Thesis (Ph.D. in Epidemiology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 83-102).

Objetivo: Construir Escala de Avaliação Funcional do Sentar e Levantar do Solo para Portadores de DMD (EAF-2) e testar sua confiabilidade intra e interexaminadores. Método: A construção da escala ocorreu em etapas: 1. Análise do movimento de sentar e levantar do solo em crianças saudáveis. 2. Análise do movimento de sentar e levantar do solo em crianças com DMD. 3. Elaboração da primeira versão da escala e do manual de instrução. 4. Avaliação por peritos e reajustes gerando a versão final. 5. Análise de confiabilidade inter e intraexaminador e correlação com a Escala de Vignos, idade e tempo de execução da atividade. Resultados: A escala abrange três fases para o sentar e cinco para o levantar, cada fase contendo itens que devem ser avaliados e pontuados. O escore pode variar de 0 a 10 para o sentar e de 0 a 15 para o levantar. Foi demonstrado muito boa repetibilidade da medida do sentar e levantar (ICC = 0,89 e 084, respectivamente) e excelente reprodutibilidade (ICC = 0,93 e 0,92, respectivamente). O Coeficiente Kappa para as 8 fases na análise interexaminadores variou de 0,77 a 1,00 (confiabilidade excelente para 5 fases e substancial para 3 fases), e na análise intra-examinador variou de 0,80 a 1,00 (confiabilidade excelente para 6 fases e substancial para 2 fases). Encontrou-se boa correlação entre as variáveis idade x Escala de Vignos (r= 0,58) e levantar x Escala de Vignos (r= 0,56), enquanto que nas variáveis restantes a correlação foi baixa.Conclusão: A EAF-2 é um instrumento de avaliação confiável que permite avaliar a atividade de sentar e levantar em portadores de DMD de forma detalhada e operacionalizada. / Objective: Construct the Scale of Functional Evaluation of Sit-and-Stand from the Ground for Patients with DMD (EAF-2) and to test its reliability intra and interexaminer. Method: The construction of the scale occurred in stages: 1. Analysis of the movement to sit and stand from the ground in healthy children. 2. Analysis of the movement to sit and stand from the ground in children with DMD. 3. Elaboration of the first version of the scale and the manual of instruction. 4. Evaluation by experts and readjustments generating the final version. 5. Analysis of Reliability inter and intra-examiner and correlation with the Vignos Scale, age and time length for the execution of the activity. Results: The scale comprehends three phases for the sitting and five for the standing, each phase with items that must be evaluated and scored. The score may vary from 0 to 10 for the sitting and from 0 to 15 for the standing. A very good repeatability of the measure of sitting as well as of standing was demonstrated (ICC = 0,89 and 084, respectively) and excellent reproducibility (ICC = 0,93 and 0,92, respectively). The Kappa Coefficient for the 8 phases in the interexaminer analysis varied from 0,77 to 1,00 (excellent reliability for 5 phases and substantial for 3 phases), and in the intra-examiner analysis varied from 0,80 to 1,00 (excellent reliability for 6 phases and substantial for 2 phases). Good correlation was found between the variable age x Vignos Scale (r= 0,58) and to stand x Vignos Scale (r= 0,56), whereas in the remaining variable the correlation was low. Conclusion: The EAF-2 is a trustful instrument of evaluation that allows to evaluate the activity of sitting and standing in people with DMD in a detailed and operationalized way.

No description available.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

Résumé de l'étude in vitro. L'objectif de cette étude in vitro était de créer un fantôme avec la même architecture musculaire (fibre, aponévrose ...) et les mêmes propriétés mécaniques que le muscle en condition passive et active. Deux fantômes homogènes ont été fabriqués avec différentes concentrations de plastisol pour simuler les propriétés élastiques du muscle en condition passive (50% plastisol) et active (70% de plastisol). Pour cela, des fils en Téflon (d = 0,9 mm) ont été insérés dans la partie supérieure du fantôme (50%) pour représenter les fibres musculaires. De plus, une feuille en matière plastique (8 x 15 cm) a également été placée au milieu du fantôme pour imiter la structure de l'aponévrose. Ensuite, des tests ERM ont été effectués à 90 Hz avec deux stimulateurs pneumatiques de différentes formes (tube en silicone, membrane circulaire) pour analyser l'effet du type du stimulateur sur la propagation des ondes. La longueur d'onde a été mesurée à partir des images phase et les propriétés élastiques (module de cisaillement) ont été calculées. Les deux fantômes (50% et 70%) ont montré des propriétés élastiques similaires à celles du muscle à l’état passif (2,40 ± 0,18 kPa) et actif (6,24 ± 0,21 kPa). Le stimulateur en forme de tube a donné des valeurs plus élevées (environ 1,2 kPa à 1,53 kPa). L'analyse du comportement des ondes a révélé un glissement le long de la feuille plastique. Ce phénomène a aussi été observé in vivo le long de l’aponévrose. L'onde a également été