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Molecular Mechanisms and Effect of Acute and Psychosocial Stress on the Intestinal Barrier Function

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Molecular Mechanisms and Effect of Acute and Psychosocial Stress on the Intestinal Barrier Function ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en DOCTORAL THESIS MOLECULAR MECHANISMS AND EFFECT OF ACUTE AND PSYCHOSOCIAL STRESS ON THE INTESTINAL BARRIER FUNCTION. IMPLICATIONS ON THE IRRITABLE BOWEL SYNDROME. Presented by MARC PIGRAU PASTOR Directed by Francisco Javier Santos Vicente, María Vicario Pérez & Carmen Alonso Cotoner Tutor Fernando Azpiroz Vidaur Thesis submitted to obtain the degree of Doctor PhD program in Medicine Department of Medicine, Medical School, Universitat Autònoma de Barcelona Barcelona, September 2018 FRANCISCO JAVIER SANTOS VICENTE, Doctor en Medicina, MARÍA VICARIO PÉREZ, Doctora en Farmacia y CARMEN ALONSO COTONER, Doctora en Medicina HACEN CONSTAR Que la tesis titulada “Molecular mechanisms and effect of acute and psychosocial stress on the intestinal barrier function. Implications on the irritable bowel syndrome.” presentada por Marc Pigrau Pastor para optar al grado de Doctor, se ha realizado bajo su dirección, y al considerarla concluida, autorizan su presentación para ser juzgada por el tribunal correspondiente. Y para que conste a los efectos firman la presente. Barcelona, Septiembre de 2018 Dr. Francisco Javier Dra. María Dra. Carmen Santos Vicente Vicario Pérez Alonso Cotoner (Directores de la tesis) Dr. Fernando Azpiroz Vidaur (Tutor de la tesis) This thesis has been possible thanks to the grant support from Fondo de Investigación Sanitaria, Instituto Carlos III, Ministerio de Ciencia e Innovación (PI08/0940) and Contrato post Formación Sanitaria Especializada Río Hortega CM10/00155. “Science is not only a disciple of reason but, also, one of romance and passion”. Stephen Hawking (1942-2018) AGRADECIMIENTOS Desde el momento en que decidí emprender el camino de realizar una tesis doctoral, sabía que no sería un camino fácil, pero viendo el resultado final, ha valido la pena. Sin duda alguna no habría sido posible realizar este camino sin la ayuda de muchas personas que han colaborado tanto en lo científico como en lo personal para superar todas las dificultades que han ido surgiendo. El inicio fue complicado, sobretodo el cambio de una visión clínica a una básica, era la misma sensación que experimenté cuando era residente de primer año, pero gracias a la ayuda y supervisión de la gente del grupo pude ir desenvolviéndome cada vez mejor hasta poder realizar este trabajo. Una de las cosas más importantes que he aprendido durante estos años ha sido que, en investigación, el trabajo en equipo facilita mucho los experimentos y al final mejora los resultados obtenidos. En primer lugar quiero expresar mi más sincero agradecimiento y admiración a mis directores, por haberme dado la posibilidad de realizar la tesis, pero lo más importante por hacerme estudiar y comprender mejor un grupo de enfermedades digestivas muy prevalentes pero poco conocidas y muy rechazadas por los médicos en general y por los gastroenterólogos en particular. A Javier, porqué a pesar de sus métodos poco ortodoxos, me ha ayudado a pensar de manera crítica y distinta tanto a nivel básico como a nivel clínico, lo cual me ha ayudado a entender la gastroenterología mejor y al fin y al cabo a atender mejor a mis pacientes. A María, por estar siempre ahí, por ayudarnos y dirigirnos desinteresadamente en todos nuestros proyectos. Y, sobre todo, porqué sin su ayuda y dirección, esta tesis no habría sido posible ya que estuvo ahí desde el inicio, cuando presenté la tesina. A Carmen por haberme animado y convencido por entrar en el grupo e iniciarme en la investigación, por todo su apoyo, su ayuda y por siempre contar conmigo, a pesar que al final no haya salido todo según lo planeado inicialmente. Al Dr. Azpiroz por haberme dado la oportunidad de quedarme en el servicio realizando la tesis y por haber sido el tutor de esta tesis. A todos los adjuntos tanto del servicio de digestivo y de endoscopias así como de otros servicios que me han formado como médico y como gastroenterólogo. A Bea y Cristina por haberme acogido cual polluelo en el laboratorio y haber dedicado su tiempo (a pesar de lo ocupadas que iban entre la consultas, experimentos y las respectivas tesis) para enseñarme toda la parte clínica y básica fundamental del laboratorio para poder ser autónomo. A Ana por haberme enseñado el rigor y la metodología a la hora de pipetear, hacer técnicas en el laboratorio, entre ellas los ELISAs y por haber