No description available.

En vue de contribuer à une meilleure compréhension des atteintes du système musculo-squelettique consécutives à une infection par un arthropod-borne-virus (arbovirus), deux arbovirus ont été étudiés : le virus du chikungunya (CHIKV) et le virus Zika (ZIKV), respectivement de la famille des Togaviridae et des Flaviviridae. Cette étude a été réalisée selon deux axes. Le premier s’intéresse aux atteintes articulaires consécutives à l’infection par le CHIKV. Nous avons mis au point un modèle d’imagerie in vivo reposant sur l’utilisation d’un virus recombinant exprimant la NanoLuciférase. Dans ce modèle, nous démontrons une persistance du signal bioluminescent dans les articulations 34 jours après infection. Par isolement des cartilages articulaires et des cellules constitutives, nous avons pu démontrer que les chondrocytes des cartilages métatarso-phalangiens sont infectés par le CHIKV de manière persistante, suggérant un rôle de réservoir de ces cellules. Les conséquences de l’infection au niveau cellulaire ont ensuite été étudiées in vitro. En utilisant des chondrocytes primaires humains, nous avons confirmé ces observations. De plus, les chondrocytes infectés produisent de nombreuses cytokines, induisant une stimulation du catabolisme du cartilage avec en particulier la synthèse de métalloprotéinases de matrice 3 et 9. De plus, l’infection par le CHIKV provoque la mort des cellules par apoptose, comme démontré par marquage TUNEL et par mesure de l’activité des caspases. Nous avons ensuite étudié les atteintes musculaires et le tropisme cellulaire du ZIKV. Dans un modèle murin, nous avons confirmé la présence de lésions musculaires, et l’utilisation de cellules musculaires primaires humaines a montré la sensibilité des myoblastes à l’infection, les myotubes étant résistants, suggérant un tropisme du ZIKV dépendant de la différenciation cellulaire. Trois souches virales ont été testées, sans relever de différences significatives en termes de cinétique d’infection, de nombre de cellules infectées et de production virale. L’infection des myoblastes entraîne la mort de ces cellules par un mécanisme caspase-indépendant. Des observations en microscopie électronique ont mis en évidence une vacuolisation du cytoplasme suite à l’infection, caractéristique d’une mort cellulaire par paraptose. Une analyse protéomique a démontré que l’infection des myoblastes par une souche asiatique conduit à une modification du protéome s’accentuant entre 24 heures et 48 heures post-infection, avec 225 protéines modulées 24 heures après infection contre 473 après 48 heures, indiquant une activation des voies de synthèse Interferon de type I et l’inhibition des capacités de synthèse des protéines. Ces résultats permettent une meilleure compréhension des atteintes du système-musculo-squelettique par les arbovirus / Musculoskeletal lesions caused by arthropod-borne-viruses (arboviruses) are invalidating for patients and remain poorly understood. In this study, we investigated the development of these manifestations after infection with two arboviruses: chikungunya virus (CHIKV) from the Togaviridae family, and Zika virus (ZIKV) from the Flaviviridae family. The first part of the study focused on arthritis following CHIKV infection. For this purpose, we developed a reporter virus expressing NanoLuciferase and performed experimental infections in a murine model. In vivo, a strong bioluminescent signal indicated viral replication and we observed persistence of the signal in the joints up to 34 days post-infection. By isolating primary murine cells from cartilage, we demonstrated the susceptibility of chondrocytes to CHIKV infection suggesting a role of reservoir of these cells. Furthermore, we investigated the consequences of the infection using in vitro models. We showed that primary human chondrocytes are also susceptible to CHIKV infection, which leads to the production of several cytokines involved in cartilage catabolism and induces apoptosis. In the second part of the study, we studied ZIKV muscular tropism and the associated lesions. Firstly, we confirmed the development of muscular lesions in a mouse model of ZIKA. Then, using human primary muscle cells we observed the infection of myoblasts but not myotubes, suggesting a differentiation-dependent tropism. We compared three viral strains and observed no significant difference in terms of replication, number of infected cells and viral production. Myoblasts infection induced a caspase-independent cells death mechanism and electronic microscope observations revealed intense vacuolization of cytoplasm, suggesting a paraptosis-like cell death. Proteomic analysis revealed that the Asian ZIKV strain modulated protein expression of infected cells with an increased effect after 48 hours. ZIKV-infection induced an important upregulation of biological processes associated with type I interferon and an inhibition of protein synthesis in the infected cells. These results provide valuable information about the mechanisms involved in the development of musculoskeletal lesions during arboviroses

