Single nucleotide polymorphisms (SNPs) have great potential to serve as important biomarkers in the clinic to identify those at increased risk for developing cancer, progressing more rapidly, and not responding to therapies. However, the clinical application of cancer-associated SNPs has proven to be more complicated than expected. One of the necessary steps will certainly be the description of the molecular and cellular mechanisms behind the observed associations. The p53 tumour suppressor pathway harbours well-described SNPs that affect p53 signalling and cancer. The aim of the work presented in this thesis was to utilise this knowledge to more efficiently characterise cancer-associated SNPs. Firstly, cancer-associated SNPs in a p53 network gene, CD44, were studied. Specifically, based on CD44’s known roles in both p53-dependent and independent signalling, it was predicted that the cancer-associated SNPs could function as biomarkers for chronic lymphocytic leukaemia progression, and for the response to anti-EGFR therapy for colorectal cancer. Indeed, supportive data is presented. Next, a methodology is presented that aims to identify cancer-associated SNPs in functional p53 binding sites using genome-wide datasets. Interestingly, a SNP is identified that dramatically influences the ability of p53 to regulate transcription of the KITLG oncogene and that associates with one of the largest risks of cancer identified to date. Intriguingly, the SNP is also shown to have undergone positive selection throughout human evolution, signifying a selective advantage, but similar SNPs are demonstrated to be rare in the genome due to negative selection, indicating that polymorphisms in p53 binding sites have been primarily detrimental to humans. Lastly, and in order to begin to explore if other polymorphic transcription factor binding motifs could be found in cancer-associated SNPs, a methodology was designed to identify SNPs in E-box transcription factor binding motifs, as they are sensitive to single base pair changes and affect cancer. Taken together, the work presented in this thesis shows how the study of how SNPs associate with, and impact upon, cancer has great potential to improve both biological knowledge and clinical outcomes.

Varicella zoster virus (VZV) is an alpha herpes virus that causes primary infection with varicella (chicken pox), establishes latency in ganglia and may later reactivate as herpes zoster (shingles). Innate immune effectors are thought to control initial viral replication, but it is the adaptive immune system, involving T cells that mediates eventual control of viraemia and the associated clinical disease. Although both CD4⁺ and CD8⁺ T cells mediate viral clearance during acute illness, memory responses are dominated by CD4⁺ T cells. We tested the hypothesis that the paucity in memory CD8⁺ T cell effectors is partly attributed to immune evasion mechanisms that are mounted by VZV. We confirmed that VZV readily down regulates cell surface HLA-A and HLA-C but spares HLA-B onVZV infected keratinocytes and VZV infected Mewo cells. Analysis of intracellular HLA protein expression and gene transcription showed global down regulation of all HLA subtypes. Further analysis showed that VZV inhibits IFN-γ mediated up regulation of HLA expression and augments IFN-γ mediated up regulation of HLA-E and CD71 expression. Furthermore, we show that acute VZV infection lowers the frequency of circulating peripheral blood myeloid dendritic cells (mDC), reduces the expression of the DC activation marker HLA-DR and impairs inflammatory cytokine secretion in blood DC populations. Inhibition of DC cytokine secretion was found to be dependent on viral replication as irradiated virus resulted only in mild inhibition of IFN-α and TNF-α secretion. Lastly, we observed that VZV infection results in increased expression of host peptides, including MHC derived leader sequences that potentially bind to HLA-E. Cell surface HLA-E is known to be a ligand for the natural killer (NK) cell inhibitory receptor CD94/ NKG2A identifying a novel mechanism of viral immune escape from NK cell surveillance. In conclusion, our data reiterates the fact that VZV targets different aspects of antigen presentation to evade the immune system with implications for pathogenesis and approaches to improved vaccination and treatment.

