Orientador: Maeli Dal Pai Silva / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T16:06:13Z (GMT). No. of bitstreams: 1 Carvalho_RobsonFrancisco_D.pdf: 1253559 bytes, checksum: e244961263e728fe00882d5f5d14b262 (MD5) Previous issue date: 2006 / Resumo: Introdução: A Insuficiência Cardíaca (IC) está associada a uma miopatia do músculo esquelético com aumento da expressão das isoformas rápidas da cadeia pesada de miosina (MHC) e alterações na matriz extracelular (MEC). Os mecanismos moleculares que controlam a expressão de MHC durante a IC ainda não foram descritos. Os fatores de regulação miogênica (MRF), uma família de fatores transcricionais que controlam vários genes músculo-específicos, podem estar relacionados com essa miopatia. As alterações da MEC podem estar associadas a um aumento na expressão de RNA mensageiro e na atividade das metaloproteinases da MEC (MMP), uma família de endopeptidases dependentes de zinco que degradam a maioria dos componentes da MEC e que são indispensáveis para a remodelação do tecido conjuntivo ao redor das fibras musculares. Objetivos: Analisar no músculo esquelético de ratos Wistar com IC induzida pela monocrotalina: 1) a expressão de RNA mensageiro para os MRF, as isoformas proteicas de MHC e a atrofia nos músculos Sóleo (SOL) e Extensor Longo dos Dedos (EDL); 2) a expressão de RNA mensageiro e a atividade das MMP nos músculos SOL, EDL e diafragma (DIA). Métodos: A expressão do RNA mensageiro para MyoD, miogenina, MRF4, MMP2 e MMP9 foi determinada por RT-PCR; as isoformas de MHC foram separadas por eletroforese em gel de poliacrilamida e a atividade das MMP, por eletroforese em gel de poliacrilaminda contendo gelatina na presença de SDS em condições não redutoras. Resultados: 1) Embora a composição de MHC do músculo esquelético de ratos com IC não tenha sido alterada, a expressão relativa do RNA mensageiro para a MyoD nos músculos SOL e EDL, e a de MRF4 no músculo SOL foi significativamente diminuída, enquanto que a expressão relativa de RNA mensageiro para a miogenina não se alterou em ambos os músculos. A diminuição na expressão relativa de RNA mensageiro para o MRF4 está associada à atrofia do SOL em resposta à IC. 2) A IC aumentou a expressão de RNA mensageiro e a atividade da MMP9 nos músculos SOL, EDL e DIA, e aumentou a expressão de RNA mensageiro da MMP2 no músculo DIA. Conclusão: Nossos resultados sugerem um potencial papel para os MRF e para as MMP na miopatia do músculo esquelético na IC / Abstract: Background: Heart failure (HF) is associated with a skeletal muscle myopathy with increased expression of fast myosin heavy chains (MHC) and extracellular matrix (ECM) alterations. The skeletal muscle-specific molecular regulatory mechanisms controlling MHC expression during HF have not been described. Myogenic regulatory factors (MRF), a family of transcriptional factors that control the expression of several skeletal muscle-specific genes, may be related to these alterations. The ECM alterations may be associated with enhanced mRNA expression and activity of matrix metalloproteinases (MMP), a family of zinc-dependent endopeptidases that degrade most ECM components and appear indispensable for the breakdown of the connective tissue surrounding muscle fibers. Objectives: This investigation was undertaken in order to examine in Wistar rat skeletal muscle with monocrotaline-induced HF: 1) potential relationships between MRF mRNA expression and MHC protein isoforms and atrophy in Soleus (SOL) and extensor digitorum longus (EDL) muscles; 2) MMP mRNA expression and their potential relationships with changes in MMP activity in SOL, EDL, and diaphragm (DIA) muscles. Methods: MyoD, myogenin, MRF4, MMP2, and MMP9 were determined by using RTPCR; MHC isoforms were separated by using polyacrylamide gel electrophoresis, and MMP activity by electrophoresis in gelatin-containing polyacrylamide gel in the presence of SDS under nonreducing conditions. Results: 1) Despite no change in MHC composition of Wistar rat skeletal muscles with HF, the mRNA relative expression of MyoD in SOL and EDL muscles and that of MRF4 in SOL muscle were significantly reduced, whereas myogenin was not changed in both muscles. This down-regulation in the mRNA relative expression of MRF4 in SOL was associated with atrophy in response to HF. 2) HF increased MMP9 mRNA expression and activity in SOL, EDL, and DIA and MMP2 mRNA expression in DIA. Conclusion: Taken together; our results show a potential role for MRF and MMP in skeletal muscle myopathy during HF / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Orientador: Maeli Dal Pai Silva / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T01:50:52Z (GMT). No. of bitstreams: 1 Barretto_JeaneMarleneFogacadeAssis_D.pdf: 16620509 bytes, checksum: f55fbc50c521ec1697bb9e56f7c70d08 (MD5) Previous issue date: 2006 / Resumo: o desenvolvimento da musculatura em animais utilizados como alimento tem importância na qualidade e no aumento da massa muscular. Esse estudo teve por objetivo avaliar as características morfológicas e o crescimento da musculatura miotomal em tilápia do Nilo (Oreochromis niloticus) submetida à dieta com suplementação de vitamina C. O experimento foi realizado a partir da primeira alimentação exógena até 60 dias. As larvas foram distribuídas em cinco tratamentos, numa densidade de 10 indivíduos/L onde foram administrados os seguintes níveis de suplementação de vitamina C/ kg de ração balanceada: T1 = sem suplementação, T2 = 250 mg, T3 = 500 mg, T4 =1000 mg, T5 = sem suplementação até o vigésimo dia do