Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [132]-134).

Extracorporeal Membrane Oxygenation (ECMO) is a life-saving therapy usedfor support in critical heart and/or lung failure. Patient’s blood is pumped viaan artificial lung for oxygenation outside of the body. The circuit is composedof a blood pump, cannulae for drainage and reinfusion, a membrane lung,tubing and connectors. Its use is associated with thromboembolic complicationsand hemolytic damage. Detailed numerical studies of two blood pumps anda lighthouse tip drainage cannula were undertaken to characterize the flowstructures in different scenarios and their link to platelet activation. The pumpsimulations were modelled according to manufacturer’s proclaimed use but alsoin off-design conditions with flow rates used in adult and neonatal patients.Lagrangian Particle Tracking (LPT) was used to simulate the injection ofparticles similar in size to platelets to compute platelet activation state (PAS).The results indicated that low flow rates impacted PAS similarly to high flowrates due to increased residence time leading to prolonged exposure to shearstress despite the fact that shear per se was lower at low flow rate. Regardingthe cannula, the results showed that a flow pattern similar to a jet in crossflowdeveloped at the side holes. A parameter study was conducted to quantifydrainage characteristics in terms of flow rate distribution across the holes wheninput variables of flow rate, modelled fluid, and hematocrit were altered. Thefindings showed, across all the cases, that the most proximal hole row drainedthe largest fraction of fluid. The effects due to the non-Newtonian nature ofblood were confined to regions far from the cannula holes and the flow structuresshowed very limited dependence on the hematocrit. A scaling law was found tobridge the global drainage performance of fluid between water and blood. / <p>QC 210906</p>

No description available.

Horizontal gene transfer transforms commensal E. faecalis into multidrug resistance (MDR) opportunistic pathogens causing diseases such as infective endocarditis (IE), septicemia, and urinary tract infections (UTI) (4,1). E. faecalis are among the top three leading causes of hospital-acquired infections and pheromone responsive plasmids (pCF10) are the most extensively characterized conjugative plasmids in E. faecalis infection (2,4). E. faecalis is a potential future public health concern because of the co-occurrence factors of antibiotic resistance and virulence traits (6)Plasmid-free commensal E. faecalis form homogenous biofilms that have a uniform distribution of the bacterial cell and a fluid-like movement (22). The introduction of the pheromone responsive plasmid pCF10 leads to the formation of heterologous rigid structures within the biofilm (22). In the current work, the timeline of biofilm tower formation was characterized. Tower formation was not observed in the commensal strain. The pCF10-containing bacteria formed a rigid base layer on day 1 and small aggregates on day 1. pCF10-containing biofilm forms heterologous towers on days two and three. Interestingly, mixed biofilms with both plasmid-containing and plasmid-free bacteria developed tower-like structures as early as day 1 and had larger resulting structures by day three. In the mixed population, we hypothesize that the induction of aggregation substance and cell clumping during plasmid transfer may further contribute to structure formation (5,10). Plasmid-free mCherry-labeled bacteria could be observed in the viscous biofilms between heterologous rigid structures; however, the rigid structures were predominantly composed of plasmid-containing cells. Occasionally, mCherry cells were observed in the rigid structures, we hypothesize that these cells represent transconjugants, where pCF10 was transferred by conjugation to mCherry-plasmid-free OG1RF. The formation of rigid structures can protect bacteria from antibiotics by reducing the penetration of the antibiotic but binding and sequestration of the antibiotic in the outer layers. Antibiotic resistance increased in the pCF10-containing biofilms as rigid structures were formed. We hypothesize that underflow, like that found in the gastrointestinal tract, the heterologous rigid structures may form protected microenvironments for sensitive regions of the biofilms. In future studies, fluorescently labeled antibiotics will be used to access the formation of protected microenvironments in biofilms underflow. Previous studies in the laboratory demonstrated that the presence of pCF10 protects E. faecalis from hydrogen peroxide oxidative stress. E. faecalis produces hydrogen peroxide. Higher levels of hydrogen peroxide can be detected in rigid structures. The presence of pCF10 is known to increase the size of heart vegetations during endocarditis and hydrogen peroxide is a known activator of platelet activation (12,19). In these studies, the presence of pCF10 increased platelet activation in pCF10 containing biofilms. Software toolboxes are currently being developed to quantitate visual observations. The role of hydrogen peroxide is supported in our preliminary experiment revealing catalase treatment reduced platelet activation. Studies are ongoing to mutate the aroc and menB gene of E. faecalis, which contribute to hydrogen peroxide production (35). We will compare platelet activation in knockout (double or single) E. faecalis and the wild-type strain. For future studies, several of the preliminary data need to be repeated to further the study. We will repeat quantitative hydrogen peroxide production in the catalase experiments. We will also finish knocking out the aroc and men B gene of E. faecalis responsible for hydrogen peroxide and then compare platelet activation to the control strain. / Microbiology and Immunology

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

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.

This work describes the development and testing of a real-time three-dimensional computational fluid dynamics simulation of thrombosis and embolization to be used in the design of blood-contacting devices. Features of the model include the adhesion and aggregation of blood platelets on device material surfaces, shear and chemical activation of blood platelets, and embolization of platelet aggregates due to shear forces. As thrombus develops, blood is diverted from its regular flow field. If shear forces on a thrombus are sufficient to overcome the strength of adhesion, the thrombus is dislodged from the wall. Development of the model included preparing thrombosis and embolization routines to run in a parallel processing configuration, and estimating necessary parameters for the model including the adhesion strength of platelet conglomerations to the device surfaces and the criterion threshold for the coalescence of neighboring thrombi. Validation of the model shows that the effect of variations in geometry may be accurately predicted through computational simulation. This work is based on previous work by Paul Goodman, Daniel Lattin, Jeff Ashton, and Denzel Frost.

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.

Introduction: Whole blood transfusions are indicated for the resuscitation of patients with hemorrhagic trauma. In Sweden, whole blood is stored for 14 days at 4°C. If possible, refrigerated blood is rewarmed to 37°C before transfusion to avoid hypothermia. Platelets contribute to hemostasis and can be activated with several pathways. During storage, shedding of platelet surface receptors takes place. Further research on how platelet storage in whole blood and rewarming before transfusion affect platelets is needed. Aim: The aim was to study how storage in whole blood and rewarming to 37°C affect platelet function, platelet activatability, and changes in platelet surface receptors. Method: Whole blood from two healthy donors was stored for 14 days. During these 14 days, two blood samples were taken on day 0, before and after leukocyte reducing filtration, 1, 3, 7, and 14. One of the two blood samples from each whole blood product was tested at room temperature and the other was rewarmed to 37°C. The blood samples were mixed with antibodies and platelet agonists and analyzed on a flow cytometer. The blood samples were also analyzed on a cell counter. Results: This study shows distinct changes in platelet spontaneous activation, platelet count, and platelet receptor shedding by increased storage time in cold-stored whole blood and even more with rewarming to 37°C. Conclusion: This study shows that during storage, spontaneous platelet activation and shedding of GPIb and GPIIb increases while platelet count decreases. All these factors are likely affecting the platelet function and hemostatic function negatively.