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

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.

Nuclear hormone receptors (NHR) constitute a superfamily of ligand inducible transcriptional activators that enable an organism to regulate development and homeostasis through switching on or off target genes in response to stimuli reflecting changes in environment as well as endocrine. NHRs include classical steroid hormone receptors (GR, AR, ER and MR) and retinoid, thyroid hormone receptors. One long-term goal of our lab is to understand the molecular mechanisms through which the transcriptional activity of NHRs is regulated. Extensive studies in the past few years have revealed that in addition to the dependence on ligand availability, the transcriptional activity of NHRs is also regulated by two types of proteins: co activators and corepressors. In the absence of ligand, many NHRs, including TR and RAR can actively repress target gene transcription with the help of corepressors, proteins that physically interact with both NHRs and histone deacetylases (HDACs). Functional interactions between NHRs and corepressors therefore lead to tightly compact and transcriptionally non-permissive chromatin structures after the removal of obstructive acetyl groups from histone tails by HDACs. On the other hand, ligand binding stabilizes NHRs in a conformation that favors interaction with proteins other than corepressors; many of these proteins are able to potentiate the transcriptional activity of NHRs through various mechanisms, such as histone acetylation, chromatin remodeling and recruitment of basal transcription machinery and are collectively termed coactivators. Two highly related corepressors, SMRT (silencing mediator of retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor corepressor), have been cloned. This research in corepressor SMRT started by a systematic study of its subcellular localization. We found that SMRT predominantly forms a specific nuclear punctuate structure that does not appear to overlap with any other well-known subnuclear domains/speckles. Although our searching for specific sequence signals that may determine the specific speckle localization of SMRT did not yield conclusive results, we discovered the colocalization of unliganded RAR and certain HDACs, including HDAC1, 3,4 and 5, in the SMRT nuclear speckles. Moreover, SMRT is likely to be the organizer of such speckles since it appears to be able to recruit other proteins into these speckles. The presence of HDAC1 in the SMRT speckles suggests a direct association between these two proteins, which has not been detected by previous biochemical analyses. Interestingly, HDAC1 point mutants that are completely defective in deacetylase activity failed to locate to SMRT nuclear speckles, while another partially active mutant maintained the colocalization. These discoveries may indicate SMRT nuclear speckles as novel nuclear domains involved in transcriptional repression. More physiologically relevant support for this hypothesis arises from study of HDAC4 and 5. HDAC4 and 5 are potent inhibitors of transcriptional activator MEF2C. Nuclear presence of HDAC4/5 can block the activation of MEF2C, which is required during muscle differentiation. Normally, HDAC4 is predominantly located in cytoplasm. However, we found that in the presence of SMRT overexpression, HDAC4 was found mostly in SMRT nuclear speckles. This accumulation enhanced HDAC4 mediated inhibition on MEF2C transcriptional activity in a transient transfection assay. SMRT overexpression also resulted in accumulation of HDAC5 in the SMRT nuclear speckles compared to the nuclear diffuse distribution in the absence of SMRT. Again, this accumulation of HDAC5 in nuclear speckles correlated with enhanced inhibition of MEF2C. Taken together, our study suggested that instead of being merely a corepressor for NHRs, SMRT might function as an organizer of a nuclear repression domain, which may be involved in a broad array of cellular processes. In contrast to the limited number of corepressors, numerous co activators have been identified; the SRC (or p160) family is relatively well studied. This family includes three highly related members, SRC-1, TIF2/GRIP1, RAC3/AIB1/ACTR/p/CIP. Similar domain structures are shared among these factors, with the most highly conserved region, the bHLH-PAS domain found within the N terminal ~400 amino acid residues. This study of RAC3 aims to identify the function of the highly conserved N terminal bHLH-PAS domain by isolating interacting proteins through yeast two-hybrid screening. One candidate gene isolated encodes the C terminal fragment of the human homologue of the yeast protein MMS19. Functional studies of this small fragment revealed that it specifically interacted with human estrogen receptors (ERs) and inhibited ligand induced transcriptional activity of ERs in the transient transfection assay. Then we cloned the full-length human MMS19 cDNA and characterized the hMMS19 as a weak coactivator for estrogen receptors in the transient transfection assay. Furthermore, when tested on separate AF-1 or AF-2 of ERs, hMMS19 specifically enhanced AF-1 but had no effect on AF-2. These results identified hMMS19 as a specific coactivator for ER AF-1.

