In the present studies, the mechanisms of estrogen regulation of Bcl-2 were investigated by analyzing the expression of different Bcl-2 promoter-driven constructs stably transfected into MCF-7 cells. An approximately 1.7 kilobase (kb) sequence which directed estrogen-dependent regulation of Bcl-2 expression in these stable MCF-7 clones was identified. Another agent which may play a role in the regulation of Bcl-2 expression is the tumour suppressor gene, p53. We investigated the effects of various mutant p53 proteins and low levels of p53 on Bcl-2 expression in MCF-7 cells. Neither MCF-7/E6 cells expressing virtually undetectable levels of p53 nor MCF-7/173L cells expressing a DNA binding domain mutant p53, showed altered E2-mediated induction of Bcl-2 mRNA or protein levels. However, MCF-7 cells expressing a truncated mutant p53 protein (Delta291) resulted in a dramatic decrease in Bcl-2 protein levels, but not mRNA levels, upon E2 treatment. These results suggest that a p53 protein lacking a carboxy-terminus may cooperate with E2 post-transcriptionally to negatively regulate Bcl-2 levels in MCF-7 human breast cancer cells. (Abstract shortened by UMI.)

Evolution from single celled to multicellular and complex organisms brings with it several challenges. Chief among these is the necessity to control cellular proliferation, differentiation and cell death. These controls ensure the proper development of anatomical structures and the cellular homeostasis of the organism. Multicellularity also entails the active elimination of cells which have outlived their usefulness or have become malignant by virtue of being oncogenic, microbially infected or otherwise dangerous. Although initiating programmed cell death, or apoptotis, is catastrophic to the affected cell, failure to do so may be detrimental to the viability of the organism as a whole. Ultimately most, if not all, apoptotic death results from the activation of a series of proteases. These powerful proteases, termed caspases, orchestrate the regulated disassembly of the apoptotic cell. In order to ensure the capacity to inhibit or prevent the inappropriate induction of cell death, several negative regulatory mechanisms have evolved. Quite recently, it was found that a novel class of anti-apoptotic proteins are direct caspase inhibitors. The mammalian inhibitor of apoptotis (IAP) family is comprised of, minimally, four core members, all of which contain three protein-protein interaction domains termed BIR domains (Baculoviral IAP repeat). It is through these domains, originally described in the baculoviral counterpart of the mammalian IAPs, that their interaction with caspases is mediated. Two of the mammalian members, HIAP1 and HIAP2, are the subject of the following investigations. First the complete genomic organization of both hiap1 and hiap2 was elucidated. Results from this study indicated that both genes lie in tandem (head to tail) on the genome (at 11q23) separated by approximately 7 kbp. Furthermore, it was revealed that it is likely that these genes, as well as that of xiap, arose by gene duplication. By comparison to the murine organization, it was also determined that these duplication events occurred prior to the divergence of the species'. It was furthermore found that both genes possess unusually long 5'UTR's. Although the presence of several possible initiation codons within of their respective 5'UTR's would preclude translation initiation by traditional scanning, no evidence was found to support the presence of a standard internal ribosome entry site (IRES) upstream of either open reading frame. Studies into the transcriptional regulation of these genes found that although hiap2 transcription was unresponsive to a variety of stimuli, that of hiap1 was markedly enhanced by agents which activate NF-kappaB. Analysis of the 5' region of hiap1 has revealed the presence of two conserved putative NF-kappaB binding sites as well as an NF-IL-6 (CIEBPP) site and a putative NF-kappaB negative regulatory element. Gel shift analysis has shown that all are able to bind their respective factors although reporter gene analysis coupled with site directed mutagenesis has shown that only the NF-kappaB and NF-IL6 sites are functional under the conditions examined. Partial characterization of a naturally occurring hiap1/hiap2 double knockout cell line derived from the Burkitt's lymphoma line BJAB was also carried out. By comparing the effect of TNFalpha on both the derivative and parental line, it was found that the presence of these genes alleviated the negative impact of TNFalpha on cellular proliferation. Furthermore, transient re-introduction of hiap1 into the derivative line or anti-sense hiap1 into the parental line partially reversed their phenotype with respect to TNFalpha response.

