O objetivo deste trabalho foi investigar o efeito da castração, tempo de confinamento, metabolismo celular e regiões do musculo sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. Oitenta e quatro bovinos (castrados e inteiros) Nelore, confinados por diferentes períodos, foram usados para conduzir estudos em músculos Longissimus dorsi e Biceps femoris. O segundo musculo foi dividido em duas porções: origem (PO) e inserção (PI). No estudo com L. dorsi, bifes foram embalados sob condições de aerobiose (PVC) e anaerobiose (vácuo) e maturados por 1, 3, 5, 7 e 9, e 1, 7, 14 e 21 dias, respectivamente. Para este músculo, nenhuma diferença na estabilidade oxidativa [tióis, carbonilas e Substancias Reativas ao Ácido Tiobarbiturico (TBARS)] e cor entre as carnes dos animais inteiros e castrados foram encontradas. Isto poderia ser explicado pela falta de diferença no status oxidativo inicial, mensurados através da atividade de enzimas antioxidantes, conteúdo de glutationa total e composição de ácidos graxos, entre as condições sexuais. Os resultados também indicaram que a oxidação dos bifes embalados a vácuo leva o dobro de dias para iniciar, em comparação aos bifes em aerobiose. No estudo com o Bíceps femoris, os animais foram abatidos com 59 e 129 dias de confinamento e os bifes da PO e PI foram maturados por 1, 30, 60 e 100 dias. Os resultados da atividade das enzimas lactato desidrogenase e citrato sintase mostraram que a PO tem metabolismo mais oxidativo (aeróbio) e a PI glicolítico (anaeróbio). A carne dos animais inteiros tiveram menor TBARS e maior luminosidade (L*), perda de peso por cocção (PPC) e força de cisalhamento (FC) em comparação aos animais castrados. A PO foi mais susceptível a oxidação proteica (menor tióis) em comparação a PI. A carne dos animais confinados por 129 dias tiveram maiores PPC e oxidação proteica (menores tióis) em comparação a carne dos animais confinados por 59 dias. Diferenças de estabilidade oxidativa entre a carne de animais castrados e inteiros confinados por menor período desapareceram quando os animais foram confinados por maior período. Valores de pH e tióis na carne dos animais castrados e inteiros foram afetados pelo tempo de maturação. Ambas as condições sexuais tiveram carne com maior valores de pH no dia 30 de maturação e este, se manteve ao longo do tempo. A PO teve maiores valores de TBARS no dia 60, PPC no dia 100 e FC nos dias 30 e 60 de maturação em comparação a PI. Foi observada uma interação entre tempo de confinamento e tempo de maturação para tióis, TBARS, metamioglobina, pH, L* e FC. Quando comparado aos animais confinados por 59 dias, os animais confinados por 129 dias tiveram: maior oxidação (maior TBARS e menor tióis) nos dias 60 e 100 de maturação; oxidação da mioglobina (metamioglobina) mais tardia, sendo o maior valor obtido no dia 100; menor luminosidade (L*) em todos os tempos de maturação; maior maciez (menor FC) aos 100 dias de maturação. Os animais confinados por 59 dias tiveram: maior oxidação proteica (menor tióis) e maciez (menor FC) aos 30 dias de maturação. De forma geral, todos os efeitos testados tais como castração, tempo de confinamento, metabolismo celular e regiões do musculo pareceram influenciar sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. / The objective of this work was to investigate the effect of the castration, time on confinement, cellular metabolism and muscle region on the protein and lipid oxidation, and other traits of beef quality. Eight-four Nellore cattle (steers and bulls), confined for different periods, were used to conduct studies in Longissimus dorsi and Biceps femoris muscles. The latter muscle was divided in two portions: origin (OP) and insertion (IP). In the study of L. dorsi muscle, steaks were packaged under aerobiosis (PVC) and anaerobiosis (vacuum) conditions and aged for 1, 3, 5, 7 and 9, and 1, 7, 14 and 21 days, respectively. For this muscle, no differences in oxidative stability [thiols, carbonyls and Thiobarbituric Acid Reactive Substances (TBARS)] and color between the meat from bulls and steers were found. This could be explained by the lack of differences in initial oxidative status, measured through the activity of the antioxidants enzymes, content of total glutathione and composition of fatty acids, between the sexual conditions. The results also indicated that the oxidation of the steaks vacuum-packaged took about twice more days to start than the steaks under aerobiosis. In the study of Biceps femoris muscle, the animals were slaughtered after 59 and 129 days on confinement and the steaks from OP and IP were aged for 1, 30, 60 and 100 days. The results of the lactate dehydrogenase and citrate synthase enzymes activity showed that the OP has a more oxidative metabolism and the IP has a more glycolytic metabolism. The meat from bulls had lower TBARS and higher lightness (L*), cooking loss (CL) and shear force (SF) in comparison with steers. The OP was more susceptible to protein oxidation (lower thiols) than the IP. Animals confined for 129 days had meat with higher CL when compared to those ones confined for 59 days. The meat from animals confined for 129 days had higher CL and protein oxidation (lower thiols) in regard to the meat from animals confined for 59 days. Differences in oxidative stability between the meat from steers and bulls confined for shorter period disappeared when the animals were confined for larger period. Values of pH and thiols in meat from steers and bulls were affected by the time of aging. Both the sexual conditions had meat with higher pH values at the day 30 of aging and this was kept across the time. The OP had higher values of TBARS at the day 60, CL at the day 100 and SF at the days 30 and 60 of aging when compared to the IP. It was observed an interaction between confinement time and aging time for thiols, TBARS, metmyoglobin, pH, L* and SF. When compared to the animals confined for 59 days, the animals confined for 129 days had: higher oxidation (higher TBARS and lower thiols) at the days 60 and 100 of aging; oxidation of myoglobin (metmyoglobin) slower, since the higher value was obtained at the day 100; lower lightness (L*) in all the times of aging; tender meat (lower SF) at 100 days of aging. The animals confined for 59 days had: higher protein oxidation (lower thiols) and tender meat (lower SF) at 30 days of aging. Overall, all the effects tested such as castration, time on confinement, cellular metabolism and muscle region seemed to influence on the protein and lipid oxidation and other traits of beef quality.

