This thesis reports on the work performed on the manipulation of micro­-particles on substrate surfaces using short laser pulses. For particles with diameters on the order of microns, the binding forces to surfaces are significantly larger than gravitational forces. To overcome these binding forces and manipulate the particles the use of femtosecond laser pulses has been investigated. Individual micro-particles (poly-divinylbenzene, glass and silver materials) with diameters around 2 um were removed from substrate surfaces (dielectric, semiconductor and metal substrates) us­ing a Ti:Sapphire laser system. The pulses produced at 800 nm had pulse lengths around 140 fs and were tightly focussed onto the surface using 5x and 10x micro­scope objectives. The peak fluence thresholds for particle removal were determined and the surfaces examined after irradiation by a scanning electron microscope and atomic force microscope to check for damage. The experimental results indicate that ablation of the substrate below the micro-particles is most likely to be respon­sible for micro-particle removal from the substrate surface when using femtosecond pulses. Ablation pits were observed for the dielectric micro-spheres on semiconductor substrates. It is also believed that ablation is responsible for the removal of other types of micro-particles from various substrates. Unlike the dielectric micro-sphere on semiconductor substrate results, the other particle-substrate combinations show a close correspondence between the removal and substrate ablation thresholds. It is believed that these results indicate the occurrence of ablation leading to the removal of the micro-particles. Calculations of the local electromagnetic fields around the micro-particles have also been carried out and the distributions used to interpret the experimental results. / Thesis / Master of Applied Science (MASc)

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Gene gun assisted micro-particle delivery system is an excellent method for the delivery of DNA into target tissue so as to carry out gene transfection in the target cells. The gene gun is primarily a particle accelerator which accelerates DNA-coated micro-particles to sufficient velocities to breach the target layer enabling the micro-particles to penetrate to a desired depth and target the cells of interest to achieve gene transfer. However, an inevitable problem in this process is the tissue/cell damage due to the impaction of the pressurized gas and micro-particles on the target. The purpose of this research is developing a new conceptual system which improves the penetration depth of micro-particles at less imposed pressure and particle injection velocity. This is achieved by applying a microneedle array and ground slide in the gene gun system, thus a study involving microneedle assisted micro-particle delivery is conducted in this work. Microneedle array is used to create holes in the target which allows a number of micro-particles to penetrate through the skin which enhances the penetration depth inside target. The ground slide is used to load a pellet of the micro-particles and prevent the pressurized gas to avoid the impaction on the target. The operation principle is that the pellet is attached to ground slide which is accelerated to a sufficient velocity by the pressurized gas. The pellet is released from the ground slide which separates into individual micro-particles by a mesh and penetrates to a desired depth inside the target. An experimental rig to study various aspects of microneedle assisted micro-particle delivery is designed in this PhD research. The passage percentage of the micro-particles and size of the separated micro-particles are analysed in relation to the operating pressure, mesh pore size and Polyvinylpyrrolidone (PVP) concentration to verify the applicability of this system for the micro-particle delivery. The results have shown that the passage percentage increases from an increase in the mesh pore size and operating pressure and a decrease in PVP concentration. A mesh pore size of 178 μm and pellet PVP concentration of 40 mg/ml were used for the bulk of the experiments in this study as these seem to provide higher passage percentage and the narrow size distribution of the separated micro-particles. In addition, the velocity of the ground slide is detected by the photoelectric sensor and shown that it increases from an increase in operating pressure and reaches 148 m/s at 6 bar pressure, A further analysis in the penetration depths of the micro-particles to determine whether they achieve enhanced penetration depths inside the target after using microneedles is carried out. A skin mimicked agarose gel is obtained from comparing the viscoelastic properties of various concentration of agarose gel in comparison with the porcine skin, which is assumed to mimic the human skin. These experiments are used to relate the micro-particle penetration depth with the operating pressure, microneedle length and particle size. In addition, a theoretical model is developed based on the experimental data to simulate the microneedle assisted micro-particle delivery which provide further understanding of the microneedle assisted micro-particle delivery. The developed model was used to analyse the penetration depth of micro-particles in relation to the operation pressure, target properties, microneedle length and particle size and density. The modelling results were compared with the experimental results to verify the feasibility of the microneedle assisted micro-particle delivery for micro-particles delivery. As expected, both experimental and theoretical results show that the micro-particles achieve an enhanced penetration depth inside target. The maximum penetration depth of micro-particles is increased from an increase in operating pressure, microneedle length, particle size and density.

