Medicine (St Vincent's) - Theses

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    Regulation of bone and fat cells by zinc-finger protein- 467
    Quach, Julie. (University of Melbourne, 2010)
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    Calcitonin inhibition of parathyroid hormone anabolic action
    Gooi, Jonathan Hsien-Yang. (University of Melbourne, 2009)
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    Investigating the miRNA-independent role of ribonuclease III enzymes in gene regulation
    Gu, Karen ( 2022)
    Ribonuclease III enzymes, Drosha and Dicer, are double-stranded RNA (dsRNA)-specific endoribonucleases that are best known for their role in microRNA (miRNA) biogenesis in mammalian cells. Accumulating evidence suggest that they play additional roles beyond miRNA biogenesis. Many of these miRNA-independent functions dependent on the cleavage activity of these enzymes. However, the cleavage targets of Drosha and Dicer have not been globally profiled, and the cleavage-mediated gene regulation has not been thoroughly investigated. To comprehensively investigate Drosha and Dicer cleavage-mediated gene regulation, I employed a high-throughput sequencing approach to profile the miRNA-independent RNA cleavage events in mouse embryonic stem (mES) cells. I found that Drosha, but not Dicer, cleaves many RNAs in mES cells. The majority of Drosha cleavage targets appear to be mRNAs rather than primary (pri)-miRNA transcripts. Although many mRNAs appeared to be cleaved by Drosha, deficiency in Drosha did not significantly alter their expression levels. Only four targets were repressed post-transcriptionally via Drosha cleavage in mES cells: Dgcr8, Rcan3, Nanos3, and Todr1. The best-known substrates of Drosha are stem-loop structures in pri-miRNA transcripts. While the structure of pri-miRNA stem-loops has been extensively studied, Drosha cleavable mRNA stem-loops remain poorly characterised. I examined the mRNA stem-loops cleaved by Drosha and found that they are typically flexible and lack sequence or structural motifs that enhance Drosha cleavage efficiency and precision. Consequently, many mRNAs are cleaved inefficiently, and the cleavage typically does not significantly affect the expression of the target gene. Nevertheless, all of them possess the essential features for Drosha cleavage: a clear single-stranded RNA (ssRNA)–dsRNA junction and a dsRNA stem longer than 21 base pairs with a small or no bulge. Finally, I investigated whether the Drosha cleavage of Myl9 and Todr1, two mRNA targets of Drosha in mouse haematopoietic stem cells (HSCs), is conserved in human HSCs. I found that Drosha represses MYL9 expression by cleaving the stem-loops in MYL9 mRNA in human HSCs. Todr1, however, is not conserved in humans. Overall, this thesis provides the first comprehensive characterisation of non-miRNA Drosha cleavage targets and identifies the most crucial features that enable Drosha processing. This study also lays the foundation for further research into the miRNA-independent functions of Drosha in mES cells.
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    Improving Diagnostics for Neuroinfectious Diseases. Bridging the Gap with Advanced Sequencing Technologies
    Ramachandran, Prashanth ( 2022)
    Infections of the central nervous system carry high morbidity and mortality. Early diagnosis and treatment are critical to prevent poor outcomes. Current diagnostic assays for certain CNS infections perform poorly, making a timely diagnosis difficult. Two neurological infections that are notoriously difficult to diagnose due to poorly performing CSF assays are neurosyphilis and tuberculous meningitis. The first part of this thesis explores the incidence and clinical characteristics of Neurosyphilis (NS) in the Top End of the Northern Territory over a ten-year period and assesses the current NS clinical diagnostic criteria. The study identified a total of 25 patients that were diagnosed with NS (9 definite, 16 probable). Dementia was the most common manifestation (58.3%), followed by epilepsy (16.7%), psychosis (12.5%), tabes dorsalis (12.5%) and meningovascular syphilis (8.3%). 63% of probable NS cases were not treated appropriately due to a negative CSF VDRL. The study demonstrated the annual incidence [95%CI] of NS was 2.47[1.28–4.31] per 100 000py in the Indigenous population compared to the non-Indigenous population 0.95[0.50–1.62] (rate ratio=2.60 [1.19–5.70]; p= 0.017). The second part of this thesis was the development of a new diagnostic test for tuberculous meningitis. 368 patients were recruited with subacute meningitis in Uganda and performed next generation sequencing of cerebrospinal fluid (CSF). We created a combined assay using metagenomic next generation sequencing (mNGS) and a machine learning classifier (MLC) created from CSF host transcriptomic data. By leveraging the specificity of mNGS and the sensitivity of the MLC, we created an assay that had high sensitivity (88.89%) and specificity (88%) for the detection of TBM and its many infectious mimics. We then used in silico predictions to assess the feasibility of such an assay in low-resource settings. We achieved comparable combined assay performance with 600,000 reads at $75/sample, a protocol that would be more amenable to performing mNGS in low-resource settings.
