[Truncated abstract] Mutations in the skeletal muscle -actin gene (ACTA1) have been shown to be one cause of a broad group of muscle disorders all termed the congenital myopathies. Over 170 different mutations have now been identified across all 6 coding exons of ACTA1 in patients presenting with muscle weakness and any one or more of the following histopathological features: nemaline rods, intranuclear rods, fibre-type disproportion, excess of thin filaments and central cores. While the identification of the causative gene has been of great comfort for affected patients and their families, with pre-natal genetic testing becoming available, the ultimate aim is to develop a therapy for these disorders. Of the therapies currently being explored for the muscular dystrophies, up-regulation of an alternative gene seemed to be one of the most promising avenues for treatment of the ACTA1 diseases. Up-regulation of utrophin, the foetal homologue of dystrophin, has been shown to be a promising therapy for the treatment of Duchenne muscular dystrophy. The main aim of my research was to determine whether up-regulation of cardiac -actin, the predominant -actin expressed in foetal skeletal muscle and in the adult heart, could be used as a therapy for the ACTA1 diseases. A proof-of-concept experiment was performed whereby skeletal muscle -actin knock-out (KO) mice (all of which die by postnatal day 9) were crossed with transgenic mice over-expressing cardiac -actin (known as Coco mice) in postnatal skeletal muscle. ... While patients that are ACTA1 nulls have been identified in a number of mainly consanguineous populations, the majority of ACTA1 mutations result in dominant disease in which the mutant protein interferes with the function of the wild-type skeletal muscle -actin. Research described in this thesis also focuses on characterizing two transgenic mouse models of dominant ACTA1 disease at the ultra-structural, cellular and functional level; this is the first step towards a proof-of-concept experiment to determine whether cardiac -actin up-regulation can dilute out the pathogenesis of dominant ACTA1 disease. It has long been noted that patients with ACTA1 disease do not have ophthalmoplegia, even in the most-severely affected individuals. Protein analysis performed on extraocular muscle (EOM) biopsies obtained from humans, sheep and pigs showed that the EOMs co-express cardiac and skeletal muscle -actin, with cardiac -actin comprising 70 % of the striated -actin pool. Thus we propose that sparing of the EOMs in ACTA1 disease is at least in part due to cardiac -actin diluting out the pathogenesis associated with expression of the mutant skeletal muscle -actin. This finding provides further support for the hypothesis that dilution of mutant skeletal muscle -actin in dominant ACTA1 disease by up-regulation of cardiac -actin may be a viable therapy for this group of devastating muscle diseases. The research contained herein has advanced the understanding of the pathobiology of skeletal muscle -actin diseases and provides strong evidence in support of cardiac -actin up-regulation as a promising therapy for these diseases.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 194-227).

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 79-89). Also available on the World Wide Web.

Thesis (Ph.D.)--University of Western Sydney, 2007. / A thesis presented to the University of Western Sydney, College of Health and Science, School of Natural Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.

University, Diss., 2000--München.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 September 20

