Medicine (St Vincent's) - Theses
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ItemRegulation of bone and fat cells by zinc-finger protein- 467(University of Melbourne, 2010)
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ItemCalcitonin inhibition of parathyroid hormone anabolic action(University of Melbourne, 2009)
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ItemFunctional interactions between c-MYC and mTORC1 in preclinical models of haemopoietic malignancy(University of Melbourne, 2008)
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ItemThe assessment of the coronary microcirculation using the index of microvascular resistance in patients with ischaemic heart disease( 2013)Despite improvements in medical therapy, ischaemic heart disease (IHD) remains a significant cause of morbidity and mortality in western societies. The link between epicardial stenosis and myocardial ischaemia is well documented, yet improvements in outcome have come from a focus on myocardial perfusion rather than just restoration of epicardial flow. Myocardial perfusion is comprised of collateral flow; epicardial flow and microcirculatory flow yet the key regulator of this process is the coronary microcirculation. Thus focusing on the coronary microcirculation may provide a further pathway for improving outcomes in patients with ischaemic heart disease. However, factors affecting the coronary microcirculation, in particular the effect of percutaneous coronary intervention, have not extensively studied. Furthermore, the role of the coronary microcirculation in defining key relationships in coronary physiology is also unresolved. There are a variety of methods that can be utilized to assess the coronary microcirculation both invasive and non-invasive. Each method has its own potential advantages and disadvantages but invasive methods would seem intuitively more useful amongst patients presenting to the catheter laboratory. The Index of Microvascular Resistance (IMR) is a novel invasive technique that has been extensively validated in both in-vivo and in-vitro models. The principle aim of this thesis is to use the index of microvascular resistance to examine the coronary microcirculation in patients with ischaemic heart disease. Specifically I will examine the predictors of IMR and the influence of the collateral circulation on IMR in patients with stable angina. I will also examine the predictors of microcirculatory dysfunction following PCI and look at factors both clinical and biochemical, effecting IMR measured following PCI. I will also use IMR to define controversial relationships in coronary physiology. Specifically I will examine the relationship between coronary flow reserve and fractional flow reserve and also the ability of the microcirculation to vasodilate in patients with NSTEMI. A variety of patients with IHD were recruited into the study. 80 patients with stable angina, 50 patients with NSTEMI and 40 patients with STEMI were included in the analysis. Coronary Physiological measurements were taken pre and following PCI in the culprit vessel and where possible, in an angiographically normal reference vessel. Bloods were taken at the time of the procedure and sequentially every 6 to 12 hours up to 24 hours following percutaneous coronary stenting. In summary, the main findings of this thesis are: i) Epicardial Stenosis does not increase microvascular resistance when collateral flow is accounted for. ii) The resting status of the coronary microcirculation is a key predictor of post PCI coronary microvascular function. iii) The resting status of the coronary microcirculation is an independent predictor of periprocedural myocardial infarction. Specifically localized impairment of the coronary microcirculation as determined by the relative pre IMR ratio was a predictor of periprocedural myocardial infarction. iv) The ability of the coronary microcirculation to vasodilate is preserved in selected patients with NSTEMI. This property is directly correlated with the baseline level of IMR and myocardial injury and suggests that the use of diagnostic tests that rely on hyperemia may be suitable for use in selected patients with NSTEMI. v) The discordance between fractional and coronary flow reserve is not explained by variations in microvascular resistance. The findings of this PhD are novel and have implications for the prediction of myocardial injury caused by percutaneous coronary intervention (PCI). Specifically that targeting a high IMR prior to stenting may allow for further improvements in outcome in elective PCI. I have also clearly shown (in the largest studied cohort to date) that the measurement of collateral flow is mandated when calculating microvascular resistance to avoid overestimation. Furthermore, that maximal hyperemia is possible in high acuity patients with non-ST segment myocardial infarction, potentially allowing the use of diagnostic tests such as fractional flow reserve that rely on an intact microcirculation. This may have far reaching clinical implications but warrants confirmation in a large prospective, randomised study.
