Two key proteins, LexA and RecA, are involved in regulation of the SOS expression system in bacteria. LexA and RecA act as the transcriptional repressor and inducer of the SOS operon, respectively. LexA downregulates the expression of at least 43 unlinked genes and activated RecA interacts with the repressor LexA and therefore, LexA undergoes self-cleavage. The ability of the LexA protein to dimerize is critical for its ability to repress SOS-regulated genes in vivo, as the N-terminal domain (NTD) alone has a lower DNA-binding affinity without the C-terminal domain (CTD) and the components for the dimerization of LexA are located in the CTD. Two antiparallel β-strands (termed β-11) in the CTD at the dimer interface of LexA are involved in the dimerization. LexA interacts with the active form of RecA in vivo during the SOS response. It was determined experimentally that monomeric and non-cleavable LexA binds more tightly to RecA and is resistant to self-cleavage. Therefore, we reasoned that if we can produce such LexA mutants we would be able to stabilize the LexA and active RecA complex for crystallization. Therefore, in this experiment, we attempted to make a non-cleavable and predominantly monomeric LexA that interacts intimately with RecA. We produced four single mutations at the dimer interface of the non-cleavable and NTD-truncated mutant of LexA (∆68LexAK156A) in order to weaken the interactions at the interface. The predominant forms of LexA mutants and the affinities of interaction between the mutant LexA proteins and RecA were examined. ∆68LexAK156AR197P mutant was found as predominantly monomeric at a concentration of 33.3 μM both by gel filtration chromatography and dynamic light scattering (DLS) experiments. It also bound RecA more tightly than wild-type LexA. Another mutant, ∆68LexAK156AI196Y, was also found as predominantly monomeric at a concentration of 33.3 μM by DLS. Both these proteins were subjected to crystallization with wild-type RecA protein. We were able to produce some predominantly monomeric LexA with good binding affinity for RecA; however, we were unsuccessful in co-crystallization.

Two key proteins, LexA and RecA, are involved in regulation of the SOS expression system in bacteria. LexA and RecA act as the transcriptional repressor and inducer of the SOS operon, respectively. LexA downregulates the expression of at least 43 unlinked genes and activated RecA interacts with the repressor LexA and therefore, LexA undergoes self-cleavage. The ability of the LexA protein to dimerize is critical for its ability to repress SOS-regulated genes in vivo, as the N-terminal domain (NTD) alone has a lower DNA-binding affinity without the C-terminal domain (CTD) and the components for the dimerization of LexA are located in the CTD. Two antiparallel β-strands (termed β-11) in the CTD at the dimer interface of LexA are involved in the dimerization. LexA interacts with the active form of RecA in vivo during the SOS response. It was determined experimentally that monomeric and non-cleavable LexA binds more tightly to RecA and is resistant to self-cleavage. Therefore, we reasoned that if we can produce such LexA mutants we would be able to stabilize the LexA and active RecA complex for crystallization. Therefore, in this experiment, we attempted to make a non-cleavable and predominantly monomeric LexA that interacts intimately with RecA. We produced four single mutations at the dimer interface of the non-cleavable and NTD-truncated mutant of LexA (∆68LexAK156A) in order to weaken the interactions at the interface. The predominant forms of LexA mutants and the affinities of interaction between the mutant LexA proteins and RecA were examined. ∆68LexAK156AR197P mutant was found as predominantly monomeric at a concentration of 33.3 μM both by gel filtration chromatography and dynamic light scattering (DLS) experiments. It also bound RecA more tightly than wild-type LexA. Another mutant, ∆68LexAK156AI196Y, was also found as predominantly monomeric at a concentration of 33.3 μM by DLS. Both these proteins were subjected to crystallization with wild-type RecA protein. We were able to produce some predominantly monomeric LexA with good binding affinity for RecA; however, we were unsuccessful in co-crystallization.

