The intestinal microbiota is a massive and complex community, essential to the human host for good health and well-being. However, this population has been associated with gastrointestinal disease, and remains poorly understood. The aim of this study was to develop and validate DNA-based assays for the intestinal microbiota and to apply these methodologies to faecal samples collected from healthy volunteers and patients with gastrointestinal disease. Over 250 faecal samples were analysed using temporal temperature gradient gel electrophoresis (TTGE) and real time PCR. Validated assays had high sensitivity and reproducibility. Healthy individuals displayed a high level of temporal stability during short term studies (≤ 6 weeks) and long term studies (1-4years). Analysis of faecal samples provided by identical and fraternal twins demonstrated an influence of host genetics over the composition of the predominant bacteria in children. Two intervention studies, bowel lavage and the Atkins' diet, were carried out to monitor the impact of environmental change on the population's stability in healthy volunteers. Following bowel lavage, microbial populations rapidly recovered to control densities, however the stability of the population was disturbed. Introduction of the Atkins' diet, led to a significant change in the composition of the microbial population. A preliminary study of the intestinal microbiota in disease groups was undertaken. Significant differences were detected between inflammatory bowel disease groups and controls. Cluster analysis in these patients indicated a potential association between the composition of the predominant bacterial population and disease localisation. The studies reported here demonstrate that the faecal microbiota in healthy individuals is a highly stable population under the influence of both host genetics and environmental variables, however the population present in patients with inflammatory bowel disease exhibits differences compared to healthy controls.

Orientador: Cristiano Gallina Moreira / Banca: Waldir Pereira Elias Nunes / Banca: Carla Raquel Fontana Mendonça / Resumo: A sinalização química é o mecanismo através do qual bactérias patogênicas interagem com o hospedeiro e sua microbiota, de modo a promover a regulação dos seus mecanismos de virulência. O estudo da sinalização química, em bactérias entéricas, do sistema de 2-componentes QseBC via autoindutor-3 (AI-3), epinefrina (Epi) e norepinefrina (NE), tem aberto perspectivas para desvendar novos mecanismos. Escherichia coli O104:H4 possui características fenotípicas clássicas de E. coli enteroagregativa (EAEC) e se apresenta positiva para toxina de Shiga, encontrada em cepas de E. coli enterohemorrágica (EHEC). A presença dessa toxina pode levar o hospedeiro ao desenvolvimento de complicações mais graves, como a síndrome hemolítica urêmica (SHU). Dessa forma, essa combinação se torna altamente perigosa e patogênica a humanos, conforme observado no surto epidêmico em 2011 na Europa. O presente estudo teve como objetivo investigar a sinalização química e os mecanismos de patogenicidade em EAEC O104:H4 C227-11, já descritos em EAEC e EHEC, bem como buscar mecanismos ainda não elucidados na literatura. Comparada com cepas protótipos de E. coli diarreiogênicas, os resultados demonstraram que a cepa C227-11 possui um fenótipo de adesão e formação de biofilme acentuados. Em meio ácido, apresentou mais robustez na sobrevivência e maior motilidade em relação à EAEC 042. Também foi possível observar que o nível de expressão gênica para qseC apresentou-se semelhante ao de EHEC e exerce um importante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chemical signalling is the mechanism through which pathogenic bacteria interact with the host microbiota, in order to promote regulation of virulence mechanisms. The study of chemical signalling in two-component system QseBC in enteric bacteria, by autoinducer3 (AI-3), epinephrine (Epi) e norepinephrine (NE), has opened perspectives to discover new mechanisms. Escherichia coli O104:H4 has classic phenotypic characteristics of enteroaggregative E. coli (EAEC) and presents itself positive for Shiga toxin, which is found in enterohemorrhagic E. coli strains (EHEC). This toxin may lead the host to the development of more serious diseases, such as the Hemolytic Uremic Syndrome (HUS). Therefore, this combination is highly dangerous and pathogenic to humans, as observed during the epidemic outbreak in Europe in 2011. This study aimed to investigate chemical signalling and mechanisms of pathogenicity in EAEC O104:H4 C227-11, already described in EAEC and EHEC, as well as mechanisms still not elucidated in literature. Compared to prototype strains of diarrheagenic E. coli, the results have shown that C227- 11 has accentuated phenotype of adhesion and biofilm formation. In acid medium, it presented more strength of survival and more motility than EAEC 042. In addition, gene expression level in qseC was found similar to EHEC and it holds an important participation in activation of virulence factors expression, highlighting, thereby, the possibility of its participation in related mechanisms. Through in vivo tests, it was noticed considerable variation of microbiota, compared to C227-11, just as significant differences among other EAEC that mostly did not present great alterations during analysed days, with predominance of Bacteroidetes over Firmicutes Phylum. The conclusion was that its large profile of adhesion and its elevated tolerance... (Complete abstract click electronic access below) / Mestre

No description available.

