Les neurones sont des cellules spécialisées dans différentes fonctions, comme le contrôle des mouvements. La transmission de l’information au sein de l’axone est comparable au trafic de voitures d’une autoroute : les informations transitent dans plusieurs directions avec parfois des bouchons. Lorsque les informations n’arrivent plus à transiter, les patients peuvent souffrir de paraplégies spastiques à caractère héréditaire (PSHs), des maladies neurodégénératives. Cette thèse étudie le rôle joué par la spastine, une protéine impliquée dans certaines PSHs, et responsable de l’entretien des autoroutes. Nos résultats montrent que l’expression d’une forme mutée ou l’absence de production de la spastine,entraîne une perturbation spécifique des routes de transport et du trafic de certains composés essentiels à la survie des neurones. Dans l’ensemble, nos résultats aident à définir es effets pathophysiologies de la spastine afin de développer de nouvelles pistes de traitements. / Central motor neurons are specialized cells involved in the control of voluntary movements in human. The transmission of information along the axon of these neurons is comparable to car trafficking in a highway: information travel in both direction and sometimes traffic jam events occur. When the transit of information are slowed or impaired, patients may suffer of neurodegenerative diseases as hereditary spastic paraplegia (HSPs). In this thesis we investigated the role of spastin, a protein implicated in HSP and the maintenance of these highways. Our results showed that depletion of spastin, or expression of its mutants, lead to a disturbance of both the integrity of these « road » and the traffic of cellular components primary involved in axonal survival and growth. These results would help to elucidate the pathophysiological mechanisms implicated in the onset of this pathology and maybe in developing proficient therapeutic strategy for HSP patients.

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Nitroalkane oxidase (NAO) catalyzes the oxidation of neutral nitroalkanes to aldehydes and ketones with oxygen consumption and the production of hydrogen peroxide and nitrite. The enzyme is a flavoprotein from the fungus Fusarium oxysporum. The active site base, Asp402, abstracts one proton from the substrate to give a carbanion which then attacks the flavin adenine dinucleotide (FAD). The three dimensional crystal structure of NAO shows that Arg409 is 3.6 Å from Asp402. When Arg409 is mutated to Lys, the rate constant for proton abstraction decreases 100-fold. The three-dimensional structure of NAO also reveals the existence of a tunnel which extends from the protein exterior and terminates at the FAD N5 atom and the residues Asp402 and Phe401. We mutated amino acids in the tunnel into tryptophan, phenylalanine and leucine. The L99W, S276W and S276A enzymes showed the biggest decreases in both kcat and kcat/Km; these amino acids are closest to the FAD molecule and the active site. Mutation of amino acids farther away from the active site showed very small changes in the kinetic parameters. Ser276 is hydrogen bonded to Asp402 in the wild-type enzyme. When this amino acid is mutated to alanine or tryptophan, k3, the rate constant for proton abstraction, decreases around 35 fold. Asp402, Arg409 and Ser276 constitute a catalytic triad in the active site of nitroalkane oxidase, and both Arg409 and Ser276 are important for positioning Asp402 and catalysis.

Complete nucleotide sequences have been derived for the Sabin type 3 vaccine, P3/Leon/l2a1b, and its neurovirulent progenitor, P3/Leon/37 (Stanway et al., 1983, 1984). These studies revealed 10 base substitution mutations which must account for the attenuated and temperature sensitive phenotypes of the vaccine. Complete DNA copies of the genomes of P3/Leon/12a1b and P3/Leon/37 were constructed, each within the prokaryote vector, pAT 153. The full-length cDNA clones were shown to be infectious following transfection of human epithelial cells. Virus rescued from the cDNA clone of P3/Leon/12a1b could not be distinguished from reference Preparations of the Sabin type 3 vaccine in standard assays for neurovirulence and temperature sensitivity. By the same criteria, virus rescued from the cDNA clone of P3/Leon/37 was shown to be identical to the parental strain. To determine the genetic basis for the attenuated phenotype, a series of inter-strain poliovirus recombinants were constructed via cDNA in-vitro. Attenuation results from the concerted effect of two independent point mutations. The first is a C-U substitution at position 472 in the 5' non-coding region of the viral genome. The second is a C-U substitution at position 2034 which results in a serine to phenylalanine substitution in VP3. The VP3 mutation confers a temperature sensitive phenotype. This appears to be the only temperature sensitive mutation in the vaccine.

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