Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death.

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB(2) knockout mice and were not prevented by CB(1/2) antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB(1/2) receptors.

The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways.

BACKGROUND: Activation of Fas (CD95) by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adaptor protein Fas-associated death domain protein (FADD). However, Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response. RESULTS: Under conditions in which proliferation of CD3-activated human T lymphocytes is increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP (FLICE-like inhibitory protein). Fas-recruited FLIP interacts with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2. CONCLUSIONS: We provide evidence that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but that it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathways.

Multi-scale systems analysis of plant development : beyond cellular signaling and tissue morphodynamics

Les plantes sont essentielles pour les sociétés humaines. Notre alimentation quotidienne, les matériaux de constructions et les sources énergétiques dérivent de la biomasse végétale. En revanche, la compréhension des multiples aspects développementaux des plantes est encore peu exploitée et représente un sujet de recherche majeur pour la science. L'émergence des technologies à haut débit pour le séquençage de génome à grande échelle ou l'imagerie de haute résolution permet à présent de produire des quantités énormes d'information. L'analyse informatique est une façon d'intégrer ces données et de réduire la complexité apparente vers une échelle d'abstraction appropriée, dont la finalité est de fournir des perspectives de recherches ciblées. Ceci représente la raison première de cette thèse. En d'autres termes, nous appliquons des méthodes descriptives et prédictives combinées à des simulations numériques afin d'apporter des solutions originales à des problèmes relatifs à la morphogénèse à l'échelle de la cellule et de l'organe. Nous nous sommes fixés parmi les objectifs principaux de cette thèse d'élucider de quelle manière l'interaction croisée des phytohormones auxine et brassinosteroïdes (BRs) détermine la croissance de la cellule dans la racine du méristème apical d'Arabidopsis thaliana, l'organisme modèle de référence pour les études moléculaires en plantes. Pour reconstruire le réseau de signalement cellulaire, nous avons extrait de la littérature les informations pertinentes concernant les relations entre les protéines impliquées dans la transduction des signaux hormonaux. Le réseau a ensuite été modélisé en utilisant un formalisme logique et qualitatif pour pallier l'absence de données quantitatives. Tout d'abord, Les résultats ont permis de confirmer que l'auxine et les BRs agissent en synergie pour contrôler la croissance de la cellule, puis, d'expliquer des observations phénotypiques paradoxales et au final, de mettre à jour une interaction clef entre deux protéines dans la maintenance du méristème de la racine. Une étude ultérieure chez la plante modèle Brachypodium dystachion (Brachypo- dium) a révélé l'ajustement du réseau d'interaction croisée entre auxine et éthylène par rapport à Arabidopsis. Chez ce dernier, interférer avec la biosynthèse de l'auxine mène à la formation d'une racine courte. Néanmoins, nous avons isolé chez Brachypodium un mutant hypomorphique dans la biosynthèse de l'auxine qui affiche une racine plus longue. Nous avons alors conduit une analyse morphométrique qui a confirmé que des cellules plus anisotropique (plus fines et longues) sont à l'origine de ce phénotype racinaire. Des analyses plus approfondies ont démontré que la différence phénotypique entre Brachypodium et Arabidopsis s'explique par une inversion de la fonction régulatrice dans la relation entre le réseau de signalisation par l'éthylène et la biosynthèse de l'auxine. L'analyse morphométrique utilisée dans l'étude précédente exploite le pipeline de traitement d'image de notre méthode d'histologie quantitative. Pendant la croissance secondaire, la symétrie bilatérale de l'hypocotyle est remplacée par une symétrie radiale et une organisation concentrique des tissus constitutifs. Ces tissus sont initialement composés d'une douzaine de cellules mais peuvent aisément atteindre des dizaines de milliers dans les derniers stades du développement. Cette échelle dépasse largement le seuil d'investigation par les moyens dits 'traditionnels' comme l'imagerie directe de tissus en profondeur. L'étude de ce système pendant cette phase de développement ne peut se faire qu'en réalisant des coupes fines de l'organe, ce qui empêche une compréhension des phénomènes cellulaires dynamiques sous-jacents. Nous y avons remédié en proposant une stratégie originale nommée, histologie quantitative. De fait, nous avons extrait l'information contenue dans des images de très haute résolution de sections transverses d'hypocotyles en utilisant un pipeline d'analyse et de segmentation d'image à grande échelle. Nous l'avons ensuite combiné avec un algorithme de reconnaissance automatique des cellules. Cet outil nous a permis de réaliser une description quantitative de la progression de la croissance secondaire révélant des schémas développementales non-apparents avec une inspection visuelle classique. La