Tese(doutorado)-Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2011. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-06-23T16:27:42Z No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-07-27T20:22:46Z (GMT) No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) / Made available in DSpace on 2012-07-27T20:22:46Z (GMT). No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) Previous issue date: 2011 / A aquicultura tem crescido significativamente nas últimas décadas devido ao aumento da demanda de pescado no mundo e à estagnação do setor pesqueiro. Porém, este crescimento depende do desenvolvimento de novos pacotes tecnológicos que visem o aumento da produtividade. Uma alternativa é a manipulação genética (transgenia), sendo que o hormônio do crescimento (GH) tem sido o principal alvo das pesquisas com peixes transgênicos. Entretanto, está comprovado que o excesso de GH acarreta uma série de efeitos adversos devido a sua ação pleiotrópica. A ativação do eixo somatotrófico de forma tecido-específica e independente do excesso de hormônio circulante pode contornar estes problemas. Neste contexto, o objetivo desta tese foi superexpressar o gene do receptor do GH (GHR) no tecido muscular esquelético do zebrafish (Danio rerio) e estudar os efeitos desta manipulação sobre os mecanismos envolvidos na regulação gênica do crescimento muscular. A linhagem transgênica estável obtida expressa o GHR especificamente no tecido muscular 100 vezes mais do que os não transgênicos. Estes transgênicos não apresentaram aumento significativo no crescimento, provavelmente devido à queda na expressão do fator de crescimento tipo insulina I (IGF-I). Esta queda foi, provavelmente, relacionada à ação dos principais moduladores da sinalização do GH (SOCS1 e 3), os quais apresentaram-se aumentados nos transgênicos. Ainda, foi observada uma queda na expressão das principais proteínas musculares estruturais (Acta1, myhc4 e mylz2), o que explica a ausência de hipertrofia nos transgênicos. Por outro lado, o aumento na expressão dos principais fatores reguladores miogênicos (myod, myf5 e myog) explica a hiperplasia observada nas análises histológicas. Para verificar como a superexpressão do GHR ativou a transcrição dos fatores reguladores miogênicos (MRFs) e, por consequência a hiperplasia, foram estudados os possíveis mecanismos envolvidos neste processo. Dentre estes, tanto a via proliferativa (MEK/ERK) quanto à via relacionada com a síntese protéica (PI3K/Akt), não tiveram alteração na expressão de seus genes. Entretanto, foi observado aumento na expressão das proteínas de transporte para o núcleo (importinas 1, 3 e 1), podendo-se concluir que a ativação dos MRFs está relacionada ao transporte do GHR para o núcleo das células musculares. Desta forma, pode se concluir que hiperplasia e hipertrofia seguem duas vias de sinalização intracelular distintas, ambas desencadeadas pelo GH, mas reguladas por mecanismos diferentes. / Aquiculture practice has been significantly increasing during the last decades due to the fish rising demand and to fishery activity stagnation. However, such increase depends on new technological packages development aiming to productivity rises. Genetic manipulation is an alternative, once growth hormone (GH) has been the main target on transgenic fish researches. Nevertheless, it has been proved that GH excess leads to many adverse effects due to its pleiotropic action. The somatotropic axis activation in a tissue-specific manner and independent on the circulating hormone excess may bypass these problems. Following these ideas, the present thesis objective was overexpressing GH receptor’s gene (GHR), in zebrafish (Danio rerio) skeletal muscular tissue, and studying such manipulation effects over the mechanisms involved in muscular growth gene regulation. The stable transgenic lineage obtained expresses GHR, specifically in muscular tissue, 100 times more than non-transgenic. These transgenic did not present significant growth, possibly due to a gene expression fall in insulin-like growth factor I (IGF-I). The mentioned fall is, probably, related to GH main signaling modulators (SOCS1 and 3) action, which were increased in transgenic. Also, a gene expression fall from the main structural muscle proteins (Acta1, myhc4 and mylz2) was observed, explaining the transgenic hypertrophy absence. However, the main myogenic regulatory factors (myod, myf5 and myog) expression rising explains the observed hyperplasia in histological analysis. Intending to verify how GHR overexpression has activated the myogenic regulatory factors (MRFs) transcription and, consequently, the hyperplasia, the mechanisms possibly involved in this process were studied. Among these, even the proliferative pathway (MEK/ERK) or the pathway related to protein synthesis (PI3K/Akt), did not presented gene expression altering. However, a gene expression rise in transporting proteins into nucleus (importins 1, 3 and 1) was observed, which may be understood as a correlation between MRFs activation and GHR transport into muscular cell’s nucleus. Therefore, it may be understood that hyperplasia and hypertrophy follow two distinct intracellular signaling pathways, both triggered by GH, but regulated by different mechanisms. These data may be important for aquiculture new transgenic lineages development.

