Dissertations / Theses on the topic 'Instestin'

Les cytokines favorisent la défense et l'homéostasie de l'intestin. Parmi les cytokines clés de l'intestin, l'interleukine-22 (IL-22) est produite par les cellules immunitaires et cible principalement les cellules épithéliales. l'IL-22 protège l'intestin contre les agents pathogènes en induisant l'expression de peptides antimicrobiens et en favorisant la réparation des tissus. Une dysrégulation de cette cytokine peut conduire à des maladies inflammatoire de l'intestin et au cancer. Au cours du développement, le tube digestif post-embryonnaire est colonisé par des micro-organismes commensaux qui favorisent la maturation de l'intestin et de son système immunitaire. Cependant, il est encore incertain si et comment l'IL-22 joue un rôle dans le développement et la maturation de l’intestin au cours de cette période critique. Le poisson-zèbre nous permet de visualiser et de manipuler les processus physiologiques in vivo dès le début du développement en raison de son développement externe et de sa transparence.Au cours de ma thèse de doctorat, j'ai cherché à décrypter la fonction de l'IL-22 dans le développement de l'intestin. Mes données montrent que l'il22 est exprimée dans les cellules épithéliales intestinales des larves avant que les lymphocytes exprimant l'IL-22 n'apparaissent dans l'intestin. J'ai identifié les cellules entéroendocrines (EEC), un sous-type de cellules épithéliales intestinales, comme la principale source d'il22 chez les larves. De plus, j'ai révélé la conservation de la voie de signalisation de l'IL-22, sa régulation transcriptionnelle par la détection des microbes et sa fonction antibactérienne dans l'intestin. Mes données les plus récentes suggèrent que l'expression d’il22 peut également être induite par l'activation de Trpa1, un récepteur connu pour reconnaître les métabolites du tryptophane chez le poissons-zèbre et la souris. Enfin, j'ai découvert un nouveau rôle de l'IL-22 dans la modulation de la motilité intestinale chez les poissons-zèbres. Mécaniquement, une dysbiose a été observée chez les il22 mutants, ainsi que des défauts potentiels dans la production de métabolites dérivés des bactéries dans l'intestin. De plus, un dysfonctionnement a été constaté dans la fonction des EEC, caractérisé par une expression hormonale dysrégulée. Il a été intéressant de constater que le mutant exprime des niveaux plus bas de 5-HT, une hormone importante régulant la motilité intestinale chez les mammifères et les poissons-zèbres. Nous avons démontré que l'administration de celle-ci est capable de rétablir la motilité intestinale, ce qui signifie que 5-HT est suffisante pour restaurer une motilité intestinale correcte. Enfin, j'ai découvert la conservation du défaut de motilité intestinale chez les souris en bas âge, ce qui suggère la conservation de cette nouvelle fonction de l'IL-22 chez les mammifères tôt au cours du développement. Dans l'ensemble, ce projet contribue à une meilleure compréhension de la manière dont les cytokines orchestrent le développement et la maturation de l'intestin au cours des premières étapes de la vie<br>Cytokines promote gut defense and homeostasis. Among key gut cytokines, interleukin-22 (IL-22) is produced by immune cells and primarily targets epithelial cells. IL-22 protects the gut from pathogens by inducing anti-microbial peptides expression and promoting tissue repair. Dysregulation of this cytokine can lead to inflammatory bowel disease and cancer. During development, the post-embryonic gut is colonized by commensal microorganisms that promote maturation of the gut and its immune system. Nevertheless, whether and how cytokines such as IL-22 play a role in gut organ development and maturation during this critical time window remains unclear. The zebrafish allows us to visualize and manipulate physiological processes in vivo since early development due to its external development and transparency.During my PhD, I wanted to decipher the function of il22 in the developing gut. My data show that il22 is expressed in larval gut epithelial cells before il22-expressing lymphocytes appear in the gut. I identified enteroendocrine cells (EECs), a gut epithelial cell subtype, as the main source of il22 in larvae. Furthermore, I revealed conservation of the IL-22 signaling pathway, its transcriptional regulation by microbe sensing, and its antibacterial function in the gut. My latest data suggest that il22 expression can also be induced by the activation of Trpa1, a receptor known to recognize tryptophan metabolites in zebrafish and mice. Finally, I found a novel role of IL-22 in modulating gut motility in zebrafish. Mechanistically, dysbiosis was observed in il22-/-, along with potential defects in the production of bacteria-derived metabolites in the gut. Surprisingly, we found that the microbiota from wild-type larvae was able to restore the gut motility impairment of il22-/-, highlighting the important role of the microbiota in il22-mediated regulation of this process. Furthermore, an impairment was found in EECs function, which are known to be sensitive and respond to microbial cues. The dysregulation of EECs was characterized by abnormal hormone expression, specifically reduced levels of serotonin (5-HT), a critical hormone regulating gut motility in both zebrafish and mammals. External administration of 5-HT successfully rescued the gut motility phenotype of il22-/-, indicating that 5-HT is sufficient to restore proper gut motility. Finally, I found conservation of the gut motility defect in early life mice, suggesting the conservation of this novel IL-22 function in mammals. Altogether, this project contributes to a better understanding of how cytokines orchestrate gut development and maturation during the early stages of vertebrate life

Les cellules mésenchymateuses sont des cellules non-hématopoïétiques, non-épithéliales et non-endothéliales présentes dans tous les organes. Elles contribuent à la structure des organes en synthétisant la matrice extracellulaire (MEC). En plus de leur rôle dans la synthèse de la MEC, un nombre croissant d’études au cours de ces vingt dernières années s’accordent sur le fait que les cellules mésenchymateuses (CM) jouent un rôle essentiel dans l’homéostasie de l’intestin. Cependant, le manque de marqueurs et d’outils spécifiques pour étudier ces cellules ont empêché une meilleure compréhension de leurs fonctions. La plupart des CMs intestinales expriment podoplanin (aussi connu sous le nom gp38), un marqueur des CMs des ganglions lymphatiques. En utilisant de nouveaux marqueurs et modèles murins pour étudier les CMs qui expriment le récepteur de la lymphotoxin-ß (LTßR), un récepteur impliqué dans l’interaction avec les cellules du système immunitaire, nous avons identifié différentes sous-population de CMs gp38+ avec des fonctions spécifiques dans l’homéostasie et l’immunité intestinale. En traçant génétiquement les CMs gp38+LTßR+ nous avons identifié un lignage spécifique de fibroblastes sous-épithéliaux qui expriment PDGFRα, appelé par la suite LTßR-SF. Nous avons montré que les ce lignage possède une fonction unique dans les premières semaines après la naissance. En effet, les LTßR-SFs se développent au moment du sevrage des souris de manière indépendante de la colonisation microbienne où ils promeuvent la maturation de la barrière intestinale, y compris la différentiation des cellules caliciformes et des cellules de Paneth qui protège l’intestin des bactéries, ainsi que le développement des cellules dendritiques CD103+CD11b+, qui régulent la tolérance intestinale. Les LTßR-SFs expriment un set de gènes essentiels pour l’homéostasie de l’épithélium et la régulation du système immunitaire tels que bmp4, sfrp3, dll1, vcam, aldh1a3 et cox1. Enfin, nous avons montré que la voie de signalisation de PDGFRα est nécessaire pour l’acquisition de ces fonctions par les LTßR-SFs. En conclusion, nos résultats montrent qu’une sous-population de CMs gp38+ joue un rôle essentiel dans le développement de la barrière intestinale au moment du sevrage en coordonnant la maturation de l’épithélium et du système immunitaire via la voie de signalisation de PDGFRα<br>Mesenchymal cells (MCs) are non-immune, non-epithelial and non-endothelial cells present in all organs. They contribute to the organ’s structure by producing extracellular matrix (ECM). In addition to their role as ECM-producing cells, increasing evidence suggests that they play an active role in intestinal homeostasis. However, the lack of specific markers and tools to investigate MCs hindered a deeper understanding of their function(s). In the intestine, a major fraction of MCs expresses podoplanin (also known as gp38) a marker for MCs in lymphoid organs. Using new markers and reporter mice to study MCs that express the Lymphotoxin-ß Receptor (LTßR), a receptor involved in the crosstalk with immune cells, we identified distinct subsets of gp38+ MCs with specific functions in intestinal homeostasis and immunity. Inducible lineage tracing of gp38+LTßR+ MCs identifies a specific lineage of PDGFRα+ mesenchymal cells located closely to epithelial cells. We show that the lineage of PDGFRα+ subepithelial fibroblasts derived from LTßR+ progenitors (LTßR-SF) has a unique function in the first few weeks after birth. Indeed, this specific lineage develops around weaning independently of microbial colonization, and promotes maturation of the epithelial barrier, including differentiation of Paneth and goblet cells, involved in bacterial protection, and development of CD103+CD11b+ dendritic cells, a specific subset of immune cells involved in intestinal tolerance. LTßR-SF overexpress a set of genes essential for epithelial homeostasis and immune regulation, including bmp4, sfrp3, dll1, vcam, aldh1a3 and cox1. We further show that PDGFRα signaling in LTßR-SF is required to imprint both epithelial and immune function. Taken together, our results show that a subset of gp38+ MCs derived from LTßR+ progenitors plays an essential role in intestinal barrier development at weaning, by coordinating immune and epithelial maturation through PDGFRα signaling

