Cancer remains a problem worldwide as one of the leading causes of morbidity and mortality. Many cancer patients experience recurrence and ultimately death due to treatment failure or the development of chemoresistance. The concept of chemoresistance however is complex, recent studies have highlighted that cellular structure and extra-cellular composition, mechanics and structure play a role in the development of chemoresistance. The mechanical properties of cells impact their architecture, migration patterns, intracellular trafficking and many other cellular functions. Studies have also revealed that cellular mechanical properties are modified during cancer progression. We investigated these mechanical properties and changes to them by using a malignant melanoma cell line (WM1158) and a chemoresistant malignant melanoma cell line (SK-MEL29). Malignant melanoma was the cell line of choice as it is one of the most prominent types of cancer known to develop chemoresistance. The aim of this study was to identify the effects of chemotherapeutic drug exposure on the mechanical properties and cytoskeletal composition of drug sensitive and drug resistant malignant melanoma cells. To achieve this, a combination of Multiple particle tracking microrheology (MPTM), quantitative RT-PCR and Western blotting techniques were utilised to demonstrate changes in cytoskeletal elements that are responsible for cellular mechanics. MPTM was developed as an approach to map intracellular mechanical properties of living cells and track the intracellular particles by Brownian motion to establish a viscoelastic model and compare it with the power-law approach. A quantification of the MPTM allowed capturing of the cell stiffness using the mean squared displacement (MSD) of cell under different conditions. The cytoskeletal elements actin and β-tubulin were analysed in qRT-PCR and Western blot as they form the key elements governing a cell’s mechanical stability and response to mechanical stimuli. The findings from this study revealed cell stiffness decreases as cancer progress, thereby cells become stiffer. The same pattern was evident for chemoresistant malignant cells and revealed that they had a loss of elasticity in comparison to their counter non-resistant malignant cells. With regards to protein levels and mRNA expression, the chemotherapeutic drug affected the cytoskeleton causing cells to undergo morphological changes which, however, was not seen in chemoresistant cells. The results from this study indicated that measuring mechanical properties of cells provides an efficient marker for cancer diagnosis and deeper understanding of cancer mechanobiology.

Cell migration plays an important role in numerous normal and pathological processes. The physical mechanisms of adhesion, protrusion/extension, contractions, and polarization can regulate cell migration speed, persistence time, and downstream effects in paracrine and endocrine signaling. Methods for understanding these biophysical and biochemical responses to date have been limited to the use of external forces acting on mechanotransductive receptors. Additionally, as the use of magnetic nanoparticles for cell tracking and cell manipulation studies continues to gain popularity, so does the importance of understanding the cellular response to mechanical forces caused by these magnetic systems. This thesis work utilizes superparamagnetic iron oxide nanoparticles and static magnets to induce an endogenous magnetic force on the cell membrane. This cell manipulation model is used to better understand the mechanobiological responses of mesenchymal stem cell to SPIO labeling and endogenous force generation. Directionally persistent motility, cytoskeletal reorganization, and altered pro-migratory cytokine secretion is reported in this thesis as a response to SPIO based cell manipulation.

