Distrofia muscular de Duchenne é uma desordem neuromuscular causada pela mutação ou deleção do gene da distrofina, a qual é ligada ao cromossomo X. Estudos recentes têm demonstrado o importante papel da distrofina no SNC, sendo sua deficiência relacionada com uma variedade de anormalidades na função do SNC, como comportamento e disfunção cognitiva. Os modelos animais mais adequados para esses estudos são os que apresentam o quadro clinico mais semelhante ao da DMD encontrada em humanos, como cães Golden Retriever com distrofia muscular (GRMD). Por não haver ainda estudos a respeito do SNC de animais GRMD, o objetivo deste trabalho foi analisar a morfologia do encéfalo dos GRMD e o de animais não distróficos, através de análise macroscópica, utilizando métodos de medição e registro fotográfico, e análise microscópica, utilizando a técnica de coloração de violeta cresil modificada. Entretanto, usando a metodologia proposta, não foi possível verificar diferenças significativas no encéfalo quando comparados os animais distróficos e os não distróficos, o que está em concordância com a literatura para a DMD usando os mesmos parâmetros. Em tempo, existe uma variação individual na morfologia do encéfalo do cão, independente de serem animais do grupo de distróficos ou controles. Outras técnicas devem ser aplicadas a fim de elucidar as consequências da ausência total ou parcial da distrofina no SNC / Duchenne muscular dystrophy is a neuromuscular disorder caused by the mutation or deletion of the dystrophin gene, which is linked to chromosome X. Recent studies have shown the important role of dystrophin in the CNS, and its related defect with a variety of abnormalities in the function of CNS, such as behavior and cognitive dysfunction. The most suitable animals models for these studies are those with the most similar clinical picture to DMD found in humans, as Golden Retriever dogs with muscular dystrophy (GRMD). There are no further studies on the GRMD animal CNS, and the aim of this study was to analyze the morphology of the brain of GRMD and not dystrophic animals through macroscopic analysis using measurement and photographic registration methods, and microscopic analysis using the modified cresyl violet staining technique. However, using the proposed methodology, we could not find significant differences in the brain when comparing the dystrophic animals and non-dystrophic, which is in agreement with the literature for DMD using the same parameters. In time, there is individual variation in dog brain morphology, whether they are animals of the dystrophic group or controls. Other techniques should be applied in order to elucidate the consequences of the total or partial absence of dystrophin in the CNS

Orientador: Humberto Santo Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T16:30:29Z (GMT). No. of bitstreams: 1 Barbin_IsabelCristinaChagas_M.pdf: 1008389 bytes, checksum: 5452120032625931602b36f22c3e2986 (MD5) Previous issue date: 2009 / Resumo: A Distrofia Muscular de Duchenne (DMD) é uma miopatia caracterizada pela ausência de distrofina, uma proteína estrutural da membrana plasmática. A ausência da distrofina faz com que a fibra muscular esquelética e cardíaca sofram alterações funcionais seguida de necrose. Com o avançar da idade cerca de 40% dos pacientes desenvolvem cardiomiopatia devido à progressiva fibrose miocárdica (FM) e vão a óbito por esta razão. Portanto, tratamento que impeça a progressão da FM é de importância em pacientes com DMD. O camundongo mdx, modelo animal da distrofia, também apresenta progressiva FM e por isso tem servido de modelo experimental para a cardiomiopatia na DMD. Parte da FM em camundongos mdx tem sido atribuída à deficiência em óxido nítrico. Desta forma, terapias baseadas na administração exógena de óxido nítrico têm sido aventadas e a L-arginina, substrato para óxido nítrico sintase tem se revelado uma candidata para tal. Esse trabalho tem como objetivo avaliar o efeito da administração de longo tempo da L-arginina sobre a progressão da FM em camundongos mdx. Os animais foram tratados com L-arginina durante 60 semanas. Os corações foram retirados e preparados para avaliação histomorfométrica da FM através de coloração com tricrômico de Masson. Lâminas coradas com