Дисертації з теми "Myotonia; Muscle"

Abstract Functional defects in the muscle specific chloride channel ClC-1 result in reduced chloride conductance and electrical hyperexcitability, which in turn impairs muscle relaxation and leads to myotonia. The gene CLCN 1 codes for ClC-1 in humans, and mutations in CLCN 1 cause the disease known as myotonia congenita. Worldwide over 80 mutations in CLCN1 have been described, but only three were found in patients in Northern Finland. These included two missense mutations and a nonsense mutation. The behavior and localization of the normal and mutated ClC-1 mRNA and protein were analyzed in muscle cell cultures. In intact muscle the ClC-1 protein was seen in the sarcolemma, but after myofiber isolation the protein was located intracellularly. Sarcolemmal localization was restored when myofibers were electrically stimulated or treated with a protein kinase C inhibitor. When mutated ClC-1 proteins were examined in a myofiber cell culture system, retardation in the ER was observed with the two missense mutations. The nonsense mutation did not have an effect on the transport from the ER to the Golgi elements, but the mutated ClC-1 was degraded more rapidly than the wild type ClC-1, at least in myotubes. Both retardation and degradation of the mutated ClC-1 are likely to result in too few channels present at the plasma membrane of the muscle cell to maintain normal physiological function. A very strict quality control in muscle cells was observed. The behavior and survival of multinuclear skeletal muscle cells is dependent on innervation and muscle activity, and the balance between the phosphorylation and dephosphorylation pathways modulates the function of muscle chloride channels
Tiivistelmä Lihasspesifisen kloridikanavan ClC-1:n toiminnalliset virheet johtavat alentuneeseen kloridin johtumiseen solukalvon läpi ja lihassolun ylieksitoitumiseen. Tämän seurauksena lihaksen rentoutuminen vaikeutuu ja havaitaan myotoniaa, lihasjäykkyyttä. Pohjoissuomalaisesta potilasmateriaalista tautiin johtavia geenimutaatioita löytyi kolme erilaista. Poikkeuksellista havainnoissa on erilaisten mutaatioiden vähyys, mikä on tyypillistä suomalaiselle tautiperinnölle. Yhteensä tämän kloridikanavan mutaatioita on julkaistu yli 80 erilaista. Tutkiessamme normaalin ja mutatoidun ClC-1 lRNA:n ja proteiinin käyttäytymistä ja sijaintia lihassoluviljelmissä. Havaitsimme eron lihasleikkeiden ja eristettyjen myofiibereiden välillä. Lihasleikkeissä ClC-1 paikantui solun pinnalle sarkolemmalle, mutta eristetyissä myofiibereissä lähinnä solun sisälle. Stimuloimalla eristettyjä myofiibereitä sähkövirralla tai käsittelemällä proteiini kinaasi C inhibiittorilla, saimme kloridikanava-proteiinin siirtymään takaisin solun pinnalle. Proteiinitasolla kuljetuksessa on havaittavissa eroja. Aminohappomuutokseen johtavat pistemutaatiot aiheuttivat proteiinin jäämisen endoplasmiseen kalvostoon, kun taas ennenaikaisen stop-kodonin johdosta lyhentynyt proteiini kuljetetaan eteenpäin Golgin laitteeseen. Myotuubeissa tämä lyhentynyt proteiini kuitenkin hajotettiin nopeammin kuin normaali kloridikanavaproteiini. Sekä kuljetuksen hidastuminen että nopeampi hajotus johtavat tilanteeseen, jossa lihassolun solukalvolla on liian vähän kloridikanavia ylläpitämään lihaksen normaalia fysiologista toimintaa. Monitumaisten lihassolujen laaduntarkkailu havaittiin vielä monitahoisemmaksi kuin yksitumaisilla. Monitumainen lihassolu on riippuvainen hermoärsytyksestä ja lihasaktiivisuudesta. Lisäksi fosforylaatioon liittyvä signalointi on tärkeää ClC-1 proteiinin oikealle paikantumiselle lihassolussa

This thesis examines clinical characteristics, molecular genetic aspects and electrophysiological features of two muscle channelopathies - myotonia congenita (MC) and Andersen-Tawil syndrome (ATS). MC is a muscle stiffness disorder caused by mutations in the skeletal muscle chloride channel gene CLCN1. A detailed genotype-phenotype analysis was undertaken in an initial MC cohort (22 families). A screening strategy for genetic testing was developed and applied to a larger cohort (303 cases). Twenty-three novel mutations and a high proportion of dominant MC predominantly due to four novel mutations clustered in exon 8 were observed. These four mutations were studied in vitro using two-electrode voltage-clamp methods in Xenopus laevis oocytes. Loss of function and clear dominant-negative effect in CO-expression experiments were demonstrated. The Xenopus oocyte expression system was also used to study the non-genomic effect of sex hormones on CLC-1 channels. It is shown that both testosterone and progesterone rapidly and reversibly inhibit wild-type CIC-1 channels by causing a prominent rightward shift in the voltage dependence of their open probability. In contrast, 17 β-estradiol causes only a small shift. These results suggest a possible mechanism to explain how the severity of myotonia congenita may be modulated by sex hormones. The potential modifying effect of the myotonic dystrophy genes DMPK and ZNF9 on the phenotype of MC was investigated. Allele sizes for these genes were measured in more than 400 patients with suspected non-dystrophic myotonia. Four individuals were identified with an intermediate size allele of DMPK and ten individuals tested positive for myotonic dystrophy type 2. ATS is characterised by the triad of periodic paralysis, cardiac arrhythmias and dysmorphic features. A UK cohort with ATS is presented with detailed phenotype-genotype correlation. Novel mutations were found and unusual clinical features including renal tubular defect, CNS involvement, dental and phonation abnormalities were observed.

Myotonic dystrophy (DM) is the most common inherited neuromuscular disorder of adult life. The genetic defect for DM was identified as an unstable CTG trinucleotide repeat found in the 3' untranslated region (UTR) of a serine threonine p&barbelow;rotein k&barbelow;inase, DMPK. Normal individuals possess 5--35 CTG repeats, typical adult DM patients have repeat sizes ranging from 80 to 1000 while cDM patients have from 1000 to several thousand CTG repeats. This discovery provided a molecular basis to account for large variability of penetrance and age of onset in DM. Work in our laboratory progressed from ascertaining mRNA levels in patient tissues to testing the hypothesis that overexpression of DMPK might cause features of DM. Transgenic mice expressed the human DMPK mRNA and protein in the appropriate tissues and had many features of DM including type I fibre atrophy, central nuclei and ringed fibres. Induction of expression resulted in about three fold higher levels of CTG 99 mRNA over CTG 11 mRNA at 48 hours post induction. Levels of cell death were assayed following induction and CTG 99 cell lines showed a marked level of cell death while CTG 11 cell lines did not. The presence of CTG repeats within mRNA therefore appears to be very problematic for the cell. Furthermore, we found that patient amniocytes and myoblasts are susceptible to staurosporine induced cell death. Taken together, this data suggests that myoblasts expressing the DMPK 3' UTR are prone to cell death in an expression level and repeat length dependent manner. In addition, patient cells were found to be susceptible in much the same way. These experiments also revealed that myogenin levels in vivo were reduced in transgenic embryos compared with wild type embryos suggesting that expression of the DMPK 3' UTR in vivo inhibits accumulation of myogenin and perhaps myogenesis. In adult mice there was consistent muscle atrophy in CTG 91 but not in CTG 11 mice despite much lower expression levels of the CTG 91 transgene. Together, these results indicate that expression of the DMPK 3' UTR by itself may inhibit myogenesis in vivo and contribute to pathological features of DM-like muscle atrophy. (Abstract shortened by UMI.)

Les Dystrophies Myotoniques de type 1 (DM1) et 2 (DM2) sont des maladies neuromusculaires autosomiques dominantes causées par l’expansion anormale de séquences microsatellites C(C)TG situées respectivement dans la région 3’UTR du gène DMPK et le premier intron du gène ZNF9. Les ARNs mutés contenant les expansions de répétitions sont retenus dans le noyau des cellules sous formes d’agrégats riboprotéiques et séquestrent les protéines de liaison à l’ARN de la famille MBNL conduisant à des dérégulations de l’épissage alternatif associés aux symptômes cliniques. Bien que différentes approches thérapeutiques pour la DM soient en développement, il n’existe à ce jour aucune thérapie. Dans ce travail de thèse, nous avons développé une stratégie innovante de thérapie génique basée sur une protéine MBNL1 modifiée. Ce dérivé MBNL∆ agit comme un leurre pour libérer les facteurs MBNL endogènes anormalement séquestrés par les ARN mutés, et restaurer leurs fonctions. Ainsi l’expression de MBNL∆ dans des modèles DM1 permet la correction des anomalies moléculaires et phénotypiques. Nous avons également réalisé différentes optimisations de cet outil dans le but d’accroître ces capacités fonctionnelles. Enfin, nous avons développé un système d’autorégulation basé sur un « senseur d’épissage » dans le but de contrôler l’expression protéique d’un transgène. La preuve de concept de ce système a été faite à l’aide de MBNL∆ et validée dans des modèles DM1. En conclusion, mon travail de thèse a permis d’établir le potentiel d’une approche de thérapie génique pour la DM de type « leurre » basée sur l’ingénierie de protéine de liaison à l’ARN
Myotonic dystrophy (DM) is an autosomal neuromuscular disease encompassing two distinct forms, type 1 (DM1) and type 2 (DM2), which are caused by abnormal microsatellite expansions of C(C)TG repeats in the 3’UTR of the DMPK and first intron of ZNF9 genes, respectively. Mutant RNAs carrying expanded repeats are retained in the nucleus as riboprotein aggregates that abnormally sequester MBNL splicing factors leading to alternative splicing misregulations associated with clinical symptoms. Although various therapeutic approaches for DM are under development, there is no effective therapy available so far. In this study, we designed a novel gene therapy strategy with the use of an engineered MBNL RNA-binding protein derivative that acts as a CUGexp-decoy to release sequestered endogenous MBNL factors and restore their proper functions. Expression of the decoy results in the correction of DM1-associated features in both in vitro and in vivo models of the disease. Subsequent optimization processes were applied to the engineered decoy and the most potent derivate that increases its functional capacity was selected for further therapeutic application. Additionally, we developed an autoregulatory system based on a splice-sensor strategy to control transgene product expression and provided a proof-of-concept of its efficacy in both in vitro and in vivo systems. In conclusion, my work establishes the potency of gene therapy treatment for DM and support the use of the decoy-based approach as an alternate or complementary therapeutic intervention for DM

