Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. 71-80. Publié aussi en version électronique.

Thèse (Ph. D.)--Université Laval, 2004. / Titre de l'écran-titre (visionné le 29 novembre 2004). Bibliogr.

Regensburg, Univ., Diss., 2004.

La protéine hétérotrimérique laminine-111 permet le lien entre la matrice-extracellulaire et l’intégrine α7β1 du sarcolemme, remplaçant ainsi dans les muscles dystrophiques, des liens normalement assurés par le complexe de la dystrophine. L’injection de laminine-111 dans des souris mdx a permis, entre autre, l’augmentation de l'expression de l'intégrine α7β1, d’empêcher les bris du sarcolemme lors de la contraction musculaire, de restaurer un niveau normal de la créatine kinase sérique, ainsi que d’augmenter la résistance et la force dans les muscles déficients en dystrophine. Ces résultats suggèrent que l'augmentation de la laminine-111 est un potentiel traitement pour la DMD. Les chaines β1 et γ1 de la laminine sont déjà exprimées dans le muscle humain adulte, mais la chaine α1 de la laminine (Lamα1) est exprimée uniquement pendant le stade très précoce 16 cellules de l'embryogenèse. Nous avons donc développé une méthode alternative à l’injection répétée de Laminine-111 en induisant l'expression endogène du gène LAMA1, afin de reformer le complexe trimérique α1β1γ1, la laminine 111. Ceci a été réalisé avec une technologie récente, le système CRISPR/Cas9, dont la Cas9 a été désactivée (dCas9) puis couplée à un domaine d’activation de la transcription, le VP160 (dCas9-VP160). L’utilisation d’un ou plusieurs ARN guides (ARNg) a permis de cibler le promoteur du gène LAMA1. L’ARNm de Lamα1 (qRT-PCR) ainsi que la protéine (immunohistochimie et immunobuvardage) n’ont pas été détecté dans le contrôle négatif, des myoblastes murins (C2C12). Cependant, une expression significative a été observée dans ces myoblastes transfectés avec des plasmides codant pour dCas9-VP160 et un ARNg. L’analyse protéique in vivo, dans des muscles de souris électroporés avec le même plasmide, a démontré une forte augmentation de la chaine α1 de la laminine. Des augmentations plus importantes de l’ARNm de Lamα1 ont été observées en utilisant 2 ARNg, suggérant un effet synergique. L’augmentation de l’expression de Lamα1 par le système de CRISPR/Cas9 devrait être étudiée d’avantage afin de vérifier si cette stratégie pourrait s’avérer efficace dans des cas de myopathies. / Laminin-111 protein complex links the extra-cellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by dystrophin complex. Laminin-111 injection in mdx mouse increased expression of integrin α7β1, stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased Laminin-111 is a potential therapy for DMD. Laminin β1 and γ1 chains are expressed in adult human muscle but laminin α1 (LAMA1) gene is expressed only during embryogenesis. We thus developed an alternative method to Laminin-111 protein repeated administration by inducing expression of the endogenous LAMA1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the LAMA1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. LAMA1 mRNA (qRT-PCR) and proteins (immunohistochemistry and western blot) were not detected in the control C2C12 myoblasts. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 a gRNA. Larger synergic increases were observed by using 2 or 3 gRNAs. Increased expression of LAMA1 by the CRISPR/Cas9 system will have to be further investigated to verify whether this could be a treatment for several myopathies.

No description available.

