluidity and increase calcium (Ca+2) influx, rise levels of intracellular calcium lead to the activation of calcineurin phosphatase (CnA). Although, the main calcineurin target are Nuclear Factor of Activated T-Cells (NFAT), its contribution to the PD is very poorly understood. NFAT proteins directly regulate the expression of genes involved in the control of cell death by apoptosis, as well as genes involved in the inflammatory process. Apoptosis and inflammation are known to be key events in neurodegeneration, which is triggered upon a remarkable increase in intracellular Ca+2. Therefore, the main goal of the present project is to evaluate the involvement of NFAT in the neurodegenerative process induced by aggregates of α-synuclein. Our initial results show that only oligomers of α-synuclein were able to mediate increase in LDH release in primary cultures of dopaminergic neurons and this effect was partially reversed when these cultures are pretreated with cyclosporin, an inhibitor of calcineurin. In addition we examined the involvement of monomers, oligomers and fibers in the synaptic events. We found that only oligomers and fibers showed to be capable of causing a decrease in Synapsin I, a protein involved in pre-synaptic events. These results suggest that the NFAT may have a possible role in the process of neurodegeneration mediated by α-synuclein aggregates. INBEB, CNPq, FAPERJ M18 - ACTION OF ALKALOID (+)-PHYLANTIDINE ON Leishmania (Leishmania) amazonensis AND THE HOST CELL.

1,2* - MORAES, L. S. M.; 2,3- RODRIGUES, A. P. D.; 1,2 - FARIAS, L.H.S.; 1,2 - SILVA, B.J.M.; 4 - DONZA, M.R.H.; 4 - GUILHON, G.M.S.P.; 1,2 - SILVA, E. O.

1 - Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, 2 - Instituto Nacional de Ciência e Tecnologia

em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro. 3 - Laboratório de Microscopia, Instituto Evandro Chagas, Secretaria de Vigilância em

Saúde, Ministério da Saúde, Belém, Pará. 4- Programa de Pós-graduação em Química,

Instituto de Ciências Exatas e Naturais, Universidade Federal do Pará, Brazil, Universidade Federal do Pará.

"Leishmaniasis is antropozoonotic disease caused by parasites of the genus Leishmania. These parasites proliferate primarily within mammalian macrophages and promote a diversity of clinical manifestations ranging from self-healing cutaneous lesions to fatal visceral leishmaniasis. The treatment available is chemotherapy, but is limited by toxicity and requires a long term treatment. The use of natural products from plants currently plays an important role and source as antileishmania agent. (+)-Phylantidine, is an alkaloid extracted from stem of Margaritaria nobilis of the family Phyllanthaceae. Thus, the aim of this study is evaluated the effects of (+)-phylantidine on promastigotes forms of Leishmania (Leishmania) amazonensis and host cell. Antiproliferative activity of promastigotes forms was observed when parasites were treated with 50, 100 e 200 µg/mL, with reduction of 82.50%, 73.75% and 88.75%, respectively when compared with non-treated parasites. In the period of 96 hours was observed an IC50 of 56.34 µg/mL. As the reference drug amphotericin B was used and observed reduction of 100% in parasites treated with 0,1 µg/mL for 96 hours. The treatment with 100 and 200 µg/ml of (+)-phylantidine promoted morphological alterations in the promastigote. Transmission Electron Microscopy (TEM) analysis showed alterations in the membrane, flagellar pocket and increases in the numbers of acidocalcisome. No citotoxic effect in mouse peritoneal macrophages was detected after treatment with phylantidine. However, ultrastructural analysis by Scanning Electron Microscopy (SEM) revealed typical activated cell morphology in treated macrophages, with alteration in cell volume, an increase in cytoplasmic projections and spreading ability. These results demonstrated that alkaloid seems to be important not only for host cell but also to promastigotas growth inhibition. Thus, (+)-phylantidine could be an alternative source for Leishmaniasis treatment. Keywords: Leishmania (Leishmania) amazonensis; (+)-phylantidine; ultrastructural alterations; host cell viability." CAPES, CNPq/UFPa, UFPA, INBEB, FAPERJ, MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ. M19 - RELAÇÕES FUNCIONAIS ENTRE PRPC E O SISTEMA CALICREINA CININAS: (1)- ESTUDOS ENZIMÁTICOS.

1-Vellasco L; 1- Nascimento CR; 2-Vieira T; 2-Lima Silva J; 1-Mariante R; 1-Linden R; 1-Scharfstein J 1 Instituto de Biofísica Carlos Chagas Filho, UFRJ; 2 Instituto de

ioquímica Médica Leopoldo de Meis. O sistema calicreína – cininas (SCC) é uma rota proteolítica iniciada pelo contato do FXII com polímeros negativamente carregados, como o dextran sulfato (DXS). A forma ativa do FXII cliva e ativa a calicreína plasmática, protease envolvida na liberação de bradicinina (BK) através da clivagem do cininogênio de alto peso molecular (HK). A ativação desta via apresenta impactos trombogênicos por ativação da via intrínseca da coagulação e inflamatórios, em consequência da liberação de BK, C5a entre outros mediadores. Polifosfatos e heparina, liberados em grânulos de mastócitos e plaquetas ativadas são ati adores end genos do FXII. Foi sugerida ainda a ati a o elas T’s liberadas or neutr filos. ati a o do CC or T’s sugere um a el dos neutr filos na regulação positiva de mecanismos tromboinflamatórios. Estudos demonstram a expressão da proteína príon celular (PrPc) na superfície de neutrófilos, sendo esta modulada por estímulos inflamatórios (Mariante et al., 2012). Considerando os achados que mostram a ligação da PrPc purificada à heparina (Vieira TC et al., 2010; e dados não publicados), hipotetizamos que a PrPc fosse capaz de mitigar a interação de ativadores da via de contato (poly P, DNA, heparina) com FXII, modulando negativamente a ativação do SCC. Com este propósito, investigamos o efeito da PrPc na ativação do SCC



utilizando substratos fluorescentes que mimetizam o HK. Consistente com nossa hipótese, observamos menor hidrólise do substrato em resposta ao DXS e heparina na presença de PrPc purificada. Neutrófilos de animais selvagens versus animais PrPc-KO foram incubados com DXS-FITC e a ligação da molécula fluorescente à PrPc foi analisada. As análises de citometria não indicaram diferenças na endocitose de DXS-FITC entre as células selvagem e PrPc-KO. Novos abordagens serão realizadas para determinar se PrPc pode mitigar o impacto da ativação intravascular do SCC sobre o perfil funcional de neutrófilos. CNPq, FAPERJ, INBEB M20 - INVESTIGATION ON THE BEHAVIOR OF TRICHOMONAS TENAX

1,2- RIBEIRO, L. C. S.; 3- SANTOS, C.; 2-BENCHIMOL, M. 1- Laboratório de ultraestrutura celular, Universidade Santa Úrsula; 2- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 3- Instituto

Nacional de Metrologia, Qualidade e Tecnologia, INMETRO T. tenax has been considered a commensal of the human mouth found under conditions of poor oral hygiene or associated with periodontal diseases. There are some controversies concerning the pathogenicity of this protozoan. T. tenax is very similar to Trichomonas vaginalis, a parasite that inhabits the human genitourinary tract and known to induce damage to various mammalian cells. Currently, there is a discussion whether T. tenax would be a genetic variant of T. vaginalis. The aim of this study was to investigate the capacity of T. tenax to provoke damage to mammalian cells and also to compare their cytotoxicity with T. vaginalis. For this, interaction assays of both protozoa with host cells, such as MDCK, HeLa and 3D spheroids were performed with the ratio of 5:1 in different periods of time. The samples were examined by scanning electron microscope (SEM) and the cytotoxic effects of the protozoa were analyzed by viability assays such as the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Trypan blue. Our results indicated some similarities between both parasites, such as (1) during the interaction T. tenax showed different forms; (2) T. tenax and T. vaginalis were able to adhere on mammalian cells and both provoked injury; (3) both protozoa presented cytotoxic effects on monolayer cells, as seen by MTT and Trypan blue experiments. Interestingly, T. tenax provoked damage to 3D spheroids of oral cells. Take together, these results indicated that T. tenax could be a parasite in vitro, not a commensal, and further experiments are necessary to better confirm its pathogenicity in vivo. CNPq, FAPERJ, AUSU, PRONEX, INBEB M21 - EFEITO DAS MICROPARTÍCULAS DE EPIGALOCATEQUINA-3-G (EG G) N GREG α-SINUCLEINA

1-FERNANDES, L.;1-BRAGA, C.;1-PALHANO,F.;1-FOGUEL,D. 1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

A Doença de Parkinson (DP) é caracterizada pela morte de neurônios dopaminérgicos na substância nigra e pela presença dos Corpos de Lewy que são inclusões protéicas intracelulares, majoritariamente, compostos por agregados amil ides de α-sinucleína α-sin). Esses agregados são caracterizados pelo alto conteúdo de folhas-β e or serem insolúveis. As terapias utilizadas no tratamento da DP, atualmente, são apenas paleativas e alguns compostos têm sido testados na tentativa intervir no curso da doença. Dentre os compostos estudados como estratégia para prevenir e tratar a DP temos o epigalocatequina-3-galato (EGCG). EGCG é uma catequina, sendo o composto bioativo mais abundante na folha do chá verde. Essa molécula é um antioxidante que quando oxidado transforma-se em quinonas capazes de reagir com outras quinonas,

formando polímeros de EGCG. Interessantemente, sob condições controladas in vitro, o EGCG polimeriza na forma de micropartículas homogêneas com 1-2um de diâmetro (EGCG bead). Foi demonstrado que o EGCG possui um potente efeito inibidor na agregação de proteínas amilóides, sendo capaz de interferir no processo de formação desses agregados. O mecanismo de ação do EGCG ainda não é conhecido, no entanto, sugere-se que há necessidade do EGCG sofrer oxidação e auto-polimerizar para exercer sua ação. Neste trabalho, nosso objetivo foi avaliar o efeito dessas micropartículas de GCG GCG bead , o idado, em inibir a agrega o da α-sin. Quando incubada sob condições de agrega o na resen a do GCG bead, a roteina α-sin permanece com estrutura secundária randômica, enquanto a amostra sem EGCG bead apresentou alto conteúdo de folhas-β. s centrifuga o e an lise or -PAGE, encontramos a maior arte da α-sin na fração solúvel quando incubada com EGCG bead, enquanto o controle se mostrou agregado. sses resultados sugerem ue GCG bead é ca a de ligar α-sin e inibir a formação de agregados amilóides. INBEB,CAPES M22 - INTRACELLULAR AGGREGATION OF P53 PROTEIN IN HUMAN GLIOBLASTOMAS 1,2-PEDROTE, M.M.;1,2-COSTA, D.C.F.; 1,2--OLIVEIRA, G.A.P.; 3-LIMA, F.R.S.; 1,2-SILVA,

J.L. 1- Instituto de Bioquímica Médica Leopoldo De Meis, Universidade Federal do Rio de

Janeiro; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro; 3 - Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro. Introduction:

Glioblastoma Multiforme (GBM) is the most common and aggressive brain tumor in humans. According to World Health Organization (WHO), it is classified as a grade IV glioma. This feature is correlated to a high invasiveness from the tumor to the adjacent normal nervous tissue. p53 is a tumor suppressor protein which plays an essential role in preventing the development of cancer by inducing cell cycle arrest or apoptosis in response to DNA damage. Mutations in TP53 gene is frequently associated to a high probability of cancer development. Therefore it is a good target against cancer. Mutations in p53 core domain normally affect protein stability, which may trigger to a strong tendency toward aggregation. Studies from our group have show that p53 aggregation in a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation, typical of a prionoid behavior. Furthermore, these aggregates are present in tissue biopsies of breast cancer especially in more aggressive ones. Objective: The main goal of this study is to evaluate whether p53 aggregates may be present in human glioblastoma cells and participate during tumorigenic process. Material and Methods: Primary GBM cultures obtained from deceased patients and T98G and U87MG cell lines were evaluated against p53, oligomer and fiber antibodies and imaged by confocal fluorescence microscopy. Results and Discussion: Our preliminary data shows greater colocalization between p53 and amyloid fibers as compared to oligomers. This behavior was observed in three GBM primary cultures. We observed a major distribution of p53-oligomer colocalization in perinuclear regions and a diffuse and citosolic distribution for p53-fiber colocalization. These cells also revealed a strong labeling of p53 in nucleolus and weak nuclei localization. Conclusions: In conclusion, our data suggest the presence of p53 oligomers and amyloid fibers in human glioblastoma cells. Further investigation will be conducted to address the participation of these p53 aggregates in other cell lines and to the tumor progression and aggressiveness. INBEB, Faperj and CNPq



M23 - HEME STIMULATES NA+/K+ ATPASE ACTIVITY TROUGH HYDROGEN PEROXIDE GENERATION IN LEISHMANIA AMAZONENSIS

1,2-ROCCO-MACHADO, N. 1,2-COSENTINO-GOMES, D. AND ROCCO-MACHADO, N. COSENTINO-GOMES, D. AND 1,2-MEYER-FERNANDES, J.R.

1-Instituto de Bioquímica Medica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 2-Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Centro de Ciências da Saúde, Universidade Federal do

Rio de Janeiro, Rio de Janeiro, RJ, Brazil Introduction: A recent work of our group has shown the activation of a Na+/K+ ATPase in L. amazonensis, through a signal transduction cascade involving the presence of heme and PKC activity. Heme is an important biomolecule with a pro-oxidant and signaling capacity. Recently, hydrogen peroxide (H2O2) has been considered an important second messenger, being able to stimulate PKC activity in several models. Our goal in this work is to investigate the role of heme-dependent hydrogen peroxide generation on Na+/K+ ATPase activity of L. amazonensis. Materials and methods: H2O2 released by L.. amazonensis intact cells was determined by the Amplex red oxidation method; The Na+/K+ activity was calculated as the difference between 32Pi released in the absence, and in the presence, of 1mM ouabain; For PKC assay was used the Kinase-Glo luminescent kit and the PKC specific substrate neurogranin. Results and discussion: Our results shows that increased concentrations of heme stimulated H2O2 generation in a dose dependent manner, reaching its maximum at 2,5 μM and Na+/K+ ATPase was stimulated by increasing concentrations of H2O2, reaching its maximum at 0.1 µM. In order to investigate the role of PKC in this signaling pathway, we verified the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and inhibitor, calphostin C. Both had no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2 and in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. Conclusion: The results suggest that PKC is activated by H2O2 generated in the presence of heme, thus activating the Na+/K+ ATPase. We are now investigating where this H2O2 is produced. INBEB, FAPERJ, CAPES, CNPQ M24 - EFFECTS OF KOJIC ACID ON HUMAN NEUTROPHILS AND DURING INTERACTION WITH LEISHMANIA (LEISHMANIA) AMAZONENSIS IN VITRO

1,2-FRADE, P. C. R.; 1,2-COSTA, J. P.; 2,3-RODRIGUES, A. P. D.; 1,2-FARIAS, L.H.S.; 1,2-SILVA, E.O.

1-Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará; 2- Instituto Nacional de Ciência e Tecnologia

em Biologia Estrutural e Bioimagem , Universidade Federal do Rio de Janeiro; 3- Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, Secretaria de

Vigilância em Saúde do Ministério da Saúde. Neutrophils are phagocytes involved in the primary immune responses to eliminate pathogens. Leishmania is a protozoan responsible for the one of the most important infectious diseases in the world and chemotherapy is potentially useful, but besides the high cost, these drugs are toxic and require a long period of treatment. Therefore the search for new drugs is of great interest. In this work, we have used kojic acid (KA), a secondary metabolite synthesized by some species of fungi from Aspergillus, Penicillium and Acetobacter genera. KA has several applications, being used as a food additive, cosmetics, antitumor agent, macrophage activator and anti-leishmanial agent. Thus, this study was designed to determine the effects of KA- induced human neutrophils activation and interaction with Leishmania (Leishmania) amazonensis. Human neutrophils were obtained from buffy coats donated from Hemocenter Fundation of Para State. Cells isolation were performed using HISTOPAQUE® 1077-density-gradient.

Neutrophils were treated for 1 hour with 50 μg/mL of KA. No cytotoxic effect was observed on the cells treated with 10-200 μg/mL of KA for 1 hour and 1-24 hours with 50 µg/mL of KA by colorimetric MTT assays. Treated neutrophils had a higher capacity to phagocytose Leishmania parasites and decreased the number of intracellular parasites a long time. Subsequently, was assessed neutrophil oxidative burst induced by KA (reactive oxygen species-ROS, myeloperoxidase activity and nitric oxide-NO), which are important leishmanicidal agents. Treated cells demonstrated microbicide activity that occurred through ROS production, detected by Nitro Blue Tetrazolium (NBT) assay and CellROX® green dye; increase myeloperoxidase (MPO) activity, however NO production was not detected. In conclusion, these results show that KA is involved in neutrophil activation might be via ROS production suggesting a possible control mechanism for Leishmania infected neutrophils. INBEB, CNPq, Capes. M25 - AÇÃO PARÁCRINA DAS CÉLULAS-TRONCO MONONUCLEARES DERIVADAS DA MEDULA ÓSSEA (CDMO) MEDIADA POR ÁCIDO LISOFOSFATÍDICO (LPA) NA PREVENÇÃO DA I/R RENAL

MATTOS, P.; GONSALEZ,S.R.; VIEYRA,A.; LARA,L.S.; EINICKER-LAMAS,M. 1 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2 -

Instituto de Ciências Biomédicas Devido a sua capacidade de diferenciação, as CDMO são elementos-chave para a terapia regenerativa. No entanto, seus efeitos benéficos não são restritos a recuperação tecidual, mas também por seus efeitos parácrinos mediados pelo LPA. O objetivo deste estudo é caracterizar o efeito do tratamento com CDMO no modelo de I/R renal avaliando a modulação sobre a autotaxina (enzima produtora de LPA) e sobre os receptores de LPA. Ratos Wistar machos (~260 g) foram divididos nos grupos controle, I/R e I/R+CDMO (n=5). Após a coleta de sangue e urina para a avaliação da função renal, os animais foram eutanasiados e os rins removidos para os ensaios bioquímicos. Em relação à função renal foi observado nos grupos I/R e I/R+CDMO: (1) aumento do volume urinário, sem alteração no peso e no consumo de água; (2) a diminuição do ritmo de filtração glomerular e da carga filtrada de Na+; (3) diminuição da concentração sérica de K+. No entanto, o tratamento com CDMO preveniu o aumento do nitrogênio uréico plasmático e da proteinúria e promoveu o aumento da excreção urinária de K+. A concentração sérica de Na+ e o pH urinário não foram modificados nos três grupos. A análise por Western blot detectou que o tratamento com CDMO preveniu o aumento do conteúdo proteico dos receptores de LPA1 e LPA3 promovido pela I/R e diminuiu a abundancia de autotaxina. Estes dados indicam que o tratamento com CDMO previne parcialmente a perda da função renal através de sua ação tubular e não glomerular. Este evento pode ser mediado pela normalização dos níveis proteicos do receptor de LPA e pela queda da produção local deste lipídio pela diminuição do conteúdo de autotaxina. CAPES-PROBITEC,FAPERJ, CNPq-DECIT M26 - ESTUDO DA RELAÇÃO ENTRE O CICLO DE VIDA E O FENÓTIPO MAGNÉTICO NA ESPÉCIE DE BACTÉRIA MAGNETOTÁTICA MAGNETOVIBRIO BLAKEMOREI

1- LEÃO,P.E.; 1- ABREU, F.; 2- BAZYLINKI, D.A.; 1- LINS, U. 1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2-

School of Life Sciences, University of Nevada Las Vegas, USA "Bactérias magnetotáticas são capazes de se alinhar as linhas do campo geomagnético (CGM) utilizando-as para se guiar no gradiente de oxigênio encontrado em ambientes aquáticos. Esta propriedade é possível devido à biomineralização de nanopartículas



magnéticas no interior da célula chamadas magnetossomos. Os magnetossomos se alinham em cadeia na porção central da célula. Neste arranjo ocorre à soma dos momentos magnéticos das partículas e a cadeia funciona como um dipolo magnético: norte e sul. Este dipolo está fixo no corpo celular por interações proteicas, assim o torque exercido sobre a cadeia pelo CGM é transferido para a bactéria magnetotática, fazendo com que ela fique orientada em relação ao CGM. O mecanismo que associa esta orientação com a aerotaxia, que guiará o sentido de natação bacteriano, chama-se magneto-aerotaxia. Para o funcionamento do modelo de magneto-aerotaxia é preciso existir a diferenciação entre dois fenótipos baseados na orientação do dipolo magnético: Tipo sul (TS), onde o momento magnético está localizado de forma antiparalela ao flagelo celular e direção de nado e Tipo norte (TN), onde o momento magnético está localizado paralelamente a direção de ando. Segundo o modelo e a observação de amostras ambientais, o TS predomina no HS e o TN no HN. Neste trabalho estudamos como este fenótipo é preservado ao longo das gerações utilizando a bactéria magnetotática Magnetovibrio blakemorei como modelo. O inóculo contendo células sem magnetossomos foi adquirido após o cultivo em meio sem ferro. Com o inóculo, foram feitos três cultivos: dois sob a influência de um campo magnético artificial (CMA) de mesma intensidade e direção, porém sentidos opostos em cada condição, e outra condição controle, onde não houve influência de um CMA. Realizamos a quantificação de cada fenótipo nos tempos:10min/1h/3h/6h/12h/24h/36h/48h através de filtração e microscopia de fluorescência. A microscopia eletrônica de varredura será utilizada para observação da cadeia de magnetossomos e do flagelo no momento da divisão celular. Resultados preliminares sugerem que os cristais sintetizados de novo já tem uma polaridade pré-definida e que o ciclo celular bacteriano auxilia na preservação do fenótipo dominante na população." FAPERJ, CNPq, CAPES M27 - ESTUDO DOS EFEITOS DAS MUTAÇÕES L81N, I88N, L81N-I88N E L50S NA ESTABILIDADE DA PROTEÍNA CAPSÍDICA POR FLUORIMETRIA E DINÂMICA MOLECULAR

1,2-Stoque, R.M.; 1,2-Gomes, D.E.B.; 1- Pascutti, P.G.; 1-Borges, R.S.M. 1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Divisão de Metrologia Aplicada às Ciencias da Vida, Instituto Nacional de Metrologia e

Qualidade "A dengue é uma arbovirose reemergente, transmitida ao hospedeiro humano pela picada de fêmeas do mosquito Aedes aegypti. Estimativas da OMS apontam para cerca de 50 a 100 milhões de casos de infecção por dengue a cada ano e 2,5 bilhões de pessoas vivam em áreas com risco de infecção. No Brasil segundo o Ministério da Saúde houve um aumento de 521.830 casos. Devido à inexistência de uma vacina e de drogas antivirais, a principal forma de combate é o controle populacional do vetor, que tem se mostrado ineficiente, tornando-se necessário um estudo mais aprofundado do vírus para um melhor controle da doença. O vírus da dengue é formado por um envelope composto pela proteína do envelope (E), de membrana (M) e a bicamada lipídica, e um capsídeo formado pela proteína capsídica (C). Quando madura, essa proteína possui 100 resíduos de aminoácidos, positivos e hidrof bicos em sua maioria. la é formada or uatro α-hélices ligadas por pequenos loops, com massa molecular de 12kDa que prontamente se dimeriza em solução, sendo sugerido que essa seja a unidade funcional do capsídeo. No presente trabalho, as proteínas contendo mutações simples, já descritas como comprometedoras para a funcionalidade da proteína, foram expressas com sucesso em E. coli. Os dímeros formados por proteínas contendo mutações simples (L50S e I88N)

apresentaram menor estabilidade se comparados àqueles formados pela proteína C selvagem, sugerindo um papel importante destes resíduos hidrofóbicos para a estabilidade do dímero. Simulações de Dinâmica Molecular foram então realizadas com as proteínas mutantes L81N, I88N, L81N-I88N, e L50S, com a finalidade de entender o efeito destas mutações na estrutura e estabilidade das proteínas. Os resultados mostraram que as mutações propiciam a entrada de água no core hidrofóbico do dímero, sem alterar significativamente a estrutura secundária dos monômeros, mas sim afetando suas estrutura terciária e quaternária." FAPERJ, CNPq, CAPES M28 - ESTUDO DE COMPLEXOS DAS CISTEÍNO-PROTEASES PLASMODIAIS FALCIPAÍNA-2 E FALCIPAÍNA-3 PARA O DESENHO DE FÁRMACOS ANTIMALÁRICOS

LEITE, R. G. G.1,2; PASCUTTI P. G.1,2 "1- Laboratório de Biologia Computacional, Diretoria de Metrologia Aplicada às Ciências

da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia - INMETRO; 2- Laboratório de Modelagem e Dinâmica Molecular, Instituto de Biofísica Carlos Chagas

Filho, Universidade Federal do Rio de Janeiro - UFRJ." "A malária é considerada um dos mais graves problemas de saúde pública mundial e sua distribuição depende de fatores como temperatura, umidade e chuva. Os parasitas causadores da doença são protozoários pertencentes ao gênero Plasmodium sp. No Brasil as espécies P. vivax e P. falciparum são as responsáveis por, respectivamente, 15% e 85% dos casos, e esta última pela maioria das mortes. O parasita é altamente adaptável resultando na crescente resistência à terapia combinada à base de artemisina (tratamento de primeira linha), porém esta estratégia está ameaçada devido ao aparecimento de parasitas resistentes no sudeste asiático. Através da conclusão do genoma do P. falciparum, quatro cisteíno-proteases do tipo papaína puderam ser identificadas, caracterizadas e isoladas. Porém, as falcipaínas 2 e 3 (FP2/FP3) foram descritas como as principais hemoglobinases presentes no vacúolo digestivo do parasita. Isso se comprova interrompendo o gene da FP2 que leva a um acúmulo de hemoglobina não degradada. Neste trabalho, estamos estudando dois inibidores diferentes de falcipaínas, selecionados a partir dos resultados obtidos de trabalhos anteriores, que exibiram melhores valores de área de contato, ligações de hidrogênio e energia livre das ligações com cada enzima. Foi encontrado um terceiro inibidor promissor contra a FP3 devido ao seu baixo valor de energia livre de ligação, ligando-se fortemente a FP3. Simulações de Dinâmica Molecular indicaram uma grande flexibilidade das enzimas. Como perspectivas, novas simulações estão em curso, assim como o ancoramento dos inibidores nas enzimas para avaliarmos suas interações, focalizando-se no sítio ativo. Também estão sendo realizadas alterações nas estruturas dos inibidores a fim de desenvolvermos compostos mais seletivos e específicos para as falcipaínas." PRONAMETRO, CAPES. M29 - INVOLVEMENT OF TRANSCRIPTION FATOR NFAT IN Z EI ER’S ISE SE

1 - REQUIÃO, R.D.; 1 - FERREIRA, S.; 2 - ROBBS, B.K; 1 - FOGUEL, D. 1 – Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro. 2 –

Faculdade de Odontologia de Nova Friburgo, Universidade Federal Fluminense (FOUFF), Nova Friburgo.

