Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Philip R. Hardwidge / Diarrheagenic Escherichia coli (E. coli) strains are common etiological agents of diarrhea. Diarrheagenic E. coli are classified into enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC or enterohemorrhagic E. coli [EHEC]), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffuse-adherent E. coli (DAEC), and adherent invasive E. coli (AIEC). In addition to encoding toxins that cause diarrhea, diarrheagenic E. coli have evolved numerous strategies to interfere with host defenses. In the first project, we identified an ETEC-secreted factor (ESF) that blocked TNF-induced NF-[kappa]B activation. One of the consequences of TNF-induced NF-[kappa]B activation is the production of pro-inflammatory cytokines that help to eliminate pathogens. Modulation of NF-[kappa]B signaling may promote ETEC colonization of the host small intestine. In this study, we fractionated ETEC supernatants and identified flagellin as necessary and sufficient for blocking the degradation of the NF-[kappa]B inhibitor I[kappa]B[alpha] in response to TNF[alpha]. In the second project, we attempted to identify an ETEC cAMP importer. ETEC diarrhea leads to cAMP release into the lumen of the small intestine. cAMP is a key secondary messenger that regulates ETEC adhesin expression. We hypothesized that a cAMP importer is present in ETEC, accounting for its hypersensitivity to extracellular cAMP. We used Tn5 transposome-mediated mutagenesis to construct a mutant library and screen for cAMP-hyporesponsive mutants. However, none of the 17,956 mutants we screened were cAMP-hyporesponsive. In the third project, we focused on gut microbiota and the T3SS effector NleH. We used the mouse-specific pathogen C. rodentium and transplanted performed microbiota between different mouse strains. We evaluated microbiota populations as a function of infection with WT and [Delta]nleH C. rodentium strains before and after microbiota transplantation. Microbiota transfer altered the resistance to WT C. rodentium infection in C57BL/10ScNJ mice and the NleH effector promoted host resistance to C. rodentium.

In recent years, the association between inflammatory bowel diseases (IBDs) and gut microbiota has been extensively studied in adults using post-treatment cohorts of patients. However, microbial composition and functional interplay between host genetics and microorganisms in newly diagnosed early IBD onset remain poorly defined. Using colonoscopic mucosal washes to collect mucosal-luminal microbiota from different intestinal locations, we studied the gut microbiome in a large number of children with either Crohn’s disease (CD) or ulcerative colitis (UC). Although no significant difference in the diversity was evident between the gut microbiota of IBD-affected and control children, the microbiome of IBD subjects is characterized by an increased abundance of potent hydrogen sulfide (H2S) producers and decreased abundance of beneficial butyrate producers. Microbiota and proteomic profiling revealed that the abundance of Atopobium parvulum, a potent H2S producer, was associated with increased CD severity and a concurrent reduction in the expression of the host H2S detoxification pathway. Gnotobiotic and conventionalized colitis-susceptible interleukin-10-deficient (Il-10-/-) mice showed that A. parvulum induces severe colitis, a phenotype requiring the presence of the gut microbiota. In addition, administration of bismuth, an H2S scavenger, prevented A. parvulum-induced colitis in Il-10-/- mice. Our findings have identified A. parvulum as a major mediator of inflammation severity. We also reveal an imbalance between the H2S production and detoxification in the gastrointestinal tract of pediatric IBD patients. Altogether, our findings provide new avenues for diagnostics as well as therapies to treat IBD.

Orientador: Raphael Lucio Andreatti Filho / Coorientador: Adriano Sakai Okamoto / Banca: Alessandre Hataka / Banca: Ana Angelita Sampaio / Resumo: A microbiota intestinal das aves exerce papel fundamental no desenvolvimento de seu potencial produtivo por garantir o bom funcionamento do trato gastroentérico e do sistema imunológico a ele intimamente ligado. Por isso diversos aditivos já foram utilizados para favorecer o desenvolvimento de uma microbiota saudável, entretanto, ainda não temos informações sobre o perfil desejável para ela. Sendo assim, nos propusemos a utilizar a Eletroforese em Gel com Gradiente de Temperatura (TGGE), para avaliar a microbiota do ceco de aves alimentadas com dietas acrescidas de halquinol, ácido caprílico e Lactobacillus, e posteriormente desafiadas com Salmonella Enteritidis (SE). Aos 10, 20, 30 e 42 dias de vida foram quantificadas as imunoglobulinas IgA, IgM, IgY e interleucinas 17 e 8, avaliada a colonização cecal de SE (UFC/mL), observação histopatológica de tonsila cecal, e esses resulatados relacionados com a microbiota cecal avaliada pelo método TGGE. O tratamento com ácido caprílico foi igualmente eficaz ao halquinol na diminuição da colonização por SE, seguidos do grupo tratado com Lactobacillus. As concentrações de Il-8 e IgM demonstraram relação com a maturação e desenvolvimento imune local e sistêmico das aves, porém, não identificamos relação com o aumento da complexidade da microbiota, entre os diferentes trtamentos e nos grupos desafiados. No presente estudo também n"ao foi possível reproduzir a técnica de TGGE / Abstract: The intestinal microbiota of birds plays a fundamental role in the development of their production potential to ensure the proper functioning of a gastrointestinal tract and immune system closely linked to it. So many additives have been used to encourage the growth of a healthy microbiota, however, we still have no information about the desired profile for her. So we set out to use the Gel Electrophoresis with Temperature Gradient (TGGE) to assess the cecal microbiota of birds fed diets plus halquinol, caprylic acid and Lactobacillus, and subsequently challenged with Salmonella Enteritidis (SE). At 10, 20, 30 and 42 days old were quantified immunoglobulins IgA, IgM, IgY and interleukins 17:08 assessed cecal colonization IF (CFU / ml), cecal tonsil histopathological observation, and those related to resulatados cecal microbiota evaluated by TGGE method. The treatment with caprylic acid was equally effective in reducing the halquinol colonization by IF, followed by the group treated with Lactobacillus. IL-8 and IgM concentrations shown maturation and compared with the local and systemic immune development of the poultry, however, we do not identify relationship with the increased complexity of the microbiota between different trtamentos and challenged groups. In this study it was also not possible to reproduce the TGGE / Mestre

