4 Year PhD Programme in the CIMR
Current students on the CIMR 4 Year PhD Programme
2011 Intake
Tom O’Loughlin
Rotation I Supervisor: Paul Lehner
Rotation II Supervisor: Folma Buss
Paloma Navarro
Rotation I Supervisor: David Rubinsztein
Rotation II Supervisor: Scottie Robinson
Francesca Nice
Rotation I Supervisor: Ken Smith
Rotation II Supervisor: Tony Green
Anne Streeter
Rotation I Supervisor: Jim Huntington
Rotation II Supervisor: Bertie Gottgens
2010 Intake
Tim Newton
Using mutant serpins to investigate sterol metabolism in proteostasis
Proteostasis refers to eukaryotic protein homeostasis, which must be carefully regulated within a cell. The endoplasmic reticulum (ER) is integral to maintaining proteostasis as it provides a protein-folding environment for transmembrane and secreted proteins. If these proteins do not fold correctly they can polymerise and aggregate, which can lead to cell death.
This project will investigate the effect of perturbing sterol metabolism on the ER, in particular how this alters the ability of the ER to deal with unfolded or polymerogenic protein. This link will be studied with mutants of the neuroserpin protein, which underlie the autosomal dominant dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). We will use small compounds that inhibit cholesterol metabolism and see if this alters cell survival and how well proteins fold. We will also investigate whether these misfolded proteins alter cholesterol metabolism.
Radu Rapiteanu
Cellular and viral regulation of a novel target of HIV1-Vpu
Human immunodeficiency virus (HIV) is the retrovirus which causes AIDS. Vpu is an accessory protein of HIV known to downregulate the main HIV receptor on immune cells and enahnce virion release. Using 'plasma-membrane profiling', a novel proteomic method developed in the Lehner laboratory, we aim to identify plasma membrane proteins whose expression is affected by viral genes. We have recently discovered that lymphocyte cell surface levels of TOV3, a novel viral target, decrease substantially in the presence of Vpu. My initial work will therefore focus on understanding how Vpu downregulates TOV3
Stephanie Polderdijk
Structure and function of protein C inhibitor
Protein C inhibitor (PCI) is a member of the serpin superfamily of protease inhibitors. It was first identified as the physiological inhibitor of activated protein C. However, it has been shown that PCI can inhibit a wide range of proteases both inside and outside of coagulation. Its targets include thrombin, factor (f) Xa, the fVIIa:tissue factor complex and fXIa. Its function is regulated by the binding of cofactors such as heparin and thrombomodulin. In this project, we will use structural and biochemical studies to study how function and specificity of PCI are determined by PCI itself and by cofactor binding.
2009 Intake
Karen Angus
Supervisor: Gillian Griffiths
An assessment of the centrosome in cytotoxic T lymphocyte function
Cytotoxic T lymphocytes (CTL) recognise virally infected or tumour target cells. CTL then remove these target cells from our bodies via polarised secretion of cytotoxic mediators towards the target cells, thus inducing cell death. The centrosome of the CTL is very important for this process. It moves to and docks at the immunological synapse which is formed at the point of cell:cell contact between the CTL and target. In this way the centrosome directs the polarisation and subsequent secretion of the cytotoxic mediators towards the target. It is unknown, however, how the centrosome actually focuses towards the immunological synapse or how it physically docks at the CTL membrane.
This research project will use live cell imaging to track the centrosome through the CTL upon target recognition. In addition, I will investigate the proteins involved in centrosome docking at the immunological synapse. The lab has noted that a similar process of centrosome docking at the plasma membrane occurs in other systems, such as cilia formation. Thus, we have identified candidate centriole proteins that will be depleted from CTL allowing us to then assess whether the centrosome can still dock upon target cell recognition and whether the CTL are still capable of fulfilling their function.
Simon Gilbert
Supervisor: Ken Smith
An investigation into the secretory machinery of Plasma Cells
Nicola Hodson
Supervisor: Scottie Robinson
Characterisation of novel machinery involved in clathrin-mediated endocytosis
Clathrin-coated vesicles (CCVs) are a major class of transport vesicle which act to selectively transport proteins between membrane compartments within the cell. They are responsible for receptor-mediated endocytosis at the plasma membrane and the sorting of proteins at the TGN and endosomes. The aim of my PhD is to identify novel components of the clathrin-mediated endocytic pathway through the validation and characterisation of hits which have emerged from a recent genome-wide siRNA screen carried out by the Robinson Lab here at the CIMR. Top hits from this screen included well-characterised machinery of clathrin-mediated endocytosis, but approximately one third of top hits were unknown proteins. The first stage of this project will involve validation of the top hits to eliminate false positives and several high-throughput assays will be carried out in order to group the validated hits according to phenotype. Finally the hits will be narrowed down to a manageable number which will be investigated in greater detail. Their exact contribution to clathrin-mediated endocytosis will be investigated using a range of techniques including comparative proteomics of clathrin-coated vesicles and plasma membranes, immunofluorescence, live cell imaging and electron microscopy.
Richard Timms
Supervisor: Paul Lehner
Developing a genetic screen in haploid human cells to identify novel components involved in the presentation of major histocompatibilty complex class I molecules
Eszter Zavodszky
Supervisor: David Rubinsztein
Identification and characterization of novel modulators of autophagy
A common feature of many neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, is the aggregation of a mutant toxic protein. Previous work has shown that induction of autophagy, a bulk cellular degradation pathway, ameliorates disease phenotypes in cell and animal models of neurodegenerative disease. Nevertheless, current autophagy-modulating agents are still imperfect, often resulting in undesirable side-effects such as immunosuppression. Additionally, the growing body of research on the topic reveals autophagy to be an increasingly complex pathway, which is regulated by a host of processes, proteins, and molecules. Through my PhD research, I hope to gain a better understanding of mammalian autophagy and thus contribute to the development more effective treatments for neurodegeneration.
