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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Collective dynamics of astrocyte and cytoskeletal systems
(2024) Mennona, Nicholas John; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Advances in imaging and biological sample preparations now allow researchersto study collective behavior in cellular networks with unprecedented detail. Imaging the electrical signaling of neuronal networks at the cellular level has generated exciting insights into the multiscale interactions within the brain. This thesis aims at a complementary view of the general information processing of the brain, focusing on other modes of non-electrical information. The modes discussed are the collective, dynamical characteristics of non-electrically active, non-neuronal brain cells, and mechanical systems. Astrocytes are the studied non-neuronal brain cells, and the cytoskeleton is the studied dynamic, mechanical system consisting of various filamentous networks. The two filamentous networks studied herein are the actin cytoskeleton and the microtubule network. Techniques from calcium imaging and cell mechanics are adapted to measure these often overlooked information channels, which operate at length scales and timescales distinct from electrical information transmission. Structural, astrocyte actin images, microtubule structural image sequences, and the calcium signals of collections of astrocytes are analyzed using computer vision and information theory. Filamentous alignment of actin with nearby boundaries reveals that stellate astrocytes have more perpendicularly oriented actin than undifferentiated astrocytes. Harnessing the larger length scale and slower dynamical time scale of microtubule filaments relative to actin filaments led to the creation of a computer vision tool to measure lateral filamentous fluctuations. Finally, we adapt information theory to the analog calcium (Ca2+) signals within astrocyte networks classified according to subtype. We find that, despite multiple physiological differences between immature and injured astrocytes, stellate (healthy) astrocytes have the same speed of information transport as these other astrocyte subtypes. This uniformity in speed persists when either the cytoskeleton (Latrunculin B) or energy state (ATP) is perturbed. Astrocytes, regardless of physiological subtype, tend to behave similarly when active under normal conditions. However, these healthy astrocytes respond most significantly to energy perturbation, relative to immature and injured astrocytes, as viewed through cross-correlation, mutual information, and partitioned entropy. These results indicate the value of drawing information from structure and dynamics. We developed and adapted tools across scales from nanometer scale alignment of actin filaments to hundreds of microns scale information dynamics in astrocyte networks. Including all potential modalities of information within complex biological systems, such as the collective dynamics of astrocytes and the cytoskeleton in brain networks is a step toward a fuller characterization of brain functioning and cognition.
Simulating membrane-bound cytoskeletal dynamics
(2023) Ni, Haoran; Papoian, Garegin A.; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The cell membrane defines the shape of the cell and plays an indispensable role in bridging intra- and extra-cellular environments. The membrane, consisting of a lipid bilayer and various attaching proteins, mechanochemically interacts with the active cytoskeletal network that dynamically self-organizes, playing a vital role in cellular biomechanics and mechanosensing. Comprehensive simulations of membrane-cytoskeleton dynamics can bring insight in understanding how the cell mechanochemically responds to external signals, but a computational model that captures the complex cytoskeleton-membrane with both refined details and computational efficiency is lacking. To address this, we introduce in this thesis a triangulated membrane model and incorporate it with the active biological matter simulation platform MEDYAN ("Mechanochemical Dynamics of Active Networks"). This model accurately captures the membrane physical properties, showing how the membrane rigidity, the structure of actin networks and local chemical environments regulate the membrane deformations. Then, we present a new method for simulating membrane proteins, using stochastic reaction-diffusion sampling on unstructured membrane meshes. By incorporating a surface potential energy field into the reaction-diffusion sampling, we demonstrate rich membrane protein collective behaviors such as confined diffusion, liquid-liquid phase separation and membrane curvature sensing. Finally, in order to capture stretching, bending, shearing and twisting of actin filaments which are not all available with traditional actomyosin simulations, we introduce new finite-radius filament models based off Cosserat theory of elastic rods, with efficient implementation using finite-dimensional configurational spaces. Using the new filament models, we show that the filaments' torsional compliance can induce chiral symmetry breaking in a cross-linked actin bundle. All the new models are implemented in the MEDYAN platform, shedding light on whole cell simulations, paving way for a better understanding of the membrane-cytoskeleton system and its role in cellular dynamics.
