Analyse du métabolome par chromatographie liquide couplée à la spectrométrie de masse : application à la recherche de biomarqueurs indirects d’induction enzymatique

This work is the result of a research partnership between the CEA and Les laboratories Servier. It deals with the characterization of biomarkers of metabolic enzyme induction in rat biofluids using MSbased metabolomics. The first part of this work included methodological developments regarding theacquisition and the processing of metabolic fingerprints. A tool based on autocorrelation matrices wasthen implemented to reduce the redundancy of data generated with mass spectrometry and subsequently accelerate the isolation of discriminating variables. The next step consisted in the evaluation of the combined use of Kendrick mass defects and methylene selectivity as an alternative visualization tool for large data set, which would rely on compound chemical structures. Finally, the last part of the work was dedicated to the identification of discriminating signals raised up by ametabolomic global approach from rat biofluids collected before and after an induction assay.Issue d’un partenariat de recherche entre le CEA et les laboratoires Servier, cette thèse avait pour objectif d’évaluer l’approche métabolomique par chromatographie liquide couplée à la spectrométrie de masse (LC-MS) pour l'identification de marqueurs indirects de l'induction dans les espèces de toxicologie. Le travail de thèse a débuté par l’optimisation de la méthode d’acquisition des empreintes métaboliques tant sur le plan analytique que dans le domaine du traitement des données brutes. Un outil reposant sur les matrices d’auto corrélation a alors été développé afin de s’affranchir d’une partie de la redondance du signal obtenu par spectrométrie de masse. Dans un troisième temps, les indices de Kendrick couplés à la sélectivité méthylène ont été appliqués à l’étude de composés biologiques en spectrométrie de masse haute résolution afin de proposer une méthode alternative de visualisation des données offrant une aide à l’identification des variables. Enfin, dans une dernière partie, les efforts se sont portés sur l’identification des composés endogènes modifiés au cours du protocole d’induction

Metabolome analysis using liquid chromatography coupled to mass spectrometry : application to biomarker characterization of metabolic enzyme induction

Issue d’un partenariat de recherche entre le CEA et les laboratoires Servier, cette thèse avait pour objectif d’évaluer l’approche métabolomique par chromatographie liquide couplée à la spectrométrie de masse (LC-MS) pour l'identification de marqueurs indirects de l'induction dans les espèces de toxicologie. Le travail de thèse a débuté par l’optimisation de la méthode d’acquisition des empreintes métaboliques tant sur le plan analytique que dans le domaine du traitement des données brutes. Un outil reposant sur les matrices d’auto corrélation a alors été développé afin de s’affranchir d’une partie de la redondance du signal obtenu par spectrométrie de masse. Dans un troisième temps, les indices de Kendrick couplés à la sélectivité méthylène ont été appliqués à l’étude de composés biologiques en spectrométrie de masse haute résolution afin de proposer une méthode alternative de visualisation des données offrant une aide à l’identification des variables. Enfin, dans une dernière partie, les efforts se sont portés sur l’identification des composés endogènes modifiés au cours du protocole d’induction.This work is the result of a research partnership between the CEA and Les laboratories Servier. It deals with the characterization of biomarkers of metabolic enzyme induction in rat biofluids using MSbased metabolomics. The first part of this work included methodological developments regarding theacquisition and the processing of metabolic fingerprints. A tool based on autocorrelation matrices wasthen implemented to reduce the redundancy of data generated with mass spectrometry and subsequently accelerate the isolation of discriminating variables. The next step consisted in the evaluation of the combined use of Kendrick mass defects and methylene selectivity as an alternative visualization tool for large data set, which would rely on compound chemical structures. Finally, the last part of the work was dedicated to the identification of discriminating signals raised up by ametabolomic global approach from rat biofluids collected before and after an induction assay

Analyse du métabolome par chromatographie liquide couplée à la spectrométrie de masse (application à la recherche de biomarqueurs indirects d'induction enzymatique)