tratamento, e com suplementação de 500 mg do vigésimo primeiro ao sexagésimo dia de experimento. Foram realizadas cinco repetições por tratamento com distribuição randômica. Cinco peixes de cada tratamento foram anestesiados com Ms-222 - SIGMA, sacrificados e a biometria foi efetuada após 20, 40 e 60 dias de experimento. Após isso, amostras de músculo foram congeladas em n-hexana resfriada em nitrogênio líquido e cortes histológicos foram submetidos à coloração HE (para análise da morfologia geral da musculatura), à reação mATPase, após pré incubação ácida (para a análise das características da ATPase miofibrilar) e à reação NADH-TR (para análise do metabolismo oxidativo das fibras). Aos 20 e 60 dias do experimento, cinco amostras de cada tratamento foram submetidos às reações imunohistoquímicas MyoD e miogenina (fatores de transcrição miogênica - para avaliar o grau de proliferação e diferenciação das celulas precursoras miogênicas). Também foram coletadas amostras de músculo para fixação em formalina neutra tamponada, processadas para serem embebidos em paraplast e os cortes histológicos foram submetidos à reação imunohistoquímica PCNA (Antígeno Nuclear de Proliferação Celular). A suplementação com os maiores níveis de vitamina C e após o período de restrição promoveu maior ganho de massa corpórea. O crescimento muscular em todos os tratamentos ocorreu principalmente por hiperplasia. Durante o experimento não foram observadas alterações morfológicas e histoquímicas nas fibras musculares. A suplementação de 500mg vit C kg-1da ração após a restrição por 20 dias promoveu a proliferação e a diferenciação das células precursoras miogênicas 500mg vit C kg-1 da ração foi o nível de suplementação apropriado para a miogênese e o desenvolvimento da Tilápia / Abstract: Muscle development in animais utilized for food has importance to muscle quality and mass increase. The aim of this study was evaluate the morphologic and the growth characteristics of the miotomal muscle in Nile tilapia (Oreochromis niloticus) submitted to a vitamin C supplemented diet. The experiment was carried through from the first exogenous feeding up to 60 days. The larvae were distributed into five treatments, in aquaria at 10/L density. The levels of vitamin C supplement/kg of ration were: T1=no supplement, T2=250mg vit C kg-1 of diet, T3=500mg vit C kg-1of diet, T4= 1000mg vit C kg-1 of diet, T5= no supplement until 20 days and 500mg vit C kg-1 of diet on days 20 to 60. There were 5 repetitions for each treatment. After 20, 40 and 60 days, 5 fish from each treatment were anaesthetized with MS-222 - SIGMA, sacrificed and weighed. Muscle fragments were cooled in liquid nitrogen. Histological sections were submitted to the following reactions: Haematoxilin-Eosin (for analysis of the muscle morphology and to determine muscle fibers diameters); Nicotinamide Adenine Dinucleotide Tetrazolium Reductase (NADH-TR), for muscle fibre metabolic evaluation (oxidative and glycolitic); and miofibrillar ATPase (mATPase), in pH 9.4, after acid preincubation to study myosin ATPase characteristics. After 20 and 60 days, muscle samples from five fish of each treatment were immersed in n-Hexane cooled in liquid nitrogen and histological sections were submitted to the MyoD and myogenin immunohistochemical reaction to evaluate cell proliferation and differentiation. Another five fish per treatment were embedded in Paraplast and histological sections were submitted to PCNA (Proliferating Cell Nuclear Antigen) immunohistochemical reaction to evaluate the degree of cell proliferation per treatment. During the experiment, ali treatments increased in weight and length. Weight differences were significant only on day 60. Supplementation with high vitamin C levels and after a period of restriction promoted an increase in body mass. Muscle fiber morphology, metabolic and myosin ATPase characteristics were normal and similar in all treatments. Muscle growth in all treatments was predominantly by hyperplasia. Proliferation and differentiation of myogenic precursor cells was higher with 500mg vit C kg-1 of supplemented diets throughout the experiment. Restriction of this supplementation for 20 days did not harm differentiation and still promoted myogenic precursor cell proliferation. The appropriate levei of vitamina C to myogenesis and the development of the Tilapia was 500mg vit C kg-1 of the ration / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientador: Maeli Dal Pai Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T13:18:40Z (GMT). No. of bitstreams: 1 Almeida_FernandaLosiAlvesde_M.pdf: 1065788 bytes, checksum: 0b390cd2e5f289613db5ad2ca384e439 (MD5) Previous issue date: 2007 / Resumo: Nos peixes, o crescimento do tecido muscular ocorre por hipertrofia e/ou hiperplasia a partir da proliferação e diferenciação de mioblastos adultos ou células miossatélites, processos regulados pela expressão diferencial dos fatores de regulação miogênica (MRFs). O objetivo desse trabalho foi avaliar os mecanismos de crescimento muscular hiperplasico e hipertrofico e a expressão do MRF MyoD, na musculatura branca do pacu (Piaractus mesopotamicus), durante o crescimento. Exemplares juvenis (n=5) e adultos (n=5) de pacu foram anestesiados, sacrificados e determinados o peso corporal (g) e o comprimento total (cm). Fragmentos musculares brancos da região dorsal de cada exemplar, em cada fase estudada, foram congelados e imersos em nhexano congelado em nitrogenio liquido. Cortes histológicos (10 µm), obtidos em criostato, foram submetidos à  coloração