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L'îlot de multirésistance aux antibiotiques, Salmonella Genomic Island 1 (SGI1) : variabilité, diffusion inter - espèces et implication dans la virulenceTargant, Hayette 27 September 2010 (has links) (PDF)
Les salmonelles sont l'une des premières causes d'infections bactériennes d'origine alimentaire. Depuis le début des années 1990, l'isolement de salmonelles multirésistantes aux antibiotiques a considérablement accru avec l'émergence des souches épidémiques Salmonella Typhimurium DT104 qui sont, pour la majorité, résistantes à l'ampicilline, le chloramphénicol, la streptomycine, les sulfamides et les tétracyclines. Les gènes codant ces résistances sont regroupés sur un intégron complexe de classe 1 nommé In104, localisé lui-même sur un îlot génomique de 43 kb désigné Salmonella Genomic Island 1 (SGI1). Depuis sa première identification chez S. Typhimurium DT104, SGI1 a été identifié à travers le monde chez plusieurs sérovars de Salmonella, et plus récemment chez Proteus mirabilis. Chez ces souches, la multirésistance aux antibiotiques est liée, soit à l'îlot SGI1 dans sa forme initialement décrite, soit à des variants de SGI1 correspondant à la structure initiale de SGI1 comportant des modifications au niveau de l'intégron complexe In104. L'îlot génomique Salmonella Genomic Island 1 (SGI1) représente une préoccupation importante car le phénotype de multirésistance qu'il confère aux souches bactériennes est souvent responsable d'échecs thérapeutiques pouvant entrainer des complications importantes, voire la mort. Dans ce contexte, le travail de thèse a été centré sur l'enjeu sanitaire majeur représenté par cette diffusion épidémique du clone S. Typhimurium au cours des années 1990 chez l'homme et les bovins. Les travaux entrepris dans le cadre de la thèse ont eu, en premier lieu, l'objectif d'apprécier l'évolution moléculaire de SGI1 dix années après l'émergence de ces souches en élevage bovin, puis d'évaluer la diffusion de SGI1 chez des souches naturelles appartenant à d'autres genres bactériens que Salmonella. Il a ainsi été dressé un bilan de la multirésistance aux antibiotiques chez les souches de S. Typhimurium isolées de bovins malades en France de 2002 à 2007 et une recherche de la présence de SGI1, chez d'autres espèces bactériennes que Salmonella, et par sondage à partir de leurs phénotypes de résistance, a été mise en œuvre. Les résultats obtenus ont indiqué un faible pouvoir évolutif de SGI1 qui semble en contradiction avec les capacités moléculaires majeures de recombinaison et de transfert démontrées tant in vitro qu'in vivo. Les études menées ont toutefois permis la première description d'un nouveau variant, nommé SGI1-T, qui résulte d'une recombinaison intramoléculaire. Le deuxième grand objectif de la thèse a été de contribuer à une meilleure connaissance du rôle que pourrait avoir SGI1 dans la virulence bactérienne. Une première stratégie de modélisation expérimentale (salmonellose systémique murine) a ainsi été conduite, qui visait à comparer le pouvoir virulent in vivo de souches isogéniques ne se distinguant que par la présence ou l'absence de SGI1. Une seconde approche a été également menée, qui a consisté en une évaluation du rôle de SGI1 dans la formation de biofilms, l'organisation en biofilms favorisant une meilleure colonisation bactérienne, qui peut constituer à son tour un élément d'efficacité du pouvoir virulent final. Les résultats obtenus ont confirmé le rôle positif de SGI1 dans la formation de biofilms, et plus généralement son implication dans la signalisation cellulaire du Quorum Sensing.
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Stress Response In Salmonella And Its Role In PathogenesisLahiri, Amit 07 1900 (has links)
Chapter: 1
Introduction
Genus Salmonella is a Gram-negative rod shaped facultative anaerobic bacteria that can survive inside the host macrophages and cause persistent infection. Salmonella Typhimurium, Salmonella Typhi and Salmonella Enteritidis are the serovars, which belong to the Salmonella enterica species. S. Typhi causes typhoid fever in humans. S. Typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice and serves as a good model system to study Salmonella pathogenesis. Salmonella infection occurs via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18+ macrophages present in the intestinal lumen or via a forced entry in the non phagocytic enterocytes. Upon entry Salmonella resides in an intracellular phagosomal compartment called the Salmonella containing vacuole (SCV). The SCV only transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7. It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position.