NOD2 is an intracellular innate immune receptor expressed in dendritic cells and gastrointestinal epithelial cells. Polymorphisms in the NOD2 gene convey a strong predisposition to Crohn’s disease (CD), a form of inflammatory bowel disease. Understanding the function of NOD2, and in what way it is aberrant in the presence of NOD2 polymorphisms, would confer a valuable paradigm for understanding Crohn’s pathogenesis. CD is thought to arise both from defects in the gut mucosal barrier and from a dysregulated Th17 immune response to commensal gut flora. Aberrant expression of IL-23 is present in both human CD and in murine models of colitis. Wild-type NOD2 contributes to NFκB activation and pro-inflammatory cytokine production on recognition of its cognate ligand, a function that is lost in CD-associated mutations. How the predominantly loss-of-function CD-NOD2 contributes to the pro-inflammatory response present in Crohn’s is not yet understood. In this thesis a set of experiments is described that aim to shed light on the function of NOD2, firstly through identification of negative regulators of immune activation that are dependent on NOD2 for their expression. This work identifies the microRNAs that are expressed following NOD2 triggering in human dendritic cells. Specifically, up-regulation of the miR-29 family was found to be dependent on wild-type NOD2 function. A number of novel miR-29 targets and their functional consequences are presented, including the cytokine subunits IL-12p40 and IL-23p19, directly linking NOD2 polymorphisms and aberrant IL-23 expression. Secondly, a project aiming to identify components of the NOD2 signalling complex (or signalosome) is described. To this end I employed a model system that involved tagging NOD2, and stable expression in a human cell line. These clones were validated for expression and function before an immunoprecipitation protocol was optimised. Mass spectrometry analysis of these samples identified the known NOD2-interacting protein Erbin.

The chromosome constitution of human cleavage stage embryos has been extensively investi-gated using a variety of techniques, revealing high levels of aneuploidy and mosaicism. However, the final phase of preimplantation development, the blastocyst stage has received relatively little attention mostly because it is only recently that embryo culture has become sufficiently well optimised to reliabley generate blastocysts. One of the aims of this study was to examine blastocyst cytogenetics, characterising the extent and variety of aneuploidy and, where possible, determining the origin of the abnormalities detected. Both the frequency of aneuploidy and the incidence of mosaicism were significantly lower in the 52 embryos generated by 20 patients that had successfully undergone the first cellular differentiation, producing trophectoderm (TE) and inner cell mass (ICM). Valuable tools for the detailed chromosomal analysis of blastocysts, used in both research and clinical contexts, were comparative genomic hybridization (CGH) and array CGH (aCGH). However, validation of these methods, especially aCGH, was required in order to verify accuracy. A low error rate and a low misdiagnosis risk were demonstrated. The morphology of 1397 embryos at the cleavage and blastocyst stages from 229 patients was evaluated in relation to their chromosomal complement. The results obtained during this part of the project showed that, in general, there is little correlation between cleavage stage morphology and chromosome status. A weak link between morphology and aneuploidy, however, was found for embryos at the blastocyst stage. Chromosomally normal female embryos had a tendency to grow faster than male embryos at the cleavage stage and therefore tended to achieve superior morphological scores, whereas the trend was reversed at the blastocyst stage. Abnormal embryos carrying types of aneuploidy compatible with formation of a clinically recognised pregnancy had morphologies indistinguishable from those of euploid embryos. This study also aimed to utilise aCGH for the preimplantation genetic diagnosis (PGD) of imbal-ances due to structural chromosome rearrangements (e.g. translocations) in 39 carriers, a total of 139 embryos were assessed. The data obtained revealed that carriers of Robertsonian translocations are at increased risk of aneuploidy affecting additional chromosomes not involved the translocation, a phenomenon known as an interchromosomal effect (ICE). Finally, the clinical outcomes of 300 patients undergoing preimplantation genetic screening (PGS) using aCGH, for various different indications, were evaluated at both the cleavage (795 embryos) and blastocyst stages (1097 embryos). The pregnancy rate following cleavage stage biopsy was significantly lower than following blastocyst stage biopsy. The miscarriage rate was significantly reduced following PGS for patients with recurrent miscarriages. This work provided promising data supporting the clinical use of comprehensive chromosome analysis for the screening or diagnosis of preimplantation embryos and also yielded scientifically useful information concerning the frequency and nature of aneuploidy at the final stage of development before implantation.

