This thesis presents the first randomised controlled trial (RCT) to investigate whether supplemented probiotic bacteria-"live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (Sanders, 2008)-affect mood and other psychological outcomes in people presenting with low mood. Seventy-nine participants with at least moderate symptoms of depression were randomised in a double-blind manner to receive either a probiotic preparation containing Lactobacillus helveticus and Bifidobacterium longum or a matched placebo for eight weeks. The RCT phase was followed by an open label extension in which all participants were offered the active study product for a further 8 weeks. Participants were followed up at 6 months post-study. Based on the existing evidence from gut-brain axis research, and on models linking depression with inflammation, immune activation, low vitamin D levels, and the gut microbiota (outlined in Chapters 1 and 2), it was hypothesised that: the overall sample would have elevated levels of inflammatory biomarkers and low levels of vitamin D at baseline, and that this would be associated with scores on psychological and irritable bowel syndrome (IBS) outcome measures; that group differences (active treatment versus placebo) would be observed in scores on psychological outcome measures after eight weeks of probiotic intervention; that group differences would also be observed in blood levels of proinflammatory cytokines, hsCRP, vitamin D and BDNF, and scores on a measure of gut function/IBS, and that levels of these variables may predict or impact on treatment response; and that group differences would be observed on outcome measures at the point of the 6-month follow-up between those who continued to take the probiotic and those who discontinued probiotic use. In total, 58 of the 77 participants who provided baseline blood samples (75%) had at least one marker of inflammation elevated outside the normal reference range at baseline. Baseline vitamin D was approaching the deficient level, displayed a seasonal pattern, and was associated with severity on one measure of cognition. No significant differences were found between the active treatment and placebo groups on any psychological outcome measure, the measure of gut function or in the level of any blood-based biomarker in the randomised phase. Baseline vitamin D level was found to moderate treatment effect on several outcome measures. The results of the open label extension supported the lack of efficacy observed in the randomised phase, and also allowed for the comparison of efficacy over intervention periods of varying durations. The results of the follow-up at 6 months post-trial indicated that, while mean scores on psychological outcome measures remained lower than baseline, there was regression on some outcome measures after the study. When the participants who replied to the 6 month follow-up questionnaire were divided into groups based on their self-reported dominant treatment since the trial (probiotics/nutrition, standard treatment or no treatment) there was no difference in mood or other psychological outcomes among the groups at 6 months. The current trial found no evidence that this probiotic formulation is effective in treating the symptoms of depression or IBS, or in moderating the levels of inflammatory and other biomarkers in a sample recruited with moderate depression. This finding does not support the theory proposed in several narrative reviews which suggests probiotics as a possible intervention for depression and other mental health outcomes, but is supported by the systematic review of human probiotics studies presented in Chapter 3 which found overall limited evidence of probiotic efficacy for psychological outcomes. Future studies in the area should attempt to further broaden this field, in particular by recruiting samples with mild and/or non-chronic depression for interventional studies, or by approaching probiotics as a preventative or adjuvant treatment strategy for depression.

Terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrosequencing were used to characterize the microbiota of the tonsil of the soft palate of 126 unfit and 18 healthy pigs. The T-RFLP analysis method was first optimized for the study of the pig tonsil microbiota and the data compared with culture-based identification of common pig pathogens. Putative identifications of the members of the microbiota revealed that the phyla Firmicutes, Proteobacteria and Bacteroidetes were the most prevalent. A comparison of the T-RFLP analysis results grouped into clusters to clinical conditions revealed paleness, abscess, PRRS virus, and Mycoplasma hyopneumoniae to be significantly associated with cluster membership. T-RFLP analysis was also used to select representative tonsil samples for pyrosequencing. These studies confirmed Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria to be the core phyla of the microbiota of the tonsil of the soft palate of pigs. / OMAFRA Animal Health Strategic Investment

Submitted by Caroline Nishisaka (csayurii@hotmail.com) on 2018-06-04T15:38:30Z No. of bitstreams: 2 Dissertação_Caroline_Sayuri_Nishisaka_UFSCar.pdf: 10342020 bytes, checksum: 83fe90894363834291d0f68de806c3ab (MD5) Carta comprovante da versao final de teses e dissertações.pdf: 204209 bytes, checksum: 74667e4b6b3923d66bce39462bb4538f (MD5) / Approved for entry into archive by Milena Rubi ( ri.bso@ufscar.br) on 2018-06-06T17:30:18Z (GMT) No. of bitstreams: 2 Dissertação_Caroline_Sayuri_Nishisaka_UFSCar.pdf: 10342020 bytes, checksum: 83fe90894363834291d0f68de806c3ab (MD5) Carta comprovante da versao final de teses e dissertações.pdf: 204209 bytes, checksum: 74667e4b6b3923d66bce39462bb4538f (MD5) / Approved for entry into archive by Milena Rubi ( ri.bso@ufscar.br) on 2018-06-06T17:30:45Z (GMT) No. of bitstreams: 2 Dissertação_Caroline_Sayuri_Nishisaka_UFSCar.pdf: 10342020 bytes, checksum: 83fe90894363834291d0f68de806c3ab (MD5) Carta comprovante da versao final de teses e dissertações.pdf: 204209 bytes, checksum: 74667e4b6b3923d66bce39462bb4538f (MD5) / Made available in DSpace on 2018-06-06T17:31:01Z (GMT). No. of bitstreams: 2 Dissertação_Caroline_Sayuri_Nishisaka_UFSCar.pdf: 10342020 bytes, checksum: 83fe90894363834291d0f68de806c3ab (MD5) Carta comprovante da versao final de teses e dissertações.pdf: 204209 bytes, checksum: 74667e4b6b3923d66bce39462bb4538f (MD5) Previous issue date: 2018-05-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Agriculture is responsible for 80% of nitrous oxide emissions on earth's atmosphere, the most potential greenhouse gas among the three main gases (CO2, CH4 and N2O). Citriculture and silviculture play an important role on Brazilian economics due to the high demand of orange and wood products for exportation. Thus, in order to get a better understand about the role of those cultures on N2O emission, this research aims to evaluate N2O emissions from Citrus sinensis and Eucalyptus spp. crops, adjacent forests of each agriculture field were used as control. Citrus and eucalyptus sites are located in Sorocaba and Salto de Pirapora municipality, both in the state of Sao Paulo, Brazil. In each agriculture field and adjacent forest, eight static chambers were randomly installed, and gas sample were collected during dry and wet region season. Soil samples were collected in each field of study from five random sampling points, for further physicochemical, molecular analysis and microcosm incubation. Gas samples were collected from microcosm after moisture adjustment in 70% of field capacity. All gas samples were analyzed by gas chromatography technique. The number of copies of genes related to denitrifying process (nirK, nirS e nosZ) was determined by quantitative real time PCR. Seasonality had significant (P<0.05) influence on N2O emission in field, and N2O fluxes in incubated soil. It was observed positive correlation among dissolved organic carbon (DOC) concentration on soil, soil and plant residue total carbon and nitrogen content and N2O emissions. High nirK, nirS and nosZ gene abundance was found on soil collected during wet season, comparing to those collected on dry season, in both field and microcosm, which was correlated with DOC content and soil moisture. However, there were not significant differences on genes abundance comparisons between cultivated soils and adjacent