Haemophilus influenzae (Hi) é uma bactéria Gram negativa dependente dos fatores de crescimento NAD e hemina. O sorotipo b (Hib) é causador de doenças invasivas de importância clínica. É uma bactéria anaeróbia facultativa, e se adapta em devido à versatilidade do seu arsenal metabólico e assim, sendo capaz de causar infecção em nichos com e sem oxigênio no hospedeiro. O objetivo deste trabalho foi estudar o comportamento cinético de Hib em anaerobiose, aerobiose e a interface entre as duas condições, utilizando diferentes aceptores de elétrons (fumarato, DMSO, nitrato e oxigênio). Nitrato possui o maior potencial de redução padrão, porém o nitrito gerado de sua oxidação inviabilizou o crescimento de Hib. DMSO apresentou o melhor crescimento celular em frascos agitados, frente a fumarato de sódio. Ensaios em biorreator demonstraram que em anaerobiose, com fumarato de sódio como aceptor, succinato é o metabólito principal formado. Acetato foi produzido com todos os aceptores testados, seja em anaerobiose ou aerobiose, majoritário na última condição. Lactato de sódio e glicerol foram totalmente consumidas em aerobiose e lentamente em anaerobiose, sem esgotar nenhuma das fontes de carbono. / Haemophilus influenzae (Hi) is a Gram-negative bacterium and require growth factors for its development (hemin and NAD). Serotype b (Hib) is an important human pathogen for public health. It is a facultative anaerobic bacterium, which is allowed to adapt in several host niches due to its metabolic versatility with oxygen or its absence. The aim of this work is to study the growth kinetic of Hibs in anaerobiosis and aerobiosis, in the presence of different final electron acceptors (fumarate, DMSO, nitrate and oxygen). Nitrate has the major reduction potential among the electron acceptors tested, however Hib did not growth. In the presence of DMSO, Hib has the better growth. Anaerobic essay carried out in bioreactor showed that in the presence of fumarate as electron acceptor, succinate was the major metabolite produced. Acetate was produced in the presence of all electron acceptors tested, in both conditions (anaerobiosis and aerobiosis. Lactate and glycerol were entirely consumed in aerobiosis as carbon source and in anaerobiosis, the consumption of both carbon sources were not expressive.

O objetivo deste trabalho foi investigar o efeito da castração, tempo de confinamento, metabolismo celular e regiões do musculo sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. Oitenta e quatro bovinos (castrados e inteiros) Nelore, confinados por diferentes períodos, foram usados para conduzir estudos em músculos Longissimus dorsi e Biceps femoris. O segundo musculo foi dividido em duas porções: origem (PO) e inserção (PI). No estudo com L. dorsi, bifes foram embalados sob condições de aerobiose (PVC) e anaerobiose (vácuo) e maturados por 1, 3, 5, 7 e 9, e 1, 7, 14 e 21 dias, respectivamente. Para este músculo, nenhuma diferença na estabilidade oxidativa [tióis, carbonilas e Substancias Reativas ao Ácido Tiobarbiturico (TBARS)] e cor entre as carnes dos animais inteiros e castrados foram encontradas. Isto poderia ser explicado pela falta de diferença no status oxidativo inicial, mensurados através da atividade de enzimas antioxidantes, conteúdo de glutationa total e composição de ácidos graxos, entre as condições sexuais. Os resultados também indicaram que a oxidação dos bifes embalados a vácuo leva o dobro de dias para iniciar, em comparação aos bifes em aerobiose. No estudo com o Bíceps femoris, os animais foram abatidos com 59 e 129 dias de confinamento e os bifes da PO e PI foram maturados por 1, 30, 60 e 100 dias. Os resultados da atividade das enzimas lactato desidrogenase e citrato sintase mostraram que a PO tem metabolismo mais oxidativo (aeróbio) e a PI glicolítico (anaeróbio). A carne dos animais inteiros tiveram menor TBARS e maior luminosidade (L*), perda de peso por cocção (PPC) e força de cisalhamento (FC) em comparação aos animais castrados. A PO foi mais susceptível a oxidação proteica (menor tióis) em comparação a PI. A carne dos animais confinados por 129 dias tiveram maiores PPC e oxidação proteica (menores tióis) em comparação a carne dos animais confinados por 59 dias. Diferenças de estabilidade oxidativa entre a carne de animais castrados e inteiros confinados por menor período desapareceram quando os animais foram confinados por maior período. Valores de pH e tióis na carne