Attaining an understanding of the mechanisms underpinning development has been amongst the cardinal scientific challenges of our age. The transition from a single cell organism to the level of complexity evidenced in higher eukaryotes has been facilitated by the advent of intricate developmental networks involving a plethora of factors that synergise to allow for precise spatio-temporal expression of the proteins present in higher organisms. Development is often portrayed as a domino like cascade of events stemming from relatively uncomplicated origins that go on to branch out and form associations and interactions amongst multitudinous actors that will inexorably lead towards a higher state of order. Transcription factors occupy a central position within this tapestry of interactions. They regulate expression of the various required proteins and they provide the cues for the developmental events that will eventually shape an organism. These factors frequently remain unknown until some occurrence causes developmental processes to fail and inadvertently focus attention on the factors that facilitate development. Myotonic dystrophy is a useful paradigm of such a developmental dysfunction that has led to the discovery of a transcription factor integral to both muscle development and gonadogenesis in both Drosophila and higher eukaryotes.

Myotonic dystrophy (DM) is the most common form of inherited neuromuscular disease in adults and is characterized by progressive muscle wasting and myotonia. The mutation responsible for DM has been identified as the amplification of apolymorphic (CTG)$\rm\sb{n}$ repeat in the 3$\sp\prime$ untranslated region of a gene encoding a serine/threonine kinase (DMK). We have produced a polyclonal rabbit antibody preparation against a fusion protein encoding C-terminal amino acids 471-629 of the human DMK gene. This antibody specifically detects products of both full length and truncated human DMK genes expressed in bacteria and in insect cells. On immunoblots, we observed protein species of $\sim$74 and 82 kDa in human and rodent cardiac muscle and skeletal muscle, as well as rodent ependyma and choroid plexus. By immunofluorescence, DMK was found to localize postsynaptically at the neuromuscular junction of skeletal muscle, at intercalated discs of cardiac tissue and at the apical membrane of the ependyma and within the choroid plexus. We have also detected 2-3 species ($\sim$45-50 kDa) in brain tissue. Neuroanatomical evidence suggests synaptic localization for DMK in rodent cerebellum, hippocampus, midbrain and medulla. These results indicate that DMK may have a role in intercellular communication. Finally, we have demonstrated that DMK is present in adult and congenital DM tissues and that its distribution is no different than that observed in normal controls.

La Dystrophie myotonique de type 1 (DM1) est une maladie neuromusculaire affectant notamment le muscle squelettique avec la présence d’une myotonie et d’une atrophie progressive et causée par une expansion anormale de triplets CTG dans le 3’UTR du gène DMPK. L’expression des ARN mutés induit la perte de fonction du facteur d’épissage MBNL1 et conduit à la réexpression de formes fœtales de certains transcrits dans les tissus adultes de patients DM1. J’ai développé un modèle de cellules musculaires exprimant de manière conditionnelle de 960 répétitions CTG interrompues, afin d’identifier des nouveaux mécanismes impliqués dans la dysfonction musculaire. Suite à l’expression ciblée d’ARN-960CUG dans des myotubes, une analyse du transcriptome montre la présence d’un certain nombre de fonction/processus biologiques typiques de la DM1. En revanche, l’induction de voies non associées à la DM1 et l’absence de phénotype suggèrent que notre modèle n’est pas approprié pour l’étude des mécanismes moléculaires. J’ai fait aussi une étude de l’impact du défaut d’épissage d’ATP2A1 (SERCA1), présent chez les patients DM1, sur la fonction musculaire. J’ai utilisé une approche antisens afin de favoriser l’exclusion de l’exon 22 d’Atp2a1 dans un muscle contrôle, conduisant à la réexpression de l’isoforme fœtal Serca1b. Chez la souris sauvage adulte, ce défaut provoque un ralentissement de la contraction et une perte de masse musculaire. Chez le poisson-zèbre, cette modification d’épissage provoque une altération de la locomotion. L’ensemble de ces résultats indique que la réexpression de Serca1b affecte la fonction musculaire et pourrait contribuer aux symptômes musculaires dans la DM1
Myotonic Dystrophy Type 1 (DM1) is a neuromuscular disease that affects mainly the skeletal muscle with the presence of myotonia and progressive atrophy and is caused by abnormal CTG expansion in the 3'UTR of the DMPK gene. The expression of the mutated RNA induces the loss of function of the MBNL1 splicing factor and leads to the re-expression of fetal isoforms of certain transcripts in the adult tissues of DM1 patients. In order to identify new mechanisms involved in muscle dysfunction, I developed a model of muscle cells conditionally expressing 960 interrupted CTG repeats. Following the targeted expression of RNA-960CUG in myotubes, transcriptome analysis shows that despite the presence of functions/biological processes typical of DM1, the induction of non-DM1 associated pathways and the absence of phenotype suggest that this model is not appropriate for this study of molecular mechanisms. I also did a study of the impact of the ATP2A1 (SERCA1) misplicing, present in DM1 patients, on the muscular function. I used an antisense approach to promote the exclusion of exon 22 from Atp2a1 in the muscle of two animal models, leading to the reexpression of the Serca1b fetal isoform. The re-expression of Serca1b in the muscle of adult wild-type mice leads to a slowing contraction and a loss of muscle mass. In zebrafish, this modification on Atp2a1 splicing causes an alteration on the locomotion. All of these results indicate that reexpression of Serca1b affects muscle function and may contribute to muscle symptoms in DM1

La dystrophie myotonique de type 1 (DM1), la maladie neuromusculaire la plus fréquente chez l’adulte, est causée par une expansion de CTG dans la partie 3’ non traduite du gène codant pour la protéine dystrophy myotonic protein kinase, DMPK. La forme adulte de la maladie est caractérisée, notamment, par une faiblesse musculaire, de la myotonie, des cataractes et des troubles cardiaques. La forme congénitale, la forme la plus sévère de la maladie, est caractérisée par de l’hypotonie, une détresse respiratoire aiguë et un retard psychomoteur important. La régénération des fibres musculaires est déficiente dans la forme adulte de DM1, tandis qu’un retard de maturation du système musculaire est observé dans la forme congénitale. A ce jour, les mécanismes moléculaires par lesquels l’expansion de CTG affecte le développement ou la régénération du système musculaire sont inconnus. Des travaux ont démontré que le sérum de mères ayant un enfant atteint de la forme congénitale de la DM1 contient un facteur soluble inhibant la différenciation terminale des myoblastes. Il est encore indéterminé si ce facteur est produit par la mère ou sécrété par le fœtus. Nous avons émis l’hypothèse qu’un facteur soluble était sécrété par les myoblastes prélevés de patients atteints de forme congénitale de la DM1 qui inhibe la différenciation terminale des précurseurs des cellules musculaires. Les résultats obtenus démontrent que les myoblastes DM1 sécrètent un facteur soluble de masse moléculaire inférieure à 30 kDa inhibant la différentiation myogénique. Cet effet est associé une diminution spécifique de la myogenin. L’effet de ce facteur soluble est proportionnel à la longueur de l’expansion des CTG. L’identification et la caractérisation du facteur soluble sécrété spécifiquement par les myoblastes DM1 permettra une meilleure compréhension des mécanismes moléculaires responsable des défauts de développement du système musculaire observés dans la forme congénitale, et des défauts de régénération des fibres musculaires observés dans la forme adulte de la DM1. L’identification de ce facteur soluble est présentement en cours.
Myotonic dystrophy (DM1), the most common form of inherited neuromuscular disease in adults, affects 1 in 8000 individuals worldwide. DM1 is an autosomal dominant muscular dystrophy with very variable symptom presentations. Adult onset DM1 is primarily characterized by myotonia, muscle wasting and weakness, but also affects a number of organs and tissues. One characteristic of the disease is the presence of a severe congenital form (CDM1), which differs from the adult form. CDM1 is characterized by a delay in the development of skeletal muscles. This form is associated with hypotonia, respiratory complications and mental retardation. DM1 is caused by the expansion of an unstable CTG trinucleotide repeat in the 3’untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. To date, the mechanisms by which the DM1 mutation affects skeletal muscles development or regeneration are unknown. A previous study demonstrated that serum produced by mothers of children with congenital myotonic dystrophy inhibits myogenic differentiation. In this study, we hypothesized that CDM1 myoblasts secrete a soluble factor that blocks myogenic differentiation. We provide evidence that this soluble factor is produced by DM1 and CDM1 myoblasts which may be involved in their deficiency to fuse. The inhibitory effect is proportional to the length of the CTG repeat expansion. In addition, the delay in muscle differentiation is associated with a specific reduction in myogenin gene expression. We believe that the DM1 mutation triggers the expression of a soluble factor, which is able to block myogenic differentiation. The identification of this soluble factor is presently under investigation.

