Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "December, 2008." Includes bibliographical references (leaves 257-286). Online version available on the World Wide Web.

Made available in DSpace on 2016-03-07T19:20:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-09-30. Added 1 bitstream(s) on 2016-03-07T19:24:51Z : No. of bitstreams: 1 000858004.pdf: 1989014 bytes, checksum: 48779343a2d8732bc458afeae78c1a5d (MD5) / O cão Golden Retriever Muscular Dystrophy (GRMD) é o melhor modelo natural da distrofia muscular de Duchenne (DMD) com manifestações genotípicas e fenotípicas semelhantes à doença humana. Como nos afetados pela DMD, a proliferação progressiva de tecido conectivo no endomísio das fibras musculares ocorre em paralelo ao curso clínico da doença nos animais GRMD. Estudos indicam a relação dos mastócitos com a deposição de tecido fibroso devido a liberação de seus mediadores que ativam os fibroblastos. O objetivo desse trabalho foi o de avaliar a expressão dos mastócitos e sua possível relação com as lesões musculares e fibrose em cães GRMD de diferentes idades. Foram utilizados fragmentos de quatro grupos musculares esqueléticos (masseter, diafragma, tríceps braquial e bíceps femoral) de dez cães entre dois a oito meses de idade sendo seis afetados e quatro controles. As amostras foram processadas por técnicas usuais de histologia, coradas com hematoxilina e eosina, azul de toluidina para verificar a expressão dos mastócitos e tricrômio de Azan para a evidenciação e quantificação da fibrose. Em todos os grupos musculares dos cães GRMD o infiltrado de mastócitos apresentou aumento significativo comparado ao grupo controle. O número médio de mastócitos diminuiu com a idade dos cães GRMD. Todos os grupos musculares apresentaram aumento significativo na quantidade de tecido colagenoso em relação ao grupo controle. A deposição de tecido fibroso diminuiu de acordo com a idade dos animais. Dos resultados afere-se que o aumento da degranulação dos mastócitos ocorreu em cães GRMD mais jovens (2 meses) fato que potencializou o aumento na deposição de tecido fibroso nas fibras musculares que foi diminuindo gradativamente com o tempo e a idade dos animais. Outros estudos são necessários para esclarecer o papel dos mastócitos na patogênese da fibrose, a fim de contribuir no desenvolvimento de novas... / Golden Retriever Muscular Dystrophy (GRMD) is the best natural model of Duchenne muscular dystrophy (DMD) with genotypic and phenotypic manifestations similar the human disease. As in to DMD patients, the progressive proliferation of connective tissue in the endomysium of the muscle fibers is parallel to the clinical course of disease in GRMD animals. Studies indicate the relationship of mast cells with the deposition of fibrous tissue due to the release of mediators that activate fibroblasts. The aim of this study was to evaluate the expression of mast cells and its possible relation with muscle injury and fibrosis in GRMD dogs at different ages. It was used fragments of four skeletal muscle groups (masseter, diaphragm, triceps brachial and biceps femoris) of ten dogs between two and eight months age, six affected and four controls. Samples were processed by usual techniques of histology, stained with hematoxylin-eosin and toluidine blue, to verify the expression of mast cells, and Azan trichrome for evaluation and quantification of fibrous. In all muscle groups of dogs GRMD the infiltration of mast cells increased significantly compared to control group. The average number of mast cells decreased with the age of GRMD dogs. All muscle groups showed a significant increase in the amount of collagenous tissue in the control group. The deposition of fibrous tissue decreased with the age of the animals. Possibly, the increase of mast cell degranulation and mediator release would occur in younger GRMD dogs (2 months), fact that potentiated the increase deposition of fibrous tissue in muscle fibers which was gradually decreasing over time and age of the animals. Other studies are needed to clarify the role of mast cells in the pathogenesis of fibrosis in order to contribute to the development of new therapeutic approaches and improve the life expectancy of humans and dogs affected by muscular dystrophy

