Genetics - Theses
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ItemThe genetic and morphological characterisation of the zebrafish intestinal mutant setebos(University of Melbourne, 2010)Colorectal cancer (CRC) arises by mutation of tumour suppressors and oncogenes that deregulate the genetic programmes that control intestinal turnover and homeostasis. The zebrafish intestinal epithelium shares extensive phenotypic and genetic homology with the mammalian intestine, providing a genetically tractable model organism for the discovery and characterisation of novel genes required for intestinal development. Increased knowledge of the genetic control of intestinal development may reveal new therapeutic avenues for the treatment of CRC. The Liverplus mutagenesis screen identified zebrafish embryos defective in endoderm organ morphogenesis, including the intestinal mutant setebos, described herein. This study reports the positional cloning of the setebos mutant locus, identifying a premature stop codon as the responsible mutation in the gene nucleolar protein 8 (nol8). Microinjection of wildtype nol8 mRNA rescued setebos developmental abnormalities whereas morpholino oligonucleotide-mediated knockdown of Nol8 phenocopied setebos morphology in wildtype embryos, providing functional proof that nol8 is the mutated locus underlying the setebos phenotype. This study demonstrated that Nol8 is essential for zebrafish survival and that nol8 mRNA is widely expressed in actively proliferating tissues of the zebrafish embryo at 72 hours post fertilisation (hpf), when developmental abnormalities in setebos mutants first become apparent. Northern blot analyses revealed that setebos mutants accumulate an intermediate ribosomal RNA (rRNA) orthologous to mammalian 36S at the expense of downstream 32S rRNA; defects predicted to result in reduced biogenesis of 28S rRNA. Bioanalyzer analyses confirm that setebos mutants are preferentially impaired for 28S biogenesis. 28S, 5.8S and 18S rRNA molecules combine with ribosomal proteins to form functional ribosomes, which are essential for translation and cellular growth. Defects in ribosome biogenesis have been shown to activate the tumour suppressor Tp53, resulting in cell cycle arrest. Tp53 is activated in the setebos mutant, resulting in upregulation of pro-cell cycle arrest genes, including the N-terminally truncated isoform of Tp53, ?113tp53. While this provides a mechanism for the hypoplasia observed in the tissues of the setebos embryo, including the intestinal epithelium, craniofacial cartilages, eyes, liver and pancreas, inactivation of Tp53 signalling in the setebos mutant failed to obviously alter setebos morphology. Nevertheless, activation of Tp53 signalling was found to significantly extend survival of setebos embryos, identifying an important biological role for Tp53 in cells undergoing ribosomal stress. Multiple human syndromes have been linked to abnormal ribosome biogenesis (ribosomopathies), and several predispose to cancer. Zebrafish haploinsufficient for 17 different ribosomal proteins also display increased cancer susceptibility (Amsterdam et al. 2004; Lai et al. 2009). Tp53 is inactivated in these zebrafish tumours, suggesting that inactivation of Tp53 may be a key event in tumour formation in these animals. In summary, setebos provides an ideal in vivo model to further characterise the link between defective ribosome biogenesis, Tp53 signalling and cancer. Since disruption of Nol8 function preferentially impairs the growth of several highly proliferative embryonic zebrafish tissues in the absence of Tp53 signalling, inhibition of Nol8 may provide a therapeutic avenue for the treatment of Tp53 mutant tumours.
