Theses and Dissertations from UMD
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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 Effects of Antigen Valency and CpG ODN on B Cells(2007-03-27) Arunkumar, Nandini; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)B cells express toll-like receptor 9 (TLR9), that recognizes microbial DNA containing unmethylated cytosyl guanosyl (CpG) sequences, induces innate immune responses and facilitates antigen-specific adaptive immunity. Studies indicate that in addition to stimulating innate immunity, TLR9 ligands can induce apoptosis in TLR9 expressing cancer cells. To understand the mechanism for TLR9-induced apoptosis, we compared the effects of CpG containing oligodeoxynucleotides (CpG ODN) on mouse primary, splenic B cells and a mouse lymphoma B cell line, CH27. CpG ODN stimulated the proliferation of primary B cells but inhibited cell proliferation and induced apoptosis in CH27 lymphoma B cells in a sequence-specific, TLR9-dependent fashion. While CpG ODN induced sustained activation of NF-B and increase in c-myc protein levels in primary B cells, NF-B activation was transient in the lymphoma B cells. These data suggest that the differential effects of CpG DNA on primary and lymphoma B cells occur due to differences in NF-B activation. The CpG ODN-induced impaired NF-B activation in the lymphoma B cells results in an imbalance between NF-B and c-myc activities, inducing apoptosis in TLR9-expressing B lymphoma cells. The B cell antigen receptor (BCR) binds to antigens in their native form. The BCR can distinguish subtle differences in antigen structure and trigger differential responses. Here, we analyzed the effects of antigen valency on the functions of the BCR using three different antigen systems - anti-BCR antibody -based antigens, phosphorylcholine (PC)-based antigens, and hen egg lysozyme (HEL)-based antigens. While both paucivalent and polyvalent antigens induced the redistribution of surface BCR into microdomains, polyvalent antigen-induced BCR microdomains persisted. Significantly, this trend was consistently observed in all three antigen systems studied. Ganglioside GM1, tyrosine-phosphorylated proteins and phosphorylated ERK colocalized with BCR microdomains, suggesting these function as surface signaling microdomains. Co-receptor, CD19 and MHC class II molecules, but not CD45 and transferrin receptor, concentrated in the BCR surface microdomains. Prolonged BCR caps were also concomitant with a reduction in BCR movement to late endosomes/lysosomes. Thus, antigen valency influences B cell responses by modulating the stability of BCR-signaling microdomains and BCR-mediated antigen transport.Item The M167 mu+kappa Immunoglobulin Transgene Increases Susceptibility to Pristane-Induced Plasmacytomas In BALB/c Mice(2005-04-20) Williams, Kimberly Gayle; Potter, Michael; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The goal of this research was to investigate the role of Ig transgenes (Tgs) in plasma cell tumor development as a useful means of examining potential antigen-specific B cell precursors. The Ig Tg M167mk (V1 mu plus Vk24) on a genetically PCT-susceptible BALB/c background was used to determine if B cells expressing anti-phosphorylcholine (PC) B cell receptors are more susceptible than non-Tg littermates to pristane induction of plasma cell tumors. The M167mk Tg mice, which express the anti-PC M167 BCR on >97% of B cells, developed a higher percentage of plasma cell tumors (63%) compared to non-Tg BALB/cAnPt littermates (35%), p<0.01, and had a reduced latent period from 240 to 200 days. Serologic and nucleic acid analysis of Igs expressed by Tg tumors revealed co-expression of the M167-id V1-mu/Vk24 transgenes and a rearranged endogenous VH-alpha/Vk Ig. In an attempt to learn something of the natural history of the PCT precursor B cell, the M167mk transgene and endogenous VH/VL Ig mRNA from Tg PCT cells were sequenced for evidence of somatic hypermutation, a hallmark of T-cell dependent germinal center activity. M167mu and k Tg mRNA transcripts revealed no evidence of mutation of the transgenes; however, sequencing of endogenous Ig mRNA from M167mk Tg PCTs showed that 80% contained somatic hypermutation (SHM) and 20% had germline Ig genes. PCTs from control groups, BALB/c non-Tg littermates and C.B20 mice, also contained 80% SHM and 20% germline Ig genes. M167mk Tg mice have an increased (MZ) B cell population (15-25% B220+), a hyper-activated TI responsive B cell phenotype, compared to non-Tg WT mice (5-10% B220+). These data suggest that PC-responsive M167mk B cells are a PCT-susceptible subset, as the M167mk Tg BCR generates B cell clones that are responsive to bacterial PC antigen that likely enhances overall proliferation of B cells during PCT development, thereby increasing the chances of a neoplastic B cell clone to expand and increasing the susceptibility of M167mk Tg mice to the induction of PCTS.