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.

Transfection of newt blastema mesenchyme using the techniques of lipofection and direct gene transfer

The regenerating amphibian limb provides a useful system for studying genes involved in the establishment of positional information. While a number of candidate genes that may playa role in pattern formation have been identified, their function in vivo is unknown in this system. To better ascertain the role of these genes, it would be useful to be able to alter their normal patterns of expression in vivo and to assess the effects of this misexpression on limb pattern. In order to achieve this, a method of introducing a plasmid containing the eDNA of a gene of interest into a newt blastema (a growth zone of mesenchymal progenitor cells) is needed. Unfortunately, most commonly used transfection techniques cannot be used with newt blastema cells. In this study, I have used the techniques of lipofection and direct gene transfer to introduce plasmid DNA containing reporter genes into the cells of a regenerating newt limb. The technique of lipofection was most effective when the blastema cells were transfected in vitro. The optimal ratio for transfection was shown to be 1:3 DNA:Lipofectin (W/w) , and an increase in the amount of DNA present in the mixture (1:3 ratio maintained) resulted in a corresponding increase in gene expression. The technique of direct gene transfer was used to transfect newt blastema cells with and without prior complex formation with Lipofectin. Injection of plasmid DNA alone provided the most 3 promising results. It was possible to introduce plasmid DNA containing the reporter gene ~-galactosidase and achieve significant gene expression in cells associated with the injection site. In the future, it would be interesting to use this technique to inject plasmid DNA containing a gene which may have a role in pattern formation into specific areas of the newt blastema and to analyze the resulting limb pattern that emerges.

Remembering Lessons Learned: knowledge management techniques for building generational memory

A presentation made at the CAUT Librarians Conference in Ottawa, Ontario in October 2005.

Perceptions of intergroup bias : the roles of social projection and meta-stereotypes

Underlying intergroup perceptions include processes of social projection (perceiving personal traitslbeliefs in others, see Krueger 1998) and meta-stereotyping (thinking about other groups' perceptions of one's own group, see Vorauer et aI., 1998). Two studies were conducted to investigate social projection and meta-stereotypes in the domain of White-Black racial relations. Study 1, a correlational study, examined the social projection of prejudice and 'prejudiced' meta-stereotypes among Whites. Results revealed that (a) Whites socially projected their intergroup attitudes onto other Whites (and Blacks) [i.e., Whites higher in prejudice against Blacks believed a large percentage of Whites (Blacks) are prejudiced against Blacks (Whites), whereas Whites low in prejudice believed a smaller percentage of Whites (Blacks) are prejudiced]; (b) Whites held the meta:..stereotype that their group (Whites) is viewed by Blacks to be prejudiced; and (c) prejudiced meta-stereotypes may be formed through the social projection of intergroup attitudes (result of path-model tests). Further, several correlates of social projection and meta-stereotypes were identified, including the finding that feeling negatively stereotyped by an outgroup predicted outgroup avoidance through heightened intergroup anxiety. Study 2 replicated and extended these findings, investigating the social projection of ingroup favouritism and meta- and other-stereotypes about ingroup favouritism. These processes were examined experimentally using an anticipated intergroup contact paradigm. The goal was to understand the experimental conditions under which people would display the strongest social projection of intergroup attitudes, and when experimentally induced meta-stereotypes (vs. other-stereotypes; beliefs about the group 11 preferences of one's outgroup) would be most damaging to intergroup contact. White participants were randomly assigned to one of six conditions and received (alleged) feedback from a previously completed computer-based test. Depending on condition, this information suggested that: (a) the participant favoured Whites over Blacks; (b) previous White participants favoured Whites over Blacks; (c) the participant's Black partner favoured Blacks over Whites; (d) previous Black participants favoured Blacks over Whites; (e) the participant's Black partner viewed the participant to favour Whites over Blacks; or (£) Black participants previously participating viewed Whites to favour Whites over Blacks. In a defensive reaction, Whites exhibited enhanced social projection of personal intergroup attitudes onto their ingroup under experimental manipulations characterized by self-concept threat (i.e., when the computer revealed that the participant favoured the ingroup or was viewed to favour the ingroup). Manipulated meta- and otherstereotype information that introduced intergroup contact threat, on the other hand, each exerted a strong negative impact on intergroup contact expectations (e.g., anxiety). Personal meta-stereotype manipulations (i.e., when the participant was informed that her/ his partner thinks s/he favours the ingroup) exerted an especially negative impact on intergroup behaviour, evidenced by increased avoidance of the upcoming interracial interaction. In contrast, personal self-stereotype manipulations (i.e., computer revealed that one favoured the ingroup) ironically improved upcoming intergroup contact expectations and intentions, likely due to an attempt to reduce the discomfort of holding negative intergroup attitudes. Implications and directions for future research are considered.

Leisure connections : a case study to understand facilitation techniques with survivors of trauma

Leisure-based therapy is a potentially effective approach to supporting survivors of trauma in their healing. The purpose ofthis qualitative case study was to describe the recreation therapist's facilitation techniques of Leisure Connections, a unique leisurebased psycho-educational group for survivors of trauma, and explore how the facilitation was experienced by participants. Qualitative case study design, following the methods of Yin (1994) was used. One two week, three session Leisure Connections group was observed. Six participants completed the Group· Therapy Alliance Scale (pinsof & Catherall, 1986) and reflection cards. In-depth, semi-structured interviews were conducted with the recreation therapist and four participants. Six themes emerged describing group leader interventions, recreation therapist's actions, recreation therapist's preparation and reflections, group members' experience of a therapeutic alliance, group cohesion, and prior influences and assumptions. Therapeutic alliance and group cohesion were influenced by the recreation therapist's group leader interventions (drawing out, processing, protecting) and actions. The context of the group within a therapeutic community milieu was an important influence.

Strategies and Techniques for Teaching Children with Learning Disabilities: A Case Study of the Spring Reading Program

This case study explored strategies and techniques in order to assist individuals with learning disabilities in their academic achievement. Of particular focus was how a literacy-based program, titled The Spring Reading Program, utilizes effective tactics and approaches that result in academic growth. The Spring Reading Program, offered by the Learning Disabilities Association of Niagara Region (LDANR) and partnered with John McNamara from Brock University, supports children with reading disabilities academically. In addition, the program helps children increase their confidence and motivation towards literacy. I began this study by outlining the importance of reading followed by and exploration of what educators and researchers have demonstrated regarding effective literacy instruction for children with learning disabilities. I studied effective strategies and techniques in the Spring Reading Program by conducting a qualitative case study of the program. This case study subsequently presents in depth, 4 specific strategies: Hands-on activities, motivation, engagement, and one-on-one instruction. Each strategy demonstrates its effectiveness through literature and examples from the Spring Reading Program.