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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ItemNo Preview AvailableUsing Artificial Intelligence to Improve the Diagnosis and Treatment of Cancer( 2022-12)Cancer is a complex and heterogeneous disease driven by the accumulation of mutations at the genetic and epigenetic levels—making it particularly challenging to study and treat. Despite Whole-genome sequencing approaches identifying thousands of variations in cancer cells and their perturbations, fundamental gaps persist in understanding cancer causes and pathogenesis. Towards this, my PhD focused on developing computational approaches by leveraging genomic and experimental data to provide fundamental insights into cancer biology, improve patient diagnosis, and guide therapeutic development. The increased mutational burden in most cancers can make it challenging to identify mutations essential for tumorigenesis (drivers) and those that are just background accumulation (passenger), impacting the success of targeted treatments. To overcome this, I focused on using insights about the mutations at the protein sequence and 3D structure level to understand the genotype-phenotype relationship to tumorigenesis. I have looked at proteins that participate in two DNA repair processes: primarily non homologous end joining (NHEJ) along with eukaryotic homologous recombination (HR), where missense mutations have been linked to many diverse cancers. The molecular consequences of these mutations on protein dynamics, stability, and binding affinities to other interacting partners were evaluated using in silico biophysical tools. This highlighted that cancer-causing mutations were associated with structure destabilization and altered protein conformation and network topology, thus impacting cell signalling and function. Interestingly, my work on NHEJ DNA repair machinery highlighted diverse driving forces for carcinogenesis among core components like Ku70/80 and DNA-PKcs. Cancer-causing mutations in anchor proteins (Ku70/80) impacted crucial protein-protein interactions, while those in catalytic components (DNA-PKcs) were likely to occur in regions undergoing iii purifying selection. This insight led to a consensus predictor for identifying driving mutations in NHEJ. While when assessing the functional consequences of BRCA1 and BRCA2 genes of HR DNA repair at the protein sequence level, this methodology underlined that cancer-causing mutations typically clustered in well-established structural domains. Using this insight, I developed a more accurate predictor for classifying pathogenic mutations in HR repair compared to existing approaches. This broad heterogeneity of cancers complicates potential treatment opportunities. I, therefore, next explored the properties of compounds potentially active against one or various types of cancer, including screens against 74 distinct cancer cell lines originating from 9 tumour types. Overall, the identified active molecules were shown to be enriched in benzene rings, aligning with Lipinski's rule of five, although this might reflect screening library biases. These insights enabled the development of a predictive platform for anticancer activity, thereby optimizing screening libraries with potentially active anticancer molecules. Similarly, I used compounds' structural and molecular properties to accurately predict those compounds with increased teratogenicity early in the drug development process and prioritize drug combinations to augment combinatorial screening libraries, potentially alleviating acquired drug resistance. The outcomes of this doctoral work highlight the potential benefits of using computational approaches in unravelling the underlying mechanisms of carcinogenesis and guiding drug discovery for designing more effective therapies. Ultimately, the predictions generated by these tools would improve our understanding of the genotype-phenotype association, enabling promising patient diagnosis and treatment.
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ItemReaction hijacking tyrosyl-tRNA synthetase as a new anti-infectives strategy( 2022)Malaria is a deadly disease of humans, with Plasmodium falciparum responsible for the most cases. Disappointingly, drug resistance is observed against current front-line therapies; thus, new drugs with novel mechanisms are urgently needed. ML901, a nucleoside sulfamate derivative, has been shown to possess good antimalarial efficacy and to specifically target P. falciparum tyrosyl-tRNA synthetase (PfYRS). PfYRS is a pivotal enzyme that participates in the protein synthesis pathway, in which tyrosine-charged tRNA is formed. ML901 appears to target PfYRS via a novel reaction hijacking mechanism in which PfYRS catalyzes the synthesis of a Tyr-ML901 adduct, which in turn poisons the enzyme. Human YRS is not susceptible to the reaction hijacking mechanism. This project sought to understand the molecular basis for the potency and specificity of ML901 and to determine if reaction hijacking could be exploited more widely. All YRS sequences harbor a conserved motif, referred to as “KMSKS”, in a loop that is reported to change conformation to facilitate ATP binding and the aminoacylation reaction. Sequence alignment across species reveals that most pathogenic parasites, including P. falciparum, possess a KMSKS motif, whereas higher eukaryotes possess an equivalent KMSSS motif. Structural analysis revealed that the motif in human YRS is part of a flexible (unstructured) loop while the equivalent loop is structured in PfYRS. Here we examined the role of the second lysine (K250) in determining loop flexibility and activity of PfYRS as well as the susceptibility of the mutant enzyme to reaction hijacking. Surprisingly, the X-ray crystal structure of recombinant PfYRS harboring the K250S mutation (PfYRSK250S) showed that the KMSSS loop is even more stable than the wildtype KMSKS loop. PfYRSK250S was found to consume substantively less ATP in the initial activation step. However, the weakly active PfYRSK250S is still susceptible to reaction hijacking by ML901. This study shows that the flexibility of the loop is not determined simply by the K250. Moreover, it shows that K250 plays an important role in enzymatic mechanism. Further investigations are required to understand the important factors that contribute to the particular susceptibility of PfYRS to reaction hijacking by ML901. Broad specificity nucleoside sulfamates, such as adenosine sulfamate (AMS), have previously been shown to have inhibitory activity against Gram-positive and Gram-negative bacteria. The equivalent of the KMSKS motif in the Escherichia coli YRS sequence is KFGKT. Here we explored the possibility that bacterial YRS might also be susceptible to inhibition via a reaction hijacking mechanism. A screen of a range of bacterial species revealed that AMS inhibits growth of E. coli and Enterococcus faecium. Targeted mass spectrometry confirmed the production of a range of amino acids adducts upon treatment in E. coli with AMS. Recombinant EcYRS was purified and expressed and shown to be inhibited via the reaction hijacking mechanism by AMS. These data suggest that bacterial amino acyl tRNA synthetases may be exciting new targets for reaction-hijacking nucleoside sulfamates.