hematoxilina-eosina para avaliação da morfologia e morfometria das fibras musculares brancas. Foi calculado o menor diametro de 100 fibras musculares brancas em cada animal de cada fase estudada. As fibras musculares foram distribuídas em classes, na dependência do seu diametro (<20, 20-50, >50 µm), para avaliar o grau de crescimento hipertrófico e hiperplá¡sico da musculatura. A expressão do MRF MyoD na musculatura branca foi analisada por Reação em Cadeia da Polimerase apos Transcrição Reversa (RT - PCR). Todos os produtos visualizados em gel de agarose a 1% foram clonados e sequenciados. A morfologia da musculatura dos exemplares juvenis e adultos foi semelhante, apresentando um padrão em mosaico caracterizado por fibras de diferentes diâmetros. Nos exemplares juvenis, foi observado um predomínio de fibras com diametro menor que 20 µm, caracterizando intensa hiperplasia. Nos exemplares adultos, houve o predomínio de fibras musculares com diâmetro maior que 50 µm, caracterizando intensa hipertrofia da musculatura. A expressão do RNAm para o gene MyoD foi significativamente maior na fase juvenil, se comparada com a fase adulta. Foi obtida a sequencia consenso parcial do gene MyoD (338 pares de bases) expresso na musculatura branca do pacu. Essa sequencia apresentou similaridade com as sequencias de MyoD de varias especies de vertebrados, incluindo peixes teleósteos. A expressão diferencial do MRF MyoD, observada nas fases de crescimento juvenil e adulta do pacu, possivelmente seja responsavel pelas diferenças observadas no padrão de crescimento, com a hiperplasia predominando nos juvenis e a hipertrofia, nos adultos / Abstract: Skeletal muscle growth in fish occurs by hypertrophy and hyperplasia and is dependent of the proliferation and differentiation of myogenic progenitor cells, events regulated by the diferential expression of the myogenic regulatory factors (MRFs). The aim of this study was to analyze the hyperplasia and hypertrophy processes and the MRF MyoD expression in the white muscle in pacu (Piaractus mesopotamicus) during growth. Juvenile (n=5) and adult (n=5) fishes were anaesthetized, sacrificed and the weight (g) and the total length (cm) were determined. White muscle samples from dorsal region of each sample, in each growth phase, were collected and and immersed in n- Hexane cooled in liquid nitrogen. Transverse sections (10 µm thick), obtained in a cryostat, were stained with Haematoxilin-Eosin to morphological and morphometric analysis. We calculated the smallest diameter from 100 white muscle fibres per animal in each group. White muscle fibers were grouped in three classes: <20, 20-50 and >50 µm to evaluate hypertrophy and hyperplasia in pacu white skeletal muscle. MyoD gene expression was determined by using RT-PCR. All PCR products visualized in 1% agarose gels were cloned and sequenced. Juvenile and adult pacu fish skeletal muscle showed similar morphology, with mosaic pattern characterized by fibers with different diameters. The great number of muscle fibers with diameter inferior 20 µm observed in juvenile fish confirms the active hyperplasic process. In adult fish, most fibers were over 50 µm diameter and denote the more intense muscle fiber hypertrophy. MyoD mRNA level in the juvenile fish was higher compared to adult fish. A consensus partial sequence for MyoD gene (338 bases pairs) was obtained. This sequence showed similarity with various vertebrate species, including teleost fishes. Differential expression of MyoD gene observed in white muscle of pacu possibly is related to differences in growth patterns during the phases analysed, with predominance of hyperplasia in juveniles and hypertrophy in adult fish / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Orientador: Maeli Dal Pai Silva / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T19:10:37Z (GMT). No. of bitstreams: 1 Aguiar_DaniloHenrique_D.pdf: 1937310 bytes, checksum: 7d4d18b54f38b44a48ac5bd8892e54d5 (MD5) Previous issue date: 2008 / Resumo: Nos peixes, o conhecimento dos fatores que controlam o crescimento muscular e a análise das proteínas miofibrilares, é importante para entender a dinâmica do crescimento, a plasticidade e as adaptações musculares, principalmente, em espécies com grande valor comercial como a tilápia do Nilo (Oreochromis niloticus). No presente estudo, utilizou-se a tilápia do Nilo em quatro estágios: alevinos de 35 dias (0.65g ± 0.08); juvenis de 60 dias (13.67g ± 1.35); adultos de 90 dias (73.18g ± 4.70) e adultos de 190 dias (349.76g ± 34.62). Em cada estágio, fragmentos musculares foram coletados e submetidos às seguintes análises: morfométrica, para caracterizar o crescimento muscular hiperplásico e hipertrófico no músculo branco; imunohistoquímica, para analisar a expressão dos fatores de regulação miogênica MyoD e miogenina e a expressão da proteína PCNA no músculo branco; histoquímica da ATPase miofibrilar (mATPase) e à eletroforese em gel de poliacrilamida ¿ duodecil sulfato de sódio (SDS-PAGE) para observar as características da mATPase e da cadeia pesada da miosina nos músculos branco e vermelho, respectivamente. Os resultados indicaram que a expressão de MyoD e miogenina foi similar em alevinos, juvenis e adultos de 90 dias, porém, em adultos de 190 dias a expressão de miogenina foi maior do que a de MyoD. A expressão do PCNA, em cada estágio, foi mais acentuada do que MyoD e miogenina com picos no estágio de alevinos e adultos de 90 dias. A expressão de MyoD e miogenina nos estágios de alevinos, juvenis e adultos de 90 dias, mostrou que a