In this thesis, we describe the fundamental aspects to the development of molecularly-defined tailor-made scaffolds for the delivery of FVIII-expressing endothelial cells in the treatment of Haemophilia A. In particular, scaffolds prepared by chemical crosslinking of type I collagen, and growth factor incorporation is discussed. The general strategy was to prepare tailor-made biomaterials as a specific microenvironment to enable cells to produce FVIII and secrete this coagulation factor into the blood stream. As an initial step, pure materials of known concentrations were combined to develop two forms of collagen scaffolds: an injectable hydrogel that may be formed in situ in the presence of cells and a capsule into which the cell-housing hydrogel can be injected. Next, scaffolds were crosslinked using natural- genipin- and synthetic- carbodiimide (EDC)- chemicals. Crosslinking resulted in collagenase-resistant scaffolds. The tissue response to scaffolds was evaluated following subcutaneous implantations in mice. Crosslinked scaffolds maintained their integrity and supported the formation of new extracellular matrix and neovascularization during tissue remodeling. Collagen scaffolds loaded with fibroblast growth factor 2 significantly enhanced FVIII production during long-term encapsulation. Potential applications of these scaffolds for ischemic models are discussed in more detail.

Zebrafish is able to regenerate its fins following amputation. We are interested in elucidating the cellular and molecular events that are underlying regeneration of the bones of the fin rays. Previous studies from our laboratory showed that genes involved in the Hedgehog Hh pathway are re-expressed during fin regeneration. In addition, gain and loss of function experiments previously showed that Hedgehog signalling might be involved in regenerate outgrowth and osteoblasts differentiation and patterning. Two members of the hedgehog gene family are expressed during fin regeneration: ihha (Indian Hedgehog) is expressed in the newly differentiated osteoblasts, while shha (Sonic Hedgehog) is expressed in a small domain of the basal epidermal layer in the differentiation zone. To understand the specific role of the shha-expressing cells, we precisely monitored these cells during the process of branching morphogenesis, using the transgenic line 2.4shha:GFPABC#15. We subsequently defined 3 stages that are successively taking place during the formation of the branches in the regenerating fin ray. We also assessed the expression profile of genes that might be involved in potential cross-talks between HH/FGF signalling during fin ray regeneration. Shh has been shown to play a role during branching morphogenesis in different organs that branche, through inducing cell proliferation. We analysed the cell proliferation profile during branching morphogenesis in the regenerating fin rays, and we suggest that shha-expressing cells might regulate the proliferation rate during branching morphogenesis. Previous results obtained from laser ablation experiments, where the transgenic line 2.4shha:gfp:ABC#15 was used to specifically but transiently ablate the shha-expressing cells using a laser beam showed that the transient absence of the shha --expressing cells from the regenerating ray results in a branching delay. Based on this finding, along with our results from cell proliferation analysis, and the time course analysis of the shha --expressing cells during branching morphogenesis, we propose a model for branches formation during fin regeneration, in which the splitting of shha ---expressing cells govern branches formation. We also elaborated a new approach based on the generation of a transgenic line expressing the nitroreductase in the shha-expressing cells, through which we aim to conditionally ablate these cells in the fin regenerate by addition of the prodrug, metronidazole. This will allow us to maintain the absence the shh-expressing cells long enough to observe the long term ablation effects and to better understand the role of these cells during caudal fin regeneration.

The cortex is formed through a series of well-organized processes by which neural progenitor cells proliferate, commit to different cell fates and differentiate to neuronal and glial cell types. These complex processes are regulated by a wide range of intrinsic and extrinsic factors, which include members of the bone morphogenetic protein (BMP) family and their receptors. Accumulating evidence suggests that BMP7, a factor clinically used to repair bone fracture and kidney failure, is involved in brain development by contributing to both neural induction and cell differentiation. In this study, the role of BMP7 in cortical neurogenesis was examined during embryonic development in mice. Specifically, neuronal differentiation was enhanced in cortical cultures following treatment with low concentration recombinant human BMP7, whereas higher concentrations enhanced glial differentiation. Comparative analysis of BMP receptor expression during cortical development indicated that activin receptor type IIB (ActRIIB) expression is significantly higher than other BMP receptors during neurogenesis, whereas it is significantly down-regulated following the onset of gliogenesis. Furthermore, the neutralization of ActRIIB in cortical cultures reduced levels of neuronal differentiation, suggesting its involvement in BMP7-induced cortical neurogenesis. Lastly, to further the study of BMP7, its signaling pathway and the downstream nuclear targets, lentiviral systems were developed and optimized to allow BMP7 overexpression and ActRIIB knockdown in the cortex. Similar to the commercially available recombinant BMP7, cortical neurogenesis was enhanced in the presence of lentiviral-derived BMP7.

