Purpose: Morocco is undergoing a nutrition transition, characterised by increasing prevalence non-communicable diseases (NCD), including obesity. In that context, it is crucial to focus on fruit and vegetable (F&V) intake as they may have a preventive effect on weight gain and NCDs. Objectives: The objectives of the present work were: to develop an objective measure of F&V intake and to provide a holistic understanding of factors that may influence F&V consumption, such as socio-demographic and psychosocial factors. Methods: The target population was Moroccan women (20-49 years), living in the urban area of Rabat-Salé. This PhD involved three different studies: the first was based on focus groups that yielded qualitative data of women’s views of F&V; the second study involved validating a quantitative F&V Food Frequency Questionnaire (FFQ); the third a cross sectional population survey-which incorporated findings from studies 1 and 2 to assess dietary intake and the factors influencing F&V consumption. Results: Validation analyses suggested that the quantitative FFQ developed was reliable and valid to measure F&V intake. The mean F&V intake was 213g per day. Women with higher education, higher economic status and better knowledge scores ate significantly larger amounts of F&V than others. Processed food consumption was inversely associated with vegetable intakes. In terms of psychosocial factors, the strongest predictor of intention to eat fruit was control beliefs. Normative beliefs were the strongest predictor of intention to eat vegetables. Intention was the strongest predictor of both fruit and vegetable consumption. Conclusion: The data collected gave an overview of the amount of fruit and vegetables consumed by urban Moroccan women, and enabled a better understanding of the determinants of fruit and vegetable intake. As a consequence, data sheds light on possible avenues for policies and nutrition interventions to focus on in Morocco, in order to increase fruit and vegetable consumption.

Telomeres are specialised structures that that cap the ends of chromosomes; and prevent the natural end of a chromosome from being recognised as a double stranded DNA break. Telomeres erode with ongoing cell division ultimately leading the induction of replicative senescence that provides a proliferative lifespan barrier. In the absence of a functional DNA damage response, telomeres are prone to end-fusions, creating dicentric chromosomes that can initiate breakage fusion–bridge cycles. Evidence from studies in well-defined mouse models of cancer, and also human tumours, have shown that telomere dysfunction may be a key event in the progression of disease via the increasing genomic instabilities that arise from telomere fusion events. The key aim of this thesis was to test the hypothesis that telomere dysfunction occurs during the progression of breast cancer and that this can drive the large-scale genomic rearrangement frequently observed in this disease. Technology development was attempted in the hope to allow single-molecule telomere fusion detection of more complex mutational structures, with limited success but possible future potential. Telomere length analysis was carried out using high resolution single molecule PCR strategies (STELA) in a panel of DNA samples derived from invasive ductal carcinoma. Telomere length data analysed alongside clinical data received for all samples was used for the potential utility of telomere length as a potential prognostic indicator in breast cancer. A key finding of this work was to demonstrate the use STELA combined with statistical tests to show that short telomere length is highly significant in terms of prognosis in breast cancer. Telomere length stratification could thus potentially be used as a method of defining new breast cancer subtypes in terms of severity.

