CUROP - Academic Poster Design Workshop 2014

Cardiff Undergraduate Research Opportunities Programme (CUROP):

Poster Design Workshop4th September 2014

Dr Nathan Roberts

Dr Iain Mossman

CUROP Co-ordinators

CUROP Poster Session

• 17 October 2014• Main Building, VJ Gallery (foyer)• Two sessions, either side of lunch

Poster Design Tips and Principles

Why posters?

• Help you get your work across in a very

concise way

• Common dissemination tool in many

disciplines – a good skill to have

• A research output that we can use


The poster session

• A busy, noisy room with up to 50 other posters

• Specialist and non-specialist viewers

• People look at posters for an average of 90

seconds. May decide in 10 seconds how much they

want to engage at all

• Make it visually interesting and easy to understand

at a glance


• Powerpoint is often the easiest option– Design Tab, Page Setup – customise the size of

the poster• A2 = 59.4cm x 42cm• A1 = 84.1cm x 59.4cm• A0 = 118.9cm x 84.1cm

– In most instances landscape is better than

portrait.

• Other options for design, include Adobe

Illustrator/Photoshop and InDesign – but require

software and know-how


• No matter what size you choose – no more than 1000 words!

• Fonts:– Sans Serif fonts (Arial, Helvetica, Calibri)

are easier to read, and should be used for headings

– Serif fonts (Times New Roman etc.) are fine for body text.

– 22 point font size is a minimum.• Instead of using words, think of ways in

which you can use photographs, cartoons or illustrations to illustrate your concept.


• Where to print?

– University: Graphic Services (Bute), Media Resources (Heath Park)

– Print Centre, Students Union. Use code CUROP2014 for discount


Elements of the poster

Title



Title

IntroductionBlah blah Blah blah Blah blah Blah blahBlah blah Blah blah Blah blah Blah

ConclusionBlah blah Blah blah Blah blah Blah blahBlah blah Blah blah Blah blah Blah



Title



MethodsBlah blah Blah blah Blah blah Blah blahBlah blah Blah blah Blah blah Blah

Findings

Images

Graphs

Acknowledgements, references, contact details



Title




Findings

Images

Graphs


Include CUROP!


Colours and backgrounds

Title




Findings

Images

Graphs


Include CUROP!

Example Posters

Exercise

• In groups, think about what is good and bad about the design of some example posters.

• Be ready to share an example of one good thing and one bad thing about the poster!

Introduction Method• 44 sites across upland Wales (Fig. 3)

sampled• Water-PAM (Walz, Germany) used to take

approx. 60 readings at each site• Readings calibrated using a light curve

that was obtained from each site• Water samples collected for DNA and

nitrate analysis.• Habitat survey conducted• Analysis of water chemistry

(pH,temperature, conductivity and % O2) using a portable multiparameter meter (HANNA instruments, USA) for further analysis of nitrates concentration in stream water.

Results, conclusion and Future Directions• Establish nitrate concentrations in rivers and streams across upland Wales.

• Practical implications include evaluating the effect of pesticide run off from farm land• Future work would involve continued assessment on a biannual basis. Developing biofilm as a important early bio-indicator of water

quality. Providing vital information to reduce and predict pesticide run off.• Thus reducing the harmful effect of pesticide on a river biofilm(4).• Reducing this negative effect suggest that that biodiversity and the economically valuable ecosystem services provided by rivers and

streams , which are dependent on biofilm and correct nitrate levels can be sustainably maintained.

Biofilms are made up from a cluster of bacteria, algae, cyanobacteria and protozoa, which are held in a polysaccharide matrix(1). Biofilms , as well as having medical implications are a essential component to the river ecosystem. The levels of biofilm have been shown to be affected by water temperature, , nutrient levels and pesticides(2). Through analysis of biofilm levels we are able to predict the corresponding levels of algae biomass, functional chlorophyll A and nitrates. Such factors are key to British river biodiversity and to the maintenance of ecosystem services, a industry which is currently valued at £200 billion (3).

