Orientador: Maeli Dal Pai Silva / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T02:52:20Z (GMT). No. of bitstreams: 1 Almeida_FernandaLosiAlvesde_D.pdf: 2894265 bytes, checksum: 520fa8f4b513d918c8eeeaeef4851198 (MD5) Previous issue date: 2011 / Resumo: Nos peixes, o crescimento muscular ocorre por hipertrofia e hiperplasia a partir da proliferação e diferenciação das células satélites, processos regulados pela expressão de fatores de transcrição e de crescimento. Os objetivos desse trabalho foram: (1) avaliar a morfologia, morfometria e expressão gênica da MyoD, miogenina e IGF-I na musculatura branca do pacu (Piaractus mesopotamicus) com 45, 90, 180, 400 dias pós-eclosão (dpe) e adultos (n=8) e (2) avaliar a expressão gênica da MyoD, miogenina e miostatina na musculatura branca e vermelha de pacus adultos (n=8). Em todos os grupos, fragmentos da musculatura branca foram dissecados e congelados em nitrogênio líquido. Nos adultos, também foram coletados fragmentos de músculo vermelho. Cortes histológicos (10 ?m) da musculatura branca, obtidos em criostato, foram corados com hematoxilina-eosina para avaliação da morfologia e morfometria. Em cada animal, foi determinado o menor diâmetro de 100 fibras musculares brancas que foram distribuídas em classes, na dependência do seu diâmetro (<20 ?m, 20-50 ?m, >50 ?m), para avaliar a hiperplasia e hipertrofia. A expressão gênica foi analisada por reação em cadeia da polimerase após transcrição reversa em tempo real. A morfologia da musculatura branca foi semelhante em todos os grupos. No grupo 45 dpe, a alta freqüência de fibras brancas com diâmetro <20 ?m indica intensa hiperplasia; nos adultos, a alta freqüência de fibras com diâmetro > 50 ?m indica intensa hipertrofia. Nos grupos 90, 180 e 400 dpe, a alta freqüência de fibras com diâmetro entre 20 e 50 ?m indica a ocorrência de hiperplasia e hipertrofia. A expressão gênica da MyoD e miogenina foi semelhante nos grupos 45, 90, 400 dpe e adultos, aumentando significativamente nos animais com 180 dpe; nos grupos 180 dpe e adultos, essa expressão foi similar. Nossos resultados sugerem que a alta expressão de MyoD e miogenina no grupo 180 dpe está relacionada com a proliferação e diferenciação de células satélites, respectivamente, contribuindo para a hiperplasia e hipertrofia. Nos adultos, essa expressão está controlando a hipertrofia. A expressão gênica de IGF-I foi semelhante nos grupos 45 e 90 dpe, diminuindo nos animais com 180 dpe e adultos; os grupos 45, 90 e 400 dpe apresentaram expressão similar. Nos grupos 45 dpe e adultos, a expressão de IGF-I está relacionada com a proliferação de células satélites durante a hiperplasia e hipertrofia, respectivamente; nos animais com 90, 180 e 400 dpe, essa expressão está controlando ambos os mecanismos de crescimento. Entre os músculos branco e vermelho dos animais adultos, a expressão de MyoD, miogenina e miostatina foi semelhante. No músculo branco, a expressão de MyoD e miogenina está relacionada com a proliferação e diferenciação das células satélites, respectivamente, durante a hipertrofia. Na musculatura vermelha, essa expressão é responsável pela manutenção do fenótipo das fibras musculares. A expressão de miostatina, nas fibras brancas e vermelhas, está relacionada com a regulação do crescimento e manutenção da massa muscular constante nesses compartimentos / Abstract: In fish, skeletal muscle growth occurs by hypertrophy and hyperplasia and is dependent of the proliferation and differentiation of satellite cells, events regulated by the expression of transcription and growth factors. The purposes of this study were: (1) to evaluate the morphology, morphometry and MyoD, myogenin and IGF-I gene expression in white skeletal muscle from pacu (Piaractus mesopotamicus) at 45, 90, 180, 400 days post- hatching (dph) and adult (n=8) and (2) to analyze the MyoD, myogenin and myostatin gene expression in white and red muscles of adult pacu (n=8). In all groups, white skeletal muscle fragments were dissected out and frozen in liquid nitrogen. In adults, red muscle fragments were also collected. Transverse sections (10 ?m) from white muscle fragments, obtained in a cryostat, were stained with haematoxilin-eosin