Theses and Dissertations from UMD
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Item MULTISCALE MEASUREMENTS OF ELECTRICAL & MECHANICAL CELLULAR DYNAMICS(2023) Alvarez, Phillip; Losert, Wolfgang; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation focuses on the study and measurement of coupled electrical and mechanical responses in mammalian cells, tissues, and organs. Cellular biophysics often studies forces and their impact on biochemical pathways. These forces can be electrical, resulting in neuronal action potentials or cardiac cell contractions, or mechanical, driving e.g., a cell’s ability to recognize physical probing or surface texture. These forces and their responses, though, are frequently coupled through interlinked cellular mechanisms which result in emergent responses that take both electrical and mechanical signals into account. One challenge in capturing these emergent responses is that they occur on multiple scales, from the intracellular scale to the organ scale, limiting the ability of commercial microscopes to image these responses simultaneously. In this work I use surface texture, optical imaging, and multiscale-capable image analysis algorithms across these scales to elicit and measure electrical and mechanical responses. To image emergent responses from electrical and mechanical coupling, I developed two custom microscopes that can image at multiple length scales and timescales simultaneously. The Multiscale Microscope can capture slow intracellular mechanical dynamics concurrently with fast tissue scale electrical dynamics, while the BEAMM microscope links fast tissue scale electrical dynamics with both intracellular mechanical dynamics and slower organ-scale mechanical and electrical responses. Finally, I describe ongoing and future studies which exploit these new capabilities for multiscale measurements of electrical and mechanical dynamics.Item Comparison of NF-kB Regulation in Naive and Anergic Primary CD8+ T Lymphocytes(2011) Clavijo, Paul Esteban; Frauwirth, Kenneth; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Due to the cytotoxic potential of CD8+ T cells, maintenance of CD8+ tolerance is critical. A major mechanism of peripheral tolerance in T lymphocytes is the induction of anergy, a refractory state caused by T lymphocyte activation in the absence of costimulation. Hallmarks of anergy are decreased IL-2 secretion and decreased proliferation. Here we used a T cell receptor transgenic mouse model to determine whether there are defects in the NF-κB signaling pathway in CD8+ T lymphocytes rendered anergic in vivo. In the anergic cell population, decreased NF-κB-mediated gene transcription and NF-κB p65 subunit DNA binding activity were observed. These changes were not due to inhibition of early NF-κB activation events, including IκBα degradation and NF-κB p65 subunit nuclear translocation, which occurred normally in anergic T lymphocytes. Nor were they related to defective phosphorylation of p65 at Ser536 in the cytoplasm or Ser276 in the nucleus, as p65 was phosphorylated at these residues in both naïve and anergic T lymphocytes with similar kinetics. However, the anergic CD8+ lymphocytes failed both to phosphorylate the NF-κB p65 subunit at Ser311 an event implicated in the recruitment of histone acetyl-transferase molecules such as CBP and p300, and to acetylate p65 at Lys310. Both of these posttranslational modifications have been shown to be critical for the positive regulation of NF-κB transcriptional activity. Thus, our results suggest that defects in key phosphorylation and acetylation events in p65 underlie defective NF-κB transactivation capacity and resultant lack of T cell function observed in anergic CD8+ T lymphocytes. Taken together these data provide a novel mechanistic explanation of how NF-κB p65 subunit is regulated in anergic CD8+T lymphocytes leading to defective NF-κB transcriptional activity and suggest that recruitment of CBP/p300 and p65 DNA binding in vivo is abrogated in anergic T lymphocytes.Item Ribosomal Protein L11: A Cog in the Nanomachine(2011) Rhodin, Michael Hoover Johannes; Dinman, Jonathan D; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Comprised of two major subunits of both rRNA and proteins, the ribosome is a biological nanomachine, acting as the central player in the process of protein translation. Recent advances in molecular imaging have enabled the visualization of the disparate functional centers within the ribosome, leading to the question of how these critical regions coordinate their actions and communicate with each other. This work examines the essential ribosomal protein