No description available.

Los peces carnívoros presentan baja capacidad para utilizar carbohidratos provenientes de la dieta y controlar los niveles de glucosa en sangre. En comparación con los mamíferos, estos animales tras la ingesta de glucosa o de dietas con alto contenido en carbohidratos, muestran una hiperglucemia mas prolongada. La alanina aminotransferasa (ALT) constituye un nexo de interacción entre el metabolismo de aminoácidos y el de carbohidratos al catalizar la reacción de la transaminación reversible entre L-alanina y 2-oxoglutarato para formar piruvato y L-glutamato. Estudios previos de nuestro grupo indicaron la presencia de tres isoformas ALT en dorada (Sparus aurata): las isoenzimas citosólicas cALT1 y cALT2 y una isoforma mitocondrial, mALT. En hígado de dorada, la expresión de cALT2 incrementa en situación de gluconeogénesis mientras que cALT1 predomina durante el período postprandial para la utilización de los nutrientes de la dieta. El objetivo general del presente estudio es comprender a nivel molecular los efectos metabólicos derivados de la inhibición de ALT en peces para ayudar a establecer nuevas aplicaciones biotecnológicas orientadas a mejorar la utilización de los nutrientes de la dieta. Así, en acuicultura, identificar los efectos metabólicos asociados a la modulación de la actividad ALT constituye un punto de interés para conocer si es posible efectuar una sustitución parcial de las proteínas de la dieta por carbohidratos u otros nutrientes, a fin de reducir el coste de la producción en acuicultura y disminuir la eutrofización de las aguas del entorno. Nuestros estudios muestran que la inyección intraperitoneal de doradas con nanopartículas del complejo pCpG-siRNA-quitosán resultó adecuada para promover la expresión de un siRNA para bloquear la expresión de cALT1 en hígado de Sparus aurata. La inyección intraperitoneal de nanopartículas de pCpGsi1sh1-quitosán promovió la silenciación de cALT1 a nivel de mRNA y actividad enzimática en hígado de dorada. Por otra parte, hemos analizado la inhibición postranscripcional de la actividad ALT in vivo e in vitro con el compuesto aminooxiacetato (AOA) y analizado los cambios promovidos en metabolitos y enzimas clave en el metabolismo intermediario de carbohidratos y proteínas en hígado de Sparus aurata, tras la ingesta del AOA con dietas de diferente composición. In vitro, el AOA ejerce una inhibición dependiente de dosis sobre la actividad ALT hepática citosólica y mitocondrial. In vivo, el AOA se comportó como inhibidor de la actividad ALT citosólica hepática, pero no de la mitocondrial. Una exposición a largo plazo a AOA promovió un aumento de la actividad piruvato quinasa en el hígado de dorada, independientemente de la composición de la dieta suministrada a los peces. Los estudios de 1H-RMN mostraron que la inclusión de AOA en la dieta promueve una disminución en los niveles hepáticos de alanina, glutamato y glucógeno. Adicionalmente, los análisis de 2H-RMN indicaron una tasa de renovación más alta de alanina en el hígado de los peces alimentados con una dieta con un contenido alto en carbohidratos y bajo en proteínas y que el AOA disminuye el enriquecimiento de alanina en 2H independientemente de la composición de la dieta. Los estudios derivados de esta tesis indican que la inhibición dependiente de AOA de la actividad de la ALT citosólica podría contribuir a aumentar el uso de nutrientes por carbohidratos de la dieta de Sparus aurata. / Carnivorous fish have low ability to utilize dietary carbohydrates and controlling blood glucose levels. Compared with mammals, these animals after ingestion of glucose or diets high in carbohydrates show a more prolonged hyperglycemia. Alanine aminotransferase (ALT) links carbohydrate and amino acid metabolism through catalysing the reversible transamination between L-alanine and 2-oxoglutarate to form pyruvate and L-glutamate. Previous studies from our group indicated the presence of three isoforms ALT bream (Sparus aurata): cALT1 cytosolic isoenzymes and cALT2 and a mitochondrial isoform, mALT. In the liver os Sparus aurata cALT2 expression increases gluconeogenesis situation prevails while cALT1 during the postprandial period for the utilization of dietary nutrients. The overall objective of this study is to understand at