In human microbiome analysis, sequencing of bacterial 16S rRNA genes has revealed a role for the gut microbiota in maintaining health and contributing to various pathologies. Novel community analysis techniques must be evaluated in terms of bias, sensitivity, and reproducibility and compared to existing techniques to be effectively implemented. Next- generation sequencing technologies offer many advantages over traditional fingerprinting methods, but this extensive evaluation required for the most efficacious use of data has not been performed previously. Illumina libraries were generated from the V3 region of the 16S rRNA gene of samples taken from 12 unique sites within the gastrointestinal tract for each of 4 individuals. Fingerprint data were generated from these samples and prominent bands were sequenced. Sequenced bands were matched with OTUs within their respective libraries. The results demonstrate that denaturing gradient gel electrophoresis (DGGE) represents relatively abundant bacterial taxa (>0.1%) beta-diversity of all samples was compared using Principal Coordinates Analysis (PCoA) of UniFrac distances and Multi-Response Permutation Procedure (MRPP) was applied to measure sample cluster strength and significance; indicator species analysis of fingerprint bands and Illumina OTUs were also compared. The results demonstrate overall similarities between community profiling methods but also indicate that sequence data were not subject to the same limitations observed with the DGGE method (i.e., only abundant taxa bands are resolved, unable to distinguish disparate samples). In addition, the effect of stochastic fluctuations in ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? differ for DGGE and next-generation sequencing. I compared pooled and individual reactions for samples of high and low template concentration for both Illumina and DGGE using the combined V3-V4 region of the 16S rRNA gene, and demonstrated that template concentration has a greater impact on reproducibility than pooling. This research shows congruity between two disparate molecular methods, identifies sources of bias, and establishes new guidelines for minimizing bias in microbial community analyses.

Humboldt-Univ., Diss (Nicht für den Austausch)--Berlin, 2007.

NY, Cornell Univ., Diss.--Ithaca, 2004. / Kopie, ersch. im Verl. UMI, Ann Arbor, Mich. - Enth. 3 Beitr.

NJ, Univ., Dep. of Economics, Diss.--Princeton, 2005. / Kopie, ersch. im Verl. UMI, Ann Arbor, Mich. - Enth. 3 Beitr.

Zugl.: Potsdam, Universiẗat, Magisterarbeit.

Univ., Diss.--Bamberg, 2004.

Les N-acyl homosérine lactones sont des molécules du quorum sensing impliquées dans la communication interbactérienne mais elles sont également capables d'intéragir avec les cellules eucaryotes. Rechercher ces molécules dans le contexte des maladies inflammatoires chroniques intestinlaes (MICI) et plus particulièrement dans le cadre de l'étude des conséquences de la dysbiose sur les voies de l'inflammation intestinale est séduisant. En utilisant la spectrométrie de mase, nous avons mis en évidence pour la première fois des AHLs dans l'écosystème intestinal humain, et plus particulièrement une nouvelle AHL, 3-oxo-C12 :2, qui est prédominante. Cette AHL est corrélée à la normobiose, est perdue au cours des MICI et exerce un effet protecteur sur les cellules épithéliales intestinales. En effet, la 3-oxo-C12 :2 exerce un effet anti-inflammatoire in vitro sur les cellules Caco-2 sans augmenter la perméabilité paracellulaire. De plus, les premiers résultats in vivo montrent que la 3-oxo-C12 est également capable d'influencer la composition du microbiote intestinal des souris. Ces résultats ouvrent de nombreuses perspectives notamment dans la recherche de traitements écologiques au cours des MICI. / Quorum sensing molecules N-acyl-homoserine lactones (AHLs) involved in bacterial communication network are also able to interact with eukaryotic cells. Searching for these molecules in the context of inflammatory bowel diseases (IBD) and more precisely when studying consequences of dysbiosis on gut inflammation pathways is appealing. Using mass spectrometry, we identified for the first time AHLs in human intestinal ecosystem, and among them a new AHL, 3-oxo-C12:2 which is prominent. This AHL correlates with normobiosis, is lost IBD and exerts protective effect on gut epithelial cells. In fact, 3-oxo-C12:2 exerts anti-inflammatory effect in vitro on Caco-2 cells without increased paracellular permeability. Furthermore, first results from in vivo experiments show that 3-oxo-C12:2 is also able to influence mice gut microbiota composition. These results open multiple perspectives especially on new ecological treatments in IBD.