Over the past decade several converging lines of evidence have highlighted the importance of post-transcriptional events in skeletal muscle. This level of regulation is controlled by multi-functional RNA-binding proteins and trans-acting factors. In fact, several RNA-binding proteins are implicated in neuromuscular disorders including myotonic dystrophy type I, spinal muscular atrophy and amyotrophic lateral sclerosis. Therefore, it is necessary to examine the impact of RNA-binding proteins during skeletal muscle development and plasticity in order to understand the consequences linked to their misregulation in disease. Here, we focused on the RNA-binding protein Staufen1, which assumes multiple roles in both skeletal muscle and neurons. We previously demonstrated that Staufen1 is regulated during myogenic differentiation and that its expression is increased in denervated and in myotonic dystrophy type I skeletal muscles. The increased expression of Staufen1 initially appeared beneficial for DM1 since further elevating Staufen1 levels rescued key hallmarks of the disease. However, based on the multi-functional nature of Staufen1, we hypothesized that Staufen1 acts as a disease modifier in DM1. To test this, we investigated the roles of Staufen1 in skeletal muscle biology and their implications for disease. Our data demonstrated that Staufen1 is required during the early stages of muscle development, however its expression must remain low in postnatal skeletal muscle. Interestingly, the overexpression of Staufen1 impaired myogenesis through the regulation of c-myc translation. Since the function of c-myc in oncogenesis is well described, we investigated the role of Staufen1 in cancer biology. In particular, we determined novel functions of Staufen1 in rhabdomyosarcoma tumorigenesis, thus providing the first direct evidence for Staufen1’s involvement in cancer. Moreover, based on Staufen1’s role in myogenic differentiation and in myotonic dystrophy type I, we generated muscle-specific transgenic mice to examine the impact of sustained Staufen1 expression in postnatal skeletal muscle. Staufen1 transgenic mice developed a myopathy characterized by histological and functional abnormalities via atrogene induction and the regulation of PTEN mRNAs. In parallel, we further investigated Staufen1-regulated alternative splicing and our data demonstrated that Staufen1 regulates multiple alternative splicing events in normal and myotonic dystrophy type I skeletal muscles, both beneficial and detrimental for the pathology. Collectively, these findings uncover several novel functions of Staufen1 in skeletal muscle biology and highlight Staufen1’s role as a disease modifier in DM1.

Introduction : les myopathies centronucléaires (CNM) sont des maladies rares caractérisées par une faiblesse musculaire et des fibres musculaires à noyaux centraux. Elles sont divisées en 3 formes : XLCNM (mutations dans MTM1), ADCNM (mutations dans DNM2) et ARCNM (mutations dans BIN1). Les protéines respectives sont la myotubularine (une phosphatase à phosphoinositides), la dynamine 2 (une large GTPase), et l’amphiphysine 2/BIN1 (une protéine capable de remodeler les membranes). Leur implication dans le trafic membranaire et le remodelage des membranes suggère une connexion fonctionnelle entre ces trois protéines. Résultats : la mise au point d’une nouvelle technique de diagnostic génétique par Western-Blot chez les patients XLCNM a montré une diminution quasi-totale de la concentration de MTM1 chez la plupart des patients. L’étude fonctionnelle des mutations dans BIN1 a mis en évidence qu’elles sont responsables soit d’une perte de tubulation membranaire soit d’une anomalie de la conformation 3D de la protéine pouvant être responsable d’une altération du recrutement de la dynamine 2. Une collaboration avec l’équipe du Dr N. Charlet-Berguerand, nous a permis de montrer que la perte de l’exon 11 de BIN1 est responsable d’un défaut de tubulation membranaire dans les cellules musculaires de patients dystrophiques. Enfin, des expériences d’interaction protéique ont montré que MTM1 et BIN1 sont capables d’interagir directement entre elles, via le domaine SH3 de BIN1. Conclusion : toutes ces données suggèrent que ces 3 protéines sont impliquées dans une voie commune dans le muscle squelettique et qu’elle pourrait avoir un lien avec la formation/le maintien des tubules-transverses
Introduction : Centronuclear Myopathies (CNM) are rare congenital diseases characterized by muscle weakness and muscular fibers with abnormal central nuclei. There are three different forms : the X-linked form XLCNM (mutations in MTM1), the autosomal dominant form ADCNM (mutations in DNM2) and the autosomal recessive form ARCNM (mutations in BIN1). Corresponding proteins are respectively myotubularin (a phosphoinositides phosphatases), dynamin 2 (a large GTPase), and amphiphysine 2/BIN1 ( a protein involved in membranes remodeling). A functional connection between these three proteins is suggested by their implication in membrane trafficking and membrane remodeling. Results : a new method of genetic diagnosis by western-blot in XLCNM has been developed. This method shows a near-total diminution of the MTM1 concentration. A functional study of BIN1 mutations shows that these mutations are responsible either of membrane tubulation diminutions or an abnormal 3D conformation of BIN1. This misconformation could corrupt the recruitment of DNM2. A collaboration with Dr N. Charlet-Berguerand team demonstrates that the abnormal splicing of BIN1 exon 11 is responsible of a defect of membrane tubulation function in muscular cells of dystrophic patients. Finally, experiences of protein interaction show that MTM1 and BIN1 are able to interact together directly via the SH3 domain of BIN1. Conclusion : all these data suggest that these 3 proteins are involved in a same pathway in the skeletal muscle. This pathway could be linked with the formation/maintenance of T-tubules

La dystrophie myotonique de type 1 (DM1) est une maladie multisystémique dominante qui représente la myopathie la plus fréquente chez l’adulte. Le muscle squelettique est particulièrement affecté : il s’atrophie et perd 1 à 3 % de sa force maximale par année. Les interventions cliniques sont sécuritaires en DM1 et certaines études rapportent même des augmentations de force musculaire. Toutefois, les dosages optimaux et les mécanismes physiologiques expliquant ces gains demeurent inconnus. Afin d’élucider ces questions, ce mémoire se divise en deux volets. Le premier volet (objectif 1) présente une revue systématique de type scoping review, qui résume les connaissances portant sur l’effet des interventions cliniques sur le muscle squelettique chez les personnes affectées par la DM1 et qui identifie les manques d’évidences scientifiques à ce sujet. Le second volet (objectif 2) étudie l’effet de l’exercice excentrique aigu sur les voies de signalisation de synthèse et de dégradation protéique dans le muscle squelettique. Pour ce second volet, 10 hommes atteints de DM1 ont accepté de participer à une séance unique d’exercice excentrique et de subir une biopsie musculaire avant et après l’exercice. La revue systématique rapporte que des gains de force sont possibles chez des individus atteints de DM1, cependant les résultats rapportés sont très hétérogènes. De plus, il existe de grandes lacunes au sujet de la compréhension des mécanismes physiologiques sous-jacents. Les résultats obtenus dans la réalisation du second volet de ce mémoire démontrent également une grande hétérogénéité dans les réponses observées chez les patients atteints de DM1. Par contre, ceux-ci suggèrent que, malgré le défaut génétique, les mécanismes impliqués dans l’hypertrophie semblent similaires à ceux rapportés chez le sujet sain. L’ensemble de ces connaissances aidera à guider les professionnels de la santé dans la prescription d’exercice à cette population dans le but d’améliorer la qualité de vie des personnes atteintes.

