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Background Post mastectomy breast reconstruction is an elective procedure offered to women as part of surgical treatment for breast cancer. Choosing to have a reconstruction, or not, and when, is a timely and challenging decision for women in an emotive environment. Decision support interventions (DSIs) have proved beneficial in enabling decision making with patients and clinicians in similar situations of clinical equipoise. The goal of the DSI is to facilitate a decision that is reflective of the patient's own priorities and preferences. However, the implementation of DSIs into routine clinical practice has proved challenging. The thesis reports on the development of a post mastectomy breast reconstruction DSI and on the feasibility of implementing a DSI in the clinical environment. Methods Women's decision making about breast reconstruction was examined through a literature review and a thematic analysis of interviews with breast cancer survivors. A collaborative development framework and iterative process were utilised to design the breast reconstruction DSI. The breast reconstruction DSI was introduced into the clinical environment in a feasibility study to identify potential barriers and facilitators to implementation. A case study approach, including the decisional conflict scale and qualitative methods, examined the perceptions and experience of the patients and clinicians using the DSI in a breast cancer clinic. Results The Option Grid DSI was published on-line as part of the Option Grid Collaborative. In feasibility testing, its flexibility and accessibility were positive features in terms of its implementation. The content and format facilitated the comparison of options. The timing of when it was introduced impacted its effect on patient decision making. Conclusions The support of local stakeholders was significant in the development of the DSI. The Option Grid DSI can be integrated into routine clinical practice, and gave some consultations more structure. Patient participants in the feasibility study found it constructive in facilitating their decision making. Further testing to explore the timing of the DSI and its impact on a wider audience is warranted.

This thesis contributes to the state of understanding about the value of latent health and fatality risk reductions, focussing on the effects of context and latency on the Value of Preventing a Statistical Cancer Fatality (VSLCAN) relative to road accident fatalities. The conceptual, methodological and empirical contributions are derived from two stated preference studies. The studies are designed to explore how the VSLCAN is driven by the context effect, which includes dread of the cause ‘cancer’ and the effects of illness prior to fatality; and the latency (delay) effect which depends upon time preferences and risk preferences. Study 1 develops a Risk-Risk survey protocol, and the resulting central tendency and regression analysis verify that the context of cancer increases the VSL and that latency decreases it. The relativity between VSLCAN and the road accident VSL is then summarised into a simple relationship where the offsetting influences of context and latency are parameterised. This novel tool has the potential to enhance the comparability and evaluation of a wide range of existing and future VSL studies involving context and latency effects through the elicitation of key underlying parameters such as the context premium and effective discount rate. As such it represents a significant methodological contribution. Study 2 focusses directly on two aspects of the latency effect. These relate to risk and time preferences, explored in Studies 2a and 2b respectively. Delayed outcomes are inherently risky, so the exploration of latent outcomes requires controlling for risk preferences. Study 2a develops a theoretical and empirical framework for eliciting risk aversion proxies in the domain of health, which have not previously been fully developed in the literature. The method extends the classic Holt-Laury risk preference elicitation framework into a new domain- health risks- and the method is implemented successfully in Study 2. This chapter therefore makes both conceptual and methodological contributions through clarifying the utility theoretic basis of a health risk aversion measure and then developing a way to elicit such a measure in surveys. Study 2b uses the novel VSLCAN:VSL relationship developed in Study 1 to elicit exponential discount rates from Risk-Risk data comparing latent cancer and road accident risks. Regression analysis performed on these rates on a sample and individual level, provides strong evidence to suggest that a non-standard (sub-additive) discounting model is the most descriptively accurate discounting assumption for this sample. It provides the first evidence regarding sub-additive discounting in the domain of health and fatality risk.

