Flow-mediated dilation (FMD) can be used to assess the risk of atherosclerosis; however, an inverse relationship between vessel size and FMD has been identified using reactive hyperemia (RH) to create a shear stress (SS) stimulus in human conduit arteries. RH creates a transient and uncontrolled SS stimulus that is inversely related to baseline arterial diameter. It is therefore unclear whether differences in FMD between groups with non-uniform artery sizes are indicative of differences in vascular health or due to the creation of a greater SS stimulus in smaller vessels. Unlike RH, exercise can effectively create sustained and controlled increases in conduit artery SS. The purpose of this study was to compare the FMD responses of two differently sized upper limb arteries (brachial (BA) and radial artery (RA)) to matched graded levels of SS. Using exercise, three distinct sustained shear rate stimuli were created ((SR)=blood flow velocity/vessel diameter; estimate of SS) in the RA and BA. Artery diameter and mean blood flow velocity were assessed with echo and Doppler ultrasound respectively in 15 healthy male subjects (19-25yrs). Data are means ±SE. Subjects performed 6-min each of adductor pollicis and handgrip exercise to increase SR in the RA and BA respectively. Exercise intensity was modulated to achieve uniformity of SR between the RA and BA at three SR targets (40s-1, 60s-1, 80s-1). Three distinct SR levels were successfully created (steady state exercise: 39.8±0.6s-1, 57.3±0.7s-1, 72.4±1.2s-1; p<0.001 between SR levels). The %FMD at the end of exercise was greater in the RA vs. BA (SR40 RA: 5.4±0.8%, BA: 1.0±0.2%; SR60 RA: 9.8±1.0%, BA: 2.5±0.5%; SR80 RA: 15.7±1.5%, BA: 5.4±0.7%; p<0.001). The mean slope of the within-subject FMD-SR dose-response regression lines was significantly greater in the RA (RA: 0.33±0.04, BA: 0.13±0.02; p<0.001) and a strong within-subjects relationship between FMD and SR was observed in both arteries (RA r2: 0.92±0.02; BA r2: 0.90±0.03). These findings suggest that the response to SS is not uniform across differently sized vessels, which is in agreement with previous RH studies. Future research is required to investigate the potential mechanisms that mediate the functional differences observed between differently sized vessels. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2010-09-22 11:01:26.028

Shear stress is the frictional force exerted on the vascular wall by blood flowing through an artery. It is a major regulator of endothelial cell function, which is essential for vasoprotection and local regulation of vascular tone. Using handgrip exercise (HGEX) to increase shear stress is an increasingly popular method for assessing brachial artery (BA) endothelial cell function via flow-mediated dilation (FMD, dilation which increases with improved endothelial function). However, different exercise duty-cycles (ratio of handgrip relaxation to contraction in seconds) produce different patterns of BA shear stress with different anterograde and retrograde flow magnitudes. PURPOSE: To determine the impact of HGEX duty-cycle on BA %FMD while maintaining a constant mean shear stress. METHODS: N=16 healthy males. BA diameter (BAD) and blood velocity (BV) were assessed via echo and Doppler ultrasound. Shear stress was estimated as shear rate (SR=BV/BAD) and reported as mean SR during the last minute of baseline (target 10 s 1) and each minute of HGEX (75 s-1). Subjects performed 3 six minute HGEX trials on each of 2 separate days (like trials averaged). Each trial was one of 3 randomly ordered HGEX duty-cycles (1:1, 3:1, 5:1). %FMD was calculated as the increase in BAD from baseline to the end of HGEX and at each minute (subset N=10) during HGEX. RESULTS: Data are means ± SD. As intended, mean SR was similar between duty-cycles (main effect, p=0.835), despite significant differences in anterograde and retrograde SR (P<0.001). There was no impact of duty cycle on blood pressure (p=0.188) or heart rate (p=0.131) responses. End exercise %FMD (4.0 ± 1.3%, 4.1 ± 2.2%, 4.2 ± 1.4%, p=0.860) and minute by minute %FMD (main effect p=0.939; interaction, p=0.545) were also not different between duty-cycles. CONCLUSION: Distinct HGEX duty-cycles create markedly different shear stress patterns in the BA. However, duty cycle had no impact on %FMD magnitude suggesting that mean shear stress is the most important stimulus for FMD in the BA. Using a 5:1 duty cycle may yield the best vessel image and diameter measurement quality due to the long period of arm stability between contractions. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2012-09-06 14:31:50.467

