Orientador: Humberto Santo Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T08:31:35Z (GMT). No. of bitstreams: 1 Perez_PaulaSpanopoulos_M.pdf: 4055081 bytes, checksum: 1297b13fc85bb6918c3785c93466dff5 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Orientador: Maria Julia Marques / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T07:55:15Z (GMT). No. of bitstreams: 1 Matsumura_CintiaYuri_D.pdf: 2096053 bytes, checksum: 36802787c48fa671b02e82fdc5d9de40 (MD5) Previous issue date: 2012 / Resumo: Na Distrofia Muscular de Duchene (DMD) e em seu modelo experimental, camundongos mdx, a ausência ou disfunção da proteína distrofina leva a degeneração muscular. Acredita-se que a patogênese da DMD esteja relacionada à fragilidade do sarcolema, ao estresse mecânico e ao maior influxo de íons cálcio na fibra muscular resultante do funcionamento anormal de canais iônicos, como os canais de cálcio ativados por estiramento. O conhecimento das proteínas envolvidas na degeneração/regeneração muscular e na proteção a mionecrose da fibra muscular distrófica é essencial para a caracterização das distrofias musculares, bem como para o estabelecimento de métodos diagnósticos e de tratamentos preventivos ou terapêuticos. No presente trabalho estudamos os mecanismos de proteção a degeneração muscular, sendo dois os objetivos. No primeiro, identificamos proteínas envolvidas na proteção a mionecrose e nos processos de degeneração/regeneração muscular através do estudo proteômico dos músculos afetados ou não pela distrofia muscular em camundongos mdx e em animais controle. No segundo, verificamos a participação dos canais de cálcio ativados por estiramento na degeneração da fibra muscular através do seu bloqueio pela estreptomicina nos diferentes músculos distróficos de mdx. Adicionalmente, verificamos os potenciais efeitos secundários da estreptomicina na estabilidade do sarcolema e no tamponamento e sinalização de cálcio. Quanto ao estudo proteômico, nossos resultados sugerem que a diferença constitutiva dentre músculos afetados e não afetados pela degeneração são fundamentais para proteção a mionecrose, permitindo a manutenção da homeostase de cálcio e melhor resposta ao estresse mecânico e oxidativo. As proteínas galectina-1, anexina A1 e proteína 1 de interação com Reticulon-4 são possíveis biomarcadores para a distrofia muscular, uma vez que participam de diferentes processos celulares. Quanto a terapia farmacológica verificamos que os músculos distróficos mais afetados, como o diafragma, possuem maior quantidade de canais de cálcio ativados por estiramento e a estreptomicina atenuou a degeneração dos músculos distróficos. Diferente do efeito secundário de outros bloqueadores de canais de cálcio, a estreptomicina não alterou as proteínas do complexo distrofina-glicoproteína (beta-distroglicana e alfa-sintrofina), bem como as relacionadas ao cálcio (calsequestrina e calmodulina). Nossos resultados abrem novas perspectivas para o desenvolvimento de terapias farmacológicas relacionadas a proteínas envolvidas na degeneração muscular e na proteção a mionecrose, assim como para o desenvolvimento de métodos diagnóstico e de acompanhamento das distrofinopatias pelos marcadores moleculares / Abstract: In Duchenne Muscular Dystrophy (DMD) and in the mdx mice model of DMD, the lack of dystrophin leads to muscle degeneration. The pathogenesis of DMD is related to sarcolemmal fragility, mechanical stress and increased influx of calcium in muscle fibers, due to dysfunction of ion channels, such as the stretch-activated calcium channels. The knowledge of the proteins related to muscle degeneration/regeneration and to the protection against myonecrosis is essential to better characterize the muscular dystrophy and to establish new diagnostic tools, in addition to new preventive or therapeutic treatments. In the present study, we addressed new mechanisms of muscle protection by focusing on two main objectives. First, to identify proteins related to the protection agaisnt myonecrosis and to muscle degeneration/regeneration by performing a comparative proteomic study of spared and affected muscles of mdx and control mice. Second, to verify the involvement of stretch-activated calcium channels in dystrophic muscle degeneration by performing a drug therapy with a stretch-activated calcium channels blocker, streptomycin, in muscles that are differently affected by the lack of dystrophin, in the exercised-mdx mice. Furthermore, we were interested to see whether the stretch-activated calcium channels blocker would have additional effects on sarcolemal stability and on calcium buffering and signaling, as previously reported for other calcium channels blockers. The proteomic study suggests that constitutive differences among