Micro-RNAs (miRNAs) are a class of non-coding RNA which post-transcriptionally regulate gene expression. Epstein-Barr Virus (EBV) transforms resting B-cells in vitro to establish continuously proliferating lymphoblastoid cell lines (LCLs) and is aetiologically linked to lymphomas. Little is known about the contribution of miRNAs to the transformation of B cells. We initially examined the regulation of the oncogenic miR-155, which is highly expressed in Hodgkin’s lymphoma but was reportedly absent in Burkitt’s lymphoma. We found that miR-155 was up-regulated by EBV-LMP1 expression, and that a reported defect of miR-155 processing in Burkitt’s lymphoma was a misinterpretation of data. Next, to identify cellular miRNAs and genes modulated during EBV-induced transformation, we compared the expression profiles of resting B cells and B cells either infected with EBV or stimulated to proliferate with CD40L and IL4. This revealed that a large proportion of miRNAs and genes differentially regulated by EBV and not by CD40L/IL4 were modulated by EBV interaction with its CD21 receptor complex, but these changes were maintained or amplified in LCLs; and included a set of tumour suppressor genes down-regulated by EBV. In addition, bioinformatics analysis indicated that EBV modulates the expression of multiple miRNAs predicted to target the same cellular genes.

During lytic cycle replication EBV expresses at least three genes; BNLF2a, BILF1 and BGLF5, which individually act to inhibit efficient processing and presentation of CD8+ T cell epitopes. This thesis sets out to assess the relative contribution of these potential immune-modulating proteins to the evasion from CD8+ T cells at different stages of EBV lytic cycle. Lentiviral vectors for shRNAs were used to silence expression of these individual viral genes in EBV-transformed B-cells, which were then probed with CD8+ T cell effector clones of specificities for epitopes derived from the three phases of the EBV lytic cycle; allowing us to determine the contribution each immune evasion gene makes towards the inhibition of antigen presentation during lytic cycle. Cells replicating viruses lacking BNLF2a were more efficiently recognised by CD8+ T cells specific for immediate early and early expressed antigens relative to those lacking BGLF5 and BILF1. Conversely, cells lacking the expression of BILF1 were better recognised by CD8+ T cells specific for early and late lytic antigens. These data suggest that whilst the role BNLF2a plays in interfering with antigen presentation diminishes as lytic cycle progresses (IE>E>>L), BILF1 plays a more active role with the progression of lytic cycle (IE<E<<L).

Intracellular signalling cascades are mediated by a plethora of receptors, enzymes, adaptors and small molecules. The Protein Tyrosine Phosphatase (PTP) Shp2 is a highly conserved enzyme involved in a myriad of cellular processes including growth, differentiation and apoptosis. Shp2 is multi-domain protein composed of two SH2 domains in tandem, a PTP catalytic domain and a C-terminal tail containing multiple phosphorylation sites and a proline-rich region. The majority of biophysical research has utilised X-ray crystallography to study interactions and effects of mutations at the structural level. To gain a further understanding of Shp2 ligand binding and perturbations caused by disease relevant mutations, a structural investigation was performed with Nuclear Magnetic Resonance (NMR) spectroscopy and Small-angle X-ray Scattering (SAXS) in solution. The NMR signals from the backbone of both SH2 domains were assigned and residue-level interactions and differential SH2 domain specificities with peptides from the novel receptor G6b-B were delineated. In addition, the E76K point mutation that causes Noonan Syndrome and leukaemia was found to have increased conformational dynamics, the first experimental evidence of this phenomenon at the structural level.

