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Dystrophin is the product of the Duchenne Muscular Dystrophy gene locus, whose absence results in progressive skeletal muscle breakdown. Despite considerable work on the localisation of dystrophin and its associated complex, its role in muscle function remains unclear. In the light of the structural and mechanical instability of the dystrophic membrane, the idea was tested that dystrophin might impart membrane integrity and strength by anchoring membrane proteins and/or delineating the surface into specialised subcellular functional domains. Specifically, because dystrophin shows high sequence, structural and spatial similarities to the cytoskeletal protein spectrin; and because spectrin is proven to sterically restrict protein lateral diffusion through a subplasmalemmal network; the capacity of dystrophin to act as a 'molecular fence' to membrane diffusion was studied by comparing lateral mobility of membrane glycoproteins by fluorescence photobleach recovery in mdx and normal tissue. Secondly, as dystrophin has been proven to interact directly with proteins of the dystrophin associated glycoprotein complex in vivo, experiments addressed whether specific binding and immobilisation of the complex by dystrophin at the membrane was essential for function. Finally, given the homology of dystrophin and spectrin, the presence of dystrophin at the neuromuscular junction, and the importance of spectrins in immobilisation of voltage gated sodium channels in the nervous system, the role of dystrophin in regulating voltage gated sodium channel distribution at the neuromuscular junction was investigated. The results show that membrane glycoproteins were immobile in the presence and absence of dystrophin, suggesting dystrophin is not an essential molecular fence component. Alternatively, viability may have been the major influence on protein and lipid diffusion in these fibres and suggestions are made as to how this may be recognised and overcome for subsequent investigation. Three novel exon specific anti-dystrophin peptide antibodies were generated during the work that will be useful for studies into Duchenne muscular dystrophy in general, and dystrophin revertant fibres in particular.

Wearable robotics for assistance and rehabilitation are not yet considered commercially mainstream products, and as a result have not yet seen advanced controls systems and interfaces. Consequently, the available technology is mostly adapted from systems used in parallel technologies, rather than custom applications intended for human use. This study concerns itself with the design and development of a custom control system for a 2-degree of freedom powered upper extremity orthosis capable of driving elbow flexion/extension 135º and humeral rotation 95º . The orthosis has been evaluated for use as both a long-term assistive technology device for persons with disabilities, and as a short-term rehabilitative tool for persons recovering injury. The target demographics for such a device vary in age, cognitive ability and physical function, thus requiring several input parameters requiring consideration. This study includes a full evaluation of the potential users of the device, as well as parameter considerations that are required during the design phase. The final control system is capable of driving each DOF independently or simultaneously, for a more realistic and natural coupled-motion, with proportional control by pulse-width modulation. The dual-axis joystick interface wirelessly transmits to the 1.21 pound control pack which houses a custom microcontroller-driven PCB and 1800 milliamp-hour lithium-ion rechargeable battery capable of delivering 4 hours of running time. Upon integration with the 2 DOF orthosis device, a user may complete full range of motion with up to 5 pounds in their hand in less than 7 seconds, providing full functionality to complete acts of daily living, thus improving quality of life.