Alzheimer's disease (AD) is a neurodegenerative disease and the primary cause of dementia in elderly. It is characterized by a progressive loss of cognitive function and it's main symptom is the loss of recent memory. The responsible factor for the clinical



manifestation of the disease is the progressive loss of synapses and neurons, especially in the hippocampus region. An important feature of AD is the presence of amyloid plaques, found in the extracellular region of patients' brains, composed mostly of the peptide beta-amyloid (Aβ). Exposed neurons to Aβ display higher levels of intracellular calcium, which could lead to the activation calcineurin A (CnA). CnA is a Ca2+/calmodulin dependent phosphatase and one of it's main targets is the transcription factor NFAT. Inhibition of NFAT leads to a prevention of both spine loss and dendritic simplification. The primary objective of the project is to define the molecular mechanisms of gene control by the transcription factor NFAT that may be involved in the AD. We already demonstrated that mice primary neuron culture treated with Aβ oligomers display NFAT translocation to the nucleus that can be completely reversed by ciclosporin A (CsA), an inhibitory drug of the NFAT activation pathway, suggesting an activation of this transcription factor. We also have evidence that these primary cultures treated with Aβ peptides have a smaller amount of dendritic spines and pre-synaptic terminals (in relation to the post-synaptic terminals), suggesting a decrease of synapses, which is also reversed by CsA. Preliminary Real Time PCR results revealed that cultures treated with Aβ oligomers show higher levels of PAK7 expression, a gene related to cytoskeleton organization, and this expression is reversed by CsA. Based on these results, we postulate that Aβ peptides are promoting a decrease of spines and synapses through activation of the calcineurin/NFAT pathway through a PAK7 overexpression. CNPq M30 - CARACTERIZAÇÃO ESTRUTURAL DA PROTEÍNA TWA1

1-ARAUJO, T. S.; 2-CIRAUQUI, N.; 1- ALMEIDA, M. S. 1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Faculdade

de Farmácia, Universidade Federal do Rio de Janeiro. Este trabalho tem como objetivo a determinação da estrutura tridimensional da proteína Twa1, uma proteína nuclear de 228 aminoácidos, identificada em 2003, através de um sistema de duplo-híbrido em levedura tendo como "isca" a proteína RanBPM. A análise da sequência primária da proteína Twa1 mostra que a mesma possui três domínios conhecidos: o domínio LisH, o domínio CTLH e o domínio CRA, dos quais os dois últimos permanecem com função e estrutura desconhecidas. O domínio LisH é frequentemente encontrado em proteínas envolvidas na segregação de cromossomos e migração celular. Além disso, estudos mostram que este domínio está envolvido na formação de homodímero. A Twa1 e RanBPM formam, em conjunto com outras proteínas, um complexo denominado Complexo CTLH, que apresenta atividade enzimática E3 ubiquitina ligase. A ubiquitinação de proteínas é uma importante forma de modificação covalente que regula diversos processos biológicos. Para realização deste trabalho três diferentes construções da proteína Twa1 foram produzidas em Escherichia coli sendo elas: Twa1 selvagem, Twa 1-212 (ausência de 16 aminoácidos do c-terminal que são sensíveis a proteinase K) e Twa1 66-209 (compreende os domínios CTLH e CRA). Neste estudo, realizamos a caracterização estrutural das diferentes construções através de ensaios de Gel Filtração, Dicroísmo Circular, Fluorescência Intrínseca do Triptofano, Proteinase K e Ressonância Magnética Nuclear. Os resultados obtidos mostram que as proteínas produzidas apresentam um enovelamento parcial e com conte do de estrutura secund ria de α-hélice. No momento estamos buscando melhorar as condições de produção da proteína recombinante Twa1 para que possamos determinar sua estrutura tridimensional por RMN em solução ou cristalografia por raios-X. INBEB, FAPERJ, CNPq.

M31 - INVESTIGAÇÃO IN SILICO DO MECANISMO DE INIBIÇÃO DA PROSTAGLANDINA E MICROSSOMAL SINTETASE I (MPGES-1).

1-FROES, T.Q. (PG), 1- SOARES, D.M.(PQ), 1- CASTILHO, M.S.(PQ) 1- Facculdade de Farmácia, Universidade Federal da Bahia

A prostaglandina E microssomal sintetase I (mPGES-1) é a última enzima responsável pela síntese de PGE2, principal prostanoíde envolvido na resposta inflamatória, a partir de PGH2 usando glutationa como cofator. Dessa forma ela é considerada um alvo promissor para o desenvolvimento de antiinflamatórios mais seletivos e seguros que os AINEs e os inibidores seletivos de COX-2. Embora dezenas de inibidores de mPGES-1 tenham sido descritos, não existem dados estruturais que comprovem o modo de ligação para nenhum deles. Por essa razão, empregamos técnicas de similaridade química e modelos farmacofóricos para determinar o provável modo de interação de 5 classes de inibidores na estrutura cristalográfica de mPGES-1. Inicialmente 127 inibidores da mPGES-1 foram sobrepostos na bis-fenil-glutationa (análogo do cofator)

-octil glucosídeo (análogo do substrato) complexados com a mPGES-1 (PDB: 4AL1). A seguir, as 5 moléculas de melhor sobreposição foram utilizadas para gerar modelos farmacofóricos, os quais foram avaliados quanto a capacidade de diferenciar inibidores verdadeiros de falsos positivos. Em ambos os casos nenhum falso positivo foi reconhecido, mas o modelo gerado a partir da bis-fenil-glutationa recupera um número

-octil glucosídeo (108). Esses dados sugerem que a maior parte dos inibidores podem se ligar tanto no sítio da gluationa quanto no sítio do substrato, o que está de acordo com dados recentes da literatura. Visando corroborar essa hipótese realizamos o acoplamento molecular de inibidores rígidos da mPGES-1 (derivados de fenantreno) na cavidade que seria ocupada pelo cofator e o substrato. Os 5 inibidores de maior pontuação foram utilizados para a construção de um novo modelo farmacofórico, o qual apresenta 2 doadores ligação de hidrogênio, 1 aceptor de ligação de hidrogênio e 5 grupos hidrofóbicos ou aromáticos. Esse modelo é superior aos anteriores pois reconhece 124 inibidores. INBEB, CAPES, CNPq. M32 - EFFECT OF EXTRACTS SCHINUS TEREBINTHIFOLIUS AND PUNICA GRANATUM ON MAYARO VIRUS REPLICATION.

1,3- SALLES, T. S.; 1- MENESES, M. D. F.; 1- GUIMARÃES, T. ;2- KUSTER, R. M.; 3- SOARES, M. R.; 1- FERREIRA D. F.*

1- Laboratório de Interação Vírus Célula, Departamento de Virologia, Instituto Microbiologia Prof. Paulo de Góes. Centro de Ciência e Saúde, Universidade Federal do

Rio de Janeiro; 2- Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro; 3- LAMMP, Departamento de

Bioquímica, Instituto de Química, Centro Tecnológico, Universidade Federal do Rio de Janeiro. *[email protected]

Mayaro virus (MAYV) is an arbovirus belonging to the genus Alphavirus, Togaviridae family which causes a disease known as Mayaro fever presenting symptoms similar to the classic dengue fever. MAYV is endemic in tropical and subtropical regions on the borders of the Amazon Basin. The Brazilian popular medicine explores the anti-inflamatory properties of four substances extracted from the plants Schinus terebinthifolius (known as mastic) and two substances Punica granatum (known as Pomegranate). In this work we tested the antiviral effect of these substances against the Alphavirus MAYV. The toxicity of these substances in VERO cells was tested by the incorporation of neutral red after 24h of treatment. The CC50 of each substance was determined. For evaluation of the antiviral effect, cells were infected for 1 h with Mayaro virus using a multiplicity of infection of 0,1. Treatment of cells was carried out


mailto:*[email protected]


for 24 hours with increasing concentrations. The viral production was quantified by TCID50 method and it was observed that the substances exhibit antiviral activity. The selectivity index (ratio CC50/IC50), was determined and the values were greater than 7 for all six substances. We also tested the substances for virucide properties, adsorption impairment, pretreatment, immunofluorescence and transmission electron microscopy (TEM). Our results showed more than 95% virucidal effect for the partitions acetate, flavonoids mastic and Crude Extract of pomegranate. Mastic oil and acetate of pomegranate did not present virucidal effect. Virucidal effect was also visible by immunofluorescence images. Was also observed in the TEM images damage in viral particles treated with substances. We conclude that some of the partitions in our substances act directly on the virus particle, and we are now assaying for this interaction at the molecular level. INBEB, FAPERJ, CAPES. M33 - EXPLORING BIOTECHNOLOGICAL FEATURES OF HYDROLYTIC XANTHOMONAS AXONOPODIS PV. CITRI ENZYMES 1- QUEIROZ, V.L.; 1- SCHULTZ, L.G.; 1- MARIÑO, M.; 1- CYPRIANO, D.Z.; 1- LEITE, H.C.; 1-

TASIC, L. 1- Laboratório de Química Biológica, Instituto de Química, Universidade Estadual de

Campinas "The search for renewable energy resources has become increasingly important as a sustainable alternative to petroleum derived fuels. Brazil is the biggest producer of oranges in the world, with 50% of the orange fruit used for obtaining juice and the other 50%, comprising bagasse, is currently dried and processed into pellets for livestock feed. Orange bagasse has high levels of carbohydrates, high susceptibility to enzymatic hydrolysis and low costs; moreover, its use does not compete with food production. Such features make the orange bagasse a promising carbon source for second-generation ethanol production. Previous studies have shown that whole enzymatic extract from Xanthomonas axonopodis pv. citri (Xac), a potent pathogen responsible for the citrus canker disease, was capable of hydrolyzing orange bagasse that was later fermented to produce second-generation ethanol. The aim of this work is to improve the efficiency of hydrolysis of orange bagasse for the production of second-generation ethanol through the study of hydrolases purified from Xanthomonas axonopodis pv. citri. Herein, we compared the structures of our target enzymes to the ones already crystallized and combined these results with peptide-signal prediction tools to construct primers for cloning of these enzymes. Physicochemical characterization and substrate kinetics will be done after successful enzyme purification. We expect that cocktail of purified Xac enzymes can provide higher yields

of fermentable sugars from orange bagasse and, consequently, higher yields of ethanol-2G in fermentation processes. INBEB M34 - MOLECULAR CLONING AND EXPRESSION OF DISINTEGRINS FROM THE VENOM OF BOTHROPS JARARACA

1 - DAVID, V.; 1 - GUERRERO, T.; 1 - GERALDO, R. B.; 3 - ALBANO, R. M.; 2 - WERMELINGER, L. S.; 1 - ZINGALI, R. B.

1Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, RJ, Brazil; 2 Departamento de Análises Clínicas e Toxicológicas, UFRJ, RJ, Brazil; 3 Departamento de Bioquímica, UERJ,

RJ, Brazil; Introduction: Snake venoms contain several components, including disintegrins that are a family of small peptides able to bind to integrin receptors. These integrins are glycoproteins that play a fundamental role in regulating physiological and pathophysiological processes, such as in arterial thrombosis. Disintegrins have been used to develop new drugs some are now used in treatment of human diseases. Two disintegrins isolated from Bothrops jararaca, in our laboratory, called jarastatin (JARC) and jararacin (JAST), have the ability to inhibit platelet aggregation. In this study, we aim to clone the rJARC and rJAST disintegrins from the venom of Bothrops jararaca using the vector pET-15b, aiming to have enough amount of peptide to structural studies, once the last vector pET-32a was unsuccessful. Materials and Methods: The disintegrins were amplified from the cDNA library using Bothrops jararaca gland. Then, an agarose gel 1.5% was performed and the DNAs were extracted and digested with restriction enzymes. The products of these reactions were subcloned into Pet-15b and transformed in bacteria Escherichia coli BL21. After that, E. coli was coated on agar, containing ampicillin and chloramphenicol, and incubated at 37°C for 16h. The colonies obtained were grown in circle growth medium to verify the integrity of the plasmid by enzymatic digestion and the expression. Results and Discussion: Amplification of the cDNA was confirmed by the profile of agarose gel, showing bands corresponding to 243 bp of inserts of disintegrins. After transfection of the plasmid the bacteria growth was observed as colonies on the plates. In addition, the integrity of the plasmid was confirmed to rJAST through the agarose profile gel that showed bands of 243 bp and the expression was successful, while rJARC showed several random bands. Conclusions: It was possible to clone the rJAST successfully. New trials in different conditions are required for cloning of rJARC. FAPERJ, CNPq and Capes



Doutorandos D1 - BONE MARROW MONONUCLEAR CELL THERAPY AFTER GLOBAL CEREBRAL ISCHEMIA IN RATS

1- RAMOS, AB., 1- BARRIA, AC., 2- NEVES, G., 2- CINTRA, WM., 1- MENDEZ-OTERO, R. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-

Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro. "Introduction: The pyramidal neurons of the hippocampal CA1 layer are essential for cognitive functions and selectively destroyed after global cerebral ischemia. Bone marrow mononuclear cell (BMMC) therapy has shown positive results in preclinical models of focal cerebral ischemia, but has not been studied in transient global ischemia. Aim: The aim of our study was to investigate whether BMMC treatment could reduce the neurodegeneration and the inflammation observed in CA1 layer of ischemic animals. Methods: Transient forebrain ischemia was performed by four vessels occlusion method. To establish the time course of CA1 neurodegeneration CA1, the rats were transcardially perfused 3, 7, 14, 21 and 90 days after ischemia (DAI). To analyze the effect of BMMC therapy in neurodegeneration, neuronal survival and reactive microgliosis in CA1 layer, ischemic animals received 3x107 BMMC 3 DAI, in the left carotid, and were sacrificed different days after ischemia. For quantification of neuronal degeneration, survival and reactive microgliosis, we counted Fluoro-Jade C, NeuN and ED-1 positive cells in CA1 layer, respectively. Results: We observed a greater number of FJC positive cells in CA1 layer of ischemic animals 7 DAI when compared with the others days. The time course of neuronal survival shows a reduction of pyramidal neurons 7 DAI and in the days after. In the analysis of the effect of BMMC in neurodegeneration and neuronal survival, we observed a significant number reduction of FJC positive cells and increase of NeuN positive cells in CA1 of animals injected with BMMC and sacrificed 7 DAI. Furthermore, we observed a lower number of ED-1 positive cells in ischemic animals treated with BMMCs compared with ischemic animals that received only saline. Conclusion: We suggest that the BMMC therapy in transient global ischemia has a neuroprotective effect in CA1 layer that was followed by reduction of microgliosis in this region." CNPq, Pibic, FAPERJ D2 - TRATAMENTO SISTÊMICO COM GUANOSINA EM UM MODELO DE LESÃO NO NERVO ÓPTICO

SILVA-JUNIOR, A.J.; MESENTIER-LOURO, L.A.; ZAVERUCHA-DO-VALLE, C.; MENDEZ-OTERO, R.; SANTIAGO, M.F.

Instituto de Biofísica Carlos Chagas Filho Optic nerve lesions can lead from partially loss of vision to complete blindness. These types of lesions affect especially the retinal ganglion cells (RGCs). One potential therapy for this case is the administration of the nucleoside guanosine. The guanosine treatment has demonstrated neuroprotective effects in several models. This work invesgated the putative neuroprotective effect of systemic administrations of guanosine in a model of optic nerve lesion. Lister-Hooded rats were submitted to crushing of the optic nerve, followed by intraperitoneal injection of guanosine or vehicle. The treatment was evaluated in three distinct protocols. In Protocol 1the groups received just only one guanosine injection (7,5 mg/kg). In Protocol 2, the animals received a daily injection of

guanosine for seven days (7,5 mg/kg). In Protocol 3, the animals received 1 injection every 12 hours for seven days (60 mg/kg). The experimental groups used in Protocols 1 and 2 were evaluated fourteen days after surgery, while the groups were analyzed after seven days. The RGC survival was assessed by Brn3a immunostaining in whole mount retinas. The number of RGCs in the fellow contralateral eye was used as control. There was no significant difference in the number of RGCs in guanosine groups when compared to the vehicle groups in all experimental protocols tested. The number of extended axons beyond the lesion site in guanosine-treated animals was similar to the vehicle-treated ones. We conclude that the guanosine systemic treatment using these doses and therapeutic windows was not effective to promote the survival and axonal extension in RGCs after optic nerve lesion. INBEB, FAPERJ, CNPq, CAPES D3 - STRUCTURAL STABILITY OF MS2 VIRUS-LIKE PARTICLES DISPLAYING FOREIGN PEPTIDES.

1- VICENTE, A.C.S.; 1- BARROSO, S.P.C.; 1- OLIVEIRA, E.G.; 1- OLIVEIRA, G.A.P.; 2- PEABODY, D.S.; 3- FERREIRA, D.F.; 4- CORDEIRO, Y.; 5- DE MESQUITA, J.F.; 1- SILVA, J.L.

& 1- OLIVEIRA, A.C. 1- Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis,

Universidade Federal do Rio de Janeiro; 2- Department of Molecular Genetics and Microbiology, University of New Mexico; 3- Instituto de Microbiologia Prof. Paulo de

Góes, UFRJ; 4- Faculdade de Farmácia, UFRJ; 5- Universidade Federal do Estado do Rio de Janeiro.

Virus-like particles (VLPs) can be considered as dense arrays of one or more repetitive subunits of a protein and this characteristic confers highly advantageous properties for their use as vaccines platforms. The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. Here, we describe a platform for vaccine development based on the VLPs of bacteriophage MS2. Peptides representing the V3 loop of HIV gp120, the ECL2 loop of the HIV coreceptor, CCR5, and the epitope Flag were inserted into a surface loop of MS2 coat protein. The effects of insertion of these peptides on the structural stability of MS2 VLPs were assessed by dynamic light scattering, small-angle x-ray scattering (SAXS), intrinsic and extrinsic fluorescence and circular dichroism (CD). We analyzed the morphology of VLPs by transmission electron microscopy. In addition, we predicted the structure of the coat protein containing the different inserts. The VLPs displaying the Flag, V3 and ECL2 peptides on their surfaces showed a different structure and lower stability than VLP formed by the native coat protein. CD measurements indicate no significant changes in secondary structure between VLPs under high concentration of urea. SAXS data show that the effect of 3 hours of pressurization was not able to promote the VLPs disassembly. Our results demonstrate that the VLPs assembled from coat protein containing peptides insertions behave slightly differently from the ones assembled from native coat protein, however they showed structural stability under most of the conditions used, suggesting that these particles are very promising for application as a vaccine platform. CAPES-FAPERJ-CNPq-PRONEX-INBEB D4 - ENVOLVIMENTO DA VIA AMPK-eNOS-N κB N P PE NEUROPROTETOR DO SILDENAFIL (VIAGRA®) EM MODELO DESMIELINIZAÇÃO

1- NUNES, A.K.S; 2-RAPÔSO C; 1 - OLIVEIRA W.H; 1-BARBOSA K.P; 1-ROCHA S.W.S, 2-CRUZ-HOFLING M.A; 1-PEIXOTO C.A.

1-Laboratório de Ultraestrutura - Instituto de Pesquisas Aggeu Magalhães - FIOCRUZ; 2-Departamento de Histologia e Embriologia, Universidade Estadual de Campinas –

UNICAMP



Recentemente, foi demonstrado que o sildenafil (Viagra®) tem papel neuroprotetor, inibindo a inflamação e desmielinização no cerebelo. No entanto, os mecanismos de neuroproteção ainda são desconhecidos. O AMPK é uma proteína reguladora do metabolismo lipídico e glicídico e sua ativação modula processos inflamatórios, possivelmente através da ativação da eNOS. O fator de transcrição nuclear NFkB desempenha um papel importante na regulação da expressão de mediadores inflamatórios. Estudos têm mostrado que a sinalização através da AMPK pode suprimir a ativação do NFkB, controlando a inflamação. O presente trabalho utilizou ressonância magnética (RM) e investigou se a via AMPK-eNOS-NFkB está envolvida na neuroproteção, em modelo de desmielinização induzido por cuprizona. Cinco camundongos machos (C57BL/6) foram separados por grupo e tratados por quatro semanas: cuprizona (CPZ) 0.2% misturada na ração; CPZ na ração associada à administração de sildenafil (25 mg/kg) por via oral, começando concomitantemente (sild-T0) ou 15 dias (sild-T15) após o início da CPZ. Os controles receberam ração e água puras. Os animais foram submetidos a análises por RM e os cerebelos foram processados para western blotting. A análise por RM mostrou que, após a ingestão de CPZ, os animais apresentaram redução do corpo caloso e dilatação dos ventrículos cerebrais, indicando perda de estrutura. Os animais do grupo sild-T0 apresentaram recuperação parcial da estrutura do corpo caloso, bem como diminuição da dilatação dos ventrículos, o que não foi observado no grupo T15. A expressão de AMPK inativa aumentou e os níveis de eNOS diminuíram após a administração de CPZ. Apenas o tratamento com sildenafil (T0) reverteu esses efeitos, aumentando significativamente a expressão da eNOS e diminuindo os níveis de AMPK. De forma semelhante, a expressão de NFkB total foi aumentada após a ingestão de CPZ. Sildenafil (T0 e T15) diminuiu significativamente os níveis de NFkB. Esses dados indicam que os efeitos neuroprotetores do sildenafil envolvem a via AMPK-eNOS- FκB, sendo esta droga potencialmente útil no tratamento de doenças desmielinizantes. INBEB, PAPES, FACEPE, CNPq, CAPES D5 - A TIME-DEPENDENT NEUROINFLAMMATORY RESPONSE AFTER ACUTE RESTRAINT STRESS

Oliveira-Poleto, A.L.; Guercio, G.D.; Panizzutti, R. 1- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro

"Stress comprises a series of physiological events that prepare the organism for a challenge. Acute stress induces cognitive deficits, which is accompanied by inflammatory response in some brain structures. In a previous study we found that acute stress decreases the levels of the neuromodulator D-serine in the frontal cortex but not in the hippocampus. Therefore, our aim was to investigate whether the inflammatory response is important for the stress- induced decrease in D-serine levels. To this end, we used imunohistochemistry to evaluate microglial activation and astrogliosis in after 90 or 180 min of acute restraint stress protocol in C57Bl/6 male adult mice. We observed increased microglial activation, in the frontal cortex but not in the hippocampus, after 90 min of stress. This increase in microglial activation in the frontal cortex was not observed after 180 min of stress. The astrogliosis measured using GFAP in the frontal cortex was not affect after 90 min of stress but it was reduced after 180 min of the stress. In the hippocampus astrogliosis was reduced after 90 min of stress and increased after 180 min of stress. These results indicate that our experimental model is capable to induce different profiles of inflammatory response in frontal cortex and hippocampus. Further analyses must be made to understand if this process is involved in the stress-induced decrease in D-serine levels, and if anti-inflammatory interventions could ameliorate the cognitive deficits induced by acute stress."

CNPq, Faperj D6 - MODULATION OF THE ECTO-3’NU E I SE I I Y EIS NI Z NENSIS BY 5’NU E I ES.

1-FREITAS-MESQUITA, A. L., 1- GOMES, M.T., 2-VIEIRA, D. P., 2-LOPES, A. H. C. S., 1- MEYER-FERNANDES, J. R.

1- Instituto de Biquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro; 2- Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do

Rio de Janeiro INTRODUCTION: Leishmania amazonensis parasites are intracellular protozoa and the etiological agent of cutaneous leishmaniasis. The flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites and develop into amastigotes inside macrophages. During this lifecycle, membrane interactions between parasites and hosts are crucial for its survival, from both physiological and immunological viewpoints. Cytoplasmic membranes contain enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ecto-enzymes, can be measured using intact living cells. Leishmania species present an ecto-en yme that hydroly es both e tracellular 3’-nucleotides and nucleic acids. The hydrolysis of 3’ MP generates adenosine that lays im ortant roles in immunological events and in the acquisition of purines, since trypanosomatids are unable to synthesize purines de novo. This work aimed to investigate and characterize the modulation of ecto-3’nucleotidase acti ity from . ama onensis by 5’nucleotides. M TERIAL E METHODS: Ecto-3’nucleotidase acti ity was determined colorimetrically by measuring the content of generated phosphate after one hour of reaction. The reaction medium contained buffer solution, 3 mM 3’ MP, . ama onensis romastigotes and increasing concentrations of different 5’nucleotides. R U T I CU I : Micromolar concentrations of guanosine, 5’GMP, G P and GTP inhibited ecto-3’nucleotidase activity, which was not observed when other nucleotides were tested. The analysis of kinetic parameters indicated that in the resence of 5’GMP the a arent Km and Vma for 3’ MP hydrolysis were lower than control alues, which suggests the occurrence of an uncom etiti e inhibition. It is already known that 3’ MP increases macro hage infection by parasites. We obser ed that 5’GMP was able to attenuate the increase of interaction romoted by 3’ MP. C C U I : tracellular nucleotides are intimately involved in the regulation of ecto-3’nucleotidase acti ity. The determination of these regulatory mechanisms is very important, since this enzyme seems to play an important role in the establishment of Leishmania infection. CNPq, CAPES and FAPERJ (INCT – INBEB) D7 - INTERFERENCE OF MYOSIN VA OR VB FUNCTION DISRUPTS HUMAN RHINOVIRUS 14 REPLICATION

1,2- Real-Hohn, A.; 3- Provance Jr., D.W.; 2- Salerno, V.P.; 1- Gomes, A.M.O. 1- Instituto de Bio u mica Médica eo oldo e Meis, UFRJ - scola de duca o F sica

e Desportos, UFRJ; 3- Centro de Desenvolvimento Tecnológico em Saúde, Fiocruz Human rhinovirus infections are the major cause of the common cold. Following cell entry, the virus releases its RNA genome into the cytoplasm and commandeers the resources of the cell for replication. The mechanisms for translocation of the virus genome to the viral replication center in the perinuclear region remain unknown. ndogenous myosin V’s are e cellent candidates since they are in ol ed in both R transport and intracellular trafficking from the periphery to the cell center. Dominant negative expression vectors, immunofluorescent localization and immunoprecipitation studies all suggest that both myosin Va and Vb function contribute to viral replication. Furthermore, the results suggest that each participates at different stages in the cycle



with myosin Va early and myosin Vb late. Together, the data provide greater insight into the cellular resources utilized by HRV-14 to propagate, which have implications in the processes involved with Poliovirus, Hepatitis A virus and Foot-and-mouth disease virus. INBEB, FAPERJ, CNPq, Capes, FINEP, PRONEX D8 - NMR STRUCTURE OF Q4D059, A CONSERVED AND SPECIFIC HYPOTHETICAL PROTEIN FROM KINETOPLASTIDS

LOPEZ-CASTILLA, A.; D' ANDREA, E.; MENEZES, R.; PIRES,. JR Instituto de Bioquímica Médica Leopoldo de Meis

In a structural genomics context, here we report the expression, purification and structural characterization of Q4D059 (Uniprot entry), an 86 residue-hypothetical protein from Trypanosoma cruzi, essential in Trypanosoma brucei and also conserved in Leishmania major, protozoan parasites of the kinetoplastid order. These parasites are the causal agents of the neglected diseases: Chagas, Sleeping Sickness and Leishmaniases respectively and had recently their genomes fully sequenced. Currently, there are no vaccines to prevent or treat these infections and available drugs are inadequate because of resistance and toxicity. Q4D059 shows low-sequence homology with mammal proteins, and because of its essentiality demonstrated in T. brucei, it is a potential target for anti-parasitic drugs. NMR and structural data is desirable facilitating drug screening and providing insights on Q4D059 function, since primary sequence homology searches only match trypanosome hypothetical proteins of unknown function. We solved the structure of Q4D059 by standard solution NMR techniques. It is composed by a parallel/anti-paralell three-stranded -sheet packed against two almost parallel oriented -helices. The structure is well defined by ca. 9 NOEs per residue and a backbone RMSD of 0.45 ± 0.19 Å was obtained for the 15 lowest-energy structures out of 200 calculated. We also investigated the dynamics by 15N R1, R2 relaxation rates and 1H-15N NOE measurements. The structure is overall rigid except for residues from 9 to 11 undergoing rapid motion in the ps-ns timescale, and V78 with motion in the µs-ms timescale. A search for structural homologs using the program DALI identified the subdomain IIB of the human heat shock protein 70kDa with the best Z-score (2.9); suggesting an evolutionary relationship between Q4D059 and an ATP-binding domain. CAPES, CNPq, FAPERJ – Brazil D9 - LIPIDOMIC PROFILING OF THE AMAZONIAN FISH

1- BARBOSA, B. S.; 2- VAL, A. L.; 1- TASIC, L. 1- Laboratório de Química Biológica, Universidade Estadual de Campinas; 2- Laboratório

de Ecofisiologia e Evolução Molecular, Instituto Nacional de Pesquisas da Amazônia "The importance of phospholipids (PL) to human health is mainly related to the significant quantities of fatty omega-3 acids in their structures. Characterization of the lipids in their intact form is scarce and remains challenging due to the complexity and specificity of these biomolecules. Opposed to the proteomics and genomics, lipidomics is not based on information that can predict the number and composition of individual lipid molecules present in an organism. Also, quantity and type of lipids may vary due to many factors, such as alimentation habits, still poorly understood. Thus, this work involves the investigation of lipids and phospholipids (PL) isolated from the dorsal muscle and/or liver from nine Amazonian fish species. All fish samples were collected during two distinct periods of the Amazon River in 2013, flood and drought, and belong to groups with different feeding habits. At least three samples from the same specie were analyzed. Using classical Bligh-Dyer technique, total lipids were isolated, then fractionated and studied applying liquid nuclear magnetic resonance spectroscopy.

