Peripheral arterial occlusive disease (PAOD) is characterized by buildup of atherosclerotic plaque in peripheral arteries that leads to an occlusion that can interrupt the supply of blood to the peripheral tissue, causing downstream tissue ischemia/hypoxia. PAOD is estimated to affect over 200 million patients worldwide. Current surgical revascularization treatments can be effective in about half of the patient population, leading to a significant number of patients with no treatment options beyond pharmacological intervention and lifestyle modification. The decrease in blood flow downstream of the occlusion leads to increased blood pressure gradient in the microvasculature, specifically in vessels that connect arterial trees (known as collaterals), which will structurally enlarge and increase blood flow to the downstream ischemic/hypoxic tissue. Targeting this process, known as arteriogenesis, can provide a potential treatment option for patients suffering from PAOD by redirecting blood flow around an occluded artery and therefore supplying hypoxic tissue with blood. In order to enhance this process, cellular transplantation has been used but the current cell types explored have not been successful in enhancing arteriogenesis. Myoblasts, proliferative muscle progenitor cells, mediate muscle regeneration, and promote angiogenesis (the growth of new capillaries to supply hypoxic tissue). Preliminary data indicates that myoblasts also promote arteriogenesis in obese mice, making them an attractive therapeutic candidate. However, the methods used in the preliminary studies limited our ability to confirm those findings and characterize the cell therapy candidate. Specifically, we lacked a reproducible and optimized method to isolate myogenic cells and characterize these cells during in vitro culture and after in vivo transplantation. Therefore, the 1st Aim of this study was to optimize the isolation to obtain the highest number possible of satellite cell-yielding myofibers by modification of enzymatic and mechanical digestion of extensor digitorum longus muscle. Modifications to this methodology increased myofiber yield by more than 150%. The 2nd Aim was to optimize the expansion of satellite cell-derived myoblasts by modification of culture media supplements to promote cell expansion while minimizing maturation. bFGF and SB 203580 supplementation improved cell proliferation and prevented myogenic cell maturation during 7-days of in vitro culture. The 3rd Aim was to develop a process for evaluating the quantity and identity of isolated myogenic cells before and after transplantation. This was achieved by implementing an immunofluorescent transcription factor labeling protocol to determine cell identity and a live/dead cell viability assay to determine cell viability and quantity. All 3 aims were integrated into a proof-of-concept pilot study on a hindlimb ischemic BALB/c mouse model. While myoblast transplantation failed to increase collateral arteriogenesis in this model, the process developed in this project provides a reproducible framework for future studies on myoblast-enhanced arteriogenesis. Further research on the effects of myoblast transplantation on arteriogenesis may facilitate the development of new therapies that improve the prognosis of patients with PAOD.

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Dock1 (aussi nommé Dock180) est le membre prototypique de la famille Dock d’activateurs des petites GTPases de la famille Rho. Dock1 agit au sein d’une voie de signalisation conservée au cours de l’évolution et des études génétiques ont démontré que les orthologues de Dock1, myoblast city (mbc) chez la drosophile et Ced-5 chez le nématode, activent Rac dans divers processus biologiques. Notamment, mbc est un important régulateur de la fusion des myoblastes lors de la formation des fibres musculaires de drosophile. Mbc est aussi essentiel à la migration collective d’un groupe de cellules, appelées cellules de bordures, lors de leur migration dans la chambre de l’oeuf suite à l’activation de récepteurs à activité tyrosine kinase (RTK). La migration collective des cellules de bordures récapitule certains des événements observés lorsque des cellules tumorales envahissent le tissu environnant lors de la formation de métastases. Chez les mammifères, des études réalisées en lignées cellulaires suggèrent que Dock1 est aussi un régulateur du cytosquelette lors de la migration cellulaire. De plus, certaines études ont démontré que la voie Dock1/Rac agit en aval de RTKs lors de l’invasion de cellules de glioblastome. Néanmoins, les fonctions in vivo de Dock1 chez les mammifères demeurent méconnues et le but de cette thèse est d’identifier et de caractériser certaines de ses fonctions. Guidés par la fonction de mbc, nous démontrons dans