to evaluate muscle morphology and morphometry. In each animal fiber cross-section diameter (?m) was determined by measuring 100 white muscle fibers which were grouped into three diameter classes (<20?m, 20-50?m, and >50?m) to evaluate the hyperplasia and hypertrophy. Gene expression was analyzed by reverse transcription quantitative real-time polymerase chain reaction. White muscle morphology was similar at all groups. The high frequency of <20?m diameter white fiber in 45 dph indicates intense hyperplasia; the high frequency of >50?m diameter fiber in adults shows intense hypertrophy. In 90, 180, and 400 dph groups the high frequency of 20-50?m diameter fibers indicates the occurrence of hyperplasia and hypertrophy. MyoD and myogenin gene expression was similar in 45, 90, and 400 dph and adult fish, peaking at 180 dph; in 180 dpe and adult groups this expression was similar. Our results suggest that the high MyoD and myogenin expression at 180 dph is related to the satellite cells proliferation and differentiation, respectively, during hyperplasia and hypertrophy. In adult fish, this expression is controlling the hypertrophy. The IGF-I gene expression was similar in 45 and 90 dpe stages, decreasing in 180 dpe and adult fish; the 45, 90 and 400 dpe groups have showed similar expression. In 45 dpe and adult groups, IGF-I expression is related to satellite cells proliferation during hyperplasia and hypertrophy, respectively; in 90, 180 and 400 dpe fish, this expression is controlling the both muscle growth mechanims. In red and white muscles from adults the the MyoD, myogenin and myostatin gene expression was similar. In white muscle, the MyoD and myogenin expression is related to the satellite cells proliferation and differentiation, respectively, during hypertrophy. In red muscle, this expression is responsible to the maintenance of fiber-type phenotype. Myostatin expression in white and red muscles is related to the growth regulation and maintenance of constant muscle mass in these compartments / 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

Myostatin (GDF-8) is the chief chalone in skeletal muscle and negatively controls adult skeletal muscle growth. The role of myostatin during overload-induced hypertrophy of adult muscle is unclear. We tested the hypothesis that overloaded adult rodent skeletal muscle would result in reduced myostatin protein levels. Overload-induced hypertrophy was accomplished by unilateral tenotomy of the gastrocnemius tendon in male adult Sprague-Dawley rats followed by a two-week period of compensatory overload of the plantaris and soleus muscles. Western blot analysis was performed to evaluate changes in active, latent and precursor myostatin protein levels. Significant hypertrophy was noted in the plantaris (494 ± 29 vs. 405 ± 15 mg, p < 0.05) and soleus (289 ± 12 vs. 179 ± 37 mg, p < 0.05) muscles following overload. Overloaded soleus muscle decreased the concentration of active myostatin protein by 32.7 ± 9.4% (p < 0.01) while the myostatin precursor protein was unchanged. Overloaded plantaris muscle decreased the concentration of active myostatin protein by 28.5 ± 8.5% (p < 0.01) while myostatin precursor levels were reduced by 17.5 ± 5.9% (p < 0.05). Myostatin latent complex concentration decreased in the overloaded soleus and plantaris muscle by 15.0 ± 5.9% and 70.0 ± 2.3% (p < 0.05), respectively. These data support the hypothesis that the myostatin signaling pathway in overloaded muscles is generally downregulated and contributes to muscle hypertrophy. Plasma concentrations of total and active myostatin proteins were similar in overloaded and control animals and averaged 8865 ± 526 pg/ml and 569 ± 28 pg/ml, respectively. Tissue levels of BMP-1, an extracellular proteinase that converts myostatin to its active form, also decreased in overloaded soleus and plantaris muscles by 40.4 ± 12.9% and 32.9 ± 6.9% (p < 0.01), respectively. These data support the hypothesis that local, rather than systemic, regulation of myostatin contributes to the growth of individual muscles, and that an association exists between the extracellular matrix proteinase BMP-1 and the amount of active myostatin in overloaded muscles.

