Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that acts as a molecular link between the coagulation and fibrinolytic cascades. TAFI can be activated by thrombin and plasmin but the reaction is enhanced significantly when thrombin is in a complex with the endothelial cofactor thrombomodulin (TM). The in vitro properties of TAFI have been extensively characterized. Activated TAFI (TAFIa) is a thermally unstable enzyme that attenuates fibrinolysis by catalyzing the removal of basic residues from partially degraded fibrin. The in vivo role of the TAFI pathway, however, is poorly defined and very little is known about the role of different activators in regulating the TAFI pathway. In the present study, we have constructed and characterized various TAFI mutants that are resistant to activation by specific activators. Based on peptide sequence studies, these mutants were constructed by altering key amino acid residues surrounding the scissile R92-A93 bond. We measured the thermal stabilities of all our mutants and found them to be similar to wild type TAFI. We have identified that the TAFI mutants P91S, R92K, and S90P are impaired in activation by thrombin or thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The TAFI mutants A93V and S94V were predicted to be resistant to activation by plasmin but this was not observed. The triple mutant, DVV was not activated by any of the aforementioned activators. Finally, we have used in vitro fibrin clot lysis assays to evaluate the antifibrinolytic potential of our variants and were able to correlate their effectiveness with their respective activation kinetics. In summary, we have developed activation resistant TAFI variants that can potentially be used to explore the role of the TAFI pathway in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 11:44:37.191

Despite a recent focus on understanding cumulative load, researchers still prefer to analyze the data using mean and peak values. At the same time, technological advancements have provided biomechanists with methods of collecting large amounts of data pertaining to joint loading. Waveform analysis offers another option that will become increasingly relevant, as wireless data collection devices become common place and provide access to waveforms from many hours of recording. The overall objectives of this research were to demonstrate some of the limits of current methods of biomechanical analysis, and introduce an alternative, and secondly, to propose a wireless system for use in field-based studies. An exploratory study using Functional Data Analysis (FDA) was completed on experimental lifting data. The results demonstrated that FDA can elucidate subtle differences in the curve shape outside of the peak areas typically used for statistical analysis that were attributed to fatigue. These findings support the need for a better understanding of how workers change their movement strategies as time progresses throughout the length of the workshift. To achieve this type of knowledge, a wireless data collection device utilizing inertial motion sensors (IMS) was introduced and validated for field use in the remaining three chapters. The IMS units in conjunction with an anthropometric model were tested against a traditional link segment model recorded in a gold-standard, video system. Testing that occurred in the entire reach space volume yielded errors as low as 5% for the lumbar moment, but errors also greatly exceeded 50% RMS error in some cases. Three hand switch alternatives were tested for their potential to provide external hand force timing and duration information to the link segment model, but none were found to be perfectly suitable in the current configuration. In conclusion, a wireless system based on IMS units has the potential to provide long-term data collection, but the development of the calibration routines and complexity of the underlying model must be improved. FDA was shown to have good potential for identifying subtle differences in curve shapes, and may become useful when long-term field-based data are readily available with the proposed IMS system. / Thesis (Ph.D, Kinesiology & Health Studies) -- Queen's University, 2009-04-21 09:01:22.968

Stair negotiation has been identified by older adults as one of the most challenging locomotor tasks, one that is associated with a high risk for falls and serious injury. Currently lacking is a comprehensive understanding of the lower limb during stair negotiation in an older adult population. It has been identified that more research is needed to determine key determinants of difficulty and safety on stairs. The objective of this thesis was to investigate lower limb kinematics and kinetics during stair negotiation and evaluate the impact of handrail use on stair ambulation in young adults, older adults, and older adults with a fear of falling (FOF). The four studies that make up this dissertation provide a detailed picture of the lower limb joint kinematics and kinetics during stair ambulation, as well as provide insight into the role of handrail use and FOF in performance of stair negotiation. Specifically, in the first study principal component analysis (PCA) was used, of the scores generated from the PCA models four principal component (PC) scores were identified that could be used to correctly classify 95% of young and older adults. The second study provided a comprehensive data set of lower limb joint kinematics and kinetics during stair negotiation. The third study identified comparable centre of pressure velocities (VCOP) between young adults and older adults during stair negotiation with and without a handrail. Whereas older adults with FOF demonstrated reduced VCOP during stair negotiation without a handrail and further reduced VCOP when using the handrail. Furthermore, no significant difference in lower limb moments during stair negotiation with and without a handrail were found in older adults, as was similar for older adults with FOF. However, a gait assessment revealed that older adults with FOF demonstrated differences from ‘normal’ gait patterns during stair negotiation with and without a handrail. These studies provide a comprehensive normative dataset of the lower limb joint kinematics and kinetics during stair negotiation, as well as provide insight into the role of handrail use and fear of falling in performance of stair ambulation. It is important to appreciate the nature and extent of normal age-related adaption and compensatory strategies to identify unique patterns of movement due to the superimposition of pathology. / Thesis (Ph.D, Kinesiology & Health Studies) -- Queen's University, 2010-09-24 14:50:22.242

Described here are detailed kinetic studies probing the structural elements which are crucial for the catalytic activity of dinuclear Zn(II) complexes towards phosphate diester cleavage. First, two sets of dinuclear Zn(II) complexes (a member with and without a bridging oxyanion linker group) were synthesized and their ability to promote the cyclization of 2-hydroxypropyl-p-nitrophenyl phosphate, a common model for RNA, was compared. Kinetic studies indicated that the complexes without the oxyanion linker were more active in promoting the cyclization in methanol under pH controlled conditions at 25 degrees. Quantitative energetics analysis shows that the rate reduction is attributable to a decrease in the second-order rate constant for the cyclization reaction, which adds 3.7 and 6.5 kcal/mol of activation energy to the respective reactions mediated by the complex with the oxyanion linker. Secondly, we have investigated a series of dinuclear Zn(II) complexes that incorporate various substituents including hydrophobic and hydrogen-bonding ones. Analysis of the data at the pH optimum for each reaction indicates that the presence of the H-bonding groups and alkyl groups provides similar increases (at least an order of magnitude) of the kcat terms over the unfunctionalized complex. There is also no clear trend that H-bonding groups or the alkyl groups provide stronger binding to the substrate than the parent complex. We also describe here the preparation and kinetic analysis of a series of solid supported transition metal catalysts for the cleavage of P=O chemical warfare simulants and P=S pesticides. We report a kinetic study of a 1,10-phenanthroline:Zn(II) complex immobilized on macroporous polystyrene which is capable of accelerating the cleavage of G-agent and V-agent simulants in methanol at neutral and ambient temperature by up to one hundred thousand-fold. The materials are recoverable and can be recycled at least ten times. We have also devised a methodology for simple immobilization of an ortho-palladated dimethylbenzylamine complex on macroporous polystyrene and amorphous silica gel. We report the catalyst preparation and a detailed kinetic study of their catalysis of the methanolysis of five P=S pesticides at neutral and ambient temperature. The polymeric catalysts give over billion-fold acceleration compared to the uncatalyzed background reaction at the same pH. / Thesis (Ph.D, Chemistry) -- Queen's University, 2010-11-23 12:18:46.936

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