dos animais castrados e inteiros foram afetados pelo tempo de maturação. Ambas as condições sexuais tiveram carne com maior valores de pH no dia 30 de maturação e este, se manteve ao longo do tempo. A PO teve maiores valores de TBARS no dia 60, PPC no dia 100 e FC nos dias 30 e 60 de maturação em comparação a PI. Foi observada uma interação entre tempo de confinamento e tempo de maturação para tióis, TBARS, metamioglobina, pH, L* e FC. Quando comparado aos animais confinados por 59 dias, os animais confinados por 129 dias tiveram: maior oxidação (maior TBARS e menor tióis) nos dias 60 e 100 de maturação; oxidação da mioglobina (metamioglobina) mais tardia, sendo o maior valor obtido no dia 100; menor luminosidade (L*) em todos os tempos de maturação; maior maciez (menor FC) aos 100 dias de maturação. Os animais confinados por 59 dias tiveram: maior oxidação proteica (menor tióis) e maciez (menor FC) aos 30 dias de maturação. De forma geral, todos os efeitos testados tais como castração, tempo de confinamento, metabolismo celular e regiões do musculo pareceram influenciar sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. / The objective of this work was to investigate the effect of the castration, time on confinement, cellular metabolism and muscle region on the protein and lipid oxidation, and other traits of beef quality. Eight-four Nellore cattle (steers and bulls), confined for different periods, were used to conduct studies in Longissimus dorsi and Biceps femoris muscles. The latter muscle was divided in two portions: origin (OP) and insertion (IP). In the study of L. dorsi muscle, steaks were packaged under aerobiosis (PVC) and anaerobiosis (vacuum) conditions and aged for 1, 3, 5, 7 and 9, and 1, 7, 14 and 21 days, respectively. For this muscle, no differences in oxidative stability [thiols, carbonyls and Thiobarbituric Acid Reactive Substances (TBARS)] and color between the meat from bulls and steers were found. This could be explained by the lack of differences in initial oxidative status, measured through the activity of the antioxidants enzymes, content of total glutathione and composition of fatty acids, between the sexual conditions. The results also indicated that the oxidation of the steaks vacuum-packaged took about twice more days to start than the steaks under aerobiosis. In the study of Biceps femoris muscle, the animals were slaughtered after 59 and 129 days on confinement and the steaks from OP and IP were aged for 1, 30, 60 and 100 days. The results of the lactate dehydrogenase and citrate synthase enzymes activity showed that the OP has a more oxidative metabolism and the IP has a more glycolytic metabolism. The meat from bulls had lower TBARS and higher lightness (L*), cooking loss (CL) and shear force (SF) in comparison with steers. The OP was more susceptible to protein oxidation (lower thiols) than the IP. Animals confined for 129 days had meat with higher CL when compared to those ones confined for 59 days. The meat from animals confined for 129 days had higher CL and protein oxidation (lower thiols) in regard to the meat from animals confined for 59 days. Differences in oxidative stability between the meat from steers and bulls confined for shorter period disappeared when the animals were confined for larger period. Values of pH and thiols in meat from steers and bulls were affected by the time of aging. Both the sexual conditions had meat with higher pH values at the day 30 of aging and this was kept across the time. The OP had higher values of TBARS at the day 60, CL at the day 100 and SF at the days 30 and 60 of aging when compared to the IP. It was observed an interaction between confinement time and aging time for thiols, TBARS, metmyoglobin, pH, L* and SF. When compared to the animals confined for 59 days, the animals confined for 129 days had: higher oxidation (higher TBARS and lower thiols) at the days 60 and 100 of aging; oxidation of myoglobin (metmyoglobin) slower, since the higher value was obtained at the day 100; lower lightness (L*) in all the times of aging; tender meat (lower SF) at 100 days of aging. The animals confined for 59 days had: higher protein oxidation (lower thiols) and tender meat (lower SF) at 30 days of aging. Overall, all the effects tested such as castration, time on confinement, cellular metabolism and muscle region seemed to influence on the protein and lipid oxidation and other traits of beef quality.

