School of Botany - Theses
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ItemDissecting antibiotic targeting in the malaria parasite Plasmodium falciparum(University of Melbourne, 2008)
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ItemNo Preview AvailableBiology of the mitochondrion of the malaria causing parasite Plasmodium falciparum(University of Melbourne, 2005)In this thesis, I examine the biology of the mitochondrial organelle of the malaria-causing parasite Plasmodium falciparum. Mitochondria are eukaryotic organelles derived through endosymbiosis from proteobacteria, a process that probably occurred early in eukaryotic evolution. Mitochondria are typically associated with energy generating functions, but are known to perform a wide range of other functions. Although a known drug target, the functions of the P. falciparum mitochondrion are largely unknown. In this thesis, I provide bioinformatic evidence that this organelle is capable of electron transport and catabolism of organic compounds through an unusual tricarboxylic acid cycle. I also identify proteins that probably function in several biosynthetic processes, including haem biosynthesis, coenzyme Q biosynthesis and iron-sulfur cluster biosynthesis. I provide evidence that proteins from several of these pathways and processes localise to the mitochondrion. I also examine the morphology, division and segregation of mitochondria in asexual stages of P. falciparum parasites. I show that the mitochondrion associates with the apicoplast (plastid) organelle during segregation, and identify proteins that may be involved in organellar division in P. falciparum.
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ItemPlastid targeting and the pyruvate dehydrogenase complex in the malaria parasite Plasmodium falciparum(University of Melbourne, 2003)The phylum Apicomplexa consists of obligate parasites that cause severe diseases in humans and livestock. The genera Plasmodium, Toxoplasma and Eimeria, for example, are the causative agents of malaria, toxoplasmosis, and coccidiosis. Apicomplexan parasites contain a relict plastid which has been termed �apicoplast�. Chapter 1 of this thesis presents an extensive literature review covering the evolutionary origin of the apicoplast, its division during the cell cycle, protein targeting and import, its metabolic function, and the effect of drugs on this organelle. Thus, it is argued that the apicoplast originated from the engulfment of a once free-living organism of the red algal lineage. Differences in organellar division between the apicomplexan plastid and chloroplasts of plants are highlighted. The review describes how the vast majority of the apicoplast proteome is encoded in the nuclear genome, and how the products of these nuclear genes are post-translationally targeted to the organelle via the secretory pathway courtesy of a bipartite N-terminal leader sequence. Finally, it summarises what is known to date about the metabolic pathways located within the apicoplast including fatty acid, isoprenoid and heme synthesis, and reviews the action of compounds that inhibit these functions. A study exploring the particular amino acid characteristics of apicoplast-targeting transit peptides from the malaria parasite Plasmodium falciparum is presented in Chapter 2. A novel algorithm (�PlasmoAP�) recognising this particular amino acid bias was developed which � in conjunction with the existing bioinformatic tool SignalP � was able to distinguish between apicoplast-targeted and non-apicoplast proteins encoded in the nuclear genome of P. falciparum. Site-directed mutagenesis altering charge-related amino acid features in a model transit peptide severely disrupted organellar targeting in vivo, confirming the biological significance of features embedded in PlasmoAP. In addition, putative Hsp70- or DnaK-binding sites are shown to be abundant in plasmodial plastid transit peptides, and the removal of predicted Hsp70-binding sites from a plasmodial transit peptide led to disruption of transit peptide function in vivo. Finally, the high AT-content of P. falciparum DNA is presented as a major driving force shaping the particular amino acid composition observed in plasmodial transit peptides. The pyruvate dehydrogenase complex (PDHC) is one member of a family of 2-oxo acid dehydrogenase complexes (ODHCs), and a concise introductory review in Chapter 3 provides an overview of these enormous multi-subunit enzyme complexes that occupy central roles in the metabolism of mitochondria and plastids. Several ODHCs were identified in the genomes of five Plasmodium species and of Toxoplasma gondii, indicating that these parasites contain one pyruvate dehydrogenase complex (PDHC) in the apicoplast, as well as one ?-ketoglutarate dehydrogenase complex (KGDHC) and one branched-chain ?-ketoacid dehydrogenase complex (BCKDHC) in the mitochondrion. The four genes encoding a complete PDHC in P. falciparum were confirmed through sequencing of cDNA clones, and 10 introns were identified in the E2 subunit sequence. Phylogenetic analyses and the apparent presence of bipartite N-terminal leaders are presented as strong circumstantial evidence arguing that the P. falciparum PDHC is located in the apicoplast. Chapter 4 describes the recombinant expression of the constituent subunits of the PDHC identified in P. falciparum. Primary sequence comparisons and high enzymatic activity measured for the recombinantly expressed catalytic domain of the PDHC subunit E2 indicate that the parasite PDHC genes encode functional enzymes. Polyclonal antibodies directed against the plasmodial PDHC subunits E1?, E1?, and E2 were generated in rabbits, and Western blot data suggests that these proteins are expressed in vivo. The thesis closes by drawing overall conclusions from the PhD research presented and by providing an outlook for its future implications and ongoing related work.
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ItemEstablishing and elucidating conditional genetic manipulation of the malaria parasite, Plasmodium falciparum( 2013)Methods to alter gene function in Plasmodium are barely out of infancy, with much growth occurring within the past five years via adaptation of protocols established in other species. In this thesis, a method where the parasite’s innate ability to control gene expression in response to environmental stimuli, and a synthetic small molecule regulated protein stability system were applied to change the phenotypic expression of introduced transgenes. In chapter 2, we aim to control activity of a temperature sensitive mutant FLP recombinase protein. Our goal was to induce FLP activity and thus change the open reading frame of a GFP expressing plasmid to mCherry using single site recombination. Dual transgenic parasites were placed in a variety of conditions to favour FLP activity and to prevent negative selection. No evidence of conditional genetic recombination or phenotypic change from green to cherry was observed. Direct expression of FLP during a short period of drug selection produced a mixed population of GFP and mCherry genotypes. The activity of FLP is inefficient and was deemed unsuccessful for our goal of raising transgenic parasites poised for conditional genetic recombination. A homogenic population of mCherry genotypes by successful recombination of the GFP ORF was achieved after drug-cycled expression of evolved FLP (FLPe). Chapter 3 focuses on turning the Plasmodium specific phenomenon of altering rRNA expression profiles in response to fluctuations of temperature and glucose concentration into a tool suitable for conditional gene expression. We copied the promoter region of the S2-Type rRNA gene and measured its ability to express luciferase in altered environmental conditions. A low level of basal activity was observed during the intra-erythrocytic developmental cycle, with expression peaking at levels similar to the weak chloroquine transporter (CRT) promoter during the trophozoite stage. The S2-Type rRNA promoter region did not produce a notable increase of luciferase transcription or activity in response to environmental change. Use of the S2-Type rRNA promoter to express FLP at the enzymatic favourable temperature of 26°C did not result in genetic recombination. The tools developed are capable of expressing transgenes, however their ability to conditionally alter the gene product is limited.