Das Chemokine SDF-1α und sein Rezeptor CXCR4 sind in eine Vielzahl biologischer Prozesse, wie der Organogenese, der Hämatopoese und der Immunantwort involviert. Die Entdeckung des alternativen SDF-1α-Rezeptors CXCR7 bewirkte eine erneute Untersuchung der Funktion des SDF-1-Systems in diesen Vorgängen. CXCR7 ist in der Lage, je nach Gewebe- oder Zelltyp, als \'Scavenger\'-Rezeptor, Modulator des CXCR4 oder selbstständig aktiver Rezeptor zu agieren. In dieser Arbeit wurde untersucht, inwiefern beide Rezeptoren im Verlauf der Entwicklung der Muskulatur exprimiert werden, welche Aufgabe sie dabei übernehmen und ob sich Rückschlüsse auf die Muskelregeneration daraus ableiten lassen. Hierfür erfolgten in vitro-Untersuchungen an C2C12-Zellen und die anschließende Analyse der Expression von CXCR4, CXCR7 und SDF-1α in der sich entwickelnden Gliedmaßenmuskulatur von Wildtyp- und mdx-Mäusen. Die Untersuchung von C2C12-Zellen zeigte in allen Differenzierungsstadien eine detektierbare Menge von CXCR4 und eine zunehmende Expression des CXCR7. Die Behandlung der Zellen mit SDF-1α führte zu einer Phosphorylierung von Erk1/2 und PKCζ/λ und hemmte gleichzeitig deren Differenzierung. Nach einer Blockierung des CXCR4 mit seinem pharmakologischen Antagonist AMD3100 oder nach Hemmung der Expression durch spezifische siRNA blieb die Aktivierung des Signalweges aus und der hemmende Einfluss des SDF-1α auf die Differenzierung verschwand vollständig. Im Gegensatz dazu blieben nach der pharmakologischen Blockierung oder durch siRNA vermittelten Expressionshemmung des CXCR7 alle SDF-1α induzierten Effekte vollständig erhalten. Eine Untersuchung des Signalweges am dritten Tag der Differenzierung zeigte keine Aktivierung von Erk1/2. Ebenso blieb Erk1/2 nach einer Hemmung der Expression des CXCR4 unphosphoryliert. Im Gegensatz dazu fand nach einer Hemmung der Expression des CXCR7 mit spezifischer siRNA erneut eine Aktivierung des Signalweges statt. Weiterhin konnte in vivo festgestellt werden, dass die Expression des CXCR4 in der Muskulatur während der embryonalen Entwicklung am höchsten ist und bereits kurz nach der Geburt stark abnimmt, wenn die sekundäre Muskelentwicklung abgeschlossen ist. Die Expression des CXCR7 hingegen steigt perinatal an und bleibt bis zum Erwachsenenalter bestehen. Zusammengefasst zeigen diese Ergebnisse, dass CXCR4 aktiv das Signalgeschehen von SDF-1α in der Myogenese vermittelt und somit zur Differenzierungshemmung von Myoblasten beiträgt. CXCR7 hingegen wirkt als passiver SDF-1α-Scavenger und induziert somit durch eine Modulierung der extrazellulären SDF-1α-Konzentration die Differenzierung. In Übereinstimmung mit der Rolle des SDF-1α-Systems bei der Muskelentwicklung, konnte eine kontinuierliche SDF-1α- Expression im Bindegewebe um pränatale und im Endomysium von postnatalen und adulten Muskelfasern festgestellt werden. Diese SDF-1α-Expression stieg ebenso wie die CXCR4-Expression bei der Analyse der Muskulatur von dystrophin-defizienten Mäusen an und zeigte somit, dass dieses System auch für die Proliferation von Muskelvorläuferzellen in der regenerativen Muskulatur eine wichtige Rolle spielt. Bemerkenswerter Weise hatte diese Muskeldystrophie keinen Einfluss auf die Expression des CXCR7 und beeinflusst vermutlich dessen Funktion über einen anderen Mechanismus. Durch die offensichtlich enge Kontrolle von Muskelentwicklung und Regeneration durch CXCR4, CXCR7 und deren Liganden SDF-1α, bilden diese ein vielversprechendes therapeutisches Ziel für bestimmte Muskelerkrankungen. / The chemokine, SDF-1α, and its receptor, CXCR4, are assumed to control a multitude of biological processes such as organogenesis, haematopoesis, and immune responses. The previous demonstration that SDF-1α additionally binds to the chemokine receptor, CXCR7, currently urges a re-evaluation of the function of the SDF-1 system in these processes. In fact, depending on the tissue and cell type, CXCR7 either acts as a scavenger receptor, a modulator of CXCR4 or an independent active receptor. This thesis is dedicated to answer the following questions: Are both SDF-1α receptors expressed during muscle development? What is the actual function of these receptors during myogenesis? Is there a role of the SDF-1 system in muscle regeneration? To adress these issues both in vitro studies with the myoblast cell line, C2C12, as well as in vivo analyses on the expression of CXCR4, CXCR7 and SDF-1α in developing and regenerating limb muscles have been performed. At all stages of differentiation, C2C12 cells exhibited measurable amounts of CXCR4. In addition, in the course of differentiation C2C12 cells showed increasing expression levels