When treating patients with cancer, the ability to predict a patient’s response to treatment is an important tool to allow therapy to be tailored for best outcome. Therefore, the need exists for a test to forecast a patient’s response using a sample that is readily accessible, and provides an accurate reflection of a patient’s response to a disease or treatment. Profiling biological fluids, such as plasma or urine, has gained considerable interest in recent years. This is because these fluids are readily available and are expected to provide an accurate representation of a patient’s response to treatment. As such, much effort has been put into finding biomarkers or prognostic indicators. Abnormal protease activity has been linked to the progression of cancer due to their involvement in several processes vital to the survival and proliferation of the disease. These include metastasis, resistance to apoptosis and angiogenesis, amongst others. In addition, dysregulated protease activity has been linked to poor response to chemotherapy as well as tumour regrowth following radiotherapy. Therefore, an increased understanding regarding the activity of a patient’s proteases may provide the clinician with more information as to how best to treat the patient. Therefore, monitoring protease activity has been suggested as a potential marker to predict a patient’s response to cancer treatment. Most enzyme activity assays are currently performed by fluorescence spectroscopy. However, these workflows suffer from limited sensitivity and linear range. Therefore, an alternative, more sensitive assay is required. Mass spectrometry (MS) is a highly sensitive analytical technique routinely used to quantify changes in biological systems. As such, MS has the potential to be used in enzyme activity assays. This study illustrates the development of a novel MS based method to monitor the activity of target enzymes in plasma; specifically asparaginyl endopeptidase (AEP) and caspase-3 using mass spectrometry. These enzymes have been linked to poor chemotherapeutic response in childhood leukaemia and tumour regrowth post-radiotherapy respectively. This project will describe the development and optimisation of each stage of a five step sample preparation and analysis method. This includes the design of enzyme substrates designed to be cleaved by the target enzyme, whilst reducing the effect of other enzymes acting on this substrate, how best to enrich samples for these target peptides, as well as determining the best MS technique to monitor these peptides. In addition, this project describes a comparison between this assay and an existing fluorescence assay when monitoring AEP activity in biological samples such as plasma and whole cell lysates. The application of this method in quantifying caspase-3 activity in plasma samples is also examined.

Les muscles squelettiques, responsables des mouvements volontaires tels que la locomotion ou le maintien de la posture, représentent environ 40% de la masse corporelle.Cette masse musculaire est maintenue par plusieurs voies de signalisation qui régulent entre autres l’équilibre entre la synthèse et la dégradation des protéines myofibrillaires. En ciblant la voie de signalisation Akt/mTOR, la myostatine est un régulateur négatif de la myogenèse. Elle inhibe la différentiation myogénique et le renouvellement cellulaire. Parmi les différents facteurs moléculaires extracellulaires qui régulent la myostatine, les protéines GASP (Growth and differentiation factor Associated Serum Protein) ont été décrites comme des antagonistes de son activité. L’Unité de Génétique Animale a développé plusieurs stratégies qui permettent d’appréhender les mécanismes moléculaires qui régissent le(s) rôle(s) des protéines GASP au cours du développement musculaire. Ainsi, la création de la lignée murine appelée surGasp-1-20 a permis de montrer que la surexpression de Gasp-1 entraîne un phénotype hypermusclé associé à une hypertrophie des myofibrilles. Une analyse de l’expression génique dans des myoblastes dérivés des cellules satellites montre une surexpression de la myostatine corrélant avec une absence d’hyperplasie chez les souris surGasp-1-20. Des études similaires actuellement en cours pour la