Androgen deprivation therapy (ADT) such as bicalutamide (BCA) has become the mainstay of treatment for locally-advanced prostate cancer (PCa). Despite initial remissions, ADT resistance almost inevitably occurs with progression to metastatic castrate-resistant PCa. Hypoxia is a common hallmark of many solid tumours and is associated with treatment failure and malignant progression; androgen withdrawal has been shown to induce profound hypoxia in androgen-sensitive tissue. This prompted an investigation into the effect of ADT on tumour oxygenation and malignant progression in PCa. Androgen-dependent luciferase-expressing PCa xenografts (LNCaP-luc) were implanted in SCID mice. When tumours reached -150mm3 mice were treated daily with SCA (2 or 6mg/kg). A reduction in tumour oxygen was observed within 24hrs (Oxylite™ oxygenelectrode); this continued to a nadir of 0.1 % oxygen at day 3 or 7 respectively. Tumours remained profoundly hypoxic until day 14-21 when oxygen levels began to rise, concomitant to time-dependent remodelling of the tumour vasculature (dorsal skin fold model). By day 28, BCA-treated xenografts were more malignant and showed greater metastatic spread to the lungs. Gene expression changes during BCA treatment of LNCaP xenografts were investigated using qPCR arrays; significant differences were found in the expression many genes involved in angiogenesis, invasion and metastasis, apoptosis resistance and the PI3K1AktimTOR signalling pathway. Informed treatment regimens combining SCA with a unidirectional hypoxia activated prod rug (AQ4N and its novel analogue OCT1002; 50mg/kg, day 7) or an Akt inhibitor (30mg/kg t.i.w) resulted in a reduction in tumour growth and metastatic spread to the lungs. When the anti-angiogenic VEGF-inhibitor (B20.4.1.1; 5mg/kg, day 14) was combined with bicalutamide, this blocked the revascularisation associated with BCA alone. This study shows that BCA-induced hypoxia induces critical changes in the tumour microenvironment which cause modified gene expression and drives malignant progression. Targeted therapeutic regimens, informed by this knowledge, may improve treatment outcomes of androgen-dependent PCa.

Tuberculosis (TB) has plagued the human race for centuries. Despite being largely a curable disease, over 3500 people die from the disease each day. Mycobaeterium . tuberculosis, that causes TB, is particularly resilient. This is due, in part, to its lipid-rich cell wall, containing mycolic acids (MAs) as a major component. MAs are long fatty acids with functional groups of precise stereochemistry. The MA composition in the cell wall has been shown to affect properties such as cell wall permeability and virulence. The functional group stereochemistry is key in the interaction with different components of the host immune system. It has been shown that MA structure determines its conformational preference. In this work, a selection of diverse computational methods is employed to study different aspects of MA conformation. The precise stereochemical effects of the cyclopropane functional groups in model compounds with short alkyl chains were investigated, through quantum chemistry. Whole MAs were simulated using molecular dynamics in the gas phase and in polar and nonpolar solvents, in order to investigate folding patterns of MAs as a function of their structure together with the external environment. Lastly, a coarse grained (CG) model of an Alpha-MA was produced and used to simulate a MA monolayer. The monolayer is a good representation of the cell wall packing of MAs and could be correlated with existing monolayer experimental data. Together, the results presented here confirm the functional groups as folding points in MAs. Larger systems that are closer to the natural MA environment are essential as they can be compared with experimental data and will be useful as a tool in combating TB. However, the integrity of the larger CG system relies on the flow of information from the smaller, more accurate computational methods through to the CG model, and hence these approaches should be continued in parallel in future work.

No description available.

No description available.

