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International audience; The availability of the sequences for whole genomes is changing our understanding of cell biology. Functional genomics refers to the comprehensive analysis, at the protein level (proteome) and at the mRNA level (transcriptome) of all events associated with the expression of whole sets of genes. New methods have been developed for transcriptome analysis. Serial Analysis of Gene Expression (SAGE) is based on the massive sequential analysis of short cDNA sequence tags. Each tag is derived from a defined position within a transcript. Its size (14 bp) is sufficient to identify the corresponding gene and the number of times each tag is observed provides an accurate measurement of its expression level. Since tag populations can be widely amplified without altering their relative proportions, SAGE may be performed with minute amounts of biological extract. Dealing with the mass of data generated by SAGE necessitates computer analysis. A software is required to automatically detect and count tags from sequence files. Criterias allowing to assess the quality of experimental data can be included at this stage. To identify the corresponding genes, a database is created registering all virtual tags susceptible to be observed, based on the present status of the genome knowledge. By using currently available database functions, it is easy to match experimental and virtual tags, thus generating a new database registering identified tags, together with their expression levels. As an open system, SAGE is able to reveal new, yet unknown, transcripts. Their identification will become increasingly easier with the progress of genome annotation. However, their direct characterization can be attempted, since tag information may be sufficient to design primers allowing to extend unknown sequences. A major advantage of SAGE is that, by measuring expression levels without reference to an arbitrary standard, data are definitively acquired and cumulative. All publicly available data can thus be stored in a unique database, facilitating whole-genome analysis of differential expression between cell types, normal and diseased samples, or samples with and without drug treatment. SAGE data are readily amenable to statistical comparisons, allowing to determine the level of confidence of the observed variations. A major limitation of SAGE is that, because each analysis is obligatory performed on the whole set of expressed genes, it can hardly be performed on multiple samples, for example in kinetics studies or to compare the effects of large numbers of drugs. To overcome this limitation, high-throughput detection of a subset of mRNAs is more rapidly performed by parallel hybridization of mRNAs on arrays of nucleic acids immobilized on solid supports. From this point of view, a SAGE platform is a powerful instrument for selecting the most informative subset of genes, assembling them to design microarrays dedicated to a specific problem and calibrating measurement by comparison with a standard cell model for which SAGE data are available. This approach is an attractive alternative to strategies based exclusively on pangenomic arrays. A very large amount of SAGE data are already available and the problem is now to extract their biological meaning. Knowledge on metabolic pathways is already organized so that its successful integration in a SAGE platform can be undertaken. For other cell components and pathways, the problem lies on the lack of controlled vocabulary to describe gene activities, starting form a clear definition of the concept of biological function itself. Progress in gene and cell ontology is expected to facilitate computer-based extraction of biological knowledge from existing and forthcoming SAGE data.

Insects represent 85% of the animals. They have adapted to many environments and play a major role in ecosystems. Many insect species exhibit phenotypic plasticity. We here report on the mechanisms involved in phenotypic plasticity of different insects (aphids, migratory locust, map butterfly, honeybee) and also on the nutritional size plasticity in Drosophila and the plasticity of the wing eye-spots of the butterfly Bicyclus anynana. We also describe in more detail our work concerning the thermal plasticity of pigmentation in Drosophila. We have shown that the expression of the tan, yellow and Ddc genes, encoding enzymes of the melanin synthesis pathway, is modulated by temperature and that it is a consequence, at least in part, of the temperature-sensitive expression of the bab locus genes that repress them.La plasticité phénotypique chez les insectes.Les insectes représentent 85 % des animaux. Ils se sont adaptés à de nombreux environnements et jouent un rôle majeur dans les écosystèmes. De nombreuses espèces d’insectes montrent de la plasticité phénotypique. Nous présentons ici les mécanismes impliqués dans la plasticité phénotypique chez différents insectes (les pucerons, le criquet migrateur, le papillon carte géographique, l’abeille ainsi que la plasticité nutritionnelle de la taille chez la drosophile et la plasticité des ocelles sur les ailes du papillon Bicyclus anynana). Nous décrivons également plus en détail nos travaux sur la plasticité thermique de la pigmentation chez la drosophile. Le froid induit une pigmentation abdominale plus foncée chez les femelles drosophiles. Nous avons montré que l’expression des gènes tan,yellow et Ddc, codant des enzymes de la voie de synthèse des mélanines, est modulée par la température et que c’est une conséquence, au moins en partie, de l’expression sensible à la température des gènes du locus bab qui les répriment.

RNA has not said its last word with the rise of a new RNA family, circular RNAs (circRNAs). Discovered 25 years ago, circRNAs were initially considered as splicing byproducts. Today it appears that 14% of human genes produce circRNAs, whereas more than 100 000 different circRNAs are expressed. They are produced from coding genes through an alternative splicing mechanism called backsplicing, where an acceptor site is linked with a donor site located downstream. Nuclear circRNAs regulate transcription and splicing of their linear isoform. Cytoplasmic circRNAs, which are predominant, either sequester miRNAs or RNA binding proteins, or are translated via internal initiation mechanisms. CircRNAs may constitute a powerful biotechnogical tool for protein synthesis, as their translation is stable over time. In addition, exogenous circRNAs generate less immune response than their linear counterparts. We will also discuss in this review their biotechnological potential and their roles in pathological processes.L’ARN circulaire nous joue-t-il des tours ?L’ARN n’a pas dit son dernier mot… avec l’émergence des ARN circulaires (circARN). Quatorze pour cent des gènes humains produisent en effet des circARN par un mécanisme d’épissage alternatif : le rétro-épissage. Chez l’homme, plus de 100 000 circARN différents ont ainsi été répertoriés. Dans le noyau, ils régulent la transcription ou l’épissage des ARNm, alors que, dans le cytoplasme, ils séquestrent des miARN et des protéines, ou sont traduits par un mécanisme d’initiation interne de la traduction. Ces circARN constituent en fait un outil biotechnologique performant car leur traduction est très stable dans le temps, et les circARN exogènes induisent moins de réponses immunitaires que les ARNm linéaires. Dans cette revue, nous discuterons, après les avoir décrits, du rôle des circARN dans différents processus pathologiques et de leur utilisation en biotechnologie.