Diabetes tipo 1 (DM1) e uma doenca autoimune que se inicia devido a defeitos na tolerancia imunologica a auto-antigenos, resultando na destruicao autoimune das celulas pancreaticas em individuos geneticamente suscetiveis. Os receptores NOD-like (NLRs) sao receptores intracelulares responsaveis pelo reconhecimento de padroes moleculares associados a patogenos (PAMPs) e padroes moleculares associados ao dano (DAMPs). Estudos recentes tem demonstrado que os receptores NOD1 e NOD2 desempenham um importante papel na ativacao da imunidade inata contra patogenos e na regulacao da imunidade adaptativa, uma vez que sua ativacao leva a producao de citocinas relacionadas a diferenciacao de linfocitos T auxiliares produtores de IL-17 (Th17). Porem, a importancia desses receptores no DM1 ainda e incerto. Nesse sentido, investigamos o papel dos receptores NOD1 e NOD2 na patogenese do DM1, com enfoque na diferenciacao de linfocitos Treg/Th17/Th1 e na plasticidade desses subtipos celulares. Nossos resultados mostram que camundongos deficientes de NOD2, mas nao NOD1 ou RIP2, sao resistentes ao DM1, como comprovado por menor incidencia, hiperglicemia, diminuicao do infiltrado inflamatorio e normalizacao dos niveis de insulina quando comparado aos controles. Foi observado tambem que animais NOD2-/- tiveram uma reducao da populacao de linfocitos Th17, Tc17, Th1 e T citotoxicos nos linfonodos pancreaticos, o que correlaciona com a inibicao da producao de IL-23p19 e IFN- no pancreas. Em paralelo, foi evidenciado o aumento do numero de celulas T reguladoras, macrofagos do perfil M2 nos linfonodos pancreaticos e elevada producao de IL-10 no pancreas de animais NOD2-/-. Alem disso, foi observado que animais NOD2-/- apresentaram uma menor populacao de linfocitos T duplo-positivos (Foxp3+RORt+ e IL-17+IFN+). Posteriormente, foi detectado menor producao de IL- 1, IL-6, IL-23p19 e IL-12p40 por celulas dendriticas de animais deficientes de NOD2. De forma interessante, foi observada a translocacao de bacterias para os linfonodos pancreaticos de animais diabeticos. Adicionalmente, animais tratados com antibioticos tornaram-se resistentes ao DM1, o que nos fornece indicios da contribuicao da microbiota intestinal na inducao da doenca. Por fim, comprovamos alta expressao genica de NOD2 nos linfonodos pancreaticos e no pancreas na fase inicial (pre-diabetica) em outro modelo de DM1, utilizando camundongos NOD (nonobese diabetic mice). Portanto, nossos dados indicam que a ativacao do receptor NOD2 por componentes bacterianos da microbiota intestinal induz a producao de citocinas pro-inflamatorias com subsequente diferenciacao/conversao de linfocitos do perfil Th17/Th1 e progressao do DM1. Dessa forma, estes dados apontam o bloqueio do receptor NOD2 como uma potencial terapia imunomoduladora para o DM1 em humanos. / Type 1 diabetes is an autoimmune disease that precipitates due to defects in the self tolerance to auto- antigens, resulting in the autoimmune destruction of the pancreatic cells in genetically susceptible individuals. NOD-like (NLRs) receptors are intracellular receptors responsible for the recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Recent studies have shown a role of NOD1 and NOD2 receptors in the innate immune response against pathogens and in the adaptive immune response, since its activation leads to the generation of cytokines related to the differentiation of IL-17-producing T helper cells (Th17). However, the role of these receptors in T1D remains elusive. Therefore, we investigated the role of NOD1 and NOD2 receptors in the pathogenesis of T1D, focusing on the differentiation of Treg/Th1/Th17 lymphocytes and in the plasticity of these subtypes. Our data demonstrate that NOD2-/- mice, but not NOD1-/- or RIP2-/-, are resistant to T1D, as shown by the lower incidence, hyperglycemia, less insulitis and normal insulin production when compared to wild type mice. It was also observed that NOD2-/- mice have a reduction in the Th17, Tc17, Th1 and cytotoxic T lymphocyte population within the pancreatic lymph nodes (PLNs), which correlates with the inhibition of IL-23p19 and IFN production in the pancreas. In parallel, there was an increase in Treg cells, M2 macrophages in the PLNs and IL-10 production in the pancreatic tissue of NOD2-/- mice. Also, NOD2-/- mice presented a downregulation of Foxp3+RORt+ and IL-17+IFN+ double-positive T cells. Later, it was shown that IL-1, IL-6, IL-23p19 and IL-12p40 production was downregulated in mice deficient to the NOD2 receptor. Interestingly, we observed a bacterial translocation to the pancreatic lymph nodes in diabetic mice, what could be triggering NOD2 activation, thus contributing to T1D development. As expected, mice pre-treated with antibiotics failed to become diabetic, suggesting a possible role of the gut microbiota in the development of the disease. Lastly, we observed a higher relative expression of NOD2 in the PLNs and pancreas of pre-diabetic mice, using another mouse model of the disease, the nonobese diabetic (NOD) mouse. Collectively, our data suggest that components from the gut microbiota are capable of translocating to the PLNs, thus triggering the activation of NOD2, which in turn induces the production of proinflammatory cytokines related to the differentiation of Th1/Th17 cells, thus contributing to T1D development in a mouse model of the disease. Therefore, the blockade of NOD2 appears as an interesting therapeutical target in the treatment of type 1 diabetes in humans.