La pyocyanine (PYO) est une phénazine de couleur bleu-vert, produite spécifiquement par la bactérie pathogène opportuniste Pseudomonas aeruginosa (Pa). La toxicité aérobie de la PYO envers les cellules de mammifères, les levures et les bactéries a été décrite de longue date, mais la compréhension des mécanismes d’action est encore lacunaire, en particulier en conditions de limitation en O2 (conditions rencontrées dans le contexte infectieux). De plus, il a récemment été montré que la PYO peut apporter des effets bénéfiques pour la souche productrice en hypoxie. Au cours de ce travail, nous avons réexaminé les effets de la PYO sur un large panel de bactéries dont son propre producteur (Pa) ainsi que sur un modèle cellulaire eucaryote Saccharomyces cerevisiae exposées à différentes tensions en O2. Nos données suggèrent que la toxicité aérobie de la PYO envers S. cerevisiae est multifactorielle, impliquant à la fois une interaction avec le complexe III de la chaîne respiratoire et l’induction d’un stress oxydatif. Pour la première fois, nous avons mis en évidence une toxicité de la PYO exacerbée en anaérobiose chez un eucaryote (S. cerevisiae). Le mécanisme d’action impliquerait le PYO radical. Nous avons également montré que la PYO peut inhiber la croissance aérobie et anaérobie des microorganismes concurrents, plus particulièrement S. aureus en bloquant le complexe III de la chaîne respiratoire. A l’inverse, la PYO peut stimuler la respiration de Pa surtout dans les conditions mimant le contexte infectieux (hypoxie, vie ralentie). Le complexe III et/ou les oxydases terminales cbb3 serait impliqué favorablement. En conclusion, la PYO jouerait à la fois un rôle de poison hypoxique mais aussi un rôle de navette redox bénéfique pour la survie et la virulence de Pa en hypoxie. / Pyocyanin (PYO) is a blue-green phenazin, specifically produced by the opportunistic bacterium Pseudomonas aeruginosa (Pa). Aerobic toxicity of PYO toward mammalian cells, yeast and bacteria has been known for a long time, but the understanding of its mechanisms of action remains unclear, especially in conditions of limited O2 (conditions encountered during infection). In addition, it has recently been shown that PYO can bring benefits to the producer strain under hypoxia. In this study, we reexamined the effects of PYO toward a large panel of bacteria including its own producer Pa as well as a model of eukaryotic cells Saccharomyces cerevisiae exposed to different oxygen tensions. Our results suggest that the aerobic toxicity of PYO toward S. cerevisiae is multifactorial: involving both interaction with the respiratory chain at the level of complex III and induction of oxidative stress. For the first time, we have shown that PYO exerts an increased toxicity toward the eukaryotic cell, S. cerevisiae under anaerobiosis. The mechanism could involve the production of PYO radical. We have also shown that PYO can inhibit the aerobic and anaerobic growth of competing microorganisms, especially S. aureus by blocking the complex III of the respiratory chain. Conversely, PYO can stimulate the respiration of Pa, in mainly in conditions similar to those encountered during infection (hypoxia, slowed growth). The complex III and/or the cbb3 oxidases could be favorably involved. To conclude, PYO could act as a hypoxic poison as well as a redox shuttle beneficial for the survival and the virulence of Pa under hypoxia.

Les populations humaines sont exposées à de très nombreux polluants. Il apparaît donc essentiel d'évaluer la toxicité de ces molécules au sein des compartiments récepteurs. En milieu urbain, de nombreux polluants (principalement hydrocarbures et métaux lourds) s'accumulent sur les surfaces imperméables (routes, toitures, parkings, bâtiments…). Ils sont remis en suspension et drainés par les eaux de pluie jusque dans les bassins d'infiltration. Ces structures ont été construites afin de réinfiltrer les eaux de ruissellement dans les nappes phréatiques sous-jacentes et de les détoxifier. Ces molécules toxiques sont capturées et accumulées au niveau des sédiments fins constitutifs des bassins, où elles peuvent atteindre des concentrations très élevées. Malgré cette forte contrainte, quelques rares invertébrés vivent dans les sédiments pollués des bassins d'infiltration, ce qui sous-entend des adaptations métaboliques, physiologiques et/ou comportementales spécifiques. L'un des plus répandus est l'oligochète aquatique Limnodrilus hoffmeisteri. Ce ver tubificidé construit des galeries (activité de bioturbation) dans les sédiments où il favorise l'activité des microorganismes sur la minéralisation de la matière organique et le recyclage des nutriments. Cette espèce présente donc un rôle essentiel dans le fonctionnement des bassins d'infiltration, et donc sur la qualité de l'eau des nappes phréatiques. Elle est aujourd'hui considérée à la fois comme une espèce sentinelle de l'état de santé de son biotope et comme un ingénieur des écosystèmes. Le premier objectif de ce travail de thèse a été de mettre en évidence les réponses écophysiologiques permettant à L. hoffmeisteri de survivre dans ces biotopes particulièrement anthropisés. Pour cela, nous avons exposé/élevé cet organisme pendant 1, 3 ou 6 mois dans des sédiments pollués issus de 3 bassins d'infiltration et un sédiment issu d'un milieu non urbanisé (sédiment témoin très peu pollué), en laboratoire. Nous avons ensuite analysé sur ce ver la survie, la consommation d'oxygène, l'état des réserves énergétiques, les concentrations de métaux lourds bioaccumulés, le stress oxydant engendré par les polluants (niveau de peroxydation lipidique) et enfin les mécanismes de protection associés (activités des principales enzymes antioxydantes). Le deuxième objectif a été de déterminer l'impact des polluants urbains sur les métabolismes aérobie et anaérobie chez L. hoffmeisteri. Pour cela, nous avons mesuré différents paramètres témoignant de