Genetics - Theses
Permanent URI for this collection
Search Results
1 - 10 of 74
-
ItemStudies on minor non-metrical skeletal variants in the mouse and man(University of Melbourne, 1970)This thesis consists of papers presenting the results of studies on the genetical, developmental and anthropological aspects of minor non-metric al variants in man and the house mouse. The work is mainly on variants of the skeleton, particularly the cranium, but includes a limited discussion of published data on minor non-metrical variants of the muscular and vascular systems. Each study is based on a number of variants, and, where applicable, single measures have been obtained to express the overall difference in skeletal variability between populations or the overall effect on skeletal variability of certain environmental factors. Investigations into the role of genotype and environment in the determination of minor skeletal variants in mice and man indicate that most of them are under some genetic control but that maternal physiology and other non-genetic factors may influence the frequency of individual variants. Data presented here (Publication 1) on 25 minor skeletal variants in inbred strains of mice and their hybrids suggest that genotype is more important than environment in determining skeletal variability. Although the frequency of a few individual variants was found to be significantly affected by certain non-genetic factors, when many variants were considered together the environment had no overall significant effect. In contrast, large differences, due mainly to genetic factors, were observed between inbred strains and hybrids. Further studies on inbred strains of mice and hybrids (Publication 2) indicate that stabilizing mechanisms operate during the formation of the skeleton. For most of the 29 bilateral minor non-metrical variants studied , the frequency of asymmetrical mice (i.e., those with the variant present on only one side) was less than expected on the assumption that the number of mice with the variant present on both, one or neither sides depends solely on the frequency of the variant on each side. This tendency for the development of the skeleton to be canalized against asymmetry has been described as a form of morphogenetic homeostasis. The same phenomenon has been observed for bilateral minor non-metrical variants in man (Publication 3) for the skeletal, muscular and vascular systems (based on data published by Danforth in 1924) and for the skeletal system of Australian Aborigines. Studies on inbred strains of mice (e.g., Publication l) indicate that genotype plays the major role in determining the frequency of minor non-metrical variants. If these findings can be extrapolated to man, minor non-metrical variants may be of use in anthropological work. A general survey of skeletal variation, based on 30 such variants, was carried out on Aboriginal crania from many parts of Australia (Publication 4). Regional differences in the pattern of cranial morphology were observed which appear to culminate in two extreme populations: one in the north and north-west of the continent, the other in south-eastern Australia. These results were considered in relation to some current theories on the origin and ethnic composition of the Australian Aborigines.
-
ItemCellular characterisation of the insulin-regulated aminopeptidase (IRAP) in the brain(University of Melbourne, 2006)Central infusions of AT4 ligands, including Angiotensin IV and LW-Hemorphin 7, enhance memory acquisition, retention and retrieval in fear-conditioned memory tasks and spatial learning. A single acute dose of an AT4 ligand is also sufficient to reverse memory deficits caused by scopolamine treatment, perforant pathway lesions and other forms of experimentally induced amnesia models. The robust effects of these ligands on learning and memory processes are thought to be mediated by binding to the catalytic site of the insulin regulated aminopeptidase (IRAP). I RAP is also found in muscle and adipose cells, where the enzyme is found colocalised with GLUT4, the insulin regulated glucose transporter, in specialised vesicles. In response to insulin stimulation, IRAP traffics with GLUT4 from an intracellular location to the plasma membrane, from where GLUT4 mediates insulin regulated glucose uptake. The N-terminal domain of IRAP is believed to interact with multiple proteins to regulate the intracellular tethering, trafficking and possibly recycling of these vesicles. It is unknown how AT4 ligands, via binding to the catalytic