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    Quality of endoscopy in the detection and management of gastrointestinal pathology
    Yang, Linda Seung-Won ( 2022)
    Endoscopy is essential in gastrointestinal medicine by allowing direct mucosal visualisation, histologic confirmation of pathology, and therapeutic procedures. Defining, measuring, and maintaining the quality of endoscopy is paramount in optimal patient care and cost-effective resource utilisation. Quality assurance in the field of colonoscopy is well-established, with adenoma detection rate and withdrawal time already implemented as key quality indicators. However, the quality of gastroscopy is under-investigated despite its importance. The studies in this thesis examined the current quality of endoscopic practice in Australia and explored methods of improvement. In addition, the use of artificial intelligence was reviewed to guide future research in the quality of modern endoscopy. This thesis identified that the current quality of gastroscopy was suboptimal based on international society guidelines. However, a simple educational intervention on endoscopists improved quality of gastroscopy which resulted in an increased detection of clinically significant pathology. Provision of up-to-date endoscopic procedures is another important aspect of quality assurance. The uptake of novel endoscopic techniques also requires more widespread awareness and better access. Endoscopy provides synergistic benefit in a multidisciplinary setting by improving accuracy of disease assessment. The role of artificial intelligence in endoscopy is increasingly recognised. This will be most beneficial in specific pathologies and patient cohorts where challenges in endoscopic imaging remain, such as dysplasia surveillance in inflammatory bowel disease. With continued advancement in endoscopic technology, future studies on quality need to adapt to the changes in clinical practice for effective measurement and assurance of quality.
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    The HBV-STOP study, a prospective study of nucleot(s)ide analogue discontinuation in non-cirrhotic Hepatitis B e-antigen negative chronic hepatitis B patients who have achieved long term virological suppression
    Hall, Samuel Anthony Lachlan ( 2022)
    It is estimated that 257 million people have chronic hepatitis B (CHB). HBV infection is endemic throughout Asia, sub-Saharan Africa, Polynesia, as well as in indigenous populations in North America, New Zealand and Australia. Nucleot(s)ide analogues (NA) are standard treatment for HBeAg-negative chronic hepatitis B (CHB). The two first-line NA, entecavir (ETV) and tenofovir disoproxil (TDF), are both potent antiviral agents which effect durable viral suppression, reduce hepatic necro-inflammation, fibrosis progression and reduce risk of cirrhosis, liver failure and hepatocellular carcinoma (HCC). However, HBsAg clearance, or functional cure, is rare, and long-term treatment is recommended. Finite therapy for individuals with HBeAg-negative CHB has been identified as an area of unmet need by expert organisations because of concerns regarding cost, long-term side effects and the risk of the development of drug resistance with indefinite treatment. In the past decade, there has been increasing interest in whether NA therapy can be safely stopped in a subset of patients. In 2012, Hadziyannis and colleagues published a landmark study evaluating clinical outcomes after stopping NA treatment in a small cohort of non-cirrhotic patients with HBeAg-negative CHB who were long-term responders to adefovir monotherapy. All patients experienced early virological relapse. Virological relapse was associated with biochemical relapse in 76% of patients, with antiviral therapy resumed in 15/33 patients. Eighteen patients remained off-treatment through five years of follow-up and all achieved a sustained response, defined by serum HBV DNA level < 2,000 IU/mL and normal serum ALT level. HBsAg loss was observed in 72% (13/18) of patients who maintained a sustained response off treatment by the end of 5 years of follow-up. HBsAg loss was associated with lower HBsAg level at the time adefovir was stopped, and was more common among participants who did not restart treatment. Among those patients who did not restart NA therapy, baseline ALT was higher in those who achieved HBsAg loss, suggesting a role for immune-mediated cytolysis of HBV-infected hepatocytes. Stopping treatment was safe, with no episodes of liver decompensation reported. Since this initial study, there have been a number of reports of clinical outcomes after stopping long-term NA therapy in patients with HBeAg-negative CHB. The studies have been heterogeneous, often retrospective in design, and with considerable variation between protocols for ethnicity of cohort, inclusion / exclusion of people with cirrhosis, duration of follow-up, as well as criteria for re-starting NA therapy. There remain important questions about the rate and outcome of virological and biochemical relapse after stopping NA treatment, the safety of this approach, and the rates of HBsAg