In recent years, substantial evidence has been generated demonstrating the importance of the immune system in preventing and controlling the growth of many cancers, including colorectal adenocarcinomas. In particular, populations of tumour-specific effector T cells appear to play a crucial role in restricting the generation and expansion of transformed neoplastic cells. However, the fact that tumours continue to grow in the presence of a seemingly intact immune system suggests that these responses are often inadequate. CD4+Foxp3+ T regulatory cells (Tregs) have been shown to play a key role in modulating the immune system by keeping immune responses to self-antigens in check, thereby preventing autoimmunity. These cells also appear to be employed by tumours to protect against recognition and eradication, and have been demonstrated to impinge upon the anti-tumour immune response in humans. Furthermore, it appears that the tumour microenvironment facilitates the development of highly immunosuppressive T cells, which may also allow subsequent tumour progression. In colorectal cancer, the relationship between Tregs and tumour progression is less clear – despite their well-documented ability to impinge on anti-tumour immune responses, increased tumour infiltrates have also been associated with prolonged survival. In this thesis, the phenotype and function of CD4+ T cells derived from PBMC, colon and tumour samples were analysed for suppressive markers by FACS, and anti-tumour responses by IFN-γ ELISpot. CRC patients with more advanced tumours responded to fewer epitopes and generated a significantly weaker epitopespecific T cell response to the oncofoetal antigen, 5T4 than healthy donors. Human depletion experiments both in vitro and in vivo indicated suppression by Foxp3+ regulatory CD4+ T cells. These cells were found in abundance amongst tumourinfiltrating lymphocytes; however, another equally prominent population of IL-10 and TGF-β-producing CD4+Foxp3- T cells were found to be >50-fold more suppressive. Thus, a major caveat to cancer immunotherapy is the suppressive tumour microenvironment, which contributes to the selective decline of measurable antitumour CD4+ T cell responses as tumours progress. These responses were enhanced in metastatic CRC patients by depleting regulatory T cells. It is hoped such findings will augment our understanding of how anti-tumour CD4+ T cells are activated and conversely regulated, with the intention of designing better treatment for patients with colorectal cancer.

CLL is a malignant disease of B-cells characterised by a heterogeneous clinical outcome. Some patients require an early treatment and have low survival time, while others never need treatment. Many prognostic markers have been established and used to help predict the clinical course of CLL. Despite advances in understanding the biology of CLL, the molecular differences underlying the variable clinical outcome of CLL are not yet fully understood. The hypothesis of this study was that the heterogeneous outcome of CLL could be driven, in part, by the aberrant expression of proteins in the two forms of CLL. Therefore, this study aimed to identify these proteins using proteomics approaches. In an attempt to achieve this goal, four steps were performed. Firstly, a cellular fractionation method was developed to extract cellular proteins into two different fractions (NP40 fraction for cytosolic protein enrichment and SDS fraction for nuclear protein enrichment). Secondly, extracted proteins were subjected to qualitative proteomics analysis using 2D nano-LC and MALDI TOF-TOF mass spectrometry in order to identify CLL proteins. Integrating the identified proteins (n=900) with previously published transcriptome of CLL cells and normal B-cells highlighted 20 proteins with preferential expression in CLL cells - some of which were linked to human cancer. Thirdly, iTRAQ technology coupled with 2D nano-LC and MALDI TOFTOF mass spectrometry was used to measure the relative expression of proteins in different CLL samples. This workflow identified 15 altered proteins in the two forms of CLL and detected 14 proteins with variable expression. Finally, six proteins were selected for investigation in an additional CLL cohort. Of these proteins thyroid hormone receptor-associated protein 3 (TR150), T-cell leukaemia/lymphoma protein 1A (TCL-1) and S100A8 showed association with poor prognosis CLL and early requirement for treatment. Additionally, myosin-9 exhibited reduced expression in poor prognosis CLL samples.! ! Overall, this study identified proteins with potential importance in CLL prognosis and pathology. These proteins merit investigation in a larger CLL cohort to further confirm their relevance to CLL. In addition, this study showed the usefulness of combining cellular fractionation with proteomics and transcriptomics to identify proteins with potential role in CLL.