SOARES, Fábio Alves. Análise da freqüência de anorretocele em mulheres adultas com evacuação obstruída, comparando com a paridade e idade, utilizando cinedefecografia e eletromanometria anorretal. 2006. 66 f. Dissertação (Mestrado em Cirurgia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2006.<br>Submitted by denise santos (denise.santos@ufc.br) on 2014-02-19T13:42:07Z No. of bitstreams: 1 2006_dis_fasoares.pdf: 2441204 bytes, checksum: 165a38ea8594ecff8508c2b7a88eb873 (MD5)<br>Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2014-02-19T13:42:47Z (GMT) No. of bitstreams: 1 2006_dis_fasoares.pdf: 2441204 bytes, checksum: 165a38ea8594ecff8508c2b7a88eb873 (MD5)<br>Made available in DSpace on 2014-02-19T13:42:47Z (GMT). No. of bitstreams: 1 2006_dis_fasoares.pdf: 2441204 bytes, checksum: 165a38ea8594ecff8508c2b7a88eb873 (MD5) Previous issue date: 2006<br>The aim of this study is to analyse the frequence of anorectocele in adult women with obstructed defecation accordind to parity and age by means of cinedefaecography and anorectal eletromanometry. Forty-five adult women complaining of obstructed defecation were evaluated, with mean age of 46.3 years (23-72) and mean SCCC-C score of 13.3 points (6-23). Fifteen (33.3%) patients were nulliparous, seven (15,6%) primiparous and 23 (51,1%) multiparous, with mean parity per patient of 2.8 (0-11), considering only vaginal deliveries. Eighteen (60%) had a history of episiotomy, fourteen (46,7%) delivered macrossomic children and two (6,7%) had history of forceps-assisted delivery. Anal hipertony was verified in fourteen (31,1%) patients, while anal hipotony was present in eight (17,8%). Anismus was identified in thirteen (28,9%) patients. Anorecoceles were demonstrated in 34 (75,6%) patients, with mean size (TAR) of 24,8 mm (0-64). Thirty-six (80%) patients presented excessive perineal descent (DPM), rectal mucosal prolapse (PM) in 17 (37,8%) and rectoanal intussusception (IRA) in twelve (26,7%). There were no correlations between anorectocele and anal hipertony (p = 0,7171), anismus (p = 0,4666), IRA (p= 0,6991), PM (p = 0,2279), parity comparing nulliparous and multiparous patients (p = 1,000), episiotomy (p = 1,0000), forceps assistance (p = 1,0000) and delivery of macrossomic children (p = 1,0000). There were also no correlation between TAR and PMR (p =0,0883), PVM (p = 0,7327), parity (p = 0,4987) or age (p = 0,8603). There were correlations between anorectocele and DPM (p = 0,0275), score of SCCC-C (p =0,0082) and anal hipotony (p = 0,0141). In conclusion, anorectocele frequence is high and doesn’t correlate to parity, age but correlates to anal hipotony, DPM andconstipation.<br>O objetivo é avaliar a freqüência e o tamanho de anorretocele em mulheres adultas com evacuação obstruída, correlacionando-os com paridade, idade e parâmetros clínicos, utilizando cinedefecografia e eletromanometria. Foram avaliadas 45 mulheres adultas, com idade média de 46,3 anos (23-73) e sintomas de evacuação obstruída, com escore médio de 13,3 (6-23) pontos, segundo o Sistema de Classificação da Cleveland Clinic para Constipação (SCCC-C). Os parâmetros avaliados foram idade, dados obstétricos, escore do, SCCC-C, dados manométricos e achados de cinedefecografia.. Quinze (33,3%) pacientes eram nulíparas, 7 (15,6%) primíparas e 23 (51,1%) multíparas, com média de 2,8 (0-11) partos vaginais por paciente. Dezoito (60,0%) pacientes haviam sido submetidas a parto vaginal com episiotomia, sendo verificado feto macrossômico em 14 (46,7%) e aplicação de fórcipe em duas (6,7%) . Foi observada hipertonia esfincteriana em 14 (31,1%) e hipotonia em 8 (17,8%) pacientes. Foi identificado anismus em 13 (28,9%) pacientes. Foram demonstradas anorretoceles em 34 (75,6%) pacientes com tamanho (TAR) médio de 24,8 mm (0 - 64). Foram verificados descenso perineal móvel acentuado (DPM) em 36 (80%) pacientes, prolapso mucoso (PM) em 17 (37,8%) e intussuscepção reto-anal (IRA) em doze (26,7%). Não houve correlação entre anorretocele e hipertonia esfincteriana (p = 0,7171), anismus (p = 0,4666), IRA (p = 0,6991), PM (p= 0,2279), paridade comparando-se nulíparas e multíparas (p =1,000), episiotomia (p = 1,0000), uso de fórcipe (p = 1,0000), parto de feto macrossômico (p = 1,0000). Não houve correlação entre TAR e PMR (p = 0,0883), PVM (p = 0,7327), paridade (p = 0,4987) e idade (p = 0,8603). Houve correlação entre anorretocele e DPM (p = 0,0275), escore de constipação do SCCC-C (p = 0,0082) e hipotonia esfincteriana (p = 0,0141). Conclui-se que a freqüência de anorretocele foi elevada, não se correlacionou com paridade e idade, associando-se com hipotonia esfincteriana, DPM e constipação.