The mechanisms by which phagocytes kill the virulent Salmonella are not completely understood, however the role of nicotinamide-adenine dinucleotide phosphate (NADPH) phagocytic oxidase system has been strongly implicated. The generation of reactive oxygen species (ROS) occurs via a membrane-bound flavocytochrome b558, consisting of two phagocytic oxidase components (gp91phox and p22phox) and four cytosolic components, p40phox, p47phox, p67phox, and a GTP-binding Rac protein. Further, professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella.
Chapter:2
Resistance to host Nitrosative stress in Salmonella by quenching L-arginine.
Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of L-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate L-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor aminoguanidine reverts the attenuation in arginase blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of L-arginine. In the whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner.
Chapter:3
Hrg (hydrogen peroxide resistant gene), a LysR type transcriptional regulator confers resistance to oxidative stress in Salmonella
LysR type transcriptional regulators are one of the key players that help bacteria adapt to different environments. We have christened STM0952, a putative LysR type transcriptional regulator in Salmonella enterica serovar Typhimurium as the hydrogen peroxide resistance gene (hrg). By generating a knock out of the hrg gene, we demonstrate that the hrg mutant serovar Typhimurium is sensitive to oxidative products of the respiratory burst, specifically to hydrogen peroxide. The hrg mutant is profoundly attenuated in the murine model of infection and shows decreased intracellular proliferation in macrophages. It was also found to induce increased amount of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild type. An overproducing strain of this gene showed a survival advantage over the wild type Salmonella under hydrogen peroxide induced stress condition. Microarray analysis suggested the presence of a Hrg regulon, which is required for resistance to the toxic oxidative products of the reticulo-endothelial system.
Chapter:4
Importance of the host oxidative stress in antigen presentation and its modulation by Salmonella: Role of TLR
Synthetic CpG containing oligodeoxynucleotide TLR-9 agonist (CpG ODN) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. We report that Salmonella Typhimurium growth can be inhibited by the CpG ODN treatment in the murine dendritic cells. This inhibitory effect was shown to be mediated by an increased reactive oxygen species (ROS) production. We further show that the CpG ODN treatment of the dendritic cells during Salmonella infection leads to a ROS dependent increased antigen presentation. In addition, TLR-9 signaling inhibitor was able to inhibit the CpG ODN mediated increased antigen presentation, ROS production and pathogen killing. These data indicate that CpG ODN can improve the ability of the murine dendritic cells to contain the growth of the virulent Salmonella through ROS dependent killing and could as well be used as an effective adjuvant in vaccines against Salmonella infection.
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Anti-salmonella adhesion activity of Saccharomyces boulardii ; Effects of of Ginkgo biloba on activities of Cytochromes P-450 /Mohutsky, Michael A. January 2002 (has links)
Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 207-232).
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Small Proline Rich Protein-2 Expression and Regulation in the Caco-2 model of Intestinal Epithelial Differentiation along the Crypt-Villus AxisHui, Patrick J.H. 28 April 2008 (has links)
Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs).
Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium.
SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells.
We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-04-25 12:39:06.427 / This work was funded by the CIHR GIDRU Training Grant and Aid in Research from Crohn's and Colitis Foundation of Canada
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Analysis of regulatory systems in two different gram⁻ bacteria /Adams, Curtis W. January 1998 (has links)
Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [119]-139).