Orientador: Maeli Dal Pai Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:12:18Z (GMT). No. of bitstreams: 1 Souza_RodrigoWagnerAlves_M.pdf: 2186081 bytes, checksum: 2a1578159471f249151f3b90ca0b76fb (MD5) Previous issue date: 2010 / Resumo: O treinamento físico pode promover adaptações benéficas ao músculo esquelético. Entretanto, o treinamento de alta intensidade associado com um tempo insuficiente de recuperação, similar às condições de sobretreinamento, pode ocasionar efeitos prejudiciais. Os fatores reguladores miogênicos (MRFs) e o fator de crescimento IGF-1 são importantes reguladores da massa muscular no treinamento físico. Neste contexto, testamos a hipótese que o treinamento de alta intensidade com curto tempo de recuperação, poderia influenciar a morfologia, as isoformas da cadeia pesada de miosina (MHC), e a expressão dos MRFs MyoD e Miogenina e do IGF-1, no músculo esquelético de ratos. Ratos Wistar machos (200-250g) foram divididos em 4 grupos: treinado 8 semanas (T8), controle 8 semanas (C8), treinado 12 semanas (T12) e controle 12 semanas (C12). Os grupos T8 e T12 realizaram um programa de treinamento resistido de alta intensidade (5 dias/semana), envolvendo sessões de saltos em uma cuba contendo água. Ao término de cada período, os animais foram sacrificados e o músculo plantar retirado e submetido às análises morfológica e histoquímica, análises das MHCs e expressão gênica da MyoD, Miogenina e IGF-1. Do início ao final do experimento todos os grupos aumentaram o peso corporal, no entanto, o grupo T12 foi estatisticamente menor em relação ao C12. Com relação à área de secção transversal, observou-se uma redução das fibras IIC e IIAD no grupo T8 e IIA e IID no grupo T12 em relação aos seus controles. O grupo treinado por 12 semanas apresentou um aumento da frequência das fibras IIBD e redução nas frequências das fibras I, IIA e IID, em relação ao grupo controle; esses dados ainda foram corroborados pela redução das isoformas MHCI e MHCIIa e aumento da MHCIIb. A MHCIId não mostrou diferença significativa. A expressão gênica do grupo T12 apontou uma diminuição da MyoD e um aumento do IGF-1 comparado com o grupo C12; já, a expressão da Miogenina foi semelhante entre os grupos. Estes dados mostram que o modelo utilizado, semelhante às condições do sobretreinamento, promoveu a atrofia muscular e a transição das fibras musculares para uma atividade contrátil mais rápida. Estes fatos podem estar associados a uma menor atividade das células satélites. Em adição, o aumento da expressão do IGF-1, decorrente do treinamento, pode ter ocorrido na tentativa de prevenir o processo atrófico. / Abstract: Physical training can promote beneficial changes in skeletal muscle. However, the high-intensity resistance training associated with insufficient recovery time may cause harmful effects. Myogenic regulatory factors (MRFs) and the growth factor IGF-1 are important mediators of muscle mass during physical training. In this context, we tested the hypothesis that high-intensity resistance training with short recovery time, similar to overtraining conditions, could influence the morphology, the myosin heavy chain (MHC) isoforms and the expression of MRFs MyoD and myogenin, and IGF-1 in skeletal muscle of rats. Male Wistar rats (200-250 g) were divided into 4 groups: trained 8 weeks (T8), control 8 weeks (C8), trained 12 weeks (T12) and control 12 weeks (C12). T8 and T12 groups were subjected to a high-intesnsity resistance training program (5 days/week), involving jumps sessions into water, carrying progressive overload equivalent to percentage of body weight. At the end of each period the animals were sacrificed and the plantaris muscles were removed and submited to morphological and histochemical analysis, myosin heavy chain (MHC) analysis and the gene expression of MyoD, Myogenin and IGF-1. From beginning to end