Introducción. Actualmente la intervención de Hartmann es una alternativa válida en el tratamiento de patologías de colon izquierdo o recto en pacientes ASA IV, con peritonitis fecaloidea, desnutridos, inmunodeprimidos o con inestabilidad hemodinámica. Se emplea en casos de riesgo de dehiscencia anastomótica, de recidiva tumoral o de incontinencia anal. Sin embargo, los factores relacionados con la decisión de reconstruir el tránsito intestinal no están tan bien establecidos. El objetivo principal del estudio consiste en determinar si existen factores que pueden predecir la reconstrucción intestinal tras una intervención de Hartmann. El análisis de las complicaciones de estas intervenciones y la determinación de los factores predictivos de ausencia de reconstrucción del tránsito intestinal pueden ayudar a realizar una precisa selección de los pacientes y ofrecer información preoperatoria individualizada sobre los resultados más probables de la intervención. Material y métodos. Estudio observacional, retrospectivo y longitudinal que incluye todos los pacientes sometidos a intervención de Hartmann desde Enero de 1999 hasta Diciembre 2014 en un hospital terciario universitario. No se excluyó ningún paciente candidato a una posible reconstrucción del tránsito intestinal. Las variables recogidas fueron clasificadas en 1) específicas del paciente: edad, sexo, IMC, riesgo ASA, índice de Charlson, incontinencia anal; 2) específicas de la enfermedad: tipo de patología (benigna vs maligna), diagnóstico principal, estadio tumoral, grado de contaminación peritoneal, 3) específicas del tratamiento: indicación de procedimiento de Hartmann, transfusión perioperatoria y postoperatoria, procedimiento quirúrgico principal, tipo de cirugía (programada vs urgente), tipo de cirujano (general vs colorrectal), longitud del muñón rectal, clasificación Clavien- Dindo, reingresos, causas de no reversión de la intervención de Hartmann. Se realizó un estudio descriptivo de la muestra. Se evaluó la asociación estadística entre las variables cualitativas con la prueba de 2 o prueba exacta de Fisher. Se compararon grupos mediante los tests U de Mann-Whitney o Kruskal-Wallis en función del tipo de variables. Se realizó un análisis univariante y multivariante mediante una regresión logística binaria. Además se realizó un árbol de clasificación y regresión. Por último, se elaboraron las curvas COR de cada modelo y se compararon entre sí mediante la prueba de DeLong. Resultados. Se incluyeron 533 pacientes consecutivos a los que se realizó un procedimiento de Hartmann. 110 (20,6%) pacientes fueron intervenidos para la reconstrucción intestinal. La edad media fue de 71,7 años. En el análisis multivariante se obtuvieron como factores independientes predictivos de presentar mayor probabilidad de reconstrucción del tránsito la edad inferior a 69 años, el grado ASA I ó II, la indicación de intervención de Hartmann por dehiscencia anastomótica y la altura del muñón rectal por encima del promontorio sacro o a la altura del mismo. Sin embargo, los factores independientes predictivos de una menor probabilidad de reconstrucción fueron la incontinencia anal, estadio IV, recibir transfusión postoperatoria o realizar intervención de Hartmann de forma programada. Del árbol de clasificación se deduce que un paciente de edad inferior a 69 años, que presente baja comorbilidad, con un muñón rectal en el promontorio o por encima de éste y que no haya requerido transfusión tendrá un 85% de probabilidad de reconstrucción intestinal. Discusión. La identificación de factores predictivos de restauración de la continuidad intestinal puede servir para aconsejar a los pacientes y ayudar a los cirujanos a escoger la mejor opción, tanto antes de la realización de una intervención de Hartmann, como en el momento de indicar su reconstrucción. Conclusión. Edad, riesgo ASA, indicación de procedimiento de Hartmann, longitud del muñón rectal, incontinencia anal, estadio tumoral, transfusión postoperatoria y cirugía programada pueden predecir la reconstrucción de la intervención de Hartmann.<br>Introduction. Hartmann´s procedure is still a valid alternative in the treatment of pathologies of the left colon or rectum in patients having an ASA score IV, feculent peritonitis, malnutrition, immunosuppression or with hemodynamic instability. Hartmann´s procedure is mainly indicated in cases of high risk of anastomotic leakage, local tumor recurrence or anal incontinence. However, the factors related to the decision to restore intestinal continuity are not well established. The main objective of this study was to determine predictive factors of Hartmann´s reversal. The analysis of the morbidity of those interventions and the identification of predictive factors of intestinal transit reconstruction could ease an accurate selection of patients and give individualized preoperative counselling providing information on the most likely outcomes of the intervention. Material and methods. A retrospective observational study in which all consecutive patients that underwent Hartmann´s procedure from January 1999 to December 2014 in a tertiary University Hospital were included. No patient with a possible intestinal continuity restoration was excluded. The data collected were classified into 1) patient-specific: age, sex, body mass index, ASA score, Charlson index, anal incontinence; 2) disease-specific: type of disorder (benign vs malignant), main diagnosis, tumor stage, degree of peritoneal contamination, 3) treatment-specific: period of years of surgery, indication of Hartmann´s procedure, perioperative and postoperative transfusion, main surgical procedure, type of surgery (elective vs urgent), type of surgeon (general vs colorrectal), length of the rectal stump, Clavien-Dindo classification, readmission rate, causes of nonreversal Hartmann´s procedure. A descriptive analysis was performed. The 2 test or the Fisher exact test were used for categorical variables. Comparisons between groups were made using Mann-Whitney U test or Kruskal-Wallis test for continuous variables, where appropriate. Univariate and multivariate binary logistic regression model were used. Further, a classification and regression tree was performed. Finally, COR curves of each model were elaborated and compared with the DeLong test. Results. A total of 533 consecutive patients underwent Hartmann’s procedure. 110 (20,8%) patients underwent Hartmann´s reversal procedure. Mean age was 71,7 years. Multivariate analysis showed that the independent predictors of higher probability of intestinal transit reversal were age lower than 69 years, ASA grade I or II, indication of HP for anastomotic leak and the rectal stump above or at the sacral promontory. However, the independent factors related to a reduced probability of intestinal reconstruction following HP were anal incontinence, stage IV, postoperative transfusion or elective Hartmann's intervention. From the classification tree it is deduced that a patient below 69 years of age who presents low comorbidity, with a rectal stump at or above the promontory and that did not require perioperative transfusion would have 85% of probability of intestinal transit reconstruction. Discussion. Identification of predictive factors of intestinal continuity restoration may help surgeons to inform the patient and to choose the better option, both before performing a Hartmann procedure, and at the time of indicating reconstruction of intestinal continuity. Conclusion. Age, ASA, indication of Hartmann’s procedure, length of rectal stump, anal incontinence, tumor stage, postoperative transfusion and elective surgery can predict Hartmann’s reversal.

Made available in DSpace on 2012-05-07T14:40:41Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 000015.pdf: 28187182 bytes, checksum: 7c5d1bdc37db0b9acc0900ca0cd85dce (MD5) Previous issue date: 2008<br>O Bacillus sphaericus (Bsp) é uma bactéria entomopatógena eficiente para o controle de Culex quinquefasciatus, um importante vetor da filariose e arboviroses. O fator larvicida do Bsp é a toxina binária (Bin) e a sua ação em C. quinquefasciatus depende da ligação ao receptor Cqm1. A ausência deste receptor no epitélio intestinal é o principal mecanismo de resistência à toxina Bin. Larvas resistentes a esta toxina, são susceptíveis ao Bsp IAB59 que, além da Bin, produz as toxinas Cry48Aa e Cry49Aa. O principal objetivo deste estudo foi caracterizar os efeitos de toxinas do Bsp nas células do epitélio intestinal de C. quinquefasciatus, utilizando como modelos larvas de uma colônia susceptível a todas as toxinas estudadas (S), de uma colônia resistente à toxina Bin (R2362) e de uma colônia resistente à Bin e Cry48Aa/Cry49Aa (RIAB59). Na primeira etapa, larvas não tratadas das colônias S e R2362 disseccionadas 30 min, 4, 6 e 48 h após a muda para o 4° estádio foram fixadas e processadas para microscopia eletrônica de transmissão (MET). A avaliação morfológica do epitélio intestinal mostrou que células de larvas R2362, ao final do 4° estádio, são caracterizadas por um intenso acúmulo de inclusões lipídicas, sugerindo que a ausência da a-glicosidase Cqm1 pode estar envolvida com alterações no metabolismo. Para caracterizar os efeitos causados pelas toxinas no epitélio intestinal, as larvas foram disseccionadas 1 e 6 h após o tratamento e processadas para MET. A avaliação ultra-estrutural da ação da toxina Bin nas células do epitélio intestinal mostrou que os principais efeitos em larvas S foram a vacuolização citoplasmática e destruição de microvilosidades. Estes foram observados exclusivamente em células que possuem o receptor Cqm1, demonstrando que esta molécula é essencial para mediar a ação da toxina Bin. Em células de larvas das colônias S e R2362, susceptíveis à Cry48Aa/Cry49Aa, o principal efeito destas toxinas foi a vacuolização mitocondrial, e este parece estar associado à Cry48Aa que possui estrutura de 3 domínios típica de toxinas da família Cry. Efeitos similares aos da toxina Bin também foram observados e parecem resultantes da ação da Cry49Aa que possui homologia com toxinas do tipo binária. Os dados mostram que a Cry48Aa/Cry49Aa possui uma ação complexa nas células e seu sítio de ligação é diferente do Cqm1. Combinações da toxina Bin com as toxinas Cry11Aa e Cyt1Aa do Bti também provocaram alterações em células de larvas R2362, desprovidas do receptor Cqm1. A combinação Bin/Cry11Aa causou efeitos similares aos induzidos pela toxina Cry48Aa/Cry49Aa, e sugerem que as toxinas de 3 domínios, Cry11Aa e Cry48Aa, podem mediar os efeitos das toxinas Bin e Cry49Aa, respectivamente, nos modelos estudados. A combinação Bin/Cyt1Aa provocou efeitos drásticos como a perda precoce de microvilosidades e a lise celular, característica da toxina Cyt1Aa que apresenta ação citolítica. Os resultados deste trabalho contribuem para o entendimento do modo de ação de toxinas do Bsp, bem como do efeito sinergístico de suas toxinas com aquelas do Bti