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Atividade mutagênica em solos sob a influência de rejeitos de carvãoSilva Júnior, Flavio Manoel Rodrigues da January 2008 (has links)
O solo é um compartimento ambiental altamente complexo e, em razão do crescimento populacional acelerado e da intensa atividade industrial e agrícola, tem sofrido com a contaminação de substâncias de origens diversas. Entre as principais atividades que contribuem para a perturbação da qualidade dos solos estão as atividades de geração de energia pela queima de combustíveis fósseis, como o carvão mineral. Dentre as substâncias presentes nos rejeitos do carvão queimado em usinas termelétricas estão compostos orgânicos e metais pesados que interagem com o material genético, produzindo mutações e acarretando prejuízos em nível de organismo (como as neoplasias), mas também em níveis maiores de organização biológica (como a perda de diversidade genética em populações). Estudos de avaliação do potencial mutagênico em amostras de solo são escassos, principalmente investigando a ação de mutágenos de origem inorgânica. Além disso, estudos de mutagênese em solos sob a influência de rejeitos de carvão têm sido pouco abordados na literatura. Desta forma, os objetivos deste trabalho foram: (i) testar um protocolo para avaliação de atividade mutagênica em extratos inorgânicos e orgânicos de solo; (ii) avaliar a presença e o perfil dos compostos mutagênicos em solos sob a influência de cinzas de carvão e (iii) investigar as rotas ambientais para dispersão dos compostos mutagênicos na área de estudo. Para atingir estes objetivos, processos de extração de compostos orgânicos e inorgânicos de solo foram testados quanto a sua eficiência em extrair da amostra compostos potencialmente mutagênicos frente a linhagem de Salmonella typhimurium que detecta erro no quadro de leitura (TA98). Os protocolos mais eficientes foram utilizados para os testes com diferentes amostras de solo sob a influência de rejeitos de carvão utilizando diversas linhagens, na ausência e presença de ativação metabólica (fração S9 mix). Os resultados de mutagênese associados aos resultados da caracterização química de compostos orgânicos e metais indicaram que os contaminantes presentes nas cinzas de carvão podem se dispersar com facilidade para áreas adjacentes. Essa abordagem de estudo permitiu relacionar a presença de determinadas classes de compostos com diferentes danos no DNA e inferir distintas rotas ambientais de dispersão para esses compostos. A realização deste trabalho ressalta a importância de estudos em matrizes ambientais complexas, em especial solos contaminados, que buscam integrar os dados de mutagênese e utilizá-los sob uma perspectiva ecológica. / Soil is a highly complex environmental compartment that has suffered contamination by substances from multiple sources mainly due to fast population growth and intense industrial and agricultural activity. Among the main activities that affect soil quality are power generation activities that use fossil fuels, such as mineral coal. Among the several compounds present in coal ashes that are employed in coal-fired power plants are substances that interact with the genetic material, causing mutations and/or damage at the individual level (like neoplasias) but also at higher levels of biological organization (like loss of genetic diversity in populations). There are few studies on the mutagenic potential of soil samples, especially those that investigate the action of mutagens from inorganic sources. Moreover, studies of mutagenesis in soils under the influence of coal-fired power plants have rarely been reported in literature. Thus, this work aimed at: (i) testing a protocol for the evaluation of mutagenic activity in inorganic and organic extracts from soil samples; (ii) evaluating the presence and the profile of mutagenic compounds in soils under the influence of coal ashes and (iii) investigating environmental routes of dispersion of mutagenic compounds in the study area. In order to achieve these aims, extraction processes for inorganic and organic compounds were tested concerning their efficiency to extract compounds potentially mutagenic to the specific Salmonella typhimurium strain that detects frameshift mutagens (TA98). The most efficient protocols were further utilized for the tests with different soil samples under the influence of coal-fired power plant using several strains, in absence and presence of metabolic activation (S9 mix fraction). Mutagenesis results associated with the chemical characterization of the organic compounds and metals indicated that contaminants present in coal ashes can be easily dispersed to adjacent areas. This approach allowed relating the presence of certain classes of compounds to specific damages in DNA and inferring distinct dispersion routes for these compounds. This work highlights the importance of studies concerning complex environmental matrices, specially contaminated soils, which seek the integration of mutagenesis data and their use from an ecological perspective.
