Quorum sensing enables bacteria to communicate with bacteria of the same or different species, and to modulate their behavior in a cell-density dependent manner. Communication occurs by means of small quorum sensing signaling molecules (QSMs) whose concentration is proportional to the population size. When a QSM threshold concentration is reached, certain genes are expressed, thus allowing control of several processes, such as, virulence factor production, antibiotic production, and biofilm formation. Not only many pathogenic bacteria are known to produce QSMs, but also QSMs have been identified in some bacteria-related disorders. Therefore, quantitative detection of QSMs present in clinical samples may be a useful tool in the investigation and monitoring of bacteria-related diseases, thus prompting the use of QSMs as biomarkers of disease. Herein, we have developed and utilized whole-cell biosensing systems and protein based biosensing systems to detect QSMs in clinical samples, such as, saliva, stool, and bowel secretions. Additionally, since bacteria are responsible for food spoilage, we employed the developed biosensing systems to detect QSMs in food samples and demonstrated their applicability for early identification of food contamination. Furthermore, we have utilized these biosensing systems to screen antibacterial compounds employed for food preservation, namely, generally regarded as safe (GRAS) compounds, for their effect on quorum sensing.

Background: IBS is the most common functional gastrointestinal disorders and affects approximately 10-20 % of the general population and is widespread in all societies and socio-economic groups. Although the disorder does not have a life-threatening course, it still seriously affects the patients in their everyday life. Aim: The general aims of this thesis were to estimate the occurrence of irritable bowel syndrome in the general population and to achieve a better understanding of present treatment of this disorder and impact on every-day life in those suffering from IBS. Material and methods: The LIPS study comprises two parts. Part I was a retrospective register study where the data collection was based on computerised medical records at three selected Primary Health Care centres in a defined region. Part II was a population based case-control study. The identified IBS cases from part I constitute the cases, while their control groups were randomly selected from the population census register in the same area as the cases. Data in part II were collected by means of a postal questionnaire to cases and controls. The study was conducted in Linköping, a city located in the south-east of Sweden with 135 000 inhabitants. Results: The female IBS patients reported lower influence on planning their work and working hours as well as fewer opportunities to learn new things at their work compared to their controls, even after adjustments in multiple logistic regressions for potential confounders like; mood, sleeping problems and perceived health. The female IBS patients had considerably lower HRQOL in all dimensions compared to their controls, even when compared to male patients. Younger female IBS cases (18-44 years) reported lower mental health on the SF-36 scale than the older IBS female cases (p=0.015). In the multivariate analysis these variables, lack of influence on planning the work, family history of IBS, anxiety and sleeping disturbance displayed an association with being diagnosed with IBS in women. In men, lack of influence on working pace, family history of IBS was associated with an IBS diagnosis.The consultation incidence of IBS in part I was 3.4 (95% CI 3.20-3.70) per 1000 person-years for all IBS cases, among females; the incidence rate was 4.6 per 1000 person-years (95% CI 4.16-4.97) and males; 2.3 per 1000 person-years (95% CI 2.01-2.59). The dominating pharmacological treatment prescribed for abdominal complaints were fibre and bulking laxatives agents as well as acid suppressive drugs. These variables had an independent impact on the probability of a follow-up consultation; diagnosed co-morbidity besides the IBS diagnosis, rectoscopy ordered and laboratory tests ordered. Conclusions: IBS patients identified in primary care are significantly affected in their working-life compared to individuals in the general population. Especially female IBS-patients report lower decision latitude at work and they also appear to have a particularly impaired psychosocial functioning in their every day life and impaired HRQOL. Factors associated with IBS diagnosis among females are anxiety as well as family history of IBS and lack of co-determination at work. The incidence rate of IBS was 3.4 per 1000 person-years which increased with age and with an overrepresentation of females. IBS patients did not appear to be heavy utilisers of primary care and those who attended were treated by their GP without further consultation. The strongest predictors for having a follow-up consultation were diagnosed co-morbidity, rectoscopy and laboratory tests ordered / Figure on page 8 reprinted from Lancet 360(9332), Nicholas, J Talley and Robin Spiller, "Irritable bowel syndrome: a little understood organic bowel disease?", pp. 555-564, Copyright 2006 with permission from Elsevier. On the day of the public defence of the doctoral thesis, the status of articles III and IV was Submitted.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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.

No description available.

