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Introduction: A hallmark of the human aging process is the gradual loss of muscle mass and strength, a phenomenon commonly referred to as sarcopenia. Although sarcopenia is likely the result of both a reduction in fiber size and a loss of muscle fibers, it has become abundantly clear that sarcopenia is primarily characterized by the reduction in type II fiber size. The mechanism(s) driving this fiber type-specific loss still remain largely unknown, but it has become evident that the dysregulation of a muscle-specific stem cell population called satellite cells (SC) plays an important role in its progression. Luckily, older adults still retain the ability to activate and expand their SC in response to exercise; what remains unclear is which modality of exercise is optimal for promoting SC contribution to skeletal muscle remodelling. We sought to test the hypothesis that both resistance exercise (RE) and high-intensity interval training (HIIT) would stimulate greater expansion of SC when compared to traditional aerobic exercise (AE). Furthermore, we also sought to determine potential extrinsic/intrinsic factors that might contribute to regulating the differences in SC behavior across the different modalities of exercise Methods: Sedentary older men (n=22; 67 ± 4 yr; BMI: 27.0 ± 2.6 kg•m-2 [mean ± SEM]) were randomly assigned to complete an acute bout of either RE (3 sets of leg extensor and press, 95% 10RM, n = 7), HIIT (10 x 1min, 95% maximal heart rate [HRmax], n= 8) or AE (30min, 55-60% HRmax. n = 7). Muscle biopsies were obtained before and at 24h and 48h following each exercise bout, while blood samples were taken before and at 24h after exercise. The SC response was analyzed using immunofluorescent microscopy and whole-muscle mRNA analysis, while the variability in exercise-induced muscle damage (EIMD) was estimated by analyzing serum samples for creatine kinase (CK) concentration. Results: The muscle SC content increased 45% (p < 0.001) and 52% (p = 0.001) relative to baseline at 24h and 48h respectively following RE, while HIIT stimulated a significant increase of 33% (p = 0.007) at 48h. Fiber type-specific analysis further revealed that both RE and HIIT were capable of inducing significant increases in both type I and type II-specific SC by 48h post-exercise. Further analysis of individual SC responses also revealed a correlation (r = 0.737, p < 0.001) between the change in SC content across the acute time-course and the relative change in creatine kinase (CK) activity levels 24h post-exercise. The lack of any notable SC expansion following AE was to some degree explained by insufficient changes to cell cycle and myostatin (MSTN)-related genes. Conversely, RE produced the greatest reduction in MSTN mRNA over the 48h time-course while inducing a noticeable decrease of 124% in SMAD3 (a mediator of MSTN signaling) mRNA at 48 post-exercise. Conclusions: These data illustrate that RE is the most potent stimulator of acute SC activity in older men, while also showing for the first time that of HIIT can induce a SC response in the same population. The positive relationship between the changes in SC content in response to exercise and CK activity levels suggests that the level of muscle damage induced by an exercise type may influence the magnitude of the subsequent SC response. The magnitude of each SC response is also likely influenced by unique changes in MSTN signaling that follow each modality of exercise. / Thesis / Master of Science in Kinesiology

INTRODUCTION: Following damaging stimuli, skeletal muscle exhibits coordinated interplay between intra- and extra-cellular processes resulting in satellite cell (SC) recruitment. SCs are known to play a central role in muscle plasticity post-injury by differentiating into myoblasts (MBL) and fusing with damaged tissue to donate myonuclei. Yet, their role within skeletal muscle remodeling through paracrine signaling remains to be fully elucidated. Thus, the purpose of this project was two-fold: 1) develop an in vitro model of MBL intercellular communication following myotube damage and 2) to determine if MBL proximity alone is adequate for improving tissue repair and reducing cellular stress during recovery. METHODS: C2C12 myotubes were exposed to 1 hour of electrical pulse stimulation (EPS) with 15Hz pulse for 5s and 5Hz pulse for 5s, separated by a 5s break. Myotubes were then introduced to non-electrically stimulated (NS) MBL adhered to a porous cell insert to allow paracrine signaling and samples were collected at varying timepoints post-EPS. RESULTS: EPS induced Z line sarcomeric disorganization and creatine kinase release into the cell culture media, which was mitigated in MBL+ groups (p<0.05). A significant main effect of MBL exposure was observed in EPS myotubes where MBL+ myotubes had greater Hsp70 gene expression, calpain 3 protein and gene expression, and t-ACC, p-ACCSer79, t-ULK, p-ULKSer555 protein expression than MBL- myotubes when recovering from EPS (p<0.05). A main effect of time was observed where B-dystroglycan and p-mTORSer2448 protein expression decreased in the EPS myotubes, and myotube diameter only decreased in the MBL+ condition (p<0.05). CONCLUSION: MBL signaling to damaged myotubes is evident and may increase catabolic processes through upregulating contraction-mediated protease activity and autophagy, as well as increase ATP generation through oxidative phosphorylation during regeneration. / Thesis / Master of Science (MSc) / When muscle damage occurs, whether through rigorous exercise or physical trauma, the muscle relies on a specific group of stem cells to help repair itself. These stem cells, termed satellite cells, can migrate to specific sites of muscle damage, differentiate into myoblasts, and donate nuclei and genetic material to the injured muscle. This increase in nuclear content helps the muscle synthesize more protein to rebuild and regenerate and promotes muscle growth. However, when the satellite cell becomes dysfunctional, as seen in aging muscle and certain genetic conditions, the muscle struggles to repair itself in response to damage and cannot grow in response to exercise. Satellite cell biology has clearly defined the role of nuclear donation in muscle function, however very little is known about how this stem cell ‘talks’ to the muscle through signaling molecules. As such, this thesis elucidates the effect of myoblast signaling on electrically stimulated damaged immature muscle fibers, otherwise known as myotubes, by preventing myoblast-myotube physical interactions in cell culture experimentation. Interestingly, the data presented here demonstrate that myoblast exposure to damaged myotubes may increase muscle protein breakdown as myotube diameters are reduced in size acutely post-damage, likely resulting from the increase in protease and autophagy protein expression markers. Additionally, myoblast exposure to damaged myotubes may increase mitochondrial fatty acid oxidation to generate energy, which is the fuel of choice during muscle regeneration.

