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Advances in tumour biology have led to studies of new biological markers, which may predict treatment response and clinical outcome independently of standard clinicopathological variables. An immunohistochemical analysis of a panel of molecular markers was studied, in a series of anorectal tumours, treated with 5'fluorouaracil (5FU) chemoradiation. 240 tumour samples from the ACT I anal cancer trial were analysed. Patients were randomised to radiation alone (RT) or concurrent mitomycin/5FU/RT (CMT). On multivariate analyses tumour stage, treatment response, CD34 (vascularity), thymidine phosphorylase (TP) and p53 expression were independent predictors of clinical outcome. Tumour stage and p53 expression were associated with a poorer response to treatment in both randomisation arms. Increasing cyclin A and decreasing bcl-2 predicted for an improved response to radiation alone whilst CD34, tumour stage and TP expression predicted for an improved survival in the CMT arm. Archived tumour samples from 60 patients with locally advanced rectal carcinoma were also studied. On multivariate analyses resection margin, nodal stage, treatment response, EGFR and p53 expression were independent predictors of clinical outcome. Increasing TP and cyclin A expression were associated with an improved response to chemoradiation. The response of TP and thymidylate synthase (TS) to radiation was investigated in two colon carcinoma cell lines. TP activity significantly increased in response to a single and fractionated RT dose and TS significantly decreased. This may explain the lack of prognostic significance of TS and confirms that capecitabine, which is activated by TP in tumour cells, is a rational agent to combine with radiation. In conclusion survival and treatment response in anorectal carcinomas can be predicted independently of standard clinicopathological characteristics. They can also predict survival between different therapeutic modalities, which may lead to improved chemoradiation strategies. Tailoring the treatment to the individual may become a future possibility.

Bloom syndrome is a rare autosomal recessive disease caused by loss of function mutations in the BLM gene and characterized by telangiectatic erythema, proportionaJ dwarfism, immune deficiency, infertility, type II diabetes, and a predisposition to many types of cancer. It is unclear how germline mutations in BLM that encodes a helicase protein lead to the highly tissueMspecific changes and disease states associated with BS. In addition, previous works have demonstrated the various roles of BLM in DNA replication and repairs; however, there is currently no mechanism to acutely disable BLM function in cells in order to identify synthetic lethality with other DNA repair factors in an effort to determine how the pathological features of BS might driven by the loss of a single helicase alone. This work aims to develop the fist selective and patent BLM inhibitor that could be used to characterize the role of BLM in gene and rnicroRNA expression as well as the dual effects ofBLM and other DNA damage response proteins such as NIP45. 8

Dendritic cells (DCs) exhibit the capacity to recognise and phagocytose dying cells and invading pathogens and to present antigens from this internalised material on their surface. The recognition of these antigens by T cells can induce specific cytotoxic responses against other cells expressing the same antigen. These characteristics suggest the potential for DCs to be used a Cancer vaccine, due to their ability to stimulate an immune response against tumour cells. To ensure that cytotoxicity, rather than tolerance to the presented antigens is induced, co- stimulatory molecule expression on a DC is necessary. These molecules provide additional signals to T cells to ensure that only antigens presented in the appropriate environment are capable of inducing cytotoxic responses. This thesis investigates the hypothesis that poly (I:C), a synthetic dsRNA analogue, may induce an inflammatory form of apoptosis, releasing tumour antigens and inflammatory mediators capable of maturing DCs, for use as a Cancer vaccine. It has previously been shown that the administration of poly (I:C) induces apoptosis and the release of inflammatory cytokines in intestinal epithelial cells'. Results presented here demonstrate apoptosis, cytokine release and the up regulation of mRNA encoding several inflammatory factors in poly (I:C) treated tumour cells. Internalisation of apoptotic cells by DCs is shown and DCs cultured in supernatants from poly (I:C) treated tumour cells express co-stimulatory and MHC molecules, indicating their potential to stimulate T cells. Results indicate that poly (I:C) treated tumour cells may provide a more efficient method for loading DCs with antigens and inducing DC maturation than the loading of DCs with irradiated tumour material

