Background: Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although the effects as a single agent are limited. This investigation determined whether the Histone Deacetylase (HDAC) inhibitor SAHA, or inhibitors of the histone methyltransferases G9a and EZH2 (BIX 01294 and GSK343) and Nuclear export inhibitor (NEI) LMB, enhance TRAIL-induced apoptosis and overcome TRAIL resistance in both suspension culture, and 3D cell culture as a model of solid disseminated MM lesions that form in bone. Methods: The effects of TRAIL sensitizers and/or TRAIL treatment were investigated in both suspension cultures and in an alginate-based 3D culture model. Apoptosis was detected by assessment of nuclear morphology using Hoechst 33342/PI staining. TRAIL-resistant cells were generated by acute exposure of TRAIL sensitive cells to TRAIL followed by the selection of TRAIL-resistant cells (TRAILR). Apoptotic effects in quiescent cells (labelled as PKH26Hi) were also determined. Subsequently, an investigation was undertaken to identify potential mechanisms of action of these agents when used alone and in combination with TRAIL. Results: TRAIL significantly induced apoptosis in a dose-dependent manner in OPM2, RPMI 8226, NCI-H 929, U266, JJN3 human MM cell lines and ADC-1 plasma cell leukaemia cells. All epigenetic modifiers and NEI synergistically enhanced TRAIL responses in several lines and responses were potentiated in 3D culture. Interestingly, TRAILR cells were sensitive to BIX 01294 and LMB; however, TRAIL responses in cells that had been selected for TRAILR were not further enhanced by SAHA and GSK343. Quiescent PKH26Hi cells were resistant to dual therapy. Mechanistically, TRAIL and TRAIL sensitizers induced apoptosis via both extrinsic and intrinsic pathways in addition to decreasing the expression of oxidative enzyme catalase. Conclusions: Inhibitors of HDAC, EZH2 and G9a and NEI are potent sensitisers of TRAIL responses both in suspension, and crucially in 3D cell culture, which may mimic physiological aspects of bone metastases. These agents may be a therapeutic option in combination with TRAIL and may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance, and not in quiescent cells.

Cancers of the breast and colorectum are two of the most frequently diagnosed malignancies worldwide and despite improvements in treatment and survival, are leading causes of death. Reproductive factors, such as age at menarche, childbirth and age at menopause have been consistently linked to risk of developing breast cancer while exposure to exogenous hormones through use of oral contraceptives (OC) and hormone therapy (HT), are associated with increased risk of breast cancer but a reduced risk of colorectal cancer. These observations support the hypothesis that variation in endogenous hormone pathways contribute to breast and colorectal cancer development. However, while a substantial body of literature has established the significance of reproductive factors and exogenous hormone use in the initiation of these tumours, data on their association with mortality among breast and colorectal cancer patients are limited. To further knowledge on the role of reproductive factors and exogenous hormone use on the natural history of breast and colorectal cancer, a comprehensive assessment of these factors in relation to breast and colorectal cancer development as well as survival was undertaken among participants of the European Prospective Investigation into Cancer (EPIC). Specifically, among 150,251 women defined as postmenopausal at study baseline, the association of age at first and last menstruation, history of pregnancy, number of full-term pregnancies, the age at birth of the first and last child, breast feeding, accumulative breast feeding duration, and ever use of oral contraceptives and hormone therapy was investigated in relation to (i) breast cancer incidence, (ii) all-cause and breast cancer-specific mortality amongst breast cancer patients, (iii) colorectal cancer incidence and (iv) all-cause and colorectal cancer-specific mortality among colorectal cancer patients. Statistical analyses were conducted using Cox proportional hazards modelling with adjustment for established breast and colorectal cancer risk factors. In multivariable analyses, a later age at menarche was associated with associated with 11% reduced risk of breast cancer ( hazard ratio [HR] ≥15 vs 12 years = 0.89, 95% confidence intervals [CI]= 0.80-1.00; P-trend=0.0001); older age at menopause was found to be associated with increased risk of breast cancer (>55 vs ≤50 HR=1.27, 95% CI=1.10-1.47; P-trend < 0.0001); further giving birth to at least one child significantly reduced the risk of developing breast cancer by 16% (HR=0.84, 95% CI= 0.77-0.91) and reduced the risk of all-cause and breast cancer-specific death among breast cancer patients by 37% (HR=0•63, 95%CI: 0.46-0.86) and 45% (HR= 0.55, 95% CI= 0.37-0.82) respectively, compared to nulliparous women. Women with >4 children were at 26% reduced risk of breast cancer expressing oestrogen receptors (ER) compared to women who had one child (HR=0.74, 95%CI: 0.64-0.86; P-trend=0.002). Breast cancer patients who expressed the ER and had at least one pregnancy were at 57% reduced risk of death compared to nulliparous patients who expressed ER (HR= 0.43, 95%CI: 0.26-0.71). The use of exogenous hormones in the form of oral contraceptives and postmenopausal hormones was found to reduce the risk of developing colorectal cancer in postmenopausal women by 15% (OC users vs non-users HR= 0.85, 95%CI: 0.76-0.95) and 14% (HT users vs non-users HR= 0.86, 95% CI= 0.0.79-0.96). However, it seems that hormone therapy users have 28% higher risk of fatal colorectal cancer compared to non-users (HR=1.28, 95%CI: 1.01-1.61). The results of this large-scale prospective investigation indicate that reproductive factors and use of exogenous hormones influence the development of breast and colorectal cancer and may also be associated with survival among patients with these malignancies. If confirmed in other studies, these findings may be further explored in prognostic modelling and may help inform treatment and surveillance of high-risk groups.