Increased calcium-activated calpain proteolysis in the sarcolemma membrane is thought to be a primary mechanism in the pathophysiology of Duchenne Muscular Dystrophy (DMD). However, few studies have tested this possibility prior to the overt signs of the dystrophy. The purpose of this study was to test the hypothesis that there is greater calpain content and total relative calpain activity in membranes obtained from dystrophic (mdx; mdx:utrophin-deficient (mdx:utrn-/-)) compared to wildtype (wt) mouse skeletal muscles during maturation at ages 7- and 21-d,and at a post-maturation age of 35-d. Calpain activity was determined as the calcium-dependent cleavage of the flurogenic substrate SLY-AMC, and content was determined by Western analysis with an anti-calpain antibody. There were several intriguing findings: 1. There was an inverse relationship between calpain content and relative activity in the whole muscle in both wt and mdx mice from age 7- to 35-d: calpain content decreased, and relative calpain activity increased as the mice aged. This suggests a similar role for calpain in both genotypes, which might relate to specific maturation processes, possibly up to age 21-d. Although the inverse relation was evident at 35-d, the targets for calpain in mdx compared to wt likely differed. 2. The increased relative calpain activity in the membrane fraction of mdx mice at age 35-d (26.73 Arbitrary Units, (AU)) compared to that of age 7- (4.9AU; p<0.05) and 21-d (8.74AU; p<0.05) is temporally related to degeneration and regeneration processes, and may also indicate activation of apoptosis, in mdx muscles at this age. 3. At age 7-d, there were no significant differences in either calpain content or relative calpain activity in all subcellular fractions for wt and mdx mice. This result might suggest similar calpain distribution and activities that are related to the regulation of muscle maturation and differentiation in both genotypes. (Note:data were not obtained for the mdx:utrn-/- mice at age 7-d because of insufficient animals). 4. At age 21-d, there was greater relative calpain activity in the myofibrillar supernatant fraction in mdx (15.13AU) than wt mice (1.18AU; p<0.05). This could indicate calpain's role in the initiation of myofibrillar protein turnover and the proteolysis of submembranous networks in the mdx muscles. 5. At age 21-d, greater calpain content in the mdx (1.40ìg) compared to wt (0.23 ìg; p<0.05) membrane fraction might suggest a broader distribution of calpain along membranes that contributes to the onset of dystrophy in the mdx muscles. 6. At age 35-d, there was greater calpain content in the mdx:utrn-/- compared to the wt membrane (0.48ìg vs 0.13 ìg), cytosolic (0.88ìg vs 0.30ìg), and myofibrillar supernatant (0.49ìg vs 0.17ìg; p<0.05 ) fractions This increased content and broad distribution across several subcellular fractions may reflect degeneration and regeneration processes, and potentially activation of apoptosis, in the mdx:utrn-/- muscles. These data suggest that calpain activity contributes to dystrophic pathophysiology mainly in the membrane fraction of mdx skeletal muscles at age ~21-d, but appears to contribute later at 35-d and in more subcellular fractions in mdx:utrn-/- skeletal muscles. / Master of Science

