<p>Complications from cardiovascular disease, such as a heart attack or a stroke, represent the leading cause of death in the United States and many developed and developing countries. Atherosclerosis is the primary pathology underlying cardiovascular disease. It is caused by an increase in endothelial cell (EC) permeability, leading to the buildup of cholesterol and lipids which form the atherosclerotic plaque. Accelerated aging in regions of atherosclerosis contribute to the development and progression of the disease. The global hypothesis that motivated this research was that activation of deacetylase antioxidant regulator, Sirtuin1 (SIRT1), improved function in senescent endothelial cells ECs by increasing the integrity of cell-cell junctions. In turn, this led to elevated EC permeability, a decline in the response to shear stress, and elevated traction forces.</p><p>Aging of hCB-ECs significantly increased permeability due to changes in tight junction protein localization and phosphorylation. Activation of SIRT1 significantly reduced permeability in aged hCB-ECs and restored junction integrity. ECs under flow also exhibited changes in cell junctions with increasing age. Aged hCB-ECs were less responsive to shear stress, exhibiting lower levels of atheroprotective genes, KLF2 and eNOS. Activation of SIRT1 in aged hCB-ECs restored the response to shear stress by altering localization and phosphorylation of adherens junction protein, VE-cadherin. The endothelial glycocalyx is a layer of proteoglycans and glycoproteins on the surface of ECs that is important in maintaining EC barrier function. In aging ECs, the glycocalyx was thinner and less dense. However, activation of SIRT1 restored the structure of the glycocalyx, suggesting that the positive effect of SIRT1 on elevated permeability in aged hCB-ECs may also be due to restoration of the glycocalyx. Aged hCB-ECs also exhibited elevated traction forces for measurements done with single cells, cell clusters (2 to 3 cells), and cell monolayers (20 to 30 cells). The elevated traction forces correlated with altered actin localization and increased actin filament thickness. Activation of SIRT1 reduced traction forces and decreased actin stress fiber thickness in aged hCB-ECs, suggesting that the effects of SIRT1 on cell-cell junctions alters binding of junction proteins to the actin cytoskeleton and generation of cell traction forces. Together, these results implicate an important role for SIRT1 in regulating permeability and mechanotransduction in aging endothelium.</p> / Dissertation

No description available.

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.

This thesis intends to utilize biomechanics to study the endothelial biomechanical response in a static hyperglycemic microenvironment. Hyperglycemia is a diabetic condition with abnormally high levels of glucose in the bloodstream. The effects of hyperglycemia over time lead to vascular complications resulting in patients being more prone to cardiovascular diseases. Current studies have focused on the molecular mechanisms affected by hyperglycemia; however, the mechanical mechanisms by which hyperglycemia causes vascular structural and functional changes are understudied. Therefore, to study the effects of hyperglycemia in the endothelium, Human Umbilical Vein Endothelial Cells (HUVEC) were cultured under three glucose conditions: normal glucose (4 mmol/l D-glucose), high glucose (30 mmol/l D-glucose), and an osmotic control (4 mmol/l D-glucose + 26 mmol/l D-mannitol). To evaluate the biomechanical response, we used traction force microscopy and monolayer stress microscopy to measure the cell-substrate tractions and cell-cell intercellular stresses. For the RMS tractions, HUVEC monolayers exposed to high glucose decreased by 10%, while the osmotic control decreased by 17% compared to the normal glucose. HUVEC monolayers exposed to high glucose produced average normal stresses that were 53% lower than monolayers exposed to normal glucose, while the osmotic control decreased by 51% compared to the normal glucose. For the maximum shear stresses, HUVEC monolayers exposed to high glucose decreased by 20%, while the osmotic control decreased by 14% compared to the normal glucose. To conclude this study, we report that hyperglycemia lowers the biomechanical response in the endothelium compared to normal glucose conditions. These results will contribute to understanding the specific role hyperglycemia has on endothelial mechanics and its role in the progression and development of cardiovascular diseases in diabetic patients.

L’originalité du sujet de thèse, initié lors du stage de M2R consiste à mesurer les propriétés de mobilité des molécules d’adhérence de cellules mécaniquement contrôlées. Le contrôle des propriétés géométriques et mécaniques du substrat seront fixées grace a l'utilisation d'une lamelle de verre comprenant des motifs de matrice extracellulaire. Nous utiliserons plusieurs techniques de mesures de mobilités, permettant d'accèder à des échelles temporelles d'étude différentes ; La FCS permettant d'accèder au dynamique rapide ; Le FRAP pour accèder au dynamique lente. / The originality of the project, initiated during the M2 internship, consist to measure the mobility of adhesive molecules of cells mechanically controlled.This control will be fixed thanks to a coverslip with adhesive protein pattern. We will next use different technics of mobility measurement to have information about different time scale : FCS for fast dynamics, FRAP for slow dynamics.