Beckwith Wiedemann syndrome (BWS) and Birt-Hogg-Dube syndrome (BHD) are two examples of genetic conditions that are associated with an increased risk of renal neoplasia (Wilms tumour and renal cell carcinoma (RCC) respectively). BWS is a model imprinting disorder characterised by overgrowth, developmental defects, predisposition to embryonal tumours and results from disordered expression of imprinted genes on chromosome 11p15.5. There is an association between the use of assisted reproductive technologies (ART) and BWS. BHD is an autosomal dominantly inherited condition characterised by cutaneous fibrofolliculomas, lung cysts predisposing to spontaneous pneumothorax and an increased lifetime risk of RCC and is caused by germline mutations in the FLCN gene. Both BWS and BHD show phenotypic variation in their manifestation. The clinical and molecular genetic investigations described in this thesis aimed to uncover factors influencing variation in phenotypic expression in these two conditions. Conclusions: Phenotypic variation in BWS can result from locus heterogeneity, epigenetic and environmental modifiers. Phenotypic variation in BHD may reflect allelic heterogeneity and the presence of genetic modifiers.

This thesis applies integrative and systemic approaches to gene expression experiments measuring responses to environmental stress. Methods were developed to identify systematic differences in response strength, functional pathway activation, and gene regulatory network structure. Results in three wild killifish populations revealed high population variability at the level of individual genes, consistent with the killifish’s genetic diversity and ability to adapt rapidly to anthropogenic pollution. Despite gene level diversity, modular network structures, patterns of pathway activation, and patterns of gene expression canalization were conserved in the three populations, demonstrating that gene regulatory networks are preserved by selective processes and may constrain killifish adaptation. The presence of arsenic during killifish acclimation to osmotic shock systematically reduced the magnitude of gene expression responses, and reduced coordination between genes that respond to osmotic shock. Results in the water flea suggested that cadmium tolerance is associated with systematically larger gene expression responses to cadmium stress, and greater network coordination among genes that respond to cadmium. In summary, environmentally responsive gene regulatory networks 1) shape the efficacy of biotic and abiotic stress responses, 2) are targeted by toxic effects, and 3) are shaped by selective forces.

Signal transduction pathways are mainly depending on phosphorylation events, which are controlled by the activity of phosphatases and kinases. Although kinases have been widely studied, however, much less is known about the contribution of phosphatases to the regulation of cell signalling pathways. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is involved the regulation of a number of receptor tyrosine kinases (RTKs) including platelet-derived growth factor receptor (PDGFβR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. The differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP) was analysed. A significant change in abundance of phosphorylation on 270 phosphorylation sites from 205 proteins was associated with the lack of LAR phosphatase activity. Gene ontology analysis revealed an enrichment of LAR-mediated phosphorylation events on proteins involved in many signalling transduction pathways including those regulating the actin cytoskeleton, cell adhesion, endocytosis and cell metabolism. Analysis of putative kinases upstream of LAR-dependent phosphorylation events revealed a role for LAR in regulating signalling through mTOR and JNK. In summary, this thesis identifies an important role for LAR phosphatase in the regulation of signal transduction, cell adhesion and cell metabolism.

Previous studies of molecular prognostic markers following resection for exocrine pancreatic cancer have produced conflicting results. The aim of this study was to undertake a comprehensive analysis of potentially useful markers in a large multicentre patient population and compare these markers with standard pathological prognostic variables. Formalin fixed, paraffin-embedded specimens of pancreatic ductal adenocarcinoma were analysed from 157 patients (100 men and 57 women with a median [range] age of 60 [33-77] years) who had undergone pancreatectomy. Immunhistochemistry was used to detect expression of p16\(^{INK4}\), p53, p21\(^{WAF1}\), cyclin D1, c-erbB-2 and c-erbB-3. In a selected number of p53 positive and negative staining cases, mutational analysis was undertaken using DNA obtained from microdissected specimens. Mutations in codons 12 and 13 of the K-ras oncogene were detected by SSCP and sequencing following DNA extraction and amplification by PCR. The median [range] survival post-resection was 12.5 [3-83] months. Abnormalities of p16\(^{INK4}\), p53, p21\(^{WAF1}\), cyclin D1, c-erbB-2 and c-erbB-3 expression were found in 87%, 41%, 75%, 72%, 33% and 57% of cases, respectively. There was no significant correlation between expression of any of these markers and patient survival. K-ras mutations were found in 73 (75%) out of 97% cases with amplifiable DNA. The presence of K-ras mutation alone did not correlate with survival, but there were significant differences in survival according to the type of K-ras mutation (p=0.0007). Reduced survival was found in patients with GaT, cGT and GcT K-ras mutations compared to GtT, aGT and GaC mutations. In conclusion survival was associated with the type of K-ras mutation but not the expression of p16\(^{INK4}\), p53, p21\(^{WAF1}\), cyclinD1, c-erbB-2 and c-erbB3.