The STAC3 gene is a functionally undefined gene predicted to encode a protein containing two SH3 domains and one cysteine-rich domain. In this study, we determined the potential role of the STAC3 gene in proliferation and differentiation of bovine satellite cells. We isolated satellite cells from skeletal muscle of adult cattle and transfected them with STAC3 small interfering RNA (siRNA) or scrambled siRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. We assessed the differentiation status of bovine satellite cells by quantifying the expression levels of myogenin and myosin heavy chain genes, and by quantifying fusion index. STAC3 knockdown stimulated mRNA and protein expression of myogenin, and myosin heavy chain 3 and 7, and increased fusion index of bovine satellite cells. These data together suggest that STAC3 inhibits differentiation of bovine satellite cells into myotubes. To determine the underlying mechanism, we identified and validated AP1?1 as a STAC3-interacting protein by yeast two-hybrid screening and co-immunoprecipitation. In C2C12 cells, STAC3 knockdown decreased the expression level of AP1?1 protein. In bovine satellite cells, STAC3 knockdown increased the membrane localization of glucose transporter 4 (GLUT4) and glucose uptake. These data together suggest the following mechanism by which STAC3 inhibits differentiation of bovine satellite cells: STAC3 increases AP1?1 stability in cells; a high level of AP1?1 keeps GLUT4 from translocating to the plasma membrane; reduced membrane localization of GLUT4 impedes glucose uptake; and restricted glucose uptake inhibits differentiation of satellite cells into myotubes. / Master of Science

Thoroughbred racehorses undergo strenuous exercise which often leads to the occurrence of exercise-induced pulmonary hemorrhage (EIPH), in which capillaries rupture within the alveoli in the lungs causing bleeding. Severe cases of EIPH lead to epistaxis and may result in fatality. Presently, the loop diuretic furosemide is the only medication approved to mitigate the effects of EIPH. Often regarded in the racing industry as "performance enhancing" due to 4% weight loss ensued by its diuretic effect, it is unknown what effects furosemide may have on muscle recovery. Therefore, the objective of this study was to determine the effects various doses of furosemide may have on equine satellite cell (eqSC) myogenesis and metabolism. Mitotic index was increased (P<0.05) as a result of treatment with 100 µg/mL furosemide, a 10-fold pharmacological dose, in comparison to vehicle, but was not different (P>0.05) compared to the physiological dose of 10 µg/mL furosemide. Average cell number decreased (P<0.05) in the excess furosemide group compared to all other groups. Pax7 expression did not differ (P>0.05) between groups. Expression of the differentiation transcription factor myogenin, and embryonic sarcomeric myosin heavy chain decreased (P<0.05) when cells were treated with 100 µg/mL furosemide. Fusion index and myotube area decreased (P<0.05) as a result of treatment with excess furosemide. Glycogen concentration in myotubes was lower (P<0.05) following treatment with 100 µg/mL furosemide, while IGF-1 was unsuccessful in rescuing the effects of furosemide. Excess furosemide decreased expression of muscle creatine kinase while increasing expression of phosphoglucomutase 1, glycogen synthase 1, and glycogen branching enzyme 1 (P<0.05). Excess furosemide decreased basal oxygen consumption rate (OCR) and increased OCR after addition of oligomycin (P<0.05). Excess furosemide did not affect myotube glycolysis rates in vitro. In conclusion, furosemide inhibits muscle differentiation and oxidative metabolism in eqSCs. / Master of Science / Thoroughbred racehorses often bleed from the lungs as a result of high-intensity exercise. This condition can oftentimes be fatal depending on severity. Furosemide, is used in the industry to reduce blood pressure within the lungs during racing to prevent bleeding. Furosemide, a diuretic given four hours prior to a race, causes a horse to excrete up to 4% of its body weight. This effect of furosemide decreases the weight a horse must carry during a race, thus allowing the horse to run faster. Therefore, deemed as a performance enhancing drug due to its effects on the kidney, to our knowledge, no research has been conducted on what effects furosemide might have on muscle generation. High-intensity exercise causes massive muscle damage and therefore must be repaired to prepare for the next bout of exercise. Muscle generation is called myogenesis. Stem cells, or satellite cells, that lie within the muscle become activated, recognizing the need for muscle repair. Satellite cells divide, increasing in cell number and then fuse together, forming new muscle fibers. Satellite cells undergo different types of metabolism depending on their state