protéine GASP-2 devraient permettre de préciser son rôle dans le contexte musculaire. Les protéines GASP sont également définies comme des inhibiteurs composés hétérotypiques caractérisés par plusieurs domaines inhibiteurs pouvant moduler l’activité de différentes protéases. Parmi ces différents domaines,le second domaine Kunitz de GASP-2 a été précédemment décrit comme pouvant inhiber la trypsine. Dans ce travail, nous avons pu montrer que les deux protéines entières conservent cette capacité d’inhibition. Nos résultats indiquent cependant que GASP-1 et GASP-2 présentent une différence de spécificité due à la composition du second domaine Kunitz et non à l’environnement moléculaire présent dans chacune des protéines. Enfin, nous proposons un modèle structural du second domaine Kunitz impliqué dans la dualité fonctionnelle dans l'inhibition anti-trypsine de GASP-1 et GASP-2. / Skeletal muscles, responsible for voluntary movements such as locomotion or posture maintenance, represent about 40% of body mass. This muscle mass is maintened by several signaling pathways that regulate , among other things, the balance between synthesis and degradation of myofibrillar proteins. By targeting the Akt/mTOR pathway, myostatin is anegative regulator of myogenesis. It inhibits myogenic differentiation and cell turnover. Among the various endogenous molecular factors that regulate myostatin, proteins GASP (Growth and differentiation factor Associated Serum Protein) have been described as antagonists of its activity. The Animal Genetics Unit has developed several strategies to understand themolecular mechanisms that govern the role (s) of GASP proteins during muscle development.Thus, the creation of the transgenic mouse line named surGasp-1-20 has shown that overexpression of Gasp-1 results in a hypermuscular phenotype associated with myofibril hypertrophy. An analysis of gene expression in myoblasts derived from satellite cells showed overexpression of myostatin correlating with an absence of hyperplasia in Gasp-1-20 mice.Similar studies currently underway for the protein GASP-2 should clarify its role in the muscular context. Proteins GASP are also defined as compound heterotypic inhibitors characterized by several inhibitory domains that can modulate the activity of different proteases. Among these different modules, the second Kunitz domain of GASP-2 was previously been described asable to inhibit trypsin. In this work, we have shown that the two whole proteins conserve this capacity of inhibition. However, our results indicate that GASP-1 and GASP-2 exhibit a difference in specificity due to the composition of the second Kunitz domain and not to the molecular environment present in each of the proteins. Finally, by modeling, we propose a structural model of the second Kunitz domain of GASP-1 and GASP-2 implicated in the antitrypsin inhibition specificity

Les maladies inflammatoires chroniques de l'intestin (MICI) se caractérisent par une inflammation sévère de l'intestin grêle et du côlon et comprennent la maladie de Crohn (MC) et la rectocolite hémorragique (RCH). Les MICI sont des maladies complexes faisant intervenir des facteurs génétiques : certains senseurs bactériens, l'autophagie et le stress du réticulum endoplasmique. Un défaut de barrière de l'épithélium digestif est également fortement impliqué dans la physiopathologie du processus inflammatoire. La fonction barrière de l'épithélium digestif est assurée par plusieurs types cellulaires, synthétisant entre autres, des peptides antimicrobiens (PAM) et des mucines. Dans les MICI, une augmentation de la perméabilité intestinale et une perte de muco-sécrétion ont été décrites. Les protéases jouent un rôle fondamental dans la digestion du bol alimentaire mais également dans le maintien de l'homéostasie intestinale en activant ou dégradant divers motifs moléculaires, ou in induisant des signaux spécifiques aux cellules par l'activation de quatre récepteurs : les PARs (Protease-Activated Receptor). Dans les MICI, un excès d'activité protéolytique de type trypsine est observé. L'origine de cette activité est théoriquement attribuée aux cellules immunitaires, à une surproduction pancréatique ou au microbiote, mais les cellules épithéliales