formation de pôle de phloèmes en structure répétée et espacée entre eux d'une longueur constante illustre les bénéfices de notre approche. Par ailleurs, l'exploitation approfondie de ces résultats a montré un changement de croissance anisotropique des cellules du cambium et du phloème qui semble en phase avec l'expansion du xylème. Combinant des outils génétiques et de la modélisation biomécanique, nous avons démontré que seule la croissance plus rapide des tissus internes peut produire une réorientation de l'axe de croissance anisotropique des tissus périphériques. Cette prédiction a été confirmée par le calcul du ratio des taux de croissance du xylème et du phloème au cours de développement secondaire ; des ratios élevés sont effectivement observés et concomitant à l'établissement progressif et tangentiel du cambium. Ces résultats suggèrent un mécanisme d'auto-organisation établi par un gradient de division méristématique qui génèrent une distribution de contraintes mécaniques. Ceci réoriente la croissance anisotropique des tissus périphériques pour supporter la croissance secondaire. - Plants are essential for human society, because our daily food, construction materials and sustainable energy are derived from plant biomass. Yet, despite this importance, the multiple developmental aspects of plants are still poorly understood and represent a major challenge for science. With the emergence of high throughput devices for genome sequencing and high-resolution imaging, data has never been so easy to collect, generating huge amounts of information. Computational analysis is one way to integrate those data and to decrease the apparent complexity towards an appropriate scale of abstraction with the aim to eventually provide new answers and direct further research perspectives. This is the motivation behind this thesis work, i.e. the application of descriptive and predictive analytics combined with computational modeling to answer problems that revolve around morphogenesis at the subcellular and organ scale. One of the goals of this thesis is to elucidate how the auxin-brassinosteroid phytohormone interaction determines the cell growth in the root apical meristem of Arabidopsis thaliana (Arabidopsis), the plant model of reference for molecular studies. The pertinent information about signaling protein relationships was obtained through the literature to reconstruct the entire hormonal crosstalk. Due to a lack of quantitative information, we employed a qualitative modeling formalism. This work permitted to confirm the synergistic effect of the hormonal crosstalk on cell elongation, to explain some of our paradoxical mutant phenotypes and to predict a novel interaction between the BREVIS RADIX (BRX) protein and the transcription factor MONOPTEROS (MP),which turned out to be critical for the maintenance of the root meristem. On the same subcellular scale, another study in the monocot model Brachypodium dystachion (Brachypodium) revealed an alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis. In the latter, increasing interference with auxin biosynthesis results in progressively shorter roots. By contrast, a hypomorphic Brachypodium mutant isolated in this study in an enzyme of the auxin biosynthesis pathway displayed a dramatically longer seminal root. Our morphometric analysis confirmed that more anisotropic cells (thinner and longer) are principally responsible for the mutant root phenotype. Further characterization pointed towards an inverted regulatory logic in the relation between ethylene signaling and auxin biosynthesis in Brachypodium as compared to Arabidopsis, which explains the phenotypic discrepancy. Finally, the morphometric analysis of hypocotyl secondary growth that we applied in this study was performed with the image-processing pipeline of our quantitative histology method. During its secondary growth, the hypocotyl reorganizes its primary bilateral symmetry to a radial symmetry of highly specialized tissues comprising several thousand cells, starting with a few dozens. However, such a scale only permits observations in thin cross-sections, severely hampering a comprehensive analysis of the morphodynamics involved. Our quantitative histology strategy overcomes this limitation. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with an automated cell type recognition algorithm, it allows precise quantitative characterization of vascular development and reveals developmental patterns that were not evident from visual inspection, for example the steady interspace distance of the phloem poles. Further analyses indicated a change in growth anisotropy of cambial and phloem cells, which appeared in phase with the expansion of xylem. Combining genetic tools and computational modeling, we showed that the reorientation of growth anisotropy axis of peripheral tissue layers only occurs when the growth rate of central tissue is higher than the peripheral one. This was confirmed by the calculation of the ratio of the growth rate xylem to phloem throughout secondary growth. High ratios are indeed observed and concomitant with the homogenization of cambium anisotropy. These results suggest a self-organization mechanism, promoted by a gradient of division in the cambium that generates a pattern of mechanical constraints. This, in turn, reorients the growth anisotropy of peripheral tissues to sustain the secondary growth.

