Objectives: Hot flushes and night sweats (HF/NS) in menopausal women are well-documented; psychological measures and interventions for managing these symptoms have been developed for women. Experiences of HF/NS in men with prostate cancer, which occur due to hormone treatment, are currently under-researched. Thus, this study involves a preliminary qualitative exploration of HF/NS cognitive appraisals and behavioural reactions reported by a sample of these men. Black men of Afro-Caribbean descent, white British/English and Irish men were included in order to consider possible differences relating to ethnicity. Methods: Semi-structured, in-depth interviews were conducted with 19 men (14 white British/English, 1 white Irish and 4 black British) who were receiving hormone treatment for prostate cancer and experiencing HF/NS. Framework analysis was used to explore HF/NS experiences, generate and categorise emergent themes and explore associations between themes. Results: Ethnicity-related thematic analysis was limited by the small sample of black British men recruited. However, analysis across all men identified a core superordinate theme labelled ’cognitions about HF/NS’, along with eight other themes. There were men who held beliefs about the impact of HF/NS on their masculinity, experienced shame and embarrassment due to concerns about HF/NS salience and perceptions by others and experienced feelings of powerlessness over HF/NS; powerlessness was associated with beliefs about the fatal consequences of discontinuing treatment. Cognitive appraisals (e.g. those associated with perceived control and embarrassment) influenced subsequent coping strategies. Thematic findings supported those identified in previous literature exploring HF/NS in male and female populations. Novel themes highlighted possible influences on HF/NS experiences, including beliefs associated with prostate cancer, general self-perceptions and potential socio-cultural influences. Conclusions: A range of men’s cognitive-behavioural experiences were generated from this qualitative exploration. An index of cognitive-behavioural themes was generated which could be used to inform future research into this under-researched field.

Cancer prevalence is an important epidemiological measure of the disease burden. It is defined as the number of people in a given population who are alive at a specified point in time (the index date) and who have previously been diagnosed with cancer. It may be expressed as either a count or as a proportion of the population. Members of the prevalent population are known as 'cancer survivors' and the time spent as such is known as 'cancer survivorship'. Complete prevalence includes all survivors regardless of when they were diagnosed, whereas N-year limited duration prevalence includes only those who have received at least one cancer diagnosis in the N years prior to the index date. In the United Kingdom (UK), addressing the needs of cancer survivors is a high priority for the Department of Health, as well as for voluntary sector organisations, and the need for further research into cancer survivorship has been highlighted. Despite this, in recent years little study has focused on cancer prevalence in the UK. The aims in preparing this thesis were to provide up-to-date estimates of cancer prevalence in the UK, to describe levels of acute health service utilisation among cancer survivors in different temporal phases of survivorship and to provide projections of future cancer prevalence. National cancer registry data for the UK were analysed, together with National Health Service hospital activity data for England. It was found that there are currently around two million cancer survivors in the UK, a figure far higher than previously thought. Levels of acute in-patient health service utilisation were, however, generally low among cancer survivors who had survived at least five years and who were not in the final year of their life. A discrete time model for projecting cancer prevalence was derived and used to project cancer prevalence in the UK from 2009 to 2040 under various different scenarios of future cancer incidence and survival. It was shown that in the coming decades cancer prevalence is likely to increase substantially. This thesis contains a detailed description of cancer prevalence and aspects of cancer survivorship in the UK which highlights the need for adequate planning to meet the many and varied needs of those diagnosed with cancer.

