Genetics - Theses
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ItemComparative analysis of dimorphic growth in the pathogenic fungus Penicillium marneffei( 2013)Fungal infection is a major cause of mortality in immunocompromised individuals and an escalating health threat in the 21st century. With very little known about the mechanisms behind its virulence, Penicillium marneffei is a dimorphic opportunistic fungal pathogen of humans. At 25°C P. marneffei grows as multicellular nonpathogenic hyphae that through differentiation produces asexual conidia, the infectious agent. Infection begins with the inhalation of conidia, followed by a dimorphic switch of these conidia to pathogenic yeast cells upon entering the 37°C environment of the host. In the host P. marneffei resides within immune phagocytic cells, where it then grows as a fission yeast. The objective of this study was to identify differences between P. marneffei hyphal and yeast cells. For this purpose, firstly extensive microarray-based expression profiling data was analysed, identifying a variety of differentially expressed genes that were organized into clusters based on function and spatiotemporal expression patterns. One key activity highlighted in the data related to the acquisition of the essential trace metal iron. Genes involved in high-affinity iron assimilation systems, their regulation and the production of key iron acquisition secondary metabolites (siderophores) were characterised in both hyphal and yeast cells. It was found that compared to hyphal cells, yeast cells rely more heavily on iron acquisition through high-affinity reductive iron assimilation. Also, at least under rich iron conditions there is a yeast cell-specific upregulation of genes involved in the biosynthesis of extracellular siderophores, via the de-repression of transcriptional regulation by SreA. Secondly, metabolite profiling and ‘footprint’ analyses of hyphal and yeast cells were performed. This snapshot of P. marneffei metabolism revealed the metabolite differences between the two cell types and pointed to the utilisation of specific pathways in each growth form. Further, 13C-labeled glucose experiments revealed an increase in specific carbon metabolic pathways in yeast cells, particularly the TCA cycle and the pentose phosphate cycle, indicative of responses to stressful environments. By combining data derived from multiple molecular approaches, key metabolic networks were reconstructed and results highlight that P. marneffei hyphal and yeast cells have definitively different developmental and nutritional profiles, reminiscent of their mode of reproduction and likely adaptations to the environmental niche they inhabit.
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ItemA genetic investigation of congenital defects in alpacas( 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.
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ItemGlacial refugia of southern Australian echinoderms: past responses to climate change in the temperate marine environment( 2013)Distributions of organisms are known to vary in response to glacial and interglacial climate cycling. While this process has been widely studied in terrestrial and aquatic environments, comparatively few have focused on marine invertebrates. The bulk of studies focus specifically on intertidal organisms, fish species and Northern Hemisphere coastlines, with fewer still addressing the dynamics of subtidal organisms and the Australian coastline. In this thesis, I address the question of population responses to glacial dynamics in a number of endemic southern Australian shallow-water echinoderm species, with the goal of expanding our understanding of the phylogeography of the region. The study region extends from Western Australia to northern New South Wales and includes Tasmania. The study is overall a comparative one, utilising multiple species and multiple genetic markers in order to track population dynamics over various time scales. Samples of all target species are initially screened for the presence of cryptic species and deep mitochondrial lineages, with the result that a high number of additional species are discovered and the initial sample size of target species is reduced. As intermittent barriers to gene flow are present on the southern coast of Australia, and as these vary in response to climate cycling, each species complex is investigated in terms of the timing of lineage production, examining the relationship of younger, allopatric lineages to older, sympatric lineages in the presence of intermittent barriers to gene flow. A large-scale comparative phylogeography of the southern coast is undertaken, examining intraspecific variation and congruent genetic breaks across ten species. A moderate level of phylogeographic congruence is apparent across these species and in this region. There was high support for all proposed biogeographic breaks. Finally, more recent dynamics are investigated by examining the distribution of intraspecific diversity and population expansion since the Last Glacial Maximum. These results suggest that, while expansions on the southern and western coasts tend to pre-date the Last Glacial Maximum, the south-eastern region of Australia is quite active in regard to post-LGM expansion.
