Obesity is a major health problem worldwide and its effects on the cardiovascular system are well documented. It also leads to the development of metabolic disease such as insulin resistance and diabetes. There is evidence that obesity leads to an increased risk of developing numerous malignancies. Indeed, obese individuals diagnosed with malignancy tend to have poorer outcomes in terms of survival. A possible explanation for this is through the action of obesity-related cytokines (adipokines). These may play a role in either propagating, or perpetuating carcinogenesis and I explored the role of one particular adipokine: chemerin in prostate cancer. Prostate cancer cell lines (PC3 & LNCaP) were used for cell proliferation, migration, invasion and apoptosis assays. Western blot analysis and qRT-PCR techniques were used to evaluate the effects of chemerin on levels of key intracellular agents of carcinogenesis (bcl-2, p53, ERK and AKT) as well as novel, pro-cancerous genes such as anterior gradient 2 (AGR2). Serum samples were obtained from adult men with prostate disease to evaluate whether chemerin is associated with body parameters. Chemerin exerts positive effects on cellular proliferation and migration as well as inhibition of apoptosis in prostate cancer cells. These effects may be mediated by increased expression of the oncogene: bcl-2. Bcl2 expression was elevated in both cell lines after 24 hours stimulation with chemerin at increasing doses. Chemerin also appeared to cause activation of ERK and AKT pathways in prostate cancer cells as well as increased expression of the pro metastatic AGR2 gene at both the mRNA and protein level. An ELISA demonstrated chemerin behaving as an adipokine in adult men with prostate disease in keeping with previously published data. Chemerin certainly appears to play a role in prostate carcinogenesis, at least at the cellular level.

This thesis is focused on the development of Raman micro-spectroscopy for label free imaging and discrimination of stem cells. The thesis is divided into six chapters. Chapter 1 gives an overview of the existing techniques used for molecular analysis of cells, with emphasis on methods that allow non-invasive label-free imaging. A literature review of the main relevant applications of Raman micro-spectroscopy for imaging cells was also included. Chapter 2 discusses the basic theoretical principles of Raman scattering and design of Raman micro-spectrometers. The practical aspects related to the design of an optimised Raman micro-spectrometer are presented in Chapter 3 along with experimental characterisation of its performance. The chapter concludes with examples of Raman spectral maps of endothelial cells. Chapter 4 and Chapter 5 present experimental results obtained by Raman micro-spectroscopy for molecular analysis of live neural and mesenchymal stem cells. In these investigations Raman spectroscopy was used to identify, image and quantify spectral markers for label-free discrimination between glial cells and their neural progeny. The potential of Raman micro-spectroscopy to measure timecourse molecular changes of individual bone nodules was demonstrated in Chapter 5. Future work and final conclusion are discussed in Chapter six of this thesis.

This thesis explores the dynamics of optomechanical systems, which use radiation pressure to couple together optical and mechanical modes. Such systems display dynamics ranging from the quantum to the classical, with a variety of applications including ground state cooling and precision measurements. In this thesis two different geometries are presented for such a system in the form of the ‘reflective’ and ‘dispersive’ systems. Different aspects of the dynamics are investigated numerically and analytically. Firstly the reflective system is introduced, which consists of a cavity formed from a fixed and a moveable mirror. The optical frequency of the cavity couples linearly to the moveable mirror’s position. This geometry is explored as the cavity is driven by a laser, revealing a range of dynamical states in the mirror as the drive frequency is varied. An alternative geometry is presented in the form of the dispersive optomechanical system. Two fixed mirrors with a partially transmitting membrane at the centre provide a cavity supporting two optical modes, that couple approximately linearly or quadratically to the membrane position, depending on where the membrane is fixed. The system is explored in both linear and quadratic coupling regimes. Quadratic coupling is explored for a single optical mode by selecting a high tunnelling rate through the membrane. The dynamics of the membrane are explored via a similar set of techniques to those applied to the reflective system. Linear coupling for two optical modes is explored in the regimes of blue and red detuning. First resolved sideband cooling is explored, providing an alternative approach ground state cooling (which has been explored for the reflective case). Finally, strongly driving the system over a range of coupling strengths induces classical behaviour, extending from limit cycle oscillations to chaotic motion.

