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The data of this thesis show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF protein, thereby modulating the MAPK signaling pathway and cell migration / Die Daten dieser Doktorarbeit zeigen nun eine unerwartete Rolle für XIAP und c-IAPs. Durch ihren Einfluß auf den Proteinumsatz von C-RAF modulieren sie die MAPK-Kaskade und somit die Wanderung von Zellen.

Hybridization within Sorghum bicolor (L.) Moench has been the primary means of creating genetic diversity needed for sorghum crop improvement. While significant variation exists within S. bicolor, there are several traits that can be improved and potential opportunities to improve S. bicolor if secondary and tertiary germplasm pools could be accessed. Recently, the discovery of the iap (Inhibition of Alien Pollen) mutant and its introgression into more breeding-amenable genetic background has facilitated the development of S. bicolor germplasm with genetic diversity not previously seen within Sorghum. The key to producing this variation is the homozygous recessive mutant gene iap which removes an important reproductive isolation barrier to hybridization. Development of a S. bicolor accession (Tx3361) containing the mutant allele iap and ms3 has allowed introgression of genomic regions from divergent sorghum species into S. bicolor. Given the success with divergent sorghum species, there is a real interest in assessing the potential of this mutant to facilitate intergeneric hybridization. The objective of this study was to determine the range and effectiveness of the iap mutant to allow pollen tubes of Poaceae species outside of the genus Sorghum to grow into S. bicolor pistils. Accessions from the genera Zea, Miscanthus, Pennisetum, and Sorghastrum were used as pollen donors onto Tx3361 and fluorescent microscopy was used to determine the distance through the pistils that foreign pollen tubes grew. Results indicate high levels of pollen tube growth into the ovaries of S. bicolor pistils for two accessions of Pennisetum ciliare (L.) Link and four accessions of Zea mays L. Pollen tubes of other accessions tested did grow to the ovary but in very small numbers. While the recovery of embryos was not attempted in this study, the results indicate that there is potential for hybridization, but the specific pollinator within a species is critical in this attempt.

Repetitive sequences, including transposable elements, represent approximately half of the mammalian genome. Epigenetic mechanisms evolved to repress these potentially deleterious mobile elements. However, such elements can be variably silenced between individuals – so called ‘metastable epialleles’. The best known example is the Avy locus where an endogenous retrovirus (ERV) of the intracisternal A-particle (IAP) class was spontaneously inserted upstream of the agouti coat colour gene, resulting in variable IAP promoter DNA methylation, variable expressivity of coat phenotype, and environmentally modulated transgenerational epigenetic inheritance within genetically identical individuals. It is not known whether the behaviour exhibited by the ERV at Avy represents a common occurrence throughout the genome or is unusual. Taking a genetic approach in purified cell populations, I have conducted a systematic genome-wide screen of murine metastable epialleles. I have identified over 100 murine IAPs with properties of metastable epialleles. Like Avy, each exhibits a stable epigenetic state within an individual but epigenetic variability between individuals. Methylation levels are locus-specific within an individual, suggesting cis-acting control. The same screening strategy was applied for identification of metastable epialleles associated with other types of LTR-retroelements. However, many of identified candidates showed no inter-individual methylation variation upon experimental validation. These results suggest that IAPs are the dominant class of ERVs capable of acquiring epigenetic states that are variable between genetically identical individuals. I have conducted an analysis of IAP induced initiation and termination of transcription events using de novo assembled transcriptomes generated for B and T cells. 143 IAPs have been identified to overlap de novo assembled transcripts. 33 IAPs are metastable epialleles. Several of them show an inverse correlation between LTR promoter methylation and adjacent gene expression. In addition, I have shown that metastable epialleles have a characteristic pattern of histone modification and are flanked by the methylation sensitive binding factor CTCF, providing testable hypotheses concerning the establishment and/or maintenance of the variable methylation state. My findings indicate that metastability is, in general, specific to the IAP class of ERVs, that only around 1% of these elements have this unusual epigenetic property and that the ability to impact transcription, such as at agouti in Avy, is not a ubiquitous feature of these loci.