site of IRAP, promote such robust memory enhancing effects. One hypothesis is based on the role of IRAP in regulating the trafficking of GLUT4 containing vesicles in muscle and adipose, suggesting that the enzyme may be present in the brain in an analogous system. In this way, modulation of IRAP activity by centrally administered AT4 ligands may result in enhanced glucose uptake into neurones. This hypothesis is supported by the findings that exogenous glucose administration promotes an equally wide range of cognition enhancing effects as demonstrated for AT4 ligands. Therefore to investigate whether IRAP location and function in the brain is analogous to that characterised in muscle and adipose cells, the studies described in this thesis aimed to: 1) Map and characterise the cellular expression of IRAP in the rodent brain 2) Determine the subcellular localisation of IRAP in neurones 3) Determine if IRAP is associated with the facilitative glucose transporter in neurones as it is in muscle and adipose 4) Investigate if IRAP may be involved in neuronal glucose uptake Using a highly specific in-house antibody raised against the unique intracellular tail of IRAP, the studies in this thesis utilised an immunohistochemical approach to characterise IRAP in the brain. Firstly, IRAP immunoreactivity was visualised in specific nuclei throughout the brain. In particular, IRAP was highly expressed in cognitive associated areas, such as the medial septum, cerebral cortex and hippocampus. IRAP immunoreactivity was also abundant in many motor and motor associated nuclei. Dual label immunohistochemistry demonstrated IRAP was exclusively expressed in neurones and was partially associated with cholinergic neurones and their projection, supporting a role for IRAP in cognitive processes. The pattern of IRAP immunoreactivity within neurones was punctate and vesicular, throughout the cell soma and extending into proximal dendrites, indicating a predominantly post-synaptic localisation. At a subcellular level, IRAP was localised to vesicles containing VAMP2, but not small synaptic vesicles at nerve terminals. A clear overlap between IRAP and markers of the Trans Golgi network and endosomal membranes was evident, although IRAP localisation was not limited to these membranes. These findings were confirmed by electron microscopy studies, that visualised IRAP specific electron-dense precipitate associated with neurosecretory vesicles and cisterns of the rough endoplasmic reticulum and golgi apparatus. In summary, these findings demonstrated the subcellular location of I RAP in neurones is analogous to that characterised in muscle and adipose. The association between IRAP expression and the different facilitative glucose transporters present in the brain was examined. IRAP immunoreactivity was found to coincide specifically with GLUT4, such that in some brain regions the subcellular localisation of I RAP and GLUT4 appeared to overlap completely. Three patterns of co-localisation were mapped and quantitated with a high degree of co-localisation noted in cognitive associated nuclei, moderate co-localisation in motor associated nuclei while low co-localisation was noted in the hypothalamus and cerebellum. In areas of high co-localisation I RAP and GLUT4 appear to be localised to the same intracellular vesicles. This subcellular co-localisation is suggestive of a role for I RAP in regulating GLUT4 activity in cognitive associated regions such as the hippocampus. Finally the role of IRAP in neuronal glucose uptake was investigated by testing the effect of AT4 ligands on hippocampal glucose uptake. Both Ang IV and LVV-H7 significantly enhanced stimulated glucose uptake specifically in areas where I RAP and GLUT4 are co-localized. Furthermore, glucose uptake was visualised to occur in the pyramidal neurones that contain colocalised I RAP and GLUT4. These results strongly suggest a role for I RAP in neuronal glucose uptake. Taken together, these studies have demonstrated that IRAP is present in the brain in localisation analogous to that found in muscle and adipose. The presence of IRAP in vesicles with the regulated glucose transporter, GLUT4, in neurones involved in cognitive processing suggests I RAP and GLUT4 may mediate inducible glucose uptake in neurones, possibly in response to heightened energy demand in neurones. Therefore the hypothesis investigated in this thesis is confirmed. Additionally these results suggest a potential molecular mechanism that may underlie the cognition enhancing effects of AT4 ligands.