forests. In conclusion, the seasonality has influence in both N2O emission and denitrifying bacteria abundance present in citrus- and eucalyptus-cultivated soils, mainly through the effects on soil and plant residue physicochemical characteristics, especially those related to COD and soil moisture. / A agricultura é responsável por 80 % do óxido nitroso (N2O) emitido para a atmosfera terrestre, o qual é considerado, dentre os principais gases causadores do efeito estufa, aquele com maior potencial de aquecimento global. A citricultura e a silvicultura são culturas agrícolas que apresentam importante papel na economia brasileira, devido a alta demanda de exportação de produtos derivados da laranja e da madeira. A fim de entender como essas culturas influenciam na emissão do N2O, esse trabalho teve como objetivo avaliar as emissões de N2O a partir de solos cultivados com Citrus sinensis e Eucalyptus spp. Solos florestais adjacentes às áreas agrícolas foram utilizados como controle. As áreas cultivadas com citros e eucalipto estão localizadas nos municípios de Sorocaba e Salto de Pirapora, respectivamente, ambos no estado de São Paulo, Brasil. Em cada área agrícola e florestal, oito câmaras estáticas foram aleatoriamente instaladas e amostras de gases foram coletadas durante o período seco e úmido. Amostras de solo foram coletadas em cada área de estudo a partir de cinco pontos amostrais, as quais foram submetidas a análises físico-químicas, moleculares e incubação em microcosmo. Amostras de gases também foram coletadas a partir dos ensaios de incubação do solo em microcosmos após ajuste da umidade em 70 % da capacidade de campo. Todas as amostras de gases foram analisadas pela técnica de cromatografia gasosa. O número de cópias de genes relacionados com o processo de desnitrificação (nirK, nirS e nosZ) foi determinado por PCR quantitativo em tempo real. A sazonalidade mostrou efeito significativo (P<0,05) nas emissões de N2O em campo, e nos fluxos de N2O determinados a partir do solo incubado. Foi mostrada correlação positiva entre a concentração de carbono orgânico dissolvido (COD) no solo, o teor de carbono e nitrogênio total em solos e resíduos vegetais e as emissões de N2O. Maior abundância dos genes nirK, nirS e nosZ foi encontrada nas amostras de solo coletadas no período úmido em comparação com aquelas coletadas no período seco, tanto em campo como em microcosmo, a qual foi correlacionada com a concentração de COD e a umidade do solo. Contudo, não houve diferença significativa na abundância de tais genes quando comparadas amostras de solos cultivados e florestais adjacentes. Tomados conjuntamente, os resultados permitiram concluir que a sazonalidade influencia tanto a emissão de N2O quanto a abundância de bactérias desnitrificantes presentes em solos cultivados com citros e eucalipto, mediante, principalmente, os efeitos nas características físicas e químicas do solo e dos resíduos vegetais, especialmente aqueles relacionados com o COD e umidade do solo. / FAPESP: 2016/15289-4 / FAPESP: 2017/17441-0

Periodontitis is a multifactorial inflammatory disease which can result in a complete loss of teeth. Its main cause is the accumulation of bacteria from the dental plaque followed by massive reaction of the host immune system. It was proved that the composition of oral microbiome (OM) differs in periodontally healthy individuals and patients with periodontitis. This work aims to solve specific parts of the long-term project concerning the taxonomic composition of the OM of periodontally healthy individuals and patients with chronic and aggressive form of the disease. The OM was characterized based on 16s rDNA sequencing. It is evident from the results that the shift in the OM composition occurs prior the development of clincal signs of the disease and that the precise dental care can significantly postpone or even avoid the onset of the disease. When comparing the OM composition in individuals with chronic and aggressive periodontitis, no remarkable differences were detected to explain the faster progress of the aggressive form of the disease. This work also aimed to compare the results obtained by 454 pyrosequencing and Illumina. Both sequencing methods were found to provide statistically comparable results. Illumina MiSeq thus can be employed to build on the former results of the long-term study...