Na presente dissertação objetivou-se avaliar a reatividade vascular e a repercussão do pré-tratamento com sinvastatina (SINV, 20mg/kg, p.o.), sob esse parâmetro, em artéria mesentérica superior (AMS) e pulmonar (AP) de ratos Wistar submetidos a 45 minutos de isquemia e 2 horas de reperfusão intestinal (iIR). Nas artérias avaliou-se a função dependente e independente do endotélio e a contração induzida pela fenilefrina agonista seletivo <font face=\"Symbol\">a1-adrenérgico, em preparações com e sem endotélio e na presença ou ausência de inibidores da NOS e da iNOS (L-NAME e 1400W, respectivamente). Pode-se concluir que a iIR modificou a reatividade vascular em AP, porém não em AMS. Na artéria pulmonar, a iIR causou disfunção endotelial por meio de ativação da iNOS, a qual levou à diminuição da resposta vasodilatadora dependente do endotélio e à hiporreatividade contrátil. Além disso, confirmando a segunda hipótese, uma única dose de sinvastatina antes da iIR previne as alterações de reatividade vascular, o que demonstra o efeito benéfico desse fármaco nessa doença.<br>This work aimed to evaluate the vascular reactivity and the effect of pre-treatment with simvastatin (SIMV, 20mg/kg, p.o.), under this parameter, in the superior mesenteric artery (SMA) and pulmonary artery (PA) of rats subjected to 45 minutes of ischemia and 2 hours of reperfusion (iIR). In these arteries, it was evaluated the endothelium-dependent and -independent function and the contraction induced by phenylephrine the selective <font face=\"Symbol\">a1-adrenergic agonist, in preparations with and without endothelium and at the presence or absence of non-selective NOS and of iNOS inhibitors (L-NAME and 1400W, respectively). It can be concluded that the iIR modified vascular reactivity in PA, but not in SMA. In the pulmonary artery, the iIR caused endothelial dysfunction via activation of iNOS, which led to decreased endothelium-dependent vasodilator response and to contractile hyporeactivity. Moreover, confirming the second hypothesis, a single dose of simvastatin prior to iIR prevents changes in vascular reactivity, which shows the beneficial effects of this drug in this disease.

Cette thèse de doctorat s'est déroulée dans le cadre d'un projet européen FP7 (7th Framework Program) MSCA (Marie Sklodowska-Curie action) ITN (Initiale Training Network) EID (European Industrial Doctorates) appelé DISCo (a multidisciplinary Doctoral Industrial School on novel preventive strategies against Escherichia coli infections) coordonné par Mariagrazia Pizza et co-coordonné par Mickaël Desvaux. Ainsi, ce doctorat s'est déroulé pour moitié en Italie au centre de recherche GSK (GlaxoSmithKline) sur le site de Sienne sous la supervision de Roberto Rosini et la direction de Fabio Polticelli de Universita degli Studi Roma Tre. L'autre moitié de la thèse s'est déroulée en France à l'INRA, centre Auvergne-Rhône-Alpes sur le site de Theix sous la direction de Mickaël Desvaux et Grégory Jubelin comme co-encadrant. Cette thèse de doctorat participe au développement de nouvelles stratégies préventives aux infections aux E. coli pathogènes intestinaux (InPEC), en particulier E. coli entérohémorragiques (EHEC), par une stratégie vaccinale. Dans ce contexte, une approche de vaccinologie inverse a été mise en œuvre pour identifier de nouveaux antigènes candidats qui ont ensuite été délivrés par la technologie GMMA (Generalized Modules for Membrane Antigens). Par ailleurs, un domaine épitope potentiel chez les autotransporteurs, i.e. l'autochaperon, a été caractérisé par analyse des séquences protéiques et modélisation structurale<br>Enterohemorrhagic E. coli (EHEC) are a major cause of large outbreaks mainly affecting developed countries. From 1982 to 2002, a total of 350 E. coli O157 outbreaks were reported in the United States. EHEC infection causes diarrheal disease often associated with clinical complications like hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although efforts focused on hygiene have been implemented in the food supply chain to reduce the risk of the foodborne E. coli O157 infection, outbreaks caused by this pathogen are still common. In addition, antibiotic-based therapy is discouraged for their potential undesirable effect in releasing shiga-toxin from the bacteria. Among non-antibiotic preventing strategies, vaccine development is warranted, still nowadays a licensed vaccine specific for human use against EHEC is not available. In this study, we used the Reverse Vaccinology approach applied on the EHEC O157:H7 genome to select new potential vaccine candidates. We identified a panel of 24 of potential protein antigens and we successfully expressed three of them in Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing these vaccine candidates resulted to be immunogenic, raising a specific antibody response for two of the selected antigens. In particular, immunization with MC001 candidate was able to reduce intestinal EHEC O157:H7 colonization lowering the bacterial count in feces, colon and ceacum tissues in mice. This candidate was found to be homologue to the Salmonella Typhimurium Lipid A deacylase enzyme (LpxR) and to our knowledge this study was the first report describing it as vaccine candidate. Also, gene distribution and sequence variability analysis showed that MC001 was mainly present and conserved in EHEC O157:H7 and in some EPEC. Given the high genetic variability among and within these pathotypes, the identification and inclusion of this conserved candidate in a vaccine might cover against major intestinal pathogenic strains. Furthermore, because it has been showed that during the infection process some autotransporters, as MC021 can be reactive, we also analysed molecular determinant with an important role for their proper secretion and folding, namely the autochaperon (AC) domain. It appeared the AC is a common feature of autotransporters but strictly associated with passenger domains exhibiting a –helix fold. Their exposition at the bacterial cell the surface further positions the AC as a potential antigenic target and/or development of new treatments. These findings further provide new research directions for the development of non-antibiotic preventive strategy against InPEC in human but also animal

