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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    Optimisation of Prime editing and its application in Fanconi anaemia
    Liu, Lu ( 2023-09)
    Fanconi anemia (FA), resulting from germline mutations in DNA repair genes of the FA/BRCA pathway, is the most prevalent inherited bone marrow failure disorder. The only approved treatment for FA is a hematopoietic stem cell (HSC) transplant from a healthy donor, but severe post-transplantation complications are common and, in some cases, lethal. To overcome these risks, in vivo or ex vivo gene editing of autologous HSCs to correct the underlying mutation has been proposed. As the defining DNA repair defect of FA cells precludes homologous recombination-dependent gene editing, we have implemented in vitro prime editing in primary HSCs from a novel mouse model with an editable human FA patient mutation (FancL-TATdel). Mice that are homozygous for the FancL-TATdel allele display abnormalities consistent with human FA, including reproductive defects, hypersensitivity to the DNA damaging agent mitomycin C, and reduced hematopoietic stem and progenitor (LSK+) cell numbers in older mice. We utilized HOXA9 immortalized myeloid cells derived from FancL-TATdel mice, or an EGFP reporter mouse, to optimize conditions for the delivery of prime editing enzymes. Using an mRNA-encoded Prime editor, we accomplished highly efficient gene editing (0.5%) and restoration of mitomycin C resistance. We next established a protocol to expand HSCs (up to 1.5 x10^5/mouse) from either FancL-TATdel adult bone marrow or fetal liver cells in serum-free media. In these in vitro expanded HSCs, we demonstrated similar editing efficiency (0.5%) with higher resistance to mitomycin in vitro following selection (44% gene editing). We also achieved high levels of in vitro editing in other cell types, such as primary and immortalized fibroblasts. In all cases, restoration of just a single allele can restore normal FA/BRCA1 pathway function. Overall, our findings illustrate that a causative FA mutation associated with loss of the FA/BRCA pathway does not prohibit prime editing-mediated gene correction. Corrected cells behave as wild-type in all in vitro assays tested, forming a basis for in vivo transplantation experiments. The FancL-TATdel mouse model is the first model of FA with an editable human patient mutation and will be a useful pre-clinical model to further explore the potential of gene editing in the treatment of bone marrow failure syndromes.
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    The role of activin and follistatin in ischaemia-reperfusion-induced acute and chronic kidney injury
    Fang, Doreen Yann Pei ( 2023-05)
    The incidence of acute kidney injury (AKI) and chronic kidney disease (CKD) is rising and presents a major economic burden in Australia and globally. AKI and CKD, once regarded as two distinct syndromes, are now increasingly recognised as interconnected syndromes and likely to promote one another. An episode of AKI, even with biochemical recovery, increases the risk of future CKD. Likewise, underlying CKD is an important risk factor for AKI. Both AKI and CKD are associated with increased morbidity and mortality, including major cardiovascular events, progression to kidney failure requiring kidney replacement therapy (KRT), and premature death. Kidney ischaemia-reperfusion injury (IRI) is one of the leading causes of AKI and predisposes to the development of CKD. In kidney transplantation, IRI is an inevitable event and has a negative impact on both short and long-term graft survival. Regardless of the initial trigger of injury to the native or transplanted kidneys, fibrosis is the characteristic pathological change and final common pathway of progressive CKD. To date, available therapies slow the progression of fibrosis but there remains no effective clinical treatment to prevent or reverse kidney fibrosis. Therefore, attenuation of IRI to reduce the severity of AKI and subsequent development of CKD and kidney failure is an important area of research. Activin A has been implicated as a key driver of inflammation and fibrosis. Similar roles are now emerging for activin B. Follistatin (FS) is an activin-binding protein that counters the action of activins. This thesis examined the role of activin A and B in ischaemia-reperfusion (IR)-induced inflammation and fibrosis, and the therapeutic potential of FS in mitigating AKI and CKD. Using mouse models of kidney IRI, we demonstrated a significant increase in activin A and B levels, albeit with different kinetics, following kidney IR, with an associated increase in the expression of pro-inflammatory cytokines and evidence of AKI. FS treatment given pre-ischaemia reduced activin levels and inflammation, and attenuated IR-induced AKI. FS treatment also attenuated IR-induced kidney fibrosis when given in the early period post-IR on day 3 to 5. Together, these findings suggested that activin A and B are likely involved in promoting IR-induced AKI as well as CKD. The activin antagonist FS is a promising therapy in reducing the severity of AKI and subsequent development of CKD.
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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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    Necroptosis in kidney ischemia-reperfusion injury
    Pefanis, Aspasia ( 2023)
    The kidneys are particularly susceptible to ischemia-reperfusion injury (IRI), which occurs when the blood supply is temporarily reduced and then restored. Kidney IRI is the most common form of acute kidney injury (AKI), which often progresses to kidney fibrosis, causing significant patient morbidity and mortality with escalating healthcare costs. There are no effective therapies to prevent AKI or the subsequent progression to kidney fibrosis following IR. This may in part be due to limited understanding of the exact molecular mechanisms that cause kidney injury and fibrosis following IR. Emerging research has identified the involvement of regulated cell death pathways such as necroptosis in kidney IRI. Necroptosis is triggered by recruitment of the intracellular kinases RIPK1 and RIPK3 and activation of the pseudokinase MLKL. Active MLKL causes cell death by plasma membrane rupture, with release of intracellular contents driving “necroinflammation”. RIPK1 and RIPK3 may also have a cell death independent role in the development of kidney fibrosis. In this thesis we performed a detailed analysis of necroptosis in kidney IRI. Using a mouse model of unilateral kidney IRI, we showed that necroptosis contributes to kidney injury following moderate (18 min) but not more severe (20-24 min) ischemia. We identified a shift in cell death mechanism from necroptosis to apoptosis with increasing severity of IRI; apoptosis was also more prominent in necroptosis-defective Mlkl-ko mice subjected to IR. Using a time course study, we explored the kinetics of kidney injury, inflammation and necroptosis following moderate ischemia. Early inflammation and activation of the necroptosis pathway resulted in tubular cell death, driving ongoing necroinflammation. In the absence of necroptosis (i.e. in Mlkl-ko mice), early inflammation and subsequent kidney injury were reduced, indicating that necroptosis and the associated inflammation are important mediators of kidney damage following IR. In a therapeutic study, we assessed the capacity of small molecule inhibitors of RIPK1, RIPK3 and MLKL to attenuate AKI following IR. Prophylactic or therapeutic inhibition of RIPK1 with Nec-1s protected against kidney IRI, whereas prophylactic inhibition of RIPK3 with GSK872 was not protective. This is consistent with results obtained in knockout mouse studies and confirms a role for necroptosis in kidney IRI. Finally, we investigated the role of RIPK1 and RIPK3 in the development of kidney fibrosis following IR. Treatment with Nec-1s or GSK872 from days 3-9 after IR (i.e. after AKI was established) reduced kidney fibrosis at day 28. This is consistent with previous studies in other mouse models suggesting that RIPK1 and RIPK3 can mediate kidney fibrosis independent of their role in necroptotic cell death. In summary, this thesis established a mouse model to definitively demonstrate the role of necroptosis in kidney IRI, and identified specific necroptosis pathway components as treatment targets to reduce both AKI and kidney fibrosis following IR. Our results support the further exploration of necroptosis inhibitors to prevent and treat kidney IRI, ultimately aiming to reduce the significant burden of kidney disease.
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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.