Neste trabalho realizou-se a avaliação de pacientes com doenças inflamatórias intestinais (DII) - doença de Crohn (DC) e retocolite ulcerativa idiopática (RCUI) quanto às variáveis psicológicas estresse e modos de enfrentamento utilizado frente à situação de doença. Investigaram-se, ainda, as relações entre estas variáveis e diferentes aspectos clínicos das DII. Os grupos de pacientes com DC e RCUI foram compostos por 25 integrantes cada, que foram comparados com igual número de controles doentes (CD) e saudáveis (CS), sendo estes últimos selecionados dentre os acompanhantes dos pacientes. Empregaram-se instrumentos padronizados e validados de avaliação psicológica: escala de eventos vitais e inventário de enfrentamento. Os resultados mostraram que proporções elevadas e semelhantes (64 80%) dos integrantes dos quatro grupos apresentaram-se sob estresse intenso, ocorrendo, porém, diferenças entre os grupos quanto aos tipos de eventos vitais estressantes experimentados. O estresse intenso se mostrou maior e associado significativamente à idade mais avançada na DC e à doença em atividade na RCUI. Quanto aos modos de enfrentamento, na DC e na RCUI, verificaram-se proporções semelhantes e significativamente superiores à do grupo CD de casos utilizando a estratégia de confronto (40% vs. 12%; p=0,01). Na DC, a proporção de pacientes utilizando a estratégia de resolução de problemas foi significativamente maior que na RCUI (96% vs. 80%; p=0,05). Na RCUI, a proporção de pacientes utilizando a estratégia de fuga/esquiva (96%) foi significativamente maior (p=0,05) que nos grupos CD (80%) e CS (76%). A utilização das várias estratégias de enfrentamento parece ser afetada, na DC, por sexo, escolaridade e estado de atividade da doença. Na RCUI, houve efeito das variáveis: escolaridade, estado de atividade e duração da doença e estresse intenso. Estes dados indicam a ocorrência de estresse intenso nos pacientes, independente do tipo de doença e, também nos acompanhantes. A DC e a RCUI se diferenciam quanto às estratégias de enfrentamento mais utilizadas e pelos fatores que as afetam, o que deve ser levado em consideração no estabelecimento de medidas de intervenção psicológica necessárias ao cuidado integral ao paciente. / This work aimed at assessing emotional stress and coping strategies in patients with inflammatory bowel diseases (IBD): Cohns disease (CD) and ulcerative colitis (UC), as well as studying the relationships between this psychological variables and demographical and clinical characteristics. Groups of patients with CD and UC (N=25) were compared with equal number of subjects from two control groups: healthy caregivers (HC) and patients with other digestive diseases (DC). Validated and standardized psychological tools, such as a stressful live events scale and a ways of coping questionnaire were utilized. Results showed that substantial proportions (64 80%) of subjects pertaining to each of the four groups were under severe stress, with a number of inter-group differences regarding the most quoted stressful event. Severe stress was associated with older age in CD patients, and with disease activity in UC patients. Regarding coping strategies, IBD patients showed an increased proportion of utilization of confrontation (40% vs. 12%; p=0.01), when compared to the DC group. In the CD group, there was a higher proportion of patients utilizing the problem solving strategy, than in the UC group (96% vs. 80%; p=0.05). In the UC group, the proportion of patients utilizing the avoidance strategy (96%) was significantly greater (p=0.05) than in both DC (80%) and HC (76%) groups. In IBD patients, coping styles seemed to be affected by sex (only in the CD group), educational level and disease activity, as well as by disease duration and severe stress (only in the UC group). Our data therefore show that severe emotional stress occurs not only in IBD and other digestive disease patients but also in their caregivers. CD and UC patients show different coping styles, which seems also to be affected by distinct demographical and clinical variables. These findings should be taken into account when designing psychological interventions, which are needed for a more comprehensive health care.