Perception, signaling and molecular basis of oviposition-mediated plant responses.

Eggs deposited on plants by herbivorous insects represent a threat as they develop into feeding larvae. Plants are not a passive substrate and have evolved sophisticated mechanisms to detect eggs and induce direct and indirect defenses. Recent years have seen exciting development in molecular aspects of egg-induced responses. Some egg-associated elicitors have been identified, and signaling pathways and egg-induced expression profiles are being uncovered. Depending on the mode of oviposition, both the jasmonic acid and salicylic acid pathways seem to play a role in the induction of defense responses. An emerging concept is that eggs are recognized like microbial pathogens and innate immune responses are triggered. In addition, some eggs contain elicitors that induce highly specific defenses in plants. Examples of egg-induced suppression of defense or, on the contrary, egg-induced resistance highlight the complexity of plant-egg interactions in an on-going arms race between herbivores and their hosts. A major challenge is to identify plant receptors for egg-associated elicitors, to assess the specificity of these elicitors and to identify molecular components that underlie various responses to oviposition.

The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning.

In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1-PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.

egr-4, a target of EGFR signaling, is required for the formation of the brian primordial and head regeneration in planarians

During the regeneration of freshwater planarians, polarity and patterning programs play essential roles in determining whether a head or a tail regenerates at anterior or posterior-facing wounds. This decision is made very soon after amputation. The pivotal role of the Wnt/β-catenin and Hh signaling pathways in re-establishing anterior-posterior (AP) polarity has been well documented. However, the mechanisms that control the growth and differentiation of the blastema in accordance with its AP identity are less well understood. Previous studies have described a role of Smed-egfr-3, a planarian epidermal growth factor receptor, in blastema growth and differentiation. Here, we identify Smed-egr-4, a zinc-finger transcription factor belonging to the early growth response gene family, as a putative downstream target of Smed-egfr-3. Smed-egr-4 is mainly expressed in the central nervous system and its silencing inhibits anterior regeneration without affecting the regeneration of posterior regions. Single and combinatorial RNA interference to target different elements of the Wnt/β-catenin pathway, together with expression analysis of brain- and anterior-specific markers, revealed that Smed-egr-4: (1) is expressed in two phases - an early Smed-egfr-3-independent phase and a late Smed-egfr-3-dependent phase; (2) is necessary for the differentiation of the brain primordia in the early stages of regeneration; and (3) that it appears to antagonize the activity of the Wnt/β-catenin pathway to allow head regeneration. These results suggest that a conserved EGFR/egr pathway plays an important role in cell differentiation during planarian regeneration and indicate an association between early brain differentiation and the proper progression of head regeneration.

Hes1 is a critical but context-dependent mediator of canonical Notch signaling in lymphocyte development and transformation.

