Theses and Dissertations from UMD
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Item BLANKET AND PATTERNED REPROGRAMMING OF AZOPOLYMER NANORIDGES AND APPLICATIONS TO CELLULAR BIOPHYSICS(2024) Abostate, Mona Hamdy Abdelrahman; Fourkas, John J; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The objective of this project is to tailor nanotopographies previously fabricated on large areas through photomodification. The original master patterns consist of nanoridges created using conventional lithography. Using an azopolymer as a photoresponsive material, replicas of the original master were prepared using soft lithography. The entire surface of the azopolymer nanoridges underwent photomodification using a 532 nm laser with varying polarizations and durations, in a process referred to as blanket reprogramming. This process resulted in controllable widening, buckling, or removal of the nanoridges due to photoisomerization and subsequent mass migration of the azopolymer. To replicate the reprogrammed surfaces, a molding procedure was employed using an acrylatic resin. The blanket reprogramming process was monitored in situ during exposure through diffraction of another reading laser beam. Cellular behaviors can be modulated in various biological contexts through interactions with their surroundings. The relationship between nanotopography and cell behavior is crucial, and has a wide range of biological consequences and medical applications. For example, nanotopography is employed to design antibacterial surfaces, preventing the adhesion of bacteria and biofilm formation, thereby reducing the risk of infections associated with medical devices. Nanostructured surfaces can inhibit the migration of cancer cells, offering insights into potential therapeutic strategies. Nanotopography is also used in nerve-regeneration scaffolds to guide neurite outgrowth, aiding in the repair of damaged neural tissue. We investigated the response of MCF10A breast epithelial cells to buckled acrylic nanoridges replicated from a master of azopolymer ridges photomodified by laser. The nanoridges became buckled after exposure to 532 nm light polarized parallel to the ridges. The impact of buckling on the dynamics and location of actin polymerization was investigated, as well as the distribution of lengths of contiguous polymerized regions. Azopolymers, known for their biocompatibility, have been employed by various research groups to create nanotopographies on which cells are plated and imaged. We conducted experiments using a spinning-disk confocal fluorescence microscope, testing exposure wavelengths ranging from 405 nm to 640 nm. Our objective was to assess the feasibility of live-cell imaging on azopolymer nanotopographies without inducing surface alterations. Our findings revealed the capability of live-cell imaging at high frame rates across a wide range of wavelengths. This result stands in contrast to prior studies, in which the selection of fluorescent dyes compatible with these materials was limited to those excited in the red spectrum and emitting in the near-infrared. I demonstrate that different patterns can be created through patterned reprogramming of the azopolymer nanoridges. A periodic arrangement of light strips was projected perpendicular to the ridges, thereby projecting an amplitude grating onto the azopolymer nanoridges. The spacing of this pattern can be adjusted by altering the mask or adjusting the magnification of the optical system. Furthermore, varying the direction of light polarization expands the potential for creating a wider variety of designs. Different types of reprogramming motifs can be implemented by projecting patterns at angles that are not perpendicular to the substrate, by tilting the incoming laser beam away from the horizontal. Various intriguing patterns, such as repeating curves, were observed, dependent on both the angle of the incident light and the direction of light polarization relative to the direction of the ridges.Item Mechanisms by which the actin cytoskeleton switches B cell receptor signaling from the activation to the attenuation mode(2022) Bhanja, Anshuman; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell-mediated humoral immune response is critical in fighting off invading pathogens and potentially harmful foreign substances. B cells detect antigens through the B cell receptor (BCR). The binding of cognate antigen to the BCR induces a signaling response, a critical initiation and regulatory step for B cell activation and differentiation. The actin cytoskeleton has been shown to play essential roles in BCR signaling. When encountering membrane-associated antigens, actin amplifies signaling by driving B cell spreading and BCR clustering, while promoting signal attenuation by causing B cell contraction. This signal attenuation is essential for curtailing the activation of autoreactive B cells. However, the mechanism by which the actin cytoskeleton