L'intérêt de l’utilisation des vecteurs viraux comme le Adeno-Associated Virus recombinant (rAAV) dans la recherche pour le traitement des maladies génétiques a conduit à une évolution rapide des méthodes de production d'AAV au cours des deux dernières décennies (Ayuso et al., 2010). Leur large biodisponibilité in vivo et leur efficacité à long terme dans les tissus postmitotiques en font de bons candidats pour de nombreuses applications de transfert de gènes. En plus, la spécificité du traitement peut être augmentée lorsque le sérotype correct est choisi pour cibler un tissu spécifique. Parmi les méthodes de production actuellement utilisées, la tri-transfection de cellules embryonnaires humaines rénales 293 (HEK293) reste la plus populaire pour l'échelle de recherche; Et la production de rAAV médiée par des baculovirus pour des échelles plus importantes. L'importance croissante des vecteurs viraux dans l'application pratique de la thérapie génique exige l'amélioration des processus de production, en particulier en ce qui concerne les rendements et la pureté du produit final. Mon travail au cours de ces quatre années a été axé sur deux points principaux: (1) améliorer les processus biotechnologiques employés dans la production de rAAV pour la recherche et les échelles d'étude préclinique et (2) tester in vitro et in vivo les applications pour le rAAV dans le l’édition de genome. L'édition de gènes médiée par des nucléases spécialement conçues offre de nouveaux espoirs pour le traitement de plusieurs maladies héréditaires monogéniques. Récemment découvert, le système CRISPR Cas9 (Clustered Regular Interspaced Short Palindromic Repeats) fournit des outils importants nécessaires pour corriger les mutations par homologie. Notre modèle canonique est la souris mdx, un modèle animal naturel de la dystrophie musculaire de Duchenne (DMD). Les mutations DMD, qui conduisent à l'absence de protéine dystrophine, entraînent une myopathie progressive et fatale. Plusieurs stratégies, allant des stratégies pharmacologiques aux stratégies de saut-d’éxon, ont tenté de renverser le phénotype et ralentisser la progression de la maladie, mais les résultats ne sont pas encore satisfaisants. Ce nouvel et puissant outil d'édition de génome peut être vectorisé par rAAV. Les résultats de la première partie ont été publiés en 2015 et 2016 et seront présentés sous la forme d'articles et pour la deuxième partie, je présenterai les résultats préliminaires et les perspectives du travail qui se poursuivra dans le laboratoire. / The interest of recombinant Adeno-Associated Virus (rAAV) vectors for research and clinical purposes in the treatment of genetic diseases have led to the rapid evolution of methods for AAV production in the last two decades (Ayuso et al., 2010). Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. In addition, the specificity of the treatment can be increased when the right serotype is chosen to target a specific tissue. Among the production methods currently in use, tri-transfection of human embryonic kidney 293 (HEK293) cells remains the most popular for research scale; and rAAV production mediated by baculoviruses for larger scales. The increasing importance of viral vectors in the practical application of gene therapy demands the improvement of production processes, especially when it concerns the yields and purity of the final product. My work during these four years was focused in two main points: (1) improve biotechnological processes employed in rAAV production for research and pre-clinical study scales and (2) test in vitro and in vivo the applications for rAAV in the field of genome editing. Gene-editing mediated by engineered nucleases offers new hopes for the treatment of several monogenic inherited diseases. Recently discovered, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas9 system provides important tools needed to correct by homology-directed repair mutations. Our canonical model is the mdx mouse, a naturally occurring animal model of Duchenne Muscular Dystrophy (DMD). DMD mutations, which lead to the absence of the protein dystrophin, results in a progressive and fatal myopathy. Several strategies, from pharmacological to exon-skipping strategies, have attempt to revert the phenotype and slow down the disease progress, however results are not yet satisfactory. This new and powerful genome editing tool can be vectorized by rAAV. Results for the first part were published in 2015 and 2016 and will be presented in the form of articles and for the second part I will present preliminary results and perspectives for the work that will be continued in the lab.