l’objectif no 1 un rôle essentiel pour ce gène au cours du développement embryonnaire grâce à la caractérisation d’une souris Dock1 knock-out. Des défauts sévères de fusion des myoblastes sont observés en absence de l’expression de Dock1 et ils contribuent à la réduction de la masse musculaire des souris mutantes. De plus, nous avons constaté une contribution du gène Dock5, un membre de la famille Dock proche de Dock1, au développement des fibres musculaires. Dans l’objectif no 2, nous avons observé que des hauts niveaux d’expression de DOCK1 corrèlent avec un mauvais pronostic chez les patientes atteintes de cancer du sein possédant une forte expression du gène codant pour le RTK HER2. Une surexpression ou une amplification du locus codant pour le récepteur HER2 est associée à près de 20% des cas de cancer du sein. Les cancers de ces patientes développent fréquemment des métastases et sont associés à un mauvais pronostic. Des études biochimiques ont révélé que DOCK1 interagit avec le récepteur HER2 dans des cellules de cancer du sein. De plus, DOCK1 est essentiel à l’activation de RAC et à la migration cellulaire induite par HER2 dans ces cellules. L’utilisation d’un modèle de cancer du sein médié par HER2 chez la souris combiné avec l’inactivation du gène Dock1 dans les glandes mammaires, nous a permis d’identifier Dock1 et Rac comme de nouveaux effecteurs de la croissance tumorale et de la formation de métastases régulées par l’oncogène HER2. Nous concluons que l’utilisation de différents modèles de souris knock-out pour le gène Dock1 nous a permis d’identifier des fonctions clés de ce gène. Tout comme son orthologue mbc, Dock1 joue un rôle important lors du développement embryonnaire en régulant notamment la fusion des myoblastes. Nos études ont également contribué à démontrer un important degré de conservation des mécanismes moléculaires de fusion entre les espèces. De plus, DOCK1 agit en aval du RTK HER2 et son expression dans les cellules épithéliales de glandes mammaires contribue au développement tumoral et à la formation de métastases induits par cet oncogène. / Dock1 (also known as Dock180) is the prototypical member of the Dock family of Rho GTPase activators (RhoGEFs). Genetic studies in Drosophila and C. elegans have demonstrated that Dock1 orthologues act upstream of the Rac GTPase to activate it during various biological processes. Myoblast city (mbc), Dock1 ortholog in the Drosophila, is an important regulator of myoblast fusion during muscle fiber formation. Moreover, mbc regulates the collective migration of a cluster of border cells downstream of the activation of some tyrosine kinase receptors (RTKs). Migration of border cells is often view as a model for studying the invasive migration of cancer cells during metastasis development. Work done in cell lines also suggests that Dock1 is an important cytoskeletal regulator that controls cell migration. The Dock1/Rac pathway was also shown to act downstream of some RTKs to promote the invasion of glioblastoma cells. Yet, the in vivo functions of Dock1 in mammals are still poorly understood and the identification and characterization of some of these functions is the main objective of my thesis. Guided by the function of mbc, in Aim #1 we revealed that Dock1 is essential to embryonic development by characterizing a Dock1 knock-out mouse model. A deficiency in myoblast fusion was observed in Dock1-null embryos which led to a reduction in their muscle mass. Furthermore, we uncovered a contribution of the other Dock1-related GEF, Dock5, to myofiber development. In Aim #2 a correlation between high level of DOCK1 expression and a poor prognosis in HER2+ breast cancer patients was revealed. Amplification or overexpression of the HER2 receptor tyrosine kinase is associated with near 20% of breast cancer cases. The presence of this genetic abnormality correlates with a poor prognosis and the development of metastasis. Biochemical and in vitro studies led us to identify that DOCK1 interacts with HER2 and is essential to HER2-mediated RAC activation and migration. The use of a HER2 breast cancer mouse model with Dock1 inactivation in the mammary gland led us to identify DOCK1-RAC signaling as novel effectors in HER2-mediated tumor growth and metastasis. We conclude that the use of Dock1 mouse models allowed us to identify some of the key functions regulated by this gene in vivo. Much like its ortholog mbc, Dock1 is essential to embryonic development and regulates myoblast fusion. Our study also reveals important degree of conservation of the mechanisms that regulate fusion between species. In addition, DOCK1 acts downstream of the HER2 RTK in mammary epithelial cells where it contributes to the progression of breast cancer pathology and the formation of metastasis induced by this oncogene.