O objetivo deste estudo foi avaliar o efeito do uso noturno ou diurno da ankle-foot orthosis (AFO) na biomecânica da marcha de pacientes com DMD. Foram avaliados 20 pacientes deambuladores, do Ambulatório de Miopatias Infantis do CER do HCFMRP-USP, com diagnóstico de distrofia muscular de Duchenne (DMD), com idades entre 4 e 12 anos. Foi realizada a avaliação inicial (Av1) em todos os pacientes e, 7 pacientes foram reavaliados após 6 meses (Av2). Na Av1, os pacientes foram agrupados conforme o uso da órtese: grupo sem órtese (SO; n=7), grupo órtese noturna (ON; n=7), grupo órtese diurna (OD; n=6). Na Av1 e na Av2 foram obtidos dados de massa corporal, altura, composição corporal pela bioimpedância elétrica, escore funcional pela escala medida da função motora, amplitude passiva de movimento articular, força muscular isométrica pelo dinamômetro Handheld e avaliação biomecânica da marcha, na velocidade habitual do paciente. Os pacientes que faziam uso da órtese diurna foram avaliados sem e com órtese, sendo denominados grupos ODs e ODc, respectivamente. Os dados foram analisados de três formas: duas transversais e uma longitudinal. Nas análises transversais, foram realizados dois procedimentos: (i) comparando dados dos grupos SO x ON x ODs; (ii) comparando dados dos grupos SO x ON x ODc. Nestas, foi utilizado o teste ANOVA, considerando um nível de significância de 5%. Na análise longitudinal, foi realizada a análise descritiva comparando os dados obtidos na Av1 e Av2, individualmente para os 7 pacientes reavaliados. Transversalmente, o grupo ODc apresentou maiores picos do ângulo de dorsiflexão e do momento dorsiflexor, menor ângulo de flexão plantar e menor geração de potência de tornozelo (p<0,05) que o grupo SO. Porém, ao caminhar sem a AFO (grupo ODs) estes resultados não foram observados (p>0,05). Em relação ao grupo ON, o grupo ODc obteve menores picos do ângulo de flexão do quadril, de absorção de potência de quadril, do ângulo de flexão plantar e maior pico do momento dorsiflexor (p<0,05), sendo que ao retirar a AFO (ODs) essas diferenças não foram observadas (p>0,05). E ainda, o grupo ON obteve maior pico do ângulo de flexão do joelho e menor momento flexor de quadril (p<0,05) em relação ao grupo ON. Na comparação dos dados entre os grupos SO e ON, o grupo ON obteve maior pico do ângulo de flexão do joelho e maior absorção de potência de quadril (p<0,05). Na análise longitudinal individual foi observado que os 2 pacientes que iniciaram precocemente e mantiveram o uso noturno da AFO apresentaram na Av2 maior velocidade da marcha, maiores momentos extensor de quadril e flexor plantar e maior geração de potência de tornozelo, contrariamente aos paciente que interromperam o uso (noturno ou diurno) da AFO. Conclui-se que o uso diurno da AFO acarretou alterações positivas na biomecânica da marcha, minimizando compensações típicas da DMD na articulação do tornozelo. O uso noturno da AFO, quando iniciado precocemente, também afetou positivamente a marcha dos pacientes. Assim, sugere-se o início precoce e contínuo do uso diurno e noturno da AFO aos pacientes com DMD. / The aim of this study was to evaluate the effect of the ankle-foot orthosis (AFO) during nocturnal or daytime usage of the gait biomechanics in patients with Duchenne Muscular Dystrophy (DMD). Twenty ambulant patients from the Myopathies Infant Ambulatory of CER - HCFMRP-USP, were diagnosed with DMD between the ages of 4 and13 years and were evaluated. The initial evaluation (Ev1) was performed in all patients, and 7 patients were reevaluated after 6 months (Ev2). In Av1, patients were grouped according to orthosis use: group without orthosis (NoO, n = 7), group with nocturnal orthosis (NiO, n = 7), group with daytime orthosis (DO, n = 6). In Ev1 and Ev2 data were obtained according to the weight, height, body composition (bioelectrical impedance), functional score (Measure scale of motor function), passive joint range of motion, isometric muscle strength (dynamometer Handheld) and biomechanical gait analyses (usual velocity for the patient). Patients who used the daytime orthosis were evaluated with and without bracing, respectively. The data were analyzed in three ways; the first two were cross-sectional and the other one was longitudinal. In the cross-sectional analyzes, an exploratory analysis of the data from each evaluation was performed, and subsequently, the variables were compared between groups, considering the means and standard deviations. ANOVA test was used, and it was considered a significant level of 5%. In the longitudinal analysis, the description of the data obtained in the evaluation 1 compared to the data obtained in the evaluation 2 was individually performed in the 7 patients who were reevaluated. A cross-sectional analysis compared the data between NoO x NiO x DO groups considering the gait analysis data from the DO group without the orthosis (barefoot), being named DOno. The other cross-sectional analysis compared the data between NoO x NiO x DO groups considering the gait analysis data from the OD group with orthosis, being named DOwith. In individual longitudinal analysis, it was observed that patients who had started early and kept the nocturnal usage of AFO which has been already showed, in six months, an increment of gait velocity, hip extensor and plantar flexor moments and also the increment of ankle power generation, which is the opposite of the patient who has discontinued the AFO usage (daytime or nocturnal). In the cross-sectional analyzes it was observed that, compared to the NoO group, the DOwith group had a higher dorsiflexion angle peak and higher dorsiflexor moment peak (p<0.05). However, when they walked without the device these results were not maintained. There was no difference (p>0.05) between DOno and NoO groups for the kinematic parameters. And, the DOno group had lower plantar flexor moment maximum peak than the SO group (p>0.05). It was concluded that AFO daytime use cause positive changes in gait biomechanics, minimizing typical compensation of DMD in the ankle joint. The night use of AFO, when started early, also positively affected the gait of patients. Thus, it is suggested early prescription of daytime and nocturnal usage of AFO for DMD patients.

