Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 84-92).

Made available in DSpace on 2016-06-02T20:18:09Z (GMT). No. of bitstreams: 1 1788.pdf: 1563222 bytes, checksum: d55e992347a7bcc9f276f927b6f940f4 (MD5) Previous issue date: 2008-05-07 / Financiadora de Estudos e Projetos / Esta pesquisa é o resultado de quatro anos dedicados ao doutorado. Apresenta caráter pioneiro em vários aspectos. Primeiro, este trabalho utilizou a metodologia de Branched de DNA (bDNA) para análise de expressão gênica em tecidos biológicos. Inicialmente, a técnica que era destinada para cultura de células, foi adaptada para homogenatos de tecido. Primeiramente, validamos a metodologia mediante o uso do protocolo QuantiGene® Reagent Systems Evaluation Program (Genospetra). Mediante este protocolo, vários ensaios experimentais foram realizados, e resultou na adequação metodológica que a empresa utilizou para padronizar os procedimentos para análise com homogenatos de tecidos animais. Atualmente, nosso protocolo faz parte do manual do Sistema de Reagentes QuantiGene Panomics® que está sendo utilizado pela própria empresa para análise da expressão gênica com homogenatos de tecido muscular. Segundo, tendo em vista a existência de poucos trabalhos na literatura que investiga o efeito do laser λ=514 nm durante a regeneração do músculo esquelético, decidimos realizar esta investigação para fins de desenvolvimento do laser de argônio de baixa intensidade para fins terapêuticos. Terceiro, consiste no primeiro trabalho que investiga três comprimentos de onda de laser de baixa intensidade sobre a expressão de genes durante o processo de regeneração muscular in vivo. Ao final desse estudo obtivemos resultados relevantes que deram origem a dois manuscritos submetidos em revistas internacionais, em anexo. Para apresentação desta tese, os resultados obtidos foram organizados em capítulos que correspondem aos dois artigos apresentados a seguir.

La voie de signalisation calcineurine/NFAT (Nuclear Factor of Activated T-cells) est très largement impliquée dans plusieurs aspects du développement et des pathologies du muscle squelettique. De nombreuses études ont souligné son implication dans la spécification des fibres musculaires adultes. Cependant, son rôle dans l’établissement du phénotype musculaire au cours de l’embryogenèse reste peu connu. Il faut noter que certains travaux suggèrent une implication de NFATc3 dans le nombre de fibres musculaires alors que l’isoforme NFATc2 pourrait être impliquée dans la taille des fibres musculaires. Les facteurs myogéniques (MRF) de la famille MyoD (incluant MyoD, Myf5, MRF4 et la myogénine) sont des facteurs de transcription à domaine bHLH (basic-Helix-Loop-Helix) clés pour la myogenèse squelettique. L’étude de leurs partenaires protéiques est essentielle pour bien comprendre la régulation de l’expression des gènes musculaires. Un nouveau paradigme de la transcription a été démontré (Armand et al. 2008), où une protéine NFAT pourrait coopérer spécifiquement avec un MRF : en effet, l’isoforme, NFATc3, coopère avec MyoD pour activer l’expression de la myogénine au cours de la somitogenèse. Ce rôle est spécifique du complexe NFATc3/MyoD puisque les souris mutantes myod (-/-) ; nfatc2 (-/-) ne présentent aucune modification de l’expression de la myogénine au cours de la somitogenèse. L’objectif de ce travail est d’étudier l’implication du complexe NFATc2/MyoD dans la myogenèse chez la souris. Nous montrons que l’isoforme NFATc2 coopère avec MyoD pour induire spécifiquement l’expression de la chaine lourde néonatale la myosine (MHCneo), in vitro aussi bien qu’in vivo. Cette coopération est spécifique et cruciale puisque les souris double mutantes myod (-/-) ; nfatc2 (-/-) meurent à la naissance avec une réduction spectaculaire de l’expression de la MHCneo, principale isoforme de la myosine exprimée à la naissance, alors que l’expression de cette chaine lourde de la myosine n’est pas affectée chez les simples mutants. Par des analyses d’immunoprécipitation de la chromatine et de retard sur gels, nous avons montré que les isoformes NFATc1 et NFATc3 se lient de manière préférentielle au gène codant la chaine lourde embryonnaire de la myosine (MHC emb). Ce travail, qui met en évidence pour la première fois le rôle de la voie de signalisation calcineurine/NFAT dans la construction précoce de la fibre musculaire au cours de l’embryogenèse, montre aussi l’implication spécifique du complexe NFATc2/MyoD dans le contrôle du nombre de fibres musculaires au cours de l’embryogenèse. / Cell biology

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

No description available.