l'activité mitochondriale (activités de la chaîne respiratoire mitochondriale et de l'ATP synthétase) ainsi que les concentrations des principaux produits terminaux du métabolisme anaérobie chez des vers exposés aux mêmes sédiments (4) pollués ou témoin. Cette étude a démontré que les contaminants induisent une transition partielle du métabolisme aérobie vers les voies anaérobies suite à un dysfonctionnement mitochondrial, et a aussi révélé que certains produits terminaux du métabolisme anaérobie (succinate et propionate) constituent des marqueurs pertinents de la pollution urbaine. Enfin, le troisième objectif de cette thèse a été d'évaluer l'impact d'une pollution d'origine anthropique sur l'activité d'ingénierie de L. hoffmeisteri (i.e. sur son rôle dans le fonctionnement des bassins d'infiltration). Pour cela, nous avons mesuré pendant 1 mois le comportement de fouissage de ce ver bioturbateur par tomographie aux rayons X dans des microcosmes contenant des sédiments pollués ou non. De plus, des mesures régulières des flux de nutriments, d'oxygène dissous, de CO2 et de CH4 au cours de l'expérimentation ont permis d'évaluer l'influence du taux de contamination sur le recyclage des nutriments dans nos systèmes expérimentaux. Ces mesures ont été effectuées à la fois dans des systèmes colonisés par L. hoffmeisteri, mais aussi dans des systèmes sans faune, afin de quantifier précisément le rôle de ces organismes ingénieurs / Human populations are exposed to numerous pollutants. It is now necessary to evaluate the toxicity of urban contaminants in receptor ecosystems. In anthropized areas, many pollutants (mainly hydrocarbons and heavy metals) accumulate on the impervious surfaces (roads, parks, buildings, rooftops…). During a rainfall event, these compounds are re-suspended and drained up to stormwater infiltration basins. These structures were built to detoxify and to infiltrate runoff water to underlying groundwater. Toxic compounds are captured and accumulated in the fine sediment layer of the infiltration basins, where their concentrations may achieve important concentrations. Despite this harsh constraint, a few invertebrates inhabit stormwater basin sediments and have developed specific metabolic, physiological and/or behavioural adaptations. One of the most spread is the oligochaeta Limnodrilus hoffmeisteri. This tubificid worm burrows galleries (bioturbation activity) in sediments where it enhances the organic matter mineralization and the nutrients recycling. This species has an essential role in the infiltration basin functioning and on groundwater quality. L. hoffmeisteri is considered both as a sentinel species of ecosystem health and an engineer species. The first aim of this work was to highlight the ecophysiological responses that allow L. hoffmeisteri to survive in these harsh conditions. We exposed this organism during 1, 3 or 6 months to polluted sediments (from 3 infiltration basins), under laboratory conditions. Then, we measured the survival, the oxygen consumption, the energy body stores, the oxidative stress induced by urban pollutants (through the lipid peroxidation level), and the antioxidant defence mechanisms (the activity of the antioxidant enzymes) in L. hoffmeisteri. The same analyses were realized on worms incubated 1 to 6 months in a sediment from a non-urbanized environment (considered as a low-polluted/control sediment). The second objective of the present work was to determine the impact of urban pollutants on aerobic and anaerobic metabolisms in L. hoffmeisteri. We measured several mitochondrial parameters (the mitochondrial respiratory chain activity and the ATP production rate) and anaerobic end product concentrations in worms exposed to the 4 same sediments (polluted or not). This study demonstrated that urban pollutants induced a shift from aerobic to the anaerobic metabolism, linked to a mitochondrial dysfunctioning. Moreover, this study also showed that two anaerobic end products (succinate and propionate) constitute relevant biomarkers of urban pollution. Lastly, the third goal of this thesis was to evaluate the impact of an anthropic pollution on the engineering activity of L. hoffmeisteri (i.e. its role in the infiltration basin functioning). To this end, we measured during 1 month the burrowing activity of this tubificid worm using X-ray tomography, in microcosms containing slightly or highly polluted sediments. We measured nutrients fluxes, dissolved oxygen, CO2 and CH4 concentrations during the experiment to determine the influence of the pollution rate on nutrients recycling. These measurements were also realized in microcosms with or without worms, to quantify the functional role of engineer organisms

Vibrio cholerae, l’agent étiologique du choléra, est une bactérie adaptée à l’environnement aquatique et, dans le cadre infectieux, à la colonisation du petit intestin humain. L’environnement intestinal est un milieu a priori hostile aux bactéries externes, à cause de son environnement anaérobie et de la présence des effecteurs du système immunitaire. Capable de produire un biofilm dans ces deux niches écologiques, V. cholerae est capable de persister dans des conditions défavorables et de résister aux molécules antimicrobiennes, y compris les peptides antimicrobiens (PAM). J’ai d’abord étudié la résistance aux antimicrobiens de V. cholerae dans des conditions expérimentales représentatives de l’intestin. Ensuite, j’ai caractérisé, quantitativement et qualitativement, la résistance à la polymyxine B (PmB) des biofilms de V. cholerae, exposés à des concentrations sous-inhibitrices ou létales de ce PAM. Nos résultats suggèrent que la résistance aux PAM de V. cholerae est influencée par la disponibilité de l’oxygène dans le milieu. Je propose également que des concentrations létales de PmB peuvent stimuler un mécanisme de résistance exclusif aux biofilms matures. Les différences soulevées par nos