La mécanotransduction est une propriété essentielle au développement des tissus, leur homéostasie et leur physiopathologie. La voie de signalisation YAP (Yes-Associated Protein) est apparue comme un régulateur particulièrement important de la mécano-réponse. Un défaut de mécanosensibilité défectueuse, associant une signalisation aberrante de la voie YAP, a récemment été rapportée dans des myoblastes humains de patients souffrant de dystrophie musculaire congénitale liée à des mutations du gène de la lamine (L-CMD) (Bertrand et al., 2014). La L-CMD est une forme grave de dystrophie musculaire à début précoce. Mon projet de doctorat visait à disséquer les défauts de la mécanosensibilité de cellules précurseurs du muscle présentant la mutation ΔK32. Mes résultats ont montré que les myoblastes mutants ΔK32 présentaient des un défaut de contact cellule-cellul, attestant d’anomalies de transmission des forces mécaniques entre cellules. Contrairement à ce que l’on observe dans les cellules contrôles à confluence, la voie YAP reste activée dans les myoblastes mutants ΔK32. Cela s‘est traduit par une activité transcriptionnelle accrue de YAP et une localisation nucléaire persistante de YAP dans les myoblastes ΔK32. La suractivité de YAP dans les myoblastes mutants ΔK32 n'était pas liée à une altération de la voie Hippo, voie de signalisation canonique qui régule YAP. La signalisation YAP défectueuse a été associée à une désorganisation de différents sous-ensembles du cytosquelette d'actine, incluant l'actine supranucléaire, l'actine basale et les fibres d'actine de la jonction cellule-cellule. La formation et la maturation de jonctions cellule-cellule était défectueuse dans les myoblates ΔK32, et les expressions protéiques des deux principales cadhérines, M et N-cadhérins, étaient significativement réduites à confluence. De plus, les myoblastes mutants ΔK32 présentaient une perte accrue de contact cellule-cellule pendant la migration, responsable d’une perte de la migration collective dans les cellules mutantes. Enfin, nous avons rapporté une augmentation de l'activité transcriptionnelle de la signalisation Smad 1/5/8 dans les myoblastes mutants ΔK32. En conclusion, ces résultats de thèse suggèrent que les défauts de mécanosensibilité dans les myoblastes mutants ΔK32 affectent la capacité des myoblastes à former des contacts cellule-cellule et à migrer collectivement. Ces défauts de mécanosensibilité peuvent contribuer à la physiopathologie de la L-CMD. / Mechanotransduction is critical for tissue development, homeostasis and diseases. YAP (Yes-Associated Protein) signaling has emerged as a particularly important regulator of the mechano-response. A defective mechanosensing response, including aberrant YAP signaling, has been recently reported in human myoblasts from patients suffering from LMNA related congenital dystrophy (L-CMD) (Bertrand et al., 2014). L-CMD is a severe early-onset form of muscular dystrophies caused by mutations in A-type lamins. My PhD project aims to further dissect mechanosensing defects of immortalized muscle precursor cells which carry the L-CMD causing ∆K32 mutation. My results showed that ∆K32 mutant myoblasts had a defective translation of mechanical forces at cell-cell contact sides. ∆K32 mutant myoblasts failed to inactivate YAP in high cell-cell contact conditions, as attested by an increased transcriptional activity of YAP and a persistent nuclear localization. YAP overactivity in ∆K32 mutant myoblasts was not related to an impaired activation of the Hippo signaling pathway. Defective YAP signaling was associated with a disorganization of different subsets of the actin cytoskeleton, including the supranuclear actin, the basal actin and the actin fibers at cell-cell junction. The formation of mature cell-cell contacts in ∆K32 myoblats was defective, and the protein expressions of both M- and N-cadherins were significantly reduced in high cell-cell contact conditions. Moreover, ∆K32 mutant myoblasts showed an increased loss of cell-cell contact during migration, which caused a shift from a sheet-like to a single cell migration pattern. Finally, we reported an increased transcriptional activity of mechanosensitive Smad 1/5/8 signaling in ∆K32 mutant myoblasts. Taken together, these results suggest that mechanosensing defects in ∆K32 mutant myoblasts affect the ability of myoblast to form cell-cell contacts and to migrate collectively. These mechanosensing defects may contribute to the pathophysiology of L-CMD.