Made available in DSpace on 2017-07-10T18:13:29Z (GMT). No. of bitstreams: 1 Milton Cezar de Moura.pdf: 472197 bytes, checksum: ab2bbd7e5f98658de30016d4f0740ac7 (MD5) Previous issue date: 2011-08-11 / This study aimed to evaluated the use of probiotics Bacillus cereus var. Toyo and Bacillus subtilis C-3102 added to the diet for juvenile Nile tilapia strain of GIFT (Genetically Improved Farmed Tilapia) in order to verify the colonization of the gut epithelium and water, the influence on the bacterial microflora, the production performance, the indexes of body composition, the chemical composition and parameters of water quality. Two experiments were conducted: culture in cages with young individuals by 127 days (E1) and culture in ponds with adults by 201 days (E2). There was used the Center for Environmental Research in Aquaculture (CPAA/Toledo-PR) twenty ponds with 8.4 m3 of water and cages with useful volumes of 0.175 m3. The fishes from each experiment were randomly assigned to four treatments with five replicates. The treatments consisted of diets for each phase of commercial cultivation, added 0.5% B. cereus var. Toyoi (BC), 0.5% B. subtilis C-3102 (BS), 0.5% of the combination of two probiotics (BC+BS) and without addition probiotics (SP). In experiment E1, it was found that B. cereus var. Toyo and B. subtilis C-3102 colonized the gut epithelium of fishes and water culture, but did not influence the other parameters (P > 0.05). Survival ranged from 80% to 90% and feed conversion of 1.69 ± 0.29 to 1.96 ± 0.94 for treatments BS and SP. The CP levels ranged from 13.08% to 13.78% for BC and BC+BS and vicerosomatic index ranged from 9.71% to 10.9% for BS and BS+BC, respectively. In experiment E2 was also observed that the probiotics colonize the intestines and water cultivation, but did not influence the other parameters (p > 0.05). In this experiment, the indexes of production performance were influenced by the average temperature of the water that stood at 20.7ºC and is below the optimal range for the species. Weight and specific growth of the fishes ranged from 324.81 g day-1 and 398.51 g day-1, and 0.62% day-1 to 0.73% day-1, respectively, for BC and BS. Feed conversion ranged from 2.40 ± 0.24 to 2.92 ± 0.41 for BS and BC and survival ranged from 84.5% to 86% for BC and SP. The probiotics B. cereus var. Toyo and B. subtilis C-3102 used individually and in combination colonized the gut epithelium of fishes and cultivation water, however, did not influence the gut microflora, the production performance, the indexes of body composition, the chemical composition and parameters of water quality. / O presente objetivou avaliar a utilização dos probióticos Bacillus cereus var. Toyoi e Bacillus subtilis C-3102 adicionados à dieta para juvenis de tilápia do Nilo da linhagem GIFT (Genetically Improved Farmed Tilapia), a fim de verificar a colonização do epitélio intestinal e água do cultivo, a influência sobre a microflora bacteriana, o desempenho zootécnico, os índices de composição corporal, composição centesimal e os parâmetros da qualidade da água. Realizaram-se dois experimentos: cultivo em tanques rede com indivíduos jovens por 127 dias (E1) e cultivo em viveiros com indivíduos adultos por 201 dias (E2). Utilizou-se no Centro de Pesquisa em Aquicultura Ambiental (CPAA/Toledo-PR), vinte viveiros escavados com 8,4 m3 de água e tanques rede com volumes úteis de 0,175 m3. Os peixes de cada experimento foram distribuídos aleatoriamente em quatro tratamentos com cinco repetições. Os tratamentos foram constituídos de rações comerciais para cada fase de cultivo, adicionadas de 0,5% de B. cereus var. Toyoi (BC), 0,5% de B. subtilis C-3102 (BS), 0,5% da combinação dos dois probióticos (BC+BS) e sem adição de probióticos (SP). No experimento E1, verificou-se que os probióticos contendo B. cereus var. Toyoi e B. subtilis C-3102 colonizaram o epitélio intestinal dos peixes e a água do cultivo, mas não influenciaram os demais parâmetros analisados (p > 0,05). A sobrevivência variou de 80% a 90% e a conversão alimentar de 1,69 ± 0,29 a 1,96 ± 0,94 para os tratamentos BS e SP. Os níveis de PB variaram de 13,08% a 13,78% para BC+BS e BC e o índice vicerosomático variou de 9,71% a 10,9% para BC+BS e BS, respectivamente. No experimento E2, observou-se também, que os probióticos colonizaram o intestino e a água do cultivo, mas não influenciaram os demais parâmetros (p > 0,05). A temperatura média da água ficou em 20,7ºC, estando abaixo da faixa ótima para a espécie. O peso e o crescimento específico dos peixes apresentaram variação de 324,81 g dia-1 e 398,51 g dia-1; e 0,62 % dia-1 a 0,73 % dia-1, respectivamente para BC e BS. A conversão alimentar variou de 2,40 ± 0,24 a 2,92 ± 0,41 para BS e BC e a sobrevivência variou de 84,5% a 86% para BC e SP. Os probióticos B. cereus var. Toyoi e B. subtilis C-3102 utilizados individualmente e combinados realizaram a colonização do epitélio intestinal dos peixes e da água do cultivo, contudo, não influenciaram a microflora bacteriana intestinal, o desempenho zootécnico, os índices de composição corporal e centesimal e os parâmetros de qualidade da água.