Cellular and molecular mechanisms of postembryonic muscle fibre recruitment were investigated in zebrafish (Danio rerio L.), a standard animal model for developmental and genetic studies. Distinct cellular mechanisms of postembryonic muscle fibre recruitment in fast and slow myotomal muscles were found. In slow muscle, three overlapping waves of stratified hyperplasia (SH) from distinct germinal zones sequentially contributed to a slow and steady increase in fibre number (FN) through the life span. In fast muscle, SH only contributed to an initial increase of FN in early larvae. Strikingly, mosaic hyperplasia (MH) appeared in late larvae and early juveniles and remained active until early adult stages, accounting for >70% of the final fibre number (FFN). The molecular regulation of postembryonic muscle fibre recruitment was then studied by characterising myospryn and cee, two strong candidate genes previously identified from a large scale screen for genes differentially expressed during the transition from hyperplastic to hypertrophic muscle phenotypes. Zebrafish myospryn contained very similar functional domains to its mammalian orthologues, which function to bind to other proteins known to regulate muscle dystrophy. Zebrafish myospryn also shared a highly conserved syntenic genomic neighbourhood with other vertebrate orthologues. As in mammals, zebrafish myospryn were specifically expressed in striated muscles. Zebrafish cee was a single-copy gene, highly conserved among metazoans, ubiquitously expressed across tissues, and did not form part of any wider gene family. Its protein encompassed a single conserved domain (DUF410) of unknown function although knock-down of cee in C. elegans and yeast have suggested a role in regulating growth patterns. Both myospyrn and cee transcripts were up-regulated concomitant with the cessation of postembryonic muscle fibre recruitment in zebrafish, indicating a potential role in regulating muscle growth. Furthermore, a genome-wide screen of genes involved in the regulation of postembryonic muscle fibre recruitment was performed using microarray. 85 genes were found to be consistently and differentially expressed between growth stages where muscle hyperplasia was active or inactive, including genes associated with muscle contraction, metabolism, and immunity. Further bioinformatic annotation indicated these genes comprised a complex transcriptional network with molecular functions, including catalytic activity and protein binding as well as pathways associated with metabolism, tight junctions, and human diseases. Finally, developmental plasticity of postembryonic muscle fibre recruitment to embryonic temperature was characterised. It involved transient effects including the relative timing and contribution of SH and MH, plus the rate and duration of fibre production, as well as a persistent alteration to FFN. Further investigation of FFN of fish over a broader range of embryonic temperature treatments (22, 26, 28, 31, 35°C) indicated that 26°C produced the highest FFN that was approximately 17% greater than at other temperatures. This finding implies the existence of an optimal embryonic temperature range for maximising FFN across a reaction norm. Additionally, a small but significant effect of parental temperature on FFN (up to 6% greater at 24 and 26°C than at 31°C) was evident, suggesting some parental mechanisms can affect muscle fibre recruitment patterns of progeny. This work provides a comprehensive investigation of mechanisms underlying postembryonic muscle fibre recruitment and demonstrates the power of zebrafish as an ideal teleost model for addressing mechanistic and practical aspects of postembryonic muscle recruitment, especially the presence of all major phases of muscle fibre production in larger commercially important teleost species.

Skeletal muscle satellite cells located between the plasma membrane and the basal lamina of muscle fibres, could for many years, only be studied in situ by electron microscopy. The introduction of immunohistochemistry and the discovery of molecular markers of satellite cells then made them accessible for light microscopic studies and a wealth of information is today available. Satellite cells are myogenic stem cells that can be activated from a quiescent state to proliferate for self-renewal or differentiate into myogenic cells. The satellite cells are involved in muscle growth during fetal and postnatal development and play a key role in repair and regeneration of damaged muscle fibres. The satellite cells are also essential for muscle fibre hypertrophy and maintenance of muscle mass in the adult. When the present thesis was initiated, studies on satellite cells in human skeletal muscle relied on the neuronal cell adhesion molecule (NCAM) as a marker for satellite cell identification. The results from different studies varied markedly. Therefore the aims of the present thesis were i) to develop a highly reliable method using light microscopy for satellite cell identification and quantification in biopsies of human skeletal muscle in normal and pathological conditions. A molecular marker for the myofibre basal lamina or plasma membrane to enhance the reliability of myonuclei and satellite cell identification were to be included. Furthermore unbiased morphometric methods should be used in the quantification process. ii) to evaluate which molecular markers which had been described for satellite cell and stem cell identification in different cell states (quiescence, activated or differentiated) are the most useful for studies on human skeletal muscle. iii) to further explore the function and heterogeneity of satellite cells with respect to different markers in human skeletal muscle by studying the effects of strength-training, intake of anabolic substances and pathological conditions. A new immunofluorescence method was developed where in the same tissue section, two satellite cell markers, the basal lamina and nuclei were monitored. From the evaluation of different markers it was found that both NCAM and Pax7 identified the majority of satellite cells but that both markers were needed for reliable identification. The members of the myogenic regulatory family were evaluated and by using the new method MyoD and myogenin were found to be useful markers to identify activated and differentiated satellite cells. Upon re-examination of biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects it was observed that the new results on satellite cell frequency were significantly different from those obtained when using staining for NCAM and nuclei alone. In addition three subtypes of satellite cells (94.4% NCAM+/Pax7+, 4.2% NCAM+/Pax7– and 1.4% NCAM–/Pax7+) were observed. Thus the multiple marker method gave more information about satellite cells heterogeneity in human muscle and we propose that this is more reliable than previous methods. Low numbers of MyoD or myogenin stained satellite cells were observed in both untrained and strength trained subjects. Other markers such as DLK1/FA1, a member of the EGF-like family and c-Met, the receptor for hepatocyte growth factor showed that satellite cell heterogeneity in human muscle is far greater than previously shown. Furthermore, new evidence is presented for so called fibre splitting observed in hypertrophic muscle fibres to be due to defect regeneration of partially damaged fibres.