En 1997, le caractère culard, un phénotype hypermusclé chez le bovin, est attribué à des mutations dans le gène de la myostatine (MSTN). Depuis, il a été confirmé qu’une baisse de l’activité de la MSTN conduisait à une augmentation de la masse musculaire chez d’autres espèces, y compris chez l’Homme. L’identification de ce facteur et des conséquences de son invalidation sur le développement musculaire ouvre de nombreuses perspectives en médecine humaine comme, par exemple, chez des personnes ayant eu une fonte musculaire importante suite à une immobilisation prolongée ou en conséquence du vieillissement ou d’une maladie chronique. L’objectif majeur de ce projet de recherche a consisté à évaluer de nouvelles stratégies permettant d’augmenter la masse musculaire chez la souris. Pour ce faire, nous nous sommes intéressés à une métalloprotéine de la matrice extracellulaire (MEC), la décorine (DCN), dont l’interaction avec la MSTN a été caractérisée comme étant zinc dépendante. Suite à l’injection de ce Small Leucine Rich Proteoglycan (SLRP) chez la souris dystrophique mdx et Gamma-sarcoglycan-/-, nous avons constaté une augmentation de la masse musculaire consécutive à un phénomène d’hypertrophie associé ou non à de l’hyperplasie. Des études de dose/cinétique ont montré que l’effet positif de la décorine sur la croissance musculaire était maximal 21 jours après administration. Nous avons également découvert qu’un fragment peptidique de 41 acides aminés du domaine N-terminal de la protéine DCN murine conservait une activité anti-myostatine et induisait une hypertrophie musculaire chez la souris dystrophique. Ce domaine, site de l’interaction directe entre la MSTN et la DCN, présente un motif CX3CXCX6C, caractéristique des SLRPs de classe I, dont le cluster de cystéines et son interaction avec le zinc ont été décrits comme indispensables à l’activité anti-MSTN de la DCN. Différentes études concernant les mécanismes induits lors de la séquestration de la MSTN par la DCN dans la MEC ont également été conduites afin d’expliquer les phénomènes observés chez la souris. Enfin, nous avons étudié le potentiel de la DCN pour favoriser la greffe de cellules myogéniques et développé différentes approches de thérapie génique. / In 1997, the double-muscling phenotype, a marked hypermuscularity in cattle, was related to mutations in the myostatin (MSTN) gene. Since, it was confirmed that a decrease of the myostatin’s activity drives an increase of the muscular mass in others species, including Human. The identification of this factor and the consequences of its invalidation on the muscular development open many perspectives in human medicine, as, for example, for people whom have an important muscular loss fallow up an extended immobilization or in consequence of old age or a chronic disease. The main purpose of this research project was to evaluate some new strategies permitting the increase of the muscular mass in mice. To achieve that, we investigated in detail the decorin (DCN), a metalloprotein of the extracellular matrix (ECM), interacting with MSTN in a zinc-dependent manner. After intramuscular injection of this Small Leucine Rich Proteoglycan (SLRP) in mdx and Gamma-Sarcoglycan-/- dystrophic mice, we observed a significant increase of the muscle mass conducted by hypertrophy associated or not with hyperplasia. Dose and cinetic studies showed that the positive effect of the decorin on muscular growth was maximal 21 days after administration. Furthermore, we showed that a peptide encompassing the 31-71 sequence retains full myostatin binding capacity and intramuscular injection of this peptide induces muscle hypertrophy in dystrophic mice. This direct interaction site between MSTN and DCN contains a conserved CX3CXCX6C pattern of class I SLRPs, whose cluster of cysteins and its interaction with zinc were shown to be crucial in the anti-MSTN activity of DCN. Various studies of the mechanism resulting of the sequestration of MSTN by DCN in ECM were conducted in order to explain the phenomenom observed in mice. Al last, we have studied the potential of DCN in the cellular transplantation and developped different anti-myostatin strategies of genetic therapy.

Phospholipids are the major components of cell membranes and have a wide variety of structures, shapes and properties. Different ratios of phospholipid species confer different properties to membranes and contribute to the normal function of organelles. We have previously shown that acute phosphatidylcholine (PC) biosynthesis inhibition leads to a severe form of lipid imbalance that disrupts ER morphology and structure. Furthermore, our previous studies also revealed a mechanism for ER proteostasis under conditions of lipid-imbalance-induced ER stress in yeast, whereby unfolded ER proteins are removed by lipid droplets (LDs) and targeted to the vacuole for degradation by microlipophagy. Here, we find that LDs also contribute to ER proteostasis during chemically induced ER stress. Furthermore, we find that ER stress results in an increase in ubiquitinated proteins in LDs as well as recruitment of cytosolic and ER heat shock proteins, as well as ER proteins to LDs. ER stress-induced microlipophagy does not require core ATG genes and can occur in the absence of lipid ordered microdomains (Lo) in the vacuolar membrane. Instead, we find that the ESCRT machinery is up-regulated and localizes to the vacuolar membrane in response to ER stress induced microlipophagy and that ESCRT I, II and III complexes are required for microlipophagy in response to each of these stressors. Similar to yeast, we find that lipid imbalance in skeletal muscle from CHKB CMD, new autosomal recessive CMD (Congenital Muscular Dystrophy) caused by a mutation of choline kinase beta (CHKB), results in abnormal SR/ER morphology. CHKB is the first enzyme in the de novo PC biosynthesis pathway and causes phospholipid imbalance in cell membranes similar to that observed in yeast. Besides the disruption of SR morphology, we also detect a dysfunction of the Ryanodine Receptor (RyR), the Ca2+ channels responsible for initiating muscle contraction. Specifically, we observe abnormal RyR morphology and increased association of RyRs with lipid droplets (LD) in muscle fibers from a CHKB CMD patient. Finally, we detect ER stress and pronounced UPR activation in rmd mice, a mouse model of CHKB CMD. Given these results, we propose that inhibition of PC biosynthesis leads to phospholipid imbalance in the SR, which in turn, causes RyR leakage and ER stress which lead to mitochondrial dysfunction and dystrophy in CHKB CMD.