The epidermal growth factor receptor (EGFR) plays a pivotal role in cellular proliferation, differentiation and migration and is found to be overexpressed in a variety of tumours including the basal-like subtype of breast cancer. Despite decades of basic research in the HER signalling field, and many EGFR-targeted anti-cancer drugs are used clinically, the success rate for these agents is low, particularly in terms of improvement of overall survival. In breast cancer, the clinical response rates vary between clinical studies, ranging from 6-49%, in part, depending on the type of breast cancer, the stage of the disease, and the treatment used. These marginal benefits are mainly due to insufficient patient selection, and potentially also due to an inadequate inhibition of HER signalling and tumour escape via alternate signalling routes (such as HER2/3/4/c-Met). The first objective of this work was to use an in-house semi-automated protein activity sensing/imaging technique, to interrogate the EGFR-centric subnetwork of proteins, in order to obtain better understanding of molecular factors influencing the therapeutic sensitivity of the EGFR network. To monitor EGFR activity in cells we use a CrkII-based biosensor (CrkII protein flanking with eGFP and mRFP1), which undergoes conformational changes upon phosphorylation of tyrosine-221 by EGFR and other receptor tyrosine kinase (RTK)s including c-Met and PDGFβR. This changes are detected by fluorescence lifetime imaging microscopy (FLIM) combined with fluorescence resonance energy transfer (FRET). Using the sensor, we have conducted a high-content FLIM screen utilising a library of short interfering (si)RNA consist of 533 genes interconnected with EGFR directly or indirectly, chosen from the Human Protein Reference Database. Genes that modulate the EGF-induced biosensor conformational changes were identified. Including EGFR, we identified 22 possible targets (4.1% from the siRNA library) and have selected a few for further validation to establish the biological importance of the genes identified as primary hits. The same biosensor was also transfected using a liposome-based delivery system into murine models of basal-like breast cancer; and demonstrated a significant degree of intratumoural heterogeneity in EGFR activity, as well as the effect of EGFR inhibitors in situ. In the long term, the knowledge obtained in this thesis can potentially be translated to biomarkers that are based on a better understanding of tumour cell sensitivity to EGFR inhibition and may thereby suggest new combination treatments.

Colorectal Cancer (CRC) screening is offered in the UK to individuals aged 60-75 years through the faecal occult blood test (FOBt). Uptake of CRC screening is variable but particularly low in South East (SE) London, an area with high ethnic diversity and socio-economic deprivation. Reasons for this low uptake are unclear. This thesis explores the role of psychological, social and cultural factors in the low uptake of CRC screening in SE London. Mixed methods were employed where a narrative synthesis examined the beliefs of various ethnic minority and socio-economic groups about the FOBt; a qualitative study explored the beliefs of 50 people of Black African, Black Caribbean and White British backgrounds from SE London; and a prospective questionnaire study (n=507) identified the demographic and psychological predictors of screening intentions and uptake. The narrative synthesis, interview topic guide and questionnaire were underpinned by Theoretical Domains Framework (TDF; Michie et al 2005). Findings of the narrative synthesis indicated a dearth of research examining both ethnicity and SES factors together. The qualitative study highlighted many similarities in the views of participants from the three main ethnic groups after considering SES. The survey indicated intentions and participation in CRC screening were underpinned by psychological and demographic factors, where psychological factors mediated the impact of ethnic differences. Although SES was not related to screening intentions or uptake, more deprived groups were significantly less likely to make an informed choice about screening. This thesis has bridged an important gap by examining the beliefs of Black African and Black Caribbean who have been previously neglected from research. Exploration of ethnicity and SES and the integrated use of a theoretical framework are distinct strengths of this thesis. These findings can help inform the design of interventions to influence screening uptake.

In this thesis, I sought to explore the biological mechanisms that may underlie the chemoprotective effects of broccoli consumption towards bladder cancer. In particular, I was interested in the effects of diet on the transition from superficial bladder cancer to a more invasive form of this disease. Initially, I characterised global gene expression profiles of superficial and invasive bladder carcinomas through tissues obtained from the Norfolk and Norwich University Hospital Tissue Bank. Non hierarchical cluster analyses was used to identify a suite of genes that characterised these two tumour types. Of particular interest was variation in genes involved in the synthesis of the extra cellular matrix, such as COL6A1 that was upregulated in the more invasive tumour type. This was subsequently verified by RT PCR with independent tissue samples. I then explored how the broccoli isothiocyanates, sulforaphane and iberin and their N-acetylcysteine conjugates were able to perturb gene expression in two bladder cell lines that differed in their cancer phenotype. I showed that not only were these ITCs able to upregulate phase II detoxification genes, which has previously been reported in other cell lines, but they, and their conjugates, were able to down regulate the COL61A gene. Studies on the effects of ITCs on gene expression were complemented with studies on cell migration and invasiveness. I then sought to see if any of the changes in gene expression observed in the cell lines could be observed in tissues obtained as part of a human intervention study. I designed and executed a small pilot study that enabled bladder tissue biopsies to be obtained before and after a four day intervention with broccoli. Global gene expression analyses again suggested alterations in genes determining the extracellular matrix, such as Tenascin-C. This study demonstrated the proof-of-principle that these types of intervention studies are possible, but was of an insufficient size to draw definite conclusions. Finally, I investigated in more details the expression of splice variants of Tenascin-C in relationship to bladder cancer grade. In conclusion, this study has suggested that dietary ITCs may perturb genes involved in the extracellular matrix and this may be an important component of understanding their chemopreventive activities, and has demonstrated the feasibility of human intervention studies with the analyses of target tissues as opposed to peripheral biomarkers.