Une carence en donneurs de méthyle (CDM) est fréquente dans les maladies inflammatoires chroniques intestinales (MICI) et induit un stress du réticulum endoplasmique (RE). La désacétylase SIRT1 (Sirtuine 1) régule la réponse cellulaire suite à un stress nutritionnel par le biais d’un stress du RE. Nous avons étudié pour la première fois l’impact d’une CDM sur le stress du RE et les effets de l'activation de SIRT1 dans un modèle expérimental de colite chez le rat. Pour restaurer l’homéostasie du réticulum endoplasmique, la cellule met en place la réponse UPR « Unfolded Protein Response ».Les quantités de formes actives des acteurs de l’UPR (effecteurs et régulateurs), ainsi que du facteur de transcription (HSF1) et SIRT1 ont été étudiés par western blot, immunoprécipitation et RT-qPCR sur des tissus coliques (rats et humains) et sur des lysats cellulaires. Les effets de l'activation de SIRT1 ont été étudiés in vitro et in vivo. Une CDM aggrave la colite induite par le DSS cliniquement, endoscopiquement et histologiquement. Une CDM induit un stress du RE en augmentant le niveau d’expression des formes active des acteurs de l’UPR : p-PERK, p-eIF-2a, p-IRE-1a, ATF6, XBP1-S et l’ARNm d’ATF4. Ceci est accompagné d’une diminution dramatique du niveau d’expression des protéines chaperonnes, d’une réduction du niveau d'expression de SIRT1 et d’une hyperacétylation de HSF1. L’ajout de la vitamine B12, de la S-Adénosine Methionine (SAM), ou un activateur de SIRT1 (SRT1720) réduit l'UPR in vitro. L’immunohistochimie a montré une diminution de l’expression de SIRT1 dans l'épithélium du côlon de patients atteints de MICI. Chez le rat, l’activation de SIRT1 prévient la colite en réduisant le niveau acétylé de HSF1 et en augmentant le niveau d’expression des molécules chaperonnes. L'activation de SIRT1 prévient la colite en réduisant le stress du RE causé par la CDM. Ces résultats suggèrent que SIRT1 pourrait être une cible thérapeutique dans les MICI où les CDM sont fréquentes / Methyl donor deficiency (MDD) aggravates experimental colitis in rats and decreases SIRT1 activity in cell models. Endoplasmic reticulum (ER) stress plays a key role in inflammatory bowel disease (IBD) pathogenesis. We investigated whether the influence of MDD on colitis resulted from an ER stress response triggered by decreased SIRT1 expression. The Unfolded protein response (UPR), chaperone proteins, heat-shock factor protein 1 (HSF1) and SIRT1 were examined in rats with MDD and dextran sulfate sodium (DSS)- induced colitis, in a Caco-2 cell model with stable expression of transcobalamin-oleosin chimera (TO), which impairs cellular availability of vitamin B12, and in human IBD. The effects of SIRT1 activation were studied both in vitro and in vivo. MDD aggravated DSS-induced colitis clinically, endoscopically and histologically. MDD activated ER stress pathways, with increased p-PERK, p-eiF-2a, p-IRE-1a, ATF6, XBP1-S protein and ATF4 mRNA expression levels in rats. This was accompanied by reduced SIRT1 expression level and greater acetylation of HSF1, in relation with a dramatic decrease of chaperons (BIP, HSP27 and HSP90). Adding either vitamin B12, Sadenosylmethionine, or a SIRT1 activator (SRT1720) reduced the UPR in vitro. In rats, SIRT1 activation by SRT1720 prevented colitis by reducing acetylation of HSF1 and increasing the expression of BIP, HSP27 and HSP90. Immunohistochemistry showed impaired expression of SIRT1 in the colonic epithelium of IBD patients. SIRT1 is a master regulator of ER stress and severity of experimental colitis in case of MDD. It could deserve further interest as a therapeutic target of IBD

Introdução: Colite Ulcerativa (UC) é uma Doença Inflamatória Intestinal (DII) caracterizada por uma resposta imune exacerbada, com sintomas como diarreia, perda de peso e sangue nas fezes. Apesar dos medicamentos disponíveis, a remissão da doença nem sempre consegue ser alcançada e há a necessidade de terapias alternativas. A colite induzida por DSS (Dextran Sulfate Sodium) é um modelo animal utilizado na investigação de novas terapias por sua semelhança à UC humana. DSS provoca dano à barreira epitelial do cólon, induzindo uma resposta imune exacerbada; entretanto, o exato mecanismo não está totalmente esclarecido. O Ultrassom Terapêutico (TUS) foi utilizado para tratamento de injúria renal em modelo experimental, sua ação se dá através da estimulação do nervo vago (VN) e consequente ativação da via antiinflamatória colinérgica (CAIP). Uma vez que pacientes com DII podem exibir atividade disfuncional do VN, TUS pode ser investigado como terapia alternativa. Objetivos: Investigar temporalmente o perfil clínico, proteômico, histológico e imunológico da colite aguda induzida por DSS; e determinar os efeitos de TUS na colite induzida por DSS. Métodos: No primeiro estudo, a severidade da colite foi avaliada pela administração de DSS 1-3%, observando a resposta clínica e histológica. A análise temporal de DSS 3% incluiu uma avaliação proteômica e histológica do cólon, e a resposta imune celular no baço, linfonodo mesentérico (MLN) e cólon. No segundo estudo, utilizando o modelo de DSS 2%, TUS foi aplicado no abdômen dos animais e foram observados os sintomas clínicos, dano histológico, proteômica do cólon e respostas imunes celulares no baço, MLN e cólon. Animais esplenectomizados ou knockout para a7nAChR (marcador clássico para ativação de CAIP) foram utilizados. Resultados: No primeiro estudo, observou-se que a severidade da doença foi aumentada seguindo concentrações de 1-3% DSS. A análise temporal de DSS 3% demonstrou que os macrófagos (F4/80+) se apresentam como a primeira resposta celular, seguidos por células T CD25+, CD4+ e CD8+. A piora clínica da doença correspondeu ao aumento progressivo de fatores pró-inflamatórios e dano tecidual no cólon, exceto no dia 8. Foram observados menores níveis dos marcadores de células T CD25+, CD4+ e CD8+ no MLN e/ou baço, sugerindo a ocorrência de tropismo destas células para o intestino. No segundo estudo, a aplicação de TUS diminuiu a severidade da doença através da melhora de sintomas clínicos, danos teciduais e encurtamento do cólon. A proteômica do cólon demonstrou uma resposta anti-inflamatória durante a fase de injúria (D0-7), induzindo uma resolução acelerada da doença