Microaerophilic and chemotaxic diazotrophs, azospirilla are found in close association with certain cereals such as durum wheat and maize and are active in enriching these ecological niches with the macronutrient nitrogen as ammonia. Regarded as highly pleomorphic, Azospirillum spp. are highly motile, using either a single polar flagellum when grown in liquid environments or peritrichous lateral flagella in viscous environments. Additionally, azospirilla are able to adhere onto surfaces as a biological film or aggregate cell-to-cell as nonproliferating flocculi, and these two processes having been suggested as positively affecting the survival and dispersal of the bacteria in the soil. Even though both biofilm formation and flocculation have been characterized via the presence of bacterial extracellular polysaccharides, the nature of the observed exopolysaccharides is still obscure, as are the underlying molecular mechanisms facilitating their organization. Here, we identified the optimal conditions for biofilm formation as a high C:N ratio under conditions of low aeration. Cells showed an increased preference for hydrophobic plastic rather than hydrophilic glass when the bacteria were first grown in a rich medium, TY, then were subcultured in a minimal media under these conditions. Using transposon mutagenesis, we also identified metabolic and cell-surface functions perhaps involved in the flocculation potential of these bacteria and we present an initial characterization of their contribution to this cellular differentiation process.

In this paper, we discuss the global existence of solutions for Chemotaxis models with saturation growth. If the coe±cients of the equations are all positive smooth T-periodic functions, then the problem has a positive T-periodic solution, and meanwhile we discuss here the stability problems for the T-periodic solutions.

Mast cells (MCs) are forceful multifunctional effector cells of the immune system. MCs are normally distributed throughout connective and mucosal tissues, but in several pathological conditions accumulation of MCs occur. This accumulation is probable due to directed migration of MCs and they are subjects for migration at least two different occations: 1) when they are recruited as progenitor cells from the blood into the tissue; and 2) when they as mature MCs are recruited to sites of inflammation. The aim of this study was to investigate MC migration to chemoattractants released in vivo or in vitro (body fluids collected from patients with asthma or rheumatoid arthritis and TH1- and TH2-cytokines) and to recombinant cytokines (transforming growth factor -β (TGF-β) and CCL5/RANTES). This thesis shows that bronchoalveolar lavage (BAL) fluid from asthmatic patients and synovial fluid from patients with rheumatiod arthritis contain MC chemoattractants, and that part of the chemotactic activity can be related to the presence of stem cell factor (SCF) and TGF-β. We also show that MC chemotactic activity during pollen season is significantly increased compared to before pollen season. Furthermore, we demonstrate that TGF-β isoforms, CCL5, TNF-α and IL-4 act as MC chemoattractants in a bellshaped dose- dependent manner. TGF-β proved to be an extremely potent attractant giving an optimal migratory response at 40fM and TGF-β3 being the most effective isoform. The chemokine CCL5 induced migration through interaction with the receptors CCR1 and CCR4 expressed on MC. Furthermore, we also found that TNF-α produced by TH1-lymphocytes and IL-4 produced by TH2-lymphocytes are MC chemoattractants. In conclusion, with this thesis we have identified six new human mast cell chemoattractants and provide evidence that BAL fluid and synovial fluid from patients with asthma and rheumatoid arthritis, respectivly, contain MC chemoattractants. This information provides important clues in understanding the mechanisms behind MC recruitment to sites of inflammation.

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.

The key control step in E. coli chemotaxis is regulation of CheA kinase activity by a set of four transmembrane chemoreceptors. The receptor dimers can form trimeric complexes (trimers of dimers), and these trimers can be joined by a bridge thought to consist of a CheW monomer, a CheA dimer, and a second CheW monomer. It has been proposed that trimers of receptor dimers may be joined by CheW-CheA dimer-CheW links to form an extended hexagonal lattice that may be the structural basis of the chemoreceptor patches seen in E. coli. The receptor/CheA/CheW ternary complex is a membrane-spanning allosteric enzyme whose activity is regulated by protein interactions. The study presented in this dissertation investigated intermolecular and intramolecular interactions that affect the chemotactic signal processing. I have examined functional interactions between the serine receptor Tsr and the aspartate receptor Tar using a receptor coupled in vitro phosphorylation assay. The results reveal the emergent properties of mixed receptor populations and emphasize their importance in the integrated signal processing that underlies bacterial chemotaxis. A mutational analysis of the extreme C-terminus (last fifty residues) of Tar is also presented. The results implicate the receptor C-terminus in maintenance of baseline receptor activity and in attractant-induced transmembrane signaling. They also suggest how adaptive methylation might counteract the effects of attractant binding.

Microaerophilic and chemotaxic diazotrophs, azospirilla are found in close association with certain cereals such as durum wheat and maize and are active in enriching these ecological niches with the macronutrient nitrogen as ammonia. Regarded as highly pleomorphic, Azospirillum spp. are highly motile, using either a single polar flagellum when grown in liquid environments or peritrichous lateral flagella in viscous environments. Additionally, azospirilla are able to adhere onto surfaces as a biological film or aggregate cell-to-cell as nonproliferating flocculi, and these two processes having been suggested as positively affecting the survival and dispersal of the bacteria in the soil. Even though both biofilm formation and flocculation have been characterized via the presence of bacterial extracellular polysaccharides, the nature of the observed exopolysaccharides is still obscure, as are the underlying molecular mechanisms facilitating their organization. Here, we identified the optimal conditions for biofilm formation as a high C:N ratio under conditions of low aeration. Cells showed an increased preference for hydrophobic plastic rather than hydrophilic glass when the bacteria were first grown in a rich medium, TY, then were subcultured in a minimal media under these conditions. Using transposon mutagenesis, we also identified metabolic and cell-surface functions perhaps involved in the flocculation potential of these bacteria and we present an initial characterization of their contribution to this cellular differentiation process.

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 163-175). Also available for download via the World Wide Web; free to University of Oregon users.

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The phenomenon of directed cell motion in response to external directional cues has drawn significant interest for more than a century, with the first recorded observations of bacterial chemotaxis at the end of the 19th century. Furthermore, movies generated by David Rogers while at Vanderbilt University of a peripheral blood neutrophil tracking a bacterium are a staple of any college biology class to demonstrate the phenomenon of eukaryotic chemotaxis. In just the last decade, our understanding of the biochemical mechanisms underlying the process of directed eukaryotic cell migration. As a result, several generalized processes have been identified, connecting multiple phenomena from cancer metastasis to axon guidance. Making further sense of the complex biochemical pathways requires both quantitative mathematical models and fine control over the external cellular environment. To this end, microfluidics has proven extremely useful, allowing for precise quantification of both the external environment and the cellular response.

Chemotaxis is a process whereby single- and multi-cellular organisms migrate in response to external chemical stimuli. This directed cell movement is regulated by complex signaling pathways and is implicated in embryonic development, immune response, and the metastasis of cancer cells. Dictyostelium discoideum, social amoebae with the ability to migrate and aggregate in response to chemoattractants such as cAMP, have been used as a model system to study chemotaxis. Preliminary research suggests that protein kinase (PKA) is involved in some of the signaling pathways that regulate chemotaxis. The role of PKA in chemotaxis was investigated, first, by characterizing the phenotype of PKA null cells using established cell biological and biochemical assays. Furthermore, spatiotemporal regulation of critical cytoskeletal proteins was probed in wild-type and PKA null cells using confocal fluorescence microscopy, indicating misregulation of both F-actin and Myosin II in pkaC- and pkaR- cells. Finally, preliminary work was done to lay the groundwork for experiments exploring possible PKA targets mediating TORC2 function in chemotaxis.