The X-chromosome linked transcription factor, FOXP3, is expressed by epithelial cells from several organs. In these cells it is considered a potent tumour suppressor directly regulating the expression of many important oncogenes. The chemokine receptor, CXCR4, can also direct a number of processes relevant to the development of breast cancer. These include chemotaxis towards the sole ligand for CXCR4, CXCL12, which is expressed at the most common sites of metastatic spread. This study was designed to define further the role FOXP3 plays in metastatic breast cancer, with a particular focus on its potential to regulate CXCR4. In comparison with normal primary breast epithelial cells, FOXP3 was downregulated at both transcript and protein levels in the breast cancer cell lines, MCF-7 and MDA-MB-231. In the more invasive MDA-MB-231, the remaining FOXP3 was located predominately within the cytoplasm and lacked the natural Δ3 isoform. Following stable FOXP3 overexpression in MDA-MB-231, significant decreases were observed in the expression of ErbB2, SKP2, c-MYC and CXCR4. In contrast, an increase in p21 expression led to inhibition of cell proliferation, with a greater proportion in the G1 phase of the cell cycle suggesting the induction of senescence. Specific knockdown of FOXP3 in normal breast epithelial cells with siRNA significantly increased ErbB2, SKP2, c-MYC and CXCR4, and decreased p21 expression. These cells also showed a significantly increased chemotactic response towards CXCL12, suggesting a role for FOXP3 influencing cell migration. The expression of FOXP3 and CXCR4 in normal breast tissue (n=2), and both lymph node negative (n=7) and lymph node positive (n=9) breast cancer samples was investigated by immunohistochemistry. Both epithelial and cancer cells in all the breast cancer samples showed increased CXCR4 expression in comparison to normal tissue. However, the expression of FOXP3 by epithelial or cancer cells did not appear different between all the samples examined. Results from this study are consistent with FOXP3 functioning as an important tumour suppressor in breast cancer. Indeed, the functions of FOXP3 in breast epithelium can now potentially be extended to include influencing the expression of CXCR4 and response to the pro-metastatic chemokine, CXCL12. Further studies should be directed to investigation of the molecular mechanisms involved in this influence of chemokine responsiveness by FOXP3.

Medulloblastoma is the most common malignant brain tumour of childhood. Transcriptomic profiling has revealed the existence of four core molecular subgroups (SHH, WNT, Group 3 and Group 4) with distinct clinical, pathologic and molecular characteristics. However, the specific molecular events associated with tumour development in these groups are poorly understood. DNA methylation plays a key role in epigenetic transcriptional regulation, and promoter hypermethylation leading to gene silencing is a common feature of medulloblastoma. DNA methylation profiling has identified distinct methylomic profiles associated with the four subgroups of medulloblastoma, and the wider role of DNA methylation in medulloblastoma now requires investigation. Using two high-throughput screening approaches, this project therefore undertook a comprehensive investigation into the potential role of specific DNA methylation events in the development of the distinct subgroups of medulloblastoma. Using DNA methylation profiles, which were generated for 216 medulloblastomas using the GoldenGate methylation array, the first approach identified 73 CpG methylation markers (encompassing 63 genes) which significantly distinguished Group 3 and/or Group 4 medulloblastomas. Subgroup-specific differential gene expression analysis showed that, for the majority of the methylation markers identified, there was no clear inverse association between methylation and gene expression. One gene (RHOH) was identified which showed strong evidence of epigenetic dysregulation in medulloblastomas. RHOH methylation represented a potential epigenetic event in Group 4 tumours; 51% of Group 4 medulloblastomas showed aberrant hypomethylation of multiple