na fase de recuperação (D8-14). TUS diminuiu os níveis de células T CD8+ e normalizou os níveis de células T CD25+ no cólon. Animais esplenectomizados não demonstraram melhora clínica ou histológica, enquanto animais a7nAChR KO apresentaram piora da colite experimental. Além disso, TUS aumentou os níveis de células F4/80+a7nAChR+ no intestino de animais WT DSS 2%. Conclusão: Nossos resultados demonstram que a severidade da doença depende da concentração de DSS, relacionada com as respostas clínica, proteômica e imune no modelo animal de DSS 3%; e TUS diminuiu a severidade da colite induzida por DSS presumidamente pela da estimulação do VN e consequente ativação de CAIP através do baço. / Introduction: Ulcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) characterized by uncontrolled immune response, presenting with symptoms of diarrhea, weight loss and bloody stools. Despite available treatments, UC sustained remission is not achievable and there is still the need for alternative therapies. Dextran Sulfate Sodium (DSS)-induced colitis is a mouse model used to investigate novel therapies, since it closely mimics human UC. DSS damages the colonic epithelial barrier, leading to an exacerbated immune response. However, the exact mechanism is not totally understood. Previous studies showed the use of Therapeutic Ultrasound (TUS) to prevent kidney injury in mice through stimulation of the vagus nerve (VN) and activation of the cholinergic anti-inflammatory pathway (CAIP). Since IBD patients can present with dysfunctional VN activity, TUS could be studied as an alternative therapy. Objectives: To investigate the temporal clinical, proteomic, histological and cellular immune profiles of DSS-induced acute colitis; and to determine the effects of TUS directed toward the VN and spleen in the course of DSS-induced colitis. Methods: First, we analyzed DSS-induced colitis severity by administration of 1-3% DSS, observing the clinical course and histological damage. A time course analysis was performed at 3% DSS, including colon proteomics, colon histology and immune cell responses in the spleen, MLN (mesenteric lymph node) and colon. Next, utilizing 2% DSS in drinking water, we applied TUS over the mice abdomen and analyzed clinical symptoms, histological damage, colon proteomics and immune cell responses in the spleen, MLN and colon. Splenectomized and a7nAChR (key indicator of CAIP activation) KO animals were also used. Results: In the first study, we observed worsening of the disease when increasing DSS concentrations from 1 to 3%. Time course analysis of 3% DSS revealed macrophages to be the first responders, followed by CD25+, CD4+ and CD8+ T cells. Worsening of the disease corresponded to a progressive increase in pro-inflammatory colonic factors and histological damage, except at day 8. Lower levels of CD25+, CD4+ and CD8+ T cells in MLN and/or spleen suggest an immune cell tropism to the gut. In the second study, TUS attenuated DSS induced colitis through amelioration of clinical symptoms, histological damage and colon shortening. Proteomic colon analysis demonstrated an antiinflammatory profile during the injury phase (D0-7), whilst inducing an early resolution of the disease during the recovery phase (D8-14). TUS decreased CD8+ and normalized CD25+ T cell levels in the gut. Splenectomized animals demonstrated no improved clinical and pathological outcomes, and a7nAChR KO mice presented with worsening of the disease. Furthermore, there were increased levels of F4/80+a7nAChR+ cells in the colon of 2% DSS WT mice under TUS treatment. Conclusion: Our results demonstrate that the severity of colitis is dependent on DSS concentration, correlated with clinical, proteomic and cellular immune responses on 3% DSS; and TUS significantly improved DSS-induced acute colitis presumably through stimulation of the VN and consequent activation of CAIP through the spleen.

Orientador: Sérgio Alberto Rupp de Paiva / Resumo: A Doença Inflamatória Intestinal (DII) apresenta curso variável. O tratamento adequado da fase aguda pode levar à remissão clínica da doença, cujo seguimento ocorre ambulatorialmente. Nesta condição, os pacientes mantêm hábito de vida normal e alguns ainda permanecem com inflamação. Esta situação de doença e estilo de vida pode interferir na composição corporal e no estado nutricional dos pacientes. Doença de Crohn (DC) e Retocolite Ulcerativa (RCU) são doenças heterogêneas em diversos aspectos, dentre eles na composição corporal e no estado nutricional. O objetivo do estudo foi avaliar clusters relacionados à variação do estado nutricional na DIl. Foi realizado estudo transversal com pacientes com DII, submetidos a avaliação clínica (Crohn’s Disease Activity Index (CDAI), escore de Mayo e uso de medicações), nutricional (recordatório de 24 horas, International Physical Activity Questionnaire (IPAQ), peso, estatura, Índice de Massa Corporal (IMC), absorciometria por raios-X de dupla energia (DEXA), análise de bioimpedância elétrica (BIA), força de preensão manual, teste de caminhada de 6 minutos) e laboratorial (hemoglobina (Hb), hematócrito (Ht), Proteína C Reativa (PCR), velocidade de hemossedimentação (VHS), albumina). Com base nestes parâmetros, foram diagnosticados com Desnutrição (Global Leadership Initiative on Malnutrition, GLIM) e Sarcopenia (European Working Group on Sacopenia in Older People 2, EWGSOP2). Foram realizados testes estatísticos descritivos por meio do ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Inflammatory Bowel Disease (IBD) presents a variable course. Adequate treatment of the acute phase may lead to clinical remission of the disease, which is followed in the outpatient clinic. In this condition, patients maintain normal life habit and some still remain with inflammation. Both disease and lifestyle situation may interfere with the body composition and nutritional status of the patients. Crohn's disease (CD) and ulcerative colitis (UC) are heterogeneous diseases in several aspects, including body composition and nutritional status. The aim of the study was to evaluate clusters related to the variation of the nutritional status in IBD. A cross-sectional study was performed with IBD patients, by clinical (Crohn's Disease Activity Index (CDAI), Mayo score and medications), nutritional (24-hour recall, International Physical Activity Questionnaire (IPAQ), weight, stature, Body Mass Index (BMI), dual energy X-ray absorptiometry (DEXA), electrical bioimpedance (BIA), handgrip force, 6-minute walk test) and laboratorial evaluation (hemoglobin (Hb), hematocrit (Ht), C-Reactive Protein (CRP), erythrocyte sedimentation rate (ESR), albumin). Based on these parameters, were diagnosed Malnutrition (Global Leadership Initiative on Malnutrition (GLIM) and Sarcopenia (European Working Group on Sacopenia in Older People 2, EWGSOP2). Descriptive statistical tests were performed by mean ± standard deviation for numerical variables with normal or median distribution and quartiles (Q1... (Complete abstract click electronic access below) / Doutor

