Genetics - Theses
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ItemThe role of presenilin in metal homeostasis and Alzheimer's disease( 2012)Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and there is currently no effective disease-modifying treatment available. One of the pathological features of AD is the accumulation of amyloid deposits in the brain. These extracellular amyloid deposits (or plaques) are primarily composed of misfolded amyloid beta (Aβ) peptide aggregates but also contain high levels of copper (Cu) and zinc (Zn). Both Aβ and its parent molecule, the amyloid precursor protein (APP), are metal-binding proteins. The formation of Aβ from APP is carried out by the sequential cleavage by β-secretase (BACE1) and γ-secretase. γ-secretase is a multi-protein aspartyl protease that contains the protein presenilin (PS, PSEN) as its catalytic component. Studies carried out almost two decades ago revealed that both Zn and Cu can cause Aβ to aggregate in vitro and subsequent studies in mice demonstrated that Zn and Cu augment Aβ pathology in vivo. Based on this pathological phenomenon the “Metal Hypothesis of Alzheimer’s Disease” was proposed whereby a disease-associated metal dyshomeostasis facilitates the pooling of Zn and Cu in synaptic junctions, the very site where Aβ accumulates. The flipside to this hypothesis is that as extracellular Zn and Cu levels rise, cells such as neurons can become deficient in these metals. This has important consequences, as Cu and Zn are essential co-factors for numerous cellular enzymes. Consistent with this hypothesis, there is evidence of reduced Cu/Zn dependent superoxide dismutase 1 (SOD1) activity in AD patient brains and AD animal models. However, there is still limited knowledge about the molecular mechanisms that can potentiate the Cu and Zn dyshomeostasis that is evident in AD. An inherited form of AD, called familial AD (FAD), is predominantly caused by mutations in PSEN1 and PSEN2, the genes that encode PS1 and PS2 respectively in mammals. In general, FAD mutations are associated with an increase in longer, more pathogenic forms of Aβ that are prone to aggregate. However, PS/γ-secretase cleaves numerous substrates other than APP, including Notch. Hence, PSEN1 and PSEN2 mutations that alter γ-secretase activity may also lead to loss-of-function phenotypes. In addition, PS has been demonstrated to function independently of γ-secretase, affecting cellular processes such as protein trafficking and calcium homeostasis. The primary aim of this PhD project was to determine whether PS expression could affect Cu or Zn homeostasis. Initial experiments focused on utilizing cultured PS knockout (PS KO) mouse embryonic fibroblast (MEF) cells and 64Cu and 65Zn radioisotope uptake assays. These experiments revealed that PS is required for a significant proportion of cellular Cu and Zn uptake in these cells. RNAi of PSEN1 or PSEN2 in cultured human embryonic kidney (HEK293) cells confirmed that 64Cu and 65Zn transport was affected by PS expression in multiple cell types. Interestingly, total Cu and Zn levels were unchanged in PS KO MEFs and 64Cu and 65Zn retention studies indicated that turnover of Cu and Zn was reduced in these cells. Consistent with a role for PS in Cu and Zn uptake, brain and other tissues from PS1 heterozygous knockout mice had lower Cu and Zn levels compared to littermate control mice. The activity of Cu/Zn dependent SOD1 was lower in both PS deficient cultured MEF cells and PS1-deficient mouse brain, a potential consequence of disturbed Cu and/or Zn homeostasis in these cells/tissue. Tellingly, knock-in mice that harbour a PS1 mutation that causes FAD in humans also had lower brain Cu and SOD1 activity. Cell surface biotinylation and confocal microscopy immuno-localization experiments indicated reduced expression of several Cu and Zn transporters at the cell surface of cultured PS deficient cells. A model of PS-dependent trafficking of these transporters is proposed whereby PS is required for the delivery or retention of specific metal transporters at the cell surface. In this model PS could influence trafficking at one or more sites along the biosynthetic or endocytic pathways, consistent with the roles for PS in protein trafficking that have been reported in the literature. These studies demonstrate that as a consequence of loss of PS function, either by loss of expression or mutation, Cu and Zn homeostasis is altered. This has important implications in terms of AD, and in particular the familial form of the disease, whereby altered PS function may modulate Cu and Zn re-uptake in cells and promote Cu and Zn interaction with extracellular Aβ. These novel findings should stimulate further studies in neuronal cells and thereby facilitate a better understanding of the involvement of PS in Cu and Zn homeostasis in the brain.
