Burke Honours Projects

Regulation of copper transport and homeostasis

Background:  Copper is an essential yet toxic micronutrient required as a co-factor for numerous vital enzymes. Mutation of the human ATP7A gene results in the lethal, untreatable X-linked disorder Menkes disease. Together with collaborators at the Florey Institute, we have also found members of the Ubiquitin Proteasome System (UPS) that bind and mediate cellular responses to copper. We now wish to identify which UPS genes modify copper import and export activity, with the aim of finding novel targets for therapeutic intervention in Menkes disease.

Project Aim:
To characterise the effect on copper transport and homeostasis of members of the proteasomal and lysosomal protein degradation pathways.

Techniques:  Targeted gene knockdown and overexpression in various Drosophila tissues; in vivo genetic interaction experiments; examination of gene expression patterns and protein localisation in various Drosophila tissues by conventional and confocal fluorescence microscopy; mosaic analysis to examine the phenotypic effects of lethal loss-of-function alleles; molecular cloning and generation of transgenic Drosophila strains; proteomic analysis of ubiquitinated proteins in vivo.

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Animal model of progressive neurodegeneration

Background: Loss of a key E2 ubiquitin conjugase gene in the Drosophila eye has minimal impact on eye development but results in a progressive neurodegenerative phenotype as the adult fly ages. This phenotype is typical of Drosophila models of human neurodegenerative diseases such as Huntingtons, Motor Neurone Disease and Alzheimers. We hypothesise that loss of this ubiquitin conjugase leads to pathogenic protein accumulation, eventually resulting in cell death.

Project Aims:
1) To identify the cellular basis of this phenotype by blocking or enhancing key apoptosis, autophagy, and proteostasis pathways;
2) To identify the E3 ubiquitin ligase gene(s) contributing to this degenerative phenotype and the target substrates dysregulated in the degenerating eye;
3) To screen for compounds capable of slowing or halting this age-dependent deterioration

Techniques: Targeted gene knockdown and overexpression in various Drosophila tissues; in vivo genetic interaction experiments; examination of gene expression patterns and protein localisation in various Drosophila tissues by conventional and confocal fluorescence microscopy; mosaic analysis to examine the phenotypic effects of lethal loss-of-function alleles; molecular cloning and generation of transgenic Drosophila strains; proteomic analysis of ubiquitinated proteins in vivo.

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Maintenance of glial cell function by copper homeostasis

Background:  Copper is well-characterised as a micronutrient essential for neuronal health and development. We have now found that maintaining appropriate copper levels is also needed for the proper function of glia (or astrocytes), the cells that play numerous critical support roles in the central nervous system. This raises the possibility that copper’s known role in human neurodegenerative diseases may extend to glial function.

Project Aims:
1) To determine which glial sub populations require adequate copper levels to remain functional
2) To determine the functional consequences in altered copper import, export and cellular distribution in key glial cell populations.

Techniques:  Targeted gene knockdown and overexpression in various Drosophila tissues; in vivo genetic interaction experiments; examination of gene expression patterns and protein localisation in various Drosophila tissues by conventional and confocal fluorescence microscopy; mosaic analysis to examine the phenotypic effects of lethal loss-of-function alleles; molecular cloning and generation of transgenic Drosophila strains; proteomic analysis of ubiquitinated proteins in vivo.