Rozprawy doktorskie na temat „Substrate rigidity”

Du cerveau à l’os, la rigidité et la composition de la matrice extracellulaire varient énormément et jouent un rôle dans les réponses cellulaires. La rigidité influe également sur la tension de la membrane plasmique, elle-même régulée par le trafic membranaire. Comment la rigidité et la composition du substrat peuvent réguler l'exocytose, qui à son tour régule la tension de la membrane, reste largement inconnu. Ici, j'ai utilisé l’imagerie pHluorin d’évènements uniques d’exocytose de cellules cultivées sur des substrats de rigidité et de composition contrôlée pour explorer la régulation de VAMP2 et VAMP7. J'ai développé un logiciel informatique pour identifier automatiquement les évènements de fusion, permettant une analyse rapide de données. J'ai contribué à l'étude montrant que l’exocytose VAMP7 est régulée par la kinase src, qui phosphoryle VAMP7 dans son domaine Longin (LD) (Burgo et al. JBC 2013). De plus, j’ai trouvé que la rigidité du substrat stimule l’exocytose, en présence de la laminine, de VAMP7, mais pas VAMP7 sans LD ni VAMP2. VAMP7 et VAMP7 sans LD sont par ailleurs également sensibles aux variations de la tension membranaire induites par chocs osmotiques. Enfin, j'ai identifié que LRRK1 est un partenaire du LD, et contrôle le transport rétrograde de VAMP7.Ces approches m’ont permis de révéler un nouveau mécanisme par lequel la rigidité, agissant sur la signalisation des intégrines, contrôle le transport de VAMP7 via LRRK1 et Rab21 (Wang et al. soumis). Ce mécanisme pourrait avoir un large intérêt potentiel pour comprendre la dynamique de la membrane dans des conditions normales et pathologiques, en particulier le cancer
From brain to bones, stiffness and composition of the extracellular matrix vary greatly and play a role in cell responses. Substrate rigidity also impacts plasma membrane tension, which has a close relationship with membrane trafficking. How substrate rigidity and chemistry sensing may regulate exocytosis, which in turn regulates membrane tension, is still largely unknown. Here, I used pHluorin imaging of single vesicle exocytosis in cells cultured on substrates of controlled rigidity and composition to explore the regulation of VAMP2 and VAMP7-mediated exocytosis. I developed a computer software to automatically identify fusion events thus allowing quick analysis of batch data. I contributed to the study showing that VAMP7 exocytosis is regulated by src kinase which phosphorylates VAMP7 in its Longin domain (LD) (Burgo et al. JBC 2013). I further found that VAMP7 but not VAMP7 lacking LD- or VAMP2-mediated secretion was stimulated by substrate stiffness on laminin. VAMP7 and VAMP7 lacking LD were similarly sensitive to osmotic chock-induced membrane tension changes. Finally, i showed that LRRK1, a regulator of egf receptor transport, is a partner of the LD, and controls the retrograde transport of VAMP7. These approaches allowed me to reveal a new mechanism whereby substrate rigidity, by acting on integrin signalling, enhances VAMP7 exocytosis via LRRK1- and Rab21-dependent regulation of its peripheral readily-releasable pool (Wang et al. submitted). This mechanism may have broad potential relevance for plasma membrane dynamics in normal conditions and diseases, particularly cancer

L’ingénierie tissulaire est une stratégie médicale qui repose sur la régénération de tissu par les cellules avec ou sans matériaux. Pour maîtriser cette synthèse, il faut comprendre la cellule comme une part intégrante du tissu. Hormis ses interactions biochimiques avec son support, la cellule interagit également mécaniquement avec son environnement. Elle s’accroche à ce dernier et évalue sa dureté pour adapter sa réponse biologique. Dans cette étude, j’ai développé des modèles numériques pour analyser l’influence de la rigidité du substrat sur le comportement mécanique de la cellule, sur sa structure contractile interne et les efforts qu’elle génère. En d’autres termes, j’ai essayé de comprendre comment la cellule ressent la rigidité de son environnement. De plus, au lieu de me focaliser sur les propriétés mécaniques quantitatives, j’ai cherché à développer un modèle conceptuel simplifié plus proche de la structure cellulaire
Tissue engineering is a medical strategy based on utilizing cells and materials to regenerate a new tissue. Yet, it involves intertwined interactions that allow cells to act as integrated parts of an organ. In addition to chemical reactions, the cell interacts mechanically with its environment by sensing its rigidity. Here, we used several computational models to understand how substrate rigidity affects a cell’s structure as it adheres and spreads on it. In other words we tried to understand the way a cell feels how soft or hard it surrounding is, how it affects its internal structure and the forces that transit within it. In addition, instead of focusing on mechanical properties, we developed a simplified, yet coherent conceptual understanding of the cellular structure

"Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering. "

