La myopathie centronucléaire (CNM) est un groupe de maladies génétiques caractérisées au niveau histologique par des noyaux au centre des myofibres au lieu de la périphérie. Des mutations dans trois gènes (MTM1, DNM2 et BIN1) sont associées à cette pathologie. Récemment, l’implication d’un nouveau gène a été révélée dans une myopathie congénitale, le gène PYROXD1. Cependant, la base moléculaire responsable du déséquilibre à l'intérieur de la cellule reste incertaine et la relation entre le niveau histologique et les symptômes chez les patients n'est pas comprise. De plus, aucun traitement n'est disponible pour ces maladies. Au cours de ma thèse, j'ai centré mon travail sur l'utilisation du modèle de levure S. cerevisiae pour comprendre trois protéines associées au CNM : la myotubularine Mtm1, l'oxydoréductase Pyroxd1 et la dynamine Dnm2. Ces données révèlent qu’il est possible d’utiliser une simple cellule eucaryote afin d'élucider certains aspects moléculaires de ces protéines impliquées dans des maladies humaines. / Centronuclear myopathy (CNM) is a group of genetic disorders characterized at the histological level by nuclei at the center of the myofibers instead of the periphery. Mutations in three genes (MTM1, DNM2 and BIN1) are associated with this pathology. Recently the implication of a new gene has been revealed in a congenital myopathy, the PYROXD1 gene.However, the molecular basis responsible for the imbalance inside the cell remains unclear and the relation between the histological level and the symptoms in patients is not understood. Moreover, there is no treatment available for these diseases.During my thesis I have focused my work on using yeast S. cerevisiae model to understand three proteins associated to CNM: the myotubularin Mtm1, the oxidoreductase Pyroxd1 and the dynamin Dnm2. These data reveal that it is possible to use a single eukaryote cell to elucidate some molecular aspects of these proteins implicated in human disorders.

Biotin Protein Ligase (BPL) is an essential enzyme responsible for the covalent attachment of biotin to a specific lysine residue of biotin-dependent carboxylases, transcarboxylases and decarboxylases. Due to the fundamental processes that these enzymes are involved in such as lipogenesis, amino acid catabolism and gluconeogenesis, much research has been conducted on these enzymes. Studies encompassing structural, mutational and catalytic functions of these enzymes have lead to novel drug developments for the treatment of obesity, diabetes, metabolic syndrome, bacterial and fungal infections. As BPL is required for activation of these enzymes by biotinylation, it is believed that it too could be targeted in a similar way to produce novel therapeutics. To date, the most characterised BPLs are from the Gramnegative bacteria Escherichia coli and the archea Pyrococcus hirokoshii. However minimal information is known about other forms of clinically important bacterial species or eukaryotic forms of this important enzyme. Through my candidature I have compiled a thorough literature review summarised as chapter 1: Introduction. Furthering this literature analysis, a human BPL model was generated with aid of BPL structural co-ordinates already deposited in the protein data bank (PDB), thus allowing focus on human BPL mutations that cause multiple carboxylase deficiency (chapter 2). I have solved the structure of BPL from the clinically important pathogenic bacteria Staphylococcus aureus. This was performed in several ligand-bound and non-bound states (chapters 3 and 4). A novel high-throughput assay was developed to test BPL activity. This assay allow testing of compounds that could potentially inhibit the BPL from Candida albicans (a species responsible for invasive fungal infections) (chapter 5). Large amounts of highly purified BPL from Saccharomyces cerevisiae allowed for the first structural analysis of a eukaryotic BPL (Chapter 6). The work has been summarised by a general discussion and future directions for the project (Chapter 7). / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009