RHOH promoter-associated CpG residues, which was associated with upregulated RHOH expression in Group 4 tumours. RHOH was re-expressed in 4 out of 6 methylated cell lines following treatment with the demethylating agent 5’-aza-2’-deoxycytidine (5-azaCdR). This study has thus identified a novel putative oncogenic role for RHOH in Group 4 medulloblastoma development. In the second approach, a functional epigenomics screen identified 283 genes which were upregulated in 2 or more cell lines investigated (n=10) following 5-azaCdR treatment. Assessment of DNA methylation using the Illumina 450K methylation array identified 160 CpG residues (encompassing 21 of the 283 genes) whose methylation status was consistent with expression alterations observed after 5-azaCDR, and methylation-dependent gene regulation, in cell lines. 9/160 CpG residues (6%) showed evidence of subgroup-specific differential methylation which was concordant with differential gene expression and potential epigenetic gene regulation in medulloblastoma subgroups. These 9 sites represented 5 candidate genes (ACTC1, ANXA2, FAM46A, PRPH and S100A4). Aberrant hypermethylation of multiple gene body CpG residues was associated with FAM46A silencing in non-SHH tumours, while aberrant hypermethylation of multiple promoter-associated residues was associated with ACTC1 silencing in Group 3 and Group 4 medulloblastomas. Single site v hypomethylation events associated with upregulated expression in WNT tumours were identified for ANXA2, PRPH and S100A4. This study has identified six genes with putative oncogenic or tumour suppressor roles in the development of distinct subgroups of medulloblastoma through their epigenetic dysregulation. Further work is now required to validate these findings and to assess their functional significance in medulloblastoma subgroups, as well as their potential relevance in medulloblastoma sub-classification and prognostication.

Quantum Dots (QDs) are fluorescent nanoparticles that can be used as fluorescent probes for cancer localisation and sentinel lymph node biopsy (SLNB). The current tracers for SLNB including the blue dye and radiocolloid have various drawbacks limiting their widespread use. Near Infrared (NIR) QDs can potentially replace these tracers, based on their deep tissue visibility as the biological window is transparent to NIR wavelengths. QDs can also be conjugated to specific biomolecules for targeted cancer localisation and therapy. Their main limitation is toxicity as most QDs are based on heavy metal salts. This project aims to develop a biocompatible QD for clinical application using a novel nanoparticle - Polyhedral Oligomeric Silsesquioxane (POSS) based surface coatings. Materials & Methods A novel nanocomposite polymer emulsion POSS-PCU was synthesized by integrating POSS units into poly(carbonate-urea)urethane (PCU) chains and used to encapsulate QDs based on CdTe/CdSe/ZnSe and CdTe/CdS/ZnS. Mercapto-POSS on its own was also used to coat CdTe core QDs. Characterization was performed using various techniques and in vitro toxicity was established. QDs were bioconjugated to anti-HER2 antibody and used to localise HER-2 receptors on SK-BR-3 breast cancer cells. In vivo biodistribution was determined using a tail vein injection of QDs into male Sprague-Dawley rats for 1 and 24h. Organ biodistribution was quantified by fluorescence studies and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). A live NIR imaging system was set up and NIR-QDs used to demonstrate SLN localisation in a rat model. Results POSS-PCU coated QDs showed significantly reduced toxicity and enhanced photostability on in vitro and in vivo studies. QD-anti-HER2-Antibody bioconjugate successfully localised HER-2 receptors in vitro. Biodistribution studies showed maximal uptake by the liver and spleen. NIR-QDs localised to the SLN and were visualised by the live NIR imaging system. Conclusion NIR-QDs can be used as fluorescent probes for cancer localisation and SLNB. POSS nanocomposite based surface coatings can stabilise QDs for overall reduced toxicity and enhanced photostability allowing relentless possibilities for clinical application.