Dock1 (aussi nommé Dock180) est le membre prototypique de la famille Dock d’activateurs des petites GTPases de la famille Rho. Dock1 agit au sein d’une voie de signalisation conservée au cours de l’évolution et des études génétiques ont démontré que les orthologues de Dock1, myoblast city (mbc) chez la drosophile et Ced-5 chez le nématode, activent Rac dans divers processus biologiques. Notamment, mbc est un important régulateur de la fusion des myoblastes lors de la formation des fibres musculaires de drosophile. Mbc est aussi essentiel à la migration collective d’un groupe de cellules, appelées cellules de bordures, lors de leur migration dans la chambre de l’oeuf suite à l’activation de récepteurs à activité tyrosine kinase (RTK). La migration collective des cellules de bordures récapitule certains des événements observés lorsque des cellules tumorales envahissent le tissu environnant lors de la formation de métastases. Chez les mammifères, des études réalisées en lignées cellulaires suggèrent que Dock1 est aussi un régulateur du cytosquelette lors de la migration cellulaire. De plus, certaines études ont démontré que la voie Dock1/Rac agit en aval de RTKs lors de l’invasion de cellules de glioblastome. Néanmoins, les fonctions in vivo de Dock1 chez les mammifères demeurent méconnues et le but de cette thèse est d’identifier et de caractériser certaines de ses fonctions. Guidés par la fonction de mbc, nous démontrons dans l’objectif no 1 un rôle essentiel pour ce gène au cours du développement embryonnaire grâce à la caractérisation d’une souris Dock1 knock-out. Des défauts sévères de fusion des myoblastes sont observés en absence de l’expression de Dock1 et ils contribuent à la réduction de la masse musculaire des souris mutantes. De plus, nous avons constaté une contribution du gène Dock5, un membre de la famille Dock proche de Dock1, au développement des fibres musculaires. Dans l’objectif no 2, nous avons observé que des hauts niveaux d’expression de DOCK1 corrèlent avec un mauvais pronostic chez les patientes atteintes de cancer du sein possédant une forte expression du gène codant pour le RTK HER2. Une surexpression ou une amplification du locus codant pour le récepteur HER2 est associée à près de 20% des cas de cancer du sein. Les cancers de ces patientes développent fréquemment des métastases et sont associés à un mauvais pronostic. Des études biochimiques ont révélé que DOCK1 interagit avec le récepteur HER2 dans des cellules de cancer du sein. De plus, DOCK1 est essentiel à l’activation de RAC et à la migration cellulaire induite par HER2 dans ces cellules. L’utilisation d’un modèle de cancer du sein médié par HER2 chez la souris combiné avec l’inactivation du gène Dock1 dans les glandes mammaires, nous a permis d’identifier Dock1 et Rac comme de nouveaux effecteurs de la croissance tumorale et de la formation de métastases régulées par l’oncogène HER2. Nous concluons que l’utilisation de différents modèles de souris knock-out pour le gène Dock1 nous a permis d’identifier des fonctions clés de ce gène. Tout comme son orthologue mbc, Dock1 joue un rôle important lors du développement embryonnaire en régulant notamment la fusion des myoblastes. Nos études ont également contribué à démontrer un important degré de conservation des mécanismes moléculaires de fusion entre les espèces. De plus, DOCK1 agit en aval du RTK HER2 et son expression dans les cellules épithéliales de glandes mammaires contribue au développement tumoral et à la formation de métastases induits par cet oncogène. / Dock1 (also known as Dock180) is the prototypical member of the Dock family of Rho GTPase activators (RhoGEFs). Genetic studies in Drosophila and C. elegans have demonstrated that Dock1 orthologues act upstream of the Rac GTPase to activate it during various biological processes. Myoblast city (mbc), Dock1 ortholog in the Drosophila, is an important regulator of myoblast fusion during muscle fiber formation. Moreover, mbc regulates the collective migration of a cluster of border cells downstream of the activation of some tyrosine kinase receptors (RTKs). Migration of border cells is often view as a model for studying the invasive migration of cancer cells during metastasis development. Work done in cell lines also suggests that Dock1 is an important cytoskeletal regulator that controls cell migration. The Dock1/Rac pathway was also shown to act downstream of some RTKs to promote the invasion of glioblastoma cells. Yet, the in vivo functions of Dock1 in mammals are still poorly understood and the identification and characterization of some of these functions is the main objective of my thesis. Guided by the function of mbc, in Aim #1 we revealed that Dock1 is essential to embryonic development by characterizing a Dock1 knock-out mouse model. A deficiency in myoblast fusion was observed in Dock1-null embryos which led to a reduction in their muscle mass. Furthermore, we uncovered a contribution of the other Dock1-related GEF, Dock5, to myofiber development. In Aim #2 a correlation between high level of DOCK1 expression and a poor prognosis in HER2+ breast cancer patients was revealed. Amplification or overexpression of the HER2 receptor tyrosine kinase is associated with near 20% of breast cancer cases. The presence of this genetic abnormality correlates with a poor prognosis and the development of metastasis. Biochemical and in vitro studies led us to identify that DOCK1 interacts with HER2 and is essential to HER2-mediated RAC activation and migration. The use of a HER2 breast cancer mouse model with Dock1 inactivation in the mammary gland led us to identify DOCK1-RAC signaling as novel effectors in HER2-mediated tumor growth and metastasis. We conclude that the use of Dock1 mouse models allowed us to identify some of the key functions regulated by this gene in vivo. Much like its ortholog mbc, Dock1 is essential to embryonic development and regulates myoblast fusion. Our study also reveals important degree of conservation of the mechanisms that regulate fusion between species. In addition, DOCK1 acts downstream of the HER2 RTK in mammary epithelial cells where it contributes to the progression of breast cancer pathology and the formation of metastasis induced by this oncogene.

