Genetics - Theses
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ItemIdentifying downstream targets of FILAMENTOUS FLOWER, a YABBY transcription factor that promotes organ polarity and lateral growth in Arabidopsis( 2017)Angiosperm leaves are typically polar structures with a distinct arrangement of cell types along the adaxial-abaxial (upper-lower) axis. Studies of leaf development in the model dicot plant Arabidopsis thaliana have shown that adaxial-abaxial patterning is not only associated with the formation of distinct cell types, but also triggers growth along the lateral axis leading to the formation of the leaf blade or lamina. Adaxial-abaxial patterning factors identified in a variety of plants including Arabidopsis, Antirrhinum and maize are either small regulatory RNAs or transcription factors. Although first categorized as being involved in adaxial-abaxial patterning, the YABBY (YAB) family of transcription factors is now thought to play a pivotal role in coordinating various developmental programs involved in leaf blade formation. As part of an approach to identify targets of the Arabidopsis YAB gene FILAMENTOUS FLOWER (FIL), this study generated transgenic lines with inducible FIL activity. Constitutive activation of FIL resulted in the partial abaxialisation of leaves and reduced blade growth, phenotypes that have previously been attributed to ectopic YAB expression. Further analysis showed that constitutive FIL activity increased the rate of cell cycle progression in specific regions of the developing leaf, as well as increasing sensitivity to exogenously applied auxin. The latter phenotype was inferred from increased activity of an auxin-signalling reporter, suggesting that FIL modulates auxin responses in Arabidopsis. Transcriptional profiling with microarrays was subsequently used to monitor genome-wide changes in gene expression following FIL activation. This analysis identified groups of genes that were either positively or negatively regulated by FIL and extensive testing of a subset of these genes showed that some were direct targets. On the basis of these results it is proposed that FIL functions as both a transcriptional activator as well as repressor during leaf development. Among the positively regulated genes identified as FIL targets, two are well-known abaxial patterning factors, KANADI1 (KAN1) and AUXIN RESPONSE FACTOR4 (ARF4). Given the well-defined role these factors play during leaf development, this study focused on their regulation. Analysis of mutant lines lacking activity of the leaf-expressed YABs revealed a significant reduction in KAN1 expression, but not ARF4 expression. These results confirm that FIL is a regulator of KAN1, but presumably regulates ARF4 in combination with other factors. In conclusion, this study identified direct targets of the bifunctional plant transcription factor FIL. Finding that two of these targets promote abaxial cell identity during leaf development supports the case for YABs being important polarity regulators. Given the expression pattern of the YABs, it is argued that YABs are unlikely to function upstream of KAN1/ARF4. Instead, a model is proposed in which YABs promote adaxial/abaxial patterning through a system of positive feedback loops that ultimately maintain the activity of these early abaxial patterning genes during leaf blade formation.
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ItemThe genetic basis of resistance to the Ryanodine Receptor modulator chlorantraniliprole in Drosophila melanogaster( 2017)The development of synthetic insecticides in the mid 20th century lead to a revolution in pest control. However, issues with environmental toxicity, adverse human exposure and insecticide resistance have meant new safer alternative pest control methods are required. Chlorantraniliprole belongs to a promising new class of insecticides that exert control by targeting the Ryanodine Receptor. As this class, the group 28 synthetic diamides, has a unique chemistry and a mode of action that is distinct from most insecticides, its market share has rapidly increased since it was first introduced in 2007. Here, I use genomic, transcriptomic and phenotypic analyses of the Drosophila Genetic Reference Panel (DGRP) to examine the way chlorantraniliprole interacts with an insect’s biology. This research reveals that a novel muscle- associated gene, Stretchin Myosin Light Kinase, is strongly associated with resistance in the DGRP. In addition, a co-expressed set of detoxification enzymes, under control of the Cap ‘n Collar/Keap1 pathway were found to be constitutively up-regulated in a subset of the DGRP and that their transcriptional abundance was correlated with survivorship on chlorantraniliprole. Transgenic ‘knock up’ of one of these putative detoxification enzymes, Cyp12d1, confers increased resistance to chlorantraniliprole and resistance to the related compound cyantraniliprole. Furthermore, a lab selection experiment based on a large Australian population of D. melanogaster also confirms an association between Cyp12d1 and resistance. This contributes to a growing body of evidence suggesting that cytochrome P450 enzymes play a role in chlorantraniliprole resistance. Through the quantitative genetic approaches employed, it is possible to demonstrate that the genetic architecture underpinning resistance changes with dose. Furthermore, as the DGRP was established before the introduction of chlorantraniliprole this study demonstrates that alleles of large effect are pre- existing in naïve populations and such alleles may increase in frequency as this class of insecticides become more widespread. Finally, this study illustrates the systems genetic approach offers unprecedented power to understand the biology perturbed by insecticides.
