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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ItemProteomic analysis of mHttex1 expression in Huntington’s disease( 2021)Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (Htt) gene. This sequence encodes an abnormally elongated polyglutamine (polyQ) tract within the Huntingtin (Htt) protein that is directly involved in aggregation and Htt-mediated cytotoxicity. The key pathological signature of HD is the aggregation of mutant Htt protein into punctate aggregates. However, the mechanism by which polyQ-expanded mutant Httex1 (mHttex1) causes toxicity remains elusive. Previous research has indicated that mHttex1 can exert toxicity to cell models through two distinct phases. The first is when the protein is soluble and the second is when it is aggregated into inclusion bodies, which are the major pathological signature of HD brain. I hypothesized that apoptosis is caused by an unresolved quality control mechanism that oversees mHttex1 at synthesis. The goal of this project was to develop and implement a novel proteomics strategy to specifically detect the proteins that engage with mutant Htt during protein synthesis. I compared pathogenic huntingtin (Q97) and non-pathogenic huntingtin (Q25) using a proteomics-based approach. Firstly, a self-cleaving NS3 viral protease system called TimeSTAMP was employed, which can efficiently cleave epitopes from newly synthesized proteins and be potently inhibited using a viral protease inhibitor. The goal was to inhibit the cleavage across different-time windows to “pulse” label newly synthesized Htt and at the end of the pulse steps, proteins were crossed-linked with disuccinimidyl sulfoxide (DSSO) to preserve transient interactions. We also wanted to examine the changes in the global proteome and phosphoproteome across these mutant form and wild-type counterpart. After transfection of Neuro2a cells with TimeSTAMP-Httex1 constructs of differing poly-Q length, cells were lysed using RIPA lysis buffer. Proteins were then treated with a label-free relative quantitative phosphoproteomics workflow: i.e., samples were denatured (e.g., in 8 M urea), reduced and alkylated, then subjected to tryptic digestion. Next, a phosphopeptide enrichment step was performed before samples analyzed using LC-MS/MS. However, there was no significant difference observed between pathogenic and non-pathogenic Huntingtin, indicating a lack of polyQ-length dependence. To further probe the toxicity of the pathogenic huntingtin, I investigated its protein-protein interactions using a different proteomics-based approach. After transfection of Neuro2a cells with the Q25-GFPEm or Q97-GFPEm constructs, proteins were cross-linked with disuccinimidyl sulfoxide (DSSO) and then protein interactors were pulled down using anti-GFP VHH coupled magnetic agarose beads. We also examined the changes in the global proteome and phosphoproteome across the mutant form and wild-type counterpart. I did not find any significant difference between pathogenic and non-pathogenic Huntingtin which further indicates that the result was not polyQ dependent. Determination of these mechanisms are anticipated to be important for the design of new therapeutic strategies that mitigate toxicity of soluble mHttex1.
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ItemDoxycycline has a dual mode of action against malaria parasites( 2021)Traditionally used as a broad-spectrum antibiotic, doxycycline is frequently used for malaria prophylaxis and treatment - the latter in combination with artemisinin derivatives. Its mechanism of action in Plasmodium spp. has not yet been fully elucidated, though there is substantial evidence that ribosomes in the apicoplast - a relict plastid - are the primary target, with doxycycline causing delayed death (a phenotype associated with inhibitors of apicoplast housekeeping). Inhibition of the apicoplast depletes isoprenoids, synthesised via a pathway housed in the organelle, perturbing the prenyl-dependent trafficking mechanism for haemoglobin uptake and trafficking. This same uptake of haemoglobin is required for activation of artemisinin derivatives. Here, we show that apicoplast-targeting antibiotics, such as doxycycline, reduce the abundance of the catalyst of artemisinin activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between dihydroartemisinin and these antibiotics, likely because apicoplast inhibitors reduce artemisinin activation. Separately, we identify a secondary, more immediate target of doxycycline that exists at clinically relevant concentrations. We show that supplementation with the apicoplast-derived isoprenoid precursor, isopentenyl pyrophosphate, only rescues parasites from delayed death, demonstrating independence of the first cycle target from the relict plastid. Instead, we show that doxycycline depletes mitochondrial electron transport and selectively reduces the abundance of proteins encoded by the mitochondrion in the related apicomplexan parasite, Toxoplasma gondii, suggesting that inhibition of mitochondrial protein synthesis could underpin the immediate death phenotype caused by doxycycline. These data have potential clinical significance when considering the reliance on - and widespread use of - doxycycline and other apicoplast-targeting antibiotics in malaria endemic regions. They reinforce the strategic importance of rational choice of antimalarial combinations; and lay the groundwork for further exploration of the underlying mechanisms of drug resistance in Plasmodium parasites.
