Airway remodeling characteristic of asthma involves structural changes altering the elasticity of the airway smooth muscle (ASM) microenvironment potentially leading to ASM dysfunction. This effect of matrix stiffness was investigated using a physiologically relevant 3D culture model. Characterisation of microtissue responses with regards to contractile function and gene expression were studied varying the ECM stiffness and through stimulation with epithelial cell (AEC) conditioned media. ASM microtissues were fabricated under four different loading conditions and the matrix stiffness was increased by crosslinking through non-enzymatic glycation and increasing the collagen density. Function was assessed through the use of pharmacological agents and by imaging microcantilever deflection, used to calculate force generation. Crosslinking microtissues enhanced contractile function in response to agonists; however, this effect disappeared in microtissues tethered to stiff microcantilevers suggesting a limit of contractility within this model. Remarkably, there was a differential response in ASM function where increasing the collagen density (stiffness) significantly attenuated function. Additionally, contractility was significantly enhanced when chronically stimulated with AEC media. ASM tissue in 3D culture is responsive to the microenvironment stiffness and increases contractility in the presence of a stiffer ECM. This could occur with thickening of the airway wall in asthma. Decreased contractility with increased collagen density is in agreement with previous studies where it was shown that type I collagen is pro-proliferative and attenuates the contractile phenotype. We show the models ability to quantitatively demonstrate the impact of biomechanical cues on ASM function providing provides new ways to elucidate the mechanisms of cellular remodeling.

Cells live in inherently noisy environments. The machinery that cells use to sense their environment is also noisy. Yet, cells are exquisite environmental sensors, often approaching the limits of what is physically possible. This thesis investigates how the precision of environmental sensing is improved when cells behave collectively. We derive physical limits to cells' ability to collectively sense and respond to chemical concentrations and gradients. For concentration sensing, we find that when cell populations become sufficiently large, long-range communication can provide higher sensory precision than short-range communication, and that the optimal cell-cell separation in such a system can be large, due to a tradeoff between maintaining communication strength and reducing signal cross-correlations. We also show that concentration profiles formed diffusively are more precise for large profile lengths while those formed via directed transport are more precise for short profile lengths. These effects are due to increased molecule refresh rate and mean concentration respectively. For gradient sensing, we derive the sensory precision of the well-known the local excitation-global inhibition (LEGI) model and the more recently proposed regional excitation-global inhibition (REGI) model for two and three dimensional cell cluster geometries. We find that REGI systems achieve higher levels of precision than LEGI systems and give rise to optimally sensing geometries that are consistent with the shapes of naturally occurring gradient-sensing cell populations. Lastly, we analyze the precision with which migrating cell clusters can track a chemical gradient via an individual-based and emergent method. We show that one and two dimensional clusters utilizing the emergent chemotactic method have improved scaling with population size due to differences in the scaling properties of the variance in the total polarization. By completing these studies we aim to understand the limits and precise roles of collective behavior in environmental sensing.

Therapeutics targeting the PI3K (phosphatidylinositol 3-kinase) and the Ras/MAPK (mitogen-activated protein kinases) pathways have potential as non-toxic treatments for triple-negative breast cancer due to their frequent over-activation in several forms of cancer. Interestingly, the PI3K and Ras/MAPK pathways have been shown to incite cancer dormancy behavior individually and tumorigenic behavior in unison when induced in healthy breast epithelial cells (MCF-10A) in vivo. Tumorigenesis and metastasis are heavily reliant on the specific mechanical and adhesive properties of cells, including decreased stiffness, increased mechanosensitivity, and decreased adhesion. However, the describe cellular behaviors are poorly understood for dormant cancer phenotypes. Understanding the mechanical and adhesive behaviors of MCF-10A cells as a function of PI3K and/or Ras/MAPK pathway over-activation further explores the cross-talk enabling unique dormant and tumorigenic characteristics. Cellular viscoelasticity and adhesion were measured for MCF-10A cells with PTEN (phosphatase and tensin homolog) knockout and activated KRAS (Kristen rat sarcoma viral oncogene homolog) overexpression to activate the PI3K and Ras/MAPK pathways respectively with atomic force microscopy. PTEN knockout alone has no observable influence on cell adhesion but resulted in softer cells with less organized cytoskeleton. Activated KRAS overexpression increased cell stiffness and cell adhesion regardless of PTEN expression level. Moreover, the overexpression of activated KRAS enhanced the sensitivity of cells to the substrate stiffness. The findings suggest that the cancer-associated pathways PI3K and Ras/MAPK regulate cell adhesion and mechanics to promote tumor formation and metastasis. More importantly, the results that signify mutations of different molecular pathways associated with cancer dormancy regulate cell mechanics differently suggests that cell stiffness is a biomarker that detects and differentiates different types of dormant cancers.