Les canaux Na dépendants du voltage sont responsables de la phase ascendante des potentiels d’action. Ils sont formés d’une sous-unité principale  et d’une ou plusieurs sous-unités secondaire . La sous-unité  seule est suffisante pour former un canal fonctionnel cependant, les sous-unités  modulent la location, l’expression ainsi que les propriétés fonctionnelles de la sous-unité . Ma thèse ce concentre sur 3 canaux Na neuronaux (Nav1.6, Nav1.7 et Nav1.8) ainsi qu’un canal sodique du muscle squelettique (Nav1.4). Les canaux Na neuronaux sont importants pour la propagation de l’influx électrique tout au long de l’axone. Nav1.7 et Nav1.8 sont les principales sous-unités exprimées dans les ganglions dorsaux. L’altération de l’expression et de la modulation de ces canaux suite à une lésion ou à l’inflammation, joue un rôle important dans la nociception et dans les douleurs chroniques. Nav1.6 est fortement concentré aux nœuds da Ranvier, il y tient un rôle important dans la conduction saltatoire et dans la répétition des potentiels d’action à hautes fréquences. Des mutations sur le canal Nav1.4 provoquent des canalopathies du muscle squelettique. Voici les questions qui ont guidé notre étude : 1) De quel façon les sous-unités  régulent les canaux Na neuronaux Nav1.7 et Nav1.8? 2) Quel anomalie biophysique est provoquée par la mutation M1476I, une mutation liée à l’effet fondateur sur le gène SCN4A qui provoque une myotonie douloureuse induite par le froid chez des Canadiens français? 3) Quels sont les propriétés biophysiques du courant persistant de Nav1.6? 4) Quel est le patron d’expression des sous-unités  et comment celles-ci régulent Nav1.7 dans les neurones de ganglions dorsaux? Afin de répondre à ces questions, plusieurs techniques ont été utilisées, notamment la technique du patch-clamp en configuration cellule entière et l’enregistrement des canaux unitaire sur des systèmes d’expression hétérologue, de la RT-PCR sur les cellules uniques, immunohistochimie et l’immunoprécipitations dans les neurones de ganglions dorsaux. Premièrement, nous avons utilisé la RT-PCR sur les cellules uniques sur des neurones dissociés de ganglions dorsaux pour identifier l’expression des sous unités 1-4 dans les neurones sensitifs de petits diamètres. Nos résultats indiques que les neurones expriment largement Nav1.6 et Nav1.8 et les sous unités 1-3. Pour étudier la régulation par les sous-unités , nous avons co-exprimés les canaux Na avec les sous-unités . La sous-unités 1 provoque une augmentation de la densité de courant de Nav1.8 lorsque co-exprimée dans des cellules HEK293 mais elle n’affecte pas la densité de courant de Nav1.6. Le domaine C-terminale de la sous-unité 1 est fortement impliqué dans la modulation de Nav1.8. Ces résultats proviennent de l’étude de l’effet de chimère 1/2 conservant différentes régions de la sous-unité 1 et de la sous-unité 2. Deuxièmement, nous avons étudié les anomalies biophysiques provoquées par la mutation M1471I de Nav1.4 en utilisant la technique du patch-clamp en mode configuration cellule entière sur des cellules tsA-201. La mutation provoque des effets similaires à d’autres mutations qui provoquent une myotonie aggravé par le potassium, incluant une augmentation du courant persistant, un ralentissement de la décroissance du courant, une dépolarisation de l’inactivation et une accélération de la récupération de l’état inactivé. Un abaissement de la température ralentit les cinétiques pour les canaux mutants et les canaux sauvages, mais il empire le défaut de l’inactivation de la mutation M1476I en augmentant l’amplitude du courant persistant. La mexiletine aide à soulager la myotonie causée par cette mutation en supprimant l’augmentation du courant persistant. Cependant, la mexiletine à une efficacité réduite sur le bloque utilisation-dpendant des canaux mutés M1476I et elle est associée à une récupération plus rapide du bloque provoqué par la mexiletine sur les canaux mutants. Troisièmement, nous avons caractérisé les propriétés du courant persistant de Nav1.6 en mode cellule entière et en courant unitaire dans des cellules HEK293 exprimant ce canal. Nous avons noté que le courant persistant de Nav1.6 est sensible à la composition du milieu intracellulaire et que l’utilisation de CsF au lieu de CsCl rendait ce courant rarement détectable. En substituant le CsF par du CsCl, nous avons montré que l’amplitude du courant persistant de Nav1.6 en mode cellule entière est de 3 à 5% du courant transitoire. Cette amplitude est similaire au ratio observé entre le maximum de probabilité d’ouverture et la probabilité d’ouverture du courant persistant observé en enregistrement de courant unitaire. L’occurrence de la réouverture des canaux explique le courant sodique persistant typique de Nav1.6. Finalement, nous avons utilisé une combinaison des techniques de RT-PCR sur les cellules uniques, immunohistochimie et d’immunoprécipitation pour étudier l’expression des sous-unités  dans différentes sous-population de neurones sensitifs. Les sous-unités  sont différentiellement exprimés dans la population de neurones de petits diamètres des ganglions dorsaux (2, 3) et dans la population de neurones de grands diamètres des ganglions dorsaux (1, 2). L’ARNm de Nav1.7 était significativement co-exprimé avec les sous-unités 2 et 3 dans la même population de neurones de petits diamètres des ganglions dorsaux. Ils forment un complexe protéine-protéine stable et sont colocalisés dans la membrane plasmatique des neurones. Lorsque les sous-unités 3 et 1 sont coexprimés avec Nav1.7, on observe un déplacement de la courbe d’activation et de la courbe d’inactivation ainsi qu’une augmentation marqués du courant de fenêtre. Nos données indiques une expression préférentielle des sous-unités  dans les neurones de petit et de grands diamètres ainsi qu’une régulation spécifique de Nav1.7 dans ces sous populations de neurones sensitifs.
Voltage-gated Na channels are responsible for the rising phase of action potentials, and consist of a pore-forming α subunit and one or more auxiliary β subunits. The α subunit alone is sufficient for the functional expression of Na channels, however, β subunits modulate the location, expression and functional properties of α subunits. My thesis will focus on three neuronal Na channels (Nav1.6, Nav1.7 and Nav1.8) and one skeletal muscle Na channel (Nav1.4). Neuronal Na channel are key players in the impulse propagation along axon. Nav1.7 and Nav1.8 are the main Na channels expressed in DRG neurons, and their altered expression and modulation following injury and inflammation play a major role in nociception and chronic pain. Nav1.6 is highly concentrated at nodes of Ranvier, and has a critical role not only in saltatory conduction but also in high-frequency repetitive firing. Skeletal muscle Na channel Nav1.4 is the initiator of muscle contraction. Mutations in Nav1.4 cause skeletal muscle channelopathies. Guiding questions for our investigations were: 1) How do auxiliary β subunits regulate peripheral nerve Na channel Nav1.6 and Nav1.8? 2) What is the underlying biophysical defect of M1476I, a novel founder SCN4A mutation associated with painful cold-induced myotonia in French Canadians? 3) What is the biophysical characterization of the Nav1.6 persistent current? 4) What is the expression pattern of auxiliary  subunits, and how do β subunits regulate Nav1.7 in DRG neurons? We addressed these questions by multiple approaches including patch clamp techniques for whole-cell and single-channel recordings in heterologous expression systems; immunohistochemistry, single-cell RT-PCR and immunoprecipitation in DRG neurons. Firstly, we employed single-cell RT-PCR of acutely dissociated DRG neurons to identify the expression of β1-4 subunits in small-diameter sensory neurons. Our results indicated that small-diameter DRG neurons widely expressed Nav1.6 and Nav1.8 channels and β1-β3 subunits. Co-expression studies were used to assess the regulation of Nav1.6 and Nav1.8 by β subunits. The β1 subunit induced a significant increase in the current density of Nav1.8 when co-expressed in HEK293 cells, but had no effect on that of Nav1.6. In addition, the C-terminal domain of β1 was involved in the modulation of Nav1.8 channel based on the results of experiments with β1/β2 chimeras harboring various regions of the strongly regulating β1 together with the weakly regulating β2 subunit. Secondly, we investigated the biophysical defects of M1476I mutation in Nav1.4 channels using whole-cell patch-clamp technique in tsA201 cells. M1476I mutant channel exhibited similar biophysical defects compared with other PAM-causing mutations, including an increased persistent current of Nav1.4, a slower current decay, a positive shift of fast inactivation, and an accelerated recovery from fast inactivation. Lowering the temperature slowed the kinetics for both wide-type and mutant channels, and worsened the defective fast inactivation of M1476I channels by further increasing the amplitude of the persistent current. Mexiletine helps relieve myotonia in M1476I carriers by effectively suppressing the increased persistent current, except for the use-dependent block. However, mexiletine had a reduced effectiveness on the use-dependent block of M1476I channels, and that was associated with a faster recovery from mexiletine block of mutant channels. Thirdly, we characterized the whole-cell and single-channel properties of Nav1.6 persistent currents expressed in HEK293 cells. We noted that Nav1.6 persistent current was highly sensitive to the composition of the internal solution, and persistent current was rarely detectable when CsF instead of CsCl was used. By substituting CsF for CsCl in the intracellular solution, we showed that Nav1.6 persistent current in the whole-cell configuration was 3–5% of the peak transient current. This amplitude of persistent current was similar to the ratio between peak and persistent open probability observed in the single-channel recording, indicating that the occurrence of late channel reopenings accounts for the persistent macroscopic Na current typical of Nav1.6. Finally, we employed a combination of single-cell RT-PCR, immunocytochemistry and immunoprecipitation to investigate  subunit expression in subpopulations of sensory neurons.  subunits were differentially expressed in small (2, 3) and large (1, 2) DRG neurons. Nav1.7 mRNA was significantly co-expressed with the 2 and 3 subunits in the same population of small-diameter DRG neurons. They formed stable protein-protein interactions and co-localized within the plasma membranes of neurons.When co-expressed in HEK293 cells, 3 and 1 subunits shifted activation and inactivation curves respectively and induced a marked increase in Nav1.7 window current. Our data indicated a preferential expression of  subunits in small and large DRG neurons and a subunit-specific Nav1.7 regulation in these subpopulations of sensory neurons.
Tableau d'honneur de la FÉSP