Les cellules s'adaptent en permanence à leur microenvironnement. En particulier, elles modifient leur morphologie, leur croissance, leur division et leur motilité en fonction des propriétés biochimiques et physiques de la matrice extracellulaire (MEC). Elles sont équipées de structures adhésives appelées plaques d’adhérences, permettant aux cellules d'interagir avec les protéines de la MEC via les protéines transmembranaires appelées intégrines et de détecter la nature et la rigidité de la MEC. Le signal est transduit par les protéines des plaques d’adhérences et résulte par exemple en une modification de la tension mécanique induite par l'acto-myosine. Les voies de signalisation en aval peuvent également atteindre le noyau. L'expression des gènes peut alors être modifiée, ce qui peut en retour affecter la composition des plaques d’adhérences et de la MEC pour une réponse cellulaire adaptative. Nous avons émis l'hypothèse qu'en plus des voies de signalisation, un couplage mécanique direct entre les événements se produisant à la périphérie de la cellule et le noyau pourrait participer à la transmission de signaux mécaniques. Bien que les filaments intermédiaires (FIs) aient des propriétés mécaniques extrêmement intéressantes et résistent à des charges de tension élevées, leur implication dans les voies de mécanotransduction est encore mal connue. En utilisant l'astrocyte comme modèle, en raison de sa combinaison spécifique de FIs : vimentine, GFAP, nestine et synémine, nous avons d'abord étudié l'effet de la rigidité du substrat sur la morphologie, la structure et les fonctions du noyau, ainsi que sur l'organisation des FIs autour du noyau. Nous avons ensuite étudié l’impact de l’absence de FI les changements nucléaires observés en réponse à la rigidité du substrat. En utilisant une combinaison de techniques de microfabrication, de méthodes biochimiques et de microscopie, nous avons montré que la rigidité du substrat affecte la forme, le volume du noyau, la structure de la chromatine et le recrutement des facteurs de transcriptions (YAP). Nos résultats suggèrent que les FI forment une structure en forme de cage autour du noyau d'une manière dépendante de la rigidité : un substrat plus rigide induit la formation d’une cage de vimentine et de nestine. Cette interaction avec le noyau pourrait expliquer les modifications nucléaires observées en réponse à la rigidité du substrat. Au total, les résultats obtenus au cours de notre étude permettent de mieux comprendre le rôle des filaments intermédiaires dans les réponses nucléaires aux propriétés mécaniques du substrat
Cells continuously adapt to their microenvironment. In particular, they modulate their morphology, growth, division, and motility according to the biochemical and physical properties of the extracellular matrix (ECM). Cells are equipped with adhesive structures called FAs, allowing them to interact with ECM proteins through the core transmembrane proteins called integrins and to sense the nature and the rigidity of the ECM. This information are transduced by FA proteins and lead, for instance, to changes in acto-myosin-mediated mechanical tension. Downstream signalling pathways also reach the nucleus; gene expression is then modified and may, in return, affect the composition of FAs or of the ECM proteins for adaptative cell response. Here, we hypothesized that, in addition to signalling pathways, a direct mechanical coupling between the events occurring at the cell periphery and the nucleus may participate in the transmission of mechanical cues and the regulation of nuclear functions. Although intermediate filaments (IFs) have extremely interesting mechanical properties and resist high tension load, their involvement in mechanotransduction pathways remains elusive. Using astrocyte as a model, due to its specific combination of IFs: vimentin, GFAP, nestin, and synemin, we studied first the effect of substrate rigidity on the nucleus morphology and function, and on the organisation of IFs around the nucleus. Then, we investigated the role of IFs in rigidity-induced nuclear changes. Using a combination of microfabrication techniques, biochemical and microscopy methods, we showed that substrate rigidity affects the nucleus shape, volume, and structure of the chromatin and the recruitment of transcription factor (YAP) and IFs are mediating these changes. Our results suggest that IFs form a cage-like structure around the nucleus in a rigidity-dependent manner: stiffer substrates promote the formation of a cage of vimentin and nestin. In the absence of IFs, the nuclear changes induced by rigidity are different than with IF. The nucleus increases its size in soft substrate, together with an increase in tension measured by YAP localising in the nucleus. The structure of the chromatin is changed. Altogether, the results obtained during our investigation give a better understanding of the role of intermediate filaments in the mechanosensitive nuclear responses

La migration des cellules souches de la pulpe dentaire (DPSCs) est un aspect fondamental de l’ingénierie tissulaire dentaire. L’objectif de cette thèse est de déterminer l’influence de la rigidité du substrat sur la migration des DPSCs. Dans une première partie, nous avons montré que les DPSCs sont capables de survivre et de proliférer sur des substrats de polydiméthylsiloxane (PDMS) avec un module de Young de 1,5 kPa à 2,5 MPa sans se différencier. Nous avons observé que la vitesse moyenne des DPSCs est augmentée sur les substrats de faible rigidité. De plus, la Yes-associated protein (YAP) conserve une localisation nucléaire même sur les PDMS de faible rigidité. Finalement, nous avons montré que sur un substrat comportant deux rigidités différentes, les DPSCs n’adoptent pas de direction de migration préférentielle, contrairement au phénomène de durotaxie classiquement décrit dans la littérature
Migration of dental pulp stem cells (DPSCs) is a fundamental aspect of dental tissue engineering. The objective of this thesis is to investigate the influence of substrate stiffness on DPSCs migration. In the first part of this thesis, we showed that DPSCs are able to survive and proliferate on polydimethylsiloxane substrates (PDMS) with a Young's modulus of 1.5 kPa to 2.5 MPa without differentiating themselves. We observed that the average speed of DPSCs is increased on substrates with low stiffness. In addition, the Yes-associated protein (YAP) maintains a nuclear localization even on PDMS with low rigidity. Finally, we have shown that on a substrate with two different stiffnesses, DPSCs do not adopt any preferential migration direction, unlike the process of durotaxis classically described in the literature

L’objectif de ce travail de thèse est de réaliser des substrats et des dispositifs de culture cellulaire pour des applications à grande échelle. En utilisant à la fois des techniques de lithographie conventionnelles et non conventionnelles, nous avons d'abord fabriqué des matrices denses de piliers élastomère avec un gradient de hauteur pour les études de migration cellulaire et nous avons observé un allongement cellulaire remarquable et une migration cellulaire dirigée, tout dépendant du gradient de rigidité. Les micropiliers élastomères pourraient également être organisés en gradient de hauteur oscillant, montrant des comportements cellulaire similaires. Sur la base d'une approche biomimétique, nous avons produit des nanofibres à deux côtés d'une membrane avec des trous traversants pour l’adhésion et la migration tridimensionnelles de cellules. Nos résultats ont montré qu'un tel substrat peut favoriser l'infiltration et la prolifération des cellules dans un environnement 3D. Enfin, nous avons utilisé des réseaux micropiliers de différentes hauteurs en tant que substrat de rigidité contrôlée pour la différenciation des cardiomyocytes à partir de cellules souches pluripotentes l'homme. À l'aide d'un stencil en élastomère, des embryons uniformes pourraient être obtenus et dérivés vers les cellules de ciblage sur le substrat de différentes rigidité, montrant clairement une dépendance de rigidité des substrats
The purpose of this work is to develop manufacturable cell culture substrates and devices for large scale applications. By using both conventional and non-conventional lithography techniques, we firstly fabricated dense elastomer pillar arrays with height gradient for cell migration studies and we observed remarkable cell elongation and directed cell migration, all depending on the strength of the stiffness gradient. Elastomer micropillars could also be organized in ripple-like height gradient patterns, showing similar cell behaviors. Based on a biomimetic approach, we produced nanofibers on both side of a membrane with through holes for three-dimensional cell adhesion and migration. Our results showed that such a 3D scaffold can promote the cell infiltration and proliferation. Finally, we used micropillar arrays of different height as stiffness controlled substrate for cardiomyocytes differentiation from human induced pluripotent stem cells (hiPSCs). With the help of an elastomer stencil, uniform embryoids could be obtained and derived to the targeting cells on the substrate of different stiffness, showing a clear stiffness dependence of the substrates