-
ItemCellular characterisation of the insulin-regulated aminopeptidase (IRAP) in the brain(University of Melbourne, 2006)Central infusions of AT4 ligands, including Angiotensin IV and LW-Hemorphin 7, enhance memory acquisition, retention and retrieval in fear-conditioned memory tasks and spatial learning. A single acute dose of an AT4 ligand is also sufficient to reverse memory deficits caused by scopolamine treatment, perforant pathway lesions and other forms of experimentally induced amnesia models. The robust effects of these ligands on learning and memory processes are thought to be mediated by binding to the catalytic site of the insulin regulated aminopeptidase (IRAP). I RAP is also found in muscle and adipose cells, where the enzyme is found colocalised with GLUT4, the insulin regulated glucose transporter, in specialised vesicles. In response to insulin stimulation, IRAP traffics with GLUT4 from an intracellular location to the plasma membrane, from where GLUT4 mediates insulin regulated glucose uptake. The N-terminal domain of IRAP is believed to interact with multiple proteins to regulate the intracellular tethering, trafficking and possibly recycling of these vesicles. It is unknown how AT4 ligands, via binding to the catalytic site of IRAP, promote such robust memory enhancing effects. One hypothesis is based on the role of IRAP in regulating the trafficking of GLUT4 containing vesicles in muscle and adipose, suggesting that the enzyme may be present in the brain in an analogous system. In this way, modulation of IRAP activity by centrally administered AT4 ligands may result in enhanced glucose uptake into neurones. This hypothesis is supported by the findings that exogenous glucose administration promotes an equally wide range of cognition enhancing effects as demonstrated for AT4 ligands. Therefore to investigate whether IRAP location and function in the brain is analogous to that characterised in muscle and adipose cells, the studies described in this thesis aimed to: 1) Map and characterise the cellular expression of IRAP in the rodent brain 2) Determine the subcellular localisation of IRAP in neurones 3) Determine if IRAP is associated with the facilitative glucose transporter in neurones as it is in muscle and adipose 4) Investigate if IRAP may be involved in neuronal glucose uptake Using a highly specific in-house antibody raised against the unique intracellular tail of IRAP, the studies in this thesis utilised an immunohistochemical approach to characterise IRAP in the brain. Firstly, IRAP immunoreactivity was visualised in specific nuclei throughout the brain. In particular, IRAP was highly expressed in cognitive associated areas, such as the medial septum, cerebral cortex and hippocampus. IRAP immunoreactivity was also abundant in many motor and motor associated nuclei. Dual label immunohistochemistry demonstrated IRAP was exclusively expressed in neurones and was partially associated with cholinergic neurones and their projection, supporting a role for IRAP in cognitive processes. The pattern of IRAP immunoreactivity within neurones was punctate and vesicular, throughout the cell soma and extending into proximal dendrites, indicating a predominantly post-synaptic localisation. At a subcellular level, IRAP was localised to vesicles containing VAMP2, but not small synaptic vesicles at nerve terminals. A clear overlap between IRAP and markers of the Trans Golgi network and endosomal membranes was evident, although IRAP localisation was not limited to these membranes. These findings were confirmed by electron microscopy studies, that visualised IRAP specific electron-dense precipitate associated with neurosecretory vesicles and cisterns of the rough endoplasmic reticulum and golgi apparatus. In summary, these findings demonstrated the subcellular location of I RAP in neurones is analogous to that characterised in muscle and adipose. The association between IRAP expression and the different facilitative glucose transporters present in the brain was examined. IRAP immunoreactivity was found to coincide specifically with GLUT4, such that in some brain regions the subcellular localisation of I RAP and GLUT4 appeared to overlap completely. Three patterns of co-localisation were mapped and quantitated with a high degree of co-localisation noted in cognitive associated nuclei, moderate co-localisation in motor associated nuclei while low co-localisation was noted in the hypothalamus and cerebellum. In areas of high co-localisation I RAP and GLUT4 appear to be localised to the same intracellular vesicles. This subcellular co-localisation is suggestive of a role for I RAP in regulating GLUT4 activity in cognitive associated regions such as the hippocampus. Finally the role of IRAP in neuronal glucose uptake was investigated by testing the effect of AT4 ligands on hippocampal glucose uptake. Both Ang IV and LVV-H7 significantly enhanced stimulated glucose uptake specifically in areas where I RAP and GLUT4 are co-localized. Furthermore, glucose uptake was visualised to occur in the pyramidal neurones that contain colocalised I RAP and GLUT4. These results strongly suggest a role for I RAP in neuronal glucose uptake. Taken together, these studies have demonstrated that IRAP is present in the brain in localisation analogous to that found in muscle and adipose. The presence of IRAP in vesicles with the regulated glucose transporter, GLUT4, in neurones involved in cognitive processing suggests I RAP and GLUT4 may mediate inducible glucose uptake in neurones, possibly in response to heightened energy demand in neurones. Therefore the hypothesis investigated in this thesis is confirmed. Additionally these results suggest a potential molecular mechanism that may underlie the cognition enhancing effects of AT4 ligands.
