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<p>Though pathogenic strains represent a small portion of the total variety of existing <em>Escherichia coli </em>strains, they contribute extensively to human morbidity and mortality. Disease outbreaks caused by enterohaemorrhagic <em>E. coli</em> of the serotype O157:H7 and the “Big Six” serotypes (i.e., O26, O45, O103, O111, O121 and O145) have driven the development of assays for pathogen detection. From culture-based assays requiring several days for confirmation of target organisms, to quantitative PCR (qPCR) tests that provide pathogen identification in several hours’ time, the sensitivity, specificity and speed of bacterial diagnostics have seen improvements that increased the efficacy of assays used to detect pathogens at clinically relevant levels. One relatively unexplored field of diagnostics is the use of conserved signature insertion/deletions (CSIs) as stable genetic markers for pathogen detection. This thesis presents two qPCR assays that target an <em>E. coli</em> O157:H7-specific insertion in a CSI. In a more preliminary study, an EvaGreen-based qPCR assay was developed that had a detection limit of 16 <em>E. coli</em> O157:H7 genome equivalents. An improved format of the O157:H7-specific CSI assay, using TaqMan probes, was later established. TaqMan probes are sequence-specific, while DNA-intercalating EvaGreen dye is sequence-independent. Though the TaqMan probe-based assay had a higher detection limit of 100 genome equivalents, the assay maintained detection sensitivity in presence of genetically similar (<em>E. coli</em> K-12) and dissimilar (fish sperm) DNA in excess amounts (1000-fold and 800-fold excess of target DNA, respectively), demonstrating its potential for pathogen detection in environmental samples where the presence of background flora may influence detection. These assays thus represent an exploration into the use of CSIs as diagnostic tools. This thesis also provides a guide for future developments of pathogen detection using CSIs, such as those that may be present in toxigenic species of Cyanobacteria and human pathogens, including <em>Vibrio</em> and <em>Campylobacter</em>.</p> / Master of Science (MSc)

<p> The profiles of polycyclic aromatic compounds (PAC) were compared in three separate studies involving air samples collected in urban and rural locations across Canada. In the Freelton/Pier 25 study (conducted near Hamilton, Ontario) a total of 32 NPAH were analyzed for in 12 composite air particulate samples from Freelton (a rural site) and Pier 25 (an urban site) using negative ion chemical ionization gas chromatography-mass spectrometry.</p> <p> The NPAH levels at the two sites were found to be similar except for the two samples at Pier 25. These results were consistent with the PAH levels determined previously which showed significantly increased levels at Pier 25 under the same condition when the sampling site was downwind of the urban/industrial core. NPAH may be significant contributors to mutation induction due to exposures to ambient air since the offspring of male mice from the Pier 25 site exposed to ambient air showed inherited mutation rates about 2 times greater than offspring of mice exposed at the Freelton site. NPAH are highly mutagenic and carcinogenic compounds that act via reductive metabolism and can be readily metabolized to potent reactive intermediates within all cells.</p> <p> Concentration data for a set of polycyclic aromatic compounds were obtained for samples collected during the day and night during a study in Simcoe (rural) and Toronto (urban) as well as at three sites in British Columbia as part of the Pacific 2001 study (Slocan (urban), Langley (suburban/rural) and Sumas (rural)). The conversion of these concentration data into particulate loadings data (using elemental carbon data) enabled us to perform a number of unique interpretations and analyses of the data sets. Since particulate loadings values are not affected by air dispersion it was possible to compare samples and individual PAC across a range of samples.</p> <p> Principal components analysis of the loadings data showed dramatic differences between the urban and rural sites from each study. Day-night samples at the rural sites also showed dramatic profile differences. The urban sites showed significantly less differences in profiles, consistent with lesser degree of air transformation and closer proximities to sources.</p> / Thesis / Master of Science (MSc)

