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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ItemUnderstanding the effects of Radiotherapy and Surgical injury on Local immunity and Lymphatic Architecture and Function( 2019)This thesis aims to provide insight into the pathophysiological mechanisms behind two important clinical problems encountered in patients surviving oncological treatments: 1) Infections in previously radiated sites and 2) Lymphoedema that follows surgical injury to lymphatics or radiation of nodal basins (RTx). Both infection and lymphoedema have significant impact on patient morbidity and quality of life (QOL). Lymphoedema, in particular, has a significant “psychological and social health cost”, and is associated with significant costs in providing long term conservative management to prevent symptom progression and maintenance of QOL outcomes for patients. Therefore, socioeconomic implications of these diseases certainly warrants further investigation of the disease pathophysiology and potential avenues for prevention and treatment. This thesis provides a review of knowledge regarding normal structure, function and development of the lymphatic system, lymphangiogenesis and lymphatic remodelling and how these processes relate to lymphoedema. In addition, the processes of antigen capture and transfer to lymph nodes for presentation to lymphocytes will also be addressed as a background to understanding infections in radiated sites. Subsequently, methods to investigate lymphatic morphology utilising a murine Prox1-eGFP model and confocal imaging were detailed, as was an automated segmentation and analysis method to allow high throughput analysis of lymphatic vessel morphology following sugical or RTx injury in detail. Functional techniques utilised to assess antigen presentation and fluid transport to draining lymph nodes (dLNS)have also been described in detail. We have demonstrated in this thesis that RTx significantly reduces CCL21 expression by LECs and has subsequent effects on antigen presentation from RTx tissue. These results have implications for the increased rates of infection noted after RTx. Moreover, RTx significantly impairs early wound lymphangiogenesis in vivo. While surgical injury alone induces remodelling of both lymphatic capillaries and deep collecting lymphatic vessels (CLVs) distal to sites of surgical, combined injury with RTx and Surgery markedly exacerbates these remodelling responses. By using longer time points, we have also explored possible mechanisms behind chronic lymphoedema, and have demonstrated that RTx continues to impair wound lymphatic regeneration in combination with severe surgical injury. Furthermore, the distal limb appears to demonstrate evidence of progressive remodelling at late time points following lymphatic injury. As with earlier time points, combined injury with RTx and Surgery, appears to promote exacerbated secondary lymphangiogenesis in the distal limb at later time points. Potential molecular mechanisms behind these differences between the groups have then been subsequently explored through RNA sequencing and multiple PCR analyses of total tissue mRNA. The broader implications of the findings of this thesis to clinical lymphoedema and RTx are then addressed, as well as potential future directions for research to build on the findings of this thesis.
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ItemStudies investigating the clinical impact and immunological host response to viral eradication in hepatitis C infection( 2019)Australia now has improved access to HCV treatment for individuals with all stages of liver disease. There is limited data however, documenting any long-term clinical benefit of viral eradication in those with early stage liver disease. We report on the long-term outcomes of a well-characterised cohort of CHC subjects with predominantly early stage liver disease, whom paired liver fibrosis assessments were performed more than 10 years apart. We show in a real-world setting, that both early and curative HCV treatment halts fibrosis progression. Our data supports the early treatment of all people with CHC regardless of liver fibrosis stage, to prevent long-term liver sequelae. In subjects with acute / subacute HCV infection, we identify predictors of innate immunological response in peripheral blood mononuclear cells, to differentiate spontaneous clearers to those who progress to chronic infection. Our data strongly implicates TLR4 signaling in the persistence of HCV infection. Those who developed chronic infection had higher TLR4 expression on peripheral monocytes and NK cells and increased IFN-gamma response to TLR4 stimulation. We also observed increased TLR7 responsiveness in this group. We confirm the previously noted associations of IFNL4 genotype and plasma IP-10. Our data presents TLR4 as a potential biomarker for predicting clearance. This would clinically translate to individuals presenting with acute / subacute HCV infection, being able to defer drug therapy and its associated morbidity and cost. In subjects with chronic HCV infection, we demonstrate a clear effect of direct antiviral agent (DAA) mediated viral suppression therapy on patterns of TLR signaling in subjects with chronic HCV-1. We show that peripheral monocytic TLR2, TLR4 and TLR7 signaling is down-regulated early on in treatment, with a strong trend to higher baseline TLR signaling being associated with viral clearance with the DAAs. We are the first study to demonstrate a relationship between TLR signaling activity and IFN-free therapy for HCV. We hypothesise that the HCV virus directly stimulates the TLR pathways to induce an antiviral effect, with higher TLR signaling evident among those who respond to DAA therapy. This was accompanied by a reduction in PBMC ISG expression, NK activation markers and plasma levels of inflammatory cytokines / chemokines, with restoration of the innate immune response. Despite excellent treatment options, the mechanisms responsible for viral eradication in HCV have remained poorly defined. Previous data suggest a link between innate immunity and HCV pathogenesis, as well as spontaneous viral clearance. The data suggested that antiviral therapy alone was not sufficient to clear virus and supported a key role for innate immunity contributing to viral clearance. Our results strongly implicate TLR signaling / expression in HCV viral clearance. We believe TLR agonists must be considered as a potential HCV vaccine candidate and further research in this area is required.