Made available in DSpace on 2014-06-11T19:26:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-08Bitstream added on 2014-06-13T19:33:12Z : No. of bitstreams: 1 meyer_jl_me_botib.pdf: 462690 bytes, checksum: 9ffa5e405830b8134648f0d22679350a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A endometriose é uma doença inflamatória estrógeno-dependente que afeta de 5 a 10% das mulheres em idade reprodutiva. É caracterizada pela presença de tecido endometrial fora da cavidade uterina e está associada à dismenorreia, dor pélvica e infertilidade. Os níveis de expressão dos receptores nucleares SF1 (fator esteroidogênico 1), estrógeno e progesterona estão alterados no tecido endometriótico comparado ao endométrio normal. Estudos prévios relataram que os genes codificadores dos receptores dos hormônios esteróides ESR1 (receptor de estrógeno 1), ESR2 (receptor de estrógeno 2) e progesterona (PGR) apresentam promotores alternativos que são modulados epigeneticamente por alterações na metilação do DNA, que ocorre preferencialmente nas ilhas CpG presentes nestas regiões. Em células endometriais normais foi observada uma associação entre a presença de metilação e ausência de expressão dos genes SF1 e ESR2 (receptor de estrógeno â) enquanto a perda da metilação foi correlacionada com níveis aumentados de expressão gênica na endometriose peritoneal e ovariana. Com base nestas evidências, o presente trabalho investigou o padrão de metilação dos genes PGR (promotores A e B), ESR1 (promotores A e B) e uma região intragênica ao ESR2. O promotor B do gene PGR e a ilha CpG localizada na 5’UTR do gene ESR2 foram avaliadas em 44 amostras de endometriose profunda intestinal pela técnica de MSP (Methylation-Specific Polymerase Chain Reaction). Em sete destas, também foi possível a investigação na amostra pareada de endométrio eutópico. O padrão de metilação dos promotores A e B do gene ESR1 e o promotor A do gene PGR... / Endometriosis is an estrogen-dependent inflammatory disease which affects 5 to 10% of women of reproductive age. This disease is characterized by the presence of endometrial tissue outside the uterine cavity and is associated with dysmenorrhea, pelvic pain, and infertility. Abnormal expression levels of SF1 (steroidogenic factor 1), estrogen and progesterone nuclear receptors were detected in the endometriotic tissue compared to the normal endometrium. Previous studies have reported that genes encoding the steroid hormone receptors ESR1 (estrogen receptor 1), ESR2 (estrogen receptor 2) and progesterone (PGR) are characterized by alternative promoters epigenetically regulated by DNA methylation at CpG islands co-localized in these regions. In normal endometrial cells, it was observed an association between DNA methylation and absence of expression of the genes SF1 and ESR2 (estrogen receptor â), while loss of DNA methylation was correlated with increased expression levels of these genes in peritoneal and ovarian endometriosis. Based on these findings, this study investigated the methylation pattern of the PGR (promoters A and B), ESR1 (promoters A and B) genes and an intragenic region of the gene ESR2. The promoter B of PGR gene and the CpG island mapped at 5 'UTR of the ESR2 gene were evaluated in 44 samples of intestinal deep endometriosis by MSP (methylation-specific polymerase chain reaction). In seven cases, paired samples of eutopic endometrium from the same patients were also evaluated, the methylation patterns of the ESR1 gene (promoters A and B) and the promoter region A of the PGR gene were investigated in 37 samples of endometriosis. The MSP method is based on the DNA modification by sodium bisulfite, which converts unmethylated cytosines to uracil. Subsequently, the target region... (Complete abstract click electronic access below)