References1) Diaz Villanueva, V., Font, J., Schwartz, T., Romani, A.M. (2011). Biofilm formation at warming temperature: acceleration of microbial colonization and microbial interactive effects. Biofouling 27:59-71 •2)Proia, L., Osorio, V., Soley, S., Köck-Schulmeyer, M., Pérez, S., Barceló, D., Romaní, A.M., Sabater, S.(2013). Effects of pesticides and pharmaceuticals on biofilms in a highly impacted river. Environmental Pollution 178:220-8• 3) http://nerc-duress.org/?page_id=40 [Accessed, August 2013.]•4) Hayashi, S., Jang, J.E., Itoh, K., Suyama, K., Yamamoto, H. 2011. Construction of river model biofilm for assessing pesticide effects. Arch Environ Contam Toxicol. 60:44-56.

Fig. 1) The water –PAM used that took one of 5,500 readings taken during this study.

Assessing the role of biofilm in nitrate regulation in streams and rivers in upland Wales using fluorescence

analysis

Fig. 3) Map of Wales with the 44 sites (black points) sampled.

Fig 4) Looking downstream form our site on the Honddu river, Powys

Fig. 2) Rhys Luckwell carrying out water chemistry analysis on the Conway river, near Snowden in upland Wales.

http://nerc-duress.org/?page_id=40

ANALYSIS OF CD112 CELL SURFACE EXPRESSION MODULATION BY HUMAN CYTOMEGALOVIRUS

STEALTHOGENDepartment of Infection, Immunity and Biochemistry

Cardiff University School of MedicineCardiff University

Health ParkCardiff

CF14 4XN

Year 4 MBBCh

ABSTRACT

IMMUNE EVASION BY MODULATION METHOD

CONCLUSIONS & FURTHER STUDY

ΔUS1-11 / ΔUL13-20 ENCODE GENE PRODUCTS MODULATING CD112 EXPRESSION

Human Cytomegalovirus (Human Herpesvirus 5, HHV5, HCMV) is a clinically relevant pathogen [1]. As the leading cause of infectious congenital birth defects and responsible for significant morbidity in immunocompromised patients (HIV/AIDS, post-transplant), it has been designated a target of highest priority for vaccine research by the US Institute of Medicine [2]. HCMV encodes multiple immune evasion functions and relies heavily on these for virulence., including downregulation of cell surface membrane ligands for NK Cell receptors [3]. Recently, UL141, an HCMV gene putatively involved in immune evasion function, was shown to downregulate NK cell ligands CD155 and CD112 from cell surface membranes, attenuating NK Cell mediated responses. However, UL141 alone was unable to reproduce functional downregulation of CD112, suggesting co-operation with other viral functions is required [4]. This investigation sought to identify candidate genes for co-operative function through screening a gene block deletion mutant library for restoring downregulation function [5, 6]. Gene loci UL13-20 and US1-11 were identified as encoding functions involved in cell surface CD112 modulation. Moreover, US2 and US11 showed trend for specific role in this capacity. This study highlights these genes for further focussed research.

The Human Cytomegalovirus genome (~236kbps) encodes an estimated 164 genes with protein products. Significantly, only 45 are essential for replication in fibroblast culture, suggesting the remainder have alternative function. Indeed, many have been associated with immune evasion [3].

HCMV UL141, a gene located in the ULb/b’ cassette has been shown to downregulate cell surface expression of CD155 (Polio Virus Receptor, Nectin-Like Molecule 5) and CD112 (Herpes Entry Mediator B, Nectin-2). Both are ligands for the activating NK cell receptor CD226 (DNAM-1). Reduction in CD155 / CD112 cell surface expression results in fewer interactions with CD226, thereby inhibiting NK cell priming and reducing NK cell mediated cytotoxicity. This promotes virus survival and facilitates further replication [2, 3, 4].