hiperplasia e a hipertrofia ocorreram como resultado da proliferação e da diferenciação dos mioblastos. O aumento da expressão de miogenina em adultos de 190 dias, indicou que a diferenciação celular e a hipertrofia foi mais significativa nesse estágio. A análise da mATPase indicou, além da presença de fibras musculares vermelhas e brancas, fibras híbridas tanto no músculo vermelho como no músculo branco, ao longo do crescimento muscular da tilápia. A partir de alevinos, o músculo vermelho da região superficial mostrou a presença de cadeia pesada da miosina slow e o músculo branco, que forma a maior parte da massa muscular, cadeia pesada da miosina fast. Essas isoformas apresentaram massa molecular semelhante à cadeia pesada da miosina do tipo I do músculo sóleo de rato. No músculo branco, a partir dos alevinos, foi observada outra isoforma de miosina de massa molecular superior à cadeia pesada da miosina do tipo I do músculo sóleo de rato. No músculo vermelho a partir dos adultos, observou-se outra isoforma de miosina de massa molecular semelhante à cadeia pesada da miosina do tipo II do músculo sóleo de rato. A expressão das isoformas de cadeias pesadas da miosina no músculo estriado esquelético da tilápia do Nilo durante o crescimento, pode estar relacionada com a plasticidade fenotípica que ocorre durante o crescimento muscular e reflete na capacidade desses peixes de se adaptar às variações ambientais, importantes para a sobrevivência / Abstract: In fish, the knowledge of factors that control the muscle growth and the myofibrillar proteins analyze is important to understand the dynamic of growth, the plasticity and the muscle adaptations, mainly, in species with high commercial valuable, as the Nile tilapia (Oreochromis niloticus). In the present study, Nile tilapia into four age stages were used: 35 day alevins (0.65g ± 0.08); 60 day juveniles (13.67g ± 1.35); 90 day adults (73.18g ± 4.70) and 190 day adults (349.76g ± 34.62). In each stage, muscle fragments were collected and submitted to the following analyzes: morphometric, to characterize the hyperplastic and hypertrophyc growth in the white muscle; immunohistochemical, to analize the myogenic regulatory factors MyoD and myogenin expression, and the PCNA protein expression in white muscle; histochemical of the myofibrillar ATPase (mATPase) and electrophoresis by sodium duodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the red and white muscle to observed mATPase and myosin heavy chain characteristics, respectively. The results indicate that MyoD and myogenin expression was similar in alevins, juveniles and 90 day adults, however, in 190 day adults the myogenin was higher than the MyoD expression. The PCNA expression, in each stage, was higher than MyoD and myogenin with peaks in alevins and 90 day adults. The MyoD expression in alevins, juveniles and 90 day adults, showed that the hyperplasia and hypertrophy occurred due to the results of myoblasts proliferation and differentiation. The increased of myogenin expression in 190 day adults indicated that cellular differentiation and the hypertrophy was more expressive in this stage. The mATPase showed, beyond red and white muscle fibers, hybrid fibers in both red and white muscle during growth. From alevins, the red muscle showed slow myosin heavy chain (MHCs) and the white muscle, fast myosin heavy chain (MHCf). These isoforms had a molecular mass similar to the type I myosin heavy chain (MHCI) of soleus rat muscle. In the white muscle, from alevins was observed other myosin isoform with molecular mass superior to the MHCI of soleus rat muscle. In the red muscle, in adults, was observed other myosin isoform with molecular mass similar to the type II myosin heavy chain (MHC II) of soleus rat muscle. The expression of myosin isoforms in the skeletal muscle of Nile tilapia during growth, can be related to the phenotypic plasticity that occur during muscle growth and reflects this fish capacity to adapt to changes in environmental conditions which are important for its survival / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

A disfunção cardiovascular induzida pelo consumo crônico de etanol esta associada à formação de espécies reativas de oxigênio (ERO). A angiotensina II, via receptores AT1, é um importante formador de ERO no sistema cardiovascular. O objetivo foi avaliar a participação dos receptores AT1 nas disfunções cardiovasculares induzidas pelo consumo crônico de etanol. Ratos Wistar foram divididos em quatro grupos: Controle: recebeu água \"ad libitum\"; Etanol: recebeu solução de etanol 20% (vol./vol.); Controle+Losartan: recebeu água \"ad libitum\" e losartan (10 mg/kg) diariamente por gavagem; Etanol+Losartan: recebeu solução de etanol 20% e losartan. Foram realizadas aferições semanais da pressão arterial e freqüência cardíaca dos animais. Foram realizadas as dosagens para determinar: o nível de etanol no sangue; os níveis plasmáticos e teciduais (aorta e leito arterial mesentérico) de angiotensina I (ANG I) e ANG II; a atividade plasmática da renina; atividade plasmática e tecidual da enzima conversora de angiotensina (ECA); níveis plasmáticos de aldosterona; níveis plasmáticos do peptídeo natriurético atrial (ANP), vasopressina (AVP) e ocitocina (OT); a osmolaridade e o sódio plasmático; nitrato plasmático e tecidual; espécies reativas ao ácido tiobarbitútico (TBARS); a formação tecidual de ânion superóxido; a capacidade antioxidante total; além de verificar a expressão gênica e protéica (aorta) da via das MAPKs, via do óxido nítrico (NO), das ciclooxigenases (COX), além dos componentes do sistema renina angiontensina (SRA). A artéria aorta torácica foi isolada e foram obtidas curvas concentração-resposta para fenilefrina, acetilcolina (Ach) e nitroprussiato de sódio (NPS). O tratamento crônico de etanol aumentou a pressão arterial sistólica, diastólica e média dos animais, sem afetar a freqüência cardíaca; induziu o aumento da atividade plasmática da renina, aumento da atividade da ECA e dos níveis plasmáticos de ANG I e ANG II, sendo que tais efeitos não foram prevenidos pela administração de losartan. Não houve alteração dos níveis teciduais de ANG I e ANG II. O tratamento com etanol não alterou a osmolaridade ou os níveis plasmáticos de sódio e de aldosterona. Nos animais tratados cronicamente com etanol houve redução plasmática de AVP, OT e aumento de ANP. O losartan não preveniu estes efeitos induzidos pelo etanol. O etanol promoveu aumento dos níveis plasmáticos de TBARS, os níveis teciduais de ânion superóxido, e o tratamento com losartan preveniu tais respostas. O tratamento com etanol não alterou os níveis plasmáticos de peróxido de hidrogênio e da atividade plasmática da SOD. Houve redução dos níveis plasmáticos e teciduais do NO, alteração da capacidade antioxidante plasmática total e o tratamento com losartan preveniu esses efeitos. O consumo etanol potencializou a resposta vasoconstritora induzida pela fenilefrina em anéis de aorta com e sem endotélio. O losartan preveniu tal resposta contrátil. No entanto, o tratamento com etanol não alterou a resposta de relaxamento induzida pela Ach e pelo NPS em anéis de aorta. O etanol foi capaz de alterar a expressão gênica e protéica da via das MAPKs, via do NO-AKT, das COX, e os componentes do SRA. Portanto, conclui-se que o consumo crônico de etanol ativa o SRA sistêmico, induz estresse oxidativo sistêmico e vascular, altera a reatividade vascular, reduz os níveis plasmáticos de NO, altera a expressão gênica e protéica da via das MAPKs, do NO, das COX e os componentes do SRA, e promove alterações neuro-humorais que, em conjunto, contribuem para a elevação da pressão arterial sistêmica e alterações cardiovasculares pela ação da ANG II via receptor AT1. / The cardiovascular dysfunction induced by chronic ethanol consumption is associated with the formation of reactive oxygen species (ROS). Angiotensin II via AT1 receptors is a major maker of ROS in the cardiovascular system. To evaluate the role of AT1 receptors in the cardiovascular dysfunction induced by chronic ethanol consumption. Male Wistar rats were divided into four groups: Control: received water \"ad libitum\"; Ethanol: received ethanol solution 20% (vol./vol.); Control+Losartan: received water \"ad libitum\" and losartan (10 mg/kg) daily by gavage; Losartan+Ethanol: received 20% ethanol solution and losartan. The measurements were performed to determine: the level of ethanol in blood, plasma and tissue levels (aorta and mesenteric arterial bed) of angiotensin I (ANG I) to ANG II, plasma renin activity, plasma and tissue activity of angiotensin converting enzyme (ACE), plasma aldosterone levels, plasma levels of atrial natriuretic peptide (ANP), vasopressin (AVP) and oxytocin (OT); osmolality and sodium, plasma and tissue nitrate (NO); tiobarbitútico reactive to acid species (TBARS); tissue formation superoxide anion, total antioxidant capacity, besides verifying the gene and protein expression of MAPK pathway, the nitric oxide (NO), the cyclooxygenase (COX), in addition to the components of the renin angiotensin (RAS). The thoracic aorta was isolated and concentration-response curves for phenylephrine, acetylcholine and sodium nitroprusside were obtained. Chronic ethanol treatment increased systolic blood pressure, diastolic and mean animals, without affecting heart rate; induced increase in plasma renin activity, increased plasma and tissue levels of ACE and plasma levels of ANG I and ANG II, and these effects were prevented by administration of losartan. There were no changes in tissue levels of ANG I and ANG II. The treatment with ethanol did not alter osmolarity and plasma levels of sodium and aldosterone. In animals chronically treated with ethanol was reduced plasma AVP, OT and ANP increase. Losartan did not prevent these effects induced by ethanol. Ethanol promoted increased plasma levels of TBARS, tissue levels of superoxide anion, and treatment with losartan prevented these responses. The ethanol treatment did not alter plasma levels of hydrogen peroxide and plasma activity of SOD. There was a reduction in plasma and tissue levels of NO, alteration in total plasma antioxidant capacity and treatment with losartan prevented these effects. The consumption ethanol potentiated the pressor response induced by phenylephrine in aortic rings with and without endothelium. The losartan prevented this contractile response. However, treatment with ethanol did not alter the response of relaxation induced by Ach and SNP in aortic rings. The ethanol was able to alter the gene and protein expression via the MAPK, via the NOAKT, the COX, and the components of the RAS. Therefore, it is concluded that chronic consumption of ethanol activates the systemic RAS, induces systemic oxidative stress and vascular change vascular reactivity, reduces plasma levels of NO, alters gene and protein expression via the MAPK, NO, the COX and RAS components, and promotes neurohumoral changes that, together, contribute to the elevation of blood pressure and cardiovascular changes by the action of ANG II via AT1 receptor.