Issue d un partenariat de recherche entre le CEA et les laboratoires Servier, cette thèse avait pour objectif d évaluer l approche métabolomique par chromatographie liquide couplée à la spectrométrie de masse (LC-MS) pour l'identification de marqueurs indirects de l'induction dans les espèces de toxicologie. Le travail de thèse a débuté par l optimisation de la méthode d acquisition des empreintes métaboliques tant sur le plan analytique que dans le domaine du traitement des données brutes. Un outil reposant sur les matrices d auto corrélation a alors été développé afin de s affranchir d une partie de la redondance du signal obtenu par spectrométrie de masse. Dans un troisième temps, les indices de Kendrick couplés à la sélectivité méthylène ont été appliqués à l étude de composés biologiques en spectrométrie de masse haute résolution afin de proposer une méthode alternative de visualisation des données offrant une aide à l identification des variables. Enfin, dans une dernière partie, les efforts se sont portés sur l identification des composés endogènes modifiés au cours du protocole d induction.This work is the result of a research partnership between the CEA and Les laboratories Servier. It deals with the characterization of biomarkers of metabolic enzyme induction in rat biofluids using MSbased metabolomics. The first part of this work included methodological developments regarding theacquisition and the processing of metabolic fingerprints. A tool based on autocorrelation matrices wasthen implemented to reduce the redundancy of data generated with mass spectrometry and subsequently accelerate the isolation of discriminating variables. The next step consisted in the evaluation of the combined use of Kendrick mass defects and methylene selectivity as an alternative visualization tool for large data set, which would rely on compound chemical structures. Finally, the last part of the work was dedicated to the identification of discriminating signals raised up by ametabolomic global approach from rat biofluids collected before and after an induction assay.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

IL-15 is an essential mediator of peripheral NK-cell homeostasis

International audienceSeveral distinct classes of surface receptors can, on ligand binding, transmit signals that modulate the survival, proliferation, and apoptosis of peripheral B, T, and natural killer (NK) cells. At the population level, dynamic changes in lymphocyte cell numbers are strictly regulated to maintain a steady state, a process referred to as homeostasis. Although several studies have investigated the signals that regulate B- and T-cell homeostasis, little is known about the mechanisms that control the survival and proliferation of peripheral NK cells. Using an adoptive transfer system, we have investigated the role of gammac-dependent cytokines, in particular interleukin 7 (IL-7) and IL-15, and major histocompatibility complex (MHC) class I molecules in peripheral NK-cell homeostasis. We observed that IL-15 plays a dominant role in the survival of peripheral NK cells, via maintenance of the antiapoptotic factor Bcl-2. IL-15 availability, however, also plays an important role because endogenous NK cells in the recipient mice influence the behavior of adoptively transferred NK cells. Finally, although NK cells bear functional inhibitory Ly49 receptors for MHC class I molecules, the presence or absence of specific ligands on host cells did not influence the survival or homeostatic expansion of donor NK cells

Metabolomic investigation of the response of the model plant Arabidopsis thaliana to cadmium exposure: Evaluation of data pretreatment methods for further statistical analyses.

International audienceUnderstanding the metabolic answer and the adaptation of plants towards heavy metal exposure opens the way to future phytoremediation of polluted sites. For this reason, we studied the impact of cadmium, a toxic heavy metal, on the metabolome of the model plant Arabidopsis thaliana. The analytical methodology (liquid chromatography coupled with mass spectrometry) that was used within the framework of a designed experiment conducted on A. thaliana cells exposed to cadmium generated an important volume of data. Multivariate statistical analyses appeared relevant to compare the metabolic fingerprints in order to isolate and identify some discriminating metabolites. Three types of data pretreatment, i.e., reduction of dimensionality, bucketing and automatic processing by the MetAlign™ software were compared for efficiency in extracting the information. The pretreated data were then subjected to multivariate statistical analysis by principal component analysis (PCA) and partial least square regression (PLS). Finally, an OSC (Orthogonal Signal Correction)-PLS2 approach performed on kinetic and dose ranging studies allowed to visualize time- and cadmium dose-induced changes on the metabolism of A. thaliana cells

Viral-based rodent and nonhuman primate models of multiple system atrophy : Fidelity to the human disease

Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel oligotrophic AAV, Olig001, to overexpress aSyn specifically in striatal oligodendrocytes of rats and nonhuman primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation of pS129+ and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histological analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons. Furthermore, a robust inflammatory response similar to MSA was produced in Olig001-aSyn NHPs, including microglial activation, astrogliosis, and a robust infiltration of T cells into the CNS. Taken together, oligodendroglial-specific viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so desperately needed for MSA