The primary purpose of this study is to perform accurate dynamic measurements on a scaled roller rig designed and constructed by Virginia Tech and the Federal Railroad Administration (VT-FRA Roller Rig). The study also aims at determining the effect of naturally generated third-body layer deposits (because of the wear of the wheel and/or roller) on creep or traction forces. The wheel-rail contact forces, also referred to as traction forces, are critical for all aspects of rail dynamics. These forces are quite complex and they have been the subject of several decades of research, both in experiments and modeling. The primary intent of the VT-FRA Roller Rig is to provide an experimental environment for more accurate testing and evaluation of some of the models currently in existence, as well as evaluate new hypothesis and theories that cannot be verified on other roller rigs available worldwide. The Rig consists of a wheel and roller in a vertical configuration that allows for closely replicating the boundary conditions of railroad wheel-rail contact via actively controlling all the wheel-rail interface degrees of freedom: angle of attack, cant angle, normal load and lateral displacement, including flanging. The Rig has two sophisticated independent drivelines to precisely control the rotational speed of the wheels, and therefore their relative slip or creepage. The Rig benefits from a novel force measurement system, suitable for steel on steel contact, to precisely measure the contact forces and moments at the wheel-rail contact. Experimental studies are conducted on the VT ��" FRA Roller Rig that involved varying the angle of attack, wheel and rail surface lubricity condition (i.e., wet vs. dry rail), and wheel wear, to study their effect on wheel-rail contact mechanics and dynamics. The wheel-rail contact is in between a one-fourth scale AAR-1B locomotive wheel and a roller machined to US-136 rail profile. A quantitative assessment of the creep-creepage measurements, which is an important metric to evaluate the wheel-rail contact mechanics and dynamics, is presented. A MATLAB routine is developed to generate the creep-creepage curves from measurements conducted as part of a broad experimental study. The shape of the contact patch and its pressure distribution have been discussed. An attempt is made to apply the results to full-scale wheels and flat rails. The research results will help in the development of better simulation models for non-Hertzian contact and non-linear creep theories for wheel-rail contact problems that require further research to more accurately represent the wheel-rail interaction. / MS / Rail vehicles are supported, steered, accelerated, and decelerated by contact forces acting in extremely small wheel-rail contact areas. The behavior of these forces is quite complex and a broad interdisciplinary research is needed to understand and optimize the contact mechanics and dynamics problem. Key industry issues, such as control of Rolling Contact Fatigue (RCF), maximizing wheelset mileages, and minimizing the impact of rolling stock on the infrastructure, are directly related to the interaction at the wheel-rail contact. The Rig consists of a wheel and roller in a vertical configuration that allows for closely replicating the boundary conditions of railroad wheel-rail contact via actively controlling all the wheel-rail interface degrees of freedom: angle of attack, cant angle, normal load and lateral displacement, including flanging. The Rig has two sophisticated independent drivelines to precisely control the rotational speed of the wheels, and therefore their relative slip or creepage. The Rig benefits from a novel force measurement system, suitable for steel on steel contact, to precisely measure the contact forces and moments at the wheel-rail contact. The primary purpose of this study is to perform accurate dynamic measurements on a scaled roller rig designed and constructed by Virginia Tech and the Federal Railroad Administration (VT-FRA Roller Rig). Experimental studies are conducted on the VT – FRA Roller Rig that involved varying the angle of attack, the wheel and rail surface lubricity condition (i.e., wet vs. dry rail), and the wheel wear to study their effects on wheel-rail contact mechanics and dynamics. The wheel-rail contact is in between a one-fourth scale AAR-1B locomotive wheel and a roller machined to US-136 rail profile. A quantitative assessment of the creep-creepage measurements, which is an important metric to evaluate the wheel-rail contact mechanics and dynamics, is presented. A MATLAB routine is developed to generate the creep-creepage curves from measurements conducted as part of a broad experimental study. The shape of the contact patch and its pressure distribution have been discussed. An attempt is made to apply the results to full-scale wheels and flat rails. The research results will help in the development of better simulation models for non-Hertzian contact and non-linear creep theories for wheel-rail contact problems that require further research to more accurately represent the wheel-rail interaction.