La dystrophie myotonique de type 1 (DM1) ou maladie de Steinert est la maladie génétique neuromusculaire la plus commune avec une incidence de 1/8000 à travers le monde. Cette maladie multisystémique touche particulièrement les muscles squelettiques (myotonie, faiblesse et perte musculaires) et le coeur qui présente des symptômes variés comme des troubles de la conduction et des arythmies. La DM1 est causée par une expansion instable de répétitions CTG dans la région 3’ non traduite du gène DMPK. Les individus sains possèdent entre 5 et 37 répétitions CTG tandis que les patients DM1 portent entre 50 et plusieurs milliers de répétitions. Il est bien établi que les expansions de répétitions non codantes forment des foci dans les noyaux musculaires où elles séquestrent le facteur d'épissage MBNL1. Toutefois, l'implication de la stabilisation et l'accumulation de CUGBP1 hyperphosphorylé par la PKC dans la maladie est un sujet controversé dans la communauté DM1. Dernièrement, en plus de la rupture de l'équilibre entre MBNL1/CUGBP1, plusieurs mécanismes ont été mis en cause dans la pathogenèse de la DM1. Parmi eux, l'expression perturbée de facteurs de transcription, la maturation altérée de miARNs, l'activation de kinases... chacune de ces altérations menant au final à une perturbation du transcriptome. Afin d'étudier l'effet de la toxicité des répétitions sur les phénotypes et lestranscriptomes, nous avons généré trois lignées de Drosophile inductibles et site-spécifiques exprimant 240, 600 et 960 répétitions de triplets. Nous avons travaillé en parallèle sur une lignée atténuée pour mbl (orthologue de MBNL1) et deux lignées gain de fonction bru -3 (orthologue de CUGBP1). Exprimées dans les muscles somatiques, les répétitions CTG conduisent à une mobilité réduite, le fractionnement des fibres musculaires, une réduction de leur taille et une altération du processus de fusion des myoblastes de manière dépendante de Mbl et Bru-3. En outre, l'expression des répétitions cause une hypercontraction musculaire dépendante de Mbl et due à un mauvais épissage de dSERCA. L'analyse transcriptionnelle comparative réalisée sur les muscles larvaires des différentes conditions pathologiques montre que l'atténuation de mbl reproduit 70-82% des dérégulations transcriptomiques des larves DM1 alors que le gain de fonction bru-3 représente 32-53% des altérations transcriptomiques des lignées DM1. Ainsi Mbl est un facteur clé des dérégulations observées dans les muscles somatiques des lignées DM1. Au contraire, les analyses physiologiques effectuées sur les coeurs adultes suggèrent que Bru-3 est un facteur clé dans la mise en place des phénotypes cardiaques. En effet, d'une part, l'atténuation de mbl dans le coeur cause une cardiomyopathie dilatée, un symptôme rarement diagnostiqué chez les patients. D'autre part, les lignées gain de fonction bru-3 et DM1 présentent de la fibrillation qui évolue avec l'âge ou la taille des répétitions vers un phénotype qui rappelle l'insuffisance cardiaque chez les patients
Myotonic Dystrophy Type 1 (DM1) or Steinert's disease is the most common genetic neuromuscular disorder affecting 1 out of 8000 people worldwide. This multisystemic disease affects particularly the skeletal muscles (myotonia, muscle weakness and wasting) and the heart, which can exhibit various symptoms like conduction disturbances and arrhythmia (auricular fibrillation and flutter). DM1 is caused by an unstable CTG repeat expansion in the 3' non-translated region of the DMPK gene. In healthy individuals, the number of CTG repeats ranges from 5 to 37 whereas DM1 patients carry from 50 to thousands repeats. It is well established that when expanded non-coding repeats aggregate into foci within muscle nuclei and sequester the MBNL1 splicing factor. However, the involvement of the stabilization and accumulation of CUGBP1 following PKC hyper-phosphorylation in the disease is a controversial matter in the DM1 community. Lately, in addition to the disruption of the balance between MBNL1/CUGBP1, several mechanisms were identified as part of the DM1 pathogenesis. Among them, transcription factors perturbations, altered maturation of miRNA, kinases activation… each of them leading eventually to transcriptomic alterations. In order to investigate the effect of toxic repeat expression on phenotypic and transcriptomic alterations, we generated three inducible site-specific Drosophila lines expressing 240, 600 and 960 triplet repeats. We worked in parallel on a mbl (MBNL1 orthologue) knocked-down line and two bru-3 (CUGBP1 orthologue) gain of function lines. When expressed in somatic muscles, CTG repeats lead to altered motility, fiber splitting, reduced fiber size and affected myoblast fusion process in a Mbl and Bru-3 dependent manner. In addition, toxic repeats cause fiber hyper-contraction in a Mbldependentmanner due to dSERCA mis-splicing. Comparative transcriptional profiling performed on larval muscles of different conditions show that mbl attenuation reproduces 70-82% of DM1 transcriptomic deregulations whereas bru-3 gain of function represents 32-53% of transcritomic alterations. Thus Mbl appears as a key factor of transcripts deregulations observed in DM1 muscles. On the contrary, physiologic analyses performed on adult hearts suggest that Bru-3 is a key factor for cardiac phenotypes. Indeed, on one hand, mbl attenuated flies display dilated cardiomyopathy, a symptom barely diagnosed in patients. On the other hand, bru-3 gain of function line and DM1 lines display fibrillation, which evolves withage or repeat size into a phenotype reminiscent of heart insufficiency in patients

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Made available in DSpace on 2016-05-17T20:16:41Z (GMT). No. of bitstreams: 1 Adriano Silvio dos Santos.pdf: 1418434 bytes, checksum: 4ac5ef78eb225e6693c107993f6ee978 (MD5) Previous issue date: 2015-02-24
Snakes venom of the Bothrops species induces a local inflammatory reaction, characterized by pain, edema, leukocyte migration and can be accompanied by tissue necrosis. The use of antivenom performs the function of neutralizing the greatest possible amount of circulating venom, thus minimizing its systemic effects, but its action does not extend to local manifestations, and thus require the use of another therapeutic option to control this reaction. The low level laser therapy (LLLT) is used as an alternative treatment in cases of muscle injury due to its biological effects, such as analgesics, anitinflamatory and healing. In a previous study of our lab it was found that LBP can enhance the viability of C2C12 muscle cells after the addition of B. jararacussu venom in the medium and that this effect of LBP is related to protection of the cell membrane. In the present study we analyzed the effect of LBP in the cell monolayer integrity, viability of muscle cells, exposed to injury by myotoxins BthTX - I - and BthTX - II isolated from Bothrops jararacussu venom. Cells received BthTX – I (75 μg / mL) and were immediately irradiated with LLLT Aluminum Indium Gallium Phosphate and Aluminium Gallium Arsenide, the wavelengths (λ) 685nm and 830 nm, power density 4 J/cm2, 100mW of power, total energy 1,3 J, application time of 13 and 35 seconds per point and the cells were incubated for 15, 30 and 60 minutes. The results demonstrated that BthTX – I affect cell viability in a dose dependent manner, but did not change cell integrity. The concentration of 75 μg/mL was chosen for the experiments with LBP. LLLT caused an significant increase in cell viability in all the analyzed period of time and in the λ 685 nm and 830 nm against Bothrops I toxin, however in the LBP λ 685 nm against Bothrops toxin II was effective only at 15 min, while the LBP at λ 830 was effective at 15 and 60 min. The LLLT was not able to change the LDH release at all times and wavelength used. Thus, LBP was able to protect C2C12 muscle cells against the miotoxic effect of isolated myotoxins isolated from B. jararacussu venom. Therefore, the results suggest that LLLT can be considered an effective therapeutic tool in patients bitten by snakes.
O veneno das serpentes do gênero Bothrops induz uma reação inflamatória local intensa, caracterizada por dor, formação de edema, migração leucocitária, podendo ser acompanhada por necrose tecidual. A utilização do soro antibotrópico desempenha a função de neutralizar a maior quantidade possível do veneno circulante, minimizando assim seus efeitos sistêmicos, porém sua ação não se estende às manifestações locais, sendo assim necessário o uso de outro recurso terapêutico para o controle dessa manifestação. A laserterapia de baixa potência (LBP) é uma alternativa de tratamento em situações de lesão muscular, devido a seus efeitos biológicos, tais como analgésicos, antinflamatórios e cicatrizantes. Em trabalhos anteriores realizados em nosso laboratório, verificou-se que o LBP foi capaz de aumentar a viabilidade de células musculares C2C12, após a adição do veneno de B. jararacussu e que esse efeito do LBP é relacionado a uma proteção da membrana celular. Assim, o objetivo deste trabalho foi analisar o efeito do LBP em células musculares C2C12 submetidas à lesão por miotoxinas (BthTX - I e BthTX - II) isoladas do veneno da serpente Bothrops jararacussu quanto a: viabilidade, descolamento celular e liberação da enzima LDH. As células receberam a BthTX – I e BthTX – II na dose 75 μg/mL e foram imediatamente irradiadas com LBP Índio Gálio Alumínio Fósforo e Arseneto de Gálio Alumínio, nos comprimentos de onda (λ) 685 nm vermelho e 830 nm infra-vermelho, de forma pontual, tempo de aplicação de 13 s e 35 s respectivamente e as células foram incubadas por 15, 30 e 60 minutos. Os resultados demonstraram que a BthTX - I e BthTX - II afetou a viabilidade celular de forma dose-dependente, sendo escolhida a dose 75 μg/mL para a realização dos experimentos com o LBP, porém não foi capaz de causar alterações na integridade. O LBP causou aumento significativo na viabilidade celular, em todos os tempos analisados no λ 685 nm e 830 nm frente à BthTX - I, entretanto o LBP no λ 685 nm e λ 830 frente a BthTX - II foi efetivo somente no tempo de 15 e 60. O LBP não foi capaz de diminuir a liberação de LDH em todos os tempos analisados e com os dois λ utilizados. Desta forma, verificou-se que o LBP foi capaz de proteger as células musculares C2C12 contra o efeito miotóxico das miotoxinas isoladas do veneno B. jararacussu e que esta proteção está relacionada ao efeito protetor a nível mitocondrial. Ainda, os resultados obtidos sugerem que o LBP pode ser considerado uma ferramenta terapêutica eficaz em pacientes picados por serpentes.

