Biochemistry and Pharmacology - Theses
Permanent URI for this collection
Search Results
1 - 10 of 163
-
ItemHexosamine-dependent growth and virulence in Leishmania major(University of Melbourne, 2010)
-
ItemInsight into the early stages of protein folding from structural models of the unfolded state(University of Melbourne, 2007)
-
ItemThe evolution of the structure and function of transthyretin-like protein(University of Melbourne, 2007)
-
ItemFunctional roles of serum amyloid P component in amyloid diseases(University of Melbourne, 2006)
-
ItemFunctional roles of serum amyloid P component in amyloid diseases(University of Melbourne, 2006)
-
ItemThe mammalian grip domain proteins : a family of golgins localised to subdomains of the trans-Golgi network(University of Melbourne, 2006)
-
ItemThe mammalian grip domain proteins : a family of golgins localised to subdomains of the trans-Golgi network(University of Melbourne, 2006)
-
ItemInvestigating the Epitranscriptome of Plasmodium falciparum( 2019)Recent years have seen an increasing identification and awareness of post-transcriptional modifications of RNA. Nucleotide modifications have long been known to be important in diverse non-coding RNAs, but post-transcriptional modifications of mRNA are now also recognised as playing an important role in regulation of gene expression. One of the most abundant mRNA modifications is N6-methyladenosine (m6 A), the presence of which guides mRNA metabolism including maturation, nuclear export, translation and decay. In model eukaryotes, m6A is produced by the action of METTL-family methyltransferases and recognised by a family of YTH proteins. These enzymes play a key role in cellular differentiation and development. I have identified several putative METTL methyltransferases and several YTH reader proteins in the Plasmodium falciparum genome, and have investigated the m6A modification in the transcriptome of P. falciparum. I have characterised two methyltransferase proteins, Mettl3 and Mettl14-like proteins and two reader YTHDC1-like proteins (PfYTHDC1-a and PfYTHDC1-b)in P. falciparum through localisation, overexpression experiments and knock-sideways studies. Initial studies using epitope tagging and GFP fusion proteins revealed a predominantly nuclear localisation for both the methyltransferase and reader proteins, with some signal in the cytoplasm. Knock-sideways studies revealed complete inducible mis-localisation by 24 hours for PfMETTL3 and within 7 hours for PfYTHDC1-a. Additionally, under induced mis-localisation, both transfectant lines underwent morphological changes as evident by microscopy, with clear spots within the digestive vacuole, changes not seen in the 3D7 parental line. A pilot RNA-seq study was conducted, wherein differential transcript expression was analysed as a means to characterise the impact of perturbing m6A modifications by overexpressing and disrupting METTL methyltransferases. Future experiments should analyse the distribution of m6A in mRNA by nanopore direct-RNA sequencing. Additional investigations are also warranted on the influence of the METTL and YTHDC1-like proteins on splicing patterns in the parasite as a means to test the role of these modifications on proliferation and differentiation.
-
ItemNeuroinflammation, microglia and the cell biology of Alzheimer's Disease( 2019)The pathology of Alzheimer’s disease (AD) is characterised by progressive accumulation of misfolded proteins, which form senile plaques and neurofibrillary tangles, and chronic inflammation in the brain associated with inflammatory mediators by the activation of innate immune responses. There has been considerable interest in the role of neuroinflammation in directly contributing to the progression of AD. Studies in mice and humans have identified a role for microglial cells, the resident innate immune cells of the CNS, in AD. Activated microglia are a key hallmark of the disease and the secretion of pro-inflammatory cytokines by microglia may result in a positive feedback loop between neurons and microglia, resulting in ongoing low-grade inflammation and associated neurotoxicity. The underlying mechanisms however are poorly understood. Here the role of microglia was investigated, especially their link to ApoE – the strongest risk factor for late onset Alzheimer’s disease – and the relationship between microglia, neurons and neuroinflammation. Target replacement mice were used, where the human ApoE2, ApoE3 or ApoE4 allele replaces the mouse ApoE allele. Microglia were activated in a two- step setup. Initially cells were primed with LPS, followed by a secondary stimulus, such as ATP or Ab. The system was used to characterise the cytokines secreted by activated microglia and to assess the impact of conditioned medium from stimulated and Ab treated microglia on neuronal morphology. The first results Chapter (Chapter 3) established the system – mouse microglia were isolated from brains of neonatal mice and characterised by CD11b staining. Microglia from all three ApoE genotypes were directly compared and the data from ELISA and mass spectrometry revealed an enhanced pro- inflammatory response by ApoE4 microglia and the least efficient at internalizing amyloid b. Chapter 4 analysed the impact of conditional medium from the microglia ApoE variants on neurons and the results showed an ApoE-dependent effect on dendrite morphology. Conditioned media from immunostimulated and Ab microglia were incubated with cortical neurons from wt animals. Both the dendrite length and number of dendrites were significantly reduced in neurons treated with conditioned medium from ApoE4 microglia. TNFa was identified as a major cytokine and was responsible for modifying neuron morphology in cell assays. Neutralising the cytokine, with an anti-TNFa antibody abrogated the majority of morphological changes induced by the conditioned media from activated microglia. Hence the data suggests that TNFa may have a major role in mediating neuroinflammation. The third results Chapter (Chapter 5) compared aspects of macrophage function with microglia. Here it was shown, that microglia do not require SNX5 for macropinocytosis, and most likely utilise peripheral mediated macropinocytosis as the main form of macropinocytic internalisation. A major finding was the ability of sodium chloride to augment a pro-inflammatory response not only by immunostimulated macrophages by also microglia. Inhibition of the p38 MAPK signalling pathway partially ameliorated the NaCl- induced inflammatory responses in both macrophages and microglia, together with high levels of secreted IL-1b, indicating activation of the NLRP3 inflammasome. Overall the studies highlight a role for APoE4 allele to promote an enhanced inflammatory response by microglia cells.
-
ItemDelayed death by plastid inhibition in Plasmodium falciparum( 2019)Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the ‘delayed death’ effect. The molecular basis of delayed death is a long-standing mystery in parasitology and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that DV disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.