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dc.contributor.authorWishart, Thomas Malcolmen
dc.date.accessioned2018-03-29T12:20:58Z
dc.date.available2018-03-29T12:20:58Z
dc.date.issued2006
dc.identifier.urihttp://hdl.handle.net/1842/29425
dc.description.abstracten
dc.description.abstractThe aim of this thesis is to advance our understanding of the mechanisms controlling nerve degeneration, by examining the molecular interactions of the Wldˢ gene and its chimeric protein product. Wallerian degeneration is normally complete within twenty-four to forty-eight hours of axotomy, but in the mouse mutant C57B1/Wldˢ the process is delayed by up to three weeks. The Wldˢ mutation comprises an 85Kb tandem triplication of genomic region containing the complete sequence for Rbp7 (retinoid binding protein 7), Nmnatl (nicotinamide mononucleotide adenyltransferase) and the N-70 amino acids of Ube4b (a ubiquitin ligase). At the boundaries of this in frame tandem triplication, a chimeric "fusion" gene is formed comprising the N-70 Ube4b linked to Nmnat. The protein product localises to nuclei. Transgenic expression of the chimeric gene has previously been shown to be sufficient to reproduce the Wldˢ nerve-degeneration phenotype in both mice and rats.en
dc.description.abstractThe main objectives in my research were; 1. To develop a rapid and effective method for determining the copy number of Wldˢ alleles in mutant and transgenic Wldˢ mouse lines. 2. To map the regional distribution and sub cellular localisation of the Wldˢ chimeric protein product within the nervous system. 3. To test the hypothesis that the expression of the constituent components of the Wldˢ chimeric gene is sufficient to alter mRNA levels of other genes that may themselves be downstream effectors of the Wldˢ neuroprotective phenotype.en
dc.description.abstract1. A rapid and cost effective method for genotyping was developed using quantitative real time PCR on genomic DNA from the spontaneous Wldˢ mutant mouse and transgenic lines. This method allows the determination of Wldˢ copy number (specifically a region of the N10-Ube4b portion of the Wldˢ gene) for genotyping and calculating insertion number in transgenic lines.en
dc.description.abstract2. A highly specific antibody generated against the Wld-18 peptide confirms that the Wlcf protein product is localised to neuronal nuclei. Mapping of the Wldˢ protein product in the CNS showed that the neuronal distribution is not uniform either in the spontaneous mutant or the transgenic models. The intranuclear distribution varied between and within cell types; some neurones showed intense, particulate staining, others showed fine speckling, while others had large nuclear inclusions that varied in shape. Cerebellar granule cells in the Wldˢ mouse have a consistent expression pattern, with about 90% of cells containing at least one large nuclear inclusion of Wldˢ protein. The expression of Wldˢ - containing inclusions in these cells increased with postnatal age in vivo and also in vitro in primary cultures and cell lines. The inclusions persist, evidently without detriment to the animals as they mature. Coimmunostaining and/or co-transfection studies in the HEK293 cell lines suggested that the Wldˢ protein co-localises with certain transcription factors including HDAC5; and members of the SMRT family; and a member of the ubiquitin proteasome system, VCP.en
dc.description.abstract3. Microarray analysis and quantitative real time PCR of Wldˢ compared with wildtype C57B16 mouse cerebellar mRNA indicated differences in expression levels for at least 11 genes outside the Wldˢ locus. Some of these genes were down-regulated, but others were up-regulated. The greatest differences were strong down-regulation of Pttgl and strong up-regulation of a homologue of Edrl. Transfection of human (HEK293) cells with a Wldˢ-eGFP construct mimicked changes shown in Wldˢ mouse mRNA levels. The sub-cellular distribution of Wldˢ protein is dependent on both the Nmnat-1 and Ube4b regions of the protein and both are required to target Wldˢ protein to discrete intranuclear foci. The induced changes in Pttgl mRNA levels, but not the Edrl-like transcript, were partly mimicked by transfecting HEK293 cells with the Nmnat-1 component of the chimeric gene, or by exogenous administration of NAD at a concentration of ImM. This effect was blocked by sirtenol, an inhibitor of the nuclear transcription factor Sirtl. Transfection with the N10-Ube4b component of the Wldˢ gene partly mimicked up-regulation of the Edrllike transcript but had no effect on expression levels of Pttg-1 mRNA. Wldˢ-positive cerebellar granule cells show a protective phenotype, but Pttgl-KO peripheral nerves do not.en
dc.description.abstractTogether, these studies suggest that the Wldˢ gene product functions as a bidirectional regulator of gene expression, driving up and down the transcription of several specific genes via more than one intra-nuclear signalling pathway. The summed effects of these variations in mRNA levels may be required for full expression of the Wldˢ neuroprotective phenotype. Identification of the genes and proteins ultimately responsible may open up new possibilities for understanding and treatment of the consequences of injury to the nervous system, whether induced by trauma or by neurodegenerative disease.en
dc.publisherThe University of Edinburghen
dc.relation.ispartofAnnexe Thesis Digitisation Project 2018 Block 17en
dc.relation.isreferencedbyAlready catalogueden
dc.titleCellular and molecular properties of the neuroprotective Wldˢ geneen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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