A synthetic pathway towards the cap-structure of 2,2,7-trimethylguanosine containing a methylene modified triphosphate bridge have been investigated. The modification to the triphosphate bridge is hoped to slow down cap degradation and give the connected  oligunucleotide an increased lifetime. This could result in an better understanding of nuclear transport of oligonucleotides and could thereby helping to develop new treatments for different diseases. The synthesis relies on a coupling reaction between the 2,2,7-trimethylguanosine 5’phosphate and 2’-O-methyladenosine with a 5’-pyrophosphate where the central oxygen has been replaced by a methylene group. The reaction pathway consists of 9 steps of which 8 steps have been successfully performed. The last step, which includes a coupling reaction, was attempted but without successful identification and isolation of the cap-structure, and will need further attention. The reaction has been performed in a milligram scale with various yields. / Presentation utförd

La dystrophie musculaire de Duchenne (DMD) est une pathologie récessive liée au chromosome X qui résulte d’une mutation sur le gène de la dystrophine aboutissant à l’absence complète de la protéine. Elle correspond à la plus fréquente des dystrophies musculaires et reste aujourd’hui sans traitement curatif. L’UMR a fait la preuve de concept de l’administration systémique d’une population de cellules souches adultes résidentes du muscle, les cellules MuStem canines chez le chien dystrophinopathe, modèle cliniquement pertinent de la DMD. L’objectif de la thèse a consisté à caractériser la population humaine (hMuStem) en terme de phénotype, myogénicité, immunomodulation et de contexte immunologique d’administration in vivo. La population hMuStem se compose de progéniteurs myogéniques précoces d’origine mésenchymateuse-périvasculaire. Elle se définit par une forte capacité proliférative, une oligopotence et une participation à la régénération musculaire après administration dans un muscle lésé. Elles présentent des propriétés immunomodulatrices en interagissant avec l’immunité adaptative et innée par inhibition de la prolifération lymphocytaire et du complément via un ensemble de molécules de surface et/ou de facteurs sécrétés. Enfin, un traitement immunosuppressif restreint à la période d’injection in vivo de la population allogénique s’avère nécessaire mais suffisant pour éviter une réaction immune de l’hôte. L’ensemble de ces résultats aboutit à une meilleure compréhension de l’identité et des modalités d’action de la population MuStem. / Duchenne Muscular Dystrophy is a X-linked recessive disorder that results from mutation in the dystrophin gene leading to a total lack of the protein. It is the most frequent muscular dystrophy with no curative treatment. The lab made a proof of concept of the systemic delivery of a muscle-derived adult stem cell population called MuStem cells in dystrophic dog, the clinically relevant DMD model. The aim of my Ph.D. was to characterize the human population (hMuStem) in terms of phenotype, myogenicity, immunomodulation and immunological context of in vivo delivery. hMuStem cell population is composed of myogenic progenitors with mesenchymal/perivascular imprint. It exhibits a high proliferative capacity, an oligopotency and a participation to muscle regeneration after transplantation into injured muscle. It displays immunomodulatory properties by interacting with adaptive and innate immunity with inhibition of lymphocyte proliferation and complement thanks to expression of surface molecules and/or secreted factors. At last, an immunosuppressive regimen restricted to the allogeneic injection period is necessary but sufficient to avoid host immune response. Collectively, these results allow a better understanding of identity and action modalities of MuStem cell population.