investigations mettent en perspective l’importance d’adapter les conditions expérimentales aux caractéristiques réelles de l’environnement infectieux lors des études de résistances aux antimicrobiens. / Vibrio cholerae, the etiological agent of cholera, is a bacterium that is adapted to the aquatic environment and, in the infectious setting, to the colonization of the small intestine. The intestinal environment is at first glance hostile to external bacteria, due to its anaerobic conditions and the presence of the effectors of the immune system. Capable of producing a biofilm in its two ecological niches, V. cholerae is quite capable of persisting in unfavorable conditions and of resisting antimicrobial molecules, including antimicrobial peptides (AMPs). I first investigated the antimicrobial resistance of V. cholerae under experimental conditions representative of the intestine. Then, I characterized, quantitatively and qualitatively, the antimicrobial resistance of V. cholerae biofilms to Polymyxin B (PmB), at sub-inhibitory or lethal concentrations of this AMP. Our results suggest that resistance to PmB in V. cholerae is influenced by oxygen availability in the medium. I also propose that lethal concentrations of PmB may promote a resistance mechanism exclusive to mature biofilms. The differences raised by our investigations put into perspective the importance of adapting laboratory conditions to the actual features of the infectious environment when investigating antimicrobial resistance.

L'acide succinique est une molécule linéaire, bi-fonctionnelle qui possède de nombreuses applications alimentaires, chimiques et pharmaceutiques etc. Les connaissances de la régulation des voies métaboliques d’organismes d’intérêt industriel, le génie génétique et le génie des procédés ont permis à des microorganismes recombinants (C. glutamicum) de produire jusqu'à 100 g.L-1 de succinate avec des rendements intéressants. C. glutamicum est largement connu comme l'un des meilleurs producteurs industriels de nombreux acides aminés (glutamate, lysine etc.). Cependant, des études de C. glutamicum ont démontré sa capacité à produire plusieurs acides organiques (succinate, lactate, acétate, etc.). Au cours de ce travail, nous avons supprimé le gène ldhA de C. glutamicum en utilisant le plasmide pk19mobsacBΔldhA. Nous avons démontré que la délétion de ce gène n’avait pas d’incidence sur la capacité de croissance de la bactérie. Par ailleurs, nous avons étudié les effets de l’oxygénation sur la réponse physiologique de C. glutamicum 2262ΔldhA à travers des expériences de cultures en fioles en verre lisses en imposant différentes conditions de kLa. Les résultats ont montré que des faibles kLa (<33 h-1) favorisaient la production d’acides organiques tandis que les kLa élevés amélioraient surtout l’accumulation de la biomasse. Nous avons également mis en œuvre un procédé de production très efficace avec une phase de transition aérobiose-anaérobiose basée sur la régulation de la concentration en oxygène dissous. Avec ce procédé, 327 mM de succinate avec un rendement de 0,94 mole par mole de glucose ont pu être produits avec le mutant ΔldhA. En outre, nous avons vérifié l’efficacité de ce nouveau procédé en l’appliquant à la souche sauvage qui normalement produit 10 fois plus de lactate que de succinate. Ces résultats ont montré une production de 793 mM (94 g.L-1) de succinate et 785 mM (71 g.L-1) de lactate. Ils soulignent ainsi, l'importance de la phase de transition aérobiose-anaérobiose lors des procédés de production de succinate par des bactéries aérobie facultatif. Enfin, des expériences en système bi-étagé ont montré que C. glutamicum 2262 pouvait s’adapter très facilement aux gradients et hétérogénéités en oxygène dissous dans les cultures à grande échelle / Succinic acid is a linear and bi-functional molecule that has several practical applications including food, chemical and pharmaceutical industries. Thanks to increased knowledge on metabolism and pathway regulation of industrially relevant organisms, to the development of performant genetic tools and process engineering, recombinants strains (Escherichia coli, Corynebacterium glutamicum etc.) have been reported to be able to produce up to 100 g.L-1 with interesting yields (> 1.5 mole per mole glucose). C. glutamicum is well known as one of the best industrial producers of numerous amino acids (glutamate, lysine etc.). However, recent studies of C. glutamicum revealed its capability to produce several organic acids (succinate, lactate, acetate, etc.). In this work, we have deleted the ldhA gene of C. glutamicum by using a plasmid vector pk19mobsacBΔldhA. We demonstrated that the mutant and the wild type presented similar growth kinetics with maximal growth rate of about 0.7 h-1. We studied also the effects of oxygenation on C. glutamicum 2262 ΔldhA through cultures at different kLa and it appeared that lower kLa (<33 h-1) favored organic acids production wile higher favored bacterial growth. Furthermore, we designed a tri-phasic process with transition phase by regulation of dissolved oxygen concentration which resulted in the production of 327 mM of succinic acid with a yield of 0.94 mole per mole glucose. The application of the designed process to C. glutamicum 2262 wild type that normally produces lactate with a lactate to succinate production ratio up to 13.3 mol.mol-1, resulted in succinate concentration up to 793 mM (94 g.L-1) and 785 mM (71 g.L-1) of lactate. The succinate production yield was 1.1 mole per mole glucose and acetate production was negligible. These results underlined the importance of aerobic to anaerobic transition in succinate production processes of facultative aerobes and the necessity to engineer not only the microorganism but also the process. Finally, scale-down study have demonstrated the robustness of C. glutamicum against the oxygen gradients in bioreactor

