Nuclear magnetic resonance spectroscopy (NMR) resonances from lipids and metabolites in tumours are associated with tumour grade and treatment response. The origin of NMR lipid signal is mainly considered to be cytoplasmic lipid droplets (LDs). The aim of this study is to investigate the lipid species of LDs in nervous system tumour cells and identify potential lipidic or metabolic markers in treatment response. NMR spectroscopic analysis revealed that the LDs contain phosphatidylcholine, cholesterol and cholesterol ester with saturated, mono-unsaturated and polyunsaturated fatty acid species. Both saturated and unsaturated lipids are accumulated into LDs in cancer cell death. It is shown that Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and Uridine diphosphate N-acetylglucosamine galactosamine (UDP-GalNAc), the main donors of glycosylation, in parallel with 1H NMR detected lipids, increased in apoptotic cancer cells.LDs in nervous system cancer cell lines contain specific lipid species. To the best of our knowledge, it is the first study mechanistically links UDP-GlcNAc and UDP-GalNAc to cancer cell death.

Iron is ubiquitous in the human body, fulfilling many crucial roles. However, accumulating evidence implicates excess iron as a carcinogen. This study aimed to further investigate the role of iron and haem in breast carcinogenesis and the potential utility of chelators in therapy. We demonstrate that the transport machinery for iron and haem is dysregulated in breast cancer, with import being promoted and export down-regulated to allow the accumulation of intra-cellular iron. In vitro studies demonstrate that, in contrast to benign cells, malignant cells are capable of importing haem as well as ionic iron. Both iron and haem stimulate aggressive behaviour in malignant cells, up-regulating viability, proliferation, adhesion, migration and invasion. These changes are abrogated by iron chelation. In addition, the expression profile of iron and haem transporters is shown to favour intra-cellular accumulation even when iron is plentiful and import would be expected to be down-graded. Overall this study suggests that breast cancer may be due to an inappropriate expression profile of iron and haem transporters, leading to excess intra-cellular iron which drives a malignant cell phenotype. In addition the action of chelators to downgrade malignant behaviour implies a potential therapeutic role.

Although knowledge of metabolic adaptations in cancer has increased dramatically, little is known about the spontaneous adoptive adaptations of cancer cells to changing conditions in the body. This is particularly important for chronic lymphocytic leukaemia (CLL) cells which continually circulate between different microenvironments in the blood, bone marrow and lymph nodes. To study such metabolic adaptations, a nuclear magnetic resonance (NMR) based approach; capable of monitoring real-time metabolism in primary CLL cells was developed. Using this setup, this thesis demonstrates fast, reversible metabolic plasticity in CLL cells during transition from normoxic to hypoxic conditions, associated with elevated HIF-1α dependent glycolysis. This work also demonstrates differential utilisation of pyruvate in oxygenated and hypoxic conditions where in the latter, pyruvate was actively transported into CLL cells to protect against oxidative stress. Moreover, real-time NMR experiments provided initial evidence that CLL metabolism in hypoxia correlates with stage of disease, adding significant relevance of our method for patient stratification. Additionally, to further investigate alterations between normoxic and hypoxic metabolism, Metabolic Flux Analysis (MFA) was carried out using primary CLL cell extracts, revealing modifications in pyruvate carboxylase (PC) activity and the pentose phosphate pathway (PPP). Despite the recent advent of promising new agents, CLL currently remains incurable and new therapeutic approaches are required. Understanding CLL cell adaptation to changing oxygen availability will permit the development of therapies that interfere with disease aetiology. This study makes several significant contributions towards this goal. Moreover, the findings may be relevant to all migratory cancer cells, and may have importance for the development of strategies to prevent cancer metastasis.

As a result of improvement in survival after childhood cancer, there are now increasing numbers of long-term survivors of childhood cancer living in the United Kingdom and across Europe. Specific groups of these childhood cancer survivors experience substantial excess risks of adverse health and social outcomes. Using the population-based British Childhood Cancer Survivor Study (BCCSS) the following areas were investigated: (I) The proportion of survivors on regular long-term hospital follow-up using risk stratification levels of care developed by the BCCSS in partnership with the National Cancer Survivorship Initiative. (2) The risks of adverse health and social outcomes using record-linkage and a self-reported questionnaire to assess which survivors of central nervous system tumours were at excess risk compared to the general population. (3) The risk of hospitalisation due to cerebrovascular conditions among childhood cancer survivors by electronic record linkage with Hospital Episode Statistics. Using the European PanCareSurFup cohort, the excess risks of genitourinary subsequent primary neoplasms were investigated among five-year survivors of childhood cancer. This thesis quantifies the risks experienced by childhood cancer survivors in four areas and provides an evidence-base for risk stratification by healthcare professionals caring for survivors.

