Investigation of single tryptophan proteins encapsulated in TEOS-derived sol-gel matrices by fluorescence spectroscopy /

In the work reported here, optically clear, ultrathin TEOS derived sol-gel slides which were suitable for studies of tryptophan (Trp) fluorescence from entrapped proteins were prepared by the sol-gel technique and characterized. The monitoring of intrinsic protein fluorescence provided information about the structure and environment of the entrapped protein, and about the kinetics of the interaction between the entrapped protein and extemal reagents. Initial studies concentrated on the single Trp protein monellin which was entrapped into the sol-gel matrices. Two types of sol-gel slides, termed "wet aged", in which the gels were aged in buffer and "dry-aged", in which the gels were aged in air , were studied in order to compare the effect of the sol-gel matrix on the structure of the protein at different aging stages. Fluorescence results suggested that the mobility of solvent inside the slides was substantially reduced. The interaction of the entrapped protein with both neutral and charged species was examined and indicated response times on the order of minutes. In the case of the neutral species the kinetics were diffusion limited in solution, but were best described by a sum of first order rate constants when the reactions occurred in the glass matrix. For charged species, interactions between the analytes and the negatively charged glass matrix caused the reaction kinetics to become complex, with the overall reaction rate depending on both the type of aging and the charge on the analyte. The stability and conformational flexibility of the entrapped monellin were also studied. These studies indicated that the encapsulation of monellin into dry-aged monoliths caused the thermal unfolding transition to broaden and shift upward by 14°C, and causedthe long-term stability to improve by 12-fold (compared to solution). Chemical stability studies also showed a broader transition for the unfolding of the protein in dry-aged monoliths, and suggested that the protein was present in a distribution of environments. Results indicated that the entrapped proteins had a smaller range of conformational motions compared to proteins in solution, and that entrapped proteins were not able to unfold completely. The restriction of conformational motion, along with the increased structural order of the internal environment of the gels, likely resulted in the improvements in themial and long-term stability that were observed. A second protein which was also studied in this work is the metal binding protein rat oncomodulin. Initially, the unfolding behavior of this protein in aqueous solution was examined. Several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM) were examined; F102W, Y57W, Y65W and the engineered protein CDOM33 which had all 12 residues of the CD loop replaced with a higher affinity binding loop. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca^^ < Tb^"^. During thermal denaturation, the transition midpoints (Tun) of Y65W appeared to be the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W apparently made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which probably acted through the disruption of hydrogen bonds at the Cterminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinatenumber (i.e. Tb^*) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability. Y57W and CE)OM33 OM were chosen for further studies when encapsulated into sol-gel derived matrices. The kinetics of interaction of terbium with the entrapped proteins, the ability of the entrapped protein to binding terbium, as well as thermal stability of these two entrapped protein were compared with different levels of Ca^"*^ present in the matrix and in solution. Results suggested that for both of the proteins, the response time and the ability to bind terbium could be adjusted by adding excess calcium to the matrix before gelation. However, the less stable protein Y57W only retained at most 45% of its binding ability in solution while the more stable protein CDOM33 was able to retain 100% binding ability. Themially induced denaturation also suggested that CDOM33 showed similar stability to the protein in solution while Y57W was destabilized. All these results suggested that "hard" proteins (i.e. very stable) can easily survive the sol-gel encapsulation process, but "soft" proteins with lower thermodynamic stability may not be able to withstand the sol-gel process. However, it is possible to control many parameters in order to successfully entrap biological molecules into the sol-gel matrices with maxunum retention of activity.