intestinales semblent également être une source majeure de protéases. L'objectif de mon projet de thèse visait à étudier l'impact des principales voies impliquées dans les MICI sur l'homéostasie des protéases épithéliales et le rôle de celles-ci dans la déstabilisation de la fonction de barrière. Nos résultats ont confirmé un excès de protéases à sérine dans les cellules épithéliales de patients atteint de MC ou de RCH. In vitro, sur des monocouches de cellules Caco-2, l'induction de l'autophagie diminuait la libération apicale de protéase de type trypsine, alors que le senseur bactériens NOD2 n'avait aucun effet. A l'inverse, une stimulation du Stress du réticulum endoplasmique (SRE) par la Thapsigargin, induisait une libération accrue de protéases actives de type trypsine au pôle apical des cellules. [...] / Crohn's disease (CD) and Ulcerative colitis (UC) are two forms of Inflammatory Bowel Disease (IBD), a chronic inflammatory pathology affecting the digestive tract. Patients suffer from relapsing flares, diarrhea, abdominal pain and bleeding. Although the molecular mechanisms of IBD are poorly understood, recent data suggest that IBD occurs in genetically predisposed individuals developing an abnormal immune response to intestinal microbes after, being exposed to specific environmental triggers. Genetic studies have reported more than 170 polymorphisms susceptible to be involved in IBD pathogenesis. The strongest associations have highlighted three main pathways altered in IBD including bacterial sensing (NOD2, CD), autophagy (ATG16L1 and IRGM, CD) and endoplasmic reticulum stress (ER-Stress) (XBP1, UC). The role of intestinal barrier function is also strongly implicated in IBD pathogenesis, and is modulated by factors present in the lumen derived from microbiota, food or at a molecular level, by factors such as proteases. In IBD pathophysiology, the inflammatory process is characterized by impaired intestinal biology including disruption of tight junctions and leaky gut, decreased amount of Paneth and Goblet cells, and translocation of luminal antigens triggering inflammation. Previous studies have demonstrated an increased level of active serine proteases in the stools and tissues of IBD patients, supposing that proteases originate from infiltrated immune cells, pancreatic secretion or microbiota. However, our team has reported that intestinal epithelial cells are a major source of serine proteases, in particular trypsin-like enzymes, are released by a stressed epithelium in pathogenic context such as irritable bowel syndrome. In this project, we aimed at better understanding whether the three main pathways involved in IBD (Nod2, autophagy, ER-stress) could be linked to an epithelial release of trypsin and reciprocally, if epithelial trypsin is able to induce or modulate these three IBD pathways. We confirmed that trypsin-like activity was significantly higher in biopsies from UC and CD patients compared to healthy controls. In Caco-2 monolayers cultured in transwells, secreted trypsin-like proteolytic activity remained stable upon NOD2 stimulation but decreased under autophagy induction. Thapsigargin (Tg) stimulation a well-known ER-stress inducer, enhanced the apical release of trypsin-like activity in Caco2 cells. [...]

Le fractionnement au pressurage, étape clé de la méthode champenoise, et le type de pressurage, conditionnent de façon significative la composition biochimique du moût et du vin. Le pressurage est fractionné en séparant les premiers moûts extraits, qui constituent la "cuvée", et qui proviennent de la pulpe, partie la plus riche en sucre et en acides (tartrique et malique), des volumes suivants, appelés "tailles", qui sont aussi riches en sucre, en sels minéraux (potassium notamment) et en matières colorantes mais moins acides. Les moûts ont des caractéristiques analytiques bien spécifiques. L’évolution de la composition des moûts au cours du pressurage et la mesure des principaux paramètres analytiques permettant de juger de la qualité de l’extraction ont fait l’objet de quelques études (Valade et Blanck, 1989; Blouin, 1998) sur la base d’un fractionnement volumétrique répondant à un cahier des charges champenois. Toutefois, aucune étude portant sur un large nombre