With recent research being conducted in categorizing and analyzing the human microbiome, evidence has now linked the human microbiome to a range of diseases. Dysbiosis of the human gut microbiome exists in colon cancer, obesity, and Clostridium difficile infections. The use of fecal microbiota transplants has been proven effective in treating recurrent C.difficile infections by restoring gut microbiota. More needs to be done to establish fecal microbiota transplants procedures, effectiveness, and safety. Once established, fecal microbiota transplants may play a role in modulating other diseases linked to human gut microbiome dysbiosis.

The Indian River Lagoon (IRL) FL, USA, is an Estuary of National Significance due to its economic and high biodiversity. Microbial populations are understudied in the IRL despite their numerous ecological services. A two-year, nineteen-site Lagoon-Wide Survey (LWS) was conducted to provide the first 16S rRNA amplicon sequencing data on the microbiome of the sediment in the IRL and determine how the microbiome changed in response to environmental and anthropogenic factors. The most influential variables that explained the variability between microbiomes were porewater salinity, total organic matter (TOM), and copper (Cu). These results correlated with some of the anthropogenic pressures the IRL faces such as freshwater discharges from St. Lucie Estuary (SLE), trace metal contamination, and the accumulation of fine-grained, highly organic sediment known as “IRL muck” (muck). Research then focused on determining the microbial differences between three sets of sample types: sediment from the IRL versus the SLE; sediment that had three muck characteristics versus those with zero; and high TOM sediment that had high Cu versus high TOM sediment that had low Cu. Differentially abundant prokaryotes between sample types were determined with novel indicator analysis techniques. One technique tested the effectiveness of an indicator list to separate samples based upon the product of the sensitivity and specificity of partitioning around medoids clustering in comparison to metadata classifications. The other technique allowed for the tracking of changes in the entire indicator microbiome. These new indicator analysis techniques were created using the original LWS data and tested to determine how sediment microbiomes responded during two opportunistic surveys: dredging of muck from an IRL tributary (Eau Gallie River) and Hurricane Irma. These studies have filled the knowledge gap regarding the unknown microbiome of the IRL and how sediment microbiomes respond to extreme events such as dredging and a hurricane. They also led to the development of new indicator analysis techniques that can be used by to track changes in the entire indicator microbiome. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

abstract: First year college students have been identified as a vulnerable population for weight gain and the onset of overweight and obesity. Research regarding the gut microbiome has identified differences in the microbial composition of overweight and obese individuals compared to normal weight individuals. Dietary components like dietary fibers, act as prebiotics, or fermentable substrate, that the gut microbiota use for metabolic functions including the production of short-chain fatty acids. The objective of this longitudinal, observational study was to assess changes in the gut microbiota over time in relation to changes in fiber consumption in healthy college students at a large a southwestern university (n=137). Anthropometric and fecal samples were collected at the beginning and end of the fall and spring semesters between August 2015 and May 2016. Both alpha, within sample, diversity and beta, between sample, diversity of participant gut microbes were assessed longitudinally using non-parametric pairwise (pre-post) comparisons and linear mixed effect (LME) models which also adjusted for covariates and accounted for time as a random effect. Alpha and beta diversity were also explored using LME first difference metrics and LME first distance metrics, respectively, to understand rates of change over time in microbial richness/phylogeny and community structure. Pre-post comparisons of Shannon Diversity and Faith’s PD were not significantly different within participant groups of fiber change (Shannon diversity, p=0.96 and Faith’s PD, p=0.66). Beta diversity pairwise comparisons also did not differ by fiber consumption groups (Unweighted UniFrac p=0.182 and Bray Curtis p=0.657). Similarly, none of the LME models suggested significant associations between dietary fiber consumption and metrics of alpha and beta diversity. Overall, data from this study indicates that small changes in fiber consumption among a free-living population did not have an impact on gut microbial richness, phylogeny or community structure. This may have been due to the low intake (~15 g/d) of fiber. Further study is needed to fully elucidate the role that fiber plays in the diversity and composition of the gut microbiota, especially when delivered from a variety of food sources rather than fiber supplements. / Dissertation/Thesis / Masters Thesis Nutrition 2020