The dual specificity Cdc25 phosphatases regulate mitosis and are expressed in eukaryotes. Cdc25 phosphatases have an active site motif, HCX5R, in common with other phosphatases. However, unlike the classical tyrosine phosphatases, they can dephosphorylate phospho-threonine in addition to phospho-tyrosine and have a much shallower active site. Increased expression of Cdc25 is correlated with poor prognosis in a range of cancers. In particular, increased expression of Cdc25C has been associated with prostate cancer making this protein an attractive target for drug discovery. However, drug discovery for these proteins has been hampered due to the shallow nature of the active site, difficulty in identifying specific inhibitors and toxicity. The thesis aim was to structurally and biochemically characterize Cdc25C using a range of techniques which include NMR, SAXS and X-ray crystallography in order to aid future drug design. The Characterisation of the Cdc25C full length revealed the regulatory domain to be intrinsically disordered and flexible. Construct and solution conditions were optimised to improve the solubility of the Cdc25C catalytic domain. Although, the \(^1\)H, \(^1\)\(^5\)N HSQC was well dispersed backbone assignments proved to be refractory. From a panel of inhibitors tested a few were shown to bind via WaterLOGSY and \(^1\)H-\(^1\)\(^5\)N HSQC.

Ataxia-telangiectasia (A-T) is a rare neurodegenerative disorder caused by mutations in the ATM gene which has a central role in the cellular response to DNA double strand breaks, cell cycle checkpoint control and initiation of the intrinsic pathway of apoptosis. Ataxiatelangiectasia is classified as an immunodeficient disorder with patients commonly showing lymphopenia and abnormalities in immunoglobulin production. They also have a high incidence of leukaemia and lymphoma at young ages. I used multicolour flow cytometry and immunological assays to characterise lymphocyte subsets in a group of 18 A-T patients and analysed the sensitivity of A-T lymphoblastoid cell lines to CD95-mediated apoptosis. I also investigated the potential role for ATM in immune surveillance via DNA damage-induced upregulation of NKG2D ligands. My results confirm a deficiency in naive T and B cells as well as high expression of the death receptor CD95 on all lymphocyte subsets excluding NK cells which together may explain the lymphopaenia in A-T. Analysis of the sensitivity of A-T LCLs to CD95-mediated apoptosis showed increased sensitivity of these cells to apoptosis but there was no evidence for a role of ATM in regulating either CD95 or cFLIP expression. Consistent with this was the increased sensitivity to CD95-mediated apoptosis of T cell prolymphocytic leukaemia (T-PLL) cells. The cause of the tumour is primary loss of ATM activity (either germline loss similar to A-T LCLs or somatic loss) allowing chromosome translocations with malignant potential, as a result of a defect in immune system gene rearrangements. An immediate consequence for A-T patients is an immunodeficiency that is not progressive, but may be described as ‘congenitally aged’. Immunodeficiency per se is not the cause of cancer in A-T but both immunodeficiency and cancer are consequences of the same basic ATM defect affecting the lymphoid system. There was no evidence of a role for ATM in NKG2D ligand upregulation following DNA damage.

In addition to its essential roles in the maintenance, replication and transcription of the EBV genome, EBNA1 has more recently been shown to influence the transcription of cellular genes and to modulate the activity of key cellular signalling pathways. EBNA1 has previously been shown to abrogate TGFβ signalling in carcinoma cell lines, although the exact mechanism remains to be elucidated. This study has endeavoured to further dissect this observation, whilst revealing a novel function for EBNA1 in the activation of the closely-related BMP signalling pathway. The observed abrogation of canonical TGFβ signalling is now proposed to be the result of an EBNA1-mediated induction of negative regulators, while a concomitant increase in secreted TGFβ, in combination with a shift towards linker region phosphorylation of the R-Smad proteins in EBNA1-expressing cells conceivably constitutes a method of promoting pro-tumourigenic TGFβ-mediated effects, such as cellular migration. In addition, results demonstrating the activation of BMP signalling upon the expression of EBNA1, and in EBV-positive cell lines, are also indicative of a role in the promotion of migration, and potentially metastasis. In this way, EBNA1 may mediate effects that contribute to the development of EBV-associated tumours.