Made available in DSpace on 2015-05-14T16:53:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-25Bitstream added on 2015-05-14T16:58:53Z : No. of bitstreams: 1 000828470_20170225.pdf: 199599 bytes, checksum: f6c77a439fbc6e7e44ae17d8d9aeb1aa (MD5) Bitstreams deleted on 2017-03-03T11:01:23Z: 000828470_20170225.pdf,. Added 1 bitstream(s) on 2017-03-03T11:02:37Z : No. of bitstreams: 1 000828470.pdf: 3674849 bytes, checksum: e6dae8050533e1481744350024c0cc97 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A Doença Inflamatória Intestinal (DII) engloba, fundamentalmente, duas doenças distintas: a Doença de Crohn (DC) e a Colite Ulcerativa (CU), ambas caracterizadas por uma inflamação crônica do intestino, com períodos de exacerbação seguidos de intervalos prolongados de remissão dos sintomas. Apesar da DII ser objeto de pesquisa há várias décadas, a sua etiologia ainda é desconhecida e um único agente ou mecanismo isolado não parecem ser suficientes para produzir ou desencadear a doença. A dificuldade em se tratar de forma efetiva todos os pacientes com DII em função da baixa resposta aos fármacos associada a diversos efeitos colaterais corrobora a busca de novas fontes de compostos úteis no tratamento e prevenção da DII. Entre as várias estratégias disponíveis, a pesquisa de plantas medicinais se mostra promissora. Bidens pilosa L., família Asteraceae, é uma erva daninha com ampla distribuição e ocorrência em diversos países, sendo conhecida no Brasil como picão-preto. Apesar de ser considerada uma erva daninha sem importância econômica tem sido utilizada em várias populações com finalidade medicinal e sua aplicação em diversas enfermidades pode ser devido a sua composição química rica em phytol e ácido graxos poli-insaturados (ácido palmítico, ácido oleico ácido linoleico), compostos com efeitos anti-inflamatórios e/ou antioxidantes. No presente trabalho o extrato supercrítico de Bidens pilosa L. foi capaz de reduzir os níveis de mediadores pró-inflamatórios, como MPO, IL-1β e TNF-α além de modular fatores anti-inflamatórios como IL-10 tanto no protocolo preventivo quanto no curativo. Modulou também a expressão gênica de genes relacionados à inflamação e proteção da mucosa, como HSP70 e MUC. Além das alterações bioquímicas pôde-se notar uma melhora histológica em amostras de cólon analisadas por microscopia eletrônica. Associada a alterações estruturais ocorreu um ... / Inflammatory Bowel Disease (IBD) comprises basically two distinct diseases: Crohn's disease (CD) and Ulcerative Colitis (UC), both characterized by chronic inflammation of the intestine, with exacerbation periods followed by long intervals of remission of symptoms. Despite being subject of research for decades, its etiology is still unknown and a single agent or isolated mechanism is not enough to produce or trigger the disease. The difficulty in effectively treat all patients based on the poor response to drug and in the several side effects that the treatments presents, supports the search for new sources of compounds useful in the treatment and prevention of IBD. Among the various strategies available, the research of medicinal plants shows promise. Bidens pilosa L., Asteraceae, is a weed with wide distribution and occurrence in some countries and is known in Brazil as picão-preto. Although it is considered a weed without economic importance, has been used in various populations with medical purpose and its application in various diseases may be due to its chemical composition rich in phytol and polyunsaturated fatty acid (palmitic acid, oleic acid and linoleic acid) compounds known to have anti-inflammatory and/or antioxidants effects. In the present study the supercritical extract of Bidens pilosa L. was able to reduce the levels of pro-inflammatory mediators, such as MPO, IL-1β and TNF-α associated with modulation of anti-inflammatory factors such as IL-10, both in the preventive and curative protocols. Also modulates gene expression of genes related to inflammation and mucosal protection, such as HSP70 and MUC. In addition to the biochemical changes might notice a histological improvement in colonic samples analyzed by electron microscopy. Together with structural changes was an increase in the production and secretion of mucin, mucosal protective agents. Thereby, B.pilosa extract is an important source of compounds which may ...

During the last two or three years, the three largest hospitals in Stockton (St. Joseph’s, Dameron and San Joaquin General Hospitals) have modified their procedures to include the search for Campylobacter. On the other hand, the incidence of Yersinia in California is unknown, as most clinical laboratories do not specifically culture for it. There have been only a few reports of Yersinia in this state (personal communication of Dennis Ferrero, Director of the San Joaquin County Public Health Laboratory). This study has a threefold purpose: to investigate the incidences of Campylobacter and Yersinia, especially in relation to the incidence of Salmonella and Shigella, to study the biochemical and antibiogram characteristics of enteric isolates, particularly the “newer” pathogens, and to review the state of routine culture methods and assess their adequacy.