Codon usage bias is due to a combination of biased mutation and selection effects. Isochore-related GC mutational bias has been shown to be the dominant cause of tissue-specific codon bias in adult human tissues and limited evidence for translational selection has been shown. This thesis is a comprehensive evaluation of the relative contribution of selection and mutation to variation in codon usage among different mouse embryonic tissues. Through a detailed analysis of tissue-specific codon usage in relation to gene expression and gene location of mouse embryonic tissue-specific genes, I found that translational selection is partially responsible for some differences in codon usage among tissue-specific genes. This observation indicates profound impact of selection favouring codon-anticodon adaptation. The characteristic GC biases of tissue-specific gene sets are shown not to be caused by their clustering on the same isochore. Tissue-specific genes are no more clustered on the genome than randomly selected genes. In mouse, the usage of some G-ending codons decreases with increasing GC bias, while all other G and C-ending codons increase. To understand this counterintuitive observation, we generate a continuous-time Markov chain model of GC-biased synonymous substitution which explains qualitative usage patterns of all codons, including non-linear and sometimes non-monotone codon usage in isoleucine, arginine and leucine. This effect is universal, extending to mouse and human genes as well as plant and prokaryotic genomes. This work enriched our understanding of the codon-anticodon adaptation theory and extended it to the level of tissue-specific genes. The result suggests the possibility of tissue-specific tRNA pools mediating tissue-specific codon-anticodon adaptation. The methods developed in the thesis can be easily extended to characterize this previously little explored facet of codon-anticodon adaptation.

Prostaglandin (PG) E2 is highly produced in the kidney and regulates sodium reabsorption, which is driven by the Na +/K+-ATPase (NKA) pump. It has been reported that high salt affects the activity of NKA in the cortical collecting duct. The current study addresses the role of PGE2 in NKA modulation and we hypothesize that PGE2 is acting via the EP4 receptor to increase NKA protein levels in hypertonic conditions. We demonstrated by immunoblotting an increase in NKA steady state levels of protein and activity in hypertonic conditions. Hypertonicity also stimulates cyclooxygenase (COX)-2 and PGE 2 production and modulates EP receptor mRNA expression. Involvement of PGE2 in NKA regulation was determined by COX inhibition (indomethacin and ibuprofen) and EP receptor antagonism (L-161 982 (EP4), SC51089 (EP1)). With the exception of L-161 982, no change in NKA protein levels was observed, suggesting that NKA regulation is independent of PGE 2 in high salt. Our results also indicated that PGE2 is not involved in cell survival and that L161 982 increases apoptosis, which can explain the decrease in NKA levels in high salt.

Nuclear hormone receptors (NHRs) are ligand-inducible transcription factors that regulate the activity of a wide variety of target genes. The glucocorticoid receptor (GR) is a NHR that is found predominantly in the cytoplasm prior to ligand binding. Nucleo-cytoplasmic shuttling has been proposed to play an important role in the regulation of NHR function by allowing for continuous communication between the nuclear receptor and cytosolic signalling pathways. To study shuttling in a setting that resembles the cellular environment I developed a fluorescence recovery after photobleaching (FRAP) technique using randomly occurring multinucleated Cos7 cells. We found little if any shuttling of the liganded receptor 4 h after photobleaching, which was consistent with the slow rate of redistribution following ligand withdrawal but not consistent with the previously described rapid export that is believed to underlie nucleocytoplasmic shuttling of GR. This persistent nuclear localisation of the liganded receptor was confirmed using two independent assays. However, experiments performed in cells fused by treatment with polyethylene glycol (4000) confirmed earlier reports that liganded GR shuttles rapidly between nucleus and cytoplasm in a manner that is dependent upon direct binding of GR to calreticulin. In contrast, disruption of calreticulin binding did not abrogate the slow export of GR observed by FRAP. Further, redistribution of the receptor to the cytoplasm following ligand withdrawal was not dependent on calreticulin binding. Our results suggest that cell fusion activates a specialised mechanism of GR export that is dependent on calreticulin, and that under normal cellular conditions NHRs may not be able to respond rapidly to cytoplasmic signalling pathways. Additionally, our findings demonstrate that nucleocytoplasmic shuttling as suggested by cell fusion-based assays may not always reflect trafficking behaviour in a normal cell. Within the course of these studies, we noted that the slow rate of GR nuclear export is accelerated upon mutation of the GR nuclear localisation sequence 1 (NL1). This suggested that in addition to mediating nuclear import of GR by binding with importin alpha, the NL1 sequence might function to retain GR in the nucleus. We determined that the hinge region of GR, which includes the NL1 sequence, was sufficient to efficiently repress export mediated by the CRM1-dependent HIV Rev nuclear export sequence (NES). (Abstract shortened by UMI.)