Colorectal cancer is one of the most prevalent malignancies worldwide with approximately 1 million cases diagnosed each year, and 500 000 deaths. If diagnosed early over 95% of patients would benefit from curative surgery. Large scale randomised studies have demonstrated a reduction in mortality with mass screening programmes. Current methods employed for screening for colorectal cancer involved the faecal occult blood test. This investigation maintains an average sensitivity for the diagnosis of colorectal cancer with poor compliance from the population. This study aimed to investigate and validate new biomarkers for the diagnosis of colorectal cancer. BORIS is a paralogue of the transcription factor CTCF. BORIS is found to be expressed normally in spermatocytes in the testis, however aberrant expression has been found to play a part in tumour development. This study set out to analyse the role of BORIS as a potential biomarker for colorectal cancer. BORIS expression in the leukocytes of colorectal cancer patients was assessed via both Western blotting and immunocytochemistry. Furthermore its expression was compared III colorectal cancer tissue and matched normal tissue by immunohistochemistry. Minimal positive expression was demonstrated for BORIS in A the leukocytes, with only two out of sixty patients showing positive expression by both methods. Similar findings were noted in the analysis of tissue samples, although significantly higher immunoreactivity was identified in the colorectal cancer tissue in comparison with the normal group. This study concluded BORIS has a limited role as a biomarker for colorectal cancer. A smaller further study set out to identify significant proteins as possible biomarkers in the leukocytes of colorectal cancer patients via two dimensional gel electrophoresis.

Prostate cancer is a very complex disease, which we still do not fully understand. It is the second most common cause of cancer- related death in men and therefore, it is very important to investigate the mechanisms of prostate tumorigenesis and drug development. Membrane proteins represent a valuable source of potential drug targets due to their intimate involvement in a wide variety of disease states including but not limited to cancer. In our study, we identified five drug targets, namely; EpCAM, TACSTD2, FAM3C, NCEHl and TMEM109 abundantly present in the membrane fraction of prostate cancer cell lines using proteomic approaches and validation of these proteins by other biological techniques. The cumulative data from our study indicate that global levels of these five candidates either at mRNA or protein levels are more present in prostate cancer cell lines and tissues than their normal counterparts. We also found by ICC/IHC/IF that these five drug targets are subcellularly localized in the cytoplasmic membrane region of prostate cancer cells and on the basolateral surfaces of prostate tumour tissues. Taking into account the link between T ACSTD2 and F AM3C to clinicopathological data of prostate tumour tissues stained and the MTT survival assay carried out on prostate cancer cell lines, our data suggest that overexpression ofTACSTD2 and FAM3C leads to poor prognosis in prostate cancer patients. We also tried to study the relationship between our drug targets, CTCF and BORIS. Our preliminary data shows a significant effect of CTCF and none of BORIS on our drug targets in only DU145 cell lines. Suggesting these to be cell line specific and CTCF may play a role in the transcriptional regulation of our target genes. Although, recently some of these proteins have been widely explored, their function and the role they play in cancer development still remain elusive.

This study describes the development of a quantitative tumour proteomics methodology and its implementation to study a type of breast cancer known as triple negative. The . quantitative proteomics technique deyeloped in the course of the study uses label-free mass spectrometry and size-based protein separation combined with bioinformatics and allows unlimited number of tumour samples to be analysed and compared in a highly automated manner. The methodology was applied to identify candidate biomarkers and drug targets for potential new therapies for triple-negative breast cancer. Another set of experiments builds on some of these findings, the surprising overexpression of interferon gamma-regulated genes in the lymph node-metastatic group of the analysed tumours, and examines the effect of interferon gamma on the water soluble and membrane-bound proteins of the metastatic triplenegative breast cancer cell line MOA MB231. A number of soluble and membrane proteins are identified to be affected by interferon gamma - notably proteins known to be involved in metastasis and calcium ion binding and calcium -regulated proteins. In a parallel line of investigation the effect of one of the proteins found to be overexpressed in lymph nodemetastatic triple-negative tumours, (074, on the proteome and phosphoproteome of another breast cancer cell line, M(F7 is examined. Overexpression of (074 is shown to cause sustai[1ed activation of the ERK1/2 module and also phosphorylation of the focal adhesion kinases FAK1/2. The activation of ERK1/2 is then validated by immunochemical techniques and shown to be synergistically regulated by calcium ions since calcium and (074 overexpression result in the strongest activation of the mitogen-activated protein kinase module. Thus, the quantitative phosphoproteomics studies again pinpoint calcium homeostasis, together with inflammation and mechanisms that are regulated by cytokines as important factors that contribute to the aggressiveness of triple-negative breast cancer.

