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.

Oesophageal adenocarcinoma (OAC), a cancer with dismal prognosis, has been increasing rapidly in incidence over the last 30 years, nowhere more so than in the UK. Intriguingly, it is a disease predominantly among white males, but there is a paucity of data from England. In performing a range of epidemiological studies, it has been confirmed that OAC has risen five-fold in the West Midlands, UK, five times more common among men, and predominantly a disease among Caucasians. A reduced incidence of OAC was identified among subjects with prostate cancer, suggesting a protective effect of anti-androgen therapy. Examination of a general practice database revealed a negative association with aspirin, non-steroidal anti-inflammatories and statins with OAC, and a positive association with inhaled steroids, increasing number of drugs with a side effect of reducing the lower oesophageal sphincter, and drugs used for asthma/COPD. Finally, a region wide case-control study, confirmed the positive association seen with increasing body mass index, waist circumference, smoking and reflux symptoms, with negative associations seen with a diet high in fruit and vegetables. This work has identified potentially modifiable risk factors that may be employed to reduce the incidence of oesophageal adenocarcinoma, and better help stratify those most likely to benefit from endoscopic surveillance.

Human papillomavirus (HPV) E2 is essential for transcriptional regulation of viral oncoprotein expression and the replication and persistence of episomal HPV genomes. Episomal persistence is mediated by tethering of viral genomes to host cell chromosomes during mitosis. Previous work demonstrated that interaction of E2 with the cellular DNA helicase ChlR1 is necessary for viral genome tethering. Therefore, disruption of this interaction is a potential therapeutic target for persistent HPV infections. To investigate the use of fragment-based drug discovery in the development of novel inhibitors of the E2-ChlR1 interaction, a fragment library was screened to identify those that bind E2 and several hits were identified. Concurrently, the interaction between HPV16 E2 and ChlR1 was characterised and shown to be a direct protein-protein interaction. The binding sites within E2 and ChlR1 were mapped and this information was used to identify a mutant E2 protein unable to bind ChlR1 (E2-Y131A). E2-Y131A was functionally characterised. HPV16 genomes encoding E2 wild type and Y131A were transfected into primary human keratinocytes to study the differentiation-dependent virus life cycle. Mutant genomes failed to establish genome maintenance, providing strong evidence that the interaction between HPV16 E2 and ChlR1 is necessary for the persistence of HPV infection.

Introduction: Paediatric low grade brain tumours present diagnostic and prognostic challenges, providing a need for better non-invasive imaging characterization. The value of \(^1\)H Magnetic Resonance Spectroscopy (MRS) performed on 5 scanners in the diagnosis and prognostication of an extensive bi-centre cohort of low-grade gliomas is investigated. Methods: Single voxel MRS was performed routinely in children with brain tumours at the Birmingham Children’s Hospital and Queen’s Medical Centre. Histopathological features were semi-quantified and in vitro \(^1\)H NMR used to study pilocytic astrocytoma cell lines. Magnetic Resonance Spectroscopic Imaging (MRSI) and texture analysis of MR images were performed. Results: MRS detects differences between subgroups of low grade brain tumours in children and between tumours of the same histology. High myo-inositol and glycerophosphocholine and low phosphocholine are markers of good prognosis. Histological correlates for MRS metabolites have been identified and paediatric pilocytic astrocytoma cell lines (‘typical’, metastatic and recurrence) have been discriminated. The value of MRSI in answering clinical questions has been demonstrated. Texture analysis achieved high accuracy in the diagnosis of paediatric posterior fossa tumours. Conclusion: Advanced MR techniques have a significant role in the study of paediatric brain tumours, and promising results from MRS, MRSI and texture analysis are reported here.

Heterogeneous blood vessels are created by angiogenesis and vasculogenesis in colorectal cancer (CRC). Assessing endothelial activities had promising applications. However no marker has translated to clinical care. Hence a multifactorial assessment or ‘endotheliome’, including angiogenic activity, the ‘angiome’, was proposed. I tested that circulating cellular and plasma biomarkers determined outcome(s). Flow cytometry quantified circulating endothelial cells (CECs, displaced from blood vessels) and endothelial progenitor cells (EPCs, for vasculogenesis). Plasma markers measured by ELISA were: von Willebrand factor (vWf, for endothelial damage/turnover), soluble E-selectin (adhesion in tumour migration), vascular endothelial growth factor (VEGF) and angiogenin (for the ‘angiogenic switch’). All markers were prospectively quantified in 154 CRC participants before treatment and compared to non-cancer controls. They were tested against the tumour’s histopathology and repeated after surgery +/- adjuvant therapy. CECs and EPCs were highest in CRC and correlated to VEGF only. Angiogenin was diagnostic of CRC and vWf predicted metastasis. All markers fell after surgery but inconsistently after adjuvant treatment. Lower CD34+CD45- cells identified responders to anti-angiogenic therapy. Models incorporating CEC, EPC, angiogenin and CRC stage predicted progression within 2 years better than CRC stage alone. In summary, the endotheliome and angiome are determinants of outcomes and may aid decisions on therapeutic strategies.