of development. For example, proliferating cells require glucose for energy, while cells fusing together forming myotubes, require oxidative metabolism for long-lasting energy. Therefore, the objective of this study was to determine the effects furosemide might have on muscle formation and metabolism. The excess furosemide dose (100 µg/mL) decreased cell proliferation. The expression of regulatory factors responsible for forming myotubes at different stages of muscle development are decreased when cells were treated with the defined excess furosemide dose. Furosemide decreased the ability of satellite cells to generate myotubes. Glycogen concentration was also decreased as a result of excess furosemide treatment. Gene expression of enzymes involved in glycogen synthesis were increased from treatment with our excess furosemide dose. No effect of furosemide was seen on glycolysis, whereas oxidative metabolism suffered as a result of treatment with excess furosemide. In conclusion, furosemide does indeed affect muscle generation and oxidative metabolism.

Made available in DSpace on 2016-06-02T20:21:36Z (GMT). No. of bitstreams: 1 6302.pdf: 1873865 bytes, checksum: 9751d8d17780f8f77d9e24a60cc67c6f (MD5) Previous issue date: 2013-09-20 / Financiadora de Estudos e Projetos / Epigenetics can be defined as the study of heritable changes in phenotype without the occurrence of changes in nucleotide sequence. Epigenetic modifications occur by the chemical changes in DNA and their associated proteins, such as DNA methylation and histone acetylation, respectively. Meat tenderness is a trait of great interest worldwide, resulting in the development of the beef livestock sector to produce meat of quality. It is worth to highlight the role of the calpain/calpastatin system on tenderness. Calpain encoded by the CAPN1 gene plays a role in proteolysis posmortem by cleaving proteins from muscle fiber. The calpastatin encoded by the CAST gene, on the other hand, acts controlling this cleavage by blocking the action of calpain. In addition, this system is also involved in myoblast differentiation into myotubes during embryogenesis. This system controls the proteolysis of proteins that constitute the cytoskeleton and the plasma membrane. To mimic the myotube formation during embryogenesis and to study the epigenetic control of CAPN1 and CAST gene expression, satellite cell cultures established from bovine muscle were kept undifferentiated (negative control) or were induced to differentiate by incubation with culture medium containing 2% fetal bovine serum in the absence (positive control) or after treatment with epigenetic modifiers like 5-Aza- 2'-deoxycytidine (Aza, DNA demethylating) for 48 h at 10 &#956;M, and Trichostatin A (TSA, histone acetylating) for 24 h at 50 nM. The results showed no differences (p>0.05) in myoblast rate fusion between Aza, TSA and positive control groups, but there were differences (p>0.05) when these groups were compared to the negative control, which showed lower fusion rates. There was no difference (p>0.05) in cell viability among the four groups, showing that Aza and TSA were not cytotoxic at the used concentrations. Concerning the gene expression, the gene CAST was more expressed (p<0,05) in the positive control group than the negative one; but no differences were seen in the expression (p>0,05) between positive control, 5-Aza-2 - deoxycytidine and Trichostatin A groups. For the CAPN1 gene, no difference was seen in the expression (p>0,05) between negative and positive control groups, but the CAPN1 gene was more expressed (p<0,05) in the 5-Aza-2 -deoxycytidine and Trichostatin A treatments relative to the positive control group. When the expression ratio of CAPN1/CAST were compared between treatments, was seen more expression (p<0,05) in the positive control group both comparing with negative control group and with 5-Aza-2 -deoxycytidine and Trichostatin A treatments. We may conclude that the treatments with the epigenetic modifier agents didn't affect both the bovine myoblast differentiation into myotubes and the CAST gene expression, but did in the CAPN1 gene expression and so did in the expression ratio of CAPN1/CAST. / Epigenetics can be defined as the study of heritable changes in phenotype without the occurrence of changes in nucleotide sequence. Epigenetic modifications occur by the chemical changes in DNA and their associated proteins, such as DNA methylation and histone acetylation, respectively. Meat tenderness is a trait of great interest worldwide, resulting in the development of the beef livestock sector to produce meat of quality. It is worth to highlight the role of the calpain/calpastatin system on tenderness. Calpain encoded by the CAPN1 gene plays a role in proteolysis posmortem by cleaving proteins from muscle fiber. The calpastatin encoded by the CAST gene, on the other