L’ingénierie du tissu musculaire squelettique vise à reconstituer in vitro un tissu fonctionnel aussi physiologique que possible dans le but de mieux comprendre la myogenèse, l’impact de mutations génétiques et tester des médicaments. Ces dernières années, différents modèles de tissus musculaires tridimensionnels ont été développés. Toutefois, l’utilisation prépondérante de cellules murines et la taille de ces modèles restreint leur pertinence pour les études de pathologies humaines et le criblage pharmacologique. Dans le cadre de ce travail de thèse, nous avons donc développé différents modèles de tissus musculaires humains micrométriques pour répondre à ces limitations. Dans un premier temps, nous avons conçu et optimisé par microfabrication une plateforme caractérisée par la présence de microcanaux. Nous avons ainsi généré des tissus musculaires multicouches alignés présentant une organisation proche du muscle natif à partir de myoblastes murins immortalisés C2C12 puis de myoblastes humains immortalisés. Nous avons ainsi montré l’influence de la topographie et de la concentration cellulaire sur l’alignement des myotubes et la maturation du tissu musculaire. Dans un second temps, nous avons développé une plateforme constituée de micropuits contenant chacun deux micropiliers permettant d’analyser la contractilité des tissus. Des microtissus musculaires 3D standardisés ont ainsi été générés avec cette plateforme à partir de myoblastes murins, et de myoblastes C2C12 électroporés avec un gène muté ou non de la desmine. Par la suite, des microtissus ont été générés à partir de myoblastes humains. L’importance du choix de la matrice dans la formation des microtissus et les bénéfices d’une coculture de myoblastes et fibroblastes dans la stabilité des tissus ont ainsi été mis en évidence. La géométrie de micropiliers a aussi été optimisée afin de générer et comparer des microtissus composés de myoblastes isolés de patients sains et malades (dystrophie musculaire de Duchenne). Une preuve de concept démontrant la possibilité d’utiliser cette technologie pour tester des thérapies chimiques et géniques a été établie. Nous avons en effet suivi en temps réel les effets de l’inhibiteur de la kinase Rho-associée Y-27632 sur la contractilité des microtissus, ainsi que la transduction d’un gène rapporteur fluorescent modèle par les cellules composant les microtissus. Les résultats de ce travail de thèse démontrent le potentiel de cette technologie pour l’étude des processus fondamentaux de la myogenèse, l’évaluation des effets fonctionnels de mutations patient-spécifique et le criblage de thérapies chimiques et géniques. / Skeletal muscle tissue engineering aims to build functional and physiological tissues in vitro in order to better understand myogenesis, to investigate the impact of genetic mutations and to screen potential therapies. Over the past few years, bi- and tridimensional models of muscle tissue have been developed, but most of these models are based on the use of murine cells and require large amounts of cells, thus limiting their relevance to study pathologies of human muscles and drug screening assays. Here we aimed at developing different models of human muscle microtissues to address these issues. By using microfabrication techniques, we first engineered a microgrooved platform we used to generate aligned multilayered skeletal muscle tissues from murine C2C12 myoblasts and human immortalized myoblasts. We showed the impact of topography and cell density on the maturation and myotube alignment. We then fabricated a microdevice, consisting of microwells containing two micropillars allowing an easy access to the contractility of muscle tissues. We engineered microtissues from C2C12 and C2C12 myoblasts electroporated with a mutated gene of desmin, and showed some limitation of this technique of transduction. Finally, we generated microtissues from human myoblasts. We investigated the role of the extracellular matrix in the tissue formation and evidenced the benefits of coculturing myoblasts and fibroblasts on the stability of muscle microtissues. Furthermore, we optimized the geometry of the micropillars to engineer and compare microtissues composed of human myoblasts isolated from healthy and diseased (Duchenne muscular dystrophy) patients. A proof of concept of the potential of this technology for screening chemical and gene therapies was established. We were indeed able to analyze in real time the effects of the Rho-associated kinase-inhibitor Y-27632 on the tissue contractility, as well as the transduction of a model fluorescent reporter gene. Altogether, the results of this work demonstrate the potential of this technology to study fundamental muscle biology, examine functional effects of patient-specific mutations or screen chemical and gene therapies.