L’objectif de cette thèse est d'étudier l'influence d'hydrogels de faibles rigidité sur l’organisation de la chromatine de cellules épithéliales PtK2 et cancéreuses SW480. Sur des hydrogels mous, la chromatine de PtK2 se structure en hétérochromatine. Les hydrogels très mous conduisent à la nécrose. Sur ces substrats, l'euchromatine, maintenue par inhibition de HDAC, guide la cellule en quiescence. Ces cellules se divisent après transfert sur surfaces rigides. Un processus de dissémination métastatique est développé en cultivant des cellules cancéreuses sur des hydrogels très mous (E20) et des surfaces rigides (verre). Les cellules meurent lors du 1er passage sur E20. Au 2ème passage sur E20, leur survie, motilité et pourcentage en hétérochromatine augmentent. Au 3ème passage, la survie et la motilité progressent cependant le pourcentage en hétérochromatine diminue. Du 1er-2ème passage, les cellules répondent à un processus de dissémination « hétérochromatine­ dépendant » , du 3ème-4ème passage à un processus « euchromatine-dépendant »
The aim of this thesis is to investigate the influence of soft hydrogels on the chromatin plasticity of epithelial PtK2 and cancer cells SW480. On soft hydrogels, the chromatin of PtK2 cells is organized in heterochromatin. The very soft hydrogels direct the cell death by necrosis. On these substrates, the euchromatin maintained by inhibition of HDAC guides the cells into quiescence. These cells transferred on stiff substrate enter in mitosis. A process of metastatic dissemination is developed from cancer cells grown on very soft hydrogels (E20) and stiff surfaces (glass). On the 1st seeding on E20, cells die. The 2nd seeding on E20 shows that cell viability, motility and heterochromatin percentage increase. On the 3rd seeding on E20, survival and motility continue to increase while the heterochromatin percentage decrease. From the 1st- 2nd E20 seeding, cells respond to a heterochromatin-dependent process of metastatic dissemination and from the 3rd-4th E20 seeding to an euchromatin-dependent process

La myopathie myofibrillaire est une maladie neuromusculaire à évolution lente caractérisée par de graves troubles musculaires causés par des mutations dans le gène codant pour des protéines du cytosquelette. L'un des gènes affectés en relation avec le développement de la MFM est DES. Des mutations dans le gène de la desmine entraînent des myopathies des muscles squelettiques et cardiaques. Cependant, les évènements qu'elles entraînent et qui sont à l’origine des phénotypes pathologiques cardiaques restent mal connus. Mon objectif est de créer un modèle in vitro de MFM basé sur des cellules souches pluripotentes humaines afin d'étudier le rôle des mutations spécifiques dans la desmine sur le développement et la fonction des cellules cardiaques. Pour atteindre cet objectif, en collaboration avec les docteurs A. Behin, K. Wahbi et la société Phenocell, nous avons généré des iPSC à partir des cellules sanguines périphériques de patients souffrant d'une forme de cardiomyopathie induite par une mutation de la desmine. Les lignées iPSC générées contenant les mutations du gène codant la desmine ont permis d’étudier le rôle d’une mutation dans la spécification et la fonction des cardiomyocytes. La bioénergétique mitochondriale, la structure cellulaire et la fonction contractiles ont été évaluées au niveau cellulaire. En conclusion, il convient de noter que les mutations de la desmine conduisent à une désorganisation des structures des sarcomères dans les cardiomyocytes et à une perturbation de l'expression des protéines mitochondriales. Ce qui conduit à une altération des fonctions de la mitochondrie. Ces données permettent d’améliorer notre compréhension des mécanismes moléculaire qui sous-tendent le développement de la MFM
Myofibrillar Myopathy is a slowly progressive neuromuscular disease characterized by severe muscular disorders caused by mutations in the gene encoded cytoskeletal proteins. One of the genes described in connection with the development of MFM is DES. Mutations in the desmin gene lead to skeletal and cardiac muscles myopathies. However, the cardiac pathological consequences caused by them remain poorly understood. My objective is to create an in vitro human stem cell model of MFM to specifically investigate the role of patient-specific mutations in desmin on cardiac lineage development and function. To achieve that objective, in collaboration with Drs. Behin and K. Wahbi and Phenocell, we generate patient-specific iPSC from peripheral blood cells of the patient suffering severel form of desmin-deficient cardiomyopathy. The generated iPSC lines carrying DES gene mutations enable a powerful examination of the role of desmin mutation on cardiomyocyte specification and function. Bioenergetic, structural, and contractile function will be assessed in a single cell. In conclusion, it should be noted that desmin mutations lead to a disorganization of sarcomere structures in cardiomyocytes and to a perturbation of mitochondrial protein expression. This leads to a distortion of functions in the mitochondria. These data facilitate the understanding of the molecular pathway underlying the development of desmin-related myopathy. And the system we have created could also allow us to better evaluate the correlation between the desmin genotype and phenotype in terms of effect on the heart