Whole genome sequencing is increasingly used in patients with genetic diseases to diagnose the mutations responsible for the phenotype. However, for a large proportion of sequenced genomes, none of the genes associated with the phenotype have a coding mutation. In these cases, it is possible that a non-coding mutation, located in a cis-regulatory region, modifies the expression of a gene involved in the disease. Despite the existence of methods for annotating and predicting regulatory sequences on the basis of biochemical and epigenetic properties, it remains difficult to define objective criteria for effectively selecting candidate mutations from the millions of non-coding variants present in each patient. In addition, the target genes of these regulatory sequences are generally not known, making it difficult to associate a non-coding mutation with the patient's phenotype.I propose here a supervised learning strategy using random forests, adapted to complex and heterogeneous data sets, to classify and select non-coding mutations deregulating genes responsible for diseases. A notable innovation of my approach is to take into account data of associations between non-coding regions and target genes. In addition, I propose a method for extracting the biological rules identified by the model for each mutation evaluated, allowing an informed selection of candidate mutations.I discuss the functional properties identified by the learning model, based on examples of non-coding variations associated with Mendelian diseases. I also illustrate the potential of this method by analyzing 255,106 de novo variants identified by complete sequencing in 1,902 children with autism spectrum disorders, in whom no pathogenic coding mutations have been identified. This method thus makes it possible to prioritize mutations, the most promising of which become experimentally testable hypotheses to confirm their involvement in the development of the diseases in question. Thus, for whole genome sequencing projects of patient cohorts, a systematic application of our method would contribute to a better understanding of the mechanisms regulating gene expression, and to an improvement in patient diagnosis.; Le séquençage de génome complet est utilisé de façon croissante chez les patients atteints de maladies génétiques pour diagnostiquer les mutations responsables. Cependant, pour une grande proportion de génomes de patients séquencés, aucun gène associé au phénotype ne présente de mutation codante. Dans ces cas, il est possible qu’une mutation non-codante, localisée dans une région cis-régulatrice, modifie l'expression d'un gène impliqué dans la maladie. Malgré l’existence de méthodes pour annoter et prédire des séquences régulatrices sur la base de propriétés biochimiques et épigénétiques, il reste difficile de définir des critères objectifs pour sélectionner efficacement des mutations candidates parmi les millions de variants non-codants présents chez chaque patient. De plus, les gènes cibles de ces séquences de régulation ne sont généralement pas connus, si bien qu’il est difficile de croiser une mutation non-codante avec le phénotype du patient. Je propose ici une stratégie d’apprentissage supervisé par forêts aléatoires, adaptée aux jeux de données complexes et hétérogènes, pour classer et sélectionner des mutations non-codantes dérégulant des gènes responsables de maladies. Une innovation notable de mon approche est de prendre en compte des données d’associations entre régions non-codantes et gènes cibles. Par ailleurs, je propose une méthode d’extraction des règles biologiques identifiées par le modèle pour chaque mutation évaluée, ce qui permet une sélection éclairée des mutations hiérarchisées. Je discute les propriétés fonctionnelles identifiées par le modèle d'apprentissage, à partir d'exemples de variations non-codantes associées à des maladies mendéliennes. J’illustre également le potentiel de cette méthode notamment par une analyse de 255 106 variants de novo identifiés par séquençage complet chez 1902 enfants souffrant de troubles du spectre autistique, et chez lesquels aucune mutation codante pathogénique n’a été identifiée. Cette méthode permet ainsi de hiérarchiser des mutations, dont les plus prometteuses deviennent des hypothèses testables expérimentalement pour confirmer leur implication dans le développement des maladies considérées. Ainsi pour les projets de séquençage génome-complets de cohortes de patients, une application systématique de notre méthode contribuerait à une meilleure compréhension des mécanismes de régulation de l’expression des gènes, et à une amélioration du diagnostic des patients.

Gene expression in higher eukaryotes is regulated through the involvement of transcription start site (TSS)-proximal (promoters) and -distal (enhancers) regulatory elements. Enhancer elements play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. Here, we discuss recent advances in high-throughput approaches to identify and characterize enhancer elements, from the well-established massively parallel reporter assays to the recent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based technologies. We discuss how these approaches contribute toward a better understanding of enhancer function in normal and pathological conditions.

Polycomb Group proteins (PcG) are repressive epigenetic factors essential for development and involved in numerous cancer processes, yet their modes of action and recruitment to specific genomic loci are not fully understood. Recently, it has been shown that the PcG protein recruitment is a dynamic process, contrary to what was foreseen in the initial hierarchical model. In addition, EZH2, a key PcG protein, can be associated to transcribed genes, challenging the former function of PcG proteins as transcriptional repressors. Furthermore, the dual role of EZH2, which can act as an oncogene or a tumor suppressor depending on the cellular type, illustrates the functional complexity of PcG proteins.

International audience; Human endogenous retroviruses (HERV) represent a large part of our genome and the few elements that have retained a potential of expression still remain "dormant" in physiological conditions. In some instances, they can be awakened by environmental factors activating their expression. The best studied conditions of HERV activation are infections caused by microorganisms such as viruses of the Herpesvirus family. This activation can thus lead to the expression of pathogenic proteins such as envelope proteins belonging to the HERV-W and HERV-K families, respectively involved in Multiple Sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Endogenous retroviral proteins can also acquire a physiological function beneficial for humans. This is the case of Syncytin-1 from the HERV-W family, that is involved in placenta formation.

