Genetics - Theses

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    Studies on minor non-metrical skeletal variants in the mouse and man
    Kellock, Wendy Lorraine. (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.
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    The mammalian stonin proteins : from translation to degradation
    Wall, Adam Alexander. (University of Melbourne, 2009)
    Recent work has centred around the role of stonin 2 in clathrin-mediated endocytosis (CME) of the integral synaptic vesicle protein synaptotagmin I. Without a mode of internalisation of this protein, synaptic vesicles that are formed from the plasma membrane are fusion-incompetent and therefore no neurotransmitter can be released (Mohrmann, 2008). Stonin 2 was first identified on the basis of homology with the Stoned B gene of Drosophila melanogaster. The stoned B protein is encoded in the second open reading frame (ORF) of a dicistronic transcript that encodes another protein involved at the synapse, named stoned A. It was in 1973 that the locus was first identified in a screen for temperature sensitive paralytic mutants. These mutants were shown to have defective nervous systems and had aberrant synaptic vesicle retrieval at the plasma membrane of the neuromuscular junction (NMJ). Like stonin 2, stoned B interacts with synaptotagmin I through its C-terminal domain that shows significant homology to the ?2 subunit of the clathrin adaptor protein complex AP-2. Stonin 2 is not the only stoned B homologue in mammalian genomes, with another protein named stonin 1 showing equal protein similarity. In this report, the stonin 1 antibodies that had previously been utilised (Arnott 2004) were re-characterised and it was concluded that they were only useful to detect expressed recombinant protein. Affinity purified stonin 2 antibodies, created in this project, were successfully utilised to detect both endogenous stonin 2 and recombinant expressed-tagged stonin 2 for both Western blot and immunocytochemistry, which required a novel antibody purification method to be created to achieve a greater resolution. After analysis of expressed tagged stonin proteins in the neuroendocrine PC12 cell line, it was suspected that both proteins were being rapidly degraded. This led to the discovery that both stonin 1 and 2 were being targeted for degradation by the ubiquitin proteasome system that appears to be neuroendocrine specific. Stonin 2 is additionally cleaved by an unknown protease to produce C-terminal fragments that are the predominant protein species in PC12 cells. The analysis of the stonin 2 protein fragmentation pattern compared to the endogenous banding pattern led to the discovery that protein variation is also achieved through initiation of translation at internal start sites. Although the specific methionine residues were not characterised, at least two protein species were identified that were the result of internal initiation. These protein species may be regulated through the use of a short upstream open reading frame that overlaps the predicted stonin 2 ORF start codon. The internal start sites for translation initiation would give rise to N-terminally truncated protein species that would lack one or two critical AP-2 binding motifs that may modulate the function of stonin 2. The final series of experiments presented in this thesis analyse the localisation and protein-protein interactions of the stonin proteins through immunocytochemical analysis of exogenously expressed-tagged stonin proteins during inhibition of the ubiquitin proteasome system. This lead to the finding that when stonin 1 is bound to internal membranes it is predominantly bound to internal structures that can be motile. Under these conditions, it was also shown that stonin 1 could bind synaptotagmin I, although it is unlikely to occur at the plasma membrane. Membrane-bound Stonin 2 is found predominantly sub-cortically or at the plasma membrane. The sub-cortical localisation may be at sites that vesicles are, or will be, attached to the plasma membrane. Stonin 2 can be part of a protein complex that involves AP-2, synaptotagmin I and the fission molecule dynamin I. These results provide new insight into the role that both stonin 1 and 2 play in the cellular trafficking network. The protein variation identified in Stonin 2 adds further complexity to the myriad of regulatory events that can occur to modulate synaptic plasticity.