Post-transplant lymphoproliferative disease (PTLD) is a life-threatening complication after solid organ, bone-marrow and haematopoietic stem cell transplantation. While early diagnosis is known to predict encouraging outcomes, no specific markers exist for the early detection of PTLD. Epstein-Barr virus (EBV) infection is considered to be an important risk factor for PTLD. However, the correlations between EBV DNA loads and the onset of PTLD are inconclusive. The aim of this study was to identify potential biomarkers for the early onset of PTLD. To accomplish this, we examined EBV and cellular gene expression patterns in (i) the context of EBV lytic induction in a lymphoblastoid cell line (LCL) model system, (ii) PTLD versus non-PTLD transplant patients. In addition we examined the contributions of lytic virus replication vs. latent infection to the onset of PTLD. We used real-time two-step RT-PCR assays with SYBR green detection to analyse EBV gene expression (76 lytic, 12 latent [n = 88 genes]), following 12-O-tetradecanoyl-13-phorbol acetate/sodium butyrate (TPA/NaB) induction of Raji cells. We identified 22 highly induced EBV genes (>90-fold) at 24 hrs post-induction. Using genome-wide Affymetrix microarrays, we identified cellular genes that were highly differentially expressed and regulated during the EBV lytic induction phase, 112 of which were regulated specifically by EBV, and 14 chosen for further study. We developed and validated real-time two-step TaqMan RT-PCR assays for clinical validation. Altogether, we evaluated 17 cellular and 14 EBV candidate genes in whole blood from paediatric solid organ transplant (SOT) recipients and adult umbilical cord transplant (UBCT) recipients, with and without PTLD. We detected a higher number and level of expression of EBV latent and lytic genes in PTLD patients, notably, BALF5, EBNA-LP and LMP-1, and in association with viral loads. Further, we detected latency III and more varied EBV latent gene expression patterns in PTLD. We also detected four cellular genes (CXCL9, CXCL10, CDC2, and CHI3L1) that were differentially expressed in PTLD vs. non-PTLD patients. To conclude, these findings suggest a basis for the likely microenvironment in which PTLD could develop. Further evaluation of candidate EBV and cellular markers could facilitate a more specific diagnosis of PTLD.