L11, located in the central protuberance of the large subunit. L11 maintains connections with the 5S rRNA, H84 of the 25S rRNA, comes in close proximity to the T-loop of the bound peptidyl tRNA, and shares an intersubunit bridge with small subunit protein S18. L11 was found to have a critical dynamic loop which samples the occupancy status of the P-site pocket of the ribosome and communicates this information through H84. L11's intersubunit bridge (the B1b/c bridge) mediates an intersubunit communication network from the decoding center to the peptidyl transferase center of the ribosome. L11 is also involved in proper subunit joining. Mutations in L11 were found to have effects on A- and P-site tRNA binding, translational fidelity, and growth and viability of yeast cells.Item The activation of memory B cells to generate high affinity antibody responses in vitro and in vivo(2011) Richard, Katharina; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Immunological memory is the hallmark of the adaptive immune system. The humoral branch of the immunological memory is mediated by memory B-cells (mB). Memory B cells are marked by longevity, expression of antibodies with high affinity, and ability to generate robust antibody responses upon reencountering pathogens. However, requirements for the activation of mB cells and the induction of humoral memory responses are not well understood. This thesis examines the role of Toll-like receptors (TLRs) in mB activation using an immunized mouse model. TLRs are a family of receptors that recognize common molecular patterns of microbial pathogens and stimulate innate immune responses. Our study found that mouse mB expressed TLR9 and 4, and responded to their agonists in vitro by differentiating into high affinity IgG secreting plasma cells. However, TLR agonists alone were not sufficient to activate memory B cells in vivo. Antigen was required for the clonal expansion of antigen-specific memory B cells, the differentiation of mB cells to high affinity IgG secreting plasma cells, and the recall of high affinity antibody responses. The Ag- specific B cells that had not yet undergone isotype switching showed a relatively higher expression of TLR4 than memory B cells, which was reflected in a heightened response to its agonist, but in both cases of TLR4 and 9 yielded mostly low affinity IgM secreting plasma cells. When immunized together with the antigen, TLR agonists not only boosted the antigen-specific titers, but also increased affinity and isotype switching of the immunoglobulin. Thus, while TLR agonists alone are unable to activate mB in vivo, they can enhance humoral memory responses induced by the antigen.Item CHARACTERIZATION OF THE TWO VACCINIA VIRUS MATURE VIRION-SPECIFIC PROTEINS, A26P AND A25P(2010) Howard, Amanda Rachel; Dinman, Jonathan; Bernard, Moss; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Poxviruses produce two morphologically distinct infectious particles, mature virions (MVs) and extracellular virions (EVs). During replication, some MVs differentiate and become wrapped with cellular membranes, transported to the periphery, and exported as EVs. Some orthopoxviruses, e.g., cowpox virus (CPXV), form large, discrete cytoplasmic inclusions called A-type inclusion bodies (ATIs) within which MVs are embedded by a process called occlusion. ATIs are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) that fail to form ATIs. VACV does encode a functional A26p, which along with the ATIp is required for occlusion. A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV and regulates occlusion. Additionally, little is known about the formation of ATIs and how MVs become embedded within them. Here, experiments show that A26p is incorporated into MVs by the A27p-A17p complex and interacts with A25p, a truncated form of the CPXV ATIp. Restoration of the full-length ATI gene is sufficient for VACV ATI formation and the occlusion of MVs. A26p directly interacts with ATIp, and this interaction, as well as the A26p-A27p interaction, are required for occlusion. The data demonstrates that ATI mRNAs are transported out of viral factories (VFs) and translated in the cytoplasm. ATIs enlarge both by new protein synthesis and by coalescence, which requires microtubules. ATIs do not nucleate around MVs but rather MVs move along microtubules to embed within ATIs. Taken together, the data suggest a model for occlusion in which MVs move along microtubules to ATIs that are translated from mRNAs in the cytoplasm. At the ATIs, A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane. Although the specificity of A26p for MVs suggested A26p might regulate