Curs Politehnica
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Transcript of Curs Politehnica
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Teoria celulara:
Toate formele de viata sunt formate din una sau mai multe celule
Celulele se formeaza din celule pre-existente (diviziunea celulara)
Celula este cea mai mica forma de viata
Evenimente majore in evolutia biologiei celulare
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TIPURI DE CELULE
Unitatea structurala si functionala a organismulul viu
Activitatea biochimica a celulei se desfasoara la nivelul organitelor
Celule conective
Celule epiteliale
Celulele transportatoare de gaze
Fara nucleu la maturitate
Celule motrice
Celule „rezervor”
Celule ale sistemulu imunitar
Celule nervoase
Celule sexuale
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1. Membrana Plasmatica
2. Citoplasma (80% apa)
a Contine organite celulare
3. Nucleu
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Membrana plasmatica (MP)
Consta intr-un bistrat lipidic
MP este fluida, fosfolipidele sunt capabile sa se deplaseze in bistrat
Regiunea
hidrofila
Regiunea
hidrofoba
Regiunea
hidrofilica
Regiunea hidrofobica
fosfat
glicerol
Acid gras
X
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www.highlands.edu
I. Bistratul lipidic
Structura membranei plasmatice
Proteinea) Locatie
-periferice
-integrale (transmembranare)
b) Functie
- transport (canale)
-enzimatica
- structurala (citoschelet)
-recunoastere (receptori)
colesterol
este strabatut de o varietate de proteine, lipide si glicoconjugati
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In celula animala:
Colesterolul regleaza fluiditatea si flexibilitatea MP
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a) Transport- proteine transmembranare care formeaza canale
selective pentru anumiti ioni sau molecule (transport pasiv/activ)
b) Semnalizare- proteine transmembranare care au situsuri de
expuse pe fata extracelulara, pentru legarea specifica a unor
molecule ( hormoni, molecule de semnalizare= „mesager
chimic”). Legarea ligandului poate determina modificarea
confomatiei receptorului, ducand la initierea unei serii de reactii
in celula
c) Strucural- proteine care formeaza matricea extracelulara si
elementele citoscheletului (in interiorul celulei). Mentin forma
celulei, stabilizeaza localizarea unor proteine membranare, rol inmotilitate si interactii intercelulare.
II. Proteine
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d) Activitate enzimatica- proteine transmembranare care catalizeaza
o reactie enzimatica, sau un compex de reactii intr-o anumita cale
metabolica
e) Jonctiuni intercelulare- proteine transmembranare care
interactioneaza apropiind celulele unele de altele; CAMs (cell
adhesion molecules)- importante in migrarea celulara.
f) Recunoastere intercelulara- glicoproteine transmembranare
sunt tinte specifice pentru receptori localizati pe alte celule
II. Proteine
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Lanturile glucidice sunt atasate de proteine (glicoproteine) sau lipide membranare
(glicolipide). In numeroase cazuri sunt tinte pentru recunoasterea intercelulara.