loss in prospective follow-up. The answers will inform decisions about patient selection for this strategy in clinical practice. Therefore, as part of my thesis, I have planned to perform a prospective multi-centre study to evaluate clinical outcomes after stopping NA therapy in non-cirrhotic individuals with HBeAg-negative CHB after 96 weeks of follow-up, in addition to a meta-analysis of prior NA cessation studies in HBeAg-negative non-cirrhotic individuals. In addition, NA cessation studies provide a unique opportunity to study the immunology of acute hepatitis flares that occur in the setting of HBV reactivation after stopping NA therapy. ALT flares are preceded by a rise in serum HBV DNA and as HBV is a non-cytopathic virus, it is thought that liver injury is primarily immune-mediated. However, the literature characterizing acute flares of HBV is sparse because flares are difficult to predict and most are asymptomatic. Innate immune pathways may participate in ALT flares either directly or in a bystander fashion. To further investigate the role of innate immunity in the host response to HBV infection in the setting of hepatitis flares, as the final component of my thesis, I have also planned to examine the longitudinal expression and activity of TLRs on peripheral monocytes and markers of peripheral NK cell activity in patients experiencing severe hepatitis flares in the setting of HBV reactivation after NA discontinuation by using samples from patients in our prospective multi-centre study investigating clinical outcomes of stopping NA therapy in non-cirrhotic individuals with HBeAg-negative CHB in order.
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    Defining mechanisms by which EphrinB2 in osteocytes controls bone strength and material quality
    Blank, Martha Alexandra ( 2022)
    Bone strength is determined by bone mass, geometry, and material quality. Mechanisms determining bone mass have been well-studied but what maintains material quality remains poorly understood. We have previously provided evidence that EphrinB2 in osteocytes may mediate one pathway supporting bone material quality. Knockdown of EphrinB2 in osteocytes led to lower bone strength without any difference in bone mass or geometry. The bone strength defect originated from greater levels of collagen and mineral in cortical bone suggesting that EphrinB2 in osteocytes limits their accrual. In this thesis, I investigate the cellular and molecular downstream effects of EphrinB2-deletion in osteocytes in vitro and assess differences in the bone material quality due to absence of EphrinB2 in osteocytes in vivo and in vitro. Since our initial data suggested that greater autophagy in osteocytes may cause the greater mineral content I sought to determine the role of EphrinB2 in autophagy. I showed that EphrinB2-Fc treatment limits autophagosome numbers and that stimulation of autophagy can increase mineralisation in osteocytes in vitro. However, further investigation showed that EphrinB2-deficiency in osteocytes leads to lower lysosome content and cis-Golgi apparatus fragmentation compared to control cells. This suggests that autophagosomes accumulate in EphrinB2-deficient osteocytes due to impaired degradation by lysosomes. Thus, I suggest that EprhinB2 deletion in osteocytes limits lysosome formation. To address the low lysosome content in EphrinB2-deficient osteocytes, I next investigated the potential dysregulated pathways supporting lysosome formation in these cells, including the transcription factor EB (TFEB) and mTOR activity specifically mTORC1. I found that EphrinB2-deficiency in osteocytes resulted in lower Tfeb mRNA and TFEB protein levels, possibly caused by greater mTORC1 activity, an inhibitor of TFEB activity. Proteomics analysis additionally showed lower levels of bone material-degrading enzymes such as cathepsins and matrix metalloproteinases, which are direct downstream targets of TFEB. However, Tfeb knockout in osteocytes did not mimic the greater mineral levels observed when EphrinB2 was absent in osteocytes suggesting that the downregulation of TFEB is not the primary mechanism by which EphrinB2 deletion leads to mineral accrual. Since the bones of mice with EphrinB2 deficient osteocytes exhibit lower bone strength due to a defective material, and my data showed lower levels of bone matrix-degrading enzymes, I next sought to determine the effects of EphrinB2-deficiency in osteocytes on the bone matrix. I found that, when cultured in a collagenous matrix, absence of EphrinB2 in osteocytes led to less contraction of their collagenous environment compared to control cells. This could explain additional observations I made in vivo, where bones with EphrinB2-deficiency in osteocytes exhibited thicker and less parallel-oriented collagen fibre bundles. When I assessed crystal length in cortical bone in vivo I observed no differences between mice with EphrinB2-deficient osteocytes and controls. This suggests that the origin of the lower bone strength caused by the deficiency of EphrinB2 in osteocytes may be due to a gradual formation of a defective collagenous matrix which provides more binding