wrapping, I did not detect an effect of either the deletion of A26 or occlusion on the production of EVs.Item ROLE OF ACID SPHINGOMYELINASE IN ICAM-1/NHE1-DEPENDENT ENDOCYTOSIS: IMPLICATIONS IN LEUKOCYTE TRANSMIGRATION(2010) Serrano, Daniel; Muro, Silvia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Engagement of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells (ECs) by anti-ICAM coated beads generates vesiculization via cell adhesion molecule (CAM)-mediated endocytosis, a clathrin-/caveolae-independent pathway involving Na+/H+ exchanger 1 (NHE1). ICAM-1 itself plays a key role in transendothelial migration (TEM) of leukocytes, particularly via the transcellular route. This involves endothelial endocytic vesicles that coalesce into transmigration pores, through which leukocytes transmigrate without disrupting EC junctions. The contribution of CAM-mediated endocytosis to the formation of docking sites and vesicular structures supporting TEM was explored in this study. Results show that the ICAM-1/NHE1-dependent CAM-mediated pathway associates with acid sphingomyelinase and ceramide. This supports plasmalemma deformability and cytoskeleton rearrangement, bridging these events to the formation of endothelial docking structures and vesicles involved in leukocyte transmigration.Item Characterization of Atg6 function in autophagy and growth control during Drosophila melanogaster development(2010) Hill, Jahda Hope; Wu, Louisa P; Baehrecke, Eric H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The tumor suppressor Beclin 1 mitigates cell stress by regulating the lysosomal degradation pathway known as autophagy. This process involves formation of intracellular double-membraned vesicles, known as autophagosomes, which engulf proteins and damaged organelles and fuse with lysosomes, where the contents are degraded. It is unclear whether the function of Beclin 1 in autophagy is related to cell transformation in beclin 1+/- animals. Using the fruit fly, Drosophila melanogaster, I investigated the function of the Beclin 1 ortholog Atg6 in autophagy and growth control. Through transgenic experiments, I found that Atg6, like Beclin 1, induces autophagy by functioning in a complex consisting of the lipid kinase Vps34 and the serine–threonine kinase Vps15. I also generated a strong loss of function mutant, Atg61, and found that Atg6 is required for development. Atg6 mutant animals contained an excess of blood cells, which surrounded melanotic tumors prior to death. At the cellular level, Atg6 is required for autophagy and endocytosis, and cells lacking Atg6 accumulate high levels of the endoplasmic reticulum stress protein Hsc3 and the adaptor protein p62. I also showed that Atg6 mutant cells displayed mis-regulated nuclear localization of NF κB proteins, transcription factors whose downstream targets include regulators of innate immunity. Significantly, my results suggest that Atg6 may regulate growth independent of its function in autophagy, as mosaic loss of Atg6 in the eye resulted in over-representation of Atg6 mutant cells, a phenotype not shared by other autophagy gene mutant mosaics. Finally, through a collaborative effort, our lab identified a novel function for Atg6 in regulation of epithelial cell polarity. This finding is significant, as epithelial tumor cells are known to lose polarity during metastasis. Our studies have provided a significant contribution to the Beclin 1 field, by providing the first characterization of a Drosophila Atg6 mutant, and demonstrating its function in novel cellular processes.Item Functional analysis of ESAT-6 and EspB, two virulence proteins secreted by the ESX-1 system in Mycobacterium marinum(2010) Smith, Jennifer Ann; Briken, Volker; Gao, Lian-Yong; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm) are able to persist inside host cell macrophages by modulating the phagosome environment. ESX-1 is a specialized secretion system that is required for virulence. Two of the proteins secreted by ESX-1 are ESAT-6 and EspB. They are codependent for secretion and are important virulence effectors, though their specific functions are not known. Mm is able to escape from the phagosome into the host cell cytosol where it can initiate actin-based motility. Mm escape is dependent on a functional ESX-1 system. I show that the ESAT-6 protein is able to form pores in host cell membranes which may play a role in Mm escape from the phagosome. I also dissect the Mm EspB protein and show that cleavage of EspB is required for growth inside RAW cells, virulence in zebrafish, and for modulating ESAT-6 secretion. The resulting C-terminal 11 kDa fragment is sufficient