Genetics - Theses
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ItemInvestigation of the trafficking and copper-responsive phosphorylation of the Menkes (ATP7A) copper translocating p-type ATPASE(University of Melbourne, 2008)The Menkes copper-translocating P-type ATPase (MNK; ATP7A gene) is a pivotal copper transport protein in copper homeostasis, functioning in systemic copper (Cu) absorption, vectorial copper transport across the blood brain barrier, detoxification via efflux and supply of copper to cuproenzymes in the secretory pathway. MNK is a polytopic protein and the importance of its function is underscored by the fatal neurodegenerative disorder, Menkes Disease, caused by mutations in the ATP7A gene. The function of MNK is regulated by copper-responsive trafficking between the trans-Golgi network (TON) and the plasma membrane. This occurs in most non-hepatic tissues of the body. MNK also undergoes kinase-dependent phosphorylation in a time and copper dose-dependent manner. The precise molecular mechanisms for MNK trafficking remain unknown. The elucidation of mechanisms of trafficking copper-transporting ATPases will ultimately offer therapeutic approaches for regulating Cu levels in many important biomedical contexts. Cu supply to the brain is emerging as an effective treatment for some cases of Alzheimer�s disease; inhibition of copper-dependent angiogenesis in cancer chemotherapy and macular degeneration is needed; and the role of MNK in cisplatin resistance in cancer chemotherapy may be treated by suppression of MNK function. The copper-responsive trafficking of copper-ATPases and its regulation by signalling may also provide an exciting paradigm for understanding molecular interactions involved in controlling function of membrane transport proteins through changes in subcellular localisation. This thesis presents the first identification of phosphorylation sites for a mammalian copper-translocating P(1B)-type ATPase, using cultured CEO cells (non-polarized) and cultured polarised MDCK epithelial as model systems. Eight in vivo phosphorylation sites were identified in cgATP7A protein expressed in cultured cell CEO cells. Twenty one in vivo sites were identified in human MNK expressed in polarized MDCK cells, and included seven homologous cgATP7A sites. All sites are located within N-terminal and C-terminal cytosolic domains and a subset of these are shown to be copper-responsive. At least three of these C-terminal sites (Serines 1463, 1469 and 1480) are essential for copper-responsive post-Golgi trafficking, and specifically, for exocytosis. The loss of phosphorylation mutants S1463A, S1469A and S1480A all disturb the trafficking process. Under copper stimulation the S1463A and S1496A mutants inefficiently exit the TON and become trapped in endosomal compartments. The S1480A mutant also affects trafficking, where confocal fluorescence imaging shows diffuse, vesicular staining. This suggests that Serine 1480 may be required fusion of vesicles with larger endosomal compartments of the cell surface. A model is proposed in which MNK phosphorylation affects multiple trafficking steps in the membrane transport route between the TON and the plasma membrane. These findings provide new insights into the mechanism of Cu-responsive trafficking of MNK and evidence in this thesis also indicates that the same regulatory mechanisms may be at play in the 60% homologous Wilson Cu P-Type ATPase. A relationship between copper homeostasis and cell signalling has been established and in combination with other MNK studies, this demonstrates that intracellular copper delivery and efflux can be influenced by intracellular copper concentrations and exogenously controlled by other signalling components.