switches BCR signaling from amplification to attenuation was unknown. My thesis research examined the mechanisms by which actin reorganization transitions B cells from spreading to contracting and B cell contraction switches BCR signaling from amplification to attenuation, using mouse splenic B cells, a functionalized planar lipid bilayer system, and total internal reflection fluorescence microscopy. Our results show that branched actin polymerized by Arp2/3 is required for B cell transition from spreading to contraction after driving B cell spreading. Ubiquitously expressed Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP), but not the haematopoietically specific WASP, activates the branched actin polymerization and generates inner actin foci from lamellipodial actin networks, by sustaining their lifetime and centripetal movement. N-WASP-dependent inner actin foci are necessary for recruiting non-muscle myosin II, creating an actomyosin ring-like structure at the periphery of the membrane contact region to drive B cell contraction. B cell contraction primarily increases the BCR molecular density in individual BCR-antigen clusters, measured by the peak fluorescence intensity. Inhibition of B cell contraction by Arp2/3 inhibitor and B cell-specific N-WASP knockout (cNKO) reduced the increasing rates of BCR molecular density. Increased molecular density caused by B cell contraction leads to decreases in the levels of phosphorylated BCR, the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. However, the levels of total Syk and SHIP-1 have a different relationship with BCR density in individual clusters: Syk does not decrease until a high threshold of BCR density, which can be achieved only by contracting B cells, but SHIP-1 consistently reduces with the increase in BCR molecular density. Inhibiting B cell contraction by cNKO reduces the molecular density of BCR clusters but does not affect the relationship of the Syk and SHIP-1 levels with BCR molecular density in clusters. Taken together, our results suggest that the actin cytoskeleton reorganizes from the lamellipodial branched actin networks to centripetally moving actin foci, enabling actomyosin ring-like structure formation, through N-WASP-activated Arp2/3. Actomyosin-mediated B cell contraction attenuates BCR signaling by increasing receptor molecular density in individual BCR clusters, which causes the dissociation of both stimulatory and inhibitory signaling molecules. My thesis research results reveal a novel negative regulatory mechanism for BCR signaling, an essential checkpoint for generating pathogen-specific and suppressing self-reactive antibody responses.Item Integration and Competition in Immune Cell Models(2022) Bull, Abby L; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cell motility plays an integral role in most biological processes. One principle of motility is the protrusions and retractions of cellular membranes called pseudopods. The physical force behind pseudopod formation is actin polymerization. As the physical driver, actin polymerization integrates the cells’ upstream biochemical signaling cascades and turns the signals into action as the combined output and readout of the state of the cell. Actin polymerization not only occurs within pseudopods but propagates throughout a cell in waves that can be understood and modeled as excitable media.This dissertation focuses on actin wave dynamics in the context of directed cell mi- gration using both experimental and numerical techniques. The directional guidance of cell migration is essential in physiological processes including embryonic development, cancer metastasis, and wound healing. In this thesis, I analyze how immune-like cells are guided by external stimuli that are common in wound environments: electric fields, chemical gradients, and surface texture. A focus of this work is on the emergent excitable wave behavior of actin, and the pseudopods they generate, in simplified, in vitro environments. Actin waves have been previously shown to respond to and be guided by the topography of an underlying substrate. Additionally, static quantifications of actin filaments during or after electric field stimulation have shown that filaments become asymmetrically distributed within the cytoplasm. In neutrophils, the combination of cues leads to higher control of cell motility by guiding the internal actin waves. Using an optical flow algorithm, I quantify the actin waves on multiple length scales to ascertain the role of each guidance cue in affecting cell motion. I find that the waves preferentially polymerize near and travel along the nanoridges. Actin waves nucleate preferentially on the cathode side and reorient the cell’s axis of polarity (i.e., the position of the dominant pseudopod). The second example of competing guidance cues involves studying the collective motion of cells in response to cell-cell signal relay in competition with surface topography. I