hematoxilina e eosina (H&E) serviram para observação histológica e quantificação da densidade de células inflamatórias. Corações de camundongos mdx de mesma idade que aqueles tratados com L-arginina serviram como controle. Nossos resultados mostram que a área média de FM nos camundongos que receberam L-Arginina não foi diferente (p> 0,05) da dos camundongos do grupo controle (29.5 ± 2.5 % para controle vs 31.4 ± 2 % para L-arginina). De outro lado, a densidade de células inflamatórias foi significativamente inferior nos camundongos que receberam L-arginina comparados ao controle (169.3 ± 6.7 células/mm2 no controle vs 102 ± 6 células/mm2 em L-arginina). Baseado nisto, conclui-se que a administração por longo tempo de L-arginina não é capaz de prevenir a FM sugerindo que o uso da L-arginina pode não ser eficaz na prevenção da FM em DMD. Durante a realização do trabalho verificou-se que a administração de Larginina aumenta a já conhecida susceptibilidade dos camundongos mdx desenvolverem tumores. Cerca de 40% dos camundongos mdx que receberam L-arginina por longo tempo apresentaram tumores cuja análise histopatológica, incluindo-se a expressão de MyoD revela serem rabdomiossarcomas. Uma análise mais detalhada mostra que a maioria deles é do tipo embrionário e um do tipo alveolar. Esses resultados são importantes uma vez que esse protocolo poderá ser empregado para indução desse tipo de câncer e consequentemente servir como modelo experimental para o mesmo. / Abstract: The Duchenne Muscle Dystrophy (DMD) is a myopathy characterized by dystrophin absence, which is a structural protein of the plasma membrane. The dystrophin absence leads to functional alterations followed by necrosis in the skeletal and cardiac muscle fiber. As the patients grow older, about 40% of them develop cardiomyopathy due to progressive myocardial fibrosis (MF), and eventually die because of this. Therefore, a treatment that avoids the MF progression is very important to DMD patients. The mouse mdx, animal model of dystrophy, also presents progressive FM and thus, has been used as an experimental model for cardiomyopathy in DMD. The MF in mice mdx has been partially attributed to the deficiency of nitric oxide. This way, therapies based on the exogenous administration of nitric oxide have been quoted and the L-arginine, which is a substrate of the synthase nitric, has been revealed to be a strong candidate to these therapies. The aim of this current project is evaluating the effect of long-term administration of L-arginine on the progression of MF in mice mdx. The animals were treated with L-arginine for 60 weeks. The hearts were taken out and prepared for histomorphometric evaluation of the myocardial fibrosis through the colouring with Masson's trichrome. Slides stained with hematoxilin and eosin (H&E) were used for histological observation and quantification of the density of inflamatory cells. Hearts of mice mdx at the same age of those ones treated with L-arginine were used as control. Our results show that the average area of MF in mice which received L-arginine wasn't different (p>0,05) from the area of the control group (29,5 ± 2,5% for the control vs 31,4 ± 2% for the L-arginine). On the other hand, the density of inflamatory cells was significantly smaller in mice which received L-arginine compared to the control (169.3 ± 6.7 cells/mm² in the control vs 102 ± 6 cells/mm² in L-arginine). Based on this, we can conclude that the long-term administration of L-arginine is not enough to prevent the MF, suggesting that the use of L-arginine may not be effective to prevent the MF in DMD. During the research achievement, we could verify that the L-arginine administration increases the already known susceptibility of mice mdx of developing tumors. About 40% of the mice mdx which received L-arginine for a long time presented tumors whose histopathological analysis, including the MyoD expression, reveals them to be rhabdomyosarcomas. A further analysis reveals that most of them is the embrionic kind and one is the alveolar kind. These results are important since this protocol can be used to induce this kind of cancer and consequently serve as an experimental model for it. / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

