Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

Understanding the Integration of Living Skills Through the Context of Health and Physical Education: A Case Study of Educators’ Experiences

The research presented is a qualitative case study of educators’ experiences in integrating living skills in the context of health and physical education (HPE). In using semi-structured interviews the study investigated HPE educators’ experiences and revealed their insights relative to three major themes; professional practice, challenges and support systems. Professional practice experiences detailed the use of progressive lesson planning, reflective and engaging activities, explicit student centered pedagogy as well as holistic teaching philosophies. Even further, the limited knowledge and awareness of living skills, conflicting teaching philosophies, competitive environments between subject areas and lack of time and accessibility were four major challenges that emerged throughout the data. Major supportive roles for HPE educators in the integration process included other educators, consultants, school administration, public health, parents, community programs and professional organizations. The study provides valuable discussion and suggestions for improvement of pedagogical practices in teaching living skills in the HPE setting.

One of Us or One of Them: A Generic Qualitative Study on the Efficacy of the Funds of Knowledge Strategy to Reduce Teacher Ethnocentrism

The purpose of this study was to examine whether English a Second Language (ESL) instructors’ ethnocentrism could be reduced using multicultural education (MCE) principles. There were three focus group discussions and a Likert scale questionnaire. The findings demonstrated that while ESL instructors were conscious of systemic barriers, media stereotypes, and bullying, more diversity training is required in order to improve teachers’ attitudes, responses, and instructional strategies regarding integration issues due to the increasing diversity of learners present in classrooms today. The findings of the study also demonstrated that MCE principles could be used to effectively raise the awareness of ESL instructors when dealing with integration and assimilation issues. When immigration, human rights, and multicultural policies were examined critically, ESL instructors were able to improve their cross-cultural skills in the classroom to be more inclusive towards diverse ethnic groups by giving learners greater opportunities to express themselves. As a result, learners’ knowledge, experience, and skills were validated in the classroom leading to a more meaningful learning experience.