The obtained results are very interesting and contribute to better understanding of biosynthetic pathways for Amazonian fish phospholipids (PL), whose structures and types of linked fatty acids are very different compared to other fish lipidomics. We also expect that clinical trials and subsequent inclusion into the human diet that are underway (collaborative work with the Faculty of Medicine, UNIFESP) can bring new insights in treating neurodegenerati e diseases such as Parkinson’s, l heimer’s, and/or untington’s." INBEB, FAPESP, PRP- UNICAMP D10 - DEVELOPMENT OF NANORADIOPHARMACEUTICAL FOR BONE METASTASIS

1,2- PATRICIO, B. F. D. C.; 2- SANTOS-OLIVEIRA, R.; 2- WEISSMÜLLER, G. 1- Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Centro de

Ciências da Saúde, Universidade Federal do Rio de Janeiro. 2- Laboratório de Nanorradiofármaco, Instituto de Engenharia Nuclear, Rio de Janeiro.

"Introduction: Cancer is a problem of worldwide concern and future perspectives are worrisome. After non-melanoma skin cancer, the two types of highest incidence are the breast and prostate. This ones lead with high frequency to bone metastasis. The treatment with radiopharmaceutical stands out a palliative treatments witch improves the quality of life and increase survival time. The 153Sm-EDTMP shows 70-80% of patients with a clear improvement. The major disadvantage of this radiopharmaceutical is the requirement of multiple doses. The nanopharmacology is a growing branch promising properties such as controlled, prolonged and sustained release of the active ingredient, reduction of the required dose for therapeutic effect and the toxic effects. Objectives: In order to circumvent the deficiencies of the radiopharmaceutical 153Sm-EDTMP, this work has the objective to enclosure the EDTMP in polymeric nanocapsules of PLA/PVA. Methods: Nanoparticles: They were produced by the method of double emulsion using poly-lactic acid (PLA) and poly-vinyl alcohol (PVA) as polymers and EDTMP as the drug. Characterization of the nanoparticles: The morphology and dimension of the nanoparticles were obtained by atomic force microscopy (AFM) using Peak Force Tapping Mode (PFQNM)®. Results: The nanoparticles showed a medium dimension 250nm, spherical shapes and a heterogeneous surface showed only in the adhesion and elastic images obtained with PFQNM. Conclusion: The nanoparticles were successfully manufactured by the double emulsion methodology and this new mode of analysis by AFM allows the evaluation of the nanoparticles physical properties." INBEB, CNPq D11 - PATHOLOGICAL IMPLICATIONS OF NUCLEIC ACIDS INTERACTIONS WITH PRION PROTEINS: IN VITRO AND IN VIVO APPROACHES Bruno Macedo¹, Mariana P. B. Gomes¹, Natália C. Ferreira1, Matheus Tempone¹, Priscila S. Ferreira², Felipe Campos², Fernanda Neves¹, Julia Clarke², Jerson L. Silva², Claudia P.

Figueiredo1 e Yraima Cordeiro¹ 1 - Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro,

Brazil; 2 - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

INTRODUCTION: Prion proteins (PrPs) are involved in lethal neurodegenerative diseases that affect humans and other animals. So far, many issues remain unclear about PrP physiopathological role. The misfolding of its normal cellular form (PrPC) into an abnormal form (PrPSc) characterizes the central event of prion diseases. Some studies have suggested nuclear localization for PrPC and functions derived from its interaction with nuclear components. Having a common place to cross-talk, nucleic acids (NAs) are



intriguing PrP molecular partners, and have been proposed as cofactors in the protein misfolding process. OBJECTIVES: Clarify the biological relevance of PrP-NA interactions. MATERIALS AND METHODS: We investigated the binding of several oligonucleotides (RNA and DNA) with the recombinant murine PrP (rPrP) through isothermal titration calorimetry (ITC) and spectroscopic assays. We performed different cellular assays to evaluate cytotoxicity and cell viability, working with two cell lineages (neuroblastoma - N2a - and kidney cells – HK2). We also investigated interaction of a well-known neurotoxic human prion peptide (PrP106-126) with the oligonucleotides. RESULTS AND DISCUSSION: We found that interactions of rPrP or PrP106-126 with NAs can lead to different aggregates and neurotoxic species, depending on the NA molecule. Interestingly, the N2a cells morphology changed in the presence of the cytotoxic PrP-NA complexes, indicating that they are affecting cell signaling. The preliminary in vivo results of the intracerebral inoculation of a rPrP-DNA complex in healthy mice (C57BL/6) indicate significant impairment of memory consolidation, although there was no apparent locomotor dysfunction in these mice through extensive behavioral tests. CONCLUSIONS: Our results show the NA ability of binding to rPrP, induce conformational changes, modulate protein aggregation and cytotoxicity in vitro and to induce neurological damages in vivo. Understanding the structural, cellular and in vivo effects observed for PrP and NAs interactions may enlighten the still obscure prion physiopathology. FAPERJ, CAPES, CNPq D12 - VÍRUS DA INFLUENZA HUMANA INATIVADO POR PRESSÃO HIDROSTÁTICA: INVESTIGANDO UM CANDIDATO PARA VACINA UNIVERSAL

1-DUMARD, C.H.; 1-Félix, A.L.; 1-SOUZA-SANTOS, P.; 1-BARROSO, S.P.C.; 2-NICO, D. 1- SILVA, J.L.

1-Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Instituto de

Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro A gripe é uma doença inflamatória causada pelo vírus da influenza, que se divide nos gêneros A, B e C. Estima-se que anualmente ocorram de 3 a 5 milhões de casos de doença grave, e entre 300 a 500 mil mortes. A vacinação é a forma mais efetiva para prevenção da doença e suas complicações. Os principais modelos de vacina se baseiam em proteínas virais purificadas ou em vírus atenuados. As vacinas baseadas em proteínas purificadas raramente geram complicações, mas também geram uma resposta imunológica mais pobre oferecendo pouca proteção contra os diversos subtipos. A vacina atenuada gera uma resposta imunológica mais eficiente, com produção de anticorpos de mucosa, no entanto pode gerar mais reações adversas e apresenta restrições para ser administrada em grupos de risco. Em nosso trabalho testamos um novo modelo de vacina baseado na inativação por pressão do vírus da influenza humana H3N2. Camundongos apresentaram resposta imunológica serológica e de mucosa ao receberem duas doses de vacina. Além disso, estes animais se mostraram eficientes ao produzirem citocinas após a vacinação. Também observamos resposta de anticorpos de memória nos animais avaliados 3 meses após vacinação. Animais situados nos grupos de risco também foram capazes de gerar anticorpos. Resposta de anticorpos in vitro contra a hemaglutinina (principal glicoproteína viral) foi induzida contra diversos subtipos de antígenos virais, indicando um largo espectro de proteção. A vacina inativada por pressão hidrostática mantém as atividades das glicoproteínas virais sendo capaz de se ligar e entrar na célula. Por apresentar muitas das etapas do ciclo infeccioso preservada sem, no entanto apresentar infecciosidade, este modelo pode ser a base de um modelo de vacina capaz de gerar resposta

imunológica contra os diversos subtipos sendo ao mesmo tempo segura para indivíduos em grupos de risco. INBEB, FAPERJ, CAPES, CNPq D13 - ANALYSIS OF CARBOHYDRATE EXPRESSION ON THE SURFACE OF INTESTINAL EPITHELIUM OF Lutzomyia longipalpis and Lutzomyia antunesi (DIPTERA, PISYCODIDAE) OF PARA STATE.

1-OLIVEIRA, D. M. S.; 1,2-FARIAS, L. H. S; 1,2-SILVA, B. J. M ;1-SILVA, E. O1. 1-Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências

Biológicas, Universidade Federal do Pará, Brazil.; 2-Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro,

Ilha do Fundão, Brasil Leishmaniasis are worldwide diseases that occur in 98 countries. In Brazil cases of American Visceral Leishmaniasis (AVL) and Cutaneous Leishmaniasis (CL) has been registrated in all State Federation including Pará State. Lutzomyia (Lutzomyia) longipalpis is the putative vector of Leishania infantum chagasi the etiological agent of AVL while Lutzomtya (Nyssomyia) antunesi is the proven vector of Leishmania (Viannia) lindenbergi suspected of causing CL. The specificity parasite-sandflies interaction is mediated by leishmania surface molecules, as Lipophosphoglycan (LPG), which is recognized by receptors of midgut epithelium. However in permissive sand flies as L. longipalpis the interaction is mediated by a lectin-like activity on the parasite surface and the presence of glycoproteins containing N-acetyl- galactosamine (GalNAc) of epithelial insect midgut. Thus, the aim of this work was to compare the expression of glycoprotein containing GalNAc in the midgut of L. longipalpis obtained from colony and forest and glycoprotein of the midgut of L. antunesi collected from forest area of Cametá Municipality of Pará. Phlebotomines were dissected in paraformaldehyde 4% and each midgut was sectioned longitudinally. After that, incubated with FITC conjugated Helix pomatia lectin (HPA-FITC) and Concanavalin A (Con-A-FITC) which are specific for GalNAc and mannose, respectively. Lysates of seven midguts of each phlebotomine species from colony and forest were analyzed by SDS-PAGE followed by western blotting with HPA and Con-A . All midgut lysates from both samples displayed peptides ranging from 35 to 75 kDa and 51 kDa. These bands react with HPA and CON-A, respectively, suggesting presence of GalNAc and mannose residues in the midgut of the sand flies. These results indicated that glycoprotein containing GalNAc and mannose residues occurs in both species studied indicating the presence of permissive sand flies to the development of different leishmanias species in endemic areas for AVL and CL of Para state CAPES, CNPq/UFPa, INBEB/FAPERJ, MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ and Evandro Chagas Institute. D14 - ANTI-INFLAMMATORY EFFECTS OF DIETHYLCARBAMAZINE ON LUNG INJURY INDUCED BY MONOCROTALINA IN MICE

1,3-RIBEIRO, E.L.;1,3- FRAGOSO, I.T.; 1,3-SILVA, A.K.S; 3,4-DONATO, M.A.M.; 1,3; GOMES, F. O.S.; 2,3-OLIVEIRA, A.C.; 3- SILVA, B.S.; 1,3 PEIXOTO, C. A.

1.PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE - PE - BRASIL; 2.GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS,

UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE - PE - BRASIL; 3.LABORATÓRIO DE ULTRAESTRUTURA, INSTITUTO AGGEU MAGALHÃES, FIOCRUZ, RECIFE - PE - BRASIL. 4.

FACULDADE INTEGRADA DE PERNAMBUCO, RECIFE-PE-BRASIL; Background: Monocrotaline (MCT) is a pyrrolizidine alkaloid produced by Crotalaria genus, which causes liver damage, modest pulmonary fibrosis and immunotoxicity effects in animals and mainly employed to establish a model of pulmonary dysfunction.



Clinical reports have described favorable results with the use of diethylcarbamazine (DEC) in bronchial asthma. This drug has an important anti-inflammatory role since it interferes with arachidonic acid metabolism. In the present study, we investigated the efficacy of oral DEC treatment in mice model of injury pulmonary induced monocrotaline. Methods and Results: C57/BL6 male mice, were used in all experiments. MCT solution intraperitoneal injection (600mg/kg) was administered once per week (7, 14 or 21 days). Six groups (n=10): control; MCT7; MCT14; MCT21, MCT14/DEC14. (50mg/Kg per day of DEC from 1 to day 14), MCT21/DEC21 (from 1 to day 21). Bronchoalveolar lavage fluid were collected for imunoassay (Griess reaction) and lung tissues for light microscopy (H.E.), immunohistochemistry (COX-2, MCP-1, CD24, F4/80, IL-6, α smooth muscle actin and eNOS) and western blot (COX-2, iNOS and NFkB). Pulmonary sections of the group control exhibited preserved morphological characteristics. MCT7 group revealed alveolar exudates, interstitial edema with thickening of the alveolar septae, perivascular edema, and inflammatory cellular Infiltrates; MCT14 group presented had an inflammatory infiltrate such as macrophages in plexiform lesion in the pulmonary arteries and emphysema; and MCT21 group showed a decrease of injury and the infiltration of PMNs. Nitrite and nitrate levels, were significantly increased in MCT7, MCT14 and MCT21 BALF comparing to the control group. In contrast, MCT14/DEC and MCT21/DEC groups showed significantly reduction of the nitrite and nitrate levels. MCT7, MCT14 and MCT21 groups revealed significant COX-2, MCP-1, CD24, F4/80, IL-6, iNOS and NFkB higher expression, whereas these expression were significantly reduced after treatement with DEC (p<0,05). On the other hand, MCT7, MCT14 and MCT21 groups presented a reduced expression of eNOS and α smooth muscle actin , after treatment with DEC the expression of these proteins returned to normal levels (p<0,05). Conclusion: In this study, treatment with DEC attenuated MCT-induced pulmonary dysfunction, reducing the lung damage. FACEPE, INBEB e CNPq D15 - SÍNTESE E AVALIAÇÃO DE GUANIL HIDRAZONAS E OXIMAS AROMÁTICAS COMO INIBIDORAS E REATIVADORAS DA ACETILCOLINESTERASE

1 - PETRONILHO, E. C.; 1- KITAGAWA, D. A. S.; 2- CASTRO, N. G.; 2- SILVA, F. M. R.; 1- PINTO, A. C.; 1- FIGUEROA-VILLAR, J. D.

1- Grupo de Química Medicinal, Departamento de Química, Instituto Militar de Engenharia, Praça Gen. Tibúrcio, 80, Praia Vermelha, 22.290-070, Rio de Janeiro-RJ, Brasil; 2- Instituto de Ciências Biomédicas, Laboratório de Farmacologia Molecular, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 – CCS, Ilha do

Fundão, 21941-902, Rio de Janeiro-RJ, Brasil. Os agentes químicos representam uma grave ameaça ao mundo moderno. Dentre eles se destacam os organofosforados (OP) neurotóxicos, devido a sua grande periculosidade, baixo custo e fácil manufatura. Os OP inibem a acetilcolinesterase (AChE), uma importante enzima responsável pela hidrólise do neurotransmissor acetilcolina (ACh), causando uma síndrome colinérgica e morte por insuficiência respiratória. Para reativar a AChE são usadas as oximas catiônicas, mas nenhuma delas é eficaz contra todos os agentes OP, demonstrando a necessidade de desenvolver novos reativadores que sejam potentes e com baixa toxicidade. Alguns estudos sugerem a avaliação de diferentes nucleófilos, como as guanil hidrazonas e a dependência da atividade biológica com a acidez. A AChE também está envolvida na Doença de Alzheimer (DA), onde un tratamento é a inibição da AChE, o que permite aumentar a quantidade de ACh, permitindo melhorar os impulsos nervosos e memória. Com esse objetivo, neste trabalho foram sintetizadas uma série de guanil hidrazonas e oximas aromáticas que possuem grupos eletroatratores, para avaliar o efeito destes grupos no processo de reativação e inibição da AChE. Estes compostos foram testados como

inibidores e reativadores da AChE de Electrophorus electricus inibida com etilparaoxon, utilizando como referência positiva a pralidoxima (2-PAM), através do método de Ellman e RMN. Os compostos obtidos somente mostraram boa ação inibitória da AChE. INBEB, CNPq, CAPES, FAPERJ, MINISTÉRIO DA DEFESA D16 - MODULATION OF HEPATIC CU(I)-ATPASE (ATP7B) ACTIVITY BY INSULIN AND GLUCAGON 1,2- HILÁRIO-SOUZA, E.; 3- CUILLEL, M.; 3- CHARBONNIER, P.; 3- MINTZ, E.; 1,2- VIEYRA,

A.; 1,2- LOWE, J. 1- Instituto de Biofísica Carlos Chagas Filho (IBCCF), UFRJ, Rio de Janeiro, Brazil. 2-

Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem (INBEB), UFRJ, Rio de Janeiro, Brazil. 3- Comissariat l’ nergie tomi ue C , Grenoble,

France. "Molecular studies of copper active transport are essential to understand the copper-related diseases (Menkes and Wilson diseases). A number of studies suggest the involvement of insulin in copper balance, although the cellular and molecular mechanisms are not yet established. The main objective of this study was to investigate whether insulin or glucagon modulates the Cu(I)-ATPase (ATP7B) activity and intracellular localization in hepatocytes. In HepG2 cells, ATP7B activity was around 40 nmol Pi x mg-1 x min-1. When they were treated with 0.1 µM insulin, ATP7B activity was increased by 100% and the treatement with 0.1 nM glucagon, ATP7B activity was inhibited by 90%. The maximum effect was observed at 5 minutes for both hormones. After treatment with 0.1 µM insulin and 10 µM H89, a permeable PKA inhibitor, ATP7B activity was similar to that in the presence of insulin alone. In the presence of 0.1 nM glucagon and 10 µM H89, ATP7B activity was the same as in the control condition. Therefore insulin inhibited and glucagon stimulated PKA signaling pathway, leading to the modulation of ATP7B activity. To analyze whether these hormones also modulates ATP7B intracellular localization, WIF-B9 cells were used because they have a well-defined bile canaliculi. ATP7B was localized in the Golgi complex and it was not observed any difference after 24 h treatment with 0.1 µM insulin or 0.1 nM glucagon. When ATP7B was already at the canaliculus (treatment with 1 µM CuCl2 for 2 h) and WIF-B9 cells were treated with different concentrations of insulin, it was observed a change in the localization of ATP7B from the canaliculi towards the Golgi complex. For the first time it was shown that hormones which are essential for the energetic metabolism could regulate the activity of ATP7B, but they do not have any influence in the protein localization." CAPES, CNPq, FAPERJ D17 - BRADYKININ DOWN-MODULATES HUMAN IL-6 PRODUCTION BY P.GINGIVALIS INFECTED GINGIVAL FIBROBLASTS: AN INNATE RESPONSE PATHWAY REGULATING TH17/TH1-POLARIZATION IN A BUCCAL MODEL OF PERIODONTIS.

1- Ramos-Junior, E.S., 1- Nascimento,C.R. , 1- Morandini, A.C. , 1- Scharfstein, J 1-INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO - Universidade Federal do Rio de

Janeiro Recently we demonstrated that Porphyromonas gingivalis, a Gram-negative bacterium that causes periodontitis, activates the kinin system via the cysteine protease gingipain. Considering the role of the kinin B2 receptor (BK2R) in inflammation and healing, the purpose of this study was to evaluate the contribution of BK2R to the pathogenesis of periodontitis. Using an in vitro model with human gingival fibroblasts (HGF) here we showed that BK down-modulates ERK/1/2 and IL-6 production in P.gingivalis-infected HGF in BK2R-dependent manner. Consistent with this, we found that addition of



purified High Molecular Weight Kininogen (HMWK) down-regulated IL-6 in P.gingivalis-infected HGF. Using a model of buccal P. gingivalis infection we then studied whether TH-cell responses (anti-P.gingivalis) were modulated by BK2R. Recall assays performed with submandibular T cells (7 d p.i.) from WT infected mice showed that the bacteria-induced profile of secreted cytokines was dominated by INF-G, accompanied by significant IL-17 responses. Interestingly, TH1/TH17 cytokines were upregulated by bacteria-specific T cells of infected BK2R-KO mice. Considering that the IL-6/IL-17 axis is involved in periodontal pathology, the findings that BK2R dampens IL-6 production by HGF suggest that TH1/TH17 responses might be modulated by kinins proteolytically released in the infected oral mucosa. INBEB, FAPERJ, CNPq D18 - AMYLOID FIBRILS TRIGGER THE RELEASE OF NEUTROPHIL EXTRACELLULAR TRAPS AND ARE DIGESTED BY ELASTASE

1-ESTEFANIA AZEVEDO, 2-ANDERSON GUIMARAES-COSTA; 1,4-CAROLINA BRAGA ; 3-JEFFERY KELLY, 2-ELVIRA SARAIVA; 1-FERNANDO PALHANO; 1-DEBORA FOGUEL.

1-Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brazil; 2-Instituto de Microbiologia Paulo de Góes, UFRJ, Brazil; and 3-The Scripps Research Institute,

California, USA, 4-Polo de Xerem, Caxias, Rio de Janeiro, Brazil. Amyloidoses are a group of diseases characterized by accumulation of amyloid fibrils in different tissues, which are produced by the aggregation of soluble proteins. We asked whether neutrophil extracellular traps (NETs) could play a role on this disease since neutrophil elastase was observed on amyloid deposits of different systemic amyloidoses. Initially, we tested NET induction incubating human blood neutrophils from healthy donors with three different amyloid fibrils and their soluble proteins (alpha-synuclein, Sup35 and transthyretin). NET-DNA was quantified in culture supernatants with the Picogreen dsDNA kit. We demonstrate that amyloid fibrils, but not soluble proteins, induce NETs release, and transthyretin induces NETs formation in a dose dependent manner. Immunofluorescence staining with anti-elastase antibody and DAPI confirmed these results. Importantly, we show that NET-associated elastase digested amyloid fibrils into oligomeric species, which were toxic for BHK-21 cells, as evidenced by the staining with live/dead reagents (calcein/ethidium homodimer). Immunohistochemical analyses of amyloidotic human tissues revealed the presence of NETs, strengthening the evidence for the participation of neutrophils in amyloid diseases. Our results reveal that amyloid fibrils induce NET release that amyloid fibrils are digested by NET-associated elastase generating toxic peptides. FAPERJ, CNPq D19 - EVALUATION OF ACUTE AND LONG-TERM EFFECTS OF PSEUDOMONAS AERUGINOSA LUNG INFECTION IN COGNITIVE AND NEUROINFLAMMATORY PARAMETERS

1- MAGNO, F.; NASCIMENTO, D. O.; ALEXANDRE, P.C.B.; CUNHA, M.G.A.T.; REIS, P.A.; BOZZA, P. T.; CASTRO-FARIA-NETO, H. C.; 2- BOZZA, F.A.

1- Instituto Oswaldo Cruz, Fiocruz; 2- Instituto de Pesquisa Clínica Evandro Chagas, Fiocruz

Sepsis is a severe medical condition characterized by systemic inflammatory response secondary to infection, which progress to multiple organ dysfunction and death. It is the leading cause of death in Intensive Care Units worldwide. Its most common source of infection is the lung with high lethality rate. Cognitive impairment is a significant consequence of sepsis among survivors. The encephalopathy associated with systemic inflammation is not well understood so it is important the development of clinical relevant models to help understand this sequelae. In this study we evaluated acute

inflammatory markers and established a long-term consequence in a murine model of severe pneumonia. C57/BL6 mice were submitted to intratracheal instillation of 105 colony forming units of Pseudomonas aeruginosa (PA). 6 and 24 hours later the bronchoalveolar fluid, the lungs and the brain were collected for cell migration, protein, myeloperoxidase, cytokine, western-blotting, thiol and histological analysis. The survival rate was observed during 7 days after the stimuli. These animals received one dose of antibiotic, 6 hours after pneumonia induction. Cognitive damage was evaluated through the freezing test. Our results showed expressive neutrophils recruitment and myeloperoxidase levels in the lungs of PA infected mice. These animals showed a significantly increase in IL-6, KC, proteins, IL1 , MCP-1 levels in the lung and brain. Histological analysis showed an intense cell infiltrate in lung tissue and survival rate was extensively lower in PA infected mice. Infected animals showed reduced expression of PSD95 pos-synaptic protein and lower thiol levels in the brain. Infected mice had a loss of aversive memory 13 days after stimuli and it remains 50 days later. We demonstrated acute inflammatory response to Pseudomonas aeruginosa lung infection and indicated that this pneumonia model can cause irreversible cognitive impairment. Our results reveal an experimental model for the encephalopathy study associated to systemic inflammation. CNPq, Faperj, Fiocruz D20 - MODELOS FARMACOFÓRICOS TRIDIMENSIONAIS DE INIBIDORES DA PTERIDINA REDUTASE DE LEISHMANIA MAJOR

1,2-LEITE, F. H. A.; 2-CASTILHO, M. S. 1- Programa de pós-graduação em Biotecnologia, Universidade Estadual de Feira de

Santana; 2- Faculdade de Farmácia, Universidade Federal da Bahia. Segundo a OMS, Leishmaniose é a segunda protozoose mais importante, em termos de mortalidade e prevalência. O repertório de fármacos disponíveis para o tratamento dessa parasitose é limitado e, geralmente, apresenta baixos índices de eficácia e segurança. Embora os protozoários do gênero Leishmania sejam auxotróficos para folatos, inibidores de diidrofolato redutase (DHFR) são pouco eficazes contra esse parasito. A baixa suscetibilidade se explica pela presença da enzima pteridina redutase (PTR1) que atua como via alternativa para a redução de ácido fólico. Diante desse cenário, o objetivo deste estudo é a construção de modelos farmacofóricos que auxiliem na identificação de inibidores que tenham afinidade por ambos os alvos terapêuticos. Assim, 6 inibidores de DHFR e 9 inibidores da PTR1 de L. major, previamente descritos na literatura, foram selecionados para a construção de modelos farmacofóricos, os quais foram avaliados quanto a sua capacidade de diferenciar inibidores conhecidos de moléculas com propriedades físico-químicas semelhantes, mas que não tem atividade sobre as enzimas (falsos positivos). Adicionalmente, os modelos foram avaliados quanto a sua habilidade de explicar a relação entre a estrutura química e a atividade biológica de inibidores de DHFR (Ki: 0,13-363,1μM e de PTR1 Ki: 16,1-436,0 μM n o utili ados ara constru o dos modelos farmacof ricos. 3 modelos apresentaram valores de sensibilidade e especificidade adequados (AUC>0,7) para discriminar moléculas ativas das inativas. Adicionalmente, a análise desses modelos revela que moléculas com afinidade por PTR1 apresentam 3 pontos hidrofóbicos (2 deles no anel pteridina), dois grupos aceitadores de ligação de hidrogênio (grupos NH2 exo-cíclicos) e 4 grupos aceitadores de ligação de hidrogênio (3 deles no anel pteridina). Moléculas com afinidade por DHFR também apresentam pontos hidrofóbicos e aceitadores de ligação de hidrogênio no anel pteridina, contudo o número de disposição do outros pontos farmacofóricos é diferente. A partir dessas informações é possível planejar inibidores com afinidade frente a ambos os alvos terapêuticos. FAPESB



D21 - TOLL-LIKE RECEPTOR 4 ACTIVATION PROMOTES CARDIAC ARRHYTHMIAS BY DECREASING THE TRANSIENT OUTWARD POTASSIUM CURRENT (ITO) THROUGH AN IRF3-DEPENDENT AND MYD88-INDEPENDENT PATHWAY.

1- MONNERAT-CAHLI, G.; 2- ALONSO, H.; 2- GALLEGO, M.; 1-ALARCON, M.L.; 3- BASSANI, R.A.; 2-CASIS, O.; 1- MEDEI, E.