A Distrofia Muscular de Duchenne (DMD) é uma doença genética neuromuscular hereditária, ligada ao cromossomo X, sendo encontrada em humanos do sexo masculino. Esta doença muscular é descrita em outras espécies. O modelo de estudo pré-clínico GRMD (Golden Retrievier Muscular Dystrophy) apresenta sintomas clínicos fenotipicamente característicos da DMD em humanos e, por esta razão, tem sido amplamente utilizado como modelo de estudos pré-clínicos. O objetivo da presente pesquisa foi avaliar o tecido muscular, no modelo canino distrófico, ao longo da gestação. Quatro fêmeas, portadoras do gene distrófico, foram inseminadas com sêmen fresco de cães distróficos. No 25&ordm; dia, pós-inseminação, as fêmeas foram submetidas a exames de ultrassonografia para confirmar a gestação. As fêmeas gestantes passaram por uma ovariosalpingohisterectomia (OSH) para a retirada dos embriões e fetos nos seguintes períodos gestacionais: 28&ordm; , 33&ordm; , 38&ordm; e 42&ordm; dias. Em seguida fragmentos de tecido muscular foram analisados macroscopicamente e microscopicamente. Para verificar expressões proteicas, amostras de tecido foram submetidas a técnicas imunológicas, e PCR para distrofina, miostatina, e utrofina. Aos, 33&ordm; e 38&ordm; dias de gestação, no grupo distrófico, foram observadas características teciduais que corroboram com desenvolvimento tardio do tecido muscular. Os resultados para detecção proteica sugerem que, a distrofina, miostatina e utrofina foram expressas igualmente nos grupos controle e distrófico, durante todos os períodos do desenvolvimento gestacional analisado. Por fim, os dados sugerem que animais distróficos apresentam músculo sadio durante a fase gestacional, o que pode ser benéfico para testes farmacológicos em idade precoce. / Duchenne Muscular Dystrophy (DMD) is a hereditary neuromuscular genetic disease linked to the X chromosome, being found in male humans. This muscle disease is described in other species. The pre-clinical GRMD (Golden Retrievier Muscular Dystrophy) study model presents phenotypically characteristic clinical symptoms of DMD in humans and,for this reason, has been widely used as a model for preclinical studies. The aim of the present study was to evaluate the muscular tissue, in the dystrophic canine model, throughout the gestation. Four females, carriers of the dystrophic gene, were inseminated with fresh semen from dystrophic dogs. On the 25th day, post-insemination, the females were submitted to ultrasonography to confirm the pregnancy. The pregnant females underwent an ovariosalpingohisterectomy (OSH) for the removal of the embryos and fetuses in the following gestational periods: 28&ordm;, 33&ordm;, 38&ordm; and 42&ordm; days. Then fragments of muscle tissue were analyzed macroscopically and microscopically. To verify protein expression, tissue samples were submitted to immunological techniques, and PCR for dystrophin, myostatin, and utrophin. At the 33 and 38th days of gestation, tissue characteristics were observed in the dystrophic group, which corroborate the late development of muscle tissue. The results for protein detection suggest that dystrophin, myostatin and utrophin were also expressed in the control and affected groups, during all periods of the gestational development analyzed. Lastly, the data suggest that dystrophic animals present healthy muscle during the gestational phase, which may be beneficial for pharmacological tests at an early age.

Les myopathies d’origine génétique sont des pathologies musculaires en rapport avec des anomalies génétiques. Les myopathies sont à l’origine d’un handicap physique majeur et affectent souvent la fonction respiratoire et parfois le cœur. Nous nous sommes intéressés à la caractérisation myocardique de 4 types de myopathies d’origine génétique à l’aide de l’échocardiographie Doppler : myopathie de Duchenne, sarcoglycanopathies, MELAS syndrome et maladie de Pompe.Nous avons analysé la fonction cardiaque dans 2 modèles murins de dystrophies musculaires: la souris mdx et la souris sgca null. En clinique, nous avons analysé la fonction cardiaque des sujets atteints de myopathie de Duchenne, de sarcoglycanopathies, de MELAS syndrome et de maladie de Pompe en échocardiographie Doppler.Dans les modèles animaux, nous avons retrouvé des anomalies myocardiques chez la souris mdx et chez la souris sgca null. Chez l’homme, l’atteinte myocardique est sévère chez les sujets atteints de myopathie de Duchenne et certains patients présentent un asynchronisme ventriculaire soulevant les indications éventuelles de resynchronisation myocardique. Les sujets atteints de gamma sarcoglycanopathies présentent de façon significative des anomalies de contraction du ventricule gauche comparativement aux sujets atteints d’alpha-sarcoglycanopathies. La fonction ventriculaire droite et gauche est préservée chez les sujets atteints de maladie de Pompe. Les sujets atteints de MELAS présentent des hypertrophies du ventricule gauche. L’analyse génétique retrouve une corrélation significative entre le taux d’hétéroplamie et la survenue d’événements cliniques. / Muscular dystrophies are genetic neuromuscular disorders that affect skeletal muscle. We sought to assess heat involvement in four genetic muscular disorders : Duchenne muscular dystrophy, sarcoglycanopathies, MELAS and adulte Pompe disease. In animal models, we sought to assess, using Echocardiography Doppler, mdx mice and sgca null mice. Myocardiac abnormalities were found in mdx mice and sgca null mice. Clinical studies found severe cardiac impairment in Duchenne muscular dystrophies and ventricular asynchrony was found in patients with severe heart failure. Patients with gamma sarcoglycanopathy have significant alteration of left ventricular function in comparison with patients with alpha sarcoglycanopathy. Left and right ventricular function were preserved in patients with Pompe disease. Left ventricular hypertrophy was found in patients with MELAS. Genetic analysis disclosed significant correlation between heteroplasmy and significant clinical events.

Thèse (Ph. D.) -- Université Laval, 1997. / Légendes sur f. en regard des ill. Bibliogr.: f. [334]-364. Publié aussi en version électronique.