A Distrofia Muscular de Duchenne (DMD) é uma patologia neuromuscular causada pela mutação ou deleção do gene da distrofina, localizado no cromossomo X, levando a degeneração muscular ao longo da vida do paciente. A doença também tem sido associada a déficit cognitivo e falta de habilidade comportamental. Pesquisas com células neurais de pacientes com DMD poderiam ajudar a elucidar os sintomas neurológicos associados. Neste trabalho, através de células-tronco pluripotentes induzidas (iPSC) derivadas da polpa de dente decíduo esfoliado (SHED) de pacientes com DMD modelamos a DMD produzindo células neurais vivas in vitro. A expressão da distrofina foi verificada durante e após a diferenciação neuronal e nos ensaios de imunofluorescência, mostrando que essa proteína está presente em células do SNC. Na análise gênica através do qPCR, a Dp71 e a Dp140, isoformas da distrofina, apresentavam uma expressão menor do que os controles. Além disso, as análises das sinapses baseada na colocalização de marcadores pré e pós-sinápticos (Sinapsina1 e Homer 1) revelaram que os neurônios dos pacientes com DMD tinham menor quantidade de sinapses que os controles, reforçando o papel da distrofina no SNC. Logo, a expressão de genes relacionados a plasticidade sináptica revelou 10 genes alterados nos neurônios dos pacientes DMD, sugerindo que a mutação no gene da distrofina possivelmente altera a plasticidade sináptica e pode estar envolvida na habilidade cognitiva destes pacientes. Desta forma, com base nos nossos achados, a modelagem neuronal de DMD é factível e pode auxiliar a elucidar os mecanismos da fisiopatologia da doença / The Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by a mutation or deletion of the dystrophin gene located on the X chromosome, leading to muscle degeneration throughout the patient\'s life. The disease has also been associated with cognitive impairment and lack of behavioral skill. Research on neural cells from patients with DMD could help to elucidate the neurological symptoms associated. In this work, through induced pluripotent stem cells (iPSC) derived from dental pulp exfoliated (SHED) of patients with DMD model the DMD producing living neural cells in vitro. The dystrophin expression was observed during and after neuronal differentiation and immunofluorescence assays, showing that this protein is present in CNS cells. In gene analysis by qPCR, the Dp71 and Dp140, isoforms of dystrophin, had a lower expression than controls. Furthermore, based on analysis of synapses colocalization pre and postsynaptic markers (Synapsin1 and Homer 1) showed that neurons of DMD patients had lower number of synapses controls, supporting a role for dystrophin in the CNS. Finally, the expression of synaptic plasticity related genes wasfound in 10 genes altered in neurons of DMD patients, suggesting that the mutation of the dystrophin gene possibly alters synaptic plasticity and may be involved in cognitive ability of these patients. Finally, based on our findings, neuronal modeling DMD is feasible and may help elucidate the mechanisms of pathophysiology of the disease

Orientador: Maria Julia Marques / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T19:42:06Z (GMT). No. of bitstreams: 1 Machado_RafaelVentura_D.pdf: 18399994 bytes, checksum: 2db06c9364e5b215f2cf4f91753c4d19 (MD5) Previous issue date: 2011 / Resumo: Na distrofia muscular de Duchenne e no camundongo mdx a proteína distrofina está ausente ou é expressa de forma não funcional. Com isso, o complexo distrofina-glicoproteínas se desorganiza, a fibra muscular se torna frágil durante os ciclos de contração e relaxamento muscular, as concentrações intracelulares de cálcio e radicais livres se elevam, resultando em necrose da célula. A inflamação mediada por células do sistema imunológico e citocinas pró-inflamatórias, como o TNF-alfa, é um evento importante diretamente relacionado com a progressão da doença. O ácido eicosapentaenóico (EPA) é um ácido graxo poli-insaturado ômega-3 que promove benefícios em doenças inflamatórias em humanos. No presente trabalho analisamos os efeitos do EPA no estágio inicial da distrofinopatia do camundongo mdx. Camundongos mdx com 14 dias de idade receberam 300 mg/kg/dia de EPA por 16 dias. Os grupos controle mdx e C57BL10 receberam