Skeletal muscle injuries and muscle degenerative diseases pose significant challenges to the healthcare. Surgical interventions are restricted due to tissue availability, donor site morbidity and alterations to tissue biomechanics. Current cell-based therapies are hindered by low survival and long-term engraftment for the transplanted cells due to the lack of appropriate supportive microenvironment (cell niche) in the injured muscle. Therefore, there is a critical need for developing strategies that provide cellular and structural support in the regeneration of functional muscle. In the present work, a bioengineered cell niche mimicking the native skeletal muscle microenvironment has been developed for skeletal muscle regenerative engineering. It is hypothesized that the bioengineered scaffolds with appropriate structural and cell instructive properties will support myoblast alignment and function, as well as promote the myogenic responses in clinically relevant skeletal muscle injuries. The current work utilized a three-pronged approach to design biomaterial scaffolds to aid in skeletal muscle regeneration. In the first part, aligned poly(lactide-co-glycolide) (PLGA) fiber scaffolds mimicking the oriented muscle fiber microenvironment with fiber diameters of 335±154 nm (nanoscale), 1352±225 nm (microscale) and 3013±531 nm (microscale) were fabricated and characterized. Myoblasts were found to respond to fiber diameter as observed from the differences in cell alignment, cell elongation, cell spreading area, proliferation and differentiation. <i>In vivo</i> study demonstrated the potential of using microscale fiber scaffolds to improve myogenic potential in the <i>mdx</i> mouse model. In the second part, we designed, synthesized, and characterized an implantable glycosaminoglycan-based composite hydrogel consisting of hyaluronic acid, chondroitin sulfate and polyethylene glycol (HA-CS) with tailored structural and mechanical properties for skeletal muscle regeneration applications. We demonstrated that HA-CS hydrogels provided a suitable microenvironment for <i>in vitro</i> myoblast proliferation and differentiation. Furthermore, <i>in vivo</i> studies using a volumetric muscle loss model in the mouse quadriceps showed that HA-CS hydrogels integrated with the surrounding host tissue and facilitated <i>de novo</i> myofiber generation, angiogenesis, nerve innervation and minimized scar tissue formation. In the third part, we investigated the effects of PC12 secreted signaling factors in modulating C2C12 myoblast behavior. We showed that PC12 conditioned media modulated myoblast proliferation and differentiation in both 2D culture and 3D aligned electrospun fiber scaffold system in a dose dependent manner. We also demonstrated the biomimetic HA-CS hydrogel system enabled 3D encapsulation of PC12 cells secreting signaling factors and promoted survival and proliferation of myoblasts in co-culture. Further proteomics analysis identified a total of 2088 protein/peptides from the secretome of the encapsulated PC12 cells and revealed the biological role and overlapping functions of nerve secreted proteins for skeletal muscle regeneration, potentially through regulating myoblast behavior, nerve function, and angiogenesis. These set of experiments not only provide critical insight on exploiting the interactions between muscle cells and their microenvironment, but they also open new avenues for developing advanced bioengineered scaffolds for regenerative engineering of skeletal muscle tissues.<br>

No description available.