a molecular level the resulting metabolic effects from the inhibition of ALT in fish to help establish new biotechnological applications aimed to improve the use of dietary nutrients. Thus in aquaculture, identifying metabolic effects associated with ALT activity modulation is a point of interest to know if it is possible to perform a partial replacement of dietary proteins by carbohydrate or other nutrients, to reduce the cost of Aquaculture production and reduce eutrophication of the environment. Our studies show that intraperitoneal injection of a chitosan-pCpG-siRNA nanoparticles complex to Gilthead seabream proved to be suitable for the expression of a siRNA to silence the expression of liver cALT1 in Sparus aurata. Intraperitoneal injection of pCpGsi1sh1-chitosan nanoparticles complex promoted cALT1 silencing at the mRNA level and a decrease of liver enzyme activity of Gilthead seabream. In the present study amino-oxyacetate (AOA) was used to evaluate its effect on liver ALT activity of the carnivorous fish Sparus aurata. Moreover, the derived metabolic effects on metabolites and other key enzymes of glycolysis, gluconeogenesis and the pentose phosphate pathway were also studied. A dose-effect-dependent inhibition of AOA on hepatic cytosolic and mitochondrial ALT activity was observed in vitro. In vivo, AOA behaved as an inhibitor of hepatic cytosolic ALT activity. A long-term exposure to AOA increased pyruvate kinase activity in the liver irrespective of the composition of the diet supplied to fish. 1H NMR studies showed that inclusion of AOA to the diet decreased the hepatic levels of alanine, glutamate and glycogen. Moreover, 2H NMR analysis indicated a higher renewal rate for alanine in the liver of fish fed with a high-carbohydrate/low-protein diet, while AOA decreased alanine 2H-enrichment irrespective of the diet. The present study indicates that AOA-dependent inhibition of the cytosolic ALT activity could help to increase the use of dietary carbohydrate nutrients by the Sparus aurata fish.

This thesis presents the cloning, purification, crystallization, and structural studies of two unknown proteins from Mycobacterium tuberculosis, and of an aminotransferase from Mycobacterium smegmatis. Structural knowledge of these proteins is of highest interest for structure-based drug design, which is one of the approaches that can be used in order to fight tuberculosis (TB). The structure of the conserved hypothetical protein Rv0216 was refined to a resolution of 1.9 Å. The structure exhibits a so-called double hotdog-fold, similar to known hydratases. However, only parts of the hydratase active site are conserved in Rv0216, and no function could be assigned to the protein. Several Rv0216-like protein sequences were found in a variety of actino- and proteobacteria, suggesting that these proteins form a new protein family. Furthermore, other hotdog-folded proteins in M. tuberculosis were identified, of which a few are likely to be hydratases or dehydratases involved in the fatty acid metabolism. The structure of Rv0130 exhibits a single hotdog-fold and contains a highly conserved R-hydratase motif. Rv0130 was shown to hydrate fatty acid coenzyme A derivatives with a length of six to eight carbons. The Rv0130 active site is situated in a long tunnel, formed by a kink in the central hotdog-helix, which indicate that it can utilize long fatty acid chains as well. A number of previously predicted hotdog-folded proteins also feature a similar tunnel. The structure of branched chain aminotransferase (BCAT) of M. smegmatis was determined in the apo-form and in complex with an aminooxy inhibitor. Mycobacterial BCAT is very similar to the human BCAT, apart for one important difference in the active site. Gly243 is a threonine in the human BCAT, a difference that offers specificity in inhibition and substrate recognition of these proteins. The aminooxy compound and MES were found to inhibit the mycobacterial BCAT activities. The aminooxy compound inhibits by blocking the substrate-pocket. A second inhibitor-binding site was identified through the binding of a MES molecule. Therefore, both the MES-binding site and the substrate-pocket of M. smegmatis BCAT are suggested to be potential sites for the development of new inhibitors against tuberculosis.

