Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

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No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EfeitoUsoDiferentes.pdf: 239802 bytes, checksum: 5b39de29c260f7db7fb4293678fffc0e (MD5) Previous issue date: 2008 / Na conservação de alimentos para animais, os inoculantes microbianos são empregados na busca da melhoria do padrão de fermentação de silagens, por meio do estímulo ao desenvolvimento populacional dos microrganismos benéficos deste processo conservativo, como acontece para as bactérias produtoras de ácido lático, em detrimento a inibição dos microrganismos indesejáveis, tais como leveduras e clostrídios. O estudo proposto avaliou o efeito do uso de diferentes inoculantes microbianos à fresco e liofilizados utilizando a cultura do sorgo [Sorghum bicolor (L.) Moench], como matéria-prima para ensilagem, a fim de indicar a possibilidade do emprego de inoculantes microbianos desenvolvidos no nosso país. Foram realizados dois experimentos, em um mesmo silo, Experimento 1 (tratamentos com inoculantes liofilizados, na região superior do silo) e Experimento 2 (tratamentos com inoculantes à fresco, na região inferior do silo), com cinco tratamentos e três repetições por silo, sendo os tratamentos caracterizados como controle (sem inoculante), inoculante microbiano comercial (IC) e distintos inoculantes confeccionados à partir de bactérias láticas isoladas da planta de sorgo: Lactobacillus plantarum, Lactobacillus paracasei e Lactobacillus rhamnosus. Quando da ensilagem, foram utilizados três silos experimentais de madeira, que foram abertos em distintos períodos, ou seja, 1, 3 e 28 dias após a ensilagem. Foi utilizado o delineamento experimental em parcelas subdivididas no tempo, no qual os três períodos de abertura foram às parcelas e os cinco tratamentos as subparcelas, em delineamento inteiramente casualizado. No experimento 1, os teores de fibra em detergente neutro (FDN) e fibra em detergente ácido (FDA) sofreram efeito dos inoculantes microbianos no 3º e 28º dia de abertura dos silos, obtendo menores valores nos tratamentos IC e LPP (L. plantarum + L. paracasei. No experimento 2, os teores de FDN, apresentaram efeito no 28º dia de abertura do silo, demonstrando que os tratamentos IC e LPP diferiram entre si, sendo estatisticamente iguais aos demais. A combinação dos isolados microbianos liofilizados de L. plantarum e L. paracasei mostrou potencial para uso prático, pois foi tão efetivo quanto o tratamento IC. / In the conservation of animal feed, the microbial inoculants are used in pursuit of improving the standard fermentation of silages, by encouraging the population development of the beneficial microorganisms in this process conservative, as the bacteria that produce lactic acid, rather than using inhibition of undesirable microorganisms such as yeasts and clostridia. The proposed study evaluated the effect of using different microbial inoculants to using fresh and freeze-dried sorghum [Sorghum bicolor (L.) Moench], as raw material for silage, to indicate the possibility of using microbial inoculants developed in the Brazil country. Were conducted two experiments in the same silo, Experiment 1 (treatment with freeze-dried inoculants in the upper region of the silo) and Experiment 2 (treatment with inoculants to cool in the lower silo), with five treatments and three replicates per silo the treatments being characterized as a control (without inoculum), commercial inoculant (IC) and the different inoculants made from lactic acid bacteria isolated from the sorghum plant: Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus rhamnosus. When silage were used three experimental silos of wood, which were opened at different periods, 1, 3 and 28 days after ensiling. We used the experimental design in split plot in time, in which the three periods were open on plots and subplots the five treatments in a completely randomized design. In experiment 1, the levels of neutral detergent fiber (NDF) and acid detergent fiber (ADF) had the effect of microbial inoculants on the 3rd and 28th day of opening the silos, getting lower values in treatments IC and LPP (L. plantarum + L. paracasei. In experiment 2, NDF, had an effect on the 28th day of opening of the silo, showing that the IC and LPP treatments differed significantly, being statistically equal to others. The combination of freeze-dried microbial isolates of L. plantarum and L. paracasei showed potential for practical use because it was so effective as treatment failure.

The subject of the thesis is study of possible applications of isoform of glutamic acid polymer (-PGA). The theoretical part is focused on the properties of this biopolymer and potential applications in various areas. Producers and mechanisms of biosynthesis are also mentioned. In the experimental part, the polymer was firstly characterised by following methods: FT-IR spectroscopy, TGA, DSC and SEC-MALS. Its isoelectric point, antimicrobial activity and solubility in various solvents were also determined. The biopolymer was also precipitated by divalent cations and its interaction with oppositely charged CTAB surfactant was studied. The main experimental study was researching the effect of -PGA on viability of Saccharomyces cerevisiae and Lactobacillus rhamnosus under stress conditions by flow cytometry. The performed stresses included ethanol exposure, high temperature and freezing stress, in which its effects were compared to conventional cryoprotectants. The cells of the mentioned microorganisms were also stressed osmotically and exposed to model gastrointestinal juices - gastric, pancreatic and bile. The protective effects of -PGA on the cells were recorded in ethanol stress on Lactobacillus rhamnosus. Its excellent cryoprotection properties were confirmed and its protective effect of gastric juice exposure on Saccharomyces cerevisiae cells was also observed. At the end of the experimental part, -PGA/alginate beads suitable for encapsulation of probiotic bacteria and -PGA/chitosan nanoparticles for encapsulation of biologically active substances.

Increasing evidence implicates the presence of bacteria in intrauterine tissues as an important risk factor for spontaneous preterm labor. Epigenetic alterations of innate immunity genes may increase the mother’s sensitivity to subclinical levels of bacteria. This study examined the presence of TET2, TLR-2, and TLR-9 in intrauterine tissue, and evaluated whether epigenetic alterations of these genes, as well as IL-8, changed their expression in human decidual tissue and a macrophage cell culture. Immunohistochemicalstaining was used to detect the presence of these proteins in intrauterine tissue. Gene expression changes were evaluated in stimulated monocytes and macrophages. Fluorescence immunohistochemistry was used to track translocation of TET2 in stimulated monocytes and macrophages. Secreted IL-8 concentration was detected with ELISA. Decidual expression of TET2, TLR-2, and TLR-9 increased in the order TNL < TL < sPTL < iPTL. This study found that TET2, TLR-2, TLR-9, and IL-8 are regulated by epigenetic mechanisms. This study was the first to report activation of TET2 involves its translocation from the cytosol to the nucleus in macrophages.