Dissertação de mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
As células estaminais mesenquimais (MSCs) são células estaminais adultas, multipotentes, capazes de se auto renovar e diferenciar em diferentes tipos celulares dentro das linhagens de origem mesenquimal. A colheita de células estaminais mesenquimais é feita a partir de tecidos mesenquimais e também de tecidos extra embrionários. Estes últimos constituem uma boa fonte de MSCs, sendo estas mais naïve e com maior potencial de proliferação do que MSCs de tecidos adultos, características que fazem com que MSCs da matriz do cordão umbilical sejam um tipo celular muito apelativo para experiências de reprogramação. A geração de células estaminais pluripotentes induzidas (iPSCs), nomeadamente a partir de MSCs, tem sido documentada na literatura por diferentes autores, no entanto sempre associada a uma baixa eficiência. É sabido que células estaminais pluripotentes e os seus núcleos têm propriedades elásticas distintas daquelas apresentadas por células diferenciadas e células estaminais adultas (e os seus respectivos núcleos). A partir destas observações colocámos a hipótese de que, através da modulação da rigidez de MSCs, poderíamos aumentar a eficiência do processo de reprogramação usando um vector lentiviral que codifica factores de pluripotência. O núcleo está mecanicamente acoplado a elementos do citoesqueleto através do complexo LINC (ligante do nucleoesqueleto ao citoesqueleto) e desta forma as forças mecânicas vindas da matriz extracelular podem ser transmitidas através do citoesqueleto até ao núcleo. Dependendo da rigidez do substrato, o núcleo está sob maior ou menor tensão, sendo eventualmente possível modular o seu módulo elástico se as células forem plaqueadas em plataformas com diferentes graus de rigidez. Com este trabalho demonstrámos que ao plaquear MSCs em substratos com distintos graus de rigidez, é possível torná-las mais propícias a uma total reprogramação. Para além disto, verificou-se também um aumento na expressão de genes de pluripotência devido apenas ao facto de MSCs serem mantidas em cultura em substratos específicos. Ao analisarmos o estado de compactação da cromatina, bem como a área nuclear tornou-se evidente o efeito que a rigidez dos substratos tem sobre as células. Assim sendo, os rácios do conteúdo eucromático e heterocromático dos núcleos, bem como a área nuclear mostraram ser modulados, o que sugere que os núcleos sofrem mecanomodulação. Foram também observadas diferenças no que ao tamanho das Adesões focais (FAs) e à organização das fibras de actina diz respeito. Tomando em conjunto todos os nossos resultados, verificou-se que é possível melhorar o processo de reprogramação através da modulação da rigidez do substrato, e que o mecanismo por detrás desta melhoria pode estar intimamente relacionado com a mecanomodulação do núcleo. Este progresso na geração de iPSCs humanas, recorrendo a substratos de rigidez definida, dá indícios de que os protocolos habituais de reprogramação celular em plataformas convencionais de cultura de células podem ser substancialmente melhorados plaqueando as células nesses mesmos substratos. Deste modo, aumentado a eficiência e a cinética da geração de células estaminais pluripotentes induzdas, estudos futuros poderão explorar a utilização de vectores de reprogramação não integrativos, considerados mais seguros para possíveis aplicações clínicas.
Mesenchymal stem cells (MSCs) are multipotent adult stem cells able to self-renew and differentiate into several cell types within mesenchymal origin, which can be collected from adult mesenchymal tissues, and also from extra-embryonic tissues. The latter constitute a good source of MSCs, being more naïve and having a more proliferative potential than MSCs from adult tissues, features that make umbilical cord matrix MSCs an appealing cell type for the generation of induced pluripotent stem cells (iPSCs). The generation of human iPSCs, namely from human MSCs, has been reported, although with a low efficiency. It is known that pluripotent stem cells and their nuclei possess distinct elastic properties from differentiated and adult stem cells (and respective nuclei). We hypothesize that, by modulating the rigidity of MSCs, it may be possible to enhance the reprogramming efficiency using a lentiviral vector encoding pluripotency factors. The nucleus is mechanically coupled to cytoskeletal elements by the LINC (Linker of Nucleoskeleton to Cytoskeleton) complex, thus mechanical forces from the extracellular matrix can be transmitted through the cytoskeleton to the nucleus. Depending on substrate stiffness, the nucleus is under more or less tension, eventually being possible to modulate its rigidity by culturing the cells on platforms with distinct degrees of stiffness. Here we demonstrated that MSCs plated on substrates with distinct range of rigidity showed different degrees of efficiency to fully reprogram. Moreover, it was shown that maintaining MSCs on specific substrates enhanced the expression of pluripotency genes. The effect of substrate rigidity on the cells was evident when chromatin compaction and nuclear area were analyzed. Thus, nuclear euchromatic and heterochromatic content ratios and area could be modulated, suggesting that nuclei were subjected to mechanomodulation. Differences were also observed in what concerns the size of Focal adhesions (FAs) and the assembling of actin stress fibers. Taken together, our results suggest that it is possible to improve the reprogramming process by modulating the substrate rigidity, and that the mechanism responsible for this improvement could be intimately related with the mechanomodulation of the nuclei. The enhanced generation of human iPSCs cells using substrates with defined stiffness indicates that the current cell reprogramming protocols can be substantially improved by seeding the cells on such substrates. Thus, by improving the efficiency and kinetics of iPSCs generation, future strategies may be further explored using non integrative reprograming delivery strategies, considered safer for putative future clinical applications.

Cell response to substrate rigidity is an emerging field with implications in processes ranging from embryological development to the pathogenesis of disease states such as cancer or fibrosis, in which changes in tissue mechanical properties may inform cellular behavior. It may also serve as a valuable tool in tissue engineering, where materials must be chosen to best influence desired cell phenotype. This thesis describes novel substrates with patterned mechanical properties and their effects on mesenchymal stem cell (MSC) and macrophage behavior. Though substrate rigidity has previously been shown to guide MSC differentiation in two dimensions on unpatterned substrates, differentiation in response to substrates with patterned mechanical properties and in three dimensions has never been demonstrated. Unfortunately, all systems currently used to study these phenomena are limited in their ability to combine spatial patterning of rigidity with cell encapsulation and 3D culture. By altering polymer molecular weight and concentration and using defined mixing and photolithographic patterning techniques, I have developed poly(ethylene glycol) diacrylate hydrogels with tunable rigidity patterned in distinct patterns and gradients. These hydrogels are highly biocompatible and may be crosslinked and patterned under conditions that allow cellular encapsulation and 3D culture. Using these hydrogels, I have shown spatial control over cellular behavior including patterned MSC differentiation in three dimensions in response to substrate rigidity. The potential to drive differentiation in 3D using the mechanical properties of the substrate is particularly exciting, as it bypasses the difficulties of spatially restricting the growth factors currently used to guide progenitor cell differentiation in vitro. In the future, these substrates may be used to engineer tissues with complex architecture in three dimensions.

Cells have long been known to sense and respond to mechanical stimuli in their environment. In the adoptive immune system particularly, cells are highly specialized and responsible for detecting and eliminating pathogens from the body. T cell mechanosensing is a relatively new field that explores how force transmission in cell-cell interaction elicits both inter- and intra-cell signaling. Owing to recent advances in genetic manipulation of T cells, it has emerged as new tool in immunotherapy. We recently demonstrated human T cell activation in response to mechanical rigidity of surfaces presenting activating antibodies CD3 and CD28. The work in this dissertation highlights new progress in the basic science of T cell mechanosensing, and the utilization of this knowledge toward the development of a more specialized expansion platform for adoptive immunotherapies. Human T cells are known to trigger more readily on softer PDMS substrates, where Young's Modulus is less than 100 kPa as compared to surfaces of 2 MPa. While the range of effective rigidities has been established, it is important to explore local differences in substrates that may also contribute to these findings. We have isolated the rigidity-dependence of cell-cell interactions apart from material properties to optimize design for a clinical cell expansion platform. Though PDMS is a well understood biomaterial and has found extensive use in cellular engineering, a PA gel substrate model allows for rigidity to be tuned more closely across this specific range of rigidities and provides control over ligand density and orientation. These rigidity-based trends will be instrumental in adapting models of mechanobiology to describe T cell activation via the immune synapse. In what is generally accepted as the clinical gold-standard for T cell expansion, rigid (GPa) antibody-coated polystyrene beads provide an increase in the ratio of stimulating surface area-per-volume, over standard culture dishes. Herein we describe the development of a soft-material fiber-based system with particular focus on maintaining mechanical properties of PDMS to exploit rigidity-based expansion trends, investigated through atomic force microscopy. This system is designed to ease risks associated with bead-cell separation while preserving a large area-to-volume ratio. Exposing T cells to electrospun mesh of varying rigidities, fiber diameters, and mesh densities over short (3 day) and long (15 day) time periods have allowed for this system's optimization. By capitalizing on the mechanisms by which rigidity mediates cell activation, clinical cell expansion can be improved to provide greater expansion in a single growth period, direct the phenotypic makeup of expanded populations, and treat more patients faster. This technology may even reach some cell populations that are not responsive to current treatments. The aims of this work are focused to identify key material properties that drive the expansion of T cells and optimize them in the design of a rigidity-based cell expansion platform.