This thesis documents the genetic characterization of the X-Linked Inhibitor of Apoptosis (XIAP) gene, and the subsequent discovery and characterization of a novel gene, called the Testis-specific IAP (TsIAP). Genomic library screening and bulk sequencing was used to isolate and sequence the genomic region that encompasses the XIAP gene. Following this, we screened cDNA libraries, as well as performed RT PCR and rapid amplification of cDNA ends (RACE) reactions to elucidate the full length cDNA sequence and intron/exon boundaries of XIAP. The XIAP gene spans a 33 kb region of chromosome X and is encoded in seven exons. The transcript is 10 kb in size, containing very large 5' and 3' untranslated regions. Four separate regions of the genome cross-react with probe derived from XIAP coding region sequence. These 'pseudogenes' were sequenced and characterized. Three of the pseudogenes appeared to be derived from retrotransposition events as they contained partially conserved copies of XIAP lacking any introns. One of these pseudogenes turned out to maintain a viable open reading frame and encoded a novel protein with expression limited to the testis. We employed a similar library screening strategy to clone and sequence the novel testis-specific IAP gene. FISH analysis was performed to localize the gene to chromosome 19q13.4. The TsIAP gene was found to encode a 2kb mRNA with an open reading frame encoding a region highly homologous to the carboxy-terminal end of the X-linked IAP. Attempts were made to find a murine homologue of TsIAP with no success, and later work published by another group confirmed that the TsIAP gene is a recent evolutionary event and that no murine homologue exists. Given the unusually long 5' UTR present in the TsIAP gene, we tested whether the mRNA could produce a protein by using Western blot analysis on both overexpressed TsIAP in cell culture, as well as endogenous expression in testicular protein extracts. A band was detected in both cases migrating at approximately 26kDa, the predicted size of the TsIAP protein. Having confirmed that the novel gene we isolated is capable of being expressed, we proceeded to characterize its functional properties. TsIAP was able to protect against apoptosis, however, only against a limited subset of apoptotic triggers when compared to its parent gene XIAP. TsIAP could not protect against the chemotoxic drugs etoposide or adriamycin, however, it showed a strong protective effect against BaX-induced apoptosis. Surprisingly, against Bax-induced apoptosis, TsIAP was a more potent inhibitor than the corresponding region of XIAP. Smac (second mitochondrial activator of caspases) is an important inhibitor of XIAP function in the context of the mitochondrial pathway of apoptosis induced by Bax. We determined that Smac was unable to bind to the TsIAP protein in pull-down experiments and thus could not reverse the protective effects of TsIAP in a Bax induced apoptosis model.

The vertebrate Dlx homeodomain-containing genes are generally organized as three convergently transcribed bigene clusters. Paired genes share highly conserved overlapping expression patterns in the forebrain, the pharyngeal arches, sensory placodes and limb/fin buds. Little is known about how the Dlx genes are targeted to their sites of expression, or what particular roles individual genes play in development. In the pharyngeal arches, Dlx genes are expressed in a nested manner, which defines proximodistal identity. We identified two distinct cis-acting enhancers, I12a and I56i situated between Dlx1/2 and Dlx5/6 pairs respectively that regulate expression in the mandibular arch. In transgenic animals, the two enhancers targeted reporter gene expression to distinct populations of mesenchymal cells in the mandibular arch. Both enhancers responded to epithelial signaling cues, such as FGF8 and BMP4, similar to endogenous Dlx genes. Therefore, the combinatorial arch expression is achieved through interaction between signaling factors and intrinsic cellular factors. To investigate the individual roles of the paired Dlx1/2 gene in patterning the pharyngeal arches, we silenced zebrafish dlx1a and dlx2a using morpholino antisense oligonucleotides. Loss of dlx2a function in zebrafish embryos resulted in reductions of arch neural crest markers as well as perturbation of neurogenic and chondrogenic derivatives. Loss of dlx1a resulted in malformations of cartilage elements indicating a role in arch patterning. To explore further the evolution of the Dlx gene family, we characterized the Distal-less homologue, Odidll, in the protochordate species, Oikopleura dioica. Odidll is not part of a bigene cluster suggesting either that a paralogue was lost or that the tandem duplication event occurred subsequent to the species' divergence. Odidll, was expressed in ectodermally-derived tissues suggesting a role that may have been observed in a common chordate ancestor. These results contribute to our understanding of Dlx regulation, function and evolution.