Previously, colleagues in the Rubinsztein laboratory performed a Drosophila melanogaster screen to search for modifiers of expanded huntingtin toxicity (the toxic species in Huntington's disease). Among the hits in this screen, we have selected genes with human orthologues to test their effects on autophagy in a mammalian cell system. I am using a variety of assays to monitor autophagy and investigate the mechanism of action of particular genes of interest.
2008 Intake
Maja Choma
Supervisor: Margaret Robinson
In search of cell machinery contributing to the activity of HIV-1 Nef
Nef is a multifunctional protein encoded by the HIV that is crucial for successful infection of host cells and progression into fully developed Acquired Immuno-Deficiency Syndrome. One of its functions is the ability to down-regulate Major Histocompatibility Complex class I (MHC I) molecules away from the plasma membrane, which allows the infected cells to carry on undetected by the immune system of the host and thus is an important part of the immunoevasion mechanisms of the virus. Although this function has been known for many years the precise mechanism and the host proteins used by Nef remain unclear.
In my project I am working to uncover the cell machinery that contributes to Nef's ability to down-regulate MHC I and to do this I will draw on the strengths of the Robinson lab. A major part of my project is a whole genome screen with a library of short interfering RNA (siRNA). I hope to find a number of proteins involved in Nef mediated down-regulation of MHC I. To achieve this I am employing a wide range of cell biology tools and assays, including fluorescence microscopy, FACS sorting assays and comparative proteomics profiling.
Maria Mascarenhas
Supervisor: Katrin Ottersbach
Haematopoietic Stem Cell Emergence
Haematopoieis is the process by which haematopoietic stem cells proliferate and/or differentiate to give rise to all mature blood cells in the organism. During embryonic development, this process occurs in different foetal haematopoietic organs, including the aorta, gonads and mesonephros region (AGM), the site of origin of haematopoietic stem cells (HSCs). Like all other cellular processes, haematopoieis is regulated by a number of factors and signalling pathways. Gene expression analyses of AGMs at the time of HSC emergence have identified several genes, the expression of which was upregulated at this stage which implies their relevance in this process. The main objective of this project is to investigate the role of some of the upregulated genes and their regulators in the regulation of AGM haematopoiesis. Specifically, their effect on the self-renewal capabilities of the stem cell progenitors of the AGM and their influence on the commitment of stem cells to a particular blood cell will be investigated.
Aoife Murray
Supervisor: Lucy Raymond
Investigation and Functional Analysis of Unique Sequence Variants in X-linked Mental Retardation
Intellecutal Disability (ID) affects 1-3% of the population, with many more affected males than females. This has led to much research being focused on the X-chromosome and many monogenic X-linked ID (XLID) conditions have been described. However, XLID is extremely heterogeneous and the underlying genetic cause of disease remains unknown in many of these patients.
The Genetics of Learning Disability (GOLD) study was established in an effort to identify genes on the X-chromosome that may be involved in intellectual disability. A cohort of families with undiagnosed XLID were recruited and the coding region of the X-chromosome was sequenced in over 200 patients. A number of unique genetic variants including truncating and splicing variants, insertions, deletions and missense variants were identified in the cohort, giving rise to a large number of candidate genes.
Determination of pathogenicity is realtively straightforward for truncations, splice variants and indels which usually have a significant impact on protein structure and function. However, distinguishing between pathogenic missense variants and rare polymorphisms is more difficult and requires functional analysis the variant protein.
My PhD project will focus on the functional analysis of some of the 312 unique of missense variants identified in an effort to identify new genes and mutations leading to XLID. In order to select the most promising candidate genes to study we first made use of algorithms that aim to predict the effect of an amino acid substitution on protein structure and function. In addition to this linkage analysis and sequencing were carried out to confirm co-segregation of the variant with disease in each family. This work allowed us to rule out 70% of the missense variants as the cause of disease, as they did not co-segregate with disease or fell outside the defined linkage regions. From the remaining variants, a subset will be chosen for functional analysis after more detailed literature and database searches into the known functions, expression and localisation of candidate genes.
Jennifer Warren
Supervisor: Paul Luzio
The late acting function of Vps20
The multi-vesicular body (MVB) trafficking pathway is important for the downregulation of cell surface receptors and proteins from the plasma membrane. In this pathway there are a number of endosomal sorting complex required for transport (ESCRT) proteins, which function in the sorting of cargo. One of these proteins, vacuolar protein sorting (Vps) 20 has been shown to be crucial for sorting in yeast. However, in HeLa cells Vps20 is not required for downregulation of virally ubiquitinated MHC class I, in contrast to other ESCRT proteins which are required. Nevertheless when Vps20 is depleted there is clustering of MVBs, swelling of lysosomes and extensive tethering between MVBs and lysosomes. This suggests that Vps20 has a role in the fusion of MVBs with lysosomes.
This research project aims to investigate the late acting function of Vps20 in delivery of MVBs to lysosomes. To do this I will be using a recently developed rapid depletion technique to study the development of morphological changes in mammalian cells upon depletion of Vps20. I will also try to identify the components which make up the tethers that are seen between MVBs and lysosomes.