碩士
國立成功大學
醫學工程研究所碩博士班
96
Both biochemical and mechanical properties of the microenvironment are equally important for cell growth and functional expression. Cell adhesion not only plays a critical role in cell physiology functions including morphology, spreading, migration, and differentiation, but also a critical step in the tissue engineering approach for maintenance of tissue structure and cell integration of biomaterial. The adhesion between cell-ECM is affected by intracellular regulations and extracellular environment, and is strengthened through focal adhesions. The focal adhesion kinase (FAK) at focal adhesions (FAs) is the major part for cell binding to the extracellular matrix (ECM). The substrate rigidity is playing an important factor for various cell behaviors, such as apoptosis, differentiation, and cancer invasion. In present study, we plate MDCK epithelial cell on glass surface, 1kPa, and 30kPa polyacrylamide gels, respectively. The FAK expression for substrate rigidity sensing and adhesion force management will be sty cytodetachment device and fluorescent microscopy. We hypothesize that FAK is the rigidity sensor in response to different rigidity of substrates. Different expression levels of FAK by gene Overexpression (FAK-WT) and knock-out (FRNK) will be also used to probe the function of FAK in rigidity sensing. The results indicate that cell spreading area, adhesion force, and work are increasing with increasing substrate rigidity. Except for cell area and normalized adhesion force, all parameters reach significantly different between FAK-WT and FRNK cells. FAK can enhance cell spreading and increase the normalized adhesion force on 30kPa and 1kPa gels. On three kinds of substrate rigidity, FAK can enhance adhesion force. We define a ratio of affecting index (AI) between FAK-WT, control and FRNK. Control, respectively. The AI ration means the effect of FAK on different substrate rigidity, the higher value of AI and the stronger effect of FAK. The AI ratio of adhesion force in glass dish and 30kPa gel are approximate the same, and the highest value occurs in 1kPa gel. The AI ratio of cell area is almost no difference between three kinds of substrate rigidity. Moreover, the affecting index of FAK-WT is inversely proportional to the substrate rigidity, but proportional to the substrate rigidity in FRNK cell. We suggest that the effect of FAK is weaker on stiffer substrate than on softer matrix.

碩士
國立成功大學
醫學工程研究所碩博士班
98
Most cells are anchorage dependent. They require a surface to attach in order to migrate, proliferate, and differentiate. It is known several chemical factors can dramatically affect those functions of cells. Nevertheless, physical factors like stiffness and surface morphology of the substrata are also thought to influence cell behaviors. The purpose of this study was to measure the cell migration under different stiffness and structure of the substrata. Human melanoma cells (A2058) and human epidermal melanocytes (HEMn) were used for this study. The polydimethylsiloxane (PDMS) with ratios of silicon elastomer/curing agent concentration at 10:1 and 20:1 were fabricated for soft substrata. The Young’s modulus of PDMS substrata with different ratios were measured by material testing system (MTS). PDMS samples were made into five forms, including flat, 6 μm cones, 6 μm grooves, 1 μm cones and 1 μm grooves by standard micro-electro-mechanical-systems (MEMS). Glass dish was used as control substrate. After cells were seeded on glass, flat PDMS, flat-top cone, or long groove PDMS for 12 hours to establish stable attachment, the cell images were taken by optical microscopy every 5 minutes for 6 hours. The speed and direction of cell migration were calculated by the coordinate of each cell on the images. The modulus for PDMS with a ratio of 10:1 and 20:1 measured by MTS were 3.04 ± 0.26 and 1.44 ± 0.13 MPa respectively. For both cell types, parallel migration along the pattern occurred on the groove surface and the directional migration increased with pattern line width; however, both cell types did not show any preferred migration orientation n the cones and flat substrata. The frequency of A2058 cells moving along the groove decreased with smaller line width. Flat-top cone was the best surface morphology to influence A2058 cell area. In contrast, the cell area of HEMn cells appeared uniform to variation in surface morphology and substrate rigidity. The speeds of HEMn cells were almost faster on all substrata when compared to the speed of A2058 cells. The migration speeds of both cell types on glass were the lowest and significantly faster on soft PDMS when compared to the stiff PDMS for most types of patterning substrate. The highest speeds of A2058 and HEMn cells were 0.91 ± 0.06 and 1.02 ± 0.11 μm/min was obtained on the soft 6 μm cones and soft 1 μm grooves respectively. In conclusion, this study demonstrated that A2058 cells were more sensitive to the changes of surface morphology and substrate rigidity than HEMn cells.