En el patró fibroestenosant de la malaltia de Crohn els fibroblasts intestinals augmenten en número contribuint al desenvolupament de la fibrosi. Un dels principals promotors de la proliferació d’aquestes cèl•lules és el bFGF. En el present estudi la fracció rica en tocotrienol ha demostrat tenir efectes antiproliferatius en fibroblasts intestinals humans independentment de la procedència dels fibroblasts. Els tocotrienols redueixen tant la proliferació basal com la induïda per bFGF en fibroblasts. L’apoptosi de les cèl•lules efectores en el desenvolupament de la fibrosi sembla ser el principal mecanisme de la seva resolució. Els tocotrienols instauraren un procés complert d’apoptosi en els fibroblasts. A diferència del que passa amb la majoria d’agents proapoptòtics, els tocotrienols provoquen l’activació tant de la via extrínseca com de la via intrínseca de l’apoptosi ja que activa les caspases 8 i 9. Sorprenentment, l’addició de ciclosporina A (CsA), un conegut inhibidor de la via intrínseca de l’apoptosi, bloqueja completament el procés d’apoptosi induït per FRT. Això demostra que tot i l’activació de la caspasa 8 i per tant, de la via extrínseca, en el tractament amb tocotrienols la predominant és la via intrínseca. L’autofàgia té un paper molt rellevant en l’aclariment de cossos apoptòtics. Els tocotrienols han demostrat eficàcia en la inducció d’autofàgia en fibroblasts, ja que provoca la maduració de la proteïna LC3 i l’aparició de vacuoles autofàgiques en el citoplasma d’aquestes cèl•lules. Un cop més la CsA reverteix aquest procés, i per tant inhibeix l’autofàgia, demostrant que aquest procés requereix la participació de la mitocòndria. Les patologies que cursen amb desenvolupament de fibrosi estan associades a alts nivells de TGF-β que resulta en el reclutament de fibroblasts al lloc de la lesió i a un increment en la producció de matriu extracel•lular. A nivell intracel•lular, la fosforilació d’Smad3 és el pas clau que regula les accions del TGF-β. Els tocotrienols han mostrat eficàcia en la disminució dels nivells intracel•lulars d’Smad3 fosforilada induïda per TGF-β. La fosforilació d’Smad3 té efectes oposats en la regulació de l’expressió gènica. Regula positivament l’expressió de TIMP-1, en canvi, regula negativament l’expressió de MMP-3. Tot i ser efectes oposats en quant a expressió gènica, aquests dos fenòmens afavoreixen l’acumulació de matriu extracel•lular. L’exposició a tocotrienols fa que es reverteixi aquest estat profibrogènic ja que indueix l’expressió de MMP-3 però no afecta als nivells de TIMP-1. La síntesi de MEC i especialment la síntesi de COL1, COL3 i COL5 està incrementada en la fibrosi intestinal, aquest fet està directament relacionat amb l’activitat de TGF-β en la zona afectada. Els tocotrienols interfereixen en la síntesi de col•lagen. A més, els tocotrienols disminueixen la fosforilació d’Smad3 induïda per TGF-β, un fet clau en la síntesi de matriu extracel•lular. Aquests resultats permeten hipotetitzar un potencial antifibrogènic dels tocotrienols. Per a confirmar aquest potencial “in vivo” ens calia disposar d’un model experimental de fibrosi intestinal. A diferència del que succeeix amb la colitis, en la que es disposa d’un gran nombre de models animals, hi ha pocs models experimentals específicament dissenyats per a reproduir la fibrosi intestinal. Aquest fet, ha limitat molt el desenvolupament de noves estratègies de tractament antifibrogènic en l’intestí. Mitjançant l’administració intracolònica repetida a dosis baixes de l’haptè TNBS vam poder establir fibrosi intestinal en la rata de manera rellevant i reproduïble. En aquest model de fibrosi intestinal, els animals que van rebre la dosi més alta de tocotrienols provada en aquest estudi van mostrar nivells disminuïts en paràmetres que mesuren la inflamació, això és, es va disminuir l’índex d’activitat de la malaltia i la pèrdua de pes causada pel TNBS així com també va reduir l’expressió de TNF-α. / Excessive fibroblast expansion and extracellular matrix deposition are key events for the development of bowel stenosis in Crohn’s disease patients. Tocotrienols are vitamin E compounds with proven in vitro antifibrogenic effects on rat pancreatic fibroblasts. We aimed at investigating the effects of tocotrienols on human intestinal fibroblast proliferation, apoptosis, autophagy, and synthesis of ECM. Intestinal fibroblasts isolated from patients with Crohn’s disease were treated with tocotrienol-rich fraction from palm oil. Tocotrienols significantly reduced proliferation, enhanced cell death by promoting apoptosis and autophagy. Fibroblast apoptosis, but not autophagy, was prevented by the pan-caspase inhibitor zVAD-fmk, whereas both types of cell death were prevented when the mitochondrial permeability transition pore was blocked by cyclosporin A, demonstrating a key role of the mitochondria in these processes. TRF diminished procollagen type I and laminin γ production these cells. Transforming growth factor-β plays a key role in matrix deposition during fibrosis. Treatment of intestinal fibroblasts with tocotrienol rich fraction prevents Smad3 phosporylation induced by transforming growth factor-β and reduces collagen type 1 and 3 production by these cells. Besides, in a rat model of intestinal fibrosis, treatment with tocotrienols reduces diarrhea, rectal bleeding and weight loss, as well as levels of colonic tumor necrosis factor-α and vimentin expression demonstrating that this compound has anti-inflammatory effects in vivo.