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 160 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Le gène CDKN1A a été initialement décrit comme une cible transcriptionnelle de la protéineoncosuppressive p53. Son produit, p21Cip1 (p21), supprime l’activité des kinases dépendantes descyclines et de la protéine PCNA, ce qui en fait un puissant inhibiteur du cycle et de la proliférationcellulaires. En outre, p21 est fréquemment inactivée dans les cancers épithéliaux. Or, la progressionde ces tumeurs est associée à l’altération de l’organisation tissulaire, au processus invasif et à ladissémination métastatique. Ces phénomènes résultent des modifications cytosquelettiquesconduisant à la transformation des propriétés d’adhésion et de migration cellulaires. Pourtant, le rôlede p21 dans la dynamique cytosquelettique des cellules épithéliales humaines n’a jamais été adressé.Nous montrons ici que p21 contribue à l’adhésion et la migration normale des cellules épithélialesmammaires non transformées. Nos résultats montrent également que l’inactivation de p21 provoquela suppression de l’adhésion focale et des fibres de stress. Ce phénotype est caractérisé parl’inactivation de la GTPase Rho et l’activation de la cofiline, facteur de dépolymérisation de l’actine. Enoutre, la suppression de p21 provoque une désacétylation des microtubules associée à unedéstabilisation microtubulaire globale. La réduction de l’instabilité dynamique, par inhibition de ladésacétylase HDAC6, restaure partiellement l’étalement cellulaire et l’adhésion focale altérés parl’inactivation de p21. L’ensemble de nos données démontre que la régulation de la dynamiquecytosquelettique par p21 est nécessaire au contrôle de l’adhésion des cellules épithéliales humainesnon tumorales. / CDKN1A gene was initially identified as a target of the tumor suppressor p53. The ability to inhibitcyclin-dependant kinase and PCNA activities confers to its product, p21Cip1 (p21), strong antiproliferativeproperties. Moreover, p21 is frequently inactivated in epithelial cancers. Progression ofthese malignancies is also associated with disorganized tissue architecture, invasion and metastaticdissemination. These processes involve major cytoskeletal reorganization associated withtransformation of adhesion and migration properties. Still, the role of p21 in cytoskeletal dynamics innormal epithelial cell has never been addressed. Here we show that p21 contributes to normaladhesion and migration in untransformed human mammary epithelial cells (HMEC). We alsodemonstrate that p21 inactivation in HMEC suppresses focal adhesion and stress fiber assembly. p21depletion is also associated with inactivation of Rho GTPase and activation of the F-actin severingfactor cofilin. In addition, p21 silencing provokes microtubule hypoacetylation associated withincreased dynamic instability. We find that forced microtubule stabilization by HDAC6 inhibitionpartially restore cell spreading and focal adhesion in p21-depleted cells. Collectively, our data showthat regulation of cytoskeletal dynamics by p21 is required for adhesion control in untransformedhuman epithelial cells.

The cyclical interaction between the force-generating protein myosin and actin is the mechanism responsible for muscle contraction among all muscle types. Cardiac muscle contraction is tightly controlled to ensure that blood pumps effectively and efficiently from the heart to peripheral organs. Mutations in various cardiac proteins can lead to cardiac dysfunction and a number of cardiomyopathies. The first part of this dissertation studies two disease-linked mutations in the regulatory light chain of the cardiac myosin molecule, D166V and K104E, and assesses the kinetic and mechanochemical effects of the mutations via the in vitro motility assay. The data show that D166V mutant myosin force generation is reduced compared to wild type, and exogenous phosphorylation of the mutant light chain rescues force generation. In contrast, the K104E mutation showed no deficit in force production but exhibited increased calcium sensitivity of activation. These results are consistent with contractile defects associated with cardiomyopathies caused by various mutation-induced changes to protein function and mechanism of interaction. The second part uncovers the actin-binding mechanism of one of the chief muscle regulatory proteins tropomyosin. In cardiac and skeletal muscle, tropomyosin and troponin modulate muscle contraction. Tropomyosin binds along the length of actin filaments and blocks myosin-binding sites. Following an excitatory stimulus, calcium binds troponin and causes tropomyosin to shift its position on actin, allowing myosin to bind. The precise mechanism of how tropomyosin monomers with low actin affinity bind to form a stably bound, high affinity chain is unknown. By directly observing fluorescently labeled tropomyosin binding to actin filaments, it was shown that tropomyosin molecules bind randomly along the actin filament. Subsequent monomer binding, and formation of tropomyosin end-to-end bonds, increases the probability of sustained chain growth by decreasing the probability of detachment prior to additional monomer binding. Tropomyosin molecules added to the growing chain at approximately 100 monomers/(μM*s). Different tropomyosin isoforms segregate to distinct functional and structural regions of cells. The last chapter presents data that show spatial segregation of two different tropomyosin isoforms on actin filaments. This suggests that tropomyosin sorting in cells is, at least partly, an intrinsic property of the binding mechanism.

