This work is centred on the development of Raman spectroscopy methods for investigating supported lipid bilayers (SLBs). These nanoscale, biological structures have found wide application as models of cellular membranes in many areas of scientific research. They consist of phospholipid molecules that self-organise into bilayer structures containing phase-separated microdomains, which play an important role in many biological processes. SLBs are well-defined and stable under a variety of conditions, allowing characterisation with a broad range of physical methods. However, many of these techniques provide purely a visualisation of the surface or disturb the bilayer with labelling. Raman spectroscopy can offer a non-invasive chemical and structural analysis of SLBs and microdomains. A Raman microspectroscopy (RMS) system with integrated atomic force microscope (AFM) has been developed and characterised for studying SLBs. This experimental setup combines the benefits of Raman spectroscopy with the high spatial resolution of confocal microscopy. Furthermore, the incorporation of AFM makes it possible to directly correlate chemical information and spatial features. Experiments are carried out to determine the capabilities of this system for investigating SLBs. A variety of substrates are considered for this application and only prolonged expose to high laser powers is found to have any effect on the Raman spectrum of lipids. However, a single SLB cannot be detected with RMS, so focus turns to employing scatteringenhancing techniques. Surface-enhanced Raman spectroscopy (SERS) substrates formed by nanosphere lithography (NSL) are developed to be used with the combined AFM-Raman system. Simultaneous topographical imaging and highsensitivity chemical mapping of molecular monolayers deposited across these substrates reveals the distribution and magnitude of electric field enhancement that they can provide. These measurements are supported by calculations and finite element method (FEM) simulations. Then similar experiments are performed on substrates covered with a bilayer of fatty acid molecules. Considering the close similarities between these molecules and phospholipids, this demonstrates the potential of combined AFM and SERS with NSL substrates for detecting SLBs and imaging the phase-separated microdomains they form. Finally, functionalised AFM probes are developed for tip-enhanced Raman spectroscopy (TERS) using dielectrophoresis (DEP). This phenomenon is generated within a conductive AFM setup to guide nanoparticles towards an AFM probe to cluster and grow at its tip apex. This growth is monitored with force spectroscopy and a variety of imaging parameters. The probes are then analysed with scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDX) to confirm the accumulation of nanoparticles on the tip both physically and chemically. The TERS activity of these functionalised probes is investigated with the combined AFM-Raman system, which demonstrates an enhancement of scattering when the tip apex of the probe and the laser are aligned.

The study has further shown that IL-1[Special character omitted] is a potential regulator of TEM-8 expression in tumours and that IL-1[Special character omitted] significantly induced the formation of capillary-like tubules from the HECV cells, accompanied by an increase in TEM-8 expression. Where as, elimination of TEM-8 by way of ribozyme transgene significantly decreased the formation of capillary-like tubules and the mobility of HECV cells. Moreover we have demonstrated that the vW domain together with trans-membrane domain of TEM-8 are the structural domain that are responsible for the tubule forming action of TEM-8, using expression construct in varying cell lines.

The major aim of this thesis was to perform the first ever proteomics study on bladder cancer exosomes. Initially, exosomes were isolated from urine specimens but hypervariable yields and poor sample quality made proteomics analysis challenging. As an alternative approach, exosomes were isolated from HT1376 bladder cancer cells. Exosomes were purified by ultracentrifugation on a sucrose cushion, and preparations verified as high quality by immunoblotting, flow cytometry and electron microscopy. For global proteomics analysis, the sample was solubilised using SDS and DTT and subjected to LC-MALDI-TOF/TOF MS. We identified 353 proteins with high confidence and 63 of these have not previously been identified in other proteomics studies on human exosomes. Overrepresentation analysis demonstrated that the proteome was consistent with that of other exosomes with significant overlap with exosomes of carcinoma origin. Comparisons with the Gene Ontology database also highlighted strong associations with carcinoma of the bladder and other sites. A GeneGo generated protein interaction network highlighted c-Myc as a major node of protein interaction within this dataset. Several MS-identified proteins were confirmed as genuinely exosomally expressed using a combination of immunoblotting, flotation on continuous sucrose gradients, and flow cytometry. Expression was also verified in exosomes from a variety of sources, including urine. The data will aid our understanding of exosome biogenesis and function and may inform the development of urine exosome-based clinical tools in bladder cancer.

