Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Tyrosine is involved in many biological processes including protein synthesis. This dissertation is focused on two different aspects: tyrosine catabolism and tyrosine derivative metabolism. Tyrosine undergoes degradation via tyrosine aminotransferase (TAT). Deficiency of TAT leads to some disease conditions or tyrosinemia type II. TAT has been characterized in several species, including humans. Mouse tyrosine aminotransferase was used as a model protein for the tyrosine catabolism portion of this study. Characterization of TAT included its expression in a bacterial expression system, purification using various chromatographic techniques, crystallization under different conditions, and its kinetic analysis, and molecular dynamics simulations. Based on sequence, structure, and kinetic data we have shown that mouse TAT behaves like human TAT. Our crystallization studies added new insights into the mechanism of TAT by shedding light on involvement of a disulfide bond in the regulation of mTAT. Molecular dynamics analysis provided perspective on the differences (preferences) in the substrate specificities of mouse and Trypanosome cruzi TAT. Tyrosine is a precursor of several key neurotransmitters. These neurotransmitters must be regulated in order to function properly. The hypothetical N-acetyltransferases from Aedes aegypti were used as model proteins for investigation of tyrosine derivative metabolism. We found nine potential arylalkylamine N-acetyltransferase (AANAT) genes in Ae. aegypti. Phylogenetic analysis suggests that these Ae. aegypti AANATs (AeAANATs) can be further divided into three clusters. Phylogenetic analysis suggests that insect AANATs may have different functions as compared with the mammalian AANATs, for which function is specific to circadian rhythm regulation. PCR amplification indicates that eight of the nine putative AeAANATs are expressed in the mosquito. Expression of the eight putative AeAANATs and substrate screening of their recombinant proteins against dopamine, octopamine, tyramine, epinephrine, tryptamine, 5-hydroxytryptamine, and methoxytryptamine established that five of the eight putative AeAANATs are true AANATs. The discontinuous expression profiles of AeAANAT genes were studied in detail. Six of the AeAANATs were expressed in the head before and after blood feeding, suggesting their potential role in neurotransmission inactivation. Down-regulation of these genes after blood feeding suggests that blood feeding or factors related to blood feeding impact on the regulation of these genes. Kinetic studies determined that two AeAANAT proteins are highly efficient in mediating the acetylation of dopamine and 5-hydroxytryptamine. Substrate analysis of AeAANATs supports the notion that acetylation of arylalkylamines is vital to the biology of mosquito species, and that these genes emerged in response to specific pressures related to necessities for biogenic amine acetylation. / Ph. D.

L’obésité, liée à la résistance à l’insuline, au diabète de type 2 et aux maladies cardiovasculaires, est un problème de santé majeur de notre société. Nous avons démontré que la protéine tyrosine phosphatase Shp1, dont l’expression est significativement augmentée dans les tissus cibles de l’insuline chez les souris obèses, est un régulateur de l’homéostasie du glucose dans le foie et le muscle. Shp1 est impliqué dans la modulation de l’expression et de l’activité du récepteur nucléaire PPARγ dans le foie. Nos recherches ont porté sur la caractérisation de Shp1 dans les adipocytes, la signalisation de l’insuline et le transport du glucose soit en sous-exprimant ou en exprimant de façon constitutive Shp1 dans les cellules adipeuses 3T3-L1. L’état physiologique des cellules a été caractérisé par la mesure de la coloration Oil-Red-O, des triglycérides, de l’expression de PPARγ et de ses gènes cibles et de leurs protéines, de la réponse à l’insuline par le transport du glucose ainsi que l’expression et de la phosphorylation des protéines impliquées dans la signalisation de l’insuline. La diminution de l’expression de Shp1 a entraîné un délai lors du début de la différenciation mesurée par l’expression retardée de PPARγ et certains de ses gènes cibles, mais n’a pas beaucoup affecté le phénotype des cellules complètement différenciées. Bien que la réduction de Shp1 ait augmenté la phosphorylation d’Akt stimulée par l’insuline, le transport de glucose n’a pas été modifié dans ces cellules, et l’expression de glut4 a légèrement diminué. L’expression constitutive de Shp1 a entraîné une forte diminution des niveaux de PPARγ, inhibant totalement la différenciation, l’expression de glut4 et le transport du glucose. Nos données suggèrent que Shp1 joue un rôle important dans les adipocytes comme régulateur de l’adipogenèse par la modulation de l’expression et l’activité de PPARγ et par la régulation de la signalisation de l’insuline. / Obesity, which is causally linked to the development of insulin resistance, type 2 diabetes (T2D) and cardiovascular disease (CVD), is a major health issue in our society. We have demonstrated that the protein tyrosine phosphatase Shp1, whose expression is significantly increased in insulin target tissues of obese mice, is a regulator of glucose homeostasis in liver and muscle. Shp1 is implicated in the modulation of expression and activity of the nuclear receptor PPARγ in liver. Here, we describe the characterization of Shp1 in adipocytes by analyzing its role in adipocyte differentiation, insulin signaling and glucose transport by either knocking-down or constitutively expressing Shp1 in 3T3-L1 adipose cells. The physiological state of the cells was characterized by measuring Oil-Red-O staining, triglyceride content, expression of PPARγ and its target genes and their proteins, insulin response by glucose uptake and the expression and phosphorylation of proteins involved in insulin signaling. Knockdown of Shp1 led to a retardation in the onset of differentiation as measured by delayed expression of PPARγ and some of its target genes, but did not much affect the phenotype of fully differentiated cells. Although reducing Shp1 increased insulin-stimulated Akt-phosphorylation, glucose transport was not changed in these cells, and glut4 expression was slightly decreased. Constitutive expression of Shp1, resulted in a strong decrease of PPARγ levels thereby totally inhibiting differentiation, glut4 expression and glucose transport. Our data suggest that Shp1 plays an important role in adipocytes both by acting as a regulator of adipogenesis through modulation of the expression and activity of PPARγ and by regulating the insulin signaling pathway.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 122-137).

Thesis (Ph. D.)--Cornell University, December, 1997. / Vita. Includes bibliographical references (leaves 96-113).

Proefschrift--Utrecht. / Title also in English. Summary in English. Vita. Includes bibliographical references.

Proefschrift--Utrecht. / Title also in English. Summary in English. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Illinois State University, 2006. / Title from title page screen, viewed on June 8, 2007. Dissertation Committee: Kevin A. Edwards (chair), Craig Gatto, Wade A. Nichols, Samuel Galewsky, John C. Sedbrook. Includes bibliographical references (leaves 218-245) and abstract. Also available in print.

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