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ItemNitenpyram resistance in Drosophila melanogaster( 2012)The neonicotinoid insecticides are effective in controlling a variety of insect pests in the field. They target the nicotinic acetylcholine receptor, a cation channel in the central nervous system. After years of usage, high levels of resistance to these insecticides are beginning to emerge. This requires greater understanding of the activity of these insecticides and the function of these channels. The bulk of this study made use of the model organism, Drosophila melanogaster, and the well-defined family of ten nicotinic acetylcholine receptor subunits encoded by its genome. Mutations and null alleles of insect nicotinic acetylcholine receptors have been described but, thus far, all of these are homozygous viable. This is surprising given the high level of conservation of these genes. Part of this study examines viability with loss of function of individual subunits through the use of stably integrated RNAi constructs. Ubiquitous knockdown of any one gene had a significant effect on mortality prior to the adult stage. Knockdown of Dα1, Dα5 or Dβ2 produced the most severe increases in mortality. However, genomic deletions of either Dα1 or Dβ2 were seen to be viable. Deletion of Dα5 resulted in a completely penetrant larval lethality phenotype with all the hallmarks of a possible role in hormonal control of development. Further studies of knockdown flies highlighted possible roles in mating for Dα1, Dα2, Dα6 and Dβ1. RNAi knockdown of individual receptors was also used to investigate the possible involvement of each subunit in mortality induced by the neonicotinoids. RNAi knockdown larvae were exposed to nitenpyram and survival to adulthood scored. Resistance was seen for knockdown of known nitenpyram targets Dα1 and Dβ2, confirming this approach provides enough sensitivity to reveal nitenpyram targets. Resistance was also seen upon knockdown of either Dα3 or Dβ1. In addition to this, knockdown of either Dα6 or Dβ3 produced hyper-sensitivity to nitenpyram. Results from this study are juxtaposed to published associations of nicotinic acetylcholine receptor subunit resistance alleles to propose likely components of the neonicotinoid binding sites. The neonicotinoid insecticides are known to bind at the acetylcholine binding site, which occurs at a cleft formed between two subunits in the mature receptor. The information gained from published work and the RNAi experiments using nitenpyram suggested two likely interfaces that could make up the insecticide binding site. Homology modeling based on the structure of the Aplysia californica acetylcholine binding protein was used to generate three dimensional structures of the two likely interfaces. Binding of several neonicotinoids was simulated and produced a similar binding orientation in a Dα1/Dβ2 interface. Comparison of imidacloprid binding to acetylcholine binding and imidacloprid resistance mutations strongly supports the model presented here. The final part of this study is the genetic mapping of a novel resistance mechanism to nitenpyram isolated in a field collected strain. A previously identified strain of D. melanogaster collected at Cape Tribulation, Australia, had been shown to carry a resistance factor on the third chromosome. P-element mapping and screening of molecular markers identified a resistance locus on chromosome 3L between cytological bands 68c4 and 68d2 that contains 50 genes. The known functions and expression patterns of these genes are considered and possible resistance mechanisms are discussed.
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ItemExpression and function of five ligand-gated chloride channel genes of Drosophila melanogaster( 2012)Annotation of the genome of the vinegar fly, Drosophila melanogaster, has identified 12 ligand-gated chloride ion channel (LCCH) subunit genes. The ligands for the receptors produced from these LCCH subunit genes are the neurotransmitters GABA (γ amino butyric acid), glutamate, histamine and glycine. The ligands of some subunits are yet to be discovered. The GABA-gated LCCH subunits have sequence homology with vertebrate GABAA receptors that have vital functions in nervous system development and neurotransmission. The Rdl gene of D. melanogaster encodes a LCCH subunit. This gene came to prominence when it was found that a naturally occurring allele, in D. melanogaster and many insect pest species, conferred high level target site resistance to the insecticide dieldrin. Subsequently the Rdl (Resistance to DieLdrin) gene and its product (RDL) have been intensively investigated. The Rdl gene has been associated with sleep, learning and memory, epileptic seizure and phenotypes due to defects in Drosophila Fragile X mental retardation gene function. There are four other D. melanogaster genes that been reported to encode subunits that assemble into GABA-gated cation channels (Lcch3 and Grd) or have the GABA-binding consensus sequences found in vertebrate GABA receptor subunits (CG8916 and CG12344). These other genes are also likely to play important functional roles in neurotransmission and/or development but, to date, there has been a striking lack of research focused on elucidating their functions. In this thesis two approaches are combined to examine the function of these genes. In the first of these, the expression of the Rdl, Lcch3, Grd, CG8916 and CG12344 genes is examined using in situ hybridization. Further, the cloned endogenous promoters for the five genes are used to drive the expression of the green fluorescent protein (GFP) using the GAL4/UAS system (Chapter 2). The spatial and temporal expression patterns for these genes provide some clues to their function. The second approach uses RNA interference to knockdown gene expression to generate mutant phenotypes indicative of gene function (Chapter 3). A number of phenotypes are described here – defects in larval forward locomotion and wing inflation, necrotic tissues and lethality. The links between these phenotypes and the function of the five genes are carefully considered. While it is not the major focus of this thesis, the applications of the research described are considered. The RDL protein has been targeted with a range of insecticides. This thesis identifies two other GABA-gated cation channels (GRD and LCCH3) as potential insecticide targets. Given the similarities between the five GABA-gated LCCH subunits and their human counterparts, some fruitful lines of research that may positively impact the further study of human biology and health are identified. Most importantly, the research reported here provides a foundation of knowledge and reagents that will be of great value in an ongoing systematic analysis of the role that these five GABA-gated LCCH subunits play in insect development and neurotransmission.
