Anatomy and Neuroscience - Theses
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ItemInvestigating gene and isoform expression quantification in human brain using nanopore single-cell long-read technologies( 2023-09)Alternative splicing (AS) is a fundamental mechanism that enables a single gene to generate multiple RNA products, referred to as isoforms. AS is a widespread phenomenon and is most abundant in the human brain. Nearly all multi-exon genes undergo AS giving rise to numerous isoforms that can vary in their form, function and expression profiles. The significance of RNA isoforms in the human brain is underscored by their influence over cell states and function and their regulatory role in developmental processes and disease. Despite the importance of isoforms, our comprehension of their intricate expression profiles, and their impact on development and disease has long been constrained by the limitations of short-read sequencing. However, with the advent of long-read sequencing technologies, such as Nanopore sequencing, a new frontier has emerged for studying RNA isoforms. This technology offers a more detailed examination of the isoform landscape and provides an opportunity to explore isoform structures, expression dynamics, and their role in shaping brain development and disease. Recent advances have coupled long-read sequencing with single-cell sequencing, providing an unprecedent opportunity to explore isoforms in individual cell types and along developmental trajectories. This combination of technologies provides an avenue to explore isoforms and their related role in brain development and disease at single-cell resolution. However, as is the case for any new and emerging technologies, these methods encounter several challenges often the result of error prone long reads. Using highly accurate matched short reads is one approach that has been used to combat these limitations, but this adds considerable complexity and expense in both generating and processing the data, impacting the accessibility of single-cell long-read sequencing. In this thesis I aim to combat single-cell long-read sequencing limitations and make these technologies more usable and accessible. To achieve this, I firstly co-developed and benchmarked “BLAZE”, a tool for accurate identification of single-cell barcodes without the need for matched short-read sequencing. Building on this foundation I implemented a new and novel long-read only method for single-cell sequencing and show that this method is capable of exploring the complex developmental process of neurogenesis at single-cell resolution, including identifying cell types, plotting developmental trajectories and identifying novel isoforms implicated in human brain development. Finally, I explore factors that may limit the usability and effectiveness on Nanopore single-cell tools including the impacts of RNA degradation and showcase methods to mitigate these effects. Taken together, these advancements improve the utility of Nanopore sequencing and enable a comprehensive investigation of isoforms involved in brain development and disease.
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ItemEarly neuronal and glial cell changes in diabetic retinopathy(University of Melbourne, 2010)
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ItemModulation of the intermediate-conductance calcium-activated potassium (IKca) channel by protein kinase a (PKA)(University of Melbourne, 2010)
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ItemNeural plasticity and gene-environment interactions in the PLC-?1 knockout mouse(University of Melbourne, 2007)
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ItemNovel therapies for nonalcoholic fatty liver disease( 2023-08)Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease worldwide. The hallmark of NAFLD is hepatic steatosis, which occurs when the delivery, storage, and de novo synthesis of lipids outweighs lipid catabolism through oxidation or secretion. Despite the common progression of NAFLD to more severe forms of liver disease, and the contribution of NAFLD to systemic metabolic dysfunction, insulin resistance and type 2 diabetes (T2D), there are currently no approved drugs for the treatment of NAFLD. The successful development of pharmacotherapies is likely to require innovative combination therapies targeting multiple pathways, or therapies targeting novel pathways that are dysregulated in the pathogenesis of NAFLD. The aim of this thesis was to gain a more comprehensive understanding of the molecular mechanisms underpinning dysregulated lipid metabolism and to test novel therapies for the treatment of NAFLD. The first study within this thesis investigated a novel combination therapy for the treatment of NAFLD. Increased fatty acid uptake and de novo lipogenesis (DNL) are hallmarks of NAFLD. In this regard, we investigated dual targeting of the rate limiting enzyme of DNL, Acetyl-CoA carboxylase (1), with inhibition of the fatty acid transporter fatty acid translocase (FAT/CD36). While we showed that ACC inhibition was effective for reducing hepatic steatosis in mice with NAFLD, CD36 inhibition unexpectedly exacerbated steatosis. Further, in mice lacking hepatic CD36, ACC inhibition was not as effective at lowering liver triglyceride content. These