Orientador: Julieta Rodini Engrácia de Moraes / Banca: Harumi de Castro Sasahara / Banca: Paula Fratini / Resumo: O cão Golden Retriever Muscular Dystrophy (GRMD) é o melhor modelo natural da distrofia muscular de Duchenne (DMD) com manifestações genotípicas e fenotípicas semelhantes à doença humana. Como nos afetados pela DMD, a proliferação progressiva de tecido conectivo no endomísio das fibras musculares ocorre em paralelo ao curso clínico da doença nos animais GRMD. Estudos indicam a relação dos mastócitos com a deposição de tecido fibroso devido a liberação de seus mediadores que ativam os fibroblastos. O objetivo desse trabalho foi o de avaliar a expressão dos mastócitos e sua possível relação com as lesões musculares e fibrose em cães GRMD de diferentes idades. Foram utilizados fragmentos de quatro grupos musculares esqueléticos (masseter, diafragma, tríceps braquial e bíceps femoral) de dez cães entre dois a oito meses de idade sendo seis afetados e quatro controles. As amostras foram processadas por técnicas usuais de histologia, coradas com hematoxilina e eosina, azul de toluidina para verificar a expressão dos mastócitos e tricrômio de Azan para a evidenciação e quantificação da fibrose. Em todos os grupos musculares dos cães GRMD o infiltrado de mastócitos apresentou aumento significativo comparado ao grupo controle. O número médio de mastócitos diminuiu com a idade dos cães GRMD. Todos os grupos musculares apresentaram aumento significativo na quantidade de tecido colagenoso em relação ao grupo controle. A deposição de tecido fibroso diminuiu de acordo com a idade dos animais. Dos resultados afere-se que o aumento da degranulação dos mastócitos ocorreu em cães GRMD mais jovens (2 meses) fato que potencializou o aumento na deposição de tecido fibroso nas fibras musculares que foi diminuindo gradativamente com o tempo e a idade dos animais. Outros estudos são necessários para esclarecer o papel dos mastócitos na patogênese da fibrose, a fim de contribuir no desenvolvimento de novas... / Abstract: Golden Retriever Muscular Dystrophy (GRMD) is the best natural model of Duchenne muscular dystrophy (DMD) with genotypic and phenotypic manifestations similar the human disease. As in to DMD patients, the progressive proliferation of connective tissue in the endomysium of the muscle fibers is parallel to the clinical course of disease in GRMD animals. Studies indicate the relationship of mast cells with the deposition of fibrous tissue due to the release of mediators that activate fibroblasts. The aim of this study was to evaluate the expression of mast cells and its possible relation with muscle injury and fibrosis in GRMD dogs at different ages. It was used fragments of four skeletal muscle groups (masseter, diaphragm, triceps brachial and biceps femoris) of ten dogs between two and eight months age, six affected and four controls. Samples were processed by usual techniques of histology, stained with hematoxylin-eosin and toluidine blue, to verify the expression of mast cells, and Azan trichrome for evaluation and quantification of fibrous. In all muscle groups of dogs GRMD the infiltration of mast cells increased significantly compared to control group. The average number of mast cells decreased with the age of GRMD dogs. All muscle groups showed a significant increase in the amount of collagenous tissue in the control group. The deposition of fibrous tissue decreased with the age of the animals. Possibly, the increase of mast cell degranulation and mediator release would occur in younger GRMD dogs (2 months), fact that potentiated the increase deposition of fibrous tissue in muscle fibers which was gradually decreasing over time and age of the animals. Other studies are needed to clarify the role of mast cells in the pathogenesis of fibrosis in order to contribute to the development of new therapeutic approaches and improve the life expectancy of humans and dogs affected by muscular dystrophy / Mestre