No description available.

Salmonella enterica sérovar Typhi (Typhi) est une bactérie pathogène spécifique à l’homme. Typhi est l’agent étiologique de la fièvre typhoïde chez l’humain, causant plus de 16 millions de nouveaux cas par année et plus de 600 000 morts. Il a été démontré que pour causer une infection systémique, Salmonella doit nécessairement survivre dans les macrophages de l'hôte. Paradoxalement, S. enterica sérovar Typhimurium, très apparenté à Typhi (près de 90 % d’homologie), n’a pas la capacité de se disséminer dans l’organisme humain et peut infecter plusieurs espèces animales. Nous avons antérieurement identifié 36 gènes uniques à Typhi (absents chez Typhimurium) situés sur 15 régions différentes et exprimés sélectivement lors de l’infection de macrophages humains. Ainsi, l’une de ces régions a suscité notre attention, soit la région sty4217-4222 et plus particulièrement le produit du gène sty4221, une aminotransférase hypothétique. Ce dernier gène est d’intérêt dû à l’homologie qu’il détient avec une hémolysine connue (Hly) produite par Treponema denticola, possédant elle-même une activité d’aminotransférase. Chez T. denticola, Hly dégrade la cystéine et produit du H2S qui est toxique pour l’hôte. Notre hypothèse est que la spécificité d’hôte et la capacité de produire une infection systémique de Typhi sont dues à l’expression de gènes qui ne se retrouvent pas chez d’autres salmonelles. Le but de cette étude était donc de caractériser le gène sty4221 quant à son activité hémolytique, cytotoxique et tenter de déterminer son rôle dans la virulence de cette bactérie. Le gène sty4221 a été cloné sous le contrôle d’un promoteur inductible à l’arabinose et exprimé par E. coli. L’activité hémolytique du clone a été déterminée par simple observation sur gélose sang. Ce clone a également permis d’observer l’effet cytotoxique du surnageant de culture sur différentes lignées cellulaires, par quantification de la relâche de LDH. Le gène sty4221 a été muté chez la souche sauvage de Typhi, ISP1820, l’implication pathogénique du gène a ainsi pu être étudiée. Des tests de phagocytose, d’invasion et de survie dans des macrophages humains ont été effectués, ainsi que des tests d’adhésion et d’invasion sur des cellules HeLa. Par ailleurs, une première tentative de purification de la protéine a été entreprise. En somme, nous savons maintenant que STY4221 a des propriétés hémolytiques, augmentées par la présence de cystéine. De plus, STY4221 a un effet cytotoxique sur les macrophages THP-I, mais aucun effet sur les HeLa. Or, sty4221 ne semble pas impliqué dans les étapes d’adhésion, d’invasion, de phagocytose ou de survie. La caractérisation de sty4221 permettra sans doute d’approfondir nos connaissances sur les toxines trouvées uniquement chez Typhi. / Salmonella enterica serovar Typhi (Typhi) is a human restricted pathogen causing typhoid fever, a systemic infection. Annually, at least 16 million new cases with 600, 000 associated deaths are reported. It has been demonstrated that Salmonella has to survive in the macrophages of its host, in order to produce a systemic disease. This ability to cause a disseminated infection in human is unique to Typhi. Our laboratory had isolated 36 genes that were unique to Typhi (absent from Typhimurium’s genome), and that were expressed during human macrophages infection. One of these genes, sty4221, a putative aminotransferase, was of high interest since it shares sequence similarities with a known hemolysin (Hly), which also possesses an aminotransferase activity. That hemolysin is produced by Treponema denticola, it catabolizes cysteine and produces H2S, a toxic metabolite for the host. Our hypothesis is that host specificity and the ability to cause a systemic infection might be explained by the expression of genes that are not found in other salmonellas. The goal of this study was to characterize the gene sty4221, in terms of hemolytic and cytotoxic activity and to determine its role in virulence. The sty4221gene has been cloned in a vector under an arabinose inducible promoter and transformed in a strain of E. coli. The hemolytic activity has been investigated on blood-agar medium. To evaluate the cytotoxicity of the STY4221 protein on human cultured cells, direct observation by photonic microscopy was done. The cytotoxicity activity on human cultured cells has been quantitatively measured with a lactate dehydrogenase release assay. Moreover, the sty4221 gene has been deleted in order to study its implication in the infection and the survival within human macrophages and for adhesion/invasion on epithelial. Protein purification was also attempted. We now know that protein STY4221 has a hemolytic activity that is enhanced by cysteine. Also, we proved that the expression of sty4221 has a cytotoxic effect on THP-I macrophages, but not on epithelial HeLa cells. Meanwhile, sty4221 does not seem to be important during adhesion, invasion, infection nor survival. The characterization of protein STY4221 might extend the list of known exotoxin of Typhi.