A perda ou falha de um órgão ou tecido é um dos problemas mais severos da saúde humana. A engenharia de tecidos, definida como o cultivo e adesão de células humanas in vitro em um scaffold ou arcabouço, surge como uma alternativa viável para reposição de órgãos e tecidos. Estas células proliferam, migram e se diferenciam num tecido específico enquanto produzem os componentes de matriz extracelular (ECM) necessários para criar este tecido. A obtenção de scaffolds fibrosos a partir da blenda polimérica de Poli (Ácido Láctico-co-Glicólico) (PLGA) e Poliisopreno (PI) é proposta como uma alternativa à engenharia de tecidos moles. Este material foi processado como estrutura fibrosa por meio de métodos de gotejamento (FD) e electrospinning (FS). Caracterização físico-química foi aplicada à blenda e às fibras geradas. Também foi averiguada a viabilidade das fibras em culturas de mioblastos murinos, fibroblastos dérmicos humanos, condrócitos bovinos e hepatocarcinomas. Nota-se que o processo de obtenção da blenda não apresentou alterações na estrutura química dos polímeros, sendo apontada também a imiscibilidade entre eles. A ductilidade do material foi apontada como efeito da presença de PI na blenda, embora esta composição apresente similar molhabilidade entre a mistura e os polímeros puros. As fibras geradas por electrospinning geraram um scaffold com menor porosidade do que as fibras obtidas por gotejamento, mesmo apresentando um diâmetro menor e uma orientação paralela entre fibras. As fibras obtidas por gotejamento apresentaram fibras emaranhadas de maior diâmetro, mas maior tammanho de poros, gerando scaffolds de maior porosidade. As propriedades mecânicas de ambos scaffolds indicam sua aplicação enquanto substitutos de tecidos moles. Ensaios de viabilidade celular condenaram o uso das fibras FS, uma vez que estas apresentaram solvente residual no interior da fibra, causando indesejada lise celular. As fibras FD apresentaram resultados de adesão e proliferação adequados para mioblastos, fibroblastos e condrócitos, porém os resultados foram considerados impróprios para hepatócitos. / The lost or failure of an organ or tissue is one of the most severe problems in human health. Tissue engineering, defined as the seeding and adhesion of human cells in vitro over a scaffold, arises as an viable alternative for reproduction of organs and tissues. These cells proliferate, migrate and differentiate into a specific tissue while producing extracellular matrix components. The obtaining of fibrous scaffolds from a polymeric blend of Poly (Lactic-co-Glycolic Acid) (PLGA) and Polyisoprene (PI) is proposed as an alternative to soft tissue engineering. This material was processed as a fibrous structure through dripping (FD) and electrospinning (FS) methods. Physical-chemical characterization was applied to the blend and to the generated fibres. Fibres viability was also observed for murine myoblasts, human dermal fibroblasts, bovine chondrocytes and hepatocellular carcinoma cultures. It was noticed that the blending process didn't have any influence over polymer's chemical structure, being observed the immiscibility between the raw materials. Blend's ductile behaviour was pointed out as an effect of PI presence, although this mixture presents similar wettability to the one presented by these raw polymers. Fibres obtained by electrospinnig generated a scaffold with smaller porosity, even presenting fibres with smaller diameter and a parallel organized topography. The fibres obtained by dripping presented a tangled structure of thicker fibres, but assembling a scaffold with higher porosity and inner space. Mechanical properties of both scaffolds indicate their applicability as soft tissue substitutes. Cell viability assays condemn the use of FS fibres, seen that they present residual solvent trapped into the fibre, causing undesirable cell lysis. On the other hand, FD fibres presented positive adhesion and proliferation results for myoblasts, fibroblasts and chondrocytes cell lines, however the results were consider inappropriate for hepatocytes.

