Genetics - Theses
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ItemIdentification and analysis of factors influencing intracellular growth of the pathogenic fungus Talaromyces marneffei( 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.
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ItemMolecular studies of gene regulation in Aspergillus nidulans( 1985)The amdA regulatory gene of Aspergillus nidulans controls the expression of both the acetamidase (amdS) gene and a gene, designated aciA, which encodes a 42 000 dalton polypeptide. The cloning and analysis of the aciA gene is described in this thesis. The aciA gene is a single copy gene approximately 2kb in size and encodes two messenger RNA species of 1.45 and 1.55kb•in size. Both of these aciA transcripts are regulated in an identical fashion. The aciA gene is demonstrated to be induced in the presence of the carbon source acetate and evidence is presented which indicates that it may also be subject to carbon catabolite repression. None of the other regulatory circuits which are known to control amdS expression affect the expression of the aciA gene. The 5' non-coding region of the aciA gene was sequenced and both the primary and secondary structures within this region are compared with the corresponding regions of the amdS gene and a cis-acting mutant of the amdS gene which increases the amdA-mediated regulation of this gene. The aciA gene possesses a canonical TATA box and also contains a number of other sequences which are similar to sequences found in the 5' non-coding region of the amdS gene. The most interesting of these is a 23bp purine-rich region which is similar to a purine-rich region occurring in approximately the same position in the amdS gene. This sequence is found to be duplicated in the amdS mutant which is subject to increased regulation by the amdA gene. Experiments in which A. nidulans was transformed with multicopy plasmids containing DNA fragments from the aciA gene have indicated that the aciA sequence responsible for the titration of the amdA gene product is located in the 5' non-coding region of this gene. A model for the regulation of the aciA gene by the amdA gene, acetate and carbon catabolite repression is presented. The possible significance of small repeated sequences within the purine-rich regions of aciA and amdS is also discussed.
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ItemCytochrome P450 gene expression in Drosophila melanogaster( 2008)Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised. In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability. In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations. This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.