Un nombre grandissant d’associations entre diverses pathologies humaines et dysbiose intestinale (définie ici comme altération de la composition du microbiote par rapport à sa composition habituelle) sont observées. Parmi les facteurs qui pourraient induire la dysbiose, les bactériophages (dit phages), sont des candidats pertinents par leur fonction prédatrice.L’objectif de la thèse a été de déterminer si les prophages de souches bactériennes du microbiote intestinal humain ont un impact négatif sur la stabilité de leur hôte dans l’intestin. Pour cela, nous avons utilisé des souris sans germes primo-colonisées avec la souche Escherichia coli LF82, puis inoculées soit avec Faecalibacterium prausntizii A2-165, soit avec Roseburia intestinalis L1-82, deux souches appartenant aux espèces dominantes du microbiote intestinal humain. Chacune de ces souches possède deux prophages dans son génome, Lagaffe et Mushu pour F. prausnitzii, Jekyll et Shimadzu pour R. intestinalis. L’impact des prophages a également été étudié lors d’une inflammation intestinale induite au DSS.Pour la combinaison F. prausnitzii/E. coli, aucun des deux prophages de F. prausnitzii n’a d’activité délétère pour son hôte bactérien chez la souris, même durant une inflammation induite au DSS. Afin de mieux caractériser l’ensemble des prophages présents chez cette espèce, une analyse bio-informatique effectuée sur 15 souches de F. prausnitzii a permis de constater que la prévalence de Mushu et Lagaffe était faible, mais aussi de découvrir une remarquable richesse phagique : au total, 18 espèces de prophages répartis en nouveaux 8 genres viraux ont été décrits. Une étude in silico de l’abondance de ces phages dans les viromes intestinaux humains a révélé que des phages du genre ‘Lugh’ et ‘Epona’ sont plus souvent présents et/ou abondants dans les viromes de patients des maladies inflammatoires chroniques de l’intestin (MICI) que chez les individus sains. Sachant que les patients atteints de MICI ont une population appauvrie de F. prausnitzii dans leur microbiote, ces observations suggèrent une activité accrue de ces phages pendant la maladie : ils pourraient déclencher ou aggraver la baisse de population de F. prausnitzii dans les patients, participant ainsi à l’aggravation des symptômes des MICI.Avec la combinaison R. intestinalis/E. coli., aucune variation de population ou effet délétère du phage Jekyll n’a pu être observé. En revanche, la population du phage Shimadzu est loin d’être stable. Dans toutes les souris, et même en l’absence d’un traitement au DSS, un mutant virulent de Shimadzu émerge, appelé Shi-vir. Ce mutant lyse massivement la population intestinale de R. intestinalis, menant à un effondrement de la population hôte. La population bactérienne remonte ensuite à son niveau initial grâce à l’émergence de mutants bactériens résistants à l’infection. Cette résistance a essentiellement pour origine l’acquisition d’un espaceur associé au système CRISPR-Cas de type IIC de R. intestinalis, et dirigé contre le phage Shimadzu. Cependant, l’acquisition de cet espaceur ne peut se faire sans qu’une sous-population de R. intestinalis soit préalablement guérie du prophage Shimadzu, sans quoi un tel espaceur tuerait la bactérie.J’ai ainsi démontré qu’un prophage peut déstabiliser sa population hôte dans l’environnement intestinal et créer des dysbioses intestinales transitoires. La pression de sélection qui résulte de l’infection par le phage Shi-vir a permis l’accélération de l’évolution de l’hôte bactérien.En conclusion, une fraction des phages tempérés du microbiote intestinal pourrait avoir un impact négatif sur la stabilité de sa population hôte dans l’environnement intestinal, soit parce le ratio phage/bactérie augmente dans cet environnement (cas des phages Lugh et Epona de F. prausnitzii), soit parce qu’il évolue vers la virulence (cas de Shi-vir chez R. intestinalis) et induit une dysbiose transitoire<br>A growing number of associations is observed between various human pathologies and intestinal dysbiosis, here defined as an alteration of the microbiota composition. Among the potential factors inducing dysbiosis, bacteriophages, called phages, are relevant candidates by their predatory function.The aim of the thesis was to determine whether prophages of bacterial strains from the human gut microbiota have a negative impact on the stability of their host in the gut environment. We studied this question by using germ-free mice colonized first with Escherichia coli strain LF82, then inoculated with two bacterial strains belonging to dominant species of the human intestinal microbiota, Faecalibacterium prausnitzii strain A2-165 or Roseburia intestinalis strain L1-82. Each of these strains has two prophages in its genome, Lagaffe and Mushu for F. prausnitzii, Jekyll and Shimadzu for R. intestinalis. The impact of these prophages was also studied during intestinal inflammation using DSS (Dextran Sulfate Sodium)-induced colitis in mice.In mice colonized with F. prausnitzii and E. coli , prophages of F. prausnitzii did not have any deleterious activity for the bacterial host, even during DSS-induced inflammation. In order to better characterize prophages of the F. prausnitzii species, a bioinformatic analysis carried out on 15 strains of F. prausnitzii highlighted that the prevalence of Mushu and Lagaffe was low. However, this analysis revealed also an enormous diversity of phages and we described 18 species of prophages divided into 8 new proposed genera. An in silico study of their abundance in 173 human intestinal viromes revealed that the phage genera 'Lugh' and 'Epona' were more present and/or abundant in viromes of Inflammatory Bowel Disease (IBD) patients compared to healthy subjects. Given that IBD patients have lower populations of F. prausnitzii in their microbiota compared to healthy subjects, our observations suggest an increased activity of these phages during disease. They may trigger or worsen population decline of F. prausnitzii in patients, participating thus to the aggravation of IBD symptomsIn mice colonized with R. intestinalis and E. coli, we did not observe variation of Jekyll population or deleterious effect of this phage on its host. In contrast, the Shimadzu population was not stable. Indeed, even in the absence of DSS treatment we observed in all mice the emergence of a virulent mutant of Shimadzu, called Shi-vir. This mutant massively lysed R. intestinalis, leading to a collapse of the bacterial host population. Then this population rose back to its original level thanks to the emergence of bacterial mutants resistant to the viral infection. This resistance was mainly due to the acquisition of a spacer associated with the CRISPR-Cas type IIC system of R. intestinalis, directed against the Shimadzu phage. However, acquisition of this spacer could not be observed unless the Shimadzu prophage was cured from the strain, showing that this spacer would kill the Shimadzu lysogen.I have shown therefore that a prophage can destabilize its host population in the intestinal environment and create transient intestinal dysbiosis. I have also highlighted that the selection pressure imposed by an ex-temperate phage infection, the Shi-vir phage, has allowed an acceleration of its host evolution.Overall, this work establishes that a fraction of the temperate phages present in intestinal microbiota may impact negatively bacterial population stability, either because the phage/bacteria ratio increases (for the Lugh and Epona phages de F. prausnitzii), or because the temperate phage evolves towards virulence (case of the Shi-vir mutant on R. intestinalis), and induces a transient dysbiosis

Los mastocitos (MC) pueden participar en la respuesta a microorganismos mediante diversos receptores de reconocimiento de patrones (PRRs). Luego de su activación a través de estos receptores, los MC pueden orquestar una respuesta mediante la secreción de mediadores inmunológicos como las citocinas. Entre éstas, la interleucina 10 (IL-10) es una citocina importante por sus características inmunomoduladoras, así como también, por su capacidad de regular la expresión de proteasas en MC. Adicionalmente, gracias a la existencia de modelos murinos modificados genéticamente, como los ratones IL-10 deficientes (IL-10-/-), que desarrollan colitis de forma espontánea, es posible investigar el rol potencial de la IL-10 en la respuesta de MC frente a la activación con antígenos de diversos microorganismos. Por otra parte, el uso de este modelo animal permite investigar la influencia de esta citocina sobre la composición de la microbiota intestinal. Este trabajo ha explorado el rol funcional de la IL-10 en mastocitos diferenciados in vitro, así como los efectos de la deficiencia de IL-10 sobre la composición de la microbiota y la expresión de factores relacionados con la respuesta inmune, antes (6 semanas) y al inicio (20 semanas) de la colitis. Para este propósito, se ha caracterizado el efecto de la deficiencia de IL-10 sobre el fenotipo mastocitario y tras su activación vía PRRs. Adicionalmente, se evaluó el efecto que produce la carencia de IL-10 sobre la composición de la microbiota, la expresión de TLRs y citocinas proinflamatorias, así como la producción de IgA luminal, en las mismas etapas y tras el tratamiento con antibióticos. Los resultados obtenidos indicaron que la deficiencia de IL-10 produjo distintos efectos dependiendo del fenotipo mastocitario, de la edad y del tipo de ligando PRR. Así, en ausencia de IL-10, MC de tipo mucosa (MLMC) mostraron una menor expresión de TLR4 y NOD2 a las 6 semanas y TLR7 a las 20 semanas. Además, ambos fenotipos de MC (mucosa y conectivo), mostraron una menor secreción de IL-6 y TNFα tras la activación de TLR2 La activación de TLR4 y TLR7 en MLMC generó una menor secreción de IL-6 a las 6 semanas, mientras MLMC secretaron menos TNFα a las 20 semanas. Finalmente, tras la estimulación de NOD2 no se observó secreción de citocinas en ninguno de los fenotipos mastocitarios. Por otra parte, se observó que en animales IL-10-/- existen factores que potencialmente favorecerían el desarrollo de colitis. Así, los ratones IL-10-/- a las 6 semanas mostraron representantes del filo Verrucomicrobia y una menor abundancia relativa de los taxa Rikenellaceae y Lachnospiraceae. Mientras que a las 20 semanas en los ratones IL-10-/- se observaron microorganismos del filo TM7, una menor expresión de IL-1β, IL-6, TLR6, -7 y -8, y un incremento de TNFα e IgA. Adicionalmente, el uso de antibióticos antes del inicio de la colitis indujo una disminución en la diversidad y una reestructuración de la microbiota, junto con una disminución en la expresión de TLRs, citocinas y menor producción de IgA luminal. En resumen, estos hallazgos proveen nuevas perspectivas sobre la función de los MC y la IL-10 en la interacción microorganismo-huésped. Muestran cómo la ausencia de IL-10 puede afectar la composición de la microbiota y la expresión de factores asociados a la respuesta inmune. Y sugieren que la modificación temprana de la microbiota mediante la utilización de antibióticos en individuos genéticamente susceptibles podría alterar la progresión de la colitis.<br>Mast cells (MC) can participate in the response to microorganisms by various pattern recognition receptors (PRRs). After their activation through these receptors, MC can orchestrate a response by secreting immunological mediators such as cytokines. Among these, interleukin 10 (IL-10) is an important cytokine due to its immunomodulatory characteristics, as well as its ability to regulate the expression of MC proteases. Additionally, thanks to the existence of genetically modified murine models, such as IL-10 deficient (IL-10-/-) mice that develop colitis spontaneously, it is possible to investigate the potential role of IL-10 in MC response to activation with antigens of different microorganisms. On the other hand, the use of this animal model allows investigating the influence of this cytokine on the composition of the intestinal microbiota. This work has explored the functional role of IL-10 in differentiated MC in vitro, as well as the effects of IL-10 deficiency on the composition of the microbiota and the expression of factors related to the immune response, before (6 weeks) and at the onset (20 weeks) of colitis. For this purpose, the effect of IL-10 deficiency has been characterized on MC of different phenotype and after its activation via PRRs. Additionally, the effect produced by the lack of IL-10 on the microbiota composition, the expression of TLRs and proinflammatory cytokines, as well as the production of luminal IgA, in the same stages and after antibiotics treatment was evaluated. The results obtained indicated that the IL-10 deficiency produced different effects depending on the MC phenotype, age and type of PRR ligand. Thus, in the absence of IL-10, mucosal-like MC (MLMC) showed lower expression of TLR4 and NOD2 at week 6 and TLR7 at week 20. In addition, both MC phenotypes (mucosa and connective), showed a lower secretion of IL-6 and TNFα after TLR2 activation. The TLR4 and TLR7 activation in MLMC generated a lower secretion of IL-6 at week 6, while MLMC secreted less TNFα at week 20. Finally, after NOD2 stimulation, no cytokine secretion was observed in any of the MC phenotypes. On the other hand, it was observed that in IL-10-/- animals there are factors that potentially favor the development of colitis. Thus, IL-10-/- mice at week 6 showed representatives of Verrucomicrobia phylum and a lower relative abundance of Rikenellaceae and Lachnospiraceae taxa. Meanwhile at week 20 in IL-10-/- mice, microorganisms of the phylum TM7 were observed, as well as, a lower expression of IL-1β, IL-6, TLR6, -7 and -8, and an increase of TNFα and IgA. Additionally, the use of antibiotics before the development of colitis induced a decrease in diversity and a restructuring of the microbiota, together with a decrease in TLRs and cytokines expression, and a lower production of luminal IgA. In summary, these findings provide new insights on the role of MC and IL-10 in the host-microorganism interaction. They show how the IL-10 deficiency can affect the microbiota composition and the expression of factors associated with the immune response. And they suggest that early modification of the microbiota through the use of antibiotics in genetically susceptible individuals could alter the colitis progression.

