The commensal intestinal microbiota contributes substantially to intestinal development in the early neonatal period by mechanisms that are not yet elucidated but could contribute to novel strategies to improve intestinal health. A series of gnotobiotic experiments using isolator-reared caesarian section-derived piglets inoculated at 1 d of age with selected bacteria and euthanized at 14 or 15 days of age were performed to investigate intestinal morphology, inflammation and digestive function. In Experiment 1, piglets were maintained germfree (GF), mono-associated with Escherichia coli (EC), mono-associated with Lactobacillus fermentum (LF) or conventionalized with sow feces (CV). Increased (P<0.05) gene expression of Fas ligand (FasL) and tumor necrosis factor (TNF?) in EC and CV as compared to LF and GF pigs coincided with increased apoptotic and proliferative activity. Toll-like receptors (TLR) 2, 4 and 9 were differentially regulated (P<0.05) by colonizing species. In Experiment 2 using the same animals as Exp. 1, increased turnover of brush border enzymes was indicated by reduced (P<0.05) specific activity of aminopeptidase N (APN) and lactase (LPH) and increased expression of APN in CV and EC as compared to GF and LF pigs. Reduced enzyme activity to gene expression ratio corresponded with an in vitro assay of microbial inactivation of APN. In Experiment 3, probiotic Lactobacillus sp., L3777, and Bifidobacteria sp., B5445, did not induce expression of inflammatory cytokines in mono-association but di-association with E. coli increased (P<0.05) inflammatory and anti-inflammatory mediators and resulted in a high rate of sepsis (50%) relative to E. coli mono-association. Induced expression of inflammatory cytokines by commensal bacteria through TLR and other means, appear to play a substantial role in microbially-induced enterocyte turnover. Enterocyte immaturity did not account for reduced enzyme activity associated with inflammation as increased expression of APN in response to microbial colonization was observed, suggesting a host response pathway enabling effective competition with the intestinal microbiota for available peptide nutrients. Probiotic bacteria were relatively benign in mono-association but may have facilitated increased translocation of <i>E. coli</i> in di-association. Gnotobiotic animal models are essential to demonstrate outcomes of host response characterized by communication among numerous cell types, although are of significant technical difficulty.

The commensal intestinal microbiota contributes substantially to intestinal development in the early neonatal period by mechanisms that are not yet elucidated but could contribute to novel strategies to improve intestinal health. A series of gnotobiotic experiments using isolator-reared caesarian section-derived piglets inoculated at 1 d of age with selected bacteria and euthanized at 14 or 15 days of age were performed to investigate intestinal morphology, inflammation and digestive function. In Experiment 1, piglets were maintained germfree (GF), mono-associated with Escherichia coli (EC), mono-associated with Lactobacillus fermentum (LF) or conventionalized with sow feces (CV). Increased (P<0.05) gene expression of Fas ligand (FasL) and tumor necrosis factor (TNF?) in EC and CV as compared to LF and GF pigs coincided with increased apoptotic and proliferative activity. Toll-like receptors (TLR) 2, 4 and 9 were differentially regulated (P<0.05) by colonizing species. In Experiment 2 using the same animals as Exp. 1, increased turnover of brush border enzymes was indicated by reduced (P<0.05) specific activity of aminopeptidase N (APN) and lactase (LPH) and increased expression of APN in CV and EC as compared to GF and LF pigs. Reduced enzyme activity to gene expression ratio corresponded with an in vitro assay of microbial inactivation of APN. In Experiment 3, probiotic Lactobacillus sp., L3777, and Bifidobacteria sp., B5445, did not induce expression of inflammatory cytokines in mono-association but di-association with E. coli increased (P<0.05) inflammatory and anti-inflammatory mediators and resulted in a high rate of sepsis (50%) relative to E. coli mono-association. Induced expression of inflammatory cytokines by commensal bacteria through TLR and other means, appear to play a substantial role in microbially-induced enterocyte turnover. Enterocyte immaturity did not account for reduced enzyme activity associated with inflammation as increased expression of APN in response to microbial colonization was observed, suggesting a host response pathway enabling effective competition with the intestinal microbiota for available peptide nutrients. Probiotic bacteria were relatively benign in mono-association but may have facilitated increased translocation of <i>E. coli</i> in di-association. Gnotobiotic animal models are essential to demonstrate outcomes of host response characterized by communication among numerous cell types, although are of significant technical difficulty.

