The N-terminus of BRCA1 is clinically important as inheritance of a mutation in this region correlates to an increased risk is breast and ovarian cancer. Whilst this is fairly clear, what specific mutations do and the aetiology of the disease is not clear. This thesis investigates N-terminal BRCA1 mutations using both in vitro and cell-based methods with a focus on DNA repair, mainly double-strand break and DNA crosslink repair. The use of chemotherapy agents is used with specific mutations to look at the individual phenotypes these create to each drug or radiation. This thesis also provides evidence on haploinsufficient or dominant negative effects of N-terminal mutations. Overall, the N-terminus of BRCA1 can affect DNA repair and increase genome instability that may lead to tumour development.

CD4+ T-cells play a pivotal role within the immune response, and multiple studies have highlighted their importance in anti-tumour immunity. TCR gene transfer is a successful method of specifically redirecting T-cell specificity. We have therefore investigated the anti tumour potential of EBV-specific MHC class II restricted T-cells, generated by this approach. We have identified and cloned a DR52b-restricted TCR, specific for an EBNA2 derived peptide (PRS), which is expressed in Post-Transplant Lymphoproliferative Disease (PTLD) and some other EBV-associated malignancies. We have shown that the TCR is functional in both CD4+ and CD8+ T-cells, with transduced T-cells specifically recognising the PRS-peptide with a high avidity. Transduced T-cells have been shown to proliferate, produce multiple cytokines and have direct cytotoxic capacity in response to physiological levels of EBNA2 processed and presented by EBV-infected B-cells. Additionally to this direct response, CD4+ T-cells retain helper functions. Importantly, transduced T-cells have shown hints of tumour control in vivo. Results from this study highlight that TCR gene transfer with EBV-specific MHC class II restricted TCRs can generate polyclonal T-cells with functional capacity against virus infected cells. PRS specific TCR gene transfer may thus be useful in rapid generation of T cells for treatment of PTLD. Given the importance of CD4+ T-cells for anti-tumour responses, this study also highlights the potential for using TCR gene transfer to target these cells towards other MHC class II-positive tumours.

γδ T-cells are unconventional lymphocytes hypothesised to act at the interface between innate and adaptive immunity. Emerging evidence indicates γδ T-cells play important, nonredundant roles in lymphoid stress surveillance during infection and tumourigenesis, and γδ T-cell-focussed immunotherapy trials suggest their potential exploitation in cancer immunotherapy. However, the molecular basis of γδ TCR/ligand recognition is poorly understood. In this thesis I first focussed on ligand recognition by the LES TCR, which is derived from a Vδ2-negative T-cell and mediates TCR-dependent recognition of CMVinfected cells and tumour cell lines by binding to Endothelial Protein C Receptor (EPCR). After producing LES TCR in a conformationally correct form, I used mutagenesis to map the LES TCR binding site on EPCR. Importantly, EPCR was recognised independently of bound lipid, suggesting it acts as a stress ligand rather than an antigen presenting molecule, and highlighting the importance of TCR-extrinsic factors in recognition. Secondly, I determined an NMR structure of Skint-1, a selecting ligand for mouse skin-resident DETC γδ T-cells that play important roles in immunoregulation and tumour immunosurveillance. This emphasised structural features unique to Skint-1, and suggested interaction with a separate ligand, such as the DETC TCR. Collectively, these studies improve understanding of γδ T-cell recognition.

ATM germ-line mutations have been identified in a proportion of patients with chronic lymphocytic leukaemia (CLL) but their role in the development of this tumour remains unknown. In the course of this study it was established that ATM pathogenic mutations were increased among patients with chromosome 11q deletion/loss of one ATM allele when compared to healthy control individuals but not in those who did not acquire this deletion in their leukemic clone. The results indicate that ATM germ-line heterozygosity does not play a role in CLL but may influence disease progression through complete ATM loss and clonal expansion. The analysis of the clinical outcome among CLL patients from phase III LRF UK CLL4 trial identified a distinctive subgroup with a particularly poor prognosis. Those patients had bi-allelic inactivation of ATM gene and showed significantly reduced progression-free survival (PFS) compared to those with ATM wild-type or mono-allelic ATM defects. Furthermore, they had equally poor PFS as those with mono- and bi-allelic TP53 abnormalities. The clinical implication of this finding might include re-consideration of the treatment strategies for this particular subgroup of patients. The improved understanding of the biological background of progressive and resistant CLL tumours, such as those with TP53 or ATM defects, results in a development of further targeted treatment strategies. The assessment of their efficacy and toxicity could be facilitated by CLL xenograft models. The optimization strategies overcoming the limitations of existing xenograft model were investigated during the course of this study. The results showed that partial depletion of patient CD3+, CD4+ or CD25+ cells prior to injection into NOG mice can prolong the engraftment of CLL cells within xenogenic microenvironment hence, providing expanded period of time for testing new therapeutic agents.

