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Les protéines PilB, nDsbD et DsbE1 de Neisseria meningitidis : caractérisation enzymatique, fonctionnelle et structurale / PilB, nDsbD and DsbE1 proteins from Neisseria meningitidis : enzymatic, functional and structural characterizationSelme-Roussel, Laure 09 November 2010 (has links)
Les espèces Neisseria gonorrhoeae et Neisseria meningitidis, sont des bactéries pathogènes obligatoires de l'Homme, qui ont acquis différents mécanismes de défense pour détecter et combattre le stress oxydant généré par les mécanismes de défense de l'hôte lors de l'infection. La protéine PilB périplasmique, ferait partie de ces mécanismes et serait de ce fait associée à leur pathogénicité. PilB est composée de trois domaines : un domaine N-terminal (Nter) à activité disulfure oxydoréductase, et les domaines central et C-terminal à activité Méthionine Sulfoxyde Réductase (Msr) respectivement de classe A et B. L'étude des domaines isolés de PilB avait montré que le domaine Nter réduit sélectivement le domaine MsrB. Par ailleurs, le domaine Nter présente un repliement de type DsbE. Les DsbE sont des disulfure oxydoréductases périplasmiques impliquées dans la maturation des cytochromes c. En particulier, la DsbE1 de N. meningitidis a été identifiée par le Dr Adeline Gand lors de son doctorat.Lors de ma thèse, l'étude des protéines PilB de N. meningitidis et de Fusobacterium nucleatum m'a permis de montrer que : 1) la sélectivité de réduction du domaine Nter pour le domaine MsrB n'est pas conservée, 2) la sélectivité de réduction des domaines Nter observée sur les domaines isolés n'est pas retrouvée sur les PilB entiers ; et 3) dans tous les PilB, la réduction du domaine MsrB par le domaine Nter peut se faire selon un mécanisme intramoléculaire. De plus, nous avons étudié in vivo l'effet de la délétion du gène pilB sur la survie d'une souche de N. meningitidis en présence d'agents oxydants. D'autre part, le domaine N-terminal de la protéine DsbD (nDsbD) de N. meningitidis a été identifié comme étant le réducteur périplasmique de PilB et de la DsbE1 de N. meningitidis. Enfin, la caractérisation de l'activité apocytochrome c réductase de la DsbE1 de N. meningitidis a été complétée par des approches in vitro et in vivo chez N. meningitidis / The Neisseria gonorrhoeae and Neisseria meningitidis species are human obligatory pathogenic bacteria, which acquired various defense mechanisms to detect and fight oxidative stress generated by mechanisms of host defense during infection. The periplasmic PilB protein, specific to these bacteria, would be part of such mechanisms and would be associated with their pathogenicity. PilB is composed of three domains: an N-terminal domain (Nter) with disulfide oxidoreductase activity, and central and C-terminal domains with Methionine sulfoxide reductase activity (Msr) of A and B class respectively. The study of isolated domains of PilB showed that the Nter domain selectively reduced MsrB domain. Moreover, this Nter domain presents a DsbE-fold. The DsbE are periplasmic disulfide oxidoreductases involved in the maturation of cytochrome c. In particular, Dr. Adeline Gand identified the DsbE1 from N. meningitidis during his PhD. During my PhD, the study of PilB proteins from N. meningitidis and Fusobacterium nucleatum allowed me to show that: 1) the selective reduction of Nter domain for the MsrB domain is not conserved, 2) the selective reduction of Nter domains observed on the isolated domains is not found in entire PilB, and 3) in all PilB, the MsrB domain reduction by Nter domain could be an intramolecular mechanism. Moreover, we studied the in vivo effect of the pilB gene deletion on the survival of a strain of N. meningitidis in the presence of oxidants. And, the N-terminal domain of DsbD protein (nDsbD) from N. meningitidis was identified as the reducing partner of periplasmic PilB and DsbE1 of N. meningitidis. Finally, the characterization of apocytochrome c reductase activity of DsbE1 N. meningitidis was complemented by in vitro and in vivo approaches in N. meningitidis
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The Effects of MsrA and MsrB in Anoxia Tolerance in Aging Drosophila melanogasterUnknown Date (has links)
Drosophila melanogaster tolerates several hours of anoxia (the absence of
oxygen) by entering a protective coma. A burst of reactive oxygen species (ROS) is
produced when oxygen is reintroduced to the cells. ROS causes oxidative damage to
critical cellular molecules, which contribute to aging and development of certain agerelated
conditions. The amino acid, methionine, is susceptible to oxidation, although this
damage can be reversed by methionine sulfoxide reductases (Msr). This project
investigates the effect of Msr-deficiency on anoxia tolerance in Drosophila throughout
the lifespan of the animal. The data show that the time for recovery from the
protective comma as well as the survival of the animals lacking any Msr activity