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ItemDevelopment of an extracellular vesicle-based therapeutic for wound healing.( 2022)Chronic wounds are defined as wounds that fail to progress through the normal stages of wound healing and stay unhealed for longer than 4 weeks. Such wounds affect 1-2 % of the population are characterized by the presence of phenotypically abnormal cells and disruptions in the inflammatory and proliferative phases of normal wound healing. Current treatment options have been ineffective as they focus on wound management and do not adequately address the underlying physiological causes. For example, platelet-derivatives have been tested due to their rich growth factor content but have yielded varying efficacies. To date, chronic wounds are responsible for a lower-limb amputation every 30 seconds globally and no new treatments have been approved for use by the FDA since 1994, highlighting the need for a new and effective treatment modality. Although platelets are well-known for their role in coagulation following an injury, they are also potent inducers of tissue regeneration. More recently, platelets have been shown to exert these regenerative properties through the release of extracellular vesicles (EVs). Platelet-derived EVs (pEVs) are spherical vesicles surrounded by a lipid bilayer enclosing a myriad of bioactive molecules. pEVs represent a new therapeutic approach as they are not only enriched in numerous growth factors and nucleic acids but also possess unique biophysical properties that are advantageous as biologic medicines. However, the clinical development of EVs as a whole, has been hampered due to the lack of a suitable isolation method. For example, high-speed differential centrifugation is currently the most-widely used method for isolation of EVs and results in their aggregation and loss of functional activity. An ideal isolation method for the clinical manufacture of EVs would have to be effective, scalable, and amenable to Good Manufacturing Practice (GMP) without compromising the structural and functional integrity of the EVs isolated. To address this, Exopharm Ltd has developed a novel isolation technology based on ion-exchange chromatography (IEX) called Ligand Exosome Affinity Purification (LEAP) that could overcome the scalability and purity challenges posed by other methods. However, the use of LEAP to isolate pEVs had not been previously tested. Hence, the first aim of this study was to induce the release of pEVs from platelets using cold-activation and to isolate the resulting pEVs using LEAP. A proliferation assay using dermal fibroblasts was developed to assess LEAP-isolated pEVs for functional activity. pEVs isolated using LEAP were found to significantly induce fibroblast proliferation. pEVs were also characterized for size and morphology and were found to adhere to guidelines set by the International Society of Extracellular Vesicles (ISEV). The second aim of this study was to conduct proteomic and transcriptomic analyses on pEVs from cold-activated platelets and isolated using LEAP to characterize their content and identify key proteins and nucleic acids that positively modulate wound healing process. Proteomic analyses through mass spectrometry revealed the presence of growth factors such as IGF and TGF-b that induce pro-regenerative and -angiogenic properties in recipient cells. Transcriptomic analyses through RNA sequencing reported that pEVs are enriched for microRNAs such as mir-21 and mir-126 that have been previously found to be downregulated within chronic wounds. Hence, content characterization provided first evidence that pEVs from cold-activated platelets and isolated using LEAP contain proteins and microRNAs important for cellular functions involved in healthy wound healing. The third aim of this study was to assess the functional activity of pEVs in in vitro assays. pEV-treatment was found to significantly induce proliferation of dermal fibroblasts and bone-marrow derived mesenchymal stem cells, both cell types important for generating new tissue following wounding. pEVs were also shown to increase the migratory capacity of fibroblasts and angiogenic capacity of dermal endothelial cells. Hence, pEVs from cold-activated platelets were found to positively influence multiple cell processes that are necessary for the proliferative phase of wound healing, that are often impaired in chronic wounds. Lastly, the use of pEVs as therapeutic for wound healing was assessed in a Phase I clinical trial to observe safety and biological activity following administration. One participant was successfully enrolled in the study and was administered autologous pEVs following a skin punch biopsy. No adverse events were reported and pEV-treatment was deemed to be safe and well-tolerated. Furthermore, histological studies of wound tissues procured at Day 7 following treatment showed increased presence of macrophages, keratinocytes and proliferating cells within pEV-treated tissue compared to the untreated control. This study was the first-in-human application of pEVs from cold-activated platelets and support the clinical development of pEVs as a next-generation therapeutic for chronic wounds.