Mechanical forces are fundamental regulators of biology. Individual cells can detect environmental forces and transform them into intracellular biochemical actions, which impact gene expression, metabolism, and differentiation. In turn, this phenomenon of “mechanotransduction” at the cellular level affects tissue- and organ-level function and can shape disease progressions. Tools that enable researchers to genetically harness mechanotransduction would therefore be powerful for developing of novel tissue engineering and cell therapy technologies. However, synthetically engineering mechanotransduction in cells has remained difficult. In this thesis, we control how cells respond to molecular forces by engineering modular mechanosensitive receptors. Using a structured-guided approach, we engineered force-sensitive protein domains that, when inserted into synthetic Notch receptors, vary the input-output relationship between mechanical force and cellular action. We demonstrate that the mechanical strength of these domains can be systematically tuned through mutagenesis. We show that our synthetic mechanoreceptors enable the design of signaling networks where tensile forces in the environment are recorded as measurable and specifiable biochemical responses, such as myogenic differentiation in mouse embryonic fibroblasts. We then present additional technologies for modulating the Notch mechanoreceptor’s endogenous mechanical strength, ligand-mediated activation, and protease-regulated activation. Taken together, this dissertation introduces a mechanogenetic framework for synthetically controlling mechanotransduction in mammalian cells, informs the design of future synthetic force-sensitive pathways, and provides valuable tools for the study of Notch signaling in development and disease. / 2022-09-30T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / The aging US population is experiencing a growing incidence of osteoporosis, characterized by increased fracture risk and low bone mass. In skeletal tissue, canonical Wnt signaling is a critical regulator of bone mass, and dysregulation of the Wnt pathway has been implicated in numerous skeletal displasias. Some components of the Wnt signaling pathway have a clear role in bone homeostasis, particularly in the response of bone to altered mechanical environment. Other pathway components are more poorly defined. One important intracellular signal transduction node in the Wnt cascade is β- catenin, which modulates gene expression and cell-cell junctions, among other functions. During periods of disuse, β-catenin is degraded, leading to inhibition of Wnt targets. Here, I characterize the role of β-catenin in bone during a disuse challenge, using a genetic mouse model expressing an inducible constitively-active mutant form of β- catenin in the osteocyte population. I hypothesize that prevention of β-catenin degradation during disuse will prevent the bone wasting effects of mechanodeprivation. As a second goal, I focus on upstream (membrane-bound) modulation of Wnt. Here, I investigate the low-density lipoprotein receptor-related receptor 4 (Lrp4), in the regulation of bone mass and mechanotransduction. I generated an Lrp4 knockin mouse model harboring a missense mutation found among human patients with abnormally high bone mass. I hypothesize that the mutation compromises sclerostin action on bone cells. Understanding how each of these components of the Wnt signaling pathway interact, may lead to novel therapeutic targets for treatment of bone diseases.

The aortic valve (AV) functions in arguably the most demanding mechanical environment in the body. The AV experiences fluid shear stress, cyclic pressure and mechanical strain in vivo. Recent evidence has shown the progression of degenerative aortic valve disease (AVD) to be an active cellular mediated process, altering the conception of the AV as a passive tissue. AVD has shown a strong correlation with altered hemodynamics and tissue mechanics. Aortic valve endothelial cells (AVECs) line the fibrosa (aortic facing) and ventricularis (left ventricle facing) surfaces of the valve. AVECs sense and respond to circulating stimuli in the blood stream while maintaining a non-thrombogenic layer. AVEC activation has been implicated in the initiation and progression of AVD, but the role of cyclic strain has yet to be elucidated. The hypothesis of this dissertation is that altered mechanical forces have a causal relationship with aortic valvular endothelial cell activation. To test this hypothesis 1) the role of in vitro cyclic strain in regulating expression of pro-inflammatory adhesion molecule was elucidated 2) cyclic strain-dependent activation of side-specific aortic valve endothelial cells was investigated 3) a novel stretch bioreactor was developed to dramatically increase the ability to correlate valvular endothelium response to physiologically relevant applied planar biaxial loads. The results from this study further the field of heart valve mechanobiology by correlating AVEC physiological and pathophysiological function to cellular and tissue level strain. Elucidating the AVEC response to an altered mechanical environment may result in novel clinical diagnostic and therapeutic approaches to the initiation and progression of degenerative AVD. Furthermore, a cardiovascular health outreach program, Bulldogs for Heart Health, has been designed and implemented to combat the startling rise in childhood obesity in the state of Mississippi. It is the hope that these results, novel methods, and outreach initiatives developed will significantly impact the study of the mechanobiology of the aortic valve endothelial cell and potential treatment and prevention of cardiovascular disease.

Human vocalization has always been considered in the context of communication. In recent years, mechanical forces have been shown to have a wide variety of effects on biological systems. Since the vocal cords produce measurable vibrations to the surrounding tissue, including the thyroid, I hypothesize that vocalization could mechanically stimulate thyroid epithelial cells. The data presented in this dissertation highlight some of the important factors for thyroid hormone synthesis. I was able to show that mechanical stimulation provided from vocalization has a similar response as hormonal stimulation on the thyroid. I also demonstrated that this response is dependent on the dosage of the oscillation. Insight into thyroid stimulation via mechanical forces may be useful for a range of medical issues spanning from hypothyroidism to astronauts travelling in space.