Parmi les canalopathies du muscle squelettique, dues à des mutations du canal Nav1. 4, on distingue 2 grands phénotypes cliniques : les Myotonies et les Paralysies Périodiques. Ce travail de thèse porte sur la physiopathologie moléculaire et le lien avec le phénotype des patients de ces canalopathies. Plus particulièrement, il consista à explorer les conséquences biophysiques de 2 mutations : la R1132Q (Paralysie Périodique Hypokaliémique ou HypoPP), et la Q270K (Paramyotonie Congénitale ou PC). Nos résultats montrent une augmentation de l’inactivation rapide et lente des canaux induite par la mutation R1132Q. Ces données reflètent l’importance de cette caractéristique commune à tous les mutants responsables d’HypoPP, mais ne permet pas d’établir le lien avec le phénotype. La mutation Q270K, localisée à proximité de la L266V, se différencie cliniquement par la présence de faiblesses musculaires, non présents pour l’autre mutant, en plus de la myotonie. Dans les 2 cas, l’inactivation rapide est modifiée de façon identique. En revanche, la mutation Q270K entraine également une diminution de l’inactivation lente, non retrouvée pour la L266V. On a testé l’action du froid sur 3 mutants responsables de PC (Q270K, S804F et T1313A). Le froid augmente l’inactivation lente des canaux sauvages et de façon plus ou moins importante celle des mutants. L’altération induite par les mutants est dynamique et fonction de la température, démontrant une relation directe entre la diminution de l’inactivation lente et les paralysies. Pour conclure, ce travail confirme l’hypothèse établie sur l’incrimination de l’inactivation rapide dans la myotonie et de l’inactivation lente dans les paralysies.

La dystrophie myotonique de type 1 (DM1), est une maladie génétique héréditaire affectant environ 1/8000 personnes. Les patients souffrant de DM1 développent essentiellement des troubles musculaires tels qu’une faiblesse et une atrophie musculaire. La cause de la DM1 est expliquée par la mutation du gène "DMPK". Lors de ma thèse, j’ai pu démontrer que l’épissage de l’ARNm BIN1 était altéré dans le muscle DM1. En effet, l’exon 7 de BIN1, qui est absent du muscle normal, est exprimé de façon aberrante chez les patients DM1. En utilisant un modèle murin, j’ai prouvé que l’expression forcée de l’exon 7 de BIN1 altérait simultanément la structure et la fonction du muscle. Nous avons notamment observés une diminution de la taille des fibres musculaires et une augmentation de la faiblesse musculaire, comparé à des souris normales. Par conséquent, ce travail aidera à la compréhension du mécanisme de la maladie et pourrait expliquer les causes de la faiblesse musculaire et de l’atrophie
Myotonic dystrophy of type 1 (DM1), is an inherited genetic disease affecting around 1 in 8000 person. Patients suffering from DM1 develop essentially muscle disorders such as muscle weakness, muscle loss and atrophy. The cause of DM1 is explained by the mutation of a gene called “DMPK“.During my thesis, I discovered that the alternative splicing of BIN1 mRNA was altered in the muscle of DM1 patients. Indeed, the BIN1 exon 7, which is normally absent in healthy muscle, is aberrantly expressed in DM1 muscle. By using a mouse model, I found that the forced expression of BIN1 exon 7 was responsible of the alteration of both muscle structure and function. Notably, we found a decrease in muscle fibers area (atrophy) and an increase of muscle weakness, compared to wild-type mice. Therefore, this work will help in the understanding of the disease mechanism and could explain the causes of muscle weakness and atrophy, which have never been elucidated to this date

Les ARN hélicases DDX5 et DDX17 sont des protéines " multi-tâches ", elles sont impliquées dans de nombreuses étapes de la régulation du métabolisme des ARNs dont la transcription, l'épissage et la dégradation des ARNs. Lors de processus biologiques complexes tels que la myogénèse, les programmes d'expression génique sont profondément modifiés. Durant mon travail de thèse, j'ai contribué à montrer que DDX5 et DDX17 sont des protéines orchestratrices de la différenciation en coordonnant de manière directe et dynamique plusieurs niveaux de régulation génique. DDX5 et DDX17 contrôlent l'activité du facteur de transcription MyoD, régulateur majeur de la myogénèse ainsi que des microARNs spécifiques du muscle miR-1 et miR-206. Ceux-ci ciblent et régulent en retour l'expression de DDX5 et DDX17 mettant en place une boucle de rétro-contrôle négative induisant la diminution d'expression de ces deux protéines au cours de la différenciation. Enfin, cette diminution d'expression permet la mise en place d'un programme d'épissage participant à l'acquisition de phénotypes morphologiques des cellules différenciées. D'un point de vue mécanistique, il apparaît qu'un sous-groupe des événements d'épissage régulés durant la différenciation est contrôlé par la coopération de DDX5 et DDX17 avec le facteur d'épissage hnRNP H/F. D'autre part, DDX5 a aussi été impliqué dans un contexte pathologique du muscle. Cette hélicase interagit avec la mutation responsable de la Dystrophie Myotonique de type 1 (DM1). Durant ma thèse, j'ai produit des résultats préliminaires suggérant un rôle de DDX5 dans la mise en place des défauts d'épissage observés dans cette pathologie

Résumé: But : Les objectifs étaient de 1) décrire les profils de force musculaires aux membres inférieurs (MIs) et les capacités aux déplacements des personnes présentant les phénotypes adulte classique (DM1-AC) et adulte tardif (DM1-AT) de la dystrophie myotonique de type 1 (DM1), et 2) d’explorer l’influence de la faiblesse des MIs sur les capacités aux déplacements dans cette population. Méthode : Cette étude consiste en une analyse secondaire de données issues d’une plus large recherche qui visait à identifier les déterminants de la participation sociale et de la qualité de vie de personnes atteintes de DM1 (n = 158 DM1-AC et n = 42 DM1-AT). La force de quatre groupes musculaires des MIs a été mesurée à l’aide du bilan musculaire manuel (BMM) et du bilan musculaire quantitatif (BMQ) par dynamométrie manuelle. Les capacités aux déplacements ont été évaluées à l’aide de tests standardisés (échelle d’équilibre de Berg, vitesse de marche et Timed Up & Go). Résultats : Le phénotype DM1-AT présente moins de faiblesse et d’incapacités que le phénotype DM1-AC (p < 0,001 – 0,020). Le BMM ne détecte pas de faiblesse chez le phénotype DM1-AT mais des pertes de force au BMQ de 12 % à 20 % ont été identifiées chez ce phénotype, excepté pour les fléchisseurs du genou, entrainant des limitations aux déplacements chez 22 % à 48 % de ces individus. Dans le phénotype DM1-AC, l’atteinte musculaire était légèrement plus importante en distal qu’en proximal. Selon ces résultats, les phénotypes DM1-AC et DM1-AT présentent des portraits distincts et les données relatives à chacun devraient être analysées séparément. Une progression générale de la faiblesse au BMQ et des scores aux tests fonctionnels a été observée en fonction des cotes de l’échelle Muscular Impairment Rating Scale (MIRS). Un déficit de force au BMQ (excepté pour les fléchisseurs du genou) et des incapacités fonctionnelles ont aussi été observés dès les premières cotes de la MIRS. Finalement, les dorsifléchisseurs de la cheville et les extenseurs du genou semblent être de bons indicateurs de la fonction des membres inférieurs en DM1. Conclusion : Cette étude a permis de dresser un portrait des atteintes de la force musculaire aux MIs et des capacités fonctionnelles liées aux déplacements pour chacun des phénotypes DM1-AC et DM1-AT de la DM1, ainsi que d’explorer la contribution de la faiblesse des groupes musculaires évaluées sur les capacités aux déplacements dans cette population. Ces résultats contribueront à mieux déterminer les cibles d’évaluation et d’interventions en réadaptation et à mieux définir le processus d’évaluation dans le contexte des essais thérapeutiques à venir.
Abstract: Purpose: The purposes of this study were 1) to describe lower limbs muscle strength and mobility capacities, and 2) to explore the respective contribution of lower limb muscle weaknesses on mobility in the adult and late-onset phenotypes of myotonic dystrophy type 1 (DM1). Methods: This study is a secondary analysis of part of the results of a larger study, whose purpose was to identify social participation and quality-of-life determinants in 200 DM1 patients (158 adult and 42 late-onset). The strength of four lower limb muscle groups was assessed using manual muscle testing (MMT) and handheld dynamometry quantitative muscle testing (QMT). Mobility capacities were assessed using standardized tests (Berg balance scale, 10 Meter Walk Test and Timed Up & Go). Results: Although the late-onset phenotype showed less weaknesses and mobility limitations than the adult phenotype (p <0.001-0.020), and although MMT showed no weakness in the late-onset phenotype, quantitative strength losses of 12-20% were measured in this phenotype, with the exception of the knee flexors. These weaknesses led to mobility limitations in 22-48% of participants with the late-onset phenotype. In the adult phenotype, muscle strength impairment was slightly more important distally than proximally (2-2.5/10 and 5.8-8.2% for MMT and QMT, respectively) (p <0.001-0.002). According to those results, the adult and late-onset phenotypes show different profiles of lower limb impairment, and should not be pooled for data analysis. A general progression of quantitative muscle weakness and of mobility scores was observed according to the Muscular Impairment Rating Scale (MIRS) classification. Quantitative weaknesses, with the exception of the knee flexors, and mobility limitations were observed from the first MIRS grades. QMT is therefore definitely a more effective tool for measuring weakness in DM1. Finally, ankle dorsiflexors and knee extensors seem to be good indicators of lower limb function in DM1. Conclusion: This study allowed a better characterization of lower limb weaknesses and mobility limitations in the adult and late-onset phenotypes of DM1, and explored the contribution of lower limb weaknesses on mobility capacities in this population. These results will be useful for developing more specific rehabilitation programs and for optimizing the evaluation of these impairments in the context of the upcoming therapeutic trials. Keywords: Myotonic dystrophy type 1, phenotypes, muscle strength, mobility capacities, lower limbs, explanatory variables, physiotherapy.