Changes that occur in malignant cells can take a variety of forms and include changes in cellular glycosylation. Aberrant glycosylation is considered to be a universal feature of malignant transformation and tumour progression. Mucins are major carriers of altered glycans in most carcinomas, and mainly belong to the MUC family, with MUC1 considered one of its most prestigious affiliates, as it is upregulated and aberrantly glycosylated in the majority of breast and many other adenocarcinomas. The large domain of the extracellular region of MUC1 is made up of tandemly repeated amino acids that form a scaffold for the attachment of the Olinked glycans. The aberrant glycosylation of MUC1 observed in breast cancer results in the expression of simple, unbranched sugars that are often sialylated and this is partly due to the upregulation of the sialyltransferase, ST3Gal-I. These consistent and conserved changes suggest that aberrant glycosylation is advantageous for the tumour, and data generated by this thesis suggest that indeed this is the case. Attention has been focused on MUC1 as a target molecule for the immunotherapy of cancer. To investigate specific MUC1 tumour-associated glycans, recombinant MUC1 glycoproteins carrying specific and defined O-linked glycans were purified from engineered CHO cells. The immune responses to these MUC1 glycoforms were analysed using wild-type and MUC1 transgenic mice. Different tumour-associated glycoforms elicited different immune responses, with MUC1 carrying the disaccharide known as T inducing a cellular response in MUC1 Tg mice, while the MUC1-ST glycoform was incapable of inducing an immune response in MUC1 Tg mice or even wild-type mice. To determine whether the changes in glycosylation, observed in breast carcinomas, confer an advantage to cancer cells, we generated transgenic mice over-expressing ST3Gal-I in the mammary gland and other epithelial tissues, and crossed these with mice that develop spontaneous mammary tumours. The tumours that developed from the ST3Gal-I mice appeared significantly earlier than the controls. Thus overexpression of ST3Gal-I in the mammary gland promotes tumorigenesis in this model, and the MUC1-ST glycoform formed by the action of ST3Gal-I on the T antigen is not immunogenic and may even inhibit an immune response.

The main subjects of my thesis can be divided into three related areas. Firstly, determination of the mechanisms of tumoriogenesis associated with a recently identified, recessively inherited syndrome, AfrTZ-associated polyposis (MAP). MAP results from defective base excision repair (BER) caused by bi-allelic germline mutations in the human Mut-Y homologue (MUTYH, MYH) and leads to the development of colorectal adenomas and cancer. My work includes: further characterisation of the MAP phenotype completion of screening for mutations in other BER enzymes (OGG1 and MTH1) in individuals with multiple colorectal adenomas determination of the genetic pathway(s) involved in MAP tumorigenesis through studying loss of heterozygosity (LOH) and chromosomal abnormalities with array comparitive genomic hybridisation. A mouse model of MAP was developed as part of this thesis in order to study the development of intestinal adenomas from their earliest stages, and to evaluate the impact of environmental modification and chemopreventative therapies. Secondly, I determined to identify novel predisposition genes for the multiple adenoma phenotype. Up to fifty percent of individuals with multiple (5-100) colorectal adenomas (MCRAs) have no germline mutation in known predisposition genes (APC and MYH), but probably have a genetic origin. In these cases I determined to identify novel predisposition genes for the MCRA phenotype utilising various techniques. These included somatic screening of the adenomas for mutational signatures, expression array analysis of lymphoblastoid cell lines from these individuals and candidate gene approaches. Finally, I investigated the clonal origins of colorectal adenomas through studying adenomas from familial adenomatous polyposis (FAP), attenuated FAP (AFAP) and sporadic cases. In this thesis I developed a novel technique which utilises somatic APC mutations as clonal markers. This approach found 100% of FAP adenomas to be polyclonal and 100% of AFAP adenomas to be clonal in their origin.

In humans, loss of function mutations in the Lkb1 serine-threonine kinase are associated with Peutz-Jegher syndrome, an inherited cancer predisposition syndrome. Roles for Lkb1 have been described in various processes, including cell cycle regulation, apoptosis and cell polarity. Comparatively little is understood of the role of Lkb1 in regulating epithelial cell polarity. The establishment and maintenance of apicobasal polarity is a fundamental process that occurs at different stages throughout development. The Lkb1 kinase has been shown to play a conserved role in regulating epithelial cell polarity in C. elegans, Xenopus, Drosophila and mammals. The primary focus of this thesis has been to further characterise the polarity phenotype and mechanisms by which Lkb1 may regulate epithelial polarity in Drosophila. To study the role of Lkb1 in the control of epithelial polarity, I generated loss of function clones in the Drosophila neuroepithelium, the eye. Immunohistochemical and biochemical analysis of Ikh1 clones in the eye reveal that Lkb1 is required for the restriction of apical, junctional and basal determinants to their appropriate domains, for the correct formation of adherens junctions, and for the maintenance of photoreceptor cell morphology. I further demonstrate that in the absence of Lkb1, the beta-catenin homologue Arm and the polarity determinant Par-1 accumulate, and additionally, that Par-1 shows reduced phosphorylation at a site that regulates its localisation and activity. Genetic interactions assays provide further evidence that the pleiotropic effects of Ikhl loss of function may be mediated through the misregulation of Par-1 and Armadillo. I have also conducted a small-scale modifier EMS screen in Drosophila to isolate components of the Lkb1 pathway. We have screened approximately 9,500 flies, and recovered a number of mutants, which can now be mapped and identified using a multi-tiered approach. The role of Lkb1 in regulating cell polarity is conserved from Drosophila to humans. Thus, a deeper understanding of the mechanisms underlying the Lkb1 mediated regulation of epithelial polarity may make a valuable contribution to, and provide a strong basis for progress in the treatment of Peutz-Jegher syndrome.

