Thèse de doctorat : Biologie cellulaire et moléculaire : Paris 12 : 2005. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 150 réf.

Reproduction de : Thèse de doctorat : Biologie : Lyon 1 : 2006. / Titre provenant de l'écran titre. Bibliogr. f. 191-209.

Duchenne muscular dystrophy (DMD) is characterized by necrosis and progressive loss of muscle fibers. DMD patients have a mutation in the gene encoding dystrophin, a large membrane-associated cytoskeletal protein on the cytoplasmic side of the sarcolemma. Gene therapy using fully deleted adenoviral vectors shows great potential for the eventual treatment of DMD and other genetic diseases. These vectors are less immunogenic than their predecessors and have the capacity to carry large DNA inserts such as the full-length dystrophin (12 kb). However, the lack of viral genes results in a weakened and subsiding (short) transgene expression in muscle. Findings in the lung and liver have shown the adenoviral E4 region, in particular E4 open reading frame 3 (ORF3) to contribute to the maintenance of transgene expression. We constructed an adenovirus in which E4 ORF3 was reintroduced into a fully-deleted adenovirus along with full-length dystrophin (AdCBDysORF3). Dystrophin levels produced by AdCBDysORF3 were found to be not sustained in mdx mice, dropping significantly by day 90. However, expression levels did increase when AdCBDysORF3 was complemented with other viral proteins such as EIB. Likewise, increasing the expression of the primary adenovirus receptor (CAR) in muscle also resulted in a higher initial dystrophin expression in myofibers.

Duchenne muscular dystrophy (DMD) is an X-linked lethal condition associated with high morbidity and mortality. There is currently no cure for this disease. Several gene-based therapeutic approaches for treating DMD are currently under development but all are dependent on the knowledge of the causative dystrophin gene mutation. A combined mutation detection approach consisting of a quantitative PCR based analysis and DNA sequencing of the dystrophin gene resulted in a mutation etection rate of 96% in the New South Wales (NSW) DMD cohort. The proportion of exon duplication mutations was twice that generally reported for similar patient opulations. The clinical utility of the combined mutation protocol for DMD carrier testing clarified the carrier status of an additional one-third (33%) of female relatives compared to a conventional approach of biochemical, pedigree and linkage studies. The generally accepted view that two-thirds of mothers of isolated cases of DMD are themselves mutation carriers is challenged. Although this assumption is valid for duplication and DNA sequence mutations, it is not valid for deletion mutations in the NSW cohort. The incidence of new cases of DMD in the New South Wales population was educed from approximately 1 in 3594 live male births to 1 in 6022 live male births over a 25 year period, indicative of a significant effect of the combination of genetic counselling and improved methods of carrier detection over that period. In a study of a cohort of boys with DMD, who had both psychological and mutational analysis, it was shown that mutations affecting the shorter, C-terminal isoforms of dystrophin are associated with decreased mean intellectual function. A hypothesis is presented that mutations within the long 5' untranslated region of the Dp140 isoform are unlikely to significantly affect expression of this brain-expressed isoform. During the course of studying the NSW DMD cohort a family was identified which exhibited X-linkage and a unique clinical presentation involving episodes of severe and prolonged muscle weakness. A novel variant in the pyruvate dehydrogenase E1 alpha subunit (PDHA 1) was identified. The phenotypic effect of this variant is not proven but a body of evidence implicates this as likely to be causative of the observed phenotype.

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr.: f. 224-254. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thèse (M.Sc.)--Université Laval, 2009. / Bibliogr.: f. [76]-97. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. 80-88. Publié aussi en version électronique.