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Background: The myotonic dystrophy (MD) is a multisystem neuromuscular disease that can affect the respiratory muscles and heart function, and cause impairment in quality of life. Objectives: Investigate the changes in respiratory muscle strength, health-related quality of life (HRQoL) and autonomic modulation heart rate (HR) in patients with MD. Methods: Twenty-three patients performed assessment of pulmonary function, sniff nasal inspiratory pressure (SNIP), the maximal inspiratory (MIP) and expiratory (MEP) pressure, and of HRQoL (SF-36 questionnaire). Of these patients, 17 underwent assessment of heart rate variability (HRV) at rest, in the supine and seated positions. Results: The values of respiratory muscle strength were 64, 70 and 80% of predicted for MEP, MIP, and SNIP, respectively. Significant differences were found in the SF-36 domains of physical functioning (58.7 ? 31,4 vs. 84.5 ? 23, p<0.01) and physical problems (43.4 ? 35.2 vs. 81.2 ? 34, p<0.001) when patients were compared with the reference values. Single linear regression analysis demonstrated that MIP explains 29% of the variance in physical functioning, 18% of physical problems and 20% of vitality. The HRV showed that from supine position to seated, HF decreased (0.43 x 0.30), and LF (0.57 x 0.70) and the LF/HF ratio (1.28 x 2.22) increased (p< 0.05). Compared to healthy persons, LF was lower in both male patients (2.68 x 2.99) and women (2.31 x 2.79) (p< 0.05). LF / HF ratio and LF were higher in men (5.52 x 1.5 and 0.8 x 0.6, p <0.05) and AF in women (0.43 x 0.21) (p< 0.05). There was positive correlation between the time of diagnosis and LF / HF ratio (r = 0.7, p <0.01). Conclusions: The expiratory muscle strength was reduced. The HRQoL was more impaired on the physical aspects and partly influenced by changes in inspiratory muscle strength. The HRV showed that may be sympathetic dysfunction in autonomic modulation of HR, although with normal adjustment of autonomic modulation during the change of posture. The parasympathetic modulation is higher in female patients and sympathetic tends to increase in patients with longer diagnosis
Introdu??o: A distrofia miot?nica (DM) ? uma doen?a neuromuscular multissist?mica que pode afetar a musculatura respirat?ria e a fun??o card?aca, e ocasionar preju?zos na qualidade de vida. Objetivos: Investigar as altera??es na for?a muscular respirat?ria, qualidade de vida relacionada ? sa?de (QVRS), e modula??o auton?mica da freq??ncia card?aca (FC) em pacientes com DM. M?todos: Foram avaliados 23 pacientes quanto ? fun??o pulmonar, press?o inspirat?ria nasal sniff (SNIP), press?es respirat?rias m?ximas (PIm?x e PEm?x), e QVRS (question?rio SF-36). Destes, 17 realizaram avalia??o da variabilidade da frequ?ncia card?aca (VFC) em repouso, nas posturas supina e sentada. Resultados: Os valores da for?a muscular respirat?ria foram de 64, 70 e 80%predito para PEm?x, PIm?x, e SNIP, respectivamente. Foi encontrada diminui??o significativa nos dom?nios do SF-36 capacidade funcional (58.7 ? 31,4 vs. 84.5 ? 23, p<0.01) e disfun??o f?sica (43.4 ? 35.2 vs. 81.2 ? 34, p<0.001) comparado a valores de refer?ncia. A an?lise de regress?o linear mostrou que a PIm?x explica 29% da vari?ncia na capacidade funcional, 18% na disfun??o f?sica e 20% na vitalidade. A VFC mostrou que, da postura supina para a sentada, o espectro AF diminuiu (0.43 x 0.30) e o espectro BF (0.57 x 0.70) e a raz?o BF/AF (1.28 x 2.22) aumentaram, com p<0.05. Comparado a valores de refer?ncia, BF foi inferior (p<0.05) tanto nos pacientes homens (2.68 x 2.99), como nas mulheres (2.31 x 2.79). A raz?o BF/AF e o espectro BF foram maiores nos homens (5.52 x 1.5 e 0.8 x 0.6), e o espectro AF, nas mulheres (0.43 x 0.21), com p<0.05. Houve correla??o significativa positiva entre tempo de diagn?stico e raz?o BF/AF (r= 0.7, p< 0.01). Conclus?es: Indiv?duos com DM t?m for?a muscular expirat?ria diminu?da. A QVRS mostrou-se mais prejudicada em rela??o a aspectos f?sicos e parcialmente influenciada por varia??es na for?a muscular inspirat?ria. Pode haver disfun??o simp?tica na modula??o auton?mica da FC, com ajuste normal da postura supina para a sentada. A modula??o parassimp?tica ? superior em pacientes mulheres e a modula??o simp?tica tende a aumentar nos pacientes com maior tempo de diagn?stico

碩士
中山醫學大學
生物醫學科學學系碩士班
100
The purpose of this study was to investigate the effect of KCNQ (potassium channel, voltage-gated, KQT-like subfamily) openers in preventing myotonia caused by anthracene-9-carboxylic acid (9-AC, a chloride channel blocker). Myotonia is a neuromuscular disorder and characterized by the membrane hyperexcitability and slow relaxation of muscles after a contraction. An animal model of myotonia can be elicited in murine skeletal muscle by 9-AC treatment. Retigabine, flupirtine and lidocaine can inhibit the increased twitch amplitude (0.1 Hz stimulation) and reduce the tetanic fade (20 Hz stimulations) observed in the presence of 9-AC. Furthermore, the prolonged twitch duration of skeletal muscle was also inhibited by retigabine, flupirtine or lidocaine. Lamotrigine (an anticonvulsant drug) has a lesser effect on the muscle twitch amplitude, tetanic fade and prolonged twitch duration as compared with other three medicines. In experiments using intracellular recordings, retigabine and flupirtine clearly reduced the firing frequencies of repetitive action potentials induced by 9-AC. Furthermore, we found that the rising slope of action potentials could not be reduced by the addition of retigabine and flupirtine. These data suggested that KCNQ openers prevent the myotonia induced by 9-AC, at least partly through enhancing potassium conductance in skeletal muscle. Taken together, these results indicate that KCNQ openers are potential alternative therapeutic agents for the treatment of myotonia.

ClC-1 is the major skeletal muscle chloride channel and is essential for re-establishing the resting membrane potential of muscle cells after an action potential has occurred. Many mutations throughout the CLCN1 gene, which codes for the CIC-1 protein, have been demonstrated via characterisation in heterologous expression systems, to be causative mutations for either Dominant Myotonia Congenita or Recessive Generalised Myotonia. Recently, increasing numbers of myotonic mutations have been found in the carboxyl tail of CIC-1, which demonstrates its importance as a domain that is essential for the normal function of CIC-1 channels. Previous studies in our laboratory defined a region of 18 amino acids in the immediate post D13 segment of rat CIC-1, essential for the expression of functional channels.
thesis (PhDBiomedicalScience)--University of South Australia, 2002.

Clinical studies reported side effects of muscular spasms and muscle stiffness following the administration of clofibrate, a drug once used to treat hyperlipidaemia in patients. Experiments with clofibrate and its analogues in animal models showed it produced these myotonic symptoms in muscle by reducing the chloride conductance of the muscle membrane. The effects of 2-(4-chlorophenoxy)propionic acid, an analogue of clofibric acid, was assessed on the rat ClC-1 channel (rClC-1). Racemic 2-(4-chlorophenoxy)propionic acid shifted the voltage dependence of rClC-1 activation to more depolarising potentials, a mechanism accounting for myotonic symptoms previously reported. Experiments with resolved enantiomers revealed that the effects recorded were due exclusively to S-(–) 2-(4- chlorophenoxy)propionic acid. The R-(+) enantiomer was ineffective at the concentrations tested. Further experiments with the compound at differing Cl- concentrations in the extracellular solution suggested that S-(–) 2-(4-chlorophenoxy)propionic acid altered the gating of ClC-1 by decreasing the affinity of the binding site where Cl- normally acts to ‘gate’ the channel. Similarities in the effects reported for most dominant mutations in the CLCN1 gene that lead to myotonia congenita and 2-(4-chlorophenoxy)propionic acid prompted experiments that introduced these point mutations in the human ClC-1 (hClC-1) gene to compare their mode of action to that of the drug. These mutations, F307S and A313T, predominantly altered the slow, or common, gate of the channel. Conversely, the effect of 2-(4-chlorophenoxy)propionic acid was predominantly on the fast gating process of hClC-1. A macroscopically similar effect therefore, can be produced by two different modes of action. Results suggested that both drug and mutations exert their action by affecting the transition of the channel from its closed to open state subsequent to Cl- binding. Investigation of the interaction between rClC-1 gating and a further 25 compounds structurally related to clofibric acid identified a number of compounds effective at shifting the open probability of fast gating to depolarising potentials. Fewer were identified that influence slow gating. Some compounds affected both gating processes, however, none were identified which influenced slow gating alone. Ability to displace the voltage dependent activation of the fast gate appeared to depend largely on the lipophilicity of the molecules tested, indicating the importance of hydrophobic interactions between drug and channel protein.
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Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Clinical studies reported side effects of muscular spasms and muscle stiffness following the administration of clofibrate, a drug once used to treat hyperlipidaemia in patients. Experiments with clofibrate and its analogues in animal models showed it produced these myotonic symptoms in muscle by reducing the chloride conductance of the muscle membrane. The effects of 2-(4-chlorophenoxy)propionic acid, an analogue of clofibric acid, was assessed on the rat ClC-1 channel (rClC-1). Racemic 2-(4-chlorophenoxy)propionic acid shifted the voltage dependence of rClC-1 activation to more depolarising potentials, a mechanism accounting for myotonic symptoms previously reported. Experiments with resolved enantiomers revealed that the effects recorded were due exclusively to S-(–) 2-(4- chlorophenoxy)propionic acid. The R-(+) enantiomer was ineffective at the concentrations tested. Further experiments with the compound at differing Cl- concentrations in the extracellular solution suggested that S-(–) 2-(4-chlorophenoxy)propionic acid altered the gating of ClC-1 by decreasing the affinity of the binding site where Cl- normally acts to ‘gate’ the channel. Similarities in the effects reported for most dominant mutations in the CLCN1 gene that lead to myotonia congenita and 2-(4-chlorophenoxy)propionic acid prompted experiments that introduced these point mutations in the human ClC-1 (hClC-1) gene to compare their mode of action to that of the drug. These mutations, F307S and A313T, predominantly altered the slow, or common, gate of the channel. Conversely, the effect of 2-(4-chlorophenoxy)propionic acid was predominantly on the fast gating process of hClC-1. A macroscopically similar effect therefore, can be produced by two different modes of action. Results suggested that both drug and mutations exert their action by affecting the transition of the channel from its closed to open state subsequent to Cl- binding. Investigation of the interaction between rClC-1 gating and a further 25 compounds structurally related to clofibric acid identified a number of compounds effective at shifting the open probability of fast gating to depolarising potentials. Fewer were identified that influence slow gating. Some compounds affected both gating processes, however, none were identified which influenced slow gating alone. Ability to displace the voltage dependent activation of the fast gate appeared to depend largely on the lipophilicity of the molecules tested, indicating the importance of hydrophobic interactions between drug and channel protein.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