1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil. 2-Departamento de Fisiología, Facultad de Farmacia, Universidad del País Vasco, Vitoria, Spain. 3-Departamento de Engenharia Biomédica – FEEC/Unicamp, Campinas,

Brasil Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll like rece tors T R’s lay an im ortant role in cardiac com lications. In the resent study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias. Also the signaling pathway involved in these phenomena was studied. Action potentials, the presence of cardiac arrhythmias and transient outward K+ current Ito were recorded in Wistar rat’s hearts after 4h e osure to the T R4 agonist ultrapure Lipopolysaccharide (LPS - 1µg/ml). TLR4 stimulation in vitro promotes a cardiac electrical remodeling that leads to cardiac action potential prolongation which evokes arrhythmic events such as delayed after depolarization (DAD's) and triggered activity. The perfusion of LPS (1 μg/ml) during 30 minutes did not modify Ito. Conversely, after 24h of LPS incubation Ito was reduced, with no changes in the biophysical properties of the current. Major changes in Ca2+ cycling were not observed in ventricular myocytes after 24 h exposure to LPS; however, extrasystolic activity was present in a considerable number of cells (25%). Neither the blockade of Interleulink-1 receptor-associated kinase 4 nor nuclear factor kappa B (NF-kB) prevented the LPS effect on Ito. However, interferon regulatory factor 3 (IRF3) inhibition prevented the effect of TLR4 activation on Ito. Activation of TLR4 induced longer AP duration and evoked DAD's and triggered activity because of a reduction in Ito. The mechanism involved is MyD88-independent and IRF3-dependent. INBEB, FAPERJ, CNPq, DECIT D22 - POTENCIAL DAS CÉLULAS TRONCO DA MEDULA ÓSSEA EM LESÃO MIOCÁRDICA RADIOINDUZIDA 1-RAMOS,IP; 1-ANDRADE,CBV; 2-SUHETT,G; 3-SALATA,C; 4-CANARY PC; 1-BRASIL,GV;1-

GOLDENBERG,RCS 1-Laboratorio de Cardiologia Celular e Molecular do Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-Hospital Albert Einstein;3-Laboratório

de Ciências Radiológicas, Universidade do Estado do Rio de Janeiro "Introdução: No Brasil, as estimativas do INCA para o ano de 2013, apontam a ocorrência de aproximadamente 518.000 casos novos de câncer, sendo o câncer de mama (CM) feminino o mais incidente deles entre as mulheres, com uma estimativa de aproximadamente 53 mil novos casos. Aproximadamente 50% das pacientes para CM recebem radioterapia (RT) para tratamento de CM. Um paciente com neoplasia, que se submete ao tratamento para câncer corre o risco substancial de deterioração da saúde cardiovascular. A terapia celular parece promissora no tratamento de doenças crônicas e degenerativas, dentre elas a cardiomiopatia radioinduzida, posto que os atuais recursos terapêuticos são insuficientes. Materiais e Métodos: Ratas Wistar, com 3 meses, foram divididas em 2 grupos, irradiados (G0) e irradiados e tratados com células-tronco de medula óssea (G1) . Os animais foram eutanasiados 3 meses após o término do tratamento. Antes da eutanásia

foi realizado ecocardiograma. Após a eutanásia foi retirado o ventriculo esquerdo para as técnicas de RT-qPCR (VEGF e Pro Colágeno I) e histologia para picrosirius. Resultados/Discussão: Após 45 dias da irradiação foi observado um aumento na fração de ejeção entre os animais do grupo G1 em relação ao grupo G0. Houve um aumento na expressão gênica de VEGF e diminuição de Pro Colágeno I nos animais do grupo G1 em relação aos do grupo G0. Demonstrando que a deposição de colágeno no coração dos animais tratados com células-tronco de medula óssea foi reduzida o que foi corroborado com os resultados obtidos por picrosirius em comparação com o grupo só irradiado. O aumento da expressão do VEGF no grupo G1 é importante, pois demonstra que pode estar ocorrendo um aumento da neoangiogênese no coração irradiado e tratado, o que não foi observado no grupo G0. Para confirmação desses dados será realizada imunohistoquímica para colágeno tipo I e CD31." CNPq, FAPERJ, CAPES, INBEB D23 - CARACTERIZAÇÃO MORFOLÓGICA E BIOQUÍMICA DA COSTA DE TRICOMONAS

1,2 – DE ANDRADE ROSA, I; 3,4-DE SOUZA W; 1,5- BENCHIMOL B 1- Laboratório de Ultraestrutura Celular, Universidade Santa Úrsula, Rio de Janeiro, Brasil 2- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de

Janeiro, Brasil 3- Laboratório Hertha Meyer, Universidade Federal do Rio de Janeiro, Brazil 4- Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de

Janeiro, Brasil 5-Instituto Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Brasil

"Tritrichomonas foetus é um protista parasito causador da tricomonose bovina, uma doença sexualmente transmissível que acarreta diversos problemas reprodutivos. Embora seja considerado um organismo primitivo, este parasito exibe um citoesqueleto incomum e complexo, formado por diversas estruturas ainda pouco estudadas. Entre estas destacam-se a pelta e o axóstilo, formados por microtúbulos, os filamentos parabasais e a costa que possuem composição proteica desconhecida. A costa trata-se de uma estrutura peculiar por apresentar uma organização diferente de outros constituintes de citoesqueleto. É considerada uma fibra estriada formada por bandas claras e escuras disposta de forma alternada e bastante regular. Desta forma, esta estrutura despertou interesse também na área de metrologia por poder ser usada como uma ferramenta de medidas em escalas nanométricas. Assim, realizamos o fracionamento celular para obter uma fração enriquecida de costas que foi caracterizada morfologicamente por contrastação negativa. Três regiões distintas foram classificadas como cabeça, pescoço e corpo, de acordo com o sua forma e padrão de bandeamento. A rápida transformada de Fourier nos auxiliou a comprovar a periodicidade da costa e a revelar a presença de sub-bandas também com padrões regulares. Com o intuito de caracterizar as proteínas que compõem a costa de T. foetus, a fração de costas foi submetida a eletroforeses uni- e bidimensionais, onde as bandas e spots foram identificados por espectrometria de massa. Ao todo, 50 proteínas hipotéticas de T. foetus, sem domínios conservados descritos, foram identificadas. Juntos, nossos resultados demostraram que a costa é uma estrutura periódica com divisões e subdivisões regulares, o que a torna uma boa ferramenta para medidas em escalas nanométricas. Além disso, verificamos também que sua composição proteica é totalmente distinta, sendo necessários mais estudos que avaliem a localização e função destas proteínas na estrutura da costa." INBEB, AUSU, Inmetro, CNPq, FAPERJ, CAPES, PRONEX.



D24 - METABOLOMIC PROFILING OF ANIMAL SERUM SAMPLES UNDER CORYNEBACTERIUM PSEUDOTUBERCULOSIS INFECTION

1- PONTES, J. G. M.; 1- BALLOTTIN, D. P. M.; 1- TASIC, L.* 1- Universidade Estadual de Campinas

"A Gram-positive and pathogenic bacterium, Corynebacterium pseudotuberculosis affects mainly caprines and sheep causing caseous lymphadenitis, the disease responsible for the enormous losses in agribusiness, since there is not effective treatment for this disease and infected animals end up being euthanized. Our objective was to evaluate and compare the metabolic changes caused in infected animals by Corynebacterium pseudotuberculosis among seropositive, seronegative and asym tomatic animal’ blood serum sam les, so that later we can better understand the mechanism of infection and the disease through in silico studies. In this work we will be presenting the first results of analyzes that have been made using 1H NMR recorded on a Bruker Avance III 600 MHz spectrometer and SEM-EDS. Metabolomic profile changes account to the chemical shift region from 0 to 2 ppm and change in signal intensities in the region between 6 to 8 ppm referring to signals related to aminoacids and proteins.” CNPq and INBEB D25 - KOJIC ACID INDUCES, IN VITRO, HUMAN MONOCYTES DIFFERENTIATION INTO MACROPHAGES THROUGH AUTOPHAGY COSTA, J.P. ¹,2*, SILVA, B. J. M 1,2*, FRADE, P.C.R1,2 , RODRIGUES, A. P. D. 1,2,3, FARIAS,

L.H.S. 1,2, NASCIMENTO, J.L.M. 4, SILVA, E.O. 1,2 1Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências

Biológicas, Universidade Federal do Pará, Belém, Brazil. 2Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brazil 3Laboratório de Microscopia, Instituto Evandro Chagas, secretaria

de vigilância em saúde, ministério da saúde, Belém, Pará, Brazil. 4Laboratório de Neuroquímica, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém,

Pará, Brazil. Monocytes are mononuclear phagocytes, present in the peripheral blood, that are able to differentiate into macrophages and dendritic cells. Macrophages play a specific role in the inflammatory process, being essential for the innate response. Given the important role of monocytes/macrophages in the immune response, this study aimed to investigate whether kojid acid, a natural product produced by some species of fungi, is able to promote differentiation of human monocytes into macrophages in vitro through differential expression of surface proteins and autophagic process. Monocytes were isolated from peripheral blood collected in EDTA obtained from healthy human volunteers (Blood collection center, Belém, Para, Brazil) and separated using Histopaque®-1077. Cytotoxicity to the host cell was assessed by colorimetric MTT assays. Autophagy was observed through the formation of autophagic vacuoles by Transmission Electron Microscopy and expression of the lysosomal protein LC3b by Immunofluorescence. The treatment of monocytes with 50 μg/mL KA for 48h induced morphological alterations, such as an increase in cell area, numerous cellular projections and increased organelle number. In addition, flow cytometry analysis showed increased labeling LC3b and was observed the presence of autophagic vacuoles in the cytoplasm indicating autophagic process in cells treated. Immunofluorescence of F4/80 and flow cytometry analysis of F4/80, CD11b and CD11C protein revealed that KA induces the differentiation of monocytes into macrophages in vitro. The cell viability of the monocytes was also maintained following KA treatment. No significant production of reactive oxygen species was detected by CellRox in treated cells. In conclusion, these results support the hypothesis that kojic acid, a natural alternative molecule, may be able to induce, in vitro, monocyte differentiation into macrophage.

INBEB D26 - THE ORIGIN OF LYSOSOMES RELATED ORGANELLES OF TRYPANOSOMA CRUZI TRYPOMASTIGOTES 1,2- JULIANA CUNHA VIDAL, 1,2-CAROLINA DE LIMA ALCANTARA, 1,2,3-WANDERLEY DE

SOUZA, 1,2- NARCISA LEAL DA CUNHA-E-SILVA 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; 2-Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e

Bioimagens, Rio de Janeiro, Brazil. 3-Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia (Inmetro), Rio de

Janeiro, Brazil. "Trypanosoma cruzi is the causative agent of Chagas disease. The parasite presents three life cycle stages: trypomastigotes, the infective forms, amastigotes, the proliferative forms found in the cytoplasm of the vertebrate host cells and epimastigotes that proliferate in the invertebrate host. In nature, triatomine bugs pick up trypomastigotes together with the blood of infected hosts. Parasites travel along the insect gut without leaving gut lumen and in their way undergo two differentiations: from trypomastigotes to epimastigotes and, after proliferating, again to trypomastigotes. The latter step, called metacyclogenesis is of particular interest, as the parasites acquired the ability of infecting humans. Metacyclic trypomastigotes are layed down together with triatomine feces at the local of the insect bite, penetrate human cells, scape early from parasitophorous vacuole to the cytoplasm, where they differentiate into amastigotes. Summarizing this complicate story, T. cruzi alternates between vertebrate and invertebrate hosts by means of alternating between infective and proliferative forms. Using previously established protocols, our laboratory is able to reproduce in vitro the complete T. cruzi life cycle1. Epimastigotes present a very efficient endocytic pathway that begins with a specialized structure, the cytostome-cytopharinx complex, a deep invagination of the cell surface, sustained by stable microtubules, that delivers cargo-containing vesicles to a tubule-vesicular network and subsequently to reservosomes, the final endocytic compartment2. Reservosomes are able to store and digest macromolecules, present an acidic character, maintained by an unusual P-type H+ATPase, concentrate cruzipain, the most important T. cruzi protease, its natural inhibitor chagasin, and other hydrolases as well. Despite these features, reservosomes were not considered genuine lysosomes because they lack classic lysosome markers3. Differently from epimastigotes, the endocytic pathway of amastigotes and trypomastigotes was not described yet. In fact, after many unsuccessful experiments, data from our group and from others indicate that trypomastigotes are unable to uptake macromolecules from medium. Nevertheless, trypomastigotes and amastigotes also present cruzipain, chagasin, and P-type H+ATPase concentrated in acidic organelles placed between kinetoplast and nucleus. Few years ago, we proposed to classify these compartments as lysosome related organelles (LROs)4. The present study aimed to investigate the origin of metacyclic trypomastigotes LROs. We have preloaded epimastigote reservosomes with uncoated fluorescent beads before inducing metacyclogenesis. The rate of metacyclogenesis did not change compared to control (not loaded) parasites: after 48h of differentiation we have obtained 53% of trypomastigotes. Surprisingly, 25% of them contained beads between kinetoplast and nucleus, consistent with the localization of LROs. These parasites were the first register of trypomastigotes containing endocytic cargo. In a similar experiment, using 10 nm gold-labeled transferrin (Tf-Au) as tracer for electron microscopy, we observed spherical organelles containing Tf-Au in parasites at the end of metacyclogenesis. We are now



characterizing these compartments by immunocytochemistry. Our observations are a strong indication that LROs from metacyclic trypomastigotes originate directly from reservosomes of epimastigotes." CAPES, CNPq, INBEB D27 - ANG-(3-4) INHIBITS RENAL NA+-ATPASE IN HYPERTENSIVE RATS THROUGH A MECHANISM THAT INVOLVES DISSOCIATION OF ANG II RECEPTORS HETERODIMERS AND PKA 1,2- DIAS, J.; 1,2,3- FERRÃO, F.M.; 1,2- AXELBAND, F.; 4- CARMONA, A.K.; 2,3- LARA, L.S.;

1,2- VIEYRA, A. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-

Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem; 3- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 4-

Departamento de Biofísica, Universidade Federal de São Paulo The physiological roles of ANG-(3-4) (Val-Tyr), a potent ANG II-derived peptide, remain largely unknown. The present study: (i) investigates whether ANG-(3-4) modulates ouabain-resistant Na+-ATPase resident in proximal tubule cells, and (ii) verifies whether its possible action on pumping activity -considered the fine tuner of Na+ reabsorption in this nephron segment- depends on blood pressure. ANG-(3-4) inhibited Na+-ATPase activity in membranes of spontaneously hypertensive rats (SHR) at nanomolar concentrations, with no effect in Wistar-Kyoto rats (WKY) or on (Na++K+)-ATPase. PD123319 (10-7 M) and PKA(5-24) (10-6 M), an AT2R antagonist and a specific PKA inhibitor respectively, abrogated this inhibition, indicating that AT2R and PKA are central in this pathway. Despite the lack of effect of ANG-(3-4) when assayed alone in WKY rats, the peptide (10-8 M) completely blocked stimulation of Na+-ATPase induced by 10-10 M ANG II in normotensive rats through a mechanism that also involves AT2R and PKA. Tubular membranes from WKY rats had higher levels of AT2R/AT1R heterodimers, which remain associated in 10-10 M ANG II and dissociate to a very low dimerization state upon addition of 10-8 M ANG-(3-4). This lower level of heterodimers was that found in SHR, and heterodimers did not dissociate when the same concentration of ANG-(3-4) was present. Oral administration of ANG-(3-4) (50 mg/kg body mass) increased [Na+]ur and UNaV with a simultaneous decrease in systolic arterial pressure in SHR, but not in WKY rats. Thus, the influence of ANG-(3-4) on Na+ transport and its hypotensive action depend on receptor association and on blood pressure. CNPq, CAPES, FAPERJ, FAPESP, INBEB D28 - INTERAÇÃO PARÁCRINA ENTRE CÉLULAS RENAIS E CÉLULAS ESTROMAIS MESENQUIMAIS: ESTUDO DOS MEDIADORES LIPÍDICOS 1,2- Santanna, J.F; 1,2- Andrade, A.S ; 1,2- Lindoso, R.S ; 1- Luna Gomes,T ; 1,2- Vieyra,

A.R ; 1- Bandeira-Melo, C. ; 1,2- Einicker-Lamas, M 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-

Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural de Bioimagem (INBEB) Células Mononucleares de Medúla Óssea (CDMO), em especial Células Mesenquimais (CM) possuem importante papel no reparo do tecido renal. Sabe-se que CDMO são mobilizadas a locais de lesão e podem atuar através da secreção parácrina. Assim, acredita-se que eicosanóides possam estar associados a repostas renoprotetoras observadas durante interação parácrina entre células renais e CM pós lesão. CDMO/CM foram co-cultivadas por 3 h com células HK-2 através de poços Millicell, possibilitando a troca de moléculas bioativas por ambos os tipos celulares. A lesão foi induzida em células HK-2 por depleção de soro 24 h e de ATP por Antimicina A (10 µM) 30 min. A biogênese de corpúsculos lipídicos, um dos principais sítios de síntese dos eicosanóides,

foi avaliada por microscopia ótica após coloração com Tetróxido de Ósmio 1,5 %. Eicosanóides foram dosados por Ensaio Imuno- Enzimático de competição do sobrenadante das co-culturas e das culturas individuais de Hk-2 ou CDMO/CM. Como resultados, eicosanóides são modulados nas diferentes condições experimentais. Níveis de prostanóides totais decaem em cultura de células HK-submetidas a lesão e são cerca de 5 x maiores em condições de interação parácrina com CDMO. Níveis de prostaglandina E2 acompanham o mesmo padrão obsevado nos prostanóides totais. Um aumento marcante de prostaglandina D2 é observado em condições de co-cultura de HK-2 com CM. Corpúsculos lipídicos sofrem alterações nas HK-2 tanto em número, aumentado pós lesão e pós co-cultura com CM; quanto em diâmetro, aumentado pós lesão e reduzido em co-cultura com CM. O número de corpúsculos lipídicos em CDMO também é aumentado pós co-cultura com HK-2. Concluindo que níveis de diferentes prostanóides são regulados durante condições de lesão química e interação parácrina, assim como seus sítios de síntese. Sendo possível que respostas de pró-sobrevivência desencadeadas nas células renais em períodos de co-cultura possam estar relacionadas a ambas alterações. FAPERJ, CNPQ, INBEB, PROBITEC, CAPES. D29 - BRADYKININ AND C5A ANAPHALATOXIN FUEL INTRACELLULAR GROWTH OF TRYPANOSOMA CRUZI IN MACROPHAGES

1-ALMEIDA, L.N.; CORDOVIL, T.; OLIVEIRA, A.C.; SCHARFSTEIN, J. 1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro

Involving two zymogens (FXII and PK) and the co-factor high molecular weight kininogen (HK), the contact pathway of coagulation is a surface-assembled complex that promotes pericellular proteolysis in neutrophils and macrophages. We have previously linked bradykinin release in T. cruzi-infected tissues to development of Th1-protective immunity. Conversely, we have recently demonstrated that T. cruzi trypomastigotes may take advantage of the transient formation of inflammatory edema (fueled by kinins, endothelin and C5a) to invade heart cells that naturally overexpress their cognate GPCRs (BKRs, ETRs and C5aRs). Consistent with this, we have recently found that mice deficient in BK1R display reduced intracardiac parasitism and are refractory to chronic myocarditis/fibrosis (Andrade et al., abstract enclosed). Prompted by these findings, here we investigated the impact of GPCR signaling (BK2R, BK1R, C5aR) on intracellular outgrowth of T. cruzi amastigotes in macrophages. To this end, we added specific antagonists of C5aR (A8B), BK2R (HOE-140) or BK1R (DAL8-BK) to monolayers of WT or BK1R-/- bone-marrow derived macrophages (BMDM), pre-treated with IFN-γ and infected 20 hours earlier with Dm28c T. cruzi. Measurements of the number of intracellular amastigotes/100 cells at 72 h p.i. revealed that parasite load was markedly reduced by each of these GPCR blockers (44%, 47% and 46%, respectively; P<0.001). Pharmacological specificity of the host protective effect was confirmed in BK1R-/- macrophages, which showed reduced amastigote outgrowth upon treatment with HOE-140 or A8B, but not with the BK1R antagonist. Intriguingly, the increased resistance conveyed by these GPCR blockers was not due to increased production of NO or ROS. These in vitro observations are consistent with the hypothesis that generation of infection-promoting GPCR ligands (BK, C5a, Des-Arg-BK) in inflammatory exudates might favor parasite persistence in chronic inflamed tissues. INBEB, FAPERJ, CNPq D30 - WHAT ARE THE GENERAL EVOLUTIONARY TRENDS OF AN OLD PROTEIN RECRUITED FOR A NEW FUNCTION? 1,2 - FARIA, L.M.; 1 - PASCUTTI, P.G.; 2 - QUATTROCCHIO, F.M.



1 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2 - Department of Genetics, Vrije Universiteit Amsterdam. "One new member of the P-type-ATPase superfamily of transporters (PH5) is part of a complex required for the building up of the proton gradient across the tonoplast of the central vacuole, in petal cells. PH5 is strongly related to plasma membrane ATPases of plants and yeast (Verweij et al., 2008). In this work we intend to investigate, by colocalization experiments and analyses of different protein fusion constructs, some evolutionary steps involved in the evolution of these P-H+-ATPases from plasma membrane to the tonoplast. PH5 belongs to the plant-specific P3A subfamily of H+-ATPases and has H+ pumping activity when expressed in yeast. However, PH5 is different from the other P3A-ATPases characterized until now (e.g. Arabidopsis AHA2), which are plasma-membrane H+ pumps, because it resides in the tonoplast (Verweij et al., 2008). Because PH5 differs in only a few amino acids from other P3A-ATPases, those few changes must have been sufficient to alter its location and to assume a new function during evolution. A set of constructs was generated in which different domains of PhAHA2 and PH5 were exchanged and the resulting chimeras were fused to GFP. These protein chimeras were then expressed in petunia petal cells together with the marker for the plasma membrane RFP-SYP122. The study of the localization of these fusion proteins gives a first indication of which domains contributed, during evolution, to the acquisition of new characteristics by the ancestor of PH5 and to its recruitment for a new function. Furthermore, normal modes analysis of PH5, PhAHA2 and the chimeras was carried out as an attempt to address the evolutionary directions of their structural changes. One of the points that has to be clarified now is whether or not amino acid changes have been selected in order to allow proteins to follow a particular single normal mode direction. CNPq, CAPES D31 - ISQUEMIA E REPERFUSÃO MIOCÁRDICA: ESTUDO DOS FATORES CARDIOPROTETORES DO PRECONDICIONAMENTO ISQUÊMICO: AVALIAÇÃO FUNCIONAL E PROTEÔMICA DE FATORES HUMORAIS COM ATIVIDADE CARDIOPROTETORA

1-MACIEL ,L; 1,2-VERISSIMO-DA-COSTA, G. C; 1-OLIVEIRA, D.F; 1-BAPTISTA G; 1,2-BISCH, P. M; 1-NASCIMENTO J.H.

1-Laboratório de Eletrofisiologia Cardíaca Antonio Paes de Carvalho / 2- Laboratório Multidisciplinar de Proteomica - Instituto de Biofísica Carlos Chagas Filho - Centro de

Ciências da Saúde - Universidade Federal do Rio de Janeiro O precondicionamento isquêmico (PCI) é um mecanismo endógeno de cardioproteção contra as in urias do infarto. PCI ode ser indu ido “distância”, em outros tecidos, sugerindo a liberação de ativadores humorais. Objetivos: Identificar e caracterizar fatores cardioprotetores liberados no PCI e elucidar as vias envolvidas nesta cardioproteção. Métodos: Corações de ratos Wistar machos foram perfundidos em aparato de Langendorf. Grupos experimentais, Controle: Submetidos a 30 min. de isquemia e 60 min. de reperfusão (I/R); PCI: Submetidos a 3 ciclos de 5 min. de isquemia e reperfusão, antes do I/R; Efl_pci: Perfusão do Efl-pci antes da I/R; Efl_fracionado: Perfusão das frações do Efl_pci (cutoff <3kDa; 3-5 kDa; 5-10 kDa; 10-30 kDa; 30-50 kDa; >50 kDa), antes da I/R. Perfusão das frações cardioprotetoras com bloqueadores: Canais de K + sens eis a TP, glyburide 10 μM ou 5 100 μM J K- T T G490 10 μM PKC ueleritrina 10 μM Rece tores ara adenosina - A1 (DPCPX 20 nM) e opióides (Naloxone 10 nM). A determinação da área de infarto foi por coloração com TTC (1%). O Efl_pci foi analisado por SDS-Page, e em LC-MS/MS, ESI-Q-Tof para identificação dos fatores humorais. Resultados: As frações <3 kDa e entre 5 e 10 kDa, apresentaram efetiva recuperação pós I/R. A cardioproteção pelas frações <3 kDa e entre 5 e 10 kDa

foi inibida pela glibenclamida, 5HD, queleritrina, AG490, DPCPX e naloxone. A análise em SDS-PAGE mostrou proteínas em diversas faixas de peso molecular. A analise em LC-MS/MS, identificou 987 proteínas. Onde 60 proteínas são descritas em vias de cardioproteção. Das quais, 8 possuem tamanho menor que 10 kDa. Conclusão: Fatores humorais, com atividade cardioprotetora, possuem peso molecular <3 kDa e entre 5 e 10 kDa. Esta cardioproteção foi antagonizada por bloqueadores para vias pré-descritas como cardioprotetoras. Análise proteômica do Efl_pci mostrou a existência de proteínas atuantes na cardioproteção. CNPq, FAPERJ, CAPES D32 - CARACTERIZAÇÃO ESTRUTURAL DE CADEIAS POLIPEPTÍDICAS NASCENTES USANDO RESSONÂNCIA MAGNÉTICA NUCLEAR EM SOLUÇÃO

1 - VAZQUEZ, L; 2 - ALMEIDA, M. S.; 3 - WÜTHRICH, K 1,2 - Instituto de Bioquímica Média de Meis/ INBEB; 3 - The Scripps Research Institute

"Proteínas incorretamente dobradas não tem um tempo de meia vida suficiente dentro de um organismo, o que de outra forma que pode levar a doenças de proteínas mal enoveladas, como a doença de príon, malde Alzheimer, câncer ou doença de Parkinson. Os ribossomos sintetizam cadeias de polipeptídeo, e durante a sua síntese, as proteínas nascentes têm a capacidade de se auto estruturar. A exigência de ribossomo para a realização da conformação específica do peptídeo nascente ainda é controversa. Com o melhor do conhecimento, alguns estudos já indicaram que polipeptídeos nascentes, ainda ligados ao ribossomo, podem dobrar na conformação nativa, após a sua cadeia completa ser quase alcançada. No entanto, nenhum destes estudos mostra o calculo da estrutura 3D e do vínculo deste polipeptídeo nascente com o complexo ribossomal, por que os obstáculos das técnicas de se trabalhar com um grande complexo, intrinsecamente reduz a quantidade de sinais de RMN, e limita a proposta nete sentido. RMN em solução, é uma técnica de escolha para caracterização este processo uma vez que muiot provavelmente a cadeia nascente polipéptido é muito dinamica, até atingir certo ponto de crescimento. Em face das dificuldades da configuração experimental dos estudos anteriores e o desafio que pode surgir a partir da solubilidade dos intermediários dobráveis, nós concebemos um protocolo em que a proteína alvo pode ser produzida de forma recombinante, em diferentes fases de alongamento, na proção c-terminal fundidos para um aumento da solubilidade e de expressão (SET). Essas construções serão pequenas o suficiente (c. 9kDa para GB1 SET e 0,5-10 kDa para os polipeptídeos alvo) para permitir que as suas estruturas 3D determinadas para frente sejam bem estabelecidos através de tripla ressonância pelo métodos de espectroscopia de RMN." INBEB, FAPERJ, CNPq D33 - ATIVIDADE PROMISSORA DA ESTEROL HIDRAZONA H3 CONTRA SPOROTHRIX SCHENCKII E SPOROTHRIX BRASILIENSIS.

1-BORBA-SANTOS, L.P.; 2- ISHIDA, K.; 3,4- VISBAL, G.; 5- RODRIGUES, A.M.; 5- DE CAMARGO, Z.P.; 6- LOPES-BEZERRA, L.M.; 1- ROZENTAL, S.