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ItemThe cell biology of the FcRn-albumin recycling system( 2021)Human serum albumin (HSA) is the most abundant protein in plasma and has an exceptionally long circulatory half-life of around three weeks in humans. The enhanced half-life properties of HSA result from the selective interaction with the neonatal Fc receptor (FcRn) in acidic endosomes, which protects endocytosed albumin from lysosomal degradation and mediates recycling back to the plasma membrane. Endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about FcRn-albumin cell biology in physiologically relevant primary cells and the spatiotemporal aspects of the FcRn-albumin interaction within intracellular endosomes. My studies have used cell biological and biophysical approaches to examine FcRn-albumin interactions and trafficking in primary macrophages and endothelial cells. Here, I used two independent biophysical approaches to visualise the intracellular receptor-ligand interactions within globular endosomes and tubular transport carriers of primary macrophages. Firstly, fluorescence lifetime imaging microscopy (FLIM) of Foerster resonance energy transfer (FRET) and secondly, raster image correlation spectroscopy (RICS) to monitor the diffusion kinetics of single fluorescent-labelled HSA molecules. Based on these analyses, I identified an interaction between FcRn and albumin within intracellular endosomes, and emerging tubules, in human FcRn-expressing macrophages. Furthermore, I detected a higher population of immobile, FcRn-bound wildtype HSA molecules within the lumen of endosomal structures compared to the non-FcRn binding rHSAH464Q mutant. My findings revealed the kinetics of FcRn-albumin binding within endosomal structures for recruitment into transport carriers for recycling. To investigate FcRn-albumin cell biology in physiologically relevant primary endothelial cells, I established cell lines of primary human vascular endothelial cells from the outgrowth in culture of blood endothelial precursors known as blood outgrowth endothelial cells (BOECs). My observations show that these endothelial cell lines internalised fluorescent-labelled HSA efficiently via fluid phase macropinocytosis. Intracellular HSA molecules co-localised with FcRn in endosomal structures potentially allowing the interaction of the receptor with its ligand. Wildtype HSA, but not the non-FcRn binding rHSAH464Q mutant, was sorted into FcRn-positive tubular transport carriers, that are likely to mediate recycling of endocytosed HSA back to the plasma membrane. These findings support the proposed contribution of vascular endothelial cells to albumin homeostasis in vivo. Understanding the underlying mechanisms of FcRn-albumin cell biology and the contribution of different cell types to albumin homeostasis is important for the design and generation of half-life extended albumin fusion proteins for the treatment of serum protein-related diseases such as hemophilia A (HemA). Despite exhibiting enhanced pharmacological properties, to date, very few albumin fusion protein therapeutics have been approved for the treatment of human patients. In particular for HemA, the treatment using recombinant coagulation factor VIII (FVIII) products is aggravated by the frequent development of inhibitory antibodies against FVIII in HemA patients which subsequently have to undergo highly expensive and burdensome immune tolerance induction protocols. In this study, I have established an imaging flow cytometry-based antigen uptake assay to investigate the internalisation of FVIII-Albumin fusion proteins by FVIII-specific B cells expanding the knowledge about how albumin fusion proteins might contribute to immune tolerance induction towards FVIII in vivo. Additionally, I established two in vitro protocols which, in combination, allow the generation of high numbers of FVIII-specific regulatory T cells. These antigen-specific Tregs have the potential to suppress immune responses against recombinant FVIII in vivo and represent an alternative approach to facilitate immune tolerance towards FVIII in HemA patients. In summary, this thesis has revealed the fundamental aspects of FcRn-albumin cell biology and trafficking in primary macrophages and endothelial cells, and potential strategies for immune tolerance induction using FVIII-Albumin fusion proteins in the context of HemA.