Although canonical Notch signaling regulates multiple hematopoietic lineage decisions including T cell and marginal zone B cell fate specification, the downstream molecular mediators of Notch function are largely unknown. We showed here that conditional inactivation of Hes1, a well-characterized Notch target gene, in adult murine bone marrow (BM) cells severely impaired T cell development without affecting other Notch-dependent hematopoietic lineages such as marginal zone B cells. Competitive mixed BM chimeras, intrathymic transfer experiments, and in vitro culture of BM progenitors on Delta-like-expressing stromal cells further demonstrated that Hes1 is required for T cell lineage commitment, but dispensable for Notch-dependent thymocyte maturation through and beyond the beta selection checkpoint. Furthermore, our data strongly suggest that Hes1 is essential for the development and maintenance of Notch-induced T cell acute lymphoblastic leukemia. Collectively, our studies identify Hes1 as a critical but context-dependent mediator of canonical Notch signaling in the hematopoietic system.

JNK3 maintains expression of the insulin receptor substrate 2 (IRS2) in insulin-secreting cells: functional consequences for insulin signaling.

We have recently shown that silencing of the brain/islet specific c-Jun N-terminal Kinase3 (JNK3) isoform enhances both basal and cytokine-induced beta-cell apoptosis, whereas silencing of JNK1 or JNK2 has opposite effects. While it is known that JNK1 or JNK2 may promote apoptosis by inhibiting the activity of the pro-survival Akt pathway, the effect of JNK3 on Akt has not been documented. This study aims to determine the involvement of individual JNKs and specifically JNK3 in the regulation of the Akt signaling pathway in insulin-secreting cells. JNK3 silencing strongly decreases Insulin Receptor Substrate 2 (IRS2) protein expression, and blocks Akt2 but not Akt1 activation by insulin, while the silencing of JNK1 or JNK2 activates both Akt1 and Akt2. Concomitantly, the silencing of JNK1 or JNK2, but not of JNK3, potently phosphorylates the glycogen synthase kinase3 (GSK3β). JNK3 silencing also decreases the activity of the transcription factor Forkhead BoxO3A (FoxO3A) that is known to control IRS2 expression, in addition to increasing c-Jun levels that are known to inhibit insulin gene expression. In conclusion, we propose that JNK1/2 on one hand and JNK3 on the other hand, have opposite effects on insulin-signaling in insulin-secreting cells; JNK3 protects beta-cells from apoptosis and dysfunction mainly through maintenance of a normal IRS2 to Akt2 signaling pathway. It seems that JNK3 mediates its effects mainly at the transcriptional level, while JNK1 or JNK2 appear to mediate their pro-apoptotic effect in the cytoplasm.

Lipoxin A₄ prevents the progression of de novo and established endometriosis in a mouse model by attenuating prostaglandin E₂ production and estrogen signaling.

Endometriosis, a leading cause of pelvic pain and infertility, is characterized by ectopic growth of endometrial-like tissue and affects approximately 176 million women worldwide. The pathophysiology involves inflammatory and angiogenic mediators as well as estrogen-mediated signaling and novel, improved therapeutics targeting these pathways are necessary. The aim of this study was to investigate mechanisms leading to the establishment and progression of endometriosis as well as the effect of local treatment with Lipoxin A4 (LXA₄), an anti-inflammatory and pro-resolving lipid mediator that we have recently characterized as an estrogen receptor agonist. LXA₄ treatment significantly reduced endometriotic lesion size and downregulated the pro-inflammatory cytokines IL-1β and IL-6, as well as the angiogenic factor VEGF. LXA₄ also inhibited COX-2 expression in both endometriotic lesions and peritoneal fluid cells, resulting in attenuated peritoneal fluid Prostaglandin E₂ (PGE₂) levels. Besides its anti-inflammatory effects, LXA₄ differentially regulated the expression and activity of the matrix remodeling enzyme matrix metalloproteinase (MMP)-9 as well as modulating transforming growth factor (TGF)-β isoform expression within endometriotic lesions and in peritoneal fluid cells. We also report for first time that LXA₄ attenuated aromatase expression, estrogen signaling and estrogen-regulated genes implicated in cellular proliferation in a mouse model of disease. These effects were observed both when LXA₄ was administered prior to disease induction and during established disease. Collectively, our findings highlight potential targets for the treatment of endometriosis and suggest a pleotropic effect of LXA₄ on disease progression, by attenuating pro-inflammatory and angiogenic mediators, matrix remodeling enzymes, estrogen metabolism and signaling, as well as downstream proliferative pathways.