Resistance to endocrine therapy in breast cancer is associated with poor prognosis. Cell models of acquired tamoxifen resistance have implicated altered growth factor receptor signalling, especially the ErbB family of receptor tyrosine kinases, in development of the accompanying aggressive phenotype. Microarray analysis of an in vitro wtMCF-7 based model of acquired tamoxifen resistance (‘Tam-R’) identified upregulation of CD44, a transmembrane glycoprotein, known to interact with ErbB receptors and influence breast cancer progression. We investigated the hypothesis that CD44 overexpression in Tam-R cells can modulate ErbB activity and promote an adverse phenotype. CD44 gene overexpression was validated by RT-PCR and its protein expression determined by Western blotting and immunocytochemistry. CD44 contribution to intracellular signalling and phenotype of Tam-R cells (migration, invasion and growth), both endogenous and in response to hyaluronan (HA), was determined using Western blotting, immunocytochemistry and functional assays including wound healing, Boyden chamber migration, Matrigel™ invasion and growth assays in the presence or absence of siRNA-mediated CD44 knockdown. Interactions between CD44 and ErbB receptors were investigated using immunofluorescence and immunoprecipitation. CD44 was overexpressed at gene and protein level in Tam-R versus wtMCF-7 cells and whilst this did not influence the endogenous phenotype of Tam-R cells, it enhanced their sensitivity to HA as evidenced by HA-induced MAPK, EGFR and HER2 activation and increased migration. HA-induced migration was attenuated following treatment with the MAPK inhibitor PD098059, gefitinib as well as trastuzumab. CD44 was found to associate with HER2 and HER3 at the cell surface whilst HA stimulation appeared to modulate ErbB dimerisation patterns. Our data suggest that CD44 overexpression sensitises tamoxifen-resistant cells to HA thereby modulating ErbB dimerisation and enhancing migration. These observations may have importance in vivo where the tumour microenvironment can provide a rich source of HA to promote the progression of tamoxifen-resistant tumours.

This thesis examines factors influencing the outcomes of patients receiving multidisciplinary stage-directed treatment for oesophagogastric cancer. The hypotheses tested were: 1.The TNM7 staging system is a more accurate prognostic tool for oesophageal cancer (OC) than TNM6. 2.Use of CT-PET upstages a significant number of patients with occult metastases. 3.OC recurrence patterns differ following definitive chemoradiotherapy (dCRT) and surgery, but overall recurrence rates and survival are comparable for advanced stage disease. 4.An involved circumferential resection margin (CRM+) following oesophagectomy is associated with poorer survival and its incidence can be reduced with neoadjuvant chemoradiotherapy. 5.Early enteral nutrition improves clinical outcomes following upper GI cancer resection. 6.Centralisation of oesophagogastric cancer (OGC) surgery in S.E. Wales is feasible and associated with improved clinical outcomes. Reclassification with TNM7 resulted in stage re-categorisation of 11.9% of OC patients. Multivariate analysis indicated only TNM7 prognostic group to be independently and significantly associated with survival. CT-PET upstaged OC M stage in 24.0% of patients. Loco-regional OC recurrence was commoner after dCRT (p<0.0001) but distant recurrence commoner after surgery (p=0.001). Disease-free survival was better after surgery for stage I (p=0.069) and II (p=0.011) but comparable with dCRT for stage III (p=0.878) and IV (p=0.710). CRM+ occurred in 38.0% of all OC patients, and 62.4% of pT3 patients. Multivariate analysis revealed lymphovascular invasion (p<0.0001) and CRM+ (p=0.002) were independently and significantly associated with disease-free survival. Multivariate analysis revealed EUS T stage (p<0.0001) and neoadjuvant chemoradiotherapy (p<0.0001) were independently associated with CRM+. Early enteral nutrition (EEN) was associated with reduced hospital stay (p=0.023) and less operative morbidity (p=0.044) than control management, due to fewer wound infections (p=0.017), chest infections (p=0.036) and anastomotic leaks (p=0.055). Following centralisation, OGC critical care (p<0.0001) and total hospital stay (p=0.037) were significantly reduced. Serious operative morbidity (Dindo-Clavien grade III+) decreased from 33.3% to 16.7% (p=0.066).