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ItemMolecular mechanisms regulating chemotherapy resistance in ovarian carcinomas( 2013)Epithelial ovarian cancer is the second most common and most lethal gynaecological malignancy. Due to lack of early diagnostic modalities this aggressive form of cancer is frequently diagnosed once it is at an advanced stage with a low 30% five year survival rate. Following primary cytoreductive surgery, ovarian cancer patients are treated with adjuvant or neo-adjuvant systemic administration of platinum and taxane based chemotherapy as part of first line chemotherapy regimens. A positive response to this form of treatment is seen in the majority of cases, with patients enjoying a short remission period. However, treatment is rarely curative and in nearly all cases within 16-22 months there is an emergence of fatal chemoresistant recurrent disease. It is believed that this phenomenon of chemoresistance and recurrence is attributed to the activation of specific cell signalling pathways associated with cancer cell survival and the emergence of cancer stem cell-like populations within the chemotherapy treated residual ovarian tumours. In order to increase the low 30% five year survival rate for this distressing disease, an understanding of the molecular processes that govern the enrichment of cancer stem cell-like cells and the activation of pro-survival signalling pathways in chemotherapy surviving residual cells is needed, as these are the ultimate source of the recurrent disease. Hence with the aim of elucidating such molecular processes, this thesis aimed to answer the following two critical questions: 1. Do first line taxane based chemotherapies facilitate the activation of pro-survival pathways such as the JAK2/STAT3 pathway which is involved in the enrichment of cancer stem cell-like characteristics and cancer cell survival in epithelial ovarian cancer? 2. Does targeting the JAK2/STAT3 pathway with small molecule inhibitors enhance the efficacy of current taxane based therapies? To answer the above question, this thesis diligently characterised the cancer stem cell-like profiles that emerged in response to taxane based chemotherapy of ovarian cancer cell lines as well as ovarian tumour cells isolated from the ascites of advanced stage ovarian cancer patients. Moreover, this thesis assessed the effects of inhibiting the JAK2/STAT3 pathway on chemoresistance and enrichment of cancer stem cell-like phenotypes. Finally, using an in vivo mouse xenograft model, this thesis validated the demonstrated results obtained from in vitro experimentations. The results of this thesis demonstrate that treatment with taxane and platinum based therapies does indeed activate the JAK2/STAT3 pathway in both ovarian cancer cell lines as well as tumour cells isolated from the ascites fluid of advanced stage ovarian cancer patients. Moreover, this thesis is the first to demonstrate that the activation of the JAK2/STAT3 pathway in response to taxane based therapies coincides with the enrichment of cancer stem cell-like phenotypes which was shown to contribute to cancer cell survival and the development of a significantly larger tumour burden in mice. Furthermore, this thesis is the first to demonstrate that inhibiting the JAK2/STAT3 pathway reduces the emergence of cancer stem cell-like populations and enhances the efficacy of current taxane based therapies resulting in the significant reduction of tumour burden in mice. The results demonstrated in this thesis have important implications for ovarian cancer patients who are being treated with taxane and or platinum based first line therapies. These findings warrant further pre-clinical investigation and may be used as a platform for the development of adjuvant therapies used in combination with the gold standard therapy that is currently offered to ovarian cancer patients.
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ItemThe doctor as moral agent, with reference to the distinction between killing and "letting die"( 2007-07)In the bioethics literature, arguments about the nature of the distinction between killing and “letting die” seem irresolvable. There is a disparity between the dominant (consequentialist) opinion on this issue and that of the medical profession. No previous studies have investigated how doctors who work with the dying understand the distinction in the medical context. The aim of my research was to explore the moral reasoning of these clinicians in relation to this question. A focused ethnographic study involved thirty Melbourne doctors (thirteen palliative care physicians, nine oncologists, six intensivists, and two advocates of physician-assisted suicide) of whom eighteen were male and twelve female, with an age range from 31 to 77 years. Half had a religious belief (Jewish or Christian) and half were atheist/agnostic. (For complete abstract open document)
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ItemSmall animal models of Gal-mediated and xenograft rejection( 2004-11)Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.