Conventional magnetic resonance imaging (MRI) is essential for the management of childhood brain tumours. However, it is increasingly being supplemented with functional techniques such as magnetic resonance spectroscopy (MRS). This thesis investigates how pre-treatment single voxel MRS can aid in diagnosis and surveillance of paediatric brain tumours and identify prognostic biomarkers. Data from multiple centres, scanners from three leading manufacturers and field strengths of 1.5 T and 3 T are incorporated. MRS was analysed using TARQUIN software with metabolite peaks fitted using a simulated basis set to provide metabolite concentrations. Univariate and multivariate statistical tests were used to compare variables. Multi-scanner spectroscopy detected significant differences in common and rare paediatric brain tumours. Diagnostic metabolite profiles were able to confirm tumour on follow-up imaging. Elevated creatine and total choline determined good prognosis in medulloblastoma. Myo-inositol and citrate aided in the characterisation of diffuse pontine gliomas (DIPG). While conventional MRI was unable to identify prognostic markers for DIPG, elevated taurine was found to be significantly associated with a better prognosis. The results encourage the use of MRS as an adjunct to conventional MRI in routine clinical practice. For future studies, accurate assignment of biomarkers will be determined in tumour tissue using in vitro high-resolution spectroscopy methods.

Thyroid cancer is responsible for more deaths than all other endocrine cancers combined, and its incidence is rising. In contrast to other cancers, thyroid carcinomas rarely display mutations in p53. p53 is a potent tumour suppressor, and serves to inhibit cell transformation. Interestingly, the thyroid gland is also sensitive to the effects of ionising radiation, where WT p53 would normally play a protective role. However, there is evidence to suggest that aberrant expression of cellular oncogenes can result in the functional inactivation of WT p53. PBF is a proto-oncogene found to be overexpressed in thyroid tumours. PBF overexpression causes transformation in vitro and tumourigenesis in vivo. Work within this thesis describes the functional relationship between PBF and p53 in thyroid cancer. We provide evidence for a direct interaction between PBF and p53 in thyroid cancer cells, and our data demonstrate that oncogenic expression of PBF adversely affects p53 homeostasis by increasing its degradation, with PBF potentially serving as a co-factor in a complex with p53 and HDM2. Surprisingly, PBF had no effect on p53-mediated transcription using focused microarrays. Nonetheless, overexpression of PBF conferred a significant survival advantage following DNA damage, indicating that PBF might potentially facilitate neoplastic growth and tumourigenesis. Besides regulation of transcription, p53 performs a wide range of biological functions. PBF may therefore serve to promote p53 inactivation independently of its role as a transcription factor. Overall, these data indicate that oncogenic expression of PBF may result in a novel mechanism of p53 inactivation, where PBF binds to p53 and accelerates its degradation in complex with HDM2. These events cause an increase in cell survival following DNA damage, thereby potentially promoting tumourigenesis in thyroid cancers expressing WT p53.