Experimentation showed HCMV ΔUL141 (derived from strain Merlin, Cardiff) was associated with enhanced susceptibility to NK cell cytolysis. However, when UL141 was re-introduced as a transgene, a resistant phenotype was rescued, suggesting an immune evasion function for this gene. Subsequently, UL141 was found to be associated with downregulation of CD155 and CD112, the former targeted for proteasomal degradation. Interestingly, when expressed alone in cells, neither UL141 or the protein product (gpUL141) were able to downregulate CD112 cell surface expression, although downregulation of CD155 was preserved. This suggested UL141 required co-operation with further HCMV genes (present in the HCMV ΔUL141 clone) to modify the expression of CD112 [4].

The aim of this investigation was to use a library of HCMV block deletion mutants (knockout for specific gene loci), to screen for modulation of CD112 cell surface expression. Once identified, the individual genes involved would be determined by rescuing their function in cells currently infected with block deletion mutants. Using a model of co-infection, transgenes were delivered by replication deficient adenovirus vectors and CD112 expression analysed.

Human Cytomegalovirus Block Deletion Mutant Library ScreenHCMV block deletion mutants were obtained from a pre-constructed library. Human Fibroblasts (HF) were infected with block deletion mutants at multiplicity of infection = 10 (MOI=10) for 2 hours, then incubated at standard conditions for 48 hours. Cells were then harvested and analysed by flow cytometry. Immunohistochemistry staining was performed as described previously [1]. 1o Antibodies: Control Ig (mIgG), Mouse IgG 1:500 (Santa Cruz, SC-2025); Anti-CD155 Mouse D171 IgG 1:500 (Abcam, AB3142); Anti-CD112 Mouse IgG2b 1:50 (Santa Cruz, SC65333); Anti-MHC Class I W632 IgG2a 1:10000 (Serotec, MCA81EL). 2o Antibody: Goat Anti-Mouse Fab fragments AF647 (Molecular Probes, A21237).

Flow CytometryFlow cytometry was performed using a BD Accuri C6 flow cytometer [1].Optimisation of Co-Infection ProtocolRecombinant adenovirus vectors expressing green fluorescent protein (RAd-GFP) and RAd-Control were prepared as previously described. For the timecourse assay, Human Foteal Foreskin Fibroblasts preferentially expressing the Coxsackie and Adenovirus Receptor (HFFF-CAR) were infected with RAd’s (MOI=5) for 2 hours and incubated under standard conditions for 24 hours and 48 hours, then analysed using fluorescence microscopy (Lecia DMIRBE microscope) and western blotting. For virus titration, HFFF-CARs were infected with RAd’s at MOI=5 and MOI=10 for 2 hours and incubated for 48 hours under standard conditions, then analysed by spectrophotometry (BMG Omega FluStar) and western blotting. For co-infection optimisation, HF’s were co-infected at the same time with HCMV (MOI=50) and RAd’s (MOI=10) for 2 hours, then incubated under standard conditions for 48 hours. Cells were analysed by flow cytometry [1, 3]. WB antibodies: Control Anti-Actin Rabbit IgG + HPO 1:10000 (Sigma, A2066); 1o Anti-GFP Rabbit IgG 1:5000 (Santa Cruz, SC-8334); 2o Goat Anti-Rabbit IgG + HPO 1:10000 (BioRad, 170-6515).

US1 – US11 ScreenHuman Fibroblasts were co-infected with HCMV Deletion Mutant (MOI=10) and RAds-US1-US11 (MOI=50) for 2 hours, then incubated at standard conditions for 48 hours. Cells were harvested and analysed by flow cytometry as per Block Deletion Mutant Library Screen.

CONFIRMATION OF TRANSGENE EXPRESSION IN MODEL OF CO-INFECTION

US2 AND US11 MODULATE CELL SURFACE CD112 EXPRESSION IN HUMAN FIBROBLASTSPrevious study showed HCMV UL141 was unable to modulate cell

surface expression of CD112 when expressed alone in cell culture. We hypothesised a co-operative interaction by UL141 with other viral protein(s) was required for functional downregulation of CD112. To screen for putative interacting function, we analysed cell surface CD112 expression in Human Fibroblasts infected with HCMV containing block deletions of known gene loci (excluding UL141). Positive results were considered a greater than 50% increase in CD112 expression relative to control.