depends on how quickly the coma is induced by the anoxic conditions. Insight into
the roles(s) of Msr genes under anoxic stress can lead us to a path of designing
therapeutic drugs around these genes in relation to stroke. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection
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Exigências de metionina + cistina e treonina para manutenção de aves /Bonato, Melina Aparecida. January 2010 (has links)
Orientador: Nilva Kazue Sakomura / Banca: Robert Mervyn Gous / Banca: Izabelle Auxiliadora Molina de Almeida Teixeira / Resumo: A mantença pode ser definida como o estado em que o animal se encontra em equilíbrio de nitrogênio, no qual a ingestão de N é igual à soma das perdas, permanecendo constante o conteúdo de N do corpo. E esta pode ser definida com base em estudos de metabolismo com aves adultas improdutivas, pelo fato das exigências totais de aminoácidos dessas aves estarem associadas apenas às perdas inevitáveis (mantença), não incluindo necessidades específicas de aminoácidos para o crescimento e/ou produção. Porém, há alguns problemas na determinação da exigência de mantença: primeiro é como comparar a mantença entre genótipos de diferentes tamanhos à maturidade, o segundo é como comparar a mantença entre animais de um mesmo genótipo em diferentes estágios de crescimento, e o terceiro é como lidar com a variação no conteúdo de gordura corporal, uma vez que não existe demanda de aminoácidos para a manutenção das reservas lipídicas. Assim, as diferenças de valores entre níveis de exigências para mantença de aminoácidos encontrados na literatura, tem sido a diretriz para o desenvolvimento de novos estudos, visando à obtenção de metodologias padronizadas e estimativas de valores condizentes com as necessidades das aves. Este estudo teve como objetivo estimar as exigências de metionina+cistina e treonina digestíveis para a mantença de aves adultas utilizando e comparando galos de diferentes pesos e composições corporais / Abstract: The maintenance can be defined as the state where the animal is in nitrogen balance, in which the intake of N is equal to the sum of the losses, stabilizing the N content in the body. And this can be defined based on studies of metabolism in adult birds unproductive, because the total amino acid requirements of these birds are associated only to the inevitable losses (maintenance), not including the specific amino acids for growth and/or production. However, there are some problems in determining the requirement for maintenance: first is to compare the maintenance among genotypes of different sizes at maturity, the second is like comparing the maintenance of animals of the same genotype at different stages of growth, and the third is how to deal with the variation in body fat content, since there is no demand for amino acids for the maintenance of lipid reserves. Thus, differences in values between levels of requirements for maintenance of amino acids found in the literature has been the guideline for the development of new studies aiming to produce standardized methodologies and estimates of amounts consistent with the needs of birds. This study aimed to estimate the methionine+cystine and threonine digestible for the maintenance of adult birds using and comparing roosters of different weights and body composition / Mestre
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Applications of hypervalent iodine reagents : from enantioselective copper-catalysed arylation-semipinacol cascade to methionine functionalisation for peptide macrocyclisationLukamto, Daniel Hartoyo January 2018 (has links)
The unifying theme of this thesis is the exploitation of the reactivity of aryliodonium salts as electrophile transfer reagents. In the first part of the thesis, diaryliodonium salts are employed as arylation reagents for the enantioselective copper-catalysed arylative semipinacol rearrangement (SPR) of various tertiary allylic alcohols. This cascade reaction is a rare example of asymmetrically activating SPR using carbon electrophiles. Different substrate classes - including dihydropyran, indene and dihydronaphthalene moieties - are converted to enantioenriched beta-aryl spirocyclic ketones in excellent yields and enantioselectivities, and often as a single diastereomer. These are in turn useful functional handles for transformations into other moieties, including further rearrangements via Baeyer-Villiger oxidation. In the second part of this thesis, a two-step process for the macrocyclisation of native peptides via a non-natural linkage is developed. This study exploits previous work conducted in the group on the use of aryliodonium salts as methionine-selective diazoacetate transfer reagents. The functionalised methionine is in turn used for an intramolecular rhodium-catalysed insertion into tryptophan. Eventual translation onto solid-phase enables facile access into various macrocyclic peptides.