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ItemLocal adaptation in two species of Carex sedges from the Australian Alps( 2013)The Australian alpine ecosystem is restricted to 0.15% of the Australian continent and 60% of its vegetation is endemic. Projections show that a 0.2°C-1°C increase from 1990 would lead to a reduction in the elevation range of the alpine-subalpine area of 2 to 21% by 2020 and an increase of 2-4°C would lead to the total disappearance of the Australian alpine climate by 2100. Although plants in this area are adapted to survive very cold climate, their persistence is possible if local adaptation to the changing environment occurs. My project uses a combination of genetic molecular work (genome scans with microsatellite markers) and quantitative work (measures of altitudinal variation in plant morphology, reciprocal transplant experiments and a crosses experiment) to assess the presence of local adaptation in alpine Carex species and to ascertain whether these species have potential for adaptive shifts to a changing environment. Carex hebes and C. breviculmis are two alpine species commonly found in tall alpine herbfield and sod tussock grassland communities in the Bogong High Plains in Victoria. Despite growing in similar habitats, the two species have contrasting geographical ranges. I found that both species reproduce sexually but none of the populations were at Hardy-Weinberg equilibrium, due to a high level of inbreeding. An increase in inbreeding with elevation was found in C. hebes populations. Despite this, there was a high level of genetic diversity along all transects and a high level of gene flow between transects for both species. These results suggest the ability for both species to respond to a changing environment through adaptive shifts; however if inbreeding depression occurs in both species it may result in lower fitness and a decline in population size. Both species showed variation in their growth forms with elevation. Plants from both species had shorter leaves at high elevation compared to lower elevation sites. Carex breviculmis plants had a larger circumference at high elevation compared to plants at low elevation. Results from reciprocal transplant and common garden experiments showed that for C. breviculmis, plants from high and low elevation may have been subjected to directional selection pressures leading to genetic divergence of leaf length while morphological variation observed for C. hebes was due to environmental effects. A cogradient pattern was observed at one site for C. breviculmis showing that this species might have the potential to adapt its morphology to changing environmental conditions through plastic responses and heritable shifts. No evidence of local adaptation was found in term of reproduction or survival on both species. Results from a crossing experiment showed the presence of maternal effects, and to a lesser extent paternal/genetic effects for C. breviculmis leaf length as well as a low but significant level of heritable variation. Maternal environmental effects along a gradient may be adaptive if the environment experienced by parents is also experienced by the next generation, and they may facilitate adaptation by influencing the rate and direction of evolutionary change in response to directional selection. The sire effects across elevation and presence of heritable variation within population may also allow for evolutionary genetic shifts across generations, provided that population growth rates and the strength of selection pressures acting on populations promote evolutionary changes. These results show that species inhabiting the same environment can have different patterns of morphological variation through heritable/ divergence and plastic responses which can affect a population’s response to a changing environment. The results can be used to assist conservation and management strategies for these two species.
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ItemGenetic regulation of cell migration and mesenchymal to epithelial transitions in the Drosophila midgut( 2013)Cell migration and epithelial to mesenchymal plasticity are important processes in animal development and disease. In Drosophila embryogenesis, the formation of the midgut provides an opportunity to study these processes. The midgut is formed by the migration of two patches of cells towards one another from the poles of the embryo, using the visceral mesoderm as a substrate. Once the cells have completed their migration, they undergo a mesenchymal to epithelial transition to form two epithelial sheets of cells. Currently, only the extracellular matrix adhesion proteins, the Integrins, and the Integrin ligand LamininA have been shown to be required for midgut migration. The aim of this thesis therefore was to use the midgut system to identify new genes involved in the formation of this tissue. I have first identified the gene responsible for the phenotype of embryos homozygous for a deficiency stock, Df(2L)30A-C, in which the midgut does not form due to a migration failure. As this stock deletes approximately 50 genes, deficiencies in the region were screened for the migration phenotype. Following this analysis, mRNA expression patterns revealed midgut expression for hoi-polloi, and subsequent mutant analysis provided further evidence that this is the gene responsible for the migration phenotype. Hoi-polloi is homologous to a core spliceosome component, and while a tissue specific role for this gene is difficult to envisage, it opens up exciting possibilities of the role of the spliceosome in developmental events. I have also found that Netrins, well known as chemoattractants, play a role in midgut migration and mesenchymal to epithelial transitions. The two Drosophila netrins, netrinA and netrinB, are expressed in the visceral mesoderm, but also localise to internal puncta in midgut cells. Loss of both netrins delays migration and disrupts polarity. The DCC orthologue Frazzled, a known netrin receptor, is expressed in midgut cells and is also required for migration. Frazzled localises to the cortex and to endocytic vesicles, and is required for internalisation of Netrins. Finally, a potential link was found between Netrin and Integrin signaling. While Netrin and Frazzled have a well-established role in migration and axon guidance, this is the first study to suggest a role for these genes in mesenchymal to epithelial transitions, and thus shows the use of the Drosophila embryonic midgut system for studying these processes.