hand, acts controlling this cleavage by blocking the action of calpain. In addition, this system is also involved in myoblast differentiation into myotubes during embryogenesis. This system controls the proteolysis of proteins that constitute the cytoskeleton and the plasma membrane. To mimic the myotube formation during embryogenesis and to study the epigenetic control of CAPN1 and CAST gene expression, satellite cell cultures established from bovine muscle were kept undifferentiated (negative control) or were induced to differentiate by incubation with culture medium containing 2% fetal bovine serum in the absence (positive control) or after treatment with epigenetic modifiers like 5-Aza- 2'-deoxycytidine (Aza, DNA demethylating) for 48 h at 10 &#956;M, and Trichostatin A (TSA, histone acetylating) for 24 h at 50 nM. The results showed no differences (p>0.05) in myoblast rate fusion between Aza, TSA and positive control groups, but there were differences (p>0.05) when these groups were compared to the negative control, which showed lower fusion rates. There was no difference (p>0.05) in cell viability among the four groups, showing that Aza and TSA were not cytotoxic at the used concentrations. Concerning the gene expression, the gene CAST was more expressed (p<0,05) in the positive control group than the negative one; but no differences were seen in the expression (p>0,05) between positive control, 5-Aza-2 - deoxycytidine and Trichostatin A groups. For the CAPN1 gene, no difference was seen in the expression (p>0,05) between negative and positive control groups, but the CAPN1 gene was more expressed (p<0,05) in the 5-Aza-2 -deoxycytidine and Trichostatin A treatments relative to the positive control group. When the expression ratio of CAPN1/CAST were compared between treatments, was seen more expression (p<0,05) in the positive control group both comparing with negative control group and with 5-Aza-2 -deoxycytidine and Trichostatin A treatments. We may conclude that the treatments with the epigenetic modifier agents didn't affect both the bovine myoblast differentiation into myotubes and the CAST gene expression, but did in the CAPN1 gene expression and so did in the expression ratio of CAPN1/CAST. / Epigenetics can be defined as the study of heritable changes in phenotype without the occurrence of changes in nucleotide sequence. Epigenetic modifications occur by the chemical changes in DNA and their associated proteins, such as DNA methylation and histone acetylation, respectively. Meat tenderness is a trait of great interest worldwide, resulting in the development of the beef livestock sector to produce meat of quality. It is worth to highlight the role of the calpain/calpastatin system on tenderness. Calpain encoded by the CAPN1 gene plays a role in proteolysis posmortem by cleaving proteins from muscle fiber. The calpastatin encoded by the CAST gene, on the other hand, acts controlling this cleavage by blocking the action of calpain. In addition, this system is also involved in myoblast differentiation into myotubes during embryogenesis. This system controls the proteolysis of proteins that constitute the cytoskeleton and the plasma membrane. To mimic the myotube formation during embryogenesis and to study the epigenetic control of CAPN1 and CAST gene expression, satellite cell cultures established from bovine muscle were kept undifferentiated (negative control) or were induced to differentiate by incubation with culture medium containing 2% fetal bovine serum in the absence (positive control) or after treatment with epigenetic modifiers like 5-Aza- 2'-deoxycytidine (Aza, DNA demethylating) for 48 h at 10 &#956;M, and Trichostatin A (TSA, histone acetylating) for 24 h at 50 nM. The results showed no differences (p>0.05) in myoblast rate fusion between Aza, TSA and positive control groups, but there were differences (p>0.05) when these groups were compared to the negative control, which showed lower fusion rates. There was no difference (p>0.05) in cell viability among the four groups, showing that Aza and TSA were not cytotoxic at the used concentrations. Concerning the gene expression, the gene CAST was more expressed (p<0,05) in the positive control group than the negative one; but no differences were seen in the expression (p>0,05) between positive control, 5-Aza-2 - deoxycytidine and Trichostatin A groups. For the CAPN1 gene, no difference was seen in the expression (p>0,05) between negative and positive control groups, but the CAPN1 gene was more expressed (p<0,05) in the 5-Aza-2 -deoxycytidine and Trichostatin A treatments relative to the positive control group. When the expression ratio of CAPN1/CAST were compared between treatments, was seen more expression (p<0,05) in the positive control group both comparing with negative control group and with 5-Aza-2 -deoxycytidine and Trichostatin A