Orientador: Louis Bernard Klaczko / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T14:49:11Z (GMT). No. of bitstreams: 1 Martins_AyanadeBrito_M.pdf: 905842 bytes, checksum: 4ad0bbbbe049319318ad9fdd6e725632 (MD5) Previous issue date: 2010 / Resumo: Elementos citoplasmáticos egoístas (ECEs) sao bactérias que apresentam transmissão predominantemente vertical e se mantém na população hospedeira através do aumento relativo da produção de fêmeas infectadas. Nesses casos a seleção natural favorece mecanismos de manipulação da reprodução do hospedeiro (feminização, partenogênese, mortalidade precoce de machos e incompatibilidade citoplasmática) e, por isso, os ECEs sao denominados parasitas reprodutivos. A mortalidade precoce de machos (em inglês early male-killing) refere-se a morte da prole masculina devida a presença de um ECE e seus agentes causadores sao denominados agentes androcidas. No caso de indutores de feminizarão, partenogênese e incompatibilidade citoplasmática, o próprio mecanismo de manipulação resulta no aumento relativo na produção de fêmeas infectadas, o que pode explicar a manutencao desses elementos na população hospedeira. Entretanto, para agentes androcidas, o mecanismo de manipulação não resulta automaticamente no aumento da produção de fêmeas infectadas. Uma das hipóteses para explicar a manutencao de agentes androcidas e a realocação de recursos: a morte precoce dos machos libera recursos preferencialmente para as suas irmãs. Dado que não sao conhecidos os mecanismos que explicam a manutencao de agentes androcidas em espécies de Drosophila, este mestrado teve como objetivos estimar a prevalência de agentes androcidas em populações de D. melanogaster e analisar fatores que podem estar envolvidos com a manutencao desses agentes nessas populações: (i) presença de outros organismos transmitidos verticalmente; (ii) evidencias de realocação de recursos em populações naturais e em linhagens no laboratório; e (iii) efeitos em diferentes componentes do valor adaptativo. A prevalência do fenótipo androcida em populações de D. melanogaster, estimada pela contagem de proles, variou entre próxima a 0 e 17,7% e esteve fortemente associada a presença de Spiroplasma, detectada por PCR. As razoes sexuais das proles de fêmeas infectadas foram heterogêneas, o que sugere variação na expressão do fenótipo androcida. Não foi detectada associação entre o agente androcida Spiroplasma e Wolbachia, outro ECE que coinfecta populações de D. melanogaster. Foram encontradas evidencias consistentes com a hipótese de realocação de recursos em D. melanogaster: (i) em experimentos no laboratório, fêmeas infectadas por agente androcida apresentaram menor tempo de desenvolvimento do que fêmeas não infectadas; (ii) e fêmeas do campo infectadas produziram mais filhas em um repique de quatro dias no laboratório. Não houve diferença na produção de filhas entre fêmeas infectadas e não infectadas de uma estirpe padrão do laboratório. E possível que o efeito de Spiroplasma em populações naturais esteja associado a composição genética da população hospedeira. / Abstract: Selfish cytoplasmic elements (SCEs) are maternally inherited bacteria which increase the net production of infected females. Due to a genetic conflict between the SCE and the host genomes, different mechanisms of reproductive manipulation (feminization, parthenogenesis, male-killing and cytoplasmic incompatibility) are favored through natural selection. For feminization, parthenogenesis and cytoplasmic incompatibility, the reproductive manipulation by itself results in a greater net production of daughters by the infected females, which may explain the persistence of these elements in the host population. However, this net difference does not hold for male-killer infected and uninfected females. One of the mechanisms that has been proposed to explain the adaptiveness of the male-killing trait is the resource reallocation from dead males to female hosts. Considering that it is still unclear how male-killers persist in Drosophila populations, the present study aimed to assess the male-killer prevalence in D. melanogaster populations and to analyze different factors which may explain their persistence in these population: (i) interaction with other vertically transmitted elements; (ii) evidence supporting the resource release hypothesis in natural populations and in laboratory strains; and (iii) direct fitness effects. The incidence of the male-killing phenotype in D. melanogaster, obtained counting the laboratory raised broods of collected females, ranged from close to 0 to 