Allergic asthma and atopic dermatitis are diseases mainly resulting from the activation of Th2 cells, that produce cytokines favouring IgE production and eosinophilia but also of Th1 cells, that contribute to inflammation chronicity. Lymphocyte recruitment and retention of Th cells in target organs are 2 key events for asthma and atopic dermatitis pathogenesis. While lymphocyte migration is regulated by chemokines and lipid mediators such as leukotrienes and prostaglandins, factors involved in lymphocyte retention and survival within inflammatory tissues remain poorly understood. Recent works show that, in allergic diseases, there is an increased expression of fractalkine/CX3CL1 and its unique receptor CX3CR1 and that this chemokine does not act as chemoattractant. In allergic asthma, CX3CR1 expression regulates Th2 and Th1 cell survival in the inflammatory lung, while, in atopic dermatitis, it regulate Th2 and Th1 cell retention into the inflammatory site. Use of peptides blocking fractalkine binding to its receptor is currently tested in the treatment of asthma and atopic dermatitis.

Bipolar disorder is a severe and complex multifactorial disease, characterized by alternance of acute episodes of depression and mania/hypomania, interspaced by euthymic periods. The etiological determinants of bipolar disorder yet, are still poorly understood. For the last 30 years, chronobiology is an important field of investigation to better understand the pathophysiology of bipolar disorder.We conducted a review using Medline, ISI Database, EMBase, PsyInfo up to January 2015, using the following keywords combinations: "mood disorder", "bipolar disorder", "depression", "unipolar disorder", "major depressive disorder", "affective disorder", for psychiatric conditions; and "circadian rhythms", "circadian markers", "circadian gene", "clock gene", "melatonin" for circadian rhythms. The search critera was presence of word in any field of the article.Quantitative and qualitative circadian abnormalities are associated with bipolar disorders both during acute episodes and euthymic periods, suggesting that these altered circadian rhythms may represent biological trait markers of the disorder. These circadian dysfunctions were assessed by various validated tools including polysomnography, actigraphy, sleep diaries, chronotype assessments and blood melatonin/cortisol measures. Other altered endogenous circadian activities have also been reported in bipolar patients, such as hormones secretion, core body temperature or fibroblasts activity. Moreover, these markers were also altered in healthy relatives of bipolar patients, suggesting a degree of heritability. Several genetic association studies have also showed associations between multiple circadian genes and bipolar disorder, such as CLOCK, ARTNL1, GSK3β, PER3, NPAS2, NR1D1, TIMELESS, RORA, RORB, and CSNK1ε. Thus, these circadian gene variants may contribute to the genetic susceptibility of the disease. Furthermore, the study of the clock system may help to better understand some phenotypic aspects like the mechanisms of pharmacological treatments used in bipolar disorder, in particular lithium carbonate.Several clinical, physiological and genetic data suggest that circadian rhythms dysregulations are involved in the pathophysiology of bipolar disorder. The circadian model has led to the development of new therapeutic strategies such as chronotherapeutics or Inter Personal Social and Rhythms Therapies. Further studies are needed in this promising research field to keep exploring the relationship between these circadian markers, genes and the clinical aspects of the disease.

International audience; INTRODUCTION: Diffuse malignant mesothelioma (MMD) is a rare disease. The diagnosis is difficult and needs an antibody panel. The tumor suppressor gene BRCA1 associated protein 1 (BAP1) is involved in several cancers, including MMD. Loss of BAP1 expression is correlated with BAP1 somatic or constitutional genetic defects. Our work assesses the value of integrating BAP1 in the panel of antibodies used for the diagnosis of MMD. MATERIALS AND METHODS: Immunohistochemical techniques were performed on cytological and histological specimens of MMD and adenocarcinoma pleural metastasis. RESULTS: Of the 26 patients with MMD and the 24 patients with adenocarcinoma pleural metastasis, loss of BAP1 expression was observed in 11 (48%) and one adenocarcinoma (6%) on cytological specimens and in 12 MMD (48%) and in one adenocarcinoma (5%) on biopsy specimens. The concordance between immunocytochemistry and immunohistochemistry was 100%. The specificity of BAP1 was 100% on cytological and biopsy specimen for the diagnosis of malignancy in case of mesothelial proliferation. DISCUSSION AND CONCLUSION: Loss of BAP1 expression is an indicator of MMD in a context of mesothelial proliferation. This immunohistochemistry could be integrated in the panel of immunostaining used for MMD diagnosis, either on histological or cytological samples. Furthermore, loss of BAP1 expression guides the patient to an oncology genetic counseling in order to eliminate a MMD developed as part of a constitutional genetic defect

A new technology, called CRISPR, derived from the immune system of bacteria, uses a Cas9 nuclease and a guided RNA complementary to a 20 nucleotides sequence of a gene to induce double strand DNA breaks. This permits to modify specifically the targeted gene in plant, animal and human cells. Variants of the technique also permit to reduce or increase the expression of a selected gene. This technology may thus be used not only to understand the role of a gene but also to develop therapies for hereditary and acquired diseases.

Galectins constitute a family of soluble animal lectins defined by their evolutionary conserved carbohydrate recognition domain and their affinity for β-galactosides containing glycoconjugates. Each galectin is characterized by a specific spatio-temporal distribution and a unique set of ligands and molecular partners. Interestingly, galectins are found both extracellularly and intracellularly and modulate various cellular processes. Knock-out mutant mice for galectins-1, 3 or 7 are viable but display a wide range of defects under various stress conditions. Indeed, galectins are multifunctional proteins involved in cell-cell and cell-extracellular matrix interactions, organization of membrane domains, cell signalling and also in intracellular trafficking, apoptosis, regulation of cell cycle. Galectins represent potential therapeutic targets, especially in the context of cancer and inflammatory diseases.