Regulation of innate immunity by signaling pathways emerging from the endoplasmic reticulum.

The innate immune system has evolved the capacity to detect specific pathogens and to interrogate cell and tissue integrity in order to mount an appropriate immune response. Loss of homeostasis in the endoplasmic reticulum (ER) triggers the ER-stress response, a hallmark of many inflammatory and infectious diseases. The IRE1/XBP1 branch of the ER-stress signaling pathway has been recently shown to regulate and be regulated by innate immune signaling pathways in both the presence and absence of ER-stress. By contrast, innate immune pathways negatively affect the activation of two other branches of the ER-stress response as evidenced by reduced expression of the pro-apoptotic transcription factor CHOP. Here we will discuss how innate immune pathways and ER-signaling intersect to regulate the intensity and duration of innate immune responses.

TLR5 signaling stimulates the innate production of IL-17 and IL-22 by CD3(neg)CD127+ immune cells in spleen and mucosa.

In adaptive immunity, Th17 lymphocytes produce the IL-17 and IL-22 cytokines that stimulate mucosal antimicrobial defenses and tissue repair. In this study, we observed that the TLR5 agonist flagellin induced swift and transient transcription of genes encoding IL-17 and IL-22 in lymphoid, gut, and lung tissues. This innate response also temporarily enhanced the expression of genes associated with the antimicrobial Th17 signature. The source of the Th17-related cytokines was identified as novel populations of CD3(neg)CD127(+) immune cells among which CD4-expressing cells resembling lymphoid tissue inducer cells. We also demonstrated that dendritic cells are essential for expression of Th17-related cytokines and so for stimulation of innate cells. These data define that TLR-induced activation of CD3(neg)CD127(+) cells and production of Th17-related cytokines may be crucial for the early defenses against pathogen invasion of host tissues.

Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex.

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

Epigenetic alterations and constitutive Wnt signaling are early events in the progression from Barrett's esophagus to esophageal adenocarcinoma