Grupele de sange ABO sunt determinate de gruparile glucidice atasate de antigenul H
prezent pe suprafata hematiilor- recunoscute de anticorpi
III. Glucide/oligozaharide/carbohidrati
Enzima A : N-acetilgalactozamintransferaza
Enzima B : Galactosiltransferaza
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https://www.youtube.com/watch?v=gFuEo2ccTPA
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CURS II
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1. Membrana Plasmatica
2. Citoplasma (80% apa)
a Contine organite celulare
3. Nucleu
Structura celulei
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Permeabilitatea membranei plasmatice
classes.midlandstech.edu
a) Difuzia simpla-trecerea
moleculelor
liposolubile direct
prin bistratul
lipidic (CO2, O2)
b) Difuzia facilitata de
transportori- proteine care
isi schimba conformatia
dupa legarea ligandului
(glucoza)
c) Difuzia facilitata
de canale proteice
(ioni selectati pe
baza de marime si
sarcina electrica)
d) Osmoza-difuzia
apei printr-un canal
strict specific-
(aquaporin) sau
direct prin bistratul
lipidic
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Liza celulara
https://www.youtube.com/watch?v=OYoaLzobQmk
Sare pe melci
Injectarea substantelor in organism
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Component E.coli Celula
mamifere
apa 70 70
Ioni 1 1
metaboliti 3 3
proteine 15 18
ARN 6 1.1
ADN 1 0.25
lipide - 2
fosfolipide 2 3
polizaharide 2 2
Volumul
total al
celulei
2x10-
12cm3
4x10-9cm3
Compozitia chimica a unei celule
20%
70%
apa
E coli
ARN
Prot
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GENA
Transcript ARN(ARNm)
Transcriptia
ADN
Translatie
Proteina
(1)
(104 copii)
(105 copii)
(TOT-109 copii)
Transmiterea informatiei genetice in celula
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În funcţie de comportamentul în mediul apos, aminoacizii suntclasificati astfel:
•
1) – aminoacizi cu grupări R nepolare: Ala, Val, Leu, Ile,Met, Pro, Phe, Trp
• 2) – aminoacizi cu grupări R polare, dar neîncărcate electric:Gly, Ser, Thr, Asn, Gln, Tyr, Cys
• 3) – aminoacizi cu grupări R polare, dar încărcate electric la pH fiziologic
- încărcare pozitivă: Lys, His, Arg
- încărcare negativă: Asp, Glu.
Structura proteinelor
Reactia de condensare intre doi AALegatura peptidica
În funcţie de capacitatea de sinteza a celulei, aminoacizii
sunt clasificati astfel
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Forma proteinei- determinata de
secventa de aminoacizi
Legaturipeptidice
•In principiu-un nr.nelimitat de conformatii
•Lantul adopta conformatia stabila
•Majoritatea proteinelor se pliaza spontan
in conformatia stabila
•Plierea este determinata de distributia
lanturilor laterale polare si nepolare
Conformatie pliata in
mediu aposPolipeptid nepliat
Resturi
nepolare
Resturi
polareMiez hidrofob
(Resturi nepolare)
Legaturi de H intre resturile polare
si moleculele de apa
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Structura proteinelor
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Structura 3D nu se poate prezice din structura primara
Structura 3D- difractie de raze X a cristalelor de proteina sau prin RMNPeste 100 paterne de pliere descoperite
sheet(structuri pliate )-
extensiv in proteinele globulare
Model IgG1(anticorp) Model mioglobina
Structuri secundare comune in proteine
alpha helix- keratina, domeniile TM
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Denaturarea proteinelor
• http://www.sumanasinc.com/webcontent/anisamples/nonmajorsbiology/proteinstructure.html
Codul genetic
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CURS III
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CITOSCHELETUL
Asigura :-forma celulei
-modificarea formei celulare
-motilitatea celulei
Microscopie fluorescenta
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COMPONENTELE CITOSCHELETULUI
Microfilamente Filamente Intermediare Microtubului
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Polimerizarea actinei
Monomer ActinaG Legare ATP Polimerizare Actina F
Microfilamente
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MICROFILAMENTE
Un filament are doua lanturi de actina polimerizata
Microscopie electronica
Polaritate polimerizare/depolimerizare
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• Microfilamentele sunt prezente in intreaga celula, dar mai concentrate in
apropierea MP
• Sunt importante in miscarea, contractia, adeziunea celulara
• Pot fi identificate in celula prin reactia cu faloidina- un compus fluorescent
care leaga doar actina F
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MICROFILAMENTE IN ADEZIUNEA CELULARA
Focar de adeziune
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FILAMENTE INTERMEDIARE (FI)
Compozitia proteica a filamentelor intermediare depinde de specie si variaza
in functie de tipul de tesut
In celule epidermale si axoni neuronali, FI sunt de cel putin 10 ori mai abundentedecat microfilamentele sau microtubulii
Principala functie este structurala, de sprijin mecanic pentru membrana plasmatica
cand celulele sunt in contact intre ele, sau cu matricea extracelulara
Spre deosebire de microfilamente si microtubuli, FI nu participa la motilitatea
celulara
FI sunt extrem de stabile, raman intacte chiar dupa extractii in detergent sau
solutii saline, in timp ce microfilamentele si microtubulii depolimerizeaza in
subunitatile constituente.