Two gene loci were associated with significantly increased CD112 cell surface expression, corresponding to US1-11 and UL13-20 (relative staining signal increase of 502% and 168% vs. control respectively) (Figure.1.). This suggested HCMV encoded at least two further products involved in cell surface CD112 modulation.

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Figure.1. ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface expression relative to HCMV control. Human Fibroblasts (HF’s) were infected with HCMV block deletion mutants (MOI=10) and analysed by flow cytometry 48h p.i. A: Cells were gated for HCMV infection. B, C: HCMV control infected cells downregulated CD155 and CD112 relative to mock infected cells. D: GFP fluorescence was used to select cells infected with HCMV block deletion mutants, since these viruses encode a gene for GFP. E-J: ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface staining relative to HCMV control. Modulation of CD155 did not reach the 150% threshold for any HCMV block deletion mutant. Isotype matched Mouse IgG (cIg) was used for staining control.

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Figure.2. RAd and HCMV co-infection proves viable technique for assessment of transgene expression and function. Human Fibroblasts preferentially expressing the Coxsackie and Adenovirus Receptor (HFFF-CAR) were infected with RAd-GFP for 2h. RAd-GFP mediated GFP expression visualised by fluorescence microscopy at A-C: 24h p.i. and D-F: 48h p.i. G: Total GFP expression was similar at both 24h and 48h p.i. as assessed by western blot. H-J: Similar GFP expression and fluorescence intensity was detected at RAd-GFP MOI=5 and MOI=10. HF’s were co-infected with RAd-GFP (MOI=25, MOI=50) and HCMV (strain Merlin) for 2h and analysed by flow cytometry 48h p.i. K: Cells were gated and L-N: HCMV infection validated by downregulation of CD155, CD112 and MHC Class I. O: Cells were gated for GFP expression and P-R: importantly, RAd-GFP infection did not appear to substantially alter expression of stained cell surface proteins. S-U: Confirmation of RAd-GFP and HCMV infection was achieved by observation that GFP expressing cells has simultaneously downregulated cell surface markers of HCMV infection.

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Figure.3. HF’s were co-infected with RAds-US1-US11 (MOI=50) and HCMV deletion mutant ΔUS1-11 (MOI=10) for 2h and analysed by flow cytometry 48h p.i. A: Live cells were selected and B-C: HCMV infection validated by downregulation of CD155 and CD112. D: Cells co-infected with RAd-US1-US11 were selected by gating for GFP expression. E-J: Two RAds, corresponding to US2 and US11 showed trend towards recovering CD112 downregulation function of HCMV, reaching significance threshold of 80% relative staining vs. control HCMV. CD155 expression showed no similar trend. Isotype matched Mouse IgG (cIg) was used as staining control.


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CE

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CO

UN

T

FL4 FL4 FL4

US

1-11

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d C

ontr

ol

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US

3

US

6

US

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US

8

US

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US

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US

11

100%

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US

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RA

d C

ontr

ol

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US

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US

6

US

7

US

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US

9

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80%

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1-11

RA

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100%

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E F G

cIg cIg

cIg cIg cIg

Mock Mock

HCMV HCMV HCMV

RAd + HCMV

HCMV

RAd + HCMV

HCMV

US2US11

US2US11

US2US11



CD155 CD112

CD155 CD112cIg

RE

LA

TIV

EE

XP

RE

SS

ION

(%)