SUMMARY Barrett's esophagus (BE) is an acquired condition in which the normal squamous epithelium in the distal esophagus is replaced by a metaplastic columnar epithelium, as a complication of chronic gastroesophageal reflux. The clinical significance of this disease is its associated predisposition to esophageal adenocarcinoma (EAC). EAC is a highly lethal disease. Better understanding of the pathogenesis of columnar metaplasia and its progression to cancer might allow the identification of biomarkers that can be used for early diagnosis, which will improve the patient survival. In this study, an improved protocol for methylation-sensitive single-strand conformation analysis, which is used to analyze promoter methylation, is proposed and a methylation-sensitive dot blot assay is described, which allows a rapid, easy, and sensitive detection of promoter methylation. Both methods were applied to study the methylation pattern of the APC promoter in histologically normal appearing gastric mucosa. The APC promoter showed monoallelic methylation, and because the methylated allele differed between the different gastric cell types, this corresponded to allelic exclusion. The APC methylation pattern was frequently altered in noimal gastric mucosa associated with neoplastic lesions, indicating that changes in the pattern of promoter methylation might precede the development of neoplasia, without accompanying histological manifestations. An epigenetic profile of 10 genes important in EAC was obtained in this study; 5 promoter genes (APC, TIMP3, TERT, CDKN2A and SFRP1) were found to be hypermethylated in the tumors. Furthermore, the promoter of APC, TIMP3 and TERT was frequently methylated in BE samples from EAC patients, but rarely in BE samples that did not progress to EAC. These three biomarkers might therefore be considered as potential predictive markers for increased EAC risk. Analysis of Wnt pathway alterations indicated that WNT2 ligand is overexpressed as early as the low-grade dysplastic stage and downregulation by promoter methylation of the SFRP1 gene occurrs already in the metaplastic lesions. Moreover, loss of APC expression is not the only factor involved in the activation of the Wnt pathway. These results indicate that a variety of biologic, mostly epigenetic events occurs very early in the carcinogenesis of BE. This new information might lead to improved early diagnosis of EAC and thus open the way to a possible application of these biomarkers in the prediction of increased EAC risk progression. RESUME L'oesophage de Barrett est une lésion métaplasique définie par le remplacement de la muqueuse malpighienne du bas oesophage par une muqueuse cylindrique glandulaire, suite à une agression chronique par du reflux gastro-esophagien. La plus importante signification clinique de cette maladie est sa prédisposition au développement d'un adénocarcinome. Le pronostic de l'adénocarcinome sur oesophage de Barrett est sombre. Seule une meilleure compréhension de la pathogenèse de l'épithélium métaplasique et de sa progression néoplasique permettrait l'identification de biomarqueurs pouvant être utilisés pour un diagnostic précoce ; la survie du patient serait ainsi augmentée. Dans cette étude, un protocole amélioré pour l'analyse de la méthylation par conformation simple brin est proposé. De plus, une technique d'analyse par dot blot permettant une détection rapide, facile et sensible de la méthylation d'un promoteur est décrite. Les deux méthodes ont été appliquées à l'étude de la méthylation du promoteur du gène APC dans des muqueuses gastriques histologiquement normales. Le promoteur APC a montré une méthylation monoallélique et, parce que les allèles méthylés différaient entre les différents types de cellules gastriques, celle-ci correspondait à une méthylation allélique exclusive. La méthylation d'APC a été trouvée fréquemment altérée dans la muqueuse gastrique normale associée à des lésions néoplasiques. Ceci indique que des changements dans la méthylation d'un promoteur peuvent précéder le développement d'une tumeur, et cela sans modification histologique. Un profil épigénétique des adénocarcinomes sur oesophage de Barrett a été obtenu dans cette étude. Cinq promoteurs (APC, TIMP3, TERT, CDKN2A et SFRP1) ont été trouvés hyperméthylés dans les tumeurs. Les promoteurs d'APC, TIMP3 et TERT étaient fréquemment méthylés dans l'épithélium métaplasique proche d'un adénocarcinome et rarement dans l'épithélium sans évolution néoplasique. Ces trois biomarkers pourraient par conséquent être considérés comme marqueur prédicatif d'un risque accru de développer une tumeur. L'analyse des altérations de la voie Wnt a montré que WNT2 est surexprimé déjà dans des dysplasies de bas-grade et que la dérégulation de SFRP1 par méthylation de son promoteur intervenait dans les lésions métaplasiques. Une perte d'expression d'APC n'est pas le seul facteur impliqué dans l'activation de cette voie. Ces résultats montrent qu'une grande diversité d'événements biologiques, principalement épigénétiques, surviennent très tôt lors de la carcinogenèse de l'oesophage de Barrett. Ces nouveaux éléments pourraient améliorer le diagnostic précoce et rendre possible l'application de ces biomarqueurs dans la prédiction d'un risque accru de développer un adénocarcinome sur un oesophage de Barrett.

Combined transcriptomic and proteomic studies reveal non-canonical signaling functions in the jasmonate pathway