1-IBCCF, UFRJ, Rio de Janeiro, Brasil; 2-ICB, USP, São Paulo, Brasil; 3-IVIC, Caracas, Venezuela; 4-INMETRO, Rio de Janeiro, Brasil; 5-UNIFESP, São Paulo, Brasil; 6-UERJ, Rio

de Janeiro, Brasil. "Sporothrix schenckii sensu stricto e Sporothrix brasiliensis são descritas como as espécies mais virulentas do complexo Sporothrix schenckii, que agrupa fungos termo-dimórficos causadores da esporotricose. O objetivo deste trabalho foi avaliar a atividade antifúngica da esterol hidrazona 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3), um inibidor da enzima esterol metiltransferase da via de síntese do ergosterol, contra isolados de S. schenckii e S. brasiliensis.



A concentração inibitória mínima (CIM) para anfotericina B, itraconazol e H3 foi determinada para 32 isolados (16 de cada espécie) na forma filamentosa e de leveduras com base nos protocolos adaptados M38-A2 e M27-A3 do CLSI. A susceptibilidade também foi avaliada segundo as concentrações fungicidas mínimas (CFM), ensaios de “Checkerboard” e “Time kill”. s efeitos nas le eduras decorrentes dos tratamentos foram avaliados por microscopia eletrônica de varredura e transmissão; e o acúmulo de lipídios neutros e alterações na atividade mitocondrial foram avaliados através da marcação com BODIPY 493/503 e MitoTracker Red CMRos e análise por citometria de fluxo. Adicionalmente, foram determinadas a citotoxicidade em células de mamífero e a atividade hemolítica de H3. H3 apresentou maior atividade antifúngica do que anfotericina B e itraconazol frente ás duas espécies na forma filamentosa e de leveduras, com CIMs variando de 0.015 a 0.5 μg/m e 0.03 a 0. 5 μg/m , res ecti amente. nsaios de “Time kill” demonstraram ue H3 possui atividade fungistática. Combinações de H3 com itraconazol apresentaram efeito sinérgico contra S. brasiliensis. Leveduras de S. schenckii tratadas por 96 horas com concentrações sub-inibitórias de H3 apresentaram acúmulo de lipídios neutros, aumento da espessura da parede celular e interferência na conversão da levedura para a forma filamentosa. Por outro lado, leveduras de S. brasiliensis tratadas apresentaram atividade mitocondrial reduzida, inchaço mitocondrial e diminuição da camada microfibrilar. H3 apresentou baixa citotoxicidade e efeito hemolítico em células de linhagem contínua LLC-MK2 e hemácias humanas, respectivamente; e grande seletividade fúngica. Juntos estes resultados refletem a grande atividade antifúngica in vitro de H3. Mais esforços serão empregados para determinar seus mecanismos de ação e sua atividade in vivo em modelo experimental de esporotricose." INBEB, FAPERJ, FAPESP, CAPES, CNPq D34 - ANTILEISHMANIAL ACTIVITY OF THE CROTOXIN DERIVED FROM CROTALUS DURISSUS TERRIFICUS SNAKE VENOM.

1,2 - Farias, L.H.S.; 2,3 - Rodrigues, A. P. D.; 4 -Sampaio, S. C.; 1,2 -Silva, E.O. 1-Laboratório de Parasitologia/ Laboratório de Biologia Estrutural, UFPa; 2- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade

Federal do Rio de Janeiro, Ilha do Fundão, Brazil; 3-Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, Pa. 4-Laboratório de Fisiopatologia, Instituto

Butantan, SP. American Tegumentary Leishmaniasis (ATL) is a parasitic disease, widely spread in most countries of Latin America, and caused by different species of the genus Leishmania. This protozoan is an obligate intracellular parasite that developed mechanisms to subvert the microbicidal activity of macrophages, such as inhibition of superoxide and nitric oxide (NO) production. The chemotherapy is one of the most effective treatments for this disease. The antileishmanial drugs available are in general toxic, expensive and require long-term treatment. Thus, the development of new natural products to treat leishmaniasis has become a priority. Ophidian toxins are natural sources of bioactive products with therapeutic properties already described. Therefore, we consider analyze the activity of crotoxin (CTX), a dimeric protein and the main neurotoxic component of Crotalus durissus terrificus snake venom, against promastigotes of Leishmania (L.) amazonensis and macrophages. The toxin significantly decreasing of 32,5% on the growth of promastigotes at 1,2µg/mL and 24,9% at 4,8µg/mL after 96 hours of treatment (IC50= 22,86µg/mL). The colorimetric assay (MTT), that measures metabolic viability of mitochondria, showed that this compound presented no cytotoxic effects against macrophages. Interestingly, CTX treated macrophages presented a significant higher capacity to metabolize the MTT substrate (mean= 59,78% ±3,31, higher) when

compared with untreated control, indicating intense mitochondrial activity. Furthermore, we analyze the capacity of host cell to produce reactive oxygen species (ROS) when treated with CTX, using CellRox green® (Molecular Probes) labeling. It was observed that treated macrophages presented intense production of ROS (mean= 35,95% ±2,76, higher) when compared with untreated cells, indicating the capacity of CTX to stimulate the microbicide response of host cell. These results demonstrated that CTX inhibits the growth of parasites and does not have cytotoxic effects on the host cell. Taken together, our results demonstrated that this compound could be a promising antileishmanial agent. CAPES, UFPA, CNPq, INBEB, FAPERJ D35 - PKC EPSILON STIMULATES CU(I)-ATPASE ACTIVITY OF ATP7B FROM PORCINE LIVER

1,2- CARDOSO, L.H.D.; 1,2- VIEYRA, A.; 1,2- LOWE,J. 1 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2 -

Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem "Copper is essential to several organisms, although in high concentrations it is extremely toxic. Therefore, copper levels in the organism must be finely regulated. ATP7B is one of the two mammalian copper-transporting ATPases, and it is essential to hepatic copper excretion into the bile. Previous studies from our laboratory have demonstrated that PKA inhibits ATP7B activity. The aim of this work is to determine the involvement of PKC on the modulation of ATP7B activity. Golgi-enriched membrane preparations were obtained from pig liver, and different PKC isoforms (alfa, epsilon, zeta) were detected by western blotting. Cu(I)-ATPase activity was measured using a colorimetric assay to detect inorganic phosphate. The PKC activator PMA (10-8 M) stimulated Cu(I)-ATPase activity by 55%, while PKC inhibitor, calphostin C (10-8 M) decreased activity by 40%. Addition of lambda phosphatase (80 U/mL) after pre-incubation with PMA decreased ATP7B activity to the same levels of lambda phosphatase alone, showing that a kinase-mediated phosphorylation step occurs in the modulation of ATP7B activity, which was later confirmed by a kinase-mediated phosphorylation assay. The inhibition of phospholipase C by U73122 (10-10 M) decreased ATP7B activity. Using PMA combined with the Ca2+ chelator EGTA, ATP7B activity increased, indicating the involvement of a novel PKC, not stimulated by Ca2+. ATP7B activity decreased in the presence of PKC epsilon specific inhibitor, showing that this novel PKC isoform is responsible for the stimulation of ATP7B activity. Enzyme kinetics assays demonstrated that PKC signaling pathway changed Vmax, but not the affinity for ATP and copper. Catalytic phosphorylation assays did not present any changes in ATP7B phosphorylation or dephosphorylation steps, indicating that the change occurs in a step after ATP7B dephosphorylation. In conclusion, signaling pathways that activate phospholipase C and PKC epsilon may stimulate ATP7B activity, consequently increasing active copper transport which could change the hepatic copper metabolism." FAPERJ, CNPq, INBEB D36 - EFEITO CARDIOPROTETOR DO PROPRANOLOL NA INSUFICIÊNCIA CARDÍACA ESTABELECIDA EM RATOS WISTAR ADULTOS DESNUTRIDOS CRONICAMENTE.

Mendes, L.V.P.1; Oliveira-Pinto, L.M.2; Nascimento, J.H.M.2; Vieyra, A.2,3; Cunha, V.M.N.1; Lara, L.S.1,3 Mendes, L.V.P.1; Oliveira-Pinto, L.M.2; Nascimento, J.H.M.2;

Vieyra, A.2, 1-Instituto de Ciências Biomédicas - UFRJ; 2-Instituto de Biofísica Carlos Chagas Filho – UFRJ; 3-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem.



A desnutrição crônica promovida em ratos wistar (DBR-CR) modifica a hemodinâmica cardíaca estabelecendo um quadro de insuficiência cardíaca (IC). O objetivo deste trabalho foi determinar se o tratamento com propranolol (30 mg/Kg) previne o desenvolvimento da IC, avaliando a sinali a o β-adrenérgica. Após o desmame, ratos Wistar foram divididos em: Controle e Controlep (alimentados com dieta convencional sem e com tratamento com propranolol, respectivamente); DBR-CR e DBR-CRp (desnutridos sem ou com tratamento, respectivamente). Após o sacrifício (13 semanas), os corações foram utilizados para ensaios de Langendorff e bioquímicos (n=5, por grupo; procedimentos aprovados pelo CEUA da UFRJ; DFCICB030). Foi observada a diminuição da pressão ventricular esquerda máxima (PDmáx) do grupo DBR-CR comparado ao controle (45 %); bem como a do índice de contratilidade (+dP/dTmax) (40 %) e a do índice de relaxamento (-dP/dTmáx) (30 %). Houve redução do Emax do isoproterenol (143±15 vs 184±11 %). Foi observado deslocamento para a esquerda da curva de Frank-Starling, e a redução da capacitância venosa em 27%. O tratamento com propranolol promoveu: (1) a manutenção da PDmáx; (2) o aumento da +dP/dTMax e da -dP/dTMax e (3) redução em 60% do Emáx do isoproterenol. Em relação as proteínas cinases, o tratamento reverteu a diminuição da razão entre atividade e expressão da proteína cinase A (PKA) observado no grupo DBR-CR, identificando-se aumento do conte do roteico de PKCα isoforma rotetora da IC e diminui o de PKCε en ol ida no desenvolvimento da IC). Estes dados indicam que a redução da contratilidade e da complacência, estabelecida no grupo DBR-CR está associado a dessensibilização da via β-adrenérgica. O tratamento com propranol preveniu a redução da função hemodinâmica cardíaca, re-estabelecendo a ia de sinali a o β-adrenérgica. CNPq D37 - THE ENDOCANNABINOID SYSTEM AS A POTENTIAL REGULATOR FOR Na+ HOMEOSTASIS IN KIDNEY CELLS 1, 2- SAMPAIO, L. S., 1, 2- TAVEIRA-SILVA, R., 1, 2- VIEYRA, A., 3- DI MARZO, V.,2- REIS, R.

A. M., 1, 2- EINICKER-LAMAS, M. 1- Instututo Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem. 2-

Instituto de Biofísica Carlos Chagas Filho – UFRJ, 3- Endocanabinnoid Research Group, Istituto di Chimica Biomolecolare, CNR, Pozzuoli

"The endocannabinoid system (ECS) is classically formed by cannabinoid receptors, endogenous lipids, enzymes for the synthesis and degradation. ECS is primarily involved in the modulation of rapid responses in various cellular models, including regulating the intracellular concentration of ions. The aim of this study was to identify the ECS in renal proximal tubule and evaluate the role of cannabinoids on the sodium active primary transport. For this purpose, LLC-PK1 cell line was grown at 90% confluence on DMEM+FBS 10%. To evaluate the presence of ECS, immunofluorescence and PCR were performed. The measure of activity of the Na+-K+ATPase was by colorimetric method. cAMP was assayed by the [3H] cAMP incorporation. Our data show that main enzymes and receptors of the ECS are expressed in LLC-PK1. Treatment with Win-55-212-2 (WIN), CB1/2 agonist, increases Na+-K+ATPase activity at 1 or 30min of incubation. Hemopressin (HP), CB1 inverse agonist, increased the activity with 1min, but decreased in 30min of treatment. Addition of AM251, CB1 antagonist, reverted the effect of both in 30min, but had no effect upon 1min. Pretreatment with Capsazepine, TRPV1 antagonist, abolished the effect of WIN or HP in 1min. Pretreated with PTX not showed difference on WIN or HP effect. The cAMP level increased 30min after addition of HP and was inhibited by AM251. The H-89, PKA inhibitor, returned the HP effect and Calphostin C, PKC inhibitor, blocked the WIN action. Our data suggests

that in LLC-PK1 there are different pathways of ECS regulated the Na+-K+ATPase between CB1/TRPV1, cAMP/PKA and PKC" INBEB, CAPES, CNPQ, FAPERJ D38 - ESTUDO DOS EFEITOS DO METILMERCÚRIO SOBRE OS CANAIS DE POTÁSSIO Kv4.3, hERG e KCNQ1/KCNE1 EM CÉLULAS HEK-293 1-SANTOS, M.C.P.; 2-GALLEGO, M.; 1-MEDEI, E.; 2-CASIS, O.; 1-NASCIMENTO, J.H. 1 – Laboratório de Eletrofisiologia Cardíaca Antonio Paes de Carvalho, IBCCF, UFRJ – RJ - Brasil 2 – Departamento de Fisiologia, Escola de Farmácia, UPV/EHU - Pais Vasco - Espanha "INTRODUÇÃO: O metilmercúrio (MeHg) é um organomercurial encontrado em peixes e outros animais aquáticos da Região Amazônica em áreas expostas à contaminação mercurial. Estudos epidemiológicos têm mostrado que a exposição humana ao MeHg pode contribuir para o desenvolvimento e progressão de desordens cardiovasculares, tais como aumento do risco de arritmias cardíacas e morte súbita. Atualmente é bem aceito que mudanças (preponderantemente diminuição) nas correntes repolarizantes de potássio são uma das principais causas destes eventos arrítmicos. OBJETIVO: Investigar os efeitos do MeHg sobre as correntes iônicas repolarizantes de potássio: Kv4.3; hERG e KCNQ1/KCNE1, envolvidas diretamente na repolarização celular, em células HEK-293. MATERIAIS E MÉTODOS: Para o estudo eletrofisiológico, foi utilizado o método de Patch-clamp na configuração Whole-cell. Assim, protocolos de voltagem específicos foram utilizados para registro das correntes de potássio permeadas pelas seguintes subunidades: Kv4.3, hERG e KCNQ1/KCNE1. Células HEK-293, foram transfectadas com as subunidades acima descritas. Uma curva dose-resposta com as seguintes concentrações de MeHg foi realizada: 0 nM; 0,01 nM; 0,1 nM; 1 nM visando analisar as mudanças nas correntes iônicas correspondentes. RESULTADOS: O MeHg promoveu redução na amplitude de todas as correntes iônicas estudadas (p<0,05). A cinética de ativação e inativação de Kv4.3 e de ativação e desativação de hERG não sofreu alterações com a utilização do MeHg. Entretanto, a corrente permeada pelas subunidades KCNQ1/KCNE1 apresentou mudança na dependência de voltagem da ativação, tendo seu V1/2 deslocado para potenciais mais positivos. CONCLUSÃO: Os resultados apresentados mostram que o MeHg é capaz de promover mudanças nas correntes repolarizantes semelhantes àquelas classicamente encontradas em corações com remodelamento elétrico e maior risco de arritmias cardíacas." CNPq, Ciência sem Fronteiras D39 - SYNTHESIS, CHARACTERIZATION AND EVALUATION OF XANTHENEDIONES AS TYROSINASE ACTIVATORS

1 FERREIRA, M.S.; 2 PIRES, D.A.T.;1 FIGUEROA-VILLAR, J.D. 1- Departamento de Química, Instituto Militar de Engenharia; 2- Instituto de Química,

Universidade de Brasília Tyrosinase is the enzyme responsible for production of melanin. The appearance of melanoma, the most serious form of skin cancer, can be related to disorders of this enzyme. Therefore is important to develop tyrosinase inhibitors to avoid skin cancer. One of the tested compounds as inhibitors of this enzyme were tetraketones. However, these compounds only display relatively limited inhibition activity. We discovered using NMR spectroscopy that some of these tetraketones suffer cyclization in solution at room temperature, forming the respective xanthenedione. For this reason, we considered the possibility of these xanthenediones displaying an opposite effect of tetraketones with tyrosinase, acting as activators. Therefore, seven of these compounds were synthesized in two steps: reaction of dimedone with benzaldehydes, forming tetraketones, followed by cyclization using p-toluenesulfonic acid at 100 oC, leading to



good yield (83 to 94%). The structure of these compounds were determined by NMR and IR spectroscopy and tested as inhibitors or activators of tyrosinase using as substrates L-tyrosine or L-Dopa by UV spectroscopy. The obtained results confirmed that six xanthenediones are activators of this enzyme. The two more effective tyrosinase activators were para-methoxyphenyl xanthenedione and phenyl xanthenedione, which increased the activity of the enzyme 89.88 and 172.56%, respectively. In function of these results we consider necessary the preparation of new tetraketones or similar compounds which could not be cyclized, a condition that could lead to better anticancer activity. INBEB, CNPq e CAPES D40 - TLR2 KNOCKOUT (TLR2-/-) ATTENUATE STREPTOZOTOCIN-INDUCED CARDIAC ELECTRICAL REMODELING IN GENDER NON-SPECIFIC MANNER

Micaela López Alarcón, Gustavo Monnerat-Cahli, Gabriel Manso, Guilherme Brasil, Emiliano Medei.

IBCCF Several studies have shown that inflammation plays a key role in diabetes. Cardiomyopathy is one of the main causes of death in this disease. The present study investigates whether Toll like receptors (TLR) 2 regulates cardiac electrical activity in diabetic mice and whether its effects is genderspecific. Methods and Results: Diabetes was induced by 5 consecutives low doses of streptozotocin (STZ) in C57BL/6 mice from both sexes. The animals were divided in eight groups (female and male): WT; WT+STZ; TLR2 -/- and TLR2-/-+STZ. Eight weeks after diabetes induction, metabolic and biometric parameters were analyzed. The cardiac electrical function was analyzed by ECG and cardiac AP. The TLR2 knockout did not prevent the hyperglycemia neither preserved biometrics alterations in both sexes. Despite the chronic hyperglycemic state, the TLR2-/-+STZ preserves in both sexes the ECG and action potential parameters analyzed. In addition the TLR2-/-+STZ prevented the plasmatic increase of IL-1beta and TNF-alpha induced by diabetes. The direct stimulation of either TLR2/1 or TLR2/6 by its specific agonists promotes a cardiac electrical remodeling, prolonging the AP duration. Cardiac function assess by MRI was similar among groups. Conclusion: The data obtained with the agonist upon cardiac electrical activity and the plasmatic cytokines demonstrated that, at least, two different mechanisms could be involved inTLR2-/- preserve cardiac electrical remodeling in hyperglycemic condition. FAPERJ, CNPq, INBEB D41 - ROLE OF CCR2 IN LEARNING PROCESS AND MEMORY CONSOLIDATION 1-TEIXEIRA-DA-CUNHA, M.G.; 1-ALVES-JÚNIOR, 1-S.C; CHAVES, D.; 1-ALBUQUERQUE, D.; 2-RAJAO, M.; 3-GUERCIO, G.; 1- ’ VI , J.C.P. 3-PIAZINUTI, R.; 3-MENDEZ-OTERO, R.; 1-

REIS, P.A.; 1-BOZZA, P. T. ; 1-BOZZA, F.A.; 1-CASTRO-FARIA-NETO, H.C.; 1-GOMES, R.N. 1-Instituto Oswaldo Cruz, FIOCRUZ; 2- INCA; 3- UFRJ

The CCR2 is a chemokine receptor coupled to G protein. Its major ligand is the CCL2, in wich is correlated with neuroinflammatory response in infection process, brain trauma, Alzheimer, multiple sclerosis. But recent studies demonstrated that both CCR2 and CCL2 are expressed constitutively in important places of the central nervous system, such as in hippocampus. The CCL2 increases the neuronal intracellular calcium and act as a neuron-excitatory mediator in neurons of the hippocampus. Any studies showed the role of CCR2 in memory consolidation under normal physiological conditions. To clarify this point, we analyzed the role of CCR2 in learning process and in memory consolidation of short time memory and longtime memory. For this we used CCR2 deficient mice, and submitted this animals to water maze test to evaluate the

contextual memory, to passive avoidance test to analyze the aversive memory, to open field and PPI to examined both motor response and reflex response. We observed that the absence of CCR2: impaired the memory consolidation of contextual memory in comparison with wild type animals, increased the number of trials that the animals require to learn in passive avoidance test, decreased the consolidation of short time memory and longtime memory. The cognitive dysfunction is not associated to motor or reflex impaired response. This decreased consolidation of memory was correlated with lower expression of MAPK p-42-44 phosphorylated 1 hour after training in passive avoidance test and decreased expression of mature BDNF in hippocampus 24h hours after training in passive avoidance test. In CCR2 deficient mice we observed decreased co-localization of PSD95 and Synaptophysin. These data suggest that the CCR2 is important to function of synapsis in hippocampus, to neuronal function involved with learning process and to sustentation of the memory of contextual and aversive memory. IOC - FIOCRUZ, FAPERJ D42 - STUDIES BY MOLECULAR DYNAMICS AND GENERALIZED SIMULATED ANNEALING OF ANTIMICROBIAL PEPTIDE DERMADISTINCTIN K Batista, Moema M..1 Fernandes,T cio V. .1, antoro, Marcelo M.3† Pascutti, Pedro

G.1,2 1Lab. de Modelagem e Dinâmica Molecular, IBCCF-UFRJ, RJ, 2INMETRO - Instituto

Nacional de Metrologia, Qualidade e Tecnologia, RJ, Brazil. Dep de Bioquímica-ICB-UFMG, MG, 3 (deceased in march 10, 2014)

"INTRODUCTION: Dermadistinctin (DD K) is an antimicrobial peptide from Phylomedusa distincta frog skin. It has an α-helix structure that appears only in biomembrane or in presence of amphipathic cosolvent. The aim of this work was to study conformational-dynamics-structure of DD K peptide in a solvent used to stabilize peptides (2,2,2-trifluorethanol-TFE/water mixture) by molecular dynamics (MD) simulations and generalized simulated annealing (GSA). METHODOLOGY: The structural properties of this peptide have been simulated in TFE/water mixture using GROMACS 4.5.5 and Gromos53a6 force field. DD K peptide was fitted in a cubic box solvated with SPC (single point charge) water model and TFE (50%/50%). 5 ions of chloride were added to this system for balance of charge. The pressure used was 1 atm and the temperature was 293,15 K. The reference structure was taken from PDB website by code: 2JX6. The GSA program was developed in the LMDM at UFRJ to predict peptides structures. It randomly searches for the global minimum in energy hyper-surface. We could build a hydrophobic environment to DD K by MD simulation and GSA algorithm where this peptide could fold. RESULTS: We observed a coil structure in the first 10 residues that in MD simulations presented a transition to β-sheet. This structure change suggests an alternative mechanism for DD K insertion in membrane. GSA results did not show this transition. We also studied the statistic of the solvent radial distribution around each peptide residue. As result ALA, GLU, ILE, LEU, TRP were solvated in TFE and LYS, GLY were solvated in water. When native contacts were calculated, 80% of conformations were next the reference structure in GSA and MD simulations. MD simulations in TFE/water mixtures were important to study DD K dynamics, showing standard of conformation that could help to understand the biological activity of the peptide in biomembranes." INBEB, FAPEAM, INMETRO, FAPERJ D43 - IDENTIFICATION OF PROMISING ANTI-PRION DRUG CANDIDATES THROUGH EX-VIVO, IN VITRO AND IN SILICO METHODOLOGIES



NATALIA C. FERREIRA1, WESLEY A. CONCEIÇÃO1, BRUNO MACEDO1, CLARICE S. MACHADO1, ALESSANDRA MASCARELLO2, LOUISE D. CHIARADIA-DELATORRE2,

ROSENDO A. YUNES2, RICARDO J. NUNES2, ANDREW G. HUGHSON3, JASON HOLLISTER3, GERALD S. BARON3, BYRON CAUGHEY3, YRAIMA CORDEIRO1

1- Departamento de Biotecnologia Farmacêutica, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil. 2- Departamento de Química, Universidade Federal de Santa Catarina, Santa Catarina, Brasil. 3- Laboratory of

Persistent Viral Diseases, Rocky Mountain Laboratories, National Institutes of Health, Montana, United States of America.

Introduction: Transmissible spongiform encephalopathies (TSEs) are infectious neurodegenerative disorders that do not have a symptomatic, curative or prophylactic treatment. TSEs arise after the conversion of the cellular prion protein (PrPsen) which is soluble, into the scrapie isoform (PrPres) that aggregates and accumulates in the brain. Although many compounds have been effective in vitro none worked in vivo, mainly because of their inability to cross the blood-brain-barrier (BBB). Screening in prion-infected cell culture (ScN2a) is commonly used to identify new anti-prion compounds. Material and methods: In this work we tested 200 aromatic chemical compounds in neuroblastoma cell infected with RML, 22L and CWD strains. Real-time quaking induced conversion (RT-QuIC) assay tested the ability of these compounds to inhibit the conversion in a cell-free system. Confocal microscopy verified if the active compounds change PrP cellular location. In silico predictors were used to deduce about pharmacokinetic and pharmacodynamic properties of these molecules. MTT reduction assay allowed verifying their cytotoxicity in neuroblastoma cells. Molecular docking provided structural models and binding affinities for the interaction between PrP and the most promising compounds. Results: 47 compounds were able to inhibit in more than 50% the amount of PrP in ScN2a cells infected with RML strain. Many compounds were also effective against 22L and CWD strains. Three compounds were effective in the RT-QuIC when brains from hamster and a patient infected with CJD were used as seed. We found that some compounds induce the internalization of PrP in neuroblastoma cells, decreasing the availability of PrP in the cell surface to be converted into PrPres. These compounds were not cytotoxic and the 5 most promising ones were predicted as non-mutagenic and able to cross the BBB. Conclusions: We identified new anti-prion drug candidates which are effective in vitro and ex-vivo, and posses pharmacokinetic properties that support their drugability. CNPq, FAPERJ, INBEB D44 - A MEMBRANOTROPIC HEPATITIS C VIRUS PEPTIDE INTERACTS WITH MODEL MEMBRANES AND HUMAN CELL SURFACE AND GAINS HELIX CONTENT 1-ALVES, N.S.; 1-MENDES, Y.S.; 2-SOUZA, T.L.F.; 1-BIANCONI, M.L.; 1-CARVALHO, C.A.M.;

3-ANOBOM, C.D.; 1-VALENTE, A.P.; 1-SILVA, J.L.; 1-GOMES, A.M.O; 1-OLIVEIRA, A.C. 1-Instituto de Bioquímica Médica Leopoldo de Meis, Univerisdade Federal do Rio de

Janeiro; 2-Faculdade de Farmácia, Univerisdade Federal do Rio de Janeiro; 3-Instituto de Química, Univerisdade Federal do Rio de Janeiro

The Hepatitis C virus (HCV) is the major cause of viral hepatitis, becoming a worldwide health problem. HCV is an enveloped RNA virus, and the glycoproteins, E1 and E2, mediate cell entry and membrane fusion. Unfortunately, the localization of a fusion peptide remains unknown. The structural characterization of HCV fusion mechanisms is ery im ortant for the de elo ment of anti− CV strategies. ere we characteri e the interaction of an E2 membranotropic peptide, HCV421-445, with different membrane models. Peptide-membrane interactions and changes resulting from these interactions were evaluated by circular dichroism (CD), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), dynamic light scattering (DLS), two-photon

excitation microscopy and nuclear magnetic resonance (NMR). ITC data show that peptide-micelle interaction is endothermic and is favored by micelle charge. This membrane-charge dependence is also observed by DSC, when changes in transition temperature are only observed when phosphatidylglycerol is present. HCV421-445 induces vesicle aggregation in a pH-dependent manner, as observed by DLS. Two-photon microscopy reveals that FITC-labeled peptide binds to HepG2 membrane and remains at cell surface for at least 30 minutes. CD and NMR analysis show that the peptide is unstructured in solution and gains helix content when in the presence of micelles. This helix could be stabilized by a helix stabilization motif, GXXXG. Our results indicate that HCV421-445 is able to perturb lipid membranes, and becomes structured, a common feature of fusion peptides. As this peptide may be important for cell fusion, these studies are important to better understand the HCV fusion mechanisms, which could help in the rational development of new antiviral therapies. Capes, CNPq, FAPERJ, PRONEX, INBEB D45 - POTENCIAL TERAPÊUTICO DE CÉLULAS-TRONCO PLURIPOTENTES HUMANAS EM MODELO RENAL DE ISQUEMIA E REPERFUSÃO

1, 2- SIGNORETTI, P. V.P.; 2- FERNANDES, A. M.; 2- ASSIS, J. L.; 1- VIEYRA, A.; 1- VALVERDE, R. R. F. H.; 2- LAMAS, M. E.