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Methionine Metabolism in Fasciola HepaticaAyer, Carol Theresa 01 January 1990 (has links)
5'-Deoxy-5'-methylthioadenosine (MTA) is derived from s-adenosylmethionine (AdoMet) during the synthesis of the polyamines spermidine and spermine. Methionine can be regenerated from MTA by one of two mechanisms. In mammalian cells and some microorganisms, MTA is degraded to adenine and 5-methylthioribose-1-phosphate (MTR-1-P) via MTA phosphorylase. In certain other microbes, however, MTA is catabolized in two steps; first to adenine and 5-methylthioribose (MTR) via MTA nucleosidase followed by conversion of MTR to MTR-1-P via MTR kinase.
This study was to demonstrate the presence of MTA nucleosidase or MTA phosphorylase in both redia containing cercariae and adult Fasciola hepatica Linnaeus, 1758. If MTA nucleosidase was present, it was wanted to determine if MTR kinase was also present.
The phosphate-dependent cleaving activity of MTA phosphorylase was demonstrated in the cell-free extracts of adult Fasciola hepatica along with an unidentified MTR metabolizing activity. Redia containing cercariae showed MTA nucleosidase and MTR kinase activity.
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The Catalytic Aspartic Acid Shows a Role in Substrate Positioning in 5-methylthioribose KinaseDawson, Karen 25 July 2012 (has links)
Methionine is involved in many cellular processes, several of which produce a feedback inhibitor. 5-methylthioribose (MTR) kinase, one protein involved in the removal of this inhibitor, has a protein kinase fold with conserved kinase motifs and several unique MTR binding motifs. Site-directed mutagenesis and characterization of the Bacillus subtilis enzyme was performed to probe the role of one motif. Active site D233 mutants show an activity profile similar to other protein kinase-like enzymes, suggesting a common mechanism that does not require a catalytic acid. An ordered sequential binding mechanism, with nucleotide binding first, was seen in wild type MTR kinase. Binding studies of the mutant proteins suggest that hydrogen bonding is important for MTR binding. The structures of the mutant proteins also show more differences in MTR binding than nucleotide binding. Overall, D233 is important for increasing the nucleophilicity of MTR, and ensuring its correct position in the active site.
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The Catalytic Aspartic Acid Shows a Role in Substrate Positioning in 5-methylthioribose KinaseDawson, Karen 25 July 2012 (has links)
Methionine is involved in many cellular processes, several of which produce a feedback inhibitor. 5-methylthioribose (MTR) kinase, one protein involved in the removal of this inhibitor, has a protein kinase fold with conserved kinase motifs and several unique MTR binding motifs. Site-directed mutagenesis and characterization of the Bacillus subtilis enzyme was performed to probe the role of one motif. Active site D233 mutants show an activity profile similar to other protein kinase-like enzymes, suggesting a common mechanism that does not require a catalytic acid. An ordered sequential binding mechanism, with nucleotide binding first, was seen in wild type MTR kinase. Binding studies of the mutant proteins suggest that hydrogen bonding is important for MTR binding. The structures of the mutant proteins also show more differences in MTR binding than nucleotide binding. Overall, D233 is important for increasing the nucleophilicity of MTR, and ensuring its correct position in the active site.
