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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    ESOTAXIS: IDENTIFYING THE FACTORS THAT INFLUENCE NANOTOPOGRAPHIC GUIDANCE OF THE DYNAMICS AND ORGANIZATION OF THE ACTIN CYTOSKELETON AND OTHER MOLECULES INVOLVED IN DIRECTED CELL MIGRATION
    (2024) Hourwitz, Matt; Fourkas, John T.; Losert, Wolfgang; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Directed migration is a crucial capability of cells in developmental and immunological processes. Defects in cell migration can lead to negative health outcomes. Cell motion depends on the organization and dynamics of internal components, especially the actin cytoskeleton, and the extracellular environment. Microscale and nanoscale topographical cues, with at least one dimension that is much smaller than most cells, can bias cell motion over long distances, due to the guidance of the organization and dynamics of the cytoskeleton and other molecules and assemblies within the cell. In this work, I describe a technique to reproduce patterned nanotopographic substrates for use in the study of esotaxis, the guided organization and dynamics of the actin cytoskeleton and other cellular components in response to nanotopographic cues. The guidance of actin drives directed cell motion along a pattern with dimensions much smaller than the cell. The dimensions of the nanotopography determine the extent to which cellular components are guided. Differences in the physical properties of the plasma membrane and the actin cytoskeleton among cell lines will influence the extent of guidance by nanotopography. Asymmetric patterns can accentuate the distinctions in esotactic responses among cell lines and drive contact guidance in different directions. The cytoskeletal response to nanotopography is a local phenomenon. A cell in contact with multiple nanotopographic cues simultaneously will show distinct organization of actin in the different regions of the cell. The importance of local actin dynamics requires an analysis method, optical flow, that can identify and track the distinct cytoskeletal motions in different parts of the cell. The formation of adhesions attached to the extracellular matrix is a characteristic of the migratory behavior of many types of cells and these adhesions are credited with allowing the cell to sense and interact with the underlying substrate. Actin can sense nanotopographic cues without the widespread availability of adhesive ligands. Although adhesion to the substrate strongly increases the extent of cell spreading and migration on nanoridges, epithelial cells can align with and migrate along nanotopography even with a dearth of adhesive cues. Therefore, actin is a supreme sensor of nanotopography that can drive directed cell migration.
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    MODULATION OF INTRACELLULAR EXCITABLE SYSTEMS THROUGH PHYSICAL MICROENVIRONMENTS
    (2022) Yang, Qixin; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Cells can sense various external cues, such as chemoattractants, electric fields (EFs), and the physical properties of their surroundings. This ability is essential to maintain normal physiological processes, and malfunctions may lead to severe diseases, such as cancer and autoimmune disease. Recent studies have observed that waves of colocalized actin, associated with its upstream signaling molecules, drive cell directional migration. Furthermore, a coupled signal transduction excitable system – cytoskeleton excitable system (STEN-CEN) has been revealed to regulate wave formation in non-neural cell types. The system display hallmarks of excitability, such as refractory periods, all-or-none type responses, and wave behavior. It is traditionally believed that different cells employ different migrational strategies. However, recent studies find that changing the states of STEN-CEN leads to the transition of migrational modes within a single cell type. This dissertation uses molecular experiments, computer-vision quantification techniques, and modeling to understand how cells sense different external cues. Numerous studies have shown that there exist multiple, parallel signal pathways that sense certain external stimuli, which suggests that the external signal is not sensed by a single molecule. Here I investigate the possibility that STEN-CEN waves act as a sensing unit for external cues. To overcome the challenge that wave dynamics are coupled with cell motion in normal-sized cells, I electro-fused tens of Dictyostelium discoideum (D. d) cells together to form giant cells. In giant cells, waves are no longer localized at the cell perimeter. The larger basal membrane area provides the opportunity to study subcellular dynamics. I recorded the dynamics of F-actin and phosphatidylinositol (3,4,5)-triphosphate (PIP3), which are indicative of CEN and STEN, respectively. To decouple STEN and CEN further, I applied a variety of chemical perturbations to suppress/activate STEN and CEN separately. Because the wave properties characterize the stage of STEN-CEN, I developed a series of quantification tools to measure wave area, duration, and speed. I collaborated with theorists to create a reaction-diffusion system model that recreates the experimental results. The dissertation focuses on studying the role of STEN-CEN waves in sensing nanotopography and EF. CEN is found to sense nanotopographical cues directly, forming long-lasting F-actin puncta at ridges in the absence of STEN. At the same time, STEN is essential for long-range wave behaviors for macro-domain nanoridge sensing. STEN and CEN cooperate in the sensing of EF signals. According to quantitative studies of wave properties, nanotopography changes the dimensionality and lifetime of waves, whereas EF can alter the activation thresholds of the intracellular systems. In summary, this thesis shows that the excitable biomechanical and biochemical wave systems act as the sensing units for the physical properties of the extracellular environment. As a result, cells can dynamically sense their surroundings and coordinate intracellular processes to migrate under guidance from external cues.
