Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery. The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined. The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

p53 functions as an important tumour suppressor through its ability to regulate a number of important cellular processes such as cell cycle arrest, apoptosis, DNA repair, senescence, and angiogenesis. An in vivo genetic modifier screen performed using Drosophila melanogaster resulted in the identification of D. melanogaster brain tumour (brat) as a putative modifier of of the p53 small eye phenotype. Mammalian homologs of brat are members of the tripartite motif family that contain a c-terminal NHL domain. We focus on elucidating the in vivo role of one such homolog, BERP, through the generation and characterization of a classical gene-deletion mouse mutant. We report that BERP-deficient mice exhibit enhanced learning/memory, increased fear, impaired motor coordination, and increased resistance to PTZ -induced seizures. Electrophysiological and biochemical studies show a decrease in mIPSC amplitude along with a decrease in cell surface expression of gamma2 subunit-containing GABA A receptors in the brains of BERP-deficient mice. In addition, no effect of genotype is apparent when examining BERP mRNA levels in the brain. This suggests that the decreased cell surface expression of gamma2 subunit-containing GABA A receptors is likely a posttranscriptional phenomenon and supports the possibility that BERP may be involved in the intracellular trafficking of GABA A receptors. In investigating the possible relationship between BERP and p53, we identify the presence of a transcriptionally competent p53 response element within the first intron of the human BERP genomic locus and demonstrate that the BERP expression is up regulated in a p53-dependent manner both in vitro and in vivo. These results support the interpretation that BERP is a novel p53-regulated gene and suggest a new role for p53 in the regulation of GABA A receptor trafficking and epileptogenesis.

One-carbon metabolism is an essential process that is conserved from yeast to humans. Glycine stimulates the expression of genes in one-carbon metabolism, whereas its withdrawal causes repression of these genes. The transcription factor Bas1p and the metabolic enzyme Shm2p have been implicated in this regulation. I have shown that Bas1p physically interacts with Shm2p through co-immunoprecipitation. Using chromatin immunoprecipitation (ChIP), I have also shown that the interaction between Bas1p and Shm2p occurs at the promoter of two genes in the one-carbon metabolism regulon and that the binding of Shm2p requires Bas1p. Using a yeast-two hybrid system, I have systematically truncated Bas1p from the C-terminal end to find a region responsible for the interaction with Shm2p. My data suggest that Shm2p is directly bound to Bas1p at the promoters of glycine regulated genes where it regulates the transcriptional activity of Bas1p in response to changes in glycine levels.

One-carbon metabolism is an essential process that is conserved from yeast to humans. Glycine stimulates the expression of genes in one-carbon metabolism, whereas its withdrawal causes repression of these genes. The transcription factor Bas1p and the metabolic enzyme Shm2p have been implicated in this regulation. I have shown that Bas1p physically interacts with Shm2p through co-immunoprecipitation. Using chromatin immunoprecipitation (ChIP), I have also shown that the interaction between Bas1p and Shm2p occurs at the promoter of two genes in the one-carbon metabolism regulon and that the binding of Shm2p requires Bas1p. Using a yeast-two hybrid system, I have systematically truncated Bas1p from the C-terminal end to find a region responsible for the interaction with Shm2p. My data suggest that Shm2p is directly bound to Bas1p at the promoters of glycine regulated genes where it regulates the transcriptional activity of Bas1p in response to changes in glycine levels.

p53 functions as an important tumour suppressor through its ability to regulate a number of important cellular processes such as cell cycle arrest, apoptosis, DNA repair, senescence, and angiogenesis. An in vivo genetic modifier screen performed using Drosophila melanogaster resulted in the identification of D. melanogaster brain tumour (brat) as a putative modifier of of the p53 small eye phenotype. Mammalian homologs of brat are members of the tripartite motif family that contain a c-terminal NHL domain. We focus on elucidating the in vivo role of one such homolog, BERP, through the generation and characterization of a classical gene-deletion mouse mutant. We report that BERP-deficient mice exhibit enhanced learning/memory, increased fear, impaired motor coordination, and increased resistance to PTZ -induced seizures. Electrophysiological and biochemical studies show a decrease in mIPSC amplitude along with a decrease in cell surface expression of gamma2 subunit-containing GABA A receptors in the brains of BERP-deficient mice. In addition, no effect of genotype is apparent when examining BERP mRNA levels in the brain. This suggests that the decreased cell surface expression of gamma2 subunit-containing GABA A receptors is likely a posttranscriptional phenomenon and supports the possibility that BERP may be involved in the intracellular trafficking of GABA A receptors. In investigating the possible relationship between BERP and p53, we identify the presence of a transcriptionally competent p53 response element within the first intron of the human BERP genomic locus and demonstrate that the BERP expression is up regulated in a p53-dependent manner both in vitro and in vivo. These results support the interpretation that BERP is a novel p53-regulated gene and suggest a new role for p53 in the regulation of GABA A receptor trafficking and epileptogenesis.

Identification of the mechanisms by which genes are regulated in eukaryotes is one of the principal challenges of modern biology. The emergence of genome sequencing has facilitated the marked expansion of experimental and computational approaches designed to address this challenge. Integrating and assessing this information remains a major scientific endeavor that requires new and innovative application of technology. Furthermore, our limited understanding of the mechanisms of gene regulation in eukaryotes has undermined our ability to understand the role of genetics in gene regulation. Regulatory variants are thought to be responsible for a considerable amount of the heterogeneity within our population and to be fundamental determinants of health. New experimental approaches offer the opportunity to effectively identify markers of disease susceptibility in gene regulatory regions but the discovery of the molecular mechanism of dysregulation remains difficult and time-consuming. It is here where computational approaches are required to prioritize candidate regulatory variants. To do so requires the development of an extensive control set from which characteristic signals can be identified. This thesis introduces novel approaches for discovering, utilizing, comparing and visualizing regulatory element predictions in completed genomes. This thesis also introduces novel bioinformatics infrastructure for curating regulatory element and variant datasets, and introduces the largest-available, open-access dataset of functional regulatory variants hand-curated from literature. This dataset is used to identify signals which discriminate functional variants from other variants in the promoter regions of human genes using regulatory and population genetics-based computational approaches.