The expression of cancer testis antigens (CTAgs) is normally restricted to spermatogenic cells of the testis but is also present in many cancers including testicular germ cell tumours (TGCTs). CTAg-specific T cell responses have been identified in patients with other solid tumours, and here we identified CTAg-specific T cells in TGCT patients. MAGEA-family specific T cell responses were detected in 21/49 patients with a magnitude of up to 0.149% of total peripheral blood mononuclear cells. Responses to multiple MAGEA antigens were frequently detected in seminoma patients irrespective of tumour stage. Conversely, NSGCTT patients only developed responses towards MAGEA3, which were strongly associated with disease progression. Longitudinal analysis revealed that the magnitude of MAGE-specific immune responses diminished over time by up to 95%, which correlated with the removal of tumour antigens. MAGE-specific CD8 T cells demonstrated cytotoxic potential against endogenously presented antigen. Tumour infiltrating T cells were antigen experienced, recently activated, oligoclonal populations; many of which expressed the inhibitory molecules, TIM-3 and PD-1. Proliferation and cytokine secretion was suppressed in vivo but was rescued with in vitro stimulation, indicative of an exhausted T cell phenotype. Our findings have significant implications in the development of novel CTAg-specific immunotherapy in patients with cancer.

This thesis investigated and examined the frequency and associations of post endoscopy gastro-intestinal cancers. Oesophagogastroduodenoscopy (OGD) and colonoscopy are the investigation of choice for diagnosing upper gastrointestinal cancers (UGIC) and colorectal cancers (CRC) respectively. Evidence suggests there are a proportion of patients who have had negative OGD or colonoscopy examinations prior to their later UGIC or CRC diagnosis. These events are commonly termed post-OGD upper gastrointestinal cancers (POUGIC) and post colonoscopy colorectal cancers (PCCRC). This body of work utilised a wide range of health data, each with its unique advantages and disadvantages. A primary care data base was used to investigate the frequency of POUGIC and its associations in the UK in a case-control study. The incidence of POUGIC and PCCRC in England was investigated with risk factors identified, provider and temporal variations examined using national hospital administration data. The incidence of POUGIC in the Midlands and its association with patient, endoscopy and endoscopist factors was investigated using regional secondary care endoscopic records with cancer registry data linkage. The findings in this body of work could be used to guide further research into reducing the incidence of POUGIC and PCCRC with the aim for earlier cancer diagnosis.

Ad5 and Ad12 inhibit ATR-dependent Chk1 phosphorylation. Ad5 E4orf3 promotes the relocalization of the MRN complex in order to inhibit Chk1 activation during infection, whereas Ad12 E4orf3 is unable to inactivate MRN by this method. Here we show that Ad12 inhibits Chk1 phosphorylation by targeting TopBP1, Timeless and Tipin for degradation. We have determined that Ad12 E4orf6 associates with the cellular Cul2-containing ubiquitin ligase to promote TopBP1 degradation. We have shown that Ad5 and Ad12 differentially activate Cullin-containing ubiquitin ligase complexes during infection. Furthermore, we have also determined that Ad12 E4orf3 promotes the degradation of Timeless and Tipin in an Ad12E1B-55k/E4orf6-independent, and Cul2-dependent fashion, during infection. Previous research from our laboratory identified WDR62 as possible E1B-55K interacting protein. The second aim of this study was to expand our current knowledge of this protein and determine its role during infection. Here we show that E1B-55K interacts with WDR62 in vivo and colocalizes with it at centrosomes. We also provide evidence to show that WDR62 functions in the cellular DNA damage response. Indeed, cells depleted of WDR62 by RNA interference resulted in a UV-sensitive phenotype, defects in ATR activation and G2/M checkpoint control, as well as displaying supernumerary centrosome during mitosis.