of CXCR7. Treatment of the cells with SDF-1α resulted in the phosphorylation of Erk1/2 and PKCζ/λ and subsequently blocked their myogenic differentiation. Following inactivation of CXCR4 either by its antagonist, AMD3100, or by specific siRNA, SDF-1α failed to activate both pathways and in addition no longer inhibited the myogenic differentiation of C2C12 cells. By contrast, inactivation of CXCR7 remained without effects on SDF-1α-induced cell signalling and resulting inhibitory effects on myogenic differentiation. Interestingly, SDF-1α also failed to activate Erk1/2 signalling in differentiated C2C12 cells. Cell signalling in differentiated C2C12 cells was, however, restored following inhibition of CXCR7 expression, but not following inhibition of CXCR4 expression. The in vivo analysis further revealed that in limb muscles expression of the CXCR4 is highest during embryonic development with a decrease in expression levels shortly after birth when secondary muscle development is completed. Vice versa, expression levels of CXCR7 increased perinatally and remained high into adulthood. In summary, these findings unravel that CXCR4 actively mediates SDF-1α-signalling during myogenesis. The findings further provide evidence that CXCR7 acts as a scavenger receptor during myogenesis which controls myogenic differentiation by modulating extracellular SDF-1α concentration. In further agreement with a major role of SDF-1α in muscle development, SDF-1α is continously expressed by the endomysium of postnatal and adult muscle fibers. Moreover, expression of SDF-1α as well as CXCR4 is massively increased in muscles of dystrophin-deficient mice further implying that the SDF-1 system plays an equally important role during muscle development and regeneration. The pivotal role of SDF-1α in muscle development and regeneration points to the SDF-1 system as a promising therapeutical target for certain muscle diseases.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-11-20T17:29:47Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Amanda de Paula Lima.pdf: 1103885 bytes, checksum: c825dfd59f4730fd1cd0b63b5796cb44 (MD5) / Made available in DSpace on 2018-11-20T17:29:47Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Amanda de Paula Lima.pdf: 1103885 bytes, checksum: c825dfd59f4730fd1cd0b63b5796cb44 (MD5) Previous issue date: 2018-09-10 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / A presente pesquisa foi realizada com o objetivo avaliar os efeitos da suplementação combinada de lisina e metionina sobre o desempenho produtivo e expressão de genes relacionados com o crescimento muscular em alevinos de tilápia do Nilo revertidos sexualmente. Trezentos e trinta e seis alevinos de tilápia do Nilo (peso inicial 0,90 ± 0,01 g) foram distribuídos em 24 aquários de 70L com sistema contínuo de fluxo de água (2,0 L/min), em um delineamento inteiramente casualizado com quatro tratamentos e seis repetições. Foram elaboradas quatro dietas isoproteicas (~330,50 g/kg de proteína bruta) e isocalóricas (~18,59 MJ/kg) sem suplementação de lisina e metionina (Controle), suplementada com lisina (Lys), suplementada com metionina (Met) e suplementada com lisina e metionina (Lys+Met) durante oito semanas. Os peixes foram alimentados manualmente, quatro vezes por dia até saciedade aparente. Peixes tratados com as dietas Lys e Met apresentaram maior peso corporal e taxa de crescimento específico em relação aos peixes mantidos com as demais dietas. Peixes tratados com dieta Lys apresentaram maior taxa de eficiência proteica em comparação aos peixes mantidos com as outras dietas. O índice hepatostomático e a gordura corporal foram menores nos peixes alimentados com as dietas Met e Lys+Met em comparação aos peixes tratados com a dieta controle. O consumo, sobrevivência, umidade, proteína bruta, cinzas corporais e a expressão do mRNA da miostatina não foram influenciados pelas dietas. Peixes que receberam dieta Lys+Met apresentaram maior nível de expressão de mRNA da MyoD em comparação aos peixes que receberam a dieta controle, mas nenhum efeito da suplementação isolada de lisina e metionina foi observada. Em conclusão, a suplementação combinada de lisina e metionina melhora o desempenho produtivo e aumenta a expressão de mRNA de MyoD e miogenina e reduz conteúdo de gordura corporal de alevinos de tilápias do Nilo. / This work was carried out with the objective of evaluating the effects of the combination of lysine and methionine on the performance of growth and expression of genes related to muscle growth in sexually reversed Nile tilapia fingerlings. Fish (n = 336, initial weight 0.90 ± 0.01 g) were randomly distributed into 