Identification of the mechanisms by which genes are regulated in eukaryotes is one of the principal challenges of modern biology. The emergence of genome sequencing has facilitated the marked expansion of experimental and computational approaches designed to address this challenge. Integrating and assessing this information remains a major scientific endeavor that requires new and innovative application of technology. Furthermore, our limited understanding of the mechanisms of gene regulation in eukaryotes has undermined our ability to understand the role of genetics in gene regulation. Regulatory variants are thought to be responsible for a considerable amount of the heterogeneity within our population and to be fundamental determinants of health. New experimental approaches offer the opportunity to effectively identify markers of disease susceptibility in gene regulatory regions but the discovery of the molecular mechanism of dysregulation remains difficult and time-consuming. It is here where computational approaches are required to prioritize candidate regulatory variants. To do so requires the development of an extensive control set from which characteristic signals can be identified. This thesis introduces novel approaches for discovering, utilizing, comparing and visualizing regulatory element predictions in completed genomes. This thesis also introduces novel bioinformatics infrastructure for curating regulatory element and variant datasets, and introduces the largest-available, open-access dataset of functional regulatory variants hand-curated from literature. This dataset is used to identify signals which discriminate functional variants from other variants in the promoter regions of human genes using regulatory and population genetics-based computational approaches.

Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is a fungal pathogen of maize that causes ear rots and stalk rots worldwide. In addition, it produces a group of mycotoxins called fumonisins when the fungus colonizes maize and maize-based products. Fumonisin B1 (FB1), the predominant form occurring in nature, can cause detrimental health effects in animals and humans. Several efforts were made to study the host and pathogen factors that contribute to the production of fumonisins. Using the available genomic resources, three genes with a potential role in FB1 regulation and development were identified. The genes are GBP1, GBB1 and GAP1. This research describes molecular characterization of these genes with respect to regulation of FB1 and development in F. verticillioides. GBP1 is a monomeric GTP binding protein with similarity to DRG and Obg sub-classes of G-proteins. GBB1 encodes heterotrimeric GTP binding protein β subunit. GAP1 is a GPI (Glycophosphotidylinositol) anchored protein, which belongs to a family of cell wall proteins. Targeted deletion and complementation studies indicated that GBP1 is negatively associated with FB1 biosynthesis but had no effect on conidiation in F. verticillioides. GBB1 plays an important role in regulation of FB1 biosynthesis, conidiation and hyphal growth, but not virulence. GAP1 is associated with growth, development and conidiation but not in positive regulation of FB1 or pathogenicity. The outcome of this study revealed new molecular genetic components that will help scientists better understand signal transduction pathways that regulate FB1 biosynthesis and conidiation in F. verticillioides.

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. These P. aeruginosa infections can be extremely difficult to treat due to the high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It was demonstrated here that the psrA gene, encoding a transcriptional regulator, was up-regulated in response to sub-inhibitory concentrations of antimicrobial peptides. Compared to wild-type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic super-susceptibility to polymyxin B, a last resort antimicrobial used against multi-drug resistant infections, and indolicidin, a bovine neutrophil antimicrobial peptide; this super-susceptibility phenotype correlated with increased outer membrane permeability. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and normal swarming motility, phenotypes that could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to wild-type by microarray analysis, demonstrating that 178 genes were up or down-regulated by greater than 2-fold (P ≤0.05). Dysregulated genes included those encoding known PsrA targets, the type III secretion apparatus and effectors, adhesion and motility genes and a variety of metabolic, energy metabolism and outer membrane permeability genes. This indicates that PsrA is a central regulator of antimicrobial peptide resistance and virulence. P. aeruginosa containing a mutation in the PhoQ sensor kinase-encoding gene was highly attenuated for persistence in a rat chronic lung infection model. In addition, the polymyxin B hyper-resistant phoQ mutant displayed reduced type IV pili-dependent twitching motility and was less cytotoxic towards human bronchial epithelial cells, indicating that the virulence defect observed could be due at least in part to these phenotypes. Using microarrays it was further demonstrated that PhoQ regulates a large number of genes that are PhoP-independent and that the phoQ mutation leads to up-regulation of PhoP- and PmrA regulated genes as well as other genes consistent with its virulence phenotypes.

Murine cytochrome P450 2A5 (CYP2A5) is an interesting enzyme for its unique regulation and its involvement in liver injury caused by various well-known pathological conditions or hepatotoxins. It has been reported that CYP2A5 is upregulated following exposure to chemical hepatotoxins and during pathophysiological conditions in which the levels of most Cytochrome P450s are either unchanged or down-regulated. Recently bilirubin has been identified as the first endogenous substrate for CYP2A5 and it has been suggested that CYP2A5 plays a major role in bilirubin clearance as an alternative mechanism to BR conjugation by UGT1A1. This study investigated the mechanisms of gene regulation and cytoprotective role of CYP2A5 in response to bilirubin treatment in liver. Our results demonstrate that bilirubin induces CYP2A5 expression at the mRNA and protein levels by increasing CYP2A5 transcription via a mechanism that involves Nrf2 activation. Furthermore, our results suggest that induced CYP2A5 plays a cytoprotective role against bilirubin toxicity by directly lowering the cellular levels of bilirubin and by inhibiting caspase-3 activation.