Glioblastoma multiforme (GBM) are the most aggressive and devastating tumours of the brain and are essentially incurable. They are defined by diffuse invasion of the surrounding brain parenchyma along preexisting structures like the vasculature. Glioma stem cells (GSC) are thought to be largely responsible for tumour recurrence following treatment due to their high resistance to therapy, their ability to recapitulate tumours from single cells and their marked invasive potential. Here we show, that normal neural stem cell in the subventricular zone are compartmentalised by endothelial ephrinB2 and their proliferation limited through activation of p53 in an Eph signalling dependent manner. GSCs however evade both compartmentalisation and proliferation inhibition and are able to invade perivascularly. Intravital imaging, coupled with mechanistic studies in vitro revealed that upregulation of ephrinB2 in highly aggressive, mesenchymal GSCs enables escape from endothelial compartmentalisation through homotypic forward signalling. Surprisingly we also find that that ephrinB2 reverse signalling promotes tumourigenesis by mediating anchorage-independent cytokinesis through activation of RhoA. In preclinical models using human GSCs we show, that inhibition of ephrinB2 by RNA silencing or with ephrinB2-blocking antibodies strongly suppresses tumourigenesis of established glioblastoma by inducing cell-cycle arrest and blocking GBM/vascular interactions. Thus, ephrinB2 is an oncogene and represents an attractive candidate for anti-GBM therapies aimed at eradicating the GSC compartment by targeting both glioma invasion and proliferation.

MicroRNAs (miRNAs) are short, non-protein coding RNAs. These small-RNAs can regulate gene expression by either inhibiting translation or promoting mRNA degradation, they are also involved in diverse physiological and pathological events. Previous data from our group, showed that phenobarbital (PB, a CAR activator), induces dysregulation of the hepatic miRNAome in rat livers. In this study, we aim to determine if non-genotoxic rodent carcinogens can deregulate miRNA expression. Thus, the liver miRNAome associated with a novel chemical (SYN) and the non-genotoxic rodent carcinogen PB was explored at different doses and time points using high-throughput sequencing of small-RNAs (miRNA seq), a powerful tool for discovery and profiling of miRNA expression, providing a deeper understanding of molecular toxicology. Male Han Wistar rats were treated with PB (50 or 1000 ppm) or SYN (50, 500, and 3000 ppm) in the diet for up to 28 days. Control animals were fed regular diet. Rats were sacrificed after 1, 3, and 28 days. Total RNA was isolated from frozen livers and library constructs were generated with the Illumina platform. The miRanalyzer algorithm and DESeq were used for bioinformatics (for known and novel miRNAs) and statistical analysis. Around 200 known miRNAs were detected per sample. Three key miRNAs related to the epithelial-to-mesenchymal transition (EMT), were detected at the 3rd day –miR-200s, miR 30b, and miR-21. Consequently downregulation of targets related to these RNAs and EMT (ZEB1/2, Snail-1, PTEN, and E-cadherin) was seen. These findings suggest activation of a homeostatic response in EMT control in response to PB/SYN treatment, presumably in order to preserve the epithelial nature of hepatocytes. Analysis of putative novel miRNAs sequences determined three candidates homologous to miR-4488 and a putative isomiRNA of miR-6215. The miRNA-seq approach enables a more detailed exploration of miRNAs and their response to hepatotoxicants, such as PB, helping elucidate underlying pathway perturbations.