de paramètres, dont les propriétés moussantes, n’avait été entreprise avant ce travail de thèse.Une contamination du raisin par le champignon pathogène Botrytis cinerea (pourriture grise) et l’effet de la maturité du raisin, autres paramètres clés dans l’élaboration du Champagne, ont un impact sur la qualité et la composition des moûts et des vins de base et notamment sur les compositions protéique, polysaccharidique et oligosaccharidique. Les essais ont été réalisés avec 2 pressoirs différents : un pressoir pneumatique industriel (capacité 8000 kg) et un pressoir de laboratoire (capacité 6 kg). Ce travail a été réalisé sur deux cépages : Pinot meunier (millésimes 2013 et 2015) et Chardonnay (millésimes 2014 et 2015). Les analyses suivantes ont été réalisées sur les moûts et vins de base : (i) paramètres œnologiques classiques des moûts et des vins, (ii) isolement et analyse des polysaccharides et oligosaccharides solubles des vins de base, (iii) quantification et identification des protéines solubles des vins de base, (iv) mesure de l’activité protéasique des vins de base (impact de Botrytis cinerea), (v) comparaison de la composition des moûts et des vins issus de raisins sains de deux millésimes : 2013 (pressoir industriel) et 2014 (pressoir de laboratoire).La connaissance de l’état sanitaire et l'optimisation du choix de la date des vendanges en fonction de la maturité sont des outils à la disposition de l’œnologue qui lui permettent d’améliorer la qualité des moûts produits au cours du pressurage et par conséquent celle des vins qui en sont issus. A la suite de cette approche, il serait intéressant de voir dans quelles conditions ces paramètres pourraient être reproduits à grande échelle pour une application industrielle. / Press fractioning is a key step in the Champagne method, and the type of pressing will significantly determine the biochemical composition of the juice and the wine. The first pressed juice obtained in the fractioned pressing cycle, called the “cuvée”, is rich in sugar and acids (tartaric and malic). The second pressed juice, called the “tailles”, is as rich in sugar, mineral salt (potassium in particular) and colorant materials as the first one but less acidic. Must has specific analytical characteristics. The evolution of composition in the must during the pressing cycle and the measure of current analytical parameters, allowing the understanding of grapes extraction, have led to little studies in the Champagne region (Valade et Blanck, 1989; Blouin, 1998). These studies have followed the changes between the Cuvée and the Tailles, according to the rules applied for Champagne production. Nevertheless, no study had considered a large number of parameters, including the foaming properties, before this thesis.The contamination of grapes by the pathogenic Botrytis cinerea (gray mold) and the effect of grape maturity, which are other key parameters in the elaboration of Champagne, have an impact on the quality of must and base wine, especially on proteins, polysaccharides and oligosaccharides. The essays were carried out in two different presses: an industrial automatic press (capacity 8000 kg) and a laboratory press (capacity 6 kg). This study was made on two grape varieties: Pinot meunier (vintages 2013 and 2015) and Chardonnay (vintages 2014 and 2015). The following analyzes were effectuated on must and base wine: (i) determination of current oenological parameters of must and base wine, (ii) isolation and analysis of soluble polysaccharides and oligosaccharides in base wine, (iii) analysis, quantification and identification of soluble proteins in base wine, (iv) quantification of protease activity in base wine (the impact of Botrytis cinerea), (v) comparison of composition of must and base wine from healthy grapes of two vintages: 2013 (industrial press) and 2014 (laboratory press).The knowledge of the sanitary state and the optimization of the harvest date are tools used by oenologist to improve the quality of must, obtained during the pressing cycle, and therefore of wine elaborated from them. Following this study, it could be interesting to examine in which conditions these parameters may be reproduced at a bigger scale for forward industrial applications.