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Atividade mutagênica em solos sob a influência de rejeitos de carvãoSilva Júnior, Flavio Manoel Rodrigues da January 2008 (has links)
O solo é um compartimento ambiental altamente complexo e, em razão do crescimento populacional acelerado e da intensa atividade industrial e agrícola, tem sofrido com a contaminação de substâncias de origens diversas. Entre as principais atividades que contribuem para a perturbação da qualidade dos solos estão as atividades de geração de energia pela queima de combustíveis fósseis, como o carvão mineral. Dentre as substâncias presentes nos rejeitos do carvão queimado em usinas termelétricas estão compostos orgânicos e metais pesados que interagem com o material genético, produzindo mutações e acarretando prejuízos em nível de organismo (como as neoplasias), mas também em níveis maiores de organização biológica (como a perda de diversidade genética em populações). Estudos de avaliação do potencial mutagênico em amostras de solo são escassos, principalmente investigando a ação de mutágenos de origem inorgânica. Além disso, estudos de mutagênese em solos sob a influência de rejeitos de carvão têm sido pouco abordados na literatura. Desta forma, os objetivos deste trabalho foram: (i) testar um protocolo para avaliação de atividade mutagênica em extratos inorgânicos e orgânicos de solo; (ii) avaliar a presença e o perfil dos compostos mutagênicos em solos sob a influência de cinzas de carvão e (iii) investigar as rotas ambientais para dispersão dos compostos mutagênicos na área de estudo. Para atingir estes objetivos, processos de extração de compostos orgânicos e inorgânicos de solo foram testados quanto a sua eficiência em extrair da amostra compostos potencialmente mutagênicos frente a linhagem de Salmonella typhimurium que detecta erro no quadro de leitura (TA98). Os protocolos mais eficientes foram utilizados para os testes com diferentes amostras de solo sob a influência de rejeitos de carvão utilizando diversas linhagens, na ausência e presença de ativação metabólica (fração S9 mix). Os resultados de mutagênese associados aos resultados da caracterização química de compostos orgânicos e metais indicaram que os contaminantes presentes nas cinzas de carvão podem se dispersar com facilidade para áreas adjacentes. Essa abordagem de estudo permitiu relacionar a presença de determinadas classes de compostos com diferentes danos no DNA e inferir distintas rotas ambientais de dispersão para esses compostos. A realização deste trabalho ressalta a importância de estudos em matrizes ambientais complexas, em especial solos contaminados, que buscam integrar os dados de mutagênese e utilizá-los sob uma perspectiva ecológica. / Soil is a highly complex environmental compartment that has suffered contamination by substances from multiple sources mainly due to fast population growth and intense industrial and agricultural activity. Among the main activities that affect soil quality are power generation activities that use fossil fuels, such as mineral coal. Among the several compounds present in coal ashes that are employed in coal-fired power plants are substances that interact with the genetic material, causing mutations and/or damage at the individual level (like neoplasias) but also at higher levels of biological organization (like loss of genetic diversity in populations). There are few studies on the mutagenic potential of soil samples, especially those that investigate the action of mutagens from inorganic sources. Moreover, studies of mutagenesis in soils under the influence of coal-fired power plants have rarely been reported in literature. Thus, this work aimed at: (i) testing a protocol for the evaluation of mutagenic activity in inorganic and organic extracts from soil samples; (ii) evaluating the presence and the profile of mutagenic compounds in soils under the influence of coal ashes and (iii) investigating environmental routes of dispersion of mutagenic compounds in the study area. In order to achieve these aims, extraction processes for inorganic and organic compounds were tested concerning their efficiency to extract compounds potentially mutagenic to the specific Salmonella typhimurium strain that detects frameshift mutagens (TA98). The most efficient protocols were further utilized for the tests with different soil samples under the influence of coal-fired power plant using several strains, in absence and presence of metabolic activation (S9 mix fraction). Mutagenesis results associated with the chemical characterization of the organic compounds and metals indicated that contaminants present in coal ashes can be easily dispersed to adjacent areas. This approach allowed relating the presence of certain classes of compounds to specific damages in DNA and inferring distinct dispersion routes for these compounds. This work highlights the importance of studies concerning complex environmental matrices, specially contaminated soils, which seek the integration of mutagenesis data and their use from an ecological perspective.