High grade serous (HGS) ovarian cancer and pancreatic ductal adenocarcinoma (PDAC) are poor prognosis neoplasms that are characterised by high levels of genomic instability, DNA damage repair (DDR) defects, and drug resistance which ultimately results in poor survival rates. This study aims to use markers of genomic instability, in the form of copy number variations (CNVs) and gene expression changes, to identify DDR gene aberrations which contribute to poor patient prognosis and drug resistance in HGS ovarian cancer and PDAC, and which may be exploited for therapeutic benefit. Bioinformatic analyses of HGS ovarian cancer patient datasets (TCGA, Genome Institute of Singapore) were used to determine whether gene pairs co-amplified together, where one of the partners is a DDR gene, associated with poor patient prognosis. RECQL, a DNA helicase, was found to be co-amplified with 6 genes from the 3q12 locus, CPOX, MINA, TMEM45A, TOMM70A, CLDND1 and PTPLB, and all of these co-amplifications were found to significantly associate with reduced PFS in ovarian cancer patients. Functional validation, using siRNA to inhibit gene expression, was carried out to assess the role of these genes in regulating survival. Using a cell line (PEO4) model of chemotherapy resistant HGS ovarian cancer, no induction of apoptosis or changes in long term survival was observed upon silencing of any of the genes. In comparison, gene silencing of RECQL and PTPLB was found to induce an apoptotic response in PDAC cell lines (Panc1 and MiaPaCa2). Long term clonogenic assays also revealed that TMEM45A, TOMM70A and CLDND1 are regulators of Panc-1 cell survival. Together this indicates that in PDAC, the survival advantages of RECQL co-amplification with the 3q12 chromosomal region may be related to the effects of more than one gene. However, the PTPLB:RECQL co-amplification may be a driver of PDAC cell survival. Bioinformatic analyses were also carried out on HGS ovarian cancer and PDAC patient datasets (TGCA and ICGC) as well as cell line datasets (Sanger Institute) aimed at determining single genes within DDR pathways whose aberrations in copy number and gene expression impacts upon patient survival or drug response to cisplatin or gemcitabine. 6 genes, TSTA3, RECQL4, ESRP1, NBN, SUMO3 and EP300, were identified that affected one of the above parameters, of which, EP300 functionally validated as a potential therapeutic target. SiRNA-mediated silencing of this was found to induce apoptosis in cell line models of HGS ovarian cancer (SKOV3) and PDAC (Panc-1). In addition, loss of EP300 increased the apoptotic response of SKOV3 cells to treatment with cisplatin, gemcitabine, doxorubicin, paclitaxel and the DNA-PKcs inhibitor NU7441. In Panc1 cells, only response to gemcitabine and paclitaxel was significantly increased with EP300 loss. Mechanistic studies to define how EP300 regulates SKOV3 cell survival found that EP300 gene silencing induced dysregulated DNA damage recognition, and cell cycle arrest at the G2/M phase. RPPA proteomic analysis identified Wee1, Plk1, cyclin B, MAPK1 and histone 3 as potential mediators of the cell cycle arrest observed. Many changes to apoptotic signalling were also observed after loss of EP300, although this counter-intuitively, increased the levels of anti-apoptotic proteins, such as Bcl-xl, Bcl-2 and pS136 Bad. Also, pro-survival Akt activity, determined as phosphorylation at S473, was reduced by EP300 gene silencing. This indicates a novel link between p300 and Akt-mediated cell survival of HGS ovarian cancer. Overall, this study has combined in silico analyses of patient and cell line datasets with functional in vitro validation to identify RECQL and EP300 as potential therapeutic targets for HGS ovarian cancer and PDAC. Future work to validate these in additional cell line models, including those with defined genomic backgrounds, will further progress these as novel therapeutic strategies.