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Agradecemos a compreensão. on 2017-07-28T13:12:00Z (GMT) / Submitted by Joana Gabriela Desiderato null (desideratojg@gmail.com) on 2017-07-28T13:22:09Z No. of bitstreams: 1 Dissertação_Joana_Gabriela_Desiderato.pdf: 2639616 bytes, checksum: a7f3656945cfe28767c4a7ff73745655 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-08-01T16:56:02Z (GMT) No. of bitstreams: 1 desiderato_jg_me_jabo.pdf: 2639616 bytes, checksum: a7f3656945cfe28767c4a7ff73745655 (MD5) / Made available in DSpace on 2017-08-01T16:56:02Z (GMT). No. of bitstreams: 1 desiderato_jg_me_jabo.pdf: 2639616 bytes, checksum: a7f3656945cfe28767c4a7ff73745655 (MD5) Previous issue date: 2017-07-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A indústria sucroalcooleira gera elevado número de resíduos de origem da biomassa lignocelulósica que apresentam grande potencial para produção de biocombustíveis, em particular o etanol de segunda geração. Uma das formas promissoras para a desconstrução da biomassa lignocelulósica é através da utilização de consórcios bacterianos que produzem enzimas altamente específicas com a capacidade de quebrar a estrutura da lignocelulose. Porém, para a otimização desse processo é importante entender as funções metabólicas presentes nesses consórcios degradadores de biomassa. Portanto, esse estudo objetivou identificar e classificar a composição de uma comunidade bacteriana proveniente de consórcio oriundo de solo contendo bagaço de cana-de-açúcar em decomposição e avaliar sua capacidade metabólica através de sequenciamento genômico de alto rendimento e análises de bioinformática. Esse consórcio foi cultivado durante vinte semanas, sendo que amostras de DNA foram extraídas para sequenciamento a cada sete dias. O sequenciamento da subunidade 16S do operon ribossomal (16S rRNA) foi realizado utilizando a plataforma Ion PGM™, enquanto que o sequenciamento do DNA total foi realizado na plataforma HiSeq 2500, Illumina®. Os resultados do sequenciamento do 16S rRNA indicam 5 diferentes famílias bacterianas ao longo das 20 semanas, sendo Burkholderiaceae (73%) e Rhodanobacteraceae (24%) as mais abundantes. Análises do potencial funcional do consórcio realizadas através dos programas PICRUSt e STAMP, indicam o enriquecimento da função de transportadores, incluindo transportadores do tipo ABC, principalmente na primeira semana, sugerindo um provável papel na metabolização do material lignocelulolítico presente no meio. Os dados gerados pelo sequenciamento total do metagenoma foram montados (“de novo” assembly) e permitiram recuperar a sequência genômica de um dos organismos mais abundantes presentes no consórcio (24%). Esse genoma foi finalizado e circularizado, apresentando 4.758.639 pb e GC% de 65,25, e similaridade de 99% da sequência do 16S rRNA e 90,77% de identidade de suas sequências codificadoras com o genoma da bactéria Dyella jiangningensis SBZ3-12 (Rhodanobacteraceae). A anotação realizada através da plataforma RAST, indicou 4.194 genes codificadores de proteínas, dentre os quais 36 Glicosil Hidrolases potencialmente envolvidas com a degradação da biomassa lignocelulósica. Portanto, esse estudo permitiu a elucidação da diversidade microbiana e perfil metabólico de um consórcio bacteriano, revelando um novo genoma pertencente ao gênero Dyella, potencialmente relacionado com o processo de degradação da biomassa lignocelulósica. Em linhas gerais, os resultados obtidos também corroboraram o potencial que ferramentas de sequenciamento de alto rendimento, metagenômica e bioinformática apresentam para o estudo de comunidades microbianas com potencial biotecnológico, mostrando-se como uma valiosa alternativa para a investigação de novos alvos para a pesquisa em bioenergia. / The sugarcane ethanol industry generates a number of residues related to the lignocellulosic biomass which exhibit potential for the production of biofuels, in particular second generation ethanol. One of the promising ways for the deconstruction of lignocellulosic biomass to valuable products is through the use of bacterial consortia. These consortia encode a specific set of enzymes capable to metabolize and decompose the lignocellulose structure. However, to optimize this process, the metabolic functions present in a biomass-degrading consortia must be well known. Therefore, this study aimed to unravel the taxonomic composition, and to evaluate the metabolic profile of a bacterial consortium from a sugar-cane derived soil containing decomposing straw, through high-throughput genomic sequencing and bioinformatics analyzes. The selected consortium was cultivated in laboratory for twenty weeks, and DNA samples were extracted for sequencing every seven days. The 16S subunit of the ribosomal operon (16S rRNA) and total DNA sequencing were performed through the Ion PGM™ (Thermo Fischer) and HiSeq 2500 (Illumina) platforms, respectively. The 16S rRNA sequencing indicate at least 5 different bacterial families during the 20 weeks of cultivation. The Burkholderiaceae (73%) and Rhodanobacteraceae (24%) are the most abundant. Functional analyses, indicate an enrichment of