La maladie de Crohn (MC) et la colite ulcéreuse (CU) sont des maladies inflammatoires chroniques du tube digestif qu’on regroupe sous le terme maladies inflammatoires de l’intestin (MII). Les mécanismes moléculaires menant au développement des MII ne sont pas entièrement connus, mais des études génétiques et fonctionnelles ont permis de mettre en évidence des interactions entre des prédispositions génétiques et des facteurs environnementaux - notamment la flore intestinale – qui contribuent au développement d’une dérégulation de la réponse immunitaire menant à l’inflammation de la muqueuse intestinale. Des études d’association pangénomiques et ciblées ont permis d’identifier plusieurs gènes de susceptibilité aux MII mais les estimations de la contribution de ces gènes à l’héritabilité suggèrent que plusieurs gènes restent à découvrir. Certains d’entre eux peuvent se trouver dans les régions identifiées par des études de liaison génétique. L’objectif de mon projet de doctorat était d’identifier un ou des facteurs de risque génétique dans la région chromosomale 19p (identifiée comme région de liaison IBD6) et de le/les caractériser au niveau fonctionnel. Nous avons d’abord entrepris une cartographie d’association de la région 19p. À la suite du génotypage successif de deux cohortes indépendantes, nous avons identifié un SNP intronique et quatre SNP codants dont un non-synonyme, rs8108738, tous localisés dans le gène microtubule associated serine threonine kinase gene-3 (MAST3) et associés aux MII. Peu d’information fonctionnelle sur MAST3 était disponible. Par contre MAST2, une protéine encodée par un gène de la même famille, régule l’activité du facteur de transcription inflammatoire NF-kappaB. Nous avons confirmé l’implication de MAST3 dans l’activité de NF-kappaB via un knockdown de MAST3 et des essais gène-rapporteur. Pour poursuivre la caractérisation fonctionnelle de MAST3, nous avons choisi une approche non ciblée pour étudier les effets de la variation des niveaux d’expression de MAST3 sur la cellule. C’est-à-dire que nous avons créé un 1er modèle cellulaire de surexpression du gène MAST3 dans les cellules HEK293 et analysé l’expression pangénomique endogène. La validation de l’expression génique dans un 2e modèle cellulaire de knockdown et de type cellulaire différent (THP1), nous a permis d’identifier et de contrer les effets non-spécifiques dus aux niveaux non-physiologiques. Notre étude d’expression a mené à l’identification d’un groupe de gènes dont l’expression est régulée par MAST3. Ces gènes sont majoritairement impliqués dans des fonctions immunitaires (cytokines pro-inflammatoires, régulateurs de NF-kappaB, migration cellulaire, etc.) et une forte proportion est régulée par NF-kappaB. Nous avons évalué l’importance du groupe de gènes régulés par MAST3 dans la présentation clinique des MII à travers des études d’expression dans des biopsies intestinales de patients atteints de CU. Nous avons constaté que l’expression de ces gènes est significativement supérieure dans les régions enflammées par rapport aux régions saines de la muqueuse intestinale des patients atteints de CU. Globalement, les résultats de nos études suggèrent que le facteur de risque aux MII MAST3 agit via la voie du facteur de transcription NF-kappaB pour influencer l’expression d’un groupe de gènes impliqués dans l’inflammation intestinale typique des MII. Chaque étude génétique sur les MII a le potentiel d’orienter les recherches fonctionnelles vers de nouvelles voies biologiques causales. Le dévoilement des mécanismes moléculaires sous-jacents à ces voies permet d’augmenter les connaissances sur le développement de ces maladies vers une compréhension plus complète de la pathogenèse qui permettra d’optimiser le diagnostic et le traitement de ces maladies. / Inflammatory bowel diseases (IBD) refer to different chronic inflammatory diseases of the digestive tract mainly Crohn’s disease (CD) and ulcerative colitis (UC). Mechanisms leading to the pathogenesis of IBD are not completely understood, but genetic and functional studies have highlighted interactions between genetic predispositions and environmental factors, such as the intestinal microbiota, as contributors to the deregulation of the immune response that leads to inflammation of the intestinal mucosa. Genome-wide and targeted association studies have identified several IBD susceptibility genes. However, estimations of the contribution of these genes to heritability of the disease suggest that more are to be discovered. Some of these genes may be in previously identified IBD linkage regions. The objective of my doctoral project was first, to identify risk factors in the 19p chromosomal region (IBD6), previously identified following a genome-wide linkage study, then to characterise them functionally. We first performed a comprehensive association mapping study of the 19p region. Our two-stage genotyping strategy led to the identification of one intronic SNP and four coding SNP –including one non-synonymous SNP, rs8108738 – all located in the microtubule associated serine threonine kinase gene 3 (MAST3) and associated to IBD. Very limited functional information on MAST3 was available at that time. However MAST2 (a gene in the same family as MAST3), is involved in the regulation of inflammation master switch, transcription factor NF-B. We confirmed the involvement of MAST3 in the modulation of NF-B via a knockdown of MAST3 and gene reporter assays. In order to further characterize the function of MAST3, we chose a non-targeted approach to study the effects of the modulation of MAST3 levels on the cell. More specifically, we created a 1st cell model of MAST3 overexpression