An optimized method for the chiral resolution of enantiomers of amino acids in bacterial supernatants is reported. This LC-MS/MS method is performed using a chiral Teichoplanin LC column and does not require sample clean up or chemical derivitization. This method allows for the determination of the relative amounts of the D and L enantiomers of 20 proteinogenic amino acids. The detection limits and response factors for the 20 amino acids were determined. Calibrations over three orders of magnitude showed least squares coefficient values (R^2) greater than 0.996 for eighty percent of the amino acids and greater than 0.992 for the remainder. The amino acids and their enantiomers were identified based on their retention times and their unique Multiple Reaction Monitoring (MRM) transitions for each amino acid. L-Aspartic acid-2,3,3-d3 was used as the internal standard. Cultures of Sinorhizobium meliloti (a nitrogen-fixing soil bacterium) were grown on minimal media; thus, all amino acids were biosynthesized by the bacterium. After centrifugation, supernatants were freeze dried, reconstituted in a small volume of methanol/water with internal standard and injected onto the LC column. The amino acids detected in the bacterial supernatant and the concentrations of the enantiomers were reported as the L and D isomers respectively: arginine [L, 12.6 ± 3.1 μg/L; D, 10.1 ± 3.2 μg/L], serine [L, 7.2 ± 1.16 μg/L; D, n.d.], threonine [L, n.d.; D, 11.2 ± 2.7 μg/L] and valine [L, 15.5 ± 4.3 μg/L; D, 11.3 ± 3.7 μg/L], where the term n.d. means below detection limit. The limits for detection for all amino acids ranged from 1.3 μg/L - 5.1 μg/L. In media with no added phosphate, the amino acid profiles changed somewhat under these stress conditions. Arginine was no longer detected while alanine and proline were now observed; the concentrations of the amino acids were: alanine [L, 7.7 ± 1.2 μg/L; D, 13.4 ± 2.5 μg/L], proline [L, n.d.; D, 8.63 ± 1.3 μg/L], serine [L, 7.6 ± 1.2 μg/L; D, n.d.], threonine [L, n.d.; D, 10.2 ± 3.2 μg/L] and valine [L, 11.6 ± 2.3 μg/L; D, 10.1 ± 3.1 μg/L]. These data represent the mean values of three independent bacterial growth experiments conducted over a 3 month period; the data came from the analysis of five separate aliquots from each growth experiment. The percent standard deviation for these data ranged from 15% to 33% and averaged 24%. Under both the normal and stressed growth conditions of S. meliloti produced the L enantiomer of serine, the D enantiomer of threonine and racemic valine. While racemic arginine was observed under normal growth conditions, levels were below detection under stressed conditions; under stress conditions only the D enantiomer of proline was observed while alanine was found in 1:2, L:D ratio. / Thesis / Master of Science (MSc)

<p> We aimed to examine the satellite cell response and the potential of HGF signaling in mediating satellite cell activation and proliferation. To achieve this, we determined the time course of satellite cell activation and expression of HGF, HGFA, HAI-l, HAI-2 and the MRFs in skeletal muscle, as well as HGF protein in the blood, before and over five days following an acute bout of eccentrically-biased exercise. Eight recreationally active participants (20.6 ± 2.1 y; 180.5 ± 5.2 cm; 81.4 ± 9.8 kg) were recruited for the study. Subjects were required to perform 300 eccentric contractions involving the quadriceps femoris muscles at 180 °/s, over a 60° range of motion with a randomly selected leg. A baseline biopsy (PRE) was taken from the opposite leg. Muscle and blood samples were taken before the exercise (PRE) and at 4 h (T4), 24 h (T24), 72 h (T72) and 120 h (T120) following the exercise. The exercise protocol resulted in an increase in the number of satellite cells (N-CAM labeled cells), expressed both relative to myofiber number and relative to total myonuclei, between PRE and T4 which was sustained over the time course (p<0.001). Further increases in N-CAM labeled cells, expressed relative to myofibre cross-section, were observed between T4 and T24 (p=0.01) and between T4 and T72 (p=0.002). Myf5 mRNA expression increased significantly from both PRE and T4 by T24 (p=0.04). MyoD mRNA increased significantly from PRE by T4 (p=0.02). Myogenin mRNA increased significantly at T24 versus PRE (p=0.02). No significant change was observed over time for MRF4. HGF protein increased significantly in serum from baseline (PRE) to T4 (p=0.04). Active HGF protein was detected in skeletal muscle at rest (14.4±1.3 avg IDV/actin avg IDV) and tended to increase from PRE to T24 (p=0.12). HGFA protein increased significantly from PRE to T24 (p=0.04). HAI-2 increased significantly from PRE at T72 (p=0.03) and T120 (p=0.04). HAI-1 protein increased significantly from PRE to T24 (p=0.02). HAI-2 (32 kDa) increased significantly from baseline (PRE) by T24 (p=0.03), and also by T72 and T120 (p=0.02). HAI-2 (28 kDa) protein showed no significant change over time HGF, HGFA, HAI-1, and HAI-2 transcripts were undetected over the time course. We conclude that a single bout of high-intensity exercise is sufficient to activate satellite cells, which may involve both a local and systemic response to exercise-induced injury. Furthermore, we propose that HGF signaling plays an important role in the regulation of satellite cells in the post exercise period.</p> / Thesis / Master of Science (MSc)