The Inhibitor of Apoptosis (IAP) proteins are a highly conserved group of anti-apoptotic proteins that regulate various pathways, particularly those that affect proliferation and cell death. Smac mimetics compounds (SMCs) are IAP antagonists that induce the degradation of two IAPs, cellular IAP 1 (cIAP1) and cellular IAP 2 (cIAP2). cIAP1 and cIAP2 are negative regulators of the alternative NF-κB pathway, which is critical to the regulation, activation, proliferation, and survival of immune cells. Consequently, SMCs can affect immunological responses by providing co-stimulatory signals for antigen-presenting cells or promoting proliferation and activation of T cells. Due to their potent immunomodulatory properties, SMCs are an ideal candidate for new vaccine adjuvants. I sought to demonstrate the potential of SMCs as a vaccine adjuvant by evaluating SMCs effects on dendritic cells (DCs). I demonstrated that SMC treatment of bone marrow derived dendritic cells (BMDCs) induces degradation of both cIAP1 and cIAP2 and leads to activation of the alternative NF-κB signalling pathway. Furthermore, SMC treatment led to upregulation of proteins associated with DC maturation, as well as secretion of pro-inflammatory cytokines. Despite the activating effects elicited by SMCs in vitro, the use of SMCs as an adjuvant for peptide vaccination failed to prevent tumour growth. Further work to determine the best use of SMCs as adjuvants in vivo needs to be done to explore the potential of this class of drugs. Thus, these findings will guide the use of SMCs in adjuvant vaccine therapies for robust protective immunity.

Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. Previous studies have demonstrated that the sexually transmitted pathogen, Neisseria gonorrhoeae, can induce or inhibit host cell death. N. gonorrhoeae is a mucosal pathogen and, in females, initiates infection in epithelial cells of the ectocervix and endocervix. Mucosal epithelial cells of the female genital tract are the first line of defense, and thus their cellular fate can alter the early immune response to invading pathogens such as N. gonorrhoeae. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In the present study, we demonstrate that N. gonorrhoeae stimulation induces a transient increase in cIAP2 protein levels in human cervical epithelial cells. High intracellular protein levels were observed during early N. gonorrhoeae stimulation and were followed by a marked intracellular decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes, which are nano-sized vesicles that carry protein and coding RNA as cargo from one cell to another. We also observed that depletion of cIAP2 in N. gonorrhoeae stimulated cells resulted in cell death resembling necroptosis, an inflammatory form of cell death. Furthermore, inhibition of cIAP2 led to an increase in interleukin-1β production. Exosomes have been found to have important roles in cell communication during microbial infection. Here, we demonstrate that N. gonorrhoeae induces exosome production and alters exosome content. We also demonstrate that exosomes elicit cytokine responses in uninfected naïve cells. Collectively, these studies highlight an additional mechanism for epithelial cells to orchestrate the immune response in the female genital tract during N. gonorrhoeae infection.

In over twenty years of operations the Canadian Innovation Centre has evaluated, through its Inventor's Assistance Program (IAP), the commercial potential of over 12,000 early stage inventions. Prior to 1989, the Canadian Innovation Centre (CIC) used a version of the Preliminary Innovation Evaluation System (PIES), developed by Gerald Udell at the Oregon Innovation Center in 1974, to evaluate the commercial potential of early stage inventions. Since 1989, the CIC has used a modified version of PIES in their evaluation process. I first estimate the ability of this program's analysts to forecast the probability that an invention will become commercialized. I also estimate a model to predict the probability that an invention will become commercialized based on the IAP's evaluation of several underlying early stage characteristics of the invention. I find that such a statistical model is based on a limited set of variables and predicts future commercial success almost as well as the IAP's forecast of success. I then use factor analysis to determine if the ratings provided by the CIC evaluation service are representative of the underlying theoretical variable structure of PIES or their modified version. Factor analysis is applied to two distinct periods that are separated by a distinct alteration of the theoretical variable structure in 1989. While I find that the factor analysis provides evidence that the post 1989 theoretical structure does provide interpretation of some of the dimensions in the ranking variables, when a combination of the post 1989 and the pre 1989 structure are examined interpretability of the extracted factors is significantly improved. Finally, I compare the model estimated on the underlying early stage characteristics with a model estimated on the extracted factors. When the predictive accuracy of the two models is compared, I find that both procedures produce models that predict almost equally well. The models and the IAP perform better than R&D managers' predictions of their own R&D projects' successes. The thesis provides recommendations for the assessment and maintenance of evaluation models for inventions, innovations and R&D projects.

