Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 114-124).

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 121-133).

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Nov 30

Proefschrift Rijksuniversiteit Groningen. / Met lit. opg. - Met samenvattingen in het Nederlands en Engels.

The enzymes phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) were studied in the yeast Sporobolomyces roseus (Kluyver and van Neil). Cells grown on a glucose-salts medium were ground with alumina, and the cell-free buffer extract was fractionated with ammonium sulfate. Enzyme activity was assayed by measuring spectrophotometrically the cinnamic acid and p-coumaric acid produced from phenylalanine and tyrosine respectively. Further attempts at purification resulted in the inactivation of the TAL. Although the two enzymes were not separated by the purification procedures used, there is some evidence that the deamination of phenylalanine and tyrosine are catalyzed by different proteins, and not by a single enzyme with wide specificity. TAL appears to be precipitated by lower concentrations of ammonium sulfate than is PAL. The pH curves of the two enzymes are different. The specific activities of the two enzymes can be changed relative to one another in the cell by changing the medium upon which the cells were grown. The rates of production of the two enzymes vary independently during the growth of the cells. While the proteins are probably distinct, the production and activity of each enzyme seem to be under common control. Peak production of both enzymes occurs during late logarithmic-early stationary phase in the growth of a batch culture. Replacement media containing either phenylalanine or tyrosine stimulate the production of both PAL and TAL. Similarly, media containing cinnamic acid or p-coumaric acid repress the formation of the enzymes. Studies using labelled substrate show that both products inhibit the action of both enzymes. / Science, Faculty of / Botany, Department of / Graduate

Interest in brain catecholamines has grown considerably in the last few years in view of their possible role as neurotransmitters. This investigation deals primarily with the enzyme tyrosine hydroxylase which is thought to be the rate limiting step in catecholamine synthesis. Using tyrosine hydroxylase measurements and catecholamine depletion techniques,attempts were made to determine the site of increased synthesis of catecholamines in animals exposed to cold. Brain, heart and spleen do not appear to be the organs involved in this change. Adrenals may be of significance but the results were suggestive rather than conclusive. Tyrosine hydroxylase distribution in brain was determined in various regions of rat, rabbit and cat brain, and activity was shown to be highest in the caudate, septal area, nucleus accombens, and anterior perforating substance, with much lower activities in other regions such as hippocampus, amygdala, hypothalamus, thalamus, cortex, cerebellum and brain stem. Using these distribution studies as indications of normal tyrosine hydroxylase activity in areas of rat brain, and electrolytic lesion techniques, studies were carried out to determine noradrenergic and dopanergic pathways in brain. Catecholamine fibers from their origin in the midbrain were traced in the midbrain and diencephalon to the caudate and septal area, and the relative positions of each group of fibers determined along their course. / Medicine, Faculty of / Graduate

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.

No description available.

No description available.

Protein phosphorylation is utilized universally as a mechanism of signal transduction. However, the use of tyrosine phosphorylation by bacteria has been a matter of dispute. Conventional wisdom dictated that "prokaryotic phosphorylation" was typified by phosphorylation of histidine and aspartate residues of proteins, while "eukaryotic phosphorylation" was characterized by modification of serine, threonine, or tyrosine residues. Increasing numbers of reports have emerged challenging the traditional view of "prokaryotic" and "eukaryotic" phosphorlyation. One of the strongest links unifying prokaryotic and eukaryotic protein phosphorylation to date is IphP, a genomically-encoded dual-specificity protein phosphatase from the cyanobacterium Nostoc commune UTEX 584 bearing the active-site signature sequence of eukaryotic tyrosine-specific and dual-specificity protein phosphatases. The catalytic properties and substrate specificity of IphP were examined in detail. The enzyme was able to discriminate among a variety of exogenous peptides and proteins. Kinetic studies revealed that IphP favors protein / peptide substrates over low molecular weight compounds. Heparin effected IphP activity in a substrate-dependent manner. Enzyme activity toward casein (P-Ser) and MAP kinase (P-Thr/P-Tyr) was stimulated in the presence of the polyanion, whereas activity was inhibited by heparin toward other protein substrates. Both stimulation and inhibition by heparin were dose-dependent. The ability to stimulate IphP activity toward select substrates was attributed to the ability of heparin to recruit the enzyme and substrate to the same microenvironment. To facilitate future genetic studies examining the role of tyrosine phosphorylation in cyanobacteria, we searched for evidence of protein tyrosine phosphorylation in Anabaena PCC 7120. In a collaborative effort with the laboratory of Dr. Potts, tyrosine phosphorylated proteins were identified in Anabaena utilizing several approaches, including comparative labelling with alpha- vs gamma-32P-ATP, phosphoamino acid analysis, and selective hydrolysis with a tyrosine specific protein phosphatase. Together, these data unequivocally demonstrate the presence of tyrosine-phosphorylated proteins in Anabaena PCC 7120. Extracts of Anabaena PCC 7120 were examined for protein tyrosine phosphatase activity. An apparent PTP activity was detected, partially purified, and characterized. The protein phosphatase was ~38kDa by SDS-PAGE and sucrose density gradient centrifugation and displayed dual-specificity protein phosphatase (DSP) activity in vitro. The enzyme was localized to the periplasm and was thus assigned the title PAD, for Periplasmic Anabaena DSP. Periplasmic phosphoproteins of ~120 and 55 kDa that had been radiolabelled in vitro were dephosphorylated by partially purified PAD. PAD activity varied in vivo ~5-fold in a rhthymic, seemingly diurnal manner. Periplasmic proteins, including the 55kDa protein, were labelled in vivo and the degree of radiolabel incorporated into these proteins varied inversely with PAD activity. / Ph. D.