in HEK293 cells and analysed the resulting genome-wide endogenous gene expression. Validation in a 2nd cell model consisting of a knockdown of MAST3 in THP1 cells, allowed to identify non-specific gene expression due to non-physiological MAST3 levels. Our expression study led to the identification of a group of genes whose expression is modulated by MAST3. These genes are mainly involved in immune functions (pro-inflammatory cytokines, NF-B regulation, cellular migration, etc) and a majority is regulated by transcription factor NF-B. We evaluated the importance of this MAST3-regulated gene set in the clinical manifestation of IBD through an expression study on biopsies of UC patients. We found that the expression the MAST3-regulated gene set was significantly enriched in inflamed region of the intestinal mucosa of UC patients compared to healthy region. Taken together, the results of our study suggest that IBD risk factor MAST3 acts on the NF-B pathway to influence the expression of a group of genes involved in intestinal inflammation typical of IBD. Every genetic study on IBD has the potential to lead functional research towards new causal biological pathways. The unravelling of the molecular mechanisms underlying these pathways aims to improve the comprehension of the pathogenesis of IBD and hopefully will allow for optimization of diagnostic and treatment of these diseases.

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.

Orientador: Alba Regina Monteiro Souza Brito / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T00:26:41Z (GMT). No. of bitstreams: 1 Ferreira_AndersonLuiz_D.pdf: 1659590 bytes, checksum: a72e12af76082feafe5a240c07b04574 (MD5) Previous issue date: 2009 / Resumo: Apesar da amplitude de possibilidades terapêuticas, ainda não existe um tratamento ideal para as doenças inflamatórias intestinais (DII), com perfil adequado de eficácia e segurança. Por essa razão, é de grande interesse estudar agentes com poucos efeitos colaterais, que preferencialmente façam parte da dieta, no tratamento/prevenção dessas doenças. Neste contexto, surgiu o interesse em estudar uva, fruta com alto teor de flavonóides e fibras dietéticas, ambas benéficas à saúde. Sabe-se que substâncias antioxidantes e ácidos graxos de cadeia curta (AGCC), principalmente o ácido butírico proveniente da fermentação de fibras dietéticas, podem estar envolvidas na prevenção e tratamento de doenças intestinais. O ácido butírico é utilizado como substrato para a reparação do tecido inflamado e inibe fatores pró-inflamatórios; portanto, quantificar a produção desse ácido é imprescindível, dado que quanto maior for sua produção, maior será a eficiência da dieta para a recuperação do tecido. Para tanto, foi realizada uma análise in vitro que demonstrou que a concentração de 0,5 g de Uva foi a que produziu maiores quantidades de butirato na análise por cromatografia de íons. Após a escolha da concentração da fruta, seguiu-se o estudo in vivo utilizando-se a dose de 0,5 g.kg-1. A princípio, verificou-se a ação antioxidante avaliando os níveis de GHS além das atividades das enzimas antioxidantes GSSG-Rd, GSH-Px e SOD em ratos com colite induzida por ácido trinitrobenzenosulfônico (TNBS). Os animais tratados com Uva apresentaram altos níveis de GSH; as atividades da GSSG-Rd, GSH-Px permaneceram próximas às do grupo sem colite. A atividade da SOD, que também foi drasticamente diminuída no grupo TNBS, foi restabelecida no grupo tratado. O Tratamento com Uva foi capaz de exercer efeito antinflamatório intestinal, já que os animais tratados apresentaram menor escore macroscópico da lesão, além de diminuir alguns mediadores pró-inflamatórios, como (COX-2, NF-_B e molécula de adesão intestinal MadCAM-1) e a atividade da MPO. A diminuição do dano cólico pela Uva pode ser explicada, em parte, pela diminuição da produção de interleucinas pró-inflamatórias (IL-6 e IL-12) que funcionam como fatores quimiotáxicos da inflamação. Por outro lado, a Uva aumentou a produção da citocina antinflamatória IL-10, sendo que esse aumento foi maior que os valores normais obtidos para o grupo salina. Conclui-se, que a Uva mostrou habilidade em modular enzimas antioxidantes, além de aumentar a produção de IL-10 o que, provavelmente, reduziu a produção de citocinas pró-inflamatórias e, consequentemente, a expressão de mediadores inflamatórios. / Abstract: Despite the range of therapeutic possibilities, there is still no ideal treatment for inflammatory bowel disease (IBD), with the right profile of efficacy and safety. For this reason, it is of great interest to study agents with little side effect, which preferably form part of the diet and that treats / prevents such diseases. In this context, emerged the interest in studying Grape, fruit with high content of flavonoids and dietary fiber, two compounds that improve the healthy. It is known that antioxidants and short chain fatty acids (SCFA), especially butyric acid, obtained from the fermentation of dietary fiber, may be involved in the prevention and treatment of intestinal diseases. The butyric acid is a substrate for the repair of inflamed tissue and inhibits pro-inflammatory factors; for that reason, to quantify the production of this acid is mandatory, given that the higher the production, the greater the efficiency of the diet for tissue recovery. Therefore, an in vitro analysis showed that the concentration of 0.5 g of Grape was the one that produced the greatest amount of butyrate in the analysis by ion chromatography. After choosing the concentration of the fruit, the next step was an in vivo study where was used the dose 0,5 g.kg-1. At first, was checked the antioxidant action through the analysis