UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
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Item The Effect of Glycemic Condition on Substrate Stiffness-Mediated Mechanosensitivity in Macrophages(2023) Johnson, Courtney Dashawn; Aranda-Espinoza, Helim; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Diabetes is a disease that plagues over 463 million people globally. About 40 million of these patients have Type 1 diabetes, and the global incidence is increasing up to 5% per year. Type 1 diabetes is characterized by the body's immune system targeting the pancreas with antibodies, leading to a disruption in insulin production. While existing treatments, such as exogenous insulin injections, are both successful and popular, the exorbitant insulin costs and need for meticulous administration are driving factors for alternative long-term solutions to glucose dysregulation caused by diabetes. Encapsulated islet transplantation (EIT) is a tissue-engineered solution to address the challenges raised by diabetes. Donor islets are encapsulated in a semi-permeable hydrogel, allowing the diffusion of oxygen, glucose, and insulin but preventing leukocyte infiltration. Despite its successes in small animal models, EIT is still far from commercialization due to the requirements of long-term systemic immunosuppressants and consistent immune rejection from the foreign body response. While most published research has focused on tailoring the characteristics of the capsule material to promote clinical viability, many studies have had a limited scope centered on biochemical changes. Current mechanobiology studies on the effect of substrate stiffness on the function of leukocytes, especially macrophages – primary foreign body response orchestrators, show promise in tailoring a favorable response to tissue-engineered therapies such as EIT. However, before successful integration into the EIT design, it is imperative to determine the impact of external glucose concentrations on substrate stiffness-mediated mechanosensitivity. Glucose plays a critical role in macrophage functionality, impairing or enhancing their function in certain situations. Immunometabolism literature demonstrates that decreasing or inhibiting the uptake of glucose via impairing glycolysis can result in a significant decline in functionality in macrophages. Patients suffering from diabetes experience dysregulation in glycemic maintenance, ranging from hypo-, normo-, and hyperglycemic conditions. As a result, it is imperative to assess whether these changes in external glucose conditions will affect macrophage mechanosensitivity in response to EIT biomaterials to use substrate stiffness as a design parameter for EIT effectively This project investigates the role of glycemic conditions on macrophage mechanosensitivity, considering substrate stiffness factors including morphology, phenotype, phagocytic and inflammatory functionality. These parameters aim to mimic the early stages of foreign body response, particularly the initiation and acute inflammation phases. This work demonstrates that glycemic condition significantly influences the severity of substrate stiffness-mediated mechanosensitivity in reference to macrophage phagocytosis and pro-inflammatory functionality. This study serves to advance the understanding of macrophage functionality, bridging the fields of mechanobiology and immunometabolism. Understanding the role of glycemic conditions on substrate stiffness-mediated mechanosensitivity will assist in EIT design to enhance the clinical viability of the therapy and prevent immune rejection by pericapsular fibrotic overgrowth.Item Quantitative Phenotyping of Brain Endothelial Cell-Cell Junctions for Physiological and Pathophysiological Applications(2019) Gray, Kelsey M; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The integrity of endothelial cell-cell junctions is required for the maintenance of normal physiological processes. The expression of junctional proteins is particularly important in the endothelial cells of the blood-brain barrier (BBB), the cellular unit that protects the brain via regulated transport between the peripheral blood and the central nervous system. Dysfunction of the BBB is linked with decreased junctional protein localization and is implicated in several diseases including Alzheimer’s disease and multiple sclerosis. On the other hand, the tight junctions of the BBB impede the delivery of medications targeting the brain. Therefore, understanding the key players driving junction stability could hold significant promise for therapeutic discovery and drug delivery applications. Despite this, the mechanisms underlying junction disruption aren’t fully understood. While several studies have linked different junction protein patterns with altered barrier function, the quantification of this parameter remains limited due to the lack of efficient measurement techniques. Here, we aimed to investigate the influence of junction phenotype on brain endothelial barrier properties. To accomplish this, we developed the Junction Analyzer Program (JAnaP) to semi-automatically calculate edge-localization protein phenotypes. Application of the JAnaP to measure the junctional proteins VE-cadherin and ZO-1 in different physiological and pathophysiological conditions revealed that discontinuous junctions contribute more to barrier permeability compared to continuous, linear junctions. Continuous junctions were also increased in endothelial cells with decreased contractility, mediated biochemically or by lowered subendothelial matrix stiffness. Finally, breast cancer cell secreted factors increased immature adherens junctions, likely through VEGF signaling, but minimally affected tight