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ItemNicotinic acetylcholine receptors; an examination of expression and insecticide interactions in Drosophila melanogaster( 2012)Nicotinic acetylcholine receptors (nAChRs) are complex transmembrane proteins that belong to the Ligand-gated ion-channel (LGIC) super-family. They are responsible for cholinergic synaptic transmission in the central nervous system (CNS), a function conserved from worms to humans. The insect nAChR is a pentamer of α subunits, or a heteromer of α and β subunits and 10 subunits have been reported in Drosophila. Vast diversity is generated through different subunit assembly, RNA editing and alternative splicing. Thousands of subtle and noticeable pharmacological and electrophysiologically diverse receptors could be assembled. In insects, nAChR’s are targets of insecticides used to control pests. Chapter two describes work on the characterization of nAChR subunit genes in the central nervous system (CNS) of an embryo and larval D. melanogaster stages through in-situ hybridization, Fluorescent in-situ hybridization (FISH) and enhancer studies. Expression of 7 of the nAChR subunits (Dα1, Dα2, Dα3, Dα5, Dα6, Dα7 and Dβ2) was observed in CNS of embryo and larval CNS tissues. Beside the CNS, expression was also observed in other tissues, such as the ring glands (Dα1, Dα5, Dα7 and Dβ2) suggesting a role for these in the developmental biology of Drosophila. Salivary gland expression was observed for Dα7 subunit while larval fat body and adult hemolymph expression was observed for the Dβ3 subunit gene suggesting novel roles for these nAChR subunits. Building on the expression of these individual nAChR subunits, co-localization was also observed for Dα1/Dα2 and Dα1/Dβ2 subunit genes in larval CNS using FISH. In the third chapter a new approach was taken using RNAi as a tool for predicting insecticide resistance before it happens and finding new insecticide targets. Ten of the nAChR subunit genes were knocked-down using RNAi lines in the CNS of Drosophila. Results suggest that Dα6 is the only subunit targeted by the insecticide spinosad. Also individual knockdown of the Dα1 and Dβ3 subunits show significant sensitivity to spinosad, suggesting some form of compensation mechanism for these nAChR subunits. Conclusions from this work were that RNAi is an excellent tool in Drosophila (due to the availability of RNAi lines) in predicting resistance to insecticides, and prior testing of compounds could assist with better management of resistance development in insect pest species as insecticide targets are commonly conserved among insect species. The fourth chapter examines a negative cross-resistance of spinosad and nitenpyram resistant strains and by using mixtures of these insecticides to detect possible synergistic interactions. Negative cross-resistance was confirmed in earlier studies carried out by T. Perry (2005); a nitenpyram resistant mutant Dα1ems1 was observed to be sensitive to spinosad and a spinosad resistant mutant Dα6ems6 showed sensitivity to nitenpyram insecticide. My work using a number of mixture ratios found significant synergism between nitenpyram and spinosad insecticides at a ratio of 75 to 1. This synergistic ratio was found to be effective against the target site resistant mutants of nitenpyram and spinosad and also against a metabolic resistance mechanism to nitenpyram, indicating that mixtures can overcome both metabolic and target site resistances. My discussion chapter (Chapter 5) evaluates the expression studies and possible functions associated with the expression patterns observed in a particular tissues/life stage of Drosophila. It examines the advantages of some of our techniques such as RNAi as a fast method of predicting resistance. The implications of the negative cross-resistance relationship between spinosad and nitenpyram insecticides and the use of these two in mixtures are discussed with reference to resistance management and touches on future directions and ideas of practical implications of this in the field.