La pathologie Tau est une lésion neuronale commune à plus d’une vingtaine de maladies neurodégénératives. Elle correspond à l’agrégation des protéines Tau anormalement modifiées. Les mécanismes moléculaires impliqués dans l’agrégation de Tau demeurent encore mal compris. Toutefois, parmi les différentes hypothèses étiologiques, celle d’une dérégulation de l’épissage alternatif de Tau nous intéresse tout particulièrement. Ici, nous considérons la dystrophie myotonique de type 1 (DM1) comme maladie « modèle » pour étudier cette relation, puisqu’elle présente à la fois une dérégulation de l’épissage alternatif de Tau et des agrégats Tau. La DM1 est la forme adulte la plus fréquente de dystrophie musculaire. Il s’agit d’une maladie héréditaire à transmission autosomale dominante caractérisée par des répétitions CTGn>50 instables localisées dans la région 3’UTR du gène DMPK. Les mécanismes impliqués supposent un gain de fonction toxique des ARN mutés conduisant à une modification de l’épissage alternatif de nombreux transcrits parmi lesquels Tau. Dans ce contexte, nos objectifs étaient 1) de caractériser le défaut d’épissage de Tau dans le cerveau de plusieurs cas DM1 2) de modéliser ce défaut d’épissage afin d'identifier les facteurs trans-régulateurs impliqués et 3) de proposer une approche visant à restaurer un épissage normal. Le défaut d’épissage de Tau a été observé dans tous les cas analysés. Celui de l’exon 10, en revanche, n’a été rapporté que chez deux cas, qui, de façon intéressante, présentaient également une augmentation de l’expression des protéines CELF, décrites comme protéines régulatrices de l’épissage de Tau. Outre les protéines CELF, nous nous sommes également intéressés à MBNL1. MBNL1 est un facteur d’épissage jouant un rôle essentiel dans la physiopathologie de la DM1 où il a été décrit comme séquestré dans les foci. Peu de choses sont connues sur MBNL1 dans le cerveau et sur son rôle sur l’épissage alternatif des transcrits neuronaux. Ici, nous montrons que le niveau d’expression cérébrale de MBNL1 ne varie pas entre les cas DM1 et contrôles. En revanche, nous montrons que son épissage alternatif est dérégulé dans le cerveau. Notre étude de relation entre la structure et la fonction de la protéine suggère que ce changement d’épissage favorise sa séquestration dans les foci en modifiant sa localisation nucléaire, son activité de facteur d’épissage et ses propriétés d’oligomérisation. Le changement d’épissage de MBNL1 n’influence pas celui de Tau. Cependant, sa perte de fonction reproduit un profil d’épissage similaire à celui observé dans les cerveaux DM1. De plus, nous montrons que la surexpression de MBNL1, en présence des répétitions CTG suffit à restaurer un épissage normal de Tau et de plusieurs autres transcrits dérégulés dans la DM1. Enfin, des expériences complémentaires réalisées avec des protéines tronquées non fonctionnelles en tant que facteur d’épissage suggèrent que la restauration d’un profil d’épissage normal dans la DM1 serait due à la saturation des sites de liaisons CUG, ce qui permettrait de libérer les protéines MBNL1 séquestrées. Ces constructions semblent donc présenter un potentiel intérêt pour inverser les changements d’épissage observés dans la DM1 et sont actuellement en cours d’études. / Tau pathology is a brain lesion common to more than twenty neurodegenerative disorders. It consists of the abnormal aggregation of the microtubule-associated protein Tau into neurofibrillary tangles. Mechanisms underlying Tau aggregation are not fully understood yet. However, among the different etiological hypothesises, the one of a relationship between Tau mis-splicing and Tau aggregates particularly interests us. Here, we proposed a disease model, being myotonic dystrophy type I (DMI), in which Tau mis-splicing and Tau aggregate occur. DM1 is the most common adult form of muscular dystrophy. It is an inherited autosomal disorder characterised by a dynamic instable CTG repeats (over 50) in the 3’UTR of DMPK gene. DM1 pathogenesis is suggested to result from a RNA toxic gain of function whereby mutant transcripts modify the splicing machinery activity leading thus to a mis-splicing of several pre-mRNA targets including Tau. In this context, our objectives were to 1) characterize Tau mis-splicing in several DM1 brain patients 2) Model it and identify the trans-regulating splicing factors likely involved and 3) Propose a therapeutic approach to reverse it. Tau mis-splicing was always observed for both exons 2 and 3 in human adult DM1 brain and consisted of a reduced inclusion. Tau exon 10 splicing was seldom mis-regulated and associated with an increase of the CELF proteins family. CELF proteins are splicing factors previously described to regulate alternative splicing of Tau exons 2, 3 and 10. In addition to the CELF proteins, we also investigated the potential role of the splicing factor MBNL1, which was shown to play an essential role in DM1 physiopathology through its sequestration by the CUG repeats. MBNL1’s brain expression was ill-defined. Here, we report that MBNL1’s expression level was not altered but its splicing modified in adult DM1 brain. In addition, we provide evidences by a relationship study between the structure and the function of MBNL1 that this mis-splicing event favored its sequestration to the foci by modifying its cell-localization, splicing activity and oligomerization properties. MBNL1 mis-splicing does not influence Tau mis-splicing. However its loss of expression reproduced the mis-splicing of Tau exons 2/3 as observed in DM1 brain. Interestingly, the overexpression of MBNL1 in the presence of the CTG repeats partially restored a normal splicing of Tau as well as several other mis-regulated pre-mRNA targets. Further experiments performed with different molecular constructs lead us to hypothezied that the reversal of the abnormal splicing events observed in DM1 was mediated by a saturation of the CUG binding sites that lead to the release of a free pool of MBNL1, recovering thus its splicing function. This work leads us to design a new molecular tool that might be of interest to reverse the pathological events observed in DM1.