for the codependent secretion of ESAT-6; while the 50 kDa N-terminal fragment seems to be somewhat dispensable for ESAT-6 secretion but is definitely required for virulence. When EspB is expressed as a full-length protein, the highly conserved WXG motif in the N-terminal fragment is involved in the codependent secretion of the two proteins since secretion is reduced when this motif is mutated. Interestingly, when the N-terminal fragment is expressed without the C-terminal fragment it can secrete independent of the ESX-1 system, indicating that the C-terminus confers specificity for EspB secretion through ESX-1. I show that the virulence function of the EspB N-terminal fragment is dependent on the secretion of ESAT-6, and EspB must be expressed in its full-length form in order to be fully functional. These results indicate that EspB function is dependent on a close association with ESAT-6. It is possible that the N-terminus is translocated into the host cell cytosol through the ESAT-6 formed pore.Item DEVELOPMENT OF TOOLS TO CHARACTERIZE PROTEIN-PROTEIN INTERACTIONS(2010) Jiang, Jiangsong; Li, Shuwei; Stewart, Richard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Protein-protein interactions (PPIs) are crucial to most biological processes and activities. Large-scale PPI screening has been applied to model organisms as well as to human cells. Two approaches have been used extensively in high-throughput PPI studies: (i) the Yeast Two-Hybrid (Y2H) assay (a bottom-up method), and (ii) the tandem affinity purification (TAP) (a top-down method). However, a close examination of both techniques revealed issues that limit their effectiveness. Thus, it is important to develop new methods that can bridge the gap between the Y2H and the TAP. In this thesis, two approaches were developed to meet this need. The first approach was a photoaffinity labeling tool, which was based on a photo-caged reactive intermediate para-quinone methide (pQM) to study protein-peptide associations. This system was developed and optimized by using the interaction between catPTP1Bm and the EGFR peptide as a test case. Highly specific protein labeling was achieved, and mass spectrometry (MS) was used to identify the crosslinked site on the target protein. Interestingly, two peptides from catPTP1Bm detected by MS were found close to the enzyme-substrate binding interface in the three-dimensional structure of the complex, which demonstrated this method might be useful for the analysis of protein complex conformation. The second approach, named "PCA plus", took advantage of a technique referred to as "Protein-fragment Complementation Assay (PCA)". A hydrolysis-deficient mutant β-lactamase (E166N) was used, which enabled interacting protein labeling in live cells. With this modification, the PCA plus method realized live cell imaging with subcellular resolution. Fluorescent microscopy and flow cytometry analysis demonstrated its potential applications. In addition, a new β-lactamase substrate was developed for the PCA plus method and was applied to enable purification, from living cells, of prey protein interacting with a bait protein. The observed enrichment of interacting partners suggested the system could be used for high-throughput PPI screening. Moreover, this method could also be useful for the characterization of low affinity and transient PPIs because of its capacity on labeling interacting protein inside cells.Item T Cell Activation Requires Glucose Retention and Use Via Mechanisms Modulated by MAPK Signaling(2010) Marko, Aimee Joy; Frauwirth, Kenneth A.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)T lymphocytes play a critical role in a cell-mediated immune response. During activation, extracellular and intracellular signals alter T cell metabolism in order to meet the energetic and biometabolic needs of a proliferating, active cell, but control of these phenomena is not well defined. The idea that metabolism can be regulated directly via receptor-mediated signals is being investigated. The T cell receptor (TCR) and CD28 pathways were stimulated and signaling was modified using chemical inhibitors of MAP kinases. Metabolic changes were monitored using assays for glucose uptake, hexokinase activity, glycolysis, Krebs Cycle, and pentose phosphate pathway. Two MAPK pathways (ERK, JNK) appear to be determining factors because their inhibition downregulated metabolism. Inhibition of another MAPK pathway (p38) causes some downregulation of these activities but the effect is not as strong as with the ERK and JNK inhibitors, suggesting that the p38 pathway is less important in regulating glucose metabolism.