Biochemistry and Pharmacology - Theses

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    Organellar translation and inhibition in Plasmodium falciparum
    Bulloch, Michaela Susan ( 2023-09)
    The malaria parasite Plasmodium falciparum has two prokaryote-derived organelles: the mitochondrion and a relic plastid known as the apicoplast. These contain their own distinct, reduced genomes which must be transcribed and translated to maintain parasite viability. The bacterial-like proteins and metabolic functions of these organelles make malaria parasites susceptible to many anti-bacterials. This study aims to investigate organellar translation in P. falciparum, including the expression of apicoplast-targeted translation enzymes, tracking the cellular consequences of apicoplast translation inhibition, and measuring active organellar protein synthesis. Aminoacyl tRNA synthetases are a family of essential enzymes required for protein translation in the cytosol, apicoplast and mitochondrion. Several of these enzymes are encoded by single genes, from which two protein isoforms are proposed to be generated by alternative translation initiation. One isoform contains an N-terminal apicoplast localisation sequence, while the other lacks this and is cytosolic. In this study we investigate the significance of the nucleotides surrounding canonical and proposed translation start sites and show that these are important for their recognition by translation machinery. Additionally, we verify one of these dual-localised enzymes - threonine aminoacyl tRNA synthetase - as the target of the potent anti-microbial agent borrelidin in P. falciparum. Most organelle translation inhibitors have a lethal, but slow phenotype, killing parasites in the cycle following their administration. This has been attributed to disruption of apicoplast translation, with parasite death due to the inability to continue synthesis of essential apicoplast-derived isoprenoid metabolites. The consequences of isoprenoid starvation has been partially characterised, implicating lipophilic prenyl and isoprene chains as important, however not all essential isoprenoid products have been identified. We therefore aimed to investigate other downstream consequences of apicoplast translation inhibitors in Plasmodium. We found that apicoplast isoprenoids are required for synthesis of the major parasite sugar anchor glycophosphatidylinositol. Following inhibition of apicoplast translation, proteins typically anchored via this glycoconjugate became untethered, resulting in parasite segmentation, egress, and invasion defects. Difficulty in detecting proteins derived from organellar genomes had made the verification of organellar translation inhibitors challenging. Here, we use a mass spectrometry approach to directly detect and measure organellar translation in P. falciparum. This has facilitated the confirmation of the anti-apicoplast mechanism of action for the clinically used anti-malarials doxycycline and clindamycin. In addition, doxycycline was determined to inhibit mitochondrial translation, which was found to affect the activity of the electron transport chain. Together, this work has confirmed both the direct mechanism of action and indirect cellular consequences of organellar translation inhibitors on P. falciparum. In verifying the essentiality of glycophosphatidylinositols for multiple processes during the asexual stages, we have highlighted the potential for designing therapies that directly target aspects of glycophosphatidylinositol maturation or their protein attachment. Furthermore, determining the secondary target of doxycycline to be the mitochondrion has important clinical implications and may influence which drugs can be safely recommended for combination treatments.
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    Hexosamine-dependent growth and virulence in Leishmania major
    Heng, Joanne Soo Ping. (University of Melbourne, 2010)
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    The evolution of the structure and function of transthyretin-like protein
    Hennebry, Sarah Catherine. (University of Melbourne, 2007)
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    Functional roles of serum amyloid P component in amyloid diseases
    Stewart, Cameron Robert. (University of Melbourne, 2006)
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    Functional roles of serum amyloid P component in amyloid diseases
    Stewart, Cameron Robert. (University of Melbourne, 2006)
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    Cellular and molecular defence mechanisms against Legionella infection
    Oberkircher, Lara Marie ( 2023-04)
    The two most prevalent causes of Legionnaires’ disease are L. pneumophila and L. longbeachae. The rising incidence of the Legionnaires’ disease over the past two decades together with the increasing prevalence of L. longbeachae in the Northern hemisphere highlights the necessity to gain a more detailed insight into the L. longbeachae-induced immune response. The aim of this thesis was to investigate the cellular and molecular mechanisms required for protection, focussing on immune cell responses and intracellular host-bacterial interactions. By combining a murine mouse model, a novel fluorescent reporter to track L. longbeachae, and cell depletion experiments, I uncovered the differential contribution of tissue-resident alveolar macrophages (AM) and infiltrating neutrophils to the defence against L. longbeachae. Early during infection, AM contained most of the bacteria. AM numbers sharply decreased during infection, which was accompanied