OBJETIVO: Pacientes com distrofia muscular de Duchenne apresentam alterações neuronais em regiões do sistema nervoso central como no giro pós-central e cerebelo. Para entender a sua influência sobre o controle motor, nós investigamos a somestesia e destreza manual, com e sem a informação visual. MÉTODO: Participaram deste estudo quarenta meninos com diagnóstico confirmado de distofia muscular de Duchenne (idade média = 9,68; DP = 2,23 anos), sem comprometimento dos membros superiores, e quarenta e nove meninos saudáveis (idade média = 8,14 anos; DP = 1,94 anos). Para avaliar a somestesia foi utilizado o teste de discriminação entre dois pontos e o teste de estereognosia, e para avaliar a destreza manual, foi utlilizado o Pick up test de Moberg com olhos abertos e olhos fechados e o teste do erro de localização. Os resultados foram submetidos pela análise de variância (ANOVA), sendo considerado estatisticamente significante p<0,05. RESULTADOS: Os resultados demonstraram que em meninos com distrofia muscular de Duchenne, a estereognosia estava comprometida em ambas as mãos (p<0,001), no entanto não houve diferença significativa no teste de discriminação entre dois pontos (p=0,313) quando comparado aos meninos saudáveis. A destreza manual estava pior nos meninos com distrofia muscular de Duchenne quando dependia de informações somestésicas, ou seja, no Pick up test de Moberg de olhos fechados e no teste do erro de localização (p<0,001). INTERPRETAÇÃO: Os resultados demonstraram que a discriminação tátil estava intacta em meninos com distrofia muscular de Duchenne, sugerindo que a via sensorial não está prejudicada. Ao contrário, tarefas que envolviam a função cerebelar, ou seja, a percepção sensorial durante o reconhecimento ativo do objeto e o controle de alcançar e agarrar, estavam prejudicadas. Abordagens terapêuticas específicas devem ser delineadas para melhorar o controle motor e, portanto, a funcionalide de pessoas com distrofia muscular de Duchenne / AIM: Neuronal alterations were reported in the postcentral gyrus and cerebellum of patients with Duchenne muscular dystrophy. To understand their influence over motor control, we investigated somesthesis and manual dexterity, with and without visual information. METHOD: Forty boys with Duchenne muscular dystrophy (mean age = 9.68; SD = 2.23 years), with no upper limb dysfunction, and 49 healthy boys (mean age = 8.14; SD = 1.94 years) participated in the study. To assess somesthesis we used the twopoint discrimination test and the six-object stereognosis test, and to assess manual dexterity, the Moberg Pick up test with the eyes opened and closed and the localization error test. The results were submitted to ANOVA, considering statistically significant a value of p<0.05. RESULTS: Boys with Duchenne muscular dystrophy had impaired stereognosis with both hands (p<0.001), but two-point discrimination did not differ between the groups (p=0.313). Manual dexterity was much worse in Duchenne muscular dystrophy boys when somesthesic information was necessary, i.e., during the Pick up test with the eyes closed and the localization error test (p<0.0001). INTERPRETATION: Tactile discrimination is intact in children with Duchenne muscular dystrophy, suggesting that the somatic pathway may not be impaired. Unlike, tasks involving cerebellar function, i.e., sensory perception during active object recognition and reach and grasp control, are impaired. Specific therapies should be delineated to improve motor control, and therefore functionality, of people with Duchenne muscular dystrophy

OBJETIVO: Pacientes com distrofia muscular de Duchenne apresentam alterações neuronais em regiões do sistema nervoso central como no giro pós-central e cerebelo. Para entender a sua influência sobre o controle motor, nós investigamos a somestesia e destreza manual, com e sem a informação visual. MÉTODO: Participaram deste estudo quarenta meninos com diagnóstico confirmado de distofia muscular de Duchenne (idade média = 9,68; DP = 2,23 anos), sem comprometimento dos membros superiores, e quarenta e nove meninos saudáveis (idade média = 8,14 anos; DP = 1,94 anos). Para avaliar a somestesia foi utilizado o teste de discriminação entre dois pontos e o teste de estereognosia, e para avaliar a destreza manual, foi utlilizado o Pick up test de Moberg com olhos abertos e olhos fechados e o teste do erro de localização. Os resultados foram submetidos pela análise de variância (ANOVA), sendo considerado estatisticamente significante p<0,05. RESULTADOS: Os resultados demonstraram que em meninos com distrofia muscular de Duchenne, a estereognosia estava comprometida em ambas as mãos (p<0,001), no entanto não houve diferença significativa no teste de discriminação entre dois pontos (p=0,313) quando comparado aos meninos saudáveis. A destreza manual estava pior nos meninos com distrofia muscular de Duchenne quando dependia de informações somestésicas, ou seja, no Pick up test de Moberg de olhos fechados e no teste do erro de localização (p<0,001). INTERPRETAÇÃO: Os resultados demonstraram que a discriminação tátil estava intacta em meninos com distrofia muscular de Duchenne, sugerindo que a via sensorial não está prejudicada. Ao contrário, tarefas que envolviam a função cerebelar, ou seja, a percepção sensorial durante o reconhecimento ativo do objeto e o controle de alcançar e agarrar, estavam prejudicadas. Abordagens terapêuticas específicas devem ser delineadas para