Pyridoxal 5’-phosphate (PLP), the active form of vitamin B6, is a cofactor for many enzymes involved in the metabolism of amino acids, amino acid derived metabolites and some amino sugars. PLP is one of the most versatile cofactors and the PLP-dependent enzymes catalyze a variety of reactions including transamination, decarboxylation, inter-conversion of L-and D-amino acids and removal or replacement of chemical groups bound at β or γ carbon of amino acids. The thesis describes the structural studies carried out on three PLP-dependent enzymes; N-acetylornithine aminotransferase (AcOAT), serine hydroxymethyltransferase (SHMT) and diaminopropionate ammonia lyase (DAPAL). Chapter 1 of the thesis begins with a brief introduction to PLP-dependent enzymes and their classification. This is followed by a review of structures of enzymes belonging to the subgroup II aminotransferases. The last section of chapter I contains a detailed description of the structures available till date for SHMT from various sources and the mutational studies carried out on SHMT. All the common experimental procedures and computational methods used for the current investigations are described in chapter II, as most of these are applicable to all structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization, and X-ray diffraction data collection. Computational methods include details of various programs used during data processing, structure determination, refinement, model building, structure validation and analysis. AcOAT is one of the key enzymes in arginine and lysine metabolism. AcOAT belongs to the fold type I (αfamily) subgroup II family of PLP dependent enzymes. Both S. typhimurium and E. coli have two genes each, one involved in the biosynthesis of arginine and another in the biodegradation of arginine. Biosynthetic AcOAT catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of L-glutamate and the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate in lysine biosynthesis. Meso-DAP and lysine, the products of lysine biosynthesis pathway, are known to function as cross-linking moieties in the peptidoglycan component of bacterial cell wall. Therefore N-acetylornithine aminotransferase could serve as a target for designing antibacterials. Chapter III gives the details of the work carried out on AcOAT. Two genes each from S. typhimurium and E. coli coding for biosynthetic and biodegradative AcOAT were cloned in E. coli, overexpressed and purified by Ni-NTA affinity chromatography. Of the four enzymes, biosynthetic AcOAT from S. typhimurium (sArgD) crystallized in the unliganded form and in the presence of the inhibitor gabaculine or one of the substrates L-glutamate, diffracted to a maximum resolution of 1.90 Å and contained a dimer in the asymmetric unit. The structure was determined by the molecular replacement method using human ornithine aminotransferase (hOAT) as the starting model. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop Lαk-βm in only one subunit. The Km and kcat/Km values determined with the purified sArgD suggested that the enzyme could accept both acetylornithine (AcOrn) and ornithine (Orn) as the substrates and had much higher affinity for AcOrn than for Orn. However, OAT accepts only Orn as the substrate. Comparison of the structurte of sArgD with T. thermophilus AcOAT and hOAT suggested that the higher specificity of sArgD towards AcOrn may not be due to specific differences in the active site residues but could result from minor conformational changes in some of them. sArgD was inhibited by gabaculine with an inhibition constant (Ki) of 7 µM and a second order rate constant (k2) of 0.16 mM-1s-1. The crystal structure of sArgD obtained in the presence of gabaculine and the spectral studies of sArgD with gabaculine suggested that the enzyme might have a low affinity for the PLP-gabaculine complex. Biosynthetic AcOAT from E. coli (eArgD) crystallized in the presence of gabaculine in hanging drop vapor diffusion method and diffracted X-rays only to a resolution of 3.5 Å. Two data sets were collected for the eArgD crystals. One of the data sets belonged to P1 (data 1) and the other to P321 space group (data 2) with a solvent content of ~70%. Data 1 was twinned and the unit cell was