Objetivo: Analisar através de metodologia molecular a microbiota fecal de recém-nascidos (RN) saudáveis, em aleitamento materno exclusivo. Materiais e métodos: Amostras fecais de dez RN foram avaliadas no 2º, 7º e 30º dias de vida (DV), através de sequenciamento do 16S rDNA bacteriano. Real-time PCR para bifidobacterias foi empregado nas amostras de 30 dias. Resultados: A diversidade bacteriana fecal aumentou do 2º para o 30º DV. E. coli predominou no 2º e 7º DV, e Clostridium no 30º DV. Usando real-time PCR, bifidobacterias foram identificadas em todas as amostras de 30 dias. Conclusão: Enterobacterias predominaram na primeira semana de vida. Aos 30 DV observou-se uma maior diversidade bacteriana, com predomínio de Clostridium.. A técnica inicial não permitiu identificar bifidobacterias.<br>Purpose: To evaluate by molecular methodology the fecal microbiota of healthy newborns, exclusively breastfed. Materials and methods: Fecal samples from ten neonates were analyzed on 2nd, 7th and 30th day of life, using 16S rDNA sequencing and real-time PCR for bifidobacteria. Results: The fecal bacteria diversity increased from the second to the 30th day of life. E. coli was predominant in the fecal samples from the 2nd and 7th day of life, and Clostridium.in the samples of the 30th day. Using real-time PCR bifidobacteria were identified in all 30th day samples. Conclusion: Enterobacteria were predominant in the first week of life. On 30th day of life a greater bacterial diversity was observed with predominance of Clostridium. The initial technique didnt allow the identification of bifidobacteria.

Le contrôle de l’homéostasie du glucose est le résultat d’un dialogue étroit entre les différents organes métaboliques par l’intermédiaire de messages nerveux et hormonaux. Parmi les mécanismes de régulation, l’incrétine GLP-1 (Glucagon-Like Peptide-1), produite et sécrétée par les cellules entéroendocrines de type L dans l’intestin en réponse à la prise alimentaire, potentialise la sécrétion d’insuline par le pancréas. Au début de ma thèse, j’ai participé aux travaux de l’équipe qui ont permis de montrer que l’activation du récepteur nucléaire Farnesoid X Receptor (FXR) diminue la production et la sécrétion de GLP-1 en réponse au glucose. Cependant, comme il existe de nombreux autres stimuli de la sécrétion de GLP-1, nous avons ensuite cherché à savoir si FXR régule également d’autres voies de sécrétion de GLP-1. En particulier, le microbiote intestinal participe au contrôle de l'homéostasie énergétique par la fermentation des fibres alimentaires, produisant dans le colon des acides gras à chaîne courte (SCFAs) qui favorisent la sécrétion de GLP-1 en se liant à leurs récepteurs membranaires FFAR2 et FFAR3. L’objectif suivant de mes travaux de thèse a donc été d’étudier le rôle de FXR dans la réponse des cellules L du colon aux SCFAs.La sécrétion de GLP-1 en réponse aux SCFAs a été évaluée ex vivo dans des explants intestinaux de souris traitées avec le GW4064, agoniste synthétique de FXR, dans des colonoïdes murins issus de souris WT et FXR KO, in vitro dans les cellules L murines GLUTag et humaines NCI-H716 activées avec le GW4064 et in vivo chez des souris WT et FXR KO après supplémentation en prébiotiques(fructanes de type inuline) pour augmenter la production de SCFAs dans le colon. L’expression des récepteurs aux SCFA FFAR2 et FFAR3 a également été examinée dans ces différents modèles et la voie de signalisation intracellulaire de type Gαq de FFAR2 a été évaluée in vitro.La sécrétion de GLP-1 induite par les SCFAs est inhibée dans les explants de côlon de souris traitées par le GW4064 et améliorée dans les colonoïdes FXR KO. L’activation in vitro de FXR inhibela sécrétion de GLP-1 en réponse aux SCFAs et aux ligands synthétiques de FFAR2, principalement en diminuant l’expression de FFAR2 et sa signalisation intracellulaire de type Gαq. Les souris FXR KO présentent une augmentation de l’expression de FFAR2 dans le côlon et des taux plasmatiques de GLP-1 augmentés lors de la supplémentation en prébiotiques.L’ensemble de mes résultats de thèse montrent donc que l’inhibition de FXR augmente la sécrétion de GLP-1 par les cellules L entéroendocrines en réponse au glucose et aux métabolites du microbiote intestinal, les SCFAs. La combinaison de l’utilisation d’antagonistes ou de dé-activateurs de FXR dans l’intestin avec une supplémentation en prébiotiques peut ainsi être une approche thérapeutique prometteuse pour stimuler l’axe incrétine dans le traitement du diabète de type 2 et des maladies du foie gras non alcoolique telles que la NASH (Non Alcoholic SteatoHepatitis)<br>The control of glucose homeostasis is the result of a close dialogue between the different metabolic organs through nervous and hormonal messages. Among the regulatory mechanisms, the incretin GLP-1 (Glucagon-Like Peptide-1), produced and secreted by enteroendocrine L cells in the intestine in response to food intake, enhances insulin secretion by the pancreas. At the beginning of mythesis, we have shown first that activation of the nuclear Farnesoid X Receptor (FXR) decreases theproduction and the secretion of GLP-1 in response to glucose. However, there are many other stimuli ofGLP-1 secretion. In particular, the intestinal microbiota participates in the control of energy homeostasisby fermentation of dietary fibers, producing short chain fatty acids (SCFAs) in the colon which promotethe secretion of the incretin GLP-1 by binding to their transmembrane receptors FFAR2 and FFAR3 inenteroendocrine L cells. We therefore investigated whether FXR could also regulate other GLP-1secretion pathways in L cell and the aim of my thesis work was then to study the role of FXR in colonicL cell response to SCFAs.GLP-1 secretion in response to SCFAs was evaluated ex vivo in intestinal biopsies from micetreated with GW4064, a synthetic agonist of FXR, in murine colonoids from WT and FXR KO mice, invitro in murine L cells GLUTag and human L cells NCI-H716 activated with GW4064 and in vivo inWT and FXR KO mice after supplementation with prebiotics (inulin type fructans) to increase SCFAsproduction in the colon. Expression of the SCFAs receptors FFAR2 and FFAR3 were also examined inthese different models and FFAR2 Gαq-signalling pathway was evaluated in vitro.SCFA-induced GLP-1 secretion is blunted in colon explants from mice treated with GW4064 and improved in FXR KO colonoids. In vitro activation of FXR inhibits GLP-1 secretion in response toSCFAs and synthetic ligands of FFAR2, mainly by decreasing FFAR2 expression and FFAR2 Gαqsignallingpathway. FXR KO mice exhibit an increased FFAR2 expression in colon and increased plasma GLP-1 levels after prebiotic supplementation.Overall my thesis results show that FXR inhibition increases GLP-1 secretion byenteroendocrine L cells in response to glucose and to gut microbiota-metabolites, the SCFAs. Thecombination of FXR antagonists with prebiotic supplementation can thus be a promising therapeuticapproach to stimulate the incretin axis in the treatment of type 2 diabetes and non-alcoholic fatty liverdiseases such as NASH (Non-Alcoholic SteatoHepatitis)