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.
Non-equilibrium Thermodynamics of Cytoskeletal Self-organization
(2021) Floyd, Carlos Shadoan; Papoian, Garegin A; Jarzynski, Christopher; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The actin-based cytoskeleton is a polymer network that plays an essential role in cell biology. By self-organizing into various local architectures, the cytoskeleton performs physiological functions that allow the cell to physically interact with its environment. It is also an example of biological active matter, consuming chemical free energy at a local scale to produce directed motion and do mechanical work. While it is well-known that cytoskeletal free energy transduction occurs, it has been a challenge to say anything quantitative about this far-from-equilibrium process due to the difficulty of making the necessary experimental measurements. This lack of methodology to quantify cytoskeletal energetics significantly hinders our understanding of the self-organization process underlying the cytoskeleton's physiological functionality. To address this research gap, we develop in this thesis an explicit computational method to quantify chemical and mechanical free energy changes during simulated cytoskeletal self-organization using the software package MEDYAN (Mechanochemical Dynamics of Active Networks). We then apply this tool in several studies to advance our understanding of the self-organization process and its thermodynamic characteristics. For instance, we analyze the thermodynamic efficiency of mechanical stress generation and the network's time-dependent dissipation rates under a range of network conditions. We also investigate the recent experimentally discovered phenomenon of cytoskeletal avalanches, which we identify in simulation as anomalous mechanical dissipation events. Our analysis clarifies the phenomenology and underlying mechanism of these avalanche events, which we propose may play an important role in cellular information processing. The in silico method developed in this thesis provides a new perspective on cytoskeletal self-organization and may be extended to investigate other biological active matter systems.
THE IMPACT OF ENGINEERED MECHANICAL CONFINEMENT ON MESENCHYMAL STEM CELL AND LUNG FIBROBLAST MECHANOBIOLOGY
(2020) Doolin, Mary; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Mechanical cues have been shown to influence cell gene expression, cell protein expression, and cell behaviors critical for homeostasis and disease progression. Cells experience the mechanical cue of confinement in vivo, such as within the extracellular matrix, and in vitro, such as within tissue engineered scaffolds. Despite its prevalence, the impact of mechanical confinement on cell fate is poorly understood. Cues from the mechanical microenvironment are primarily sensed and responded to by the cytoskeleton, which transmits forces to the nucleus and can thereby alter gene expression. The nucleus itself is also a mechanosensor, sensing external forces and again altering gene expression. Mesenchymal stem cells (MSCs) and lung fibroblasts are known to be sensitive to mechanical forces, yet the effect of mechanical confinement on these cells is unclear. In this dissertation, we investigated how mechanical confinement induced by engineered microchannels influences MSC morphology and migration. Notably, we show that confinement alters the relative contributions of cytoskeletal and contractile machinery in MSC migration in unconfined and confined spaces. We next investigated how mechanical confinement induced by microchannels influences MSC and fibroblast nucleus volume. When certain cytoskeletal machinery was inhibited, nucleus volume was altered only in MSCs in wide channels, suggesting diverging roles of the cytoskeleton in regulating nuclear deformation and migration in different degrees of confinement and in different cell types. While performing this work, we observed a lack of assays that provide precise control over the degree of confinement induced on cells, yield a large sample size, enable long-term culture, and enable easy visualization of cells over time. Therefore, we designed, created, and validated a confining micropillar assay that achieves these requirements. Using these confining micropillars, we investigated the effect of confinement on lung fibroblast to myofibroblast transition (FMT), a hallmark of idiopathic pulmonary fibrosis. Cell density was more predictive of FMT than the degree of confinement induced by micropillar arrays. These results improve our understanding of how MSCs and lung fibroblasts respond to confinement, which will aid in the rational design of MSC-based therapies and FMT-targeting therapies.