La capacité des cellules à sentir les propriétés physiques de leur environnement est un facteur déterminant de l'homéostasie tissulaire. Ainsi, la rigidité de la matrice extracellulaire (forces exogènes) et les tensions du cytosquelette (forces endogènes) coopèrent de manière fonctionnelle modulant les transformations phénotypiques. Les cellules perçoivent et transmettent des forces en développant des structures d'adhérences appelées adhésions focales. Ces adhésions sont composées de protéines transmembranaires, les intégrines, qui font le lien entre le cytosquelette et la matrice extracellulaire.La partie centrale de mon projet de thèse aborde la question du couplage des intégrines b1 et b3 dans la mécanotransduction. Les données actuelles plaident fortement en faveur d'une relation bidirectionnelle entre l'adhésion intégrine-dépendante et les forces mécaniques générées dans ce processus. Les approches génétiques classiques ont souligné le rôle majeur des intégrines b1 et b3 dans mécanosensibilité cellulaire, sans préciser leur contribution relative. Par exemple, la manière dont la modulation de l'expression de l'intégrine b3 affecte la génération des forces de traction cellulaires et la distribution des adhésions intégrines-dépendantes reste à être explorées. Dans ce travail de thèse nous avons montré que les intégrines b1 ont un rôle essentiel dans la génération de forces cellulaires, que les intégrines b1 sont régulées négativement par les intégrines b3 en affectant la distribution spatiale des intégrines b1 à travers leur capacité à lier à la fois la taline et la kindline. Et enfin, nous avons montré que les intégrines b3 régulent temporellement l'activité contractile de la cellule.J'ai également participé à deux autres études dans le cadre de collaborations avec le Pr. Holmgren et le Dr. Debili, au cours desquelles j'ai utilisé la microscopie à traction de forces comme un outil diagnostique afin d'observer l'effet des forces contractiles dans la formation de la lumen aortique et de la formation des plaquettes sanguines. J'ai ainsi pu confirmer que la protéine amotL2, reliant les fibres contractiles aux VE-cadhérines, est impliquée dans la force intercellulaire nécessaire à la formation de la lumen aortique. Et lors d'une deuxième collaboration, j'ai pu montrer que la contractilité des mégacaryocytes, via leur système actomyosine, est nécessaire pour la formation des proplaquettes. / Cell ability to sense mechanical properties of their microenvironment is crucial for tissue homeostasis which means their capacity to maintain mechanical integrity as they are submitted to external forces.Integrins have been highlighted as mechanotransducers able to form micro-scale structures called focal adhesion sites which mechanically link cells to the extracellular matrix by recruiting various adaptors. Both b1 and b3 integrins have been identified as the principal actors of tensional homeostasis. However as the resulting mechanotransduction processes are intrinsically dynamic, the respective and cooperative roles b1 and b3 integrins need to be addressed over time and space.In the present work, coupling time-resolved traction force microscopy and genetics approaches, we investigated the respective role of b1 and b3 integrins in active force generation at the single cell level. Our findings show that b1 integrins has an essential role in generation of cellular traction forces, b1 integrin-generated force is negatively regulated by b3 integrins which impacts the redistribution of b1 integrin containing adhesion through its ability to bind to talin and kindlin, b3 integrin supports min-scale temporal regulation of cellular contractile activity generated by b1 integrin. Finally, cell mechanical equilibrium relies on the ability of cells to maintain a fixed contractile moment.I also participated in two others studies in the framework of collaborations in which I used the traction force microscopy as a diagnostic tool to observe the effect of contractile forces in the formation of the aortic lumen and the formation of proplatelets. I was able to confirm that the protein amotL2 connecting the contractile fibers to VE-cadherin, is involved in intercellular forces necessary for the formation of the aortic lumen. And in a second collaboration, where I found by using traction force microscopy that the contractility of megakaryocytes via its actomyosin system, is necessary for the formation proplatelets.