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ItemActivation of islet inflammation by cytokine signalling in pancreatic beta cells: understanding the role of protein tyrosine phosphatases( 2018)Type 1 diabetes is characterised by the autoimmune destruction of insulin producing beta-cells in the islets of Langerhans of the pancreas. Immune cells release pro-inflammatory cytokines such as interferon-g (IFN-g), tumour necrosis factor-a (TNF-a) and interleukin- 1b (IL-1b) into the islet microenvironment which activate phosphorylation cascades and gene expression in beta-cells that increase their susceptibility to autoimmune attack and destruction. Protein tyrosine phosphatases (PTPs) regulate phosphorylation based signalling pathways and have previously been shown to negatively regulate IFN-g induced cell death of beta-cells in vitro. We previously showed that during immune infiltration to the islet PTPs, including PTPN1 and PTPN6, are rendered catalytically inactive through oxidation resulting in loss of signal regulation. The overall aim of this thesis is to observe if antioxidant treatment can reduce autoimmune development in the NOD/Lt mouse through reduction of oxidised PTPs and dissect the role of PTPN1 and PTPN6 in the regulation of cytotoxic signalling events in the NIT-1 beta-cell line and isolated NODPI islets in vitro. Chapter 3 studies the effect of the mitochondrial targeted antioxidant mito-TEMPO on insulitis and diabetes development in the NOD/Lt mouse. Delivery of mito-TEMPO through drinking water reduced levels of oxidised PTPs in the pancreas of NOD/Lt mice but had no effect on the development of insulitis, activity or number of CD8+ and CD4+ T- cells in the periphery in NOD/Lt mice or diabetes development in a diabetes transfer model. Chapter 4 describes the regulation of cytokine signalling in NIT-1 cells and isolated NODPI islets by PTPN1. Inhibition of PTPN1 activity reduced IFN-g, TNF-a and IL-1b induced death of NIT-1 cells in vitro. Activation of the IFN-g, TNF-a and IL-1b signalling pathways and downstream transcription of pro-inflammatory gene signatures associated with autoimmune diabetes were also reduced with PTPN1 inactivation. Furthermore, PTPN1 inhibition reduced IFN-g induced MHC-I expression on the surface of NODPI beta-cells and reduced the ability of autoreactive NOD8.3 CD8+ T-cells to destroy isolated NOD/Lt islets. These studies showed that PTPN1 is a positive regulator of cytotoxic signalling in NIT-1 cells and NODPI islets and promotes immune cell mediated death, suggesting it may be a potential therapeutic target for type 1 diabetes. Chapters 5&6 study the role of PTPN6 in cytokine signalling regulation in NIT-1 cells. PTPN6 inhibition was found to enhance TNF-a induced NIT-1 cell death independent of IFN-g and IL-1b in vitro. TNF-a induced JNK signalling was enhanced with PTPN6 inhibition which resulted in reduced anti-apoptotic BCL-2 protein expression and enhanced caspase-3 cleavage. Pan-caspase inhibition prevented TNF-a induced cell death suggesting that cells were dying through apoptosis. TNF-a induced cell death was also prevented with RIPK1 inhibition which prevented enhanced caspase-8 cleavage in PTPN6 deficient cells. Collectively these studies showed that PTPN6 negatively regulates TNF-a induced intrinsic and extrinsic apoptosis of beta-cells in vitro. Overall, the data indicate that PTPs play a nonredundant role in the regulation of cytotoxic signalling in NIT-1 cells and NODPI islets. This finding is consistent with results in other disease pathologies. The results provide a mechanistic insight into how PTPN1 and PTPN6 have opposite roles in regulating cytokine signalling, highlighting how PTPN1 antagonism and PTPN6 agonism may prove beneficial in reducing beta-cell death in vitro. Whether these results are directly translatable into in vivo models of autoimmune diabetes remains undetermined. The use of PTPN1 inhibitors or PTPN6 agonists currently under development would allow direct translation of these results.