treatments. We may conclude that the treatments with the epigenetic modifier agents didn't affect both the bovine myoblast differentiation into myotubes and the CAST gene expression, but did in the CAPN1 gene expression and so did in the expression ratio of CAPN1/CAST. / A epigenética pode ser definida como o estudo de mudanças herdáveis no fenótipo sem a ocorrência de mudanças na sequência de nucleotídeos. As modificações epigenéticas caracterizam-se por alterações químicas no DNA e em suas proteínas associadas, como a metilação no DNA e a acetilação nas proteínas histonas, respectivamente. A maciez da carne bovina tem ganhado interesse por todo mundo, e o setor pecuário tem se desenvolvido para produzir carne de qualidade que satisfaça a essa demanda. Vale destacar os papéis do sistema calpaína/calpastatina. A calpaína, codificada pelo gene CAPN1, desempenha um papel fundamental na proteólise posmortem pela clivagem de proteínas que constituem a fibra muscular. A calpastatina, codificado pelo gene CAST, por outro lado, age controlando essa clivagem pelo bloqueio da ação da calpaína. Além disso, esse sistema também está envolvido na diferenciação dos mioblastos em miotubos na embriogênese. A ação desse sistema ocorre pela proteólise controlada de proteínas que constituem a membrana plasmática e o citoesqueleto. Para reproduzir a formação de miotubos durante a embriogênese e estudar o controle epigenético na expressão dos genes CAPN1 e CAST, foram estabelecidas culturas de células satélites de músculo bovino, que foram mantidas sem diferenciação (controle negativo) ou foram induzidas à diferenciação, por meio da incubação com meio de cultivo com 2% de soro fetal bovino, na ausência (controle positivo) e após o tratamento com os agentes modificadores epigenéticos como o 5-Aza-2 - desoxicitidina (Aza; desmetilante de DNA), por um período de 48 h á concentração de 10 &#956;M, e Tricostatina A (TSA; acetilante de histona), por um período de 24 h á concentração de 50 nM. Os resultados mostraram que não houve diferenças (p>0,05) no índice de fusão dos mioblastos entre os grupos Aza, TSA e controle positivo, mas que houve diferença (p<0,05) desses grupos em comparação ao controle negativo, que apresentou menor índice de fusão. Também não houve diferença (p>0,05) nas taxas de viabilidade das células entre os grupos, mostrando que o Aza e TSA não foram citotóxicos nas concentrações usadas Em relação à expressão gênica, o gene CAST foi mais expresso (p<0,05) no grupo controle positivo em comparação ao controle negativo; mas não foram observadas diferenças de expressão (p>0,05) entre os grupos controle positivo, 5-Aza-2 -desoxicitidina e Tricostatina A. Para o gene CAPN1, não foi observada diferença (p>0,05) de expressão entre os grupos controle negativo e positivo, mas o gene CAPN1 foi mais expresso (p<0,05) nos tratamentos 5-Aza-2 -desoxicitidina e Tricostatina A em comparação ao grupo controle positivo. Quando a razão de expressão CAST/CAPN1 foi comparada entre os tratamentos, foi observada maior expressão (p<0,05) no grupo controle positivo, tanto em comparação ao grupo controle negativo, quanto aos tratamentos 5-Aza-2 -desoxicitidina e Tricostatina A. Podemos concluir que os tratamentos com agentes modificadores epigenéticos não afetaram a diferenciação de mioblastos bovinos em miotubos e nem a expressão do gene CAST, mas afetaram a expressão do gene CAPN1 e a razão de expressão CAST/CAPN1. / A epigenética pode ser definida como o estudo de mudanças herdáveis no fenótipo sem a ocorrência de mudanças na sequência de nucleotídeos. As modificações epigenéticas caracterizam-se por alterações químicas no DNA e em suas proteínas associadas, como a metilação no DNA e a acetilação nas proteínas histonas, respectivamente. A maciez da carne bovina tem ganhado interesse por todo mundo, e o setor pecuário tem se desenvolvido para produzir carne de qualidade que satisfaça a essa demanda. Vale destacar os papéis do sistema calpaína/calpastatina. A calpaína, codificada pelo gene CAPN1, desempenha um papel fundamental na proteólise posmortem pela clivagem de proteínas que constituem a fibra muscular. A calpastatina, codificado pelo gene CAST, por outro lado, age controlando essa clivagem pelo bloqueio da ação da calpaína. Além disso, esse sistema também está envolvido na diferenciação dos mioblastos em miotubos na embriogênese. A ação desse sistema ocorre pela proteólise controlada de proteínas que constituem a membrana plasmática e o citoesqueleto. Para reproduzir a formação de miotubos durante a embriogênese e estudar o controle epigenético na expressão dos genes CAPN1 e CAST, foram estabelecidas culturas de células satélites de músculo bovino, que foram mantidas sem diferenciação (controle negativo) ou foram induzidas à diferenciação, por meio da incubação com meio de cultivo com 2% de soro fetal bovino, na ausência (controle positivo) e