17.7% and was strongly associated with Spiroplasma infection, assessed by PCR. The sex ratio of female biased trains had a bimodal distribution which suggests variation in the expression of the male-killing phenotype. No evidence of positive or negative interaction between male-killing Spiroplasma and Wolbachia (other SCE coinfecting D. melanogaster) infections was found. We found evidence consistent with the resource reallocation hypothesis in D. melanogaster: (i) infected females had a shorter generation time in laboratory experiments; (ii) and field females produced more daughters in their first brood in the laboratory. No difference in number of daughter was detected between infected and uninfected females in an experiment using flies from a standard laboratory strain. The effect of male-killing Spiroplasma in natural populations may be conditioned to the host's genetic background. / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

De nombreux et divers micro-organismes vivent dans les tissus interne et externe desfeuilles des plantes, la phyllosphère. Ils influencent de nombreux traits, les interactions biotiques,le flux d’énergie, la tolérance au stress de leur hôte et en fin de compte la valeur sélectivede leurs hôtes. Il a été montré que plusieurs traits quantitatifs de plantes structurentla communauté microbienne de la phyllosphère. Ainsi des Loci de ces traits quantitatifs(Quantitative Trait Loci QTL) liés à la structure de cette communauté étaient attendus.L’objectif principal de ce travail était de rechercher des régions génomiques chez le chêne(Quercus robur L.), dont l’effet se prolonge jusqu’au niveau de la communauté, influençantainsi le microbiote de la phyllosphère. Tout d’abord, nous avons étudié la composition etle réseau d’interactions du microbiote de la phyllosphère partant un intérêt particulier àErysiphe alphitoides, un agent pathogène majeur pour les chênes. Nous avons montré quel’infection par E. alphitoides est accompagnée par des changements dans la composition dela communauté fongique foliaire, mais pas dans le composition de la communauté bactérienne.Nous avons souligné certains partenaires d’interaction d’E. alphitoides et nous avonsmontré que le réseau d’interactions microbiennes, contrairement aux résultats précédents,été dominé par des interactions positives. Ensuite nous avons effectué une analyse QTLde descripteurs de la communauté microbienne dans une population de pleins frères. Nousavons trouvé 8 QTL correspondant à des traits de la communauté microbienne: compositionfongique et bactérienne, diversité fongique, pourcentage de Erysiphe alphitoides. Troisd’entre eux sont colocalisés avec un QTL de sensibilité à E. alphitoides, suggèrent un fortdéterminisme génétique de la résistance à l’oïdium chez Q. robur. Enfin, nous présentonsles résultats préliminaires d’une étude d’association génétique et discutons nos résultatsavec une perspective évolutive. / Numerous and various microorganisms inhabit inner and outer tissues of plant leaves, thephyllosphere. They influence many plant traits, biotic interactions, energy flux, host stresstolerance and ultimately the fitness of their hosts. Many plant quantitative traits wereshown to structure the phyllosphere microbial community. Hence quantitative trait loci(QTLs) linked to the structure of this community were expected. The main objective ofthis work was to search for genomic regions in oak (Quercus robur L.), whose effect extendsto the community level, influencing the phyllosphere microbiota. First, we studied thecomposition and the interaction network of oak phyllosphere microbiota with specific focuson Erysiphe alphitoides, a major biotrophic pathogen of oak. We showed that infection byE. alphitoides is accompanied by changes in the foliar fungal community composition butnot in the bacterial community composition. We highlighted likely interaction partners ofE. alphitoides and we showed that the complex microbial interaction network, in contrastto previous findings, was dominated by positive interactions. Next we performed QTLanalysis of microbial community descriptors in a full-sib mapping population of oak. Wefound 8 QTLs for microbial community traits: fungal and bacterial composition, fungaldiversity, and percentage of Erysiphe alphitoides reads. Three of these QTLs colocalizedwith a QTL for powdery mildew sensibility, suggesting for strong genetic determinism ofpowdery mildew resistance in Q. robur. Finally we present preliminary results of a geneticassociation study and discuss our findings within an evolutionary perspective.