1- Laboratório de Físico-Química Biológica Aída Hassón Voloch, Instituto de Biofísica Carlos Chagas Filho; 2- Laboratório de Biomembrana,Instituto de Biofísica Carlos Chagas

Filho "INTRODUÇÃO: As doenças renais (DRs) representam um grave problema de saúde pública onde cerca de 50% dos pacientes não sobrevivem. Tratamentos como a diálise, aumentam a qualidade de vida dos pacientes mas tem efeito meramente paliativo e grande impacto financeiro no SUS, principal financiador do tratamento de aproximadamente 85% destes pacientes. Uma alternativa para o tratamento das DRs é o transplantes, contudo, a disponibilidade do órgão e a compatibilidade doador/receptor continuam sendo fatores determinantes e limitantes. Diante deste cenário, a utilização de células-tronco visando o reparo de órgãos lesados é sugerida como principal alternativa. Células-Tronco Embrionárias Humanas (hES) são relevantes em estratégias regenerativas principalmente por oferecerem possibilidade de reposição das células afetadas devido a sua pluripotencialidade. OBJETIVO: Diante do exposto acima, o presente trabalho teve por objetivo analisar o potencial terapêutico de hES em modelo animal de lesão renal por isquemia e reperfusão (I/R). METODOLOGIA: 1 x 106 células/100 μ foram injetadas na cápsula renal de ratos machos Wistar 1 hora antes da lesão por isquemia bilateral (30 minutos) e reperfusão (24 h) (CEUA: IBCCF148). Os animais foram acompanhados 4 e 24 horas após injeção por SPECT (CENABIO) via marcação com DMSA-Tc99m e análises para marcadores de células humanas (anti-HNA) e de morte celular além de swelling mitocondrial foram realizadas. RESULTADOS: Foi observada a melhora no parênquima renal 24 h após administração de hES. Marcação para HNA indicou a presença das hES no tecido renal isquêmico. Além disso, houve aumento de 5x na expressão de Bcl-2 (sem alteração significativa de Bax) e redução do swelling mitocondrial nos animais submetidos à injeção com as hES quando comparado aos animais isquêmicos. CONCLUSÃO: A partir desses dados preliminares, podemos sugerir que as hES parecem recuperar a função e a integridade tecidual renal neste modelo de lesão por I/R." CAPES/PROBITEC, CAPES/INL, FAPERJ D46 - EFEITO CRÔNICO DE ESTERÓIDES ANABÓLICOS SOBRE AS CORRENTES ICA,L E ITO E A MOBILIZAÇÃO DE CÁLCIO EM MIÓCITOS VENTRICULARES.

1-ARANTES, P. C.; 2-MEDEI, E.; 3- NASCIMENTO, J. H



Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro Esteroides anabólicos (EAs) são derivados sintéticos da testosterona utilizados ilicitamente por atletas e não atletas, em doses supra-fisiológicas, para promover ganho de massa muscular, força e resistência ou para fins estéticos. Causam efeitos colaterais, dentre eles alterações cardiovasculares, como hipertensão arterial, hipertrofia cardíaca, arritmias e infarto do miocárdio. Este estudo objetivou investigar o remodelamento elétrico cardíaco e sua repercussão no acoplamento excitação-contração, induzido pelo tratamento crônico com EAs. Ratos sedentários, foram tratados por 8 semanas com 10 mg/kg/sem. (i.m.) de decanoato de nandrolona (Deca) ou veículo (Ctrl). Foram usadas técnicas de ECG na derivação D2, microeletrodo intracelular e patch clamp na configuração Whole cell, para análise da repolarização ventricular e a técnica de tensão isométrica em fibra cardíaca desnuda avaliação do acoplamento excitação-contração. O grupo Deca apresentou aumento do intervalo QT e QTc (p<0,001), sem alteração da frequência cardíaca; aumento na duração do potencial de ação a 30% e a 90% (p<0,001) da repolarização máxima; e aumento da triangulação do potencial de ação (p<0,001). A amplitude de Ito nas camadas endocárdica e epicárdica do ventrículo esquerdo foi menor (p<0,05 e p<0,001, respectivamente), enquanto a amplitude da ICa,L foi maior no grupo Deca (p<0.01). Na avaliação do acoplamento excitação-contração, as medidas de tensão isométrica mostraram que o grupo Deca desenvolve maior tensão nos pCa 6,0; 5,6; e 4,8 (p<0,001) e maior sensibilidade ao Ca2+ (p<0,05). O grupo Deca apresentou menor resposta contrátil a diferentes concentrações de cafeína após tempo fixo (3 min) de carregamento de Ca2+ (p<0,001) e a 20 mM de cafeína após 1, 3 e 5 min de carregamento do reticulo sarcoplasmático (p<0,001). Concluímos que o tratamento crônico com decanoato de nandrolona causou remodelamento elétrico cardíaco nas alterações nas amplitudes de ICa,L e Ito refletiram no acoplamento excitação-contração, causando menor mobilização e maior sensibilidade ao Ca2+. Capes,CNPq D47 - ANÁLISE ESTRUTURAL DO FRAGMENTO N-TERMINAL DA ENDOSTATINA E SUA INTERAÇÃO PUTATIVA COM A INTEGRINA ΑVΒ3

1,2- TORRES, P.H.M.; 1- PASCUTTI, P.G. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-

Instituto Nacional de Metrologia, Qualidade e Tecnologia A angiogênese, ou neovascularização, é um processo fisiológico complexo que culmina com a proliferação e migração de células endoteliais e a formação de novos vasos sanguíneos. É um evento importante em processos de cicatrização e na formação do endométrio uterino, mas também pode ser estimulado por determinadas patologias como o câncer. Compostos antiangiogênicos, como a endostatina, a canstatina e o arresteno podem ser, portanto, utilizados no tratamento de pacientes acometidos por estas patologias. Muitos destes compostos, entre eles a endostatina, exercem parte de suas funções através da interação com integrinas, que desempenham papel central na migração das células endoteliais e no estabelecimento de novos vasos. Recentemente, foi demonstrado que a atividade antiangiogênica da endostatina pode ser desempenhada por um peptídeo de 27 resíduos pertencente à região N-terminal da proteína. O presente estudo tem como objetivo contribuir para o entendimento do mecanismo de ação deste peptídeo e a possível interação do mesmo com a integrina alfa-v-beta-3, bem como elucidar mecanismos envolvidos na ativação desta integrina. Para tanto, foram utilizadas técnicas de ressonância magnética nuclear, dicroísmo circular, modelagem e dinâmica molecular. Os resultados sugerem que o peptídeo assume uma conformação em grampo-beta, capaz de interagir com uma fenda existente na interface entre as subunidades da integrina alfa-v-beta-3 que apresenta

complementariedade estérica e eletrostática. Identificamos também regiões importantes para a manutenção da conformação inativa da integrina, como uma alça pertencente à 5ª lâmina do domínio beta-propeller e um grupo hidrofóbico formado por resíduos dos domínios beta-A e beta-tail. Sendo assim, propusemos que este peptídeo, bem como outras moléculas antiangiogênicas, podem exercer suas funções biológicas através de uma interação ainda não descrita na literatura e a exploração destas regiões pode, portanto, mostrar-se relevante para o desenho de fármacos inibidores de integrina. INBEB, FAPERJ, CNPq D48 - NMR-SUPPORTED STRUCTURAL GENOMIC STUDIES OF TRYPANOSOMES

1 - RACHEL SANTOS DE MENEZES; 1 - ARACELYS LÓPEZ CASTILLA; 1 - EVERTON DIAS D'ANDREA; 1 - GABRIELA PINHEIRO HEREDIA; 1 - RAQUEL DA MOTTA; 2 - CHRISTOPH RADEMACHER; 3 - ANNE DIEHL; 3 - HARTMUT OSCHKINAT; 1 - JOSÉ RICARDO PIRES

1 - 1Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2 - 2Max Planck Institute of Colloids and Interfaces , Berlin, Germany; 3 - 3Leibniz Institutute of

Molecular Pharmacology, Berlin, Germany Chagas disease, Sleeping sickness and Leishmaniasis are between the so called neglected diseases, endemic in poor countries of south-America and Africa. Sequencing of the genome of the kinetoplastids Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp databank) open up new perspectives for drug research against these diseases. In each genome ca. 20,000 genes were identified ca. 50 % coding for proteins of unknown function. Proteomic studies confirmed the expression of several of these proteins in a life-cycle dependent manner. In the present work using bioinformatics tools available we mined the tritryp databank to end up with a list of 409 proteins up to 30 kDa, conserved in kinetoplastids, without orthologues in mammals, plant or fungi, without trans-membrane regions and without homologous sequences in the PDB that should be suitable for structural studies by solution NMR. From these proteins 20 were cloned in expression plasmids, and at least 6 were expressed as soluble and folded proteins in E. coli, as evidenced by their 1H or 1H-15N HSQC nmr spectra. The expression of the remaining proteins is still being optimized. The NMR structures of four proteins were solved: Chagasin, a cysteine-protease inhibitor; FKBP12, a peptidyl-prolyl cis-trans isomerase; a protein containing a single domain of unknown function (DUF 1935), kinetoplastid specific related to the cysteine-protease Calpain; and the hypothetical protein Q4D059. Interaction studies for some of these proteins with potential ligands are also described in this work. CAPES, CNPq, FAPERJ D49 - DESENVOLVIMENTO DE FÁRMACOS ANTI-HIV: PLANEJAMENTO DE INIBIDORES PARA O VÍRUS HIV-1 NÃO-B

1-REIS, R.R.; 1-PASCUTTI, P.G. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.

"O vírus da imunodeficiência humana (HIV) afeta o sistema imunológico caracterizando a Síndrome da Imunodeficiência Adquirida (AIDS). Teve seus primeiros casos reportados nos EUA, Haiti e África Central nos anos de 1977 e 1978. Segundo dados estatísticos, esta doença matou 1,7 milhões de pessoas em 2011, tendo atualmente 34 milhões de infectados no mundo. As proteínas do vírus são expressas como longas cadeias polipeptídicas que devem ser processadas, via proteólise pela aspartil-protease do vírus, para adquirirem atividade. Dessa forma, a protease é um eficiente alvo para terapia anti-HIV, resultando em uma nova classe de fármacos, os inibidores de protease. Existem dois tipos prevalentes do vírus: HIV-1 e HIV-2. O mais comum, HIV-1, pode ser



dividido em grupos (M, N, O e P) e subtipos: A-K. A maior parte das infecções por HIV na África são causadas pelos subtipos virais A e C. No Brasil, a lista de subtipos presentes inclui os subtipos B (o principal em circulação), F (18% de prevalência), C, D, e A. O subtipo F é distribuído em todo o país e corresponde aproximadamente a 10% das amostras analisadas nas cidades do Rio de Janeiro e São Paulo. Neste trabalho, estamos estudando os subtipos A, B, C, e F da protease complexada com novos inibidores (já utilizados na clínica) e com três inibidores clássicos (ritonavir, indinavir e nelfinavir). Como resultados preliminares, temos a análise da protease por meio de simulações de dinâmica molecular que sugerem que a proteina é bastante estável. Simulações de docking molecular dos inibidores no sítio ativo dos diferentes subtipos e simulações de dinâmica molecular do complexo estão em curso visando a proposição de novos fármacos." CNPq D50 - STRUCTURAL STABILITY IN AMYLOIDOGENIC AND NON-AMYLOIDOGENIC VARIANTS OF THE TRANSTHYRETIN PROTEIN BY MD SIMULATIONS UNDER HIGH PRESSURE 1-REINALDO S. DE OLIVEIRA JÚNIOR; 2-FERNANDO L. PALHANO; 2-DEBORA FOGUEL; 1-

PEDRO G. PASCUTTI 1-Instituto de Biofísica Carlos Chagas Filho - UFRJ; 2-Instituto de Bioquímica Medica -

UFRJ INTRODUCTION: The formation of insoluble amyloid fibers is a characteristic of many diseases known as amyloidosis. The transthyretin (TTR) is a homo-tetramer protein of 55 kDa and 127 residues per monomer. Over of one hundred mutations have been described for the structure of transthyretin related to amyloid diseases such as Familial Amyloid Polyneuropathy (FAP), characterized by the deposition of amyloid aggregates in the peripheral nervous system. METHODOLOGY: The dissociation and denaturation of TTR variants have been studied in different conditions of temperature, pH and pressure. In the previous studies our group has described that the non-amyloidogenic variant as T119M has great stability under 3.0 Kbar, pH 7.5, and 1 °C compared with the amyloidogenic one. We used Gromos96_53a6 Force Field, Reaction Field (radius 1.4 nm) in electrostatic treatment; SPC/E water model, cubical box, 5 ns simulation for stabilization the hydrated layer of protein and ions. Consecutive simulations totalize 400 ns, 25 ns for each pressure step of 0,5 kbar, temperature of 1 °C, pH 3,0, and pressure enhancement from 1,0 bar ≅ 1,0 atm to 3,5 kbar. RESULTS AND DISCUSSION: Multiple and sequential Molecular Dynamics (MD) simulations of WT - and V30M-TTR dimers were performed at high pressure (up to 3,5 kbar) and in explicit water to assess the structural stability of transthyretin. The conformational space upon protein unfolding was explored, and it was identified structural changes that may lead to the amyloid assembly. The analysis of molecular properties such as secondary structure, hydrogen bonds, and solvent accessible surface area along the MD unfolding trajectories clearly demonstrate that V30M-TTR has a much higher tendency to unfold than WT-TTR. These results are in agreement with previously published experimental data on the conformational stability of dimers of several TTR variants. CNPq, FAPERJ, INBEB D51 - INSIGHTS INTO IONIZATION STATES OF THE CATALYTIC RESIDUES IN HIV-1 PROTEASE BY HYBRID QM/MM MOLECULAR DYNAMICS SIMULATIONS

1-SOARES, R. O.; 2-GONÇALVES, A. S.; 1-PASCUTTI, P. G. 1-Instituto de Biofísica, UFRJ; 2-Instituto Federal de Educação, Ciência e Tecnologia do

Espírito Santo,Unidade Guarapari

"The HIV protease (HIV-PR) is an enzyme whose catalytic residue consists of two aspartate side chains (Asp25/Asp25'). This protein is a relevant therapeutic target for antiretroviral (ARV) treatment against AIDS. Several works suggest a monoprotonation states for the aspartate residues in the active site of HIV-PR, with one of the residues charged and the other neutral or both sharing a proton. However, there are experimental evidences that HIV could be found in this protonation state, but also in other two different states. In the present work we used the combined method of quantum-mechanics and molecular-mechanics simulations (QM/MM) to investigate the functional role of protonation in HIV-PR complexed with a substrate. We investigated three different protonation states for the two aspartate catalytic residues: monoprotonation, diprotonation and deprotonation and analyzed the binding of Gag peptide substrate p2/NC (sequence of aminoacid residues SATIM/MQRGN) to the enzyme in these three different states. Our results demonstrate that the non protonation of both aspartic acids has a strong influence on the dynamic behavior of the HIV-PR flaps, making ease its opening with the greater amplitude. Beside, this work showed that the monoprotonation in one of the Asp25 residues results in the strongest interaction between these catalytic residues by sharing the proton. This indicates that the alterations in the flaps's dynamics are likely relationship as the different states of protonation on residue catalytic and we expect these findings to contribute to a better understanding of the protonation state's role in the catalytic action of HIV-PR." CNPq, FAPERJ, INBEB D52 - O TRATAMENTO COM LPA (ÁCIDO LISOFOSFATÍDICO) PREVINE OS DANOS RENAIS E A ETAPA INICIAL DA ATIVAÇÃO DO ESTRESSE DO RETÍCULO ENDOPLASMÁTICO EM RATOS WISTAR SUBMETIDO AO PROCESSO DE ISQUEMIA-REPERFUSÃO RENAL.

1-GONSALEZ, S.R.; 1-LEAL, A.C.; 2,3-EINICKER-LAMAS, M.; 1,3-LARA, L.S. 1-INSTITUTO DE CIÊNCIAS BIOMÉDICAS - UFRJ; 2 - INSTITUTO DE BIOFÍSICA CARLOS

CHAGAS FILHO - UFRJ; 3 - INSTITUTO NACIONAL DE CIÊNCIA E TECNOLOGIA DE BIOLOGIA ESTRUTURAL E BIOMAGEM – UFRJ.

A clínica carece de tratamentos eficazes na prevenção do dano renal mediado pela isquemia-reperfusão (I/R). O ácido lisofosfatídico (LPA) surge como um potencial agente terapêutico. O objetivo foi caracterizar o efeito do tratamento com LPA no modelo de I/R renal com ênfase ao transporte de Na+ e a proteção contra o estresse do retículo endoplasmático. Foram utilizados 3 grupos experimentais: controle; I/R: sofreu isquemia das artérias renais por 30 min e 24h de reperfusão; I/R+LPA: administração de LPA (1 mg/Kg) durante a isquemia. Foram realizados ensaios funcionais renais e morfológicos, Western blot, e atividade enzimática. O conteúdo de nitrogênio ureico no sangue aumentou em 241% na I/R e o LPA preveniu esse aumento, sem alteração da proteinúria. O ritmo de filtração glomerular diminuiu em 40% na I/R, retornando aos valores controle com o tratamento. Já a excreção de Na+ diminui em 79%, e não foi prevenida pelo LPA. Durante a I/R, a atividade e a expressão da NKA aumentam cerca de 50 % enquanto a atividade da NaA diminui em 46%; este efeito pode estar associado a diminuição de 40% da atividade da proteína cinase C (PKC). O tratamento com LPA previne o efeito da I/R sobre a NKA e a PKC, mas não sobre a NaA. O mecanismo de ação do LPA envolve: a recuperação exclusiva da perda de sensibilidade da NKA pela via PLC/PKC ocasionada pela I/R, a diminuição da expressão do receptor de LPA2 (50%) e a manutenção da expressão de GRP78, reduzida em 40% na I/R. O LPA protege o rim do dano glomerular, mas não do tubular, este está associada à insensibilidade da NaA ao tratamento. A manutenção da via PLC/PKC é o mecanismo central sobre as atividades ATPásicas e sobre na manutenção da homeostasia do retículo endoplasmático, evidenciado pela prevenção do aumento de GRP78.



FAPERJ, CNPq, INBEB D53 - IMPAIRMENT OF MEGAKARYOPOIESIS BY YELLOW FEVER VIRUS AND DENGUE VIRUS 1- Campos, S.P.C.; 1- Ferreira, D.L.; 1- Castro, M. G.; 1- Sanches; D.; 2- Rodrigues, M. F.;

3- Paredes, B. D.; 1- Silva, J.L.; 1- Gomes, A.M.O. & 1- Oliveira, A.C. 1 Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber -

IBqM/UFRJ; 2 Laboratório de Biologia Molecular do Câncer - IBqM/UFRJ; 3 Laboratório de Cardiologia Celular e Molecular – IBCCF/UFRJ

"Introduction: Dengue Virus (DENV) and Yellow Fever Virus (YFV) have great importance in Africa, South America and Asia and cause acute hemorrhagic fevers that are related to hemostasis dysfunction, with thrombocytopenia. The thrombocytopenia is related to the diseases severity evolution. Platelets play a crucial role in hemostasis and are cytoplasmic fragments of megakaryocytes. The megakaryocytes are derived from their hematopoietic progenitors, megakaryoblast. Each of them gives rise to various megakaryocytes and single megakaryocyte produces from 5.000 to 10.000 platelets. The processes that lead to dengue and yellow fever diseases severity evolution and how these viruses alters platelets production have not yet been elucidated. Aim: To better clarify the processes in which viral infection leads to thrombocytopenia, we aim to study the interaction between DENV and YFV with megakaryocyte progenitors, analyzing possible events of megakaryopoiesis impairment. Material and Methods: We infected MEG-01 cells (Human Megakaryoblastic cell line) with DENV-2 and YFV 17 DD in a multiplicity of infection of 1. Results and Discussion: We detected intracellular YFV proteins since 24h post infection (p.i.) by confocal microscopy, confirming infection. We analyzed the production of infectious particles by plaque assay and observed increasing production until 96h p.i., followed by decrease. We analyzed cell viability by extracellular activity of LDH and trypan blue exclusion. We observed higher LDH activity from 96h p.i. with YFV but not with DENV-2. We observed an increase in cell death from 120h p.i. for both viruses. During YFV infection, we also observed reduction on infected 4N cell population from 144h p.i. compared to control. Conclusion: Our data suggest that YFV infects and replicates in MEG-01 cells. Our data also suggest that DENV-2 and YFV can induce cell death from 120h p.i. Furthermore, YFV infections also changes differentiation profile by reducing the 4N cell population 144h p.i." CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX D54 - EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITY OF DIETHYLCARBAMAZINE (DEC) ON CARBON TETRACHLORIDE–INDUCED HEPATIC DAMAGE IN MICE 1-ROCHA,SWS*; 1-FRANÇA, MER; 1-RODRIGUES, GB; 1-BARBOSA, KPS; 1-NUNES, AKS; 3-

PASTOR, AF; 2-OLIVEIRA, AGV; 1-OLIVEIRA, WH; 1-LUNA, RL; 1-PEIXOTO, AC. 1-Laboratório de Ultraestrutura, Centro de Pesquisas Aggeu Magalhães - FIOCRUZ. 2-

Laboratório Microscopia e Microanálise, Centro de Tecnologias Estratégicas do Nordeste-MCT. 3-Laboratório de Virologia e Terapia Experimental, CPqAM- FIOCRUZ.

Pharmacological studies showed that DEC interferes in the arachidonic acid metabolism, acting as an anti-inflammatory drug. Rocha et al (2012) demonstrated that DEC can be a potential drug for the treatment of chronic inflammation induced by chronic alcoholism and as a hepatoprotective drug in reducing lesions in mice malnourished. In the present study we investigated the effect of DEC on chronic liver inflammation induced by carbon tetrachloride (CCl4). Forty C57BL/6J male mice were separated in groups (n=10): control group, DEC 50mg kg group, CCl4 group and CCl4 + DEC group. DEC (50mg/kg) was

administered in bottle for 12 days. CCl4 (0.5 ml/g) was administered for 6 weeks (2 injections/week). Liver fragments were processed for histological (HE and Sirius red), immunohistochemical, immunofluorescence and molecular analysis. Histological analyses of the CCl4 group showed large areas of extensive necrosis and fibrosis, lipid accumulation and inflammatory cell infiltration. The CCl4+DEC group exhibited reduced inflammatory process and prevented liver necrosis and fibrosis. CCl4 group increased collagen deposition, assessed by Sirius red-positive areas, which were markedly reduced in the CCl4+DEC group. Immunohistochemical and immunofluorescence analyses of the CCl4 group showed substantial COX-2, IL1β, MDA, TGFβ and αSMA immunopositivity. On the other hand, substantially reduced enzyme and cytokines expression were found in the CCl4+DEC group. The CCl4 group exhibited increased IL1β, COX-2, NFκB, IFNγ and TGFβ expressions in the western blot analysis, all of which were reduced in the CCl4+DEC group. The CCl4 group exhibited low expression of IL-10 (anti-inflammatory cytokine), whereas the CCl4+DEC group enhanced significantly the IL-10 expression. The persistence of hepatic injury in the CCl4 group was confirmed by the COX-2 and iNOS mRNA expression levels. In contrast, the CCl4+DEC group reduced significantly the mRNA enzymes expression. According to the present results, DEC is a potential alternative treatment for chronic liver inflammation induced by CCl4. INBEB, PAPES, FACEPE, CNPq. D55 - PREDICTION OF PROTEIN FOLDED IN NATIVE STRUCTURES BY GENERALIZED SIMULATED ANNEALING COUPLED TO GROMOS FORCE FIELD

1,2- FERNANDES, T.V.A.; 1,2- PASCUTTI, P.G. 1- Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro; 2-

National Institute of Metrology Quality and Technology, INMETRO "The genome mapping and sequencing advances are producing an exponential number of amino acid sequences of new proteins, and the comprehension of these protein structures becomes a crucial extension to these progresses. Currently, the three-dimensional structure of a protein is obtained by experimental techniques such as X-ray Crystallography or NMR. However, due to the limitations and high costs of these techniques, determination of three-dimensional structure of a protein is a problem that still challenges the scientists. In this sense, theoretical and computational studies have increased the understanding of the factors that drive a polypeptide sequence to its native state. This improvement is due mainly to advances in computing power in recent years. The purpose of the protein structure predictions is to provide the conformation of the native state for a given amino acids sequence. In general, it is assumed that the protein sequence is folded in a native state, or a collection of native states, which are found at (or near) the global minimum (free) energy. The goal of this work is the development of methodologies and protocols, based on the Tsallis thermostatistics, for ab initio protein structure prediction using atomistic molecular models. Thus, we use a set of 66 protein models, with length from 9 up to 60 amino acids residues in order to calculate their native structures. In this work we use an optimization method, the Generalized Simulated Annealing (GSA), coupled to the GROMOS Force Field to investigate the protein folding problem. We were able to find structures very near of the native states. In some structures such as the 1LCX, 2BP4, 1PEF, 1L2Y (PDB ID) we found deviations smaller than 3.0Å, what is considered a high resolution prediction. Furthermore, 60% of the tested models showed deviations of less than 4.0Å, which are considered good results." INBEB, FAPERJ, CNPq.