by a large influx of neutrophils that also internalized bacteria. Comparative analysis of bacterial viability revealed that neutrophils were more efficient at killing and clearing of L. longbeachae than AM. In contrast, the results presented here indicated that lung-resident AM promoted infection, most likely by serving as a replicative niche. Defence against L. pneumophila is known to require IFN-gamma but not IL-18, a strong IFN-gamma inducer. However, Il18r1–/– mice were less able to clear L. longbeachae and had significantly impaired IFN-gamma levels in the lung. Furthermore, IL-18R signalling was critical for the efficient bacterial killing by neutrophils via production of reactive oxygen species. Previous bone marrow chimera experiments in our laboratory suggested that IL-18R expression in epithelial cells was necessary and sufficient for protection against L. longbeachae. However, genetic in vivo experiments using the Cre-lox-system revealed that IL-18R+ NK cells and T cells were central players in the IL-18-dependent anti-L. longbeachae defence via IFN-gamma secretion, whereas IL-18R expression by ciliated bronchiolar epithelial cells did not confer protection. Finally, our results highlighted key mechanisms by which Legionella subverts host macrophages to form an intracellular endoplasmic reticulum (ER)-like vacuole as its intracellular replicative niche. Establishment of the Legionella-containing vacuole (LCV) by recruitment of ER-derived vesicles induces ER stress. Although the relevance of ER stress in this process is unclear, effector proteins secreted by L. pneumophila are known to inhibit onset of the ER stress response. Using the pharmacological ER stress inducer thapsigargin, we showed that ER stress induces a protective host response promoting the secretion of pro-inflammatory cytokines, limiting intracellular L. pneumophila replication, and improving host survival. Mechanistically, ER stress induced a novel non-canonical activation of the transcription factor STAT1 via the IRE1 kinase driving transcription of the IFN-gamma-induced chemokine CXCL10. These results highlighted a potential role of the host ER stress response in the initiation of a protective cellular immune response towards L. pneumophila.
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    Hyper-nutritional cell culture medium distorts the expression of anti-cancer drug targets
    Cheng, Tianhong ( 2023-07)
    In vitro cell cultures are instrumental in deciphering the mechanisms that underlie cell processes in vivo including differentiation, migration, growth and mechanics, all of which are impacted by both biochemical and biomechanical microenvironments. Evolving technologies provide continuous improvement to cell culture towards more physiologically relevant systems that better emulate in vivo conditions. However, the cell culture media as one of the fundamental elements for in vitro cell culture have been overlooked with incremental rather than the transformational improvements that are required. Nutrient availability in the tumour microenvironment is essential for metabolism, cellular processes and may be a determinant of anti-cancer drug responses. Nevertheless, the overwhelming majority of current in vitro biomedical research is conducted in unphysiological conditions, using conventional cell culture medium developed generations ago with hyper-physiological level of nutrients. In addition, most of the cell culture were in static conditions, lacking the flow/renewal of the in vivo situation to provide appropriate biochemical and mechanical cues and prevent accumulation of metabolic by-products. We therefore developed an improved physiological medium, “Melbourne Medium” (MM) with human plasma-like composition for continuous supply to multi-well cell culture environments using the custom-built RPM2 multiplexed superfusion system. Our focus here is on the imperative to understand the influence of physiological media on cellular behaviours by contrast with conventional hyper-nutritional media. Culture of non-small cell lung cancer (NSCLC) A549 cells in MM slowed proliferation, promoted an epithelial-mesenchymal transition (EMT)-like phenotype with an increased motility and induced paclitaxel resistance. Global proteomic analyses revealed expected differential expression of metabolism-related gene ontologies, but also multiple biological pathways of critical importance to the growth and spread of NSCLC. Notably, significant distortion was identified in the expression of key anti-cancer drug targets including EGFR, STAT3, TGFBI and Smad3, as well as tubulins, specifically betaII and betaIV tubulin isoforms implicated in paclitaxel resistance. The most up- and down- regulated proteins in MM, CNBP and 15-hydroxy prostaglandin dehydrogenase (15-PGDH), respectively, are each associated with lung cancer survival, the former association being discovered in this thesis study. The cell metabolism processes critical to tumour progression were perturbed by hyper-nutritional medium, as indicated by both proteomic and metabolomic analyses. For the first time, by applying the RPM2 multiplexed superfusion of media, we established that the physiological nature of MM could be maintained, while minimising accumulation of both bioactive metabolic by-products and previously unappreciated metabotoxin formation. Together, these studies have demonstrated that hyper-nutritional (conventional) media profoundly distort tumour cell functions, whereas superfusion of MM facilitated a more physiological metabolic environment, associated with improved drug target validation and drug screening.