Recently circadian clock genes have been identified in humans but information regarding their expression has remained very limited. The evaluation of circadian variations in the expression of clock genes in humans seems to be a major importance both from a fundamental point of view as a diagnostic and therapeutic perspective. In this context, several works including ours have described the fluctuation of clock genes.describing rhythmic expression of clock genes in intensive care units patients during 24h and we tried to determine the effect of the absence of synchronizers such as light/ dark cycle on these rhythms.15 patients received care in private room in intensive care units in the hospital Sahloul (5 comatous and 10 non comatous patients). For RNA isolation we used peripheral blood mononuclear cells which represent an ideal material to investigate non-invasively the human clock at the molecular level.In the present study, we noticed that clock genes mRNA exhibit a circadian expression in comatose patients, while the rhythmicity of some studied genes disappeared in non-comatose patients.The disturbance of the rhythmic fluctuation of the clock genes could be the result of the effect of surgery on some biological rhythms as it could be explained by the lack of synchronizers in intensive care units such as light/dark cycle.

Huntington's disease is a rare neurodegenerative disease caused by a pathologic CAG expansion in the exon 1 of the huntingtin (HTT) gene. Aggregation and abnormal function of the mutant HTT (mHTT) cause motor, cognitive and psychiatric symptoms in patients, which lead to death in 15-20 years. Currently, there is no treatment for HD. Experimental approaches based on drug, cell or gene therapy are developed and reach progressively to the clinic. Among them, mHTT silencing using small non-coding nucleic acids display important physiopathological benefit in HD experimental models.

Enfermer le porteur de l’information génétique dans le noyau a obligée la cellule a créé un système de transport complexe, qui permet l’export d’un ARNm du noyau au cytoplasme. Le mécanisme général de l’export des ARNm est encore mal connu, même si les facteurs principaux ont été découverts il y a longtemps. De récents progrès en microscopie nous ont permis d’étudier directement le comportement des ARNm durant le processus d’export. Durant ma maitrise, nous avons été capables de localiser et suivre des ARNm en temps réel pour la première fois chez Saccharomyces cerevisiae. Nous avons créé un gène rapporteur en mettant le gène GLT1 sous le contrôle du promoteur GAL1. Nous avons aussi marqué l’ARNm de GLT1 avec plusieurs boucles PP7. L’ARNm sera visible après l’attachement de plusieurs protéines PP7-GFP aux boucles. En utilisant la technique d’imagerie en cellules vivantes, nous sommes capable de visualiser et suivre chaque ARNm, depuis son relâchement du site de transcription jusqu’à l’export. Une fois relâché du site de transcription, l’ARNm diffuse librement dans le nucléoplasme, mais une fois à la périphérie nucléaire, il commence à « scanner » l’enveloppe nucléaire avant d’être exporté. Nous avons trouvé que le « scanning » dépend de la présence des Myosin Like Proteins (Mlp1p et Mlp2p), protéines qui forment le panier nucléaire, car suite à la délétion de MLP1 et MLP2, les ARNm n’étaient plus capable de « scanner ». Nous avons également trouvé que la partie C-terminale de Mlp1p était nécessaire au « scanning ». De plus, suite à la délétion du gène TOM1, gène codant pour une ubiquitine ligase, les ARNm ont un comportement similaire aux ARNm d’une souche ∆mlp1/mlp2, suggérant que le « scanning » permet à Tom1p d’ubiquitiner Yra1p, ce qui causera son relâchement de l’ARNm. Également, nous avons montré que les ARNm endogènes MDN1 et CBL2 scannent aussi la périphérie nucléaire. Ensemble, nos résultats suggèrent que le scanning est un processus par lequel passent tout les ARNm nucléaire lorsqu’ils se retrouvent à la périphérie du noyau, pour initier plusieurs étapes de réarrangements nécessaires à leurs export. De plus, nous avons examiné le rôle de Yhr127p, une protéine nouvellement identifiée qui se lie à l’ARN. Après avoir marqué cette protéine avec la GFP, nous avons montré qu’elle forme des foci dans le noyau et que ces derniers vont disparaitre suite à l’arrêt de la transcription. La délétion de YHR127 à conduit à une augmentation de la transcription de quelques gènes spécifiques, mais n’affecte pas la capacité de la cellule à exporter les ARNm. Nos résultats suggèrent que cette protéine joue un rôle dans la régulation de la transcription et/ou dans la stabilité de l’ARNm., In eukaryotic cells, the processes of RNA and protein synthesis are spatially separated into two distinct compartments. With this division, a complex pathway of nucleocytoplasmic RNA export has evolved, which to date remains poorly understood. Recent advances in single-molecule microscopy have enabled direct studies focused on investigating the dynamics and kinetics of RNA export. In this Master thesis, we present the first real time visualization of mRNA export in the yeast Saccharomyces cerevisiae. We first generated a GLT1 reporter under the control of the inducible GAL1 promoter, in which the GLT1 mRNA was tagged with an array of PP7 repeats and detected by exogenous PP7-GFP binding protein. Using a single-molecule live cell imaging approach, we were able to visualize and track the behavior of individual mRNAs from the site of transcription to the point of export. Interestingly, we found that once released from the transcription site, single mRNAs diffuse freely in the nucleoplasm, but once they reach the nuclear periphery, they scan the periphery before being exported to the cytoplasm. This scanning behavior was dependent on Myosin Like Proteins (Mlp1p and Mlp2p), which form the basket of the Nuclear Pore Complex (NPC), as mRNAs were not retained at the periphery and were rapidly released into the nucleoplasm in mlp1p/mlp2p double mutant cells. Specifically, we found that the C-terminal part of Mlp1p was important for scanning. Furthermore, mRNAs from cells depleted of the E3 ubiquitin ligase TOM1 had a similar phenotype to mRNAs in mlp1p/mlp2p double mutant cells, suggesting a role for scanning in the Tom1p-mediated release of Yra1p from the RNA. Lastly, we confirmed that endogenous MDN1 and CBL2 mRNAs also exhibit scanning behaviour. Taken together, our results suggest that mRNAs scanning the nuclear periphery is a general behaviour for all mRNAs to initiate the mRNA export process, allowing mRNP arrangement required for export to occur at the nuclear periphery. In addition, we investigated the role of YHR127, a newly identified RNA binding protein, in RNA biogenesis. Notably, we show that GFP-tagged YHR127p formed distinct foci in the nucleus, which were lost upon transcription arrest. Deletion of YHR127 led to an increase in transcript levels of specific genes, but not to a global accumulation of mRNAs in the nucleus, suggesting a role for this protein in regulating transcription and/or mRNA stability.