This thesis presents some experimental work in the area of manipulation of microparticles. Manipulation of both magnetic and non magnetic beads as well as microorganisms are addressed. The work on magnetic bead manipulation is focused on controlled transport and release, on a micrometer level, of proteins bound to the bead surface. Experimental results for protein transport and release using a method based on magnetization/demagnetization of micron-sized magnetic elements patterned on a modified chip-surface are presented. Special attention has been placed on minimizing bead-surface interactions since sticking problems have shown to be of major importance when protein-coated beads are used. The work with non-magnetic microparticles is focused on the dielectrophoretic manipulation of microorganisms. Preliminary experimental results for trapping and spatial separation of bacteria, yeast and non-magnetic beads are presented. The overall goal was to investigate the use of dielectrophoresis for the separation of sub-populations of bacteria differing in, for example, protein content. This was, however, not possible to demonstrate using our methods.Within the non-magnetic microparticle work, a method for determining the conductivity of bacteria in bulk was also developed. The method is based on the continuous lowering of medium conductivity of a bacterialsuspension while monitoring the medium and suspension conductivities.

A commercial valve-assisted micromixer, manufactured by Ehrfeld (Germany), was tested for its use to precipitate L-asparagine from an aqueous solution using isopropanol as antisolvent. In a first part the mixing quality provided by the micromixer was studied by means of a competitive/parallel set of reactions following the approach of Baldyga, Bourne and Walker, Canadian J. Chem. Eng. 76 (1998) 641-649. Different experiments have been implemented and interpreted considering the average of Reynolds number of the inlet streams. Results show a good mixing quality that is comparable, in terms of absolute values of conversion, with other works present in literature. The precipitation experiments that followed revealed the limitation of the micromixer. The system was instable and particles adhesion occurred inside the mixing chamber. Improvements have been realized by changing the spring tension of the valve and introducing a commercial surfactant TRITON X-100.

No description available.

Because of their good mechanical resistance at high temperature, Ni-based superalloys are used for aero-engine and land-based turbines but undergo "dry" oxidation between 900 and 1500°C. These materials are thus coated with nickel-aluminide coatings (BC). An additional thermal barrier coating (TBC) is generally applied in the hottest sections of the turbines (T>1050°C) to lower the impact of the temperature on the substrate. In the framework of the European research programme "PARTICOAT", this PhD work was focused on the growth mechanisms of a full protective coating system (BC+TBC) in a single step process, using a water-based slurry containing a dispersion of Al micro-particles to satisfy the European environmental directives. The rheological and physico-chemical characterizations showed the slurry stability up to seven days. After depositing the latter by air spraying, a tailored thermal treatment resulted in a nickel-aluminide coating (β-NiAl) similar to the conventional industrial ones but through an intermediate Al liquid phase stage. Simultaneously, the oxidation of the Al micro-particles brought aboutthe formation of a top alumina "foam" (PARTICOAT concept). After a validation step of the mechanisms involved in pure Ni substrate, the extrapolation of the process to several Ni-based superalloys (René N5 (SX), CM-247 (DS), PWA- 1483 (SX) and IN-738LC (EQ)) revealed different coating compositions and microstructures. A particular attention was therefore paid onto the effect of alloying elements (Cr, Ta, Ti) as well as their segregation in the coating. The high temperature behaviour of the coated samples has been studied through isothermal oxidation (1000h in air between 900 and 1100°C) and showed that the oxidation and interdiffusion phenomena ruled the degradation mechanisms. Besides, the electrodeposition of ceria before the application of the PARTICOAT coating allowed to strongly limit interdiffusion phenomena and stabilized the nickel aluminide coating.

Cardiovascular disease is a major cause of morbidity and mortality around the globe, which is linked to athero-thrombosis. The risk factors for atherothrombosis, thus cardiovascular disease is impaired anti-thrombotic and antiinflammatory functions of the endothelium. Thrombosis is a hallmark of cardiovascular disease/complications characterised by increased platelet activation and increased secretion of platelet micro-particles that induce angiogenesis. This study determined the role of platelet micro-particles derived microRNA in the regulation of angiogenesis and migration, with a focus on the regulation of thrombospondin-1 release by platelet micro-particles delivered Let- 7a. The role of thrombospondin-1 receptors (integrin beta-1 and integrin associated protein) and downstream caspase-3 activation were explored by Let-7a inhibition prior to PMP treatment. MicroRNA dependent modulation of proangiogenic proteins including monocyte chemoattractant protein-1 and placental growth factor, and recruitment of activating transcription factor-4 protein to their promoter regions were explored. Main findings are: 1. Platelet micro-particles induce angiogenesis, migration, and release of novel cytokine subsets specific to platelet micro-particle’s RNA content. 2. The targeting of thrombospondin-1 mRNA by platelet micro-particles’ transferred Let-7a chiefly modulate the angiogenic effect on endothelial cells. 3. The inhibition of thrombospondin-1 translation enable platelet micro-particles to increase angiogenesis and migration in the presence of functional integrin beta-1 and integrin associated protein, and reduced cleaving of caspase-3. 4. Platelet micro-particle modulate the transcription of monocyte chemoattractant protein-1 and placental growth factor in a Let-7a dependent manner. 5. Let-7a induce angiogenesis ii independent of other platelet micro-particle’s microRNAs. Platelet micro-particle derived Let-7a is a master regulator of endothelial cell function in this model, which presents an opportunity for the development of new biomarkers and therapeutic approaches in the management of cardiovascular disease. Future studies should aim to confirm these findings in-vivo.