Duchenne Muscular Dystrophy (DMD) is an x-linked neuromuscular disease that is caused by an absence of dystrophin protein, rendering skeletal muscle more susceptible to contraction-induced damage. One therapeutic strategy focuses on increasing the expression of endogenous utrophin A, a dystrophin homologue. Interestingly, slow muscle is more resistant to the dystrophic pathology and has increased utrophin A expression (Webster 1998; Gramolini 2001b). These observations led researchers to explore the therapeutic potential of stimulating the slow, oxidative myogenic program (SOMP) in the mdx context. Beneficial adaptations were seen with pharmacological activation of PPARδ and AMPK. We treated mdx mice with resveratrol (~100mg/kg/day), a putative SIRT1 activator, for 6-7 weeks and evaluated the activity of phenotypic modifiers that are known to influence the SOMP. SIRT1 activity and protein levels increased significantly, as well as downstream PGC-1α activity. There was evidence of a fibre type conversion as the treated mice had a higher proportion of the slow myosin heavy chain isoforms in both the EDL and Soleus skeletal muscles. Utrophin A protein levels showed modest, but consistent increases with resveratrol treatment. Finally, histological analysis revealed improvements in central nucleation and fibre size variability. These findings were promising, but raised the question of whether modifying the treatment regimen may result in greater therapeutic benefits. Surprisingly, we discovered that an elevated dose of 500mg/kg/day was ineffective in its promotion of the SOMP. SIRT1 was not activated and there was no change in utrophin A levels with resveratrol treatment. Taken together, this study demonstrates that resveratrol has the ability to promote the SOMP through SIRT1 and PGC-1α activation. It also highlights the importance of selecting an appropriate dose of resveratrol to maximize its effectiveness.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-18T22:38:09Z No. of bitstreams: 1 Erika Cassia Barroso Batista.pdf: 1153764 bytes, checksum: d2f3d36a962ecb178963820d00e754fa (MD5) / Made available in DSpace on 2018-06-18T22:38:09Z (GMT). No. of bitstreams: 1 Erika Cassia Barroso Batista.pdf: 1153764 bytes, checksum: d2f3d36a962ecb178963820d00e754fa (MD5) Previous issue date: 2015-12-16 / The activation, proliferation and migration of myogenic precursor cells are essential for muscle regeneration after injury, orchestrated by cells and local components, mainly inflammatory cells, especially macrophages. These are identified as a target for the treatment of muscle injuries. Laser therapy has shown good results in treatment of injuries and the ability to accelerate the migration of various cell types, but there are no reports on their effect on macrophage products influencing the migration of myogenic precursor cells. The aim of the study was to evaluate the effect of low level laser (LLL) on migration of myoblasts cultured with macrophage culture supernatants of different phenotypes. Therefore, C2C12 cells were cultured with supernatants from cultures of macrophages (J774) treated with LPS and IFN-γ (for the activation phenotype M1), IL-4 (M2a phenotype) and IL-10 and dexamethasone (M2c phenotype) and LLL irradiated at wavelengths of 660nm and 780nm (70mW; 17,5J / cm2; 14.3 s; 0,8J). Supernatants from macrophage cultures were harvested 24h after irradiation and transferred to myoblast cultures. Migration was assessed using the scratch assay and the results statistically analyzed. Myoblasts cultured with phenotype macrophage supernatants M2c and irradiated with LBP (660nm) showed higher migration capability that cultured with supernatants of M2C phenotype of macrophages after 12 hours of cultivation. There was no difference between the other groups. The LLL was able to modulate the migration of myoblasts C2C12 when M2c supernatants of macrophage phenotype / A ativação, proliferação e migração das células precursoras miogênicas são essenciais na regeneração muscular após lesões, orquestrados pelas células e componentes locais, principalmente pelas células inflamatórias, em especial os macrófagos. Estes são apontadas como alvo para o tratamento das lesões musculares. A laserterapia tem demonstrado bons resultados no tratamento destas lesões e na capacidade de acelerar a migração de vários tipos celulares, mas não existem relatos sobre seu efeito nos produtos de macrófagos que influenciam a migração de células precursoras miogênicas. O objetivo do estudo foi avaliar o efeito do laser de baixa potência (LBP) sobre a migração de mioblastos cultivadas com sobrenadantes de culturas de macrófagos de diferentes fenótipos. Para tanto, as células C2C12 foram cultivadas com sobrenadantes de culturas de macrófagos (J774) tratadas com LPS e IFN- γ (ativação para o fenótipo M1), IL-4 (fenótipo M2a) e IL-10 e dexametasona (fenótipo M2c) e irradiadas com LBP nos comprimentos de onda de 660nm e 780nm (70mW; 17,5J/cm2; 14,3 s; 0,8J). Os sobrenadantes das culturas de macrófagos foram colhidos 24h após as irradiações e transferidos para culturas de mioblastos. A migração foi avaliada por meio do ensaio de ferida e os resultados submetidos à análise estatística. Após 12h de cultivo, os mioblastos cultivados com sobrenadantes de macrófagos de fenótipo M2c e irradiados com LBP (660nm) mostraram maior capacidade de migração que os cultivados com sobrenadantes de macrófagos de fenótipo M2c não irradiados. Não houve diferença entre os demais grupos. O LBP foi capaz de modular a migração de mioblastos C2C12 quando cultivados com sobrenadantes de macrófagos de fenótipo M2c.