Alterações no mecanismo de transporte de oxigênio (O2) frequentes em inflamações, infecções, tumores, transplantes e isquemia, levam a hipóxia tecidual. Espécies reativas do O2 são produzidas e citocinas inflamatórias são liberadas engatilhando uma série de eventos, os quais são amplificados após a restituição do fluxo sanguíneo resultando em inflamação sistêmica. No presente estudo, caracterizamos a regulação da Resposta Inflamatória Aguda (AIR) após indução de isquemia e reperfusão intestinal (I/Ri) e a participação do HIF-1α neste fenótipo. Camundongos selecionados para alta (AIRmax) e baixa (AIRmin) AIR foram submetidos a I/Ri e avaliados em diferentes períodos de reperfusão (0, 1, 4 e 24h). Nossos resultados demonstraram maior sensibilidade da linhagem AIRmax frente a I/Ri, confirmada por: 1) maior mobilização de neutrófilos para circulação periférica; 2) maior adesão celular e aumento da migração granulocítica no intestino e pulmão; 3) aumento da expressão de genes de citocinas e daqueles expressos em hipóxia (Tnfa, Il1, Il6 e Hif1a); 4) Translocação Bacteriana (TB); 5) maior expressão pulmonar da proteína HIF-1α e de proteínas envolvidas em processos inflamatórios tais como S100A9, Anexina 1, Profilina 1, Tropomiosina. Por outro lado, a linhagem AIRmin foi considerada pouco responsiva aos efeitos da I/Ri. Diante do exposto, nós concluímos que a sensibilidade dos camundongos AIRmax à injuria após indução de IRi está associada ao agravamento da inflamação sistemica, a qual foi determinada pela indução de HIF-1α atrelada à expressão de proteínas pró- inflamatórias e TB, indicando o compartilhamento ou a co- segregação entre os genes envolvidos na AIR e na hipóxia. / Changes in oxygen transport mechanism (O2) frequent in inflammation, infection, tumors, transplantation and ischemia, lead to tissue hypoxia. Reactive species of O2 are produced and inflammatory cytokines are released triggering a series of events, which are amplified after blood flow refund resulting in systemic inflammation. In the present study, we characterized the regulation of Acute Inflammatory Response (AIR) after intestinal ischemia and reperfusion (I/Ri) induction and the involvement of HIF-1α in this phenotype. Mice selected for high (AIRmax) and low (AIRmin) AIR were subjected to I/Ri and evaluated in different periods of reperfusion (0, 1, 4 and 24h). Our results show sensitivity of AIRmax front line I/Ri, confirmed by: 1) higher neutrophils mobilization to peripheral circulation; 2) increase in cell adhesion and granulocyte migration in lung and intestine; 3) higher expression of cytokine genes and those expressed in hypoxia (TNFa, IL-1, IL-6 and HIF1a); 4) Bacterial Translocation (BT), 5) increase in HIF-1α pulmonary protein expression and those involved in inflammatory processes such as S100A9, Annexin 1, profilin 1 Tropomyosin. On the other hand, the AIRmin line was considered unresponsive to effects of I/Ri. We concluded that the I/Ri sensitivity of the AIRmax mice were associated with worsening of systemic inflammation, which was determined by HIF-1α induction linked to the expression of pro- inflammatory proteins and TB, indicating the share and/or co-segregation of the genes involved in AIR.