Cells sense and interact with their microenvironment to retrieve signals which include cell-matrix and cell-cell contacts. These signals account for the influence of culturing conditions and often control the local cellular phenotype and global functions of tissues. Here, I sought to understand if there is any information processed by cells in guiding cellular phenotype given the control of cell shapes and substrate rigidities. If there is, would these phenotypic changes achieve biomedical purposes? What is the strategy to engineer platforms that can handle the longstanding challenges in those fields? In this dissertation, the first chapter serves as an introduction which involves the origin of motivations, which mainly came from current challenges in biomedical researches of kidney podocytes. I have attempted to understand if it is possible to control podocyte differentiation through cell shape control which mimics their in vivo morphology. On the other hand, I have tried to reveal if it is possible that tissue stiffness can affect podocyte phenotype as a result of stiffness sensing. These two topics were rarely investigated for kidney podocytes, which is the critical component of human filtration barrier to perform renal functions. The effort that addresses the question how shape and substrate rigidity as in- formation repositories affect kidney podocytes phenotype has profound meaning in the understanding of renal physiological system and pathological mechanisms. The second chapter will focus on the methods to achieve successful long-term shape control on cells. Engineered cell-device interface using cross-linking biomaterial SU-8 plays a key role in this study. Compared with other previously used approaches summarized in this chapter, SU-8 provides various advantages both in the fabrication of micro- pattern architecture as well as its sustaining effectiveness in controlling cell shape. This approach has been proved very efficient and economic to facilitate single cell level manipulation. The chapter will describe in details the interface micro-fabrication and encountered technical challenges. The results that kidney podocytes were in good compliance with the micro-pattern proved the successful application of this technique. The third chapter will then transfer from micro-fabrication to biological experiments, which discusses in details how in intro kidney podocytes responded to their shapes by enforcing protein localization which characterizes a phenotype found in vivo. This phenotype among in vitro podocytes was further verified that it may contribute to podocytes differentiation and physiological functions. The information processed by shape was proved independent of tension-related processes and thus shape and tension could be regarded as separate contributors in cellular development. The interpretation of shape’s contribution could be referred to my previous publication in the journal of Cell: ”Decoding Information in Cell Shape”. In this study, the motifs of research were applied to other cell lines (Human vascular smooth muscle cell) as a step to generalize the ubiquity of shape’s contribution to cell differentiation. The study here was to differentiate shape and tension through investigating the difference between two major mechanosensors: β3 and β1 integrin receptors. The difference in cell phenotypes through integrin inhibition experiments demonstrated critical but unique role of integrin-based shape sensing in vitro. This chapter in dissertation covers most of the content in a previously submitted paper to Nature Cell Biology. In the fourth chapter, I further carried out a study that investigated if stiffness sensing can influence kidney podocyte phenotype. The fourth chapter will basically review the techniques in the fabrication of hydrogel-based cell culture platforms. In a similar manner to previous study using biomimetic shape for podocytes and find its phenotype, the target of this analysis was to use hydrogel-based biomimetic substrate with renal physiological stiffness and find if there is a differentiation phenotype. Since numerous materials have been reported in hydrogel studies, I will focus on the introduction to representative ones that have been most widely used. Their characteristics will be compared with the demands of kidney podocyte reasearch. Methodologies were the key to a successful research, and in this chapter I will describe in details what choices I made in choosing experimental methods that improved the efficiency and quality of cell culture platforms. A natural enzyme (microbial transglutaminase) cross-linked gelatin hydrogel was adopted here to provide ideal substrate rigidity control for podocytes. This method has demonstrated high efficiency and stability in making large cell culture surface. Moreover, it provides the hydrogel platform with an ideal range of elastic moduli suitable for renal tissue culture. The results will be discussed in detail in the fifth chapter. I successfully found a differentiation phenotype for podocytes cultured on the hydrogel platforms with a physiological stiffness. Similar phenotype, on the contrary, were not found in podocytes on platforms which were either too soft or too stiff. These resutls have formed one of my submitted paper to Scientific Report. The differentiation phenotype for kidney podocytes was characterized by up-regulation of differentiation markers. These findings were in a similar manner to a series of stem cells differentiation guided by regulated substrate stiffnesses. This phenotype of kidney podocytes was verified by microarray technique which confirmed the stiffness sensing using transcription factors. The enrichment analysis of kinases also showed significant response of Src, Fyn etc, of which the activities have been shown critical for podocytes to preserve their physiological functions. These results have successfully suggested the close relations between stiffness changes of glomeruli basement membrane (GBM) and progressive podocyte dysfunction. In summary, this dissertation covers interdisciplinary researches that decoded the information processed by cells from two separate aspects: shape and stiffness sensing. The details in each chapter cover a broader scope than the content selected for publications. Through this dissertation, readers will get in touch with the technique developed for plat- form and their applications to biomedical researches. I wish this will help people new in the field to get my hands-on experience.

The extracellular matrix (ECM) has been implicated in numerous physiological and pathogenic processes. Integrins are thought to be the primary receptors that cells use to transduce biochemical and physical signals from the ECM. Integrin - ligand binding is specific for ECM molecules and is regulated by specific protein-protein interactions that further regulate downstream cellular activity such as motility, survival, growth, and proliferation. Termed outside-in signaling, the engagement of integrins results in protein recruitment to sites of cell - ECM contacts known as focal adhesions. Focal adhesions (FAs) are central to cell spreading, motility, survival and growth and serve as both physical linkages between the ECM and cytoskeleton as well as signaling centers for a cell on 2D substrates. Termed focal adhesion-actin coupling, FAs physically link the cytoskeleton with the ECM via actin binding proteins and are involved in mechanically coupling the cell to the ECM. To date, FAs' signaling properties and FA- actin coupling have been unrelated and independent mechanisms. This study provides data that suggests the amount, or level, of focal adhesion coupling in addition to regulating traction force generation, motility events and the rigidity response, also regulates the amount of biochemical signaling towards survival, growth and proliferation. First, via a knockout cell line system I demonstrate that Integrin-Linked Kinase is involved in coupling Beta1 integrins to collagen and FAs. I then demonstrate that lack of coupling results in altered rigidity sensing, defects in spreading of the cytoplasm, lower force generation and collagen contraction, as well as altered localization and activation of MAP kinases. Specifically, when ILK null cells were plated on collagen coated glass they were unable to reinforce Beta1 integrin mediated interactions nor spread their cytoplasm or undergo contractile activity. In contrast, when ILK null cells were plated on fibronectin coated glass, ILK null cells progressed to the contractile phase of spreading and then retracted their adhesions, losing the ability to stabilize late stage Beta1 integrin mediated fibronectin interactions. Moreover, I demonstrate that actin retrograde flow regulates the localization and modification state of FA signaling molecules that regulate survival, growth, and proliferation. Secondly, via changing ECM composition and rigidity of the substrate, I demonstrate that the engagement of both Beta1 and Beta3 integrins via collagen type I and fibronectin increases focal adhesion size, focal adhesion-actin coupling, and activation of signaling molecules involved in translation, survival, growth, and proliferation. This investigation presents data that supports the idea that the degree of focal adhesion mediated ECM-cytoskeletal coupling correlates with the ability to activate signaling molecules and suggests a model in which focal adhesion-actin coupling regulates the localization and modification state of scaffold and signaling proteins that result in the modulation of survival, growth and proliferation. Finally, I propose the use of an experimentally derived metric to describe ECM-FA-actin coupling and present preliminary data that the proposed metric can also be used as a biomarker for specific disease states such as cancer.