após o tratamento com os agentes modificadores epigenéticos como o 5-Aza-2 - desoxicitidina (Aza; desmetilante de DNA), por um período de 48 h á concentração de 10 &#956;M, e Tricostatina A (TSA; acetilante de histona), por um período de 24 h á concentração de 50 nM. Os resultados mostraram que não houve diferenças (p>0,05) no índice de fusão dos mioblastos entre os grupos Aza, TSA e controle positivo, mas que houve diferença (p<0,05) desses grupos em comparação ao controle negativo, que apresentou menor índice de fusão. Também não houve diferença (p>0,05) nas taxas de viabilidade das células entre os grupos, mostrando que o Aza e TSA não foram citotóxicos nas concentrações usadas Em relação à expressão gênica, o gene CAST foi mais expresso (p<0,05) no grupo controle positivo em comparação ao controle negativo; mas não foram observadas diferenças de expressão (p>0,05) entre os grupos controle positivo, 5-Aza-2 -desoxicitidina e Tricostatina A. Para o gene CAPN1, não foi observada diferença (p>0,05) de expressão entre os grupos controle negativo e positivo, mas o gene CAPN1 foi mais expresso (p<0,05) nos tratamentos 5-Aza-2 -desoxicitidina e Tricostatina A em comparação ao grupo controle positivo. Quando a razão de expressão CAST/CAPN1 foi comparada entre os tratamentos, foi observada maior expressão (p<0,05) no grupo controle positivo, tanto em comparação ao grupo controle negativo, quanto aos tratamentos 5-Aza-2 -desoxicitidina e Tricostatina A. Podemos concluir que os tratamentos com agentes modificadores epigenéticos não afetaram a diferenciação de mioblastos bovinos em miotubos e nem a expressão do gene CAST, mas afetaram a expressão do gene CAPN1 e a razão de expressão CAST/CAPN1. / A epigenética pode ser definida como o estudo de mudanças herdáveis no fenótipo sem a ocorrência de mudanças na sequência de nucleotídeos. As modificações epigenéticas caracterizam-se por alterações químicas no DNA e em suas proteínas associadas, como a metilação no DNA e a acetilação nas proteínas histonas, respectivamente. A maciez da carne bovina tem ganhado interesse por todo mundo, e o setor pecuário tem se desenvolvido para produzir carne de qualidade que satisfaça a essa demanda. Vale destacar os papéis do sistema calpaína/calpastatina. A calpaína, codificada pelo gene CAPN1, desempenha um papel fundamental na proteólise posmortem pela clivagem de proteínas que constituem a fibra muscular. A calpastatina, codificado pelo gene CAST, por outro lado, age controlando essa clivagem pelo bloqueio da ação da calpaína. Além disso, esse sistema também está envolvido na diferenciação dos mioblastos em miotubos na embriogênese. A ação desse sistema ocorre pela proteólise controlada de proteínas que constituem a membrana plasmática e o citoesqueleto. Para reproduzir a formação de miotubos durante a embriogênese e estudar o controle epigenético na expressão dos genes CAPN1 e CAST, foram estabelecidas culturas de células satélites de músculo bovino, que foram mantidas sem diferenciação (controle negativo) ou foram induzidas à diferenciação, por meio da incubação com meio de cultivo com 2% de soro fetal bovino, na ausência (controle positivo) e após o tratamento com os agentes modificadores epigenéticos como o 5-Aza-2 - desoxicitidina (Aza; desmetilante de DNA), por um período de 48 h á concentração de 10 &#956;M, e Tricostatina A (TSA; acetilante de histona), por um período de 24 h á concentração de 50 nM. Os resultados mostraram que não houve diferenças (p>0,05) no índice de fusão dos mioblastos entre os grupos Aza, TSA e controle positivo, mas que houve diferença (p<0,05) desses grupos em comparação ao controle negativo, que apresentou menor índice de fusão. Também não houve diferença (p>0,05) nas taxas de viabilidade das células entre os grupos, mostrando que o Aza e TSA não foram citotóxicos nas concentrações usadas Em relação à expressão gênica, o gene CAST foi mais expresso (p<0,05) no grupo controle positivo em comparação ao controle negativo; mas não foram observadas diferenças de expressão (p>0,05) entre os grupos controle positivo, 5-Aza-2 -desoxicitidina e Tricostatina A. Para o gene CAPN1, não foi observada diferença (p>0,05) de expressão entre os grupos controle negativo e positivo, mas o gene CAPN1 foi mais expresso (p<0,05) nos tratamentos 5-Aza-2 -desoxicitidina e Tricostatina A em comparação ao grupo controle positivo. Quando a razão de expressão CAST/CAPN1 foi comparada entre os tratamentos, foi observada maior expressão (p<0,05) no grupo controle positivo, tanto em comparação ao grupo controle negativo, quanto aos tratamentos 5-Aza-2 -desoxicitidina e Tricostatina A. Podemos concluir que os tratamentos com agentes modificadores epigenéticos não afetaram a diferenciação de mioblastos bovinos em miotubos e nem a expressão do gene CAST, mas afetaram a expressão do gene CAPN1 e a razão de expressão CAST/CAPN1.