space for mineral crystals to nucleate and this leads to brittle bones. Having initially shown that stimulating autophagy using rapamycin can increase mineral deposition in vitro I wanted to investigate whether the stimulation of autophagy can be used as a therapeutical tool to improve bone material quality. For this I used a mouse of osteogenesis imperfecta (OI) and tested whether the removal of toxic collagen I aggregates in osteoblasts could improve the severity of the OI phenotype using the FDA-approved autophagy-inducing drug carbamazepine (CBZ). Neither short- (3 weeks) nor long-term treatment (6 weeks) improved bone size, strength, or material quality in growing OI mice. Additionally, long-term treatment with CBZ had no effect on bone material quality, but led to lower bone strength in control mice caused by lower cortical and trabecular bone mass. These data suggest that CBZ-induced autophagy does not improve the OI phenotype but inhibits osteoblast function and bone growth in young individuals. In summary, I showed that EphrinB2 deficient osteocytes have a lower lysosome content and fragmented cis-Golgi apparatus and produce lower levels of bone matrix-degrading proteins compared to control cells in vitro. Additionally, EphrinB2-deficiency in osteocytes led to less collagen arrangement in vitro and thicker and less-parallel oriented collagen fibre bundles in bones in vivo without any difference in mineral crystal length compared to control bone. This suggests that EprhinB2 in osteocytes is essential for lysosome formation and supports lysosomal enzyme production and thereby maintains collagen fibre integrity. This reveals a novel physiological role for osteocytes to gradually limit mineral and collagen accrual in bone tissue to maintain bone strength.
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    Creating a state-wide registry to drive new epidemiological, forensic and clinical insights into sudden cardiac arrest in young Australians
    Paratz, Elizabeth Davida ( 2022)
    Sudden cardiac arrest (SCA) is a leading cause of death globally, accounting for approximately half of all deaths from cardiovascular disease. As demonstrated in this thesis, in young Australians it accounts for one-quarter of all deaths and is associated with approximately 90% mortality. Despite the condition’s high mortality rate and prevalence, systematic and adjudicated data collection remains lacking. As indicated in Chapter 1, only a few registries worldwide provide comprehensive multi-source surveillance of all SCAs, and many important questions in the field of SCA remain unanswered. The substantial economic cost imposed by young SCA in Australia is calculated for the first time in this thesis and provides an economic as well as medical imperative to better understand SCA. In Chapter 2, the aims of this PhD project are established as being to design a multi-source SCA registry with comprehensive case capture and adjudication. Such a registry would enable the secondary aims of identifying epidemiological and forensic insights, as well as creating an opportunity to highlight the experiences of selected patient groups. Chapter 3 of this thesis describes in detail the process of establishing the End Unexplained Cardiac Death (EndUCD) registry including its hierarchical consent process, data flow, data quality control and governance processes. From the establishment of the EndUCD registry, large-scale epidemiological assessments have been undertaken (Chapter 4). These include delineating overarching features of the entire patient group such as causes of arrest, the role of obesity and the role of rural residence. We have also identified variations in administrative data coding that will create under-appreciation of the community burden of SCA. The registry also enabled several projects to be undertaken in close collaboration with the Victorian Institute of Forensic Medicine (Chapter 5), yielding novel forensic information in the investigation of young sudden cardiac death (SCD). These projects include identifying rates of referral and potential under-referral of young SCD for forensic investigation, investigating a novel approach of performing post-mortem coronary artery calcium scores to assist cause of death determination, and examining the diagnostic yield of post-mortem interrogation of implanted cardiac devices such as pacemakers. The EndUCD registry’s unfortunately substantial number of patients created an opportunity to examine multiple relevant clinical questions (Chapter 6). These include more targeted clinical scenarios such as the outcomes of pregnant women experiencing SCA, those with coronary artery anomalies and unusual cases such as IgG4 coronary arteritis causing SCA. Two registry participants also graciously waived their anonymity and shared their experiences in being affected by SCA. In conclusion, the EndUCD registry has been established in line with best practice globally, and successfully commenced operations. Data from this registry has enabled diverse important projects providing new insights into the management of SCA on epidemiological, forensic and clinical levels.
