Cytoskeletal tension plays an important role in numerous biological functions of adherent cells, including mechanosensing of the cell’s microenvironment, mechanotransduction, cell spreading and migration, cell shape stability, and in stem cell lineage. It is believed that for normal biological functions the cell must maintain its cytoskeletal tension stable, at a preferred set-point level, under external perturbations. This is known as tensional homeostasis. Any breakdown of tensional homeostasis is closely associated with disease progression, including cancer, atherosclerosis, and thrombosis. The exact mechanism and the relevant environmental conditions for the maintenance of tensional homeostasis are not yet fully understood. This thesis investigates the impacts of substrate stiffness, availability of functional cadherin junctions and steady shear stress on tensional homeostasis of cells and cell clusters. We define tensional homeostasis as the ability of cells to maintain a consistent level of tension with low temporal traction field fluctuations. Traction forces of isolated cells, multicellular clusters, and monolayer are measured using micropattern traction microscopy. Temporal fluctuations of the traction field are calculated from time-lapsed traction measurements. Results demonstrated that substrate stiffness, cadherin cell-cell junctions and shear stress all impact tensional homeostasis. In particular, we found that stiffer substrates promoted tensional homeostasis in endothelial cells, but were detrimental to tensional homeostasis in vascular smooth muscle cells. We also found that E-cadherins were essential for tensional homeostasis of gastric cancer cells and that extracellular and intracellular mutations of E-cadherin had domain-specific effects on tensional homeostasis. Finally, laminar flow-induced shear stress led to increased traction field fluctuations in endothelial cell monolayers, contrary to reports of physiological shear promoting vascular homeostasis. A possible reason for this discrepancy might be the limitation of our approach which could not account for mechanical balance of traction forces in the monolayers. Through the exploration of these environmental factors, we also found that tensional homeostasis was a length scale-dependent and cell type-dependent phenomenon. These insights suggest that future studies need to take a more comprehensive approach and aim to make observations of different cell types on multiple length scales, in order decipher the mechanism of tensional homeostasis and its role in (patho)physiology. / 2021-08-30T00:00:00Z

<p>The untethered transfer of energy and scalability of magnetic actuators enables functionality to an otherwise passive system. For example, wireless magnetic actuation can turn static 2D and 3D cell cultures into a more physiologically-relevant dynamic environment while limiting contamination. Moreover, indwelling catheters and implantable sensors are typically stationary devices that are notorious for their short lifespan when implanting into the body due to immune responses. Magnetic microactuators may be used for wireless actuation for in situ removal of biological materials accumulated on chronically implanted devices. In this dissertation, I will demonstrate examples of novel biomedical microdevices enabled by magnetic actuation for added functional benefits. First, I will describe a soft polymer magnetic actuator that can facilitate the study of a physiologically relevant cell culturing system. By cyclically stretching an extracellular matrix protein in a 3D cell culture, this system can elucidate the process by which breast cancer cells respond to a dynamic environment in the lungs. The fibrillar fibronectin suspended across the body of the magnetic actuator provides a matrix representative of early metastasis for 3D cell culture that has not yet been recapitulated in vitro until now. Our results demonstrate a clear suppressive cellular response due to cyclic stretching that has implications for a mechanical role in the dormancy and reactivation of disseminated breast cancer cells to macrometastases. As a second application, I will demonstrate the use of magnetic microactuators to remove biofouling on an implantable biosensor in order to prolong its functionality. The results of our work suggest that the motion of the actuator on the sensor surface can maintain biosensor signal integrity and prevents the downstream effects of the foreign body response. Additionally, I will present the design and proof of concept testing of a novel aspiration thrombectomy catheter meant to improve the engagement between the catheter and the blood clot being removed. Preliminary results demonstrate the added benefit of incorporating a microstructure in the inner diameter of the catheter meant to increase the retraction force aspiration catheters have when retrieving corked emboli at the catheter tip. </p> <p><br></p>

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No description available.