Birt–Hogg–Dubé (BHD) syndrome is a rare inherited autosomal dominant disorder first described by Birt, Hogg and Dube in 1977 (Birt et al,. 1977). BHD affects approximately 100 families worldwide. Approximately one third of diagnosed BHD patients also develop renal cell carcinoma (RCC) (Schmidt et al., 2005). BHD arises as a result of loss of function of the Folliculin protein expressed from the BHD gene, suggesting than Folliculin serves as a tumour suppressor (Vocke et al., 2005). Although it is considered that FLCN represses cell growth, the role that FLCN plays in cancer progression and/or initiation is currently unresolved. It has been observed that tumours taken from BHD patients have reduced levels of mitochondria (Yang et al., 2008). Previous studies have also suggested that aberrant levels of mTOR activity are observed in Folliculin-deficient cell lines (Baba et al., 2006; Baba et al., 2008). Aberrant levels of mTORC1 activity have been associated with increased levels of Hypoxia inducible factor transcriptional activity, decreased autophagic activity (Land and Tee, 2007; Hands et al., 2009). There are several genes within the mTOR pathways which act as Tumour Suppressors. Mutations within these genes result in inherited genetic disorders, including Tuberous, Sclerosis complex (TSC). Loss of function of TSC1/TSC2 results in TSC which is clinically similar to BHD (Gomez et al., 1999). Arbarrant levels of mitochondrial biogeneisis and HIF activity have been observed in cells deficient in TSC1 ad TSC2 (Land and Tee, 2007; Chen et al., 2008). Increased levels of mTORC1 activity have also been shown to result in decreased levels of autophagic activity in TSC2 deficient cells (Parkhitko et al., 2011). In order to characterise the tumour suppression function of Folliculin, the effects of loss of function of Folliculin on mitochondrial biogenesis and mitochondrial function, hypoxia inducible factor (HIF) transcriptional activity, and autophagic activity were investigated using multiple cell lines. The results from this study suggest that loss of function of FLCN results in increased production of ROS species. This leads to compromised mitochondrial membrane potential and decreased ATP production as a result of increased expression of uncoupling proteins in order to try and reduce the increased levels of ROS. FLCN is also phosphorylated at multiple residues by both mTORC1 and AMPK and activity of both mTORC1 and AMPK is increased in response to the depleted ATP levels as a result of increased UCP production in response to the increased ROS production observed upon loss of functional FLCN. This results in increased levels of mitochondrial biogenesis and increased levels of glycolytic activity via increased activation of HIFα proteins in order to compensate for this energy deficit. Furthermore, it appears that both mTORC1 and AMPK could drive HIF transcription and mitochondrial biogenesis through modulation of FLCN phosphorylation. ULK-mediated autophagy also appears to be upregulated upon loss of functional FLCN, possibly as a result of the increased levels of AMPK activation in order to provide a protection for these cells. This may be via the catabolising the dysfunctional mitochondria observed in cells upon loss of BHD, a process which may be exploited as a potential therapeutic target for BHD patients. Further investigations into these cellular processes may also provide clues to potential therapeutic targets for treatment of BHD patients.

The aim of this study was to investigate the role of the adipokine visfatin as a possible molecular mediator between obesity and prostate cancer. Visfatin, an adipokine that is elevated in obesity and has many proposed roles and has been linked to a variety of cancers. No data pertaining to the role of visfatin in prostate cancer existed and this was an area that this study looked to address. It is suggested that obesity is a significant risk factor for prostate cancer; in particular, aggressive disease and adipokines have been investigated as a link for this hypothesis. This study presents novel data demonstrating the expression of visfatin in the LNCaP and PC3 cell lines as well as in benign and cancerous prostate tissue at both mRNA and protein level. Furthermore visfatin is shown to have functional roles in autoregulation and promoting increased cell proliferation in PC3 cells and also showed further effects with respect to cell migration across a wound. These data gave promise to develop the study further and evaluate potential mechanisms of action including common second messenger systems such as MAPK and also other oncologically multifunctional molecules in the forms of MMP-2/-9. We then demonstrated that visfatin up-regulated MAPK phosphorylation and MMP mRNA/protein expression and more importantly MMP-2/-9 zymographic activity. This provided possible mechanisms by which visfatin may mediate a role for obesity driven aggressive prostate cancer. The study then looked to evaluate NMN (the byproduct of visfatin catalysed biosynthetic activity), as well as the visfatin inhibitor FK866 which is being evaluated as chemotherapeutic agent. Unsurprisingly NMN and FK866 had opposing actions on proliferation and FK866 was naturally proapoptotic. NMN was able to rescue the effect of FK866 on PC3 cell apoptosis. Prior studies have shown that NMN did not affect oncogenes however NMN was found to significantly reduce BAX mRNA expression in PC3 cells. The findings are consistent with other studies linking visfatin with cancer states. These novel data indicate roles for visfatin in prostate cancer and possible mechanisms linking obesity and prostate cancer.