DB C

H I J

A

FSC

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C

CE

LL

CO

UN

T

FL4 FL4

FL1

FS

C

CE

LL

CO

UN

T

FL4 FL4 FL4

US

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RA

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ol

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US

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US

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US

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US

9

US

10

US

11

100%

80%

US

1-11

RA

d C

ontr

ol

US

2

US

3

US

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US

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US

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US

10

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11

80%

100%

US

1-11

RA

d C

ontr

ol

US

2

US

3

US

6

US

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US

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US

9

US

10

US

11

100%

80%

E F G

cIg cIg

cIg cIg cIg

Mock Mock

HCMV HCMV HCMV

RAd + HCMV

HCMV

RAd + HCMV

HCMV

US2US11

US2US11

US2US11



CD155 CD112

CD155 CD112cIg

RE

LA

TIV

EE

XP

RE

SS

ION

(%)

DB C

H I J

A

FSC

SS

C

CE

LL

CO

UN

T

FL4 FL4

FL1

FS

C

CE

LL

CO

UN

T

FL4 FL4 FL4

US

1-11

RA

d C

ontr

ol

US

2

US

3

US

6

US

7

US

8

US

9

US

10

US

11100%

80%

US

1-11

RA

d C

ontr

ol

US

2

US

3

US

6

US

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US

8

US

9

US

10

US

11

80%

100%

US

1-11

RA

d C

ontr

ol

US

2

US

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US

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US

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US

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US

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US

10

US

11

100%

80%

E F G

cIg cIg

cIg cIg cIg

Mock Mock

HCMV HCMV HCMV

RAd + HCMV

HCMV

RAd + HCMV

HCMV

US2US11

US2US11

US2US11

To determine which genes within the block modulate of cell surface CD112 expression we proposed a co-infection technique, whereby fibroblasts infected with HCMV block deletion mutants would also be infected with a RAd encoding a single deleted gene. This transgene would then be expressed in target cells; rescuing function and permitting analysis of CD112 expression.

Optimisation of technique was first necessary to define conditions for best transgene expression. RAd-GFP, an adenovirus encoding green fluorescent protein was used for this calibration due to ease of protein expression analysis. A time course was performed to measure transgene expression over time, no difference was observed between 24h and 48h post infection using fluorescence microscopy and western blotting. Titration to select multiplicity of infection indicated transgene expression was similar at RAd MOI=5 and MOI=10 when assessed by spectrophotometry and western blotting. Optimal co-infection conditions were determined as 48h incubation at MOI=50 (accounting for reduced infectivity of Human Fibroblasts relative to HFFF-CAR). Analysis of cells exposed to both HCMV and RAd-GFP showed downregulation of cell surface markers typical of HCMV infection (CD155, MHC-Class I) with concomitant fluorescence in green channel, suggesting cells had been co-infected.

Having identified the US1-11 gene loci as putatively encoding function(s) involved in modulating cell surface CD112 expression, the validated co-infection technique was utilised to screen US1 – US11 individually for functional modulation of cell surface CD112 expression.

Significant downregulation of cell surface CD112 expression was defined as cell surface CD112 staining less than or equal to 80% relative to control. Cells co-infected with RAd’s US2 and US11 showed a trend suggestive of cell surface CD112 downregulation using this threshold (73% and 76% respectively). This indicated US2 and US11 encode functions involved in cell surface CD112 modulation. No significant modulation of cell surface CD155 or MHC Class-1 was observed, thus the effect was likely specific to CD112.

Screening HCMV block deletion mutants highlighted two loci associated with increased cell surface CD112 expression when removed (US1 – 11 and UL13 -20). To analyse individual genes within these blocks, a co-infection technique was developed and optimised.

Previous research has shown UL141 functionally downregulates cell surface CD112, although this effect is not seen when expressed alone intracellularly, suggesting co-operation with other HCMV proteins is required [4]. This investigation identified US2, US11 and an unknown gene in UL13-20 (UL X) as having potential role in UL141 complementation.

UL141 downregulates CD155 by targeting the protein for proteasomal degredation. It is logical to assume a similar mechanism exists for CD112 [4]. Indeed, US2 and US11 have also been demonstrated to target MHC Class-1 for proteasomal degradation, making this function even more likely [5, 6].

