Medicine (St Vincent's) - Theses
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ItemBreast cancer risk assessment and risk management:: Development of a personalised, web- based, decision support tool (iPrevent®)( 2016)Breast cancer risk is a complex interaction of environmental factors, inheritance and genetics. Awareness of her personal breast cancer risk can allow a woman to make informed decisions regarding the management of her risk, through screening or risk reduction measures such as surgery or medication. The effect of risk factors, both inherited and modifiable, on breast cancer risk is complex but mathematical models exist to estimate an individual’s risk. These models use epidemiological data to quantify the risk for an individual, based on a range of their risk factors. As these models were developed primarily for research purposes, they are not designed for ease-of-use by a range of clinicians, nor designed to be used by those unfamiliar with estimating breast cancer risk. Once estimated, a woman’s breast cancer risk must then be explained in a way that is comprehensible to the woman, together with ways to manage that risk in a similar format. If this were achieved for all women, it may allow them to make informed choices and potentially even prevent breast cancer or reduce its impact greatly. The ultimate aim of this research was to develop a user-friendly computerised, web-based breast cancer risk assessment and risk management support tool. This tool, called iPrevent©, uses the existing mathematical models to estimate individualised breast cancer risk, but using a user friendly interface. It then goes further, and provides Cancer Australia guideline-based recommendations, based on that risk, for each individual woman. It presents the risks and befits of each evidence-based intervention in a similar manner to the risk so that women can make an informed choice regarding their breast cancer prevention strategy. Before developing iPrevent©, I first examined the other possible effects of being a carrier of a mutation in breast cancer predisposition genes, as it was hypothesised that other factors such as fertility effects, could have a large bearing on any future decisions women may make, including risk reducing surgery. I then explored current behaviours to reduce risk among women at highest risk, in an attempt to understand the magnitude of the possible benefits of iPrevent© in this highest risk group. Through focus group studies, I examined the information needs of clinicians to facilitate breast cancer risk discussions. Understanding the needs of end-user clinicians of iPrevent© ensures it could meet their needs. This usability may increase uptake and use, of both the tool and breast cancer risk management strategies where appropriate. This tool, iPrevent©, is currently undergoing clinical validation studies, outside the scope of this thesis, but will shortly become freely available with the aim of increasing individual awareness of each woman’s own breast cancer risk, enabling her to manage that risk according to the evidence, Cancer Australia guidelines and her own preferences.
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ItemThe contribution of genetic variations in the region of the parathyroid hormone-like hormone, PTHLH, gene to breast cancer susceptibility( 2016)Aims: • Analyse the evidence for PTHLH and PTHrP, its protein product, playing a role in breast cancer and update the empirical definition of the gene. • Describe the 3-Dimensional structure of the PTHLH region and determine its system of regulatory interactions, including remote regulatory elements affecting PTHLH. • Integrate existing tools in addition to the novel perspectives generated above to enable a comprehensive annotation and analysis of genetic variants identified through molecular epidemiological techniques including Genome-Wide Association Studies (GWAS) and somatic DNA sequencing of tumour tissue to derive putative molecular mechanisms for the region’s involvement in breast cancer susceptibility. Methodology: • Review published studies and databases, integrating findings from diverse sources. • Acquire and analyse DNA and RNA sequencing, regulatory, expression, algorithm-inferred, and proximity-ligation data from multiple public data sources including ENCODE, ROADMAP, dbGAP, COSMIC and other to update the definition of PTHLH, and advance concepts of structure and regulatory function in the region. • Acquire and analyse primary GWAS data, performing imputation with multiple algorithms and references, and annotating associated variants with a suite of tools. • Use genome browsers including UCSC, WUSTL, and Golden Helix SVS, and their associated databases and tools, to analyse and visually integrate findings. Results: • PTHrP has multiple discretely functional segments active throughout the cell. It likely plays a bivalent and context-dependent role in cancer biology. Analysis of somatic variation in cancer suggests PTHrP may have a tumourigenic role within the nucleus. • PTHLH sits within a 1.3Mb TAD featuring multiple sub-structures that are integrated with the region’s regulatory function. There are activated chromatin hubs (ACHs) at protein-coding genes with evidence of extensive interaction between them. This is facilitated by the TAD’s structure, collocating them at the neck of the TAD. • The ACHs at MRPS35, KLHL42, and CCDC91 each monopolise a subordinate regulatory sub-net with a hierarchical structure. They each appear to act as important remote regulatory elements that integrate regulatory signals generated within their respective sub-nets, transferring them to PTHLH, and other genes, via ACH-ACH interactions. • There appear to be multiple discrete GWAS breast cancer association signals in the PTHLH region. Annotation of the associated variants suggests three particular regulatory elements may be its key drivers. In the context of the regulatory concepts developed in this thesis, the variants may affect a particular regulatory signal at multiple points in its assembly. PTHLH is the likely downstream target of this signal. • There are multiple poorly-describe coding, and non-coding, genes in the region that are also potential actors in breast cancer and should be investigated. Conclusion: • The PTHLH region is likely involved in the pathogenesis of breast cancer through the modification of PTHLH expression. There are likely to be other mechanisms in parallel that are yet to be fully described.