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Estratégias para redução da produção de acetato em cultivos de Salmonella typhimuriumFuzer Neto, José Roberto 23 February 2017 (has links)
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Previous issue date: 2017-02-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / In recent years, the application of attenuated Salmonella spp. has been investigated for development of several biotechnological products, mainly vaccines. However, the implementation of industrial processes to obtain these products depends on the development of strategies for this microorganism high-cell density cultures (HCDC). One of the HCDC’s greatest challenges is overcoming Salmonella’s metabolic limitations, as it presents a high organic acids production (mainly acetic acid) that inhibits biomass formation. In this context, this work proposes two approaches to deal with this problem and implement Salmonella’s HCDC: studying the cultivation of S. typhimurium using glycerol as carbon source to reduce the generation of organic acids; studying the cultivation of a recombinant strain of S. typhimurium expressing the enzyme acetyl-CoA synthetase (ACS) from E. coli, for improved acetate assimilation). Initially, cultures were grown in agitated flasks in minimal media for two carbon sources (glucose or glycerol) for the wild-type and the recombinant strain. After the preliminary experiments, the recombinant strain was cultivated in bioreactor operated in batch mode with minimal medium formulated with glycerol, glucose or acetic acid as carbon source, to evaluate acetate production and assimilation. The wild-type strain was cultivated in a continuous-mode bioreactor on minimal medium with glycerol at D=0.10; 0.17 and 0.22 h-1 to evaluate the S. typhimurium glycerol metabolism. During the cultivations, samples were collected and analyzed by high-performance liquid chromatography to quantify the production of organic acids and substrate consumption. To quantify the concentration of biomass, optical density (600 nm) readings of culture broth and dry cell weight measurements were performed. Agitated flasks and batch cultivations results indicated that the acetate production is reduced in medium with glycerol for both strains, and that the genetically modified cells present a lower acetate accumulation phenotype compared to the wild-type. Continuous cultures of the wild-type strain showed no acetate accumulation for a 0.1 h-1 dilution rate. At rates of 0.17 h-1 and 0.22 h-1 acetate accumulation was observed, but acetate flux was 2-fold lower than the flux reached in chemostat with glucose-formulated medium. Simulations were performed with the STM_v1.0 model using as input data the glycerol and oxygen fluxes estimated from the experimental results. Good predictions were obtained for the biomass, CO2 and acetate fluxes at the higher dilution rates. The results suggest that fed-batch culture using glycerol as carbon source along with an exponential feed to maintain a 0.1 h-1 specific growth rate as a promising strategy to obtain high cellular concentrations of wild-type S. typhimurium. The efficient acetate uptake observed for the recombinant S. typhimurium cells may allow higher values of specific growth rate to be used for this strain, resulting in a higher productivity of biomass. / Nos últimos anos, a aplicação de linhagens atenuadas de Salmonella spp. vem sendo amplamente investigada para o desenvolvimento de diversos produtos biotecnológicos, principalmente vacinas. No entanto, a implementação de processos industriais para a obtenção destes produtos depende do desenvolvimento de estratégias para o cultivo em alta densidade celular (CADC) deste microrganismo. Para isso, um dos grandes desafios a ser superado se refere às limitações metabólicas da Salmonella, uma vez que esta apresenta elevada produção de ácidos orgânicos (principalmente ácido acético) que inibem a formação de biomassa. Neste contexto, este trabalho propõe duas abordagens para lidar com este problema e implementar CADC de Salmonella: estudar o crescimento de Salmonella typhimurium em glicerol avaliando seu metabolismo como uma fonte de carbono menos propensa à geração de ácidos orgânicos; e estudar a produção destes ácidos por uma cepa de S. typhimurium geneticamente modificada para diminuir o acúmulo de acetato (super-expressão do gene acs, de Escherichia coli, que codifica a enzima acetil-CoA sintetase (ACS) responsável pela assimilação de acetato). Inicialmente foram realizados cultivos em frascos agitados em meio mínimo para duas fontes de carbono (glicose ou glicerol), para a cepa selvagem e para a cepa recombinante. Após os experimentos preliminares, foram realizados cultivos em biorreator operado em modo batelada com a cepa modificada em meio