Lesões nervosas periféricas com alterações morfofuncionais são de grande importância clínica, porque pode prejudicar a função, comprometendo a sensibilidade e/ou a motricidade do órgão alvo. Quando o nervo é lesado, o indivíduo torna-se impossibilitado de realizar suas atividades, seja profissional ou pessoal, e a partir do acidente esta situação se agrava ainda mais, pois tem início uma trajetória de sofrimento e humilhações decorrentes do tipo de assistência que passa a receber, tendo em vista, ainda, a fragilidade emocional e o abatimento moral de que passa a ser vítima. Na tentativa de reparo de lesões graves de nervos periféricos, várias técnicas têm sido utilizada, mas algumas com prejuízos funcionais para outras área do corpo, como por exemplo, quando se usa outro nervo no enxerto. Considerando que enxertos venosos tem tido bons resultados na capacidade regenerativa das fibras nervosas, e como elas são encontradas em abundância e em locais de fácil acesso cirúrgico, pensou-se em verificar se o tecido adiposo e o laser de baixa potência alterariam os resultados da reinervação, por tubulização, em músculos de contração rápida (EDL). Para isso foi utilizado 84 ratos (Rattus norvegicus) da linhagem wistar, machos, que foram divididos em 12 grupos (oito experimentais e quatro controles). Nos grupos experimentais (GE) foi utilizada tubulização de veia preenchida, ou não de tecido adiposo (GEVV e GEVG, respectivamente), com e sem tratamento de laser (GEVVL e GEVGL, respectivamente). Os grupos controles (GC) receberam os nomes de positivos (GCP) quando os animais não sofreram intervenção cirúrgica, e negativos (GCN) quando os animais foram submetidos à desnervação do nervo ciático. Todos os grupos tiveram os seus animais sacrificados em dois períodos, 45 e 150 dias, após o início do experimento. A certificação da recuperação foi feita por meio da análise dos músculos inervados por ele (EDL), comparando-os com os respectivos grupos controles. Técnicas de microscopia, Imunofluorescência (MyoD e miogenina), apoptose (Tunel), morfométricas e análise funcional do ciático, foram empregadas nesta investigação. Os resultados mostraram que aos 45 dias pós desnervação os dados dos grupos experimentais estavam mais próximos do grupo controle negativo, mas aos 150 dias eles estavam mais próximos aos do grupo controle positivo. Baseado nos dados obtidos pode-se concluir que o uso de tecido adiposo e do laser de baixa potência na técnica de tubulização do nervo ciático interferem na recuperação do músculo EDL desnervado. / The peripheral nerves injuries with morphofunctional alterations, have great clinical importance because could prejudice the function, committing the sensibility and/or the motricity of target organ. When nerve is damage, the individual becomes disabled to realize yours activities, either professional or personal, in the post accident periods, this situation aggravates each more, therefore initiate a trajectory of suffering and distressing despite of the kind of assistance that this person receives, in view of your emotional fragility and your moral discouragement that pass to be victim. In attempt to repair severe peripheral nerves lesions, many techniques had been used, but some present functional prejudices to other area of bodies, for example when other autologous nerve graft it is used. Considering that, vein graft had demonstrated good results in regenerative nerve fibers capacity, and the vein are found in abundance in many locals of chirurgic access, it thought in verify if the adipose tissue and low power laser could alter the reinnervation results, by tubulization technique, in fast twitch muscle (EDL). For this, was used 84 rats (Rattus norvegicus) wistar, male, divided in 12 groups (eight experimental and four controls). In the experimental groups (EG) was used tubulization by vein combined / or not with adipose tissue (EGV and EGVA, receptively), with or without laser treatment (EGVL and EGVAL, respectively). The controls groups (CG) was called of positives (CGP) when the animals did not subject to transaction nerve, and negatives (CGN) when the sciatic nerve was transaction in this animals. All groups had the animals scarified in two periods, 45 and 150 days post experiments beginning. The recuperation was notified by means of muscle innervated analysis (EDL), comparing with the respective controls groups. Microscope techniques, Immunofluorescence for (MyoD and Miogenin), apoptosis by (Tunel assay), morphometrics and sciatic functional analysis, were employed in this investigation. The results showed that in the 45 days post-dennervation, the data of experimental groups was nearest of negative control group (transaction sciatic nerve), but in the 150 days they was nearest to the positive control group. Based on this, could be conclude that the use of adipose tissue and low power laser used in the tubulization technique by vein in the sciatic nerve interfere in the recuperation of EDL muscle dennervated.