spared and affected dystrophic muscles are essential for muscle protection agaisnt myonecrosis, allowing a better calcium homeostasis and response to oxidative and mechanical stress. Galectin-1, annexin A1 and the protein interacting with Reticulon-4 are potencial biomarkers for muscular dystrophy, due to their involvement in different cellular processes. The drug therapy study shows that stretch-activated calcium channels participates in dystrophic muscle degeneration, showing higher levels in the mostly affected muscle, the diaphragm. Streptomycin protects the dystrophic muscles against myonecrosis, but has no further effects on other mechanisms of dystrophic muscle protection, i.e., did not improve the dystrophin-glycoprotein complex (assayed by beta-dystroglycan and alpha-syntrofin levels), nor calcium binding protein (assayed by calsequestrin and calmodulin levels). Overall, the present study opens new perspectives for the development of drug therapies to dystrophinopathies by suggesting potential new molecular markers of dystrophy (proteomic study) and by suggesting new mechanistic views to explain the differences in the response of dystrophic muscles to the lack of dystrophin (drug therapy) / 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 2018-08-21T09:08:14Z (GMT). No. of bitstreams: 1 Apolinario_LeticiaMontanholi_M.pdf: 1840325 bytes, checksum: aac9928181ab48946d6ed91bfa3a9c50 (MD5) Previous issue date: 2012 / Resumo: A Distrofia Muscular de Duchenne (DMD) é uma doença recessiva ligada ao cromossomo X. Os glicocorticoides são amplamente utilizados para o tratamento da DMD, entretanto os efeitos colaterais decorrente de seu uso contínuo motivam a busca por novas terapias farmacológicas. Ácidos graxos poli-insaturados têm sido empregados para o tratamento de várias doenças, exercendo seus efeitos através de mecanismos pouco conhecidos... O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disease that leads to myonecrosis and cardiorespiratory failure. Glucocorticoids are so far the choice treatment for DMD. However their side effects due to continuous use motivate the search for new therapies¿ The complete Abstract is available with the full electronic document / Mestrado / Anatomia / Mestra em Biologia Celular e Estrutural

Orientador: Humberto Santo Neto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T21:05:12Z (GMT). No. of bitstreams: 1 Ferretti_Renato_D.pdf: 15919724 bytes, checksum: 2e3e4ba8b562aaaaf3dde85b2e810561 (MD5) Previous issue date: 2012 / Resumo: Os músculos intrínsecos da laringe (MIL) são protegidos da mionecrose em camundongos mdx, modelo da distrofia muscular de Duchenne (DMD). A DMD caracteriza-se pela mionecrose, causada pela ausência da proteína distrofina (DIS). A DIS se une a um complexo glicoproteínas (CDG), que auxilia na estabilidade da fibra e interage com proteínas reguladoras do Ca2+. O sarcolema instável dado pela ausência da DIS, diminuição de proteínas do CDG, desregulação de canais de Ca2+ e leva ao influxo de Ca2+ exacerbado, associado à diminuição da homeostase do Ca2+ intracelular e capacidade do estresse oxidativa, resulta na mionecrose. A utrofina (Utrn), semelhante à proteína DIS, se conecta às proteínas do CDG, parece ter papel na agregação dos receptores de acetilcolina (AChRs) nas junções neuromusculares (JNM) e auxiliaria na proteção da mionecrose. Neste trabalho examinamos os níveis das proteínas do CDG (beta-distroglicana [b-DG], beta-sarcoglicana [b-SG] e alfa-sintrofina [a-SIN]), da Utrn, de canais de estoque do Ca2+ (SOCE; proteínas Orai e STIM1) e proteína relacionada ao estresse oxidativo na mitocôndria (PGC-1a) em MIL distróficos. Além disso, analisamos o padrão de distribuição da Utrn e AChRs nas JNM de MIL distróficos. A análise da b-DG, b-SG, a- SIN e da Utrn pela técnica de imunohistoquímica e western blotting, mostrou que nos MIL distróficos os níveis da b-DG e da Utrn são normais em MIL adultos comparados com controle. Com envelhecimento, os níveis de proteínas do CDG e Utrn são alterados em MIL distróficos e parece não estar relacionado a proteção da mionecrose. Com emprego de técnicas moleculares e bioquímicas para análise de canais de estoque do Ca2+ (Orai e STIM1) e proteína do estress oxidativo na mitocôndria (PGC-1a), foram observados níveis aumentados de STIM1, com similar aumento da proteína calmodulina (CaM), bem como aumento da expressão do PGC-1a em MIL distróficos. Músculos acometidos distróficos apresentaram diminuição na expressão destas proteínas. Utilizando imunohistoquimica e microscopia confocal, foi observado que o padrão de distribuição da Utrn e dos AChRs não se altera nas JNM de MIL mdx. Em fibras regeneradas tanto o padrão de distribuição da Utrn quanto dos AChRs mostraram-se alterados. Pode-se