Cette thèse, consacrée aux myopathies inflammatoires, démontre le rôle dans les maladies auto-immunes des Toll-like récepteurs (TLRs), véritable passerelle entre immunité innée et adaptative, et plus spécifiquement dans le muscle, le rôle fondamental de la cellule musculaire elle-même. Après une présentation globale des myopathies inflammatoires et des différents aspects immunopathologiques, la réponse immunitaire adaptative est abordée en rapportant notamment dans le muscle des myopathies inflammatoires une accumulation de cellules dendritiques matures, et la présence des lymphocytes Th1 et Th17, avec un profil prépondérant Th1. L’implication de l’immunité innée est démontrée in vivo par l’expression musculaire des TLR3 et 7, et des C-type lectin récepteurs, spécifique des myopathies inflammatoires. In vitro, l’activation de la voie TLR3 induit la production par les cellules musculaires d’IL6, de la βchémokine CCL20, contribuant au recrutement et à la différentiation des cellules dendritiques et lymphocytes T, et de l’IFNβ qui participe à la surexpression des antigènes HLA de classe I. Les mécanismes de régulation impliquent une balance cytokinique Th1 et Th17. Finalement, l’importance des précurseurs musculaires immatures est soulignée. Contrairement au tissu musculaire normal, une surexpression des antigènes HLA de classe I, des TLRs, des auto-antigènes et de l’IFNβ, par les précurseurs musculaires immatures, est caractéristique des myopathies inflammatoires. Le rôle central de ces cellules musculaires immatures à potentiel de régénération pourrait expliquer un défaut de réparation associé au processus auto-immun de destruction musculaire. / This thesis, devoted to the inflammatory myopathies, is demonstrating the potential role in autoimmune disorders of Toll-like receptors (TLRs), gateway between innate and adaptive immune system, and more specifically in muscular diseases the fundamental role of muscle cell it-self. After the presentation of the general clinical features and the immunopathology of inflammatory myopathies, the adaptive immune response is the subject of the second part,demonstrating the abnormal accumulation of mature dendritic cells in myositis muscle, and the presence of Th1 and Th17 cells with a predominant Th1 profile. Innate immune system is next investigated, demonstrating the overexpression of TLR3 and 7 and of C-type lectin receptors characteristic of inflammatory myopathies. In vitro, stimulation of the TLR3 pathway in human myoblasts induces the production of IL6 and of the βchemokine CCL20, which in turn participate to the differentiation and the migration of T cells and dendritic cells, and of IFNβ which contributes to HLA class I up-regulation. The expression of TLR3 is differentially regulated by Th1 and Th17 cytokines. Finally, this work strongly implicates immature myoblast precursors in the pathogenesis of inflammatory myopathies. In contrast to normal muscle tissue, myositis tissue is characterized by the overexpression of HLA class I antigens, TLR3 and TLR7, myositis autoantigens, and IFNβ, all observed in immature myoblast precursors. By focusing damage onto those cells accomplishing repair, a feedforward loop of tissue damage is induced and could explain the defective repair in muscle in addition to the autoimmune attack.

Directing differential cellular response by manipulating the physical characteristics of the material is regarded as a key challenge in biomaterial implant design and tissue engineering. In developing various biomaterials, the influence of substrate properties, like surface topography, stiffness and wettability on the cell functionality has been investigated widely. However, such study to probe into the influence of substrate conductivity on cell fate processes is rather limited. The need for such an understanding is based on the fact that specific tissues in the body are electrically active in nature, such as in brain, heart and skeletal muscle. These tissues make use of electrical conductivity as an effective cue for tissue homeostasis, development, regeneration and so on. Moreover, understanding the importance of underlying conductivity in basic biological processes is essential in developing electrically conductive biomaterials with the ability to simulate normal electrophysiology of the body by interfacing with bioelectric fields in cells and tissues. Electrical stimulation and charge conduction can regulate numerous intracellular signalling pathways, can interact with cytoskeleton proteins to modulate the morphology, increase protein synthesis and on the more can favor the ECM protein conformational changes. On these grounds, the present dissertation illustrates that persistent electrical activation influences the multipotency of hMSCs and acts like a promoter towards selective differentiation of hMSCs into neural/cardiomyogenic or osteogenic lineage. Besides, continual exposure to electric field stimulated conducting culture environments lead to growth arrest while enhancing differentiation. In total, this dissertation suggests the dominant role of conductivity in inducing my oblast differentiation and hMSc lineage commitment that involves EF stimulated in vitro culture conditions. Also, a knowledge base with qualitative and quantitative understanding of stem cells and their response to substrate physical properties and external field effect was developed through this comprehensive study. Such an improved understanding of the ability of hMSCs in sensing electrical conductivity may lead to the development of culture additives/conditions that better induce directed stem cell differentiation.