Skeletal muscle differentiation requires synergy between tissue-specific transcription factors, chromatin remodeling enzymes and the general transcription machinery. Here we demonstrate that two distinct protein arginine methyltransferases are required to complete the differentiation program. Prmt5 is a type II methyltransferase, symmetrically dimethylates histones H3 and H4 and has been shown to play a role in transcriptional repression. An additional member of the Prmt family, Carm1 is a type I methyltransferase, and asymmetrically methylates histone H3 and its substrate proteins. MyoD regulates the activation of the early class of skeletal muscle genes, which includes myogenin. Prmt5 was bound to and dimethylates H3R8 at the myogenin promoter in a differentiation-dependent fashion. When proteins levels of Prmt5 were reduced by antisense, disappearance of H3R8 dimethylation and Prmt5 binding was observed. Furthermore, binding of Brg1 to regulatory sequences of the myogenin promoter was abolished. All subsequent events relying on Brg1 function, such as chromatin remodeling and stable binding by muscle specific transcription factors such as MyoD, were eliminated. Robust association of Prmt5 and dimethylation of H3R8 at myogenin promoter sequences was observed in mouse satellite cells, the precursors of mature myofibers. Prmt5 binding and histone modification were observed to a lesser degree in mature myofibers. Therefore, these results indicate that Prmt5 is required for dimethylating histone at the myogenin locus during skeletal muscle differentiation in order to facilitate the binding of Brg1, the ATPase subunit of the chromatin remodeling complex SWI/SNF. Further exploration of the role of Prmt5 during the activation of the late class of muscle genes revealed that though Prmt5 is associated with and dimethylates histones at the regulatory elements of late muscle genes in tissue and in culture, it was dispensable for late gene activation. Previous reports had indicated that Carm1 was involved during late gene activation. We observed that Carm1 was bound to and responsible for dimethylating histones at late muscle gene promoters in tissue and in culture. In contrast to Prmt5, a complete knockout of Carm1 resulted in abrogation of late muscle gene activation. Furthermore, loss of Carm1 binding and dimethylated histones resulted in a disappearance of Brg1 binding and chromatin remodeling at late muscle gene loci. Time course chromatin immunoprecipitations revealed that Carm1 binding and histone dimethylation occurred concurrently with the onset of late gene activation. In vitro binding assays revealed that an interaction between Carm1, myogenin and Mef2D exists. These results demonstrate that Carm1 is recruited to the regulatory sequences of late muscle genes via its interaction with either myogenin or Mef2D and is responsible for dimethylates histones in order to facilitate the binding of Brg1. Therefore, these results indicate that during skeletal muscle differentiation, distinct roles exist for these Prmts such that Prmt5 is required for activation of early genes while Carm1 is essential for late gene induction.

Objective: Vestibular Evoked Myogenic Potentials (VEMP) were investigated to differentiate between parkinsonian syndromes. We correlated balance and VEMP parameters to investigate the VEMP brainstem circuits as possible origin for postural instability. Methods: We assessed clinical status, ocular and cervical VEMP (oVEMP, cVEMP) and a balance assessment (posturography, Activities-specific Balance Confidence Scale, Berg Balance Scale, modified Barthel Index) in 76 subjects: 30 with Parkinson’s disease (PD), 16 with atypical parkinsonism (AP) and 30 healthy controls. VEMP were elicited by using a mini-shaker on the forehead. Results: Patients with PD had a prolonged oVEMP n10 in comparison to controls and prolonged p15 compared to controls and AP. Patients with AP showed reduced oVEMP amplitudes compared to PD and controls. CVEMP did not differ between groups. Postural impairment was higher in AP compared to controls and PD, particularly in the rating scales. No correlations between VEMP and posturography were found. A classifier using support vector machine was able to automatically classify controls and patient subgroups with moderate to good accuracy based on oVEMP latencies and balance questionnaires. Conclusions: Both oVEMP and posturography, but not cVEMP, may be differentially affected in PD and AP. We did not find evidence that impairment of the cVEMP or oVEMP pathways is directly related to postural impairment. Significance: OVEMP and balance assessment could be implemented in the differential diagnostic work-up of parkinsonian syndromes.:1. Einleitung 2. Publikationsmanuskript 3. Zusammenfassung 4. Literaturverzeichnis 5. Anlagen 6. Darstellung des eigenen Beitrages 7. Selbstständigkeitserklärung 8. Lebenslauf 9. Danksagung

Le muscle strié squelettique est un tissu richement vascularisé. Au delà de l'apport en oxygène et en nutriments, de nouvelles fonctions des vaisseaux ont été récemment identifiées, par le biais des interactions établies entre les cellules du vaisseau (cellules endothéliales) et les cellules du muscle, en particulier les cellules souches musculaires (cellules satellites). Celles-ci interagissent étroitement avec les cellules endothéliales pour leur expansion et leur différenciation, puis avec les cellules péri-endothéliales pour leur auto-renouvellement et leur retour à la quiescence. Les vaisseaux participent ainsi au contrôle de l’homéostasie du muscle squelettique. Grâce à ces interactions, les cellules vasculaires jouent donc un rôle central dans le remodelage tissulaire après un phénomène destructif, survenant par exemple au cours d’un trauma ou d’une myopathie. Pour étudier, les mécanismes de la plasticité vasculaire au cours du remodelage tissulaire, deux situations paradigmatiques de muscle en régénération chez l’enfant : la dermatomyosite juvénile (DMJ) et la dystrophie musculaire de Duchenne (DMD) ont été étudiées. Il existe, dans ces deux pathologies une