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ItemStructure(University of Melbourne, 2010)MLK2 is a serine/threonine kinase with a N-terminal SH3 domain and a centrally located dual leucine zipper region that defines the mixed lineage kinase family. Motifs for interaction with the small Rho-like GTPases, 14-3-3 signalling adaptor proteins, as well as for nuclear import and export have been identified in MLK2 and other MLK family members, providing the capacity for a large number of diverse interactions. MLK2 extracted from over-expressing cells is catalytically active and long-term over-expression is lethal. A study of the contribution of the SH3 domain, catalytic domain and dual leucine zipper region to the overall activity of MLK2 was undertaken. This investigation utilised targeted mutagenesis to alter the coding sequence of conserved amino acids to produce non-functional and constitutively active protein domains. The SH3 domain contributes to the regulation of catalytic activity by binding within the protein C-terminal domain. Once released from auto-inhibition, the SH3 domain interacts with dynamin and is capable of activating dynamin I GTPase activity, thereby increasing the rates of dynamin mediated endocytosis. Both auto-inhibition and dynamin activation is ablated by substitution of a praline residue within the conserved binding cleft of the SH3 domain. Dim�risation via the dual leucine zipper region is required to achieve full catalytic activity. Substitution within the catalytic domain produced a kinase-inactive protein that was used throughout the study. In addition, this inactive variant also demonstrated the requirement for phosphorylation of the activation loop prior to an increase in catalytic activity. Membrane localisation of MLK2 was demonstrated to be independent of catalytic activity and SH3 domain or leucine zipper binding capacity. MLK2 is a member of the MAPKKK family capable of activating the c-jun N terminal kinases via MKK4 and MKK7. The use of kinase negative MKK4 confirmed the ability of MLK2 to signal to JNK through an alternative MKK protein. Kinase negative MLK2 was a target for JNK phosphorylation producing a pattern of phosphopeptide TLC-TLE spots with commonalities to autophosphorylated MLK2. Phosphorylation was concentrated within the C-terminal half of the protein where no large functional domains have been identified but a number of smaller motifs are found. Retrophosphorylation was proposed to have a positive effect on MLK2, sustaining activity and signalling that ultimately leads to apoptosis. Stable expression of GFP tagged MLK2 was only achieved with kinase inactive or truncated protein coding constructs, confirming the lethal effect of over-expressing active MLK2. Kinase negative MLK2 expression decreased cell doubling rates, increased the number of fine membrane projections and trafficked to the nucleus in a cell-cycle dependent manner. The CRIB domain allowed for interaction between Cdc42, Rad and MLK2. MLK2 was proposed to influence cytoskeletal remodelling in response to Cdc42 and Rad activation leading to stimulation of the JNK pathway. Finally, MLK2 was has been shown to have a role in the progression of the neurodegenerative pathology of Alzheimer�s disease. Stimulation of the JNK pathway, coupled with the ability to mediate receptor activation by modulation of rates of endocytosis theoretically places MLK2 in a key position in the response to excitory amino acid excitotoxicity, in Huntington�s disease, and to apoER2 activation in Alzheimer s disease. The characterisation of the contributions made by SH3 domain, catalytic domain and dual leucine zipper region to the overall activity of MLK2 will allow for the design and optimisation of novel therapeutic targets aimed at reducing MLK2 activity, opening up new scope for disease management.
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ItemThe roles of a novel group of plastid transporters in glutathione metabolism in arabidopsis(University of Melbourne, 2010)The tripeptide glutathione (GSH) is an important compound with a broad spectrum of functions in plants, including stress responses and is a key element of the intracellular redox buffer that protects cells from oxidation. In Arabidopsis GSH is synthesized in two enzymatic steps by GSH1, found exclusively in the plastids, forming the pathway intermediate ?-glutamylcysteine (?-EC) and then by GSH2 which is located in both plastids and cytosol. This suggests a mechanism for ?-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is believed that the cytosol may be the main compartment for GSH biosynthesis. In addition, GSH may be efficiently transported between the two compartments to maintain proper homeostasis. However, comparatively little is understood about the mechanisms that control and interconnect these two subcellular GSH pools. The identification and characterization of a novel family of transporters, the CRT Like Transporters (CLTs), has provided further insights into glutathione metabolism in Arabidopsis. The CLT1 gene was originally identified in a screen for mutants resistant to L-Buthionine-SR-sulfoximine (BSO), a synthetic compound used to specifically inhibit GSH1 and GSH biosynthesis. Two paralogues in Arabidopsis, CLT2 and CLT3, were identified and all three CLTs are localized to the plastids. Previous studies have shown the clt1clt2clt3 triple mutant is GSH-deficient in roots and cadmium-sensitive suggesting a role for the CLTs in transporting ?