The human genome is characterized by an extent of functions that act further than its genetic role. Indeed, the genome can also affect cellular processes by nongenetic means through its physical and structural properties, specifically by exerting mechanical forces that shape nuclear morphology and architecture. The balancing between two chromatin compartments with antagonist functions, namely Transcriptional and Polycomb condensates, is required for preserving nuclear mechanical properties and its perturbation is causative of the pathogenic condition Kabuki syndrome (KS) (Fasciani et al., 2020). KS is a rare monogenic disease caused by the haploinsufficiency in the KMT2D gene encoding for MLL4, a H3K4-specific methyltransferase important for the regulation of gene expression. By interrogating the effect of KMT2D haploinsufficiency in Mesenchymal Stem Cells (MSCs) we discovered that MLL4 loss of function (LoF) impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture and the cell mechanoresponsiveness during differentiation (Fasciani et al., 2020). These results suggest that altered nuclear mechanics rely on chromatin architecture and could potentially lead to changes in cell responses to external mechanical stimuli. In the present work, we investigated the role of Transcriptional and Polycomb condensates in tuning nuclear responses to different external mechano-physical conditions. To affect nuclear mechanics, we employed the use of several mechanical devices (e. g. substrate stiffness, microchannels with constrictions, and cell confinement). We found that Polycomb and Transcriptional condensates are modulated by changes in substrate rigidity in healthy conditions and that MLL4 LoF impairs the MSCs nuclear condensates-driven mechanical response. Furthermore, we observed that MLL4 LoF impacts nuclear adaptation to confined spaces by incrementing susceptibility to nuclear envelope rupture. We also showed that the increased nuclear fragility in MLL4 LoF is accompanied by an alteration of cell migratory capacity and survival rate. Altogether these findings suggest that MLL4 LoF impairs cell responses to external mechanical stimuli, shedding light on the pathological connection between the altered cell mechanoresponsiveness during differentiation and KS phenotype in terms of skeletal and cartilage anomalies.

This study was motivated by a theoretical formulation on mechanobiology of soft and hard skeletal tissue differentiation. To prove this formulation experimentally, I hypothesized that cartilaginous phenotype can be induced in vitro in a seemingly non-cartilaginous cell source from fibrous tissue. In testing this hypothesis, I have focused on cartilage as a target and fibrous tissue as an origin or the source of cell. Four different trials were pursued with one supposition in common, i.e. hydrostatic pressure is one of the main driving forces for chondroinduction in vitro. The first and second trials pertained to the influence of a relatively short and long duration cyclic hydrostatic compression on rat Achilles tendon fibroblasts. The third trial was to examine the effect of two different drugs on cytoskeletal elements of mesenchymal stem cells or mouse embryonic fibroblast lines in pellet cultures combined with the similar duration and/or frequency of cyclic hydrostatic pressure adopted in the aforesaid trials with no pharmacological agents added. Last, attempts were made to implement an advanced technique in molecular biology called 'PCR array' to further quantify expression levels of eighty four pathway-specific genes in mouse TGFbeta/BMP signaling traffic under the same physiological regimen of hydrostatic compression. Results demonstrated that transdifferentation in phenotype from tendon to fibrocartilage may have occurred in vitro in tendon fibroblasts in pellet cultures exposed to hydrostatic pressure. Experiments on the role of the cytoskeleton in mechanotransduction of the applied level of hydrostatic pressure demonstrated that disruption of microfilaments in the presence of cytochalasin-D did not significantly interfere with the anabolic effect of cyclic pressure. However, disruption of microtubule assembly by nocodazole abolished the pressure-induced stimulation in cartilage marker genes. These findings suggest that microtubules, but not microfilaments, are involved in mechanotransduction of hydrostatic pressure by mesenchymal stem cells.

No description available.

No description available.

Metastasis is the leading cause of death among cancer patients. The metastatic cascade, during which cancer cells from the primary tumor reach a distant organ and form multiple secondary tumors, consists of a series of events starting with cancer cells invasion through the surrounding tissue of the primary tumor. Invading cells may perform proteolytic degradation of the surrounding extracellular matrix (ECM) and directed migration in order to disseminate through the tissue. Both of the mentioned processes are profoundly affected by several parameters originating from the tumor microenvironment (extrinsic) and tumor cells themselves (intrinsic). However, due to the complexity of the invasion process and heterogeneity of the tumor tissue, the exact effect of many of these parameters are yet to be elucidated. ECM proteolysis is widely performed by cancer cells to facilitate the invasion process through the dense and highly cross-linked tumor tissue. It has been shown in vivo that the proteolytic activity of the cancer cells correlates with the cross-linking level of their surrounding ECM. Therefore, the first part of this thesis seeks to understand how ECM cross-linking regulates cancer cells proteolytic activity. This chapter first quantitatively characterizes the correlation between ECM cross-linking and the dynamics of cancer cells proteolytic activity and then identifies ß1-integrin subunit as a master regulator of this process. Once cancer cells degrade their immediate ECM, they directionally migrate through it. Bundles of aligned collagen fibers and gradients of soluble growth factors are two well-known cues of directed migration that are abundantly present in tumor tissues stimulating contact guidance and chemotaxis, respectively. While such cues direct the cells towards a specific direction, they are also known to stimulate cell cycle progression. Moreover, due to the complexity of the tumor tissue, cells may be exposed to both cues simultaneously, and this co-stimulation may happen in the same or different directions. Hence, in the next two chapters of this thesis, the effect of cell cycle progression and contact guidance-chemotaxis dual-cue environments on directional migration of invading cells are assessed. First, we show that cell cycle progression affects contact guidance and not random motility of the cells. Next, we show how exposure of cancer cells to contact guidance-chemotaxis dual-cue environments can improve distinctive aspects of cancer invasion depending on the spatial conformation of the two cues. In this dissertation, we strive to achieve the defined milestones by developing novel mathematical and experimental models of cancer invasion as well as utilizing fluorescent time-lapse microscopy and automated image and signal processing techniques. The results of this study improve our knowledge about the role of the studied extrinsic and intrinsic cues in cancer invasion. / Bioengineering / Accompanied by fourteen .avi files.