Made available in DSpace on 2015-03-26T13:51:46Z (GMT). No. of bitstreams: 1 01 - capa_abstract.pdf: 113068 bytes, checksum: ac8d58045328d0ccbe13706fe9c3dede (MD5) Previous issue date: 2008-09-12 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The heavy oil (10 20ºAPI) is more than half of world oil reserves, there are predictions that in 2025 are the main source of oil in the world. The difficulties in exploration this kind of oil are associated with unfavorable characteristics such as density and viscosity (100 to 10.000 cP). With the need to improve the utilization of reservoir - in view of the relatively low levels of efficiency of traditional techniques - it has expanded the research to develop new techniques for recovery, especially Microbialy Enhanced Oil Recovery (MEOR). In the recovery of oil contained in sand-pack columns, two dimensionless variables are reported as important: the capillary number and ratio of viscosity. As the increases capillary number, the residual oil decreases. This can be achieved by reducing the interfacial tension between the fluid of residual oil. Several authors have reported the tensions interfaciais ultra between biosurfactant and hydrocarbons, including heavy oil. In the present work, tested the hypothesis that the biosurfactant produced on condition of reservoir, by two isolates of Bacillus subtilis from the collection of culture of the Laboratório de Biotecnologia e Biodiversidade para o Meio Ambiente (LBBMA / DMB / UFV), are capable to remove heavy oil in sand-pack columns. Initially, the isolates of Bacillus subtilis LBBMA 155 and Bacillus subtilis subsp. spizizenii LBBMA 258 were evaluated as to the growth and biosurfactant production in response to temperature and salinity. A central composite rotatable design (CCRD) was used, with temperature and salinity being independent variable. The results were analyzed using Response Surface Methodology (RSM) and showed that the those isolates were able of growth and biosurfactant production of under anaerobic conditions. The growth was influenced by temperature and biosurfactant production was influenced by temperature and salinity. After that, the biosurfactant produced by those isolates, alone or mixed, were analyzed as the ability to remove heavy oil trapped in sand-pack columms. The injection of three volumes of porous extract biossurfactante (EB) produced by Bacillus subtilis LBBMA 155 and Bacillus subtilis subsp. spizizenii LBBMA 258 removed 13.55% and 17.42%, respectively, from residual oil (14 ºAPI). The mixed of extracts from biosurfactant produced by those two isolates showed intermediate values of DMC and recovery of residual oil reported for each. Summarize, the extracts from biosurfactant (EB) produced under anaerobic conditions by Bacillus subtilis LBBMA 155 e Bacillus subtilis subsp. spizizenii LBBM 258 are effective in recovery heavy oil nuclei of the sand-pack columms and have potencial for use Microbialy Enhanced Oil Recovery. / Os óleos pesados (10-20 ºAPI) constituem mais da metade das reservas mundiais de petróleo, havendo previsões de que em 2025 sejam a principal fonte de petróleo no mundo. As dificuldades na exploração desse tipo de óleo estão associadas a características desfavoráveis, como densidade e viscosidade (100 a 10000 cP). Com a necessidade de melhorar o aproveitamento dos reservatórios tendo em vista os níveis relativamente baixos de eficiência das técnicas tradicionais tem-se ampliado as pesquisas para o desenvolvimento de novas técnicas de recuperação, destacando-se a recuperação avançada de petróleo melhorada por microrganismos (Microbially enhanced oil recovery MEOR). Na recuperação de óleos contidos em meios porosos, duas variáveis adimensionais são relatadas como importantes: o número capilar e a razão de viscosidade. À medida que o número capilar aumenta, o óleo residual decresce. Isso pode ser obtido por meio da redução da tensão interfacial entre o fluido de arraste e o óleo residual. Diversos autores têm reportado a obtenção de tensões interfaciais ultrabaixas entre biossurfactantes e hidrocarbonetos, incluindo o óleo pesado. Neste trabalho, testou-se a hipótese de que os biossurfactantes produzidos, em condição de reservatório, por dois isolados de Bacillus subtilis pertencentes à Coleção de Culturas do Laboratório de Biotecnologia e Biodiversidade para o Meio Ambiente (LBBMA/DMB/UFV), são capazes de mobilizar óleo pesado em sistemas porosos de areia. Inicialmente, os isolados de Bacillus subtilis LBBMA 155 e BacilIus subtilis spizizenii LBBMA 258 foram avaliados quanto ao crescimento e produção de biossurfactante em resposta a variações de temperatura e salinidade. Para tanto, foi empregada a Metodologia de Superfície de Resposta (MSR) através do delineamento experimental Composto Central Rotacional (DCCR), e observou-se que esses isolados foram capazes de produzir biossurfactantes em anaerobiose, sendo o crescimento influenciado pela temperatura e a produção de biossurfactante influenciada pela temperatura e pela salinidade. Posteriormente, os biossurfactantes produzidos por esses isolados, sozinhos ou em misturas, foram avaliados quanto à capacidade de mobilização de óleo pesado retido em núcleos porosos de areia. A injeção de três volumes porosos do extrato de biossurfactante (EB) produzidos por Bacillus subtilis LBBMA 155 e Bacillus subtilis subsp. spizizenii LBBMA 258 removeram 13,55% e 17,42%, respectivamente, de petróleo residual (14 ºAPI). A mistura dos extratos de biossurfactantes produzidos por esses dois isolados mostrou valores intermediários de DMC e recuperação de petróleo residual reportados para cada um. Sinteticamente, os extratos de biossurfactantes (EB) produzidos em anaerobiose por B. subtilis LBBMA 155 e B. subtilis subsp. spizizenii LBBMA 258 são efetivos na mobilização de óleo pesado em núcleos porosos de areia e possuem potencial de utilização na recuperação avançada de petróleo melhorada por microrganismos.

