Les Rhabdomyosarcomes (RMS) sont des sarcomes qui touchent préférentiellement les enfants et les adolescents. Les RMS sont à l'origine de nombreuses métastases qui sont responsables d'une réduction importante de l'espérance de vie du malade. Une meilleure compréhension des mécanismes sous-tendant la migration et l'invasion des RMS pourrait orienter vers de nouvelles thérapies visant à enrayer le développement de métastases. La dissémination métastatique fait intervenir de nombreuses protéases dont la µ- et la m-calpaïne, cystéine-protéases, constituant avec leur inhibiteur endogène, la calpastatine, le système protéolytique neutre calcium-dépendant. Dans différents travaux, l'activité de ces calpaïnes a été montrée comme dérégulée, notamment dans le cancer du rein, de la peau ou encore les adénocarcinomes colorectaux. Une connaissance approfondie de l'impact de la dérégulation de l'activité des calpaïnes sur la dissémination métastatique pourrait en faire des cibles thérapeutiques de choix. L’étude de l’activité, de l’expression et de la localisation des différents composants du système protéolytique neutre calcium-dépendant a permis de mettre en évidence une activité dérégulée des calpaïnes dans les RMS. Cette forte activité serait due à une expression très faible de la calpastatine. L’analyse comparative des caractéristiques adhésives et cinétiques des RMS par rapport aux cellules témoins, des myoblastes humains (LHCN-M2) montre un faible taux d’adhésion associé à une vitesse de migration élevée des RMS. L’activité calpaïne présente une corrélation linéaire positive avec la vitesse de migration ; les calpaïnes se présentent donc comme marqueur de l’agressivité tumorale. L’inhibition des calpaïnes par la calpeptine réduit fortement cette vitesse. Le cytosquelette des RMS est désorganisé et ne présente pas, contrairement à celui des cellules non-tumorales, de fibres de stress. À ce niveau, les études pour tenter de discriminer le rôle de la µ-calpaïne et de la m-calpaïne, utilisant des oligonucléotides antisens, montrent que dans les LHCN-M2, la µ-calpaïne jouerait un rôle prépondérant dans la régulation de l’alpha-actine en régulant de manière négative son expression. Quant à la béta-actine, elle serait régulée, aussi de manière négative, mais, uniquement par la m-calpaïne. Dans les ARMS, la µ-calpaïne et la m-calpaïne jouent un rôle similaire en stimulant l’expression des deux isoformes. De plus, le pouvoir invasif important des RMS est fortement diminué lorsque l’activité calpaïne est inhibée. L’implication des calpaïnes tant au niveau de l’adhésion, de la migration que de l’invasion des RMS font de ces dernières une cible d’étude importante pour tenter de contrecarrer le développement de métastases. / Rhabdomyosarcoma (RMS) are a soft-tissue sarcoma commonly encountered in childhood and adolescence. RMS cells can acquire invasive behaviour and can form metastases which decrease less than 20% the patients healing. The comprehension of mechanisms that regulate cancer cells migration and invasion may be a key for development of new therapies for limiting metastasis. The metastatic dissemination implicates lots of proteases of which µ-calpain and m-calpain. Calpain activity is Ca2+-dependent and is principally regulated by calpastatin, its specific endogenous inhibitor. The deregulation of calpains has been involved in tumour invasion and metastasis in several different cell types. Then, calpains would be a good target for development of novel therapies to control metastasis. Study of calpain activity, expression, and localisation underline the deregulation of calpain activity in RMS. This high activity may be due to weak expression of calpastatin. The comparative analysis of adhesive and kinetical characteristics of RMS cells, compared to human myoblasts, LHCN-M2 cells, show a weak adhesiveness and an important migration velocity in RMS cells. The calpain activity presents a positive linear correlation with the migration velocity; so, calpain may be considered as marker of tumoral aggressiveness. The inhibition of calpain by calpeptin reduces significantly the migration velocity. The cytoskeleton RMS cells is disorganised and does not present stress fibers, contrary to LHCN-M2 cells. At this level, discrimination of µ- and m-calpain role, using antisens oligonucleotides, shows that in LHCN-M2 myoblasts, µ-calpain may negatively regulate the alpha-actin expression and that béta-actin may be positively regulated by the m-calpain. In ARMS cells, both µ- and m-calpain positively regulate alpha- and béta-actin. Moreover, the invasive behaviour of RMS cells is importantly decreased when calpains are inhibited. In summery, calpains may implicate in the anarchic adhesion, migration and increase of invasion. In this way, targeting calpain activity may represent a good strategy for limiting development of RMS tumour as well as their metastatic behaviour.