In the skeletal muscle cell membrane, the voltage gated chloride channel, CIC-1, maintains as unusually high resting membrane conductance and thereby prevents myotonic skeletal muscle disease. Protein crystallization experiments with bacterial CIC proteins, provide the information for the three dimensional (3D) structure of CIC chloride channels.
PhD Doctorate

Myotonic dystrophy type 1 (DM1) is an autosomal dominant hereditary disease caused by an alteration leading to an abnormal expansion of unstable repetitions of CTG in the 3’ untranslated region of Myotonic Dystrophy Protein Kinase (DMPK) gene. DM1 is characterized by myotonia, progressive distal muscle weakness and by multisystemic involvement namely cataracts, muscle pain, cardiac and respiratory dysfunctions, endocrine dysfunctions (insulin resistance, metabolic syndrome, dyslipidemia), cancer and alterations in the central nervous system (CNS). Patients with DM1 have a frequency of metabolic syndrome higher than in the general population. Thus, the study of the metabolome is of a great importance since it can give new insight regarding the molecular pathways affected in DM1 diseases as well as to discriminate between the different degrees of severities in patients with DM1 and may also, lead to the development of new metabolic therapeutics. Given the previously reported metabolic alterations observed in patients DM1, we considered that the evaluation of the metabolic profile of those patients of paramount importance. Therefore, we started with the literature review for summarizing the metabolic alterations previously reported in patients with DM1 and the relationship of Lipin with the metabolic alterations in DM1 (Chapter I). Essentially, the previous studies showed a clear metabolic alteration between patients with DM1 and control groups, namely, increased total cholestrol, Low-density lipoprotein, triacylglycerol, insulin and HOMA-Insulin resistance levels, increased glucose levels and low levels of high-density lipoprotein. This review also showed a potential relationship between Lipin and its association with metabolic abnormalities found in patients with DM1, namely, the metabolic roles in adipose tissue, skeletal-muscle, liver and its association with dyslipidemia and insulin resistance, which is a characteristic feature in patients with DM1. The metabolic profile of patients with DM1 then was evaluated using the ATR-FTIR spectroscopy technique, together with multivariate analysis, which is suitable for providing a (bio)chemical profile of patients with DM1 and controls. Essentially, DM1-derived fibroblast and controls were used, and the results showed a clear discrimination within DM1-derived fibroblast with different CTG repeat length and age at onset, meaning that they may have a distinct metabolic profile. This discrimination can be attributed mainly to the altered lipid metabolism in 1800-1500 region cm-1 . It was also possible to discriminate between the control groups and both DM1-derived fibroblast from Coriell institute and Centro Hospitalar do Tâmega e Sousa in 3000-2800 cm-1 region (Chapter II). Additionally, a systematic review was made to gather information of all outcome and measurements used to assess muscle strength in adult patients with DM1 (Chapter IV). The cardiac, skeletal and respiratory muscle strength was evaluated. Briefly, the systematic review showed a consistent use of echocardiography, quantitative muscle test, manual muscle test and manometry to assess cardiac, skeletal and respiratory muscle strength. The measures of choice to assess muscle strength were: (1) ejection fraction in cardiac muscle; (2) muscle isometric torque, grip strength and medical research council (0-5 points and 0-60 points) in skeletal-muscle; (3) maximal inspiratory pressure and maximal expiratory pressure in respiratory muscles. In conclusion, our results suggest that there is a need to further research the lipid metabolism of patients with DM1, not only to better characterize these patients but also to understander the underlying mechanism of lipid abnormalities and to have new insights of Lipin in DM1. FTIR spectroscopy is a valuable tool to characterize patients with DM1 severities, which is crucial for a proper diagnosis and further studies. We successfully gather the more consensual and important measures to evaluate muscle strength. The results obtained were important and useful given that they will be valuable for muscle strength evaluation in future clinical trials and observational studies, particularly to test if a drug is improving muscle strength in patients with DM1.
A distrofia miotônica tipo 1 (DM1) é uma doença hereditária autossómica dominante causada por uma alteração que leva a uma expansão anormal de repetições instáveis de CTG na região 3' não traduzida do gene da proteína quinase da distrofia miotônica (DMPK). DM1 é caracterizado por miotonia, fraqueza muscular distal progressiva e por envolvimento multissistémica, nomeadamente cataratas, dores musculares, disfunções cardíacas e respiratórias, disfunções endócrinas (resistência à insulina, síndrome metabólica, dislipidemia), cancro e alterações no sistema nervoso central (SNC). Doentes com DM1 apresentam frequência de síndrome metabólica maior do que na população geral. Assim, o estudo do metaboloma é de grande importância, pois pode fornecer novos ideias sobre as vias moleculares afetadas nas doenças DM1, bem como discriminar entre os diferentes graus de gravidade em doentes com DM1 e também pode levar ao desenvolvimento de novas terapêuticas metabólicas. Dadas as alterações metabólicas previamente descritas e observadas em doentes com DM1, consideramos que a avaliação do perfil metabólico destes doentes é de grande importância. Portanto, elaborou-se uma revisão da literatura para resumir as alterações metabólicas previamente descritas em doentes com DM1 e a relação da Lipina com as alterações metabólicas na DM1 (Capítulo I). Essencialmente, os estudos anteriores mostraram uma clara alteração metabólica entre os doentes com DM1 e os grupos controlo, nomeadamente o aumento dos níveis de colesterol total, lipoproteína de baixa densidade, triacilglicerol, insulina e resistência HOMA-insulina, o aumento dos níveis de glicose, assim como a diminuição dos níveis de lipoproteína de alta densidade. Esta revisão também demonstrou uma potencial relação entre a Lipina e a sua associação com as anormalidades metabólicas encontradas em doentes com DM1, nomeadamente os papéis metabólicos no tecido adiposo, músculo esquelético, fígado e a sua associação com a dislipidemia e a resistência à insulina, que é uma das características em doentes com DM1. O perfil metabólico dos doentes com DM1 foi então avaliado pela técnica de espectroscopia ATR FTIR, em conjunto com a análise multivariada, sendo que é adequada para fornecer um perfil (bio) químico dos doentes com DM1 e controlos. Essencialmente, fibroblastos derivados de DM1 e controlos foram utilizados, e os resultados demonstraram uma clara discriminação dentro de fibroblastos derivados de DM1 com diferentes repetições de CTG e idades de início da doença, o que significa que estes podem ter um perfil metabólico distinto. Esta discriminação pode ser atribuída principalmente ao metabolismo lipídico alterado na região 1800-1500 cm-1 . Também foi possível discriminar entre os grupos controlo e fibroblastos derivados de DM1 do Instituto Coriell e Centro Hospitalar do Tâmega e Sousa na região de 3000-2800 cm-1 (Capítulo II). Além disso, foi feita uma revisão sistemática para reunir informações de todos os resultados e medidas utilizadas para avaliar a força muscular em doentes adultos com DM1 (Capítulo IV). Foi avaliada a força muscular cardíaca, esquelética e respiratória. Resumidamente, a revisão sistemática demonstrou uma utilização consistente da ecocardiografia, teste muscular quantitativo, teste muscular manual e manometria para avaliar a força muscular cardíaca, esquelética e respiratória. As medidas escolhidas para avaliar a força muscular foram: (1) fração de ejeção para a força do musculo cardíaco; (2) torque isométrico muscular, força de preensão e conselho de pesquisa médica (0-5 pontos e 0-60 pontos) para a força do músculo esquelético; (3) pressão inspiratória máxima e pressão expiratória máxima para a força dos músculos respiratórios. Em conclusão, os resultados sugerem que há uma necessidade de estudos adicionais relativamente ao metabolismo lipídico em doentes com DM1, não apenas para caracterizar melhor estes doentes, como também para compreender o mecanismo subjacente das anormalidades lipídicas e ter novas noções sobre a Lipina na DM1. A espectroscopia FTIR é uma ferramenta valiosa para caracterizar doentes com diferentes severidades da DM1, o que é crucial para um diagnóstico adequado e para estudos futuros. Reunimos com sucesso as medidas mais consensuais e importantes para avaliar a força muscular. Os resultados obtidos foram importantes e úteis, pois serão valiosos para avaliação da força muscular em futuros ensaios clínicos e estudos observacionais, principalm
Mestrado em Biologia Molecular e Celular