melhorar o controle motor e, portanto, a funcionalide de pessoas com distrofia muscular de Duchenne / AIM: Neuronal alterations were reported in the postcentral gyrus and cerebellum of patients with Duchenne muscular dystrophy. To understand their influence over motor control, we investigated somesthesis and manual dexterity, with and without visual information. METHOD: Forty boys with Duchenne muscular dystrophy (mean age = 9.68; SD = 2.23 years), with no upper limb dysfunction, and 49 healthy boys (mean age = 8.14; SD = 1.94 years) participated in the study. To assess somesthesis we used the twopoint discrimination test and the six-object stereognosis test, and to assess manual dexterity, the Moberg Pick up test with the eyes opened and closed and the localization error test. The results were submitted to ANOVA, considering statistically significant a value of p<0.05. RESULTS: Boys with Duchenne muscular dystrophy had impaired stereognosis with both hands (p<0.001), but two-point discrimination did not differ between the groups (p=0.313). Manual dexterity was much worse in Duchenne muscular dystrophy boys when somesthesic information was necessary, i.e., during the Pick up test with the eyes closed and the localization error test (p<0.0001). INTERPRETATION: Tactile discrimination is intact in children with Duchenne muscular dystrophy, suggesting that the somatic pathway may not be impaired. Unlike, tasks involving cerebellar function, i.e., sensory perception during active object recognition and reach and grasp control, are impaired. Specific therapies should be delineated to improve motor control, and therefore functionality, of people with Duchenne muscular dystrophy

Orientadores: Maria Julia Marques, Selma Maria Michelin Matheus / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T06:49:40Z (GMT). No. of bitstreams: 1 DeCarvalho_SamaraCamacari_M.pdf: 1740331 bytes, checksum: ad4bf7c2d274ea63e20314141eeb1e83 (MD5) Previous issue date: 2012 / Resumo: A distrofia muscular de Duchenne (DMD) é uma miopatia progressiva causada pela ausência da proteína distrofina e necrose muscular progressiva. No camundongo mdx, modelo da DMD, a resposta inflamatória é exacerbada e populações distintas de macrófagos, M1 e M2, influenciam a degeneração e regeneração muscular, respectivamente, regulando a progressão da doença. Anti-inflamatórios esteróides são utilizados para a terapia farmacológica da DMD. Contudo, os efeitos colaterais decorrentes do seu uso contínuo estimulam o desenvolvimento de novas terapias farmacológicas para esta doença. No presente trabalho, verificamos os efeitos do ácido eicosapentaenóico (EPA) sobre os macrófagos M1 e M2 nos músculos bíceps braquial (BB), diafragma (DIA) e quadríceps femoral (QDR) do camundongo mdx. Camundongos mdx (14 dias de vida pós-natal) receberam 300mg/Kg de EPA diluído em óleo mineral, via gavagem, diariamente, por 16 dias. Camundongos mdx não tratados e camundongos C57BL/10 não tratados receberam óleo mineral via gavagem, pelo mesmo período. Verificamos que o EPA diminuiu a mionecrose (redução da CK no plasma) e aumentou o número de fibras com núcleo periférico, principalmente no BB e DIA. Em todos os músculos estudados, o tratamento com EPA diminuiu significativamente a área total de inflamação. Nos músculos BB e DIA, o tratamento com EPA aumentou a área de macrófagos M2. No QDR, observou-se predominância de regeneração muscular após o EPA, evidenciada por extensas áreas contendo fibras com núcleo central, em diferentes estágios de regeneração. Estes resultados sugerem que o EPA altera o balanço entre os macrófagos M1 e M2, promovendo diminuição de macrófagos M1, citotóxicos, o que pode contribuir para a proteção contra a mionecrose dos músculos distróficos / Abstract: Duchenne muscular dystrophy (DMD) is a progressive myopathy characterized by the absence of dystrophin and progressive muscle necrosis. In the mdx mice model of DMD, the inflammatory response is exacerbated and distinct macrophage populations, M1 and M2, influence muscle degeneration and regeneration, respectively, regulating the progression of the disease. Antiinflammatory steroids are the choice pharmacological therapy for DMD. However, their side effects stimulate the development of new drug therapies for this disease. In the present study, we observed the effects of eicosapentaenoic acid (EPA) on M1 and M2 macrophages in the biceps brachii (BB), diaphragm (DIA) and quadriceps (QDR) muscles of the mdx mice. Mdx mice (14 days old) received 300mg/kg of EPA diluted in mineral oil by gavage, daily, for 16 days. Untreated mdx and C57BL/10 mice received mineral oil by gavage for the same period. We observed that EPA decreased myonecrosis (reduced plasma CK) and increased the number of fibers with peripheral nuclei, mainly in the BB and DIA. In all muscles, treatment with EPA significantly decreased the total area of inflammation. In DIA and BB muscles, treatment with EPA increased the area of M2 macrophage. In the QDR, there was a predominance of muscle regeneration after EPA, with extensive areas containing fibers with central nuclei at different stages of regeneration. These results suggest that EPA affects the balance of M1 and M2 macrophages in dystrophic muscles, with a trend towards a decrease in the cytotoxic M1 phenotype, which may contribute to the protection against myonecrosis in the dystrophic muscles / Mestrado / Anatomia / Mestra em Biologia Celular e Estrutural