Die Untersuchung von Zell-Material-Wechselwirkungen ist bedeutsam für die Entwicklung innovativer Biomaterialien, wobei aus biophysikalischer Sicht der Einfluss mechanischer Eigenschaften auf das Zellverhalten, d.h., die Mechanotransduktion, von besonderem Interesse ist. Für diese Dissertation wurden humane Endothelzellen aus der Nabelschnurvene zur Adhäsion auf Polyacrylamidhydrogele (PAA-Hydrogele) gegeben, die mit einer Maleinsäurecopolymer-Beschichtung versehen waren. Für Experimente unter veränderlichen Substrateigenschaften wurden die Steifigkeit der PAA-Hydrogele und die Ligandenaffinität der Beschichtung variiert. Der erste Teil der Dissertation umfasste die Charakterisierung der beschichteten PAA-Hydrogele. Dafür wurde der Elastizitätsmodul gemessen und die Adsorption von Fibronektin untersucht. Im zweiten Teil der Dissertation wurden die PAA-Hydrogele in der Zellzugkraftmikroskopie während der initialen Zelladhäsion (2 h) verwendet. Dabei stellte sich heraus, dass zwar die finale Zellfläche unabhängig von den Substratparametern war, aber die Ausbreitung von Zellen mit zunehmender Steifigkeit und Ligandenaffinität schneller ablief. Außerdem waren der Anstieg und die Plateauwerte der Zellzugkräfte auf steiferen Substraten größer. Die Steifigkeitsabhängigkeit lässt sich aus der Dehnungsversteifung des Aktinzytoskeletts unter Wirkung einer Spannung erklären. Eine Zunahme der Ligandenaffinität führte ebenfalls zu einer schnelleren Zunahme und größeren Plateauwerten von Gesamtzellzugkräften. Diese Beobachtung kann der Zunahme von Reibungskräften zugesprochen werden. Im letzten Teil der Dissertation sollten die biophysikalischen Ergebnisse durch die Untersuchung intrazellulärer Signalprozesse zusätzlich unterlegt werden. Dafür wurde die Entwicklung von Adhäsionsstellen durch eine immunzytochemische Färbung untersucht. Obwohl diese aufgrund der technischen Herausforderungen keine umfassenden Aussagen liefern konnte, deuteten sich einige Korrelationen, z.B. eine schnellere Entwicklung der Adhäsionsstellen auf steiferen Substraten, an. Die Ergebnisse der Dissertation ordnen sich in den aktuellen Forschungsstand zur Mechanotransduktion von Zellen ein und konnten in Bezug auf die Adhäsionsdynamik neue Erkenntnisse beisteuern. Vor allem der Stellenwert dissipativer Beiträge zu Zell-Substrat-Wechselwirkungen (z.B. Ligandenreibung) wurde unterstrichen. Diese sind in der Entwicklung neuer Biomaterialien mit spezifischen viskoelastischen Eigenschaften von besonderer Bedeutung. / The investigation of cell-substrate-interactions is of great importance for the development of innovative biomaterials. The influence of thematerials mechanical properties on cells and their functions, i. e., the process of mechanotransduction, is of particular interest from a biophysical point of view. In this dissertation human umbilical cord vein endothelial cells were seeded onto polyacrylamide hydrogels which had been modified by a maleic acid copolymer coating. To tune the mechanical properties of the substrate the hydrogels’ stiffness and the affinity of the coatings to the adhesion ligand fibronectin were variied. The first part of the dissertation is concerned with the characterization of the coated polyacrylamide hydrogels. The hydrogels’ Young’s modulus was measured and the adsorption of fibronectin was investigated. In the second part of the dissertation these cell culture scaffolds were used for cell traction force microscopy during the first two hours of cell adhesion. Although maximum cell area was not influenced by substrate parameters, cell spreading was faster for higher stiffness and higher ligand affinity. Traction force increase as well as plateau forces were higher on stiff substrates. The dependence of the dynamics of area and traction force on stiffness and their respective magnitudes after saturation could be related to properties of the actin cytoskeleton under stress. The increase in ligand affinity also led to a faster increase and higher mean plateau values of the total cell force. This observation was assigned to increasing friction forces between ligands and polymer coating. In the last part of the dissertation possible correlations between cell traction forces and intracellular signalling processes were examined. The development of adhesion sites during early cell adhesion was investigated by immunocytochemical staining. Due to technical reasons no comprehensive investigation could be realized, but nevertheless some correlations were observed, such as a faster adhesion site formation with higher stiffness. The results of this dissertation add to the current state of research regarding mechanotransduction of cells and yield new findings regarding to cell adhesion dynamics. Most notably viscous contributions to cell-substrate-interactions (i.e., ligand friction) were shown to influence cell behavior. This highlights that a thorough understanding of viscous processes is of utmost significance for the development of new biomaterials with specific viscoelastic properties.