A perda ou falha de um órgão ou tecido é um dos problemas mais severos da saúde humana. A engenharia de tecidos, definida como o cultivo e adesão de células humanas in vitro em um scaffold ou arcabouço, surge como uma alternativa viável para reposição de órgãos e tecidos. Estas células proliferam, migram e se diferenciam num tecido específico enquanto produzem os componentes de matriz extracelular (ECM) necessários para criar este tecido. A obtenção de scaffolds fibrosos a partir da blenda polimérica de Poli (Ácido Láctico-co-Glicólico) (PLGA) e Poliisopreno (PI) é proposta como uma alternativa à engenharia de tecidos moles. Este material foi processado como estrutura fibrosa por meio de métodos de gotejamento (FD) e electrospinning (FS). Caracterização físico-química foi aplicada à blenda e às fibras geradas. Também foi averiguada a viabilidade das fibras em culturas de mioblastos murinos, fibroblastos dérmicos humanos, condrócitos bovinos e hepatocarcinomas. Nota-se que o processo de obtenção da blenda não apresentou alterações na estrutura química dos polímeros, sendo apontada também a imiscibilidade entre eles. A ductilidade do material foi apontada como efeito da presença de PI na blenda, embora esta composição apresente similar molhabilidade entre a mistura e os polímeros puros. As fibras geradas por electrospinning geraram um scaffold com menor porosidade do que as fibras obtidas por gotejamento, mesmo apresentando um diâmetro menor e uma orientação paralela entre fibras. As fibras obtidas por gotejamento apresentaram fibras emaranhadas de maior diâmetro, mas maior tammanho de poros, gerando scaffolds de maior porosidade. As propriedades mecânicas de ambos scaffolds indicam sua aplicação enquanto substitutos de tecidos moles. Ensaios de viabilidade celular condenaram o uso das fibras FS, uma vez que estas apresentaram solvente residual no interior da fibra, causando indesejada lise celular. As fibras FD apresentaram resultados de adesão e proliferação adequados para mioblastos, fibroblastos e condrócitos, porém os resultados foram considerados impróprios para hepatócitos. / The lost or failure of an organ or tissue is one of the most severe problems in human health. Tissue engineering, defined as the seeding and adhesion of human cells in vitro over a scaffold, arises as an viable alternative for reproduction of organs and tissues. These cells proliferate, migrate and differentiate into a specific tissue while producing extracellular matrix components. The obtaining of fibrous scaffolds from a polymeric blend of Poly (Lactic-co-Glycolic Acid) (PLGA) and Polyisoprene (PI) is proposed as an alternative to soft tissue engineering. This material was processed as a fibrous structure through dripping (FD) and electrospinning (FS) methods. Physical-chemical characterization was applied to the blend and to the generated fibres. Fibres viability was also observed for murine myoblasts, human dermal fibroblasts, bovine chondrocytes and hepatocellular carcinoma cultures. It was noticed that the blending process didn't have any influence over polymer's chemical structure, being observed the immiscibility between the raw materials. Blend's ductile behaviour was pointed out as an effect of PI presence, although this mixture presents similar wettability to the one presented by these raw polymers. Fibres obtained by electrospinnig generated a scaffold with smaller porosity, even presenting fibres with smaller diameter and a parallel organized topography. The fibres obtained by dripping presented a tangled structure of thicker fibres, but assembling a scaffold with higher porosity and inner space. Mechanical properties of both scaffolds indicate their applicability as soft tissue substitutes. Cell viability assays condemn the use of FS fibres, seen that they present residual solvent trapped into the fibre, causing undesirable cell lysis. On the other hand, FD fibres presented positive adhesion and proliferation results for myoblasts, fibroblasts and chondrocytes cell lines, however the results were consider inappropriate for hepatocytes.