In over twenty years of operations the Canadian Innovation Centre has evaluated, through its Inventor's Assistance Program (IAP), the commercial potential of over 12,000 early stage inventions. Prior to 1989, the Canadian Innovation Centre (CIC) used a version of the Preliminary Innovation Evaluation System (PIES), developed by Gerald Udell at the Oregon Innovation Center in 1974, to evaluate the commercial potential of early stage inventions. Since 1989, the CIC has used a modified version of PIES in their evaluation process. I first estimate the ability of this program's analysts to forecast the probability that an invention will become commercialized. I also estimate a model to predict the probability that an invention will become commercialized based on the IAP's evaluation of several underlying early stage characteristics of the invention. I find that such a statistical model is based on a limited set of variables and predicts future commercial success almost as well as the IAP's forecast of success. I then use factor analysis to determine if the ratings provided by the CIC evaluation service are representative of the underlying theoretical variable structure of PIES or their modified version. Factor analysis is applied to two distinct periods that are separated by a distinct alteration of the theoretical variable structure in 1989. While I find that the factor analysis provides evidence that the post 1989 theoretical structure does provide interpretation of some of the dimensions in the ranking variables, when a combination of the post 1989 and the pre 1989 structure are examined interpretability of the extracted factors is significantly improved. Finally, I compare the model estimated on the underlying early stage characteristics with a model estimated on the extracted factors. When the predictive accuracy of the two models is compared, I find that both procedures produce models that predict almost equally well. The models and the IAP perform better than R&D managers' predictions of their own R&D projects' successes. The thesis provides recommendations for the assessment and maintenance of evaluation models for inventions, innovations and R&D projects.

Monocytic cells survive HIV replication and consequent cytopathic effects because of their decreased sensitivity to HIV-induced apoptosis. However, the mechanism underlying this resistance to apoptosis remains poorly understood. I hypothesized that exposure to microbial products, translocated from the gut, may confer anti-apoptotic properties in human monocytic cells through interaction with their corresponding Toll-like receptors (TLRs). Using HIV-Vpr(52-96) peptide as a model apoptosis-inducing agent, I demonstrated that unlike monocyte-derived macrophages, undifferentiated primary human monocytes and pro-monocytic THP-1 cells are highly susceptible to Vpr(52-96)-induced apoptosis. Interestingly, monocytes and THP-1 cells stimulated with TLR-9 agonists, CpG and E.coli DNA, induced almost complete resistance to Vpr(52-96)-induced apoptosis albiet via a TLR-9 independent signaling pathway. Moreover, CpG and E.coli DNA selectively induced the anti- apoptotic Inhibitor of Apoptosis Protein-2 (c-IAP-2) and inhibition of the c-IAP-2 gene by either specific siRNAs or synthetic second mitochondrial activator of caspases (Smac) mimetic reversed CpG-induced resistance against Vpr(52-96)-mediated apoptosis. I demonstrated that c-IAP-2 was regulated by the c-Jun N terminal kinase (JNK) and the calcium signaling pathway in particular the calmodulin-dependent protein kinase-II (CaMK-II). Furthermore, inhibition of JNK and the calcium signaling including CaMK-II by either pharmacological inhibitors or their specific siRNAs reversed CpG-induced protection against Vpr(52-96)-mediated apoptosis. I also showed that CpG-induced JNK phosphorylation through activation of calcium signaling pathway.

Monocytic cells survive HIV replication and consequent cytopathic effects because of their decreased sensitivity to HIV-induced apoptosis. However, the mechanism underlying this resistance to apoptosis remains poorly understood. I hypothesized that exposure to microbial products, translocated from the gut, may confer anti-apoptotic properties in human monocytic cells through interaction with their corresponding Toll-like receptors (TLRs). Using HIV-Vpr(52-96) peptide as a model apoptosis-inducing agent, I demonstrated that unlike monocyte-derived macrophages, undifferentiated primary human monocytes and pro-monocytic THP-1 cells are highly susceptible to Vpr(52-96)-induced apoptosis. Interestingly, monocytes and THP-1 cells stimulated with TLR-9 agonists, CpG and E.coli DNA, induced almost complete resistance to Vpr(52-96)-induced apoptosis albiet via a TLR-9 independent signaling pathway. Moreover, CpG and E.coli DNA selectively induced the anti- apoptotic Inhibitor of Apoptosis Protein-2 (c-IAP-2) and inhibition of the c-IAP-2 gene by either specific siRNAs or synthetic second mitochondrial activator of caspases (Smac) mimetic reversed CpG-induced resistance against Vpr(52-96)-mediated apoptosis. I demonstrated that c-IAP-2 was regulated by the c-Jun N terminal kinase (JNK) and the calcium signaling pathway in particular the calmodulin-dependent protein kinase-II (CaMK-II). Furthermore, inhibition of JNK and the calcium signaling including CaMK-II by either pharmacological inhibitors or their specific siRNAs reversed CpG-induced protection against Vpr(52-96)-mediated apoptosis. I also showed that CpG-induced JNK phosphorylation through activation of calcium signaling pathway.