Die Untersuchung von Zell-Material-Wechselwirkungen ist bedeutsam für die Entwicklung innovativer Biomaterialien, wobei aus biophysikalischer Sicht der Einfluss mechanischer Eigenschaften auf das Zellverhalten, d.h., die Mechanotransduktion, von besonderem Interesse ist. Für diese Dissertation wurden humane Endothelzellen aus der Nabelschnurvene zur Adhäsion auf Polyacrylamidhydrogele (PAA-Hydrogele) gegeben, die mit einer Maleinsäurecopolymer-Beschichtung versehen waren. Für Experimente unter veränderlichen Substrateigenschaften wurden die Steifigkeit der PAA-Hydrogele und die Ligandenaffinität der Beschichtung variiert. Der erste Teil der Dissertation umfasste die Charakterisierung der beschichteten PAA-Hydrogele. Dafür wurde der Elastizitätsmodul gemessen und die Adsorption von Fibronektin untersucht. Im zweiten Teil der Dissertation wurden die PAA-Hydrogele in der Zellzugkraftmikroskopie während der initialen Zelladhäsion (2 h) verwendet. Dabei stellte sich heraus, dass zwar die finale Zellfläche unabhängig von den Substratparametern war, aber die Ausbreitung von Zellen mit zunehmender Steifigkeit und Ligandenaffinität schneller ablief. Außerdem waren der Anstieg und die Plateauwerte der Zellzugkräfte auf steiferen Substraten größer. Die Steifigkeitsabhängigkeit lässt sich aus der Dehnungsversteifung des Aktinzytoskeletts unter Wirkung einer Spannung erklären. Eine Zunahme der Ligandenaffinität führte ebenfalls zu einer schnelleren Zunahme und größeren Plateauwerten von Gesamtzellzugkräften. Diese Beobachtung kann der Zunahme von Reibungskräften zugesprochen werden. Im letzten Teil der Dissertation sollten die biophysikalischen Ergebnisse durch die Untersuchung intrazellulärer Signalprozesse zusätzlich unterlegt werden. Dafür wurde die Entwicklung von Adhäsionsstellen durch eine immunzytochemische Färbung untersucht. Obwohl diese aufgrund der technischen Herausforderungen keine umfassenden Aussagen liefern konnte, deuteten sich einige Korrelationen, z.B. eine schnellere Entwicklung der Adhäsionsstellen auf steiferen Substraten, an. Die Ergebnisse der Dissertation ordnen sich in den aktuellen Forschungsstand zur Mechanotransduktion von Zellen ein und konnten in Bezug auf die Adhäsionsdynamik neue Erkenntnisse beisteuern. Vor allem der Stellenwert dissipativer Beiträge zu Zell-Substrat-Wechselwirkungen (z.B. Ligandenreibung) wurde unterstrichen. Diese sind in der Entwicklung neuer Biomaterialien mit spezifischen viskoelastischen Eigenschaften von besonderer Bedeutung. / The investigation of cell-substrate-interactions is of great importance for the development of innovative biomaterials. The influence of thematerials mechanical properties on cells and their functions, i. e., the process of mechanotransduction, is of particular interest from a biophysical point of view. In this dissertation human umbilical cord vein endothelial cells were seeded onto polyacrylamide hydrogels which had been modified by a maleic acid copolymer coating. To tune the mechanical properties of the substrate the hydrogels’ stiffness and the affinity of the coatings to the adhesion ligand fibronectin were variied. The first part of the dissertation is concerned with the characterization of the coated polyacrylamide hydrogels. The hydrogels’ Young’s modulus was measured and the adsorption of fibronectin was investigated. In the second part of the dissertation these cell culture scaffolds were used for cell traction force microscopy during the first two hours of cell adhesion. Although maximum cell area was not influenced by substrate parameters, cell spreading was faster for higher stiffness and higher ligand affinity. Traction force increase as well as plateau forces were higher on stiff substrates. The dependence of the dynamics of area and traction force on stiffness and their respective magnitudes after saturation could be related to properties of the actin cytoskeleton under stress. The increase in ligand affinity also led to a faster increase and higher mean plateau values of the total cell force. This observation was assigned to increasing friction forces between ligands and polymer coating. In the last part of the dissertation possible correlations between cell traction forces and intracellular signalling processes were examined. The development of adhesion sites during early cell adhesion was investigated by immunocytochemical staining. Due to technical reasons no comprehensive investigation could be realized, but nevertheless some correlations were observed, such as a faster adhesion site formation with higher stiffness. The results of this dissertation add to the current state of research regarding mechanotransduction of cells and yield new findings regarding to cell adhesion dynamics. Most notably viscous contributions to cell-substrate-interactions (i.e., ligand friction) were shown to influence cell behavior. This highlights that a thorough understanding of viscous processes is of utmost significance for the development of new biomaterials with specific viscoelastic properties.