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ItemThe contribution of the pulmonary circulation and cardiac function to exercise intolerance in people with diabetes( 2019)Diabetes mellitus (DM), and in particular type 2 DM (T2DM), is approaching epidemic proportions globally. Relative to people without diabetes, exercise performance is reduced by approximately 20% in T2DM subjects universally, whereas impairment in type 1 DM (T1DM) is less consistently reported. Risk of cardiovascular disease (CVD) is significantly elevated in both T1DM and T2DM, and thus emphasis is placed on cardiovascular constraints to exercise capacity, including subclinical cardiac dysfunction and the entity of diabetic cardiomyopathy (DMC). A clear bidirectional association between heart failure and T2DM exists, with numerous pathophysiological mechanisms identified in animal models. Longitudinal data suggests a low prevalence of heart failure in T1DM, on the other hand, which insinuates the pathological mechanisms thought to underpin DMC may be exclusive of chronic hyperglycemia which unifies the diagnosis of T1DM and T2DM. The pulmonary microvasculature is targeted in diabetes, meanwhile, and may be an under-appreciated contributor to exercise limitation. It is thought that the lungs are protected from deleterious effects of subclinical microvascular disease by virtue of their substantial size, although the ability to assess pulmonary microvascular function during exercise has been limited. This thesis assesses exercise performance (VO2peak) in people with T1DM and T2DM, evaluates the contribution of cardiac function and the pulmonary circulation using comprehensive traditional and novel echocardiographic analyses, and investigates the effect of phosphodiesterase type 5 (PDE5) inhibitor Sildenafil on exercise capacity. VO2peak is demonstrated to be impaired in only the T2DM cohort of the study group, and independently associated with sedentary lifestyle and reduced left ventricular end diastolic volume. Comprehensive resting and exercise echocardiographic measurements of biventricular systolic function and LV diastolic function, in addition to speckle-tracking global longitudinal strain and LV twist mechanics, are normal. The novel echocardiographic assessment of pulmonary microvascular function by measuring the pulmonary transit of agitated contrast (PTAC) identifies less PTAC (low-PTAC) in DM subjects, and associations with reduced RV function, higher pulmonary artery pressures, and lower exercise capacity. Finally, Sildenafil improves a number of central hemodynamic parameters but does not improve VO2peak. Results of this thesis support the argument that in the presence of impaired exercise performance and longstanding DM, subclinical cardiac dysfunction and DMC should not be universally anticipated. Diagnosis of pulmonary microvascular dysfunction using PTAC is feasible and readily accessible, and pulmonary microvascular disease is associated with reduced exercise capacity. Nonetheless, quantification of pulmonary microvascular disease remains imperfect, and the pulmonary vasodilator Sildenafil is ineffective in improving exercise performance presumably due to the mismatch between the pathophysiology thought to underpin diabetic lung disease and the more proximal smooth muscle vascular target of Sildenafil.