La dystrophie FacioScapuloHumérale (FSHD), décrite pour la première fois en 1885 par Landouzy Dejerine, est la première dystrophie musculaire de l’adulte en France affectant entre 4000 et 5000 personnes. La destruction progressive des fibres musculaires entraîne une atrophie et une faiblesse musculaires s’aggravant progressivement, avec cependant une grande variabilité intra-familiale du degré des atteintes. Une caractéristique de l’atteinte musculaire est généralement son asymétrie. Les premières manifestations concernent souvent les muscles du visage, les muscles de l’omoplate et des muscles perihuméraux. En progressant la pathologie va toucher d’autres territoires musculaires. Dans environ 10 à 15 % des cas, à un stade évolué, les patients sont contraints d'utiliser un fauteuil roulant. En dépit d’avancées majeures dans la compréhension du locus morbide, les mécanismes exacts responsables des défauts musculaires de la FSHD ne sont toujours pas compris et il n’existe aucune thérapie. Toutefois, il existe de plus en plus de données qui permettent une implication probable du stress oxydant dans cette pathologie. L’hypothèse selon laquelle les réponses antioxydantes sont altérées dans la FSHD s’appuie sur des dérégulations d’enzymes impliqués dans le stress oxydant. Une étude prospective réalisée sur des patients FSHD et des volontaires sains nous a ainsi permis de mettre en évidence une corrélation entre le stress oxydant systémique et musculaire et leurs déficits fonctionnels musculaires. Ces données nous ont conduit à la mise en place d’un essai clinique randomisé, contrôlé, en double aveugle contre placébo, visant à évaluer les effets d’une supplémentation en antioxydants chez 54 patients atteints de FSHD pendant 17 semaines. Cet essai a ainsi permis de montrer une augmentation significative de la force et l’endurance des quadriceps corrélée à une diminution du stress oxydant et une augmentation des défenses antioxydantes chez les patients atteints de FSHD. De nombreuses caractéristiques de la FSHD pourraient être causées et/ou exacerbées par des perturbations de la production des espèces radicalaires ou une réponse non adaptée à cette production. Aussi le premier objectif de ma thèse est de mener une étude comparative des profils d’oxygénation par spectroscopie dans le proche infrarouge de patients atteints de FSHD et sains. Le second objectif est d’évaluer l’effet de la supplémentation en antioxydant sur le volume des quadriceps par IRM et leur qualité musculaire déterminée par le ratio Force/Volume musculaire du quadriceps et d’évaluer les corrélations entre ces variables, la force et le stress oxydant. Les données obtenues ont permis de montrer une réduction de la capacité oxydative lors d’une contraction isométrique volontaire des quadriceps et ont permis d’étudier l’effet de la supplémentation sur les volumes et la qualité musculaire des quadriceps. Ces augmentations sont associées non seulement à une augmentation de la force des quadriceps mais aussi à une diminution du stress oxydant et une augmentation des défenses antioxydantes. L’ensemble de ces données montrent que le stress oxydant pourrait jouer un rôle important dans la FSHD et qu’une approche antioxydante semble adaptée à cette pathologie. Des analyses plus fines sur l’action des espèces réactives de l’oxygène (ROS) et leurs sources pourraient contribuer à une meilleure compréhension des bases physiopathologiques de la FSHD. / Facioscapulohumeral muscular dystrophy (FSHD), first described in 1885 by Landouzy Dejerine, is the most common inherited skeletal muscle disease of adult life affecting 4000 to 5000 persons in France. Progressive evolution of the disease leads to progressive weakness and atrophy of muscle fibers associated to a wide variability. The pattern of muscle weakness is often asymmetrical and the rate and extent of progression may vary considerably with sudden periods of unexplained rapid disease progression. This muscle disorder is characterized by progressive muscle weakness, beginning with facial muscles and the shoulder girdle, followed by the pelvic girdle and the muscles of the lower extremities. In 10 to 15% of cases, patients need to use a wheelchair. Despite major progress in the understanding of the genetic basis of FSHD, the exact mechanisms that lead to FSHD defects are not completely understood and no curative treatment is available. However, there is growing evidence that oxidative stress may contribute to FSHD pathology. The hypothesis that oxidative stress responses might be specifically altered in FSHD is supported by the deregulation of enzymes involved in oxidative stress.A prospective study realized with FSHD patients and healthy subjects unrevealed the correlation between systemic and muscular oxidative stress and functional muscle defects. Based on these data, we organized a randomized, double-blind, placebo-controlled pilot clinical trial in order to evaluate the effects of 17 weeks antioxidant supplementation in 54 FSHD patients. This clinical trial demonstrates a significant increase in muscle force and quadriceps endurance correlated to a decrease in oxidative stress and an increase in antioxidant defense in FSHD patientsFurthermore, many FSHD features may be caused or exacerbated by perturbations in the production of free radicals or inappropriate response to such stressors. Therefore the first objective was planned to investigate muscle oxygenation patterns during and after a MVCQ by near-infrared diffuse optical spectroscopy (NIRS). The second objective is to evaluate the effect of antioxidant supplementation on quadriceps volumes by IRM and determine the muscle quality using Strength/ Volume ratio of quadriceps muscles and correlate this variables with force and oxidative stress parameters.The major findings of this study show a significant decrease in oxidative capacity during voluntary isometric contraction in quadriceps and demonstrate the effect of supplementation on muscle volume and quality. Indeed, vitamin E, vitamin C, zinc and selenium supplementation improves muscle volume and quality of both quadriceps by enhancing the antioxidant defences and reducing oxidative stress.This increase are associated to increase in strength and decrease in oxidative stress and increase in antioxidant defences. Taken together, we show that oxidative stress plays an important role in FSHD and that an anti-oxidant strategy adapted to the FSHD-specific “oxidative stress” may be a relevant therapeutic approach for these patients. Further analyses of ROS production and sources could contribute to a better understanding of the pathophysiological mechanisms implicated in FSHD.