unusually large and could accommodate ~24 molecules in the asymmetric unit where as data 2 had four molecules in the asymmetric unit. Biodegradataive AcOAT from E. coli also crystallized in presence of gabaculine in hanging drop vapor diffusion method and suffered from low diffraction quality, where as that from S. typhimurium did not yield crystals. In chapter IV, X-ray crystallographic studies on various site specific mutants of SHMT from Bacillus stereotherophilus (bs) and a detailed comparison of structural data with the biochemical results in relation to mechanism of catalysis are presented. SHMT is a member of the α-class of PLP-dependent enzymes and catalyzes the reversible conversion of L-Ser and THF to glycine and 5,10-methylene THF. 5,10-methylene THF serves as a major source of one-carbon units in the biosynthesis of nucleotides and a few amino acids. SHMT also catalyses the cleavage of β-hydroxy amino acids like L-allo-threonine, transamination, racemization and decarboxylation reactions. SHMT shows increased activity along with enhanced nucleotide synthesis and therefore is a potential target for cancer chemotherapy. The availability of structural and biochemical data on SHMT from different sources ranging from human to E. coli enabled the identification of active site residues and a more critical examination of the role of these residues in the different steps of catalysis. The important mutants studied in the present investigation are E53Q, Y51F, Y61F, Y61A, Y60A, N341A and F351G of bsSHMT. The crystal structures of all these mutants are solved in the presence of various ligands, which gave many interesting results. E53, one of the active residues, interacts with the side chain hydroxyl group of serine bound to PLP in the wild type serine complex and N10 and formyl oxygen in the wild type glycine-FTHF complex. In E53Q glycine and serine complexes, glycine carboxyl and serine side chain were in two conformations, respectively, the new conformation being stabilized by their interaction with the mutated residue Q53. The structure of E53Q-Gly complex obtained in the presence and absence of 5-formyl THF(FTHF) showed an interesting case of enzyme memory in which the final conformational state depends on the way it was obtained and suggested that E53 is crucial for FTHF/THF binding. Though the spectrum showed that FTHF binds to the mutant initially, no density was observed for FTHF in the final structure. FTHF is believed to dissociate from the active site with prolonged incubation leaving behind a few significant conformational changes. Y51, one of the highly conserved tyrosines in SHMT, has hydrogen bonding interactions with the phosphate group of PLP and the active site lysine (K226) in bsSHMT. Mutation of Y51 to F resulted in significant changes at the active site. In all the structures of Y51F complexes, the phosphate group is in two conformations and F51 has moved away from the phosphate and in turn changed the position of Y61, another tyrosine in the active site. The residue Y61 is hydrogen bonded to R357 in the internal aldimine complex of bsSHMT. Addition of glycine/serine to bsSHMT resulted in the conformational change of Y61 away from R357 and towards E53, allowing the added glycine/serine to interact with R357. Mutation of Y61 to A did not bring significant structural changes. Structures of Y51F and Y61A mutants complexed with L-allo-Thr (cleaved to Gly by the wild type enzyme) showed that L-allo-Thr was not cleaved to glycine and acetaldehyde and confirmed the biochemical observation that these two residues are essential even for the THF-independent reaction. Residues Y60 and N341 are also highly conserved residues among SHMTs. Y60 stacks over PABA ring of FTHF in the wild type glycine-FTHF ternary complex. N341 has strong hydrogen bonding interactions with N1 and N8 atoms of the pteridine ring of FTHF. Mutation of either Y60 or N341 to A destroys the binding ability of FTHF/THF to the enzyme according to the biochemical and structural observations. The residue F351 exhibits different conformations in the two subunits of wild type glycine-FTHF ternary complex and is thought to be an important residue in determining the asymmetric binding of FTHF. Mutation of F351 to G