A perda ou falha de um órgão ou tecido é um dos problemas mais severos da saúde humana. A engenharia de tecidos, definida como o cultivo e adesão de células humanas in vitro em um scaffold ou arcabouço, surge como uma alternativa viável para reposição de órgãos e tecidos. Estas células proliferam, migram e se diferenciam num tecido específico enquanto produzem os componentes de matriz extracelular (ECM) necessários para criar este tecido. A obtenção de scaffolds fibrosos a partir da blenda polimérica de Poli (Ácido Láctico-co-Glicólico) (PLGA) e Poliisopreno (PI) é proposta como uma alternativa à engenharia de tecidos moles. Este material foi processado como estrutura fibrosa por meio de métodos de gotejamento (FD) e electrospinning (FS). Caracterização físico-química foi aplicada à blenda e às fibras geradas. Também foi averiguada a viabilidade das fibras em culturas de mioblastos murinos, fibroblastos dérmicos humanos, condrócitos bovinos e hepatocarcinomas. Nota-se que o processo de obtenção da blenda não apresentou alterações na estrutura química dos polímeros, sendo apontada também a imiscibilidade entre eles. A ductilidade do material foi apontada como efeito da presença de PI na blenda, embora esta composição apresente similar molhabilidade entre a mistura e os polímeros puros. As fibras geradas por electrospinning geraram um scaffold com menor porosidade do que as fibras obtidas por gotejamento, mesmo apresentando um diâmetro menor e uma orientação paralela entre fibras. As fibras obtidas por gotejamento apresentaram fibras emaranhadas de maior diâmetro, mas maior tammanho de poros, gerando scaffolds de maior porosidade. As propriedades mecânicas de ambos scaffolds indicam sua aplicação enquanto substitutos de tecidos moles. Ensaios de viabilidade celular condenaram o uso das fibras FS, uma vez que estas apresentaram solvente residual no interior da fibra, causando indesejada lise celular. As fibras FD apresentaram resultados de adesão e proliferação adequados para mioblastos, fibroblastos e condrócitos, porém os resultados foram considerados impróprios para hepatócitos. / The lost or failure of an organ or tissue is one of the most severe problems in human health. Tissue engineering, defined as the seeding and adhesion of human cells in vitro over a scaffold, arises as an viable alternative for reproduction of organs and tissues. These cells proliferate, migrate and differentiate into a specific tissue while producing extracellular matrix components. The obtaining of fibrous scaffolds from a polymeric blend of Poly (Lactic-co-Glycolic Acid) (PLGA) and Polyisoprene (PI) is proposed as an alternative to soft tissue engineering. This material was processed as a fibrous structure through dripping (FD) and electrospinning (FS) methods. Physical-chemical characterization was applied to the blend and to the generated fibres. Fibres viability was also observed for murine myoblasts, human dermal fibroblasts, bovine chondrocytes and hepatocellular carcinoma cultures. It was noticed that the blending process didn't have any influence over polymer's chemical structure, being observed the immiscibility between the raw materials. Blend's ductile behaviour was pointed out as an effect of PI presence, although this mixture presents similar wettability to the one presented by these raw polymers. Fibres obtained by electrospinnig generated a scaffold with smaller porosity, even presenting fibres with smaller diameter and a parallel organized topography. The fibres obtained by dripping presented a tangled structure of thicker fibres, but assembling a scaffold with higher porosity and inner space. Mechanical properties of both scaffolds indicate their applicability as soft tissue substitutes. Cell viability assays condemn the use of FS fibres, seen that they present residual solvent trapped into the fibre, causing undesirable cell lysis. On the other hand, FD fibres presented positive adhesion and proliferation results for myoblasts, fibroblasts and chondrocytes cell lines, however the results were consider inappropriate for hepatocytes.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-15T14:58:15Z No. of bitstreams: 1 Vinicius de Oliveira Cardoso.pdf: 848877 bytes, checksum: fe763aa5d3e08d692e2c53a7d7bb14cb (MD5) / Made available in DSpace on 2018-06-15T14:58:15Z (GMT). No. of bitstreams: 1 Vinicius de Oliveira Cardoso.pdf: 848877 bytes, checksum: fe763aa5d3e08d692e2c53a7d7bb14cb (MD5) Previous issue date: 2015-12-02 / The skeletal muscle cells are constant target for damage the exercise, direct trauma, inflammatory processes, among others. In order to contain or reset the loss of muscle mass, begins a highly organized repair process with activation, proliferation and differentiation of satellite cells. The use of phototherapy resources that can promote faster and efficient