-EC and/or GSH between the plastid and cytosol compartments. In this study redirecting GSH1 activity or GSH biosynthesis exclusively to the cytosol by complementing the Arabidopsis gsh1 and gsh2 mutants with E. coli GSHA and GSHB, respectively, has no significant impact on phenotypes or stress resistance suggesting efficient exchange of ?-EC and GSH between the plastid and cytosol compartments. The use of redox-sensitive roGFP2 reporters in combination with confocal imaging with monochlorobimane and measurements of cytosolic thiols has demonstrated that the clt1clt2clt3 mutant is GSH-deficient in the cytosol but not the plastid in both roots and shoots consistent with a predicted role for CLTs in ?-EC and/or GSH transport. The Arabidopsis gsh2 mutant accumulates high levels of ?-EC in the cytosol. However, the combination of the clt1clt2clt3 mutations with gsh2 did not decrease ?-EC levels suggesting CLTs may not contribute to ?-EC transport in vivo. To further study the roles of the CLTs separately on ?-EC and GSH transport in planta different clt mutations were introduced into transgenic lines expressing plastid- or cytosol-targeted GSH2. These observations suggest that de novo GSH biosynthesis in wildtype Arabidopsis occurs largely in the plastids and that CLT2 and CLT1/CLT3 are mainly involved in ?-EC and GSH export, respectively, in planta.
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ItemInvestigating the mechanisms of zinc homeostasis in Arabidopsis thaliana(University of Melbourne, 2010)Zn is an element essential for all forms of life, and either Zn-deficiency or excess Zn can have serious biological consequences. In plants, Zn levels are controlled by a number of physiological and cellular mechanisms to maintain appropriate physiological concentrations of Zn. This study further characterises the HMA2 and HMA4 transporters by developing a novel technique using the Zn fluorophore Zinpyr-1. A procedure for the use of Zinpyr-1 in Arabidopsis roots was developed and validated which allowed the visualisation and relative quantification of Zn. This technique showed that both hma4 and hma2/hma4 mutants differed in the amount of Zn and its distribution compared to wildtype or hma2. This correlates to previous data showing that hma4 and hma2/hma4 are Zn-deficient in shoots but accumulate Zn in roots. Thus we can say that this Zn- deficiency in shoot occurs due to an inability to load Zn into the xylem for transport to the shoot. To investigate the regulation of Zn-homeostasis, Zn-deficient and hma2/hma4 plants were compared to wildtype in micro-array experiments to test whether plants respond systemically to Zn- deficiency. hma2/hma4 plants are Zn-deficient in shoots but over-accumulate Zn in roots, so genes co-regulated in hma2/hma4 roots and shoots, and genes co-regulated in hma2/hma4 roots and Zn- deficient wildtype roots were identified. 52 and 27 genes were identified in these comparisons respectively as regulated >2-fold in the same direction. These were considered robust as their co-regulation was observed in four measurements, two technical replicates of two biological replicates. promoter
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ItemPopulation genetics and ecology of the lucerne flea, sminthurus viridis, with applications for control(University of Melbourne, 2010)The lucerne flea, Sminthurus viridis (Collembola: Sminthuridae), is a major pest of broadacre grains and pasture throughout southern Australia. Current control methods for this pest rely heavily on the prophylactic use of broad spectrum pesticides and are unlikely to be sustainable. Development of an integrated pest management (IPM) strategy is needed. However, this requires a greater understanding of the biology, ecology and genetics of S. viridis. This thesis aims to address this by examining the population genetics and life-cycle of this pest, as well as investigating chemical and biological control methods. Laboratory pesticide bioassays were used to compare the response of S. viridis with the redlegged earth mite (Halotydeus destructor) to six broad spectrum pesticides. Sminthurus viridis displayed significantly higher pesticides tolerance than H. destructor and the tolerance differences were greater for the synthetic pyrethroids than the organophosphorous pesticides. This indicates that application rates for S. viridis should be greater than H. destructor and that organophosphates are likely to provide the most effective control of this pest. Seasonal abundance patterns and summer diapause response of S. viridis in south-eastern Australia were investigated using field and shade-house experiments. The seasonal activity agreed with previous studies and parameters predicting autumn