CueO is a multicopper oxidase and catalyses the four-electron reduction of dioxygen to water and functions to protect Escherichia coli against copper-induced toxicity. The mechanism of oxygen reduction in multicopper oxidases has been well studied, but the key structures of the reaction intermediates are not known. A combination of kinetic measurements, mutagenesis and X-ray crystallographic studies were conducted to entrap and structurally characterize the reaction states in CueO. CueO has a methionine-rich insert and a labile copper binding site, two features found only in multicopper oxidases involved in copper detoxification. The role of these features in CueO activity has been investigated. In a separate study, a simple mathematical model based on infectivity amongst clustered receptors was developed to explain the chemotactic sensitivity, response range and other key features of chemotaxis.This study describes the successful entrapment of three out of four functional states in CueO. The crystal structures of these reaction states are presented. Using single-turnover oxygen reduction kinetics that were measured using a stopped-flow device, the optical absorption features of three different fully oxidized forms of CueO were captured: the native intermediate, the resting oxidized state and another intermediate lying between them. Stopped-flow studies combined with electron transfer kinetic measurements revealed a role of the conserved residue, E506, in either the protonation of the native intermediate or the release of water molecules formed as a product of the reaction.Cu(I) and Ag(I) bound crystal structures of CueO were determined revealing three binding sites along the methionine-rich helix used by both metal ions. The labile, regulatory copper site in CueO was shown to be a Cu(I) susbtrate oxidation site. Ag(I) was shown to be a potent inhibitor of all CueO activities in vitro and copper detoxification by the cue system in vivo. The cus system was discovered to be necessary for removing Ag(I) inhibition of copper detoxification by the cue system. These results provide further insights into the role of CueO in copper detoxification and the effect of silver on the detoxification mechanism.

CD34 and its relatives, Podocalyxin and Endoglycan, comprise of a family of surface sialomucins expressed by hematopoietic stem/progenitor cells, and vascular endothelia. Recent data suggest that they serve as either pro- or anti-adhesion molecules depending on their cellular context and their post-translational modifications. We were interested in identifying Podocalyxin ligands and their cellular distribution and understanding the role of these factors in signaling, adhesion and migration. Using both a lambda phage screen assay and mass spectrometry, we identified the Na⁺/H⁺ exchanger regulatory factor-i (NHERF-l) as a selective ligand for Podocalyxin and Endoglycan but not for the closely related CD34. Furthermore, we showed that NHERF-1 is expressed by all, lineage⁻, Sca-1⁺ and c-kit⁺ (LSK) cells, which are known to express Podocalyxin and have long-term repopulating characteristics of hematopoietic stem cells. In addition, upon IL-3 stimulation of a factor dependent cell line (FDC-P 1) these proteins re-localize and co-localize in an asymmetrical pattern. By using a lentiviral based shRNA system to silence Podocalyxin and NHERF- i proteins, we observed that migration across stromal monolayer towards a CXCL12 and SCF gradient is significantly impeded in cells that lack Podocalyxin but not NHERF-1. Following in vitro stimulation with a combination of CXCL12 and SCF we observed that Podocalyxin co-associates with CXCR4. Furthermore, cells lacking Podocalyxin have decreased phospho-AKT, a key signaling molecule downstream of c-kit and CXCR4 receptors. Taken together, our data supports the conclusion that Podocalyxin co-association with CXCR4 modulates downstream signaling to efficiently regulate HSC homing.

This work begins with chemotaxis studies involving Salmonella typhimurium. Known chemical attractants (ribose, aspartic acid, etc.) and repellents (nickel chloride, sodium acetate, etc.) were tested to direct bacteria swimming patterns. It was found that high concentrations of both attractant and repellent, approximately 10% chemical in deionized (DI) water, yielded better separation results than lower concentrations, such as 1% and .1% chemical in DI water. Utilizing these attractants or repellents appropriately can allow live bacteria to be directed in a desired manner in a microfluidic device, while dead bacteria, which yield no response, can be separated into a waste reservoir. Another important aspect of bacteria separation is preconcentration, or the process of concentrating bacteria in a usable amount of liquid for further analysis in a microfluidic device. This study introduces a method of capturing Salmonella typhimurium through the use of magnetic nanoparticles (MNPs) without functionalizing them with antibody or amine coatings. Based on the work by Deng et al., MNPs were prepared in various ways to alter their diameter and surface characteristics to achieve optimal bacteria capture efficiency. A capture efficiency of approximately 94% has been achieved by altering chemical quantities in the MNP fabrication process. A macro-scale flow cell prototype was designed and characterized in order to ‘clean’ large volumes of buffer and separate the bacteria-MNP aggregates through the use of a magnetic field. Finally, intervention of bacteria is a significant topic in food safety applications. This study utilizes Fe (III) to inhibit bacteria growth. This chemical was used in the presence of Salmonella, E. coli, Staphylococcus, and Pseudomonas. Further experiments were conducted with raw chicken and lettuce contaminated with Salmonella typhimurium. Using as little as .005M Fe III in DI water, up to 5 orders of magnitude reduction in bacteria growth was seen on test plates as compared to control plates.