repair as the Low Level Laser Therapy (LLLT) has shown good results in the regeneration of skeletal muscle. The use of herbal medicines has also been described in the literature and has shown promising results with regard to tissue repair. The objective of the study was to evaluate the effects of treatment with LED on cell viability, Nitric Oxide (NO) synthesis and total protein dosage in C2C12 muscle cells cultured with Melaleuca armillaris extract in differentiation medium. We analyzed eight experimental groups: (1) Control C2C12; (2) LED 0,2J; (3) LED 2,0J; (4) LED 4,0J; (5) 1% Melaleuca; (6) Melaleuca 1% + LED 0,2J; (7) Melaleuca 1% + LED 2,0J; (8) Melaleuca 1% + LED 4,0J. Cells received Melaleuca extract at a concentration of 1.0% (v/v) and were irradiated with infrared LED (850nm, 30mW power for 7, 67 and 134 seconds at full power 0,2J, and 2,0J 4,0J, respectively) according to the group. Cell viability was assessed by MTT method, the synthesis of NO by the Griess method and total protein determination was performed using the Bradford method after periods of 1, 2 and 3 days of incubation. The results showed that cell viability increased TLED the three periods energies and tested without changing the total concentration of protein and NO. Since the herbal remedy promoted a significant reduction in cell viability compared to control small a reduction in the dosage of NO after 24 hours and no change in the protein assay. However, the combined use of the two resources, the energy value of 0.2 and showed viability 2,0J, NO and protein similar to the group treated only with 1% Melaleuca a tendency to increase in these levels in the group irradiated with 2,0J energy. However, the group associated 4,0J showed a growth recovery in cell viability in the three periods analyzed and behavior similar to nitric oxide and total protein compared to the control. In conclusion, the TLED positively modulated the proliferation of C2C12 cells without deterioration in nitric oxide synthesis and total protein, while incubation with associated or not to TLED Melaleuca needs more specific analyzes to elucidate their contribution in this context. / As células do músculo esquelético sofrem constantes lesões provocadas por exercício, traumas diretos, processos inflamatórios entre outros. A fim de conter ou repor a perda de massa muscular, inicia-se um processo de reparo altamente organizado com ativação, proliferação e diferenciação das células satélites. A utilização de recursos fototerápicos que possam promover um reparo mais rápido e eficiente como o Laser de Baixa Potência (LBP) tem apresentado bons resultados na regeneração do músculo esquelético. O uso de fitoterápicos também tem sido descrito na literatura e tem evidenciado resultados promissores no que diz respeito ao reparo tecidual. Assim, o objetivo do estudo foi avaliar os efeitos do tratamento com LED sobre a viabilidade celular, síntese de Óxido Nítrico (NO) e dosagem total de proteínas em células musculares C2C12 cultivadas com extrato de Melaleuca armillaris em meio de diferenciação. Foram analisados 8 grupos experimentais, sendo: (1) Controle C2C12; (2) LED 0,2J; (3) LED 2,0J; (4) LED 4,0J; (5) Melaleuca 1%; (6) Melaleuca 1%+LED 0,2J; (7) Melaleuca 1%+LED 2,0J; (8) Melaleuca 1%+LED 4,0J. As células receberam extrato de Melaleuca na concentração de 1,0% (v/v) e foram irradiadas com LED Infravermelho (850nm, potência de 30mW) por 7, 67 e 134 segundos, com energia total de 0,2J, 2,0J e 4,0J, respectivamente de acordo com o grupo. A viabilidade celular foi avaliada pelo método de MTT, a síntese de NO pelo método de Griess e a dosagem total de proteínas foi feita utilizando o método de Bradford após os períodos de 1, 2 e 3 dias de incubação. Os resultados demonstraram que a TLED aumentou a viabilidade celular nas três energias e períodos testados sem alterar a concentração de proteína total e NO. Já o recurso fitoterápico promoveu uma grande redução da viabilidade celular comparada ao controle, pequena redução na dosagem de NO após 24h e nenhuma alteração na dosagem de proteína. Contudo, o uso associado dos dois recursos, na energia de 0,2 e 2,0J apresentaram valor de viabilidade, NO e proteína semelhante ao grupo tratado somente com Melaleuca 1% com uma tendência no aumento destes níveis no grupo irradiado com 2,0J de energia. Entretanto, o grupo 4,0J associado evidenciou uma retomada de crescimento na viabilidade celular nos três períodos analisados e comportamento semelhante para óxido nítrico e proteína total comparados ao controle. Em conclusão, o TLED modulou positivamente a proliferação das células C2C12 sem apresentar alterações na síntese de óxido nítrico e proteína total, enquanto que a incubação com Melaleuca associada ou não ao TLED necessita de análises mais específicas para elucidar sua contribuição neste contexto.

