Theses and Dissertations from UMD
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Item Biomechanical regulation of T cells: The cytoskeleton at the nexus of force and function(2024) Pathni, Aashli; Upadhyaya, Arpita; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The adaptive immune response is a sophisticated and multi-pronged defense mechanism that provides specific and long-lasting protection against infections and cancer. Central to this response are T lymphocytes - immune cells that orchestrate the immune response and directly eliminate infected or malignant cells. T cell function is intricately linked to their cytoskeleton, a dynamic network of protein filaments, consisting of actin, microtubules, and intermediate filaments, which provides structure, facilitates movement, and regulates intracellular transport. While the biochemical aspects of T cell function have been well-studied, recent advances have highlighted how mechanical forces influence T cell behaviors such as activation, migration, and effector functions—all processes driven by dynamic cytoskeletal remodeling. However, the mechanisms by which cytoskeletal dynamics, forces and mechanical stimuli drive T cell function remain poorly understood. This dissertation investigates this interplay, focusing on cytotoxic T lymphocytes (CTLs), a subtype of T cells that directly kill infected or cancerous cells. To launch a killing response, naïve CD8+ T cells must be activated by antigen-presenting cells (APCs) in lymph nodes, following which they proliferate and differentiate into an effector CTL population. CTLs eliminate targets via a specialized interface called the immunological synapse (IS), where they release lytic granules containing cytotoxic molecules and exert cytoskeletal forces to induce target cell death. A key event in IS formation is polarization of the centrosome, or the microtubule-organizing center, facilitating directional release of lytic granules. We first examined how biochemical signals provided by APCs modulate the cellular cytoskeleton. APCs provide not only antigenic stimulation, but also co-stimulatory signals required for full activation. Inflammatory cytokines such as interleukin-12 (IL-12) act as a third signal, enhancing CTL proliferation and cytotoxicity. Our findings demonstrate that CTLs activated in the presence of IL-12 exhibit enhanced IS formation, altered actin dynamics and microtubule growth, and generate greater mechanical forces, thus highlighting how activation signals can shape T cell mechanics, dynamics and function. Next, we investigated how the mechanical properties of target cells influence CTL function. Employing a biomimetic hydrogel system that mimics the stiffness of target cells, we demonstrate that substrate stiffness modulates multiple aspects of CTL responses. CTLs interacting with stiffer substrates exhibit enhanced spreading, accelerated actin ring formation, increased contractile forces, and more efficient centrosome polarization. Mechanical cues also influence lytic granule release and the nuclear translocation of mechanosensitive transcription factors. This work underscores the importance of mechanical cues in regulating immune responses. Given that coordinated cytoskeletal interactions are crucial for T cells to effectively respond to environmental cues, we further examined this crosstalk with a focus on intermediate filaments, the third, often understudied component of the cytoskeleton. Our characterization of the vimentin intermediate filament network reveals an expansive structure complementary to and dependent on other cytoskeletal components. We study the dynamics and organization of the vimentin network and find a close association of this network with the centrosome. Our results suggest a structural role for vimentin in supporting IS formation. Throughout this work, we use advanced imaging techniques and analysis approaches to probe various facets of T cell function. By bridging immunology, cell biology, and biophysics, this research contributes to our understanding of how physical forces shape immune responses.Item Mechanical Adaptability of Ovarian Cancer Tumor Spheroids(2021) Conrad, Christina Barber; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A major obstacle in ovarian cancer treatment is the onset of ascites, an abnormal build-up of fluid in the peritoneal cavity. Using in vitro perfusion models, ascitic flow has been shown to drive epithelial-mesenchymal-transition (EMT) biomarker expression, promote epidermal growth factor receptor (EGFR) downstream signaling, and upregulate chemoresistance. Given the close ties between cell mechanics and behaviors, it is of interest to establish if mechanotransduction serves a role in cell signaling dysfunction. Here, we identified the mechanical behavior of tumor spheroids subjected to flow using Brillouin confocal microscopy, a non-contact optical method based on the interaction between incident light and microscopic mechanical waves within matter. We validated this technique by establishing a relationship with the traditionally derived Young’s modulus measured using atomic force microscopy and a parallel-plate compression device. Following characterization, we used Brillouin confocal microscopy to map mechanical properties of tumor spheroids embedded in a microfluidic chip and found that continuous flow for 7 days caused a decreased Brillouin shift (i.e., stiffness) compared to tumors in a static condition. Another physical phenomenon related to ascites is dysregulated osmolality. Maintaining cell water homeostasis is driven by the transport of water to balance solute concentration and can have direct consequences on mechanics and biochemical signaling in cells. Recently, it was demonstrated in single cells that cell volume correlated with mechanical properties; but the effects in tumor spheroids which exhibit multi-cellular interfaces has remained unclear. Here, we derived relationships between osmolality and nuclear volume, tumor cell density, and Young’s modulus, and found the correlations in spheroids resembled single cell relationships previously described in literature. Additionally, we looked at the impact of osmotic shocks on E-cadherin junctions and found aggregates formed with a unique timescale compared to morphology. Lastly, we observed reversibility of the mechanical, morphological, and molecular properties which showed the tumor’s dynamic ability to respond to physical cues. Altogether, this work demonstrated how flow and osmosis associated with ovarian cancer ascites can trigger phenotype transformations. These findings warrant future investigations into how the regulation of mechanotransduction pathways can be harnessed to prevent chemoresistance and signaling dysfunction.Item The Equilibrium Geometry Theory for Bone Fracture Healing(2008-04-29) Yew, Alvin Garwai; Hsieh, Adam H; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Models describing the impact of mechanical stimuli on bone fracture healing can be used to design improved fixation devices and optimize clinical treatment. Existing models however, are limited because they fail to consider the changing fracture callus morphology and probabilistic behavior of biological systems. To resolve these issues, the Equilibrium Geometry Theory (EGT) was conceptualized and when coupled with a mechanoregulation algorithm for differentiation, it provides a way to simulate cell processes at the fracture site. A three-dimensional, anisotropic random walk model with an adaptive finite element domain was developed for studying the entire course of fracture healing based on EGT fundamentals. Although a coarse cell dispersal lattice and finite element mesh were used for analyses, the computational platform provides exceptional latitude for visualizing the growth and remodeling of tissue. Preliminary parameter and sensitivity studies show that simulations can be fine-tuned for a wide variety of clinical and research applications.