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Effect of cereal type and commensal bacteria on availability of methionine sources and intestinal physiology in pigsMalik, Gita 21 September 2009
An investigation was conducted to determine the contribution of the gastrointestinal microbiota to variation in bioefficacy of methionine sources and the interrelationship between intestinal microbiota and cereal grain type with respect to gastrointestinal physiology. Apparent gastrointestinal absorption of DL-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA), post-weaning intestinal morphology, digestive physiology, mucin dynamics and digesta flow were studied in a series of experiments using conventional and gnotobiotic pigs. At 14 d of age, sow - reared conventional (CON) pigs and isolator - reared monoassociated gnotobiotic pigs (EF) were weaned to corn or wheat/barley based diets supplemented with MET or MHA-FA. At 24 d of age, after an overnight fast, pigs were fed experimental diet supplemented with 107 Bq of either 3H-L-MET or 3H-L-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed 3 h after consuming the meal to collect digesta and tissue samples from the stomach and along the small intestinal (SI) length. Conventional pigs fed a wheat/barley-based diet had increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal SI. Among the gnotobiotic pigs, 8 pigs (2 isolators) were monoassociated with a bacteria closely related to <i>Providencia</i> spp. and 16 pigs (4 isolators) were monoassociated with <i>Enterococcus faecium</i> (EF). Species of bacterial contaminant and diet composition did not affect residual MET or MHA-FA in digesta. Decreased (P < 0.05) apparent residual MET in digesta compared with MHA-FA in CON but not monoasscoiated pigs, along with significantly higher (P<0.05) MET associated radioactivity at 5% SI tissue suggested that microbial metabolism of MHA-FA increases its retention in small intestinal digesta and contributes in part to the lower bioefficacy of MHA-FA compared to MET. A comparison of CON and EF pigs showed that wheat/barley diets increased digesta viscosity (<i>P</i> < 0.01) and proliferating cell nuclear antigen (PCNA) expression (<i>P</i> < 0.001) and tended to decrease (<i>P</i> < 0.07) aminopeptidase N (APN) activity. Monoassociation decreased (<i>P</i> < 0.01) body weight, relative spleen weight, crypt depth, PCNA expression, caspase-3 activity, sucrase expression, total goblet cells in crypts and mucin gene expression and increased (<i>P</i> < 0.01) relative SI length, digesta viscosity, villus height, APN and sucrase activity. Interactive effects between cereal grain type and microbial status were observed only as trends (<i>P</i> < 0.1) for PCNA, Muc2, APN and sucrase suggesting these effects were mediated indirectly through microbial changes. Decreased % retained chromic oxide in digesta at all SI locations and no chromic oxide at 95% SI length in monoassociated pigs indicated slower small intestinal transit of digesta in monoassociated pigs. We successfully developed the chromic oxide microassay for estimating chromic oxide in 1/20th of original sample size (2.0 g). Results of this study indicate that microbial metabolism of MHA-FA contributes in part to the lower bioefficacy of MHA-FA compared to MET. Monoassociation had major effects on intestinal physiology whereas limited indirectly mediated effects of cereal type were observed suggesting no major influences of cereal grain type during the short early post-weaning phase.
