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.

Understanding value congruence : a sport organization case study

With the recent growth in cultural complexity, many organizations are faced with increasingly diverse employee pools. Gaining a greater understanding of the values that employees possess is the first step in effectively satisfying their needs and achieving a more productive workforce (lung & Avolio, 2000). Values playa significant role in influencing individual behaviours. It is therefore necessary to assess the qualities of employee value systems and directly link them to the values of the organization. The importance of values and value congruence has been emphasized by many organizational behaviour researchers (cf. Adkins & Caldwell, 2004; Erdogan, Kraimer, & Liden, 2004; Jung & Avolio, 2000; Rokeach, 1973); however the emphasis on value studies remains fairly stagnant within the sport industry (Amis, Slack, & Hinings, 2002). In order to examine the realities that were constructed by the participants in this study a holistic view of the impact of values within a specific sport organization were provided. The purpose of this case study was to examine organizational and employee values to understand the effects of values and value congruence on employee behaviours within the context of a large Canadian sport organization. A mUltiple methods case study approach was adopted in order to fully serve the purpose and provide a comprehensive view of the organization being examined. Document analysis, observations, surveys, as well as semi-structured interviews were conducted. The process allowed for triangulation and confirmability of the findings. Each method functioned to create an overarching understanding of the values and value congruence within this organization. The analysis of the findings was divided into qualitative and quantitative sections. The qualitative documents were analyzed twice, once manually by the researcher and once via AtIas.ti Version 4 (1998). The a priori and emergent coding that took place was based on triangulating the findings and uncovering common themes throughout the data. The Rokeach Value Survey (1973) that was incorporated into the survey design of the study was analyzed using descriptive statistics, as well as Mann-Whitney U, and Kruskal Wallis formulas. These were deemed appropriate for analysis given the non-parametric nature of the survey instrument (Kinnear & Gray, 2004). The quantitative survey served to help define the values and value congruence that was then holistically examined through the qualitative interviews, document analyses, and observations. The results of the study indicated incongruent value levels between employees and those stated or perceived as the organization's values. Each finding demonstrated that varying levels of congruence may have diverse affects on individual behaviours. These behaviours range from production levels to interactions with fellow employees to turnover. In addition to the findings pertaining to the research questions, a number of other key issues were uncovered regarding departmentalization, communication, and board relations. Each has contributed to a greater understanding of the organization and has created direction for further research within this area of study.

Order and membrane organization in chlorhexidine-lipid mixtures /

Formulations of a general bactericidal agent, chlorhexidine, mixed with a phospholipid at different concentrations are investigated using ^H NMR spectroscopy on a chain-deuterated lipid analog. Lipid-chlorhexidine formulation is known to release the drug into an aqueous medium slowly, maintaining a comparable concentration of the drug for up to four times longer than a direct aqueous solution. The NMR data does not support the proposed liposomal entrapment of chlorhexidine in lipid compartments. Complex thermal history of the lipid-chlorhexidine preparations is investigated in detail. In preparation for a counterpart measurement, using ^H NMR of deuterated chlorhexidine mixed with protonated lipid, the synthesis of a deuterated analog of chlorhexidine is performed.

Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.

Primary structure and expression studies of the dihydrofolate reductase gene (DFRI) of Saccharomyces cerevisiae