concluir que os MIL de camundongos mdx apresentam manutenção dos níveis normais de proteínas do CDG e da Utrn, bem como o aumento de STIM1, CaM e PGC-1a, poderiam auxiliar, pelo menos em parte, à proteção da mionecrose / Abstract: The intrinsic laryngeal muscles (ILM) are protected from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy (DMD). The DMD is characterized by myonecrosis, resulting from the absence of dystrophin protein. Dystrophin links the cytoskeleton to a complex of glycoprotein, the dystrophin-glycoprotein complex (DGC), which interacts with Ca2+-dependent channels for signaling and stability of the muscle membrane. In the absence of dystrophin, the sarcolemma becomes instable due to a decrease in the DGC level, deregulation of Ca2+-dependent channels, which increase Ca2+ influx, decreased Ca2+-handling and oxidative stress capacity, that result in muscle fiber necrosis. Utrophin (Utrn), similarly to the dystrophin, conects to DGC proteins, assembles the acethylcoline receptors (AChRs) at the neuromuscular junctions (NMJ) and may play a role in dystrophic muscle sparing. In this study, we examined the levels of DGC proteins (beta-dystroglycan [b-DG], beta-sarcoglycana [b-SG], alpha-syntrophin [a-SIN]), Utrn, store-operated Ca2+ channels (SOCE; STIM1 and Orai1 proteins), and mitochondrial oxidative protein (PGC-1a) in dystrophic ILM. We also examined the distribution of Utrn and AChRs in the dystrophic ILM NMJ. Immunohistochemistry and western blotting analyses of b-DG, b-SG and a-SIN, and the Utrn showed normal levels in adult dystrophic ILM compared with adult control. In aged mice, a dramatic decrease in DGC levels was observed in all dystrophic muscles compared with control. There are age-related alterations in DGC in the ILM of mdx mice, regardless of their protection against the lack of dystrophin. Using biochemical and molecular techniques to analyze SOCE proteins and mitochondrial oxidative protein (PGC-1a), we observed increased levels of STIM1, associated with increased level of calmodulin (CaM), and increased level of PGC-1a in dystrophic ILM. Dystrophic affected muscles have decreased levels of those proteins. Using molecular and biochemical methods we observed that Utrn and AChRs are fragmented only in affected muscle fibers and remaining unchanged in dystrophic MIL. We conclude that in adult dystrophic MIL the rescue of the DGC, increased levels of STIM1, CaM and PGC-1a, may be associated, at least in part, with their protection against myonecrosis / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

O objetivo deste estudo foi analisar os efeitos da suplementação com ácido ascórbico (AA) sobre os aspectos morfológicos do plexo mioentérico de camundongos mdx jovens. Foram utilizados camundongos mdx e controles C57BL/10 machos divididos em seis grupos (n=5): GC30 (Grupo controle com 30 dias de idade); GC60 (Grupo controle com 60 dias de idade); GCS60 (Grupo controle suplementado com ácido ascórbico, com 60 dias de idade); GD30 (Grupo distrófico com 30 dias de idade); GD60 (Grupo distrófico com 60 dias de idade) e GDS60 (Grupo distrófico suplementado com AA, com 60 dias de idade). Após a eutanásia, os segmentos orais do jejuno (SOJ) foram coletados e submetidos a técnicas histoquimicas de evidenciação neuronal: NADH-diaforase, NADPH-diaforase e AChE. Os resultados demonstraram um aumento significativo do peso corporal para os animais distróficos (grupos GD30 e GD60) quando comparados aos seus respectivos controles (grupos GC30 e GC60). A análise quantitativa demonstrou que os animais do grupo GD60 possui área total do intestino delgado significativamente maior que os animais do grupo GC60; Não houve diferenças significativas entre os grupos GCS60 e GDS60. Em todos os grupos de animais com 60 dias, exceto para a densidade de neurônios nitrérgicos que se apresentou maior no grupo GCS60, todos os demais parâmetros (densidade e área do perfil neuronal) foram semelhantes entre os grupos, tanto controle (GC60 e GCS60) como distrófico (GD60 E GDS60), nas duas metodologias de coloração utilizadas (NADH-d e NADPH-d). A análise qualitativa demonstrou que componentes do plexo mioentérico dos animais distróficos suplementados com AA (GDS60) evidenciados positivos a NADPH-d e AChE apresentaram manutenção dos aspectos normais do plexo mioentérico quando comparados aos animais do grupo GD60. / The aim of this study was assessment of the effects of ascorbic acid (AA) supplementation on morphoquantitative aspects of myenteric plexus of jejunum young mdx mice. Dystrophic mdx and control C57BL/10 animals were used and divided in six groups (n = 5): GC30 (control group with 30 days of age); GC60 (control group with 60 days of age); GCS60 (control group supplemented with ascorbic