In order to study mutagenicity occurring within a tumour environment, our lab has developed the Mutated murine tumour model, which is able to sensitively detect mutations at a genetically manipulated hprt locus. Prior to my work, the X-linked hprt gene in the Mutated cell lines was thought to be heterozygous based on high sensitivity to induction of mutations by ionizing radiation and the identification of 3 X-chromosomes by fluorescent in situ hybridization. In order to determine the hprt genotype and to confirm that the hprt gene was indeed heterozygous, the hprt cDNA of Mutated cells was cloned and sequenced. Three mRNA species were identified: mRNA-A corresponds to wild type hprt, mRNA-B contains a single nucleotide deletion within exon 3, and mRNA-C contains a deletion of exons 2 and 3. In order to identify the types of mutations that are generated during in vivo tumour growth, a screening method was devised to analyze mutations occurring in the wild type hprt gene. (Abstract shortened by UMI.)

Molecular analysis of Facioscapulohumeral Muscular Dystrophy (FSHD) patients has demonstrated that the disorder is associated with deletions of multiple copies of a subtelomeric 3.3 kb unit termed D4Z4, which lie at 4q35. Sequence analysis of the D4Z4 repeat has failed to identify a functional transcript; thus, it has been postulated that FSHD results from position effects on a neighboring gene(s). The purpose of our investigation was to determine the function that these repeats may hold in the pathogenesis of FSHD by evaluating their regulatory effect on reporter gene expression. To address this, we developed LacZ expression constructs containing 1 to 4 D4Z4 repeats and assessed reporter gene activity using transient and stable transfection assays in C2C12 myoblasts. Our results have revealed inconclusive evidence to support a repressive effect involving the D4Z4 repeats in either transient transfections or stable clones grown under normal or myogenic differentiation conditions. However, during differentiation it was observed that the D4Z4 repeats confer a mild fusion defect onto the multinucleated myofibres. The presence of D4Z4 not only accompanied the reduced fusion competence of myoblast cultures resulting in the formation of less myotubes, but also corresponded to the heightened incidence of deformed myotubes. While the results of these experiments do not provide irrefutable evidence, they have provided some insight towards an association between the D4Z4 repeat and repression and have increased our understanding of the role D4Z4 repeats may play in the etiology of Facioscapulohumeral Muscular Dystrophy.

Xenopus laevis oocytes are physiologically arrested at the first meiotic prophase. Re-initiation of meiosis, or oocyte maturation, is triggered in vivo by progesterone, but can also be triggered by insulin and insulin-like growth factor-1 (IGF-1) in vitro. The mechanism by which these two very different hormones regulate the same physiological process is poorly understood. Chapter 2 describes my research that contributed to the characterization of the progesterone receptor responsible for inducing oocyte maturation. We demonstrated that the Xenopus progesterone receptor (xPR) is a dual functional protein. When expressed in the heterologous COS-7 cells, OR is imported into the nucleus and functions as a progesterone-regulated transcription factor. In contrast, the endogenous OR in Xenopus oocytes is restricted in the cytoplasm and appears to mediate cytoplasmic signalling. Chapter 3 describes a functional link between the IGF-1 receptor and G-protein signalling in the control of oocyte maturation. The Xenopus homologue of GIPC, a PDZ-domain-containing protein, was identified as a binding partner for the cytoplasmic domain of the IGF-1 receptor. GIPC is known to interact with the C-terminus of a Galphai-specific GAP, RGS-GAIP. Expression of two dominant negative forms of xGIPC blocked insulin-induced MAPK activation and oocyte maturation, while full-length xGIPC synergized with human RGS-GRIP to enhance insulin signalling. This is the first demonstration that the GIPC/RGS-GAIP complex acts positively in IGF-1 receptor signalling.

Relatively little is known about the molecular mechanisms involved in the import of proteins into the nucleus. This lack of knowledge extends to he mode of nuclear entry of steroid-receptor complexes and modulation of steroid hormone action during its passage across the nuclear envelope. The main goal of this thesis was to identify the molecular machinery that interacts with the nuclear localization signals located in steroid hormone receptors. Synthetic peptides corresponding to the glucocorticoid and thyroid hormone receptor nuclear localization signals were radioiodinated and incubated with cytosol, high salt- and detergent-extracted rat liver nuclei or nuclear envelopes in the presence of a crosslinking agent. Two specifically labeled polypeptides of 60 and 76 kDa were identified with both synthetic peptides in all fractions by autoradiography after SDS-Page. The two general conclusions drawn from my data are: first, that these two specifically labeled polypeptides may act as shuttling vectors in nuclear transport; and second, the binding sites may be part of a general mechanism for nuclear entry of most nuclear proteins. Nuclear import of proteins is comprised of two steps, the first step being the binding of the protein to he outer nuclear envelope and nuclear pore complex, and the second step being the translocation of the protein through the nuclear pore complex into the nucleus. I established an in vitro system, which consist of isolated rat liver nuclei, capable of the first step but not the second step, to study glucocorticoid receptor nuclear binding. I have also demonstrated that digitonin-permeabilized cells can be used to study both steps (binding and import) of the glucocorticoid receptor translocation into nuclei in an ATP-dependent manner similar to other nuclear proteins. These in vitro models will allow us to further define the mechanisms involved in the nuclear import of the glucocorticoid receptor, particularly the hormone-induced nuclear translocation of the receptor, and to establish the role of the 60- and 76-kDa polypeptides in nuclear import. The ligand-induced nuclear translocation of the glucocorticoid receptor is dependent on the intracellular hormone concentration which is influenced by membrane binding sites for the glucocorticoid ligand. With the affinity label, dexamethasone 21-mesylate, I have identified a 45-kDa microsomal membrane site that displays the characteristics of a low affinity glucocorticoid binder. This protein may limit the amount of free hormone for the receptor and therefore blunt the action of the steroid hormone in the nucleus.