CD161 γδ T-cells have been implicated in the pathogenesis of Multiple Sclerosis however their role in health and chronic liver disease requires further exploration. In health, the majority of γδ T-cells expressed CD161 – a C-type lectin, and predominantly expressed the Vδ2 chain. The CD161+ γδ T-cells demonstrated a Th1-like pattern, expressing IFN-γ, TNF-α and Granzymes/Perforin when compared to the CD161- subset. The CD161+ γδ T-cells also expressed CCR6 and IL-18R thus also displaying a Th17-like pattern. These cells were also found in the lamina propria in the gut and rapidly expanded in the 1^st few weeks of life in the periphery. On gene array analysis, there were 409 genes expressed on the CD161+ γδ T-cells when compared to their CD161-ve counterparts including those coding for β2 receptors, CCL20, Acetycholinesterase, CCR1 and IL-18R. A potential clinical correlation to cardiac diseases was found when the upregulated genes were analysed. When the CD161+ γδ and CD161+ αβ T-cell populations were compared via gene-array, an association with a risk variant for coeliac disease was found. Thus in health, CD161+ γδ T-cells are not only a distinct subset of T-cells (confirmed by a FACS approach and gene array methods), but also the expression of CD161 may be linked to common genetic signals downstream in cell processes and disease pathogenesis, irrespective of T-cell subset population. In chronic liver disease there was a significant reduction in the periphery of CD161+ γδ T-cells in patients with chronic Hepatitis C (HCV) and an increase in patients with Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis when compared with healthy individuals. The CD161+ γδ T-cells appeared to be of a different phenotype in HCV infection. There was no overall significant localisation into of CD161+ γδ T-cells patients with chronic liver disease or specifically in HCV infection. There was however a CD161+ γδ T-cell enrichment in the liver in patients with Non-Alcoholic Fatty Liver disease. The CD161+ γδ T-cells were also found in Hepatocellular Carcinoma tissue. Overall it appears the CD161+ γδ T-cells are indeed a unique subset, playing a distinct role in health, as part of an early innate response, but also potentially involved in disease pathogenesis.

Neurological diseases are devastating illnesses that affect over one billion people worldwide. Drosophila melanogaster provides a genetically tractable system in which to study gene function and the mechanisms of pathogenesis of neurological diseases. In this study I have investigated the function of survival motor neuron (SMN), the causative gene in the neuromuscular disease spinal muscular atrophy (SMA), in growth and differentiation in Drosophila. In addition, I have used the fruit fly to investigate a putative modifier of a previously characterised Drosophila model of Parkinson's disease. Spinal muscular atrophy is an autosomal recessive neurological disease that is characterised by motor neuron loss resulting in muscle weakness. The disease is caused by the deletion or mutation of the survival motor neuron (SMN) gene. In Drosophila, SMN was found to be highly expressed in dividing tissues and a reduction in SMN levels resulted in growth defects, stem cell defects and developmental delay. SMN was also shown to regulate chromosome morphology of the endocycling nurse cells of the female germline. Therefore it appears that SMN has a role in growth control and development in Drosophila. Parkinson's disease is a common disorder that results in widespread neurodegeneration with a predilection for dopaminergic neuron loss resulting in movement defects. A defining neuropathological feature of the disease is the presence of alpha-synuclein containing inclusions. Using a Drosophila model of PD, I have shown that specific alpha-synuclein-induced phenotypes in the fly can be suppressed by the overexpression of the E3 ubiquitin ligase, Nedd4.

Intracellular hyperthermic therapy may prove to be a unique and novel approach to the management of pancreatic cancer. Utilizing the principle of photothermal destruction, selective killing of cancer cells with minimal injury to normal tissues may be possible. This dissertation investigated the role of antibody targeted metal nanoparticles and the cytotoxic effects of nonionizing radiofrequency fields in pancreatic cancer. Cancer cell death was induced by heat release from intracellular metal nanoparticles after radiofrequency field exposure. Fluorescent and gold nanoparticles were delivered with two antibodies, cetuximab and PAM-4, to pancreatic cancer cells in vitro and mouse xenografts in vivo. Selective delivery of these nanoparticles induced cell death in vitro and decreased tumor burden in vivo after whole animal RF field exposure. This occurred through both apoptosis and necrosis. In addition, activated caspase-3 was increased after antibody treatment and RF field exposure. Furthermore, although there was non-specific uptake by the liver and spleen in vivo, there was no evidence of acute or chronic toxicity in the animals. These results are in agreement with the principle that malignant cells are more thermally sensitive than normal cells or tissues. Selective intracellular delivery of metal nanoparticles coupled with whole body RF field exposure may be a beneficial therapy against micrometastases and unresectable pancreatic cancer in the future. Further studies are planned with more specific antibodies, other nanoparticles, and other cancer targets.