Muscle aging, in particular, is characterized by the reduction of tissue mass and function, which are particularly prominent in geriatric individuals undergoing sarcopenia. The age-associated muscle wasting is also associated with a decline in regenerative ability and a reduction in resident muscle stem cell (satellite cell) number and function. Although sarcopenia is one of the major contributors to the general loss of physiological function, the mechanisms involved in age-related loss of muscle homeostasis and satellite cell activity are yet poorly understood. Using a microarray-based transcriptome analysis of muscle stem cells isolated from young and physiologically aged/geriatric mice, we uncovered specific changes in the gene expression profile that highlighted key biological processes and potential molecular markers associated with satellite cell aging, which included p16INK4a. We used Bmi1-deficient mice to further explore the implications of p16INK4a up-regulation in satellite cell function. We found premature p16INK4a up-regulation in young/adult Bmi1-deficient satellite cells correlating with defects in satellite cell number, proliferation and self-renewal capacity. In addition we have identified a number of overlapping biological processes dysregulated in physiologically aged and Bmi1-deficient satellite cells, suggesting that Bmi1-dependent epigenetic regulation may underlie many of the intrinsic changes taking place in chronologically aged satellite cells. In addition, we show that Bmi1 loss causes defects of late postnatal/adult muscle growth characterized by reduced muscle mass with smaller muscle fibers, typical of atrophying senescent/sarcopenic muscle. Since p16INK4a expression is specifically up-regulated in muscle satellite cells of geriatric, sarcopenic mice and in a mouse model of accelerated senescence/sarcopenia (SAMP8), we propose that the Bmi1/p16INK4a axis might be particularly operative in muscle stem cells from the elderly. Muscle wasting is one of the physiological consequences of sarcopenia and the identification of novel factors regulating muscle growth and atrophy is of potential relevance for therapeutical applications. We have uncovered a new role for Sestrins as skeletal muscle growth promoting factors in the adult. We found Sestrins expression regulated in mouse models of skeletal muscle atrophy and hypertrophy and in human myopathies. Through a gain of function approach we show that Sestrins induce skeletal muscle growth, by activating the IGF1/PI3K/AKT pathway. / El envejecimiento del tejido muscular está caracterizado concretamente por una reducción global de la masa muscular y un empeoramiento de la función de tejido, particularmente prominentes en individuos muy viejos (geriátricos) que padecen sarcopenia. La pérdida muscular asociado a la edad, se acompaña de una reducción en la capacidad de regeneración del músculo y en una reducción del número y la función de las células madre residentes en el músculo (células satélite). Aunque la sarcopenia sea una de las causas principales de la pérdida general de función fisiológica del músculo, los mecanismos implicados en la reducción de la homeostasis muscular y de actividad de las células satélite no han sido completamente caracterizados. Mediante el análisis comparativo del transcriptoma de células madre musculares aisladas de ratones jóvenes y de ratones viejos (geriátricos), hemos encontrado cambios específicos en su perfil de expresión génica que apuntan a los procesos biológicos dominantes y a los marcadores moleculares potencialmente asociados con el envejecimiento de las células satélite, entre los que destaca p16INK4a. Por ello, hemos utilizado ratones deficientes en Bmi1 para explorar más profundamente las implicaciones de la sobreexpresión de p16INK4a en la función de las células satélite. Hemos encontrado que células satélite jóvenes del ratón Bmi1-/- presentan sobrexpresión de p16INK4a, que correlacionan con una reducción en el número de la células, y en su capacidad de proliferación y autorenovación. Además hemos identificado un grupo de procesos biológicos comunes entre las células satélite viejas y las deficientes en Bmi1, sugiriendo que la regulación epigenética mediada por Bmi1 puede ser la base de muchos de los cambios intrínsecos que ocurren en células envejecidas