The overall objective of this study was to investigate how NS0 cell proliferation is regulated in protein-free media. The hypothesis was that during the adaptation to growth factor-free media, animal cell lines start to produce their own autocrine growth factors to support proliferation, and after some time in a culture the effects of these factors are lost which results in cessation of proliferation. A chemically defined, protein-free and animal component-free medium was developed for the NS0 cells. This medium was comprised of a basal hybridoma medium to which phosphatidyl¬choline, cholesterol, β-cyclodextrin, ferric citrate and amino acids were added. A fed-batch process was then developed in this medium. The feed profile was optimised in a step-wise manner with a final feed solution containing glucose, glutamine, lipids, amino acids, vitamins, sodium selenite and ethanolamine. Specifically, supplementation with lipids (cholesterol) had a drastic effect on cell growth. Calcium, magnesium and potassium were not depleted during culture and a feed containing also iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield further. More than 8 x 106 viable cells mL-1 and 600 mg antibody L-1 was obtained in the final fed-batch. This corresponded to a 4.3-fold increase in viable cell yield and an 11.4-fold increase in product yield compared to bioreactor batch culture when the dilution of the fed-batch culture was also accounted for. The presence of autocrine growth factors in NS0 cell cultures was initially investigated by studying the effects of conditioned medium (CM). Concentrated CM had a significant positive effect on cell growth and part of this effect could be attributed to factor(s) eluting from a gel-filtration column at 20-25 kDa. In the search for cell-derived factors affecting cell growth the following proteins were identified as released/secreted by the NS0 cells; cyclophilin A, cyclophilin B, cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerise, macrophage migration inhibitory factor (MIF), β2-microglobulin, niemann pick type C2, secretory leukocyte protease inhibitor (SLPI), thioredoxin-1, TNF-α, tumour protein translationally controlled-1 and ubiquitin. Zymogram electrophoresis further identified aspartic acid, papain-like cysteine (including cathepsin L) and serine protease activity in the CM. Pro/cathepsin L, CypB, EGF, IFN-α/β/γ, IGF-I/II, leukaemia inhibitory factor, IL-6, IL-11, IL-25, MIF, oncostatin M, TGF-β and TNF-α were excluded as involved in autocrine regulation of NS0 cell proliferation. The serine protease activity was suggested to affect the cells negatively and since the serine protease inhibitor SLPI is also present in NS0 CM, a balance in serine protease activity may be crucial for optimal cell growth. Further, the receptor gp130, known to be associated with myeloma cell growth, was shown to be essential for NS0 cell proliferation as demonstrated by siRNA gene silencing. The results suggested that autocrine regulation of proliferation in NS0 cell cultures involves the receptor subunit gp130. / QC 20100920

.La consommation journalière de fructose est en augmentation croissante, jusqu'à plus de 50g par jour aux Etats-Unis et en Europe de l'Ouest. Le fructose est de plus en plus incorporé dans les boissons, les produits laitiers et les conserves, les produits cuisinés ou transformés. Le fructose peut déclencher ou aggraver les symptômes digestifs chez des volontaires sains, mais aussi dans le syndrome de l'intestin irritable. Le rôle de l'hypersensibilité viscérale dans le déclenchement des symptômes, lié à la prise de fructose a été suspecté mais n'a jamais été évalué. La prévalence de la malabsorption du fructose était mal documentée chez les patients souffrant d'un syndrome de l'intestin irritable, principalement en raison de l'hétérogénéité des méthodes diagnostiques. Le premier objectif de ce travail a été de définir, dans une population de patients souffrant d'un syndrome de l'intestin irritable, la prévalence de la malabsorption du fructose. Notre test de malabsorption du fructose a été défini par une dose de charge de 25g. Chez nos patients souffrant d'un syndrome de l'intestin irritable, 22% présentaient une malabsorption du fructose. Les patients jeunes et de sexe masculin étaient plus à risque de malabsorption du fructose. Nous avons également étudié l'association de la malabsorption du fructose avec d'autres anomalies physiopathologiques connues dans le syndrome de l'intestin irritable. Nous n'avons pas retrouvé d'association entre la présence d'une inflammation digestive et la présence ou non d'une malabsorption du fructose. En revanche, une association a été retrouvée entre malabsorption du fructose et hypersensibilité viscérale. L'efficacité du régime appauvri en fructose dans le syndrome de l'intestin irritable est connue. L'existence ou non d'une malabsorption du fructose associée pourrait être un facteur prédictif d'efficacité d'un tel régime. Le deuxième objectif de ce travail a été de déterminer si le test de dépistage de la malabsorption au fructose permettait de prédire l'efficacité du régime appauvri en