It has been well established that the fundamental behaviors of mammalian cells are influenced by the physical cues that they experience from their surrounding environment. With respect to cells in our bodies, mechanically-driven morphological and phenotypic changes to our cells have been linked to responses critical to both normal development and disease progression, including lung, heart, muscle and bone disorders, and cancer. Although significant advancements to our understanding of cell behavior have been made using 2D cell culture methods, questions regarding how physical stretch guides cell behavior in more complex 3D biological systems remain unanswered. To address these questions, we used microfabrication techniques to develop vacuum-actuated stretchers for high throughput stretching and dynamic mechanical screening of 3D microtissue cultures. This thesis contains five research chapters that have utilized these devices to advance our understanding of how cells feel stretch and how it influences their behavior in a 3D matrix. In the first research chapter (chapter 2), we characterized how stretch is transferred from the tissue-level to the single-cell level and we investigated the cytoskeletal reinforcement response to long-term mechanical conditioning. In the second research chapter (chapter 3), we examined the effects of an acute dynamic stretch and found that 3D cultures soften through actin depolymerization to homeostatically maintain a mean tension. This softening response to stretch may lengthen tissues in our body, and thus may be an important mechanism by which airway resistance and arterial blood pressure are controlled. In the third and forth research chapters (chapter 4-5), we investigated the time dependencies of microtissues cultures and we found that their behavior differed from our knowledge of the rheological behavior of cells in 2D culture. Microtissues instead followed a stretched exponential model that seemed to be set by a dynamic equilibrium between cytoskeletal assembly and disassembly rates. The difference in the behavior from cells in 2D may reflect the profound changes to the structure and distribution of the cytoskeleton that occur when cells are grown on flat surfaces vs. within a 3D environment. In the fifth and final research chapter (chapter 6), we examined how mechanical forces may contribute to the progression of tissue fibrosis through activating latent TGF-β1. Our results suggest that mechanical stretch contributes to a feed forward loop that preserves a myofibroblastic phenotype. Together these investigations further our understanding of how cells respond to mechanical stimuli within 3D environments, and thus, mark a significant contribution to the fields of mechanobiology and cell mechanics.