碩士
國立成功大學
醫學工程研究所碩博士班
95
Cell adhesion plays a critical role in cell physiology functions, which influences morphology, spreading, migration, and differentiation, etc. The adhesion is affected by intracellular regulations and extracellular environment. Most of the adhesion to extracellular matrix is derived from focal adhesions, which enhance adhesion, functioning as structural links between the extracellular matrix and the cytoskeleton that regulates intracellular tension, and direct cell function by triggering signaling pathways. 　In this study, the relationship between different substratum rigidity and cell adhesion was investigated. The sensibility and reaction to different substratum rigidity differ from kinds of cells. Several studies have shown that apoptosis would be induced while certain kinds of polarized cells were seeded on soft substrate, but not fibroblasts. To understand effects of the substrate rigidity on cell behaviors, the adhesion force was measured quantitatively between cells and collagen-coated substrates with different rigidity in two types of cells, and also cell spreading area, adhesion force, and adhesion stress were analyzed. In the experiments, the epithelia cell, (LLC-PK1), and fibroblast, (NIH-3T3), were seeded on collagen-coated polyacrylamide substrates with rigidity 1000, 10000Pa and glass surface (control group), respectively. After 6 hours of seeding, the adhesion force was measured by cytodetachment technique, and cell area was also detected. Therefore, the cell adhesion force per unit area, defined as adhesion stress, was figured out. 　The results show that the cell spreading area and adhesion force increased with increasing rigidity. The cell adhesion stress at 6 hour seeding was 825.23±215.97, 991.97±368.26, and 1231.86±359.70 Pa on substratum rigidity 1000, 10000Pa, and glass, respectively, for 3T3, and they are 911.32±254.79, 920.36±173.87, and 960.83±321.14 Pa for PK1. Also, the adhesion stress increased with increasing rigidity. The one-way ANOVA analysis shows significant differences in spreading area and adhesion force among three different rigidity substrates for 3T3 and PK1. However, the cell adhesion stress has significant difference in 3T3 but not for pk1 among different substrate. 　Besides, the detachment energy (work) was obtained by integrating the area of the adhesion force-distance curve, and the normalized work was also found by dividing cell area. For 3T3, the detachment work and the work per unit area was 3.83±1.24, 5.09±1.12, 5.55±1.84 (10-12J) and 11.55±4.39, 16.66±4.66, 12.14±3.74 (10-3 J/m2) on substratum rigidity 1000, 10000Pa, and glass. For PK1, they are 11.37±2.42, 16.70±6.56, 18.97±7.22 (10-12J) and 23.10±8.75, 22.38±12.22, 30.65±13.58 (10-3 J/m2).

碩士
國立成功大學
醫學工程研究所碩博士班
96
The cell can link another cell and ECM by adhesion molecules. The behavior of cell adhesion is influenced by the cell physiology and external environment. It plays an important role in cell morphology, motility, movement and differentiation, and cancer metastasis etc. So the ability to cell adhesion means that the cell carries out the normal physiological behavior. At present, a lot of technology can be used to quantify cell detaching force, for instance, micropipette, optical tweezers and so on. AFM has a lot of advantages, like parameters control easily, high resolution, and measuring in air. AFM combined with cell probe replacing tip function could measure the cell detaching force between cell-substrate and cell-cell. The rigidity of the substrates is considered to affect cell behavior. The rigidity sensitivity and respond of each cell is not the same. The poupose of this study is to investigate the relation between the rigidity of substrate and cell detaching force. Fibroblast cell was used to quantify cell detaching force. The contact times were set at 30, 60, 90, and 300 seconds. All the surfaces of substrates were coated with collagen type Ⅰ. The rigidity of the substrates were 1037 Pa, 10930 Pa and glass (control group). Statistics is by one-way ANOVA and Tukey HSD. Under the same contact time, the detaching force rise gradually with increasing substrate rigidity. Cell detaching force on 1037 Pa, 10930 Pa and glass substrates were 296.99±107.73, 459.69±228.51, and 620.01±211.98 pN respectively for 30 seconds contact time; the forces were 344.96±198.85, 561.84±283.58, and 1917.3±344.2 pN for 60 seconds; the forces were 347.44±125.22, 644.75±358.57, and 2519.76±685.06 pN for 90 seconds.; and the forces were 482.11±194.34, 1820.11±949.29, and 3373.45±1867.02 pN for 300 seconds. The cell detaching force among three rigidity substrates were significant different on each contact time. The detaching force increased slowly on 1037 Pa, increasing notablely from 90 to 300 seconds on 10930 Pa, and increasing notablely to be stable from 30 to 300 seconds on glass. Our results showed cell adhesion could be influenced by substrate rigidity. This cell probe technique can be further used for parameter study on cell substrate interaction in the future.

碩士
國立成功大學
生理學研究所
94
Abstract The proto-oncoprotein c-Jun is a component of the transcription factor AP-1 (activator protein-1) involved in cellular proliferation, differentiation and death. Maintenance of c-Jun protein levels plays an important role in proliferation and survival of epithelial cells. Previous studies in our lab showed that epithelial cells cultured on collagen gel developed apoptosis due to low substratum rigidity. Low rigidity-induced cell apoptosis was mediated by degradation of c-Jun, which was observed only in epithelial, but not transformed cells. The purpose of my study was to delineate the underlying mechanism whereby low substratum rigidity induced degradation of c-Jun. The low rigidity-induced degradation of c-Jun could be reversed by 26S proteasome specific inhibitors. Here we showed that Cul1, a ring-domain ubiquitin ligase, had a specific physiological role in low rigidity-induced c-Jun degradation. Low substratum rigidity induced Cul1 neddylation and enhanced Cul1-mediated polyubiquitination. Under low substratum rigidity condition, Cul1 physically interacted with c-Jun. Immunofluorescence study showed that low rigidity induced the accumulation of Cul1 in the nucleus, which was associated with degradation of c-Jun. In addition, low rigidity also triggered translocation of 26S proteasome into the nucleus. Taken together, we demonstrate that low substratum rigidity induces Cul1 neddylation which triggers c-Jun polyubiquitination and results in c-Jun degradation through ubiquitin-proteasome proteolysis in epithelial cells.