emergence were defined. A summer-diapausing egg stage was also confirmed, with diapause eggs accumulating across the season. These results suggest autumn emergence of S. viridis can be predicted for target control strategies, but late-season spraying strategies may be difficult to implement. The species status, mode of reproduction and population genetics of S. viridis were examined using microsatellite markers I developed, together with allozymes and a mitochondrial DNA marker. A single, sexually reproducing species was found with limited gene flow between Australian populations. Genetic similarity between some distant populations was also shown, indicating that wind or human-mediated long-distance dispersal occurs. These findings suggest that management strategies may be more effective when locally developed and need to consider the effects of human- mediated dispersal. The impact of the predatory mite, Bdellodes lapidaria, for controlling S. viridis and its susceptibility to several pesticides were assessed using laboratory and field experiments. Bdellodes lapidaria was more susceptible to the synthetic pyrethroids in the laboratory, but was not affected by pesticide applications in the field. In addition, no detectable predator-prey relationship was found between these species. These results indicate that B. lapidaria is not strongly susceptible to pesticides in the field and may not be an effective predator of S. viridis. Overall, this thesis contributes important knowledge of the pesticide tolerance, life- cycle, population genetics and biological control of S. viridis. These findings highlight ways to improve the effectiveness and sustainability of control methods and bring closer the development of an integrated pest management strategy for S. viridis.
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ItemPopulation genetics and ecology of the lucerne flea, Sminthurus viridis, with applications for control( 2010)The lucerne flea, Sminthurus viridis (Collembola: Sminthuridae), is a major pest of broadacre grains and pasture throughout southern Australia. Current control methods for this pest rely heavily on the prophylactic use of broad spectrum pesticides and are unlikely to be sustainable. Development of an integrated pest management (IPM) strategy is needed. However, this requires a greater understanding of the biology, ecology and genetics of S. viridis. This thesis aims to address this by examining the population genetics and life-cycle of this pest, as well as investigating chemical and biological control methods. Laboratory pesticide bioassays were used to compare the response of S. viridis with the redlegged earth mite (Halotydeus destructor) to six broad spectrum pesticides. Sminthurus viridis displayed significantly higher pesticides tolerance than H. destructor and the tolerance differences were greater for the synthetic pyrethroids than the organophosphorous pesticides. This indicates that application rates for S. viridis should be greater than H. destructor and that organophosphates are likely to provide the most effective control of this pest. Seasonal abundance patterns and summer diapause response of S. viridis in southeastern Australia were investigated using field and shade-house experiments. The seasonal activity agreed with previous studies and parameters predicting autumn emergence were defined. A summer-diapausing egg stage was also confirmed, with diapause eggs accumulating across the season. These results suggest autumn emergence of S. viridis can be predicted for target control strategies, but late-season spraying strategies may be difficult to implement. The species status, mode of reproduction and population genetics of S. viridis were examined using microsatellite markers I developed, together with allozymes and a mitochondrial DNA marker. A single, sexually reproducing species was found with limited gene flow between Australian populations. Genetic similarity between some distant populations was also shown, indicating that wind or human-mediated longdistance dispersal occurs. These findings suggest that management strategies may be more effective when locally developed and need to consider the effects of humanmediated dispersal. The impact of the predatory mite, Bdellodes lapidaria, for controlling S. viridis and its susceptibility to several pesticides were assessed using laboratory and field experiments. Bdellodes lapidaria was more susceptible to the synthetic pyrethroids in the laboratory, but was not affected by pesticide applications in the field. In addition, no detectable predator-prey relationship was found between these species. These results indicate that B. lapidaria is not strongly susceptible to pesticides in the field and may not be an effective predator of S. viridis. Overall, this thesis contributes important knowledge of the pesticide tolerance, lifecycle, population genetics and biological control of S. viridis. These findings highlight ways to improve the effectiveness and sustainability of control methods and bring closer the development of an integrated pest management strategy for S. viridis.