sensible à la présence des fils en téflon car des coupures, des trous, ont été identifiés au cours de la propagation de l’onde. Une nouvelle méthode de post-traitement a été créée pour mesurer les paramètres G' et G" à partir de tests ERM réalisés à plusieurs fréquences (60, 80, 100 Hz) et en utilisant des modèles rhéologiques. Cette méthode a été testée sur un fantôme (50%) qui n’avait pas d’inclusion. Les résultats des mesures viscoélastiques (G', G") ont été validés avec la technique HFVS (Hyper-Fréquence viscoélastique Spectroscopy). Des valeurs similaires, G' et G’’, ont été obtenues avec les deux techniques. Ce dernier résultat valide la nouvelle méthode de post-traitement pour mesurer les propriétés viscoélastiques. Résumé de l'étude in vivo. L'objectif de cette étude in vivo a été de développer des protocoles ERM pour caractériser les propriétés élastiques (module de cisaillement) des neuf muscles de la cuisse. Ces tests ont été effectués à une seule fréquence (90 Hz). Différents modules de cisaillement ont été trouvés entre les muscles. Le gracilis a révélé des propriétés élastiques plus élevées que les autres muscles. Ces différentes élasticités peuvent être dues à différentes compositions physiologiques et architecturales entre les tissus. Ensuite, les propriétés viscoélastiques des muscles ischio (ST, SM, et la BC) et du muscle Gr ont été déterminées en appliquant la nouvelle méthode de post-traitement des données (précédemment validée sur le fantôme 50%) avec des tests ERM multi fréquences (70, 90 et 120 Hz) et en utilisant des modèles rhéologiques. Les résultats ont montré que deux modèles rhéologiques, Zener et springpot, peuvent être utilisés pour mesurer les propriétés viscoélastiques des muscles à l’état passif. De plus, des résultats similaires ont été trouvés entre G "/ G ', obtenus expérimentalement à 90 Hz, et la valeur α du modèle de springpot. / Summary of the vitro studies. The objective of this in vitro study was to create a phantom witch the same muscle architecture (fiber, aponeurosis …) and mechanical properties of muscle in passive and active states. Two homogeneous phantoms were manufactured with different concentrations of plastisol to simulate the muscle elastic properties in passive (50% of plastisol) and active (70% of plastisol) muscle conditions. Moreover, teflon tubing pipes (D = 0.9 mm) were thread in the upper part of the phantom (50%) to represent the muscle fibers and a plastic sheet (8 x 15 cm) was also included in the middle of the phantom to mimic the aponeurosis structure. Subsequently, MRE tests were performed at 90Hz with two different pneumatic drivers, tube and round shapes, to analyze the effect of the type of driver on the wave propagation. The wavelength was measured from the phase images and the elastic properties (shear modulus) were calculated. Both phantoms revealed elastic properties which were in the same range as in vivo muscle in passive (2.40 ± 0.18 kPa) and active (6.24 ± 0.21 kPa) states. The impact of the type of driver showed higher values with the tube (range: 1.2 kPa to 1.53 kPa). The analysis of the wave behavior revealed a sliding along the plastic sheet as it was observed for in vivo muscle study. The wave was also sensitive to the presence of the fibers where gaps were identified. A new post processing method was established to measure G’ and G” from experimental multi frequencies (60, 80, 100 Hz) MRE (MMRE) tests and rheological models. This method was tested on the phantom (50%) made without fiber. Cross validation of the viscoelastic (G’, G”) results was made with Hyper-Frequency Viscoelastic Spectroscopy (HFVS). Both techniques showed similar range of values for G’ and G” at the same frequencies. This last result validated our new data processing for the viscoelastic measurement. Summary of the in vivo studies. The objective of this in vivo study was to develop MRE protocols to characterize the elastic properties (shear modulus) of the nine thigh muscles. These tests were performed at a single frequency (90Hz). Different shear moduli were found between the muscles. The gracilis revealed the highest elastic properties compared to all the other muscles. These different elasticities may be due to different physiological and architectural compositions between the tissues. Then the viscoelastic properties of the ischio (ST, SM, and BC) and Gr muscles were determined based on our new data-processing method (validated on the phantom 50%) using MMRE tests (70, 90 and 120Hz) and rheological models. The results revealed that two rheological models, zener and springpot, can be used to measure the viscoelastic properties in passive state. A similar trend was found between the experimental ratios G”/G’ obtained at 90 Hz and the α value of the springpot model. The present MRE muscle protocol, and the viscoelastic data base, could be used as non-invasive diagnostic tools to evaluate tissue alterations, the progression of diseases, and the effect of treatments, such as the ongoing therapeutic trials for Duchenne muscular dystrophy.