óleo mineral. EPA diminuiu a mionecrose, os níveis séricos da enzima creatinoquinase e o TNF-alfa em músculos esqueléticos distróficos. Sugere-se que o EPA tenha utilidade terapêutica nas distrofinopatias / Abstract: In Duchenne muscular dystrophy (DMD) and in the mdx murine model of DMD, lack of dystrophin leads to myonecrosis and cardiorespiratory failure. The intense inflammatory reaction, mediated by immune cells and TNF-alpha, contributes to the progressive myonecrosis. The eicosapentaenoic acid (EPA) is an omega-3 fatty acid that shows beneficial effects in inflammatory diseases. In the present study, we examined the effects of EPA on the early stages of dystrophy in mdx mice. Mdx mice (14 days old) received EPA at 300 mg/kg EPA for 16 days, while the control mdx mice and C57BL10 received vehicle. EPA treatment decreased creatine kinase and TNF-alpha levels and reduced myonecrosis. The present results support further studies with EPA as a potential therapy for dystrophinopathies / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Orientador: Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T01:04:16Z (GMT). No. of bitstreams: 1 Mauricio_AdrianaFogagnolo_M.pdf: 1763243 bytes, checksum: bb94a0eae96254b7325735da6a1f32c7 (MD5) Previous issue date: 2012 / Resumo: Na Distrofia Muscular de Duchenne (DMD) e no camundongo mdx, modelo experimental da DMD, a ausência de distrofina promove instabilidade do sarcolema e degeneração muscular progressiva. O processo inflamatório que se instala contribui de forma significativa para a fisiopatologia da doença, sendo que antiinflamatórios esteroides são amplamente utilizados para a terapia da DMD. Entretanto, em decorrência da sua ação pouco efetiva e dos efeitos colaterais outras drogas são investigadas com o objetivo de substituir o uso dos corticoides. Em estudo prévio, demonstramos que o ácido eicosapentaenoico, em uma forma altamente purificada, atenuou a mionecrose em músculos esqueléticos de camundongos mdx durante os estágios iniciais da doença. No presente trabalho, verificamos se diferentes formas de ácidos graxos disponíveis comercialmente para o consumo humano (cápsulas de ômega-3 e sementes de linhaça) também teriam efeito protetor nos músculos distróficos, principalmente nos estágios tardios da doença, em que há comprometimento do músculo cardíaco. Animais mdx jovens (14 dias de idade) receberam ômega-3 diariamente, durante 16 dias, via gavagem. Animais mdx idosos (8 meses de idade) receberam sementes de linhaça durante 5 meses. Animais controle mdx, jovens e idosos, receberam óleo mineral e nenhum complemento alimentar, respectivamente. Nos animais jovens, o ômega-3 melhorou a distrofinopatia, reduzindo a mionecrose e o processo inflamatório no músculo mais afetado, o diafragma. Nos animais idosos, a linhaça resultou em melhora da distrofinopatia no diafragma. Entretanto, a fibrose cardíaca, que caracteriza morfologicamente a cardiomiopatia dos animais idosos mdx, não foi alterada pela linhaça, provavelmente devido a diferenças de metabolismo de ácidos graxos entre os músculos esquelético e cardíaco. Sugere-se que ácidos graxos ômega-3, em formulações disponibilizadas comercialmente para uso humano, são potencialmente úteis para o tratamento das distrofinopatias dos músculos estriados esqueléticos, nos estágios inicial e tardio da doença / Abstract: In Duchenne muscular dystrophy (DMD) and in the mdx mice model of DMD, absence of dystrophin promotes instability of the sarcolemma and progressive muscle degeneration. The inflammatory process contributes significantly to the pathophysiology of the disease. Anti-inflammatory steroids are widely used for DMD therapy. However, due to its ineffective action and their side effects, other drugs are investigated in order to replace the use of corticosteroids. Previously, we have demonstrated that eicosapentaenoic acid in a highly purified form inhibit myonecrosis in skeletal muscles of mdx mice during the early stages of the disease. In the present study, we have verified whether different forms of commercially available fatty acids for human (capsules of omega-3 and flaxseed) would also have a protective effect in dystrophic muscles, especially in the later stages of the disease, when cardiac muscle is also