-
ItemSexual segregation in kangaroos : testing alternative evolutionary hypotheses(University of Melbourne, 2006)Males and females of many animal species live apart for most of the year, coming together only for mating. The causes of this sexual segregation are still debated, with most research being focused on ungulates. However, if we are to develop robust and inclusive explanations for this phenomenon we need better coverage of other vertebrate taxa. In this thesis, I investigate the occurrence of sexual segregation in Australian marsupials, and describe and quantify this phenomenon in western grey kangaroos, Macropus fuliginosus, and red kangaroos, M rufus, at Hattah-Kulkyne National Park in north-western Victoria, Australia. Exploiting some of the unique life-history characteristics of kangaroos, which are ecological analogues of ungulates, I test a number of hypotheses that have been proposed to account for the evolution of sexual segregation in ungulates. A literature review revealed that sexual segregation occurs in some Australian marsupials, as it does in some eutherians, but is most pronounced among the larger macropods, as it amongst their eutherian counterparts, the ungulates. Sexual segregation appears to be associated with body size dimorphism and birth synchrony in marsupials, as it is in ungulates: all species that exhibited sexual segregation were either dimorphic in body size, highly synchronous in breeding, or both. Species that were monomorphic, and gave birth year-round or only moderately synchronously, did not exhibit segregation. Sexual segregation occurs at the social, spatial and habitat level in western grey and red kangaroos, and the degree of social and spatial segregation is greater than that of habitat segregation in both species. The magnitude and timing of sexual segregation in these species is influenced by the synchrony and timing of their mating activity. In the synchronously breeding western grey kangaroo, segregation peaks during autumn, when females are in lactational anoestrus. In red kangaroos, which mate year round, segregation is weaker but occurs throughout the year. Intraspecific variation in body size in kangaroos, particularly amongst males, allows predictions of the four main hypotheses for sexual segregation (predation risk, forage selection, activity budget and social factors) to be tested independently of each other. I found that, in accordance with the predation risk hypothesis, female western grey kangaroos made greater use of secure habitats, large males made greater use of nutrient-rich, open habitat, while small males were intermediate between females and large males in their habitat use. Consistent with the social factors hypothesis, same-sex groups composed of females only, and males of both small and large body size, predominated. The activity budget hypothesis is an unlikely explanation for social segregation in western grey kangaroos since two assumptions of this hypothesis were not supported by my results. First, differences in body size did not result in activity budget differences, and consequently activity asynchrony: individuals of different body size did not have different transition times between resting and feeding, sex- size classes were not least synchronized when in mixed-body size groups, and synchrony was not lower in mixed-body size than same-body size groups. Second, activity asynchrony did not cause mixed-body size groups to be less cohesive than same-body size groups. Although mixed-body size groups were fused for less time than same-body size groups, synchrony was not correlated with group stability. An examination of the fission/fusion dynamics of groups of western grey kangaroos confirmed that the social factors hypothesis, and more specifically, social affinity among males, best explains social segregation in western grey kangaroos. As predicted by the male social affinity hypothesis, large males joined groups composed of other males more frequently than expected. This study contributes significantly to our understanding of the behavioural ecology of kangaroos, and more generally, identifies key factors involved in the evolution of sexual segregation in vertebrates.