Myofibrillogenesis is a complex process, which involves assembly of hundreds of structural proteins in a highly ordered manner to form the contractile structural unit of muscle, the sarcomere. Several myopathic conditions reported in humans are caused due to abnormal myofibrillogenesis owing to mutations in the genes coding for many of these structural proteins. These myopathies have highly variable clinical features and time of onset. Since their aetiology is poorly understood, it becomes imperative to have a model system to study the muscle defects. Present study proposes to employ the Indirect Flight Muscle (IFM) system in Drosophila melanogaster as a model to analyse the development/onset of some of these myopathies and resulting pathophysiology. We have carried out a systematic study on mutations in two major proteins of the sarcomere, actin and myosin, to understand the pathophysiology associated with the disease conditions and in turn gain insights into the process of myofibrillogenesis. To verify whether the human muscle phenotypes are observed in flies, we analysed the IFM for functional and structural defects categorised by the presence of aberrant sarcomeric structures. An important question that we have addressed is whether mutants of the Drosophila IFM recapitulate human conditions and whether it can serve as a good genetic model to study the developmental mechanisms of the human skeletal myopathies in vivo. Mutations of the human ACTA1 skeletal actin gene produce seven congenital myopathies – actin myopathy, nemaline rod myopathy, intranuclear rod myopathy, congenital fibre type disproportion, congenital myopathy with core-like areas, cap disease and zebra body myopathy. Four known mutations in Act88F—a Drosophila homologue of ACTA1—occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for muscle phenotypes with nemaline structures. Mutant homozygotes show phenotypes ranging from lack of myofibrils to almost normal sarcomeres at eclosion. Whereas, heterozygotes do allow myofibrillar assembly to certain extent; however, atypical structures are seen adjacent to normal sarcomeres. Aberrant Z disc-like structures and serial Z disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all the four Act88F mutants. The electron-dense structures observed in electron micrographs show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. A possible mechanism for the ‘zebra bodies’ is proposed based on this study. Analysis of IFM at early developmental stages shows that in three of the mutants, there is an abnormal myofibril assembly leading to malformed sarcomeres mirrored in the adult stages. In one of the Act88F mutants, normal myofibrils are seen post-eclosion but the IFM show activity dependant progression of muscle degeneration. All the Act88F mutants produce dominant disruption of muscle structure and function which cannot be rescued even by three copies of the wild type Act88F gene implying that the mutants are strong antimorphs. Myosin myopathies are a group of human muscle diseases with heterogeneous clinical features and are caused by mutations in the skeletal muscle myosin heavy chain. We identified two chemical mutagen generated flightless mutants, Ifm(2)RU1 and ifm(2)RU2 that map closely to myosin heavy chain gene (Mhc) region. Since there are no structural proteins predicted in the mapped region, it was likely that these two are Mhc mutations. We show that Ifm(2)RU1 and ifm(2)RU2 are indeed Mhc mutations and the molecular aberrations affect amino acid residues present in the myosin rod region. Human muscle myosin heavy chain (MyHC) mutations that cause Laing early onset distal myopathy and myosin storage myopathy occur in this domain of the protein. Even though mutations lie in the same region of myosin rod, Ifm(2)RU1 is semidominant, whereas ifm(2)RU2 is recessive. Both the mutants show IFM defects and the presence of abnormal myofibrils. Mutant myofibrillar structures can be rescued with an additional wild type Mhc gene copy. However, the restored myofibrillar structure is incapable of rescuing the flight ability of mutants. The muscle phenotypes are due to defects in thick filament assembly which manifest from the early stages of sarcomere development. The MHC protein rod region is an α-helix that forms coiled-coils which further self assemble to form thick filaments or aggregates as observed in in vitro conditions. Biophysical and biochemical analyses reveal that the coiled-coil structure of mutant rods is not affected, however the thermodynamic stability is altered in ifm(2)RU2 mutation. Interestingly, rod aggregate size and stability are not affected in mutant rods. The Drosophila MHC mutant rods were studied along with four MHC mutant rods that harbour human rod mutations to compare the molecular consequences. The Drosophila mutations do not hamper the rod structure and assembly. Therefore, the defects may arise due to altered interactions with myosin rod binding proteins. Flightin is an extensively studied myosin rod binding protein. The amount and phosphorylation status of flightin are an extremely sensitive measure of thick filament assembly. Flightin phosphorylation is affected in the mutants suggesting a functional dependence on MHC and it also indicates MHC instability. In the light of the work done, we have assessed the mutations with respect to their structure-functional implications. The acto-myosin interactions responsible for the defects are also discussed. Formation of unusual myofibrillar structures are analysed with regards to the process of myofibrillogenesis. An understanding of this entire process with the information available from IFM is reviewed in detail. The work so far has helped in understanding the manifestation of myopathies at tissue/cellular levels with insights into the plausible mechanisms of origin of the disease phenotypes. Myopathic condition may arise due to developmental or functional defects. For therapeutic considerations, the fly provides a simple test to inspect the effects of adding extra copies of the wild type gene. We conclude that the Drosophila IFM provide a good model system for the study of human ACTA1 and MyHC myopathies.