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ItemAdenosine generation and signalling in diabetes and islet transplantation( 2013)Clinical islet transplantation is a potential cure for type 1 diabetes, however, hurdles such as the instant blood-mediated inflammatory reaction (IBMIR), recurrent autoimmunity and allograft rejection restrict the effectiveness of this therapy. During the IBMIR process, thrombotic and inflammatory cascades are activated, resulting in the loss of more than 50% of islet graft mass. This poor engraftment, combined with the ongoing destruction of islets by recurrent autoimmunity and the allo-immune response, means that more than one donor per recipient is needed to achieve normoglycemia. CD39 is an anti-thrombotic and anti-inflammatory molecule, which promotes the generation of extracellular AMP and adenosine, and has been shown to mitigate IBMIR in vitro when over-expressed on islets. We hypothesised that the over-expression of CD39 on the islet surface would confer in vivo protection against chemically induced diabetes and attenuate IBMIR-mediated graft loss via adenosine-related signalling pathways. The aim of the thesis was to evaluate the impact of adenosine generation and signalling in mouse models of T cell-mediated diabetes and islet transplantation. CD39 over-expression protected against T cell mediated diabetes and this effect persisted in the presence of defective regulatory T cells. The absence or inhibition of particular adenosine receptors increased susceptibility to diabetes. Surprisingly, in the absence of an important generator of extracellular adenosine (i.e. CD73), mice were protected in this model. Finally, islets over-expressing CD39 showed prolonged graft survival in a syngeneic intraportal islet transplant model, implicating an effect of CD39 in inhibiting IBMIR. Together these results support the notion that the over-expression of CD39 promotes islet engraftment and survival. The clinical translation of these findings could lead to greater insulin independence and the need for fewer donors to achieve this.
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ItemThe role of Trig: a novel toll-like receptor induced gene, in dendritic cell function and autoimmune disease( 2013)The immune system is comprised of a complex network of cells and signalling pathways that must be tightly regulated to maintain immune homeostasis. Defective negative regulation results in enhanced immunogenicity, loss of immune tolerance and, eventually, the development of autoimmune disease. Toll-like receptors (TLRs) are an essential component of the immune system as they act as early sensors of microbial pathogens and play a critical role in linking the innate and adaptive arms of the immune response. There is also increasing evidence that aberrant TLR signalling and TLR-mediated immune responses contribute to the development of autoimmune diseases, such as type 1 diabetes (T1D), although investigating these abnormalities and the underlying genetic defects in humans is often difficult. Instead, the non-obese diabetic (NOD) mouse strain, which spontaneously develops T1D, has proven to be a useful animal model for investigating genetic variants that contribute to autoimmune disease by altering TLR-mediated immune responses. Predisposition to T1D in the NOD mouse is due to allelic variation at multiple loci across the genome. More than 30 susceptibility loci have been linked to T1D development in the NOD mouse. Positional cloning of one of these loci, termed Idd11, has led to the identification of a novel gene termed Trig (AK005651). Allelic variation for Trig is associated with T1D development and this gene is differentially expressed in immune-related tissues between diabetes-resistant and diabetes-susceptible mouse strains. Preliminary experiments also revealed that Trig is upregulated in a dendritic cell (DC) line in response to TLR9 stimulation. It was therefore hypothesised that genetic variation for Trig alters TLR-mediated immune responses that affect the development of autoimmune disease. The first aim of this thesis was to perform a preliminary characterisation of a novel mouse strain deficient for Trig. Trig-deficient mice were observed to be viable and fertile, were indistinguishable from wildtype littermates for weight and gross anatomical development, and did not develop any observable signs of ill health. Furthermore, no statistical differences between Trig-deficient and wildtype littermates were identified for immune cell number or frequency in the peripheral blood, thymus or spleen. This study indicates that Trig is not an essential gene for the basic development and viability of B6 mice housed in specific pathogen-free