The nucleotide sequence of a genomic DNA fragment thought previously to contain the dihydrofolate reductase gene (DFR1) of Saccharomyces cerevisiae by genetic criteria was determined. This DNA fragment of 1784' basepairs contains a large open reading frame from position 800 to 1432, which encodes a enzyme with a predicted molecular weight of 24,229.8 Daltons. Analysis of the amino acid sequence of this protein revealed that the yeast polypep·tide contained 211 amino acids, compared to the 186 residues commonly found in the polypeptides of other eukaryotes. The difference in size of the gene product can be attributed mainly to an insert in the yeast gene. Within this region, several consensus sequences required for processing of yeast nuclear and class II mitochondrial introns were identified, but appear not sufficient for the RNA splicing. The primary structure of the yeast DHFR protein has considerable sequence homology with analogous polypeptides from other organisms, especially in the consensus residues involved in cofactor and/or inhibitor binding. Analysis of the nucleotide sequence also revealed the presence of a number of canonical sequences identified in yeast as having some function in the regulation of gene expression. These include UAS elements (TGACTC) required for tIle amino acid general control response, and "TATA H boxes as well as several consensus sequences thought to be required for transcriptional termination and polyadenylation. Analysis of the codon usage of the yeast DFRl coding region revealed a codon bias index of 0.0083. this valve very close to zero suggestes 3 that the gene is expressed at a relatively low level under normal physiological conditions. The information concerning the organization of the DFRl were used to construct a variety of fusions of its 5' regulatory region with the coding region of the lacZ gene of E. coli. Some of such fused genes encoded a fusion product that expressed in E.coli and/or in yeast under the control of the 5' regulatory elements of the DFR1. Further studies with these fusion constructions revealed that the beta-galactosidase activity encoded on multicopy plasmids was stimulated transiently by prior exposure of yeast host cells to UV light. This suggests that the yeast PFRl gene is indu.ced by UV light and nlay in1ply a novel function of DHFR protein in the cellular responses to DNA damage. Another novel f~ature of yeast DHFR was revealed during preliminary studies of a diploid strain containing a heterozygous DFRl null allele. The strain was constructed by insertion of a URA3 gene within the coding region of DFR1. Sporulation of this diploid revealed that meiotic products segregated 2:0 for uracil prototrophy when spore clones were germinated on medium supplemented with 5-formyltetrahydrofolate (folinic acid). This finding suggests that, in addition to its catalytic activity, the DFRl gene product nlay play some role in the anabolisln of folinic acid. Alternatively, this result may indicate that Ura+ haploid segregants were inviable and suggest that the enzyme has an essential cellular function in this species.

Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells /

The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of -0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.

Mechanisms for sustainable accountability : a case study of a nonprofit educational organization

A qualitative case study of the capacity to be accountable in one nonprofit intennediary educational organization yielded an emergent conceptual framework of four mechanisms: structural, governing, communicative, and educative mechanisms to build and sustain the capacity of accountability. Drawing attention to the purposeful creation of structures that support accountability, purposeful navigation of the complex matrix of accountability relationships, and purposeful transfer of knowledge to infonn future accountability, this study calls for mindfulness in practice in broader educational contexts. Protocols to pass on knowledge gained in building the four capacities reveal a new dimension of accountability: continuity. In this model, the educative mechanism is the life force that feeds the other three mechanisms to increase accountability and sustain it over time.

Human Resource Training and National Sport Organization Managers: Examining the Impact of Training on Individual and Organizational Performance

Within sport, a tremendous amount of effort is committed to the on-the-field performance of athletes and coaches, neglecting the off-the-field performance and development of sport managers. This study examines the impact of human resource training on the performance of five Canadian national sport organizations (NSO) and their managers (N=22). Data were collected on three outcome variables (learning, individual performance, organizational performance) and three mediating variables (motivation to transfer, training design, organizational climate) at three time measures (pre-training, post-training1, post-training2). Results indicate that training improves the learning and individual performance of sport managers, as well as the organizational performance of NSOs. Varying relationships were found at each of the three time measures, demonstrating that a progression to training-related performance change exists, while providing support for three levels of analysis (individual, organizational, systemic). Implications and future research directions are discussed and highlight the need for on-going training opportunities for Canadian sport managers.