<p> Ink-jet printing onto flat paper is a widely established process. In this thesis, we make extensions to printing on target surfaces such as metals and glass, using a 5-axis orientable head. Original artwork is created using CAD, and is sampled to create the ink jet point cloud. The target surface location is registered using a standard Coordinate Measuring Machine (CMM) 5-axis touch trigger probe. The probe is then replaced with the ink jet head and the printing process is carried out. Demonstration of the system is illustrated using flat metal and glass samples, as well as rapid prototyped 3-D plastic shapes.</p> / Thesis / Master of Applied Science (MASc)

The human species have used metals for a long time and with the human population increasing the use of metals also increase. Because of their composition, metals can stay in nature long after they were released. Since the 1960s a sampling of two mosses (Pleurozium schreberi and Hylocomium splendens) have been made all around Sweden to investigate heavy metals bound in moss. Every year manufactories must submit their emissions to their responsible authority. This study aims to investigate if it is possible to use moss samples, wind direction and manufactories registered air emissions to track the transportation and spread of the air emissions. The manufactories examined were Korstaverket, Kubikenborg Aluminium AB, SCA Ortviken and SCA Östrand. The heavy metals examined were cadmium (Cd) and mercury (Hg). Data for this study are based on public information and were collected from SMHI and Länsstyrelsen Västernorrland. The statistical method used was Wilcoxon-Mann- Whitney U- test. The result showed that the heavy metal content in moss samples have increased while the air emission from the manufactories have decreased. Therefore, the conclusion is that it is not possible to trace the registered air emissions of local industries using moss samples and wind direction, based on the collected data.

In the last few years, estimators of the scale of a univariate distribution have been developed that are location-free in the sense that they do not depend on an estimate of the center of the underlying distribution. These proposed location-free estimators have generally been quite robust in terms of having a high breakdown point and can achieve a surprisingly high Gaussian efficiency. This idea has also been extended to the simple linear regression model, where typical estimators of the dispersion of the errors depend on an estimator of the regression line. The few estimators that have been developed that do not depend on a line estimator, called regression-free scale estimators, do achieve a high breakdown point but are useful mainly for data sets that have no replication at any regressor value. We propose new regression-free scale estimators that achieve a high breakdown point, can be quite efficient, and are useful when the data contain replication. Also, we propose a robust estimator of the common scale parameter in the k-sample model that reduces to an existing location-free estimator in the case of univariate data. We derive the breakdown point of this estimator as well as its maximum bias curve. Simulation results show that it can be quite efficient with Gaussian data. / Ph. D.

No / Noncontact scanning near-field optical microscope (SNOM) systems can be used to optically resolve samples in atmospheric conditions at theoretical resolutions comparable to those of transmission electron microscope and atomic force microscope systems. SNOM systems are also increasingly used to image biological samples. In this study we custom built a SNOM system with the aim of further demonstrating the potential applications of near-field optical examination of biological material. In this study we were able to image both fixed whole-cell samples in air and liquid environments and live whole-cell samples in liquids. The images acquired were of a relatively low resolution, but this work has shown that SNOM systems can be used to monitor the dynamics of living cells at subnanometric resolutions in the z axis and for fluorescent imaging of whole cells in a liquid medium.