This thesis investigates the epidemiology of tuberculosis (TB) disease in populations. It applies molecular epidemiological methods to elucidate the relative effects of HIV on TB disease following recent first infection, reactivation and recent reinfection with Mycobacterium tuberculosis (Mtb). Finally it aims to explore the impact of Antiretroviral Therapy (ART) on the incidence of TB in a population in rural Sub Saharan Africa. The data in chapters 2 and 3 is taken from a systematic literature review I performed of population based studies that reported TB molecular epidemiological data. In the subsequent chapters I analyse existing and newly collected data from the Karonga Prevention Study, set in Northern Malawi, to address the research questions. The results strongly suggest that HIV-infection increases an individual's risk of TB disease due to recent Mtb (re)infection more than through reactivation in populations with generalised HIV epidemics. The last chapter suggests that patients on ART experience a high risk of TB compared to HIV positive/ART naive patients, especially in the first months after initiating ART. Also, it appears that after the introduction of ART in 2005 TB incidence in Karonga District plateaued after declining in the previous 10 years. These findings strongly suggest that TB programmes in areas with generalised HIV epidemics should focus more of their efforts on reducing Mtb transmission. Improved collaboration between TB and ART programmes may help to reduce TB rates in the highly vulnerable ART receiving population and subsequently in the general population.

Phase III trials for new tuberculosis treatment regimens require large numbers of participants and can take over five years to complete. A surrogate marker for poor outcome (failure at end of treatment or recurrence following successful treatment), the established endpoint in such trials, could shorten trial duration and reduce trial size. Culture results after two months of treatment have shown the most promise but, prior to this research, no formal evaluation had been performed. In this thesis, culture results during treatment are evaluated as prognostic and surrogate markers for poor outcome using data on 6974 patients from twelve tuberculosis treatment randomised controlled multi-arm trials conducted in East Africa and East Asia. A strong association was found between culture results during treatment and poor outcome. Nevertheless, culture results were not good patient-specific predictors of poor outcome with low sensitivities and specificities. Existing meta-analytic methods for evaluating surrogate markers are not wholly suited to this setting of multi-arm trials with binary true and surrogate endpoints. Extending these methods, the two month culture was found to be a good surrogate marker using data from Hong Kong trials and the three month culture was found to be a good surrogate marker using data from East African trials. These results are an indication that cultures during treatment do capture some of the treatment effect. Further work is needed in understanding the differences between the Hong Kong and East African trials. The meta-analytic methods for evaluating surrogate markers in this thesis included a graphical representation that permitted a clear visual evaluation of the surrogate. Methods developed in this thesis for modelling the relationship between the treatment effects on the true and surrogate endpoints were not satisfactory. The deficiencies were not overcome with the two extensions proposed. Further work is needed in developing a more appropriate model.

More than 85% of the global burden of cervical cancer occurs in developing countries, where it is the second most common cancer among women. In Brazil alone, a total of 17,500 new cases and 3,300 deaths of cervical cancer are expected in 2012. Despite the investments in cytologybased screening in the country, the reduction of cervical cancer incidence has been less than expected. The aim of this thesis was to investigate the cost-effectiveness of alternative cervical cancer screening and HPV vaccination strategies in Brazil. This was achieved by focusing on three specific objectives: 1) To evaluate the cost-effectiveness of cervical cancer screening strategies for women presenting equivocal cytological results, 2) To evaluate the costeffectiveness of cervical cancer screening strategies for HIV-infected women, 3) To evaluate the cost-effectiveness of HPVvaccination for pre-adolescent women. An additional objective was to review and provide guidance on the use of model calibration methods in economic modelling assessments,as they are particularly important in screening and vaccination studies. The first empirical analysis found that HPV triage for women above 30 years-old presenting equivocal cytology results was likely to be very cost-effective. The second empirical analysis found that to screen HIV-infected women with HPVtesting followed by cytology annually was also likely to be very cost-effective. The third empirical analysis demonstrated that adding the quadrivalent vaccination of pre-adolescent girls to the current efforts to control cervical cancer in Brazil was very cost-effective for most of the scenarios analyzed. The vaccine was even cost saving when considering low coverage and cost of vaccination. This thesis presents findings that will inform cervical cancer screening and HPV vaccination policies in middle-income countries like Brazil and also provides guidance to help improve the standards of model calibration approaches used in cost-effectiveness analysis.