The GILZ (glucocorticoid-induced leucine zipper) protein has first been identified as a glucocorticoid-responsive gene and is now presented as a major regulator of inflammation. Expanding literature documents a role for GILZ as a mediator of the immuno-modulatory and anti-inflammatory effects of glucocorticoids, mainly through interference with key signal transduction pathways such as nuclear factor-kappa B (NF-kB) or activated protein-1 (AP-1). The TSC-22 (TGF-β-stimulated clone-22) protein is described as an apoptosis modulator and as a new tumor suppressor gene. GILZ and TSC-22, characterized by the presence of a leucine zipper domain and a TSC-box, belong to the TSC-22D (TSC-22 domain) family of proteins which comprises today 18 members. Functions of these proteins suggest that this family plays a major role in cell homeostasis and in the regulation of the immune system.

Renal fibrosis represents the final stage of most chronic kidney diseases and contributes to the progressive and irreversible decline in kidney function with accumulation of extracellular matrix components in the renal parenchyma. The molecular mechanisms governing the renal fibrosis process are complex and remain poorly understood. Recently, the profibrotic role of several microRNAs (miRNAs) has been described in kidney fibrosis. MiRNAs are a new class of, small non-coding RNAs of about 20 nucleotides that act as gene expression negative regulators at the post-transcriptional level. Seminal studies have highlighted the potential importance of miRNA as new therapeutic targets and innovative diagnostic and/or prognostic biomarkers. This review summarizes recent scientific advances on the role played by miRNAs in kidney fibrogenesis and discusses potential clinical applications as well as future research directions.

International audience; Type I interferons play a central role in the establishment of an innate immune response against viral infections and tumor cells. Shortly after their discovery in 1957, several groups have looked for small molecules capable of inducing the expression of these cytokines with therapeutic applications in mind. A set of active compounds in mice were identified, but because of their relative inefficiency in humans for reasons not understood at the time, these studies fell into oblivion. In recent years, the characterization of pathogen recognition receptors and the signaling pathways they activate, together with the discovery of plasmacytoid dendritic cells, have revolutionized our understanding of innate immunity. These discoveries and the popularization of high-throughput screening technologies have renewed the interest for small molecules that can induce type I interferons. Proofs about their therapeutic potency in humans are expected very soon.; Les interférons de type I jouent un rôle central dans la mise en place d’une réponse immunitaire innée contre les infections virales et les cellules tumorales. Peu après leur découverte en 1957, plusieurs équipes ont recherché des petites molécules capables d’induire l’expression de ces cytokines à des fins thérapeutiques. Un ensemble de composés actifs chez la souris ont ainsi été identifiés, mais en raison de leur relative inefficacité chez l’homme pour des raisons incomprises à l’époque, ces travaux ont été abandonnés et sont tombés dans l’oubli. Ces dernières années, la caractérisation des récepteurs impliqués dans la reconnaissance des pathogènes, des voies de signalisation qu’ils activent, ainsi que la découverte des cellules dendritiques plasmacytoïdes ont révolutionné notre compréhension de l’immunité innée. Ces découvertes et les nouvelles technologies de criblages à haut débit ont ravivé l’intérêt pour les petites molécules capables d’induire les interférons de type I. Les preuves de leur efficacité thérapeutique chez l’homme sont attendues très prochainement.

Normal glucose homeostasis is characterized by appropriate insulin secretion and low HbA1c. Gene expression signatures associated with these two phenotypes could be essential for islet function and pathophysiology of type 2 diabetes (T2D). Herein, we employed a novel approach to identify candidate genes involved in T2D by correlating islet microarray gene expression data (78 donors) with insulin secretion and HbA1c level. The expression of 649 genes (P < 0.05) was correlated with insulin secretion and HbA1c. Of them, five genes (GLR1A, PPP1R1A, PLCDXD3, FAM105A and ENO2) correlated positively with insulin secretion/negatively with HbA1c and one gene (GNG5) correlated negatively with insulin secretion/positively with HbA1c were followed up. The five positively correlated genes have lower expression levels in diabetic islets, whereas GNG5 expression is higher. Exposure of human islets to high glucose for 24 h resulted in up-regulation of GNG5 and PPP1R1A expression, whereas the expression of ENO2 and GLRA1 was down-regulated. No effect was seen on the expression of FAM105A and PLCXD3. siRNA silencing in INS-1 832/13 cells showed reduction in insulin secretion for PPP1R1A, PLXCD3, ENO2, FAM105A and GNG5 but not GLRA1. Although no SNP in these gene loci passed the genome-wide significance for association with T2D in DIAGRAM+ database, four SNPs influenced gene expression in cis in human islets. In conclusion, we identified and confirmed PPP1R1A, FAM105A, ENO2, PLCDX3 and GNG5 as potential regulators of islet function. We provide a list of candidate genes as a resource for exploring their role in the pathogenesis of T2D.

