No description available.

No description available.

No description available.

No description available.

Peritrichously flagellated bacteria carry out chemotaxis by modulating the frequency of switching between smooth swimming and tumbling. The tumbling frequency is controlled by a signal transduction cascade in which transmembrane receptors modulate the activity of a histidine kinase CheA that transfers phosphate to its cognate response regulator CheY. The proteins of the chemotaxis signaling cascade are localized to clusters found primarily at the poles of cells. In this work, the localization of the CheZ protein, a phosphatase that dephosphorylates CheY~P, is examined. Using a CheZ-GFP fusion protein, we show that CheZ was localized to the polar receptor patch via interaction with the short form of CheA (CheAS). Aromatic residues of CheZ near one end of the elongated CheZ four-helix bundle were determined to be critical for localization. Aliphatic residues in CheAS were also determined to be critical for CheZ localization to the receptor patch and substitution of these residues conferred a tumble bias to swimming cells. A mechanism of CheZ localization is proposed in which the CheZ apical loop interacts with a binding site formed by dimerization of the P1 domain of CheAS. The potential role of CheZ localization as a means of coordinating the rotation state of peritrichously distributed flagella is discussed.

The overall goal of this work was to develop and utilize microfluidic models for investigating bacterial chemotaxis and biofilm formation - phenotypes that play key roles in bacterial infections. Classical methods for investigating chemotaxis and biofilm formation have many limitations and drawbacks. These include being unsuitable for investigating the effect of chemorepellents, non-quantitative readouts, and not accounting for interaction between hydrodynamics and biofilm formation. The novel microfluidic model systems for chemotaxis and biofilm formation developed in this study addresses these drawbacks. Chemotaxis model system development was done in three stages. We first developed two static chemotaxis model systems - the two fluorophore chemotaxis agarose plug assay and the mu Plug assay - for rapidly determining the extent of chemotaxis in a qualitative manner. A key feature of these model systems was the incorporation of dead cells and differential labeling with green and red fluorescent proteins for partitioning the effects of movement due to fluid flow from chemotaxis. The static systems were used to rapidly screen a wide range of conditions for use in the flow-based mu Flow chemotaxis model system. The effect of four major variables - cell preparation method, gradient strength, flow rate in the device, and imaging position - that influence the chemotactic response in the mu Flow was characterized using the repellent taxis from Ni^2 gradients as the model chemoeffector. Using the mu Flow chemotaxis device, we investigated the chemotaxis of Escherichia coli RP437 to different signals that are present in the human gastrointestinal tract and are likely to be mediators of infection through their effect on chemotaxis. Our data show that the bacterial signal indole is a repellent, while the signals autoinducer-2 (AI-2) and isatin are attractants for E. coli RP437. However, cells exposed to a competing gradient of indole and either AI-2 or isatin, attracts E. coli. The ?Flow device was also used to refute a long-standing view on how the repellent Ni2 is sensed in E. coli. Our data show that only the Tar chemoreceptor is needed for sensing Ni^2 and the nickel binding protein, NikA, and the Ni^2 transport system proteins, NikB and NikC, are not required for repellent taxis from nickel. A microfluidic biofilm model was also developed in this study and used in conjunction with a mathematical model to investigate biofilm formation and quorum sensing in closed systems (where biofilm growth and hydrodynamics are interdependent). The mathematical model predictions were experimentally validated using Pseudomonas aeruginosa PA14 in a microfluidic biofilm system at various flow rates.

One major barrier that prevents cartilage integration following mosaic arthroplasty is the presence of a zone of chondrocyte death (ZCD) that is generated upon osteochondral graft harvest, which can extend up to 400 μm into the cartilaginous portion of the graft. In order for cartilage integration to occur, chondrocytes must be present at the graft periphery; however chondrocyte migration through the ZCD to the graft periphery is inhibited by the dense extracellular matrix (ECM) of cartilage. The purpose of this study was to develop a method for increasing the number of chondrocytes within the ZCD and at the periphery of a cartilage graft. This method used a combination of collagenase treatment (as a means of degrading the ECM within the ZCD) and chondrocyte chemotaxis (as a means of improving chondrocyte migration into the ZCD and to the cartilage periphery). Results indicate that treating bovine articular cartilage with 0.6 % collagenase for 10 min decreased with extent of the ZCD by approximately 35% (collagenase: 109 ± 13 μm; control: 175 ± 13 μm). Each of the chemotactic agents tested (PDGF-bb, bFGF, and IGF-I) were found to induce bovine chondrocyte chemotaxis at concentrations of 25 ng/mL in modified Boyden chamber experiments. However, in bovine articular cartilage samples that were pre-treated with collagenase (0.6% for 10 min), supplementation with 25 ng/mL of either PDGF-bb or bFGF had no apparent effect on the ZCD relative to samples treated only with collagenase (PDGF-bb: 85 ± 10 μm; bFGF: 88 ± 10 μm). Alternatively, bovine articular cartilage samples pre-treated with collagenase (0.6% for 10 min) and supplementation with 25 ng/mL IGF-I resulted in an approximately 65% reduction in the ZCD relative to samples treated only with collagenase (IGF-1: 38 ± 5 μm). Thus, treating osteochondral grafts with collagenase and IGF-1 induces chondrocyte repopulation of the zone of chondrocyte death generated by osteochondral graft harvesting, and could enhance cartilage integration after implantation. / Thesis (Master, Chemical Engineering) -- Queen's University, 2009-12-21 20:16:05.815

Directed immune cell migration plays important roles in immunosurveillance and immune responses. Understanding the mechanisms of immune cell migration is important for the biology of immune cells with high relevance to immune cell trafficking mediated physiological processes and diseases. Immune cell migration can be directed by various guiding cues such as chemical concentration gradients (a process termed chemotaxis) and direct current electric fields (dcEF)(a process termed electrotaxis). Microfluidic devices that consist of small channels with micrometer dimensions have been increasingly developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus provide powerful quantitative test beds for studying the complex guiding mechanisms for cell migration. In the research of this thesis, a PDMS-based and a glass-based microfluidic devices were developed for producing controlled dcEF and these devices were used to analyze electrotaxis of activated human blood T cells. Using both devices, we have successfully demonstrated that activated human blood T cells migrate toward the cathode of the applied dcEF. Furthermore, a novel microfluidic device was developed to configure better controlled single or co-existing chemical gradients and dcEF to mimic the complex guiding environments in tissues and this device was used to investigate the competition of chemical gradients and dcEF in directing activated human blood T cell migration.

Directed immune cell migration plays important roles in immunosurveillance and immune responses. Understanding the mechanisms of immune cell migration is important for the biology of immune cells with high relevance to immune cell trafficking mediated physiological processes and diseases. Immune cell migration can be directed by various guiding cues such as chemical concentration gradients (a process termed chemotaxis) and direct current electric fields (dcEF)(a process termed electrotaxis). Microfluidic devices that consist of small channels with micrometer dimensions have been increasingly developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus provide powerful quantitative test beds for studying the complex guiding mechanisms for cell migration. In the research of this thesis, a PDMS-based and a glass-based microfluidic devices were developed for producing controlled dcEF and these devices were used to analyze electrotaxis of activated human blood T cells. Using both devices, we have successfully demonstrated that activated human blood T cells migrate toward the cathode of the applied dcEF. Furthermore, a novel microfluidic device was developed to configure better controlled single or co-existing chemical gradients and dcEF to mimic the complex guiding environments in tissues and this device was used to investigate the competition of chemical gradients and dcEF in directing activated human blood T cell migration.

Ulm, Univ., Diss., 2008.