In cancer, epithelial cell de-differentiation is a feature of rapidly dividing cells under non-controlled growth and it often reflects a change in the gene expression pattern; however, the relationship between proliferation and alterations in cellular differentiation has not yet been identified. This work examined how changes in the characteristics of cells that discriminate their differentiated and proliferative states can be used to improve on current pancreatic cancer chemotherapeutic strategies. PepT1, a high substrate-capacity and low-affinity transporter system, has been suggested as an attractive drug delivery target for pancreatic cancer. Through a combination of immunological assays, PepT1 normally restricted to the apical surfaces in polarised intestinal epithelial cells, was shown to distribute at the cell membrane of non-polarised cancerous ductal cells. Anti-inflammatory or anti-cancer agents, like ibuprofen or gemcitabine, were conjugated to selected amino acids to enhance their uptake via PepT1. Studies with these conjugates demonstrated enhanced uptake into pancreatic cancer cells, AsPc-1 and HPAFII. Subsequent studies investigated how cell polarity that is typically disrupted in cancer can be modulated to affect the balance of epithelial differentiation versus proliferation. Pharmacological attenuation of YAP (c-Yes associated protein) using a β adrenergic agonist, dobutamine, increased functional tight junction (TJ) structures and diminished proliferation rates of two pancreatic cancer cells, AsPc-1 and HPAFII. Dobutamine also primed an apoptotic cell response. When given in combination with gemcitabine, dobutamine further reduced cell proliferation. Overall, these studies have provided support for using PepT1 as a method to target pancreatic cancer cells for the delivery of anti-cancer agents. Additionally, dobutamine was identified as a potential pharmacological agent to suppress the proliferation of pancreatic cancer cells by altering increasing cell programming that drives epithelial cell differentiation.

This work describes investigations into novel fluorescent nanohybrids based on carbon nanomaterials for the optical imaging of prostate cancer cells. The synthesis and characterisation of the different components of the nanoprobes are described within each chapter. The cellular response of as-prepared fluorophores, peptide conjugate and nanoprobes was evaluated in PC-3 cells by optical microscopy techniques for the first time. The new fluorescent conjugate synthesised showed targeted behaviour towards prostate cancer cells and the functional hybrids showed promising characteristics as synthetic scaffolds for future imaging nanomaterials. Chapter 1 describes the context of this work, including different imaging modalities applied to the diagnosis, staging and follow-up of prostate cancer. The use of bombesin as a targeting peptide for prostate cancer, and its incorporation in different imaging probes from current state-of the-art is discussed. The use of specific imaging probes as thiosemicarbazonato-based species is described, due to their current importance in tumour hypoxia detection and multimodality imaging potential. Finally, the use of single walled carbon nanotubes as biomedical scaffolds with a special interest in imaging applications is reviewed. Chapter 2 describes synthetic approaches towards novel functional unsymmetrical thiosemicarbazonato metal complexes. The synthesis of functional novel thiosemicarbazides and the formation of the thiosemicarbazone ligands is reported hereby. Several aromatic dicarbonylic starting materials (other than the known acenaphthenequinone) were explored and a panel of new zinc thiosemicarbazonato complexes were obtained and characterised spectroscopically. Chapter 3 contains the synthesis of bifunctional BODIPY derivatives which incorporate a protected amino acid residue. The synthetic approach towards new derivatives with fluorescence emission in the near-infrared region of the spectra is also reported. Chapter 4 describes the methodology towards the incorporation of the new carbon nanomaterial scaffold. The functionalisation of pristine single walled carbon nanotubes and graphene oxide to incorporate linkers in the structure, and the characterisation of the functionalised materials, are given hereby. Chapter 5 contains the synthesis and purification of the targeting peptide and the BODIPY-peptide conjugate involved as key components for the novel nanohybrids. Other nanohybrids containing the BODIPY or gallium thiosemicarbazonato species are also reported. The behaviour of these compounds in living PC-3 cells was evaluated and the cytotoxicity of a selection of compounds determined, for the first time. Chapter 6 provides a summary of the work carried out during this thesis and some proposals for future work arising from the research findings described herein. Chapter 7 contains detailed experimental details and characterisation data for the compounds described. The Appendices provide supporting spectroscopic evidence and X-ray diffraction data for the new compounds synthesised.