Cette thèse interdisciplinaire a été dédiée à la caractérisation des propriétés mécaniques de myoblastes (murins et humains) et de myotubes (murins) à l'aide de la microscopie à force atomique (AFM). En modifiant ou en inhibant la dynamique du cytosquelette (CSK) d’actine de ces cellules, nous avons pu montrer que ces propriétés mécaniques variaient. L’enregistrement de courbes de force indentation nous a permis de montrer que la présence de cellules adhérentes introduisait sur les leviers d’AFM un amortissement visqueux supplémentaire à celui d’une paroi solide, et que cet amortissement visqueux dépendait de sa vitesse d’approche et que celui-ci restait non négligeable pour les plus faibles vitesses (1μm/s). Nous avons observé que les propriétés mécaniques des précurseurs de muscles devenaient non linéaires (comportement plastiques) pour des grandes déformations (>1μm) et qu’elles dépendaient de l’état, du type de cellule et de leur environnement. En combinant des expériences d’AFM, des modèles visco-élastiques et des méthodes d'analyse multi-échelle basées sur la transformation en ondelettes, nous avons illustré la variabilité des réponses mécaniques de ces cellules (de visco-élastiques à visco-plastiques). À l'aide de courbes de force-indentation, de l’imagerie morpho-structurale (DIC, microscopie à fluorescence) et de traitements pharmacologiques, nous avons éclairé le rôle essentiel des processus actifs (dépendants de l’ATP) dans la mécanique de myoblastes, en discutant tout particulièrement ceux des moteurs moléculaires (myosine II) couplés aux filaments d’actine. En particulier, nous avons montré que les fibres de stress du cytosquelette d’actine situées autour du noyau pouvaient présenter des évènements de remodelage soudains (ruptures) et que ces ruptures étaient une mesure indirecte de l’aptitude de ces cellules à tendre leur CSK. Nous avons enfin montré qu’il était possible de généraliser cette approche à des cas cliniques humains, en l’occurrence des myoblastes primaires de porteurs sains et de patients atteints de dystrophie musculaire de Duchenne, ouvrant la voie à des études plus larges sur d’autres types cellulaires et pathologies. / This interdisciplinary thesis was dedicated to the atomic force microscopy (AFM) characterization of the mechanical properties of myoblasts (murine and human) and myotubes (murine). We reported that the mechanical properties of these cells were modified when their actin cytoskeleton (CSK) dynamics was inhibited or altered. Recording single AFM force indentation curves, we showed that adherent layers of myoblasts and myotubes introduced on the AFM cantilever an extra hydrodynamic drag as compared to a solid wall. This phenomenon was dependent on the cantilever scan speed and not negligible even at low scan velocities (1μm/s). We observed that the mechanical properties of the muscle precursor cells became non-linear (plastic behaviour) for large local deformations (>1μm) and that they varied depending on the state, type and environment of the cells. Combining AFM experiments, viscoelastic modeling and multi-scale analyzing methods based on the wavelet transform, we illustrated the variability of the mechanical responses of these cells (from viscoelastic to viscoplastic). Through AFM force indentation curves analysis, morpho-structural imaging (DIC, fluorescence microscopy) and pharmacological treatments, we enlightened the important role of active (ATP-dependent) processes in myoblast mechanics, focusing especially on those related to the molecular motors (myosin II) coupled to the actin filaments. In particular, we showed that the perinuclear actin stress fibers could exhibit some abrupt remodelling events (ruptures), which are characteristic of the ability of these cells to tense their CSK. Finally, we showed that this approach can be generalized to some human clinical cases, namely primary human myoblasts from healthy donors and patients affected by Duchenne muscular dystrophy, paving the way for broader studies on different cell types and diseases.

Muscle development involves concerted action of a repertoire of mechanisms governing myoblast proliferation, migration, fusion and differentiation. Subsequently, there are cellular events administrating proper muscle function and maintenance of muscle integrity. Chapter 1 covers what is known about muscle development, building up of mass and maintenance in vertebrates and Drosophila, highlighting the myogenic programs and factors that play a role in them. The formation of vertebrate skeletal muscles can be recapitulated in Drosophila indirect flight muscles (IFMs), making IFMs an interesting model to dissect and understand the mechanisms of muscle development and maintenance. The cellular and developmental events that occur during IFM development have been discussed in detail along with their genetic control which encompasses both cell autonomous and cell non-autonomous mechanisms. The fly resources and tools used for experimentations have been described in Chapter 2. One of the hallmark events during muscle development is myoblast fusion. Myoblasts are kept in undifferentiated state until they fuse through a balanced action of anti-differentiation and pro-differentiation factors. The swarming myoblasts are in semi-differentiated state and just prior to fusion should exit cell cycle to achieve terminal differentiation. The mechanisms of cyclin/CDK complexes and their regulation via CKI (CDK inhibitor) are known in a cell. However, tissue specific factors exerting additional control on molecules that participate in cell cycle have been proposed but have not been shown in vivo. Chapter 3 uncovers a novel role played by the transcription factor, Erect wing (Ewg) in IFM development and patterning. Despite the fact that Ewg is known to express in fusing myoblasts and nuclei of developing IFMs and has long been used as a nuclear marker for IFMs, the mechanism(s) behind Ewg‟s function has remained