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    Addressing knowledge gaps in the assessment of disease status in systemic sclerosis
    Ross, Laura ( 2022)
    Systemic sclerosis (SSc, or scleroderma) is a rare, orphan condition associated with high morbidity and mortality. There remain significant areas of unmet need in terms of the understanding of the underlying pathogenesis of the disease, how disease manifestations are identified and measured in clinical trials, as well as in clinical practice, in addition to a lack of effective, targeted therapeutics that offer improved survival and quality of life to patients. SSc is frequently a fatal disease, with a mortality risk up to seven times that of the general population and it is associated with a large burden of physical and mental ill-health, notable from the time of onset of disease. Our understanding of the disease has progressed significantly over the past 100 years, since Matsui confirmed that SSc was in fact a multi-system disease, not an exclusively dermatological condition. The importance of vascular changes and auto-inflammatory mechanisms in addition to excessive collagen deposition in the development of SSc and subsequent disease progression are now appreciated. Important pathogenic pathways that lead to the dense fibrotic extra-cellular matrix deposition and obliterative vasculopathy that characterise advanced SSc have been identified. However there is an ongoing absence of evidence to link levels and activities of cytokines and other cell mediators to specific pathogenic effects in SSc. The precise pathogenic mechanisms of specific disease manifestations remain incompletely understood and insights are often gained by working backwards through studying those individuals with advanced disease and identifying the clinical and pathogenic mechanisms that set them apart from others with mild disease. Heterogeneity, both of disease presentation and progression, is a clinical hallmark of SSc, and this has impeded efforts to both define the presence of disease and measure important changes in disease status, either in response to novel therapies in a clinical trial or in observational, longitudinal cohort studies. Important disease manifestations such as cardiac disease or gastrointestinal disease remain largely undefined and the lack of fully-validated multi-system measures of disease status have contributed to the failure of multiple randomised controlled therapeutic trials. Treatment options in SSc are limited, and it remains one of the few rheumatic diseases without a targeted treatment that alters the disease course. Optimal clinical trial design remains debated and effective recruitment strategies to identify those individuals most likely to benefit from treatment are controversial. Methodological inconsistencies remain an Achilles heel of SSc clinical trials, meaning accurate, valid measurement of disease status and comparison between study cohorts cannot be performed. In this context, this thesis aims to further our understanding of SSc by studying specific organ effects of SSc and reviewing and developing methodologically sound means of disease assessment. Specifically, this thesis will address the significant knowledge gaps that currently limit our ability to develop a global measure of disease activity in SSc. I will address the face, content and construct validity of items to measure activity within particular organ systems and will present a preliminary multi-system activity index. Additionally, I will explore SSc-associated heart involvement. This organ system was prioritised in my thesis research because of the prognostic importance of cardiac disease in SSc and the lack of understanding of the full spectrum and natural history of heart involvement, and the absence of valid methods of diagnosis. I present results that quantify the prevalence of myocardial fibrosis in SSc, evaluate the utility of commonly available cardiac investigations in the diagnosis of SSc heart involvement and the progress made in the development of classification criteria for the diagnosis of SSc-associated heart involvement.