changes were associated with an upregulation of genes and proteins of DNL, including ACC, and decreased liver triglyceride secretion ex vivo. Taken together, the results from this study indicate that dual ACC and CD36 inhibition is not an effective treatment for NAFLD. The second study within this thesis investigated novel regulators of fatty acid oxidation in vivo. Mitochondrial fatty acid oxidation requires the effective transfer of fatty acids between lipid droplets and mitochondria, yet the mechanisms which allow for this transfer are not well characterised. Recent research from our lab showed that the proteins extended synaptotagmin 1 and -2 (E-Syt1, E-Syt2) transfer fatty acids at lipid droplet and mitochondria contact sites and regulate fatty acid oxidation in cells. As such, the second aim of this thesis was to investigate the role of these proteins in lipid metabolism in vivo. We showed that short term inhibition of E-Syt1 and E-Syt2 in mice reduced hepatocyte fatty acid oxidation, and that short- term overexpression of E-Syt1 and E-Syt2 reduced hepatic triglyceride content in mice. In Drosophila, we showed that inhibition of the E-Syt2 remodelled lipid droplet storage and reduced survival in response to starvation, a setting where there is an increased reliance on fatty acid oxidation for energy production. Given these findings, we investigated the effects of long-term E-Syt1 and E-Syt2 inhibition and overexpression on hepatic lipid metabolism in mice. In mice lacking both hepatic E-Syt1 and E-Syt2, although not in singular knockout mice, hepatic steatosis was significantly increased. This was associated with reduced hepatic triglyceride secretion. In mice with long- term hepatic overexpression of E-Syt1 and E-Syt2, there was no effect on liver lipid storage or lipid metabolism. The work in this thesis extends our understanding of the molecular pathways involved in dysregulated lipid metabolism in NAFLD, and the interrelationship between these pathways. These studies support the notion that combination therapies will be required to effectively treat NAFLD in the future.
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ItemThe effect of the ablation of the SEZ6 protein family on microglial structure and function in the central nervous system( 2023-09)Microglia are the resident immune cells of the brain and are responsible for mediating the central nervous system (CNS) response to infection, injury and disease. More recently, roles for microglia in the developing brain have become evident, including maintaining tissue homeostasis, vascular control, and synaptic pruning, a normal developmental process during which synaptic connections are refined. Microglia have been shown to remove weaker synaptic connections through the involvement of the complement system, particularly the complement proteins C3 and C1q which label the synapse for microglial mediated removal. Reflecting their important role in this process, microglia are the only cells of the CNS that express complement receptor (CR) 3 and lack of C1q, C3 or CR3 results in reduced synaptic pruning during development. The Seizure-related protein 6 (SEZ6) family contain similar structural domains to those of complement regulatory proteins, namely Complement Control Protein (CCP) and CUB domains and SEZ6 proteins were recently shown to protect expressing cells against complement deposition. In genetic knockout models [SEZ6 knockout (KO) or SEZ6 triple knockout (TKO) mice lacking all three SEZ6 family members], roles for SEZ6 proteins in the development and maintenance of excitatory synapses have been identified. Preliminary data from our laboratory indicate that lack of SEZ6 proteins leads to reductions in microglial biomarker proteins in synaptosome preparations from mouse brain, suggesting that SEZ6 proteins are involved in regulating microglial-mediated synapse remodelling. This thesis will explore the role of SEZ6 proteins in regulating microglial structure and function in the brain. The aims of this work were to validate the preliminary synaptosome data and determine whether loss of SEZ6 proteins results in altered microglial expression of key proteins, including the cluster of differentiation (CD) 11B protein which, together with CD18, comprises CR3. Western analysis of CD11B protein in synaptosome preparations isolated from SEZ6 TKO showed reduced CD11B levels compared to control mice, supporting our preliminary data. Interestingly, this reduction was observed in female SEZ6 TKO mice only, with no change in synaptosome CD11B levels observed in males. In order to determine whether the decrease in synaptosome CD11B was due to a decrease in total expression in microglia, CD11B protein expression was also quantified in fluorescence-activated cell sorting (FACS)-isolated microglia. No change in total CD11B protein expression was found in either male or female SEZ6 TKO mice. However, when surface expression of CD11B and F11R were quantified via flow cytometry, a reduction in the surface expression of CD11B was found in both sexes and surface F11R expression was reduced in female SEZ6 TKO mice. Work in this chapter showed that loss of SEZ6 reduced the presence of the complement receptor CR3 subunit CD11B in synaptosomes of female mice and this may be due to altered surface expression