Increased ERα expression correlates with increased breast cancer risk, suggesting that the ER and estrogens play a role in cancer initiation and progression. Nonetheless, a clear understanding of the involvement of the ER in breast epithelium formation, carcinogenesis and cancer progression is lacking. In addition to this, the role of other estrogen-responsive receptors, such GPER-1 remains unknown. The mammary gland is comprised of milk ducts terminating in secretory units termed acini. These are comprised of a layer of epithelial cells surrounding a hollow lumen. These cells are in turn surrounded by a layer of myoepithelial cells attached to an underlying basement membrane. 3D cultures of immortalised breast cells recapitulate many of the features of acini in vivo, such as spherical growth-arrested acini with a layer of epithelial cells attached to an underlying basement membrane, surrounding a hollow lumen. We aimed to use 3D cultures of ERα, ERβ and GPER competent MCF-12A cells to assess the effects of estrogenic compounds on acini formation. MCF-12A cells were cultured in Matrigel with etOH (solvent control), E2, bisphenol A (BPA) or n-propylparaben. To assess the role of the ER or GPER-1, cells were also pre-treated with the ER and GPER -1 antagonists, as well as inhibitors of the MAPK and PI3K signalling cascades. Immunocytochemical staining using antibodies against activated caspase-3 and laminin-V was performed. Image acquisition was achieved by laser confocal microscopy. Control acini were encapsulated by a basement membrane with central cells either cleared or undergoing apoptosis. In comparison, cells treated with E2, BPA or propyparaben possessed filled lumen with little or no sign of apoptotic central cells and were larger and disorganised. Co-incubation with ER or GPER-1 antagonists, or PI3K and MAPK inhibitors reverted some of the effects of the test compounds resulting in growth-arrested, spherical acini with evidence of luminal clearing. We have successfully established an in vitro 3D model using immortalised ER competent cells to study the effects of estrogens on mammary gland formation. We have demonstrated that estrogenic compounds disrupt acini by inducing luminal filling and preventing growth arrest. Moreover, these effects seem to be mediated by both estrogen responsive receptors ER and GPER-1.

Human tuberculosis (TB) is caused by several closely related species of bacteria collectively known as the Mycobacterium tuberculosis complex (MTBC). In this thesis the identification and effect of lineage-specific genetic variation within the phylogenetic lineages of the MTBC was investigated using a combination of computational methods and high-throughput sequencing technology. Genome sequencing has now identified an extensive repertoire of single nucleotide polymorphisms (SNPs) amongst clinical isolates of the MTBC. Comparative analysis focused on the detection of all lineage-specific SNPs, providing the first glimpse of the total SNP diversity that separates the main phylogenetic lineages from each other. Bioinformatic analysis focused on SNPs more likely to contribute to functional diversity, which predicted nearly half of all SNPs in the MTBC to have functional consequences, while SNPs within regulatory proteins were over-represented. To determine whether these and other lineage- specific SNPs lead to phenotypic diversity, genome datasets were integrated with RNA- sequencing to assess their impact on the comparative transcriptome profiles of strains belonging to two MTBC lineages. Analysing the transcriptomes in the light of the underlying genetic variation found clear correlations between genotype and transcriptional phenotype. These arose by three mechanisms. First, lineage-specific changes in amino acid sequence of transcriptional regulators were associated with alterations in their ability to control gene expression. Second, changes in nucleotide sequence were associated with alteration of promoter activity and generation of novel transcriptional start sites in intergenic regions and within coding sequences. Finally, genes showing lineage-specific patterns of differential expression not linked directly to primary mutations were characterised by a striking over- representation of toxin-antitoxin pairs.

