Genetics - Theses
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ItemThe evolution of pathogenicity and isolate variation in Talaromyces marneffeiPAYNE, MICHAEL ( 2017)The opportunistic fungal pathogen of humans, Talaromyces marneffei, is one of very few pathogens in an order of over a thousand species and the only species that has the capacity to switch between two morphologically distinct growth forms (known as dimorphism). Growth at 25°C results in a saprophytic multicellular, hyphal form while infectious growth in a host occurs as a uninucleate unicellular yeast that resides within phagocytic cells of the immune system. The intracellular niche of T. marneffei differs significantly from the niches of other Talaromycetes. The identification of the mechanisms by which T. marneffei can survive and grow in this intracellular niche is a major aim of this study. Comparisons of the genomes of three closely related non-dimorphic, non-pathogenic species with the T. marneffei genome identified unique features that contribute to niche specific growth and the ability to cause disease. Most significant of these were an overall reduction in genome size and gene number in T. marneffei with substantial gene losses in families responsible for environmental interaction. These and other findings strongly indicate that T. marneffei has adapted to an intracellular host niche distinct from its saprophytic relatives. Against this background of gene loss three gene families were identified that had been significantly expanded in T. marneffei. These expanded gene families showed putative extracellular and cell surface localisation and consisted of cell wall galactomannoproteins (mpl family), aspartyl proteases (pop family) and a family of small proteins with very little functional characterisation in any species (mib family). Genes in the pop, mpl and mib families were over-represented in subtelomeric regions, under positive selection, had copy number variation in T. marneffei isolates and many had high levels of repetitive adjacent sequences including several transposon families. In the host T. marneffei grows as an intracellular pathogen within phagocytes and as such extracellular proteins interact directly with the host. Therefore another aim of this study was to characterise these expanded gene families and their role in pathogenesis. Deletion studies in pop genes revealed roles in yeast cell formation during intracellular growth, while high variability in cell-to-cell protein production for two mib genes suggested a role in cell surface variation when interacting with the host. Understanding the type and degree of variation within the population of a fungal pathogen can reveal its population structure and potential to adapt to stressors such as antifungal compounds. Genome wide variation in the T. marneffei population had yet to be examined therefore an aim of this study was to characterise the degree and type of this variation. To this end several clinical and environmental isolates of T. marneffei were examined for variation in chromosomal structure, which is a common means of generating phenotypic variation in other fungi. While no obvious abnormalities were observed, gene copy number variation in subtelomeric regions was widespread and several strains showed specific small mutations with impacts in antifungal resistance and phenotypic instability. Overall this study has revealed the genomic and genetic changes within T. marneffei and between it and other Talaromycetes. Many of these changes help to explain its unique niche as an intracellular pathogen within an almost entirely non-pathogenic clade. This research also highlights specific genes and gene families with roles in this pathogenesis and identifies potential therapeutic targets and genes involved in host interactions for future investigation.
ItemProbing insecticide biology using Drosophila melanogasterDenecke, Shane ( 2017)Insecticides are often used to control insect pests, but resistance to these chemicals arises quickly, leading to agricultural losses and public health concerns. Understanding how insects cope with insecticides is necessary when designing rational pest management strategies, but much still remains unknown regarding the fate of insecticides once inside the body. Furthermore, the genetic variation that governs an insects ability to survive insecticide exposures has not been fully described. Here, a 3 pronged approach is applied to study insecticide biology using the model insect Drosophila melanogaster. First, an acute, sub-lethal insecticide response assay was developed, which provided information complementary to that obtained from more common toxicology assays. In particular, behavioural response observed in a hyper-resistant target site mutant suggests additional target sites for the insecticide spinosad. This bioassay was then applied in a forward genetics approach to describe the genetic basis of resistance to the insecticide imidacloprid. This approach identified a variety of neuronal genes and the previously identified drug metabolizing enzyme Cyp6g1, which was explored through genetic manipulation. Finally, a reverse genetics approach was employed in order to study the effect of an ABC transporter protein Mdr65 on insecticide resistance. Removing the gene made the insects more susceptible to a subset of the insecticides tested, and this was confirmed with genetic and chemical complementation tests. These data provide information both on the genetics and kinetics of insecticide biology. Such information will help to better understand insecticide resistance and design rational resistance management strategies.