博士
國立成功大學
基礎醫學研究所
96
Most organs are soft, and the likely reason is that substratum rigidity plays important roles in regulation of cellular functions and behaviors. Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment has not been fully explored. The aim of this thesis is to understand how cells sense their physical environment. Two parts in this study are included: (1) to investigate whether and how low substratum rigidity of collagen gel modifies canonical integrin-mediated cellular behavior, and (2) to delineate the mechanosensing machinery and its mode of action for cells at substratum rigidity of soft tissue range. The fisrt part of this thesis showed that cells cultured on collagen gel exhibited higher migration capacity than those cultured on collagen gel-coated dishes. Low rigidity of collagen gel induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collagen gel-induced ERK1/2 phosphorylation by MEK inhibitors or ERK2 kinase mutant induced a rounding up of the cells and prevented collagen gel-induced cell migration. Interestingly, phosphorylated ERK1/2 induced by low rigidity was present in focal adhesion sites and the lipid rafts. MbCD (Methyl-b-cyclodextrin), a lipid raft inhibitor, inhibited collagen gel-induced ERK1/2 phosphorylation and cell migration. Overexpression of FAK C-terminal fragment (FRNK) triggered ERK phosphorylation in MDCK cells. Meanwhile, low substratum rigidity induced degradation of FAK into a 35kDa C-terminal fragment. A calpain inhibitor that partially rescued FAK degradation also prevented low rigidity-induced ERK phosphorylation. However, MbCD did not prevent low rigidity-induced FAK degradation. Taken together, we demonstrate that the degradation product of FAK induced by collagen gel triggers activation of ERK1/2, which in turn facilitates cell spreading and migration through the lipid raft. The second part of this thesis showed that low rigidity of collagen gel down-regulates b1 integrin activation, clustering and FAKY397 phosphorylation, which is mediated by delayed raft externalization. Moreover, overexpression of auto-clustered b1 integrin (V737N) but not constitutively active b1 integrin (G429N) rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer (FRET) to assess b1 integrin clustering, we have found that substratum rigidity between 58-386 pascal (Pa) triggers b1 integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of b1 integrin clustering enhances the interaction between b1 integrin, FAK and talin. Taken together, our findings provide a new insight into how soft tissues sense their physical environment and the mode of action. In conclusion, this thesis provides the key to understand the fundamental mechanisms in how cells interact with physical environment.

碩士
國立成功大學
生理學研究所
96
Our previous studies have demonstrated that low-stiffness of collagen gel-induced epithelial cells apoptosis is mediated by deregulation of AP-1 proteins and endoplasmic reticulum stress-mediated disturbance of Ca2+ homeostasis. In order to elucidate to what extent and by what mechanism low rigidity induces apoptosis, we employed polyacrylamide (PA) gel to control substratum flexibility. We confirmed that low rigidity-induced apoptosis was only observed in epithelial cells. PA gel-induced apoptosis ratio was inversely correlated with substratum rigidity, and the threshold of substratum rigidity in triggering epithelial cell apoptosis was one thousand Pascal. In addition, low-rigidity of PA gel also induced c-Jun down-regulation, which is associated with c-Jun ubiquitination similar to the results observed by collagen gel. Interestingly, when cells were cultured on polyacrylamide gel, an increase in substratum rigidity augmented levels of c-Fos, JNK, and phosphorylation of JNK, unlike what was observed in cells cultured on collagen gel. Enhancement of substratum rigidity also up-regulated levels of c-Jun as well as phosphorylated c-Jun, which was dependent on JNK activation. In addition, augmentation of c-Jun phosphorylation by phosphatase inhibitor could prevent the degradation of c-Jun at various substratum rigidity. Taken together, these results indicate that low substratum rigidity triggers epithelial cell apoptosis through downregulation of JNK-c-Jun axis. As well as c-Jun ubiquitination and degradation, polyacrylamide gel elicits distinctive signal transduction mechanism from collagen gel.

Encore en 2015, un grand nombre d’individus décèdent de pathologies du rythme cardiaque non contrôlées ou d’un manque de disponibilité de donneurs d’organes compatibles. Le génie tissulaire en créant, réparant ou améliorant la fonction des tissus est une option prometteuse afin de diminuer la mortalité associée à ces pathologies. L’objectif global de mon projet de recherche était de développer des outils et d’étudier l’impact fonctionnel des différents stimuli (mécanique et électrique) de l’environnement cardiaque dans le but de définir des conditions optimisées de culture pour la fabrication de tissu de remplacement par génie tissulaire. Cette thèse présente le développement d’un bioréacteur; un système qui optimise les conditions pour la culture cellulaire. L’efficacité du bioréacteur est validée par des expériences de culture cellulaire qui se concentrent sur la prolifération cellulaire, l’organisation cellulaire, l’expression génique et protéique de même que sur l’activité contractile spontanée. En premier lieu, nos résultats montrent, bien que la fréquence de contraction moyenne mesurée reste inchangée, une augmentation significative du nombre de cas de réentrées pour les cultures sur verre comparativement aux cultures sur Polydimethylsiloxane. Une augmentation de l’instabilité spatiotemporelle a été démontrée lorsque les cardiomyocytes étaient déposés sur un support de Polydimethylsiloxane et cette dernière corrèle avec une diminution non-significative de l’ARNm de la connexine-43 et une augmentation significative de l’ARNm pour CaV3.1 et HCN2. La culture sur Polydimethylsiloxane est également associée avec une plus forte réponse à l’isoprotérénol (β-adrénergique) et à l’acétylcholine (parasympathique). En second lieu, nous présentons les résultats du développement de notre bioréacteur en mettant l’emphase sur les caractéristiques (composantes accessibles, étirement uniaxial, électrode de carbone, stimulation biphasique) tout en validant notre approche pour optimiser les conditions de culture et améliorer la rentabilité des étapes de production du tissu de remplacement. Pour finir, nous partageons une nouvelle approche d’évaluation des caractéristiques contractiles de cellules cardiaques en culture. Nous avons développé des algorithmes qui utilisent les données de vidéomicroscopie pour valider l’impact de stimuli, évaluer l’hétérogénéité du signal enregistré et détecter des conditions favorables au développement d’arythmies.
In 2015, there are still a large number of people who die due to diseases of uncontrolled heart rhythm or due to lack of availability of compatible donor organs. Tissue engineering aim to create, repair or improve the function by different techniques. Tissue engineering is a viable option to reduce the mortality associated with many heart conditions. The overall goal of my PhD research was to study the functional impact of different stimuli in cardiac environment (mechanical and electrical stimulation) on cardiac cell cultures. This, in order to define optimized culture conditions for the production of replacement tissue using tissue engineering. This thesis presents the stages of creation and development of a bioreactor; a system that permits the culture of cardiac cells by integrating various stimuli. The optimization of culture conditions by using the bioreactor was confirmed by cell culture experiments that focus on cell proliferation, cell organization, gene and protein expression as well as on spontaneous activity. In the first place, our results show that although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 (unsignificant) and a significant increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol (β-adrenergic) and acetylcholine (parasympathetic). Secondly, we present the design of our bioreactor with an emphasis on its characteristics and by putting in perspective the relevance of our approach to optimize culture conditions and to improve profitability culture experiences and production stages of replacement heart tissue. Finally, a new approach is proposed to evaluate the characteristics of the contractile cells in culture which allows to validate the functional impact of stimuli, evaluate the heterogeneity in the beating behavior of the cells and to detect localized abnormal activity that could favour arrhythmia.