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ItemNo Preview AvailableRoles of nicotinic acetylcholine receptors in development, viability, and insecticide response, in Drosophila melanogaster( 2021)The majority of excitatory neurotransmission in the insect brain occurs via nicotinic acetylcholine receptors (nAChRs), however knowledge of which nAChR subunits may be required in specific neurons is virtually absent. The ubiquity of nAChRs in the insect brain also makes them ideal molecular targets for many neuroactive insecticides. Mutations in specific nAChR subunits can confer high levels of resistance, but the fitness costs that may be associated with resistance alleles or with low-dose insecticide exposure are not fully understood. This thesis examines two Drosophila nAChR subunits with the most severe loss of function phenotypes, with a focus on their endogenous functions and their roles in insecticide response. Chapter Two investigates the role of the Da5 subunit in larval development. Preliminary observations had indicated that loss of Da5 causes larval mortality and is associated with precocious wandering and moulting phenotypes. Here, these phenotypes are quantified and found to be associated with loss of ecdysis triggering hormone. Cell types requiring Da5 were also narrowed-down to potentially include the prothoracic gland cells, or the neurons innervating the prothoracic gland. In insecticide exposure assays, loss of Da5 was shown to not confer altered response to spinosad, suggesting that Da5 may not be contributing to the primary spinosad target. In Chapter Three, loss of the Db1 subunit is shown to result in pleiotropic consequences, including severely shortened longevity, reduced male courtship, limited locomotion, and unsuccessful wing expansion. Since wing expansion is controlled by a well-characterised hormone (bursicon) in a small and well-defined subset of neurons (the CCAP neurons), this phenotype was examined further. Removal of Db1 specifically from CCAP neurons using somatic CRISPR was sufficient to disrupt wing expansion and loss of Db1 was shown to cause loss of the hormone bursicon. Together, these experiments identify CCAP neurons as a specific subset requiring Db1 for normal function. Chapter Four extends findings from the previous chapter, by testing whether alternative Db1 alleles, and a non-Drosophila b1 subunit orthologue, can rescue the Db1 loss of function phenotypes. Db1 cDNA rescue constructs containing the amino acid replacements R81T (found in resistant populations of aphids), and R81Q (naturally occurring in insensitive arachnids), were both found to rescue all elements of the Db1 phenotype, but only R81Q was found to confer high levels of imidacloprid resistance. The aphid subunit Mpb1 was also able to rescue loss of Db1, and fully restored sensitivity to imidacloprid, revealing substantial functional conservation between the b1 subunits in these two species. This work provides a platform for studying resistance-conferring amino acid replacements in pest nAChR subunits within the Drosophila model. Understanding the endogenous roles of nAChR subunits will be essential for characterising the function of every pathway in the insect brain. By characterising the roles of Da5 and Db1, this thesis provides great insight into the fitness costs insects may endure when evolving insecticide resistance. It also reveals the developmental and behavioural pathways that may be affected when pest and non-pest species are chronically exposed to the low doses of insecticides that contaminate the environment.