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    EMERGENT NETWORK ORGANIZATION IN LINEAR AND DENDRITIC ACTIN NETWORKS REVEALED BY MECHANOCHEMICAL SIMULATIONS
    (2021) Chandrasekaran, Aravind; Papoian, Garegin A; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Cells employ networks of filamentous biopolymers to achieve shape changes and exert migratory forces. As the networks offer structural integrity to a cell, they are referred to as the cytoskeleton. Actin is an essential component of the cellular cytoskeleton. The organization of the actin cytoskeleton is through a combination of linear and branched filaments. Despite the knowledge of various actin-binding proteins and their interactions with individual actin filaments, the network level organization that emerges from filament level dynamics is not well understood. In this thesis, we address this issue by using advanced computer simulations that account for the complex mechanochemical dynamics of the actin networks. We begin by investigating the conditions that stabilize three critical bundle morphologies formed of linear actin filaments in the absence of external forces. We find that unipolar bundles are more stable than apolar bundles. We provide a novel mechanism for the sarcomere-like organization of bundles that have not been reported before. Then, we investigate the effect of branching nucleators, Arp2/3, on the hierarchical organization of actin in a network.By analyzing actin density fields, we find that Arp2/3 works antagonistic to myosin contractility, and excess Arp2/3 leads to spatial fragmentation of high-density actin domains. We also highlight the roles of myosin and Arp2/3 in causing the fragmentation. Finally, we understand the cooperation between the linear and dendritic filament organization strategies in the context of the growth cone. We simulate networks at various concentrations of branching molecule Arp2/3 and processive polymerase, Enabled to mimic the effect of a key axonal signaling protein, Abelson receptor non-tyrosine kinase (Abl). We find that Arp2/3 has a more substantial role in altering filament lengths and spatial actin distribution. By looking at conditions that mimic Abl signaling, we find that overexpression mimics are characterized by network fragmentation. We explore the consequence of such a fragmentation with perturbative simulations and determine that Abl overexpression causes mechanochemical fragmentation of actin networks. This finding could explain the increased developmental errors and actin fragmentation observed in vivo. Our research provides fundamental self-assembly mechanisms for linear and dendritic actin networks also highlights specific mechanochemical properties that have not been observed earlier.
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    THE ROLE OF THE ACTIN CROSSLINKER PALLADIN: FROM RECONSTITUTED NETWORKS TO LIVE CELLS
    (2013) Grooman, Brian; Upadhyaya, Arpita; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Biophysics is a rapidly growing area of research. New discoveries continue to show the importance of mechanical phenomenon in biological processes, even at the cellular and sub-cellular levels. The complexity of living cells, coupled with their small size makes their study particularly difficult. Palladin is an actin-crosslinker that has not yet been studied as much as other actin-crosslinkers. It localizes with alpha-actinin in stress fibers in many adult cell types. Palladin's exact purpose is still unknown. Through in-vitro studies of reconstituted actin networks we gain insight into the mechanical importance of this novel protein, and show that when partnered with α-actinin, palladin efficiently enhances the network stiffness. Pancreatic Stellate Cells are responsible for maintaining organ integrity, and their malignant counterparts are responsible for one of the most deadly forms of cancer. Interestingly, palladin is shown to be up-regulated in tumors derived from these cells. By studying the stiffness of the cells with and without palladin (via genetic manipulation) we investigate the mechanical importance of palladin in vivo. GFP labelled palladin can serve as a useful marker because it naturally localizes into a regular pattern along stress fibers. Combined with image processing, this makes tracking local strain rates within the cell possible. Pancreatic stellate cells will respond to an applied force by actively contracting their stress fibers. The dynamics of these responses are quantified by tracking the spots of palladin. Through various pharmacological manipulations we study possible signaling pathways that lead from an applied force to stress fiber contraction. Overall, this work explores the mechanical importance of palladin and also investigates the mechanical properties of tumor-associated pancreatic stellate cells, neither of which have been previously studied. Our work shows that palladin controls network stiffness in-vitro, but not in-vivo, suggesting a yet undiscovered purpose. We have also shown that pancreatic stellate cells are in the same range of stiffness as other fibroblasts, and can actively respond to external forces. All of these findings contribute to an increased understanding of the complex systems which govern the mechanical properties of living material.
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    Cytoskeletal Mechanics and Mobility in the Axons of Sensory Neurons
    (2011) Chetta, Joshua; Shah, Sameer B; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The axon is a long specialized signaling projection of neurons, whose cytoskeleton is composed of networks of microtubules and actin filaments. The dynamic nature of these networks and the action of their associated motor and cross-linking proteins drives axonal growth. Understanding the mechanisms that control these processes is vitally important to neuroregenerative medicine and in this dissertation, evidence will be presented to support a model of interconnectivity between actin and microtubules in the axons of rat sensory neurons. First, the movement of GFP-actin was evaluated during unimpeded axonal outgrowth and a novel transport mechanism was discovered. Most other cargoes in the axon are actively moved by kinesin and dynein motor proteins along stationary microtubules, or are moved along actin filaments by myosin motor proteins. Actin, however, appears to be collected into short-lived bundles that are either actively carried as cargoes along other actin filaments, or are moved as passive cargoes on short mobile microtubules. Additionally, in response to an applied stretch, the axon does not behave as a uniform visco-elastic solid but rather exhibits local heterogeneity, both in the instantaneous response to stretch and in the remodeling which follows. After stretch, heterogeneity was observed in both the realized strain and long term reorganization along the length of the axon suggesting local variation in the distribution and connectivity of the cytoskeleton. This supports a model of stretch response in which sliding filaments dynamically break and reform connections within and between the actin and microtubule networks. Taken together, these two studies provide evidence for the mechanical and functional connectivity between actin and microtubules in the axonal cytoskeleton and suggest a far more important role for actin in the development of the peripheral nervous system. Moreover this provides a biological framework for the exploration of future regenerative therapies.