TMEFF2 (transmembrane protein with EGF-like and two follistatin motifs 2) is a transmembrane protein expressed in brain and prostate and over-expressed in prostate cancer (Uchida et al. 1999; Horie et al. 2000). The role of TMEFF2 in prostate cancer is controversial. Several data indicate that TMEFF2 has cancer-promoting activity (Glynne- Jones et al. 2001; Ali and Knäuper 2007), while others suggest that TMEFF2 inhibits progression of cancer (Gery et al. 2002; Gery and Koeffler 2003). TMEFF2 is cleaved by membrane-anchored proteases, including disintegrin and metalloproteases (ADAMs) and -secretase (Ali and Knäuper 2007), but the biological meaning of TMEFF2 shedding is not known. It was hypothesized that the opposing findings describing the role of TMEFF2 in prostate cancer result from proteolytic processing of TMEFF2 by different proteases which are co-expressed with TMEFF2 in prostate cancer cells, such as the type II transmembrane serine proteases (TTSPs), prostasin and ADAMs. To support this hypothesis co-expression of TMEFF2 and serine proteases was analyzed in prostate cancer cell lines and clinical samples. The shedding of TMEFF2 by ADAMs and serine proteases was investigated using HEK293 cells expressing AP/V5 TMEFF2 or shedding resistant AP/V5 303-320TMEFF2 mutant (Ali and Knäuper 2007). The data obtained from AP activity assay and Western blot analysis of cell lysates showed that TMEFF2 is cleaved by serine proteases (matriptase and hepsin) and ADAMs (ADAM9, ADAM12). Moreover, serine proteases and ADAMs cleave TMEFF2 in different positions, generating several soluble TMEFF2 fragments. To establish the biological role of TMEFF2 processing, N-terminal TMEFF2 fragments predicted to be generated by TTSPs and ADAMs were expressed in E. coli and mammalian cells. Preliminary experiments using HEK293 and PNT2-C2 cells indicated that soluble TMEFF2 does not signal through ErbB receptors and suggested several signaling pathways that might be regulated by TMEFF2. The fate of TMEFF2 C-terminus following ectodomain shedding was examined by confocal microscopy and Western blotting, indicating that TMEFF2 cytoplasmic domain is likely degraded following the release of TMEFF2-ECD

In ER+ breast cancer initial responses to antihormones are variable, complete responses are rare and resistance is eventually acquired by many patients. It is important to model these events to discover predictive markers of antihormone outcome and so targeted strategies can be developed to maximise antihormone effectiveness. To date, most studies have employed the MCF-7 cell line which fails to represent the variability of ER+ disease. Focusing on Faslodex, the thesis objective was to use 4 cell lines in vitro encompassing ER+/HER2- (MCF-7/T47D) and ER+/HER2+ (BT474/MDA-MB-361) disease to (i) characterise the magnitude of initial antihormone response, (ii) monitor the onset of resistance by prolonged treatment and (iii) detail gene expression changes during Faslodex treatment. All models were initially growth-inhibited by Faslodex, with superior responses in HER2- lines. Microarray analysis revealed gene cohorts affected by Faslodex treatment differed between HER2+ and HER2- models. While MCF-7, BT474 and MDA-MB-361 cells acquired Faslodex resistance, this failed to develop in the T47D line, providing a model of complete-response. A filtering process identified genes involved in the varying Faslodex responses and clinical relevance was determined using the NEWEST Faslodex clinical trial dataset. Of interest was the Faslodex-induction of CXCR4, as a potential mediator of acquired resistance, while suppression of the RET signalling pathway related to improved initial response in the ER+/HER2- setting. Importantly up-regulation of DCN by Faslodex was associated with improved Faslodex response in T47D cells and also with proliferation (Ki67) fall in the NEWEST clinical trial. shRNA knockdown of DCN reduced the sensitivity of T47D cells to Faslodex and enabled development of resistance. This thesis has successfully identified novel elements of Faslodex response and resistance and further work is now required to clarify the importance of these mediators and to determine if DCN could prove a useful clinical biomarker of Faslodex response.

