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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ItemRole of purinergic signalling in the pathophysiology of antiphospholipid antibody induced miscarriages( 2017)Antiphospholipid syndrome (APS) is an autoimmune disorder in antibodies are generated against cell membrane and self-antigens (aPL-ab) hence the name Antiphospholipid disorder. This autoimmune disorder characterised by the presence of antibodies against β2Glycoprotein-1 and cardiolipin. Pathophysiology includes thrombosis ('blood clotting') and devastating pregnancy complications such as recurrent morbidity (miscarriage), low birth weight and preeclmapsia. Antiphospholipid antibodies bind to endothelial cells and platelets causing vascular complications including thrombosis ultimately leading to clot formation at the site of the injury. The key components implicated in the parthenogenesis of APS are complement cascade, Tissue factor expression, inflammation and coagulation. Increasing evidence shows extensive cross talk between inflammation and coagulation, wherein inflammation leads to activation of coagulation and coagulation considerably enhances inflammatory activity. Purinergic signalling involving catabolism of ATP “danger signal” to adenosine by orchestrated action of cell surface enzymes CD39 and CD73 has been shown to have anti-inflammatory and anti-thrombotic effects. In this project, we aimed to ascertain whether activities of CD39 and CD73are important in developing aPL-ab induced miscarraiges. We utilised mouse model of misccaraiges by passive transfusion of purified human aPL-ab to pregnant wildtype and mice that are modified for CD39 and CD73 activity. We were able to show that aPL-ab infusion in pregnant CD39 and CD73 knock out mice trigger an increase in the rate of miscarriages associated wth increased expression of tissue factor, complement deposition and elevated oxidative stress. There is also an increase in the pro-inflammatory TNF-α and IL-10 expression within the placental vasculature. In contrast to these observations, we also observed that mice over expressing CD39 were protected against aPL-ab induced miscarriages. These mice had reduced TF expression in the decidua, along with reduction in the complement C3D component deposition. Diminished lipid peroxidation and reduction in the proinflammatory TNF-αexpression was also observed in these mice. Taken together, our results provide a rationale for both perturbations in the purinergic pathway to explain disease associated with aPL-ab and for the development of endothelial cell targeted soluble CD39 as a novel therapeutic for management of APS.
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ItemImmunological and microbiological studies in post-operative Crohn's disease( 2017)Crohn’s disease is a chronic, inflammatory condition of the bowel. The aetiology of Crohn’s has not been fully elucidated, but is believed to arise from the interaction between the gut microbiota, the host immune system and environmental factors. A majority of Crohn’s disease patients will require a bowel resection as a result of disease, which causes significant morbidity and impacts on quality of life. While surgery can ameliorate clinical symptoms, the disease often initially recurs at the site of the resection. This may be sub-clinical initially, and can be identified endoscopically. This thesis investigates the immunological and microbiological characteristics of post-operative Crohn’s disease recurrence in, addressing serologic markers, the faecal microbiome and the possible contribution of Proteus species to gastrointestinal disease. Serologic markers, while disappointing for prediction of disease recurrence, did have some utility for identifying patients at the highest risk of disease recurrence. I also demonstrated lower rates of antibody positivity in Crohn’s patients who smoke for the first time, indicating that these results should be interpreted with caution in current and past smokers. I have further evaluated the faecal microbiome in the setting of post-operative disease using metagenomic techniques. This work showed that after resection for Crohn's disease, enrichment of the bacterial family Lachnospiraceae is associated with maintenance of disease remission, while patients enriched for the Enterobacteriaceae are more likely to recur. Recurrence may result from a higher abundance of enteric pathobionts such as Proteus, Klebsiella, Serratia, and Escherichia. The Lachnospiraceae are an important family of butyrate producing bacteria in the gut, and depletion of this bacterial family may perpetuate the expansion of Enterobacteriaceae via environmental perturbation and ecologic shifts. These findings indicate possible protective and pathogenic bacteria in post-operative recurrence. Finally, I addressed the potential contribution of Proteus species (from the Enterobacteriaceae family) to gastrointestinal diseases including to inflammatory bowel disease. Proteus spp. are low-abundance commensals of the human gut that harbour significant pathogenic potential. Preliminary evidence of a pathogenic role in the gut should stimulate further investigation. I have elucidated some aspects of the microbiome and host immune factors involved, in the pathophysiology of post-operative Crohn’s disease recurrence. This work should encourage further work on a unifying hypothesis for the aetiology of disease recurrence after resectional surgery for Crohn’s disease.