acid, 60 days of age); GD30 (dystrophic group 30 days of age); GD60 (dystrophic group 60 days of age) and GDS60 (dystrophic group supplemented with AA, 60 days of age). After euthanasia, the oral segments of the jejunum (SOJ) were collected and submitted to the histochemical techniques of neuronal evidencing: NADH-diaphorase, NADPHdiaphorase and AChE. The results showed a significant increase of weight in dystrophic mice (groups GD30, GD60 and GDS60) compared to its controls (groups GC30, GC60 and GCS60). The quantitative analysis showed that the GD60 group had a significantly higher small intestine total area than the GC60; no differences significant between GCS60 and GDS60 groups. All groups of animals with 60 days of age, except for the density of nitrergic neurons showed higher in GCS60 group, in all other parameters (the neuronal density and neuronal profile area) were similar between groups, controls (GC60 and GCS60) and dystrophic (GD60 e GDS60), for techniques of the NADH-d e NADPH-d.Morphological analysis of the components of myenteric plexus of dystrophic animals supplemented with AA (group GDS60) and evidenced by the techniques of NADPH-d and AChE, showed repair of the myenteric plexus aspects compared to animals of group no supplemented (GD60).

As doenças genéticas degenerativas atingem milhões de crianças em todo o mundo. Dentre essas doenças, a distrofia muscular, caracterizada como uma doença monogênica poderia ser tratada na sua origem através da terapia gênica. Assim, este estudo propõe à correção da mutação no gene da distrofina, causador da distrofia muscular através de modificações genéticas específicas. A criação de novas classes de terapêuticos que podem desencadear rearranjos no DNA genômico de maneira específica representa uma nova promessa para experimentos em terapia gênica. A tecnologia usada foi o RNA interferente (RNAi) que é utilizada para regulação da expressão gênica pós-transcricional. A Ku 70 é uma das proteínas específicas para a recombinação não homóloga, o RNAi foi usado na tentativa de atenuar a Ku70, prevalecendo então a expressão da recombinação homóloga, com intuito de corrigir a mutação gênica causadora da distrofia muscular em cães. Para tal avaliação, utilizamos linhagens de células tronco (CT) mesenquimais recentemente isoladas, oriundas de populações mononucleares da médula óssea de cães jovens afetados pela distrofia muscular, apresentando bons resultados em cultivo e caracterização. Este trabalho proporciona além da criação de uma nova terapêutica específica para a correção da distrofia muscular, o aumento do conhecimento e entendimento na indução de modificações genômicas em células, no desenvolvimento de novas classes de agentes terapêuticos moleculares que representam um grande potencial em estudos e no tratamento de várias doenças genéticas e infecciosas, degenerativas ou adquiridas. O presente trabalho apresenta métodos de isolamento e caracterização de células tronco-mesenquimais bem como a utilização de RNAi visando promover a recombinação homóloga entre o DNA transfectado e o alvo no DNA genômico. / The degenerative genetic diseases affect millions of children around the world. Among these diseases, muscular dystrophy, characterized as a monogenic disease can be treated at its source through gene therapy. Thus, this study proposes the correction of the gene that causes muscular dystrophy through genetic modification specific. The creation of new classes of therapeutics that can trigger rearrangements in the genomic DNA in a specific manner represents a new promise for gene therapy experiments. The technology will be used by RNA interference (RNAi) and that used to regulate gene expression post-transcriptional. The 70 Ku is a protein specific to the non-homologous recombination, RNAi was used in an attempt to mitigate the Ku70, then the prevailing expression of homologous recombination, aiming to correct the mutation that causes muscular dystrophy in dogs. For this evaluation, we use mesenchymal stem cell lines recently isolated populations derived from bone marrow mononuclear cells of young dogs affected by muscular dystrophy, presenting good results in characterization and culture. This work also provides the creation of a new specific therapy for the correction of muscular dystrophy, increased knowledge and understanding in the induction of genomic changes in cells in the development of new classes of molecular therapeutic agents have great potential in studies and treatment of various genetic and infectious diseases, degenerative or acquired. This paper presents methods for isolation and characterization of mesenchymal stem cells and the use of RNAi to promote homologous recombination between transfected DNA and genomic target DNA.