Introdução: As miopatias congênitas são doenças musculares genéticas caracterizadas por hipotonia e fraqueza muscular de início precoce na infância. Histologicamente são caracterizadas por alterações estruturais no músculo esquelético (corpos nemalínicos, cores ou centralização nuclear), no entanto, existem casos com alterações leves e inespecíficas, alterações mínimas, tais como, desproporção no tamanho das fibras e desarranjo na arquitetura interna das fibras (falhas focais na atividade oxidativa). Quanto ao aspecto molecular, vários genes já foram identificados em associação com os diversos subtipos, porém com grande sobreposição de achados histológicos e clínicos. Objetivo: Caracterização clínica, histológica e molecular de pacientes Brasileiros com miopatia nemalínica e com miopatia congênita com achados histológicos mínimos. Métodos: Avaliação clínica e histológica (revisão dos achados das biopsias musculares) de pacientes com diagnóstico de miopatia congênita nemalínica e com alterações mínimas, provenientes de dois centros de investigação em doenças neuromusculares da cidade de São Paulo (HC-FMUSP e UNIFESP). O estudo molecular foi realizado através de sequenciamento Sanger para os genes ACTA1, TPM3, MYH7 e SEPN1 e/ou sequenciamento de nova geração para painel de genes musculares e/ou exoma. Resultados: Foram avaliados 23 pacientes com miopatia nemalínica (20 famílias) e 22 pacientes com alterações mínimas (20 famílias). O diagnóstico molecular foi concluído em sete famílias com miopatia nemalínica, sendo quatro com variantes missense, em heterozigose, no gene ACTA1 já associadas previamente a miopatia nemalínica, e três famílias, com variantes não conhecidas, em heterozigose, no gene NEB com alta predição de patogenicidade. Na coorte de miopatias congênitas com alterações mínimas o diagnóstico molecular foi concluído em nove famílias, sendo uma com variante conhecida no gene CHRNE, descrita em miastenia congênita; duas famílias com variantes no gene TPM3, sendo uma inédita, em homozigose, e outra, em heterozigose, já conhecida; duas famílias com variantes novas, em heterozigose, no RYR1, uma no gene TTN e três famílias com variantes já conhecidas no gene no MYH7 com fenótipo de miopatia distal de Laing. A despeito da realização de sequenciamento de exoma, sete famílias ainda permanecem sem gene candidato. Conclusões: Os achados clínicos, histológicos e moleculares dos pacientes da coorte de miopatia nemalínica seguem aos padrões descritos da literatura. O estudo dos pacientes com miopatia congênita com alterações mínimas se revelou complexo e variável, tanto no fenótipo quanto no genótipo. As mutações novas no gene NEB, RYR1, TTN, TPM3 e MYH7 confirmam a importância e patogenicidade destes genes nas miopatias congênitas e ampliam o seu espectro de alterações. Diante da quantidade de genes candidatos e do tamanho de alguns genes envolvidos com essas miopatias, técnicas de sequenciamento de nova geração são de grande valor / Introduction: Congenital myopathy are a group of genetic muscle diseases characterized by hypotonia and weakness in early childhood. They are characterized by structural abnormalities in muscle biopsy (nemaline bodies, central-cores or nuclear centralization). However, it can present within mild and unspecific findings like fiber type disproportion and abnormalities on oxidative staining (minimal changes). Regarding the molecular aspects, there are many genes associated with the congenital myopathies with an important overlapping between the histological and phenotypical findings. Objectives: Clinical, histological and molecular characterization of Brazilian patients with nemaline myopathy and congenital myopathy with minimal changes. Methods: Clinical and histological evaluation (review of muscle biopsy) of patients with nemaline myopathy and congenital myopathy with minimal changes from two centers of neuromuscular diseases (HC-FMUSP e UNIFESP). The molecular study was performed using Sanger sequencing for ACTA1, TPM3, SEPN1 and MYH7 genes and/or neuromuscular panel and/or exome. Results: Twenty-three patients with nemaline myopathy (20 families) and 22 patients with congenital myopathy with minimal changes (20 families) were evaluated. The molecular diagnose were concluded in seven families with nemaline myopathy, with four families having missense, heterozygous and pathogenic ACTA1 variants and three families having unknown heterozygous and pathogenic variants in NEB gene. In the congenital myopathy with minimal findings group, the diagnose was concluded in 9 families. One presenting with a pathogenic variant in CHRNE gene previously described in congenital myasthenia, two families with pathogenic variants in TPM3, one novel homozygous and one heterozygous previously reported. Two families presented with novel and pathogenic RYR1 variants, one with novel and pathogenic TTN variants and 3 families presented with heterozygous variants in MYH7 myopathy with Laing distal myopathy phenotype. Despite the NGS realization, 7 families remain without a gene candidate. Conclusions: The clinical, histological and molecular findings of nemaline myopathy cohort follow the literature pattern. In contrast, the study for minimal change patients appear complex and variable, either on phenotype or on genotype. The new gene mutations for NEB, RYR1, TTN, TPM3 and MYH7 reinforce relevance and pathogenicity of these genes in the congenital myopathies and expand the mutation spectrum. In light of diversity of candidate genes and the size of some genes involved with these myopathies, next generation sequencing techniques have been proved essential