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ItemAnalysis of glucose homeostasis and beta cell mass in a mouse model of type 2 diabetes deficient in the pro-apoptotic protein BIM( 2017)Loss of beta cell mass is a feature of type 2 diabetes and evidence suggests that apoptosis contributes to this loss. Previously in our lab, we identified that the pro-apoptotic BH3-only proteins BIM and PUMA are required for glucose-induced apoptosis of islet cells in vitro. High concentrations of glucose induce ER and oxidative stress that activate the downstream intrinsic apoptosis of beta cells through a BIM and PUMA dependent pathway. Islets isolated from human pancreas donors had increased expression of BIM in type 2 diabetic patients compared to non-diabetic pancreas donors. This suggests that BIM may play an important role in the loss of beta cell mass in humans as well. In this project, my aim was to investigate the role of the pro-apoptotic molecule BIM in loss of beta cell mass in vivo in type 2 diabetes. I tested the hypothesis that inhibiting BIM in type 2 diabetes will enhance islet survival and improve glucose homeostasis. Chapter 2 describes the role of BIM in the type 2 diabetic Leprdb/db model by studying metabolism. Global deficiency of BIM significantly protected Leprdb/db mice by improving glucose homeostasis. Loss of BIM decreased non-fasting and fasting glucose, and improved insulin sensitivity and glucose tolerance in Leprdb/db mice. Previous data from our lab suggest that whole body BIM deletion results in increased insulin sensitivity in muscle, liver and adipose tissue. This is due to a role for BIM in mitochondrial function, and a preference for BIM-deficient cells to utilize lipids in metabolism. Therefore, to examine the effects of BIM deficiency on beta cell death, we generated mice with beta cell specific deficiency of BIM. In contrast to global knockouts, BIM deficiency only in the beta cells of Leprdb/db mice did not improve glucose homeostasis. These findings suggest that the improved metabolic phenotype of global BIM deficiency results from enhanced insulin sensitivity and not prevention of beta cell apoptosis. In chapter 3, the role of BIM in beta cell survival in type 2 diabetes was studied in Leprdb/db mice with global BIM deficiency or beta cell specific BIM deficiency. We observed a striking increase in islet size in BIM-deficient Leprdb/db mice by histology, while the beta-cell size was similar between Leprdb/dbBIM-/- and Leprdb/dbBIM+/+ mice. This suggests that the increase in islet size was due to an increase in the number of beta cells. Quantification of islet volume with optical projection tomography revealed a significant increase in islet volume in BIM-deficient mice compared with wild-type Leprdb/db mice. We detected a reduced number of TUNEL positive islets cells in BIM-deficient compared with wild type Leprdb/db islets. We also observed about 2-fold increase in proliferating beta cells in Leprdb/dbBIM-/- mice compared to that in Leprdb/dbBIM+/+ mice. However, there was no change in islet size in the Leprdb/db mice with beta cell specific deletion of BIM. These results indicate that BIM does not play a direct role in beta cell apoptosis in type 2 diabetic conditions, but its global inhibition generates a compensatory model by increasing beta cell mass, and this is likely due to the improved insulin sensitivity as a result of BIM inhibition. Last, to test the hypothesis that BIM inhibition contributes to improvement of glucose homeostasis and beta cell expansion through regulating insulin sensitivity, we generated a model with severe insulin resistance using the insulin antagonist S961. Impaired glucose homeostasis and beta cell expansion was comparable in BIM-deficient and wild-type controls after S961 treatment. This indicates that BIM inhibition could not contribute to glucose homeostasis or beta cell expansion in the total absence of insulin sensitivity. This finding supports the idea that BIM’s function in regulating diabetes is related to insulin sensitivity. Overall, my results in this thesis suggest that inhibition of BIM in beta cells only cannot prevent type 2 diabetes, however, global inhibition of BIM contributes to improvement of glucose homeostasis and beta cell expansion in type 2 diabetes through regulating insulin sensitivity.