A Distrofia Muscular de Duchenne (DMD) é uma doença recessiva ligada ao cromossomo ?X?, causada pela ausência da proteína distrofina que ocorre em vários tecidos, sendo caracterizada por uma severa disfunção da musculatura esquelética ocasionando morte prematura do paciente. Embora controverso, alguns autores reportaram que o GH (hormônio do crescimento) estaria implicado no desenvolvimento da doença e poderia ser utilizado no tratamento da mesma. Desta forma, neste estudo a dosagem sérica de IGF-I (Fator I de crescimento Similar à insulina) um peptídeo GH-dependente que regula as ações do hormônio do crescimento, foi realizada no intuito de verificar se existe correlação ou não entre o desenvolvimento da doença e a concentração sérica de IGF-I. As variações nos níveis deste hormônio foram demonstradas com o decorrer da idade sendo que, nos três primeiros meses todos os animais apresentaram comportamento semelhante com aumento dos níveis de IGF-I, porém no quarto mês os animais Distróficos benignos apresentaram redução média de 34% deste hormônio, enquanto ocorreu aumento de 1% no animal não Distrófico. Ainda, as células granulares adenohipofisárias relacionadas à produção do hormônio de crescimento em cães Golden Retriever com Distrofia Muscular apresentam-se maiores do que nos cães Golden Retriever não distróficos quanto aos seguintes parâmetros (eixo longo, área seccional e volume celular). À microscopia eletrônica de transmissão observamos que as células estudadas apresentam grânulos elétron-densos de parede dupla e distribuídos por todo o citosol. Estes grânulos apresentaram-se maiores nos animais Distróficos quando comparados aos animais não Distróficos. / The Duchenne Muscular Dystrophy (DMD) is a X linked recessive disease, caused by the absence of dystrophin which is found in a variety of tissues and characterized by a severe disfunction of the skeletal musculature that results in a premature death of the patient. Theoretically, the growth hormone (GH) is considered to be associated to the development of Muscular Dystrophy and that could be used in its treatment. Hence in this study, a IGF-I (Insulin like growth factor-I) seric dosage was performed to verify whether or not there might be a between the evolution of the disease and IGF´s seric concentration. IGF-I is a GH-peptide dependent that regulates the GH actions during the growth. Changes in IGF-I levels were recorded during the dog?s post-natal development. On the first trimester, all animals presented similar IGF-I levels, although in the fourth month, a stark 34% decrease was observed in the dystrophic animals whereas a 1% increase was seen in the healthy dog. Furthermore, the GRMD´s granule-containing cells were larger when compared to the healthy animals. The following parameters in this comparison: long axis, cross-sectional area and cell volume. The ultrastructural study showed electron-dense granules composed by a double membrane and homogeneously distributed through the cell. These granules were larger in the dystrophic animals then in healthy dogs.