Astrocytomas are the most common variety of primary tumours of the central nervous system (CNS). The incidence increases with age, peaking in patients aged 65--75 years. They are generally unresponsive to treatment, and most patients die within one year of diagnosis. Recent genetic studies of astrocytoma susceptibility syndromes, familial- and sporadic astrocytomas have led to the discovery of many genes and molecular mechanisms underlying astrocytoma oncogenesis. A few of the genes involved in inherited astrocytoma associated-syndromes (e.g., Cowden and Li-Fraumeni syndromes) are also strongly implicated in sporadic astrocytomas. However for several other well characterised genes i.e. those mutated in Tuberous sclerosis (TSC) and Neurofibromatosis (NF) any evidence for involvement in sporadic astrocytomas is much less clear. The objective of this study was to undertake a genome-wide survey of high malignancy grade astrocytomas with a view to ascertaining the distribution and prevalence of copy number aberrations, search for associations with prognosis and outcomes to treatment, and to discover possible novel pathways of oncogenesis. Thirty-two high malignancy grade astrocytomas were investigated. Twenty were available as frozen biopsies and 12 as short-term cell cultures. Genomic profiling of all 32, comprising 6 tumours of WHO Grade III and 26 of Grade IV, was achieved by the method of Comparative Genomic Hybridisation onto metaphase chromosomes. 7 of the tumours were investigated by array CGH, with one further investigated by MFISH. Two tumours did not reveal aberrations. For the other 30 tumours, data pooled from a minimum of 10 profiles of each tumour were analysed. Recurrent DNA copy number gains and losses were detected across the genome. In a number of tumours aberrations spanned loci of established candidate genes previously associated with sporadic astrocytomas, on chromosomes 7, 9p, lOq, 12q, 13q and 17p. In addition over 70% of 'sporadic' tumours appeared to have DNA-copy number aberrations implicating genes with established roles in astrocytoma-susceptibility syndromes. The chromosomal region 9q34 (site of TSC1) appeared to be under-represented in 25% of the tumours, while 16pl3 (site of TSC2) was diminished in -38%. Similarly, loci for NF1 and NF2 were involved in aberrations in -10% and 38% of the cases respectively, as were those of PMS2 (22%), APC (19%) and a number of miss- I Cytogenetic Analysis of DNA Copy Number Aberrations in High Malignancy Grade Astrocytomas match repair (MMR) genes. Eight tumours had probable loss at lp36. Several novel locations were also suggested An attempt to correlate DNA copy number alterations with survival showed that among patients with grade IV tumours, there were on average far fewer chromosome aberrations per tumour in four of the five patients surviving longer than one year after diagnosis than in those who died before this. There was some suggestion of a worse prognosis in Grade IV tumours with a specific deletion of lp36, which would agree with a previous report. The only Grade IV tumour that was studied by all three approaches showed good agreement between array and metaphase CGH. In addition, array CGH revealed small regions of loss in the region of PTEN (CHR lOq) and TP53 (CHR 17p) below the resolution of the metaphase CGH. The M-FISH revealed very large numbers of chromosomes and several translocations, and comparison of the microarray data and the MFISH data suggests possible candidate regions for breakpoints. The data are discussed in the light of previous work and with a view to the possibility that there may be some diversity in the cellular origin of astrocytomas and that haploinsufficiency may play some role in oncogenesis.