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ItemIn vivo functional characterization of nicotinic acetylcholine receptors in Drosophila melanogaster( 2018)Nicotinic acetylcholine receptors (nAChRs) are responsible for fast excitatory synaptic transmission in insect central nervous system. Their role as targets for commercial insecticides have resulted in extensive studies on their structure and pharmacological properties. However, many other aspects of their fundamental biology remain less understood. For example, what behaviours are underpinned by the activity of nicotinic acetylcholine receptors? Here, we used reverse genetics to address this question. The precise genome editing power of CRISPR/Cas9 technology was used to generate a collection of Drosophila melanogaster lines harbouring precise genomic deletions of the genes of interest, including the subunits for the nicotinic acetylcholine receptors as well as a couple of their accessory proteins. The overall strategy was to remove as much as of the genomic locus as possible by having two sgRNAs directing Cas9 to cut at the 5’ and 3’ ends of the gene’s coding sequence and relying on non-homologous end joining repair to ligate the termini together creating a deletion. In total, nine knockout strains were generated for four genes, successfully removing genomic sequences ranging from 4 to 83kb in length. For three genes, Dα4, Dα6 and DmRIC3, the same allele was recapitulated for three backgrounds. The role of nAChRs in regulating sleep behaviour in vinegar flies was investigated using null alleles of the receptor subunits. For seven of the ten subunits, flies harbouring null alleles were viable as adults for behavioural assays. All mutants showed changes in total sleep amount compared to their controls, which most strongly correlated with changes in sleep episode duration. Additionally, genotypes carrying partial deletions or point mutations displayed different sleep changes, suggesting that allelic variation within subunits can yield different phenotypes. These data confirmed a role in sleep regulation for most nAChR subunits. Furthermore, the role of the nAchR accessory proteins were considered. Lines with a deletion of the nAChR-specific chaperone DmRIC3 responded to two commercial insecticides in similar manner to loss of the subunit Dα1. Those lines also phenocopied sleep behaviour of flies lacking receptor subunits. This is the first in vivo evidence of the functional significance of DmRIC3 to nAChRs in D. melanogaster. Altogether, these results show that significant behavioural changes might be considerable fitness costs beyond viability for resistant alleles of genes with important functions in the central nervous system such as nAChRs. However, resistance could still arise from disruption to other proteins interacting and regulating nAChRs with less severe costs.
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ItemPopulation genomics and transcriptomics in the cotton bollworm, Helicoverpa armigera( 2018)Helicoverpa armigera is an agricultural pest that causes billions of dollars' worth of damage each year. As H. armigera has evolved resistance to insecticides, an understanding of resistance genes will provide useful insights into managing this pest. One approach to identify candidate genes is to scan the genome for signs of strong and recent selective sweeps. This extends the search beyond typical candidate genes (detoxifying enzymes and molecular targets) although a limitation of the approach is that the selective agent causing a sweep may not be an insecticide. Another approach is to compare the differences between lab-selected and unselected cohorts. Genes that are differentially expressed are good candidates for further investigation. Here, I present estimates of some baseline parameters such as nucleotide diversity and the extent of linkage disequilibrium to lay a foundation for detecting selective sweeps in H. armigera, and I identify a gene exhibiting the hallmarks of strong and recent selection. I also present some preliminary findings from an analysis of differentially-expressed genes between selected and unselected cohorts of H. armigera in response to a pyrethroid insecticide.
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ItemUnderstanding aphids: transcriptomics, molecular evolution and pest control( 2018)Aphids exhibit fascinating biological features including parthenogenesis, symbiosis, altruism and host-plant preference; all of which would be better understood if genetic tools and molecular biological techniques were applied to them. Aphids are also agricultural pests that vector plant viruses and new approaches to control them are required. This thesis addresses questions motivated by an interest in the biology of aphids and a desire to improve the agricultural impact of aphids. It does so through transcriptomic analyses and RNA interference (RNAi) technology. I examined the ways in which the transcriptome of aphid changes with host-plant, between tissues, within species and between species. The three-aphid species studied (the green peach aphid: Myzus persicae, the mustard aphid: Lipaphis erysimi, and the cabbage aphid: Brevicoryne brassicae) are all pests of economically important brassica crops (such as cabbage, cauliflower, mustard and canola). These data may provide insights into the way different aphid species deal with plant secondary compounds such as glucosinolates. These data also allowed me to examine the structure, function and evolution of myrosinase enzymes that have allowed some aphid species to develop an anti-predator ‘mustard bomb’. RNAi has been suggested as a way to specifically target pest that would be more ‘environmentally friendly’ than conventional insecticides. I experimentally assessed the feasibility of orally-delivered RNAi to control aphids and the potential of this technology to be developed as a functional genomic tool. RNAi was fed to aphids via artificial diets at various concentrations and with various delivery agents and via transgenic Arabidopsis thaliana plants that I created that produced dsRNA’s corresponding to aphid genes. These studies lead me to suggest that more work needs to be done to limit the effects of RNase enzymes of the aphid gut digesting orally delivered RNAi and to more carefully characterize factors that may affect within-species variation in RNAi efficacy.
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