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ItemGenetic variation in field populations of the cotton bollworm, Helicoverpa armigera (H�bner) (Lepidoptera: Noctuidae)(University of Melbourne, 2008)Within the noctuid moth genus Helicoverpa, H. armigera from the Old World and H. zea of the New World are considered as the most devastating agricultural pests, while species such as H. assulta and H. punctigera are also significant agricultural pests but are limited geographically and/or in their host plant range. Due to their morphological similarity, these Helicoverpa pest species are only reliably distinguishable through adult genitalia morphologies. Despite inflicting significant annual agricultural and socioeconomic costs associated with crop damage and with the application of pesticides in outbreak control measures, fundamental questions such as the single species status of H. armigera throughout its distribution range, its evolutionary relationship with H. zea, and the population genetic structure of H. armigera have not been adequately addressed. Difficulties in obtaining adequate sample sizes to address single species status and inter-species evolutionary relationships have hindered the progress of basic evolutionary genetic research in Helicoverpa pest species. Furthermore, despite significant efforts invested by various research groups in China and Australia in marker development, poor efficacies of microsatellite DNA markers developed to-date represented a significant underlying factor in the lack of consistent advance in population genetic research on H. armigera. In this study, the biology, ecology, population genetics, and problems associated with microsatellite DNA markers of H. armigera are critically reviewed in Chapter I. Chapter II reports on the single species status of H. armigera and of its relationships with other Helicoverpa pest species including that with the New World H. zea based on the mtDNA barcoding gene Cytochrome Oxidase I (COI). Results from the mtDNA COI phylogeny supported the single species status of H. armigera across its Australian, Asian and African distribution range, while high levels of genetic similarity between H. armigera and H. zea suggested that H. zea evolved from a H. armigera founder event around 1.5 million years ago. Chapter II also reports on the patterns of restriction fragment length polymorphisms (RFLPs) of partial mtDNA COI and Cytochrome b (Cyt b) genes produced by endonucleases BstZ17l and Hphl in the four major Helicoverpa pest species. This provides a rapid species identification PCR-RFLP technique which is highly reliable and effective irrespective of insect life stages, and offers support to conventional taxonomic differentiation based on morphological characters. Chapter III reports molecular characterisation of selected published H. armigera microsatellite DNA loci in order to identify factors affecting marker efficacies. Allele characterisation by DNA sequencing and bioinformatic analyses revealed complicating factors caused by single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) at primer binding sites, large INDELS due to Insertion Sequences, and the presence of microsatellite DNA families that potentially associated with transposable elements (TEs). The development of alternative, exon-primed, intron-crossing (EPIC) PCR markers in H. armigera is also presented in Chapter III. The robustness of the EPIC PCR markers was demonstrated through mapping family studies and cross-species amplification, with their suitability as population genetic markers determined from field H. armigera populations. Preliminary analysis of limited Indian H. armigera populations using EPIC PCR markers provided evidence of heterogeneous population substructure that may reflect the diversity of Indian cropping systems and associated geographic heterogeneity. Results from this preliminary population genetic study identified factors fundamental to the success of future H. armigera population genetic studies relevant to the complex Indian agricultural system.
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ItemA genetic investigation of copper deficiency using Drosophila melanogaster(University of Melbourne, 2008)The phenotypic consequences of copper deficiency are serious and include cardiopathy and neurological dysfunction. These are complex traits that belie simple responsive biomarkers. At present, there are no robust biomarkers for copper deficiency within the human population. This has implications for setting the Recommended Daily Intake (RDI) that will protect most of the population from the consequences of copper deficiency, which various measures place between 0.9mg of copper per day and 3.0mg/day for adult males. The best information available on the copper content of Western diets places a significant proportion of the population below all current RDIs. This incongruity suggests that marginal copper deficiency may be more prevalent than is currently appreciated. Due to the lack of informative biomarkers and the complexity of outcomes in Man, it is appropriate to interrogate the genetics of copper deficiency in order to identify its phenotypic consequences in more tractable model systems, such as Drosophila melanogaster. The aim-set of this investigation is to (1) experimentally generate copper deficiency in Drosophila melanogaster, (2) identify and characterise the phenotypic outcomes of this copper deficiency and (3) identify novel genetic components that respond to copper deficiency. This investigation has identified that the genetic manipulation of DmATP7 elicits a functional copper deficiency. The phenotypic consequences of this are a disruption to development and an adult hypopigmentation of the cuticle. This altered pigmentation has been defined as a valuable indicator of copper status within the fly, and has formed the basis of a forward genetic screen. Malvolio has been identified as an important and functional copper transporter in Drosophila. The significance of this transporter to copper homeostasis, immunity and neuronal function in both insects and humans is considered. This thesis hopes to advance the paradigm of copper homeostasis in Drosophila with the aim of developing a robust definition of copper deficiency within the complex milieu of zinc and iron homeostasis.