enigmatic. Historical perspective of Ewg has been presented in Chapter 1. One set of IFMs; dorsal longitudinal muscles (DLMs) require larval templates for their formation and the other set; dorsal ventral muscles (DVMs) form de novo. Chapter 3 shows that Ewg is required in a spatio-temporal fashion to initiate myoblast fusion process. In the absence of Ewg, the number of fusion events in a given time is reduced. In addition de novo fusion is observed in the region of DLM development just like DVM and overall IFM development is delayed resulting in an aberrant adult IFM pattern. Genetic studies undertaken reveal a requirement for Ewg in exerting a temporal control on myoblast fusion. This is achieved by down-regulating Cyclin E levels, as a result of which the myoblasts exit cell cycle at G1/S stage. Through this study the proposal for DLM development and pattern has been put forth as follows: i) appropriate progression of DLM development commences on synchronous exit of myoblasts from cell cycle. This function is facilitated by Ewg expression in fusing myoblasts assisting symmetrical DLM formation in hemithoraces. ii) DLM pattern of six muscles in each hemithorax is dependent on template survival which requires fusion of enough myoblasts and further subsequent fusion events to support the splitting of three larval templates or presumptive DLM. The muscles that develop should preserve their structural integrity for efficient functional output. Muscles perform extensive activities warranting high energy requirements. IFMs are widely utilised for thorax movements that aid flight. IFMs are exclusively oxidative in nature with upto 40% mass contributed by large mitochondria themselves. Chapter 4 describes yet another novel finding for Ewg function in IFM maintenance. Vertebrate homolog of Ewg is nuclear respiratory factor 1 (NRF1) known for its role in mitochondrial biogenesis. This prompted an investigation on the role of Ewg, if any, in mitochondrial function and IFM maintenance. In this chapter, Ewg null adult IFMs are shown to undergo degeneration. Mitochondria in these muscles show rounder and smaller phenotype. Mitochondria morphology is traced throughout Drosophila pupal DLM development and extensive fusion is observed in last one-fourth of pupal phase. In Ewg null condition transcripts of Opa1-like required for inner mitochondrial membrane fusion is found to be absent, suggesting lack of mitochondrial fusion behind the smaller mitochondrial morphology. This emerged as an intriguing problem since Ewg expression follows until sarcomerogenesis (formation of sarcomeres) initiates at mid pupal stage. Developmentally extending Ewg‟s expression beyond mid pupal stage is not observed to increase Opa1-like levels pointing an indirect regulation by Ewg. However, Opa1-like knock-down beyond mid pupal stage is not observed to result in any muscle or flight defect. It is thus proposed that Ewg expression early during muscle development helps to up-regulate Opa1-like levels needed to support mitochondrial growth and fusion. In addition, this chapter provides additional data on requirement of Opa1-like for maintenance of mitochondrial as well as muscle integrity. This is the first ever report of tissue specific temporal regulation of Opa1-like by Ewg. Chapter 3 and Chapter 4 conclude spatially segregated functional requirements of Ewg which are also mechanistically distinct. Expression in fusing myoblasts channelizes fusing myoblasts to exit cell cycle and undergo timely fusion saving the larval template, subsequent fusion assists template splitting thus forming the appropriate adult DLM pattern. On the other hand expression until mid pupal stage up-regulates Opa1-like expression, facilitating mitochondrial fusion during the late pupal stage. This as a result helps maintain structural integrity of muscles in the adult. Vertebrate skeletal muscle contains multiple muscle fibres that provide appropriate mass and size to muscles. As DLM share similarity in development to that of the vertebrate skeletal muscle, DLM organisation is studied to get insights into the mechanisms which regulate the process. Chapter 5 discusses role of nuclear number and nuclear activity in determining the DLM organisation. In order to alter nuclear number, myoblast population is reduced to amounts lesser than that of the wild type and to alter nuclear activity, two nuclear encoded genes Opa1-like and Marf , involved in inner and outer mitochondrial membrane fusion respectively have been knocked down in IFMs. First, the DLM organisation is established by comparing it to the vertebrate skeletal muscle organisation. This organisation is affected on lowering the number of myoblasts destined to fuse and form IFMs, without affecting the differentiation. On the other hand, when nuclear encoded mitochondrial fusion genes are knocked down, not only DLM organisation but their differentiation is also affected. A proposal for achieving DLM organisation has been presented which should also apply to vertebrate skeletal muscle given their developmental similarity. In conclusion, the studies decipher a novel mechanism by which a transcription factor, Ewg exerts a temporal control on myoblast fusions directly influencing progression of DLM formation, and thereby, symmetry and pattern. Moreover, Ewg is also shown here to regulate mitochondrial fusion during later pupal stages helping muscles to attain greater function and maintain structural integrity. Discovery of such regulatory mechanisms controlling mitochondrial dynamics in vertebrates can open up new avenues to understand and design new therapeutic approaches to tackle mitochondrial diseases. Additionally, myoblast fusion and hence myonuclear number and their efficient functioning are shown to be important determinants of muscle organisation. This has further implications in understanding and using stem cell science in dystrophic or atrophic or ageing related muscle loss and therapy.