fisiológicamente. Además, demostramos que la pérdida Bmi1 causa defectos en el crecimiento postnatal/adulto del músculo, caracterizado por pérdida de masa muscular con fibras más pequeñas, típico del músculo atrofiado senescente o sarcopénico. Puesto que la expresión de p16 está aumentada específicamente en el músculo de ratones viejos, sarcopénicos y en un modelo del ratón con envejecimiento (senescencia) acelerado (SAMP8), proponemos que el eje Bmi1/p16 puede actuar particularmente en las células madre musculares de los ancianos. La pérdida de masa muscular es una de las consecuencias fisiológicas de la sarcopenia y la identificación de nuevos factores que regulen el crecimiento y atrofia del músculo es de gran importancia para aplicaciones terapéuticas. Hemos descubierto un nuevo papel de las Sestrinas como factores promotores del crecimiento del músculo esquelético en el adulto. Hemos encontrado que la expresión de las Sestrinas se regula en modelos del ratón de atrofia y de hipertrofia muscular y en miopatías humanas. Mediante experimentos de ganacia de función hemos demostrado que las Sestrinas inducen el crecimiento del músculo esquelético, activando el ruta de señalización de IGF1/PI3K/AKT

Orientador: Robson Francisco Carvalho / Resumo: A irradiação pelo laser de baixa intensidade (LBI) tem sido utilizada como um método nãoinvasivo para promover ou acelerar a capacidade de regeneração muscular. No entanto, os mecanismos moleculares regulatórios pelos quais o LBI exerce esses efeitos, permanecem em grande parte desconhecidos. Nosso objetivo foi realizar uma análise de sequenciamento de RNA (RNA-Seq) em mioblastos C2C12 após LBI. Foram realizadas as taxas de viabilidade, migração, proliferação e os dados de RNA-Seq dos mioblastos C2C12, identificando 514 genes diferencialmente expressos após LBI. Em seguida, uma análise de ontologia genética e das vias dos genes diferencialmente expressos revelaram transcritos relacionadas ao ciclo celular, biogênese ribossômica, resposta ao estresse, migração celular, estrutura morfológica e proliferação de células musculares. Após, cruzamos nossos dados de RNA-Seq com dados de transcriptomas disponíveis em base de dados públicas, com dados de diferenciação miogênica que mostraram um total de 42 transcritos sobrepostos (mioblastos vs miotubos). Este conjunto de transcritos compartilhados mostrou que os mioblastos irradiados pelo LBI, possuem um perfil transcricional semelhante ao de miotubo, agrupando-se distante do perfil transcricional dos mioblastos. Concluíndo, revelamos pela primeira vez que LBI, induz a uma expressão de um grande conjunto de RNAm, que codificam proteínas reguladoras do ciclo celular que podem controlar a proliferação e diferenciação de mioblastos em mio... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Low-level laser irradiation (LLLT) has been used as a non-invasive method to promote or accelerate muscular regeneration capability. However, the regulatory molecular mechanisms by which LLLT exerts these effects remain largely unknown. Our goal was to perform a RNAsequencing (RNA-Seq) analysis in C2C12 myoblasts after LLLT. C2C12 myoblasts viability, migration, proliferation and RNA-Seq were performed, identifying 514 differentially expressed genes after LLLT. Next, gene ontology and pathway analysis of the differentially expressed genes revealed transcripts among categories related to cell cycle, ribosome biogenesis, response to stress, cell migration, morphological structure and muscle cell proliferation. After, we intersected our RNA-Seq data with transcriptomes publicly available myogenic differentiation data that showed a total of 42 overlapping transcripts (myoblasts vs myotube). This set of shared transcripts showed that the LLLT-myoblasts have a myotube-like profile, clustering away from the myoblast profile. In conclusion, we revealed for the first time that LLLT induces the expression a large set of mRNAs encoding for cell cycle regulatory proteins that may control myoblasts proliferation and differentiation into myotubes. Importantly, these set of mRNA revealed a myotube-like transcriptional profile and provided new insights to the understanding of the specific molecular changes underlying the effects of LLLT irradiation on skeletal muscle cells. / Doutor

No description available.