D56 - EVALUATION OF HOST CELL MODIFICATIONS UNDER CYST FORMATION

Tatiana C. Paredes-Santos1, 2, Marcia Attias1, 2and Rossiane C. Vommaro1,2* Universidade Federal do Rio de Janeiro (UFRJ), 2 Instituto Nacional de Ciência e

Tecnologia -Biologia Estrutural e Bioimagem(INBEB) The conversion of Toxoplasma gondii from the tachyzoite to the bradyzoite form leads to the persistence of infection in intermediate hosts, including humans. In the process, the reminiscent parasitophorous vacuole (PV) converts to an intracellular cyst presenting a cyst wall; this stage can persist for long periods in the host. The modifications of host cell organelles are poorly understood until now. Previous works have already shown that T. gondii can recruit host cell organelles and cytoskeleton filaments to the vicinity of the PV along the infection. Our aim was to investigate the reorientation of host cell organelles during spontaneous in vitrocystogenesis. The epithelial cell line LLC-MK2 infected with the cystogenic strain EGS was used as experimental model. The distribution of host cytoskeleton was evaluated by electron and fluorescence microscopy and compared to uninfected cells. Intermediate filaments of cytokeratin were observed concentrated around the cysts by electron microscopy, however, using the anti pan cytokeratin staining, it was not possible to observe this phenomenon in the fluorescence mycroscopy. Fluorescence analysis showed that the distribution of actin filaments was unaltered upon cyst formation. Furthermore, microtubules were seen surrounding the cysts, forming a cage around it both by TEM and IFA. The presence of microtubules around the cysts may be indicative that endosomal compartments are able to reach the cyst wall vicinity and in an IFA assay it was possible to observe lysosomes recruited to the border of the cysts. Besides, several profiles of endoplasmic reticulum were seen closely related to the cyst wall membrane, although mitochondria were seen not associated as shown previously for PV. These preliminary results indicate that formation of T. gondii cysts in EGS strain modifies the inner organization of host cells likely to obtain nutrients or to control host cell responses not yet determined till now. Faperj, CNPq and CAPES. D57 - CARACTERIZAÇÃO DO TRANSPORTE DE FOSFATO INORGÂNICO EM TRYPANOSOMA BRUCEI

1,3- RUSSO-ABRAHÃO, T., 2,3- SILVA-RITO, S., 2,3- MARINS-LUCENA, T., 2- ALVES-BEZERRA, 4- M., KOELLER, C.M., 2- DE PAULA, I. F., 4- HEISE, N., 2- GONDIM, K.C. E 1,2,3-

MEYER-FERNANDES, J.R. 1- Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, 2-

Instituto de Bioquímica Médica Leopoldo De Meis, Universidade Federal do Rio de Janeiro, 3- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e

Bioimagens, 4-Instituto de Biofísica Carlos Chagas Filho, Universidade Federa do Rio de Janeiro

INTRODUCTION. Trypanosoma brucei is an extracellular protozoan parasite that causes human African trypanosomiasis or "sleeping sickness ". During his supply of blood from the mammalian host an infected tsé-tsé fly (genus Glossina ) injects metacyclic trypomastigotes into the skin . The parasites enter the lymphatic system and pass into the bloodstream, where they differentiate into blood trypomastigotes, which are transported to other locations throughout the body. The tsé-tsé fly becomes infected with bloodstream trypomastigotes when it feeds on the blood of an infected mammalian host. In the fly intestine, parasites differentiate into procyclic trypomastigotes, leaving the intestine and differentiate into epimastigotes, affecting the salivary glands of the fly. During the different phases of their life cycle, T. brucei depends on exogenous inorganic phosphate (Pi), but little is known about the transport

of Pi across the plasma membrane in this organism. In addition, Pi transporters have been described in Saccharomyces cerevisiae, Plasmodium, Trypanosoma rangeli, Leishmania infantum , Trypanosoma cruzi and other microorganisms . OBJECTIVES. Investigate the kinetics of 32Pi transport, pH influence, H+ and K+ ionophores and inhibitors influence, H+:Pi cotransporter gene expression and RNAi of PHO84 gene. METHODOLOGY. RESULTS. The Pi transport is modulated by pH variation, with higher activity at acidic pH. FCCP (H+-ionophore), nigericin (K+-ionophore), valinomycin (K+-ionophore) and SCH28080 (H+, K+-ATPase inhibitor) inhibited the Pi transport, which was not inhibited by bafilomicina A1 (vacuolar ATPase inhibitor) . In addition, the Pi transport showed Michaelis- Menten kinetics. A sequence encoding a carrier of phosphate was identified in the genome of T. brucei, and expression of the gene TbPho84 was obtained. The RNAi methodology resulted in a decrease in cell growth and gene expression. CONCLUSIONS. These results confirm the presence of a Pi carrier in T. brucei, similar to Pho84 described in S. cerevisiae, which contributes to the acquisition of inorganic phosphate and may be involved in the growth and survival of procyclic forms of T. brucei. This work presents the first description of a Pi transporter - PHO84 in T. brucei, parasite responsible for many infections worldwide, especially in sub -Saharan Africa. CNPq, FAPERJ, CAPES, INBEB D58 - DNA BINDING OF FEIIIZNII COMPLEXES PROBED BY DNASE I FOOTPRINTING

1-BORTOLOTTO, T.; 2-CAMARGO, T.P.; 2-NEVES, A.; 1-TERENZI, H. 1-Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade

Federal de Santa Catarina, Santa Catarina, Brazil; 2-Departmento de Química, Universidade Federal de Santa Catarina, Santa Catarina, Brazil.

In recent years, we have demonstrated the DNA cleavage ability of a FeIIIZnII complex ([FeIII-(μ-OH)ZnIIL-H]) using the unsymmetrical donor ligand 2-[N-bis-(2 pyridylmethyl)-aminomethyl]-4-methyl-6-[ ’-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H2L-H) (1). To improve this ability, the complex ligand was specifically altered by the addition of one or two pyrene motifs that tightly bind to the DNA structure, facilitating the phosphodiester bond hydrolysis. Herein, we report an analysis of the complex-DNA binding features by 1 and by its parent complexes [FeIII-(μ-OH)ZnIIL-Pyrene] (2) containing one pyrene motif and [FeIII-(μ-OH)ZnIIL-Pyrene2] (3), using DNAse I footprinting. A 49-mer 5’-FAM-labeled (FAM is 6-fluorescein) self-complementary oligonucleotide was used as DNA probe of DNAse I footprinting assays. Footprinting assays revealed that 1 binds preferentially at an AT-rich region covering four nucleotides from T9 to A12 (A, TTAA). Two another weak binding sites are located at C16-C17 (B) and C23-T24 (C). The complex 2, showed further binding sites including an expanded binding site A including T13 (TTAAT) and a fourth binding site (D, T41-A42 and A44) that opposes to the binding site A forming a nearly complementary A/D binding site. Finally, the complex 3 showed similar results to 2, with addition of an expanded binding site B including a G15 and G18-C20 (E, GCCGGC). The presence of pyrene motifs in 2 and 3 showed enhance the binding affinity of these compounds to DNA, evidenced by the increase of apparent binding strength among binding sites A/D and B, but also expanded the range and the number of the binding sites. The addition of pyrene increases the structural size of the complexes, affecting the number of nucleotides that interact with 2 and 3 inside each binding site. Since pyrene is a general intercalator, it can acts as a “chemical anchor” allowing that and 3 interact to additional icinal nucleotides. CNPq, CAPES, MCT, FINEP, FAPESC, INBEB



Pós-doutorandos PD1 - REVEALING THE CAMPTOTHECIN MECHANISM OF ACTION IN THE TRYPANOSOMA CRUZI EPIMASTIGOTE FORM

1-Zuma, A.A; 2- Mendes, I.C.; 1-de Souza, L.C.R; 3-Elias, M.C.; 1,4,5 -de Souza, W.; 2- Machado, C.R.; 1-Motta, M.C.M

1 - Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho - UFRJ, RJ, Brasil.; 2 - Laboratório de Genética Bioquímica, Departamento de Bioquímica e Imunologia - ICB, UFMG, MG, Brasil; 3 - Laboratório Especial de Ciclo

Celular - Instituto Butantan, SP, Brasil; 4 - Instituto Nacional de Metrologia, Qualidade e Tecnologia (Inmetro); 5 - Instituto Nacional de Ciência e Tecnologia de Biologia

Estrutural e Bioimagem Chagas’ disease, one of the most neglected tropical diseases in the world, affects approximately 8 million people in Latin America. This illness is caused by Trypanosoma cruzi, a parasite that has a single nucleus and a unique mitochondrion with an enlarged portion (named kinetoplast) that harbors the mitochondrial DNA (kDNA). The DNA topology is modulated by topoisomerases that act during replication, transcription and repair reverting positive and negative supercoilings of the double-strand. Trypanosomatids topoisomerases are distinct from human enzymes, what encourages their use as targets in chemotherapeutic studies. In the present work, we evaluated the effects of camptothecin, a topoisomerase I inhibitor, considering T. cruzi epimastigote proliferation, ultrastructure, cell cycle, DNA lesions, mitochondrial activity and apoptosis. Our data showed that camptothecin caused a strong proliferation inhibition and only the lowest drug concentration used (1µM) led to a reversible effect on cell growth, ultrastructure and phosphatidylserine exposure. At ultrastructural level, treated parasites presented unpacking of the nuclear heterochromatin and the swelling of the mitochondrion. Camptothecin also promoted cell cycle arrest at G2/M and caused nuclear DNA lesions. On the other hand, some protozoa entered in early apoptosis, but they do not progress to late apoptosis, which may indicate that the parasites stay alive in a ˝senescence-like˝ state. We also obser ed that treated cells resented higher le els of reactive oxygen species and loss of mitochondrial membrane potential, what may be associated with apoptosis. Thus, this work indicates that the camptothecin mechanism of action comprises linked events that block the cell cycle, affect DNA organization and mitochondrial activity, which may result in apoptosis. CNPq, FAPERJ, INBEB PD2 - FALCIPAÍNA 2 COMO PROVÁVEL SÍTIO DE AÇÃO DA CLOROQUINA? MENEZES, C. M. S.1; FELICIANO, D. N.1,2; DAMETTO, M.1, GOMES, D. E. B.3, PASCUTTI,

P. G.1 1 Laboratório de Modelagem e Dinâmica Molecular, Instituto de Biofísica Carlos Chagas

Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro; 2 Grupo Biología Molecular, Centro Nacional de Sanidad Agropecuaria, La Habana, Cuba. 3 Laboratório de

Biologia Computacional, Instituto Nacional de Metrologia, Qualidade de Tecnologia, Duque de Caxias.

"Desde a Segunda Guerra Mundial, a cloroquina (CQ) tem sido considerada como antimalárico ideal. Contudo, o uso contínuo e indiscriminado levou ao surgimento de cepas resistentes ou multi-resistentes, em especial, do Plasmodium falciparum e P. vivax, espécies responsáveis, respectivamente, pelos maiores índices de mortalidade e

morbidade desta infecção. O mecanismo de ação da CQ é associado à interferência no processo de digestão da hemoglobina humana, durante a fase intra-eritrocítica do ciclo de vida do parasito. Este processo é classificado como cooperativo por envolver sistema enzimático múltiplo, como as cisteinil (falcipaínas 2 e 3), aspartil (plasmepsinas) e metalo proteases, e posteriormente, aminopeptidases, com o objetivo do parasito obter aminoácidos essenciais para seu crescimento, sobrevivência e reprodução. O grupo heme é liberado como sub-produto e, por sua natureza oxidativa, é subsequentemente olimeri ado β-hematina ou hemozoína. A CQ atuaria por inibição desta última etapa, via interação com o grupo heme ou com a hemozoína, impedindo a polimerização e causando a morte do parasito por estresse oxidativo, Recentemente, foi sugerido por Chugh et al. (1) mecanismo adicional em que a CQ também atuaria por inibição da falcipaína 2. A fim de verificar esta possibilidade, realizamos estudo de ancoramento molecular da CQ, do grupo heme e do inibidor clássico E-64 à estrutura da falcipaína 2 (código pdb ID 3BPF). Em auxílio à interpretação destes resultados, a superfície topográfica da protease foi avaliada com o servidor CASTP. A CQ (assim como o grupo heme, em corroboração a trabalho anterior do grupo (2)), ocupa sítios secundários aos do E-64, comprovando a hipótese de interação da CQ com a falcipaína, o que prejudicaria a atividade proteolítica desta última. Estudos de dinâmica molecular serão realizados para refinar estes resultados e validar esta hipótese. INBEB, FAPERJ, CNPq. PD3 - UNRAVELING THE INFECTION ROUTE OF MAYARO VIRUS

1- CARVALHO, C.A.M.; 1- SILVA, J.L.; 1- GOMES, A.M.O. 1- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de

Janeiro Mayaro virus (MAYV) is an alphavirus related to several sporadic outbreaks of a highly debilitating febrile illness in many regions of South America. Although highly neglected, Mayaro fever in humans is often more incapacitating than dengue and its urbanization from the Amazon region is on the verge. Alphavirus entry into target cells is supposed to occur by receptor-mediated endocytosis followed by fusion between the viral envelope and the endosomal membrane, although non-endocytic penetration of the viral genetic material into the cytoplasm without membrane fusion has also been suggested. The aim of this work was to determine the infection route of MAYV, pointing out the cell structures explored by the virus to get access to the inner of the cell. Purified virus particles were labeled with the lipophilic fluorescent probe DiD without impairment to viral infectivity and the fluorescent signals were tracked in susceptible cells transfected with endocytic markers by laser-scanning confocal fluorescence microscopy in real time. Our results show that labeled virus particles were endocytosed a few seconds after binding to receptors on the cell surface. Following DiD fluorescence dequenching at the single particle level, we could capture the moment of the fusion between the viral envelope and the endosomal membrane, that was shown to occur around 3 min post-binding, and found that it spatially correlated with both early endosome and caveosome markers. Altogether, our data indicates that MAYV entry into cells occurs by a fast endocytic mechanism that involves acidic and sterol-enriched vesicles. This work unravels important details about the entry of MAYV particles in living cells and may provide important insights to the development of antiviral strategies. CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX. PD4 - MAPEAMENTO DA TOPOLOGIA DE INTERAÇÃO DO PEPTÍDEO DA REGIÃO DO PLASMINOGÊNIO HUMANO QUE INTERAGE COM A PLASMINA DE YERSINIA PESTIS

1-Sarzedas, C.G., 1-Seraphim, A.C.V, 1-Tinoco, L.W.



1-Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro "A peste bubônica é uma doença que coloca em risco a segurança nacional e foi classificada na categoria A pelo CDC (Center for Disease Control). Desde o seu aparecimento até os dias de hoje, a peste já matou em torno de 200 milhões de pessoas. Esta doença é causada pela bactéria Y. pestis que produz na sua membrana externa a proteína plasmina (Pla), a principal responsável pela invasão da célula hospedeira pela bactéria. Após a infecção a Pla promove a clivagem (ativação) do plasminogênio humano (Plg) gerando plasmina e causando hemorragias em vários órgãos. No Plg foi identificado o peptídeo PK2: PKKCPGRVVGGCV, que representa a região onde acontece a interação com a Pla. As análises por CD, RMN e fluorescência mostraram que o PK2 livre possui uma estrutura estendida, esperada para um peptídeo cíclico devido à ponte de dissulfeto entre as cisteínas 4 e 12. Na presença da Pla, o PK2 sofre alterações estruturais com a mudança do deslocamento químico dos hidrogênios amídicos na região RVV, indicada como sendo a região de clivagem do Plg pela plasmina. Contudo essa interação não foi capaz de romper a ponte de dissulfeto, responsável por manter a estabilidade estrutural do PK2. Embora a Pla seja um barril-hidrofóbicos não sejam indicados como participantes diretos da clivagem, a presença do PK2 aumentou a intensidade de fluorescência da Pla, sugerindo que, tanto a Pla quanto o PK2, estão sofrendo modificações estruturais na interação Pla-PK2. Esses resultados serão usados para o desenvolvimento de um protocolo de análise que seja rápido e eficiente para avaliar a interação Pla-PK2 e ativação da Pla. Desta forma, estes testes poderão ser usados na busca de compostos capazes de inibir essa interação." INBEB, Capes, CNPq, Faperj PD5 - AVALIAÇÃO DE DOSES PARA INDUÇÃO DE LESÃO HEPÁTICA INDUZIDA POR RADIAÇÃO IONIZANTE.

1-ANDRADE,CBV;1-RAMOS,IP;1-FACCIOLI,LP;2-RESENDE,CMC;2- CANARY PC; 1-GOLDENBERG,RCS

1-Laboratorio de Cardiologia Celular e Molecular do Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-Hospital Clementino Fraga Filho

"Introdução: A despeito da grande capacidade regenerativa do fígado, o dano tardio para o tecido, tal como fibrose hepática radioinduzida, é inevitável. Assim, os esforços para investigar os mecanismos que levam aos efeitos deletérios tardios da radioterapia têm aumentado. A regeneração hepática tem sido estudada por muitos anos, no entanto estudos mais aprofundados sobre os mecanismos que governam os processos regenerativos ainda são pouco conhecidos e podem expandir as opções de tratamento para os pacientes com doença hepática. Desta forma, o objetivo desse trabalho é estabelecer um modelo de lesão hepática radioinduzida para posterior tratamento com células-tronco adultas.Materiais e Métodos: camundongos C57/BL-6, com peso entre 25-30 gr, foram divididos em 3 grupos: controle e irradiados com 15 Gy e 20 Gy. Os animais foram eutanasiados 30 e 60 dias após a irradiação (dpi). Antes da eutanásia foi realizada ultrassonografia hepática dos animais. Foi realizada a coleta de sangue para dosagem de enzimas hepáticas (albumina, ALT e AST) e obtidos fragmentos dos fígados para coloração de picrosirius. Resultados/Discussão: A curva de sobrevivência dos animais demonstrou que somente 6% dos animais da dose de 20 Gy chegaram a 60dpi, enquanto que o outro grupo experimental teve uma sobrevida de 98% no mesmo período. No ultrassom observou-se um aumento da ecogeneicidade do fígado em relação ao rim tanto nos animais do grupo irradiado com 15Gy quanto com 20Gy, característica de fígados com esteatose, nesses grupos também foi observado a presença de grades de fibrose, sempre quando comparados ao grupo controle. As dosagens séricas demonstraram redução significativa nos níveis de albumina e ALT

indicando que a irradiação gerou alteração funcional do fígado. Na análise histológica por picrosirius foi observado alterações na distribuição do colágeno pela matriz hepática, encontrou-se colágeno distribuído entre os hepatócitos, porém o esperado era encontra-lo somente ao redor dos vasos." CNPq, FAPERJ, CAPES, INBEB PD6 - INFLAMMATORY EDEMA ELICITED BY TRYPANOSOMA CRUZI FUELS HEART TISSUE PARASITISM THROUGH THE PROTEOLYTIC ACTIVATION OF THE MAST CELL/KALLIKREIN-KININ PATHWAY 1- Andrade D; 1-Nascimento CR, 1-Brazil G ; 2-Eponina Carvalho-Pinto C; 1-Oliveira AC; 1-Schnaider Ramos-Junior E, 1-Serra R; 1-Almeida L; 1-Cordovil da Silva T; 1-Vellasco L; 1-Vairo L; 3- Fortes F; 4-Andrade M; 5- Lannes-Viera J; 6-Juliano L; 1-Goldenberg R; 7-Sirois P; 8- Köhl J; 9-Alvarenga P; 9-Monteiro RQ; 1-Campos de Carvalho A; 1-Svensjö E and 1-Scharfstein J * first authorship should be shared between Daniele Andrade and

Clarissa Nascimento 1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio

de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutual e Bio-Imagem (INBEB), Brazil;2Departamento de Patologia, Universidade Federal Fluminense, UFF, Rio de Janeiro, Brazil;3 Centro Universitário Estadual da Zona Oeste, UENZO, Rio de

Janeiro, Brazil; 4 Faculty of Medicine, UFMG 5Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil; 6 Departament of Biophysics, UNIFESP, 7

Sherbrooke University, Canada. 8 Lubeck University, Germany, 9 Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

Chagas disease (CD) is a major cause of myocardiopathy in Latin America. We hypothesized that vasoactive kinins released in inflammatory exudates might fuel heart tissue parasitism via signaling of bradykinin GPCRs (BKRs) and endothelin receptors (ETRs). Here we show evidence that this survival strategy depends on the pathogen ability to evoke edema through the mast cell (MC)-driven activation of the kallikrein-kinin system (KKS). Using intravital microscopy, we showed that histamine potentiates parasite-induced plasma leakage, the response being conversely blunted by cromalyn- a MC stabilizer- or by inhibitors of FXIIa and PKa (KKS proteases). Guided by echocardiography, we then inoculated TCT in the left ventricle of mice and found evidence of trans-endothelial leakage of dextran-TRITC (2 h p.i.) in wt heart, but not in B2R-/- mice nor in wt mice pretreated with specific antagonists of BK2R, BK1R, or ETaR/ETbR. Heart parasitism (30 d p.i.) is decreased in MC-deficient mouse strain or in infected WT mice pretreated with cromalyn, FXIIa inhibitor or BKR or ETR antagonists, thus linking MC-KKS-driven edema to parasite infectivity. Strikingly, the mice that received a single-dose of GPCR blockers prior to intracardiac challenge were subsequently protected from myocarditis/fibrosis (30 d p.i.). We then infected wt and BK1R-/- mice via the i.p. route and found that they displayed markedly reduced intracardiac parasite load (qPCR; 14 d p.i.). Moreover, BK1R-/- mice were protected from chronic myocarditis and heart fibrosis (90 d p.i.). Extending these studies to skeletal muscles, we found that interstitial edema and T. cruzi load (3 d p.i.) in sternomastoid muscles were blunted in B6 mice pretreated with HOE-140 or R954, respectively BK2R or B1R antagonist. Combined, these results suggest that drugs that stabilize endothelial barrier through the targeting of the MC-KKS pathway might limit myocardial edema and heart parasitism, consequently relieving adverse heart remodelling in CD. CNPq, FAPERJ, INBEB PD7 - STRUCTURAL AND THERMODYNAMIC STUDIES OF THE HSP70/HSP90 ORGANIZING PROTEIN – HOP FROM LEISHMANIA BRAZILIENSIS



1,2- SANTOS, C.A.; 1- GONZAGA, M.R.; SERAPHIM, T.V.; 2,3- RAMOS, C.H.I.; 1- BORGES, J.C.

1- Grupo de Biologia Molecular e Bioquímica, Instituto de Química de São Carlos, Uni ersidade de o Paulo U P , 13560‐970, o Carlos, P, Bra il - Instituto de

Química, Universidade Estadual de Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil; 3- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem,

Brazil. "The Hsp70/90-organizing protein (Hop) is a multi-domain protein containing three tetratricopeptide repeat (TPR) motifs that cooperates with Hsp70 and Hsp90 in the protein folding in the cellular cytosol. Hop is a co-chaperone that can modulate the ATPase activity of both Hsp90 and Hsp70 and is directly associated with the quality protein control. Despite numerous studies focusing on the role of Hop is not yet fully understood how it interacts with Hsp90. From structural and biochemical studies it is known that Hop binds to the C-terminal ME(E/Q)VD motif of Hsp90. However, recent studies have shown that the Hsp90 middle domain may also interact with Hop and both C-terminal and middle domain Hsp90 interactions are via TPR2A and TPR2B present in the Hop structure. In this work the full-length Hop (LbHop) and a deletion mutant (LbHopTPR2AB) containing the TPR2A e TPR2B domains of Leishmania braziliensis were clonned, overexpressed and purified. Structural properties of the recombinant proteins were initially evaluated by circular dichroism and analytical size exclusion chromatography. Finally, thermodynamic parameters of the molecular bindings events between L. braziliensis Hsp90 (LbHsp90), previously characterized by our group, and the full-length LbHop and the deletion mutant LbHopTPR2AB, target of this study, were obtained with isothermal titration calorimetry (ITC) measurements. Our findings showed that the functional state LbHop is a monomer in solution and the stoichiometry of interaction between LbHop and LbHsp90 is one monomer of LbHop to a dimer of LbHsp90. Similar results were also observed for the Hop deletion mutant, confirming the role of TPR2A and TPR2B motifs for LbHsp90 interaction/modulation. In conclusion, our study provides novel contribution to the mechanism of interaction between Hsp90 and Hop co-chaperone, and sheds light on how this molecular chaperone system works in protozoa, especially as regards to L. braziliensis. Keywords: Hop, TPR domain, Hsp90, Leishmania braziliensis, ITC." FAPESP PD8 - THERMODYNAMIC AND CONFORMATIONAL ANALYSIS OF THE BINDING OF FEIIIZNII – PYRENE COMPLEXES TO DNA 1- NORBERTO D.R.; 1- BORTOLOTTO, T.; 1- CAMARGO, T.P.; 2- NEVES, A.; 1- TERENZI, H. 1- Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade

Federal de Santa Catarina-UFSC; 2- 2Laboratório de Bioinorgânica e Cristalografia, Departamento de Química, Universidade Federal de Santa Catarina-UFSC

Metallonucleases have a natural ability for interacting with DNA. Since binding and cleavage of DNA are at the central of action of the cellular transcription and translation, it represents an important target for therapeutic intervention and the development of diagnostic structural probes. In general, metallonucleases bind DNA in a non-covalent interaction fashion, including electrostatic or groove binding, intercalation between base-pairs, and catalysis of DNA cleavage through hydrolytic or oxidative mechanisms. Despite of the progress of recent years in this field, there are many aspects that remain unexplored. This work reports an investigation into the thermodynamic behavior of FeIIIZnII - pyrene complexes interacting with calf thymus DNA (CT-DNA) and a tentative model that aim to explain the properties used in certain families of antitumor agents was developed. The complexes FeZnLAld devoid of pyrene ligands, and FeZnLP1 and FeZnLP2, which are functionalized with one or two pyrene moieties, respectively, were

examined by isothermal titration calorimetry (ITC), and compared to UV-Vis and fluorescence s ectrosco ies. The arameters Kb, ∆ b and n were determined using a single set of site fittings and indicate that the binding has a positive enthalpy change associated to the substitutions of pyrene, with affinity values from 4.8 x104 M-1 to 1.8x106 M-1. The results show some aspects of intercalation of a planar ligand of the complex in the DNA base pairs stack, with drastic increasing in entropic contribution. The binding free energy ∆Gb was calculated according to different representations and they are in agreement with studies of other heterovalent dinuclear complexes. Our data demonstrate that FeIIIZnII complexes modified with pyrene strongly bind to DNA, and would be important in the development of novel chemical hydrolases capable of DNA targeting and cleavage. CNPq, CAPES. PD9 - LUMINAL ANG II ON CA2+ MOBILIZATION OF PORCINE PROXIMAL TUBULE KIDNEY CELLS (LLC-PK1): INCREASE OF SERCA ACTIVITY VIA AT1 AND AT2 HETERODIMERS STIMULATING PLC/PKC PATHWAY.

1,2,3- FERRÃO, F.M.; 2,3- LARA, L.S.; 1,2- DIAS J.; 4- DRUMMOND, H.A.; 5- REIS, R.I.; 5- COSTA-NETO, C.M.; 6- ZHUO, J.L.; 7- CARMONA A,K.; 1,2- VIEYRA, A.; 1,2- LOWE, J.

1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de

Janeiro; 3- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 4- Department of Physiology and Biophysics and Center for Excellence in Cardiovascular-

Renal Research, University of Mississippi Medical Center, Jackson, Mississippi; 5- Departmento de Bioquímica e Imunologia, Escola de Medicina de Ribeirão Preto,

Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil; 6- Laboratory of Receptor and Signal Transduction, Department of Pharmacology and Toxicology, University of

Mississippi Medical Center, Jackson, Mississippi; 7- Departmento de Biofísica, Universidade Federal de São Paulo, São Paulo.

Angiotensin II (Ang II) is found in renal interstitium and tubular lumen at higher concentrations than systemic Ang II. We demonstrated that Ang II facing luminal membranes of porcine proximal tubule cells (LLC-PK1) stimulate SERCA activity (10-14 M - 10-10 M), while in high concentrations (10-8 M - 10-6 M) this effect is not observed. The objective of this study was to investigate molecular mechanisms involved in Ang II –stimulated SERCA activity, evaluating its physiological relevance. Both Ang II concentrations (10-10 M and 10-6 M) stimulated AT1/AT2 heterodimers formation in LLC-PK1 cells, and the use of losartan and PD123319 demonstrated that Ang II is endocyted. This event is microtubule-dependent, but not mediated by β-arrestin and clathrin, since colchicin, but not Pitstop 2 blocked endocytosis. Co-localization of Alexa Fluor 488-conjugated Ang II with ER tracker suggests the translocation of Ang II to the reticulum after endocytosis. Calphostin C (5 × 10-8 M; PKC inhibitor) and PMA (0,1 µM e 1 µM; PKC activator) blocked and mimicked Ang II stimulation of SERCA activity, respectively. Calphostin C –sensitive PKC activity is 40 % increased at 10-10 M and 37 % inhibited at 10-6 M Ang II. Moreover, pre incubation of 10-9 M Ang II for 30 min, induced a 10 times higher and 3 times longer Ca2+ mobilization than in absence of previous incubation with Ang II. In conclusion, after endocytosis of luminal Ang II with AT1/AT2 heterodimers and translocation to the reticulum, SERCA is the target Ca2+-ATPase to regulate intracellular Ca2+ homeostasis. The PLC/DAG/PKC pathway mediates this phenomenon. The Ca2+ mobilization study indicates that increased Ca2+ intracellular store by SERCA activation, results in a more efficient response to agonist future stimulus contributing to increase fluid and solute reabsorption in renal proximal tubule. CNPq, FAPERJ, CAPES



PD10 - UNDERNUTRITION DURING FETAL LIFE IMPAIRS REPRODUCTIVE SUCCESS OF ADULT MALE RATS UNDERPINNED BY ABNORMAL Ca2+ HANDLING IN VAS DEFERENS

1,2,3- Muzi-Filho, H.; 1- Souza, A.M.; 1- Bezerra, C.G.P.; 1,4- Boldrini, L.C.; 2- Takiya, C.M.; 1- Oliveira, F.L.; 1- Nesi, R.T.; 1- Valença, S.S.; 2,3- Einicker-Lamas, M.; 2,3- Vieyra,

A.; 1,3- Lara, L.S.; 1- Cunha, V.M.N. 1- Institute of Biomedical Sciences, Federal University of Rio de Janeiro; 2- Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro; 3- National Institute of Science and Technology for Structural Biology and Bioimaging, Federal University of Rio

de Janeiro; 4- Directorate of Metrology Applied Life Sciences, National Institute of Metrology, Quality and Technology.