VIMENTIN AND CYTOKERATIN INTERMEDIATE FILAMENTS IN THE MECHANOBIOLOGY AND MALIGNANT BEHAVIORS OF CHORDOMA CELLS
(2018) Resutek, Lauren; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Chordoma, an aggressive tumor derived from notochordal remnants, is difficult to treat due to its proximity to the spinal cord and brain stem and its resistance to conventional treatments, such as radiation and chemotherapy. The development of effective treatments requires research at the molecular level, which presumably due to its rare diagnosis, is lacking for chordoma. Recent studies have identified potential targets for systemic therapy; however, there are currently no drugs approved by the Food and Drug Administration (FDA) to treat chordoma. One promising approach is to target the cytoskeleton, in order to stall progression and sensitize cells to chemotherapeutics. Similar to other cancers, chordoma cells co-express vimentin and cytokeratin intermediate filaments (IFs), which have both been found to play roles in cell mechanical properties and behaviors and their expression has been associated with cancer metastasis, chemoresistance, and poor prognosis. Therefore, we investigated the functional roles of vimentin and cytokeratin IFs in chordoma cells using RNA interference (RNAi). First, we examined whether cytoskeletal disruption by siRNA-mediated silencing of vimentin or cytokeratin-8 altered the chordoma phenotype. We determined that the vacuolated cytoplasm, a distinguishing feature of chordoma, was dependent on cytokeratin-8 IFs. Next, we examined the effects of vimentin and cytokeratin-8 knockdown on chordoma cell mechanics. We found that chordoma cell stiffness, traction forces, and mechanosensitivity to substrate stiffness were all dependent on vimentin IFs. These results suggest that vimentin, rather than cytokeratin, IFs play a predominant role in chordoma cell mechanobiology. Finally, we analyzed the roles of vimentin and cytokeratin-8 IFs in cellular behaviors associated with cancer progression. We demonstrated that chordoma cell invasion and expression of the biomarker sonic hedgehog were dependent on vimentin. Further, we found that decreasing vimentin expression in chordoma cells may increase their sensitivity to chemotherapeutics. Because mechanical cues are important determinants of cell function, we hypothesize this correlation is in part due to the newly discovered role of vimentin IFs in chordoma cell mechanobiology. These results elucidate novel roles of vimentin and cytokeratin-8 IFs in chordoma cells, which may assist in the development of effective treatments for chordoma.
The Influence of Vimentin Intermediate Filaments on Human Mesenchymal Stem Cell Response to Physical Stimuli
(2017) Sharma, Poonam; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Mesenchymal stem cells (MSCs) are increasingly being investigated as a therapeutic cell population for a variety of diseases. However, these therapies are limited by an imperfect understanding of how MSCs interact with and respond to their physical environment. Cell response to external stimuli is mediated by the cytoskeleton. Of the cytoskeletal proteins, understanding of vimentin intermediate filaments’ influence on MSC behavior is still lacking, despite increasing evidence that they are involved in many cellular processes. In this work, we investigated the influence of vimentin intermediate filaments in modulating MSC characteristics and behavior by using lentiviral shRNA transduction to decrease vimentin levels in MSCs through RNA interference. First, the contribution of vimentin intermediate filaments to the deformability of MSCs within agarose hydrogels was examined. Vimentin-deficient MSCs were found to be less deformable than control cell populations and this resistance to deformation may be due to the compensatory role of actin microfilaments. Next, to determine how vimentin affects the ability of MSCs to interact with various microenvironments, we examined cell spreading on different extracellular matrix proteins, multiple substrate stiffness’, and in response to fluid shear stress. An intact vimentin network was found to be necessary for unimpaired spreading on fibronectin, but only on stiffer substrates. Further, vimentin appears to be involved in resisting cell area changes in response to low fluid shear stress. Vimentin’s physical interaction with focal adhesions, rather than an impact at the transcriptional or translational level, may contribute to the cell spreading response observed. Finally, in the third part of this work, we examined the influence of vimentin on chondrogenic differentiation of MSC populations. Unexpectedly, we found that vimentin may not be involved in chondrogenic differentiation in late stage chondrogenic cultures. Instead, the culture condition-dependent microenvironment may have a greater impact, particularly in gene expression of matrix degrading enzymes and the αV integrin subunit. Altogether, these studies indicate a role for vimentin in the MSC response to physical stimuli. Moreover, this work furthers the dialogue surrounding MSCs’ interaction with different environments, the understanding of which will be critical for the development and evaluation of cell-based therapies.
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.
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.
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.

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