Mitochondrial diseases, defined as the diseases due to oxidative phosphorylation defects, are the most frequent inborn errors of metabolism. Their clinical presentation is highly diverse. Their diagnosis is difficult. It relies on metabolic parameters, histological anomalies and enzymatic assays showing defective activity, all of which are both inconstant and relatively unspecific. Most mitochondrial diseases have a genetic origin. Candidate genes are very numerous, located either in the mitochondrial genome or the nuclear DNA. Pathophysiological mechanisms of mitochondrial diseases are still the matter of much debate. Those underlying the tissue-specificity of diseases due to the alterations of a ubiquitously expressed gene are discussed including (i) quantitative aspect of the expression of the causal gene or its partners when appropriate, (ii) quantitative aspects of the bioenergetic function in each tissue, and (iii) tissue distribution of heteroplasmic mitochondrial DNA alterations.

Cilia are found in many eukaryotic species and share a common microtubule architecture that can nonetheless show very diverse features within one animal. The genesis of cilia and their diversity require the expression of different specific genes. At least two classes of transcription factors are involved in ciliogenesis: the RFX family, essential for the assembly of most cilia and the FOXJ1 transcription factors that are key regulators of motile cilia assembly. These two different families of transcription factors have both specific and common target genes and they can also cooperate for the formation of cilia. In collaboration with cell type specific factors, they also contribute to the specialisation of cilia. As a consequence, the identification of RFX and FOXJ1 target genes has emerged as an efficient strategy to identify novel ciliary genes, and in particular genes potentially implicated in ciliopathies.

PML/TRIM19 is the organizer of PML nuclear bodies (NB), large multiprotein structures associated to the nuclear matrix, which recruit a great number of proteins and which are implicated in various cellular processes including antiviral defense. The conjugation of PML to SUMO is required for the formation and function of PML NB. Alternative splicing from a single PML gene generates several PML isoforms (PMLI to PMLVIIb), each harboring a specific carboxy-terminal region. This variability allows each isoform to recruit different partners and thus confers them specific functions. PML gene is directly induced by interferon and certain PML isoforms are implicated in its antiviral properties, as they display intrinsic antiviral activities against RNA or DNA viruses. One isoform, PMLIV, is also implicated in innate immunity by enhancing IFN-β production during a viral infection. Here we review recent findings on PML/TRIM19 implication in interferon response and antiviral defense, at the interface between intrinsic and innate immunity.

Transposable elements (TE) represent around 40% of the human genome. They are endogenous mobile DNA sequences able to jump and duplicate in the host genome. TE have long been considered as "junk" DNA but are now believed to be important regulators of gene expression by participating to the establishment of the DNA methylation profile. Recent advances in genome sequencing reveals a higher transposition frequency and TE driven gene expression in somatic cells than previously thought. As TE propagation is deleterious and may be involved in oncogenic mechanisms, host cells have developed silencing mechanisms mainly described in germinal and embryonic cells. However, somatic cells are also proned to TE transposition and use specific mechanisms involving tumor suppressor proteins including p53, Rb and PLZF. These transcription factors specifically target genomic retrotransposon sequences, histone deacetylase and DNA methylase activities, inducing epigenetic modifications related to gene silencing. Thus, these transcription factors negatively regulate TE expression by the formation of DNA methylation profil in somatic cells possibly associated with oncogenic mechanisms.

In 2014, a phase II randomised, double blind clinical trial assessing the efficacy of cholecalciferol (vitamin D3) in patients with a cervical trauma will be set up. This trial stems from previous studies showing that vitamin D supplementation improves functional recovery in rat models of peripheral or central nerve injury. In a first series of experiments, we used a rat model of peripheral nerve trauma to demonstrate the therapeutic efficiency of vitamin D. We first demonstrated that ergocalciferol (vitamin D2) increases the number and the diameter of newly formed axons and improves the response of metabosensitive fibers from tibialis muscle, in a model of transected peroneal nerve. Then, we compared vitamin D2 and vitamin D3 and observed that the latter is more efficient. At the dose of 500 IU/kg/day, vitamin D3 induces a dramatic functional recovery. We also demonstrated that vitamin D3 increases the number of preserved or newly formed axons in the proximal end, the mean axon diameter in the distal end, neurite myelination in both the distal and proximal ends as well as the expression of genes involved in axogenesis and myelination. In parallel, we assessed the therapeutic role of vitamin D on the central nervous system. In a first study, using a rat model of spinal cord compression at the T10 thoracic level, we delivered vitamin D3 (cholecalciferol) orally at the dose of 50 IU/kg/day or 200 IU/kg/day. When compared to control animals, vitamin D-treated rats displayed, three months after injury, a significant improvement of ventilatory frequency and a reduction of H reflex indicating functional improvements at three months post-injury. In a second study, we used a rat model of cervical hemisection (C2) with a higher dose of oral vitamin D3 (500 IU/kg/day) delivered weekly, during 12 weeks. We observed an improved locomotor recovery, a reduced spasticity and a significantly higher rate of axons crossing the lesion site in treated animals. However, it must be pointed out that the functional improvement is reduced when vitamin D is provided one week after the trauma.

