Most bacteriophages release progeny virions at the end of the infection cycle by lysis of the host. Large phages with double-stranded DNA genomes use a multigene strategy based on holins, small membrane proteins, and bacteriolytic enzymes, or endolysins. Holins mediate the control of endolysin activity and thus the timing of lysis. Phages with small genomes only encode a single protein for cell lysis. There are three known unrelated single protein lysis systems: the ?X174 E protein, the MS2 L protein, and the Q? A2 protein. None of these phages encodes a cell wall degrading activity, and previous work has shown that the lytic activity of E stems from its ability to inhibit the host enzyme, MraY, which catalyzes the formation of lipid I, the first lipid intermediate in cell wall synthesis. The purpose of the work described in this dissertation was to characterize the ?X174 E-mediated inhibition of MraY using genetic and biochemical strategies. A fundamental question was why no large phages use the single gene system. This was addressed by constructing a recombinant phage, ?E, in which the holin-endolysin based lysis cassette of ? was replaced with E. ?E was compared with ? in genetic and physiological experiments, with the results indicating that the holin-endolysin system increases fitness in terms of adjusting lysis timing to environmental conditions. Using ?E, physiological experiments were conducted to characterize the interaction between E and MraY in vivo. Transmembrane domains (TMD) 5 and 9 have been identified as the potential E binding site by isolating MraY mutants resistant to E inhibition. The five Eresistant MraY mutants were found to fall into three classes, which reflect the apparent affinity of the mutant proteins for E. Finally, an assay for MraY activity employing the dansylated UDP-MurNAc-pentapeptide and phytol-P, was used to demonstrate the inhibition of MraY by purified E protein. It was determined that E is a non-competitive inhibitor for MraY in respect with both substrates. A model for E-mediated inhibition of MraY was proposed, in which E binds to TMDs 5 and 9 in MraY and thus inactivates the enzyme by inducing a conformational change.

The purpose of this study was to investigate the relative effectiveness of positive interdependence and group processing on student achievement, interaction, and attitude in online cooperative learning. All of the participants, 144 college students enrolled in one of three different courses, received initial general instruction about teamwork skills and cooperative learning at the start of the study. Participants were then randomly assigned to one of three treatment groups: positive interdependence, group processing, and no structure. The ?positive interdependence? groups received subsequent positive interdependence skills training which were then utilized in their instructional activities. The ?group processing? groups received subsequent group processing skills training for use in their instructional activities. The ?no structure? groups received no additional instructional treatment beyond the initial basic teamwork and cooperative learning training. Results indicated that there were significant differences among students in the ?positive interdependence,? ?group processing,? and ?no structure? groups with respect to their achievement scores and interactions. Participants in the ?positive interdependence? groups had significantly higher achievement than participants in either the ?group processing? groups or the ?no structure? groups. In addition, participants in the ?positive interdependence? groups and the ?group processing? groups interacted with each other to a greater extent than those in the ?no structure? groups. This study also examined the relative effectiveness of positive interdependence and group processing on types of student interaction. The results indicated that ?positive interdependence? strategies were relatively more effective than ?group processing? strategies on ?sharing and comparing of information? interactions, whereas ?group processing? strategies were relatively more effective than ?positive interdependence? strategies on ?negotiation of meaning and co-construction of knowledge? interactions. Regarding student attitude towards the experiences of cooperative learning: participation, communication resources, and online activities, there was no significant difference among any of the three groups. The overall results of this study suggest that instructors would be advised to incorporate positive interdependence strategies in their online courses to help students perceive that they should actively contribute to their online group activities. In addition, instructors are recommended to inform groups of the individual progress of each member?s activities periodically by employing group processing strategies.

Coaches in Canada are trained through the National Coaching Certification Program (NCCP) administered by The Coaching Association of Canada. The NCCP is the national standard and recently shifted its educational emphasis from the transfer of knowledge to the development of coaching competency. As a result, coaches are required to demonstrate competency in a specified sport and level prior to being awarded a coaching certification. The purpose of this study was to establish validity and reliability of an NCCP coaching competency assessment. Gymnastics Canada agreed to have their Community Sport coaching competency instrument tested for content validity, face validity, inter-rater reliability and intra-rater reliability. Their original assessment instrument was revised during content validity testing based on recommendations from five coaching context experts. Removing six items, adding one item, and revising four items for clarity modified the original 48-item instrument. An assessment of overall competency was added to the instrument to rate the coach as Beginner, Competent, Proficient, or Expert. Three certified coaches confirmed the face validity of the modified instrument. Reliability tests were conducted on the ratings provided by ten experts who observed a coach’s lesson on video. The result was a moderate level of inter-rater reliability, displayed by an Agreement Coefficient (AC1) of 0.43 and a Percent of Agreement (PA) of 72%. Nine of the ten raters assessed the coach’s performance as Competent, Proficient or Expert, while one of the ten rated the coach as Beginner. After a repeat observation of the same coaching performance, the intra-rater reliability of five raters resulted in agreement levels of Moderate (AC1 = 0.45; PA = 67%), Substantial (AC1 = 0.75, 0.77; PA = 81%, 89%), and Almost Perfect (AC1 = 0.87, 0.82; PA = 93%, 90%). The modified instrument has content and face validity. However, its usefulness is impacted by variability in rater preferences. The inter-rater reliability results attest to concerns about the consistency in assessment of a coaching certificate for Community Sport Artistic Gymnastics coaches. Suggestions to improve the reliability of the instrument include training raters to classify the coach’s competency on a standardized scale of sport-specific expertise.

The effects of electron beam (e-beam) irradiation and sanitizers in the reduction of Escherichia coli O157:H7 and Salmonella counts and attachment prevention on spinach was studied. Survival of these pathogens in spinach was observed at multiple times and temperatures. Inoculated spinach was examined by confocal microscopy to determine attachment sites and internalization of these pathogens. To determine the effectiveness of sanitizers in reducing pathogen numbers, inoculated spinach was treated with L-lactic acid, peroxyacetic acid, calcium hypochlorite, ozone, and chlorine dioxide. Inoculated spinach was exposed to e-beam irradiation and tested for counts of both pathogens immediately after irradiation treatment to determine bacterial reduction, and at 2 day intervals over 8 days to determine effects of ionizing irradiation on pathogen survival. Respiration rates were measured on spinach exposed to e-beam. The effectiveness of e-beam irradiation on the microbiological and sensory characteristics of spinach was studied. For spinach samples stored at 4�C and 10�C for 8 days, E. coli O157:H7 survived and grew significantly in samples stored at 21�C for 24 h. Confocal microscopy images provided valuable information on the attachment sites and internalization of the pathogens on spinach. The greatest reduction by a chemical sanitizer was 55�C L-lactic acid with a 2.7 log CFU/g reduction for E. coli O157:H7 and 2.3 log CFU/g reduction for Salmonella. Each dose of e-beam irradiation significantly reduced populations of both pathogens. Respiration rates of spinach increased as irradiation treatment doses increased. Total aerobic plate counts were reduced by 2.6 and 3.2 log CFU/g at 0.7 and 1.4 kGy, respectively. Lactic acid bacteria were reduced at both doses but grew slowly over the 35 day period. Yeasts and molds were not reduced in samples exposed to 0.7 kGy whereas 1.4 kGy had significantly reduced counts. Gas compositions for samples receiving 0.7 and 1.4 kGy were significantly different than controls. Irradiation did not affect the objective color or basic taste, aromatic or mouthfeel attributes of spinach. These results suggest that low dose e-beam irradiation may be a viable tool for reducing microbial populations or eliminating E. coli O157:H7 and Salmonella from spinach with minimal product damage.