While tissue-engineering approaches of heart valves have made great strides towards creating functional tissues in vitro, the instruments used, named bioreactors, cannot efficiently integrate multiple stimuli to accurately emulate the physiological microenvironment. To address this, we conceptually designed and built a bioreactor system that applied a range of mechanical tension conditions, modulated matrix stiffness, and introduced biochemical signals in a combinatorial and high-throughput manner. Proof-of-concept experiments on PAVIC-seeded hydrogels were performed to assess the independent and combined effects of tensile strain, matrix stiffness and TGF-β1 on myofibroblast differentiation by measuring α-SMA expression, a marker that indicates a disease-associated phenotype. We found that matrix stiffness and TGF-β1 significantly increased α-SMA levels (p < 0.001), while the effect of mechanical strain was only significant on soft gels (~12 kPa) without TGF-β1. This study therefore demonstrated independent and integrated effects of multiple stimuli in regulating key cellular events in the aortic valve.

"Mechanical loads play a pivotal role in the growth, maintenance, remodeling, and disease onset in connective tissues. Harnessing the relationship between mechanical signals and how cells remodel their surrounding extracellular matrix would provide new insights into the fundamental processes of wound healing and fibrosis and also assist in the creation of custom-tailored tissue equivalents for use in regenerative medicine. In 3D tissue models, uniaxial cyclic stretch has been shown to stimulate the synthesis and crosslinking of collagen while increasing the matrix density, fiber alignment, stiffness, and tensile strength in the direction of principal stretch. Unfortunately, the profound fiber realignment in these systems render it difficult to differentiate between passive effects and cell-mediated remodeling. Further, these previous studies generally focus on a single level of stretch magnitude and duration, and they also investigate matrix remodeling under a homogeneous strain conditions. Therefore, these studies are not sufficient to establish key information regarding stretch-dependent remodeling for use in tissue engineering and also do not simulate the complex mechanical environment of connective tissue. We first developed a novel in vitro model system using equibiaxial stretch on fibrin gels (early models of wound healing) that enabled the isolation of mechanical effects on cell-mediated matrix remodeling. Using this system we demonstrated that in the absence of in-plane alignment, stretch stimulates fibroblasts to produce a stronger tissue by synthesizing collagen and condensing their surrounding matrix. We then developed dose-response curves for multiple aspects of tissue remodeling as a function of stretch magnitude and duration (intermittent versus continuous stretch). Our results indicate that both the magnitude and the duration per day of stretch are important factors in mechanically induced cell activity, as evidenced by dose-dependent responses of several remodeling metrics in response to these two parameters (UTS, matrix stiffness, collagen content, cell number). In addition, we found that cellularity, collagen content, and resistance to tension increased when the tissues were mechanically loaded intermittently as opposed to continuously. Finally, we developed a novel model system that produces non-homogeneous strain distribution, allowing for the simultaneous study of strain gradients, strain anisotropy, and strain magnitude in 2D and 3D. Establishing a system that produces complex strain distributions provides a more accurate model of the mechanical conditions found in connective tissue, and also allows for the investigation of cellular adaptations to a changing mechanical environment. "

While tissue-engineering approaches of heart valves have made great strides towards creating functional tissues in vitro, the instruments used, named bioreactors, cannot efficiently integrate multiple stimuli to accurately emulate the physiological microenvironment. To address this, we conceptually designed and built a bioreactor system that applied a range of mechanical tension conditions, modulated matrix stiffness, and introduced biochemical signals in a combinatorial and high-throughput manner. Proof-of-concept experiments on PAVIC-seeded hydrogels were performed to assess the independent and combined effects of tensile strain, matrix stiffness and TGF-β1 on myofibroblast differentiation by measuring α-SMA expression, a marker that indicates a disease-associated phenotype. We found that matrix stiffness and TGF-β1 significantly increased α-SMA levels (p < 0.001), while the effect of mechanical strain was only significant on soft gels (~12 kPa) without TGF-β1. This study therefore demonstrated independent and integrated effects of multiple stimuli in regulating key cellular events in the aortic valve.

No description available.