-
ItemSexual segregation in kangaroos : testing alternative evolutionary hypotheses(University of Melbourne, 2006)Males and females of many animal species live apart for most of the year, coming together only for mating. The causes of this sexual segregation are still debated, with most research being focused on ungulates. However, if we are to develop robust and inclusive explanations for this phenomenon we need better coverage of other vertebrate taxa. In this thesis, I investigate the occurrence of sexual segregation in Australian marsupials, and describe and quantify this phenomenon in western grey kangaroos, Macropus fuliginosus, and red kangaroos, M rufus, at Hattah-Kulkyne National Park in north-western Victoria, Australia. Exploiting some of the unique life-history characteristics of kangaroos, which are ecological analogues of ungulates, I test a number of hypotheses that have been proposed to account for the evolution of sexual segregation in ungulates. A literature review revealed that sexual segregation occurs in some Australian marsupials, as it does in some eutherians, but is most pronounced among the larger macropods, as it amongst their eutherian counterparts, the ungulates. Sexual segregation appears to be associated with body size dimorphism and birth synchrony in marsupials, as it is in ungulates: all species that exhibited sexual segregation were either dimorphic in body size, highly synchronous in breeding, or both. Species that were monomorphic, and gave birth year-round or only moderately synchronously, did not exhibit segregation. Sexual segregation occurs at the social, spatial and habitat level in western grey and red kangaroos, and the degree of social and spatial segregation is greater than that of habitat segregation in both species. The magnitude and timing of sexual segregation in these species is influenced by the synchrony and timing of their mating activity. In the synchronously breeding western grey kangaroo, segregation peaks during autumn, when females are in lactational anoestrus. In red kangaroos, which mate year round, segregation is weaker but occurs throughout the year. Intraspecific variation in body size in kangaroos, particularly amongst males, allows predictions of the four main hypotheses for sexual segregation (predation risk, forage selection, activity budget and social factors) to be tested independently of each other. I found that, in accordance with the predation risk hypothesis, female western grey kangaroos made greater use of secure habitats, large males made greater use of nutrient-rich, open habitat, while small males were intermediate between females and large males in their habitat use. Consistent with the social factors hypothesis, same-sex groups composed of females only, and males of both small and large body size, predominated. The activity budget hypothesis is an unlikely explanation for social segregation in western grey kangaroos since two assumptions of this hypothesis were not supported by my results. First, differences in body size did not result in activity budget differences, and consequently activity asynchrony: individuals of different body size did not have different transition times between resting and feeding, sex- size classes were not least synchronized when in mixed-body size groups, and synchrony was not lower in mixed-body size than same-body size groups. Second, activity asynchrony did not cause mixed-body size groups to be less cohesive than same-body size groups. Although mixed-body size groups were fused for less time than same-body size groups, synchrony was not correlated with group stability. An examination of the fission/fusion dynamics of groups of western grey kangaroos confirmed that the social factors hypothesis, and more specifically, social affinity among males, best explains social segregation in western grey kangaroos. As predicted by the male social affinity hypothesis, large males joined groups composed of other males more frequently than expected. This study contributes significantly to our understanding of the behavioural ecology of kangaroos, and more generally, identifies key factors involved in the evolution of sexual segregation in vertebrates.
-