Aim: The overall aim of this thesis, using a salutogenic framework, was to develop knowledge about experiences and perceptions of living with recessive limb-girdle muscular dystrophy and its influences on health, from the affected young adults’ and their parents’ perspectives. Methods: A qualitative explorative and descriptive study design was used. Semi-structured interviews were held with 14 young adults diagnosed with recessive limb-girdle muscular dystrophy, aged 20–0 years, and 19 parents. Data analyses were conducted using content analysis (I, II, III) and phenomenography (IV). In order to mirror the interview data, the participants also answered the 13-item sense of coherence questionnaire. Findings: Recessive limb-girdle muscular dystrophy has a major impact on the affected young adults’ and their parents’ lives as the disease progresses. Health described in terms of well-being was thus perceived to be influenced, not only by physical, emotional and social consequences due to the disease and worry about disease progression but also by external factors, such as accessibility to support provided by society and other people’s attitudes. There was, however, a determination among the participants to try to make the best of the situation. The importance of being able to mobilize internal resources, having social support, meaningful daily activities, adapted environment, the young adult being seen as a person and having support from concerned professionals, including personal assistance when needed, was thereby described. Self-rated sense of coherence scores varied. Those who scored above or the same as median among the young adults (≥56) and the parents (≥68) expressed greater extent satisfaction regarding social relations, daily activities and external support than those who scored less than median. Conclusion: This thesis highlights the importance of early identification of personal perceptions and needs to enable timely health-promoting interventions. Through dialogue, not only support needed for the person to comprehend, manage and find meaning in everyday life can be identified, but also internal and external resources available to enhance health and well-being, taking into account the person’s social context as well as medical aspects.

One of the major drawbacks of using stem cell therapy to treat muscular dystrophies is the challenge of isolating sufficient numbers of suitable precursor cells for transplantation. As such, a deeper understanding of the molecular mechanisms involved during muscle development, which would increase the proportion of embryonic stem cells that can differentiate into skeletal myocytes, is essential. In conditional SOX7-/- mice, we observed that the loss of SOX7 in satellite cells resulted in poor differentiation and fusion. In vivo, we observed fewer Pax7+ satellite cells in the mice lacking SOX7 as well as smaller muscle fibers. RT-qPCR data also revealed that Pax7, MRF and MHC3 transcript levels were down-regulated in SOX7 knockdown mice. Surprisingly, when SOX7 was over-expressed in embryonic stem cells, we found that there was a defect in making muscle precursor cells, specifically a failure to activate Pax7 expression. Taken together, these results suggest that SOX7 expression is required for the proper regulation of skeletal myogenesis.