of the levels of GSH and the activity of antioxidants enzimas like GSSG-Rd, GSH-Px and SOD in rats with colitis induced by trinitrobenzenosulfonic acid (TNBS). Animals treated with Grape showed high levels of GSH, the activities of GSSG-Rd, GSH-Px remained close to the group without colitis. The activity of SOD, which was also drastically reduced in the TNBS group, was restored in the treated group. The treatment with Grape was able to exert intestinal anti-inflammatory effect, since the treated animals showed lower scores of the lesion and reduced some proinflammatory mediators such as MPO activity, COX-2, NF-_B and the intestinal adhesion molecule MadCAM-1 expression. The decrease in colonic damage can be explained, partially, by the action of the Grape that decreased the production of pro-inflammatory interleukins (IL-6 and IL-12) that act as chemotactic factors of inflammation. On the other hand, the treatment of Grape increased the production of anti-inflammatory cytokine, such as IL-10, and this increase was higher than normal values showed by the saline group. It can be concluded that the Grape showed ability to modulate the antioxidant enzymes and increased the production of IL-10. Probably, the increased of the levels of this interleukin resulted in a decrease in the production of proinflammatory cytokines and, consequently, in a decrease the expression of inflammatory mediators. / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Neste trabalho realizou-se a avaliação de pacientes com doenças inflamatórias intestinais (DII) - doença de Crohn (DC) e retocolite ulcerativa idiopática (RCUI) quanto às variáveis psicológicas estresse e modos de enfrentamento utilizado frente à situação de doença. Investigaram-se, ainda, as relações entre estas variáveis e diferentes aspectos clínicos das DII. Os grupos de pacientes com DC e RCUI foram compostos por 25 integrantes cada, que foram comparados com igual número de controles doentes (CD) e saudáveis (CS), sendo estes últimos selecionados dentre os acompanhantes dos pacientes. Empregaram-se instrumentos padronizados e validados de avaliação psicológica: escala de eventos vitais e inventário de enfrentamento. Os resultados mostraram que proporções elevadas e semelhantes (64 80%) dos integrantes dos quatro grupos apresentaram-se sob estresse intenso, ocorrendo, porém, diferenças entre os grupos quanto aos tipos de eventos vitais estressantes experimentados. O estresse intenso se mostrou maior e associado significativamente à idade mais avançada na DC e à doença em atividade na RCUI. Quanto aos modos de enfrentamento, na DC e na RCUI, verificaram-se proporções semelhantes e significativamente superiores à do grupo CD de casos utilizando a estratégia de confronto (40% vs. 12%; p=0,01). Na DC, a proporção de pacientes utilizando a estratégia de resolução de problemas foi significativamente maior que na RCUI (96% vs. 80%; p=0,05). Na RCUI, a proporção de pacientes utilizando a estratégia de fuga/esquiva (96%) foi significativamente maior (p=0,05) que nos grupos CD (80%) e CS (76%). A utilização das várias estratégias de enfrentamento parece ser afetada, na DC, por sexo, escolaridade e estado de atividade da doença. Na RCUI, houve efeito das variáveis: escolaridade, estado de atividade e duração da doença e estresse intenso. Estes dados indicam a ocorrência de estresse intenso nos pacientes, independente do tipo de doença e, também nos acompanhantes. A DC e a RCUI se diferenciam quanto às estratégias de enfrentamento mais utilizadas e pelos fatores que as afetam, o que deve ser levado em consideração no estabelecimento de medidas de intervenção psicológica necessárias ao cuidado integral ao paciente. / This work aimed at assessing emotional stress and coping strategies in patients with inflammatory bowel diseases (IBD): Cohns disease (CD) and ulcerative colitis (UC), as well as studying the relationships between this psychological variables and demographical and clinical characteristics. Groups of patients with CD and UC (N=25) were compared with equal number of subjects from two control groups: healthy caregivers (HC) and patients with other digestive diseases (DC). Validated and standardized psychological tools, such as a stressful live events scale and a ways of coping questionnaire were utilized. Results showed that substantial proportions (64 80%) of subjects pertaining to each of the four groups were under severe stress, with a number of inter-group differences regarding the most quoted stressful event. Severe stress was associated with older age in CD patients, and with disease activity in UC patients. Regarding coping strategies, IBD patients showed an increased proportion of utilization of confrontation (40% vs. 12%; p=0.01), when compared to the DC group. In the CD group, there was a higher proportion of patients utilizing the problem solving strategy, than in the UC group (96% vs. 80%; p=0.05). In the UC group, the proportion of patients utilizing the avoidance strategy (96%) was significantly greater (p=0.05) than in both DC (80%) and HC (76%) groups. In IBD patients, coping styles seemed to be affected by sex (only in the CD group), educational level and disease activity, as well as by disease duration and severe stress (only in the UC group). Our data therefore show that severe emotional stress occurs not only in IBD and other digestive disease patients but also in their caregivers. CD and UC patients show different coping styles, which seems also to be affected by distinct demographical and clinical variables. These findings should be taken into account when designing psychological interventions, which are needed for a more comprehensive health care.