use Dictyostelium discoideum cells as a model system for this work, as they migrate collectively due to signal relay. The signal relay of these cells is similar to many immune cell species. Using a combination of image analysis tools and a coarse-grained stochastic model, I find that guidance by nanoridges overrides the chemical signal relay and forces cells to migrate individually, suppressing streaming behavior. I model both the secretion and propagation of chemical signals using an excitable systems framework. This work highlights that bidirectional signals can be effective at suppressing cell-cell attraction and streaming motion. The response of immune cells to external stimuli in the wound environment is not universal. Macrophages, one of the largest immune cells, are observed to migrate away from the wound upon wound-induced electric field generation. In the third example, I study actin dynamics of M0 (resting) macrophage cells to elucidate how these cells interact with external electric fields. This cell type exhibits oscillatory actin waves at rest. With electric field stimulation, the oscillatory actin waves start to generate protrusions. Often, the protrusions begin with actin-depleted regions, indicating that contractile ele- ments are involved in conjunction with overall cell volume conservation. This thesis highlights the different methods in which actin waves integrate external cues, specifically electric fields, into cell responses that are cell-type specific.Item PHYSICAL FACTORS IN B CELL ACTIN DYNAMICS AND ACTIVATION(2016) Ketchum, Christina; Upadhyaya, Arpita; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cells adapt to their changing world by sensing environmental cues and responding appropriately. This is made possible by complex cascades of biochemical signals that originate at the cell membrane. In the last decade it has become apparent that the origin of these signals can also arise from physical cues in the environment. Our motivation is to investigate the role of physical factors in the cellular response of the B lymphocyte. B cells patrol the body for signs of invading pathogens in the form of antigen on the surface of antigen presenting cells. Binding of antigen with surface proteins initiates biochemical signaling essential to the immune response. Once contact is made, the B cell spreads on the surface of the antigen presenting cell in order to gather as much antigen as possible. The physical mechanisms that govern this process are unexplored. In this research, we examine the role of the physical parameters of antigen mobility and cell surface topography on B cell spreading and activation. Both physical parameters are biologically relevant as immunogens for vaccine design, which can provide laterally mobile and immobile antigens and topographical surfaces. Another physical parameter that influences B cell response and the formation of the cell-cell junction is surface topography. This is biologically relevant as antigen presenting cells have highly convoluted membranes, resulting in variable topography. We found that B cell activation required the formation of antigen-receptor clusters and their translocation within the attachment plane. We showed that cells which failed to achieve these mobile clusters due to prohibited ligand mobility were much less activation competent. To investigate the effect of topography, we use nano- and micro-patterned substrates, on which B cells were allowed to spread and become activated. We found that B cell spreading, actin dynamics, B cell receptor distribution and calcium signaling are dependent on the topographical patterning of the substrate. A quantitative understanding of cellular response to physical parameters is essential to uncover the fundamental mechanisms that drive B cell activation. The results of this research are highly applicable to the field of vaccine development and therapies for autoimmune diseases. Our studies of the physical aspects of lymphocyte activation will reveal the role these factors play in immunity, thus enabling their optimization for biological function and potentially enabling the production of more effective vaccines.Item The role of actin netoworks in cellular mechanosensing(2015) Azatov, Mikheil; Upadhyaya, Arpita; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.Item CELLULAR PATHWAYS INVOLVED IN EPITHELIAL-TO-MESENCHYMAL TRANSITIONS IN NEURAL CREST CELLS(2013) Li, Shen; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neural crest cells are a population of multi-potent progenitor cells in the developing vertebrate embryo that undergo an epithelial-to-mesenchymal transition (EMT) and migrate extensively to generate diverse derivatives. As such, abnormal development of neural crest cells can lead to human congenital and hereditary malformations, diseases and cancers. Both internal molecular signals and external mechanical factors play essential roles in facilitating neural