This study investigated the effects of a 60-day bed rest with or without countermeasures on muscular phenotype and post-translational modifications of the regulatory Myosin Light Chain 2 (MLC2) protein. Soleus biopsies were obtained from female subjects before and after bed rest. Control subjects were assigned only to bed rest (BR), BR+Ex subjects were submitted to combined aerobic and resistive exercises, and BR+Nut to nutritional leucine and valine diet. We determined Myosin Heavy Chains (MHC) and MLC2 composition of muscles using 1D SDS-PAGE. MLC2 phosphorylation was measured on 2D gels and O-N-Acetyl Glucosaminylation (O-GlcNAc) level of MLC2 was determined. Our results showed a slow-to-fast shift of MHC and MLC2 isoforms in BR and BR+Nut while BR+Ex combinations prevented these phenotype changes. After BR, the MLC2 phosphorylation state was increased while the global MLC2 glycosylation level was decreased. Exercises prevented the variations of phosphorylation and glycosylation observed after BR whereas nutrition had no effects. These results suggested an interplay between phosphorylation and glycosylation of MLC2, which might be involved in the development of muscle atrophy and associated changes. These findings of differential responses to exercises and nutrition protocols were discussed with implications for future prescription models to preserve muscle against long-term unloading. 540;1-2

Soon after their discovery microRNA (miRNA) emerged as central natural regulators of gene expression. Although the complex mechanisms of action and impact of miRNA on development, physiology and disease are still elusive, significant progress has been made in deciphering the roles of some miRNA in insulin secretion and action. Here we examine the close relationship existing between miRNA and glucose metabolism as well as their putative role in the pathogenesis of diabetes and their possible utility as biomarkers of this disease.

The lysosomal storage disorders (LSD) comprise a heterogeneous group of inborn errors of metabolism. The resulting enzymatic defect leads to accumulation of its substrate in the lysosome. Their clinical patterns reflect the site of substrate storage. Central nervous system involvement is often present in the younger patients affected by the most severe phenotypes. Substantial progress has been made in the pathophysiological knowledge, leading to new therapeutic options in LSD. Enzyme replacement therapy (ERT) is the dominant approach and is actually proposed in six LSD: Gaucher disease, Fabry disease, Pompe disease and mucopolysaccharidoisis (MPS) I (Hurler disease), II (Hunter disease) and VI (Maroteaux-Lamy disease). This treatment reduces lysosomal storage, and sometimes reduces, but most often limits the progression of visceral involvement and of its clinical consequences. However, ERT does not cross the blood-brain barrier and is ineffective on neurological symptoms. In the younger patients with MPS I (Hurler disease) and with selected cases of other LSD, haematopoietic stem cell transplantation remains the optimal option. Other strategies using small molecules are being explored in order to cross the blood-brain barrier. This includes substrate reduction or depletion therapies, which decrease the amount of substrate, and the use of pharmacological chaperones, which enhance the residual activity of the mutant enzyme. Miglustat is the proposed substrate reduction therapy in Niemann-Pick C disease and clinical trials are actually performed in several LSD using other substrate reduction or chaperone drugs.

Glucocorticoids exert their actions at nuclear levels through genomic mechanisms including both transcriptional activation (transactivation) and gene expression repression (transrepression). Transactivation mechanisms are mediated by transcription factors, the main one being the activated glucocorticoid receptor (GR). These mechanisms contribute to both powerful therapeutic effects of glucocorticoids on inflammatory and immune diseases, and adverse effects than can be harmful on vital functions. Non-genomic mechanisms, which act faster than genomic ones, have also been explored. They also involve the GR in different membranous and cytosolic sites. The phenomenon of glucocorticoid resistance is also complex and several different mechanisms may mediate this phenomenon. Among them are alterations in number, binding affinity or phosphorylation status of the GR, changes in capacity of cellular apoptosis, polymorphic changes or expression of proteins involved in the genomic actions of glucocorticoids. Finally, some proteins, which mediate glucocorticoid activity could be therapeutic targets for reducing glucocorticoid-induced adverse effects.

In microarray data, wide-scale correlations are numerous and increase the number of genes correlated to a test condition (phenotype, mutation status, etc.) either positively or negatively. Several methods have been developed to limit the effect of such correlations on the false discovery rate, but these may reject too many genes that have a mild or indirect impact on the studied condition. We propose here a simple methodology to correct this spurious effect without eliminating weak but true correlations.This methodology was applied to a microarray dataset designed to distinguish heterozygous BRCA1 mutation carriers from non-carriers. As our samples were collected at different times in the morning, we evaluated the effect of correlations due to circadian rhythm. The circadian system is a well-known correlation network, regulated by a small number of period genes whose expression varies throughout the day in predictable ways. The downstream effects of this variation on the expression of other genes, however, are incompletely characterized. We used two different strategies to correct this correlation bias, by either dividing or multiplying the expression of correlated genes by the expression of the considered period gene according to the sign of the correlation between the period gene and correlated gene (respectively positive or negative).We observed a linear relationship between the number of false-positive/negative genes and the strength of the correlation of the candidate gene to the test condition. BRCA1 was highly correlated to the period gene Per1; our correction methodology enabled us to recover genes coding for BRCA1-interacting proteins which were not selected in the initial direct analysis. This methodology may be valuable for other studies and can be applied very easily in case of well-known correlation networks.