Ovarian cancer is the fifth most common cancer in women in developed countries and is associated with poor survival due to late diagnoses. Strategies focusing on detecting the disease in the earliest stages and/or improving risk prediction may represent effective clinical intervention reducing disease burden. Women at the greatest risk of epithelial ovarian cancer (EOC) can be offered prophylactic risk-reducing salpingo-oopherectomy (RRSO), which is currently only offered to women with mutations in the highly penetrant susceptibility genes BRCA1 or BRCA2. Previous studies show that 46% of familial cases of EOC carry a deleterious mutation in BRCA1 (37%) or BRCA2 (9%). The residual proportion of familial risk is likely to be attributable to other genetic variants providing a rationale for identifying additional susceptibility alleles using rapid high-throughput next generation sequencing (NGS) in large samples sizes. A pilot study determines the principle of NGS in mutation detection sequencing BRCA1 gene in 12 DNA samples with known mutations. The 11bp deletion, missed in the analysis, is detected by altering the bioinformatics. The second study sequences 1506 cases and 1130 healthy controls using Fluidigm microfluidic technology and Illumina HiSeq2000 in 6 DNA repair genes (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and SLX4). 94% of the targeted region is sequenced with >30 reads. 23 cases and 1 control show a putative protein-truncating variant in 5 genes. Many missense variants are detected in cases and controls suggesting these are not pathogenic. Epidemiological data shows that women with family history and a deleterious mutation develop EOC on average 10 years younger. Interestingly, half of those women with detected mutations have no family history. A final study uses the established NGS approach to characterise the mutation prevalence in 4 known and 5 candidate EOC susceptibility genes in 2300 unaffected women from high-risk breast-ovarian cancer families. BRCA1 and BRCA2 deleterious mutations are identified in 53 and 49 women respectively. Deleterious mutations are detected in 6 additional genes, BRIP1 (n=5), RAD51C (n=3), RAD51D (n=1) PALB2 (n=5), BARD1 (n=1) and NBN (n=3). Importantly, a bioinformatics pipeline is refined to maximise variant detection sensitivity with zero false negatives where read depth is >30X. Further large case-control studies are recommended to examine the population frequencies in these novel genes. These studies demonstrate the potential of targeted NGS approaches for population-wide risk prediction and early detection of EOC.

It is well known now that changes in the cellular microenvironment contribute to tumour genesis through paracrine effects. Fibroblasts, the predominant cells of the stromal breast carcinomas, play an important role in the development and spread of cancer cells. However, the nature, molecular mediators and pathways of the crosstalk between cancer cells and their stromal fibroblasts are still not well defined. There is evidence that normal breast stromal fibroblasts (NBFs) suppress tumor growth while cancer-associated fibroblasts (CAFs) promote tumorogenesis through functional interactions with cancer cells. Little is known about the biology and the carcinogenic potential of stromal fibroblasts present in histologically normal surgical margins (TCFs). In addition, the nature, molecular mediators and pathways of the crosstalk between cancer cells and their stromal fibroblasts are still not well defined. Therefore, we first undertook gene expression analysis on five CAF/TCF pairs from breast cancer patients and three NBFs (derived from mammoplasties). This comparative analysis revealed variation in gene expression between these three categories of cells, with a TCF-specific gene expression profile. This variability was higher in TCFs than in their paired CAFs and also NBFs. Cytokine arrays show that TCFs have a specific secretory cytokine profile. In addition, stromal fibroblasts from surgical margins expressed high levels of α-SMA and SDF-1 and exhibited higher migratory/invasiveness abilities. Indirect co-culturing showed that TCF cells enhance the proliferation of noncancerous mammary epithelial cells, and the epithelial to mesenchymal transition of breast cancer cells. Moreover, TCF and CAF cells increased the level of PCNA, MMP-2 and the phosphorylated/activated form of Akt in normal breast luminal fibroblasts in a paracrine manner. Furthermore, TCFs were able to promote the formation and growth of humanized orthotopic breast tumors in nude mice. Interestingly, these TCF phenotypes and the extent of their effects were intermediate between NBFs and CAFs. Together, these results indicate that stromal fibroblasts located in noncancerous tissues exhibit a tumor-promoting phenotype, indicating that their presence post-surgery may play important roles in cancer recurrence. Moreover, we have shown that the tumor suppressor CHEK2 gene is down-regulated in CAF cells as compared to TCF cells and NBF cells in vitro at both the mRNA and protein levels and in vivo. Using specific siRNA we have shown that CHEK2-down-regulation increases the expression and the secretion of SDF-1, IL-6, VEGF-A and MMP-9 and enhances the proliferation, migration and invasion of breast cancer cells. These results present the Chk2 kinase protein as an important mediator in the cross-talk between breast carcinomas and their stromal fibroblasts.