Orientador: Maria Julia Marques / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T02:52:40Z (GMT). No. of bitstreams: 1 Pereira_ElaineCristinaLeite_D.pdf: 915105 bytes, checksum: 44cd1900344bb482d9d7f8badf08d53f (MD5) Previous issue date: 2005 / Resumo: Músculos distróficos apresentam alterações no complexo distrofinaglicoproteínas, bem como ausência da enzima óxido nítrico sintase neuronal, associada a alterações na estrutura da junção neuromuscular. No presente trabalho, estudamos as respostas dos terminais nervosos e das células de Schwann terminais após lesão nervosa, na ausência de óxido nítrico (NO). Nove dias após lesão nervosa por esmagamento, 24% das junções controle (n=200) apresentaram brotamentos ultraterminais. Na ausência de NO, o mesmo foi observado em 28.5% das junções (n=217; p>0.05 comparado aos controles; X2). Quatorze dias após a lesão nervosa, todas as células de Schwann terminais observadas formaram uma rede de processos citoplasmáticos, que se estendiam para fora do sítio sináptico. Em ausência do NO, as células de Schwann terminais não apresentaram processos fora da região juncional. Estes resultados mostram que o NO está envolvido na resposta das células de Schwann terminais à lesão nervosa. Isto sugere que a sinalização molecular entre os componentes présinápticos da junção neuromuscular pode estar prejudicada nos músculos distróficos, o que poderia influenciar a inervação e sobrevivência das novas fibras musculares obtidas por terapias celulares / Abstract: Dystrophic muscles show alterations in the dystrophinglycoprotein complex and a lack of neuronal nitric oxide (NO) synthase, associated with structural changes in neuromuscular junction. In this study, we examined the nerve terminal and Schwann cell responses after a crush lesion in NOdeficient mice. Nine days after nerve crush, 24% of the control junctions (n=200) showed ultraterminal sprouts. In the absence of NO, this frequency was 28.5% (n=217; p>0.05 compared to the controls; X2 test). Fourteen days after nerve lesion, all of the Schwann cells showed an extensive network of processes away from the synaptic site, whereas in the absence of NO, Schwann cells processes failed to extend away from the endplate. These results show that NO is involved in the Schwann cell response to nerve injury. They also suggest that presynaptic molecular signaling may be impaired in dystrophic muscles, and that this could influence the innervation and survival of newly formed myofibers generated by cellmediated therapies / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

La dystrophie musculaire de Duchenne (DMD) est la conséquence de la perte de la dystrophine, une protéine cytosquelettique indispensable au maintien mécanique et fonctionnel du sarcolemme. Notre équipe a largement étudié les entrées cationiques dans les lignées murines et a montré : 1- une augmentation anormale des influx dépendant des stocks calciques (SOCE) dans les myotubes (MT) déficients en dystrophine (dys-), 2- que les influx SOCE sont médiés par les canaux TRPC1 et TRPC4, 3- que la dérégulation des SOCE dans les MT dys- est corrigée grâce à la surexpression de l'α1-syntrophine. Au jour d'aujourd’hui, il existe peu d'éléments dans la littérature quant à la caractérisation des entrées SOCEs dans les cellules musculaires humaines et dans la DMD. Ce travail de thèse s'articule autour de deux parties : Le modèle murin, dans lequel nous avons montré un rôle indispensable de STIM1 et Orai1 dans la mise en place des entrées SOCEs et l'implication de la voie Ca2+/PLC/PKC dans l'augmentation anormale de ces entrées dans les MT murins dys-. Le modèle humain primaire, dans lequel nous avons mis en évidence : 1- une augmentation anormale des influx SOCEs dans les MT DMD et établit le profil d'expression des différentes protéines nécessaires à la mise en place de ces entrées ; 2- l'implication de la voie Ca2+/PLC/PKC dans la dérégulation des SOCEs dans les MT humains DMD et le rôle de l'α1-syntrophine dans la régulation de ces entrées dans les MT humains ; 3- la dérégulation de l'homéostasie calcique dans la DMD qui se produit par l'intermédiaire des entrées cationiques dépendantes de TRPV2 dans les cellules musculaires dystrophiques. / Duchenne muscular dystrophy (DMD) is the consequence of the loss of dystrophin, a cytoskeletal protein essential for the mechanical and functional maintenance of the sarcolemma. Our group has extensively studied store-operated cation influx (SOCE) in mouse cell lines and highlighted: 1- an abnormal increase in SOCE in dystrophin-deficient (dys-) mouse myotubes (MT), 2- That SOCE are mediated by TRPC1 and TRPC4, 3- that SOCE deregulation in dys- MT is corrected by overexpression of α1-syntrophin. As of today, there is little evidence in the literature regarding the characterization of SOCE in human muscle cells and in human DMD. This thesis work is divided in two parts : In the murine model, we demonstrated an essential role of STIM1 and Orai1 in the establishment of SOCE and highlighted the involvement of Ca2+/PLC/PKC pathway in the abnormal increase of cation entry in dystrophin-deficient mouse myotubes.In primary human model, we showed: 1- an abnormal increase of SOCE in DMD MT and established the expression profile of various proteins necessary for the implementation of this influx; 2- the involvement of Ca2+/PLC/PKC in SOCE deregulation in human DMD MT and the role of α1-syntrophin in the regulation of cation entry in human MT; 3- the deregulation of calcium homeostasis in DMD that occurs through TRPV2. This work proposes a new regulatory pathway, Ca2+/PLC/PKC, for SOCE in skeletal muscle cells and provides the first elements of the disruption of calcium homeostasis in DMD human myotubes due to the absence of SOCE's regulation by the α1-syntrophin and to the overactivation of TRPV2 channels.