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No. of bitstreams: 2 Dissertação - Joniel Mendes de Araújo - 2017.pdf: 3123838 bytes, checksum: 85b81eeeeeddff88dbab91146cde5709 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-09-28 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Crohn's disease (CD) and ulcerative colitis (RCU) are the major and most prevalent inflammatory bowel diseases (IBD). The lack of effective treatment makes the search for new drugs to treat IBD extremely important. Bixin, a carotenoid present in the species Bixa orellana L., appears as a possible candidate for presenting several biological activities among them antibacterial, antioxidant and anti-inflammatory. The objective of this study was to evaluate the intestinal anti-inflammatory effect of Bixin in experimental TNBS-induced colitis in rats. For the experimental protocol Wistar rats (n = 10) were divided into 7 groups: Saline group (without colitis), TNBS group (with colitis) and groups treated with Bixina gavage at doses of 1, 3, 6, 9 and 12mg / kg. Colitis was induced according to the procedure described by Morris et al. (1989). Euthanasia was performed by CO2 overdose followed by removal of the colon for macroscopic and histological analysis. The results demonstrated that the presence of adhesions and diarrhea was decreased in the groups treated with Bixina. The macroscopic analysis of the colon evidenced the presence of severe damage to the colonic mucosa, with edema, deep ulcerations and hemorrhage in the TNBS group. The Bixina-treated group (12 mg/kg) significantly attenuated the areas of ulcerative lesion and macroscopic score when compared to the TNBS group. Animals receiving Bixin (12 mg/kg) also had a decreased mass / length ratio of the colon compared to the TNBS group. The histological characteristics of the colon of the TNBS group showed severe signs of inflammation, necrosis and edema. However, the Bixin group (12 mg/kg) showed better cell organization and less intestinal epithelial damage when compared to the TNBS group. The results suggest a protective effect of the carotenoid Bixin against experimental colitis induced by TNBS. However, new studies must be performed to verify the mechanisms involved in this activity. / A Doença de Crohn (DC) e a Retocolite Ulcerativa (RCU) são as principais e mais predominantes doenças inflamatórias intestinais (DII). A inexistência de um tratamento eficaz, faz extremamente importante a busca por novos fármacos para tratar as DII. A Bixina, um carotenoide presente na espécie Bixa orellana L., aparece, como um possível candidato por apresentar diversas atividades biológicas dentre elas antibacteriana, antioxidante e anti-inflamatória. O objetivo deste trabalho foi avaliar o efeito anti-inflamatório intestinal da Bixina na colite experimental induzida por TNBS em ratos. Para a realização do protocolo experimental utilizou-se fêmeas de ratos Wistar (n = 10) divididas em 7 grupos: grupo Salina (sem colite), grupo TNBS (com colite) e grupos tratados por gavagem com Bixina nas doses de 1, 3, 6, 9 e 12mg/Kg. A colite foi induzida de acordo com o procedimento descrito por Morris et al. (1989). E a eutanásia foi realizada por overdose de CO2, seguida da remoção do cólon para realização das análises macroscópicas e histológicas. Os resultados demonstraram que a presença de adesões e diarreia, foi diminuída nos grupos tratados com Bixina. A análise macroscópica do cólon evidenciou a presença de danos graves na mucosa cólica, com edema, ulcerações profundas e hemorragia no grupo TNBS. O grupo tratado com Bixina (12 mg/kg) atenuou significativamente as áreas de lesão ulcerativa e o escore macroscópico quando comparado ao grupo TNBS. Os Animais que receberam Bixina (12 mg/kg) apresentaram também uma relação massa/comprimento do cólon diminuída em comparação com o grupo TNBS. As características histológicas do cólon de ratos do grupo TNBS apresentaram sinais severos de inflamação, necrose e edema. Contudo, o grupo Bixina (12 mg/Kg), apresentou uma melhor organização celular e menos danos do epitélio intestinal quando comparado ao grupo TNBS. Os resultados sugerem um efeito protetor do carotenoide Bixina contra a colite experimental induzida pelo TNBS. Contudo, novos estudos devem ser realizados para verificar os mecanismos envolvidos nessa atividade.