Previous studies in this laboratory demonstrated that the glucocorticoid receptor (GR) ligand binding domain (LBD) can enhance transcription mediated by the CCAAT/enhaner binding protein C/EBPbeta. This result suggests that the receptor can act in a non-classical manner to positively affect transcription without directly binding DNA. To study the physiological impact of the potentiation of C/EBPbeta activity by the GR LBD, we focused on preadipocyte differentiation as both GR and C/EBPbeta influence early events. Using retroviral expression of the GR LBD in 3T3 L1 cells, a 3-fold increase in preadipocyte differentiation was observed when cultures were induced to differentiate in a cocktail containing dexamethasone. The GR LBD was also able to enhance transcription mediated by DNA-bound C/EBPbeta from the C/EBPalpha promoter and its expression increased C/EBPbeta protein levels during preadipocyte differentiation. The effect of the GR LBD was accomplished through the targeted degradation of a sub-population of the transcriptional repressor histone deacetylase 1 (HDAC1), which associates with C/EBPbeta via corepressor protein mSin3A. In the absence of glucocorticoids, HDAC1 maintains the C/EBPalpha promoter in a deacetylated state. The addition of steroid to the differentiation cocktail causes the degradation of the HDAC1 found in the CIEBPbeta-associated complex, a release of mSin3A, increased acetylation of the promoter histone H4 and recruitment of RNA polymerise II leading to maximal transcription from the C/EBPalpha promoter. C/EBPbeta itself is acetylated by the histone acetyltransferases GCN5 and PCAF within lysines 98, 101 and 102. In 3T3 L1 preadipocytes where only GCNS is expressed, GCNS coprecipitates with C/EBPbeta and can be found at the C/EBPalpha promoter by chromatin immunoprecipitation. The acetylation of C/EBPbeta is essential for development of full transcriptional activation potential and for differentiation of preadipocytes. Mutation of these residues leads to loss of transcriptional potentiation by GR and a striking decrease in the number of differentiated cells in both NIH 3T3 and 3T3 L1 cultures. Following glucocorticoid treatment, the mutant C/EBPbeta remains associated with HDAC1 and is inefficient in driving C/EBPalpha transcription. Acetylation may be a required modification to prevent C/EBPbeta from repression by HDAC1.

The genomic G+C content of prokaryotes varies from approximately 23% to 77% among genomes. In contrast, among vertebrates, the variation is greatest within the same genome rather than between genomes. There has been a long-standing controversy concerning the causes of these inter- and intra-specific variations. Is it caused by natural selection, favored by the selectionists or, conversely, is it selectively neutral (the neutralist view)? In this study, we investigated the source of nucleotide compositional variation (nucleotide bias) and the consequences of the bias on protein sequence and genome evolution. Thermal adaptation is a primary example to study the effect of natural selection and has been thoroughly studied in this project. We found that both GC content and length of ribosomal RNA genes show positive correlations with optimal growth temperature in prokaryotes and these correlations are not due to phylogenetic history. The correlations are concentrated almost entirely within the stem regions of the rRNA. The rRNA loops, however, show very constant base composition regardless of temperature optima or genomic GC content. The loops were found to have very high amount of adenosine nucleotides throughout prokaryotes and eukaryotes. These results clearly demonstrated that environmental temperature is a selective force that drives rRNA gene evolution and different segments of the same gene (i.e., the stems and loops of the rRNA gene) experience differential selection, although the mutation spectrum presumably should be similar between the loops and stems. For protein coding genes, mutation and natural selection play a different role compared to the rRNA genes. The neutralist predicts mutational bias would cause protein sequence evolution, while the selectionist would predict that the protein sequence is not related to genomic GC content. To investigate these two postulations and analyze the consequences of nucleotide bias in eukaryotec genomes, we studied homologous genes and their encoded proteins in two flowering plants, Oryza sativa (rice) and Arabidopsis thaliana. While there is a relatively homogenous GC content in the Arabidopsis genes (26% to 69%), the GC content of the rice genes is very heterogeneous (27% to 90%). High GC rice genes encode proteins having a high frequency of GC-rich codons encoded amino acids, i.e., glycine, alanine, arginine and proline. (Abstract shortened by UMI.)