published_or_final_version / Biochemistry / Master / Master of Philosophy

<p>Soft tissue sarcomas (STSs) are a heterogeneous group of mesenchymal tumors comprised of >70 subtypes. An important question is how the cell of origin and the pathways to tumor development shape the broad array of STS subtypes. By forcing identical tumor-promoting mutations to different cell types in Genetically Engineered Mouse Models (GEMMs) of STS, I have a unique model system to investigate this question. In the process of performing these experiments I observed that genetic mutations are necessary, but not sufficient for rapid sarcoma formation. However, tissue injury dramatically accelerates sarcoma formation in our GEMM of STS. For my thesis, I have worked to understand how cell of origin affects sarcoma subtype and how the microenvironment in our models promotes transformation. I have observed that cell of origin plays an important, but not the only, role in defining STS subtype. Additionally, I have concluded that the microenvironment, and specifically the HGF/c-MET signaling pathway play a crucial role in promoting sarcoma development after acute tissue injury.</p> / Dissertation

The skeletal muscle has a remarkable regenerative capacity upon injury, due to the presence of the satellite cells, which remain quiescent in the tissue, but, when required, they are able to proliferate and form and/or repair myofibers. Moreover, satellite cells are important to muscle growth and maintenance. However, in many neuromuscular disorders, the amount, function, and proliferative capacity of these cells are impaired. Centronuclear myopathies (CNM) are a group of muscle diseases characterized by generalized muscle weakness and myofibers with central nuclei. The autosomal dominant form (AD-CNM) is caused by mutations in the DNM2 gene. Dynamin 2 protein is ubiquitously expressed and is involved in membrane remodeling, intracellular trafficking, and cytoskeleton dynamics. Therefore, the pathophysiological mechanisms are equally diverse e not completely understood, mainly the fact to be a muscle-specific disease. In the present Ph.D. thesis, we sought to investigate the satellite cells in the context of centronuclear myopathy. For this, we used the mouse model KI-Dnm2R465W, bearing the most frequent mutation found in human patients. Since in centronuclear myopathy there is no evident degenerative process ongoing, we induced muscle lesion by electrical shock, a protocol developed for this thesis, comparatively to cardiotoxin injection. We verified that the number of satellite cells in gastrocnemius muscle is reduced in the KI-Dnm2R465W mouse in relation to wild-type animals. As a result, the regenerative potential of the mutant mouse is decreased and the muscle is not able to fully recover. In addition, we investigated the functional consequences of two mutations, p.R465W and p.E650K, in immortalized myoblasts. We examined the myogenic potential in vitro, the migratory property, and the endocytosis capacity. We found that both mutations impact on the myogenic potential, but in different ways. We also show that both mutations impair the migratory capacity of myoblasts that justify, in parts, the alterations in their myogenic potential. Finally, we verified that the endocytosis capacity is affected in a mutation-dependent manner, which may also indirectly disturb the myogenic differentiation efficiency / O músculo esquelético possui grande capacidade regenerativa após sofrer lesões, por causa da presença das chamadas células-satélite, que permanecem no tecido em estado quiescente, mas que, na presença de uma lesão, são capazes de proliferar e formar e/ou reparar miofibras. As células-satélite são importantes para o crescimento e manutenção do músculo adulto. Porém, em diversas doenças neuromusculares, a quantidade, a função e a capacidade proliferativa destas células podem estar comprometidas. As miopatias centronucleares (CNM) são um grupo de doenças musculares caracterizadas por fraqueza muscular generalizada e o posicionamento dos núcleos na porção central da miofibra. A forma autossômica dominante (AD-CNM) é causada por mutações no gene DNM2. A proteína dinamina 2 é expressa ubiquamente e está envolvida no remodelamento de membranas, no tráfego intracelular e na dinâmica do citoesqueleto. Consequentemente, os mecanismos fisiopatológicos também são diversos e não completamente compreendidos, principalmente o fato de ser uma doença músculo-específica. Nesta tese de doutorado, buscamos investigar as células-satélite no contexto da miopatia centronuclear. Para isto, utilizamos o camundongo modelo KI-Dnm2R465W, portador da mutação mais frequente em pacientes humanos. Como na miopatia centronuclear não há um processo degenerativo em atividade, induzimos nos camundongos a lesão muscular por choque elétrico, em protocolo desenvolvido nesta tese, comparativamente a injeção de cardiotoxina. Verificamos que o número de células satélite no músculo gastrocnêmio do camundongo KI-Dnm2R465W é reduzido em relação aos animais selvagens. Em consequência disto, o potencial regenerativo do animal mutante é reduzido e o músculo não se recupera completamente. Investigamos também os efeitos funcionais de duas mutações, p.R465W e p.E650K, em mioblastos imortalizados. Examinamos o potencial miogênico in vitro, a propriedade migratória e a capacidade de endocitose. Verificamos que o potencial miogênico destas células é afetado pelas mutações, porém de maneiras distintas. Mostramos também que ambas as mutações impactam negativamente na capacidade migratória dos mioblastos, o que em parte justifica as alterações no potencial miogênico dos mesmos. Por fim, verificamos que a capacidade endocítica em mioblastos é alterada a depender da mutação, o que indiretamente também pode afetar a capacidade de diferenciação miogênica