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ItemThe potential for evolution in the rainforest restricted species : drosophila bunnanda(University of Melbourne, 2008)Most quantitative genetic studies to date have focused on model and agricultural species and ecological traits unrelated to the distributional limits of species, leading to the evolutionary paradigm that species should not be limited in their ability to adapt to environmental changes by a lack of genetic variation. However whether this assumption can be extended to specialist species is unknown. The overall aim of this study was to investigate the adaptive potential of a rainforest restricted species of Drosophila. Using a large family experiment analyzed with a powerful animal model analysis I found low levels of additive genetic variation and narrow sense heritability (h2 < 0.10) for desiccation resistance in multiple populations of the rainforest restricted Drosophila bunnanda. Despite small standard errors, both additive genetic variation and narrow sense heritability estimates for desiccation resistance were not more than two standard errors from zero suggesting this rainforest restricted species will have difficulty adapting to lower humidity levels. However, additive genetic variation and narrow sense heritability estimates for wing size and wing aspect were significantly different from zero, suggesting genetic variation for quantitative traits is not generally low in this species. Furthermore, high variation and low population differentiation in microsatellite and a mitochondrial gene further indicate population processes, such as small population size or restricted dispersal, are probably not influencing low additive genetic variance for desiccation resistance in D. bunnanda. A comparison of neutral genetic population structure and phylogeographic history between rainforest restricted and generalist species of Drosophila found ecological and geographic distribution does not influence neutral genetic structure. To explore whether the apparent evolutionary limit towards increased desiccation could be overcome, I investigated whether a population bottleneck could increase VA for desiccation resistance in D. bunnanda by creating replicate single pair bottleneck lines. I found both epistatic and dominance components released by population bottleneck increased additive genetic variance and narrow sense heritability to a significant level in some lines, but no selection response was observed.
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ItemThe evolutionary history of Elaeniine tyrannids (Aves; Tyrannidae) : patterns, mechanisms and timing of diversification(University of Melbourne, 2007)The present thesis explores the evolutionary history of Elaeniinae or elaeniid tyrant-flycatchers (Aves; Tyrannidae) in an attempt to unravel the patterns, mechanisms and timing of their extraordinary diversification. The Elaeniinae are a loosely defined subfamily of the tyrant-flycatchers (Tyrannidae), one of the largest but equally least-known bird families. Tyrant-flycatchers are distributed throughout the New World and have diversified into approximately 450 species, most of which share a conservative flycatcher bauplan with an inconspicuous colouration. What tyrant-flycatchers lack in morphological diversity, they make up for in terms of their ecological breadth and the great number of habitats and niches they have adapted to. As such, tyrant- flycatchers are a superb model group to study the speciation mechanisms that have led to the prolific and unparalleled diversity of life forms in the Neotropics. In this thesis, I tackle several discrete questions that surround the evolutionary history of elaeniid flycatchers. In Chapter 1, the phylogenetic position of the Elaeniinae within the tyrant-flycatcher family is explored, since knowledge on their systematic placement and assessment of their monophyletic status is prerequisite to any further evolutionary research. In Chapter 2,1 then provide the temporal framework for the diversification of one elaeniid genus (Myiopagis) by comparing calibrations derived from various molecular clock rates with paleogeographic data. Both Chapters 1 and 2 have important ramifications, since they provide inferences on phylogeny and the timing of diversification in tyrant-flycatchers that are then to be used in subsequent chapters. In Chapter 3,1 employ a comparative approach using molecular and vocal data to investigate the remarkable evolution of the genus Zimmerius. Chapter 4 is the first of three chapters focussed on the genus Elaenia. This chapter looks 'at habitat shifts in the evolution of the genus by reconstructing ancestral ecological parameters. These parameter reconstructions coupled with inferences about the timing of flycatcher evolution from Chapter 2 facilitate the characterisation of the mechanisms that have set Elaenia speciation in motion. Chapter 5 delves into an intriguing case of cryptic speciation in one of the better-known species of Elaenia (E. chiriquensis). I would like to dedicate this chapter to the memory of Paul Coopmans, who sadly passed away on 1 January 2007, and without whom this remarkable case of unrecognised speciation would have remained thus. This chapter draws heavily on the methodology of Chapter 3 in matching patterns of vocal differentiation with those of DNA differentiation. Paul�s sound recordings were instrumental in establishing vocal and molecular congruence. Finally, the thesis concludes with Chapter 6, in which I explore a complicated pattern of multiple mitochondrial and nuclear polyphyly in Elaenia. Using population genetic and phylogenetic methods, I demonstrate that three different factors account for polyphyly in Elaenia: faulty taxonomy, genetic introgression, and incomplete lineage sorting. Besides, differences in population histories of various taxa exacerbate the variability of genetic diversity within some of these polyphyletic lineages. Besides advancing our knowledge of relationships within the elaeniid flycatchers and putting their diversification into a temporal framework, the results of the present research indicate that elaeniid evolution has been closely dictated by geologic, tectonic and climatic events. This thesis also uncovers a surprising amount of cryptic diversity within elaeniid flycatchers and suggests that acoustic traits may be a better indicator of phylogenetic relationships than morphology. Much diversity remains to discovered and described in tyrant-flycatchers, the last big avian parcel of systematic terra incognita.
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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.
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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.
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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.
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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.
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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