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ItemThe effect of multisite phosphorylation on the regulation of human CaMKK2 activity( 2017)Ca2+/Calmodulin dependent protein kinase kinase 2 (CaMKK2) is a serine-threonine directed protein kinase that is activated by increases in intracellular calcium concentration ([Ca2+]). The enzyme plays an important role in regulating neuronal function and whole-body energy metabolism, and genome wide association studies have implicated CaMKK2 in the pathogenesis of schizophrenia and bipolar disorder. In addition to its regulation by changes in intracellular [Ca2+], CaMKK2 activity is also modulated by a range of phosphorylation events, some of which are poorly understood. This study examines the role of reversible phosphorylation on CaMKK2 activity and function. This work first characterises nine rare-variant non-synonymous single nucleotide polymorphisms (SNPs) located proximal to key regulatory sites described in the literature, assessing their effect on kinase activity and providing insight into the mechanisms underpinning CaMKK2 regulation by phosphorylation. This thesis also describes the identification of multiple cAMP-dependent protein kinase (PKA) phosphorylation sites on CaMKK2 that result in the suppression of Ca2+/CaM-dependent CaMKK2 activity and the recruitment of 14-3-3 protein, definitively linking PKA signaling to down-regulation of CaMKK2 in vitro and in cells. Finally, this thesis re-examines the phosphorylation of Ser129, Ser133 and Ser137 in the N-terminus of the enzyme, which is known to inhibit the Ca2+/CaM-independent activity of the kinase. This work concludes that cyclin-dependent kinase-5 (CDK5), previously identified as the Ser137 kinase, is unlikely to be performing this role in vivo, and provides evidence that casein kinase-1 (CK1) is likely the physiological Ser137 kinase. In addition to this, the development of an optimised protocol for the isolation of large quantities of highly purified, bacterial expressed CAMKK2 is also described, which provides the foundations to explore the regulation of CaMKK2 by multisite phosphorylation at the structural level.
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ItemProtein and lipid markers of coronary artery disease( 2017)Identification of high risk coronary lesions, plaque rupture, early myocardial ischemia and in-stent restenosis (ISR) remains a clinical challenge. We employed proteomic, lipidomic and traditional approaches to identify novel biomarkers. TMEM198 was identified as a marker of plaque rupture. 42 proteins and multiple lipids identified early myocardial ischemia. 12 proteins and 2 lipids identified ISR. Phospholipase A2 was associated with periprocedural ischemia. Combined, these experiments utilise unbiased methodologies for novel cardiac biomarker discovery.
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ItemSubstituting beta cell function in type 1 diabetes( 2017)Matching exogenous insulin dosing to the varying metabolic requirements of people with type 1 diabetes is crucial for optimising health and minimising the burden of diabetes self-care. Advances in insulin formulations, insulin delivery systems and glucose monitoring technology have resulted in improvements in glucose control and in increased automation of therapy. However, subcutaneous insulin administration is significantly limited by its non-physiological delivery. Continuous subcutaneous delivery of rapid-acting insulin analogues via pump is well established in clinical care. Despite this, pharmacokinetic and pharmacodynamic responses to small insulin pump basal rate changes—typical of those implemented in clinical practice—have not previously been established. There is additional complexity associated with exercise due to changes in insulin sensitivity, absorption and action. While blood glucose meters represent a proven technology for point glucose measurement, their use is painful, requires user initiation and does not provide predictive information. These shortcomings are in part addressed by continuous glucose monitoring technology; however, the performance of the present generation of glucose sensors has substantial limitations. Hence, maintenance of glucose homeostasis in type 1 diabetes remains a therapeutic challenge. This research investigated the utility of effectively employing insulin pump and glucose sensor technology to optimise metabolic control and improve diabetes outcomes for adults with type 1 diabetes. This thesis shows that after small insulin pump basal rate changes, there are substantial delays until changes in circulating insulin levels occur. Moreover, for small rate changes of equal magnitude, it takes longer to achieve change in circulating insulin after a rate reduction than after an increase. Adjustment of basal insulin delivery to minimise hypoglycaemia with exercise was investigated. Findings demonstrated that very large reductions in basal insulin delivery are required to achieve a timely decrease in circulating insulin for aerobic exercise; when pre-exercise glucose levels are low-normal, supplemental carbohydrate ingestion may also be necessary to avoid hypoglycaemia. In a cross-sectional study, insulin pump users were observed to have more favourable vascular health profiles than those treated with insulin injections; these differences are possibly explained by multiple factors independent of the insulin delivery modality. To improve glucose sensor performance, a novel sensor combining two distinct sensing methodologies was developed and investigated. Feasibility of the novel sensor was confirmed, and its accuracy compared favourably with glucose sensors available at the time the research was undertaken. This thesis expands the current understanding of insulin delivery via pump and glucose sensing technology for people with type 1 diabetes. Until type 1 diabetes prevention and cure are achieved, the optimisation of insulin dose adjustment in parallel with the further development of glucose sensing technology is still required to mimic healthy pancreatic beta cell function.