In this thesis, I examined the expression of CD1d on respiratory epithelial cells (REC) in human and explored its potential role in mucosal immunity in the lungs. Hitherto, there have been no published reports of CD1d expression on REC though it has been observed on other epithelial surface (notably intestinal epithelial cells). This observation, and work in my supervisor’s laboratory demonstrating CD1d-restricted natural killer T cells (iNKT) cells as early players in the lungs of influenza A virus (IAV)–infected mice prompted my interest in this area. I hypothesized that CD1d is expressed on REC and that it contributes to activation of iNKT cells in the lungs via presentation of endogenous or pathogenic glycolipids. I asked following questions – i) is CD1d expressed on REC ii) can this expression be regulated and iii) does CD1d expression on REC have a function. This thesis provides the first evidence for CD1d expression on human RECs (in cell lines and primary RECs) and also presence of alternatively spliced variants. CD1d expression was inducible by viral-associated signals in vitro and despite being non-professional antigen presenting cells, RECs can present glycolipid (α–GC) to, and activate iNKT cells in a CD1d-dependent process resulting in production of both Th1 and Th2 cytokines. Using whole genome expression profiling, I then showed that iNKT cells expressed a distinct profile of genes while in direct contact with α–GC-bound CD1d on RECs compared to cells separated by transwell membrane. Here early biological pathways were dominated by cytokine and chemokine related genes (JAK-STAT signaling pathways, cytokine-responsive elements and cytokine/chemokine genes) and apoptosis-related genes. This suggested that glycolipid-bound CD1d on REC was capable of inducing a programme of immune activation in iNKT cells. I concluded my work by examining if CD1d expression on RECs influenced its active role in immunity. Using wild type and CD1d-deficient transgenic mice challenged with IAV, I showed that CD1d expression is induced on REC in vivo after viral challenge, and in the absence of CD1d, mice showed worse outcome. RECs isolated from CD1d-deficient mice had a much stronger gene expression profile for pro-inflammatory genes. This suggested that CD1d expression on REC could have a bi-directional effect – on the RECs that expressed CD1d (preventing excessive immune-related genes activation) and on the iNKT cells that it engaged (activation, with pro-immunity effects). The thesis concludes with discussion of the potential implications of these findings and future work to examine hypotheses generated from this work.

The development of an efficacious P. falciparum malaria vaccine remains a top priority. Pre-erythrocytic vaccine efforts have traditionally focussed on two well- known antigens, CSP and TRAP, yet thousands of antigens are expressed throughout the liver-stage. The work described in this thesis aimed to assess the ability of other pre-erythrocytic antigens to induce an immune response and provide protective efficacy against transgenic parasites in a mouse model. Research undertaken in our laboratory has demonstrated the ability of prime-boost viral vectored sub-unit vaccination regimens to elicit high levels of antigen-specific T cells. Eight candidate antigens were therefore expressed individually in the viral vectors ChAd63 and MVA. Two antigens, PfLSA1 and PfLSAP2, were identified that confer greater protective efficacy in inbred mice than either CSP or TRAP. PfLSA1 was also able to induce almost complete sterile efficacy in outbred mice, suggesting this vaccine should be assessed in a clinical trial. Immune responses to the candidate antigens were also assessed in human volunteers following their first exposure to controlled malaria infection. The antigen TRAP was further characterised by epitope mapping in volunteers vaccinated with ChAd63-MVA ME-TRAP. However, no functional T cell assay exists to measure inhibition of P. falciparum liver-stage parasites. An improved murine in vitro T cell killing assay was developed, and preliminary experiments were conducted that demonstrate the potential and promise of a P. falciparum T cell killing assay. Such assays will not only allow mechanistic studies to be undertaken, but could also change the way we screen pre-clinical liver-stage vaccines.

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