Migratory cells in vivo move within a predominantly fibrous microenvironment through the action of forces. These dynamic interactions facilitate mechanosensing, critical to fundamental biological processes in pathophysiology. Naturally, the field of mechanobiology has evolved over the past several decades to decipher the role of forces in mechanotransduction using a variety of force-measurement platforms. A central challenge that has yet to be overcome in the field is connecting forces with the interplay between cell shape and ever-changing environment. Here, through design of specific fibrous architectures, a mechanobiological understanding of force feed-forward loop accounting for shape shifting of the environment and cells is developed. Using the non-electrospinning Spinneret Tunable Engineered Parameters (STEP) technique, two complementary force measurement platforms of varying physical attributes are developed to investigate how the force feed-forward loop impacts cell fate. Nanonet Force Microscopy (NFM) comprised of aligned nanonets is designed to study anisotropic cell shapes, while Crosshatch Force Microscopy (CM) comprised of orthogonal arrangement of fibers is designed to study cell bodies of broad shapes. The combination of shapes achieved on these networks recapitulate mesenchymal shapes observed in vivo, which are used to describe cell behaviors not reported before. The new findings include (i) discovery of a new biological structure, termed 3D-perpendicular lateral protrusions (3D-PLPs) which is proposed to be the missing biophysical link in the remodeling of the ECM and perpetuation of desmoplasia. Using NFM, seven discreet steps in formation of force-exerting PLPs anywhere along the cell body is documented, which allow cells to spread laterally and increase in contractility. Using a variety of fiber networks, it is shown that aligned fibers are necessary for PLP formation and suitable environments for myofibroblast activation, and (ii) a force dipole that links matrix deformability with cell contractility. Aided by machine learning, CFM automates the process of fiber feature recognition to measure forces as cells change shapes during migration and differentiate to osteogenic and adipogenic lineages. The force platforms are applied to investigate (i) the bioenergetic contributors fueling cellular migration and a surprisingly overwhelming impact of glycolytic energetic pathway over the traditionally thought mitochondrial energy production is found. However, neither pathway has substantial impact over the cellular force production, and (ii) quantitate the migratory and contractile response of enucleated cytoplasmic fragments naturally shed by cells. A peculiar contractility driven oscillatory migratory phenotype is found, capable of lasting over tens of hours, and absent in intact cells. Overall, new high spatiotemporal capabilities are developed in mechanobiology to quantitate the force-feed forward loops between cell shape and ECM in pathophysiology. / Doctor of Philosophy / Pathophysiology is the study of abnormal changes in the regular body functions of an organism that are causes or consequences of disease onset. Research in this area is mainly focused on identifying the different factors that cause and propagate the disease states such as cancer. Central to many of these processes are events such as cell migration and remodeling of their surrounding environment. The native microenvironment surrounding cells is highly complex and is composed of many classes of macromolecules, with fibrous components being one of the most important. How cells interact with these environments through application of forces and how this further regulates cellular behavior is vital to advancing our understanding of many of these pathophysiological processes. Currently, there is a lack in our understanding of how this dynamic process referred to as the "force feed-forward loop", is perpetuated. This limitation in our understanding can be attributed to the lack of an in vivo mimicking platform that captures this dynamic interaction and is capable of measuring the forces. To this end, the development of two novel single cell force measurement platforms: Nanonet Force Microscopy (NFM) and Crosshatch Force Microscopy (CFM) is presented. These platforms are fiber based systems, generated with the utilization of previously established non-electrospinning technique of Spinneret based Tunable Engineered Parameters (STEP) technique. Using NFM and CFM, forces were computed in wide range of cell shapes from anisotropic to all other spread morphologies. These platforms were applied to identify a new biological structure called perpendicular lateral protrusions and shown to have potential role in the spreading of tumor microenvironment. Furthermore, the force dynamics in physiological processes such as stem cell differentiation into fat cells or bone cells is also identified. How cellular processes such as migration and force production is fueled is also investigated and found to be not heavily reliant on the commonly understood mitochondrial activity. Finally, sub-cellular components known as cell fragments, which are devoid of nucleus, are also observed to be contractile and migratory in nature, independent of parent cell body. These platforms and findings can be further utilized to advance our current knowledge of the progression of these physiological and pathological processes and serve as diagnostic tools for the early identification of disease onset. Furthermore, based on these findings, strategies can be developed for early intervention to inhibit disease progression or devise bioengineered scaffolds for applications in tissue engineering.