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ItemMechanisms and inhibition of CD4+ T cell migration in pre-clinical and humanised mouse models of type 1 diabetes( 2017)Type 1 diabetes (T1D) is an autoimmune disease that develops when the insulin-secreting beta cells in the pancreas are destroyed. This destruction is primarily mediated by T cells, of which CD4+ T cells play a central role by controlling immune responses via the production of cytokines. The aim of this thesis was to investigate mechanisms by which CD4+ T cells migrate to the pancreatic islets and kill beta cells, to test a therapeutic method of inhibiting this process, and to develop a mouse model capable of analysing the pathogenicity of human-islet infiltrating CD4+ T cell clones. Deficiency of IFNγ receptor has been reported to prevent the adoptive transfer of CD4+ T cell mediated diabetes. In chapter three, I confirmed these findings and investigate the hypothesis that IFNγ promotes the migration of islet antigen-specific CD4+ T cells by upregulating MHC class II on islet endothelial cells (IEC), thereby providing a cognate antigen signal for diapedesis across the microvessels into the islets. IFNγ treatment of islets led to MHC class II expression on IECs and high MHC class II was detected on IECs in the early stages of insulitis. However, bone marrow chimera experiments revealed MHC class II on IECs is not required for the transfer of CD4+ T cell mediated diabetes. This work rules out antigen presentation by IECs as a putative mechanism for the homing of antigen-specific CD4+ T cells into the pancreatic islets. Cytokines that signal through the JAK-STAT pathway play a role in CD4+ T cell-dependent diabetes. In chapter four, I tested whether JAK1/2 inhibition prevents CD4+ T cell mediated diabetes to determine whether this could be a viable method of blocking cytokine signalling, and pathogenic T-cell responses, in a clinical setting. AZD1480, a JAK1/2 inhibitor, delayed diabetes induced by adoptive transfer of highly diabetogenic CD4+ BDC2.5 T cells. AZD1480 slowed the development of insulitis, and decreased the absolute numbers of leukocytes, including CD4+ T cells, in the islets and pancreatic lymph nodes (PLN), especially reflected in reduced effector-memory T cells. Combined with the recent success of our laboratory in using JAK1/2 inhibitors to abrogate CD8+ T cell mediated diabetes and induce disease reversal, we envision that JAK inhibitors could be trialled in patients at risk of developing type 1 diabetes. Autoimmunity to proinsulin is crucial in the development of diabetes in mice. However, it is unclear whether T cell responses to proinsulin are required for type 1 diabetes in humans. In chapter five, I surveyed the characteristics required to accurately model, in a humanised mouse, CD4+ T-cell responses to proinsulin seen in the pancreatic islets of a deceased organ donor who suffered from T1D. Three components were determined to be necessary: human proinsulin, HLA-DQ8 and chimeric T-cell receptors containing the human TCR variable regions that encode for proinsulin recognition. This led to the generation of a human proinsulin knockin mouse, NOD.HuPI. In this mouse, the murine Ins1 gene was ‘replaced’ with human INS. In addition, chimeric TCR constructs for several of the proinsulin-specific T-cell clones were generated and tested. These constructs are now ready for use to create TCR transgenic or retrogenic mice. This humanised mouse model will serve as a vehicle to determine which proinsulin-responding clone(s) induce diabetes pathogenesis. This information is vital for designing antigen-specific therapies to prevent and treat T1D in the future.