La dystrophie myotonique de type I (DM1) constitue la plus fréquente des pathologies musculaires héréditaires chez l’adulte. Bien qu’initialement considérée comme une maladie musculaire, la DM1 présente une atteinte neurologique très handicapante. Cette maladie autosomique dominante résulte de l’expansion anormale d’un triplet CTG dans la partie 3’UTR du gène DMPK. Un effet trans du transcrit DMPK muté entraine une dérégulation de l‘épissage alternatif dans de nombreux tissus. Cependant, les mécanismes pathologiques de la DM1 dans le cerveau restent encore peu compris. Afin de disséquer ce mécanisme, notre laboratoire a créé des souris transgéniques exprimant le transcrit DMPK avec de larges expansions CUG dans de nombreux tissus. Ces souris nommées DMSXL, recréent d’importants aspects pathologiques de la DM1, comme des anomalies du comportement et électrophysiologiques du cerveau. Elles représentent donc un excellent outil pour explorer l’effet pathologique de la mutation dans le SNC. En m’appuyant sur ce modèle, j’ai exploré dans un premier temps l’effet trans des ARNs toxiques et l’ampleur de la splicéopathie dans le SNC. De façon intéressante, certains défauts d’épissage sont régions spécifiques, et ne montrent pas d’aggravation avec l’âge des souris DMSXL. Mes résultats démontrent que les ARNs mutés sont capables de déréguler l’épissage alternatif dans l’ensemble du SNC. La région du cervelet a aussi montré des anomalies de l’épissage dans les souris DMSXL, qui, en plus, présentent des perturbations cognitives dépendantes de cette région cérébrale. Le cervelet des souris DMSXL présente aussi des déficits électrophysiologiques suggérant une dysfonction cérébelleuse et plus précisément une dysfonction des cellules de Purkinje. Dans la recherche des populations cellulaires les plus affectées dans le cervelet, j’ai démontré la présence de signes de la toxicité de l’ARN plus marqués dans la glie de Bergman, entourant les cellules de Purkinje. Pour trouver les voies moléculaires perturbées dans le cervelet, et disséquer le mécanisme derrière les anomalies observées, j’ai réalisé une approche protéomique globale et trouvé une sévère baisse de l’expression du transporteur glial de glutamate GLT1/EAAT2, suggérant une dysfonction du cervelet, en conséquence d’un possible métabolisme anormal du glutamate. L’analyse protéomique globale du cerveau des souris DM1 a aussi identifié des différences d’expression et des modifications post-traductionnelles de protéines impliquées dans la signalisation du calcium. L’étude du métabolisme des ARNm dans la DM1 a mis en évidence la dérégulation de l’épissage de gènes impliqués dans le métabolisme du calcium, soutenant l’hypothèse d’une dysfonction calcique dans le SNC. Pour étudier les conséquences de la mutation sur les variations calciques cellulaires, j’ai caractérisé un modèle cellulaire astrocytaire de la DM1. Ce modèle m’a permis de démontrer une localisation anormale du récepteur GRIN1/NMDAR1, ainsi qu’une réponse calcique anormale dans les astrocytes primaires porteurs des amplifications CTG. Malgré les avancés thérapeutiques dans le muscle, on ne sait pas à quel point les stratégies en cours de développement sont efficaces dans le SNC. Pour étudier ce problème, j’ai utilisé le modèle astrocytaire de la DM1 afin de valider in cellulo une stratégie thérapeutique qui vise à rétablir une activité normale du facteur d’épissage MBNL1 endogène. Mes travaux de thèse ont permis d’avancer dans la compréhension de la neuropathologie de la DM1. Ils ont mis en évidence pour la première fois une dysfonction du cervelet, ainsi que la possible dérégulation de la voix calcique dans le SNC. Mes résultats ont donc contribué à mieux comprendre le mécanisme de la DM1 dans le SNC, pour, à long terme, développer des approches thérapeutiques ciblant des évènements moléculaires précis. / Myotonic