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This thesis examines Escherichia coli pyruvate kinase type 1 (PK1), a regulatory enzyme core to energy metabolism. Specifically, this thesis characterises a series of mutations in PK1 that were found when populations of E. coli were evolved in a glucose-limited environment for 20,000 generations. The gene pykF, which codes for the PK1 enzyme, was found to have developed nonsynonymous mutations in all replicate populations. Although the mutations at the nucleotide level were not the same (i.e. not parallel), it is not clear whether parallel adaptation exists at the protein structure/function level. This study aimed to address this question by investigating the kinetic and biophysical properties of the wild-type and seven mutant enzymes. The recombinant wild-type PK1 enzyme used in this study was found to have steady state kinetics consistent with those previously reported. Unlike the rabbit kidney PK enzyme, E. coli PK1 was shown to have a very tight tetrameric structure (picomolar range), which was not affected by the enzyme’s substrates (PEP and ADP), or the allosteric effector (FBP), as judged by analytical ultracentrifugation with fluorescence detection. The mutated residues were highly conserved, and found to fall loosely into three groups: those at the active site (P70T, P70Q and D127N); those at the subunit interface (I264F, A301T and A301S); and at the allosteric binding site (G381A). The seven mutated PK1 enzymes were obtained by mutagenesis followed by protein purification. Steady state kinetic analysis showed that the mutated enzymes displayed a variety of functional changes, suggesting that the populations had not evolved in a parallel manner at the enzyme structure/function level. Mutations within the active site (P70T, P70Q and D127N) all showed a decrease in catalytic potency. P70 is located at the hinge connecting the A and B domains, which forms the active site. PK1-P70Q showed strong cooperative binding to PEP, similar to the wild-type enzyme, in the absence of FBP, whereas PK1-P70T had little cooperativity, suggesting changes in the active site. PK1-D127N showed severely attenuated activity, suggesting, for the first time, that this residue is essential for catalysis. Mutations at the subunit interface (I264F, A301T and A301S) all showed altered allosteric regulation, suggesting that this interface is important in the FBP allosteric response. PK1-I264F, which had lower activity, but greater affinity for PEP, displayed a decreased α-helix content (as judged by CD), indicating that a subunit interface helix that includes this residue had altered. Despite still having a similar response to FBP, PK1-G381A showed an increased affinity for PEP, which, together with an increased α-helix content, suggests that this mutation had changed the structure of the FBP binding domain. None of the mutated enzymes showed altered quaternary structure. Although the populations evolved parallel changes with respect to cell physiology, fitness, and gene expression, this study suggests, for the first time, that the populations have not evolved in a parallel way with respect to protein structure and function.

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There is currently worldwide interesti n the implementationa nd effectivenesso f the quality management movement in financial service organisations. Especially the effect Total Quality Managemen(tT QM) hasu pon transforminga n industryw hich is in a stateo f considerables tructuralc hangea, nd in which the competitioni s intense. The first stageo f the researchin volves the assessmenot f the statuso f TQM in bankingo rganisationsin the UK and Brazilian financial servicess ector.A questionnaire was used to investigate the perception of branch managers concerning the implementation and majors issues of TQM within the financial services industry in the UK and Brazil. Structured interviews with senior managers in charge of planning and developing the quality initiatives in the organisations surveyed were performed to investigatet he strategiesa ndm odelso f the TQM implementation. The collection of datai n this studyw asc onductedin two parts: (1) In 1995 the fieldwork in the UK was carried out this involved a survey questionnaire with branch managerso f fifteen 11ighS treetf inancial serviceo rganisationsa nd a set of interviewsw ithin two of the organisations urveyed. (2) In 1996 the Brazilian fieldwork was carried out. This involved the survey questionnairew ith branch managerso f ten High Street financial serviceo rganisations and a set of interviewsw ithin seveno f thoseo rganisationss urveyed. The analysis of the data obtained enable the identification of the current approachesto the implementationo f TQM; the effects of TQM in the financial services sector in the UK and Brazil; and the current strategiesa pplied and conceptualm ethods used for implementing TQM in those organisations. The second stage of the research presents the development of a TQM implementation framework employing the Quality Function Deployment (QFD) methodology. This has been designed to assist in the selection and formulation of the most comprehensiveT QM implementationa pproachf or a banking organisationsin both countries. The framework was designed as a proposal document to be used by quality practitioners,t op managerso f organisationsa nd quality managemenrte searcherws ho are implementing TQM.