ItemNitrogen utilization and its regulation in Aspergillus nidulans(University of Melbourne, 2006)The filamentous fungus Aspergillus nidulans is able to utilize a wide range of compounds as sources of nitrogen. The presence of preferred nitrogen sources (ammonium and glutamine) signals nitrogen sufficiency, and genes required for the utilization of alternative sources are not expressed. In the absence of preferred nitrogen sources regulatory proteins activate expression of these genes. This constitutes nitrogen metabolite repression, and ensures the most efficient use of available nitrogen. In A.nidulans nitrogen metabolite repression is mediated by AreA, a positively acting GATA transcription factor. This thesis describes the investigation of two genes whose expression is subject to nitrogen metabolite repression and controlled by AreA, and the characterization of a fourth AMT/MEP gene in A. nidulans. areA102 is a specific mutation of the areA gene which results in a protein with altered promoter binding specificity, and areA102 mutants grow more strongly on a range of amino acids as nitrogen sources. Mutations at the sarA locus were first isolated as suppressors of the strong growth of an areA102 strain on histidine. In this study the sarA gene was characterized and confirmed to encode an L-amino acid oxidase (LAO) with broad substrate specificity. A sarA gene inactivation abolished LAO activity and suppressed the areA102 phenotype on histidine. An areA102 mutant was found to have increased utilization and stronger growth on amino acids which are LAO substrates. Investigation of sarA dependent and independent amino acid catabolism further defined the substrate specificity of LAO, and the contribution of other catabolic pathways was assessed. In A.nidulans the LAO was found to be the sole pathway for the catabolism of some amino acids, while for others this enzyme represents only a minor pathway. A.nidulans is known to possess four ammonium permeases differentially regulated by AreA. meaA encodes a low affinity ammonium transporter responsible for the majority of ammonium uptake. mepA has been shown to encode a high affinity permease which scavenges low concentrations of ammonium during nitrogen limitation, and mepB encodes a second high affinity permease only expressed during nitrogen starvation. To confirm the role of the fourth permease in ammonium acquisition, the mepC gene was cloned and its DNA and protein sequences analysed. The MepC ammonium transporter motifs differed somewhat from the consensus, and topology predictions indicated that mepC was more structurally divergent than the other A.nidulans ammonium transporters. The mepC gene was inactivated and the deletion strain was found to be indistinguishable from wildtype and from meaA, mepA, and mepB single, double and triple deletion backgrounds. However, strains over-expressing mepC from the highly inducible xylP promoter were able to partially complement the poor growth of an meaA?; mepA?; mepB? strain, indicating that MepC is capable of transporting ammonium. In other fungal systems certain ammonium permeases act as sensors of cellular nitrogen status. MeaA, MepA, and MepB do not act as nitrogen sensors. To determine whether MepC played a role in nitrogen sensing, amdS
-
ItemNitrogen utilization and its regulation in Aspergillus nidulans(University of Melbourne, 2006)The filamentous fungus Aspergillus nidulans is able to utilize a wide range of compounds as sources of nitrogen. The presence of preferred nitrogen sources (ammonium and glutamine) signals nitrogen sufficiency, and genes required for the utilization of alternative sources are not expressed. In the absence of preferred nitrogen sources regulatory proteins activate expression of these genes. This constitutes nitrogen metabolite repression, and ensures the most efficient use of available nitrogen. In A.nidulans nitrogen metabolite repression is mediated by AreA, a positively acting GATA transcription factor. This thesis describes the investigation of two genes whose expression is subject to nitrogen metabolite repression and controlled by AreA, and the characterization of a fourth AMT/MEP gene in A. nidulans. areA102 is a specific mutation of the areA gene which results in a protein with altered promoter binding specificity, and areA102 mutants grow more strongly on a range of amino acids as nitrogen sources. Mutations at the sarA locus were first isolated as suppressors of the strong growth of an areA102 strain on histidine. In this study the sarA gene was characterized and confirmed to encode an L-amino acid oxidase (LAO) with broad substrate specificity. A sarA gene inactivation abolished LAO activity and suppressed the areA102 phenotype on histidine. An areA102 mutant was found to have increased utilization and stronger growth on amino acids which are LAO substrates. Investigation of sarA dependent and independent amino acid catabolism further defined the substrate specificity of LAO, and the