The anti-viral acyclic nucleoside monophosphate compound Cidofovir has shown efficacy in treatment of Human Papillomavirus (HPV) associated genital intraepithelial neoplasia; however, the mechanism of action of Cidofovir in this setting has not been determined. This investigation focused on modifying nucleoside analogue compounds to increase their efficacy in HPV positive cell models of disease, in addition to determining the molecular mechanism of action of Cidofovir in premalignant HPV associated intraepithelial neoplasia. ProTide modification increases the efficacy of nucleoside analogue compounds by increasing their cellular permeability. Cidofovir was not amenable to ProTide manipulation; however, ProTide derivatives of its sister compounds, Adefovir and Tenofovir, were synthesized. Parent Adefovir and Tenofovir and a range of their respective ProTide modified daughter compounds were examined for inhibition of cell growth and effect on cell size and morphology in HPV positive and negative transformed cell lines. The most effective compounds were further examined for dose response in normal HPV negative untransformed Human Epidermal Keratinocytes (HEKs) and naturally HPV immortalized short term (NHIST) cell lines cloned from vulval and vaginal intraepithelial neoplasia biopsies. ProTide analogues displayed striking increased efficacy in comparison to their parent compounds; however, they did not show specificity to transformed or HPV positive cell lines. Cidofovir did not show specificity to HPV positive cells when examined for growth inhibitory effect in HPV positive and negative cell models. A variety of molecular processes were examined to determine the mechanism by which Cidofovir inhibits cell growth in validated NHIST cell lines and HEK cells. At the concentrations investigated, Cidofovir did not cause apoptosis in HPV positive or negative cells and its growth inhibitory effect appeared likely to be associated with cell cycle arrest or senescence. The effects of radiation on the molecular response induced by Cidofovir were also evaluated as previous studies suggested Cidofovir can function as a radiosensitizer. Cidofovir combined with gamma radiation did not result in apoptosis but was associated with an augmented molecular response in NHIST cell lines. On the contrary, Cidofovir combined with gamma radiation caused a major apoptotic response in HPV negative HEKs, suggesting such a combination could result in disadvantageous effects on healthy tissue if it were used in vivo.

In this study a CD31-expressing co-culture system was used to establish whether differential CD31/CD38 signalling may contribute to the poor prognosis associated with CD38 expression in CLL. Using western blot analysis, a PKB phospho-substrate antibody was used in combination with phospho-specific antibodies to identify ribosomal protein S6 and GSK3β as key signalling molecules that were augmented following short-term CD31-expressing co-culture. CD31-expressing co-culture did not alter the phosphorylation of STAT6. However, the addition of IL-4 to the cultures was a potent mediator of this signalling pathway. This highlights the specificity of signalling molecules to different external stimuli. Multi-colour flow cytometry was employed to quantify the expression of cell surface activation markers as well as intracellular phospho-proteins. The CD31-expressing co-culture led to a significant up regulation of the activation markers CD38, CD49d and CD69. Selective pharmacological inhibition of the phosphorylation of S6, STAT6 and ERK resulted in the down regulation of activation markers. Furthermore, the inhibition of p-STAT6 and p-ERK resulted in increased levels of apoptosis, which indicates that these signalling pathways are directly involved in CLL cell survival. Multi-colour flow cytometry was also used to quantitate the levels of phospho proteins, p-S6 and p-ERK. Similar to the results observed by antibody detection following western blotting, basal and inducible levels of p-S6 and p-ERK were elevated in primary CLL cells expressing high levels of CD38. Taken together, the work carried out in this project highlights the importance of using co-culture systems to stimulate CLL cells in vitro in order to mimic some of the key stimuli encountered in vivo. The dissection of the signalling pathways activated as a result of CD31/CD38 interactions provides a rational for the poor prognosis associated with elevated CD38 expression in this disease and identified candidate therapeutic targets that might particularly benefit this group of patients.