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ItemAdenocarcinoma of the lung: an exploration of the relationships between histopathology, molecular pathology and inflammatory markers and their relationship to patient outcomes( 2017)Lung cancer remains the most common cause of cancer related death worldwide, with nearly 1.4 million deaths in 2008 globally. Adenocarcinoma is the most common type of lung cancer, and its frequency compared to other histologic subtypes is increasing. The simplicity of the label “adenocarcinoma” hides its significant pathologic and clinical heterogeneity. This thesis explores a number of clinicopathologic correlates in lung adenocarcinoma specimens obtained from patients treated at St Vincent’s Hospital in Melbourne, Australia. In 2011 the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS) and the European Respiratory Society (ERS) proposed a new classification system for pulmonary adenocarcinoma. This was subsequently adopted in the 2015 edition of the World Health Organisation Classification of Tumours of the Lung, Pleura, Thymus and Heart. Multiple groups demonstrated that the new classification had prognostic significance following resection of pulmonary adenocarcinoma independent of stage. The impact of the classification in metastatic disease was not known. This thesis found that it was possible to identify the adenocarcinoma patterns of solid with mucin, papillary, micropapillary and acinar in each specimen taken from a metastatic site and semi-quantitatively assess each component. Further, the identification of a major pattern was not prognostic, but did predict for differences in survival time for patients treated with systemic therapy. The worst outcomes were observed for patients with tumours with a major solid pattern. The major solid pattern was also found to have infrequent occurrence of activating epidermal growth factor receptor (EGFR) mutations. As this is the first time that this novel finding has been reported. Validation from other groups is required. The presence of the IASLC/ATS/ERS classification as a robust new tool with clinical relevance has led to further research to define other clinicopathologic correlates. Oncogene driver mutations in genes such as EGFR and Kirsten RAS (KRAS) are critical in selection of therapy in advanced disease. This thesis examined relationships between adenocarcinoma subtype and mutation status for patients who had resected lung adenocarcinoma. Patients with solid predominant adenocarcinoma were significantly less likely to have EGFR mutations, while KRAS mutation was a frequent event in invasive mucinous adenocarcinoma. No other significant associations were found. The findings were consistent with those recently reported by other groups from centres located in predominantly Caucasian countries. EGFR inhibition and the discovery of EGFR mutations was the starting point for a major change in the approach to treatment of advanced lung adenocarcinoma, however resistant to treatment occurs. It had been suggested that upregulation of phosphorylated STAT3 (pSTAT3) via interleukin 6 (IL6) and Janus Kinase (JAK) may be linked to EGFR mutation status in the absence of treatment with EGFR tyrosine kinase inhibitors and therefore may offer a rational target to delay resistance to such therapies. In the patient cohort studied the presence of EGFR or KRAS mutation status did not enrich for activation of IL6, JAK1 or pSTAT3 as determined by immunohistochemistry. Further, there was no clinicopathologic or prognostic correlates of note found by the IL6, JAK1 or pSTAT3 activation state. The assessment of IL6, JAK1 and pSTAT3 in the same samples and by two methods to assess positivity was a unique feature of this study. In conclusion this contributes new knowledge on the relevance of pathologic subtyping in advanced lung adenocarcinoma. It confirms and consolidates recent reports oncogene mutation status and adenocarcinoma subtype following surgical resection. It examines the IL6 / JAK1 / pSTAT3 pathway in detail in resected pulmonary adenocarcinoma. Translational research that explores why adenocarcinoma subtypes have different outcomes by treatment may allow clinicians to direct therapies differently or unlock new pathways for targeting lung adenocarcinoma with therapeutic effect.
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ItemCognitive and psychosocial functioning in genetic generalised epilepsy( 2017)Genetic generalised epilepsies (GGE) are a common, but under-studied cluster of epileptic syndromes of predominantly child and adolescent onset. The primary syndromes of GGE are childhood absence epilepsy (CAE), juvenile absence epilepsy(JAE), juvenile myoclonic epilepsy (JME), and genetic generalised epilepsy with generalised tonic-clonic seizures only (GTSCO). Important questions remain regarding: the degree of cognitive and psychopathological comorbidity, particularly in adults and in syndromes other than JME; effects of the disease on cognitive function; and psychopathology and psychosocial wellbeing in these patient groups. This thesis aimed to provide a detailed and quantitative description of cognitive function and psychopathology in GGE, assess the impact of contributing factors including subclinical epileptiform discharges on cognitive and psychopathology outcomes, and to evaluate the relationship between psychopathology and cognition. Methods employed include narrative systematic review, quantitative meta-analysis, and prospective assessment of cognitive and psychosocial functioning of a relatively large sample of people with GGE. Results indicated mild to moderately large reductions across most cognitive factors relative to that of healthy control participants and age-based normative data, with a relative weakness in long-term retrieval and memory function. Short-term memory function was not reduced relative to age-based normative data. Overall cognitive ability and memory function was negatively associated with total duration of epileptiform discharges during a 24-hour period. Approximately 50% of the sample reported elevated symptoms on a measure of psychopathology spanning six symptom types, with depression and anxiety the most common amongst these. Collectively, these findings highlight the need for increased awareness, screening and the provision of services for psychological comorbidities for people with GGE.