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.

Du fait de sa complexité, le fonctionnement du cerveau est exploré par des méthodes très diverses, telles que la neurophysiologie et les neurosciences cognitives, et à des échelles variées, allant de l'observation de l'organe dans son ensemble jusqu'aux molécules impliquées dans les processus biologiques. Ici, nous proposons une étude à l'échelle cellulaire qui s'intéresse à deux briques élémentaires du cerveau : les neurones et les cellules gliales. L'approche choisie est la biophysique, de part les outils utilisés et les questions abordées sous l'angle de la physique. L'originalité de ce travail est d'utiliser des cellules primaires du cerveau dans un souci de proximité avec l'in vivo, au sein de systèmes in vitro dont la structure chimique et physique est contrôlé à l'échelle micrométrique. Utilisant les outils de la microélectronique pour un contrôle robuste des paramètres physico-chimiques de l'environnement cellulaire, ce travail s'intéresse à deux aspects de la biologie du cerveau : la polarisation neuronale, et la sensibilité des cellules gliales aux propriétés mécaniques de leur environnement. A noter que ces deux questions sont étroitement imbriquées lors de la réparation d'une lésion. La première est cruciale pour la directionalité de la transmission de signaux électriques et chimiques et se traduit par une rupture de symétrie dans la morphologie du neurone. La seconde intervient dans les mécanismes de recolonisation des lésions, dont les propriétés mécaniques sont altérées., Les études quantitatives menées au cours de cette thèse portent essentiellement sur la phénoménologie de la croissance de ces deux types de cellules et leur réponse à des contraintes géométriques ou mécaniques. L'objectif in fine est d'élucider quelques mécanismes moléculaires associés aux modifications de la structure cellulaire et donc du cytosquelette. Un des résultats significatifs de ce travail est le contrôle de la polarisation neuronale par le simple contrôle de la morphologie cellulaire. Ce résultat ouvre la possibilité de développer des architectures neuronales contrôlées in vitro à l'échelle de la cellule individuelle. / The complex structure of the brain is explored by various methods, such as neurophysiology and cognitive neuroscience. This exploration occurs at different scales, from the observation of this organ as a whole entity to molecules involved in biological processes. Here, we propose a study at the cellular scale that focuses on two building elements of brain: neurons and glial cells. Our approach reachs biophysics field for two main reasons: tools that are used and the physical approach to the issues. The originality of our work is to keep close to the in vivo by using primary brain cells in in vitro systems, where chemical and physical environments are controled at micrometric scale. Microelectronic tools are employed to provide a reliable control of the physical and chemical cellular environment. This work focuses on two aspects of brain cell biology: neuronal polarization and glial cell sensitivity to mechanical properties of their environment. As an example, these two issues are involved in injured brains. The first is crucial for the directionality of the transmission of electrical and chemical signals and is associated to a break of symmetry in neuron morphology. The second occurs in recolonization mechanisms of lesions, whose mechanical properties are impaired. During this thesis, quantitative studies are performed on these two cell types, focusing on their growth and their response to geometrical and mechanical constraints. The final aim is to elucidate some molecular mechanisms underlying changes of the cellular structure, and therefore of the cytoskeleton. A significant outcome of this work is the control of the neuronal polarization by a simple control of cell morphology. This result opens the possibility to develop controlled neural architectures in vitro with a single cell precision.