Abstract Lipid derived signals mediate many stress and defense responses in multicellular eukaryotes. Among these are the jasmonates, potently active signaling compounds in plants. Jasmonic acid (JA) and 12-oxo-phytodienoic acid (OPDA) are the two best known members of the large jasmonate family. This thesis further investigates their roles as signals using genomic and proteomic approaches. The study is based on a simple genetic model involving two key genes. The first is ALLENE OXIDE SYNTHASE (AOS), encoding the most important enzyme in generating jasmonates. The second is CORONATINE INSENSITIVE 1 (COI1), a gene involved in all currently documented canonical signaling responses. We asked the simple question: do null mutations in AOS and COI1 have analogous effects on the transcriptome ? We found that they do not. If most COI1-dependent genes were also AOS-dependent, the expression of a zinc-finger protein was AOS-dependent but was unaffected by the coi1-1 mutation. We thus supposed that a jasmonate member, most probably OPDA, can alter gene expression partially independently of COI1. Conversely, the expression of at least three genes, one of these is a protein kinase, was shown to be COI1-dependent but did not require a functional AOS protein. We conclude that a non-jasmonate signal might alter gene expression through COIL Proteomic comparison of coi1-1 and aos plants confirmed these observations and highlighted probable protein degradation processes controlled by jasmonates and COI1 in the wounded leaf. This thesis revealed new functions for COI1 and for AOS-generated oxylipins in the jasmonate signaling pathway. Résumé Les signaux dérivés d'acides gras sont des médiateurs de réponses aux stress et de la défense des eucaryotes multicellulaires. Parmi eux, les jasmonates sont de puissants composés de sig¬nalisation chez les plantes. L'acide jasmonique (JA) et l'acide 12-oxo-phytodienoïc (OPDA) sont les deux membres les mieux caractérisés de la grande famille des jasmonates. Cette thèse étudie plus profondément leurs rôles de signalisation en utilisant des approches génomique et protéomique. Cette étude est basée sur un modèle génétique simple n'impliquant que deux gènes. Le premier est PALLENE OXYDE SYNTHASE (AOS) qui encode l'enzyme la plus importante pour la fabrication des jasmonates. Le deuxième est CORONATINE INSENSITIVE 1 (COI1) qui est impliqué dans la totalité des réponses aux jasmonates connues à ce jour. Nous avons posé la question suivante : est-ce que les mutations nulles dans les gènes AOS et COI1 ont des effets analogues sur le transcriptome ? Nous avons trouvé que ce n'était pas le cas. Si la majorité des gènes dépendants de COI1 sont également dépendants d'AOS, l'expression d'un gène codant pour une protéine formée de doigts de zinc n'est pas affectée par la mutation de COI1 tout en étant dépendante d'AOS. Nous avons donc supposé qu'un membre de la famille des jasmonates, probablement OPDA, pouvait modifier l'expression de certains gènes indépendamment de COI1. Inversement, nous avons montré que, tout en étant dépendante de COI1, l'expression d'au moins trois gènes, dont un codant pour une protéine kinase, n'était pas affectée par l'absence d'une protéine AOS fonctionnelle. Nous en avons conclu qu'un signal autre qu'un jasmonate devait modifier l'expression de certains gènes à travers COI1. La comparaison par protéomique de plantes aos et coi1-1 a confirmé ces observations et a mis en évidence un probable processus de dégradation de protéines contrôlé par les jasmonates et COU_ Cette thèse a mis en avant de nouvelles fonctions pour COI1 et pour des oxylipines générées par AOS dans le cadre de la signalisation par les jasmonates.

Characterization of Tollip in the Interleulkin-1 Receptor/Toll Like Receptors signaling pathways