affected. Young mdx mice (14 days old) received omega-3 daily for 16 days via gavage. Older mdx mice (8 months old) received flaxseed for 5 months. mdx control group, young and old, received mineral oil and no food supplement, respectively. In young mice, omega-3 inhibits the dystrophinopathy, reducing myonecrosis and inflammatory process in the most affected muscle, the diaphragm. In aged animals, flaxseed resulted in improvement of dystrophinopathy only in the diaphragm. In the dystrophic heart, flaxseed did not inhibit fibrosis, which is a feature of cardiomyopathy in older mdx mice. Possibly, differences in fatty acid metabolism between skeletal and cardiac muscles may explain these differential results. It is concluded that omega-3 fatty acids commercially available for human use are potentially useful for the treatment of skeletal muscle dystrophinopathy, during early and late stages of the disease / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

La dystrophie musculaire de Duchenne (DMD) est la conséquence de la perte de la dystrophine, protéine sous membranaire indispensable au maintien mécanique et fonctionnel du sarcolemme. Cette déficience augmenterait les influx cationiques par des microruptures de la membrane ou par la dérégulation de canaux tels que les canaux activés par l'étirement (SACs: Stretch-activated channel). Dans ce travail, les effets d'une stimulation mécanique ont été explorés sur des cardiomyocytes dans le contexte pathologique de la cardiomyopathie dilatée associée à la DMD. L'utilisation de fibres de carbone a permis de réaliser un étirement axial similaire aux conditions physiologiques de remplissage ventriculaire. Dans ces conditions, l'exploration de la topographie membranaire par la microscopie de conductance ionique à balayage n'a montré aucune évolution de la surface ni de lésion du sarcolemmel dans les conditions d'étirement. L'étude s'est donc focalisée sur l'activité de candidats moléculaires des SACs et plus particulièrement ceux appartenant à la famille des TRPs (Transient Receptor Potential) dans le dérèglement de l'homéostasie calcique induite par l'étirement. Les influx cationiques évalués par la technique d'extinction de fluorescence et l'étude de la concentration intracellulaire de Ca2+ ([Ca2+]i) grâce à la sonde Fluo8 montrent une implication des canaux TRPV2 et TRPCs. Les premiers semblent responsables d'une entrée cationique et d'une augmentation de [Ca2+]i importante dans les cardiomyocytes mdx. Les seconds, bien que responsables d'un influx, ne participeraient pas à l'augmentation de [Ca2+]i. Ces résultats révèlent que les canaux TRPV2 pourraient jouer un rôle important dans la dérégulation calcique observée dans les cardiomyocytes déficients en dystrophine. / Duchenne muscular dystrophy (DMD) is the consequence of the loss of dystrophin, a subsarcolemmal protein essential for mechanical and functional maintenances of the sarcolemma. This deficiency could increase cationic influxes by membrane microruptures or by dysregulation of channels such as stretch-activated channels (SACs). In this work, the effects of a mechanical stretch were explored on cardiomyocytes in the pathological context of dilated cardiomyopathy associated with DMD. Using carbon fibers, an homogenous axial stretch was performed to mimic physiological conditions of ventricular filling. In these conditions, exploration of membrane topography using the scanning ion conductance microscopy did not show any surface evolution or sarcolemma disruption in stretch condition. The study was thus focused on activity and identification of molecular candidates for SACs, especially the TRPs (Transient Receptor Potential) channels in the stretch-induced. Ca2+ homeostasis dysregulation. Cationic influxes assessed by Mn2+-quenching and assessment of the intracellular Ca2+ concentration ([Ca2+]i) using fluo-8 fluorescence demonstrated an involvement of TRPV2 and TRPCs channels. The first ones seem to be responsible for cationic entry and [Ca2+]i increase in mdx cardiomyocytes. The latter, though responsible for an influx, do not contribute to [Ca2+]i increase. These findings reveal that TRPV2 channels could play an important role in calcium dysregulation observed in dystrophin-deficient cardiomyocytes.

