Biochemistry and Pharmacology - Theses
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ItemHexosamine-dependent growth and virulence in Leishmania major(University of Melbourne, 2010)
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ItemInsight into the early stages of protein folding from structural models of the unfolded state(University of Melbourne, 2007)
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ItemThe evolution of the structure and function of transthyretin-like protein(University of Melbourne, 2007)
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ItemFunctional roles of serum amyloid P component in amyloid diseases(University of Melbourne, 2006)
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ItemFunctional roles of serum amyloid P component in amyloid diseases(University of Melbourne, 2006)
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ItemThe mammalian grip domain proteins : a family of golgins localised to subdomains of the trans-Golgi network(University of Melbourne, 2006)
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ItemThe mammalian grip domain proteins : a family of golgins localised to subdomains of the trans-Golgi network(University of Melbourne, 2006)
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ItemImpact of respiratory syncytial virus infection on host mitochondrial organisation and function( 2018)Respiratory syncytial virus (RSV), a leading cause of acute lower respiratory illness in infants, immunosuppressed adults and the elderly, is responsible for more deaths each year than influenza. Despite this, there are no freely available treatment options, making the development of safe and efficacious anti-RSV therapeutics a high priority. However, in order to achieve this, a deeper understanding of the RSV-host cell interaction is required. RSV infection has previously been found to induce global changes in the mitochondrial proteome and interfere with mitochondria-mediated antiviral signalling, but details of the RSV-host cell mitochondrial interaction are poorly understood. Therefore, the aim of this thesis is to explore the impact of RSV infection on host mitochondria and its role in viral pathogenesis in order to identify novel anti-RSV strategies. The results presented in this thesis reveal for the first time that RSV induces a staged, microtubule/dynein-dependent redistribution of mitochondria, concomitant with reduced mRNA levels of genes encoding mitochondrial proteins, compromised mitochondrial respiration, dissipated mitochondrial membrane potential (Δѱm), and increased generation of mitochondrial reactive oxygen species (ROS). It was also found that inhibiting mitochondrial redistribution or mitochondrial ROS production strongly suppressed RSV virus production, highlighting the RSV-mitochondrial interface as a potential antiviral target. Analysis of RSV proteins identified the matrix protein (M) is sufficient and necessary to induce mitochondrial perinuclear clustering, downregulation of mitochondrial genes, inhibition of mitochondrial respiration, loss of Δѱm, and accumulation of mitochondrial ROS in infection, while deletion and mutation studies identified its central nucleic acid-binding domain, and arginine/lysine residues 170/172 in particular, as essential for its remodelling ability in host cell mitochondria. Recombinant RSV carrying the arginine/lysine mutations in M was unable to elicit these effects on host mitochondria, and its replication in infected cells was severely impaired, underlining the importance of M-dependent effects on mitochondria to RSV infection. Importantly, clinically relevant human cell models of RSV infection were examined, highlighting the importance of RSV’s impact on host mitochondria to its infectious cycle, and its relevance to human disease. Further, inhibiting the accumulation of mitochondrial ROS in infected cells was confirmed as a viable anti-RSV approach in these systems, and work was extended to include a mouse model that showed significantly reduced RSV-related pathology as a result of treatment with a mitochondrial ROS scavenger. In summary, the studies presented in this thesis shed new light on the impact of RSV infection on host cell mitochondria by establishing the unique ability of RSV, facilitated by the M protein, to co-opt the host cell mitochondria to enhance virus production. In addition, the importance of RSV’s impact on host mitochondria for pathogenesis is explored in multiple disease models, highlighting it as a potential target for the development of anti-RSV treatments. Significantly, the studies reveal the inhibition of mitochondrial ROS levels for the first time as a viable approach to counteract RSV infection.