There is a great need for the identification of non-invasive biomarkers for early detection, prognosis and treatment efficacy of breast cancer. Peripheral white blood cells (WBCs) carrying the information related to the presence of cancer, represent an attractive source for novel biomarkers. The main aims of the study were to develop the pipeline to discover and validate novel biomarkers in WBCs of breast cancer patients using proteomic and genomic approaches, and assess these biomarkers’ utility. Using the highthroughput mass spectrometry and 2D-gel-electrophoresis, the protein profiles of the WBCs from breast cancer patients and healthy individuals were generated and compared with publicly available gene expression data from the WBCs of breast cancer patients and the information on protein profiles of the WBCs from the metastatic breast cancer patients. The shortlisted 15 genes were then validated using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). The mRNA levels of ITGA4, LCN2, CPNE3 and SERPINB1 were found to be altered significantly in the WBCs of breast cancer patients. The levels of SERPINB1 (Serpin B1, neutrophil elastase inhibitor) and CPNE3 (Copine 3, phospholipid binding protein) were assessed using Western blotting. These analyses demonstrated the association of SERPINB1 with breast cancer metastases, and suggested its potential utility as a biomarker of poor prognosis and treatment efficacy. Further quantitative validation of SERPINB1 in a larger panel of WBCs by ELISA will be required for a clinical phase in the biomarker development pipeline. No conclusive results were obtained for CPNE3 and, together with ITGA4, LCN2 and other additional candidate biomarkers (to be selected from the initial list), they will be tested further. The data generated for this study has also given insight into differences in the molecular portraits of the cells of immune system associated with breast cancer, which will need to be validated by laboratory based functional assays.

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Colorectal cancer is a major health problem, and many patients relapse despite apparently curative treatment. Local recurrence is an important factor, and is more common when cancer cells are present on the free serosal surface or circumferential resection margin. We hypothesised that detecting tumour cells in the peritoneal cavity during surgery (in peritoneal washings) or post-operatively (in drain fluids) would act as a marker for risk of local recurrence, and that the use of molecular biological techniques would allow for the sensitive detection of such cells. We collected samples of colorectal tumours, with washings from the peritoneal cavity at the start and end of surgery, and fluid from the surgical drain on the first and second postoperative day. Epithelial cells in the peritoneal samples were enriched using magnetic cell separation (MACS). Using mutation specific PCR or a Mismatch Ligation Assay we detected common mutations of K-ras, TP53, APe and BRAF in primary tumour samples, and when such a mutation was found, we studied the peritoneal samples from that patient for the same mutation. We detected 23 mutations in 22 (out of 46) tumours from 21 patients. In 16 patients, at least one of the peritoneal samples gave a positive result for the same mutant DNA. Using MACS increased the proportion of positive samples. Half of the patients with positive peritoneal samples had Dukes' stage A or B tumours. Follow up is not yet long enough to allow conclusions to be drawn on the significance of these results. We have described techniques allowing mutations to be characterised in half of colorectal tumours and have demonstrated the presence of cells with the same mutation in peritoneal samples from three-quarters of patients. Longer follow up will show whether our tests are too sensitive, or whether they provide useful information about likely local recurrence.

The genomes of higher eukaryotes are marked by distinct chromatin domains, which allow for the control of different gene expression states. It is thought that the boundaries of chromatin domains could be formed by DNA sequence elements called insulators. The paradigm HS4 insulator element is located at a boundary between the β-globin gene cluster and an adjacent condensed chromatin domain. Proteins that bind to the HS4 sequence recruit enzymes that mediate a number of histone modifications generally associated with chromatin accessibility. Inspired by yeast genetic studies, we hypothesised that H2B ubiquitination might be a key regulator of these ‘active’ marks. It was found that HS4 and another chromatin boundary at the neighbouring FOLR1 gene locus, HSA/HSB, are sites of H2B ubiquitination. The ubiquitination E3 ligase RNF20 was found to be necessary for global H2B ubiquitination and for methylation of H3K4, in a trans-histone modification pathway that is conserved from yeast to man. RNAi-mediated knockdown of RNF20 not only resulting in the depletion of H2B ubiquitination normally found at chromatin boundaries, but also disrupted their H3K4 methylation and acetylation at multiple histones. H2B ubiquitination is a master controller of the active chromatin state at the HS4 and HSA/HSB chromatin boundaries. Long term depletion of RNF20 expression leads to a compromise of the boundaries, allowing the spreading of heterochromatin into the FOLR1 and β-globin gene loci, resulting in gene silencing. This study also looked at the recruitment of factors that mediate the incorporation of the histone variant H2A.Z at chromatin boundary elements in vertebrates. It was found that insulator binding proteins control H2A.Z incorporation and acetylation.