The Epstein-Barr virus (EBV)-encoded RNAs (EBERs) are abundantly expressed in all EBV-associated malignancies, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression have not been fully determined. Abundant EBER expression has previously been demonstrated to be important for the oncogenic potential of the EBERs to be realised in epithelial cells. This study aimed to elucidate further how EBV achieves abundant EBER expression. Firstly, experimental analysis focussed on the possible direct correlation between EBV genomic copy number and levels of EBER expression. These initial experiments revealed that no direct link between EBV genomic copy number and levels of EBER expression was evident, suggesting that EBV may be influencing the cellular environment. Previously, EBV has been demonstrated to induce a variety of cellular EBER-associated transcription factors, and the remainder of the study focussed on determining the role of EBV nuclear antigen 1 (EBNA1) in inducing changes to the cellular environment to allow high levels of EBER expression. EBNA1 was found to induce the transcription of cellular RNA polymerase (pol) II and pol III transcription factors associated with EBER expression and levels of the cellular transcripts transcribed by the EBER-transcribing pol III were increased. Cell systems were generated which allowed EBNA1 to be expressed transiently in EBER-expressing cells, with such expression resulting in increased EBER expression, demonstrating directly that EBNA1 is able to increase levels of EBER expression. EBNA1 promoter binding studies were conducted, revealing EBNA1 to be present at the promoter regions of transcriptionally induced genes, affording mechanistic insight into EBNA1’s mode of action. The results of this study prove significant not only in determining how EBV expresses such high levels of EBERs, but also in that EBNA1 is shown to influence several cellular factors directly implicated in oncogenesis.

Little is known about the long term adverse outcomes following colposcopy. This thesis employs a mixed method approach to investigate the long term adverse impacts of undergoing this procedure. Following a systematic review undertaken to explore the evidence base in terms of the potential impacts of colposcopy upon psycho-sexual functioning a two stage cohort study using questionnaires was undertaken employing quantitative and qualitative data collection tools. Of particular interest was whether the level of colposcopic intervention (colposcopy, biopsy or loop excision)was associated with more pronounced levels of sexual dysfunction, higher levels ofanxiety and depression and impaired quality of life. There were no significant differences observed between women undergoing colposcopy, biopsy or loop excision for any ofthe outcome measures across both stages ofthe study. Age was the only predictor found to be associated with some of the outcomes measured. The study concludes that the level of colposcopic intervention has no impact upon outcomes measured. Factors other than undergoing colposcopy are likely to explain any problems observed in this cohort.

In Western populations the incidence of oesophageal adenocarcinoma (OAC) is increasing greater than any other malignant disease. Increasing epidemiological and experimental evidence associates iron with OAC. Our understanding of iron physiology has increased enormously in recent years due to the elucidation of transport proteins and regulatory pathways. Haem scavenging and metabolism remains to be clearly characterised although candidate import proteins have been identified. This project aimed to characterise the iron transport machinery in the progression of OAC and the effects of iron exposure in-vitro. Overexpression of iron and haem import proteins, with a repression of iron export, was identified in the progression of Barrett’s metaplasia to OAC suggesting a role for iron in disease progression. Interestingly expression of hepcidin, the hepatic peptide responsible for systemic control of iron, was identified in samples of OAC. Culturing OAC cells with iron or haem increased cell proliferation, migration and anchorage independent growth. These effects were inhibited by the addition of alginate, a naturally occurring chelator. Knockdown of LRP-1, the prime haem import candidate, confirmed the role of this transporter. Understanding the differential expression of these proteins between benign and malignant tissue may permit novel therapeutic strategies in the future.

Background: Type II endometrial carcinoma, sarcoma and carcinosarcoma account for 10% of uterine malignancies but 50% of recurrences. Survival at recurrence is poor and better prognostic markers are needed to guide therapy. The prognostic significance of the novel markers clusterin and tetraspanin CD151 were evaluated in a cohort of poor outcome endometrial malignancies, along with oestrogen receptor, progesterone receptor, p53 and human epidermal growth factor receptor 2. Immunohistochemistry profiles and survival outcome between grade 3 endometroid cancers and type 2 cancers were compared. Material and Methods: Tissue microarrays constructed from 156 poor outcome uterine malignancies, tested with immunohistochemistry and staining were correlated with clinicopathological, mortality and survival data. Results: Expression of CD151 was significantly higher in uterine papillary serous and clear cell carcinoma (USPC+CC) compared to grade 3 endometrioid carcinoma, sarcoma or carcinosarcoma. All other markers were not prognostic for survival. Except for CD151, there was no significant difference in marker positivity, age, stage or survival between G3 EEC and UPSC+CC. Conclusion: CD151 is a novel marker in type 2 cancers that may guide therapeutic decisions. These data also suggest that grade 3 EEC is better characterised as a type II endometrial cancer and may benefit from similar treatment.