Over the past decade several converging lines of evidence have highlighted the importance of post-transcriptional events in skeletal muscle. This level of regulation is controlled by multi-functional RNA-binding proteins and trans-acting factors. In fact, several RNA-binding proteins are implicated in neuromuscular disorders including myotonic dystrophy type I, spinal muscular atrophy and amyotrophic lateral sclerosis. Therefore, it is necessary to examine the impact of RNA-binding proteins during skeletal muscle development and plasticity in order to understand the consequences linked to their misregulation in disease. Here, we focused on the RNA-binding protein Staufen1, which assumes multiple roles in both skeletal muscle and neurons. We previously demonstrated that Staufen1 is regulated during myogenic differentiation and that its expression is increased in denervated and in myotonic dystrophy type I skeletal muscles. The increased expression of Staufen1 initially appeared beneficial for DM1 since further elevating Staufen1 levels rescued key hallmarks of the disease. However, based on the multi-functional nature of Staufen1, we hypothesized that Staufen1 acts as a disease modifier in DM1. To test this, we investigated the roles of Staufen1 in skeletal muscle biology and their implications for disease. Our data demonstrated that Staufen1 is required during the early stages of muscle development, however its expression must remain low in postnatal skeletal muscle. Interestingly, the overexpression of Staufen1 impaired myogenesis through the regulation of c-myc translation. Since the function of c-myc in oncogenesis is well described, we investigated the role of Staufen1 in cancer biology. In particular, we determined novel functions of Staufen1 in rhabdomyosarcoma tumorigenesis, thus providing the first direct evidence for Staufen1’s involvement in cancer. Moreover, based on Staufen1’s role in myogenic differentiation and in myotonic dystrophy type I, we generated muscle-specific transgenic mice to examine the impact of sustained Staufen1 expression in postnatal skeletal muscle. Staufen1 transgenic mice developed a myopathy characterized by histological and functional abnormalities via atrogene induction and the regulation of PTEN mRNAs. In parallel, we further investigated Staufen1-regulated alternative splicing and our data demonstrated that Staufen1 regulates multiple alternative splicing events in normal and myotonic dystrophy type I skeletal muscles, both beneficial and detrimental for the pathology. Collectively, these findings uncover several novel functions of Staufen1 in skeletal muscle biology and highlight Staufen1’s role as a disease modifier in DM1.

Adult skeletal muscles have remarkable regenerative capacity. Muscle regeneration occurs when muscle tissue experiences injury, causing a population of normally quiescent muscle-resident stem cells, called satellite cells, to become activated. The CCAAT/enhancer binding proteins known as C/EBPs are transcription factors belonging to the bZIP family. Previous work from our lab has identified C/EBPβ as an important negative regulator of myogenesis. C/EBPβ expression is localized to muscle satellite cells and is downregulated upon induction to differentiate, mirroring the loss of Pax7 expression in early myogenesis. C/EBPβ expression also negatively regulates MyoD protein expression. Leaky ribosomal scanning of the Cebpb mRNA produces three C/EBPβ isoforms: LAP*, LAP and LIP, though the individual role of each of these isoforms has not been investigated in myoblasts. This thesis focuses on determining the role of each of the C/EBPβ isoforms during skeletal muscle differentiation. Forced expression of the C/EBPβ-LIP isoform in myoblasts led to a decrease in Myf5, MyoD, and myogenin expression under differentiation conditions when compared to empty vector controls. Further, the fusion of cells was greatly reduced following differentiation. C/EBPβ-LIP expressing cells also demonstrated a growth defect, with pronounced G1 arrest and features of senescence. In contrast, myoblasts expressing the C/EBPβ-LAP isoform has impaired differentiation, though this was not as pronounced as in C/EBPβ-LIP expressing cells and proliferated normally. While LIP is not normally expressed in primary myoblasts from healthy muscle, the ratio of LIP:LAP was increased in primary myoblasts isolated from mdx mice, an animal model for Duchenne muscular dystrophy. These findings suggest that the regulation of C/EBPβ isoform expression could regulate stem cell stamina and may contribute to defects in muscle regeneration in disease.