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ItemPseudogenes and neutral evolution in Drosophila melanogaster( 2013)An understanding of the rates and patterns of neutral evolution is important for estimating divergence times and for recognising selection acting upon the genome. In order to characterise neutral evolution it is necessary to identify sequences that are evolving free from selective constraint. This has proven difficult in Drosophila, where sites such as synonymous sites, introns, and untranslated regions of genes have been shown to have selection acting upon them. Pseudogenes are inactive copies of genes that are by definition functionless, and are thus ideal candidates for the study of neutral evolution. Historically few pseudogenes have been known in Drosophila, and many sequences that have been thought to be pseudogenes have been found to be functional. The advent of the genomics era has allowed for the identification of many more potential pseudogenes. In this thesis I study these in order to identify likely genuine pseudogenes, and use these to characterise neutral evolution in Drosophila. The first step of this project was to identify a list of genuine pseudogenes. Two pseudogene datasets were used: those identified in a paper by Harrison et al., (2003), and those listed on the Drosophila genome database Flybase. A number of techniques were used to study these pseudogenes, including cDNA analysis, conservation analysis, and resequencing. The results of this study showed that many of these sequences were not genuine pseudogenes and had been incorrectly assigned due to incorrect genome annotation, unknown splicing patterns, and polymorphic inactivating mutations. In total 73 likely Drosophila melanogaster pseudogenes were identified. The technique Gene Identification by Nonsense-Mediated Decay (GINI) (Noensie & Dietz, 2001) was investigated as a possible technique for identifying new pseudogenes. This involved feeding Drosophila larvae drugs known to inhibit nonsense-mediated decay and then determining whether this resulted in an upregulation of transcripts known to contain premature termination codons (PTCs). Caffeine was found to result in the upregulation of 4 of 6 PTC-containing transcripts, most notably those with longer 3’ UTRs. This technique thus seems promising for the identification of new pseudogenes. Following this, 47 pseudogenes were resequenced in three populations. It was found that ancient pseudogenes had higher levels of nucleotide diversity than recently inactivated genes and new pseudogenes, presumably due to their time since inactivation and pseudogene population size during this time. Various aspects of pseudogene evolution were characterised including nucleotide diversity, mutation patterns, and FST, allowing us to gain a better understanding of population structure and the background mutational patterns of the genome. Finally, I investigated several hypotheses as to why the Esterase-7 gene might have a large proportion of inactivated alleles in natural populations of D. melanogaster. I concluded that the selective constraint acting on Esterase-7 has likely been relaxed in D. melanogaster relative to other species, and Esterase-7 may be in the process of becoming a pseudogene. This gives us key insights into the pseudogenisation of functional genes by mutation and genetic drift - mutations gradually occur and spread throughout the population while functional copies still remain in the population.
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ItemTargeting chromosomal instability for the specific killing of cancer cells( 2013)Chromosomal INstability (CIN), a common feature of advanced tumors, is linked to drug resistance, metastasis, relapse and lower survival rates of patients in clinical settings. CIN describes cells with the tendency to progressively gain and lose large sections of DNA. This feature is often observed in cells with defects in chromosomal segregation, a process monitored by the spindle assembly checkpoint (SAC). Using Drosophila melanogaster carrying a weakened SAC as a CIN model, a genetic screen was carried out to identify genes that induce lethality specifically in animals with CIN. From this screen the c-Jun N terminal kinase (JNK) pathway was identified as a modifier of CIN cell fate. Knockdown of the JNK pathway was found to induce apoptosis in CIN but not normal cells, via the canonical apoptotic pathway, partly independent of the key tumor suppressor p53. Knockdown of SAC induces DNA damage, and the level of damage significantly increased when JNK was concurrently knocked down, implicating DNA damage in the induction of the observed apoptosis. Evidence is presented implicating G2 phase length in the survival of CIN cells, where shortening G2 but not G1 phase in CIN cells mimics the apoptosis induced by knockdown of JNK, while lengthening G2 but not G1 phase rescues the apoptosis. Based on these observations, I propose that JNK may therefore play a role in the regulation of G2 length following chromosome missegregation.