"Introduction. Epidemiological and animal model studies show that placental undernutrition impairs reproduction performance in the adult offspring, but the underlying molecular mechanisms within the male genital tract are mostly unknown. Due to its special physiological characteristics in the transport of seminal fluid, we hypothesized that the vas deferens should be a highly sensitive target. Regarding this organ, prenatal undernutrition that induces molecular alterations can compromise reproductive success in adulthood, even with normal nutrition post-partum onwards. We have investigated whether in utero malnutrition provokes structural and molecular abnormalities that ultimately lead to diminished fecundity and fertility. Methods and Results. Male adult rats that were malnourished during their intrauterine life had a uniquely increased vas deferens weight associated to a thickening of the muscular coat, decrease of total and haploid germ cells, a marked increase in immature cells, and an accentuated decline in fertility and fecundity. At the molecular level, the vas deferens has a marked decrease in Ca2+ transport due to uncoupling of Ca2+-stimulated ATP hydrolysis and ATP-driven Ca2+ flux, downregulation of SERCA2 and coupling factor FKBP12. A vast increase in protein carbonylation (a marker of oxidative damage), and an abnormal balance between protein kinase C and cyclic AMP-dependent protein kinase, which are the main signaling components of pathways involved in the regulation of Ca2+ handling, are seen as the legacy of undernutrition in early life. Conclusion. Overall the results provide structural and molecular basis to explain, at least partly, why adult individuals experiencing undernutrition in early life have a deficient reproductive performance as adults." Financial Support. The Brazilian National Research Council (CNPq), the Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ) and the National Institute of Science and Technology for Structural Biology and Bioimaging (INBEB). PD 11 - P75NTR: STRUCTURAL ANALYSIS OF A SIGNALING PATHWAY INVOLVED IN NEURODEGENERATION.

1-HEIMFARTH, L; 1-ALMEIDA, M.S.; 1-CABRAL, K.; 1,2,3-WUTHRICH, K. ] 1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-

Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037; Institute of Molecular Biology and Biophysics,

Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland Neuronal injury or stress often results in activation of signaling pathways that are active normally only during embryonic and neonatal development. One of these signaling pathways is initiated by the p75 neurotrophin receptor (p75NTR). Studies have demonstrated that p75NTR signaling contributes to neuronal degeneration during injury and cellular stress, including the exposure to -amyloid protein. p75NTR is a Type I transmembrane receptor with an extracellular domain that contains four cysteine-rich domains (CRDs), the intracellular domain and a type II death domain, a potential G

protein activating domain. The aim of this work was the determination of the 3D structure of human p75NTR death domain ([u-13C,15N]-hp75DD) by NMR and the determination of termal denaturation curve by Dichroism Circular. The results show that the NMR structure of [u-13C,15N]-hp75DD is a globular protein with six alpha hélices. The N-terminal polypeptide segment of the hp75DD is located inside the structure and C-terminal polypeptide segment is flexible region. Furthermore, we determined the CD thermal denaturation curve for hp75DD. The CD data show a decrease in the intensity of λ max at 208 and 222 nm with increasing temperature. The hp75DD denaturation melting temperature (Tm) is 67 +/- 2,0 ºC and renaturation Tm is 72 +-1 ºC. The structural characterization of hp75DD is very important to understand the involvement of this receptor signaling pathway in cell degeneration. To solve the structure of a protein can facilitate the prediction of drug and contribute to cure of a disease. CNPq, FAPERJ, INBEB PD12 - TOXOPLASMA GONDII EGRESS: IS HOST CELL CYTOSKELETON A DEAD-LOCK?

1,2 - CALDAS, L.A.; 3 - SEABRA, S.H.; 1,2 - ATTIAS, M.; 1,2,4 - DE SOUZA, W. 1- Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos

Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil; 2- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Inbeb, Brazil; 3- Universidade

Estadual da Zona Oeste, Rio de Janeiro, Brazil; 4- Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de Janeiro, Brazil

The obligate intracellular protozoan parasite Toxoplasma gondii, capable of infecting almost every nucleated warm blooded cells, has raised medical and veterinary importance. Nevertheless, its egress from host cell is still poorly understood. Herein, we investigated this step of the protozoan cell cycle by colloidal gold and fluorescent markers labeling of actin and tubulin in infected cells. 24 hours post infection with 5 parasites per cell, samples were treated for 5 minutes with 5µM calcium ionophore in order to induce T. gondii egress. Fixation was then performed with 4% formaldehyde in PBS, pH 7.2, for 20 min followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min at room temperature. For electron microscopy, samples were probed with rabbit anti-actin antibody at a 1:100 dilution, followed by a goat-anti-rabbit antibody conjugated to 10 nm colloidal gold particles, both incubated for 1h. The monolayers were washed and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, post-fixed for 1 h with 1% OsO4 in 0.1 M cacodylate buffer pH 7.2 and 0.8% potassium ferrocyanide, dehydrated in ethanol, critical point dried in CO2, sputtered with carbon and observed in a Jeol 6340 field emission scanning electron microscope. For confocal microscopy, the monolayers were pre-incubated with 50 mM ammonium chloride and 3% BSA in PBS pH 8.0 for 45 min. The samples were then incubated with primary antibodies at a 1:100 dilution for 1 h, rinsed, and incubated with 1:400 secondary antibodies at room temperature for 1 h. After rinsing in PBS and mounting with a prolong antifade, samples were examined in a Zeiss 510 LSM 510 NLO. We observed a dual possible role for actin: as a substrate for intracellular gliding of the parasite, contributing to its scape, and also as a barrier for T. gondii during egress since escape from host cells occurred preferentially at actin free zones. As long as dynamin and actin were shown to interact directly with each other, infected cells were treated with dynasore, a potent dynamin inhibitor, and were labeled for actin and tubulin in order to observe the cytoskeleton arrangement. Dynamin seemed not to be necessary for T. gondii egress. CNPq, FAPERJ e CAPES



PD13 - KING PEN SS Rĺ E E EP RIN N HEPARINASE PARA CATÁLISE DIRIGIDA

1,2- DAMETTO, M.; 1,2- PASCUTTI, P.G. 1- Instituto de Biofisica Carlos Chagas Filho, UFRJ; 2- INMETRO, Xerem.

A heparina é usualmente utilizada como fármaco anticoagulante na prática médica, como em cirurgias cardíacas e hemodiálise. A capacidade anticoagulante da heparina está relacionada à sua ligação com antitrombina. Após ligação com a heparina, a antitrombina é ativada e interage com a trombina, inibindo-a e promovendo o processo de anticoagulação. A ligação antitrombina-heparina ocorre especificamente e com alta afinidade devido a um pentassacarídeo presente na molécula de heparina. Entretanto, menos de 1/3 das sequências de heparina obtidas nas preparações comerciais são ativas como anticoagulante. Além disso, a distribuição mundial de formulações de heparinas contaminadas em 2007 causou preocupação quanto à confiabilidade e segurança da heparina proveniente de animais. Desta forma, aumentou-se a busca por um método de baixo custo para a preparação de heparina sintética. Contudo, a síntese química heparina sintética comercial tem mais de 50 passos e um rendimento de ~ 0.1%, portanto, é um fármaco muito caro. Neste trabalho, estamos desenvolvendo uma heparinase que cliva a molécula de heparina num tamanho suficiente para que esta tenha atividade anticoagulante. A enzima deve lisar a cadeia de heparina no terminal redutor de seu pentasacarídeo reconhecido pela antitrombina, possibilitando a obtenção de formulações farmacológicas mais puras e homogêneas, e evitando o risco da presença de contaminantes. Nesta etapa inicial, simulações de docking indicaram que o pentassacarideo de heparina interage de fato com o sítio ativo da heparinase e sua posição no sitio ativo da enzima favorece a clivagem no anel de açúcar específico. CAPES, FAPERJ, CNPq PD14 - CHRONIC UNDERNUTRITION AFFECTS Na+-ATPase KINETICS AND REGULATION, SIGNALING PATHWAYS INVOLVED ANG II, KINASES AND PHOSPHATASES AND HISTONE DEACTYLASE ACTIVITY IN PROXIMAL RENAL TUBULES 1,2- SILVA, P.A.; 1,2- MUZI-FILHO, H.; 1,2- PEREIRA-ACÁCIO, A.; 1,2- MARTINS, J.F.; 1,2-

LANDIM-VIEIRA, M.; 1,2- VIEYRA, A. 1- Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro; 2- National Institute of Science and Technology for Structural Biology and Bioimaging,

Federal University of Rio de Janeiro. "Introduction. Malnutrition is an important problem of health in developing countries. Multideficient diets affect the renal tissue, leading to the appearance of renal diseases. Objectives. We aimed to investigate: (i) alterations in the Na+-ATPase affinity for Na+ and kinetics; (ii) alterations in the signaling components (kinase-mediated pathways and abundance of protein phosphatase 2A – PP2A) linked to the renin/angiotensin system; (iii) impact on histone deacetylases (HDAC). Methods. Wistar rats aged 90 days were fed from weaning with either a control (CTR) or a deficient diet (RBD). Homogenates and membranes from proximal tubules were used (i) to study the kinetics of the Na+-ATPase, (ii) to evaluate the abundance of PKA, PKC (alpha, epsilon, dzeta and lambda isoforms), PP2A, AT1R and AT2R, and (iii) to quantify the total activity of the HDACs. Results. RBD increased maximal velocity (118.0 ± 14.2 against 44.4 ± 3.9 nmol Pi.mg-1.min-1), affinity for Na+ (Na+0.5 = 4.0 ± 1.2 mM in CTR group and 0.2 ± 0.02 mM in RBD group) and phosphate liberation from the phosphorylated intermediate of the Na+-ATPase in initial cycles (about 3 times). RBD group presents, when compared to the CTR group, a reduced abundance of PKA (about 50%) and an increased abundance of 3 isoforms of PKC (50% for PKC-alpha, 90% for PKC-dzeta and 300% for PKC-lambda, without alterations in the PKC-epsilon abundance). RBD rats presented with increased abundance of AT1R (about 15%, without alterations in the AT2R abundance). RBD

reduced the activity of the HDACs in ~40%. Conclusion. These cellular and molecular alterations, associated with increased Na+ reabsorption, culminate in the onset of hypertension in the adulthood, and can be considered critical features in chronic undernutrition." The Brazilian National Research Council (CNPq), the Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ) and the National Institute of Science and Technology for Structural Biology and Bioimaging (INBEB). PD15 - NANOSCALE ELASTIC PROPERTIES OF SIMULATED LIPID BILAYERS

1- LAPIDO-LOUREIRO, P.A.; MASCARENHAS, W.F.; PASCUTTI, P.G. 1,3 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2

- Instituto de Matemática e Estatística - Universidade de São Paulo "Cell membranes are not in a tensionless state, when they perform tasks such as membrane fission, endocytosis, cell shape deformation or muscle contraction. Despite the fact that the structural properties of lipid bilayers at equilibrium have been extensively studied, much less is known about the molecular structure of lipid bilayers at nonzero lateral tension. One of the approaches able to tackle these issues is the molecular dynamics (MD) simulation technique. We carried out MD simulations of DPPC bilayer patches composed of 128 lipid molecules stretched to different areas departing from the equilibrium area, at 298, 310 and 323 K. Thousands of molecular configurations derived from extensive simulations (more than 4 μsec simulation time) were subjected to a computational geometry algorithm (2D Voronoi diagram - VD) that partitions the plane in a natural way. Using atom-level VD, we measured the area occupied by each constituent lipid atom. In this way, we were able to calculate more precise values of the areas of individual molecules and a better picture of their fluctuation properties. From the fluctuation-dissipation theorem, we calculated the area elastic constant Ka of the simulated systems. We constructed a simplified Ka versus area phase diagram that suggests that even at small area strains there are marked alterations in the elastic behavior of bilayers. At large strains and according to temperature, simulated bilayers undergo tension-induced phase transitions (either an interdigitated gel or a liquid-expanded phase), that, in vivo, would preclude membrane protein functioning or herald rupture, respectively. To our knowledge, this is the first work to use atom-level tessellations to study fluctuation properties of molecular areas of lipid bilayers. We were able to measure elastic constants of strained bilayers in a precise way, stressing the important role of MD simulations in situations hardly amenable to experimental studies." INBEB, FAPERJ, CNPq PD16 - EXTRACELLULAR VESICLES RELEASED FROM MESENCHYMAL STROMAL CELLS MODULATE MIRNA IN RENAL TUBULAR CELLS AND INHIBIT ATP DEPLETION INJURY.

1,3,4- Lindoso, R.S.; 1- Collino, F.; 2- Bruno, S.; 3,4- Araujo, D.D.; 3,4- Sant'anna, J.; 5- Tetta, C.; 2,6- Provero, P.; 7- Quesenberry, P.J.; 3,4- Vieyra, A.; 3,4- Einicker-Lamas,

M.;1- Camussi, G. 1- Department of Medical Sciences and Molecular Biotechnology Center, University of

Torino, Turin, Italy; 2- Department of Molecular Biotechnology and Health Science, Turin; 3- Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro,

Rio de Janeiro, Brazil; 4- National Institute of Science and Technology for Structural Biology and Bioimaging, Rio de Janeiro, Brazil; 5- EMEA LA Medical Board,Fresenius

Medical Care, Bad Homburg, Germany; 6- Center for Translational Genomics and



Bioinformatics San Raffaele Scientific Institute, Milan, Italy;7- Department of Medicine, the Warren Alpert Medical School of Brown University, Providence, RI, USA.

"Introduction: The extracellular vesicles (EVs) derived from human mesenchymal stromal cells (hMSC) are possible mediators of the paracrine mechanism of these cells. miRNAs are involved in the regulation of several mechanisms, including repair processes. The aim of this study was to analyze the role of EVs in renal repair, identifying the miRNAs involved in this process. Material & Methods: HK-2 cells (human renal epithelial cells) were treated with antimycin A for 1 h, promoting ATP depletion. EVs were obtained from hMSC cultured overnight in RPMI with 0.5% of BSA, submitted to a 150,000 rcf for 1 h at 4°C. Incorporation of EVs double-stained with VybrantTM Dil and YT ® R elect™ was analy ed by confocal microsco y. Proliferation was evaluated by Brdu incorporation). Cell death assay was performed by FACS analysis (MuseTM Annexin V Dead cell kit). Profiling of 365 mature miRNAs was performed by TaqMan® Arrays and qRT-PCR was used to confirm miRNAs and mRNA expression. Predicted miRNA targets were obtained from Targetscan 6.1. Results: The hMSC-EVs promote protection of renal cells after injury, leading to a significant cell death reduction. The EVs incorporation was increased after damage (increase of 100%) and the CD29, CD44 receptors were involved in this process. Several miRNAs were modulated by injury and treatment with EVs. Some miRNAs were directly transferred from EVs while others were induced or inhibited. Up-regulated miRNAs targets revealed important genes related to recovery process as apoptosis, cytoskeleton reorganization and hypoxia. The changes in expression of these genes during injury were reverted with EVs incubation. Conclusions: The results show that the paracrine actions of hMSC are in part mediated by EVs and the protective effects promoted by these vesicles were mediated by the transfer or induction of miRNAs that regulate important targets related to cell recovery." CAPES, CNPq, FAPERJ, DECIT-MS, Fresenius Medical Care, Associazione Italiana per la Ricerca sul Cancro (AIRC) and National Institutes of Health (NIH). PD17 - PRESSURE-INACTIVATED AVIAN INFLUENZA VIRUS: IMPLICATIONS FOR VACCINE DEVELOPMENT

Barroso, S.P.C.1, Nico D.2, Nascimento,D.3, Santos,A.C.V.1,Couceiro,J.N.S.S.2, Bozza,F.A.3, Souza, T.M.L.3, Sacramento, C. Q.3,Ferreira,A.M.A.2, Palatinik,C.2,

Silva,J.L.1,Oliveira, A.C.1 1Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de

Janeiro, and Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Brazil. 2Instituto de Microbiologia, Universidade Federal do Rio de Janeiro,

Brazil. 3Fundação Oswaldo Fiocruz, Rio de Janeiro, Brazil. "Introduction: Influenza virus poses a serious global health threat. Vaccination remains the primary method for preventing influenza or to avoid developing serious complications related to the disease. To induce a greater mucosal immune response, vaccines by intranasal administration are highly desired. An egg grown inactivated H3N8 whole virus vaccine was prepared using hydrostatic pressure as inactivating agent. The hemagglutinin and neuraminidase activities were assayed by hemagglutination and sialidase assay, respectively. Objective: Evaluate the immunogenic capacity of pressure-inactivated vaccine. Material and methods: To evaluate the immunogenic capacity of inactivated particles we used six-week-old female Balb/c mice. The animals were immunized intranasally at weekly intervals, with 3 doses of 100 µg/mL pressurized virus (1 TCID50, 265HAU/50 µL). On day 21, mice were challenged in intranasal route.After vaccination and infection, IgG2a, IgG1 and IgA were measured in sera, faeces and nasal wash using ELISA.To further investigate the ability of the pressure-inactivated virus in stimulating the immune system we evaluated the production of cytokines(IL-2, IL-4, IL-6,

IL-10, IL-17, IFN-γand TNF-α) in the supernatants of splenocytes and broncheoalveolar lavage (BAL). We also performed lung histopathology analysis by light microscope. Results and Discussion: In this study we show that hydrostatic pressure is able to inactivate the H3N8 avian influenza virus, while still maintaining hemaglutination and neuramidase functionality. Challenged vaccinated-animals showed no disease signs. In the same way, these animals showed less Evans blue leakage and cell counts in BAL when compared to challenged non vaccinated group. We found that the whole inactivated particles were capable to generate neutralizing antibody response in serum. After vaccination and challenge we found Th1/Th2 cytokines secretion with prevalence of IFN-γ. Vaccinated and further-challenged mice showed no lung damage. Conclusion: Our results showed that the animals present a satisfactory immune response after vaccination and are protected against challenge." CAPES, INBEB, FAPERJ, CNPq. PD18 - LONGITUDINAL EVALUATION OF SALIVARY PROFILE FROM CHILDREN WITH DENTAL CARIES BEFORE AND AFTER TREATMENT 1- FIDALGO, T.K.S.; 1- FREITAS-FERNANDES, L.B.; 2- ALMEIDA, F.C.L.; 2- VALENTE, A.P.; 1-

SOUZA, I.P.R 1- Faculdade de Odontologia, Universidade Federal do Rio de Janeiro; 2- Instituto de

Bioquímica Médica, Universidade Federal do Rio de Janeiro. The saliva is a biofluid largely used in metabolomic for assessment of local and systemic diseases. Our group was able to demonstrate salivary metabolomic signature of children with dental caries (Fidalgo et al, 2013). Thus, the aim of the current study was to investigate the changes observed for metabolites related caries caries-lesion before and after dental treatment using NMR. Saliva samples from children without caries and with dental caries before and after treatment. 1H-NMR spectra were submitted to Partial Least Squared Discriminant Analysis (PLS-DA). Streptococcus mutans and Lactobacillus sp and pH were also evaluated. As expected, caries-free children presented low levels of microorganisms when comparing to children with dental caries (p < 0.05; Mann-Whitney test). Also, after dental treatment it was observed a reduction of microorganisms (p < 0.05; Wilcoxon test) and the increase of saliva pH. PLS-DA showed a clear separation of saliva from children with caries and caries-free. In addition, after dental treatment it was observed a reduction in the levels of acetate, propionate, fatty acid, butyrate, and saccharide region. PLS-DA applied on 1H-NMR saliva spectra distinguished the metabolites related to dental caries before and after dental treatment. INBEB, FAPERJ, CNPq, CAPES PD19 - MITOSOME BEHAVIOR DURING THE LIFE CYCLE OF THE PATHOGENIC PROTOZOAN GIARDIA INTESTINALIS 1,3- MIDLEJ, V.; 1- PENHA, L.; 1- SILVA, R.; 1,2,3- DE SOUZA, W.; 1,2,3- BENCHIMOL, M. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Metrologia e Qualidade Industrial – Inmetro; 3- Instituto Nacional

de Ciência e Tecnologia em Biologia Estrutural e Bioimagens-INBEB. The mitosome is a double-membrane bounded organelle found in few unicellular eukaryotes, one of which is the human intestinal parasitic protozoan Giardia intestinalis. The discovery of mitosomes in Giardia was strongly supported by the identification of the protein machinery components responsible for iron-sulfur (Fe-S) cluster assembly (IscS and IscU proteins) in this organelle. G. intestinalis also lacks mitochondria and peroxisomes and has been considered to be among the earliest-branching eukaryotes. This flagellated protozoan grows in vitro as trophozoites and under some conditions differentiates into cysts, characterized by the absence of flagella, a rounded shape, and



the presence of a cyst wall. Using antibodies that recognize two proteins present in the mitosome, heat-shock protein 70 (mit-HSP70) and giardial chaperonin 60 (GiaCpn60), we used confocal laser scanning and electron tomography microscopy, western blots and qRT-PCR to analyze the presence and distribution of the mitosomes during both the cell cycle and the process of the trophozoite-to-cyst transformation. At early stages of the differentiation process (~12 h), there was a significant decrease in the extent of labeling in the cells and the numbers of mitosomes, which almost disappeared after 21 h, but were recovered during the cyst stage. This was confirmed by an mRNA expression analysis, thus indicating a process that modulates the formation of mitosomes during the G. intestinalis life cycle. Electron microscopy tomography, which allows for three-dimensional reconstruction, revealed the presence of both rounded and elongated mitosomes. CNPq, FAPERJ, CAPES, INBEB



Pesquisadores P1 - DANCE AND SCIENCE: STUDY UPON THE SCRIPTING AND THE CREATIVE CHOREOGRAPHIC COMPOSITION PROCESS BY THE HELENITA SÁ EARP DANCE FUNDAMENTALS BASED UPON BIOLOGICAL ORGANIZATION PATTERNS AND FORMS.

1- MEYER, A; 2- VIEYRA, A. 1- Escola de Educação Física e Desportos, Universidade Federal do Rio de Janeiro; 2-

Instituto de Biofísica Carlos Chagas Filho "This research demonstrates the scripting and the creative choreographic composition process by the Helenita Sá Earp Dance Fundamentals involved in the making of a multimedia dance spectacle which itself is based upon biological organization patterns and forms. The interactions between science and dance that occurred from the twentieth century up until the current days are then analyzed in both theoretical aspects and the utilization of bioscience themes for choreographic spectacles creation purposes. The work establishes that the Helenita Sá Earp Dance Fundamentals possesses an ensemble of epistemologically and methodically principles which are ca able of instituting body language di ersifying agents. ar ’s conce tion osits dance as wide-scoped knowledge, including a “science of dance” where its e istemology is made of an o en ty e of knowledge named “ ance Parameters”, namely Motion, Space, Shape, Dynamics and Time. In this manner, they are shown in systematic relations as references capable to generate a kind of access to body actions stimulating interdisciplinarity, benefitting the interaction between dance language and other knowledge areas. The work attests how the Helenita Sá Earp Dance Fundamentals were applied in the choreographic aestheticization of sub-molecular, molecular and cellular conformational and structural changes that are present in the copper homeostasis model which was utili ed in the creation of the “Transi es” dance show. This artistic work was resented in the “II Feira F P RJ Ci ncia, Tecnologia e Ino a o”, in the “Fe B 011”, in the Teatro Munici al de o a Friburgo and in the o ening of the “VIII Congresso Brasileiro de Farm cia omeo tica”. FAPERJ P2 - INFLUÊNCIA DO ESTADO DE PROTONAÇÃO NA PLASTICIDADE ESTRUTURAL DA CELULASE CEL9A-68 DE Thermobifida fusca

1,2- FERNANDES T.A.V.; 3- COSTA M.G. ; 3- BATISTA P.R. ; 2- PASCUTTI P.G. ; 3- CAFFARENA E.R. ; 1- GOMES D.EB.

1- Instituto Nacional de Metrologia, Qualidade e Tecnologia; 2- Universidade Federal do Rio de Janeiro; 3- Fundação Oswaldo Cruz

As celulases catalisam a hidrolise de celulose em açúcares simples, que podem ser fermentados em etanol ou outros produtos químicos. Portanto, a enorme quantidade de biomassa disponível é uma fonte em potencial para produção de biocombustíveis. Entretanto, para a produção de etanol para o uso de energia em larga escala, é necessário aperfeiçoar a conversão desta biomassa. Neste cenário, as celulases termofílicas ganham destaque por sua capacidade de clivar cadeias de açúcares em altas temperaturas. A hidrólise lignocelulósica utilizando celulases em altas temperaturas oferece várias vantagens, incluindo aumento da carga sólida devido à viscosidade reduzida, menor risco de contaminação microbiana, maior compatibilidade com os pré-tratamentos de alta temperatura, aumento da transferência massa e taxas

mais rápidas de hidrólise. Uma das celulases termofílicas mais bem estudadas é a endo-exo-celulase Cel9A-68 da bactéria Thermobifida fusca, que possui um domínio catalítico (CD) e um módulo de ligação a celulose (CBM), conectados por um linker rico em resíduos de Pro-Ser-Thr. Acredita-se que a ação conjunta dessas estruturas promove uma hidrolise eficiente das fibras de celulose. A fim de tornar este processo mais eficiente através de bioengenharia, precisamos de uma compreensão detalhada, em nível molecular, da flexibilidade intrínseca da celulase. Visando descrever esta plasticidade, em nível molecular, realizamos simulações computacionais em diversos estados de protonação (pH de 2.5 à 12), na temperatura ótima da enzima (325K). Observamos uma alta variação do potencial eletrostático (de 57.99 até -122.19 KT/e) indicando possíveis instabilidades estruturais dependente do pH. Essas oscilações na mobilidade estrutural foram observadas nos sistemas simulados com estado de protonação dos pHs abaixo do ótimo (pH = 5.5), sugerindo propostas para sítios de mutação, a fim de manter as características da flexibilidade das subunidades em temperaturas maiores e/ou em pH mais ácidos. CNPq, INBEB P3 - STRUCTURAL ANNOTATION OF SEQUENCES FROM THE ENDOPHYTIC BACTERIUM GLUCONACETOBACTER DIAZOTROPHICUS USING ASAPROT

1,2- DA SILVA, M.L. ; 2- BISCH, P. M. 1 - Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de

Metrologia, Qualidade e Tecnologia; 2 - Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro

"The gram-negative endophytic bacterium Gluconacetobacter diazotrophicus is found in plants such as sugarcane, pineapple, coffee and sweet potato. This bacterium fixes atmospheric nitrogen, produces plant growth-promoting hormones, bacteriocins and helps solubilization of zinc compounds. 3778 protein sequences were predicted in the G. diazotrophicus PAL5 genome [Refseq: NC_010125]. We investigated these 3778 proteins in order to compare the predictions made by conventional annotation and structural annotation. Conventional annotation is based on information from the primary sequences of proteins and structural annotation is based on information from the three-dimensional structure of these proteins. Structural properties of these proteins were analyzed by ASAProt workflow. ASAProt is a computational workflow for structural annotation projects. The 3D models were built and evaluated with Ramachandran plot, RMSD and BATS classification by MHOLline. Furthermore, structural domains and superfamilies of the proteins were analyzed by fastSCOP and 3DBlast. Simultaneously, the primary sequences of proteins were analyzed by conventional approaches like COG, SMART and Uniprot. We have successfully constructed 3D models with excellent stereochemistry quality for 1390 proteins. So it was possible to compare the conventional annotation and the structural annotation for 1225 sequences. Similar functions have been predicted by both approaches for 58% of these sequences. We note the importance of structural annotation in structural genomics projects, since the structural annotation might complement the conventional annotation. Moreover, we can infer function at 53 proteins before predicted as hypothetical proteins by conventional annotation." CNPq.


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