junction presentation. Thus far, the development and application of the JAnaP has revealed insights into the effects of junction patterns on barrier function, the mechanobiology of endothelial cells, and the response of brain endothelial cells to biochemical cues involved in breast cancer metastasis. Understanding the conditions driving altered junction presentation, and the resultant effects on barrier integrity, could lead to the development of therapeutics capable of traversing the BBB for delivery to the brain or for diseases associated with BBB dysfunction. Future use of this program holds significant potential for physiological and pathophysiological study in various endothelial and epithelial cell systems.Item THE ROLE OF THE MECHANICAL ENVIRONMENT ON CANCER CELL TRANSMIGRATION AND MRNA LOCALIZATION(2016) Hamilla, Susan M.; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Most cancer-related deaths are due to metastasis formation, the ability of cancer cells to break away from the primary tumor site, transmigrate through the endothelium, and form secondary tumors in distant areas. Many studies have identified links between the mechanical properties of the cellular microenvironment and the behavior of cancer cells. Cells may experience heterogeneous microenvironments of varying stiffness during tumor progression, transmigration, and invasion into the basement membrane. In addition to mechanical factors, the localization of RNAs to lamellipodial regions has been proposed to play an important part in metastasis. This dissertation provides a quantitative evaluation of the biophysical effects on cancer cell transmigration and RNA localization. In the first part of this dissertation, we sought to compare cancer cell and leukocyte transmigration and investigate the impact of matrix stiffness on transmigration process. We found that cancer cell transmigration includes an additional step, ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs and spread into the monolayer. Furthermore, the effects of subendothelial matrix stiffness and endothelial activation on cancer cell incorporation are cell-specific, a notable difference from the process by which leukocytes transmigrate. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation into the endothelium is one of the earliest steps. In the next part of this work, we investigated how matrix stiffness impacts RNA localization and its relevance to cancer metastasis. In migrating cells, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cellular protrusions. We observed that increasing stiffness promotes the peripheral localization of these APC-dependent RNAs and that cellular contractility plays a role in regulating this pathway. We next investigated the mechanism underlying the effect of substrate stiffness and cellular contractility. We found that contractility drives localization of RNAs to protrusions through modulation of detyrosinated microtubules, a network of modified microtubules that associate with, and are required for localization of APC-dependent RNAs. These results raise the possibility that as the matrix environment becomes stiffer during tumor progression, it promotes the localization of RNAs and ultimately induces a metastatic phenotype.Item Mechanobiology of T cell activation(2015) Hui, King Lam; Upadhyaya, Arpita; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cells can sense and respond to the physical environment through generation and transmission of mechanical forces from the surroundings to the cell interior and from one cell to another. This dissertation focuses on mechanosensing by T cells, key players in the adaptive immune system, which form a strong line of defense against infections by their ability to recognize foreign molecules and develop an appropriate response. T cells form close contact with an opposing antigen presenting cell upon recognition of protein fragments derived from infecting pathogens (antigens). Recent studies have shown that externally applied forces can trigger biochemical signaling in T cells. How forces are internally generated by T cells, involved in signaling and transmitted at the level of the cell interface, remains unclear. In this thesis, we investigate the molecular mechanisms of force generation by T cells and their response to forces and the stiffness of the opposing surface. We have quantitatively characterized the initial phase of T cell contact with a model of antigen-bearing surfaces. We observe that T cells spread on such substrates and that the kinetics of spreading follows a universal function, with the spreading rate dependent on actin polymerization and myosin II activity. Altering cell-substrate adhesions leads to qualitative changes in cell spreading dynamics and wave-like patterns of actin dynamics. We then used soft elastic substrates with stiffness comparable to that of antigen presenting cells, to measure the forces generated by T cells during activation. Perturbation experiments reveal that these forces are largely due to actin assembly and dynamics, with myosin contractility contributing to the development of traction forces but not its maintenance. We find that Jurkat T-cells are mechanosensitive, with both traction forces and signaling dynamics exhibiting sensitivity to the stiffness of the substrate. We further demonstrate that dynamics of the T cell microtubule cytoskeleton also participates in regulating forces at the cell-substrate interface, through the Rho/ROCK pathway which regulates myosin II light chain phosphorylation. Overall, this work highlights physical force as an essential mediator that connects stiffness sensing to intracellular signaling, which then directs gene expression and eventually the immune response in T cells.