mínimo formulado com glicerol, glicose ou ácido acético, como fonte de carbono, a fim de avaliar a produção e a assimilação de acetato. Foram realizados cultivos em biorreator em modo contínuo com a cepa selvagem em meio mínimo com glicerol com D=0,10; 0,17 e 0,22 h-1 para avaliar o metabolismo do glicerol pela S. typhimurium. Ao longo dos cultivos foram coletadas amostras do caldo e analisadas por cromatografia líquida de alta eficiência para quantificar a produção de ácidos orgânicos e o consumo de substrato. Para quantificar a biomassa produzida, foram realizadas medidas de densidade ótica (600 nm) e de massa seca. Os resultados dos cultivos em frascos agitados e das bateladas indicam que, para ambas as cepas, a produção de acetato é reduzida em meio formulado com glicerol, e que as células modificadas geneticamente apresentam um fenótipo de menor acúmulo de acetato comparadas à linhagem selvagem. Os cultivos contínuos realizados com a cepa selvagem mostraram que não houve acúmulo de acetato para a taxa de diluição de 0,1 h-1. Já nas taxas de 0,17 h-1 e 0,22 h-1, apesar de haver acúmulo, o fluxo de produção de acetato foi cerca de 2 vezes menor que o observado em quimiostato em meio formulado com glicose. Foram realizadas simulações com o modelo STM_v1.0, tendo como dados de entrada os fluxos de glicerol e de oxigênio estimados a partir dos dados experimentais. O modelo descreveu bem os fluxos de biomassa, CO2 e acetato para as taxas de diluição mais altas. Os resultados obtidos sugerem o cultivo em batelada alimentada com glicerol como fonte de carbono, e alimentação exponencial definida para manter a velocidade específica de crescimento em 0,1 h-1, como uma estratégia promissora para obter altas concentrações celulares de S. typhimurium selvagem. Para a S. typhimurium recombinante, devido à sua eficiente assimilação de acetato, valores ainda maiores de velocidade específica de crescimento poderiam ser impostos, com elevado aumento de produtividade em biomassa.
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Atividade mutagênica em solos sob a influência de rejeitos de carvãoSilva Júnior, Flavio Manoel Rodrigues da January 2008 (has links)
O solo é um compartimento ambiental altamente complexo e, em razão do crescimento populacional acelerado e da intensa atividade industrial e agrícola, tem sofrido com a contaminação de substâncias de origens diversas. Entre as principais atividades que contribuem para a perturbação da qualidade dos solos estão as atividades de geração de energia pela queima de combustíveis fósseis, como o carvão mineral. Dentre as substâncias presentes nos rejeitos do carvão queimado em usinas termelétricas estão compostos orgânicos e metais pesados que interagem com o material genético, produzindo mutações e acarretando prejuízos em nível de organismo (como as neoplasias), mas também em níveis maiores de organização biológica (como a perda de diversidade genética em populações). Estudos de avaliação do potencial mutagênico em amostras de solo são escassos, principalmente investigando a ação de mutágenos de origem inorgânica. Além disso, estudos de mutagênese em solos sob a influência de rejeitos de carvão têm sido pouco abordados na literatura. Desta forma, os objetivos deste trabalho foram: (i) testar um protocolo para avaliação de atividade mutagênica em extratos inorgânicos e orgânicos de solo; (ii) avaliar a presença e o perfil dos compostos mutagênicos em solos sob a influência de cinzas de carvão e (iii) investigar as rotas ambientais para dispersão dos compostos mutagênicos na área de estudo. Para atingir estes objetivos, processos de extração de compostos orgânicos e inorgânicos de solo foram testados quanto a sua eficiência em extrair da amostra compostos potencialmente mutagênicos frente a linhagem de Salmonella typhimurium que detecta erro no quadro de leitura (TA98). Os protocolos mais eficientes foram utilizados para os testes com diferentes amostras de solo sob a influência de rejeitos de carvão utilizando diversas linhagens, na ausência e presença de ativação metabólica (fração S9 mix). Os resultados de mutagênese associados aos resultados da caracterização química de compostos orgânicos e metais indicaram que os contaminantes presentes nas cinzas de carvão podem se dispersar com facilidade para áreas adjacentes. Essa abordagem de estudo permitiu relacionar a presença de determinadas classes de compostos com diferentes danos no DNA e inferir distintas rotas ambientais de dispersão para esses compostos. A realização deste trabalho ressalta a importância de estudos em matrizes ambientais complexas, em especial solos contaminados, que buscam integrar os dados de mutagênese e utilizá-los sob uma perspectiva ecológica. / Soil is a highly complex environmental compartment that has suffered contamination