Lesões nervosas periféricas com alterações morfofuncionais são de grande importância clínica, porque pode prejudicar a função, comprometendo a sensibilidade e/ou a motricidade do órgão alvo. Quando o nervo é lesado, o indivíduo torna-se impossibilitado de realizar suas atividades, seja profissional ou pessoal, e a partir do acidente esta situação se agrava ainda mais, pois tem início uma trajetória de sofrimento e humilhações decorrentes do tipo de assistência que passa a receber, tendo em vista, ainda, a fragilidade emocional e o abatimento moral de que passa a ser vítima. Na tentativa de reparo de lesões graves de nervos periféricos, várias técnicas têm sido utilizada, mas algumas com prejuízos funcionais para outras área do corpo, como por exemplo, quando se usa outro nervo no enxerto. Considerando que enxertos venosos tem tido bons resultados na capacidade regenerativa das fibras nervosas, e como elas são encontradas em abundância e em locais de fácil acesso cirúrgico, pensou-se em verificar se o tecido adiposo e o laser de baixa potência alterariam os resultados da reinervação, por tubulização, em músculos de contração rápida (EDL). Para isso foi utilizado 84 ratos (Rattus norvegicus) da linhagem wistar, machos, que foram divididos em 12 grupos (oito experimentais e quatro controles). Nos grupos experimentais (GE) foi utilizada tubulização de veia preenchida, ou não de tecido adiposo (GEVV e GEVG, respectivamente), com e sem tratamento de laser (GEVVL e GEVGL, respectivamente). Os grupos controles (GC) receberam os nomes de positivos (GCP) quando os animais não sofreram intervenção cirúrgica, e negativos (GCN) quando os animais foram submetidos à desnervação do nervo ciático. Todos os grupos tiveram os seus animais sacrificados em dois períodos, 45 e 150 dias, após o início do experimento. A certificação da recuperação foi feita por meio da análise dos músculos inervados por ele (EDL), comparando-os com os respectivos grupos controles. Técnicas de microscopia, Imunofluorescência (MyoD e miogenina), apoptose (Tunel), morfométricas e análise funcional do ciático, foram empregadas nesta investigação. Os resultados mostraram que aos 45 dias pós desnervação os dados dos grupos experimentais estavam mais próximos do grupo controle negativo, mas aos 150 dias eles estavam mais próximos aos do grupo controle positivo. Baseado nos dados obtidos pode-se concluir que o uso de tecido adiposo e do laser de baixa potência na técnica de tubulização do nervo ciático interferem na recuperação do músculo EDL desnervado. / The peripheral nerves injuries with morphofunctional alterations, have great clinical importance because could prejudice the function, committing the sensibility and/or the motricity of target organ. When nerve is damage, the individual becomes disabled to realize yours activities, either professional or personal, in the post accident periods, this situation aggravates each more, therefore initiate a trajectory of suffering and distressing despite of the kind of assistance that this person receives, in view of your emotional fragility and your moral discouragement that pass to be victim. In attempt to repair severe peripheral nerves lesions, many techniques had been used, but some present functional prejudices to other area of bodies, for example when other autologous nerve graft it is used. Considering that, vein graft had demonstrated good results in regenerative nerve fibers capacity, and the vein are found in abundance in many locals of chirurgic access, it thought in verify if the adipose tissue and low power laser could alter the reinnervation results, by tubulization technique, in fast twitch muscle (EDL). For this, was used 84 rats (Rattus norvegicus) wistar, male, divided in 12 groups (eight experimental and four controls). In the experimental groups (EG) was used tubulization by vein combined / or not with adipose tissue (EGV and EGVA, receptively), with or without laser treatment (EGVL and EGVAL, respectively). The controls groups (CG) was called of positives (CGP) when the animals did not subject to transaction nerve, and negatives (CGN) when the sciatic nerve was transaction in this animals. All groups had the animals scarified in two periods, 45 and 150 days post experiments beginning. The recuperation was notified by means of muscle innervated analysis (EDL), comparing with the respective controls groups. Microscope techniques, Immunofluorescence for (MyoD and Miogenin), apoptosis by (Tunel assay), morphometrics and sciatic functional analysis, were employed in this investigation. The results showed that in the 45 days post-dennervation, the data of experimental groups was nearest of negative control group (transaction sciatic nerve), but in the 150 days they was nearest to the positive control group. Based on this, could be conclude that the use of adipose tissue and low power laser used in the tubulization technique by vein in the sciatic nerve interfere in the recuperation of EDL muscle dennervated.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2001. / Vita. Bibliography: 108-125.

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.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 91-100).