Effects of sample solvent composition and injection volume on chromatographic peak profiles of methyl w- benzimidazolecarbamate and 3-butyl1-2,4-dioxo[1,2-a]-s- triazinobenzimidazole in RP-HPLC

The effects of sample solvent composition and the injection volume, on the chromatographic peak profiles of two carbamate derivatives, methyl 2-benzimidazolecarbamate (MBC) and 3-butyl-2,4-dioxo[1,2-a]-s-triazinobenzimidazole (STB), were studied using reverse phase high performance liquid chromatograph. The study examined the effects of acetonitrile percentage in the sample solvent from 5 to 50%, effects of methanol percentage from 5 to 50%, effects of pH increase from 4.42 to 9.10, and effect of increasing buffer concentration from ° to 0.12M. The effects were studied at constant and increasing injection mass and at four injection volumes of 10, 50, 100 and 200 uL. The study demonstrated that the amount and the type of the organic solvents, the pH, and the buffer strength of the sample solution can have a pronounced effect on the peak heights, peak widths, and retention times of compounds analysed. MBC, which is capable of intramolecular hydrogen bonding and has no tendency to ionize, showed a predictable increase .in band broadening and a decrease in retention times at higher eluting strengths of the sample solvent. STB, which has a tendency to ionize or to strongly interact with the sample solvent, was influenced in various ways by the changes in ths sample solvent composition. The sample solvent effects became more pronounced as the injection volume increased and as the percentage of organic solvent in the sample solution became greater. The peak height increases for STB at increasing buffer concentrations became much more pronounced at higher analyte concentrations. It was shown that the widely accepted procedure of dissolving samples in the mobile phase does not yield the most efficient chromatograms. For that reason samples should be dissolved in the solutions with higher aqueous content than that of the mobile phase whenever possible. The results strongly recommend that all the samples and standards, regardless whether the standards are external or internal, be analysed at a constant sample composition and a constant injection volume.

Women over thirty choosing education as a solution to a problem

This study explored motivations of mid-life women over 30 years old who had returned to school. It sought to fmd whether these women returned to solve a problem arising from life events, whether viewing a problem was related to internal or external motivation, whether this perception was related to having greater coping skills, and whether having greater coping was related to seeking support from internal or external sources. This study examined which emotions were most related to viewing a life event as a problem. Finally, it explored the results of previous research of mid-life women in their role as a student. Women (N==83) from three types of institutions volunteered for this study: a university (N==34), a college (N==28), and an adult education centre (N==21). Participants took home a questionnaire package - a I3-page questionnaire and consent form - that were completed and mailed back to the researcher in pre-paid envelopes. Results showed that women over 30 seek education as a solution to a life event problem. External motivation was related to a life event being a problem (p<.005). There was a significant difference in coping scores between institutions. Moods that were related to viewing a life event as problematic were: anger and depressive moods (p<. 001), fatigue and vigor (p<.O 1), and tension/anxiety (p<.05). Mid-life women students' satisfaction in this role was related to being externally motivated. These women sought support from both internal and external sources, rarely had social interactions with peers, and viewed this role as important, yet, temporary in that it will help them change their lives. Implications ofthe results suggest further exploration ofthe roles of anger and depression in motivating women over 30 to learn and finding ways of directing women to use their emotional intelligence to seek out learning.

Development and characterization of concentric capillary nebulizer used in inductively coupled plasma analysis /

A simple, low-cost concentric capillary nebulizer (CCN) was developed and evaluated for ICP spectrometry. The CCN could be operated at sample uptake rates of 0.050-1.00 ml min'^ and under oscillating and non-oscillating conditions. Aerosol characteristics for the CCN were studied using a laser Fraunhofter diffraction analyzer. Solvent transport efficiencies and transport rates, detection limits, and short- and long-term stabilities were evaluated for the CCN with a modified cyclonic spray chamber at different sample uptake rates. The Mg II (280.2nm)/l\/lg 1(285.2nm) ratio was used for matrix effect studies. Results were compared to those with conventional nebulizers, a cross-flow nebulizer with a Scott-type spray chamber, a GemCone nebulizer with a cyclonic spray chamber, and a Meinhard TR-30-K3 concentric nebulizer with a cyclonic spray chamber. Transport efficiencies of up to 57% were obtained for the CCN. For the elements tested, short- and long-term precisions and detection limits obtained with the CCN at 0.050-0.500 ml min'^ are similar to, or better than, those obtained on the same instrument using the conventional nebulizers (at 1.0 ml min'^). The depressive and enhancement effects of easily ionizable element Na, sulfuric acid, and dodecylamine surfactant on analyte signals with the CCN are similar to, or better than, those obtained with the conventional nebulizers. However, capillary clog was observed when the sample solution with high dissolved solids was nebulized for more than 40 min. The effects of data acquisition and data processing on detection limits were studied using inductively coupled plasma-atomic emission spectrometry. The study examined the effects of different detection limit approaches, the effects of data integration modes, the effects of regression modes, the effects of the standard concentration range and the number of standards, the effects of sample uptake rate, and the effect of Integration time. All the experiments followed the same protocols. Three detection limit approaches were examined, lUPAC method, the residual standard deviation (RSD), and the signal-to-background ratio and relative standard deviation of the background (SBR-RSDB). The study demonstrated that the different approaches, the integration modes, the regression methods, and the sample uptake rates can have an effect on detection limits. The study also showed that the different approaches give different detection limits and some methods (for example, RSD) are susceptible to the quality of calibration curves. Multicomponents spectral fitting (MSF) gave the best results among these three integration modes, peak height, peak area, and MSF. Weighted least squares method showed the ability to obtain better quality calibration curves. Although an effect of the number of standards on detection limits was not observed, multiple standards are recommended because they provide more reliable calibration curves. An increase of sample uptake rate and integration time could improve detection limits. However, an improvement with increased integration time on detection limits was not observed because the auto integration mode was used.