did not affect the catalytic activity. Surprisingly, in the crystal structure obtained in the presence of L-allo-Thr, the ligand did not get cleaved to glycine, though in solution, the mutant is as active as the wild type enzyme. Chapter V describes the preliminary structural studies carried out on DAPAL from E. coli and S. typhimurium. DAPAL catalyzes the α, βelimination of both L-and D-diaminopropionate (DAP). DAP is the immediate precursor of two neurotoxins 3oxalyl and 2,3-dioxalyl DAP present in Lathyrus sativus, a grain legume rich in proteins and capable of growing well in drought conditions. The presence of these two neurotoxins precludes its use as a source of protein rich food. This enzyme is present only in bacteria and few species of actinomycetes. Unlike many other PLP-dependent enzymes, DAPAL does not catalyze any side reaction and is the only enzyme known to remove an amino group from the βcarbon of the substrate. The enzymes from E. coli (eDAPAL) and S. typhimurium (sDAPAL) produced diffraction quality crystals. However, crystals of sDAPAL did not survive heavy atom soaking and eDAPAL crystals suffered from poor reproducibility and severe non-isomorphism making it difficult to obtain suitable heavy atom derivatives for structure determination. Production of selenomethionine labelled proteins for these enzymes was initiated and thin crystals were obtained for eDAPAL. Improvement of the quality of these crystals is necessary in order to solve the structure of DAPAL by MAD method.

Salmonella enterica sérovar Typhi (Typhi) est une bactérie pathogène spécifique à l’homme. Typhi est l’agent étiologique de la fièvre typhoïde chez l’humain, causant plus de 16 millions de nouveaux cas par année et plus de 600 000 morts. Il a été démontré que pour causer une infection systémique, Salmonella doit nécessairement survivre dans les macrophages de l'hôte. Paradoxalement, S. enterica sérovar Typhimurium, très apparenté à Typhi (près de 90 % d’homologie), n’a pas la capacité de se disséminer dans l’organisme humain et peut infecter plusieurs espèces animales. Nous avons antérieurement identifié 36 gènes uniques à Typhi (absents chez Typhimurium) situés sur 15 régions différentes et exprimés sélectivement lors de l’infection de macrophages humains. Ainsi, l’une de ces régions a suscité notre attention, soit la région sty4217-4222 et plus particulièrement le produit du gène sty4221, une aminotransférase hypothétique. Ce dernier gène est d’intérêt dû à l’homologie qu’il détient avec une hémolysine connue (Hly) produite par Treponema denticola, possédant elle-même une activité d’aminotransférase. Chez T. denticola, Hly dégrade la cystéine et produit du H2S qui est toxique pour l’hôte. Notre hypothèse est que la spécificité d’hôte et la capacité de produire une infection systémique de Typhi sont dues à l’expression de gènes qui ne se retrouvent pas chez d’autres salmonelles. Le but de cette étude était donc de caractériser le gène sty4221 quant à son activité hémolytique, cytotoxique et tenter de déterminer son rôle dans la virulence de cette bactérie. Le gène sty4221 a été cloné sous le contrôle d’un promoteur inductible à l’arabinose et exprimé par E. coli. L’activité hémolytique du clone a été déterminée par simple observation sur gélose sang. Ce clone a également permis d’observer l’effet cytotoxique du surnageant de culture sur différentes lignées cellulaires, par quantification de la relâche de LDH. Le gène sty4221 a été muté chez la souche sauvage de Typhi, ISP1820, l’implication pathogénique du gène a ainsi pu être étudiée. Des tests de phagocytose, d’invasion et de survie dans des macrophages humains ont été effectués, ainsi que des tests d’adhésion et d’invasion sur des cellules HeLa. Par ailleurs, une première tentative de purification de la protéine a été entreprise. En somme, nous savons maintenant que STY4221 a des propriétés hémolytiques, augmentées par la présence de cystéine. De plus, STY4221 a un effet cytotoxique sur les macrophages THP-I, mais aucun effet sur les HeLa. Or, sty4221 ne semble pas impliqué dans les étapes d’adhésion, d’invasion, de phagocytose ou de survie. La caractérisation de sty4221 permettra sans doute d’approfondir nos connaissances sur les toxines trouvées