by substances from multiple sources mainly due to fast population growth and intense industrial and agricultural activity. Among the main activities that affect soil quality are power generation activities that use fossil fuels, such as mineral coal. Among the several compounds present in coal ashes that are employed in coal-fired power plants are substances that interact with the genetic material, causing mutations and/or damage at the individual level (like neoplasias) but also at higher levels of biological organization (like loss of genetic diversity in populations). There are few studies on the mutagenic potential of soil samples, especially those that investigate the action of mutagens from inorganic sources. Moreover, studies of mutagenesis in soils under the influence of coal-fired power plants have rarely been reported in literature. Thus, this work aimed at: (i) testing a protocol for the evaluation of mutagenic activity in inorganic and organic extracts from soil samples; (ii) evaluating the presence and the profile of mutagenic compounds in soils under the influence of coal ashes and (iii) investigating environmental routes of dispersion of mutagenic compounds in the study area. In order to achieve these aims, extraction processes for inorganic and organic compounds were tested concerning their efficiency to extract compounds potentially mutagenic to the specific Salmonella typhimurium strain that detects frameshift mutagens (TA98). The most efficient protocols were further utilized for the tests with different soil samples under the influence of coal-fired power plant using several strains, in absence and presence of metabolic activation (S9 mix fraction). Mutagenesis results associated with the chemical characterization of the organic compounds and metals indicated that contaminants present in coal ashes can be easily dispersed to adjacent areas. This approach allowed relating the presence of certain classes of compounds to specific damages in DNA and inferring distinct dispersion routes for these compounds. This work highlights the importance of studies concerning complex environmental matrices, specially contaminated soils, which seek the integration of mutagenesis data and their use from an ecological perspective.
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Modelo teórico de estimativa de risco de Salmonella Enteritidis em sistema integrado de produção de frango de corte e tipagem molecular de Salmonella spp. oriundas de aves e rações submetidas a diferentes tratamentos com ácido / Theoretical model of risk assessment of Salmonella Enteritidis in broiler chicken production integrated system and molecular typing of Salmonella spp. from birds and feed submitted to decontamination with different organic acids.Oyama Rodrigues da Silva 28 September 2007 (has links)
O presente trabalho objetivou identificar os fatores de risco para a presença de S. Enteritidis no sistema de produção de frangos de corte, avaliar, qualificar e quantificar as variáveis encontradas e elaborar um modelo teórico de estimativa de risco deste sorovar em frangos criados em sistema de integração. Os dados foram obtidos de trabalhos recentes realizados por alguns autores e deram subsídios à realização de uma análise de riscos microbiológicos. Para caracterização molecular foram utilizadas 42 cepas de Salmonella isoladas de frangos e rações inoculados experimentalmente com uma cepa de S. Typhimurium. A inoculação da bactéria foi realizada na ração e a mesma tratada com diferentes concentrações dos ácidos propílico, fórmico e acético sendo, então, fornecida para consumo ad libitum até os 21 dias de idade, quando as aves foram sacrificadas. Foram obtidos diferentes perfis genéticos com o uso do ERIC e BOX-PCR, que se mostraram eficientes para discriminação das cepas em estudo. / The aim of this work was identify the risk factors for S. Enteritidis in the production system of broiler chickens, to evaluate, qualify and quantify the variables studied and to make a theoretical model of risk assessment of this serovar in broilers in integration system. Therefore, the data was obtain from works of some authors and supported the proposed model of microbiological risk analysis. For molecular characterization were included 42 Salmonella spp. strains isolated from chicks and feed experimentally inoculated with S. Tiphimurium. After inoculation of feed with the specific dose of strain, it was submitted to treatment with propilic, formic and acetic acids in several concentrations and it was given to birds ad libitum until 21 days old, when they were sacrificed. It was obtained different patterns through the ERIC and BOX-PCR techniques, which showed good discrimination power for the strains analyzed.
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