Renal disease is a major global public health issue that affects 10% of the general population with premature morbidity and mortality related to cardiovascular disease and infection. Interstitial fibrosis is a common hallmark of progressive kidney dysfunction. There remains a stubborn discrepancy in identifying which patients suffer adverse events because of their disease or resulting treatment. Investigation in patient genome variation may explain this discrepancy. Caveolin-1 is the essential structural protein for caveolae that are ubiquitously distributed in fibroblasts, endothelial and epithelial cells. They act as molecular chaperones for transcellular signaling such as degradation of the activated TGFβ-1 receptor. In this thesis, caveolin-1 single nucleotide polymorphism rs4730751 CC genotype is shown to be associated with a better outcome in renal patients for arterial stiffness, and reduced mortality from cardiovascular disease, infection, malignancy in ANCA associated vasculitis. By inducing renal models of fibrosis in caveolin-1 knockout mice, deletion of caveolin-1 leads to increased fibrosis. In conclusion, this polymorphism could be used as a marker of disease risk either in isolation or as part of a clinical risk score to counsel patients on the likely prognosis of their condition. Manipulation of caveolin-1 expression may be a therapeutic strategy in reducing renal fibrosis.

The major and minor nitroreductase enzymes of Escherichia coli, NfsA and NfsB, have both been identified as potential prodrug activating enzymes in Gene-Directed Enzyme Prodrug Therapy (GDEPT) of cancer with 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954). NfsB mutants have been selected for their ability to sensitize cells to successively lower concentrations of CB1954. In this study the crystal structures of most improved double (T41L/N71S) and triple (T41Q/N71S/F124T) mutants have been determined using X-ray crystallography. The structures help explain the improvements seen with the mutants, in particular, the structure of the triple mutant reveals a hydrogen bond between Gln41 and Thr124, explaining why this mutation has greater activity than expected from the single mutants. Crystal structures of NfsB mutants where Phe124 is replaced with either of the unnatural amino acids para-nitrophenylalanine or para-aminophenylalanine show a shift in the side chain rotamer allowing these polar groups to interact with substrates, suggesting this leads to their increased activity. For the first time, the crystal structure of NfsA is presented bound to a series of ligands, including the inhibitors succinate and fumarate, and the substrate nitrofurantoin. Another structure bound to a second FMN shows how the flexible loop may move to form a phosphate binding pocket for NADPH.

Crop wild relatives are important for agriculture due to the genetic richness they possess. They have been used in plant breeding to develop high yielding varieties; varieties with improved resistance to biotic and abiotic stresses, and enhanced nutritional content. Securing their conservation in the long-term is critical to enable the continuous development of crops’ varieties able to respond to future challenges. The work presented in this thesis is a contribution to the effort of understanding the ex situ conservation gaps of crop wild relatives, their expected response to climate change and their needs for conservation. Methods used in this thesis include species distribution modelling, gap analyses, a case study assessing the preliminary IUCN Red List categories, species distribution projections onto future climate change scenarios, and an estimation of the global value of crop wild relatives based on their likelihood of being used in plant breeding, and the contributions of their associated crops to human diets and agricultural production systems. The methods used here can be applied to more crop genepools for global conservation planning, and can also be adapted for analysis at the regional and national level. The results presented here are being used to improve the conservation of the wild relatives of 29 crops.