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- Celule epiteliale contin FI formate din keratine (peste 50 de tipuri)
- Celule mezenchimale sunt bogate in FI alcatuite din vimentina
- Celulele musculare au FI formate din desmina
- Neuronii si celulele gliale au FI compuse din peripherina si proteina fibrilara acidica gliala
(GFAP).
- FI din lamina nucleara care formeaza reteaua fibroasa de proteine pe partea interna a
membranei nucleare. Laminele participa la atasarea cromozomilor de membrana nucleara
nuclear
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DEFECTE GENETICE IN LAMINA NUCLEARA
Mutatie punctiforma la nivelul genei LMNA- codifica lamina A
>90% dintre pacienti mor din cauza complicatiilor date de ateroscleroza (stop
cardiac) inainte de varsta de 13 ani.
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MICROTUBULI (MT)
Sunt formati prin asocierea dimerilor de alfa si beta tubulina
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Polimerizarea microtubulilor incepe de la centrozom, localizat langa nucleu
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Microtubulii sunt esentiali in miscarea organitelor
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Microtubulii sunt esentiali in miscarea cililor si flagelilor
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CURS IV
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Microscopia clasica Arhitectura celulara
Metode de investigare celulara la nivel molecular
Imagini de microscopie electronica
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Molecule de interes
Ioni anorganici (Ca, Na, etc)
ARN, ADN
Macromolecule mari Proteine
-Cum identificam moleculele specifice implicate in reactii celulare ?
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-Cum identificam moleculele specifice implicate in reactii celulare ?
Importante pentru caracterizarea cailor metabolice/de semnalizare, etc in stare
normala si patologica
-Cum determinam cantitativ moleculele implicate ?
Important pentru patologii. Ex. (titrul viral in infectii)
Leziuni
hepatice
Markeri cuantificati in cursul
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-Cum aflam daca sinteza sau localizarea lor celulara se schimba ?
Important in patologii. EX. Fibroza cistica, mutatie la nivelul CFTR (cystic fibrosis
transmembrane conductance regulator) care conduce la sinteza unui canal nefucntional sau
blocarea acestuia in RE urmata de degradare.
Celula pulmonara
Normal Fibroza cistica
Acumulare de mucus „uscat”- blocheaza caile respiratorii
Infectii pulmonare
Acumulare de mucus blocheaza caile pancreatice
Po>>
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Fibroza cistica: speranta de viata 37-40 ani
Maladie genetica autozomala recesiva
Incidenta 1:2500 nasteri
Metode de detectie a proteinelor
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Metode de detectie a proteinelor
Electroforeza in gel
Suport pentru migrare (gel): poliacrilamida, agaroza
Metoda analitica de separare in camp electric a proteinelor in amestec in functie de masa lor
Structura gelului de poliacrilamida
Structura gelului de agaroza
Polimerizare
Gelificare
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Mobilitatea moleculelor biologice depinde de incarcarea electrica si de masa
Proteinele sunt supuse unui tratament care sa le asigure o incarcare electrica uniforma
astfel incat mobilitatea electroforetica sa depinda doar de marimea lor.
Denaturare termica in prezenta de detergenti (SDS)
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Electroforeza denaturanta in prezenta de agenti reducatori
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p g
Electroforeza proteinelor serice
Variante de electroforeza
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Blue Native PAGE (BN-PAGE)
Pentru separarea complexelor proteice largi in forma lor nativa. SDS este inlocuit cu Coomassie (Brilliant) Blue G-250
(Incarcat negativ)
Zymogram PAGE
Pentru identificarea unor proteine active enzimatic (colagenaze sau alte proteaze). Substratele ( gelatina sau cazaina)
sunt inglobate in gel. Migrare in conditii nereducatoare . Colorare cu Coomassie (Brilliant) Blue R-250.
Focusarea izoelectrica
Separare in gradient de pH gradient pentru separarea proteinelor in functie de punctul isoelectric (pI).