contribution of other catabolic pathways was assessed. In A.nidulans the LAO was found to be the sole pathway for the catabolism of some amino acids, while for others this enzyme represents only a minor pathway. A.nidulans is known to possess four ammonium permeases differentially regulated by AreA. meaA encodes a low affinity ammonium transporter responsible for the majority of ammonium uptake. mepA has been shown to encode a high affinity permease which scavenges low concentrations of ammonium during nitrogen limitation, and mepB encodes a second high affinity permease only expressed during nitrogen starvation. To confirm the role of the fourth permease in ammonium acquisition, the mepC gene was cloned and its DNA and protein sequences analysed. The MepC ammonium transporter motifs differed somewhat from the consensus, and topology predictions indicated that mepC was more structurally divergent than the other A.nidulans ammonium transporters. The mepC gene was inactivated and the deletion strain was found to be indistinguishable from wildtype and from meaA, mepA, and mepB single, double and triple deletion backgrounds. However, strains over-expressing mepC from the highly inducible xylP promoter were able to partially complement the poor growth of an meaA?; mepA?; mepB? strain, indicating that MepC is capable of transporting ammonium. In other fungal systems certain ammonium permeases act as sensors of cellular nitrogen status. MeaA, MepA, and MepB do not act as nitrogen sensors. To determine whether MepC played a role in nitrogen sensing, amdS
-
ItemMolecular and genetic analysis of Bt and spinosad resistance in diamondback moth, Plutella xylostella(University of Melbourne, 2005)Diamondback moth, Plutella xylostella, is a destructive global pest of nutritional and economically important cruciferous plants. Over the last 50 years, field strains have developed resistance to all major classes of insecticides used against them, including Bacillus thuringiensis (Bt) toxins and spinosyns. Molecular and genetic analysis of Bt resistance Diamondback moth is the only insect to develop open field resistance to Bt toxins. Many other insect species have been selected for Bt resistance under laboratory conditions, and in three lepidopteran pests, resistance has been genetically associated with mutations in a CrylA-binding midgut cadherin protein. Here, diamondback moth strains SCI and NO-QA were analysed. Both are resistant to CrylAa, CrylAb, CrylAc, CryIF and CrylJ however SCI was also resistant to Cry1C. AFLP linkage mapping was used to identify twenty-nine of the expected 31 linkage groups in SCI. CrylA resistance was significantly associated with a single linkage group (LG22 ?2=15.6, dfi=1, P<0.0001) and Cry 1C resistance was associated with two others (?2 =6.3 and 4.8, df=1). This data confirms different genetic mechanisms are responsible for CrylAc and CrylC resistance in this strain. Genes for lepidopteran Bt binding proteins were identified and mapped in SCI, including the 12-domain midgut cadherin, aminopeptidase N, alkaline phosphatase, glycosyl transferase, and P252-like genes, however none mapped to CrylAc or CrylC resistance associated linkage groups. Molecular analysis and interstrain complementation tests for allelism show mutations in the same genes are responsible for CrylAc resistance in SCl and the second resistant strain, NO-QA, Comparative genomics identified seven ribosomal genes that mapped to the CrylAc resistance linkage. Where possible, these single copy anchor loci were used to construct linkage maps for NO-QA and SCI. In NO-QA, 9 AFLP markers clustered around the Cry 1 Ac resistance locus were identified using bulked segregant analysis. Sequencing and inverse PCR were carried out on two of these AFLP markers and a PCR based resistance detection assay designed from one of these markers. Molecular and genetic analysis of spinosad resistance Spinosad is a novel class of insecticides that primarily targets nACh Receptors. The mechanism for field evolved spinosad resistance in diamondback moth strain Pearl-Sel (18,600 fold resistant) is hypothesised to be a single, recessive, autosomal gene. AFLP linkage mapping identified 28 linkage groups including one autosome responsible for spinosad resistance (?2=21.1, df=1, P<0.0001). Degenerate PCR identified five nAChR genes, two of which mapped to the resistance linkage group (nAChR a7-1 and a7-2). A linkage map of the resistance linkage group positioned nAChR ?7-1 more than 30 Haldane centimorgans (cM) and nAChR ?7-2 only 4.2 cM from the theoretical resistance locus, SpR- 1.