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    Characterisation of putative fatty acyl-CoA synthetases and genes involved in �-oxidation in Aspergillus nidulans
    Reiser, Kathrin. (University of Melbourne, 2009)
    Aspergillus nidulans is able to use short and long chain fatty acids as sole carbon and energy sources via the 13-oxidation pathway. This pathway occurs in both peroxisomes and mitochondria of A. nidulans. While various genes encoding the mitochondrial 13- oxidation enzymes are known, the genes encoding the peroxisomal ?-oxidation proteins have not been fully described. To investigate the first step of peroxisomal 13-oxidation two putative fatty acyl-CoA dehydrogenases, AcdA and AcdB, and two putative fatty acyl-CoA oxidases, AoxA and AoxB, were identified in the genome by blast search using the Saccharomyces cerevisiae fatty acyl-CoA oxidase FoxA and a Neurospora crassa fatty acyl-CoA dehydrogenase. The localisation of these proteins, the induction of the genes and the gene deletion phenotypes have been characterised to assess their possible involvement in ?-oxidation. The results have shown that AoxA is the major fatty acyl-CoA oxidase for peroxisomal long chain fatty acid utilisation, however, the leaky loss-of-growth phenotype of the aoxA? strain implies that there are additional peroxisomal ?-oxidation pathways. The induction of the four putative fatty acyl-CoA dehydrogenases and fatty acyl-CoA oxidases in response to fatty acids was shown to be dependent on the transcriptional regulators FarA required for the induction by short and long chain fatty acids and FarB and ScfA required for short chain fatty acid induction. This pattern of induction was observed for aoxA, while acdA, acdB and aoxB show a previously undescribed induction pattern with FarB and ScfA being required for short chain and long chain fatty acid induction and FarA for short chain fatty acid induction only. The occurrence of two induction patterns implies a more complex regulation by the three regulatory proteins. Six putative fatty acyl-CoA synthetases, FatA, FatB, FatC, FatD, FaaA and FaaB, have been identified using the S. cerevisiae fatty acyl-CoA transporters and synthetases Fat1, Fat2, Faa1, Faa2, Faa3 and Faa4. Investigation of the localisation of these proteins and phenotypes associated with the deletion of these genes showed that FaaB is likely to be the major peroxisomal fatty acyl-CoA synthetase and activates fatty acids with a wide range of chain lengths. The effect of deletion of the genes encoding these putative fatty acyl-CoA synthetases on the regulation of aoxA and acuJ was investigated to determine whether any of the putative fatty acyl-CoA synthetases were required to generate activated fatty acids for induction. It is clear that there are multiple genes contributing to fatty acid utilisation and that there is considerable redundancy. Additional genome and phylogenetics analyses have identified a variety of additional proteins, which may be involved in peroxisomal ?-oxidation pathways, as well as a potential mitochondrial fatty acyl-CoA synthetase (AN4659.3). This protein is predicted to localise to mitochondria, contains a short chain fatty acyl-CoA synthetase motif and is likely to be a synthetase activating short chain fatty acids, which are utilised in mitochondria.
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    Regulation of gluconeogenesis in aspergillus nidulans
    Suzuki, Yumi. (University of Melbourne, 2009)
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    Novel roles of cytochrome P450 genes in insect development
    Sztal, Tamar Esther. (University of Melbourne, 2009)
    Cytochrome P450s form one of the largest enzyme superfamilies. They are involved in the catalysis of a number of different chemical reactions and are involved in a wide range of biological processes. In the vinegar fly, Drosophila melanogaster, 85 P450s have been identified. This thesis uses genetic approaches to investigate the roles of specific P450s in different aspects of D. melanogaster development and reproduction. The expression patterns of all 85 P450s in the D. melanogaster y ; cn, bw; sp strain were analysed by RT-PCR and in situ hybridisation during embryogenesis. In situ hybridisation patterns were obtained from 50 out of 65 P450s expressed during embryogenesis. Many novel expression patterns were identified; Cyp28c1 was detected in the salivary glands, Cyp49a1 was detected in the hindgut and Cyp18a1 was detected in the epidermis. These results were compared with larval in situ hybridisation patterns of P450s previously characterised, to identify P450s with conserved expression throughout the life-stage. An RNAi screen was also performed. Of the 60 P450s targeted by RNAi, nine were shown to be lethal (Cyp18a1, Cyp28c1, Cyp306a1, Cyp309a1, Cyp311a1, Cyp314a1, Cyp4c3, Cyp4d2, Cyp4g1, Cyp6g2) and three showed reduced survival (Cyp318a1, Cyp4ac2, Cyp4s3). The function of Cyp301a1 was also investigated using in situ hybridisation and RNAi. Cyp301a1 was detected in the hindgut and epidermis in D. melanogaster y; cn, bw; sp embryonic and adult stages. RNAi knockdown of Cyp301a1 as well as analysis of the Cyp301a1f02301 mutant flies showed disrupted cuticle phenotypes, suggesting that Cyp301a1 has a