No description available.

Malignant melanoma currently accounts for four percent of newly diagnosed cancers in the UK. The genetic alterations that bring about melanoma are not fully understood, but several genes and pathways have been implicated. Following oncogenic activation, somatic cells undergo an irreversible cessation of proliferation (senescence), which is thought to act as a barrier to malignancy. Oncogenic BRAF mutations (commonly BRAFv600E) are found in 50-70% of melanomas. Oncogene induced senescence is evident in benign melanocytic naevi, where BRAF mutations are often found with p 16 activity. P 16 is a tumour suppressor protein and senescence mediator often defective in melanoma. The aim of this project is to understand better the role that genetic alterations have in melanoma progression. Normal, p 16-null, and immortalised p 16-null human melanocytes were transfected with vectors that can be induced to express BRAFwT or BRAFv600E. My data suggest that low levels of BRAFvE may induce proliferation and support the hypothesis that p 16 is necessary for BRAF -induced senescence. It has now been illustrated that the proliferation rate of p 16-null melanocytes is below normal in culture and can be restored in the presence of keratinocytic growth factors (endothelin 1 and stem cell factor). Additionally, the dependency exhibited by pl6-null human melanocytes is reduced by inhibiting p53 or through immortalisation with TERT, which removes a source ofp53 signalling. The AKT signalling pathway has been shown to playa major role in mediating this response, suggesting that the keratinocyte factors signal through this pathway resulting in growth and survival. The role oftelomerase in the immortalisation of cells, through extending telomeres, has previously been well characterised. It was observed that cells immortalised with hTERT were less dependent on growth factors for survival. Further transformation assays have demonstrated that telomerase not only immortalises cells, but also transforms them altering many characteristics.

Anosmin-l, encoded by KALl gene, is an extracellular matrix (ECM)-associated protein, which plays essential roles in the migration of olfactory and gonadotropinreleasing hormone (GnRH) neurons during early brain development. Loss-of-function mutations in KALl result in Kallmann syndrome, a developmental genetic disorder with delayed puberty and anosmia. However, there is little comprehension of its role in the developed brain. Since reactivation of developmental signal pathways often contributes to tumourigenesis, I investigate if anosmin-l-mediated cellular mechanisms are involved in brain tumours. Meta-analysis of public microarray data sets and expression profile analysis of patient biopsy samples revealed that KALl mRNA was significantly upregulated in increasing grades of primary brain tumours compared to normal brain and metastasised tumours. Anosmin-l enhanced motility and proliferation of glioblastoma (GBM) cells via fibroblast growth factor receptor 1 (FGFR 1) and urokinase plasminogen activator (uPA) pathways in vitro. Anosmin-l formed a complex with ~l integrin and co-localised with active ~ 1 at the leading edge of GBM cells, inducing downstream signalling pathways including focal adhesion kinase (F AK), protein kinase B (PKBI AKT) and extracellular signal regulated kinase (ERK). Knockdown of anosmin-l attenuated cellular motility and growth but induced apoptosis. Anosmin-l also modulated fibronectin-mediated cell adhesion and activated extracellular proteinases uPA and matrix metalloproteinases (MMP)-2/9. Anosmin-l showed pro-angiogenic effects in endothelial cell culture. Combined, anosmin-l may promote GBM cell proliferation, migration, invasion, adhesion and survival. In a mouse xenograft model, anosmin-l-expressing tumours grew faster and showed increased vasculature and infiltrations. To predict the localisation of anosmin-l and its interact ants within the tumour architecture, the xenograft tissues were analysed by immunohistological methods. These studies provided novel insights for the function of anosmin-l within the tumour microenvironment, contributing to the neoplastic progression. Therefore, anosmin-l is required for normal GnRH neuronal migration, but inappropriate expression in adult brain may be a potential mechanism of malignant brain tumours.