Impact d'une contamination pétrolière sur les tapis microbiens et étude de leur réponse

Ecosystèmes ancestraux et ubiquistes, les tapis microbiens sont caractérisés par une importante biodiversité et une grande richesse métabolique. Au niveau des zones côtières, ces structures bactériennes peuvent être soumises à divers types de contaminations. Du fait notamment du rôle majeur des tapis microbiens dans les cycles biogéochimiques, il apparaît essentiel de mieux comprendre l impact que peuvent avoir de telles contaminations sur ces écosystèmes. Dans ce contexte, le travail de thèse présenté vise à mieux comprendre l impact que peut avoir une pollution pétrolière sur les communautés bactériennes complexes des tapis microbiens. L essentiel de l étude est e tablie à partir d expérimentations en microcosmes réalisées avec différents types de tapis. Des approches moléculaires (T-RFLP et analyse de banques de clones) basées sur les gènes codant pour les ARN ribosomiques 16S ont tout d abord permis de suivre après plusieurs mois et jusqu à un an d incubation l impact d une pollution pétrolière sur les communautés bactériennes des tapis microbiens. Cet aspect a également été abordé à un niveau transcriptomique (ARNr 16S) afin de préciser la réponse des communautés bactériennes métaboliquement actives au sein de ces structures microbiennes. L ensemble de ces travaux a notamment permis de mettre en évidence les capacités de résilience des tapis microbiens. Par la suite, l étude de gènes codant pour des enzymes impliquées dans la dégradation d hydrocarbures (dioxygénases et benzylsuccinate-synthase) a été réalisée dans le but de préciser la réponse fonctionnelle des tapis microbiens après une contamination pétrolière. L absence de réponse apparente au niveau de ces gènes fonctionnels nous a conduit à la recherche de nouveaux gènes pouvant être impliqués après une contamination pétrolière. Ce dernier aspect a fait l objet d une approche par analyse différentielle sur les ARN et a permis de mettre en évidence certain gènes/fonctions pouvant intervenir dans la réponse des tapis microbiens après une contamination pétrolière.Ancestral and ubiquist ecosystems, microbial mats present an important biodiversity and a high metabolic richness. In coastal zone, these vertically laminated bacterial structures are exposed to diverse forms of contamination. Because they are main actors in biochemical cycles, the appreciation of contamination impact on these ecosystems is essential in microbial ecology. In this context, the present PhD study attempts specifically to better understand the impact of petroleum contamination on the complex bacterial community of microbial mats. The study is essentially based on microcosm experiments performed with different microbial mats. First, molecular approaches (T-RFLP and clone libraries analyses) based on 16S rRNA encoding genes allowed to follow during at least three months and up to one year of incubation, a petroleum pollution impact on bacterial communities of microbial mats. The study was also performed at the transcriptomic level (16S rRNA) in order to precise the response of metabolically active bacterial communities in these microbial structures. The main result of this work showed the resilience capacity of microbial mats. Afterward, the study of genes encoding for enzymes involved in hydrocarbon degradation (dioxygenases and benzylsuccinate synthase) have been performed in order to precise the functional response of microbial mats after petroleum contamination. Since the study of these functional genes could not relay the impact of petroleum we focused our study on new potentially involved genes. For this purpose, differential display approach on ARN was applied and allowed to display some gene/function potentially involved in microbial mat response after petroleum contamination.PAU-BU Sciences (644452103) / SudocSudocFranceF

Diabète de type 2 et réentraînement à l'effort (optimisation de la prescription, des effets du réentraînement sur l'utilisation des substrats énergétiques aux adaptations cellulaires musculaires)

MONTPELLIER-BU Médecine UPM (341722108) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

Effects of Heavy Fuel Oil on the Bacterial Community Structure of a Pristine Microbial Mat▿

The effects of petroleum contamination on the bacterial community of a pristine microbial mat from Salins-de-Giraud (Camargue, France) have been investigated. Mats were maintained as microcosms and contaminated with no. 2 fuel oil from the wreck of the Erika. The evolution of the complex bacterial community was monitored by combining analyses based on 16S rRNA genes and their transcripts. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analyses clearly showed the effects of the heavy fuel oil after 60 days of incubation. At the end of the experiment, the initial community structure was recovered, illustrating the resilience of this microbial ecosystem. In addition, the responses of the metabolically active bacterial community were evaluated by T-RFLP and clone library analyses based on 16S rRNA. Immediately after the heavy fuel oil was added to the microcosms, the structure of the active bacterial community was modified, indicating a rapid microbial mat response. Members of the Gammaproteobacteria were initially dominant in the contaminated microcosms. Pseudomonas and Acinetobacter were the main genera representative of this class. After 90 days of incubation, the Gammaproteobacteria were superseded by “Bacilli” and Alphaproteobacteria. This study shows the major changes that occur in the microbial mat community at different time periods following contamination. At the conclusion of the experiment, the RNA approach also demonstrated the resilience of the microbial mat community in resisting environmental stress resulting from oil pollution