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ItemMolecular Investigation of the Fanconi Anemia DNA repair pathway( 2019)Chemotherapeutic drugs often kill cancer cells by inducing toxic DNA interstrand crosslinks (ICLs) that inhibit DNA replication or transcription. Cells from patients with the genetic disorder Fanconi Anaemia (FA) are extremely sensitive to such DNA crosslinking agents. A critical step in the repair of ICLs is the biochemical modification of FANCI:FANCD2 protein by monoubiquitination. This monoubiquitination reaction is defective in 95% of FA patients, however the exact function of this process remains unclear. This thesis aims to better investigate how FANCI:FANCD2 monoubiquitination is regulated. To determine how monoubiquitinated proteins regulate the FA DNA repair pathway, several targets of monoubiquitination including FANCI:FANCD2, PCNA trimer and nucleosomes ubiquitinated by BRCA1:BARD1 were purified using Avi-ubiquitin. This thesis presents a minimal approach to purify challenging DNA repair proteins, which could potentially be applied to elucidate signalling mechanisms of other ubiquitination-mediated DNA repair pathways. Biochemical reconstitution of the FA pathway using recombinant human proteins revealed that FANCI:FANCD2 monoubiquitination is dependent on DNA and low ionic strength buffers. In addition, electrophoretic mobility shift assays revealed that monoubiquitination locks FANCI:FANCD2 on double-stranded DNA (dsDNA). Affinity pulldown of monoubiquitinated FANCI:FANCD2 confirmed that the complex remained bound to dsDNA, reflecting the monoubiquitination’s role in stabilising the FANCI:FANCD2 complex. The increased affinity of monoubiquitinated proteins on dsDNA suggests a conformational change on FANCI:FANCD2 after monoubiquitination. Using a newly developed Avi-tag-ubiquitin construct, the first successful purification of dually-monoubiquitinated FANCI:FANCD2 complex bound to DNA was reported. Strikingly, single-particle electron microscopy analysis revealed that monoubiquitinated FANCI:FANCD2 forms oligomers on dsDNA. This unexpected discovery hints that the function of monoubiquitination is to lock FANCI:FANCD2 oligomers on chromatin to initiate DNA repair. FANCI phosphorylation was previously reported to play a key role in stimulating FANCI:FANCD2 monoubiquitination. Mass spectrometry and western blot using phospho-specific antibodies revealed that ATR-mediated FANCI phosphorylation at specific serine-glutamate (SQ) clusters regulate both ubiquitination and deubiquitination of FANCI:FANCD2 complex. These results indicate a mechanistic view where FANCI phosphorylation is required for ubiquitination-mediated DNA repair which gives insights into the biology of FA pathway and potential chemosensitiser development. Collectively, the results presented in this thesis elucidate the role of monoubiquitination in locking FANCI:FANCD2 on DNA to form oligomers to initiate DNA repair. This work uncovers a new role of monoubiquitinated FANCI:FANCD2 during interstrand crosslink repair and provides new potential chemotherapeutic targets.
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ItemNovel aspects of commonly encountered pituitary adenomas in clinical practice( 2019)Clinically relevant pituitary adenomas are common in the general population occurring in 1 per 865-1470 people. Prolactinomas and non-functioning pituitary adenomas account for more than 80% of pituitary adenoma types. My thesis is composed of several studies, undertaken at a single centre specialising in the multidisciplinary care of pituitary diseases, investigating clinical issues pertinent to the management of patients with the above adenomas. The findings of these studies contribute to the understanding of outcomes in the local practice, as well as being an Australian contribution to the international experience of managing these adenomas. Firstly, I examine the issue of valvular heart disease in prolactinoma patients treated with cabergoline therapy. By studying a local cohort and by a systematic review I found that the prevalence of cabergoline-associated valvular heart disease (CAV) in prolactinoma patients to be extremely low, with just three confirmed cases amongst 1800 patients described in the literature. I have shown evidence that a simple annual cardiovascular examination is a suitable screening tool for this rare condition and made recommendations on when to consider a diagnostic echocardiogram. Secondly, I examine two clinical issues affecting patients with surgically treated non-functioning pituitary macroadenomas (NFPMAs): the issues of regrowth and recurrence, and of hormonal outcomes. In the largest Australian cohort of cases with NFPMAs to be described, it was found that residual disease is a common finding post-surgery. In cases with residual disease, regrowth occurred in 40% of these cases compared to only 12.5% in cases without residual disease, at a median of 3.6 years of follow-up. Not surprisingly, larger baseline adenoma size was a predictor of regrowth and recurrence. In multivariate analysis, the presence of residual disease and younger age (under 41 years) at presentation were independent risk factors for regrowth and recurrences. Based on this risk factor of younger age, I have recommended that these cases have lifelong radiological follow-up. Results of hormonal outcomes in cases with NFPMAs treated with surgery showed the novel finding of gender differences in hormonal outcomes, with males having a higher prevalence of multiple (more than two) hormone deficiencies (MHD) at presentation than females, and after surgery. In particular, pre-menopausal females had very few hormone deficiencies post-operatively and demonstrated a propensity to recover function. These gender subgroup differences are the first to be noted in the literature and have important clinical implications particularly for pre-menopausal females where fertility preservation is an issue. I also found that pre-menopausal females had smaller adenomas than males suggesting that they may present earlier in the natural history of the disease. In multivariate analysis, larger adenoma size remained a significant factor associated with post-operative MHD. The final part of this thesis is an exploratory study of intrasellar pressure (ISP) and how it relates to adenoma size and hormonal outcomes in cases with NFPMAs undergoing surgery. I did not find that ISP was correlated to adenoma size, but results did suggest that raised ISP may play a role in more frequent hormone deficiencies at presentation. In this study adenoma size was again noted to be showing a trend towards influencing hormonal outcomes. The findings from this study will require a larger cohort in order to better quantify how ISP and adenoma size interact to contribute to the pathophysiology of hormone deficiencies. In these three studies of NFPMAs adenoma size has emerged as an important factor influencing surgical and hormonal outcomes; smaller adenomas are more likely to be fully resected and less likely to result in hormone deficiencies. This thesis lays the foundations for advocating for the consideration of early surgical intervention of NFPMAs (when adenomas are smaller and earlier in the natural history of the disease), in order to optimise surgical and hormone outcomes. The above should now be an important clinical consideration in the multidisciplinary care of these patients.