Made available in DSpace on 2015-12-06T23:05:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2004 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundo de Auxílio aos Docentes e Alunos (FADA) / Programa de Apoio a Núcleos de Excelência (Pronex) / A distrofia muscular de Duchenne (DMD) é uma distrofia muscular que se expressa com déficit cognitivo em 20-30% dos casos. Apesar do cérebro destas crianças parecer normal, já foram encontradas alterações diversas, não explicando, porém o acometimento cognitivo. O gene da distrofina é muito grande e codifica a distrofina em várias isoformas. Deleções e duplicações em locais de ligação às isoformas distais como a Dp140, Dp71 favorecem a incidência de retardo mental. A distrofina se liga a um complexo de proteínas (CPAD), entre elas, as distroglicanas. Dentro do complexo das distroglicanas, a α-distroglicana tem sido demonstrada como importante no mecanismo, não só de estabilização da musculatura esquelética à contração e relaxamento musculares, mas também na sinaptogênese, migração neuronal e plasticidade neuronal. Seus diferentes papéis, na musculatura esquelética e no sistema nervoso central (SNC), nos levam a pensar sobre os mecanismos envolvidos em sua deficiência, bem como da distrofina, na fisiopatologia do déficit cognitivo dos pacientes com DMD. Foram avaliados 19 pacientes com DMD, sendo encontrada uma alta proporção (42,1%) com quociente de inteligência (QI) abaixo da média. Os pacientes apresentavam na época da biopsia muscular, entre 4 anos e 2 meses e 10 anos e 5 meses; e, na época das avaliações, entre 6 e 12 anos de idade. Dos 19 pacientes, 2 foram avaliados pelo Stanford-Binet. Dos 17 pacientes avaliados pelo WISC-III, 52,9% apresentaram um QI verbal menor do que a faixa média de inteligência, sendo que destes 29,4% um QI abaixo de 70. A proporção de pacientes com QI verbal dentro da faixa média de inteligência, isto é, entre a média inferior e a média superior, foi de 47%. Em nossa amostra de musculatura esquelética de biopsias de pacientes com DMD, obteve-se que, 89,5% dos pacientes apresentaram a imunoexpressão da α-distroglicana entre 0 e 25% do total das fibras musculares. Porém, um indivíduo apresentou uma imunoexpressão de 70,4%, e outro de 43,2%, ambos com performance cognitiva dentro da faixa média de inteligência, achado este não mencionado em literatura. Não houve uma relação estatisticamente significante entre os valores de QI total, verbal e de execução com a imunoexpressão da α-distroglicana em fragmentos musculares destes pacientes. A presença da α-distroglicana no cérebro, bem como da distrofina, sugere o seu papel na fisiopatologia do déficit cognitivo das crianças com DMD. Os mecanismos que envolvem a inteligência são complexos. Podemos atribuir fatores genéticos, estruturais, mas também devemos considerar os fatores ambientais e psicossociais, no caso de nossos pacientes, de grande valia no que se refere à carência de estímulos e oportunidades de aprendizado. / The Duchenne muscular Dystrophy (DMD) is a muscular dystrophy with cognitive impairment present in 20-30% of the cases. Despite of the fact that theses children’s brain looks like normal, it has already been bound a great variety of abnormalities, however not explaining the cognitive impairment. The dystrophin gene is very large and encodes several dystrophin isoforms. Deletions and duplications in linking sites to distal isoforms like Dp140, Dp71 favor mental retardation incidence. The dystrophin connects to a proteic complex (CDG), among these proteins, the dystroglicans. Within the dystroglycan complex, the α-dystroglycan, have been known as important in several mechanisms, not only at skeletal muscle stabilization at muscular contraction, but also at synaptogenesis, neuronal migration, neuronal plasticity. Its different roles, at skeletal muscle and Central Nervous System (CNS), bring us to think about the mechanisms involved when it is deficient, as well, as the dystrophin, at cognitive impairment physiopathology in DMD patients. Nineteen DMD patients were assessed, and a high proportion (42%) performed the intelligence quotient (IQ) below the average. The patients aged at the time of the muscle biopsy, between 4 and ten years, and, at the assessment time, between 6 and 12 years old. Among the 19 patients, 2 were assessed by the Stanford-Binet. Among the 17 assessed by WISC-III, 52.9% performed a verbal IQ below the average, and 29.4% below 70. The proportion of patients who performed an average verbal IQ, was 47%. In our muscle biopsies samples of DMD patients, 89.5% presented α-dystroglycan immunostaining between 0 and 25%. However, one patient presented a α-dystroglycan immunostaining of 70.4%, and the other 43.2% and who performed an average IQ, finding not mentioned in literature. There was no significant statistic relationship among total IQ, verbal IQ and execution IQ and α-dystroglycan immunostaining at these patients muscle samples. The presence of α-dystroglycan in the brain, as well as, the dystrophin, suggest their role on the pathophysiology of cognitive impairment in DMD children. The mechanisms involved in intelligence are complex. We can atribute genetic factor, structural ones, but also consider the environment and psychosocials ones, in the case of our patients, worthy if we consider the lack of stimulus and learning opportunities. / BV UNIFESP: Teses e dissertações

Duchenne Muscular Dystrophy (DMD) results from the absence of a functional dystrophin protein. Among its possible therapeutic options is the upregulation of dystrophin’s autosomal analogue, utrophin A. This can be achieved by a pharmacologically induced shift towards a slower, more oxidative skeletal muscle phenotype, which has been shown to confer morphological and functional improvements on models of DMD. Whether these improvements are a result of the utrophin A upregulation or other beneficial adaptations associated with the slow, oxidative phenotype, such as improved autophagy, has not been determined. To understand the importance of utrophin A to the therapeutic value of the slow, oxidative phenotype, we used the utrophin/dystrophin double knockout (dKO) model of DMD. We found the dKO mouse to have a similar skeletal muscle signaling capacity and phenotype to mdx mice. When treated with the adenosine monophosphate activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), both dKO and mdx mice expressed a shift towards a slower, more oxidative phenotype. In the mdx mice, this shift caused improvements in muscle fiber central nucleation, IgM penetration, damage from eccentric contractions, and forelimb grip strength. These morphological and functional benefits were not seen in the AICAR treated dKO mice. This study highlights the importance of utrophin A upregulation to the benefits of the slow, oxidative myogenic program to dystrophic mice. It confirms utrophin A as a therapeutic target in DMD and the slow, oxidative myogenic program as clinically relevant avenue towards treatment of the disease.