Made available in DSpace on 2016-01-26T12:51:20Z (GMT). No. of bitstreams: 1 marilitafalangolaaccioly_tese.pdf: 2076302 bytes, checksum: 922e6d60d30a7543a522073d3b9c0607 (MD5) Previous issue date: 2007-10-25 / Statins are used to treat dislipidemias with great tolerance, however, several side effects can occur, specially myopathies. The regular practice of physical exercise (PE) produce beneficial alteration in the lipidic profile, however can generate muscle injuries. OBJECTIVE -To evaluate the effect of PE in the lipidic profile; the efficacy of the assocciation between PE and statins in the control of the lipidic profile and evaluate the effect of the association between PE and statins in the muscular function through histological analysis. MATERIAL/METHOD- It was used 80 male Wistar rats , distributed in 8 groups, including animals submitted to a hypercholesterolemic diet (HD), simvastatin with (G1) and without (G2) PE; HD and fluvastatin with (G3) and without PE (G4); fed with comercial ration (CR) in presence (G5) and absence (G6) of PE; HD submitted (G7) or not to PE (G8). The HD was administered for 90 days, statins and practice of PE in the treadmill for 8 weeks. It was measured in the beginning (T0) and at the end (T2) of the experiment the level of total cholesterol (TC) , the fraction of cholesterol of high density lipoprotein (HDLc), triglycerides (TG), piruvic glutamic transaminase (TGP) and the fraction of the cholesterol non-HDLc (non-HDLc) was calculated. The animals were sacrificed, the soleus muscle removed for histological analysis. The paired t test and multivaried analysis were applied with a significance level of p<0,05. RESULTS â The analysis of the groups, at the T2, showed reduction in the TC level when compared G3 with groups G1 (p=0,0414), G6 (p=0,0021) and G8 (p=0,0099) and G7 with group G8 (p<0,0001). However, G1 presented elevated levels in relation to G5 (p=0,0286), G7 (p=0,0192) and G8 (p=0,0196), as well as the G2 in comparison to G7 (p=0,0115). The PE associated with fluvastatin (G3) or simvastatin (G1) has not induced a significant increase in the levels of HDLc, when compared to G4 and G2 (p>0,05). The CR, even with sedentarism (G6) kept the levels of HDLc elevated when compared to the groups remaining. Fluvastatin, independently of the presence of PE (G3 and G4) reduced the fraction of non-HDLc in comparison to G6 (p=0,0201; p=0,0315) and G1 (p=0,0271; p=0,0390, respectively). For the triglycerides levels G1 showed elevated values compared to G3 (p=0,0278), as well as TGP (p=0,0151). The main histological alteration found were fibers of different diameter, atrophics, in degeneration, splitting, edema, inflammatory infiltrate. These alterations were observed in 90% of the animals of G1, 80% of G2, 70% of G3, 30% of G4, 40% of G5 and 30% of G7. In G6 and G8 muscular fibers with preserved morphology were identified. CONCLUSION â PE influenciates in the reduction of TC levels even with DH, becoming the differential in the reduction of TC, non-HDLc and TG with the use of fluvastatin compared to simvastatin, whilst levels of HDLc resist to increase. Conversely, the increase in TGP levels are associated with HD and the use of statins, preferably simvastatin and PE practice. Moreover, PE seems to potentialize muscle injury induced by statins. / As estatinas são utilizadas no tratamento das dislipidemias, com grande tolerância, no entanto, vários efeitos colaterais podem surgir, destacando-se miopatia. A prática regular do exercício físico (EF) produz modificações favoráveis no perfil lipídico, entretanto, pode gerar lesões musculares. OBJETIVO - Avaliar a influência do EF no perfil lipídico; a eficácia da associação entre EF e estatinas no controle do perfil lipídico e avaliar o efeito da associação entre EF e estatinas na função muscular pela análise histológica. MATERIAL e Mà TODO - Foram utilizados 80 ratos machos Wistar, distribuídos em 8 grupos, incluindo animais submetidos à dieta hipercolesterolêmica (DH), simvastatina com (G1) e sem (G2) EF; DH e fluvastatina, com (G3) e sem EF (G4); alimentados com ração comercial (RC) na presença (G5) e ausência de (G6) EF; DH submetidos (G7) ou não (G8) a EF. A DH foi administrada por 90 dias, as estatinas e prática de EF em esteira rolante por 8 semanas. Foram dosados, no início (T0) e ao final (T2) dos experimentos, os níveis de colesterol total (CT), fração de colesterol da lipoproteína de alta densidade (HDLc), triglicérides (TG), transaminase glutâmico pirúvico (TGP) e calculada a fração de colesterol não-HDLc (não-HDLc). Os animais foram sacrificados, e o músculo sóleo retirado para análise histológica. Aplicaram-se os testes t-teste pareado e análise multivariada, com nível significante para p<0,05. RESULTADOS â A análise entre os grupos, no T2, mostrou redução dos níveis de CT quando comparado G3 aos grupos G1 (p=0,0414), G6 (p=0,0021) e G8 (p=0,0099) e G7 ao grupo G8 (p<0,0001). Entretanto, G1 apresentou níveis aumentados em relação a G5 (p=0,0286), G7 (p=0,0192) e G8 (p=0,0196), assim como, o G2 em comparação ao G7 (p=0,0115). O EF associado à fluvastatina (G3) ou a simvastatina. (G1) não induziu aumento significante nos níveis de HDLc, quando comparado a G4 e G2 (p>0,05). A RC mesmo com o sedentarismo (G6) manteve os níveis de HDLc elevados quando comparados aos demais grupos. A fluvastatina independente da presença de EF (G3 e G4) reduziu a fração não-HDLc em comparação a G6 (p=0,0201; p=0,0315) e a G1 (p=0,0271; p=0,0390, respectivamente). Para os níveis de TG destacou-se G1 com valores elevados em comparação a G3 (p=0,0278), assim como para TGP (p=0,0151). As principais alterações histológicas encontradas foram fibras de diferentes diâmetros, atróficas, em degeneração, splitting, edema, infiltrado inflamatório. Essas alterações foram observadas em 90% dos animais do grupo G1, 80% de G2, 70% de G3, 30% de G4, 40% de G5 e 30% de G7. Nos grupos G6 e G8 identificaram-se fibras musculares com morfologia preservada. CONCLUSà ES â EF influencia na redução dos níveis de CT mesmo com DH, tornando-se o diferencial na diminuição dos níveis de CT, não-HDLc e TG no uso de fluvastatina comparado a simvastatina, enquanto os níveis de HDLc são resistentes a elevação. Por outro lado, o aumento nos níveis de TGP associa-se com DH e uso de estatinas, preferencialmente simvastatina e prática de EF. Além disso, o EF parece potencializar a lesão muscular induzida pelas estatinas.