Diminished ovarian reserve (DOR) is one of the causes of infertility. In this prospective study, gene expression profiling (GEP) of corona radiata cells (CRC) was performed to identify genes deregulated in DOR patients.Microarray-based GEP of CRC isolated from eight women undergoing IVF was performed to identify genes differentially expressed between patients with normal ovarian reserve and DOR patients. Microfluidic-based quantitative RT-PCR assay were used to validate selected transcripts on 40 independent patients.Forty-eight transcripts were differentially expressed, including CXXC5 and FOXC1 down regulated in DOR, as well as CTGF, FSTL3, PTGS2 and SOCS2 up regulated in DOR. According to these transcripts, two DOR patients'subgroups (DOR Gr1 and Gr2) were identified. In DOR Gr2 patients, CITED2, CTGF, GAS-1, IRS2, PTGS2, SOCS2, VCAN were expressed at significantly higher levels, and CXXC5, FOXC1, GBP2 and ZMIZ1 at significantly lower level. Eleven of those genes are transcriptional targets of Estrogens and higher baseline oestradiol levels were observed in DOR Gr2 patients (P0.006).Twelve genes deregulated in CRC of DOR patients were identified, which could be involved in DOR pathogenesis. The distinction of a particular subgroup of DOR patients suggests the possibility of deregulation of estrogen response genes.

The patient's rare KEL:1,-2 phenotype was highlighted in course of a routine preoperative erythrocyte typing. Unexpectedly, her two daughters presented a KEL:-1,2 phenotype what appeared first as an apparent maternity exclusion. Flow cytometry, genotyping and adsorption-elution analyses were then performed for those three patients. KEL genotyping showed that the patient's genotype was KEL*01/KEL*02 whereas that of her daughters was KEL*02/KEL*02. By using polyclonal anti-KEL2 reagent, weak amount of KEL2 was identified on the patient's erythrocytes, a result which was confirmed by both flow cytometry and adsorption-elution assays, suggesting that patient's phenotype was in fact KEL:1,2w. These results are in favour of a weak expressed KEL*02 allele (KEL*2mod) transmission coding for a KEL2 antigen detected in some technical conditions only. Those results allowed to explain the apparent maternity exclusion based on initial KEL phenotype. This study also seems to confirm the presence of a compensatory mechanism of the KELmod allele deficient expression in heterozygote patients. A KEL phenotype retrospective study of 80,000 subjects showed a local KEL:1,-2 frequency four times lower than that described in literature. Moreover, a significant number of those individuals would in reality be KEL:1,2w, what still would decrease the real frequency of the KEL:1,2 subjects.

Aging is a complex process that is accompanied by the onset of a series of age-related diseases, including Alzheimer's disease. Aging is controlled by a combination of genetic and environmental factors. Among the genes that regulate aging, the insulin-FOXO signaling pathway plays a central role, as this pathway regulates lifespan in multiple species, such as worms, flies, and mice. In humans, exceptional longevity - being a centenarian - is also associated with genetic variation in this insulin-FOXO pathway. Recent evidence indicates that the FOXO family of transcription factors plays a key role in the self-renewal of adult and embryonic stem cells, which could contribute to tissue regeneration. Understanding the mechanisms underlying aging should help better prevent and treat age-dependent diseases.

International audience; P-TEFb stimulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA pol II and antagonizing the effects of negative elongation factors. Its cellular availability is controlled by an abundant non coding RNA, conserved through evolution, the 7SK RNA. Together with the HEXIM proteins, 7SK RNA associates with and sequesters a fraction of cellular P-TEFb into a catalytically inactive complex. Active and inactive forms of P-TEFb are kept in a functional and dynamic equilibrium tightly linked to the transcriptional requirement of the cell. Importantly, cardiac hypertrophy and development of various types of human malignancies have been associated with increased P-TEFb activity, consequence of a disruption of this regulatory equilibrium. In addition, the HIV-1 Tat protein also releases P-TEFb from the 7SK/HEXIM complex during viral infection to promote viral transcription and replication. Here, we review the roles played by the 7SK RNP in cancer development, cardiac hypertrophy and AIDS.

The function of IAP has long been limited to an inhibition of apoptosis through their capacity to bind some caspases. Since the expression of these proteins is altered in some tumor samples, IAPs are targets for anticancer therapy and many small molecules have been designed for their capacity to inhibit IAP-caspase interaction. Unexpectedly, these molecules appeared to significantly affect NF-κB activation. In this review, we will discuss the central role of cIAP1, cIAP2 and XIAP in the regulation of NF-κB activating signaling pathways.

International audience; Toll-like receptors (TLR) sense a variety of microbial products and play an important role in the mounting of innate and adaptive immune responses. TLR1 to TLR9 are common in mice and humans and recognize similar ligands in both species, with the exception of TLR8. Human TLR7 and TLR8 and mouse TLR7 detect viral single-stranded RNA and imidazoquinoline compounds, while mouse TLR8 not. Based on this discrepancy, for long time it was believed that mouse TLR8 is not functional and as a consequence the contribution of TLR8 to innate immunity remained poorly understood. Our recent studies revealed an important role for TLR8 in the regulation of TLR7-mediated autoimmunity in the mouse. This review illustrates our current understanding regarding the function of TLR8 and its potential for future clinical use for the treatment and/or prevention of various pathological conditions.

The MYH9 syndrome is a group of rare autosomal dominant platelet disorders associating in most of the cases a macrothrombocytopenia and characteristic leukocyte inclusions. Clinical features may include renal, visual, or hearing impairment. The bleeding tendency is usually moderate.We report a 28-year-old-man, with an auto-immune haemolytic anaemia associated with a MYH9 syndrome.To our knowledge, this is the first report of such an association.

CYLD deubiquitinase has been originally defined as a tumor suppressor based exclusively on genetic findings. Indeed, inactivation of CYLD in humans results in familial cylindromatosis and multiple trichoepithelioma, two pathologies characterized by the development of tumors originating specifically from the skin appendages. A set of recent publications has revealed that recurrent inactivation of CYLD occurs through diverse mechanisms in several forms of cancer, unequivocally confirming its tumor suppressor function. This property is associated with the critical role played by CYLD in negatively regulating several signaling pathway, among them the NF-κB signaling pathway.