intentado invadir constantemente mi espacio en el despacho. A Mila, por ser como una madre dentro del laboratorio y por toda la ayuda para que esta tesis fuese posible. Por cierto, aún espero que me hagas unos ELISAs. A Elo, Bruno y César por haber hecho la estancia más llevadera y por mantener todas esas discusiones tanto científicas como no científicas pero igualmente enriquecedoras. A Sara, Dory y a las Lauras por su inestimable ayuda y por hacer que el PIO fuese sólo un estudio de estrés para el sujeto y no también para quien lo realizaba. A Puri porque sin su ayuda el estudio de permeabilidades hubiese sido mucho más complicado y lento de hacer. También agradecerles a Anna y a Maite por su ayuda con las biopsias intestinales. Al resto de componentes del laboratorio tanto presentes (Marina, Cristina y Elba) como pasados por su ayuda. A Fina por toda su ayuda en la parte que se nos da peor, la burocrática. A todos aquellos que contribuyeron a que mi estancia Canadá fuese más fácil y provechosa. Al Dr. Collins por haberme aceptado en el grupo de investigación. A Premek y Elena, no sólo por todo lo que me han enseñado científicamente sino por todo el apoyo que me han brindado durante mi estancia, estar lejos de casa se hizo menos duro con unos jefes como ellos. A todos los compañeros del laboratorio pero en especial, a Inés y a Christian por facilitarme mucho la adaptación a Hamilton. A Ryan y a su familia (Wayne y Gail) que me acogieron como uno más de la familia. Y a Giada por su amistad y su ayuda tanto dentro como fuera del laboratorio y sin quien no hubiese sido posible tener una estancia tan productiva en Canadá. Quería agradecerle también a mi familia, empezando por l’àvia, mi hermano y siguiendo por mis padres por todo lo que me han dado para poder llegar a ser quien soy actualmente y por su incondicional apoyo, a pesar que a veces sea difícil entender situaciones, cómo la de irse a un océano de distancia. Finalmente, le dedico esta tesis a Ceci, las palabras no pueden reflejar todo lo que siento y agradezco que me hayas acompañado en este viaje y sufrimiento que ha sido la tesis, querría agradecerte tu paciencia y tu incondicional apoyo en todos estos años, tanto en los buenos momentos pero sobretodo en los malos. ¡GRACIAS POR TODO! ¡Te quiero! A Ceci y a mis padres. ABBREVIATIONS ABBREVIATIONS ABBREVIATIONS ABBREVIATIONS 5-HT: 5-hydroxy tryptamine ACTH: adrenocorticotropic hormone ADHD: attention deficit and hyperactivity disorder AJ: adherens junctions ANOVA: analysis of the variance AVP: arginine-vasopressin BDI: Beck’s depression Inventory BGA: brain-gut axis BMAL1: brain–muscle–arnt-like protein 1 BMI: body mass index bpm: beats per minute CB1: cannabinoid 1 receptor CGRP: calcitonin gene-related peptide CLOCK: circadian locomotor output cycle kaput CLRs: C-type lectins receptors CNS: central nervous system CP: canonical pathway CPS: cold pain stress CRH: corticotrophin-releasing hormone DAMPs: danger-associated molecular patterns DBP: diastolic blood pressure DHEA: dehydroepiandrosterone ENS: enteric nervous system F: female FC: fold change FD: functional dyspepsia FE: fractional excretion FGID: functional gastrointestinal disorders Fx: functions GALT: gut-associated lymphoid tissue GI: gastrointestinal GR: glucocorticoid receptor H-R: modified social readjustment scale of Holmes-Rahe ABBREVIATIONS HPA: hypophysis-pituitary-adrenal HPF: high power field HR: heart rate HV: healthy volunteer IBD: inflammatory bowel disease IBS: irritable bowel syndrome IBS-C: constipation-predominant IBS IBS-D: diarrhea-predominant irritable bowel syndrome IBS-SSS: irritable bowel syndrome severity score scale IELs: intraepithelial lymphocytes IgA: immunoglobulin A IPA: Ingenuity pathway analysis JAMs: junctional adhesion molecules LC: locus ceruleus LIMMA: comparative analysis and linear models for microarray data LMR: lactulose-mannitol ratio LPS: lipopolysaccharide LS: low stress M: male MBP: median blood pressure MC2-R: melanocortin type 2 receptor MR: mineralocorticoid receptor MS: moderate stress NE: norepinephrine NGF: nerve growth factor NLRs: NOD-like receptors NOD: nucleotide-binding and oligomerization domain PAMPs: pathogen-associated molecular patterns PAR2: protease-activated receptor 2 PER: periods PI-IBS: post-infectious irritable bowel syndrome POMC: pro-opiomelanocortin PPIA: cyclophilin PRRs: pattern recognition receptors PSS: perceived stress scale RCT: randomized controlled trial ABBREVIATIONS RIG-I: retinoic acid inducible gene-I RLRs: RIG-I-like receptors RT-qPCR: quantitative real time polymerase chain reaction S: stress sd: standard
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