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Le vin en tant qu’écosystème complexe est un modèle particulièrement intéressant pour l’étudie des interactions entre les microorganismes. L’interaction sans contact celluaire (interaction indirecte) entre la levure Saccharomyces cerevisae et la bactérie lactique Oenococcus oeni a un effect direct sur l’induction et l'achèvement de la fermentation malolactique (FML), une fermentation très importante pour la qualité du vin. Une souche levurienne peut être classée FML+ si elle stimule la croissance bactérienne et FML- si elle a un effet inhibiteur. Les métabolites connus qui inhibent ou stimulent la FML ne permettent pas toujours d’expliquer cette distinction phénotypique. Dans ce travail de thèse, nous avon développé un workflow multidisciplinaire qui combine l’approche métabolomique non ciblée, l’analyse classique ciblée, les statistiques et les réseaux. L’objectif premier était de dévoiler des métabolites levuriens impliqués dans l’interaction entre levures et bactéries par une comparaison directe des exométabolome des deux phénotypes.À cet effet et pour la première fois dans l’éude d’interactions inter-espèces, la Spectrométrie de Masse à Résonance Cyclotronique des Ions et à Transformée de Fourier (FT-ICR-MS) et la Chromatographie Liquide couplée à la Spectrométrie de Masses (UPLC-Q-TOF-MS) ont été combinées. Pour mieux visualiser les données à haut débit générées par les deux plate-formes, une méthode statistique non supervisée MetICA a été developpée et validée. Par rapport à l’analyse en composantes principales (ACP), cette nouvelle méthode peut réduire la dimension des données d'une façon plus robuste et fiable. Afin d’extraire des métabolites impliquées dans la distinction phénotypique, nous avons comparé différentes methodes de classification et choisi la meilleure pour chaque jeu de données. Les structures putatives de ces biomarqueurs ont été validés par la spectrométrie de masse MS/MS et leurs rôles physiologiques sur la croissance bactérienne ont été confirmées in vitro. La découverte de biomarqueurs a été complétée par l’analyse ciblée réalisées par Chromatographie en Phase Liquide à Haute Performance (HPLC). La complémentarité entre les différentes techniques métabolomiques a conduit à l’identification de nouveaux biomarqueurs de familles distinctes, comme des composés phénoliques, des sucres, des nucléotides, des acides aminés et des peptides. En outre , l'analyse des réseaux métaboliques a révélé des liens entre les biomarqueurs de levure et a suggéré des voies bactériennes influencés par l’exo-métabolome de levure.Notre workflow multidisciplinaire a révélé une réelle capacité à identifier des signatures moléculaires nouvelles et inattendues de l’interaction levure-bactérie. / As a complex microbial ecosystem, wine is a particularly interesting model for studying interactions between microorganisms. Contact-independent interactions (indirect interactions) between the yeast Saccharomyces cerevisae and the lactic acid bacterium Oenococcus oeni have a direct effect on malolactic fermentation (MLF), induction and completion, which is an important factor in wine quality. Yeast strains could be classified as MLF+ phenotype if it usually stimulates the bacterial growth or MLF- in the opposite case. The known metabolites that stimulate or inhibit the MLF cannot always explain the phenotypic distinction. In this work, a multidisciplinary workflow combining non-targeted metabolomics, targeted analysis, statistics and network was developed. The main objective was to unravel diverse yeast metabolites involved in yeast-bacteria interaction via a direct comparison of exo-metabolomes of MLF+ and MLF- phenotypes.To that purpose, and for the first time in the research of interspecies microbial interactions, two metabolomics platforms, Fourier Transform Ion Cyclotron Resonance -Mass Spectrometry (FT-ICR-MS) and Liquid Chromatography coupled with Mass Spectrometry (UPLC-Q-TOF-MS) were used in combination. To better visualize the high-throughput data generated from the two platforms, a novel unsupervised statistical method, the MetICA was developed and validated. Compared to classical principal component analysis (PCA), the new method reduced the data dimension in a more robust and reliable way. To extract metabolic features involved in the phenotypic distinction, we have compared different statistical classifiers and selected the best one for each dataset. Putative structures of these biomarkers were validated via MS/MS fragmentation analysis and their physiological roles to bacteria were confirmed in vitro. The discovery of biomarkers was complemented by targeted HPLC (high performance liquid chromatography) analysis. The complementarities between different analytical techniques led to new biomarkers of distinct chemical families, such as phenolic compounds, carbohydrates, nucleotides, amino acids and peptides. Furthermore, metabolic network analysis has revealed connections between yeast biomarkers and suggested bacterial pathways influenced by yeast exo-metabolome.Our multidisciplinary workflow has shown its ability to find new and unexpected molecular evidence of wine yeast-bacteria interaction.