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.

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.

Measurement of growth in the lichen Rhizocarpon geographicum using a new photographic technique

Lichenologists and users of lichenometry have long used calipers or photogrammetry to measure the growth of crustose lichens. Now, digital photography and popular computer software provide methodological alternatives. This thesis developed and tested a new methodology for tracking change and growth of the lichen, Rhizocarpon geographicum. Adobe Photoshop CS3 Extended software and a photographic time series (1996,2003,2006 and 2007) were used to measure thallus diameter, area, prothallus width and areolae area in 115 small R. geographicum thalli (0.53-1049.88 mm2 ). Measures of 8 diameters per thallus showed that change in diameter was highly variable and is a weak index of growth. Thallus area was a reliable measure of growth (power correlation, R2 = 0.89). Rapid, highly irregular growth occurred in small thalli «30 mm2 ), and steady, uniform growth occurred in larger thalli (>30 mm2 ). This new methodology is tedious but can potentially generate accurate and precise measures for even the tiniest of lichens.

Genetic Programming for Non-Photorealistic Rendering

This thesis focuses on developing an evolutionary art system using genetic programming. The main goal is to produce new forms of evolutionary art that filter existing images into new non-photorealistic (NPR) styles, by obtaining images that look like traditional media such as watercolor or pencil, as well as brand new effects. The approach permits GP to generate creative forms of NPR results. The GP language is extended with different techniques and methods inspired from NPR research such as colour mixing expressions, image processing filters and painting algorithm. Colour mixing is a major new contribution, as it enables many familiar and innovative NPR effects to arise. Another major innovation is that many GP functions process the canvas (rendered image), while is dynamically changing. Automatic fitness scoring uses aesthetic evaluation models and statistical analysis, and multi-objective fitness evaluation is used. Results showed a variety of NPR effects, as well as new, creative possibilities.

Returning from the Wild: Exploring Participant's Experiences of Re-entry from Extended Wilderness Trips

The experience of a strong sense of community developed while participating in extended wilderness expeditions is one of the most significant and meaningful experiences associated with taking part in this form of outdoor recreation. The experience of returning to a home community from an extended wilderness expedition is explored through the impacts associated with psychological sense of community (McMillian & Chavis, 1986; McMillian, 1996). A phenomenological approach was used to investigate the re-entry experiences of six individuals through the use of semi-structured interviews. Twelve main themes and seventeen subthemes emerged within the findings and illustrate a lack of preparation for the difficulties associated with re-entry, negative impacts associated with the experience of sense of community, and problems transferring aspects of a wilderness community into participant’s post-expedition lives.

Modeling Metal Protein Complexes from Experimental Extended X-ray Absorption Fine Structure using Computational Intelligence

Experimental Extended X-ray Absorption Fine Structure (EXAFS) spectra carry information about the chemical structure of metal protein complexes. However, pre- dicting the structure of such complexes from EXAFS spectra is not a simple task. Currently methods such as Monte Carlo optimization or simulated annealing are used in structure refinement of EXAFS. These methods have proven somewhat successful in structure refinement but have not been successful in finding the global minima. Multiple population based algorithms, including a genetic algorithm, a restarting ge- netic algorithm, differential evolution, and particle swarm optimization, are studied for their effectiveness in structure refinement of EXAFS. The oxygen-evolving com- plex in S1 is used as a benchmark for comparing the algorithms. These algorithms were successful in finding new atomic structures that produced improved calculated EXAFS spectra over atomic structures previously found.

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.