CREB, cAMP responsive element binding protein is a positive regulatory protein transcriptional factor, for genes including aromatase, an enzyme that converts androgens to oestrogens, c-fos, tyrosine hydroxylase and neuropeptides like somatostatin and enkephalin. The expression of aromatase is highly aberrant in human breast cancer and has been implicated in the disease progression. Aromatase expression in breast cancer tissue is directed mainly by promoters 1.3 and II. CRE1 and CRE2 are essential for cAMP induced promoter II activity. CRE binding protein (CREB) bound to this element (CRE) and that this interaction was enhanced in the presence of cAMP. Despite the extensive work on aromatase, little information is available on the expression and role of CREB in human breast cancer. The aim of this study was to investigate the molecular impact of CREB family of proteins on the aggressive nature of breast cancer cells and to investigate the expression pattern in breast cancer tissues in relation to tumour histopathological grade, stage, nodal status and the clinical outcome of the patients. In this study we examined the expression of CREB1 and ATFs (Activating transcription factors) in breast cancer cell lines using RT-PCR, which allowed us to design the strategy of in vitro experiments. Ribozyme knockdown technology was used to target the expression of CREB1 in a breast cancer cell line MDA-MB-231. Knockdown of CREB1 using ribozyme transgenes resulted in decrease in in vitro cell growth and invasiveness in breast cancer cells. The results presented here demonstrate that the level of CREB-1 and ATFs in breast cancer patients was elevated. The study results presented here revealed a significant link between CREB and mortality, in that high levels are associated with shorter disease free survival and interestingly we found significantly low levels of ATFs in patients with poor prognosis, metastatic disease and nodal involvement. We conclude that the level of CREB-1 and ATFs are aberrantly expressed in human breast cancer which may be associated with disease progression in breast cancer patients and has significant bearing to the clinical outcome of the patients. Over-expression of aromatase in adipose tissue surrounding breast tumour could arise through increase in both CREB expression and CREB transcriptional activity. Inhibition of CREB activity could inhibit aromatase expression and hence decrease oestrogen production in breast tissue. An understanding of the molecular mechanisms of expression of CREB, together with aromatase between non-cancerous and cancerous breast tissue at both transcriptional and translational levels may help in the design of a therapy based on suppressing aromatase expression in breast cancer tissues.

Wnt signalling components are reported to be deregulated in breast cancer but the contribution of this pathway in endocrine resistance is less clearly defined. Endocrine resistance is an important clinical challenge affecting up to a quarter of all breast cancer patients and is associated with a poorer clinical prognosis. This project focussed on exploring the role of Wnt signalling in endocrine resistant breast cancer cell models. Wnt pathway elements were deregulated in the acquired tamoxifen resistant cell line (Tam-R) compared to tamoxifen sensitive parental cells (MCF-7), with changes supportive of Wnt signalling activation in this tamoxifen resistant model apparent from Affymetrix HGU-133A gene microarray data and Western blot analysis. In contrast, Wnt signalling appeared to be suppressed based on Affymetrix data for MCF-7 cells treated with oestradiol for 10 days, with equivocal changes in MCF-7 cells treated with tamoxifen for 10 days or a faslodex resistant cell model (Fas-R). Excitingly, Tam-R cells were also more sensitive than MCF-7 cells to pharmacological manipulation of Wnt signalling. While Wnt activation using Wnt3a and LiCl did not affect cell growth or migration, inhibition of Wnt signalling usingIWP2, PNU 74654 and iCRT14 suppressed Tam-R cell growth and migration. There is mounting evidence of cross talk between Wnt and EGFR signalling in breast cancer, and EGFR activity is upregulated in Tam-R cells. The project’s findings tentatively supported cross-talk between the two signalling pathways in this model. Thus, targeting of the Wnt pathway alongside EGFR blockade was superior in suppressing cell growth and migration in Tam-R cells. The effect appeared to be more pronounced when Wnt signalling was inhibited at the nuclear level using iCRT14. Collectively, these data suggest that Wnt signalling may play an important role in tamoxifen resistance where it may offer an opportunity for more effective therapeutic intervention to control relapse and associated tumour aggressiveness.