Introdução: As miopatias congênitas são doenças musculares genéticas caracterizadas por hipotonia e fraqueza muscular de início precoce na infância. Histologicamente são caracterizadas por alterações estruturais no músculo esquelético (corpos nemalínicos, cores ou centralização nuclear), no entanto, existem casos com alterações leves e inespecíficas, alterações mínimas, tais como, desproporção no tamanho das fibras e desarranjo na arquitetura interna das fibras (falhas focais na atividade oxidativa). Quanto ao aspecto molecular, vários genes já foram identificados em associação com os diversos subtipos, porém com grande sobreposição de achados histológicos e clínicos. Objetivo: Caracterização clínica, histológica e molecular de pacientes Brasileiros com miopatia nemalínica e com miopatia congênita com achados histológicos mínimos. Métodos: Avaliação clínica e histológica (revisão dos achados das biopsias musculares) de pacientes com diagnóstico de miopatia congênita nemalínica e com alterações mínimas, provenientes de dois centros de investigação em doenças neuromusculares da cidade de São Paulo (HC-FMUSP e UNIFESP). O estudo molecular foi realizado através de sequenciamento Sanger para os genes ACTA1, TPM3, MYH7 e SEPN1 e/ou sequenciamento de nova geração para painel de genes musculares e/ou exoma. Resultados: Foram avaliados 23 pacientes com miopatia nemalínica (20 famílias) e 22 pacientes com alterações mínimas (20 famílias). O diagnóstico molecular foi concluído em sete famílias com miopatia nemalínica, sendo quatro com variantes missense, em heterozigose, no gene ACTA1 já associadas previamente a miopatia nemalínica, e três famílias, com variantes não conhecidas, em heterozigose, no gene NEB com alta predição de patogenicidade. Na coorte de miopatias congênitas com alterações mínimas o diagnóstico molecular foi concluído em nove famílias, sendo uma com variante conhecida no gene CHRNE, descrita em miastenia congênita; duas famílias com variantes no gene TPM3, sendo uma inédita, em homozigose, e outra, em heterozigose, já conhecida; duas famílias com variantes novas, em heterozigose, no RYR1, uma no gene TTN e três famílias com variantes já conhecidas no gene no MYH7 com fenótipo de miopatia distal de Laing. A despeito da realização de sequenciamento de exoma, sete famílias ainda permanecem sem gene candidato. Conclusões: Os achados clínicos, histológicos e moleculares dos pacientes da coorte de miopatia nemalínica seguem aos padrões descritos da literatura. O estudo dos pacientes com miopatia congênita com alterações mínimas se revelou complexo e variável, tanto no fenótipo quanto no genótipo. As mutações novas no gene NEB, RYR1, TTN, TPM3 e MYH7 confirmam a importância e patogenicidade destes genes nas miopatias congênitas e ampliam o seu espectro de alterações. Diante da quantidade de genes candidatos e do tamanho de alguns genes envolvidos com essas miopatias, técnicas de sequenciamento de nova geração são de grande valor / Introduction: Congenital myopathy are a group of genetic muscle diseases characterized by hypotonia and weakness in early childhood. They are characterized by structural abnormalities in muscle biopsy (nemaline bodies, central-cores or nuclear centralization). However, it can present within mild and unspecific findings like fiber type disproportion and abnormalities on oxidative staining (minimal changes). Regarding the molecular aspects, there are many genes associated with the congenital myopathies with an important overlapping between the histological and phenotypical findings. Objectives: Clinical, histological and molecular characterization of Brazilian patients with nemaline myopathy and congenital myopathy with minimal changes. Methods: Clinical and histological evaluation (review of muscle biopsy) of patients with nemaline myopathy and congenital myopathy with minimal changes from two centers of neuromuscular diseases (HC-FMUSP e UNIFESP). The molecular study was performed using Sanger sequencing for ACTA1, TPM3, SEPN1 and MYH7 genes and/or neuromuscular panel and/or exome. Results: Twenty-three patients with nemaline myopathy (20 families) and 22 patients with congenital myopathy with minimal changes (20 families) were evaluated. The molecular diagnose were concluded in seven families with nemaline myopathy, with four families having missense, heterozygous and pathogenic ACTA1 variants and three families having unknown heterozygous and pathogenic variants in NEB gene. In the congenital myopathy with minimal findings group, the diagnose was concluded in 9 families. One presenting with a pathogenic variant in CHRNE gene previously described in congenital myasthenia, two families with pathogenic variants in TPM3, one novel homozygous and one heterozygous previously reported. Two families presented with novel and pathogenic RYR1 variants, one with novel and pathogenic TTN variants and 3 families presented with heterozygous variants in MYH7 myopathy with Laing distal myopathy phenotype. Despite the NGS realization, 7 families remain without a gene candidate. Conclusions: The clinical, histological and molecular findings of nemaline myopathy cohort follow the literature pattern. In contrast, the study for minimal change patients appear complex and variable, either on phenotype or on genotype. The new gene mutations for NEB, RYR1, TTN, TPM3 and MYH7 reinforce relevance and pathogenicity of these genes in the congenital myopathies and expand the mutation spectrum. In light of diversity of candidate genes and the size of some genes involved with these myopathies, next generation sequencing techniques have been proved essential