The effect of orally administered duodenal contents on preventing diarrhea in neonatal calves challenged with enteropathogenic E. coli was studied in a 3 x 2 factorial experiment with five replications. Newborn calves received either 0 or 200 ml of intestinal fluid inoculum, obtained from older milk-fed calves, 2 h after entering the isolation facility. Colostrum was consumed following inoculum administration. The uninoculated calves received colostrum 2 h after entering the isolation facility. In compliance with the 3 x 2 factorial arrangement, two-thirds of the calves received an E. coli challenge 12 or 24 h after colostrum feeding. The remaining calves were unchallenged. Raw milk was fed at the rate of 10 percent of body weight per day. All experimental calves were observed daily for physical condition, percent dry matter of feces, urine output, rectal temperature, and dietary intake. Body weight and packed cell volume (PCV) were determined every third day. Gamma globulin per 100 ml serum was determined at 24 h of age. The inoculum was assayed microbiologically for total anaerobes, anaerobic lactobacilli, total aerobes, coliforms, and aerobic lactobacilli. Twelve calves were slaughtered at seven days of age to determine microbiological populations of the duodenal tissue and digesta. During the first six days of life calves receiving the inoculum exhibited a lower incidence of diarrhea, greater daily urine output, lower PCV, and superior average daily gain as compared to the uninoculated calves. The incidence of diarrhea and its accompanying symptoms were most severe in uninoculated calves challenged at 12 h. Rectal temperature was not affected by treatment. The differences in response to the challenge between inoculated and uninoculated calves for the complete experimental period were similar, but not as great as during the first six days of life. Serum gamma globulin at 24 h of age was abnormally low in inoculated calves. Uninoculated calves possessed normal levels of serum gamma globulin. Bacterial populations of duodenal tissue and fluid of seven day old calves were not influenced by treatment. / M.S.

Orientador: Cláudia Aparecida Rainho / Banca: Roberta Guembarovisk / Banca: Celia Regina Nogueira / Resumo: A endometriose é uma doença inflamatória estrógeno-dependente que afeta de 5 a 10% das mulheres em idade reprodutiva. É caracterizada pela presença de tecido endometrial fora da cavidade uterina e está associada à dismenorreia, dor pélvica e infertilidade. Os níveis de expressão dos receptores nucleares SF1 (fator esteroidogênico 1), estrógeno e progesterona estão alterados no tecido endometriótico comparado ao endométrio normal. Estudos prévios relataram que os genes codificadores dos receptores dos hormônios esteróides ESR1 (receptor de estrógeno 1), ESR2 (receptor de estrógeno 2) e progesterona (PGR) apresentam promotores alternativos que são modulados epigeneticamente por alterações na metilação do DNA, que ocorre preferencialmente nas ilhas CpG presentes nestas regiões. Em células endometriais normais foi observada uma associação entre a presença de metilação e ausência de expressão dos genes SF1 e ESR2 (receptor de estrógeno â) enquanto a perda da metilação foi correlacionada com níveis aumentados de expressão gênica na endometriose peritoneal e ovariana. Com base nestas evidências, o presente trabalho investigou o padrão de metilação dos genes PGR (promotores A e B), ESR1 (promotores A e B) e uma região intragênica ao ESR2. O promotor B do gene PGR e a ilha CpG localizada na 5'UTR do gene ESR2 foram avaliadas em 44 amostras de endometriose profunda intestinal pela técnica de MSP (Methylation-Specific Polymerase Chain Reaction). Em sete destas, também foi possível a investigação na amostra pareada de endométrio eutópico. O padrão de metilação dos promotores A e B do gene ESR1 e o promotor A do gene PGR... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Endometriosis is an estrogen-dependent inflammatory disease which affects 5 to 10% of women of reproductive age. This disease is characterized by the presence of endometrial tissue outside the uterine cavity and is associated with dysmenorrhea, pelvic pain, and infertility. Abnormal expression levels of SF1 (steroidogenic factor 1), estrogen and progesterone nuclear receptors were detected in the endometriotic tissue compared to the normal endometrium. Previous studies have reported that genes encoding the steroid hormone receptors ESR1 (estrogen receptor 1), ESR2 (estrogen receptor 2) and progesterone (PGR) are characterized by alternative promoters epigenetically regulated by DNA methylation at CpG islands co-localized in these regions. In normal endometrial cells, it was observed an association between DNA methylation and absence of expression of the genes SF1 and ESR2 (estrogen receptor â), while loss of DNA methylation was correlated with increased expression levels of these genes in peritoneal and ovarian endometriosis. Based on these findings, this study investigated the methylation pattern of the PGR (promoters A and B), ESR1 (promoters A and B) genes and an intragenic region of the gene ESR2. The promoter B of PGR gene and the CpG island mapped at 5 'UTR of the ESR2 gene were evaluated in 44 samples of intestinal deep endometriosis by MSP (methylation-specific polymerase chain reaction). In seven cases, paired samples of eutopic endometrium from the same patients were also evaluated, the methylation patterns of the ESR1 gene (promoters A and B) and the promoter region A of the PGR gene were investigated in 37 samples of endometriosis. The MSP method is based on the DNA modification by sodium bisulfite, which converts unmethylated cytosines to uracil. Subsequently, the target region... (Complete abstract click electronic access below) / Mestre