In the present study, three molecular methods, restriction enzyme (RE) analysis of chromosomal DNA, restriction fragment length polymorphism (RFLP) analysis of ribosomal RNA (rRNA) genes (ribotyping) and pulsed field gel electrophoresis (PFGE) were evaluated for their ability to discriminate within and between five groups of gonococcal isolates. By comparing the Re analysis and ribotyping for 100 gonococcal isolates as well as PFGE analysis for 26 of the 100 gonococcal isolates, there were twenty RE patterns, eleven ribotypes and twenty-five PFGE patterns produced. Overall, PFGE produced greater discrimination between isolates within the same A/S classes than ribotyping or RE analysis. In some cases, as with arginine-requiring strains or strains from an outbreak, several molecular methods should be tested in order to produce maximum discrimination between strains. Arginine auxotyphy is commonly encountered in Neisseria gonorrhoeae, comprising 40-60% of clinical isolates, including isolates with multiple nutritional requirement. Arginine (Arg) and uracil (U)-requiring strains are common in Canada. These AU isolates may have a deficiency affecting both the arginine and uracil biosynthesis pathways. Isolates may have a deficiency affecting both the arginine and uracil biosynthesis pathways. Isolates which require citrulline (C) alone for growth are very rare. Only 2 of 1540 of the arginine-requiring isolates in the culture collection of the NLSTD were citrulline-requiring. Strains requiring citrulline may be mutant for one of two enzymes, ornithine transcarbamoylase (OTCase), which catalyze L-citrulline formation from L-ornithine and carbamoylohosphate, or carbamoylphosphate synthetase (CPSase), which implicated in arginine and uracil biosynthesis pathways. Strains which are CPSase deficient may also be mutant in pyrimidine (U) biosynthesis. Twenty-two CU requiring isolates and two C requiring isolates were tested for OTCase and CPSase activities. The 22 CU auxotype possessed OTCase activity, but did not have CPSase activity. The 2 isolates requiring citrulline alone had CPSase activity, but did not have OTCase activity. (Abstract shortened by UMI.)

Brassica napus microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. This switch in developmental pathways has been shown to be accompanied by the induction of high levels of napin seed storage protein gene expression (DeMoor, 1992). Specific members of the napin multigene family that were expressed at this time were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), are members of the highly conserved BnmNAP subfamily of napin genes. DNA gel blot analysis, using a subfamily-specific probe, suggested that this subfamily may consist of up to 5 members. RNA gel blot analysis, also using the subfamily-specific probe, indicated that the BnmNAP subfamily was also expressed during embryo development. BnmNAP mRNA was detected as early as the globular stage of development in microsporic embryos, but not until the late torpedo/early cotyledon stage of development in zygotic embryos. A BngNAP1 promoter-$\beta$-glucuronidase (GUS) gene fusion was introduced into B. napus and Nicotiana tabacum (tobacco) plants in order to examine the spatial and temporal pattern of expression of one member of the BnmNAP subfamily. The BngNAP1-GUS construct was shown to be highly expressed in microspores that had been induced to undergo embryogenesis, but was not expressed in microspores continuing pollen development in culture. Furthermore, BngNAP1-directed GUS activity appeared to be predominantly localized in those microspores that have been shown to have the greatest potential to form embryos in culture. Fluorogenic and histochemical analysis of developing microsporic and zygotic embryos of B. napus indicated that the BngNAP1-GUS fusion was expressed as early as the globular stage of development. GUS activity was first detected in the micropylar region of the future embryonic axis and continued to spread upward during subsequent stages of development. In tobacco, GUS activity was first detected in the endosperm of seeds containing globular stage embryos. GUS activity did not begin to accumulate in tobacco embryos until the early heart stage of development, where it appeared as a band in the middle of the embryo, just under the lobes of the emerging cotyledons. This activity continued to spread outward in both directions as development proceeded. Thus the timing, but not the spatial localization, of BngNAP1-directed GUS expression was maintained in transgenic tobacco.