of this receptor on microglia, supporting a role for SEZ6 protein in modulating the interaction between microglia and synapses. To further investigate the interaction of microglia and synapses in the absence of SEZ6 proteins, morphology of microglia was analysed as morphological changes in microglia are indicative of an altered state and associated with the microglial response to changes in their environment. In addition, microglia density was analysed as it was hypothesized that a lower density of microglia might lead to synapses being less frequently contacted. Quantification of the microglial density and morphology revealed strong regional differences, particularly in the cerebellum compared to the rest of the brain. While the density of microglia remained unchanged in the absence of SEZ6 proteins, microglia were found to be hyper-ramified in the hippocampus and striatum of SEZ6 TKO mice. This finding further supports an altered state of microglia and changed interaction with synapses. Lastly, the effect of the lack of SEZ6 proteins on synaptic pruning was analysed to investigate whether lack of SEZ6 proteins affects microglial function. Anterograde tracers [cholera toxin subunit beta (CTB) linked to different fluorophores] were injected separately into the left and right eye of P9 mice and the overlap of retinal ganglion cell (RGC) terminal fields was analysed in the P11 dorsal lateral geniculate nucleus (dLGN). No significant difference in the RGC terminal overlap was observed between SEZ6 TKO and control mice but pruning effects could not be excluded due to the incomplete labelling of the dLGN, even after troubleshooting this challenging intravitreal injection procedure. Together, the reduction of CR3 subunits in synaptosomes and on the surface of cortical microglia as well as the hyper-ramification of microglial processes in the striatum and hippocampus indicates that microglia have an altered interaction with synapses in the absence of SEZ6 proteins. While a direct functional effect on developmental synaptic pruning in SEZ6 TKO mice could not be shown, these results indicate novel roles for SEZ6 proteins as regulators of microglial function.
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ItemExploring concepts of cardiomyocyte 'stiffness' in cardiac diastolic pathology( 2023-12)Background Diastolic dysfunction is a common and early feature of cardiometabolic diseases. In patients with cardiometabolic disease it is a predictor of clinical outcomes. It typically progresses silently over a period of years in the worst case scenario resulting in heart failure. Despite this, there are no in vitro diagnostics or treatments that directly target diastolic dysfunction in this population. This may reflect absence of understanding about the combination of cellular and molecular characteristics that manifest diastolic dysfunction. At the intact heart level, diastolic dysfunction can be characterized as an impaired ability to relax and increased left ventricular stiffness. Consistent functional and molecular features of cardiomyocytes which may contribute to diastolic dysfunction in cardiometabolic disease settings remain unresolved. At the cardiomyocyte level, emerging evidence suggests that intrinsic stiffness of cardiomyocytes may be an important contributor to diastolic dysfunction while at the molecular level a sugar replete heart is a characteristic feature in cardiometabolic disease. The ability for excess sugar availability to predispose to non-enzymatic and irreversible modification (glycation) of the structure and function of the myofilament proteins responsible for cardiomyocyte stiffness and relaxation remains unexplored. Importantly, these cellular and molecular characteristics may form the basis of novel diagnostic or treatment strategies for early detection of diastolic dysfunction. The aim of this Thesis was to explore and define concepts and characteristics of cardiomyocyte stiffness and the molecular features thereof, especially as it relates to cardiometabolic disease. Research questions Question 1: Does intrinsic cardiomyocyte stiffness measured in vitro contribute to in vivo diastolic dysfunction in diet-induced cardiometabolic disease? (Chapter 2) Question 2: Are human purified troponin complex and troponins enriched from rodent myocardium modified by advanced glycation end products endogenously and are these modification sites in domains of functional relevance as described in the literature? (Chapter 3) Question 3: Can a liquid chromatography coupled MS/MS approach be developed to quantify the extent of amino acid site specific carboxymethyllysine modification on cardiac troponin I. (Chapter 4) Methods Cardiometabolic disease was induced in rodents via dietary (High fat/sugar; prediabetes or Type 2 diabetes) or pharmacological (streptozotocin; Type 1 diabetes) interventions. Echocardiography was used to assess in vivo heart function in cardiometabolic disease rodent models. Cardiomyocytes were isolated from high fat/sugar fed mice and respective controls by collagenase dissociation. Glass fibers were attached (MyoTak) at cardiomyocyte longitudinal ends, and paced cardiomyocytes (2, 4Hz, 2.0mM Calcium, 37C) were subjected to progressive stretch protocol. Force development, sarcomere length, and intracellular calcium transients (Fura2AM, 5uM) were simultaneously measured (Myostretcher, IonOptix). Liquid chromatography coupled MS/MS was used to map the occurrence of Advanced Glycation End product modifications (CML, carboxymethyllysine; CEL, carboxyethyllysine; MGH1, Methylglyoxal derived hydroimidazolone) across the human troponin complex. Western blot was used to quantify total CML amounts on myocardial proteins of interest in Type 2 diabetic human and Type 1 diabetic rat myocardial samples. A novel immuno enrichment, on-bead AspN digestion, and inclusion list assisted LC MS/MS acquisition approach was developed to quantify site specific abundance of CML modifications on myocardial proteins of interest. Results Answer 1: Intrinsic cardiomyocyte stiffness is a contributing characteristic of diastolic dysfunction in diet induced cardiometabolic disease. This shows that load and stretch are important factors to consider in assessing diastolic function of cardiomyocytes. (Chapter 2) Answer 2: The human troponin complex is modified by advanced glycation end-products at 18 lysine and arginine sites. Additionally, the advanced glycation end product carboxymethyllysine was detected at 3 sites in streptozotocin-induced Type 1 Diabetic rats but not control counterparts. These modifications were detected in regions of protein protein interaction, adjacent to phosphorylation sites and in key regions of conformational change. (Chapter 3) Answer 3: A new approach for quantifying carboxymethyllysine modified protein was demonstrated. The approach employed a combination CML-modified reference library paired with specific protein immuno enrichment and yielded a set of 4 criteria to use to validate peak selection for quantification. (Chapter 4) Conclusion This Thesis reveals novel pathologic features of cardiomyocyte function in cardiometabolic disease and links them with in vivo diastolic dysfunction. New molecular mechanisms relating to disease specific post translational modification which may underly cardiomyocyte diastolic dysfunction at the contractile myofilament were discovered. Finally, novel quantitative methodology was developed to advance the translational application of these myofilament modifications as potential diagnostic markers for the early detection of diastolic dysfunction in cardiometabolic disease. Specific information pertaining to Chapter 4 of the Thesis has been omitted from this publicly published abstract for the purpose of intellectual property protection.
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ItemThe role of neurotrophic factors in osteoarthritis pain( 2023-10)Introduction: Osteoarthritis (OA) is a progressive disease of synovial joints and subchondral bone characterized by swelling, stiffness and pain. Brain-derived neurotrophic factor (BDNF) and artemin (ARTN) are neurotrophic factors that are important regulators of pain, and have recently been implicated in the pathogenesis of OA pain. This study aimed to explore roles for BDNF and ARTN in OA pain, by investigating whether the expression of BDNF and ARTN, and their receptors (TrkB and GFRa3), is altered in different tissues at different stages of OA, and whether blocking their signalling during late-stage OA can alleviate pain. Methods: The monoiodoacetate (MIA)-induced OA of the rat knee joint was used to explore roles for BDNF and ARTN signalling in OA pain. Pain behaviour was assessed using the dynamic weight-bearing apparatus to assay OA-induced changes in hindlimb weight bearing behaviour, at different stages of disease (early vs late). Histopathological alterations in the knee joint and surrounding bones were assayed using Haematoxylin and Eosin staining and scored using a modified OARSI scale. Changes in expression of BDNF/TrkB and ARTN/GFRa3 were explored using Western blot analysis of lysates from different tissues (joint, bone, and DRG), and at different timepoints of the disease (early vs late). The dynamic weight-bearing assay was used to determine if inhibiting BDNF signalling (with a peptide mimetic TrkB inhibitor) or ARTN signalling (with a sequestering antibody) could relieve pain at late-stage disease. Results: The results of this thesis highlight differential histopathological changes occurring in the early and late stages of OA, with joint involvement being prominent in early OA, and bone and cartilage involvement in late OA. BDNF expression was increased in the joint in early OA and in the bone in late OA. ARTN expression was also increased in the joint in both early and late OA and in the bone in late OA. Attempts to alleviate pain in MIA-injected animals by targeting the BDNF/TrkB and ARTN/GFRa3 signalling pathways did not yield pain relief outcomes with the therapeutic approach chosen in this study. Conclusion: Our findings suggest that altered pain behaviour in early MIA-induced OA is associated with changes in the joint not surrounding bones, while altered pain behaviour in late MIA-induced OA are attributable to the surrounding bones. Furthermore, BDNF and ARTN may contribute differentially to pain in early and late stages of MIA-induced OA through actions in joint versus bone. These findings support further investigations into the role of BDNF and ARTN signalling in OA pain and the development of novel targeted therapeutic approaches for managing OA pain.