La thérapie cellulaire est une envisagée pour traiter des pathologies cardiaques ou squelettiques basée sur la médecine régénérative. Les progéniteurs cellulaires classiquement utilisés (myoblastes ou cellules mésenchymateuses) n'ont démontré qu'une efficacité limitée. Dans ce contexte, notre laboratoire a identifié une nouvelle catégorie de progéniteurs, sur la base de leur activité enzymatique aldéhyde déshydrogénase (ALDH) mise en évidence par un substrat fluorescent, l'Aldéfluor, et en association avec le marqueur CD34. Les ALDH sont impliquées dans le métabolisme et la détoxification des aldéhydes, et constituent un nouveau marqueur des cellules souches. Ce travail de thèse a permis de mieux caractériser les progéniteurs myogéniques (ALDH+/CD34-) et non myogéniques (ALDH+/CD34+), dans différents contextes physiopathologiques. Leur présence dans différents muscles de primates humains ou non humains, leur persistance au cours du vieillissement naturel ou lors d'atteinte par la dystrophie musculaire de Duchenne (DMD) chez l'Homme et dans des modèles animaux, suggèrent une utilisation possible des cellules ALDH+/CD34- pour des développements thérapeutiques ultérieurs. L'étude phénotypique révèle que des marqueurs transmembranaires sont associés à des sous-populations de cellules ALDH myogéniques ou non myogéniques dont la comparaison permettra de proposer de meilleurs candidats de thérapie cellulaire. En parallèle, les caractérisations histologiques et cytologiques ont identifié des sous-populations exprimant des isoenzymes et les analyses d'expressiongénique réalisées ex vivo et en culture suggèrent que certaines sont impliquées dans l'homéostasie musculaires. / Cell therapy is a regenerative medicine strategy considered for the treatment of cardiac or skeletal muscle diseases. The cellular progenitors used to date (myoblasts or mesenchymal stem cells) provided mitigated success, thus mandating the identification and characterization of new categories of progenitors. Our laboratory has identified new populations of progenitors, based on their Aldehyde Deshydrogenase activity (ALDH) detectable using the fluorescent substrate Aldefluor, associated with the expression of the CD34 marker. ALDH are involved in metabolism and detoxification of aldehydes, they play important roles in cell survival and differentiation and are considered a new marker of stem cells. The present project allowed characterizing extensively the myogenic (ALDH+/CD34-) and non myogenic (ALDH+/CD34+) progenitors, in several physiopathological contexts and animal models. The presence of ALDH+/CD34- cells in distinct muscle groups in Human and non-human Primates, their persistence through natural ageing and despite the ongoing degenerative process observed in Duchenne muscular dystrophy in Human patients and animal models suggest their future use for therapeutic applications. The phenotypic characterization indicated that membrane markers are associated to myogenic or non myogenic sub-populations of ALDH cells. The comparison on their efficacies in vito and in vivo will allow proposing new candidates for cell therapy. In parallele, histological and cytological analysis identified cell populations expressing isoenzymes The analysis of gene expressions suggested that, at least, some of them are involved in muscle homeostasis in situ or in vitro.