uniquement chez Typhi. / Salmonella enterica serovar Typhi (Typhi) is a human restricted pathogen causing typhoid fever, a systemic infection. Annually, at least 16 million new cases with 600, 000 associated deaths are reported. It has been demonstrated that Salmonella has to survive in the macrophages of its host, in order to produce a systemic disease. This ability to cause a disseminated infection in human is unique to Typhi. Our laboratory had isolated 36 genes that were unique to Typhi (absent from Typhimurium’s genome), and that were expressed during human macrophages infection. One of these genes, sty4221, a putative aminotransferase, was of high interest since it shares sequence similarities with a known hemolysin (Hly), which also possesses an aminotransferase activity. That hemolysin is produced by Treponema denticola, it catabolizes cysteine and produces H2S, a toxic metabolite for the host. Our hypothesis is that host specificity and the ability to cause a systemic infection might be explained by the expression of genes that are not found in other salmonellas. The goal of this study was to characterize the gene sty4221, in terms of hemolytic and cytotoxic activity and to determine its role in virulence. The sty4221gene has been cloned in a vector under an arabinose inducible promoter and transformed in a strain of E. coli. The hemolytic activity has been investigated on blood-agar medium. To evaluate the cytotoxicity of the STY4221 protein on human cultured cells, direct observation by photonic microscopy was done. The cytotoxicity activity on human cultured cells has been quantitatively measured with a lactate dehydrogenase release assay. Moreover, the sty4221 gene has been deleted in order to study its implication in the infection and the survival within human macrophages and for adhesion/invasion on epithelial. Protein purification was also attempted. We now know that protein STY4221 has a hemolytic activity that is enhanced by cysteine. Also, we proved that the expression of sty4221 has a cytotoxic effect on THP-I macrophages, but not on epithelial HeLa cells. Meanwhile, sty4221 does not seem to be important during adhesion, invasion, infection nor survival. The characterization of protein STY4221 might extend the list of known exotoxin of Typhi.

The objective of the present work was to identify serum copper levels and aspartateaminotransferase (AST) and gamagluthamyl transferase (GGT) activity in purebred show and field raised sheep and to contribute to formation of regional reference values. Blood samples of 120 show sheep and 110 field raised animals were collected independently of age, sex and breed. Means serum levels of copper for show and field sheep 66.14 ± 9.76 and 62.01 ±16.36 μg.dl-1 (P<0.0357), respectively; AST activity was higher in show sheep than in field animals (85.01 ±40.02 vs. 65.58 ±16.19 IU, P<0.0001); GGT activity was 41.02 ±10.61 and 41.91 ±9.49 IU with no differences between groups. In conclusion, serum copper levels in all show animals were within international reference values, whereas field raised animals presented several individuals that fell bellow the lower limit. Serum activitiy of AST and GGT appear not to be related to copper metabolism in these animals. / O objetivo desse trabalho foi identificar as concentrações séricas de cobre e enzimas aspartatoaminotransferase (AST) e gamaglutamiltransferase (GGT) tanto em ovinos preparados para exposição como criados a campo, a fim de contribuir para a formação de valores de referência e auxiliar no prognóstico da saúde desses animais. Colheram-se 120 amostras de sangue de animais de exposição e 110 de animais de campo, independente de sexo, raça e idade. Os níveis séricos médios de cobre, AST e GGT dos animais de exposição e a campo foi igual a 66,14 ±9,76 e 62,01 ±16,36 μg.dl-1 (P<0,0357); 85,01 ±40,02 e 65,58 ±16,19 UI (P<0,0001); 41,02 ±10,61 e 41,91 ±9,49 UI, respectivamente. Em conclusão, as médias do elemento cobre de animais preparados para exposição mostraram-se dentro dos valores de referência e as de alguns animais mantidos a campo abaixo do intervalo fisiológico. Os níveis séricos de AST e GGT aumentados no grupo exposição parecem não estar relacionados com o acúmulo hepático de cobre.

