Currently a second year student on the MRC funded doctorial training partnership.

Education

Warwick University, M.Sc, Biomedical Interdisciplinary Research (Merit)

Summary of first dissertation

Marek’s Disease Virus (MDV), its serotypes and other alphaherpesviruses encode VP22. This protein has been shown to interact with microtubules. This association results in the stabilization and the appearance of think microtubule bundles in infected cells as well as VP22 transfections. This has been suggested in both mammalian herpes viruses like herpes simplex virus 1 and in avian herpes viruses like Marek’s Disease virus. VP22 transfection experiments with GFP show no microtubule association in both mammalian and avian cell lines, this was interrogated by treating cells with nocodazole. The GFP tag may be having an effect on the function of VP22 in transfections. Truncations of wild type MDV VP22 was made, these are N-terminal removed, C- terminal removed and both terminals removed. Using an in vitro microtubule-binding assay it has been shown that VP22 from HVT is able to directly bind to microtubules, without the need of any other microtubule associated proteins. Determining the exact dissociation constant of this association given a rough estimation of 23.5nM. From an in vitro microtubule dynamics assay with TIRF microscopy it has been determined that the VP22 association with microtubules results in the stabilization of microtubules with fewer catastrophes, faster growth rates, higher dynamicity as well as faster shrinking speeds on the plus end.

Summary of second dissertation

The assembly of human immunodeficiency virus (HIV) requires the transport and oligomerization of Gag accompanied with other viral proteins and geneomic RNA. The target for HIV-1 Gag has been established to be the plasma membrane, were assembly occurs and viral particles bud. The site of assembly for HIV-2 is not clear. By generating a replication-competent HIV-2 molecular clone that carries a Gag internal/interdomain green fluorescent protein fusion (iGFP) we can study the physiologically accurate temporal sequence of Gag localization and oligomerization during the formation of HIV-2. This recombinant HIV-2 iGFP virus has been shown to express Gag and form viral particles that bud to the supernatant. This recombinant virus shows no maturation post budding, therefore cannot be used for maturation studies. Immunofluorescence studies show a very similar pattern of localization as the iGFP plasmids. Transfection excitements with HIV-1 iGFP have shown that HIV-1 localizes to the plasma membrane and internal compartments but not late endosomes/ lysomes. HIV-2 iGFP has shown that there is no co- localization with the plasma membrane but there is some localization with internal compartments like lysosomes/late endosomes. Quantifying the speeds of the iGFP foci from live cell imaging has shown that there are two distinct populations, suggesting two different trafficking methods for Gag. Mutant analysis of HIV-1/-2 MA domains has shown that the MA domain is responsible for the localization target of Gag in HIV-1/-2. The PTAP motif found on the MA domain of HIV-2 was suggested to be involved in viral budding.

Imperial College London, M.Sc, The Molecular Biology and Pathology of Viruses (Merit)

My MSc dissertation at Imperial College London was based on examining the effects of non-structural West Nile Virus proteins on the innate immune pathways in avian species. This project involved transfecting various reporter plasmids in to avian cell lines and inducing the promoters of the intracellular innate immune pathways, in order to test the effects of the non-structural proteins on the innate immune pathways. Additionally qRT-PCR was used in order determine the effect of these non-structural proteins to individual innate immunity regulatory proteins and their mRNA expression levels. I have taken on several satellite projects during the progression of my main project and these involve; cloning the 184 gene of Fowlpox Virus and becoming familiar with the cloning process and the cloning software CLC Bio. Mapping out the translocation of IRF-7 in avian cell lines and determining the effects of Fowl Pox virus on various innate immunity regulators with western blotting

Middlesex University, BSc, Biomedical Science (1st)

I have graduated from Middlesex University with a first class honours degree in Biomedical Science, were I gained experience in many different laboratory techniques and biomedical applications. Through my undergraduate degree I have developed an understanding cancer development and conventional oncological treatments. My dissertation project involved the use of culturing rat fibroblasts that express Murine Polyoma Virus middle tumour antigen (MT), and the of use immunefluorescent staining to determine the subcellular localisation of src family kinases in the presence of MT

Background
University College London Research Technician

Was research technician at University College London working with HCMV latency and reactivation. My duties as a technician involved maintaining cell culture of both primary cell lines (human fibroblasts) and immortalised cell lines (THP1), ordering and general laboratory maintenance. In addition to my duties as a technician I had undertaken a project that aimed to understand the role of HP1 in relation to latency and reactivation and, in particular to address whether the recruitment of HP1 to the MIEP is important beyond its known role in chromatin biology. I hope to initially understand this with employing siRNA knockdown studies in the THP1 cell lines and also primary cells that are sites of HCMV latency (i.e. monocytes and CD34+ cells). Over the course of this project I will used techniques including MACS cell isolation, Western blotting, qPCR and qRT-PCR, and protein immuno- precipitation protocols.

Visting Researcher Imperial College London, Section of Virology

Was associate of Imperial College London working as a visiting researcher. This voluntary position involved working on IL15 promoter activity in avian species and designing truncated versions of the promoter region and cloning these into plasmids that have an indicator (e.g. luciferase). By transfecting these plasmids into avian cells and performing luciferase assays it was possible to determine sections of the promoter that are necessary for successful transcription.

G dot Tut at warwick dot ac dot uk