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ItemRoles of cyclin-dependent kinase substrates: cell cycle and beyond( 2015)The cyclin-dependent kinases (CDKs) are serine/threonine specific kinases that are key regulators of the cell cycle. However, several reports indicated their roles in other pathways. Therefore, it is important to identify novel CDK substrates in order to gain a better understanding of the pathways they regulate and in turn, the diseases where they are deregulated. In this thesis, I describe two substrates; Brahma (Brm), an ATPase subunit of the SWI/SNF chromatin remodeling complex and Breast Cancer Metastasis Suppressor 1 (BRMS1), a metastasis suppressor in human cancers. Brm has long been modeled to be part of the pRb/E2F complex, regulating entry into S phase of the cell cycle. In addition, several studies have indicated that Brm interacts with Cyclin E and CDKs. Here, I demonstrate that Drosophila Brm is phosphorylated in vitro by both Cyclin A and Cyclin E/CDK2. Furthermore, using Drosophila as an animal model, a phospho-mimic of Brm was able to bypass the developmental G1 arrest in the wing discs’ zone of non-proliferating cells (ZNC), indicating its role in inhibiting S phase entry. In addition, based on the phenotypes obtained from the expression of Brm phospho-mutants and the current literature, I postulate that CDK-mediated phosphorylation of Brm also has roles in maintaining genomic stability, cell signaling and expression of cell adhesion systems. Our laboratory had previously identified the regulation of Drosophila EGFR/Ras signaling by Brm-DN and its antagonism by Gem. In this thesis, I have further characterised the roles of Brm-DN and Gem in Drosophila development and have identified Rhomboid (a positive regulator of EGFR/Ras signaling) to be the target gene that is transcriptionally regulated by Brm and Gem. Finally, BRMS1 is a metastasis suppressor, better known to exert its functions by being a part of the SAP30/mSin3/HDAC complex to modulate transcription. Furthermore, it was previously reported to be in complex with RBP1, a CDK substrate, which inhibits S phase entry. In this thesis, we have found that BRMS1 is phosphorylated by CDKs both in vivo and in vitro. Using Mass Spectrometry, we have further identified the phosphorylated site to be Serine 237. Interestingly, mutation of this phosphorylation site had no impact on cell cycle progression and BRMS1’s role as a transcriptional regulator, however, it modulated BRMS1’s role in inhibiting cell migration. Taken together, this thesis confirms the CDK-mediated phosphorylation of two proteins and has expanded our understanding of the various roles that CDK substrates have beyond the cell cycle. Overall, this work provides further insights into the role of CDK substrates in cellular behaviour, tissue growth and differentiation, and the development of cancer and metastasis.
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ItemEpithelial - mesenchymal plasticity in circulating and disseminated tumour cells( 2013)Metastasis, or the spread of cancer from the site of origin to a distant site, is the leading cause of cancer-associated death. The work presented in this thesis revolves around the process of metastasis in order to understand some of the underlying mechanisms. Specifically, the focus of this project was on cancer cells moving through the blood circulation, known as circulating tumour cells (CTC), as well as cancer cells that have escaped into a distant site but are undetectable by conventional screening methodologies, known as disseminated tumour cells (DTC). Collectively, CTC and DTC are often referred to as minimal residual disease. Whilst there is an exponentially growing amount of attention on minimal residual disease research, in addition to a large push towards the use of these cells (especially CTC) clinically, their clinical utility currently remains in limbo. The detection and enumeration of CTC/DTC in cancer patients has been demonstrated to associate with worse disease-free survival and overall survival, yet a relatively small proportion of cancer patients with detectable CTC/DTC do not fit the predicted relapse and/or survival trends. Thus, there is growing emphasis in published literature to delve beyond the simple detection of CTC/DTC, and into their characterization. The belief is that this will then enable one to identify a patient who bears CTC/DTC that are of actual biological concern. Particularly, the ability of cancer cells to switch between epithelial and mesenchymal states, or even display both epithelial and mesenchymal properties, has been proposed by our lab to be important in the generation and function of CTC/DTC. This ‘flexibility’ in cellular status has been termed in our lab as epithelial – mesenchymal plasticity (EMP). This project is divided into three main sections pertaining to; (i) establishment and optimisation of CTC/DTC capture and detection/analysis protocols, (ii) the use of xenograft mouse models for studying EMP in minimal residual disease, and (iii) the study of EMP in minimal residual disease of metastatic breast cancer patients. A number of other laboratories around the world also propose a role for EMP in CTC/DTC, and several publications have come forth in very recent years attempting to look into this phenomenon. However, to the best of our knowledge, (i) the work presented in this thesis is the first characterising the EMP status of CTC/DTC in Australia, and (ii) the only of its kind (globally) utilising the MDA-MB-468 and ED03 xenograft models to study minimal residual disease using our specific experimental design and ‘in-house’ developed assays.