La Dystrophie Musculaire de Duchenne (DMD) est la plus fréquente et la plus sévère des maladies dégénératives du muscle. Elle se caractérise par une dégénérescence progressive des fibres musculaires due à l’absence de dystrophine fonctionnelle dans les muscles. Actuellement, le rôle physiologique de la dystrophine n’est pas clairement établi et il n’existe pas encore de traitement curatif pour cette maladie. La difficulté de mettre en évidence la fonction de la dystrophine et la physiopathologie de la DMD est en partie expliquée par la complexité moléculaire et cellulaire du muscle des modèles vertébrés utilisés dans les études actuelles. Notre équipe de recherche a développé un modèle de DMD chez le nématode Caenorhabditis elegans. Dans ce modèle, la mutation du gène de la dystrophine, provoque une dégénérescence progressive des muscles conduisant à une paralysie des animaux adultes. Nous utilisons ce modèle afin d’étudier la fonction de la dystrophine et les mécanismes impliqués dans la dégénérescence musculaire chez le nématode. Ce travail de thèse porte sur deux nouveaux acteurs de la dégénérescence musculaire dépendante de la dystrophine : la protéine DYC‐1 et son principal partenaire ZYX‐1. Ce travail présente la caractérisation de ces deux protéines et étudie leurs fonctions dans le muscle. Par ailleurs, ce travail de thèse présente les premiers résultats d’un projet de microscopie électronique ayant pour but de caractériser en détail les évènements subcellulaires du processus dégénératif au cours du cycle de vie du nématode dystrophique. À plus long terme, les études chez le nématode permettront de proposer de nouvelles hypothèses quant aux mécanismes moléculaires et cellulaires de la dégénérescence musculaire / Duchenne Muscular Dystrophy (DMD) is the most prevalent and one of the most severe muscular dystrophy. DMD is due to the absence of functional dystrophin in cardiac and skeletal muscle cells, this lack leads to a progressive muscle degeneration of contractile fibres. Currently, the physiological role of dystrophin is not yet clearly established and curative treatments for DMD are not yet available. The lack of knowledge about dystrophin function and DMD physiopathology can be partly attributed to the complexity of vertebrate muscle, and the absence of a simple model that emulates the human pathology. Our research team developed a model of muscle degeneration in the nematode Caenorhabditis elegans. In this model, the mutation of the dystrophin gene produces a progressive muscle degeneration leading to the paralysis of the adult worms. We use this model for investigating the role of dystrophin and the mechanisms of muscle degeneration in C. elegans. This PhD work concerns two new actors of dystrophin‐dependant muscle degeneration: The DYC‐1 protein and its main interactor ZYX‐1. This study aims to characterise these proteins and to study their muscle functions. Moreover, this PhD work presents preliminary results of an in depth characterisation of subcellular processes of muscle degeneration in dystrophic worms by electron microscopy. Our aim is to visualise first events and to observe the progression of degeneration until the death of muscle cell. These molecular and cellular approaches aims to get new insights in the mechanisms underlying muscle degeneration in order to propose new hypotheses for the understanding of DMD

L'objectif de ce travail est de développer la partie clinique de la banque de données du gène DMD, afin d'étudier l'histoire naturelle des dystrophinopathies et les corrélations génotype–phénotype, et de faciliter la sélection des patients pour les futurs essais thérapeutiques. La méthodologie créée pour le gène DMD peut être généralisée et utilisée pour d'autres banques de données dédiées à des maladies génétiques. La collecte de 70 000 données cliniques chez 600 patients avec un suivi longitudinal moyen de 12 ans permet de décrire l'histoire naturelle des dystrophies musculaires de Duchenne et de Becker et des formes symptomatiques chez les femmes. Nous avons pu préciser l'hétérogénéité phénotypique sur le plan moteur, orthopédique et respiratoire (forme sévère et forme intermédiaire de la dystrophie musculaire de Duchenne), sur le plan cardiaque (absence de corrélation entre les atteintes motrice et cardiaque, variabilité de l'atteinte cardiaque), et sur le plan cérébral (atteinte intellectuelle chez les patients avec dystrophie musculaire de Becker, troubles psychologiques des dystrophinopathies). L'utilisation de cet outil par les cliniciens et les généticiens devrait faciliter le travail de recherche clinique et la réalisation des futurs essais cliniques. Ceci nécessite maintenant de développer l'accessibilité de la banque de données et d'envisager sa pérennisation. / The objective of this work is to develop the clinical part of the French dystrophinopathy data-base, in order to study the natural history and the genotype-phenotype correlations, and to facilitate the selection of the patients for the future therapeutic trials. The methodology developed for the DMD gene can be generalized and used for the other databases dedicated to genetic diseases. The collection of 70 000 clinical data for 600 patients with an average lon-gitudinal follow-up of 12 years allows to clarify the natural history of the muscular dystrophies of Duchenne and Becker and in symptomatic females. We were able to specify the pheno-typic heterogeneity of the motor, orthopaedic and respiratory involvements (severe form and intermediary form of the Duchenne muscular dystrophy), of the cardiac disorder (absence of correlation between motor and cardiac involvements, variability of the cardiomyopathy), and of the brain function (mental deficiency in the patients with Becker muscular dystrophy, psychological disorders in dystrophinopathies). The use of this tool by the clinicians and the ge-neticists should facilitate their clinical research work and the realization of the future clinical trials. This requires now to develop the accessibility of the database and to ensure its continued existence.