The Role of Experience in the Organization and Refinement of Face Space

Adults code faces in reference to category-specific norms that represent the different face categories encountered in the environment (e.g., race, age). Reliance on such norm-based coding appears to aid recognition, but few studies have examined the development of separable prototypes and the way in which experience influences the refinement of the coding dimensions associated with different face categories. The present dissertation was thus designed to investigate the organization and refinement of face space and the role of experience in shaping sensitivity to its underlying dimensions. In Study 1, I demonstrated that face space is organized with regard to norms that reflect face categories that are both visually and socially distinct. These results provide an indication of the types of category-specific prototypes that can conceivably exist in face space. Study 2 was designed to investigate whether children rely on category-specific prototypes and the extent to which experience facilitates the development of separable norms. I demonstrated that unlike adults and older children, 5-year-olds rely on a relatively undifferentiated face space, even for categories with which they receive ample experience. These results suggest that the dimensions of face space undergo significant refinement throughout childhood; 5 years of experience with a face category is not sufficient to facilitate the development of separable norms. In Studies 3 through 5, I examined how early and continuous exposure to young adult faces may optimize the face processing system for the dimensions of young relative to older adult faces. In Study 3, I found evidence for a young adult bias in attentional allocation among young and older adults. However, whereas young adults showed an own-age recognition advantage, older adults exhibited comparable recognition for young and older faces. These results suggest that despite the significant experience that older adults have with older faces, the early and continuous exposure they received with young faces continues to influence their recognition, perhaps because face space is optimized for young faces. In Studies 4 and 5, I examined whether sensitivity to deviations from the norm is superior for young relative to older adult faces. I used normality/attractiveness judgments as a measure of this sensitivity; to examine whether biases were specific to norm-based coding, I asked participants to discriminate between the same faces. Both young and older adults were more accurate when tested with young relative to older faces—but only when judging normality. Like adults, 3- and 7-year-olds were more accurate in judging the attractiveness of young faces; however, unlike adults, this bias extended to the discrimination task. Thus by 3 years of age children are more sensitive to differences among young relative to older faces, suggesting that young children's perceptual system is more finely tuned for young than older adult faces. Collectively, the results of this dissertation help elucidate the development of category-specific norms and clarify the role of experience in shaping sensitivity to the dimensions of face space.

Social Organization on the Web: How Sex workers Rights Organizations Engage in Activism and Advocacy Work Online

The purpose of this study was to examine how sex workers rights organizations use their websites as a site of activist and advocacy work and ask (i) how do various organizations conceptualize sex work on their websites, and to what extent do they incorporate an intersectional feminist perspective? (ii) what communication strategies are used by the four organizations to target audiences in the viewing public? (iii) what audiences do the four websites target? (iv) how do the four organizations discuss successes and challenges on their websites? (v) in what ways do sex worker right organizations use websites to further their goals? The websites of Maggie’s, POWER, and Stella attempt to embrace an intersectional feminist perspective of sex work, while PACE does not. The four organizations strategically use their websites to target audiences with diverse needs, specifically through advocacy efforts in educating the general public about the legitimacy of sexual labour. Additionally, to increase the use of the websites by sex workers, using social media platforms, such as Facebook and Twitter augment the untapped potential for creating action, mobilization, interaction, and dialogue on the websites.

Best Practices of Sport For Development: A Case Study of An African Organization

Sport-for-development is the active practice of achieving social ideals through the use of sport and other traditional development programs. The purpose of this thesis was to evaluate SFD best practices from the context of an African organization development project. The case was a development organization in Zambia, Africa that was utilizing sport within its strategy. The data collection and analysis framed using Curado and Bontis (2007) MIC Matrix, the Sport For Development International Working Group’s (2007) best practices model, and B. Kidd’s (2011) Sport-in-Development Logic Model. The research supports that a SFD project is multi-faceted and should include the employment of strategic community programming on the basis of collaborative and integrative sport, health care and education. Further, the researcher found that the best practices include setting specific goals and objectives, as well as instituting regular monitoring and evaluation strategies

Map - Plan of Organization of Conquered Position

A map titled "Plan of Organization of Conquered Position". There is a legend at the top right that reads: "Parallel of Surveillance, Parallel of Resistance, Parallel of the Redoubts, Limit between Bns., Final Objective, Final Line after Counter Attack." The map is dated 17 December 1918