-
ItemNo Preview AvailableCopper homeostasis and the Alzheimer's disease amyloid precursor protein(University of Melbourne, 2005)The transition metal copper is both essential for normal cellular function and potentially highly toxic, thus it is critical that all living organisms, from bacteria to humans, have developed copper homeostasis mechanisms. Alzheimer's disease is characterised by the accumulation of amyloid-? peptide, which is cleaved from the copper-binding Amyloid Precursor Protein. The normal biological function of the Amyloid Precursor Protein is poorly understood. Amyloid Precursor Protein is a member of a multi-gene family, including Amyloid Precursor like Proteins-1 and -2. The copper-binding domain is similar among Amyloid Precursor Protein family members, suggesting an overall conservation in its function or activity. This study investigated the hypothesis that the Amyloid Precursor Protein functions in copper homeostasis and that copper levels may regulate Amyloid precursor protein gene expression. To investigate the hypothesis that Amyloid Precursor Protein has a functional role in cellular copper homeostasis, mammalian cell culture systems in both neuronal and non-neuronal cells with varied Amyloid Precursor Protein expression were characterised for copper transport utilising a radio-copper assay. Over-expression of Amyloid Precursor Protein increased intracellular copper accumulation in non-neuronal and neuronal mammalian cells. Studies utilising mouse primary neuronal cortical cultures, demonstrated a gene dosage-dependent effect of Amyloid Precursor Protein expression on increasing cellular copper levels in cells that have a genetic ablation of Amyloid Precursor-like Protein-2 expression. In contrast, overexpression of the Swedish mutant of Amyloid Precursor Protein in primary cortical neurons reduces cellular copper levels. These findings provide evidence for the role of Amyloid Precursor Protein in neuronal copper homeostasis as a copper detoxification and/or efflux protein. To investigate the hypothesis that copper may regulate Amyloid Precursor Protein gene expression, a novel cell culture system was utilised. In this system, intracellular copper levels were genetically manipulated through altered expression of the Menkes protein, a major mammalian copper efflux protein. Cells lacking the Menkes protein show high intracellular copper levels due to reduced copper efflux, while restoration of Menkes protein function by over-expression restores copper efflux ability, resulting in dramatically decreased intracellular copper levels. Data presented in this study show that depletion of intracellular copper results in significantly reduced Amyloid Precursor Protein levels and a significant reduction in Amyloid Precursor Protein gene expression. In addition. Amyloid Precursor Protein promoter analysis suggests that putative metal regulatory elements, in the region -490 to +104 of the Amyloid Precursor Protein promoter, may be involved in mediating the response to copper depletion to regulate Amyloid Precursor Protein gene expression. This demonstrates a previously uncharacterised aspect of human Amyloid Precursor Protein gene regulation and supports the hypothesis that copper can regulate Amyloid Precursor Protein gene expression. Furthermore, these data support a role for the Amyloid Precursor Protein in copper homeostasis as a copper detoxification and/or efflux protein. Overall, this thesis presents strong evidence for the role of the Amyloid Precursor Protein in copper detoxification and/or efflux and the role of copper in the regulation of Amyloid Precursor Protein gene expression. Characterisation of copper homeostasis mechanisms of Amyloid Precursor Protein in copper detoxification and/or efflux and the elucidation of the copper-regulation mechanisms of the Amyloid Precursor Protein gene may provide potential therapeutic targets towards the treatment of Alzheimer's disease.
-
ItemGenomics of insecticide resistance and xenobiotic metabolism in helicoverpa armigera(University of Melbourne, 2005)Helicoverpa armigera is a polyphagous lepidopteran that causes severe agricultural losses globally and it has a great capacity to develop resistance to insecticides. Previous research has shown that metabolic based resistance is the major cause of pyrethroid resistance in Australia and both cytochrome P450 monooxygenases and carboxylesterases have been implicated. AN02, a field strain of H. armigera exhibits a 50 fold resistance to fenvalerate in the larval stage. Resistance can be almost completely synergised by a P450 inhibitor, piperonyl butoxide, suggesting that resistance is metabolic based and likely to be mediated by P450 enzymes. The research presented in this thesis utilised two techniques, cDNA-AFLP and cDNA microarrays to identify differentially expressed genes between fenvalerate resistant and susceptible backcross progeny from an AN02 heterozygous resistant female and susceptible male. A novel cytochrome P450, CYP337B1 was found to be constitutively overexpressed in resistant individuals, and was mapped to within 1 cM of the resistance locus RFen1. Two other P450s which have previously been identified to be overexpressed in fenvalerate resistant H. armigera in Australia, CYP4G8 and CYP6B7 were also overexpressed in AN02, as were two glutathione S-transferases and one carboxylesterase. H. armigera larvae were also challenged with different xenobiotics; piperonyl butoxide, phenobarbital, esfenvalerate and jasmonic acid. Induction was studied in three separate tissues, integument, fatbody and midgut. Suites of inducible genes were identified, which may play a role in detoxifying these compounds. Notably, the three cytochrome P450s identified as constitutively upregulated in AN02 (CYP337B1, CYP4G8 and CYP6B7) were among the genes induced by esfenvalerate.