Lymphocyte migration is crucial for adaptive immunity. CCR7 and its ligands mediate the migration and positioning of T cells in lymph nodes but the underlying mechanism is complex. The research in this thesis investigated CCR7-mediated T cell migration using a microfluidics-based approach. A microfluidic method suitable for quantitative migration analysis of genetically modified lymphocyte transfectants was developed. Using this method, I demonstrated chemotaxis of Jurkat transfectants expressing wild-type or C-terminal mutated CCR7 to a CCL19 gradient, and characterized the difference in transfectant migration mediated by wild-type and mutant CCR7. The fluorescent tag allows identification of CCR7-expressing transfectants in cell migration analysis, and microscopy assessment of CCR7 dynamics in migrating cells. Furthermore, my results also showed interesting migratory behaviours of CCR7 Jurkat transfectants in a specific co-existing CCL19 and CCL21 fields. This developed method will be broadly useful for studying cell migration signalling.

<p>Many cells are remarkably proficient at tracking even shallow chemical gradients, despite tiny differences in receptor occupancy across the cell. Stochastic receptor-ligand interactions introduce considerable noise in instantaneous receptor occupancy, so it is thought that spatial information must be integrated over time to allow noise filtering. The mechanism of temporal integration is unknown. We used the mating response of the budding yeast, <italic>Saccharomyces cerevisiae</italic>, as a model to study eukaryotic gradient tracking. </p><p>During mating, yeast cells polarize and grow up a gradient of pheromone to find and fuse with opposite-sex partners. Exposure to pheromone causes polarity regulators to cluster into a tight "patch" at the cortex, directing growth toward that site. Timelapse microscopy of fluorescently-labeled polarity proteins revealed that the patch wandered around the cortex during gradient tracking. Mathematical modeling and genetic analysis suggested that fusion of vesicles near the polarization site could perturb the polarity patch and promote wandering. Wandering is decreased due to global effects from pheromone signaling as well as interactions between receptor-activated G&beta; and the exchange factor for the polarity regulator Cdc42. We found that artificially stabilizing patch wandering impaired accurate gradient tracking.</p><p>We suggest that ongoing polarized vesicle traffic causes patch wandering, which is locally reduced by pheromone-bound receptors. Thus, over time, spatial information from the pheromone gradient biases the random wandering of the polarity patch so that growth occurs predominantly up-gradient. Such temporal integration may enable sorting the low signal from stochastic noise when tracking shallow gradients.</p> / Dissertation

An important mechanism during nervous system development is the guidance of axons by chemical gradients. The structure responsible for responding to chemical cues in the embryonic environment is the axonal growth cone -- a structure combining sensory and motor functions to direct axon growth. In this thesis, we develop a series of mathematical models for the gradient-based guidance of axonal growth cones, based on the idea that growth cones might be optimised for such a task. In particular, we study axon guidance from the framework of Bayesian decision theory, an approach that has recently proved to be very successful in understanding higher level sensory processing problems. We build our models in complexity, beginning with a one-dimensional array of chemoreceptors simply trying to decide whether an external gradient points to the right or the left. Even with this highly simplified model, we can obtain a good fit of theory to experiment. Furthermore, we find that the information a growth cone can obtain about the locations of its receptors has a strong influence on the functional dependence of gradient sensing performance on average concentration. We find that the shape of the sensitivity curve is robust to changes in the precise inference strategy used to determine gradient detection, and depends only on the information the growth cone can obtain about the locations of its receptors. We then consider the optimal distribution of guidance cues for guidance over long range, and find that the same upper limit on guidance distance is reached regardless of whether only bound, or only unbound receptors signal. We also discuss how information from multiple cues ought to be combined for optimal guidance. In chapters 5 and 6, we extend our model to two-dimensions, and to explicitly include temporal dynamics. The two-dimensional case yields results which are essentially equivalent to the one dimensional model. In contrast, explicitly including temporal dynamics in our leads to some significant departures from the one-dimensional and two-dimensional models, depending on the timescales over which various processes operate. Overall, we suggest that decision theory, in addition to providing a useful normative approach to studying growth cone chemotaxis, might provide a framework for understanding some of the biochemical pathways involved in growth cone chemotaxis, and in the chemotaxis of other eukaryotic cells.

Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of schistosome-induced pathology, including granuloma formation, fibrosis and splenomegaly, is essential for understanding how schistosomes influence the immune system of the mammalian host. I report on the first whole genome microarray analysis of the murine liver and spleen during the progression of Schistosoma japonicum infection and of S. japonicum-Soluble Egg Antigen (SEA)-stimulated macrophages. My analyses of the infected liver revealed a distinct temporal relationship between the expression of chemokines and the recruitment of cells to the liver. T-cell and B-cell chemoattractants were up-regulated earlier reflecting the recruitment of these cells to the liver as illustrated by flow cytometry. The later phases of the response corresponded with peak accumulation of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs) and the expression of chemokines with activity for these cells including CCL11, members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12) and the hepatic stellate cell/myofibroblast chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14) and markers of alternatively-activated macrophages (e.g. Retnla) during this later phase provided further evidence for a role for these cells in schistosome-induced pathology. Additionally, I demonstrated that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggested the involvement of neutrophils in S. japonicum¬-induced hepatic fibrosis. The transcriptional profile of the spleen was closely related to changes in cellular composition illustrated by flow cytometry and immunohistochemistry. Significant up-regulation of genes associated with progression through the cell cycle, proliferation makers and genes involved in lymphocyte proliferation, paralleled the initial expansion of T-cells and B-cells and the increased cellularity of the spleen overtime. Accumulation of eosinophils, neutrophils and macrophages was paralleled by enhanced expression of markers for these cells and the declining proportion of B- and T-cells in the spleen over time was reflected in the decreased expression of B- and T-cell markers. Significant up-regulation of Chi3l3 and F4/80+ macrophages suggested the presence of alternatively activated macrophages in the spleen, where these cells could play an immunoregulatory role. Comparison of the liver and spleen profiles revealed divergent expression of chemokines and cell adhesion molecules. Expression of lymphocyte chemokines including the homeostatic chemokines, CXCL13, CCL19 and CCL21, were significantly up-regulated in the liver while down regulated in the spleen. Expression of chemokines with activity for eosinophils (CCL11, CCL24), neutrophils (CXCL1) and monocytes (CXCL14, CCL12) and the cell adhesion molecules VCAM1, NCAM1, PECAM1 were up-regulated in the liver while unchanged in the spleen. Chemokines up-regulated in both organs were expressed at significantly higher levels in the liver. Divergent expression of chemokines and cell adhesion molecules likely contributes to the development of a chemotactic signalling gradient that promotes recruitment of effector cells to the liver. The results of liver and spleen microarrays suggested an important role for alternatively activated macrophages in the development of schistosome-induced pathology. This led me to investigate the in vivo transcriptional profile of S. japonicum SEA-stimulated peritoneal macrophages. The transcriptional profile of these cells was characterised by up-regulation of alternatively activated macrophage makers (Chi3l3, Chi3l4, Arg1). Retnla was not significantly induced in these macrophages suggesting that the specific function of these cells may differ to those induced by S. mansoni and other parasites. Other features of the transcriptional profile of these cells included modulated expression of T-cell co-stimulatory molecules and chemokines which may confer immunomodulatory activity. S. japonicum-stimulated alternative activation of macrophages was additionally associated with deactivation of classical activation pathways and altered expression of cell surface receptors and complement components that may alter phagocytic activity. Together these data significantly enhance our understanding of the mechanisms associated with alternative activation of macrophages and provide significant insight into the role of these cells in schistosomiasis japonica. The findings presented in this thesis represent the most comprehensive description to date of the molecular mechanisms, and especially chemotactic signalling pathways, regulating the development of schistosome-induced granulomas, fibrosis, splenomegaly and alternative macrophage activation in the murine host. In summary, my data have revealed that co-ordinated gene expression of chemokines in the liver and spleen regulates the recruitment of cells to the liver during schistosome infection. My results provide additional evidence for a role for neutrophils and alternatively activated macrophages in the development of schistosome-induced pathology and provide further insight to the molecular basis of alternative macrophage activation during infection. Furthermore, my data serve to highlight clear differences in the pathogenesis of schistosomiasis mansoni and schistosomiasis japonica. Together these findings further our understanding of the systemic, local, cellular, and especially, chemokine signalling pathways that regulate the development of S. japonicum-induced pathology and offer correlative insight into the pathogenesis of other chronic inflammatory diseases where fibrosis, splenomegaly and alternative activation of macrophages are common features.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 107-114). Also available for download via the World Wide Web; free to University of Oregon users.

Ulm, Univ., Diss., 2001.

Hohenheim, Univ., Diss., 2007.