A Basis for Understanding Volunteer Coach Retention in Youth Sports

Youth sport organizations depend on volunteers to coach the teams in the organization. The purpose of this quantitative study was to develop a further understanding of volunteer coach retention in youth sport. The data was collected through a quantitative questionnaire which used close-ended and Likert-scale questions. The questionnaire collected data on the modified Model of Volunteer Retention in Youth Sports, reasons to withdraw from coaching and human resource management. There were 126 surveys collected from members of the three largest youth sport associations in the town of Aylmer, Ontario. The study found that Person-Task fit was the best predictor of volunteer coach retention as it significantly correlated to one’s intention to continue coaching (p< 0.01). Furthermore, additional reasons were found to explain withdrawal from coaching - if one’s child stops playing the sport or if coaching is too time consuming. The retention of volunteer coaches in youth sport organizations requires a multi-dimensional approach in understanding how to best retain volunteer coaches.

“You Know What I Mean?”- Language and Cultural Retention in Luso-Canadian Mothers in the Greater Toronto Area

The Portuguese community is one of the largest diasporic groups in the Greater Toronto Area and the choice of retention and transmission of language and culture to Luso-Canadians is crucial to the development and sustainability of the community. The overall objective of this study is to learn about the factors that influence Luso-Canadian mothers’ inclination to teach Portuguese language and cultural retention to their children. To explore this topic I employed a qualitative research design that included in-depth interviews conducted in 2012 with six Luso-Canadian mothers. Three central arguments emerged from the findings. First, Luso-Canadian mothers interviewed posses a pronounced desire for their children to succeed academically, and to provide opportunities that their children that they did not have. Second, five of the mothers attempt to achieve this mothering objective partly by disconnecting from their Portuguese roots, and by disassociating their children from the Portuguese language and culture. Third, the disconnection they experience and enact is influenced by the divisions evident in the Portuguese community in the GTA that divides regions and hierarchically ranks dialects, and groups. I conclude that the children in these households inevitably bear the prospects of maintaining a vibrant Portuguese community in the GTA and I propose that actions by the community in ranking dialects influence mothers’ decisions about transmitting language and culture to their children.

Perceived Deservingness and the Toleration of Human Rights Violations: The Problem and a Possible Solution

Despite general endorsement of universal human rights, people continue to tolerate specific human rights violations. I conducted a two-part study to investigate this issue. For Part I, I examined whether people tolerated torture (a human rights violation) based on the morality and deservingness of the target. Participants tolerated torture more when the target had committed a highly morally reprehensible transgression. This effect was mediated by the target’s perceived deservingness for harsh treatment, and held over and above participants’ abstract support for the right to humane treatment. For Part II, hypocrisy induction was used in an attempt to reduce participants’ toleration of the torture. Participants were assigned to either the hypocrisy induction or control condition. Unexpectedly, participants who tolerated the torture more in Part I reduced their toleration the most in the control condition, possibly because of consistency and floor effects. Limitations and implications of the findings are discussed.