Le fonctionnement des organismes vivants requiert la production deforces à grande échelle, pour des processus biologiques aussi diversque la motilité cellulaire, le développement embryonnaire, lacicatrisation ou encore la contraction musculaire. Dans de telssystèmes, les forces générées à l'échelle moléculaire par des moteursprotéiques sont transmises par des réseaux de fibres désordonnés,menant à des tensions actives à grande échelle. Les propriétésmacroscopiques passives de ces réseaux de fibres sont biencaractérisées. En revanche, ce problème de production de stress pardes unités actives microscopiques n'est pas résolu. Cette Thèseprésente une étude approfondie, par des méthodes théoriques etnumériques, de la transmission de forces dans les réseaux élastiquesde biopolymères. Je montre que la réponse linéaire, à faible force,des réseaux est remarquablement simple : elle est déterminée par laseule la géométrie des unités actives exerçant les forces. Aucontraire, lorsque les forces actives sont suffisamment importantespour provoquer le flambage non-linéaire des fibres, ces forces sontrectifiées par le réseau, et deviennent isotropiquementcontractiles. La contraction émergente qui en résulte est amplifiéepar la transmission de forces non-linéaire à travers le réseau. Cetteamplification du stress macroscopique est renforcée par le caractèredésordonnée du réseau, mais sature lorsque la densité d'unités activesest grande. Nos prédictions sont en accord quantitatifs avec desrésultats expérimentaux sur des tissus reconstitués et des réseauxd'actomyosine in vitro, et apportent un éclairage nouveau surl'influence de l'architecture microscopique des réseaux sur structuredes stress à l'échelle de la cellule et du tissu. / Large-scale force generation is essential for biological functionssuch as cell motility, embryonic development, wound healing and musclecontraction. In these processes, forces generated at the molecularlevel by motor proteins are transmitted by disordered fiber networks,resulting in large-scale active stresses. While fiber networks arewell characterized macroscopically, this stress generation bymicroscopic active units is not well understood. In this Thesis, Ipresent a comprehensive theoretical and numerical study of forcetransmission in elastic fiber networks. I show that the linear,small-force response of the networks is remarkably simple, as themacroscopic active stress depends only on the geometry of theforce-exerting unit. In contrast, as non-linear buckling occurs aroundthese units, local active forces are rectified towards isotropiccontraction, making the local geometry of force exertion irrelevant.This emergent contractility is amplified by non-linear forcetransmission through the network. This stress amplification isreinforced by the networks' disordered nature, but saturates for highdensities of active units. Our predictions are quantitativelyconsistent with experiments on reconstituted tissues and actomyosinnetworks, and that they shed light on the role of the networkmicrostructure in shaping active stresses in cells and tissue.

Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning after traditional cloning methods were not successful. The constructs were expressed in E. coli, then the recombinant proteins were purified. To determine if the GST-tagged proteins are functional, ATPase activity and tubulin polymerization assays were performed. While both GST-katanin-p60 and GST-spastin hydrolyzed ATP indicating that the ATPase domains are functional, the results of the tubulin polymerization assays were less clear and further experimentation is necessary.

Evolutionary-developmental study of membrane proteins Mgr. Stanislav Vosolsobě Abstract Using a plethora of experimental approaches for phylogenetical and functional study on several membrane signalling proteins, I brought new evidences supporting a hypothesis that the molecular evolution of protein families is a highly dynamic, not conservative, process. In DREPP family of calcium-binding peripherally-associated plasma-membrane proteins I found a broad flexibility in protein-membrane binding manners coupled with a many independent duplication of this Euphyllophyta-clade specific plant gene. In three families of auxin transporting proteins, PIN-FORMED, LAX and PILS, I showed that emergences of these proteins are uncorrelated and placed on different levels of the plant kingdom phylogenetic tree. However these proteins ensure very fundamental plant morphogenetic processes, like cell differentiation, organ formation or tropisms, with strong effects of their deleterious mutations, I found many gene radiations and losses on a all taxonomic levels in these families, evidencing that key and shared physiological processes may be realised by genes touched by a recently undergoing evolution. Evolutionary-developmental synthesis of a functional and phylogenetic data must be done with caution due to high risk of...