La dystrophie musculaire de Duchenne (DMD) est une maladie liée à l'X caractérisée par la détérioration progressive des muscles en raison de l'absence de la protéine dystrophine. Les muscles atteints chez l'homme ou dans des modèles animaux (souris mdx) présentent une fibrose, accumulation excessive de protéines de la matrice extracellulaire. Parmi les facteurs induisant la fibrose se trouvent le Facteur de Croissance de Transformation de type β (TGF-β) et le Facteur de Croissance du Tissu Conjonctif (CTGF). Ce dernier est une cible de la voie de signalisation médiée par TGF-β/SMAD et est responsable des effets profibrotiques de TGF-β. La régulation de l'expression de CTGF médiée par TGF-β dans les cellules musculaires est peu connue. Nous décrivons ici un nouvel élément de liaison SMAD situé dans la région 5’UTR du gène de CTGF, important pour l'expression de CTGF médiée par le TGF-β dans des myoblastes. De plus, nos résultats suggèrent que d'autres sites de liaison du facteur de transcription présents dans le 5’UTR du gène de CTGF sont importants pour cette expression.Par ailleurs, le Facteur de Nécrose Tumorale (TNF) est une cytokine inflammatoire présente dans les muscles atteints de DMD et est responsable de la nécrose du muscle et de l'infiltration de cellules inflammatoires. Nous montrons que l’expression du récepteur soluble TNFRI par électrotransfert (ET) dans le muscle tibialis anterior de la souris atténue l'inflammation, les dommages et la fibrose dans le muscle squelettique des souris mdx, et provoque une augmentation de la force musculaire. Par conséquent, nous proposons l'ET comme thérapie efficace anti-TNF pour le traitement de dystrophies musculaires. / The Duchenne Muscular Dystrophy (DMD) is an X-linked disease characterized by progressive damage in the muscle due to the absence of the dystrophin protein. Fibrosis, the excessive accumulation of extracellular matrix (ECM) proteins, is also present in the muscle of DMD patients and several animal models (such as the mdx mice). Among the factors that induce fibrosis are Transforming Growth Factor type β (TGF-β) and Connective Tissue Growth Factor (CTGF), the latter being a target of the TGF-β/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-β and are augmented in fibrosis tissues. Little is known about the regulation of the expression of CTGF mediated by TGF-β in muscle cells. In here, we described a novel SMAD Binding Element (SBE) located in the 5’ UTR region of the CTGF gene important for the TGF-β mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites present in the 5’ UTR of the CTGF gene are important for this expression. On the other hand, the Tumor Necrosis Factor (TNF) is an inflammatory cytokine that is present in DMD muscles and is responsible for muscle necrosis and inflammatory cell infiltration. In this study, we show that the increased expression of the soluble TNF Receptor I by electrotransfer (ET) in the tibialis anterior muscle attenuates inflammation, damage and fibrosis in the skeletal muscle of the mdx mice. In addition, we found increased muscle strength in the mdx mice. Therefore, we propose that ET could be used as an efficient anti-TNF therapy for treating muscle dystrophies.