As células-satélite musculares vêm sendo muito estudadas em diferentes pesquisas, especialmente com o objetivo de aumentar a compreensão do mecanismo de sua ação na regeneração muscular e as respectivas implicações nas diferentes miopatias, visando identificar possíveis alvos terapêuticos. Dois modelos para distrofias, os camundongos Largemy d e Lama2dy2j/J possuem um padrão de degeneração intenso e bastante semelhante, mas com diferenças na expressão de genes envolvidos na cascata de regeneração. Por isso, estes constituem interessantes modelos para o estudo de possíveis diferenças no mecanismo de ativação e atuação das células-satélite no músculo distrófico. Assim, os objetivos específicos deste projeto consistiram em: 1) isolar e caracterizar por citometria de fluxo as populações de células-satélite dos modelos Largemy d e Lama2dy2j/J em comparação com o normal C57Black6, quanto a expressão de marcadores de miogênese e de células-tronco pluripotentes; e 2) estudar e comparar o perfil de expressão gênica destas populações de células satélites através de microarray de expressão. Na caracterização fenotípica das células isoladas do músculo normal, aquelas que aderem mais precocemente (PP1) e mais tardiamente (PP2) mostram um padrão fenotípico semelhante entre si e com características mais próximas às miogênicas. Já a população de células de adesão bem mais tardia (PP6) apresentou um padrão de marcação misto, mantendo as características miogênicas, mas apresentando tambem padrão de células-tronco mesenquimais, sugerindo o fenótipo de células mais imaturas. Nos músculos distróficos, identificamos diferenças na constituição do pool de células presentes inicialmente no músculo, onde na linhagem Lama2dy2j/J há indícios de uma população em estágio proliferativo, enquanto que na linhagem Largemy d há presença de células com maior imaturidade. Os resultados da analise de expressão gênica nas populações caracterizadas se mostraram concordantes com os fenótipos celulares avaliados por citometria. Identificamos a hipo-expressão de genes ligados à regeneração e remodelamento muscular em ambos modelos distróficos. Considerando os genes diferentemente expressos somente em cada um dos modelos, os resultados sugerem ativação de proliferação celular e inibição da diferenciação na linhagem Lama2dy2j/J e distúrbios na miogênese na linhagem Largemy d. Assim, pudemos identificar vias importantes alteradas em cada um dos modelos que explicam parte das diferenças encontradas nos trabalhos anteriores / Muscle satellite cells have been widely studied, especially to understand their mechanism of action in muscle regeneration and correspondent implications in the different dystrophic processes, aiming the identification of potential therapeutic targets. Two mice models for muscular dystrophies, Largemyd and Lama2dy2j/J, have a pattern of an intense and very similar degeneration, but with differences in the expression of genes involved in the regeneration cascade. Therefore, they are interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objectives of this project are: 1) to isolate and characterize by flow cytometry, populations of satellite cells from Largemyd and Lama2dy2j/J models, as compared to normal C57Black6, evaluating the presence of myogenic and pluripotent stem cells markers; and 2) to study and compare gene expression profiles of these populations of satellite cells using microarray technique. In the phenotypic characterization of cells harvested from normal muscle, both faster (PP1) and slower (PP2) populations to adhere in culture flasks show similar phenotypic characteristics, which were closer to myogenic phenotype. On the other hand, the population of cells with very delayed adhesion ability (PP6) presented a mixed pattern, maintaining the myogenic characteristics, but associated to positive mesenchymal stem cell´s markers, suggesting a phenotype of more immature cells. In dystrophic muscles, we could identify differences in the constitution of the first pool of cells present in the Lama2dy2j/J muscle where there is evidence of a population in proliferative stage, while in the Largemyd strain, we found more immature cells. Gene expression profile in the characterized populations showed consistent concordance with the cellular phenotypes assessed by flow citometry. In both dystrophic models, we identified down-regulated genes related to regeneration and remodeling of the muscle. Considering only the genes differently expressed in each dystrophic model, data suggest the activation of cell proliferation and inhibition of differentiation pathways in Lama2dy2j/J strain and altered myogenesis in the Largemyd model. Thus, we identified important altered pathways in each of the dystrophic models that could explain most of the differences in gene expression profile in the muscle, described in our previous work