ItemIdentification and analysis of factors influencing intracellular growth of the pathogenic fungus Talaromyces marneffeiWeerasinghe, Harshini ( 2017)Fungal pathogens of animals and plants are a major concern in society with huge economic and public health consequences. The prevalence of pathogenic fungi and the emergence of new, opportunistic fungal pathogens, about which we know little, compounds the current problem. Talaromyces marneffei is a dimorphic, opportunistic pathogenic fungus that infects immunocompromised individuals. At 25 ̊C T. marneffei grows in a multinucleate hyphal form that can undergo a process of asexual development to produce conidia, which are the infectious agents. Upon inhalation, conidia reach the alveoli of the lungs where they are phagocytosed by resident phagocytes such as alveolar macrophages. The transition to 37 ̊C, which is the human host body temperature, induces the dimorphic switch to a pathogenic, uninucleate, fission yeast form. The yeast form is able to utilize macrophages as a niche from within which to avoid detection by the host immune system. T marneffei yeast must then be able to withstand macrophage-killing responses and obtain nutrients in order to proliferate inside these phagocytic cells. The objectives of this study were to determine the transcriptional response of T. marneffei to the host environment, including those induced by growth at body temperature as well as to host-derived cellular signals. This would lead to the identification of genes and pathways necessary for establishing infection. For this purpose RNAseq analysis was used to create a transcriptomic profile of T. marneffei during in vitro growth at 25°C (hyphal) and 37°C (yeast) and during murine and human macrophage infection (ex vivo). To identify pathways that are important during the establishment of the pathogenic yeast cell type, expression data for hyphal growth was compared to yeast growth in vitro and during intracellular macrophage infection. Key nutritional and cell protective pathways that show common upregulation during the yeast growth phase were identified and these included carbon and nitrogen utilization, micronutrient uptake, melanin generation and oxidative stress protection. Additionally, to separate host specific responses from temperature driven expression and identify genes that are specifically regulated during infection, macrophage infection specific transcriptional data sets were compared against the expressionprofile of T. marneffei grown at 37 ̊C in vitro. Twelve genes were chosen for phenotypic characterization using gene deletion as a way of validating the output of the screen, and these genes were shown to be important for different aspects of establishment and maintenance of T. marneffei yeast growth in macrophages. The analysis was extended for a novel gene designated msgA, encoding a guanyl nucleotide exchange factor,which was found to be essential for maintaining appropriate cell morphology, which in turn is crucial for T. marneffei occupying its macrophage niche. Overall the findings of this study revealed that T. marneffei yeast and hyphal forms have adapted specific metabolic programs tailored to the diverse environmental conditions encountered by each cell type. The identification of genes which are specifically required for establishing yeast growth during macrophage infection, uncovered components of pathways that respond to host–specific rather than temperature specific signals. Together these provide valuable insights into the initiation of infection and pathogenicity establishment in T. marneffei, and may serve to broaden our understanding of the means by which to target opportunistic fungal infections.
ItemA genetic investigation of congenital defects in alpacasJackling, Felicity Claire ( 2013)The aim of this PhD project was to understand the genetic mechanisms contributing to congenital defects in alpacas. Alpaca veterinarians report a prevalence of congenital defects much higher than any other livestock species. A reduction in genetic diversity due to mating between closely related individuals can cause congenital defects. In this study, inbreeding coefficients estimated from genomic data were compared between individuals with congenital defects and healthy individuals. Australian alpacas with congenital defects did not show significantly higher levels of inbreeding than alpacas without diagnosed defects. Therefore, high levels of inbreeding cannot explain the prevalence of congenital defects in Australian alpacas. One common congenital defect is the blue-eyed white phenotype which is characterized by solid white fleece, two blue eyes and often deafness. A genetic investigation of the blue-eyed white phenotype and the mapping of this trait form the second aim of this project. Case-control association analyses were performed and KIT was identified as the gene likely to be responsible for this trait. Two haplotypes were present in BEW (blue-eyed white) individuals and this suggested that two mutations contribute to this phenotype. Next-generation sequencing was used to identify possible causative mutations. Single nucleotide polymorphism analysis was used to refine the region which contains the mutations responsible for this trait and to examine the linkage disequilibrium in this region. The experimental results from this thesis were used to formulate a model for the genetic inheritance of the BEW phenotype in alpacas. The genetic markers examined in the study have the potential to provide a useful genetic test for breeders who view the BEW phenotype as a congenital defect which should be culled from the population. Together the aspects of this project aim to provide breeders with information on the genetic diversity of Australian alpaca stock as well as provide a genetic tool to enable the elimination of a deleterious phenotypic trait.