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Multicellular tumour spheroids (TS) are an in vitro model of avascular tumours, and have been widely used to investigate tumour growth, metabolism and hypoxia. The geometry of the TS lends itself to mathematical representation, and theoretical models of TS growth and the development of hypoxia are abundant. With some notable exceptions however, these models have been developed independently of the biological data collection process and are overwhelmingly based upon data from multiple sources. Thus, whilst mathematical modeling has the potential to help explain and guide biological experiments, without reliable data it is unlikely to live up to this expectation. In this thesis, a combination of experimental and theoretical approaches was used to characterize the relationship between proliferation, hypoxia and metabolism during the growth of TS derived from the DLD-1 human colon adenocarcinoma cell line. Experimental data were collected over the entire period of TS growth, generating a high volume of predominantly imaging data. To facilitate the extraction of quantitative information from this, a suite of image analysis software, which is readily applicable to other data sets, was developed. During growth, the DLD-1 TS maintained a macroscopic spherical geometry but at the microscale level the TS boundary was increasingly irregular, with TS disintegrating rapidly after 20 days. Immunofluorescence (IF) studies showed that hypoxia developed soon after TS initiation, followed by the characteristic onset of necrosis. Reduced proliferation was found to be concomitant with the development of hypoxia, although some cells retained proliferative capacity even under severely hypoxic conditions. Towards the end of culture, TS were primarily comprised of severely hypoxic and necrotic cells, a probable cause of disintegration. Mathematical simulation of oxygen gradients in TS using literature-based values for the maximal rate of oxygen consumption was used to estimate the partial oxygen pressure (pO₂) at which the IF marker of hypoxia was bound. Assuming a spatially-invariant rate of oxygen consumption, the model predicted that the onset of hypoxic binding occurs at pO₂ levels similar to those reported in the literature, however the onset of necrosis was overestimated. Mathematical simulations predicted that oxygen consumption decreases as TSs increase in size, supporting previous observations. The Warburg Effect, where glucose metabolism is favoured even under aerobic conditions, is a hallmark of tumours. Although development of the glycolytic phenotype during TS growth was observed in the form of an elevated activity of the lactate dehydrogenase V (LDHV) enzyme, the activity and expression of other glycolytic enzymes, such as hexokinase II (HKII), was unaltered. Whilst the spatial distribution of HKII was unrestricted throughout the TS's viable fraction, LDHV expression was elevated in regions of hypoxia, suggesting constant adaptation of tumour cells to their microenvironment. In addition to the above findings, the data generated have been collected and analysed in the context of the requirements of theoretical modelling at each step; thus, they can be used to parameterise and inform more sophisticated models of tumour metabolism.

A published meta-analysis on breast cancer and vegetables and fruit consumption was described to present a methodology used on meta-analysis in Epidemiology. Meta-analysis confirmed the association between intake of vegetables (RR=0.74; 95%CI 0.65-0.84) and, to a lesser extent, fruit and breast cancer risk (RR=0.93; 95%CI 0.79-1.09). Using this methodology, present in a peer-reviewed journal, a systematic meta-analysis on melanoma was conducted extracting RRs from published studies. Fully adjusted estimates were obtained from those studies, when available; RRs adjusted for confounders not related to sun exposure, such as naevi, were considered for sun exposure and sunburns pooled estimates. Pooled estimates were obtained for all main risk factors for melanoma: sun exposure (total, intermittent and chronic), sunburns (in childhood and in adulthood), indicators of actinic damage, family history of melanoma and phenotype characteristics. Investigation of biases and inconsistencies among studies was one of the key phases of the meta-analysis to look for patterns among studies that might explain discrepant findings. The analyses on pigmented lesions and sun exposure showed that the choice of sources of cases and controls influenced significantly the estimate. An indication of a protective effect of chronic sun exposure came from studies that did not include subjects with dermatological problems (significantly different from the other studies: p=0.01). Publication year was an important factor for total sun exposure (p=0.005). Latitude of the study seemed to be an important factor for sunburns (p=0.002) and for high density of freckles (p=0.04). Estimates for hair colour and eye colour adjusted for phenotype and/or photo-type were significantly lower than unadjusted ones (p=0.06 and p=0.06, respectively). This study highlighted how several features of study design, type of analysis, categorization of exposures, study location and populations significantly explained between-study heterogeneity.