role in cuticle formation. Cyp6g2 was the only P450 detected in the corpus allatum, the site of juvenile hormone synthesis. RNAi knockdown of Cyp6g2 is lethal at pupal stages and lethality can be rescued by over-expression of D. willistoni Cyp6g2. Cyp6g2 appears to be unique to the Drosophila genus and is highly conserved in all twelve sequenced Drosophila genomes. Cyp6g2 expression fluctuates dynamically during larval and pupal development and is roughly correlated with high JH titres. Knockdown of Cyp6g2 disrupts the expression of other genes known to be regulated by JH, suggesting that Cyp6g2 may have an essential role in juvenile hormone regulation. Interestingly, Cyp6g2 is also expressed in the adult male ejaculatory bulb and was differentially expressed during mating, indicating an additional reproductive role. The evolution of the Cyp307a genes in Drosophila was investigated. The Cyp307a genes are involved the synthesis of 20-hydroxyecdysone, an essential insect moulting hormone. In D. melanogaster, there are two Cyp307a genes; Cyp307a1, which was detected in the yolk cells of the early embryo and in the follicle cells of the female ovary and Cyp307a2, which was detected in the prothoracic cells of the ring gland. Phylogenetic and microsynteny analyses in Drosophila showed a complicated evolutionary scenario for the Cyp307a genes, invoking multiple gene duplications (and subsequent losses) in different lineages. The varied temporal and spatial expression of the Cyp307a genes in Drosophila suggested that they have undergone independent evolution and sub-functionalisation within the Drosophila genus. Finally, this thesis investigated roles of cytochrome P450s in reproduction. Using in situ hybridisation, several P450s were detected in the adult female reproductive tissues. Cyp6a19 and Cyp6a22 were highly expressed in the nurse cells whereas Cyp18a1 was detected in the follicle cells of the developing oocyte. Cyp305a1 was detected in the follicle cells covering the late stage oocyte and in the dorsal region specifying the appendage. Ubiquitous knockdown of Cyp305a1 by RNAi was lethal, suggesting it plays an essential role during development. RNAi of Cyp305a1 in the follicle cells, showed defects in nurse cell dumping and dorsal appendage formation. Several P450s were also detected in the adult male reproductive system, with Cyp312a1 expressed highly in the developing spermatocytes of the male testes. RNAi knockdown of Cyp312a1 was viable and Cyp312a1 RNAi progeny showed normal testes development. However Cyp312a1 RNAi males were less fertile, which may be due to defects in sperm maturation or protection in the female reproductive tract.
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    Zinc homeostasis in plants : identification and characterisation of new mechanisms
    Tehseen, Muhammad. (University of Melbourne, 2009)
    Zinc (Zn) is an essential element in numerous biochemical and physiological processes in plants, yet at elevated concentrations Zn can be toxic. Plants have a number of membrane Zn transporters of the ZIP, CDF and P-type ATPase classes. The P1B ATPases, HMA2 and HMA4, in Arabidopsis are essential for Zn homeostasis: an hma2; hma4 double mutant is severely Zn-deficient and can be rescued by the application of exogenous Zn (Hussain et al, 2004). This study aims to identify additional components of Zn homeostasis. First, an activation tag T-DNA (Weigel et al., 2000) was used to suppress to hma Zn-deficient phenotype. More than 14,000 transformants were isolated and found three hma2, hma4 double mutant plants showing suppression of the phenotype without Zn supplementation in the soil. Unfortunately, probably due to cross-pollination from other transgenic plants, these turned out to be contaminants containing a different transgene expressing HMA2. A complete inventory of metallochaperone-like proteins containing an HMA domain in Arabidopsis revealed a surprisingly large family of proteins (67). In this family cluster IV is notable because of the presence of conserved Asp (D) residue before the CxxC, metal binding motif, analogous to the Zn binding motif in the E. coli ZntA. Some members of this cluster were studied in more detail. HIPP20, HIPP21, HIPP22, HIPP26 and HIPP27 were able to rescue the Cd-sensitive, ycf1 yeast mutant but failed to rescue the growth of zrt1zrt2, zrc1cot1 and ?atx1 mutants. Characterisation of hipp20/21/22, triple mutant suggests the HIPPs can have a role in Cd-detoxification, probably by binding Cd. The triple mutant was more sensitive to Cd and accumulated less Cd compared to the wild-type. Also, hipp21/26 double mutant exhibited a late-bolting phenotype. Promoter-GUS reporter expression studies indicate variable expression of HIPPs in different tissues. In roots where HIPP26 is only expressed in lateral root tips, HIPP20, HIPP21, HIPP22 and HIPP25 showed strong expression in the root vasculature. Yeast two-hybrid studies indicate that none of the HIPPs tested was able to interact with the N-terminal metal binding domain of HMA2 and HMA4 and a yeast two-hybrid library screen identified a zinc finger protein, At3g62970 that strongly interacts with N-terminal domain of HMA4 but not of HMA2. This protein may have role in HMA4 turn-over via the ubiquitination pathway.