Dissertation (MSc (Human Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

The radioactive isotope K⁴² was used to measure the rate of potassium exchange in muscle from 129 strain mice. The results followed an unique course if plotted as K⁴² uptake versus (external potassium concentration ∙ time) [superscript ½], and corresponded to the result predicted for K⁴² uptake mediated by an ion-exchange compartment in the muscle. Variations in external potassium concentration did not affect the uptake rate if plotted this way, but sodium ion exerted some effect on the rate. Dystrophic mouse tissue accumulated K⁴² more rapidly than did normal tissue, and the effect of varying the external potassium concentration did not alter this rate. The effects of sodium variation were more pronounced than in normal tissue. Inulin space of muscle was measured in vivo as well as in vitro, to enable a correction for K⁴² in the extracellular space to be made. The inulin space was found to decrease with increasing muscle size, and this was thought to be related to the development of the muscle. Dystrophic muscle exhibited more of a dependance of inulin space on muscle size than did normal muscle. The suggestion was made that the dystrophic muscle membrane might be abnormally permeable to inulin. Muscles were excised and assayed by flame photometry for sodium and potassium content. They were assayed when freshly excised, and also following incubation in a variant of Locke’s solution. The muscle cations were stable for the first two hours of incubation, but after this time, intracellular sodium rose and potassium fell. Fresh dystrophic mouse muscle had lower potassium and higher sodium content than normal fresh muscle. The cation changes following incubation resembled those found for normal muscle. The changes in intracellular cations were correlated with the K⁴² uptake results, and discussed in some detail. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Duchenne Muscular Dystrophy (DMD) results from the absence of a functional dystrophin protein. Among its possible therapeutic options is the upregulation of dystrophin’s autosomal analogue, utrophin A. This can be achieved by a pharmacologically induced shift towards a slower, more oxidative skeletal muscle phenotype, which has been shown to confer morphological and functional improvements on models of DMD. Whether these improvements are a result of the utrophin A upregulation or other beneficial adaptations associated with the slow, oxidative phenotype, such as improved autophagy, has not been determined. To understand the importance of utrophin A to the therapeutic value of the slow, oxidative phenotype, we used the utrophin/dystrophin double knockout (dKO) model of DMD. We found the dKO mouse to have a similar skeletal muscle signaling capacity and phenotype to mdx mice. When treated with the adenosine monophosphate activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), both dKO and mdx mice expressed a shift towards a slower, more oxidative phenotype. In the mdx mice, this shift caused improvements in muscle fiber central nucleation, IgM penetration, damage from eccentric contractions, and forelimb grip strength. These morphological and functional benefits were not seen in the AICAR treated dKO mice. This study highlights the importance of utrophin A upregulation to the benefits of the slow, oxidative myogenic program to dystrophic mice. It confirms utrophin A as a therapeutic target in DMD and the slow, oxidative myogenic program as clinically relevant avenue towards treatment of the disease.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by mutations or deletions in the dystrophin gene. Utrophin up-regulation therapy is among the various therapeutic strategies that are being investigated to treat DMD. In this strategy utrophin, a dystrophin homologue, is up-regulated along the entire length of the sarcolemma to replace the absent dystrophin protein. Previous studies have revealed that utrophin A expression can be controlled by various transcriptional, post-transcriptional and translational mechanisms and pharmacological modulation of these pathways can stimulate its expression in muscle. In the present study we screened several FDA approved and natural pharmacological compounds that can potentially activate utrophin A expression in muscle. We found that AICAR (AMPK activator) and heparin (p38 activator) were most effective in stimulating utrophin A expression in our C2C12 muscle cell system. Next, we analyzed the effect of combining these activators on utrophin A expression in muscle cells and preclinical mdx mouse model of DMD. Our findings revealed that combinatorial treatment of AICAR and heparin instigated an additive effect on utrophin A expression both in C2C12 muscle cells and mdx mice. Further characterization of treated mdx mice revealed that combinatorial treatment of AICAR and heparin caused improvements in the dystrophic phenotype as indicated by decreased central nucleation, decreased fiber size variability and improved sarcolemmal integrity in dystrophic muscle. Together these findings established that combinatorial treatment of AICAR and heparin ameliorates the dystrophic phenotype in mdx mice and may serve as an effective therapeutic strategy for DMD.

A variety of cardiomyopathies are due to familial disease. Most are primarily associated with cardiac involvement and can lead to hypertrophic, dilated, or restrictive cardiomyopathy. Myotonic dystrophy (MD) is a multisystem disease with autosomal dominant inheritance and variable penetrance. Cardiac diseases are important causes of morbidity and mortality in MD patients. Patients with primary MD should be carefully investigated with an electrocardiogram, stress test, and an echocardiogram to identify preclinical cardiac involvement and to prevent life-threatening complications. Any new onset of atrial flutter or atrial fibrillation in a young patient without any underlying cardiac abnormality should be investigated for underlying myopathy. The authors report on a male adolescent with MD who presented with atrial flutter. The patient had been diagnosed with MD at birth. He had an impaired ejection fraction of 38% to 45%. The patient described sharp chest pain in the retrosternal area, with no radiation, that was induced by exercise.

No description available.