Over 500 million people worldwide suffer from disease associated with intestinal inflammation, including gastric cancer, inflammatory bowel disease, h. pylori infections, and numerous viral and bacterial infections. Although potentially effective therapeutics exist for many of these pathologies, delivery challenges thwart their clinical viability. The objective of this work was to develop drug delivery platforms that could target toxic immunomodulatory therapeutics to diseased intestinal tissues. To meet this objective, we developed an oral delivery vehicle for siRNA and an NF-κB inhibiting nanoparticle that reduces drug-resistance. Small interfering RNA (siRNA) represents a promising treatment strategy for numerous gastrointestinal (GI) diseases; however, the oral delivery of siRNA to inflamed intestinal tissues remains a major challenge. In this presentation, we describe a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can orally deliver siRNA to sites of intestinal inflammation, and thus inhibit gene expression in diseased intestinal tissue. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with TNFα-siRNA (TNFα-TKNs) diminish TNFα messenger RNA (mRNA) levels in the colon and protect mice from intestinal inflammation. Activation of nuclear factor-κB (NF-κB) results in the expression of numerous prosurvival genes that block apoptosis, thus mitigating the efficacy of chemotherapeutics. Paradoxically, all conventional therapeutics for cancer activate NF-κB, and in doing so initiate drug resistance. Although adjuvant strategies that block NF-κB activation could potentiate the activity of chemotherapeutics in drug resistant tumors, clinical evidence suggests that current adjuvant strategies also increase apoptosis in non-malignant cells. In this presentation, we present a nanoparticle, formulated from a polymeric NF-κB-inhibiting prodrug, that target the chemotherapeutic irinotecan (CPT-11) to solid tumors, and thus abrogates CPT-11-mediated drug resistance and inhibits tumor growth. In order to maximize the amount of NF-κB inhibitor delivered to tumors, we synthesized a novel polymeric prodrug, termed PCAPE, that releases the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) as its major degradation product. Using a murine model of colitis-associated cancer, we demonstrate that when administered systemically, CPT-11-loaded PCAPE-nanoparticles (CCNPs) are three time more effective than a cocktail of the free drugs at reducing both tumor multiplicity and tumor size.

Background: Inflammatory Bowel Disease (IBD) is a group of chronic gastrointestinal diseases with a relapsing nature. The two main types are Crohn’s disease (CD) and ulcerative colitis (UC). Both CD and UC patients experience very similar and distressing symptoms: acute abdominal pain, vomiting, malnutrition, fever, fatigue, diarrhoea and rectal bleeding. These symptoms are disabling and have a severe impact on physical and psychosocial wellbeing. Around 30% of patients suffer from moderate to severe psychological distress and have difficulties coping with the illness even in remission. However, it appears that mental health is overlooked by clinicians who often focus on physical gastrointestinal symptoms only. Mindfulness-Based Cognitive Therapy (MBCT) is evidence based, group psychological intervention that has been successful in reducing depression and anxiety scores in patients with depression while improving overall quality of life. However, MBCT has never been tested in the IBD population before. PhD question: Can MBCT be used as an adjunct therapy to IBD symptom management, for improving IBD patients' general well-being and quality of life? Aims and objectives: The overall aim of the thesis was to develop and collate the evidence for a definitive randomised controlled trial (RCT) testing the effectiveness of MBCT for patients with inflammatory bowel disease (IBD). The thesis brings together six publications. The six publications were integrated into four objectives that collectively contributed in answering the overall PhD question. Results: The findings from the first three publications highlighted the disease-related concerns and psychological needs for patients with IBD. The findings from the last three publications highlighted how feasible it is to use MBCT in IBD and emphasised the IBD patients’ perspectives about MBCT. Conclusion: The thesis concluded that a definitive RCT of MBCT for IBD patients is both feasible and acceptable.