Les protéines d’échafaudage de la famille Elmo forment des complexes avec les facteurs d’échange de nucléotides de la guanine (GEF) de la famille des protéines Dock. Globalement, le complexe Elmo/Dock est caractérisé par une régulation conformationnelle, où au niveau basal il se retrouve dans un état fermé, en raison de la présence de sites de contact bloquant la liaison des interacteurs d’Elmo et la fonction GEF de Dock qui permet l’activation de Rac1. Une fois dans un état activé, le complexe adoptera une conformation ouverte et les sites de liaison d’Elmo seront disponibles. Par exemple, il a été démontré que la liaison des GTPases RhoG ou Arl4A au domaine RBD d’Elmo induit le recrutement du complexe à la membrane cellulaire. Le domaine DHR-2 étant alors accessible, celui-ci assurera l’activation spécifique de la GTPase Rac1 afin d’induire, entre autres, le remodelage du cytosquelette d’actine. Divers processus cellulaires seront alors d clench s, tels que la migration cellulaire, la phagocytose et la fusion des cellules musculaires (nommées myoblastes). Dans le cadre de cette thèse, nous avons étudié par l’entremise de deux objectifs l’importance de la régulation conformationnelle d’Elmo. Pour le premier objectif, nous avons étudié la régulation conformationnelle d’Elmo durant la myogenèse. La formation de fibres multi-nuclées est fondamentale pour l’établissement du muscle squelettique. Durant ce processus, la fusion des myoblastes est une étape clé pour permettre le développement et la régénération musculaire. Afin d’étudier les fonctions d’Elmo in vivo dans ce contexte, nous avons généré une série de modèles de souris. Tout d’abord, via la génération de souris double knock-out pour Elmo1 et Elmo2 (Elmo1KOElmo2cKO), nous avons démontré la fonction essentielle d’Elmo durant la fusion des myoblastes embryonnaires. En effet, uniquement des myofibres mononuclées sont observées suite à l’inactivation génétique d’Elmo1 et Elmo2. Par la suite, nous avons également généré des lignées de souris knock-in, où des mutations ont été introduites dans des domaines spécifiques d’Elmo2 afin d’induire sa conformation ouverte (Elmo2EID) ou fermée (Elmo2RBD). Nous avons ainsi démontré qu’en présence d’Elmo2EID, la capacité de fusion est augmentée et des fibres musculaires plus larges sont développées. De plus, la régénération musculaire est plus efficace chez ces souris. À l’opposé, lorsqu’Elmo2 a perdu l’activité de son domaine RBD (Elmo1KOElmo2RBD), des fibres musculaires plus étroites sont retrouvées chez les jeunes souris adultes ainsi qu’une régénération du muscle moins efficace. Finalement, nous avons démontré que l’augmentation de l’activité de la voie Elmo2-Dock1-Rac1, directement via le contrôle de la régulation conformationnelle d’Elmo2, améliore les phénotypes dystrophiques retrouvés chez les souris DysferlinKO, un modèle récapitulant la dystrophie musculaire des ceintures de type 2B (LGMD2B). Ainsi, pour la première fois, nous avons établi la possibilité d’exploiter la fusion des myoblastes en tant que thérapie régénérative pour des maladies musculaires. Pour le deuxième objectif, nous avons étudié Elmo3 dans un cas clinique dans le cadre d’une collaboration internationale. Plus précisément, des mutations bi-alléliques dans le gène Elmo3 ont été identifiées chez un jeune patient atteint d’une déficience intellectuelle. En effectuant des études biochimiques et fonctionnelles, nous avons démontré que ces mutations ont un impact sur l’activation de Rac1, sans toutefois influencer l’interaction entre les protéines du complexe Elmo3/Dock1. Cette étude apporte les premières évidences de fonctions biologiques pour Elmo3. Pour conclure, nos études ont permis de souligner l’importance d’un control approprié de la régulation conformationnelle d’Elmo. En effet, en manipulant cette régulation dans un modèle de muscle squelettique, via l’introduction d’une mutation maintenant la protéine dans une conformation ouverte, cela a permis un effet positif sur la fusion des myoblastes et sur la régénération musculaire, menant à l’amélioration des phénotypes de dystrophie musculaire.   