Lactate, a metabolic end product of glycolysis in mammals, has emerged as an important energy substrate for the brain. In addition to its energetic role, lactate was shown to modulate the excitability of neurons, to have a neuroprotective role and to participate in long-term memory formation. One previous investigation from our group reported that lactate modulates 4 synaptic plasticity-associated genes and potentiates the activity of the N-Methyl-D-aspartic acid (NMDA) receptor, a major receptor type involved in glutamatergic neurotransmission. The current thesis aimed at first to extend these findings by examining genome-wide transcriptional responses to this metabolite in cortical neurons. Using ribonucleic acid(RNA) sequencing to evaluate expression changes in protein-coding genes, we found that lactate modulates robustly after 1h, 20 genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway and in synaptic plasticity in a NMDA receptor activitydependent manner and that nicotinamide adenine dinucleotide, reduced (NADH), but not pyruvate, reproduces the modulatory effects of lactate on 70% of all differentially expressed genes. In a time course experiment, genes modulated after lactate treatment for 6h and 24h were also identified; these are involved in 9 signaling pathways including circadian rhythm, drug addiction, and retrograde endocannabinoid signaling. Bioinformatics analyses indicated CREB1 and CREM as candidate master regulators of gene expression and the modulatory effect of lactate was prevented by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, indicating a role for this kinase in mediating lactate signaling. An examination of changes in dendritic spines’ morphology and density - a morphologicalcorrelate of synaptic plasticity – has shown that lactate modulated spine density changes induced by potassium chloride (KCl) and carbachol. An additional investigation described in this thesis indicated that different gut microbiota manipulations (germ-free, prebiotics, high-fat diet) regulated mRNA expression of genes involved in the Astrocyte-Neuron Lactate Shuttle (ANLS) - a metabolic cooperation mechanism between astrocytes and glutamatergic neurons. Overall, the results of this thesis help to establish a role for lactate as a signaling molecule in the brain, highlight mechanisms implicated in its signaling, and open new avenues for investigation of links between the gut microbiota and brain energy metabolism.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22334号 / 医博第4575号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Research in our lab has predicted hundreds of bacterial genes that influence nine different traits in the fruit fly, Drosophila melanogaster. As a practical alternative to creating site-directed mutants for each of the predicted genes, we created an arrayed transposon insertion library using a strain of Acetobacter fabarum DsW_054 isolated from fruit flies. Creation of the Acetobacter fabarum DsW_054 gene knock-out library was done through random transposon insertion, combinatorial mapping and Illumina sequencing. Successful mapping of transposon insertion was achieved for 6418 mutants with hits within 63% of annotated genes within Acetobacter fabarum DsW_054. Insertion sites were verified in 40 mutants through arbitrary PCR and sequencing. To test the utility of the library, genes were selected from MGWAS results on host colonization which show LPS pathway enrichment in the significant gene predicctions. Genes upstream of Lipid-A creation show significant differences in host colonization whereas downstream genes show no effect. In addition, genes were selected from MGWAS results on Drosophila starvation resistance which show Methionine/Cysteine synthesis, Cobalamin synthesis, and Biotin synthesis pathway enrichment. Under our experimental conditions we could not verify influence of these pathways on host starvation resistance. However, they do appear to influence host colonization abundance. This transposon insertion mutant library will be useful for ongoing research in our lab as well as any field studying Acetobacter species, such as other insect microbiome and fermentation research.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objective: The objective of this study was to characterize the composition of the gut microbiota in type 2 Diabetes Mellitus (T2DM) patients with adequate and inadequate metabolic control, and its relationship with fiber consumption. Results: A total of 26 patients with type 2 diabetes mellitus (T2DM) were enrolled, of which 7 (26.9%) cases had adequate metabolic control (HbA1c < 7%) and 19 (73.1%) inadequate metabolic control (HbA1c ≥ 7%). It was observed that among patients with controlled T2DM, 2 (28.6%) cases presented good intake of fiber and 5 (71.4%) cases a regular intake. In contrast, in patients with uncontrolled T2DM, 13 (68.4%) patients reported a regular intake and 6 (31.6%) a poor intake. In relation to the identification of the gut microbiota, both groups presented a similar characterization. There were differences in the population of bacteria identified in both groups, however, the results were not statistically significant. The most frequently identified bacteria in controlled and uncontrolled T2DM patients were Prevotella (71.4% vs 52.6%), followed by Firmicutes (71.4% vs 42.1%), Proteobacteria (71.4% vs 36.8%) and Bacteroidetes (57.1% vs 37.8%). On the other hand, Fusobacterium, Actinobacteria were not identified in either of the two groups of study. / Revisión por pares