souffrance musculaire associée à des cycles de nécrose/régénération. Elles se différencient par leur plasticité vasculaire et par leur évolution. En effet, la DMJ, la myopathie inflammatoire la plus fréquente de l’enfant est caractérisée par une vasculopathie avec perte en capillaires. L’évolution peut être favorable avec restitution ad integrum du muscle. La DMD est une myopathie génétique conduisant à une dégradation progressive de la force musculaire associée à une néovascularisation compensatrice. Le volet clinique/histologique incluant une analyse multiparamétrique des critères évolutifs cliniques et de réponse thérapeutique couplée à une réévaluation des données histologiques de la DMJ (analyse morphométrique des muscles DMJ) a permis de montrer qu’il existait des sous groupes phénotypiques homogènes de sévérité différente dans la DMJ. Le degré de sévérité clinique est relié à la gravité de la vasculopathie musculaire Par ailleurs, des marqueurs cliniques et histologiques simples permettant de repérer au diagnostic les patients nécessitant une escalade thérapeutique rapide (CMAS>34, atteinte gastrointestinale, fibrose endomysiale musculaire au diagnostic) ont été identifiés. Le volet cellulaire a permis l’identification in vitro des interactions cellulaires spécifiques et différentielles des myoblastes issues de patients DMD et DMJ sur les cellules endothéliales normales par l’analyse de leur rôle sur la prolifération, migration et différenciation des cellules vasculaires. Dans la DMD, les myoblastes entrainent une réponse angiogénique importante mais non efficace (néovascularisation anarchique). Dans la DMJ, les myoblastes participent efficacement à la reconstruction vasculaire notamment via la sécrétion de facteurs proangiogéniques. Ces résultats ont été renforcés par analyse transcriptomique effectuée à partir de cellules endothéliales et satellites isolées de muscles de patients confirmant le rôle central de la vasculopathie associée à un contexte inflammatoire spécifique lié à l’interféron dans la physiopathologie de la DMJ et montrant dans la DMD une dérégulation de l’homéostasie normale des interactions vaisseau-muscle avec mise en jeu d’un remodelage tissulaire non efficace. Ces données permettent d'identifier de nouvelles fonctions des cellules vasculaires dans le remodelage du muscle strié squelettique au cours des pathologies musculaires de l'enfant, et devraient ouvrir la voie à de nouvelles approches thérapeutiques. / Skeletal muscle is highly vascularised. Beyond oxygen and nutriment supply, new functions for vessels have been recently identified, through the interactions that vessel cells (endothelial cells) establish with muscle cells, particularly with muscle stem cells (satellite cells). These latter closely interact with endothelial cells for their expansion and their differentiation, then with periendothelial cells for their self-renewal and return to quiescence. During skeletal muscle regeneration endothelial cells reciprocally interact with myogenic cells by direct contact or by releasing soluble factors to promote both myogenesis and angiogenesis processes. Skeletal muscle regeneration typically occurs as a result of a trauma or disease, such as congenital or myopathies. To better understand the role of vessel plasticity in tissue remodeling, we took advantage of two muscular disorders that could be considered as paradigmatic situations of regenerating skeletal muscle in the child: Juvenile Dermatomyositis (JDM), the most frequent inflammatory myopathy and Duchenne Muscular Dystrophy (DMD), the most common type of muscular dystrophy. Although these two muscular disorders share, at the tissue level, similar mechanisms of necrosis-inflammation, they differ regarding the vessel domain. In JDM patients, microvascular changes consist in a destruction of endothelial cells assessed by focal capillary loss. This capillary bed destruction is transient. The tissue remodeling is efficient and muscle may progressively recover its function. By contrast, in DMD, despite an increase of vessels density in an attempt to improve the muscle perfusion, the muscle function progressively alters with age. We identified clinical and pathological markers of severity and predictive factors for poor clinical outcome in JDM by computing a comprehensive initial and follow-up clinical data set with deltoid muscle biopsy alterations controlled by age-based analysis of the deltoid muscle capillarization. We demonstrated that JDM can be divided into two distinctive clinical subgroups. The severe clinical presentation and outcome are linked to vasculopathy. Furthermore, a set of simple predictors (CMAS<34, gastrointestinal involvement, muscle endomysial fibrosis at disease onset) allow early recognition of patients needing rapid therapeutic escalation with more potent drugs. We studied in vitro the specific cell interactions between myogenic cells issued from JDM and DMD patients and normal endothelial cells to explore whether myogenic cells participate to the vessel remodeling observed in the two pathologies. We demonstrated that MPCs possessed angiogenic properties depending on the pathological environment. In DMD, MPCs promoted the development of establishment of an anarchic, although strong, EC stimulation, leading to the formation of weakly functional vessels. In JDM, MPCs enhanced the vessel reconstruction via the secretion of proangiogenic factors. This functional analysis was supported by the transcriptomic analysis consistent with a central vasculopathy in JDM including a strong and specific response to an inflammatory environment. On the contrary, DMD cells presented an unbalanced homeostasis with deregulation of several processes including muscle and vessel development with attempts to recover neuromuscular system by MPCs. To summarize, our data should allow the definition of new functions of vessel cells in skeletal muscle remodelling during muscle pathologies of the child that will open the way to explore new therapeutic options and to gain further insights in the pathogenesis of these diseases.