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Effect of cereal type and commensal bacteria on availability of methionine sources and intestinal physiology in pigsMalik, Gita 21 September 2009 (has links)
An investigation was conducted to determine the contribution of the gastrointestinal microbiota to variation in bioefficacy of methionine sources and the interrelationship between intestinal microbiota and cereal grain type with respect to gastrointestinal physiology. Apparent gastrointestinal absorption of DL-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA), post-weaning intestinal morphology, digestive physiology, mucin dynamics and digesta flow were studied in a series of experiments using conventional and gnotobiotic pigs. At 14 d of age, sow - reared conventional (CON) pigs and isolator - reared monoassociated gnotobiotic pigs (EF) were weaned to corn or wheat/barley based diets supplemented with MET or MHA-FA. At 24 d of age, after an overnight fast, pigs were fed experimental diet supplemented with 107 Bq of either 3H-L-MET or 3H-L-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed 3 h after consuming the meal to collect digesta and tissue samples from the stomach and along the small intestinal (SI) length. Conventional pigs fed a wheat/barley-based diet had increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal SI. Among the gnotobiotic pigs, 8 pigs (2 isolators) were monoassociated with a bacteria closely related to <i>Providencia</i> spp. and 16 pigs (4 isolators) were monoassociated with <i>Enterococcus faecium</i> (EF). Species of bacterial contaminant and diet composition did not affect residual MET or MHA-FA in digesta. Decreased (P < 0.05) apparent residual MET in digesta compared with MHA-FA in CON but not monoasscoiated pigs, along with significantly higher (P<0.05) MET associated radioactivity at 5% SI tissue suggested that microbial metabolism of MHA-FA increases its retention in small intestinal digesta and contributes in part to the lower bioefficacy of MHA-FA compared to MET. A comparison of CON and EF pigs showed that wheat/barley diets increased digesta viscosity (<i>P</i> < 0.01) and proliferating cell nuclear antigen (PCNA) expression (<i>P</i> < 0.001) and tended to decrease (<i>P</i> < 0.07) aminopeptidase N (APN) activity. Monoassociation decreased (<i>P</i> < 0.01) body weight, relative spleen weight, crypt depth, PCNA expression, caspase-3 activity, sucrase expression, total goblet cells in crypts and mucin gene expression and increased (<i>P</i> < 0.01) relative SI length, digesta viscosity, villus height, APN and sucrase activity. Interactive effects between cereal grain type and microbial status were observed only as trends (<i>P</i> < 0.1) for PCNA, Muc2, APN and sucrase suggesting these effects were mediated indirectly through microbial changes. Decreased % retained chromic oxide in digesta at all SI locations and no chromic oxide at 95% SI length in monoassociated pigs indicated slower small intestinal transit of digesta in monoassociated pigs. We successfully developed the chromic oxide microassay for estimating chromic oxide in 1/20th of original sample size (2.0 g). Results of this study indicate that microbial metabolism of MHA-FA contributes in part to the lower bioefficacy of MHA-FA compared to MET. Monoassociation had major effects on intestinal physiology whereas limited indirectly mediated effects of cereal type were observed suggesting no major influences of cereal grain type during the short early post-weaning phase.
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Characterization of Metal Binding Peptides Derived from Copper Trafficking ProteinsRubino, Jeffrey Tyler January 2010 (has links)
<p>Copper was first released into the environment as the result of the mass generation of oxygen from photosynthetic bacteria roughly 2.7 billion years ago. While it proved to be poisonous to early life on Earth, those that met the evolutionary challenge utilized the metal as a cofactor in enzymes to perform biochemically significant functions, while controlling intracellular levels of copper with a sophisticated network of trafficking proteins. Proteins and enzymes that utilize copper as a cofactor have evolved significantly different coordination environments than copper trafficking proteins, as a result of the different functions they perform. Of particular interest was characterizing the unique Cu(I) binding events observed in some of these proteins, the extracellular N-terminal regions of eukaryotic high affinity copper transport proteins (Ctr), and the bacterial periplamsic CusF protein of the CusFBCA Cu(I)/Ag(I) efflux pathway. </p><p>Model peptides corresponding to the methionine rich binding motifs (Mets motifs) were characterized in terms of Cu(I) binding affinity, stoichiometry, and metal specificity, via an ascorbic acid oxidation assay and electrospray ionization mass spectrometry. Metal induced structural features and coordination environments were elucidated with NMR, CD and X-ray spectroscopy. A series of peptides was also examined to infer the relative Cu(I) binding affinities, and susceptibility to oxidation, of methionine, histidine, cysteine residues found in copper binding motifs. The resistance of Cu+ specific peptides to metal catalyzed oxidation is also described. Attempts were also made to model the Cu(I)/Ag(I) tryptophan cation-π interaction observed in CusF.</p> / Dissertation
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