Theses and Dissertations from UMD
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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 give thesis/dissertation in DRUM
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Item Analysis of Flow-Based Microfluidic Gradient Generators for the Study of Bacterial Chemotaxis(2015) Wolfram, Christopher James; Rubloff, Gary W; Material Science and Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chemotaxis is a phenomenon which enables cells to sense concentrations of certain chemical species in their microenvironment and move towards chemically favorable regions. This behavior is best understood in the bacteria Escherichia coli, which exhibits chemotaxis towards a variety of energy sources and signaling molecules. Recent advances in microbiology have engineered the chemotactic properties of bacteria to perform novel functions, but traditional methods of characterizing chemotaxis are not sufficient for such complex applications. The field of microfluidics offers solutions in the form of gradient generators. Many of these gradient generators are flow-based, where a chemical species diffuses across a solution moving through a microchannel. A microfluidic gradient generator was explored as a chemotaxis platform. Sources of error during experimental operation and methods of mitigating this error were demonstrated, and the fundamental theory behind these devices was examined. These devices were determined to be inadequate for the study of bacterial chemotaxis.Item Developing Tools for Investigating Chemotaxis Signal Clusters in Bacillus subtilis(2012) Rogers, James Allen; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Many bacteria make use of a set of dedicated chemoreceptor proteins to control a His-Asp signaling system; this control converts environmental sensory information into instructions that regulate flagellar rotation, enabling chemotaxis. This thesis summarizes my investigations of some of the chemotaxis signaling proteins in Bacillus subtilis, particularly coupling proteins CheW and CheV. Proteins CheA, CheW, CheV, CheY, and FliM were each expressed in B. subtilis as translational fusions with either YFP or CFP. These fusion proteins were then shown to fluoresce in living bacterial cells. Motility experiments were conducted to compare the function of these fusion proteins to their wild type counterparts. This thesis proposes a series of experiments that would use these fluorescent fusion proteins to further explore the idea that these chemotaxis proteins change position when B. subtilis encounters chemostimuli.Item Individual and collective dynamics of chemotaxing cells(2011) McCann, Colin Patrick; Losert, Wolfgang; Parent, Carole A; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The study of the dynamics of interacting self-propelled entities is a growing area of physics research. This dissertation investigates individual and collective motion of the eukaryote Dictyostelium discoideum, a system amenable to signal manipulation, mathematical modeling, and quantitative analysis. In the wild, Dictyostelium survive adverse conditions through collective behaviors caused by secreting and responding to chemical signals. We explore this collective behavior on size scales ranging from subcellular biochemistry up to dynamics of thousands of communicating cells. To study how individual cells respond to multiple signals, we perform stability analysis on a previously-developed computational model of signal sensing. Polarized cells are linearly stable to perturbations, with a least stable region at about 60 degrees off the polarization axis. This finding is confirmed through simulations of the model response to additional chemical signals. The off-axis sensitivity suggests a mechanism for previously observed zig-zag motion of real cells randomly migrating or chemotaxing in a linear gradient. Moving up in scale, we experimentally investigate the rules of cell motion and interaction in the context of thousands of cells. Migrating Dictyostelium discoideum cells communicate by sensing and secreting directional signals, and we find that this process leads to an initial signal having an increased spatial range of an order of magnitude. While this process steers cells, measurements indicate that intrinsic cell motility remains unaffected. Additionally, migration of individual cells is unaffected by changing cell-surface adhesion energy by nine orders of magnitude, showing that individual motility is a robust process. In contrast, we find that collective dynamics depend on cell-surface adhesion, with greater adhesion causing cells to form smaller collective structures. Overall, this work suggests that the underlying migration ability of individual Dictyostelium cells operates largely independent of environmental conditions. Our gradient-sensing model shows that polarized cells are stable to small perturbations, and our experiments demonstrate that the motility apparatus is robust to considerable changes in cell-surface adhesion or complex signaling fields. However, we find that environmental factors can dramatically affect the collective behavior of cells, emphasizing that the laws governing cell-cell interaction can change migration patterns without altering intrinsic cell motility.Item Binding Interactions in the Bacterial Chemotaxis Signal Transduction Pathway(2008-12-08) Eaton, Anna Kolesar; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The investigation of signal transduction pathways is critical to the basic understanding of cellular processes as these pathways function to regulate diverse processes in both eukaryotes and prokaryotes. This dissertation focuses on understanding some of the biochemical events that take place in the chemotaxis signal transduction pathway of bacteria. In this system, cell-surface receptor proteins regulate a histidine protein kinase, CheA, that autophosphorylates and then transfers its phosphate to an effector protein, CheY. Phospho-CheY, in turn, influences the direction of flagellar rotation. This sequence of biochemical events establishes a chain of communication that ultimately allows the chemotaxis receptor proteins to regulate the swimming pattern of the bacterial cell when it encounters gradients of attractant and repellent chemicals in its environment. The three projects presented in this dissertation sought to fill basic gaps in our current understanding of CheA and CheY function. In the first project, I examined the nucleotide binding reaction of CheA using the fluorescent nucleotide analogue, TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate]. TNP-ATP is an effective inhibitor for CheA. By monitoring the fluorescence of TNP-ATP when it bound to CheA, I examined the affinity of the binding interaction and discovered that the two ATP binding sites of each CheA dimer exhibited negative cooperativity in their interactions with TNP-ATP. This is the first evidence of cooperativity in the histidine protein kinase superfamily. In the second project, I focused on elucidating the binding