Environ 30% des CDS prédits d'un génome code pour des protéines de fonction inconnue ou hypothétiques. La compréhension du rôle de ces protéines est donc l'un des grands challenges de la communauté scientifique.L'objectif principal de cette thèse est de comprendre la fonction de six protéines de fonction inconnue spécifiques de l'anaérobiose formant un complexe, appelé complexe ORP chez Desulfovibrio vulgaris Hildenborough (DvH). Ce système est répandu dans de nombreuses espèces anaérobies, et certaines de ses protéines possèdent des homologies significatives avec des protéines impliquées dans la division cellulaire.Des outils de microscopie dédiés à l'anaérobiose ont été développés au cours de cette thèse et ont permis, pour la première fois, l'observation du cycle cellulaire de DvH. L'étude de l'effet de l'oxygène à l'échelle de la cellule unique a montré une inhibition réversible de la division cellulaire en présence d'oxygène révélant une nouvelle stratégie impliquée dans l'aérotolérance de DvH.Chez DvH, le complexe ORP est codé par des gènes organisés en deux opérons divergents, orp1 et orp2, dont la transcription est gouvernée par l'ARN polymérase sigma54, le facteur de transcription IHF et l'activateur de transcription DVU2106.La diminution de la quantité de complexe ORP conduit à une hétérogénéité de la taille des cellules en accord avec un rôle potentiel du complexe dans le contrôle de la division cellulaire. Alors que l'absence de certaines protéines ORP n'affecte pas de manière significative la division de la bactérie en anaérobiose, la protéine DVU2109 présente une localisation dynamique au cours du cycle cellulaire et semble être essentielle chez DvH. / Up to now, approximately 30% of the predicted CDS in genomes encode for hypothetical or unknown function proteins. Understanding the role and the function of these proteins is now a major challenge for the scientific community.The main objective of this thesis is to determine the function of six proteins of unknown function specific of anaerobiosis and able to forming a multiprotein complex in Desulfovibrio vulgaris Hildenborough (DvH), named the ORP complex. This system is widely found in many anaerobic microorganisms, and some proteins of this system have significant homologies with proteins involved in cell division.Tools for microscopy in anaerobiosis have been developed during this thesis and have allowed observation, for the first time, of a complete DvH cell cycle. The study of oxygen effect on DvH at a single cell level has showed a reversible inhibition of cell division during oxygen exposure revealing a new strategy involved in DvH aerotolerance.In DvH, the ORP complex is encoding by genes organized in two divergent operons, orp1 and orp2, whose transcription is governed by sigma 54 RNA polymerase, the transcription factor IHF and the transcriptional regulator DVU2106. The decreased in the amount of ORP complex leads to heterogeneity of the cell size in accordance with a potential role of this complex in the spatio-temporal control of DvH cell division. While the absence of the majority of ORP proteins doesn't significantly affect DvH division in anaerobic conditions, the protein DVU2109 has a dynamic location during cell cycle and appears to be essential in the cell.

Soil aquifer treatment was simulated in 1 m laboratory soil columns containing silica sand under saturated and unsaturated soil conditions to examine the effect of travel length through the unsaturated zone on the removal of wastewater organic matter, the effect of soil type on dissolved organic carbon removal and also the type of microorganisms involved in the removal process. Dissolved organic carbon removal and nitrification did enhance when the wastewater travelled a longer length through the unsaturated zone. A similar consortium of microorganisms was found to exist in both saturated and unsaturated columns. Microbial concentrations however were lowest in the soil column containing silt and clay in addition to silica sand. The presence of silt and clay was detrimental to DOC removal efficiency under saturated soil conditions due to their negative effect on the hydraulic performance of the soil column and microbial growth.