Exercice physique et insulinorésistance : de la physiopathologie métabolique musculaire à l'intervention thérapeutique

L'insulinorésistance qui caractérise l'obésité et le diabète de type 2 est liée à des facteurs génétiques et environnementaux. La sédentarité joue un rôle déterminant dans le développement de l'insulinorésistance et le muscle des patients obèses ou diabétiques de type 2 présente de nombreuses anomalies qui portent sur le métabolisme des glucides et des lipides. La pratique régulière de l'exercice physique par ses effets bénéfiques sur le muscle et notamment sur la fonction mitochondriale constitue un bon moyen de prévention et une thérapeutique efficace dans l'obésité et le diabète de type 2

New insight on spatial localization and microstructures of calcite-aragonite interfaces in adult shell of Haliotis tuberculata: Investigations of wild and farmed abalones by FTIR and Raman mapping

International audienceIn the present study, we investigated the shell microstructures of the gastropod European abalone Haliotis tuberculata in order to clarify the complex spatial distribution of the different mineral phases. Our studies were carried out with a standardized methodology on thirty adult European abalone H. tuberculata (5-6 cm long) composed of 15 wild individuals and 15 individuals taken from the France Haliotis hatchery. The macroscopic (binocular) and microscopic observations coupled with Fourier Transform Infrared Spectroscopy (FTIR) and Raman vibrational analysis allowed to unambiguously detect, identify and localize calcite and aragonite. For the first time it has been shown that calcite is present in 100% of farmed and wild adult shell. The microstructural details of the calcite-aragonite interfaces were revealed by using both confocal micro-Raman mapping and Scanning Electron Microscopy (SEM) observations. Calcite zones are systematically found in the spherulitic layer without direct contact with the nacreous layer. The calcite area-nacreous layer interface is made of a thin spherulitic layer with variable thickness from a few micrometers to several millimeters. In order to contribute to a better understanding of the biomineralization process, a model explaining the hierarchical arrangement of the different phases of calcium carbonate is presented and discussed. Finally, it has been shown that these calcitic zones can be connected to each other within the shells and that their spatial distributions correspond to streaks perpendicular to the direction of length growth

Characterization of abalone Haliotis tuberculata–Vibrio harveyi interactions in gill primary cultures

International audienceThe decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments

Biomineralization markers during early shell formation in the European abalone Haliotis tuberculata

International audienc

Characterization of abalone Haliotis tuberculata-Vibrio harveyi interactions in gill primary cultures

The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a nonpathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments

Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata

International audienc

Chemical Heterogeneities within the Disordered Mineral domains of Aragonite Platelets in nacre from the European Abalone Haliotis tuberculata

International audienceSince the observation in 2005 of a disordered mineral layer at the surface of aragonite platelets in abalone Haliotis laevigata nacre, the model of the organo–mineral interface in such biomineralized tissue has been challenged. As a direct interaction between the aragonite crystalline core and the organic matrix is no longer appropriate to describe such interface, a structural description of the disordered mineral domains at the atomic level is a key for a comprehensive view of nacre ultrastructure. Here, we use European abalone Haliotis tuberculata as a model to investigate aragonite nacre through high-resolution transmission electron microscopy (HR-TEM) and multinuclear solid state nuclear magnetic resonance (ssNMR). The presence of a disordered domain around aragonite crystals is shown through HR-TEM observations similarly to H. laevigata. The structure and the ionic composition of the disordered mineral environments of H. tuberculata nacre are investigated through 1H, 13C, 43Ca, and 23Na ssNMR. Interestingly, we demonstrate that the disordered mineral domains in nacre seem to be heterogeneous in terms of structure and chemical composition and do not match with amorphous calcium carbonate stricto sensu. At least three different carbonates species are evidenced, including CO32– and HCO3– present in the same mineral domain and closely associated with rigid H2O molecules. The local disorder around these ions is found to be inhomogeneous as some CO32– possess an aragonitic environment and are rather ordered (according to the position and line width of their 13C resonance) whereas, in opposition, the chemical environment around HCO3– is highly distributed. The analysis of potential cations as counterions revealed the presence of disordered Ca2+ and the presence of Na+ closely associated with HCO3–. On the basis of these structural data, we propose an atomic-level model for the disordered domains in abalone (H.tuberculata) nacre where the protonation level of carbonate ions, the proportion of sodium ions and the local disorder are increasing from the inner to the outer part of disordered domains. These results give an unprecedented structural view at the atomic scale of such disordered mineral domains in nacre aragonite tablets