conditions. The second aim was to investigate the experimental conditions that alter expression of Trig. Trig was found to be upregulated in an immortalised DC line, primary DCs and bone-marrow-derived macrophages, after exposure to ligands that activate MyD88-dependent TLR signalling pathways. This upregulation was abrogated by interferon (IFN)γ signalling, indicating that TLR/IFNγ signalling cross-talk regulates the expression of Trig. These studies suggest that Trig might act as a negative feedback regulator of TLR-mediated immune responses. The third aim was to determine which TLR-mediated immune responses were regulated by Trig in DC subsets. A series of in vitro assays were performed to assess the capacity of TLR9-stimulated Trig-deficient DC subsets to produce cytokines, upregulate cell-surface molecules and present antigen to T cells. This revealed that Trig-deficiency leads to enhanced cytokine production in specific DC subsets. Subsequent analysis of DC subsets isolated from NOD, B6 and Idd11 congenic mice revealed that strain variation for Trig also affects TLR9-mediated cytokine production. Collectively, these findings indicate that Trig negatively regulates TLR9-mediated cytokine responses in specific DC subsets. The fourth aim of this thesis was to explore the role of Trig in systemic inflammation and autoimmune disease. The serum cytokine levels of Trig-deficient mice treated with a TLR9 agonist were similar to those of wildtype littermates, indicating that Trig is not an essential negative regulator of TLR9-mediated cytokine production under the experimental conditions tested. Instead, Trig-deficiency had a subtle affect on the onset and/or severity of three inducible models of autoimmune disease. Therefore, this thesis presents cumulative evidence that supports a role for Trig in attenuating TLR-mediated immune responses that contribute to immune dysregulation and the development of autoimmune disease.
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ItemAdipose-derived mesenchymal cell derivation, characterization and differentiation for potential use in cell replacement therapy for diabetes( 2013)Type 1 diabetes (T1D) is characterized by the loss of insulin-producing β-cells in the pancreas. T1D can be treated using cadaveric islet transplantation, but this therapy is severely limited by a lack of donor pancreas. To develop an alternative cell therapy, candidate populations were identified through epigenetic characterization of multiple tissues. Histone modification status at the promoter region of key endocrine pancreatic genes was assessed using chromatin immunoprecipitation sequencing (ChIP-seq) and validated using promoter-specific TaqMan-based quantitative PCR (qPCR). Visceral fat was identified as a tissue retaining epigenetic signatures similar to those observed in the pancreas. Human adipose-derived mesenchymal cells (AMCs) were characterized using flowcytometry, confocal microscopy, qPCR, in situ PCR and next generation sequencing technologies. Multiple transcription factor-encoding adenoviruses (e.g. Pdx1, MafA, Ngn3) were employed to determine the differentiation potential of these cells. Analysis of multiple pancreatic hormones and transcription factors in these samples demonstrated consistent differentiation. The differentiation potential was further explored using AMCs isolated from transgenic mice that express GFP under the regulation of Pdx1 (pancreatic and duodenal homeobox 1) or insulin-1 gene promoters. GFP expression was quantitated as an index of gene promoter activity during differentiation to insulin-producing cells, in the presence of various pro-differentiation small molecules. Human AMCs were exposed to a standard differentiation protocol and seen to migrate to form islet-like cell aggregates (ICAs), showing significant increases in islet hormone transcripts in vitro. These adipose-derived ICAs were transplanted into immunocompromised animals using two models of transplantation. Cells were transplanted in a Theracyte immunoisolation device into the peritoneum, and within a blood clot under the kidney capsule. Transplanted cells maintained expression of endocrine pancreatic transcription factors and did not undergo a regressive mesenchymal transition following surgery. Circulating blood samples collected from peripheral circulation of these mice, following a glucose injection, showed that differentiated and engrafted human AMCs could sense, transcribe, translate, package and secrete insulin in response to a glucose stimulus. These studies indicate that human AMCs can differentiate into insulin-producing cells in vitro and have potential for cell replacement therapy in diabetes.
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