Within this thesis the role of cognitive processes in irritable bowel syndrome (IBS) will be examined. A systematic review and meta-analysis of the rate of psychiatric comorbidity in IBS participants, relative to controls, was performed. Evidence supported heightened rates of psychiatric disorder. A novel hypothesis regarding overlapping cognitive vulnerability to IBS and psychiatric disorders was found to fit well with the pattern of comorbidity. Competing hypotheses and the potential moderators were examined. Overall, no single model of psychiatric morbidity in IBS could fully account for the results of the meta-analysis. The implications of this meta-analysis for a cognitive approach to IBS are discussed. Cognitive processes were directly investigated in two experiments. First, in a modified exogenous cueing task, which assessed attention to pain words, there was faster orienting towards, and engagement with pain words in IBS participants relative to controls. Next, participants completed a primed lexical decision task, which indexed interpretation biases by measuring response times to targets after ambiguous illness primes. Relative to controls, IBS participants’ responses were slower to target words presented after ambiguous illness primes, and demonstrated priming for targets related to the neutral meaning of the illness prime. In the second study, different IBS and healthy control participants completed an internet-based survey of autobiographical memory. Participants described and rated painful and emotional autobiographical events. IBS participants reported pain memories from a more observer perspective relative to controls, suggesting a possible coping strategy for pain content. Finally, three cognitive styles, alexithymia, rumination and self-blame, were evaluated using existing and novel self-report measures. Overall, when compared with healthy participants, IBS participants reported: less difficulty identifying feelings as indexed by the alexithymia measure; increased pain-focused rumination; and a general, negative self-blame. These results may imply a vigilance-avoidance model of cognitive processing in IBS.

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

Les maladies inflammatoires de l’intestin (MIIs, [MIM 266600]) sont caractérisées par une inflammation chronique au niveau du tube gastro-intestinal. Les deux principales formes sont la maladie de Crohn (MC) et la colite ulcéreuse (CU). Les MIIs résulteraient d’un défaut du système immunitaire et de l’épithélium intestinal. Ce dernier forme une barrière physique et biochimique qui sépare notre système immunitaire des microorganismes commensaux et pathogènes de la microflore intestinale. Un défaut dans la barrière épithéliale intestinale pourrait donc mener à une réponse immunitaire soutenue contre notre microflore intestinale. Les études d’association pangénomiques (GWAS) ont permis d’identifier 201 régions de susceptibilité aux MIIs. Parmi celles-ci, la région 1q32 associée à la MC (p<2x10-11) et à la CU (p<6x10-7) contient 4 gènes, dont C1orf106, un gène codant pour une protéine de fonction inconnue. Le re-séquençage de la région 1q32 a permis d’identifier une variante génétique rare de C1orf106 (MAF˂1%) associée aux MIIs (p=0,009), Y333F. Nous avons démontré que la substitution de la tyr333 par une phénylalanine semble avoir un effet sur la stabilité protéique de C1orf106 tel que démontré lors de l’inhibition de la synthèse protéique induite par le cycloheximide. Nous avons déterminé que C1orf106 est exprimé dans le côlon et l’intestin grêle. De plus, son expression est augmentée lors de la différenciation des cellules épithéliales Caco-2 en épithélium intestinal polarisé. Son profil d’expression correspond aux types cellulaires et tissulaires affectés dans les MIIs. De plus, C1orf106 est partiellement co-localisée avec le marqueur des jonctions serrées, ZO-1. Toutefois, son marquage reproduit parfaitement celui du marqueur des jonctions adhérentes, E-cadhérine. Les jonctions serrées et adhérentes sont localisées du côté apical de la jonction intercellulaire et sont toutes deux impliquées dans l’établissement de la barrière épithéliale. Nous avons donc testé l’impact de C1orf106 sur la perméabilité de l’épithélium intestinal. Nous avons observé une augmentation de la perméabilité épithéliale chez un épithélium intestinal formé par des cellules Caco-2 sous-exprimant C1orf106. Nos résultats suggèrent que C1orf106 pourrait être le gène causal de la région 1q32. / The Inflammatory bowel diseases (IBD, [MIM 266600]) involve chronic inflammation of the digestive tract and include ulcerative colitis (UC) and Crohn’s disease (CD). IBD may result from defects in the homeostasis of immune system and intestinal epithelium. The latter forms a physical and biochemical barrier to commensal and pathogenic microorganisms. A dysfunction in the epithelial barrier may lead to a sustained immune response against the gut flora. Genome wide association studies (GWAS) have identified 200 susceptibility regions in IBD. Among these, the 1q32 region associated with risk of both CD (p<2x10-11) and UC (p<6x10-7), contains the gene C1orf106. Our targeted re-sequencing study has identified a low-frequency variant, Y333F (p=0.009) in C1orf106, a protein of unknown function and in which tyrosine333 is predicted to be phosphorylated. We demonstrated that its substitution by a phenylalanine may have an effect on C1orf106 protein stability as shown by cycloheximide treatment experiments. Our RNA expression analyses of human tissues and cell lines demonstrated that C1orf106 is mostly expressed in the small intestine and colon. It is also detectable in monocytic cell lines but more highly expressed in colonic epithelial cell lines. Furthermore, its expression is increased by 40% during differentiation of colonic epithelial Caco-2 cells into polarized epithelium. To provide further biological context, we generated colorectal LS174T cells that stably overexpress the Y333F alleles and demonstrated that it is partially localized with ZO-1, used as a tight junction (TJ) marker. We did observe tighter colocalization with E-cadherin, a canonical marker for adherens junctions (AJ), typically located below the TJ complex. AJ and TJ play an essential role in the establishment of epithelial barrier. The localization of C1orf106 at these regions suggests its possible implication in epithelial barrier homeostasis. Using trans-epithelial measurement of ions movement across epithelium, we demonstrated an increased in permeability of an epithelium formed by C1orf106 knock-down Caco-2 cells. Our results suggest that C1orf106 could be the causal gene of the 1q32 susceptibility region.