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ItemEchocardiographic evaluation of the evolution of cardiac dysfunction in the community and risk of heart failure, using the SCReening Evaluation of the Evolution of New Heart Failure (SCREEN-HF) cohort( 2019)Heart failure (HF) is a major cause of morbidity and mortality in the community. Outcomes for HF with reduced ejection fraction (HFrEF) remain poor despite available treatments, and no medical therapy has been shown to improve survival in HF with preserved ejection fraction (HFpEF). Consequently, HF prevention is urgently required to curb the growth of HF in our communities. HF prevention requires that we improve our ability to identify individuals at high-risk of HF who may benefit from preventative measures. Although cardiovascular disease prevention has traditionally focused on risk assessment using cardiovascular risk factors, HF risk is further increased by the additional presence of cardiac structural and functional abnormalities. Cardiac dysfunction detected by echocardiography may therefore aid in improving risk stratification in HF. The SCReening Evaluation of the Evolution of New Heart Failure (SCREEN-HF) study is a community-based evaluation of individuals aged 60 years or over with cardiovascular risk factors followed longitudinally for the development of HF. Baseline transthoracic echocardiographic examinations were performed to document the asymptomatic cardiac dysfunction which preceded the development of incident HFpEF and HFrEF, and follow-up echocardiography was performed approximately four years later to document age-related cardiac changes. Using data from the SCREEN-HF cohort, this thesis investigated the clinical and echocardiographic predictors of incident HFpEF and HFrEF, in order to gain insights into HF development, and the role of echocardiography in the risk assessment of HFpEF and HFrEF. Contrasting clinical predictors of HFpEF and HFrEF provided insight into their underlying mechanisms. Risk factors associated with atherosclerosis, including smoking and abdominal adiposity, predicted only HFrEF, while pro-inflammatory conditions such as diabetes, obesity and renal dysfunction, and additionally high white cell count predicted only HFpEF. These pro-inflammatory risk factors were present approximately four years prior to clinical HFpEF, suggesting that focused risk factor management is an important strategy in HFpEF prevention. Baseline echocardiographic data from over 3000 subjects was used to investigate the role of echocardiography in HFpEF and HFrEF risk assessment. Diastolic dysfunction is an integral feature of HFpEF and diastolic dysfunction determined using current guideline algorithms was shown to predict incident HFpEF. In addition, individual echocardiographic parameters reflecting diastolic dysfunction, long axis systolic dysfunction and increased left ventricular mass predicted both HFpEF and HFrEF, while left ventricular enlargement and low left ventricular ejection fraction predicted only HFrEF. Using combined echocardiographic abnormalities, a low- and high-risk subgroup for HFpEF and HFrEF were identified, and echocardiography also assisted in differentiating between HFpEF and HFrEF risk. Older individuals are more likely affected by HFpEF than HFrEF. Using serial echocardiographic examinations from over 2300 subjects, age-related changes in cardiac structure and function were evaluated to provide insight into the tendency for older individuals to develop HFpEF. The pattern of age-related cardiac remodeling, particularly in women, was found to parallel the cardiac changes seen in HFpEF, providing one explanation for the propensity of older women to develop HFpEF. This thesis provides insights into HF development and provides evidence for the utility of echocardiography in HF risk stratification. These results serve as a platform on which echocardiography-guided management of HF risk can be further evaluated.