Human cytomegalovirus (HCMV) is an ubiquitous member of the herpesvirus family that is responsible for morbidity and mortality in immunocompromised individuals. Our understanding of the immune response to this complex pathogen is still incomplete, limiting our capacity to devise effective strategies for its control. The goal of the research described in this thesis was to use the idiotype (id) network of the immune response as a means of manipulating the response to HCMV infection. More specifically, the objectives were to generate anti-id antibodies that would mimic an epitope of the viral envelope glycoprotein complex B (gB), an immunodominant constituent of HCMV that is widely regarded as a potential vaccine candidate. The anti-id antibodies would then be evaluated for their ability to act as surrogate immunogens in stimulating an immune response to gB in naive hosts, as well as for their utility in developing improved HCMV diagnostics. Anti-id antibodies (Ab2) were induced against the gB-specific neutralizing murine monoclonal antibody CMVB1 (mAb1). Two different series of mAb2 were generated, as well as rabbit polyclonal Ab2. Extensive analyses indicated that the Ab2 were directed to idiotopes closely associated with the combining site of mAb CMVB1, and thus might mimic the original reference epitope of gB. Purified mAb2 and rabbit Ab2 were therefore prepared and used to immunize mice. Resulting data showed that all mice mounted an anti-Ab2 (Ab3) response whereas mice immunized with appropriate controls did not. The Ab3 mouse sera were then further evaluated to determine if they exhibited antigen-specific reactivity for the reference antigen, gB. Using assays previously developed to characterize the anti-HCMV activity of the Ab1 mAb CMVB1, we found no detectable antigen-specific activity in any of the Ab3 sera raised against the mAb2. However, the Ab3 sera raised against purified rabbit Ab2 did exhibit anti-HCMV activity, in a manner analogous to that of mAb CMVB1. This was manifested by binding to HCMV-infected cells, by immunoprecipitating viral proteins identified as gB, and by neutralizing viral activity in vitro. A comparison of the id profiles of the Ab1 and antigen-specific Ab3 showed that at least one idiotope was co-expressed on the Ab1 and antigen-specific Ab3 antibodies, suggesting some dominance of this recurrent id. However, the data also demonstrated that different subsets of B lymphocytes had been stimulated by gB compared to the rabbit Ab2. These results not only revealed mechanisms by which the id network may contribute to the generation of antibody diversity in the immune response, but also illustrated how subtle structural differences may influence the immune response that is induced, considerations of some relevance in the selection of id based vaccines and therapies. The mAb2 were used to develop an innovative ELISA for detecting a laboratory strain of HCMV, based on the ability of viral antigen to inhibit the specific interaction between Ab1 (mAb CMVB1) and complementary mAb2. A linear dose-response curve was obtained for HCMV between 20 and 0.6 $\times$ 10$\sp3$ PFU/ml, with 50% inhibition at approximately 3 $\times$ 10$\sp3$ PFU/ml. The principles of this immunoassay show promise not only for HCMV, but also for the rapid measurement of other infectious agents.

The detection of human papillomavirus (HPV) sequences associated with anogenital tract infections is problematic due to the abundant diversity of HPV types found in the genital mucosa, the apparent geographic variation of HPV types, the prevalence of certain types in ethnic groups and the presence of yet unknown HPV types in anogenital samples. The objective of this work was to develop a technique that would allow for the detection of all HPVs in a variety of clinical samples: diseased tissues, latent infections, and samples with limited amount of material. Because of its promising potential, the polymerase chain reaction (PCR) was selected as the method of choice. To fulfill the objective of amplifying all HPV sequences, primers were designed from regions of homology identified in the E1 ORF from a selected group of HPV types. The primers were assayed with a variety of HPV types and their utility evaluated in clinical samples. After amplification, Southern blot hybridization was performed for accuracy of typing as well as to increase the sensitivity of the assay. PCR using E1 ORF consensus primers proved to be a successful tool in the detection of HPV DNA sequences in clinical samples. The primers are capable of amplifying known and unknown HPV types in diseased tissues and tissues latently infected with HPV. Their application allowed for the identification of potential new HPV sequences.