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ItemThe significance of low level mosaicism in Fragile X Syndrome.( 2017)Fragile X Syndrome (FXS) is the most common single disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD). FXS is fundamentally caused by a trinucleotide CGG sequence repeat expansion within the 5’ untranslated portion of the FMR1 gene, to more than 200 repeats which is called Full Mutation (FM). This is associated with abnormal methylation of this gene’s promoter and silencing of FMR1 expression. To date, the type and severity of the neurocognitive phenotype has been related more closely with the methylation status than with the number of CGG repeats in the FM range. Smaller CGG expansions are called premutation (PM) (55 and 200 CGGs), grey zone (GZ) (45-54 CGGs) and normal size (<44 CGGs) alleles, and are usually associated with presence of an unmethylated FMR1 promoter, and normal FMR1 expression. There is a proportion of FXS individuals in which two or more population of cells can be found, and who are called mosaic or mosaicism. These individuals possess cells with FM and smaller CGG sizes (e.g. Normal, GZ or PM), which lead to milder forms of the FXS phenotype. This PhD explores the clinical and diagnostic significance of low level mosaicism (LLM) and DNA methylation variation amongst different CpG sites within FMR1. The first aim of this study was to define the lower limit of detection LLM of six FMR1 tests currently used in diagnostic laboratories. This was achieved using three FMR1 PCR commercial kits (AmplideXTM, X-Sense and FastFraXTM) targeting the CGG region and three methylation tests (Methylation Specific- Quantitative Melt Analysis [MS-QMA], Sequenom®EpiTYPER system [MALDI-TOF MS] and mSouthern blot) targeting methylation of Exon1/Intron1 boundary region and specific CpG sites within FMR1 promoter. The second aim was to screen a large number of males with ID and ASD of unknown etiology referred for FXS testing to determine the prevalence of mosaicism containing FM together with a normal or GZ allele (cryptic FMR1-FM) who are not being identified as a part of standard FXS testing. This was achieved using MS-QMA and the positive results were confirmed with the FMR1 tests assessed in Aim 1. The third aim of this study was to examine the association between DNA methylation of FMR1 and mRNA levels and severity of intellectual functioning impairment in individuals with a cryptic FMR1-FM identified in Aim 2 and males and females with typical FXS (only FM and PM/FM alleles). Three significant contributions to the FXS field resulting from this PhD are: i) implementation of a new highly sensitive assay for detection LLM missed by standard testing; ii) determination of the prevalence of cryptic FM-FMR1 in males with idiopathic ID/ASD referred for FXS testing; iii) clinical and molecular characterization of LLM and its methylation characterization amongst tissues and CpG sites; and demonstration that LLM levels are significantly correlated with severity of cognitive impairment and ASD as well as FMR1 mRNA levels.
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ItemThe evolution of pathogenicity and isolate variation in Talaromyces marneffei( 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.
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ItemProbing insecticide biology using Drosophila melanogaster( 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.
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ItemThe genomic basis of climate and host adaptation( 2017)Many species are currently threatened by the direct and indirect effects of anthropogenically driven climate change. The elevation of global temperatures and increase in variability in both temperature and precipitation pose a risk to biodiversity as species are pushed close to their thermal safety margins. Current predictions suggest a dramatic loss of species diversity and the contraction of geographical ranges of many species. Many ectothermic insects that cannot regulate their body temperature are likely to be threatened, particularly ecologically- restricted herbivorous insects that depend for on plants for food and that are often in phenological synchrony with their plant hosts. However, adaptive shifts in these species in response to host loss and climatic extremes may counter the effects of climate change to some extent. This highlights the importance of studying species-specific adaptation mechanisms including host interactions. This dissertation contributes to this overall aim by studying the genomic basis of climatic and host adaptation. I use Drosophila melanogaster as a model system at the intraspecific level, and Drosophila species from the repleta group as a model system for the comparative level. In assessing the genomic basis of host responses, I consider a much broader range of insect taxa. This dissertation begins with a study on the use of chromosome level sequencing of D. melanogaster populations from two ends of a thermal cline. I present genomic evidence for the role of the inversion 3R Payne in capturing alleles favourable to local climatic conditions in the non-inverted form, and therefore driving adaptation to climate change. The study further elucidates the impact of climatically important chromosomal inversions in driving higher linkage disequilibrium on the non-inverted form - potentially benefiting both karyotypes. In the second chapter, I develop a new pipeline, Orthonome, and tools for multi-species comparisons for prediction of orthologues and inparalogues with the highest accuracy and recall. Using Orthonome, I was able to identify a much greater level of conservation across Drosophilid lineages than earlier thought, amounting to nearly 33% better resolution than industry-accepted methods. I then use Orthonome in the third chapter to compare the genomes of 58 insect species – most of which are known to be agricultural pests. Testing across eight gene families, I present evidence for genomic patterns in only four gene families (P450s, CCEs, GSTs and ABCs) as being associated with polyphagy or particular host ranges. While three of them have been reported before, I find that ABC transporters present much stronger evidence than reflected in earlier studies, with feeding behaviour as well as host tissue displaying an effect on gene gain in more voracious pest species. Finally, in the last study, I use novel genomic data and evidence from the repleta group of drosophilids to carry out phylogenetically constrained analyses of genes potentially associated with host and thermal stress adaptation. My aim here is to find mutually exclusive evolutionary pathways to neofunctionalisation between stress tolerant cactophilic specialists and less tolerant generalists in the group. I also find a different adaptive response in the cactophilic species compared to the generalist species; these species show little lineage specific gene gain, suggesting an exception to current standing theories on neofunctionalisation for adaptation. I further discuss the applicability the species level and order level analyses for an overall detailed and systematic approach to identify the genomic basis of climatic and host adaptation.
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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.