La rétinite pigmentaire est une maladie héréditaire rétinienne menant à la cécité, et pour laquelle il n’existe aucun traitement. La cause la plus fréquente des formes autosomiques dominantes de la maladie est une mutation ponctuelle dans le gène de la rhodopsine (RHO) induisant la mort des photorécepteurs (PR). Pour éviter la dégénérescence des PR, la stratégie thérapeutique doit supprimer l’expression de la protéine mutante tout en restaurant celle de la protéine normale et ce à un niveau physiologique. Mon projet était de réparer le pré-ARNm RHO par la technologie SMaRT (trans-épissage (TE) médié par le splicéosome). Ceci nécessite d’introduire par transfert de gène, dans la cellule cible, un ARN exogène, appelé PTM pour molécule pre-ARNm de TE, pouvant induire un épissage en trans.Nous avons créé 20 PTM différents et obtenu un taux maximal de TE in vitro de 40% après co-transfection transitoire des constructions RHO et PTM dans les cellules HEK293T. Nous avons générés des lignées cellulaires d’expression stable de RHO normale ou mutée par transduction lentivirale. Alors que la RHO normale se localise à la membrane plasmique, la mutation induit la rétention cytoplasmique de la protéine. La transfection du PTM dans la lignée cellulaire de RHO mutée a induit du TE, capable de restaurer partiellement la localisation de la RHO réparée à la membrane.Nous avons alors testé le TE in vivo dans un modèle murin humanisé de rétinite pigmentaire. L’injection sous-rétinienne d’un AAV2/8-bRho-PTM a permis le TE in vivo, mais n’a pas suffi à prévenir la dégénérescence des PR observée par SD-OCT (technologie que nous avons améliorée au cours de ce projet). / Retinitis pigmentosa is an hereditary retinal dystrophy involving degeneration of photoreceptors leading to blindness and for which there is currently no treatment. The most frequent cause of autosomal dominant forms of the disease is a point mutation in the rhodopsin gene (RHO). Therapeutic strategy should both suppress mutant protein expression and restore that of the normal one to physiologic level to prevent photoreceptor degeneration. My PhD project was to repair RHO pre-mRNA by SMaRT (Spliceosome Mediated RNA Trans-splicing) technology. This implies to introduce by gene transfer into the target cell an exogenous RNA, called PTM for Pre-mRNA Trans-splicing Molecule. This one was able to promote a splice reaction in trans, leading to the replacement of the mutated exons. We designed 20 different PTM and obtained in vitro a maximum trans-splicing rate of 40% after transient co-transfection of PTM and RHO constructs in HEK293T cells. We then created WT or mutated RHO stable expression cell lines by lentiviral transduction. Mutation induced retention of the protein into the cytoplasm, while the WT RHO was localized to the plasma membrane. We observed that the PTM transfection in the mutated RHO cell line induced trans-splicing, which was able to partially restore localization to the plasma membrane of repaired RHO. We then tested trans-splicing in vivo in mRho+/- RHO P347S+ mice, a humanized heterozygous mouse model of retinitis pigmentosa. After subretinal injection of AAV2/8-bRho-PTM we observed that trans-splicing occurred in vivo. Unfortunately we did not observe by SD-OCT (a technology that we improve in this project) any rescue of the degenerative phenotype.

Le diagnostic des maladies génétiques est difficile à émettre. En effet, il est souvent difficile de déterminer comment une mutation va entrainer la pathologie. Le but de cette thèse est de développer des outils permettant de répondre à cette interrogation. Les mutations peuvent entrainer des anomalies à différents niveaux, différentes outils ont ainsi été développées en parallèle afin de pouvoir détecter différents types d'anomalies. Ces outils ont été développés en utilisant comme modèle une maladie génétique appartenant à la famille des myopathies, entrainant une dégénérescence des muscles des patients. Les travaux de cette thèse ont permis de confirmer le caractère délétère de certaines mutations en démontrant des anomalies dans un mécanisme appelé « épissage » qui permet la transmission de l'information contenue dans le génome. Les mutations en empêchant cette transmission vont ainsi être responsables de la maladie. / Diagnosis of genetic diseases is a difficult task. Indeed, it is often difficult to determine if mutations detected in patients will be responsible of the disease. The aim of this thesis is to develop tools allowing answering on this question. Mutations can have deleterious effects to several levels, thus different tools have been develop in parallel in order to detect different kind of abnormalities. These tools have been developed using as model a genetic disease belonging to the family of myopathy, leading to a degeneration of patients muscles. These thesis works have confirmed the deleterious effect on some mutations in a mechanism named "splicing" which allow transmission of the genome's information. Mutations preventing the transmission will thus be responsible of the disease.