Les myopathies héréditaires représentent un large groupe de pathologies neuromusculaires progressives affectant l’intégrité générale, structurelle et fonctionnelle du muscle squelettique. Elles engendrent une myriade de symptômes, ternissant qualité de vie et autonomie, et pouvant même s’avérer mortelles. La pose d’un diagnostic juste peut être difficile, entravée notamment par une faible prévalence de certaines myopathies, l’importante hétérogénéité clinique existante, et le chevauchement symptomatique des diverses formes. Malgré les avancées récentes faites dans le domaine des techniques de séquençage qui contribuent grandement au dépistage, au moins 25% des individus atteints de myopathies demeurent sans diagnostic génétique. Suivant l’investigation clinique d’un patient (Z46) atteint d’une myopathie distale à apparition tardive, l’analyse par séquençage ARN (RNA-seq) a révélé un variant non-sens homozygote de signification inconnue (VUS) à la fin de l’exon 5 du gène MLIP. Les niveaux d’expression génique altérés de « Protéine musculaire interagissant avec LMNA » (MLIP) et son partenaire « Lamine de type A » (LMNA) ont poussé à approfondir l’investigation. Davantage d’altérations -omiques furent identifiées par les techniques de RT-PCR, qPCR et WB, renforçant l’effet pathogénique du variant. Consolidant tous les résultats, le séquençage de longues lectures (LRS) a révélé un mécanisme d’épissage alternatif compensatoire de MLIP, qui tend à contourner et minimiser la production de transcrits arborant l’exon 5 muté. La présente étude vise à : 1) apporter un diagnostic génétique définitif au patient Z46, posant le variant MLIP comme causatif de la myopathie distale; 2) démontrer le pouvoir diagnostique du RNA-seq dans la résolution de ce cas complexe par l’identification et l’élucidation du VUS; 3) témoigner de l’étendue de la caractérisation transcriptomique offerte par les longues lectures du LRS. Couplé à cela, la modélisation du variant pathogénique par CRISPR/Cas9 dans une lignée cellulaire de myoblastes humains immortalisés permettra l’évaluation des impacts morpho-fonctionnels; conférant un supplément d’informations relatives aux fonctions musculaires normales et pathologiques de MLIP, faiblement caractérisées jusqu’à présent. / Hereditary myopathies represent a large group of progressive neuromuscular disorders affecting the general, structural and functional integrity of skeletal muscle. They cause a myriad of symptoms, impairing quality of life and autonomy, and can even be fatal. Making an accurate diagnosis can be difficult, hampered in particular by the low prevalence of certain myopathies, the significant clinical heterogeneity that exists, and the symptomatic overlap of the various forms. Despite recent advances in sequencing techniques that greatly assist in screening, at least 25% of individuals with myopathies remain without a genetic diagnosis. Following the clinical investigation of a patient (Z46) with a late-onset distal myopathy, RNA-sequencing (RNA-seq) analysis revealed a homozygous nonsense variant of unknown significance (VUS) at the end of exon 5 of the MLIP gene. Altered gene expression levels of ‘’Muscular LMNA-Interacting Protein’’ (MLIP) and its partner ‘’A-type Lamin’’ (LMNA) prompted further investigation. More -omic alterations were identified by RT-PCR, qPCR and WB technics, reinforcing the pathogenic effect of the variant. Consolidating all results, Long-Read Sequencing (LRS) revealed a compensatory alternative splicing mechanism of MLIP, which tends to bypass and minimize the production of transcripts carrying the mutated exon 5. The present study aims to: 1) provide a formal genetic diagnosis for patient Z46, positing the MLIP variant as causative of the distal myopathy; 2) demonstrate the diagnostic power of RNA-seq in resolving this complex case through identification and elucidation of the VUS; 3) testify to the breadth of transcriptomic characterization afforded by the long reads of LRS. Coupled with this, CRISPR/Cas9 modeling of the pathogenic variant in an immortalized human myoblast cell line will allow assessment of morpho-functional impacts; conferring additional information related to the normal and pathological muscles functions of MLIP, poorly characterized thus far.