Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

Development of a bacteriophage-based biopesticide for fire blight

Fire blight is an economically important disease of apples and pears that is caused by the bacterium Erwinia amylovora. Control of the disease depends on limiting primaly blosson1 infection in the spring, and rapidly removing infected tissue. The possibility of using phages to control E.amylovora populations has been suggested, but previous studies have. failed to show high treatment efficacies. This work describes the development of a phage-based biopesticide that controls E. amylovora populations under field conditions, and significantly reduces the incidence of fire blight. This work reports the first use ofPantoea agglomerans, a non-pathogenic relative ofE. amylovora, as a carrier for E. amylovora.phages. Its role is to support a replicating population of these phages on blossom surfaces during the period when the flowers are most susceptible to infection. Seven phages and one carrier isolate were selected for field trials from existing collections of 56 E. amylovora phages and 249 epiphytic orchard bacteria. Selection of the . /' phages and carrier was based on characteristics relevant to the production and field perfonnance of a biopesticide: host range, genetic diversity, growth under the conditions of large-scale production, and the ability to prevent E. amylovora from infecting pear blossoms. In planta assays showed that both the phages and the carrier make significant contributions to reducirig the development of fire blight symptoms in pear blossoms. Field-scale phage production and purification methods were developed based on the growth characteristics of the phages and bacteria in liquid culture, and on the survival of phages in various liquid media. Six of twelve phage-carrier biopesticide treatments caused statistically signiflcant reductions in disease incidence during orchard trials. Multiplex real-time PCR was used to simultaneously monitor the phage, carrier, and pathogen populations over the course of selected treatments. In all cases. the observed population dynamics of the biocontrol agents and the pathogen were consistent with the success or failure of each treatment to control disease incidence. In treatments exhibiting a significantly reduced incidel1ce of fire blight, the average blossom population ofE.amylovora had been reduced to pre-experiment epiphytic levels. In successful treatments the phages grew on the P. agglomerans carrier for 2 to 3 d after treatment application. The phages then grew preferentially on the pathogen, once it was introduced into this blossom ecosystem. The efficacy of the successful phage-based treatnlents was statistically similar to that of streptomycin, which is the most effective bactericide currently available for fire blight prevention. The in planta behaviour ofE. amylovora was compared to that ofErwinia pyrifoliae, a closely related species that causes fire blight-like synlptoms on pears in southeast Asia. Duplex real-time PCR was used to monitor the population dynamics of both species on single blossonls. E. amylovora exhibited a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae. The genome ofErwinia phage Ea21-4 was sequenced and annotated. Most of the 8-4.7 kB genome is substantially different from previously described sequences, though some regions are notably similar to Salmonella phage Felix 01 . Putative functions were assigned to approximately 30% of the predicted open reading frames based on amino acid sequence comparisons and N-terminal sequencing of structural proteins.

The development of norm-based coding and category-specific face prototypes : an examination of 5- and 8-year-olds' face space

The present set of experiments was designed to investigate the organization and refmement of young children's face space. Past research has demonstrated that adults encode individual faces in reference to a distinct face prototype that represents the average of all faces ever encountered. The prototype is not a static abstracted norm but rather a malleable face average that is continuously updated by experience (Valentine, 1991); for example, following prolonged viewing of faces with compressed features (a technique referred to as adaptation), adults rate similarly distorted faces as more normal and more attractive (simple attractiveness aftereffects). Recent studies have shown that adults possess category-specific face prototypes (e.g., based on race, sex). After viewing faces from two categories (e.g., Caucasian/Chinese) that are distorted in opposite directions, adults' attractiveness ratings simultaneously shift in opposite directions (opposing aftereffects). The current series of studies used a child-friendly method to examine whether, like adults, 5- and 8-year-old children show evidence for category-contingent opposing aftereffects. Participants were shown a computerized storybook in which Caucasian and Chinese children's faces were distorted in opposite directions (expanded and compressed). Both before and after adaptation (i.e., reading the storybook), participants judged the normality/attractiveness of a small number of expanded, compressed, and undistorted Caucasian and Chinese faces. The method was first validated by testing adults (Experiment I ) and was then refined in order to test 8- (Experiment 2) and 5-yearold (Experiment 4a) children. Five-year-olds (our youngest age group) were also tested in a simple aftereffects paradigm (Experiment 3) and with male and female faces distorted in opposite directions (Experiment 4b). The current research is the first to demonstrate evidence for simple attractiveness aftereffects in children as young as 5, thereby indicating that similar to adults, 5-year-olds utilize norm-based coding. Furthermore, this research provides evidence for racecontingent opposing aftereffects in both 5- and 8-year-olds; however, the opposing aftereffects demonstrated by 5-year-olds were driven largely by simple aftereffects for Caucasian faces. The lack of simple aftereffects for Chinese faces in 5-year-olds may be reflective of young children's limited experience with other-race faces and suggests that children's face space undergoes a period of increasing differentiation over time with respect to race. Lastly, we found no evidence for sex -contingent opposing aftereffects in 5-year-olds, which suggests that young children do not rely on a fully adult-like face space even for highly salient face categories (i.e., male/female) with which they have comparable levels of experience.

The Mirror Room Project: A Critical Ethnographic Program Evaluation of a Teaching Personal and Social Responsibility Based Youth Development Program

Amongst a host of other benefits, proper physical education has the possibility to create a safe place where responsibility can be transferred from the teacher/facilitator, to the student. This is especially true with an underserved population. This critical program evaluation of the program CHARM was done for the purpose of program improvement. This research was a place for participants to share their experiences of the program. The participants were 5 underserved youth, 5 undergraduate students, 3 teachers and 1 graduate student. Observations, interviews, and document analysis were used to gather data. Data was analyzed using a first level read-through, and two second-level analyses. Summaries were written, and cross-case analyses were completed. The main finding of the research was the development of a Handbook, which is a guide to running the program. Secondary findings include issues of program structure, goal setting, meaningful relationships, roles, SNAP, and an outlier in the data.

SAVOURING ABILITIES SCALE (SAS): THE DEVELOPMENT OF A STRENGTHS - BASED ASSESSMENT FOR THERAPEUTIC RECREATION PROFESSIONALS

The purpose of this study was to develop and evaluate an assessment that measured savouring leisure. The assessment items developed were reviewed for content validity by eight international therapeutic recreation (TR) educators and administrators as well as eleven TR practitioners. An exploratory factor analysis was conducted to determine the most suitable items for the assessment. The results suggest that the original three subscale design needed to be modified to five. The reliability of the total assessment is α=.84. Statistical analysis for construct validity reveal 58.52% of the variance explained, and a moderate correlation was found between this study and other savouring assessments. The implications of the shift in conceptualization are reviewed through discussing factor analysis issues, the lived experience of savouring leisure, and the impact of the content validity process. This study contributes to the ongoing dialog of savouring leisure. Recommendations for future research are discussed.