The cost of lamb mortality caused by cold exposure has been estimated at approximately 40 million dollars per year. This value is probably conservative as it does not include the cost due to the reduction in productivity in hypothermic lambs that manage to survive or the cost of reduced selection potential incurred by fewer lambs surviving until selection. The objectives of this research was to investigate whether sire-line variation exists in neonatal lamb cold tolerance and whether polymorphism in the β₃ adrenergic receptor gene can be used as a genetic marker for lamb cold tolerance and lean muscle growth. The influence of the climate, birthweight, age of dam at lambing, gender and birth rank on neonatal lamb cold tolerance was also analysed. Neonatal lamb mortality due to cold exposure was analysed in four field trials that used neonatal lamb morality from cold exposure as a predictor of neonatal lamb cold tolerance. Sire-line variation in neonatal lamb morality was observed in all trials, though it appeared that this effect was largely mediated through sire-line variation in lamb birth weight. Variation in lamb birth weight between birth rank classed was also found to be responsible for the influence of birth rank on neonatal lamb mortality due to cold exposure. The age of dam at lambing and the lamb gender was not observed to influence neonatal lamb mortality due to cold exposure. The sires from the cold tolerance study and the progeny of the lean muscle growth study were genotyped for the β₃ adrenergic receptor locus. Other studies have found evidence that a major gene exists in the catecholamine stimulation of brown adipose thermogenesis and evidence that the β₃-AR gene is a likely candidate. However, this hypothesis and the hypothesis that polymorphism in the β₃-AR gene is also linked to lean muscle growth in lambs was not confirmed in this study. So while it appears that the results were confounded by experimental design, there is evidence that influence of polymorphism in the ovine β₃ AR gene on neonatal lamb mortality and/or lean muscle growth is not sufficient to be considered a major gene effect. The implications of this experiment on the sheep industry and sheep farmers in general are huge. While completely eliminating lamb deaths due to inadequate cold tolerance is impossible, this study shows that large gains in lamb survival could be possible through selective breeding.

The cost of lamb mortality caused by cold exposure has been estimated at approximately 40 million dollars per year. This value is probably conservative as it does not include the cost due to the reduction in productivity in hypothermic lambs that manage to survive or the cost of reduced selection potential incurred by fewer lambs surviving until selection. The objectives of this research was to investigate whether sire-line variation exists in neonatal lamb cold tolerance and whether polymorphism in the β₃ adrenergic receptor gene can be used as a genetic marker for lamb cold tolerance and lean muscle growth. The influence of the climate, birthweight, age of dam at lambing, gender and birth rank on neonatal lamb cold tolerance was also analysed. Neonatal lamb mortality due to cold exposure was analysed in four field trials that used neonatal lamb morality from cold exposure as a predictor of neonatal lamb cold tolerance. Sire-line variation in neonatal lamb morality was observed in all trials, though it appeared that this effect was largely mediated through sire-line variation in lamb birth weight. Variation in lamb birth weight between birth rank classed was also found to be responsible for the influence of birth rank on neonatal lamb mortality due to cold exposure. The age of dam at lambing and the lamb gender was not observed to influence neonatal lamb mortality due to cold exposure. The sires from the cold tolerance study and the progeny of the lean muscle growth study were genotyped for the β₃ adrenergic receptor locus. Other studies have found evidence that a major gene exists in the catecholamine stimulation of brown adipose thermogenesis and evidence that the β₃-AR gene is a likely candidate. However, this hypothesis and the hypothesis that polymorphism in the β₃-AR gene is also linked to lean muscle growth in lambs was not confirmed in this study. So while it appears that the results were confounded by experimental design, there is evidence that influence of polymorphism in the ovine β₃ AR gene on neonatal lamb mortality and/or lean muscle growth is not sufficient to be considered a major gene effect. The implications of this experiment on the sheep industry and sheep farmers in general are huge. While completely eliminating lamb deaths due to inadequate cold tolerance is impossible, this study shows that large gains in lamb survival could be possible through selective breeding.