INTRODUÇÃO: Capítulo 1. A Insuficiência Cardíaca (IC) é acompanhada de uma hiperativação simpática e do sistema renina-angiotensina (SRA). As ações deletérias do SRA são atribuídas à Angiotensina II (AngII), mas a Angiotensina-(1- 7) (Ang-(1-7)), um metabólito da AngII, demonstra efeitos cardiovasculares benéficos, contrários aos da AngII. O conceito tradicional é de que as concentrações sistêmicas mediam as respostas do SRA, mas evidências emergem acerca da importância funcional do SRA local. Nesse estudo, estudou-se o SRA circulante e muscular esquelético na IC, testando-se a hipótese de que as alterações seriam diferentes nesses dois territórios, e que o treinamento físico corrigiria essas alterações. Capítulo 2. A IC é uma síndrome sistêmica, onde fatores neuroendócrinos, como a AngII, podem levar a alterações periféricas. Na musculatura esquelética, a hiperatividade do sistema ubiquitina-proteassoma (SUP) é um dos elementos que compõem um quadro de miopatia, aumentando o catabolismo muscular em direção à atrofia, e contribuindo com o agravamento da síndrome. O treinamento físico normaliza o SUP e reduz as concentrações plasmáticas de AngII na IC. Dessa forma, testamos a hipótese de que a redução do SUP mediada pelo treinamento físico na IC depende da queda das concentrações plasmáticas de AngII. MÉTODOS: Capítulo 1. Ratos Wistar, machos, foram induzidos à IC por ligadura da artéria coronária descendente anterior, ou cirurgia fictícia (Sham, SH). Os animais foram divididos em grupos mantidos sedentários, SD (SHSD, n=10 e ICSD, n=12) ou submetidos ao treinamento físico, TR (SHTR, n=10, ICTR, n=12). O treinamento físico foi realizado em esteira, a 60% do consumo máximo de oxigênio, 5 dias por semana durante 8 semanas, quando foram sacrificados para coleta de sangue e músculos (sóleo e plantar). As angiotensinas circulantes e musculares foram dosadas por HPLC. A atividade sérica e muscular da ECA e da ECA2 por fluorimetria. Os receptores AT1 e AT2 foram analisados por expressão gênica (RT-PCR) e proteica (Western Blot), e o receptor Mas por expressão gênica. Capítulo 2. Ratos Wistar, machos, foram induzidos à IC por ligadura da artéria coronária descendente anterior, ou cirurgia fictícia (Sham). Após 4 semanas, os animais Sham (n=10) constituíram um grupo sedentário saudável (SHSD) e os animais com IC (n=30) foram igualmente alocados em 3 grupos: um mantido sedentário (ICSD), um treinado (ICTR) e um treinado com as concentrações plasmáticas de AngII nos mesmos níveis dos animais do grupo ICSD (ICTRAII), mantidas através de minipump osmótica. O treinamento físico foi realizado em esteira, a 60% do consumo máximo de oxigênio, 5 dias por semana durante 8 semanas, quando foram sacrificados para coleta de sangue e músculos (sóleo e plantar). As angiotensinas circulantes e musculares foram dosadas por HPLC. A expressão gênica das enzimas ligases E3?, MuRF e Atrogin foi realizada por RT-PCR. O receptor AT1, as proteínas ubiquitinadas e as proteínas carboniladas (Oxyblot) foram quantificadas por Western Blot. A atividade da porção 26S do proteassoma foi determinada por fluorimetria. RESULTADOS: Capítulo 1. Na circulação, a atividade da ECA2 estava reduzida na IC, e o treinamento físico reduziu a atividade da ECA e restaurou a atividade da ECA2 esses animais. A concentração de AngII reduziu nos grupos treinados, e a razão Ang-(1-7)/AngII aumentou no grupo ICTR. Nos músculos, não houve alteração em relação à atividade ou expressão proteica da ECA ou da ECA2, mas a concentração de AngII estava aumentada com a IC, e normalizou com o treinamento físico. A concentração de Ang-(1-7) aumentou no músculo plantar do grupo ICTR, e a razão Ang-(1-7)/AngII apresentou forte tendência de aumento no músculo sóleo dos animais treinados. No músculo sóleo, o AT1 estava aumentado nos animais com IC, e o treinamento físico normalizou a expressão gênica e proteica desse receptor, e também aumentou a expressão gênica do receptor Mas nos grupos treinados. No músculo plantar, normalizou a expressão gênica do receptor Mas, sem alterar o AT1. Não foram encontradas diferenças significativas na expressão do receptor AT2 nos músculos estudados. Capítulo 2. O treinamento físico promoveu uma melhora da capacidade de exercício em ambos os grupos treinados. A AngII aumentou nos músculos dos animais com IC, e o treinamento físico normalizou esses valores. Na circulação, como se esperava, a AngII diminuiu apenas no grupo ICTR. A expressão do receptor AT1 aumentou no músculo sóleo com a IC e normalizou com o treinamento físico, sem diferenças entre grupos no músculo plantar. Em relação à expressão gênica das E3 ligases e na quantidade de proteínas ubiquitinadas e carboniladas, não houve diferenças entre os grupos no músculo sóleo. Já no músculo plantar, a expressão do atrogin estava aumentada nos animais com IC, e o treinamento físico reduziu a expressão tanto da atrogin quanto da E3? e da MuRF. Essa melhora foi prejudicada com a infusão de AngII. Refletindo esse cenário, a quantidade de proteínas ubiquitinadas e carboniladas estavam aumentadas na IC e reduziram com o treinamento físico, e a infusão de AngII atenuou a redução das proteínas ubiquitinadas e aboliu a diminuição das oxidadas. A atividade do proteassoma aumentou em ambos os músculos de animais com IC, e o treinamento físico reduziu a atividade nos animais treinados, sendo significativamente menor no grupo ICTRAII. CONCLUSÕES: Capítulo 1. Em modelo de IC crônica, os níveis de AngII estão aumentados na musculatura esquelética, mas não na circulação. O treinamento físico reduz os níveis plasmáticos de AngII na circulação e normaliza nos músculos. Essa redução é acompanhada de um aumento dos níveis de Ang-(1-7) ou da melhora na razão Ang-(1-7)/AngII em ambos os territórios, indicando uma atenuação da hiperativação do SRA na IC com o treinamento físico. Capítulo 2. Em modelo isquêmico de IC crônica em ratos, há uma diferença no perfil do SUP no músculo sóleo e no músculo plantar. O treinamento físico reduz a atividade do SUP e, ao menos no músculo plantar, essa melhora parece ser dependente da redução dos