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ItemBiochemical mechanisms of biomineralization and elemental incorporation in otoliths: implications for fish and fisheries research( 2018)All vertebrates have small bioinorganic “earstones” in their inner ear labyrinth that are essential for hearing and balance. While otoliths play a vital anatomical role in fish, their true value to science is as biochronometers, largely due to their unique pattern of growth. Otoliths first form in embryo and continue to grow throughout the life of an individual, with a double-banded increment composed of a calcium carbonate-rich region and a protein-rich region being deposited daily. In addition to this, they are considered to be metabolically inert, and do not undergo remodelling or resorption. Consequently, otoliths are employed in a variety of ways in fish ecology. Firstly, an individual fish’s age and growth rate can be estimated through counting increments and measuring their widths. Secondly, analysis of increment trace element:calcium ratios, such as by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), can allow for the reconstruction of environmental histories, aiding in the determination of natal origin, movement, habitat use, diet and the impacts of climate change. The utility of specific trace elements as indicators of environmental change, however, is unclear as there is considerable uncertainty as to whether a given trace element is interacting with the mineral or protein components of an increment. This uncertainty is a consequence of otolith research having been largely focussed upon either microstructure or inorganic chemistry, with very few studies on the protein-rich regions of the otolith. As a result, very little is understood about the biochemical mechanisms of biomineralization or trace element incorporation. This is important, as the mechanisms that govern otolith formation and growth underpin the assumptions made in traditional increment analyses. In this thesis, I initially undertook a systematic review of all the literature pertaining to otolith biochemistry, revealing the significant gaps that exist in otolith biochemistry as a discipline. Importantly, I determined that fewer than a score of otolith proteins had been identified – a stark contrast to the hundreds or thousands of proteins that have been identified in comparable biomineral systems such as enamel or bone. Working on black bream (Acanthopagrus butcheri), an extensively studied species endemic to southern Australia, I used size exclusion chromatography coupled with ICP-MS to determine the trace element:protein interactions in endolymph, the inner ear fluid that otoliths are submerged in, and the source of all of its constituents. In this study, I assayed 22 elements, and determined that 12 were solely present in a protein-bound form, 6 were present as free ions, and 4 were present in both forms. This allowed me to make recommendations as to their utility in environmental reconstructions. In my next study, I created a unique, multi-disciplinary workflow that combined transcriptomics with proteomics. In this study, I sequenced the transcriptome of the black bream inner ear and used this to identify proteins from the separated organic phase of otoliths and endolymph from wild caught adult black bream. This resulted in the discovery of hundreds of previously unknown proteins, providing new insights into the likely biochemical mechanisms involved in otolith formation and growth. In my final study, I tested the utility of trace element ratios in environmental reconstructions. Specifically, I compared the ability of different cluster analysis approaches to resolve spatial and temporal differences in the likely spawning and larval nursery habitats of juvenile black bream in the Gippsland Lakes, Australia. The results from my thesis have allowed me to make recommendations as to the utility of trace elements in environmental reconstructions and have revealed exciting new avenues of research that fuse ecology and biochemistry.
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ItemNew pathogenic mechanisms in SCA1 neurodegenerative disease revealed by the ataxin-1 interactome( 2018)Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease in which a marked atrophy of neurons in the cerebellum and brainstem causes coordination and movement disabilities and ultimately death within 10-20 years of symptom onset. The molecular cause is an expanded polyglutamine (polyQ) sequence in the ataxin-1 protein. The resulting accumulation of the mutant ataxin-1 (polyQ-ataxin-1) protein as distinctive nuclear bodies (NBs) has been proposed as contributing to neuronal toxicity and/or dysfunction, but little is known about the biophysical properties of these NBs and their ultimate impact on neuronal cells. The studies presented in this thesis address these issues initially with proteomics approaches in Neuro-2a neuronal cells to identify the interacting protein partners of polyQ-ataxin-1, i.e. the polyQ-ataxin-1 interactome. The results from proximity labelling and affinity purification approaches were combined to improve confidence in the resulting lists of partner proteins. Further bioinformatics analysis identified enrichment of several protein functional groups; nuclear transport proteins and RNA helicases were prioritised for further study by biochemical and advanced imaging techniques. The expression of polyQ-ataxin-1 in Neuro-2a cells was shown to disrupt the localisation of multiple nuclear transport proteins. Nuclear transporters importin-α2, importin-β1, importin- 13, Hikeshi, exportin-1, and nucleoporin NUP98 have been mislocalized and partially co- localized with ataxin-1 NBs. The observations of altered nuclear/cytoplasmic distributions of model cargo proteins were also consistent with the disruption of the processes of nuclear transport in the presence of polyQ-ataxin-1. The physical properties of the polyQ-ataxin-1 NBs were also assessed, with specific consideration of the contributions by RNA/RNA helicases. Under standard conditions these NBs showed rapid exchange of the ataxin-1 protein consistent with dynamic liquid droplets; the down-regulation of RNA helicases DDX42, DDX46, and DHX15, the decreased ATP level, or altered environmental conditions were shown to slow exchange. These results thus reveal the phase transition to a less dynamic hydrogel or fibrillar phase and emphasize the tunable dynamics of these polyQ-ataxin-1 NBs as RNA/protein droplets. Taken together, these studies have revealed new insights into the impact and regulation of polyQ-ataxin-1 made possible by the identification of new proximal or interacting partners for polyQ-ataxin-1, and thus suggest new strategies for future interventions in the treatment of SCA1 and other neurodegenerative diseases.