SUMMARY IL-1R and TLRs are key players in innate immunity and inflammation. Tollip was identified as a component of IL-1RI, TLR2 and TLR4 signaling complexes that activate NF-κB and MAP kinase pathways. Tollip was previously shown as a negative regulator of NF-κB and MAP Kinase activation. We have characterized the role of Tollip in IL-R/TLRs induced signaling by the analysis of the Tollip deficient mice. We showed that NF-κB and MAPK (p38, JNK, or ERK1/2) signaling appeared normal in Tollip deficient cells following stimulation with IL-1β, lipopolysaccharide (LPS), and other TLR ligands. Also IL-1β and TLRs ligands induced activation of immune cells was indistinguishable from wild-type cells. Strikingly, in Tollip deficient mice the production of the inflammatory cytokines, IL-6 or TNF-α was significantly reduced relative to control mice after treatment with physiological doses of IL-1β or LPS, whereas no difference was observed at high doses of stimulation with LPS or in LPS induced septic shock. Therefore, Tollip could be critical for regulation of optimal responses to IL-1β and LPS, in addition to its role as negative regulator of the signaling. We also studied the role of Tollip as an endocytic adaptor for IL-1R endocytosis. We could show that Il-1R is ubiquitinated after IL-1β stimulation, and that Tollip's CUE domain binds IL-1RI in an ubiquitin-dependent manner. We followed IL-1R internalization and Tollip localization by confocal microscopy. Consistent with a role for Tollip in sorting of ubiquitinated IL-1RI, a significant amount of Tollip was also localized at the late endosomal compartment. We could show that Tollip is required for efficient lysosomal targeting of ubiquitinated IL-1R1, In the absence of Tollip or in Tollip deficient cells reconstituted with a Tollip mutant (defective in ubiquitin binding) IL-1RI accumulates in enlarged late endosomes. In addition, Tollip was shown to interact with, another endocytic adapter, Toml, and both interact with IL-1RI. In conclusion, we showed that Tollip is required for IL-1β and LPS signaling for cytokine production. In addition we showed and that Tollip has a role as an endocytic adapter, necessary for efficient trafficking and lysosomal degradation of IL-1RI. Resumé Le récepteur à l'interleukine-1 (IL-1R) et les récepteurs "Toll-like" (TLRs) sont des acteurs cruciaux de la réponse immunitaire innée et de l'inflammation. La proteine Tollip a été identifiée comme étant un élément des complexes de signalisation, induits par les récepteurs IL-1RI, TLR-2 et TLR-4, qui mènent à l'activation de la voie des MAP kinases et de NF-κB. Dans de précédentes études, il a été montré que Tollip pouvait inhiber ces deux voies de signalisation. Nous avons voulu caractériser plus précisément le rôle de Tollip dans l'activation des voies de signalisation mitées par IL-1R/TLRs en utilisant une lignée murine déficiente pour la protéine Tollip. Ainsi, en absence de Tollip, les cascades d'activation de NF-κB et MAPK (p38, JNK, or ERK1/2) ne semblent pas affectées après stimulation avec IL-1β, lipopolysaccharide (LPS) ou d' autres ligands des TLR. La réponse des cellules du système immunitaire induite par la stimulation avec IL-1β et les ligands des TLR est également comparable entre les souris sauvages et les souris deficientes pour Tollip. Par contre, dans cette lignée murine, la production de cytokines proinflammatoires IL-6 et TNFα induite par la stimulation à dose physiologique de IL-1β or LPS, est réduite. Cependant, lors de stimulation à plus hautes doses de LPS ou pendant un choc septique induit par de LPS, cette réduction n'est pas observée. Ces résultats montrent que Tollip pourrait avoir un rôle déterminant dans l'activation optimale en réponse à l' IL-1β et au LPS qui s'ajoute à sa fonction inhibitrice des mêmes voies de signalisation. Nous avons aussi étudié le rôle de Tollip comme molécule adaptatatrice du mécanisme endocytique d'internalisation de l' IL-1RI. Ainsi, l' IL-1R est ubiquitiné après stimulation par l' IL-1β , permettant à Tollip de se lier au récepteur. Cette interaction est réalisée entre le domaine CUE de Tollip et l'IL-1R via l'ubiquitine. L'internalisation et la localisation intracellulaire de l'IL-1RI et de Tollip ont été observés par microscopie confocale. En accord avec le rôle de Tollip dans le triage et la recirculation des IL-1R ubiquitiné, une quantité importante de Tollip été détectée dans l' endosome tardif. Nous avons pu démontrer que Tollip était nécessaire pour diriger efficacement ubiquitiné vers les lysosomes. Dans des cellules déficientes pour Tollip, ou reconstituées avec un mutant de Tollip (MF/AA) incapable de lier l'ubiquitine, IL-1RI s'accumule dans des vesicules anormales de l'endosome tardif. Dans ce travail, nous avons pu confirmer et préciser la fonction de la protéine Tollip dans l' activation de la production de cytokines induites par l' IL-1p and le LPS lors de l'inflammation et découvrir son rôle d'adaptateur dans l' internalisation et l'endocytose de l' IL-1RI.