of the experiment all groups increased body weight, however, in T12 body weight was lower compared to the C12. Regarding the cross-sectional area, there was a significant reduction of the IIC fibers and IIAD in T8 group and IIA and IID in T12 compared to their controls. The group trained by 12 weeks showed an increase in the IIBD, accompanied by a reduction in the I, IIA and IID muscle fibers frequency, compared to control group; these data have been corroborated by the reduction of MHCI and MHCIIa isoforms and increased of MHCIIb isoform. The MHCIId showed no significant differences. The gene expression of the T12 group showed a decrease in MyoD and an increase in IGF-1 compared with the C12 group; already, the expression of Myogenin was similar between groups. These data show that the model used, similar to the conditions of overtraining, promoted muscular atrophy and muscle fiber transition to a faster contractile activity. These facts may be associated with a lower activity of satellite cells. In addition, increased expression of IGF-1, due to training, may have occurred in an attempt to prevent the atrophic process. / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

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Orientador: Maeli Dal Pai Silva / Tese ( doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T12:24:51Z (GMT). No. of bitstreams: 1 Carani_FernandaRegina_D.pdf: 24872848 bytes, checksum: e4b7c794fa866be614e932ec5a33eaf6 (MD5) Previous issue date: 2011 / Resumo: O pirarucu (Arapaima gigas) caracteriza-se como uma espécie promissora para a Aqüicultura, devido principalmente às suas características de rápido crescimento e rusticidade. Sua criação em regime intensivo tem obtido enorme sucesso, podendo alcançar até 10 quilos de peso corporal em apenas um ano de criação. O pirarucu é considerado hoje uma das mais importantes espécies de peixes da bacia Amazônica e, por esta razão, é primordial que se investigue os mecanismos celulares e moleculares que controlam o rápido crescimento muscular, contribuindo com novas estratégias de criação e com a manutenção da espécie. O crescimento do músculo estriado esquelético nos peixes pode ocorrer por dois mecanismos: hipertrofia e/ou hiperplasia das fibras, a partir de células satélites ou mioblastos. Esse processo é controlado por Fatores Reguladores Miogênicos (MRFs) e pelo fator de crescimento Miostatina. O objetivo do presente estudo foi avaliar as características morfológicas e o crescimento muscular hipertrófico e hiperplásico, bem como analisar a expressão gênica e protéica da MyoD, da Miogenina e da Miostatina na musculatura esquelética do pirarucu (A. gigas), em diferentes fases de crescimento. Os animais utilizados no presente estudo foram provenientes de duas pisciculturas: na primeira, foram obtidos os "alevinos" (pós-larvas; 48 g); na segunda, os animais em diferentes estágios de crescimento, divididos em quatro grupos de acordo com o peso corporal. Grupo A (50 gramas, n=7), grupo B (420 gramas, n=7), grupo C (5,5 quilogramas, n=7) e grupo D (9,1 quilogramas, n=7). As amostras musculares foram coletadas, congeladas e submetidas à coloração HE, para avaliação do padrão morfológico das fibras, e à reação para a enzima NADH-TR, para avaliar o metabolismo oxidativo das fibras. Para avaliar o padrão de crescimento hiperplásico e hipertrófico da musculatura branca, foi calculado o menor diâmetro de uma população de fibras por animal, e estas foram distribuídas em classes, na dependência do seu diâmetro. A análise da expressão gênica de MyoD, miogenina e miostatina foi feita por Reação em Cadeia da Polimerase após Transcrição Reversa (RTqPCR); para análise da expressão protéica, foi utilizado o Western Blot. A distribuição das fibras em classes de diâmetro exibiu o seguinte padrão: o grupo A mostrou a maior parte das fibras na classe 20, o grupo