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ItemTargeting lipid metabolism for prostate cancer therapy.( 2023-10)Deregulating cellular metabolism as a hallmark of cancer involves adaptation in nutrient acquisition, preferential utilisation of substrates, and transcriptional changes that alter intracellular metabolic signalling pathways. Understanding aspects of metabolism that are essential for cancer cells to sustain their uncontrolled proliferation can help to identify metabolic vulnerabilities, which can be therapeutically harnessed to limit cancer growth. Although prior studies in cells and mice have demonstrated the importance of oxidative metabolism and lipogenesis in prostate cancer growth and progression, however, the metabolic landscape of human prostate cancer remains unclear. Accordingly, this thesis aimed to elucidate the metabolic landscape of human prostate cancer and to identify targetable metabolic dependency for the development of novel therapeutic strategies for patients with prostate cancer. To define the metabolic landscape of human prostate cancer, we assessed substrate metabolism using radiolabelled (14C) and stable (13C) isotope tracing in precision-cut slices of patient-derived xenografts (PDXs). Glucose, glutamine, and fatty acid oxidation was variably upregulated in malignant compared to benign PDXs. De novo lipogenesis (DNL) and storage of free fatty acids into phospholipids and triacylglycerols were increased in malignant PDXs. There was no difference in substrate utilisation between localised and metastatic PDXs and hierarchical clustering revealed marked metabolic heterogeneity across all PDXs. Mechanistically, glucose utilisation was mediated by acetyl-CoA production rather than carboxylation of pyruvate, while glutamine entered the TCA cycle through transaminase reactions before being utilised via oxidative or reductive pathways. Blocking fatty acid uptake or oxidation with pharmacologic inhibitors was sufficient to reduce cell viability in PDX-derived organoids, whereas blockade of DNL, or glucose or glutamine oxidation induced variable and limited therapeutic efficacy. These findings demonstrate that human prostate cancer, irrespective of disease stage, can effectively utilise all metabolic substrates, albeit with marked heterogeneity across tumours. We also confirm that fatty acid uptake and oxidation are targetable metabolic dependencies in human prostate cancer. Considering the requirement of fatty acids in most tissues, targeting these processes may not be achievable. Therefore, we posited that identification of essential proteins of lipid metabolism, which are required for prostate cancer survival, would guide the development of novel strategies for treating prostate cancer. In Chapter 3, we developed a bespoke human lipid metabolism knockout library, performed CRISPR-Cas9-based functional genomics screening, and identified 63 commonly shared essential genes between LNCaP, C4-2B, and MR-49F. Having identified multiple genes within the mevalonate-dolichol-N-glycosylation biosynthesis to be essential, we subsequently validated the requirement of this pathway in Chapter 4. Deletion of genes encoding enzymes within this pathway resulted in reduced cell proliferation and anchorage-independent growth. This was partly mediated by G1 cell cycle arrest, induction of cell death and senescence. Further in vivo validation of genes within the dolichol-N-glycosylation biosynthesis (NUS1, DOLK, DPAGT1) in xenograft model demonstrated the requirement of NUS1 for tumour growth. Collectively, the work in this thesis highlights the heterogeneity and complexity of metabolic profiles in human prostate cancer. Furthermore, we have identified putative essential genes of lipid metabolism which may provide novel strategies for targeting lipid metabolism for patients with prostate cancer.