fructose sur les symptômes digestifs des patients souffrant d'un syndrome de l'intestin irritable. Les résultats de notre étude ont confirmé l'efficacité du régime appauvri en fructose dans le syndrome de l'intestin irritable. En revanche, la présence ou non d'un test respiratoire au fructose positif n'impactait pas l'efficacité du régime. Le dernier objectif de ce travail était de modéliser la malabsorption du fructose sur des modèles murins, pour permettre d'identifier les mécanismes physiopathologiques sous-jacents. La modélisation sur 3 modèles murins de malabsorption du fructose (par régime riche en fructose, par délétion des gènes codant les transporteurs du fructose GLUT5 et GUT2) permettait d'induire une hypersensibilité viscérale associée à une augmentation de la perméabilité intestinale, deux anomalies déjà rapportées dans le syndrome de l'intestin irritable. L'étude des mécanismes physiopathologiques sous-jacents a permis d'écarter l'implication d'une inflammation de bas grade qui n'était pas retrouvée chez nos souris. L'augmentation d'activité élastase dans les selles de souris avec malabsorption du fructose était associée à l'hypersensibilité viscérale. Or il a déjà été démontré que l'activité protéasique pouvait être responsable d'une hypersensibilité viscérale et d'une augmentation de la perméabilité intestinale. Les récepteurs associés à la protéase-2 sont connus pour être associés à l'hypersensibilité viscérale et l'augmentation de la perméabilité intestinale. Les résultats obtenus dans le cadre de ce travail soulignent le rôle de la malabsorption du fructose, qui entraine la survenue d'une hypersensibilité viscérale et d'une augmentation de la perméabilité intestinale, dans le syndrome de l'intestin irritable. Un régime appauvri en fructose n'améliore pas de manière ciblée les symptômes des patients souffrant d'un syndrome de l'intestin irritable avec malabsorption du fructose. / Fructose intake has increased by up to 50 g per day in the USA and Western Europe. Fructose is increasingly incorporated in beverages, dairy products and canned, baked or processed foods worldwide. Fructose has been shown to trigger or worsen digestive symptoms not only in healthy volunteers, but also in patients with irritable bowel syndrome. The involvement of visceral hypersensitivity has been suspected but has never been assessed. The prevalence of fructose malabsorption in patients with irritable bowel syndrome in Western Europe remains poorly documented, due to the heterogeneity of available tests. Therefore, the first objective of this present work was to assess the prevalence of fructose malabsorption in patients with irritable bowel syndrome. We assessed fructose malabsorption with a fructose breath test, after a 25 g load. We systematically ruled out small intestinal bacterial overgrowth which could promote false positive. In our irritable bowel syndrome patients, 22% had fructose malabsorption. Young, male patients were more likely to have fructose malabsorption. We also assessed the association between fructose malabsorption and other abnormalities. We did not observe any association between low-grade inflammation (with faecal calprotectin dosage) or fructose malabsorption. In contrast, an association between fructose malabsorption and visceral hypersensitivity was evidenced. Low fructose diet is known to improve symptoms in patients with irritable bowel syndrome. The presence of fructose malabsorption could be predictive of the efficacy of a low fructose diet. The second objective of this work was to determine if an abnormal fructose breath test was a predictor of symptomatic response to low fructose diet in irritable bowel syndrome. Our study has confirmed the efficacy of low fructose diet on irritable bowel syndrome. However, the results of the fructose breath test had no impact on its efficacy. One explanation for this result could be the presence of other abnormalities (including visceral hypersensitivity) that were not addressed only with a diet. The last objective of this work was to model fructose malabsorption in mice, in order to identify the underlying mechanisms. We used three models of fructose malabsorption (high fructose diet, invalidation of GLUT5 and GLUT2 coding gene). In these models, fructose malabsorption induced visceral hypersensitivity and increased intestinal permeability, the two abnormalities being reported in irritable bowel syndrome. In our models, there was no low-grade inflammation. Increased elastase activity in mice faeces was associated with visceral hypersensitivity. Protease-activated receptor-2 is known to be associated with visceral hypersensitivity and increases intestinal permeability. Further works are warranted to determine the involvement of protease-activated receptor-2 in fructose malabsorption-associated visceral hypersensitivity. The results of this work underlined the role of fructose malabsorption in irritable bowel syndrome, in the onset of visceral hypersensitivity and increased intestinal permeability. A low fructose diet is not helpful to improve symptoms of irritable bowel syndrome with fructose malabsorption.