dystrophy type 1 (DM1) is the most frequent inherited muscular disorder in adults. Although traditionally regarded as a muscle disease, DM1 presents debilitating neurological manifestations. DM1 is an autosomic dominant disease caused by the abnormal expansion of a CTG triplet within the 3’UTR of the DMPK gene. Many molecular aspects of the DM1 are mediated by a trans effect of the expanded DMPK transcripts, whose accumulation leads to splicing deregulation in many tissues. Despite recent progress in the understanding of DM1 pathogenesis in muscle and central nervous system (CNS), the detailed molecular disease mechanism operating in the brain is still poorly understood. In order to investigate the pathophysiology, our laboratory has generated DMSXL transgenic mice expressing DMPK transcripts containing large CUG expansions in many tissues. DMSXL mice mimic important features of the DM1, notably in the CNS, showing behaviour as well as electrophysiological abnormalities. Therefore, this mouse line represents an excellent tool to investigate the toxic effects of the mutation in the CNS. Taking advantages of this transgenic model, I have first explored the trans effect of the toxic RNA and the extent of DM1-associated spliceopathy in the CNS. Interestingly, some splicing defects were region-specific, and their severity did not increase with the age of the DMSXL mice. My data demonstrate that CUG-containing RNAs have a wide deleterious effect and deregulate alternative splicing in many areas of the CNS. In addition to splicing abnormalities in cerebellum, DMSXL mice also displayed deficits in cerebellum-dependant motor coordination. Plus, DMSXL cerebellum showed electrophysiological abnormalities, suggesting cerebellar dysfunction and more precisely Purkinje cell dysfunction. In the search for the cellular populations showing the greatest susceptibility to RNA toxicity in the cerebellum, I have found extensive foci accumulation as well as pronounced splicing defects in the Bergman glia, surrounding Purkinje cells, in DMSXL and DM1 patients cerebellum. In order to identify molecular pathways and mechanisms behind the behaviour and electrophysiological abnormalities detected, I have performed a global proteomics approach and found a severe decrease in the expression of a glial glutamate transporter GLT1/EAAT2, suggesting that DM1 causes cerebellum dysfunction, through abnormal glutamate metabolism. Global proteomic analysis of DMSXL cerebellum also identified expression and post-translational changes of several proteins involved in calcium signalling. Missplicing of different transcripts involved in calcium metabolism reinforces the idea of calcium dysfunction in the neuropathogenesis of the DM1. To study the effects of toxic RNA on calcium homeostasis and flux, I have established and characterised a brain cell model of DM1. DMSXL primary astrocyte cultures allowed me to show the mislocalisation of the glutamate receptor GRIN1/NMDAR1, as well as abnormal calcium responses to stimulation. Despite recent therapeutic advances in muscle, we do not know the CNS efficiency of the therapeutic strategies currently being developed. To address this problem, I have used the DM1 astrocyte cell model to validate in cellulo a therapeutic strategy aiming to restore the activity of the endogenous splicing factor MBNL1. My thesis work provided a significant step in the understanding of the DM1 pathology in the CNS. My results revealed for the first time signs of cerebellum dysfunction in DM1, as well as signs of calcium homeostasis deregulation in the SNC. My work contributed to better understand the pathological mechanisms of DM1, the brain pathways and cell types most susceptible to toxic RNA. In the long term, my data will contribute to the rational development of therapeutic strategies targeting precise and deleterious molecular events.