mechanism that underlies formation of the CheA:TNP-ATP complex. My results indicated a three-step mechanism, including rapid formation of a low-affinity complex, followed by two steps during which conformational changes give rise to the final high-affinity complex. This same basic mechanism applied to CheA from Escherichia coli and from Thermotoga maritima. In the third project, I turned my attention to studying the CheY phosphorylation and binding reactions using fluorescently labeled versions of CheY. The results of this final study indicated that CheY proteins labeled with the fluorophore Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] could be useful tools for investigating CheY biochemistry. However my results also brought to light some of the limitations and difficulties of this approach.Item The Role of CheV in S. typhimurium Chemotaxis(2006-12-11) Dougherty, Megan; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The chemotaxis systems of Escherichia coli and Salmonella typhimurium are thought to be virtually identical. However, recently a putative chemotaxis gene, cheV, was found to be present in S. typhimurium but not in E. coli. Sequence analysis shows that the CheV protein shares sequence similarity to both CheW and CheY. My thesis research investigated whether cheV does play a role in S. typhimurium chemotaxis. My results show that disruption of the cheV gene had no effect on S. typhimurium's swarming ability and only a minor effect on the ability of S. typhimurium to sense/respond to serine and its ability to accomplish surface motility. My results also indicate that overexpression of the cheV gene disrupts S. typhimurium's swarming ability, as well as, S. typhimurium's ability to sense/respond to serine and S. typhimurium's ability to accomplish surface motility. Overall, these results suggest that CheV may be involved in S. typhimurium chemotaxis.Item Distinguishing Modes of Eukaryotic Gradient Sensing(2005-08-25) Skupsky, Ron; Losert, Wolfgang; Nossal, Ralph J; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The behaviors of biological systems depend on complex networks of interactions between large numbers of components. The network of interactions that allows biological cells to detect and respond to external gradients of small molecules with directed movement is an example where many of the relevant components have been identified. This behavior, called chemotaxis, is essential for biological functions ranging from immune response in higher animals to the food gathering and social behavior of ameboid cells. Gradient sensing is the component of this behavior whereby cells transduce the spatio-temporal information in the external stimulus into an internal distribution of molecules that mediate the mechanical and morphological changes necessary for movement. Signaling by membrane lipids, in particular 3' phosphoinositides (3'PIs), is thought to play an important role in this transduction. Key features of the network of interactions that regulates the dynamics of these lipids are coupled positive feedbacks that might lead to response bifurcations and the involvement of molecules that translocate from the cytosol to the membrane, coupling responses at distant point on the cell surface. Both are likely to play important roles in amplifying cellular responses and shaping their qualitative features. To better understand the network of interactions that regulates 3'PI dynamics in gradient sensing, we develop a computational model at an intermediate level of detail. To investigate how the qualitative features of cellular response depend on the structure of this network, we define four variants of our model by adjusting the effectiveness of the included feedback loops and the importance of translocating molecules in response amplification. Simulations of characteristic responses suggest that differences between our model variants are most evident at transitions between efficient gradient detection and failure. Based on these results, we propose criteria to distinguish between possible modes of gradient sensing in real cells, where many biochemical parameters may be unknown. We also identify constraints on parameters required for efficient gradient detection. Finally, our analysis suggests how a cell might transition between responsiveness and non-responsiveness, and between different modes of gradient sensing, by adjusting its biochemical parameters.Item SWIMMING FOR SULFUR: ANALYSIS OF THE ROSEOBACTER-DINOFLAGELLATE INTERACTION(2005-01-06) Miller, Todd Rex; Belas, Robert; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Marine algae are some of the most productive organisms on earth, and their survival is dependent upon a diverse community of bacteria that consume algal products. The identity of these bacteria and mechanisms used to interact with their algal partner are not well understood. Recently it has been shown that -Proteobacteria of the Roseobacter clade are the primary consumers of the algal osmolyte, dimethylsulfoniopropionate (DMSP). In addition, their production and activity is highly correlated with DMSP producing algal blooms, especially those containing dinoflagellates. To understand more about this relationship, I have studied Roseobacter-dinoflagellate interactions in laboratory cultures of Pfiesteria dinoflagellates, a ubiquitous group of estuarine, heterotrophic dinoflagellates. The results show that cultures of P. piscicida and a similar dinoflagellate, Cryptoperidiniopsis sp., harbor a robust DMSP degrading bacterial community that contains members of the Roseobacter clade. One of these bacteria, Silicibacter sp. TM1040 degrades DMSP by demethylation producing 3-methymercaptopropionate (MMPA). Interestingly, this bacterium senses and actively moves toward P. piscicida cells. It is highly chemotactic toward amino acids, especially methionine, and DMSP metabolites, including DMSP and MMPA. Chemotaxis of TM1040 toward P. piscicida cells is mediated in part by the presence of these compounds in the dinoflagellates. Using a fluorescent tracer dye, this bacterium was found attached and/or within P. piscicida cells. The apparent intracellular occurence of Silicibacter sp. TM1040 requires both flagella and motility since mutants lacking motility and/or flagella are not found within the dinoflagellate, although they can be found attached. The presence of Silicibacter sp. TM1040 in axenic dinoflagellate cultures enhances dinoflagellate growth, a process that does not require the bacteria to be intracellular. The genome sequence of Silicibacter sp. TM1040 indicates that this bacterium contains a large number (20) of chemoreceptors and a full complement of flagellar and other chemotaxis genes. In addition, this bacterium contains all of the genes necessary to produce a type IV secretion system similar to the vir pilus of Agrobacterium tumefaciens. Taken together, the data suggest that Silicibacter sp. TM1040 is an attached and/or intracellular symbiont of P. piscicida. The significance of this study to microbial and algal bloom ecology is discussed.