Polymer distributed Bragg reflectors (DBRs) were prepared by spin-casting alternating layers of polystyrene (PS) and poly(vinylpyrrolidone) (PVP) from mutually exclusive (orthogonal) solvents. These all polymer photonic structures were prepared using a purpose built automated spin-coater system. Samples were prepared with targeted optical properties such as the wavelength position, intensity and bandwidth of reflection peaks. The wavelength position of the reflection peaks was controlled by the deposition spin-speed used during sample preparation. Reflectance was controlled by the number of layers deposited onto the sample. The bandwidth was increased by chirping the layers in the photonic structure. Reflection bands were measured in the UV/visible region of the spectrum using two different (transmission and reflection mode) purpose built spectrometer set-ups. Measured reflection bands had narrow bandwidths between 10nm and 20nm. Chirping these photonic structures broadened the peaks to bandwidths of ~ 50nm. A 100 layer PVP/PS DBR had a total reflectance of 93 ± 1%. The wavelength of the reflection peaks from flat DBR samples blue-shifted when measured away from normal incidence. This was reduced when corrugating a DBR by wrinkling the films with mechanical strain. The wavelength of the reflection band from a corrugated DBR remained constant when the sample was rotated. Thus improving the angular dependence of the structures. Fourier transform infra-red spectroscopy was used to measure reflection bands which were between wavelengths of 1600 nm and 2700 nm. These reflection bands had narrow bandwidths between 40 nm and 60 nm. The largest reflectance measured within the infra-red spectra was 80 ± 1% from a 50 layer PVP/PS DBR. A modified optical transfer matrix method was used to model the optical properties of the DBRs. Changes in the refractive index contrast (between 0.020 and 0.028 for 30 layer PVP/PS DBRs) were needed to fit the model to the measured UV/visible spectra. It was concluded that trapped solvent (from sample preparation) was lowering the refractive indices of the layers. The polymer-polymer interface widths of spin-cast polymer multi-layers were measured using neutron reflectivity. Each polymer-polymer interface width was less than 1nm throughout the DBR samples. The polymer multi-layer samples were measured using time of flight secondary ion mass spectrometry (TOF-SIMS). An Ar2000+ sputtering source was used to etch through the multi-layer samples. It was concluded that the thickness of spin-cast films did not change when preparing a multi-layer structure. However, other techniques, such as ellipsometry, are more suitable for measuring the thickness of films. The TOF-SIMS technique was unable to measure polymer-polymer interface widths in multi-layer samples. This was due to the sputtering beam roughening/mixing the polymers at the interfaces. It was concluded that PVP/PS DBRs could be used as inexpensive narrowband reflectors/filters. However, alternative polymer systems may be more useful for other applications which require a greater reflectance. This includes creating resonant cavities to improve the efficiency of optical devices (such as LEDS and solar cells). The results and techniques from these experiments are useful for further development in polymer photonic structures and polymer multi-layer devices.

The aims of this study were to develop novel in vitro models of Human Papillomavirus (HPV) -associated vulval and vaginal neoplasia and to use them to investigate the mechanism(s) of action of the nucleoside analogue, Cidofovir (CDV), for which a mechanism is unknown. Single cell clones were successfully isolated from heterogeneous vulval and vaginal parental populations. The clonal cell lines were characterised morphologically and in terms of cell proliferation; a range of growth rate and morphological variants were identified. Clonal lines were also characterised with respect to HPV integration state using Amplification of Papillomavirus Oncogene Transcripts (APOT) and Detection of Integrated Papillomavirus Sequences (DIPS). Cell lines were identified that represented naturally occurring episomal and integrated HPV infections. HPV gene expression analysis was also performed using quantitative real-time reverse transcription PCR (qRT-PCR) which displayed different expression profiles for each line. Patterns of HPV gene expression appeared to correlate with gene disruptions associated with integration. Characterised clonal lines were used to investigate the effects of CDV on cell viability, morphology and HPV gene expression. CDV (10 μM) reduced cell viability in all HPV-positive clonal lines and HPV negative HEK cells; viable cell counts showed that a more considerable response was detected in vulval lines compared to vaginal lines and that response in HEK lines was similar to vulval lines. Cell enlargement was observed in response to treatment in the clonal lines but not the HEK line. CDV treatment did not cause significant reduction in expression of HPV E6 or E7. mRNA sequencing confirmed HPV integration and gene expression profiles. Differentially Expressed Gene (DEG) analysis identified a large percentage of the top 20 most significant Gene Ontology (GO) categories to be involved in nucleotide synthesis and RNA polymerase activity. Gene Ontology Over Representation Analysis (GO-ORA) showed that 4 GO categories relating to cellular senescence were found in over 300 GO categories comprising the top 500 most significant gene transcripts.

Prostate cancer metastasis is a complex process, involving multiple pathways in its orchestration. Malignant cells are influenced by different growth factors from the extracellular environment which promote or inhibit cell movement and metastasis. HGF has been implicated in progression and metastasis of prostate cancer. A cell interacts with the environment through surface molecules like integrins. These interactions are further translated in to different responses through various intracellular machineries. Furthermore organization of the actin cytoskeleton is vital for many cellular functions. WAVEs are member of WASP family of proteins, which have important role in regulation of actin dynamics through regulation of actin related protein (ARP 2/3). The role of individual members of WASP family has been investigated in development and progression of different cancers. We documented the expression of different WAVE family members in various prostate cancer cell lines. Expression of WAVE-3 was effectively knocked down with the use of hammer head ribozymes. Loss of WAVE-3 expression resulted in reduced cell movement and invasion in the PC-3 cell line. These cells failed to show any significant increase in cellular movement and invasive potential following treatment with HGF. Further experiments to investigate the underlying mechanism of this phenotypic change revealed that optimum levels of phosphorylated paxillin play an important role in this change. Our study also indicates that reduced potential of invasive capability following WAVE-3 knock down, may be related to reduced availability of MMP-2 in the cellular environment.