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.

Preparing students for the transition from college to work /

The purpose of this study was to examine the perceived preparedness of college students for the transition from college to full-time employment. The study was concerned with the interest and rationale behind developing a required Exit Course for college students in order to improve the college to work transition. As well, possible content of an Exit Course was evaluated. The importance of addressing college to work transitions is highlighted by two phenomena. First, there are specific employability skills that employers in Canada are seeking in newly hired employees. Second, the provincial government in Ontario is determining college funding based on graduate employment statistics which are measured by graduate satisfaction, graduate employment, and employer satisfaction. The research concentrated on the following stakeholders involved in the transition from college to work: (a) current students, (b) recent graduates, (c) support staff who assist students in college to work transition (Career Educators), and (d) employers. Through qualitative research, including focus groups and interviews, these stakeholder groups participated in the research to determine if the Exit Course was a viable solution to facilitate the transition from college to work. Focus groups were conducted with current students, while one-on-one, semi-structured interviews were conducted with recent graduates, Career Educators, and employers. Common themes elicited from the participants included the following: (a) although students were perceived by the participants of this study to be technically prepared for employment, they were perceived to have weak job search skills and unrealistic expectations of the world of work unless they had received the benefits of a Co-operative Education experience; (b) an Exit Course was seen as a viable solution to the issues involved in college to work transition; (c) an Exit Course should be comprised of skills necessary to obtain and succeed in a job and the course should be taught by individuals with extensive qualifications in this area; and (d) there is a need to develop college and business partnerships to ensure that students are connected to employers. Educators within post secondary institutions, specifically colleges, can benefit from the information provided within this study to gain a better understanding of the perceived level of preparedness of students for the transition from college to work. Suggestions with regard to how to improve this transition were made, with specific reference to the addition of an Exit Course as one possible solution.

An examination of learning resulting from a transition from a directed to a self-directed work environment

~ This study focuses on the process of self-directed learning that individuals go through as they adapt to new work situations. This is a study of how one critical incident, specifically the transition from a traditional office structure to a home office structure, affected employees and what their learning process was as they adapted to the new environment. This study has 3 educational foundations: adult learning, self-directed learning, and the social context from which the learning will occur. Six women and 2 men were interviewed approximately 1 year following the transition. Analysis of the data revealed 5 themes of: impacts of the self-directed environment on participants' personal lives, their roles, skill set, productivity, and the physical environment; support offered by the organization, family, and office administration; personal development, specific learning needs, and personal skills; boundaries as they relate to family and work; and skill set and orientation requirements of new home office employees. The findings revealed the learning processes of the 8 participants. The learning processes of these participants were discussed within a theoretical framework of the learners, their immediate surroundings, and the larger social environment. The results indicated that the transition from a directed work environment to a self directed work environment is a complex, interrelated process. An element found throughout the theoretical framework is that of control. A second critical element is the need for participants to have a clearly defined work role and an opportunity to engage in discussion with peers and the community. Further findings reinforced the importance of climate and found that the physical environment is a key factor in a successful selfdirected work environment. The findings of this study revealed that no one factor makes an individual function successfully in a self-directed work environment, but that it is a complex interplay among the leamer, their immediate surroundings, and the social environment that will have the greatest impact on success. Recommendations are made which can be used to guide organizational leaders in facilitating employees' transition from a directed to a self-directed work environment. Additionally, recommendations are made for further research in the area of self-directed work environments.

How Ontario elementary school principals negotiate their varied work roles

This qualitative study>addresses the question of how Ontario elementary school principals negotiate their varied work roles, through interviews with and observations of 6 principals. Using inductive data analysis, principals' negotiations were divided into 5 categories: negotiating priorities, negotiating the process, negotiating constraints, negotiating the roles of others, and negotiating the self. These principals worked within these categories simultaneously, emphasizing some more than others, dependent on the circumstances. For these principals, the time they spent with people in the school and the resulting relationships that enabled them to build were a first priority, and a large part of how each principal chose to negotiate the demands of their role arose from their personality and their personal values.

Teachers' experiences and perceptions of voluntary committee work as a vehicle for leadership development

In response to a looming leadership shortage, leadership development for teachers has become an increasingly important area of interest around the world. A review of the literature identified the key components of educational leadership development programs as effective curriculum, leadership practice, relationship building, and reflection. A gap in the research was found regarding the use of voluntary committee work as a vehicle for leadership practice. The purpose of this study was to explore teachers' perceptions of their experiences within board-level committees to determine the key factors that contributed, positively or negatively, to their leadership,pevelopment. A qualitative research design was employed using semistructured interviews with 8 participants. The key findings included a list of factors perceived by teachers as either supporting or hindering their leadership development. The supporting factors were: (a) leadership practice, (b) mentors and role models, (c) relationships and networks, and (d) positive outcomes for students. The hindering factors were: (a) lack of follow through and support, (b) committee members with a careerist approach to the experience, (c) personal and political agendas, and (d) overcommitment leading to burnout. Recommendations for practice focused on strategies to enhanc~_ the committee experience as a tool for leadership development. Recommendations for theory and research suggested more research be done on each of the 8 key factors, perceptions associated with teachers choosing to follow a leadership path, and how school boards can structure the committee process as an effective leadership development tool. This study provides a starting point for educators to begin to intentionally design, develop, and deliver voluntary committee experiences as a tool for leadership development.