24 70 L aquaria with a continuous water flow system in an entirely randomized design with four treatments and six replicates. Four isoproteic (~330.50 g/kg crude protein) and isocaloric (~ 18.59 MJ / kg) diets without lysine or methionine supplementation (Control), or supplemented with lysine (Lys), methionine (Met) and lysine and methionine (Lys + Met) were elaborated. Fish were hand fed until apparent satiety. Fish fed diets Lys+Met and Met showed higher final body weight and specific growth ratio compared to fish fed other diets. The protein efficiency ratio was higher in fish diet Lys compared to fish fed other diets. Fish fed Met and Lys+Met diets showed lower hepatosomatic index and whole-body fat compared to fish fed the control diet. Feed intake, survival and whole-body moisture, crude protein, ash and mRNA expression of myostatin of fish were not affected by diets. Fish fed diet Lys+Met demonstrated higher mRNA expression level of MyoD compared to those fed the control diet. In conclusion, Nile tilapia fingerlings fed combined lysine and methionine demonstrates improved growth performance in line to higher mRNA expression of MyoD and myogenin, and also reduced body fat contents

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 53-58).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 70-79).

AN ABSTRACT OF THE DISSERTATION OF Priya Londhe, for the Doctor of Philosophy degree in Biochemistry and Molecular Biology, presented on 30th July, 2013 at Southern Illinois University Carbondale. TITLE: UNRAVELING THE MOLECULAR MECHANISMS OF THE CLASS II TRANSACTIVATOR IN SKELETAL MUSCLE MAJOR PROFESSOR: Dr. Judy Davie The inflammatory cytokine, interferon gamma, IFN-gamma orchestrates a diverse array of fundamental physiological processes and exhibits complex effects on myogenesis. IFN-gamma also induces the class II transactivator, CIITA, which is a critical mediator of IFN-gamma mediated repression and activation. The aims in my dissertation are directed towards understanding the role of IFN-gamma and CIITA in muscle. Stimulation by IFN-gamma in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. We show that the IFN-gamma induced inhibition of myogenesis is mediated by CIITA, which specifically interacts with myogenin. CIITA acts by both, repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. The IFN-gamma mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-gamma. We also show that CIITA is indispensible for the inhibition of myogenesis. To gain a mechanistic insight into the IFN-gamma induced repression of myogenesis, we have discovered that IFN-gamma and CIITA inhibit myogenesis by modifying gene regulation in a muscle cell subject to inflammation. We show that CIITA first interacts with JARID2, a non catalytic subunit of PRC2 complex, which induces a paused RNAPII, phosphorylated at serine 5 and then interacts with the catalytic subunit EZH2, in a JARID2 dependent manner. Our data show that both CIITA and IFN-gamma block myogenesis by the induction and recruitment of the PRC2 complex, which is normally silenced in a differentiating muscle cell. One of my dissertation aims sheds light on the silencing of CIITA in Rhabdomyosarcoma. Silencing of CIITA prevents the expression of MHC Class I and II genes. We have found that IFN-gamma signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells, but shows a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have also observed that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-gamma treatment, is blocked in both types of RMS cells. Further, our studies also impart a novel role for IFN-gamma and CIITA in inhibiting the IGF induced activation of muscle specific genes. Our data show that IFN-gamma does not block the signaling cascade of IGF. However, blocking exogenous IFN-gamma restores IGF activation of muscle specific genes. My dissertation also reveals an important role for the FACT complex in the early steps of gene activation through its histone chaperone activities that serve to open chromatin structure and facilitate transcription promoting muscle differentiation. We show that myogenin interacts with the FACT complex and the recruitment of FACT complex to muscle specific genes is dependent on myogenin. The final aim in my dissertation highlights the distinct binding profiles of the MRFs and E proteins during proliferation and differentiation. Our sequential ChIP assays show that MYOD, MYOG, and MYF5 co-occupy promoters. Taken together, my dissertation provides a comprehensive understanding of the molecular mechanisms during myogenesis and reveals the deleterious effects of chronic inflammation in skeletal muscle.