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Curs V
D t ti t i l i
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Detectia proteinelor prin
Western blotting
Principiu: identificarea specifica a proteinelor pe baza interactiei cu anticorpi
- -
+
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Ag imobilizat
Ac primari fata de
AgAc secundari(marcati):
Ac fata de Ac primari
Anticorpi : Proteine produse de sistemul imun ca reactie de aparare fata de infectii
Anticorp (Ac) Antigen (Ag)
Specificitatea interactiei Ag-Ac des folosita in biologia celulara
epitopi
Reactia antigen-anticorp
i i i li l li
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Anticorpi mono-si policlonali
Anticorpii recunosc
mai multi epitopi ai Ag
Ac-recunoaste
1 singur epitop
Cum migreaza in electroforeza in conditii
reduse/nereduse? Lant H-55 KDa, Lant L-25 kDa
Proteina HIV
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Policlonal
Monoclonal
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Ag
Celule B Ac
Celule B
Tumorale’’imortalizate’’
I Fuziune
AcII Expandare
III Clonarea celulelor
prin dilutie seriala
IV Selectarea clonelor pozitiveAc monoclonal
Anticorp monoclonali
Propagarea unei clone
-1 singura celula B
-Obtinere de hibridoame--celule hibride
Ac-recunoaste
1 singur epitop
Anticorpi policlonaliAg
Antiserul contine Ac
Amestec heterogen de Ac, fiecare
produs de cite o celula B secretoare
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Detectia proteinelor poate fi colorimetrica sau prin chemiluminiscenta
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Avantaje:
Foarte specifica pentru identificarea unei anumite proteineDetecteaza proteine si mai putin peptide
Poate confirma prezenta (determinare calitativa) dar si cantitatea unei proteine intr-o
proba data
Procesare minimala a probelor
Dezavantaje:
Proteina de interes trebuie sa fie cunoscuta
Anumiti detergenti pot da interferente
Nu este foarte ieftina (nu se aplica in teste de rutina).
Premise: VEGF (vascular endothelial growth factor) este un agent angiogenic,implicat in permeabilitatea vasculara
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implicat in permeabilitatea vasculara
VEGF creste in hipoxie si ischemie vasculara (reducerea fluxului sanguin)
Concluzii
experimentale??
Detectia proteinelor prin
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Detectia proteinelor prin
IMUNOFLUORESCENTA
Presupune utilizarea unui anticorp primar, monoclonal sau policlonal, fata de o proteina specifica
Anticorpul primar poate fi direct marcat cu un fluorofor
Se pot folosi anticorpi secundari marcati cu un fluorofor
Fluorescenta emisa de proba este vizualizata cu microscopul de fluorescenta
In cazul in care detectia se face prim microscopie electronica anticorpii sunt legati de un atom metalic
greu (Au)
Aplicatii: in diagnostic clinic Identificare de celule canceroase prin evidentierea unui anumit marker;
cercetare (prezenta sau absenta unei proteine, distributia celulara, modificari de sinteza si degradare
Fluorofori excitatie-emisie
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Fluorofori excitatie emisie
Izotiocianat de fluoresceina (FITC)
Evidentierea mai multor proteine in aceeasi
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Evidentierea mai multor proteine in aceeasi
proba
E id ti t i l l
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Radiatiile UV produc leziuni la nivelul ADN.
Se declanseaza un proces de reparare a leziunilorcare genereaza mutatii
Celula epiteliala mutata induce un proces de
proliferare necontrolata
cancer de piele.
Piele normala
CD95
UV
Marcare cu- anticorpi primari iepure anti- CD95
-anticorpi secundari de capra anti-IgG iepure marcati fluorescent
Clusterele de CD95 semnalizeaza celulei
declansarea apoptozei celulare.
Uniform –
suprafata celulei Clustere
Evidentierea apoptozei celulare
Evidentierea tesutului malign
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Sectiune prin tesut pancreatic, in verde celule
pancreatice tumorale- marker specific
Celule hepatice infectate cu HCV, identificarea unei
proteine virale nestructurale
Evidentierea infectiei virale