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    Molecular evolution, structure and function of Glutathione S-Transferases in the genus Drosophila
    Low, Wai Yee. (University of Melbourne, 2009)
    GLUTATHIONE S-TRANSFERASES (GSTs) are a superfamily of ubiquitous enzymes that fulfill a variety of biological functions including detoxification. They have been implicated in insecticide resistance and may have evolved in response to toxins in the niche-defining feeding substrates of Drosophila species. In this thesis, a bioinformatic approach was used to identify GST genes of the twelve Drosophila species with recently sequenced genomes. This was followed by comprehensive analyses of their molecular evolution. Gene copy number variation is mainly attributable to unequal crossing over events in the large delta and epsilon clusters. Within these gene clusters there are also GST genes with slowly diverging orthologs. This implies that they have their own unique functions or have spatial/temporal expression patterns that impose significant selective constraints. Searches for positively selected sites within GSTs identified G171K in GSTD1, an enzyme with a promiscuous use of substrates. The same radical substitution (G171K) in GSTD1 has occurred at least three times in the Drosophila radiation. As D. melanogaster GSTD1 can metabolize DDT in a DDTase reaction, the hypothesis that the G171K mutation is associated with DDT selection was tested. Sequencing of historical alleles revealed that the G171K substitution was fixed prior to the use of DDT and so, this insecticide is not the selective agent. Both population genetic and biochemical analyses suggested that the DDTase activity in GSTD1 preceded the use of DDT in the field. In an effort towards probing GSTs binding potential against insecticides and substrates, a general strategy was tested. The strategy entails the following steps: 3D structure determination by X-ray crystallography, in silico molecular docking of candidate insecticide, and in vitro insecticide binding study using NMR spectroscopy. The utility of this approach is investigated with D. melanogaster GSTD1 interaction with DDT. The crystal structure of D. melanogaster GSTD1 has been determined to 1.1 � resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. After removing seven residues of the last helix, DDT was computationally docked into the active site in an orientation favoring catalysis. 2D 15N HSQC NMR experiments of GSTD1 suggest conformational changes occur in GSTD1 upon the binding of glutathione and DDT. Furthermore the 15NH resonances of residues 117 - 121 significantly broaden upon the addition of DDT and thereby support the predicted binding site. The overexpression of D. melanogaster GstD1 has been implicated in DDT resistance, oxidative stress protection and extension of lifespan. However, a study that controls for the genetic background so that the effect of the overexpression of this gene is the sole factor responsible for a phenotype has not been performed. The GAL4/UAS ?C31 integrase system was used to constitutively overexpress D. melanogaster GstD1 and GstD2 at 2 fold and 278 fold, respectively. The paralog, D. melanogaster GstD2 was used because it is not a DDTase and thus, it served as a control for DDTase activity. Probit mortality analysis revealed that the overexpression of D. melanogaster GstD1 did not lead to an increased detoxification of DDT. The overexpression of both D. melanogaster GstD1 and GstD2 resulted in lifespan extension with median survival age of 69 days whereas the control had a median survival age of 52 days. The overexpression of D. melanogaster GstD1 but not GstD2 can lead to an increased survival in the presence of paraquat, which is an oxidative stress generator. The 4-hydroxynonenal conjugating activity of D. melanogaster GstD1 and GstD2 may be the factor responsible for the paraquat resistance and lifespan extension results.
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    Genetic variation in field populations of the cotton bollworm, Helicoverpa armigera (H�bner) (Lepidoptera: Noctuidae)
    Behere, Gajanan T. (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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    A genetic investigation of copper deficiency using Drosophila melanogaster
    Farlow, Ashley Robert. (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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    Investigation of the trafficking and copper-responsive phosphorylation of the Menkes (ATP7A) copper translocating p-type ATPASE
    Veldhuis, Nicholas Andrew. (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.