Les bénéfices attribués aux probiotiques sont nombreux : effets bénéfiques sur le microbiote intestinal, confort digestif, modulation du système immunitaire et prévention des infections intestinales. Ces effets divers et variés sont souches-spécifiques. Actuellement, de nombreuses études visent à mieux caractériser ces effets probiotiques.Ces travaux avaient pour objectif de déterminer et d’analyser les effets probiotiques de trois souches de la collection Danone : la souche de Lactobacillus paracasei CNCM I-3689, la souche de L. rhamnosus CNCM I-3690 ainsi que la souche de Bifidobacterium animalis subps. lactis CNCM I-2494. Dans un premier temps, leurs effets ont été caractérisés dans des modèles in vitro pour des propriétés anti-pathogènes, des propriétés d’immuno-modulation ainsi que pour des propriétés de protection de la barrière épithéliale intestinale. Dans un second temps, leurs effets de la protection de la barrière ont été confirmés dans un modèle murin de faible inflammation.Une analyse des mécanismes sous-jacents à ces effets à la fois chez la souche probiotique et chez l’hôte a été initiée par la construction et l’analyse fonctionnelle de banques génomiques bactériennes ainsi que par l’étude de la modulation des gènes impliqués dans le maintien de la barrière intestinale chez l’hôte. / Health benefits of probiotic bacteria are numerous: beneficial effects on the intestinal microbiota, digestive comfort, modulation of the immune system and prevention of winter infection. These diverse and various effects are strain-specific. Nowadays, numerous studies aim at better characterizing those probiotics effects.This project aimed at identifying and analyzing the probiotic effects of three strains from Danone collection: Lactobacillus paracasei CNCM I-3689, L. rhamnosus CNCM I-3690 and Bifidobacterium animalis subsp. lactis CNCM I-2494. First, their effects were assessed in in vitro models for immunomodulation properties, antipathogens activity and intestinal barrier protection. Secondly, their beneficial effects were confirmed in low-grade inflammation murine model.The analysis of the underlying mechanisms has been initiated both in the bacterial strains by the construction and the functional analysis of genomic libraries and in the host by measuring the modulation of the genes involved in the intestinal barrier maintain.

Inflammatory bowel disease encompasses a variety of heterogeneous chronic inflammatory diseases that affect the gastrointestinal tract, where Crohn’s disease and ulcerative colitis are the principal examples. The etiology of these, and many other complex human diseases, remain largely unknown and therefore pose relevant targets for novel research strategies. One such strategy is the in silico application of network theory derived methods to data sourced from publicly available repositories of e.g. gene expression data. Specifically, methods generating graphs of interconnected elements enriched by differentially expressed genes—disease modules—were inferred with data available through the Gene Expression Omnibus. Based on a previous method, the current project aimed to evaluate disease modules, combined from stand-alone inferential methods, in disease consensus modules: representing pathophenotypical motifs for the diseases of interest. The modules found to be significantly enriched by genome-wide association study inferred single-nucleotide polymorphisms, as validated using the Pathway Scoring Algorithm, were subsequently subjects for further analysis using Kyoto Encyclopedia of Genes and Genomes-pathway enrichment, and literature searches. The results of this study adheres to previous findings relating to the employed method, but lack any novelty pertaining the diseases of interest. However, the results substantiate the preceding methods’ conclusion by including parameters that increase statistical validity. In addition, the study contributed to peripheral results concerning both the methodology of consensus module methods, and the elucidation of inflammatory bowel disease etiology and disease subtype differentiation, that pose interesting subjects for future investigation.