the transporter-related function, including ABC-transporters mainly in the first week of cultivation, suggesting a probable role related with the decomposing of the lignocellulolytic material. The whole metagenome sequencing uncover the genomic sequence of one of the most abundant organisms present in the consortium (abundance ~24% of the sequenced reads). This genome was finished and circularized, exhibiting 4,758,639 bp, GC% of 65.25, and 99% of similarity of the 16S rRNA and 90.77% identity of the coding sequences to the genome of the bacterium Dyella jiangningensis SBZ3- 12 (Rhodanobacteraceae). The annotation revealed 4,194 proteins coding genes, among which 36 Glycosyl Hydrolases potentially involved with the degradation of lignocellulosic biomass. Therefore, the microbial diversity and metabolic profile of the consortium was revealed. In addition a new genome belonging to the Dyella genus potentially involved with degradation of the lignocellulosic biomass were also uncovered. Overall, the results also corroborated the potential of high-throughput metagenomic sequencing and bioinformatic aproach for the elucidation of microbial communities with biotechnological potential, proving to be a valuable alternative for the bioenergy research. / CNPq: 132532/2015-8

English Abstract Crohn's disease together with ulcerative colitis, is a type of inflammatory bowel disease (IBD) with increasing incidence and prevalence in developed countries. IBD is an immunologically mediated multifactorial disease and it's mechanism of action is still unknown. Current well- established treatment targets the inflammation with corticosteroids and immunosuppressive drugs. Apart from the intestinal inflammation, which is the primary target of the treatment, patients are characteristically afflicted with intestinal dysbiosis. Therefore, possible interventions might be an adjuvant or biological therapy. Adjuvant therapy directly aims the microbiota with probiotics, whereas the target of biological therapy is TNF-α, a pro- inflammatory cytokine excessively secreted by macrophages. The aim of this thesis is to evaluate intestinal microbiota composition changes in IBD patients with regard to courses of adjuvant and biological therapy. Bacterial diversity was analyzed using three different DNA extraction techniques. Rapid beat beating + column (RBB+C) was chosen for analyzing patient samples, as it showed the highest DNA yield and the highest DNA purity. Primarily the bacterial diversity was analyzed using degradation gradient gel electrophoresis (DGGE) with subsequent sequencing of bands of...

Colorectal cancer (CRC) is one of the most treatable cancers, with a 5-year survival rate of similar to 64%, yet over 50,000 deaths occur yearly in the United States. In 15% of cases, deficiency in mismatch repair leads to null mutations in transforming growth factor beta (TGF-beta) type II receptor, yet genotype alone is not responsible for tumorigenesis. Previous work in mice shows that disruptions in TGF-beta signaling combined with Helicobacter hepaticus cause tumorigenesis, indicating a synergistic effect between genotype and microbial environment. Here, we examine functional shifts in the gut microbiome in CRC using integrated - omics approaches to untangle the role of host genotype, inflammation, and microbial ecology. We profile the gut microbiome of 40 mice with/without deficiency in TGF-beta signaling from a Smad3 (mothers against decapentaplegic homolog-3) knockout and with/without inoculation with H. hepaticus. Clear functional differences in the microbiome tied to specific bacterial species emerge from four pathways related to human colon cancer: lipopolysaccharide (LPS) production, polyamine synthesis, butyrate metabolism, and oxidative phosphorylation (OXPHOS). Specifically, an increase in Mucispirillum schaedleri drives LPS production, which is associated with an inflammatory response. We observe a commensurate decrease in butyrate production from Lachnospiraceae bacterium A4, which could promote tumor formation. H. hepaticus causes an increase in OXPHOS that may increase DNA-damaging free radicals. Finally, multiple bacterial species increase polyamines that are associated with colon cancer, implicating not just diet but also the microbiome in polyamine levels. These insights into cross talk between the microbiome, host genotype, and inflammation could promote the development of diagnostics and therapies for CRC. IMPORTANCE Most research on the gut microbiome in colon cancer focuses on taxonomic changes at the genus level using 16S rRNA gene sequencing. Here, we develop a new methodology to integrate DNA and RNA data sets to examine functional shifts at the species level that are important to tumor development. We uncover several metabolic pathways in the microbiome that, when perturbed by host genetics and H. hepaticus inoculation, contribute to colon cancer. The work presented here lays a foundation for improved bioinformatics methodologies to closely examine the cross talk between specific organisms and the host, important for the development of diagnostics and pre/probiotic treatment.