Les acides aminés sont des molécules importantes pour la synthèse des protéines, la fourniture de substrats énergétiques ou la production de facteurs de régulation de l'activité cellulaire. L'ornithine aminotransférase {OAT} est une enzyme clef du métabolisme des acides aminés qui relie l'arginine, un acide aminé indispensable à la synthèse protéique au cours de la croissance, et le glutamate, la glutamine et l'a-cétoglutarate, qui peuvent être transformées en glucose. Nous nous sommes intéressés à la caractérisation biochimique de l'activité de l'OAT et à ses variations chez la souris, en utilisant deux contraintes physiologiques, le développement postnatal et l'exposition au froid. Au cours d'une première étude, nous avons caractérisé une activité OAT fonctionnelle dans tous les organes étudiés. Nous avons démontré l'existence d'un dimorphisme sexuel de l'activité, en faveur des femelles, dans la plupart des organes. Dans une seconde étude, nous avons montré des variations organe et sexe spécifiques de l'activité OAT au cours du développement postnatal en réponse aux besoins spécifiques en acides aminés : une régulation de l'expression de l'OAT par la testostérone et le changement de régime alimentaire lors du sevrage ; une contribution potentielle importante et insoupçonnée du muscle squelettique au métabolisme de l'ornithine. Enfin, nous avons mis en évidence que l'exposition au froid augmente ou diminue l'activité OAT de façon tissu-spécifique en liaison avec les besoins énergétiques de l'organisme. Nos résultats montrent que l'activité OAT est modulée dans les différents organes en fonction des besoins en acides aminés et en substrats. Cela suggère un rôle important du métabolisme des acides aminés dans les réponses adaptatives des organismes à leur environnement / Amino acids are important molecules for protein synthesis and energetic substrates supply. Ornithine aminotransferase {OAT} is a key enzyme of amino acids metabolism linking arginine, an essential amino acid for protein synthesis during growth, and glutamate, glutamine and alpha-ketoglutarate which could be converted into glucose. We principally focused on biochemical characterization of OAT activity and its variations in the mouse using two physiological constraints, postnatal development and cold exposition. During a first study, we characterized a functional OAT activity in all the males and females mice organs studied. We showed the existence of a sexual dimorphism of OAT activity in almost all the organs studied, with a higher OAT activity in females. ln a second study, we showed that the variations of OAT activity were organ and sex specific during the postnatal development in response to the specific needs in amino acids during the growth. We linked these variations to the regulation of OAT expression by testosterone and the change of the diet during weaning. We demonstrated an important and unsuspected contribution of skeletal muscles in pups' ornithine metabolism. Finally, we showed that cold exposition modulates OAT activity in a tissue specific way, in relation with energetic needs and the multiple roles of the substrates generated by the metabolic pathways of OAT. Our results show that in the different organs, OAT activity is modulated in function of amino acids needs, energetic substrates or detoxification needs of the organism during the different metabolic constraints. This is suggesting an important role for the amino acids metabolism in adaptive responses of organisms to their environment

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