Os recetores olfativos (ORs) são proteínas transmembranares, associadas à proteína G e que funcionam, no epitélio olfativo, como quimiosensores para a deteção de odores. A capacidade do sistema olfativo para detetar um vasto universo de compostos químicos depende, em larga medida, do fato de existirem cerca de 1000 genes de ORs no genoma dos mamíferos. No epitélio olfativo, a informação sobre os odores, recebida por esta multiplicidade de ORs, é canalizada apenas por uma via de sinalização. Quando a molécula de odor se liga ao OR, este ativa um tipo específico de proteína G, a proteína G olfativa trimérica (Golf), a qual por sua vez ativa a isoforma olfativa da adenilato ciclase (AC3), resultando num aumento de Adenosina Monofosfato Cíclica (cAMP). Um dos alvos do cAMP é um canal catiónico que, quando aberto, permite um influxo de iões sódio (Na+) e iões cálcio (Ca2+) no cílio, resultando numa despolarização da célula. Tanto a AC3 como a Golf e o canal catiónico são constituintes obrigatórios da maquinaria olfativa. Já foram descritas algumas situações, em que os ORs têm funções quimiosensoriais, fora do epitélio olfativo nomeadamente no esperma, placenta e rim. Com este projeto, pretendeu-se saber se as vias de sinalização dos ORs são usadas pelas células intestinais de forma a responder a alterações no lúmen do trato gastrointestinal. O estudo da expressão a nível do mRNA, foi desenvolvido por RT-PCR e a análise da expressão das proteínas por Western Blotting e Imunohistoquímica. Os principais constituintes da via olfativa foram identificados no intestino: os ensaios de Western Blot e Imunohistoquímica mostraram a presença dos recetores olfativos (OR19, OR63, OR600, OR620/624 e OR1496), a adenilato ciclase tipo 3 (AC3) e a subunidade α da proteína G específica do olfato (Gαolf). Os estudos de RT-PCR, embora ainda em curso, mostraram a presença de mRNA de genes que codificam para recetores olfativos e para a Gαolf. Os resultados obtidos neste projeto experimental sugerem que a cascata de sinalização da transdução olfativa pode estar ativa no intestino, uma vez que as várias proteínas que a constituem se encontram expressas neste tecido. Esta via pode regular a motilidade do intestino e funcionar como um detetor de moléculas químicas solúveis que se encontrem no lúmen intestinal, induzindo respostas celulares de acordo com as necessidades fisiológicas.<br>Olfactory receptors (ORs) are transmembrane proteins associated with G protein involved in recognition and transduction of odorant stimuli. The ability of the olfactory system to detect a broad universe of chemical compounds depends largely on the fact that there are about 1000 genes in the ORs mammalian genome. In the olfactory epithelium, the information about odors, received by this plurality of ORs is channeled only through a signaling pathway. When the OR binds to the odor molecule, it activates a specific type of G protein, the specific olfactory trimeric G-protein (Golf), which in turn activates adenylate cyclase olfactory isoform (AC3), resulting in an increase in Adenosine Monophosphate Cyclic (cAMP). One of the targets of cAMP is a cation channel which, when opened, allows an influx of sodium ions (Na+ ) and calcium ions (Ca2+) in the cilia, resulting in depolarization of the cell. Both the AC3 as the Golf and the cationic channel are mandatory components in the olfactory machinery. Olfactory-like chemosensory signaling occurs outside the olfactory epithelium, in other tissues where it also monitors the composition of fluids, in particular sperm, placenta, and kidney. So, with this project, we sought to know whether the olfactory signaling pathways are used by intestinal cells to respond to changes in the lumen of the gastrointestinal tract. The RNA analysis was performed by conventional PCR, and protein analysis by Western Blotting and Immunohistochemistry. We found that major components of olfaction, including olfactory receptors (OR19, OR63, OR600, OR620/624 e OR1496), olfactory-related adenylate cyclase (AC3) and olfactory G protein (Gαolf), are expressed in the rat bowel. These results suggest that olfactory-like chemosensory pathway might be active in bowel and could function as regulator of intestine motility and as a detector of soluble chemical molecules in intestinal lumen, therefore inducing several cellular responses, according to physiological needs.

La hipótesis de este estudio presentó la relación entre la composición y distribución de lípidos en zonas de criptas y vellosidades de mucosa intestinal bovina con la Proteína Transportadora de Acidos Grasos (PTAG). En los primeros experimentos de examinó la actividad de fosfatasa en homogeneizados obtenidos de raspados de mucosa intestinal bovina. Los raspados superficiales presentaron una actividad de fosfatasa alcalina 65 veces mayor que en los raspados profundos. Los estudios realizados en homogeneizados indicaron que la cantidad de proteína total, lípidos totales y fosfolípidos se incrementó durante la migración de los enterocitos desde las criptas (zonas profunda) hacia el ápice de las vellosidades (zona superficial), sin embargo, el colesterol y sus ésteres disminuyeron. La composición en ácidos grasos de lípidos polares y neutros (Cromatografía gaseosa computarizada) indicó un contenido mayor de acído linoleico (C18:2 n-6) y ácido araquidónico (C20:4 n-6) en fosfolípidos totales comparado con fracciones de triglicéridos y ésteres de colesterol. La relación ácido graso etilénico/saturado de fosfolípidos y ésteres de colesterol mostró un descenso de zona superficial a profunda, pero en triglicéridos los valores resultaron inversos. El índice de peroxidabilidad, calculado a partir de la composición total de ácidos grasos de los lípidos totales, fue superior en la zona superficial y cuando los homogeneizados fueron peroxidados, siempre la zona superficial presentó mayor concentración de malondialdehído. La incorporación de ácido oleico 1-14C en fosfolípidos, triglicéridos y esteres de colesterol fue mayor en los microsomas de la zona superficial que en los microsomas de la zona profunda. Al mismo tiempo, la cantidad de proteína microsomal y citosólica aumentó en el sentido de la migración vellositaria, observandose a la vez un significativo incremento de la concentración proteíca en citosoles enriquesidos en PTAG, siempre desde el punto de vista de la diferenciación enterocitaria en el sentido de la maduración del epitelio de la mucosa intestinal bovina. La composición en ácidos grasos de microsomas del intestino delgado reveló un mayor nivel de ácidos oleico (C18:1 n-9), linoleico (C18:2 n-6) y araquidónico (20:4 n-6) en microsomas de zona superficial comparados con los de zona profunda. Cuando se analizaron las diferentes clases de fosdolípidos se observó el mismo comportamiento. En los estudios finales, los citosoles fueron incubados con ácidos oleico 1-14C y linoleico 1-14C y sembrados en columnas de Sephadex G75 observándose un ligado significativamente superior en la zona superficial comparado con la zona profunda. Finalmente, fue estudiada la remoción de ácido limoneico 1-14C desde microsomas por fracciones enriquesidas en PTAG, apreciandose una remoción mayor en la zona superficial comparado con la zona profunda. De este modo, se vio que junto a una modificación de lípido y sus ácidos grasos desde las zonas de criptas hacia las zonas de ápice se manifestó una actividad mayor de ligado de ácido graso por fracciones proteicas citosólicas enriquesidas en PTAG en el sentido de diferenciación de la mucosa intestinal bovina. Los resultados comprueban la relación entre la composición y distribución de los lípidos de zonas de criptas y ápice de vellosidades de la mucosa intestinal bovina con la PTAG.<br>The hypothesis of study presented the posible relationship between the composition and distribution of lipids in crypts and villus zones of bovine intestinal mucose and the Fatty Acid Binding Protein (FABP). In the initials experiments were examined the alkaline phosphatase activity of whole homogenates prepared by differential scraping of bovine intestinal mucosa. The alkaline phosphatase activity was superior in the superficial scrapings (65 times) than in the deep scrapings. The estudies carried out in homogenates indicates that the quantity of total protein, lipids, and phospholipids increased during the migration of cell from crypt to villus tip; but the cholesterol and their esters decreased. The fatty acid composition of polar and neutral lipids from superficial scrapings (analysed by Gas Liquid Cromatography) indicated that the arachidonic (C20:4 n-6) and linoleic acid (C18:2 n-6) contents of phospholipidis were considerably higher than those present in triglycerides and cholesteryl esters. The etilenic/saturated fatty acid ratio of phospholipids and cholesteryl esters showed a decrease from superficial to deep scrapings, whereas the opposite results were observed in triglycerides. Therefore, the peroxidizability index calculated from the fatty acid composition of total lipids was significantly higher in the superficial than in the deep scraping and when the homogenates were peroxidated: superficial zones showed more malondialdehyde concentration. the incorporation of 1-14C oleic acid into phospholipids, triglycerides and cholesterol esters was higher in superficial than in deep microsomes. Beside, the quantity of total protein ofmicrosomes and cytosols increased during the migration of enterocytes from crypt to villus tip. This indicate a higher FABP concentration in superficial cytosols than in deep ones. The fatty acid composition of smal intestinal microsomes revealed thet the level of oleic (C18:1 n-9), linoleic (C18:2 n-6) and arachidonic (C20:4 n-6) acids was increased in the superficial zone in relation with deep zone. the same fatty acids in microsomes presented a similar behavior, when the fatty acid composition of different phospholipids was analysed. In the final studies, the cytosols were incubated with 1- 14C oleic and 11-14C linoleic acids and subjetcted to gel filtration on Sephadex G75 and revealed that more effective binding capacity for superficial fractions was percived. In this way, we apreciated together with a lipid modification and theirs fatty acids composition from crypst zones to villus ones, a superior binding capacity of cytosolic fractions enriched in FABP, for superficial zone than in deep one, considering the sense of the migartion of the cells in the bovine intestinal mucose. Through the evidence of these resultas is shown a real relationship between the composition and distribution of lipids in crypts and villus zones of bovine intestinal mucose and the FABP.