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.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-19T15:19:00Z No. of bitstreams: 1 Jacira Souza Ribeiro.pdf: 1136563 bytes, checksum: 6f35b1abf4cd3ca62f8560ee50069169 (MD5) / Made available in DSpace on 2018-06-19T15:19:00Z (GMT). No. of bitstreams: 1 Jacira Souza Ribeiro.pdf: 1136563 bytes, checksum: 6f35b1abf4cd3ca62f8560ee50069169 (MD5) Previous issue date: 2015-12-10 / The low-intensity pulsed ultrasound (LIPUS) has been used to promote muscle repair with better quality and in shorter time, however, there is no standardization for the parameters used in clinical practice. Thus, the aim of this study was to evaluate the effect of USPBI on the repair of skeletal muscle of rats after cryoinjury. Male Wistar rats (n=45) were divided into 3 groups: control; only injury; Injured and treated with LIPUS. The LIPUS application was performed daily, using the stationary mode, pulse 1: 4, 1 MHz frequency, intensity 0.4 W / cm2 for 3 minutes. The injured groups were euthanized at 1, 2, 3 and 7 days following injury induction. The tibialis anterior muscle (TA) was removed for morphological analysis and collagen remodeling, and the muscle sections stained with H&E and Picrosirus Red, respectively. Then, the slides were photographed and quantified using the program "Image J". The analysis of MyoD and myogenin gene expression was performed using real time PCR. The results showed that the USPBI promoted modulation of inflammatory responses with a decrease of inflammatory infiltrates after 1, 2, 3 and 7 days, and reduction of myonecrosis after 7 days, followed by an increase in the number of immature fibers after 3 and 7 days, and increase of blood vessels on days 2, 3 and 7 days. Regarding the deposition of collagen, the results showed better organization of the fibers in all experimental periods, and increased deposition of collagen fibers in the injured group and treated after 2 and 3 days. In addition, treatment with LIPUS promoted increased gene expression of MyoD reduction after 3 days and after 7 days. Regarding myogenin expression, the treated group showed increased expression after 7 days. In conclusion, the LIPUS induced positive effects on muscle repair process leading to reduced inflammation and myonecrosis, increased in the immature fibers and mature blood vessels, as well as modulation of Myod and miogenin in different periods. / O ultrassom pulsado de baixa intensidade (USPBI) tem sido utilizado por promover um reparo muscular de melhor qualidade e menor duração, porém, não há padronização quanto aos parâmetros utilizados na prática clínica. O objetivo deste estudo foi avaliar o efeito do USPBI sobre o reparo do músculo esquelético de rato após criolesão. Foram utilizados 45 ratos Wistar, machos, divididos em 3 grupos experimentais: controle; somente lesão; lesionado tratado com USPBI. A aplicação de USPBI foi realizada diariamente após indução da lesão, modo estacionário, pulsado 1:4, frequência 1 MHz, intensidade 0,4 W/cm2, durante 3 minutos. Os grupos lesionados foram eutanasiados após 1, 2, 3 e 7 dias da indução da lesão. O músculo tibial anterior (TA) foi removido para análise morfológica e de remodelamento do colágeno, sendo os cortes corados com H&E e Picrosirius Red, respectivamente. As lâminas foram fotografadas e quantificadas com auxílio do programa “Image J”. A expressão gênica de MyoD e miogenina foi obtida por PCR em tempo real. Os resultados evidenciaram que o USPBI promoveu modulação da resposta inflamatória, havendo redução do infiltrado inflamatório após 1, 2, 3 e 7 dias, e redução da mionecrose após 7 dias, seguido pelo aumento no número de fibras imaturas após 3 e 7 dias, e aumento dos vasos sanguíneos nos dias 2, 3 e 7 dias. Em relação à deposição de colágeno, os resultados evidenciaram melhor organização das fibras em todos os períodos experimentais, além de aumento da deposição de fibras colágenas no grupo lesionado e tratado após 2 e 3 dias. Além disso, o tratamento com USPBI promoveu aumento da expressão gênica de MyoD após 3 dias e redução após 7 dias. Em relação a expressão de miogenina, o grupo tratado demonstrou aumento da expressão após 7 dias. Em conclusão, o USPBI nos parâmetros utilizados induziu efeitos positivos ao processo de reparo muscular causando redução do processo inflamatório e mionecrose, aumento de fibras jovens vasos sanguíneos maduros, além de modulação de MyoD e miogenina nos diferentes períodos avaliados.