<p>Within the last decade, the popularity and interest in dry-aging have constantly increased among both consumers and producers. Dry-aging is a natural value-adding process where meat is exposed to a controlled refrigerated environment without any protective barrier during the aging process. This process leads to the development of unique flavors in the final meat product. Although the prevalence of this process is increasing, there are inconsistent reports regarding the impacts of dry-aging on meat sensory attributes, especially on the flavor aspect. Given that flavor generation is dependent on the composition and availability of flavor precursors, the presence or absence of these precursors may contribute to the inconsistency observed. Thus the main objective of the research described here was to characterize the flavor precursors in dry-aged meat and elucidate potential factors or mechanisms favoring to their production.</p> <p> To achieve this objective, metabolomics analysis was conducted in conjunction with various chemical analyses (free amino acids, fatty acids, sugar content and volatile analysis), microbiome profiling and meat quality analysis (tenderness, water holding capacity, color stability, oxidative stability, microbial attributes and sensory analysis) to identify the essential flavor precursors and their production process. In addition, similar analyses were conducted using multiple meat sources (grass-fed beef loins, cull cow beef loins and pork loins) aged by wet-aging (WA), conventional dry-aging (DA), dry-aging in bag (DWA) and UV-light dry-aging (UDA) to elucidate the impact of the different aging treatments on meat quality, sensory attributes and flavor precursor availability.</p> <p>Regardless of the meat source, the results demonstrated that dry-aging altered the meat flavor precursor compositions, primarily by increasing the presence of protein-derived precursors (e.g., free amino acids and dipeptides), especially glutamine and glutamate compounds. Additionally, nucleotide and carbohydrate-derived compounds such as adenosine and reducing sugars were greatly increased after the dry-aging process. While the fatty acid profile was minimally affected, metabolomics analysis revealed a decrease in sterol and terpenoid lipids following dry-aging, which could potentially reduce off-flavors development in the meat. Other compounds such as vitamin B and vitamin C were also detected in the dry-aged product, which potentially could contribute to the flavor development.</p> <p>Analysis of the liberation mechanisms demonstrated that dehydration played a role in increasing the concentration of the flavor precursors in the dry-aged product, potentially promoting greater (e.g., Maillard reaction) during cooking. Furthermore, microorganisms might be responsible for further increasing the availability of flavor precursors in dry-aged meat, especially free amino acids, along with the dehydration process. Microbiome profiling found that <i>Pseudomonas</i> spp. are the most prominent bacterial species in microbial communities found on dry-aged meat which could affect the precursor release in dry-aged meat. Metabolomics analysis also indicated increased glutathione metabolism during dry-aging, which could lead to the liberation of glutamine-related compounds. The analysis also identified other compounds such as porphyrin rings (iron-related) and shikimic acid (bacterial metabolism), providing further examples of how metabolomics can identify dry-aged flavor precursors and reveal other potential mechanisms related to flavor development mechanisms.</p> <p>These outcomes demonstrate that dry-aging alters meat flavor precursor composition, mainly by increasing the availability of protein-, nucleotide- and carbohydrate-derived compounds. Such results indicate that the Maillard reaction is likely be the main mechanism in flavor generation in dry-aged meat. The current results provided more insights into the dry-aging flavor development, especially highlighting important flavor precursor such as glutamate and glutamine containing products, likely to contribute to the dry-aged flavor. Future study to identify the impact of different microorganism (especially mold and yeast) on dry-aging flavor development would be of interest. Additionally, impact of different cooking process should also be studies to maximize the dry-aged flavor potential from the product.</p>

No description available.

Microorganisms are all around us. Some of them even live in our body and are essential for our healthy being. Study of microbial communities based on their genetic content has become very popular with the development of new technologies, which enable easy reading of DNA or RNA. The key role of these studies is usually to characterize significant microbial patterns of an environment. However, currently used visualization tools have many drawbacks for such analyses. The subject of this thesis is to design a R/Bioconductor package for simple creation of bipartite graphs from microbial data. This type of visualization brings many advantages for microbiome analysis. Benefits of bipartite graphs are further demonstrated by analysis of main parameters affecting computer processing of microbial data.