B, na classe 50, o grupo C, nas classes 50 e 80, e o grupo D, na classe 80. Isso indica uma alta taxa de hiperplasia das fibras nos grupos menores (A e B) e alta hipertrofia das fibras nos grupos maiores (C e D). Para a análise da expressão gênica de MyoD e miogenina no músculo vermelho e branco dos "alevinos", não houve diferença estatística; para a miostatina, houve expressão diferencial, com os maiores níveis encontrados no músculo branco em comparação com o músculo vermelho. Na avaliação da expressão de MyoD e miogenina, tanto a expressão gênica como a expressão protéica não mostraram diferença significativa. Por outro lado, a expressão gênica da miostatina foi menor no grupo A e maior nos demais, e a expressão da proteína miostatina foi maior no grupo A, diminuindo nos demais grupos. Estes resultados refletem as características de crescimento muscular da espécie e sugerem que a expressão dos MRFs e da miostatina são responsáveis pelo balanço entre a hiperplasia e a hipertrofia das fibras, contribuindo para o rápido crescimento da espécie e a manutenção das características do filé / Abstract: Pirarucu (Arapaima gigas) is a promising fish species for Aquaculture programs mainly by the fast growing feature and rusticity. Their rearing under intensive conditions generated much successful results, as they reach up to 10 kilograms in just one year. Considered one of the most important fish species from Amazon basin, it is of primary interest to investigate the cellular and molecular mechanisms that control the fast muscle growth in pirarucu, providing information for new rearing strategies and species conservation. In most fish, skeletal muscle growth occurs by two mechanisms: hypertrophy and hyperplasia, from satellite cells or myoblasts. These process are under control by Myogenic Regulatory Factors (MRFs) and by the growth factor Myostatin. The animals were obtained from two pisciculture, where we got the alevin pirarucu (n=7; 48 grams weight), and the specimens at different growth stages, divided into groups according body weight. Group A (50 grams, n=7), group B (420 grams, n=7), group C (5,5 kilograms, n=7) and group D (9,1 kilograms, n=7). Muscle samples were collected, frozen and stained with HE for morphological analysis, and submitted to NADH-TR enzyme reaction for oxidative methabolism analysis. To evaluate hyperplasic and hypertrophic muscle growth, it was measured the smallest diameter from a set of fibers, which were grouped into diameter classes. Gene expression analysis of MyoD, Myogenin and Myostatin were performed by Quantitative Polimerase Chain Reaction after Reverse Transcription (RT-qPCR); protein content analysis was by Western Blot technique. Muscle fibers distribution in classes showed the following pattern: group A showed most fibers in class 20, group B, in class 50, group C, in classes 50 and 80, and group D, in class 80. This is an indicative of high fiber hiperplasia rate in groups A and B, and high hypertrophy in groups C and D. There was no statistical difference in MyoD and Myogenin genes expression in red and white muscles of pirarucu; however, Myostatin expression showed high levels in white muscle compared to red muscle. Evaluating MyoD and Myogenin expression in white muscle of pirarucu at different growth stages, both gene and protein levels were similar. Myostatin gene expression was low in group A and high in the other groups; on the other hand, Myostatin protein was high in group A and low in the other groups. These results reflect the muscle growth characteristics in pirarucu and suggest that the MRFs and Myostatin expression are controlling the balance between hyperplasic and hypertrophic mechanisms, promoting the fast rate feature of pirarucu and their fillet quality / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

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