níveis de AngII / INTRODUCTION: Chapter 1. Heart Failure (IC) is a syndrome accompanied by a sympathetic and renin-angiotensin system (RAS) hyperactivity. The deleterious actions of RAS are attributed to Angiotensin II (AngII), but Angiotensin-(1-7) (Ang-(1- 7)), a metabolite of AngII, shows benefic cardiovascular effects opposing to AngII. The traditional concept states that the systemic concentrations are responsible for RAS actions, although increasingly evidence emerge about the functional role of local RAS. The working hypothesis was that the RAS alterations, if any, would be different on this two territories of heart failure rats, and the exercise training should correct this alterations. Chapter 2. Heart failure is a systemic syndrome in which neuroendocrine factors, such as angiotensin II (AngII), can lead to peripheral damage. In skeletal muscle, the hyperactivity of ubiquitin-proteasome system (SUP) is one of the elements composing the myopathy framework, elevating the catabolism toward atrophy, and contributing to the worsening of the syndrome. Exercise training normalizes SUP and reduces plasmatic concentrations of AngII. On this way, we tested the hypothesis that exercise training-mediated SUP deactivation is dependent on plasma falls of AngII. METHODS: Chapter 1. Male Wistar rats underwent left coronary artery ligation or Sham (SH) operation. They were allocated in sedentary, SD (SHSD, n=10 and ICSD, n=12) or trained, TR (SHTR, n=10 and ICTR, n=12) groups. The exercise training consisted in treadmill running, at 60% of maximal oxygen uptake, 5 days per week, during 8 weeks, when they were killed for blood and skeletal muscle (soleus and plantaris) collection. Angiotensin\'s concentrations were determined by HPLC. ACE and ACE2 activity were accessed in serum and muscles by fluorimetry, and by protein expression (Western Blot) in the muscles. AT1 and AT2 receptors were quantified by protein and gene (RT-PCR) expression, and Mas receptor by gene expression. Chapter 2. Male Wistar rats underwent left coronary artery ligation or Sham operation. After 4 weeks, Sham operated rats (n=10) constituted a healthy, sedentary control group (SHSD), and the heart failure rats (n=30) were equally allocated into 3 groups: sedentary (ICSD), trained (ICTR) and trained with plasma AngII at the same level of sedentary, heart failure rats (ICTRAII), kept by an osmotic minipump. The exercise training consisted in treadmill running, at 55% of maximal oxygen uptake, 5 days per week, during 8 weeks, when they were killed for blood and skeletal muscle (soleus and plantaris) collection. AngII concentrations were determined by HPLC. Gene expression of E3?, MuRF e Atrogin were performed by PR-PCR. AT1 receptor, ubiquitinated and carbonylated (oxyblot) proteins were quantified by Western Blot. Proteasomal 26S activity were determined by fluorimetry. RESULTS: Chapter 1. Heart failure reduced circulating ACE2 activity, and exercise training reduced ACE and normalized ACE2 activity in this rats. AngII concentration reduced in both trained groups, increasing Ang-(1-7)/AngII ratio on ICTR group. The studied skeletal muscles did not change activity or protein expression of ACE and ACE2, although the AngII, which was increased with heart failure, has normalized with exercise training. Absolute Ang-(1-7) concentration increased in plantaris muscle, and a strong tendency of significant increase was shown in soleus muscle of trained rats. Also in the soleus, AT1 receptor raised with heart failure, and the exercise training normalized the gene as well as protein expression of this receptor, also increasing gene expression of Mas receptor of trained groups. In plantaris muscle, exercise normalized Mas receptor in ICTR, without influencing AT1 receptor. No significant changes among groups were found in relation to AT2 receptor of the studied muscles. Chapter 2. Exercise training promoted an improvement of exercise capacity in trained groups. AngII raised in skeletal muscle of rats with heart failure, and exercise training normalized this. Circulating AngII, as expected, reduced only in ICTR group. AT1 receptor expression increased in soleus muscle of heart failure, and normalized after exercise in trained rats, without any difference among groups in plantaris muscle. Regarding E3 ligases gene expression and quantity of ubiquitinated and carbonylated proteins, there were no differences among groups in soleus muscle. Nevertheless, in plantaris muscle, atrogin expression was increased in heart failure rats, and exercise training reduced atrogin, as well as E3alpha and MuRF expression. These improvements were impaired by AngII infusion. Mirroring this scenario, the amount of ubiquitinated and carbonylated proteins increased with heart failure and reduced with exercise training, but AngII infusion lessen the reduction of ubiquitinated proteins and completely blunted the effects of exercise on carbonylated proteins. Proteasome 26S activity was increased in both muscles of heart failure rats, and exercise avoided this increase in trained rats, being significantly reduced in ICTRAII. CONCLUSIONS: Chapter 1. In a model of chronic heart failure rats, AngII levels are increased in skeletal muscle, but not in the circulation. The exercise training reduces plasma and normalizes skeletal muscle concentration of AngII. This reduction is accompanied by an increase Ang-(1-7) levels, or improvements of Ang-(1-7)/AngII ratio in both systemic and local territories, indicating an attenuation of RAS hyperactivity with exercise training in heart failure rats. Chapter 2. In a model of chronic heart failure rats, there is a difference on SUP activation profile in soleus and plantaris muscle. Exercise training reduces SUP activity and, in plantaris muscle, this amelioration seems to be, at least in part, dependent of a reduction in AngII levels