The cornea forms the anterior surface of the eye and is responsible for most of the eye’s refractive power. Injury to the outermost layer of the cornea, a non-keratinized stratified squamous epithelium, triggers a transient rise in intracellular calcium concentration that propagates radially from the wound. This calcium mobilization is initiated by the binding of nucleotides such as adenosine triphosphate (ATP), which are released from cells ruptured by the injury, to purinergic receptors (purinoreceptors) on undamaged cells near the wound. Downstream effects of this injury-induced "calcium wave" are generally thought to include the activation of signaling pathways that promote wound healing. However, the specific contributions of individual purinergic receptors to the overall wound response have in most cases not been well characterized. Purinoreceptors are classified into two broad categories: the P2Y class of G protein-coupled receptors, which act through second messengers to release calcium into the cytosol from the endoplasmic reticulum, and the P2X class of ligand-gated ionotropic receptors, which release calcium into the cytosol from the extracellular environment. Previously, our lab established the importance of the P2Y2 receptor to corneal epithelial wound healing by showing that P2Y2 activation makes a substantial contribution to the overall wound-induced calcium response, particularly in cells back from the leading edge, and promotes cell migration after injury. P2Y2 activation was also found to promote the phosphorylation of proteins involved in focal adhesions, which are multi-protein complexes that facilitate cell migration by transmitting the forces generated by the actin cytoskeleton to the extracellular environment. More recently, our lab has begun to demonstrate that P2X7 may play an equally important, yet distinct and perhaps complementary role in corneal epithelial wound healing. For instance, P2X7 was found to strongly influence the intensity of the injury-induced calcium response in cells immediately adjacent to the wound, and treatment with the P2X7 inhibitor oxidized ATP (oxATP) was shown to impair migration after injury both in vitro and in ex vivo rat corneas. Additionally, immunofluorescence of cells fixed eight hours after injury revealed an altered actin cytoskeletal architecture and localization of the focal adhesion proteins talin and vinculin in oxATP-treated cells compared to control cells. The goal of the present study was to further characterize P2X7’s role in the overall response to injury by using live cell imaging to examine actin cytoskeletal rearrangements and focal adhesion dynamics after injury under both control conditions and conditions of P2X7 inhibition. Human corneal limbal epithelial (HCLE) cells were transduced to express either actin or talin tagged with green fluorescent protein (GFP), grown into confluent monolayers, and scratch wounded in the presence or absence of oxATP. Cells at the leading edge of the wound were imaged using confocal microscopy every 10 minutes for 4 hours beginning 0.5 hours after injury. Analysis of the resulting actin-GFP movies revealed trends toward delayed extension of filopodia in oxATP-treated cells relative to control cells, as well as complex changes in the number of filopodia per cell over time. Additionally, while both groups formed lamella containing thick actin bundles that were oriented perpendicularly to the direction of migration, in oxATP-treated cells the formation of these structures was delayed. Furthermore, in oxATP-treated cells these actin bundles tended to persist once formed. This was in contrast to control cells, in which they tended to turn over to be replaced by thinner and shorter actin bundles that were oriented more obliquely relative to the direction of migration. Finally, analysis of talin-GFP movies demonstrated that focal adhesion lifespan was extended in oxATP-treated cells compared to control cells. Focal adhesions in oxATP-treated cells also exhibited a greater propensity to merge together or split apart, further suggesting impaired focal adhesion turnover. Overall, these findings suggest that P2X7 plays a critical role in promoting migration after corneal epithelial injury by coordinating rapid rearrangements of the actin cytoskeleton and turnover of focal adhesions at the leading edge.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23551号 / 生博第462号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 中野 雄司, 教授 見学 美根子, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19473号 / 理博第4133号 / 新制||理||1594(附属図書館) / 32509 / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)講師 市川 正敏, 教授 佐々 真一, 教授 山本 潤 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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Breast cancer is the most common type of cancer among women in the United States and metastasis is the leading cause of mortality in patients diagnosed with malignant breast cancer. The receptor of thromboxane A2 (TxA2), TP, is a member of the G-protein coupled receptor family. Increased expression of TP at RNA level was found to correlate with a poor prognosis in breast cancer patients; however, it is unknown how TP expression and activities are involved in breast cancer progression. Here we report that TP is expressed in breast cancer cells at both RNA and protein levels. And further, activation of this receptor elicits rapid activation of small GTPase RhoA and cytoskeleton reorganization. We also found that knockdown of TP expression or inhibition of TP activation by SQ29548, a TP antagonist, reduces tumor cell motility, reduce tumor cell extravasation from the circulatory system, and most importantly, reduce breast cancer metastasis in vivo. These data provide compelling evidence suggesting that the TxA2-TP pathway plays an important role in breast cancer metastasis.