DNA methylation helps regulate transcriptional activity and is widely studied in cancer biology. This investigation aimed to establish the significance of Human Papillomavirus (HPV) DNA methylation in HPV-associated disease both in terms of basic biology and as a potential biomarker. Assays to assess DNA methylation and gene expression were developed and evaluated. Pyrosequencing was used to assess DNA methylation of four regions of the HPV16 genome (E2, L1/L2, enhancer, promoter). Gene expression was assessed using quantitative PCR with assays for E2, E6 and E7. HPV integration was assessed using Detection of Integrated Papillomavirus Sequences (DIPS). The relationship between HPV methylation, gene expression and integration was explored in vitro and in vivo using cell cultures and clinical cohorts. A variety of sample materials were used including short term and immortal cell lines, cervical cancer biopsies, cytology samples and Vulval Intraepithelial Neoplasia (VIN) biopsies. In general, hypermethylation of the HPV genome was associated with low HPV gene expression and the presence of integrated HPV genomes. To better understand the potential clinical utility of HPV DNA methylation, the relationship between HPV DNA methylation and various stages of cervical disease was determined. The HPV genome was progressively hypermethylated with increasing severity of cervical disease and certain regions of the HPV genome were more affected than others. A longitudinal study was also performed in order to determine a relationship between HPV methylation and clinical outcome. Differences in HPV methylation among patients who had persistent HPV infection and low grade disease, persistent infection and high grade disease and patients that cleared HPV infections were observed. Throughout the study the potential application of a HPV biomarker was considered and the correct biomarker design procedures were referred to. Several of the early biomarker development steps were successfully achieved.

Cervical cancer is the second most common cancer among women worldwide. Infection with Human Papillomavirus (HPV) is essential but not the only contributing factor in cervical cancer development. HPV integration is reported to be present in over 80% of cervical cancers and disruption of HPV genome through integration leads to high levels of HPV oncogene expression. DNA damage and repair pathways are thought to induce HPV integration since HPV is detected at fragile sites in the human genome. There is controversy as to whether integration is an early or late event in cervical oncogenesis and there are no published studies to date that have investigated HPV integration using sensitive, DNA based, techniques at the nucleotide level in cervical precancers. This study aimed to test the hypothesis that integration is an early event in cervical neoplasia and episomal loss causes malignant transformation through transcription of integrated HPV. Also, this study served to pilot whether HPV integration can predict high-grade cervical disease in women with cytological abnormalities with an aim to improve current cervical screening methods. Assays to detect integration and E2 as a marker of episomal state were developed for HPV16, HPV18 and HPV45 and applied to cervical smears and biopsies from women with varying disease grades. The data presented in this thesis highlight that integration may not be essential for cervical cancer progression and different modes of disease progression may exist between young women and older women. Integration was detected at chromosome fragile sites but was more prevalent at SINE or LINE repeat elements; this implies a role for retroelements in the mechanism of integration. Finally, the data here suggest that integration induces a unique selective process in each individual and clonal selection may arise due to altered HPV oncogene expression and/or disruption to human gene expression.

LKB1 has been implicated in a wide range of cellular functions and is associated with many potential substrates in in vitro studies, however the in vivo role of LKB1 remains unclear and its precise contribution to the prevention of intestinal tumours in the hereditary Peutz-Jegers syndrome is as yet uncharacterised. Conditional deletion of LKB1 in the murine small intestine resulted in significant disruption of intestinal homeostasis, particularly that of the differentiation process, suggesting LKB1 plays a key role in intestinal differentiation and it is loss of this function that predisposes to tumourigenesis