Tesis (Doctor en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2021<br>RESUMEN Giardia lamblia es un protozoo intestinal que parasita a una gran variedad de mamíferos, incluidos los humanos, causando una enfermedad aguda o crónica, con presencia o no de síntomas. La giardiasis tiene un impacto significativo en la salud pública debido a su alta prevalencia y morbilidad, su propensión a causar brotes importantes, y a sus efectos negativos en el desarrollo de los niños infectados. El hecho de que Giardia presente características indiscutiblemente primitivas, pero a su vez elementos que reflejan funciones celulares universales de eucariotas, la convierten en un excelente modelo para abordar estudios básicos de procesos celulares e identificar mecanismos moleculares claves de patógenos unicelulares y organismos eucariotas superiores. Giardia manifiesta variación antigénica de sus proteínas de superficie llamadas VSPs (Variant-specific Surface Proteins) tanto in vitro como in vivo, y se cree que utiliza tal mecanismo de recambio para (i) confrontar variaciones del ambiente hostil que existe en la porción superior del intestino delgado del hospedador, (ii) diversificarse para poder infectar un amplio rango de hospedadores y (iii) evadir la respuesta inmune del individuo infectado. En el presente trabajo de tesis se aborda el estudio de las capacidades protectivas que le brindan las VSPs a los trofozoítos de Giardia y el rol que cumplen las mismas moldeando la dinámica de la población de parásitos durante la infección. Nuestros resultados indican que la relación parásito-hospedador es más compleja que la previamente informada y que Giardia utiliza mecanismos típicos de organismos procariotas, así como de eucariotas para su supervivencia tanto dentro como fuera del intestino del hospedador.<br>ABSTRACT Giardia lamblia is an intestinal protozoan that parasitizes a wide variety of mammals, including humans, causing an acute or chronic disease, with presence or absence of symptoms. Giardiasis has a significant public health impact because of the high prevalence and morbidity, its propensity in causing major outbreaks, and its negative effects on the development of infected children. The fact that Giardia has indisputably primitive characteristics, but at the same time features that resemble universal eukaryotic cellular functions, make it an excellent model to address basic studies of cellular processes and identify key molecular mechanisms of unicellular pathogens and higher eukaryotic organisms. Giardia manifests antigenic variation of its surface antigens called VSPs (Variant-specific Surface Proteins) both in vitro and in vivo, and it is believed that it uses such a turnover mechanism to (i) confront variations in the hostile environment that exists in the upper portion of the host´s small intestine, (ii) diversify in order to infect a wide range of hosts, and (iii) evade the immune response of the infected individual. This thesis work addresses the study of the protective capacities that VSPs provide to Giardia trophozoites and the role they play in shaping the dynamics of the parasite population during the infection. Our results indicate that the parasite-host relationship is more complex than previously reported and that Giardia uses mechanisms typical of prokaryotic organisms for its survival both inside and outside the host gut.<br>2023-04-30<br>Fil: Ríos, Diego Nicolás. Universidad Católica de Córdoba; Argentina.<br>Fil: Ríos, Diego Nicolás. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.<br>Fil: Luján, Hugo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas; Argentina.<br>Fil: Luján, Hugo Daniel. Universidad Católica de Córdoba. Facultad de Medicina. Cátedra de Química; Argentina.<br>Fil: Panzetta de Dutari, Graciela María Del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.<br>Fil: Panzetta de Dutari, Graciela María del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.<br>Fil: Argaraña, Carlos Enrique. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.<br>Fil: Argaraña, Carlos Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.<br>Fil: Barra, José Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.<br>Fil: Barra, José Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.<br>Fil: Uttaro, Antonio D. Universidad Nacional de Rosario; Argentina.<br>Fil: Uttaro, Antonio D. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario; Argentina.

Faculty of Health Scicence School of Medicine 0100107g johannes.geyer@wits.ac.za<br>The 14C and 13C labelled urea breath tests (UBT) for detecting Helico-bacter pylori infection are well established but scope for improvement exists in both to reduce some of their shortcomings. For this study, the 14C UBT investigation focussed on reducing the quantity of radioactive tracer that is administered to the subject un-dergoing this test, with the aim of lowering the radiation dose to the patient, reducing the impact to the environment and exempting the test from radioactive materials licensing. Wider acceptance, availabil-ity, affordability to lower socio-economic groups and third party medi-cal treatment payers and using readily available equipment were fac-tors considered when developing the method. The principle of the method developed is to collect larger volume breath sample, quantitatively absorbing a defined volume of extracted breath CO2 in an efficient CO2 trapping agent using a specifically de-signed apparatus and measuring the activity with a low background β-spectrometer. A reduction in the quantity of 14C labelled urea administered to the pa-tient was achieved. The method also reduced the counting error mar-gin at a lower detection limit, improving discrimination between H. py-lori positive and negative patients. iii The 13C UBT is a non-radioactive test however, it is substantially more expensive. The 13C UBT investigation aimed to determine whether commercially available un-enriched urea could be used thus reducing the cost of the 13C UBT. A simple protocol with Isotope Ratio Mass Spectrometry (IRMS) for the measurement was used as opposed to the well-established 13C UBT protocol. The principle of the 13C UBT investigation was to detect the change of the breath δ13C (13C/12C) ratio after the administration of un-enriched urea with a δ13C different to the exhaled breath. Theoretical calculations showed that an administered dose of 500mg un-enriched urea with at least a 10‰ δ13C difference may be detectable using IRMS. In vitro investigations confirmed that levels of 0.01 to 0.001‰ δ13C were detectable by IRMS. A change in the δ13C of a standard breath CO2 was confirmed for a range between 0.14 to 50% v/v mixed CO2 samples, i.e. the projected range for in-vivo investigation. Results from the in-vivo investigation however were not able to distinguish positive from negative H. pylori patients. The use of the 1000mg dose of urea appears to have caused saturation of the enzyme. It was con-cluded that some enrichment of the 13C is necessary or less urea be used.