ItemSelection and characterisation of Arabidopsis mutants resistant to a glutathione biosynthesis inhibitorMaughan, Spencer C. ( 2003)This thesis describes the investigation of GSH regulation and signaling in Arabidopsis thaliana by a genetic approach. A procedure is described to select for mutants resistant to a chemical inhibitor of glutathione (GSH) biosynthesis, L-buthionine-SR-sulfoximine (BSO). Fifteen mutants were selected and preliminary genetic examinations indicated that BSO-resistance phenotypes are often highly variable. Consequently five mutants were chosen for further genetic, molecular and biochemical characterisation. All five of these mutants were shown to have altered GSH levels. Moreover, root growth among these mutants varied indicating that different mechanisms may be conferring BSO-resistance. Molecular mapping techniques were used to assign the BSO-resistance loci of four of the mutants to chromosome regions. Two mutants (a T-DNA insertion mutant and an EMS-derived mutant) were found to map to the same position between the markers CHS1 and R89998 on chromosome 5. Subsequently, complementation tests confirmed that these mutants define a single locus. The mutated locus in the T-DNA insertion mutant was determined using molecular techniques. The gene that contained the insert encodes a putative transport protein and was designated the BSO Resistance Transporter1 (BRT1). No transcript was detected in the T-DNA insertion mutant (brt1-1) which suggests that brt1-1 is likely to be a null allele. The EMS-derived mutant (brt1-2) contained an intron splice-site consensus sequence mutation which resulted in transcripts that included an unspliced intron. The function of the BRT1 gene was investigated by phenotype testing and despite the brt1 mutants having 2-fold GSH levels they were not resistant to known oxidising agents. Northern analysis of GCS and the use of a GCS-GUS reporter line indicated that the increase in GSH levels in these mutants was not accompanied by increases in GCS transcription and translation. This suggests that BRT1 is a novel component influencing GSH metabolism. BRT1 defines a novel family of three genes in Arabidopsis. Preliminary experiments indicate that these three genes are expressed in the same tissues providing for possible a level of redundancy between them. Supporting this is a newly identified insertion mutant in a second BRT gene (brt2-1) which is also resistant to BS0. Moreover, the double mutant, brt1-1;brt2-1, exhibits increased BSO-resistance suggesting an additive effect of the mutations. Database searches revealed that there were homologues in other organisms including one from the malaria parasite (PfCRT). Mutations in PfCRT confer resistance to a widely used antimalarial, chloroquine (CQ). However the function of PfCRT is not known. The BRT1 mutant was shown to have a level of functional conservation with a malaria parasite homologue, PfCRT, and therefore further characterisation of these proteins may provide insights into GSH metabolism and perhaps CQ-resistance in the malaria parasite.
ItemThe regulation and function of the Drosophila melanogaster Cytochrome P450 gene, Cyp12d1Boey, Hui Kuang Adrian ( 2011)Cytochrome P450s are an important family of monooxygenase enzymes implicated in numerous xenobiotic detoxification events as well as in essential endogenous functions. The vinegar fly Drosophila melanogaster has 85 P450 genes; however, the large majority of them remain uncharacterised in terms of their function and regulation. Cyp12d1 is arguably the most xenobiotic inducible P450 gene in the D. melanogaster genome. It has been suggested that Cyp12d1 is an excellent candidate gene to study Drosophila xenobiotic induction pathways as it responds to a wide range of chemical inducers, indicating that it contains most if not all of the cis-regulatory elements needed for xenobiotic induction in Drosophila. Hence, the transcriptional regulation of Cyp12d1 was investigated to identify novel P450 induction pathways in D. melanogaster. Cyp12d1 basal transcriptional regulators were found in Cyp12d1 upstream and downstream regulatory regions, while enhancers for Phenobarbital and caffeine induction were located upstream. Site-directed mutagenesis experiments identified GATA family transcription factors as important Cyp12d1 midgut expression regulatory proteins. However, their role in xenobiotic induction remains unclear. Biochemical sequencing of electomobility-shift assay protein bands, and genetic RNAi screens of genes encoding other candidate transcription factors, failed to identify any other potential xenobiotic regulatory proteins. Cyp12d1 function was also investigated in this study. Cyp12d1 overexpression has been shown to confer resistance to the insecticides DDT and dicyclanil, but other functions have not been identified prior to this study. Adult Cyp12d1 functions were investigated through Cyp12d1 RNAi and overexpression studies. Cyp12d1 was found to be involved in adult longevity and oxidative stress resistance, suggesting other potential functions in addition to known detoxification functions. Cyp12d1 has been tandemly duplicated in D. melanogaster, and this duplication exists as a polymorphism in field populations. The geographical distribution of the Cyp12d1 duplication was examined in flies collected along the eastern coastline of Australia. The frequency of the duplicated Cyp12d1 gene was found to vary spatially, with flies in lower latitudes being more likely to possess the Cyp12d1 duplication and flies in higher latitudes being less likely. Cyp12d1 tissue-specific embryonic expression and mRNA transcript length was different in Cyp12d1-duplicated lines when compared to non-Cyp12d1 duplicated lines. These results indicate the Cyp12d1 duplication confers changes in Cyp12d1 expression patterns and suggest that Cyp12d1 may be involved in local adaptation to the microenvironment.