Further focussed research should aim to investigate the function of US2 and US11. Proteasome inhibition assays should be performed to analyse for reversal of downregulation in the presence of an inhibitor (e.g. MG132). Reversal of downregulation would strongly suggest a functional degredation of CD112, corroborating previous findings US2 and US11 modulate MHC Class-1 via this pathway [1, 4, 7, 8, 9].

Additional confirmation could be obtained through conjugation of US2 / US11 with a V5-Strep tag to assess subcellular localisation. Sequestration of US2 / US11 within the endoplasmic reticulum would imply early modification of target proteins during post-translational processing [6].

Furthermore, complementation assays using combinations of UL141 and US2 / US11 / ULX should prove that these proteins act in concert to modulate cell surface CD112 expression.

References1. Tomasec, P. Wang, E. Davison, A. Vojtesek, B. Armstrong, M. Griffin, C. McSharry, B. Morris, R. Llewellyn-Lacey, S. Rickards, C. Nomoto, A. Sinzger, C. Wilkinson, G. 2005. “Downregulation of Natural Killer Cell-Activating Ligand CD155 by Human Cytomgealovirus UL141”, Nature Immunology, 6(2), Pg. 181 – 188.2. Stanton, R. Baluchova, K. Dargan, D. Cinningham, C. Sheehy, O. Seirafian, S. McSharry, B. Neale, M. Davies, J. Tomasec, P. Davison, A. Wilkinson, G. 2010. “Reconstruction of the Complete Human Cytomeglovirus Genome in a BAC Reveals RL13 to be a Potent Inhibitor of Replication”, Journal of Clinical Investigation, 120(9), Pg. 3191 – 3208.3. Wilkinson, G. Tomasec, P. Stanton, R. Armstrong, M. Prod’homme, V. Aicheler, R. McSharry, B. Rickards, C. Cochrane, D. Llewellyn-Lacey, S. Wang, E. Griffin, C. Davison, A. 2008. “Modulation of Natural Killer Cells by Human Cytomegalovirus”, Journal of Clinical Virology, 41, Pg. 206 – 212.4. Prod’homme, V. Sugrue, D. Stanton, R. Nomoto, A. davies, J. Rickards, C. Cochrane, D. Moore, M. Wilkinson, G. Tomasec, P. 2010. “Human Cytomegalovirus UL141 Promotes Efficient Downregulation of the Natural Killer Cell Activating Ligand CD112”, Journal of General Virology, 91, Pg. 2034 – 2039.5. Sugrue, D. Doctor of Philosophy Thesis. 2012. “Modulation of Natural Killer Cell Response by Human Cytomegalovirus”.

6. Seirafian, S. Doctor of Philosophy Thesis. 2013. “An Analysis of Human Cytomegalovirus Gene Usage”.7. Smith, W. Tomasec, P. Aichleler, R. Loewendorf, A. Nemcovicova, I. Wang, E. Stanton, R. Macauley, M. Norris, P. Willen, L. Ruckova, E. Nomoto, A. Schneider, P. Hahn, G. Zajonc, D. Ware, C. Wilkinson, G. Benedict, C. 2013. “Human Cytomegalovirus Glycoprotein UL141 Targets the TRAIL Death Receptors to Thwart Host Innate Antiviral Defenses”, Cell Host & Microbe, 13, Pg. 324 – 335.8. Stanton, R. McSharry, B. Armstrong, M. Tomasec, P. Wilkinson, G. 2008. “Re-Engineering Adenovrisu Vector Systems to Enable High-Throughput Analyses of Gene Function”, Biotechniques, 45(6), Pg. 659 – 668.9. Nemcovicova, I. Benedict, C. Zajonc, D. 2013. “Structure of Human Cytomegalovirus UL141 Binding to TRAIL-R2 Reveals Novel, Non-Canonical Death Receptor Interactions”, PLOS Pathogens, 9(3), Pg. 1 – 14.

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