l’opposé, la présence chez l’humain de mutations dans Elmo peut également affecter l’activation de Rac1 par Dock1, contribuant ainsi à une déficience intellectuelle chez le porteur. / The scaffold proteins Elmo forms a complex with guanine nucleotide exchange factors (GEFs) of the Dock family. The Elmo/Dock complex is characterized with a conformational regulation and at the basal level, the complex is found in a closed state, owing to the presence of contact sites blocking the binding of Elmo interactors and the GEF activity of Dock for the activation of Rac1. In their activated state, the complex adopts an open conformation and Elmo binding sites will be available. For example, binding of the GTPases RhoG or Arl4A to the RBD domain of Elmo has been shown to induce the recruitment of the complex at the cell membrane. Likewise, the DHR- 2 domain of Dock being available, the GTPase Rac1 will then be specifically activated by Dock and thus induce the remodeling of the actin cytoskeleton. Various cellular processes will then be triggered, such as cell migration, phagocytosis and muscle cell (named myoblast) fusion. In this thesis, we have emphasized the importance of the conformational regulation of Elmo by achieving two objectives. For the first objective, we studied the conformational regulation of Elmo during myogenesis, i.e. during the establishment of skeletal muscle. The formation of multinucleated myofibers is fundamental for skeletal muscle. During this process, myoblast fusion is a key step to allow the development as well as the regeneration of the muscle. In order to study Elmo in this in vivo context, we generated a series of mouse models. First, through the generation of double knockout mice for Elmo1 and Elmo2 (Elmo1KOElmo2cKO), we demonstrated the essential function of Elmo during embryonic myoblast fusion. Indeed, only mononucleated myofibers are observed following the genetic inactivation of Elmo1 and Elmo2. Subsequently, we also generated knockin mouse lines, where mutations were introduced in specific domains of Elmo to induce its opened (Elmo2EID) or closed (Elmo2RBD) conformation. Thus, we have demonstrated that when Elmo2EID is expressed, the fusion capability is increased and the myofibres are larger. Moreover, muscle regeneration is more efficient in these mice. At the opposite, when Elmo2 has lost its RBD activity (Elmo1KOElmo2RBD), smaller myofibers are observed as well as a less efficient muscle regeneration. Finally, we demonstrated that increasing the Elmo-Dock1-Rac1 pathway activity, directly through the control of the conformational regulation of Elmo, leads to the improvement of the dystrophic phenotypes found in DysferlinKO mice, a mouse model of the limb-girdle muscular dystrophy type 2B (LGMD2B). Thus, for the first time, we have established the possibility of exploiting myoblast fusion as a regenerative therapy for muscle diseases. For the second objective, we studied Elmo3 in a clinical case, as part of an international collaboration. More specifically, biallelic mutations in Elmo3 gene have been identified in a young patient with intellectual disability. Through biochemical and functional studies, we have shown that the mutations have an impact on the activation of Rac1, without however affecting the interaction between the proteins of the Elmo3/Dock1 complex. This study provides the first evidence of biological functions for Elmo3. In conclusion, our study has emphasis the relevance of the proper control of the conformational regulation of Elmo. In fact, by manipulating this regulation in a skeletal muscle model, through the introduction of a specific mutation promoting the open conformation of Elmo, it promotes myoblast fusion and induce a more efficient muscle regeneration, thus improving the dystrophic phenotypes. In contrast, the presence of human mutations in Elmo can also affect the activation of Rac1 by Dock1, hence contributing to intellectual disability in the carrier.