crest cell EMT. How cells modulate their adhesion machinery and dynamically reorganize their actin cytoskeleton to respond to the mechanical features of their external environment during EMT is not well understood. To evaluate the role of the actomyosin cytoskeleton during neural crest cell EMT and migration, midbrain neural folds that contain premigratory neural crest cells were dissected out from chick embryos, explanted into chamber slides, and incubated to allow for the formation of migratory neural crest cells. Time-lapse imaging technique was used to record cell behaviors. To elucidate cellular pathways controlling EMT and migration, chemical inhibitors (blebbistatin, Y-27632, latrunculin-A, and nocodazole) that perturb molecular cascades regulating cellular structures were employed. Effects of these perturbations on neural crest cell EMT and migration were quantified in terms of the spreading rate of the explants, and vorticity of collectively moving cell groups. We observed that blebbistatin treatment reduced the overall velocity of migratory neural crest cells to negligible levels. Moreover, migratory neural cells developed rounder cell bodies, and lamellipodia were transformed into filopodia at the periphery of the extract. Y-27632 treatment led to more neural crest cells coming out from these explants within a shorter time period compared to control. Nocodazole treatment blocked neural crest cell EMT and the resumption was dose-dependent. Latrunculin-A caused cell death at a very low concentration. These results implicate roles for non-muscle myosin II, the target of blebbistatin, and ROCK, the target of Y-27632, as well as microtubules and actin filaments, in chick midbrain neural crest cell EMT and migration. Actin crosslinkers such as α-actinin and actin-associated proteins like palladin also participate in pathways affected by these cytoskeletal inhibitors through their regulation of focal adhesion formation and cytoskeletal organization, thereby modulating cell stiffness and migration. We are also documented the distribution of α-actinin and palladin in migratory neural crest cells in vivo. Collectively, our studies have provided insight into specific cellular pathways regulating neural crest cell EMT and migration and the impact on various biophysical parameters upon perturbation of these pathways.Item REGULATORY FUNCTIONS OF THE ACTIN CYTOSKELETON IN B CELL RECEPTOR SIGNALING(2013) Liu, Chaohong; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The binding of antigen (Ag) to the B cell receptor (BCR) induces the activation of intracellular signaling and the reorganization of the actin cytoskeleton. However, the function of actin reorganization and the mechanisms by which BCR signaling and actin reorganization is coupled have not been well studied. This thesis has investigated how BCR signaling regulates actin reorganization and how actin remodeling in turn influences BCR signalig. My studies show that the key stimulatory signaling molecule of the BCR, Bruton's tyrosine kinase (Btk), is critical for actin polymerization at the activation surface and BCR clustering and B cell spreading, events that are essential for signaling initiation and amplification. The key inhibitory signaling molecule, SH2-containing phosphatidylinositol-5 phasphatase (SHIP-1), is important for removal of F-actin from the activation surface, and actin-mediated B cell contraction and the formation of BCR central clusters. SHIP-1 suppresses actin polymerization by inhibiting Btk-dependent activation of Wiskott-Aldrich syndrome protein (WASP). These results suggest that BCR signaling can regulate B cell morphology and surface BCR clustering via modulationg actin dynamics. To understand the roles of actin reorganization in BCR signaling, I investigated the effects of gene knockout of the two actin regulators, WASP and its homolog, neuronal (N)-WASP. My results show that both WASP and N-WASP are required for optimal BCR clustering, B cell spreading, and BCR signaling, but they play distinct roles. WASP promotes actin polymerization, B cell spreading, BCR clustering, and signaling amplification, and N-WASP inhibits actin polymerization at the activation surface and promotes B cell contraction, BCR central cluster formation, and signaling attenuation. Importantly, B cell-specific N-WASP knockout causes increases in the levels of autoantibody. In addition, WASP and N-WASP negatively regulate each other, compete for Arp2/3, and are inversely regulated by Btk and SHIP-1. Taken together, these results demonstrate that the balance of stimulatory and inhibitory BCR signaling controls actin dynamics and organization through regulating the activities of WASP and N-WASP. Actin remodeling in turn amplifies BCR signaling activation or down regulation by modulating B cell morphlogy and the organization of surface BCRs.This research reveals a bidrectional feedback loop between BCR signaling and the actin cytoskeleton.