The slime mould Dictyostelium discoideum contains a novel cAMP-binding protein, CABPl, that is composed of two subunits. Anti-CABPl monoclonal antibodies were used to isolate a cDNA from a $ lambda$gtll expression library. In hybrid selection experiments, this cDNA was found to be complementary to mRNAs encoding both CABPl subunits and also to share some homology with two polypeptides, p34 and p31, that copurify with CABPl. Further analysis showed that the two CABPl polypeptides are identical except for an additional 37 amino acids present in the larger polypeptide. Both polypeptides are encoded by a single gene and the production of the two CABPl mRNAs is accomplished by an unusual splicing event. The p34 and p31 polypeptides are smaller but highly homologous to the two CABPl polypeptides and are probably produced in a similar fashion. The carboxyl halves of the four polypeptides show a 50% similarity to two polypeptides of a tellurium anion resistance determinant encoded on a bacterial plasmid.

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.

People with high (HSEs) and low self-esteem (LSEs) often react differently to interpersonal risk. When concerns about their relationship are salient, HSEs seek connection with their partners to quell feelings of vulnerability whereas LSEs distance themselves from their partners to minimize the impact of potential rejection. In the present research, I investigate the extent to which these regulatory dynamics reflect executive-based processes that govern broader regulatory responses. In Studies 1 and 2, HSEs exhibited stronger approach goals (i.e., a greater tendency to pursue rewards and ignore risks) in non-social domains when faced with interpersonal risk whereas LSEs inhibited approach goals and made more conservative decisions. In Studies 3 and 4, I demonstrated that HSEs’ and LSEs’ divergent regulatory responses to risk were contingent on executive control. When participants were cognitively busy, HSEs were less likely to bolster relationship evaluations in the face of interpersonal risk whereas LSEs were more likely to do so. Finally, Study 5 demonstrated that these global regulatory strategies govern HSEs’ and LSEs’ responses to non-interpersonal risk as well. HSEs were more willing to engage in risky social comparison following failure than they were when they received neutral feedback about their performance, whereas LSEs were less willing to compare themselves with others after failure. These effects did not emerge when cognitive resources were depleted. Taken together, the results suggest that HSEs’ and LSEs’ self-regulatory responses to risk are broader and more controlled than previously theorized.

In a world that is increasingly digital, the ability to communicate clearly in writing is of utmost importance. An important part of writing in both professional and academic settings is self-regulation. In academics, the use of video as a teaching tool in online environments is becoming more popular. This thesis investigated whether or not video scribed animation could be used to teach writing self-regulation strategies in an online course. Student perceptions of video scribed animation and its use in education were also examined. Students completed self-report measures of their grade goals, self-efficacy for grade achievement, and self-regulation strategy use on blogging assignments. Results showed that there were statistically significant increases in students’ environmental self-regulation and goal setting. For example, students worked in quieter environments and set more concrete, challenging goals after watching the video scribed animation that modeled self-regulation strategies. Students found the video both entertaining and educational, and indicated that it caught and sustained their attention. Treatment group students that opted not to watch the video scribed animation were more likely to have achieved their grade goals on the previous assignment than the students that watched the video.

This thesis presents the design and implementation of PMOS-based integrated charge pumps with extended voltage range and their regulation circuits in a standard process. The performance of charge pumps are evaluated by their output resistances and power conversion efficiencies. Formulas which describe the charge pump characteristics are developed and presented. Existing charge pumps are analyzed and studied to understand their limitations in generating high voltages and achieving high performance. The proposed charge pump structures are designed to use PMOS switches to alleviate the high voltage stresses across transistors by biasing their bulk independently. The voltages across transistors and capacitors are kept within the suggested voltage rating (VDD)regardless of how high the output voltage is, thus the maximum voltage range is extended and no longer limited by the breakdown voltages of the devices. The charge pump circuits only need low-voltage devices and standard processes, and can be easily integrated in a digital or mixed-signal design. The proposed charge pump regulation circuits include a voltage divider, a voltage controlled ring oscillator and a feedback operational amplifier. The regulation circuits are able to adjust the clock frequency to regulate the charge pump to a steady output voltage (set by the reference voltage) under a large range of current loads. A test chip including the proposed charge pumps and regulation circuits was fabricated in a 0.18 um digital CMOS process provided by Taiwan Semiconductor Manufacturing Company (TSMC). The proposed charge pumps were tested and demonstrated the reliable generation of output voltages up to 11.47 V using only low-voltage devices. The simulation and measurement results have been presented and compared, demonstrating the functionality and performance of the proposed circuits. / Kapik Integration, Mitacs

Messenger RNA transcripts of αs2- and к-casein are translated at 25% of the efficiency of αs1- and β-casein transcripts; however, the molecular mechanisms governing the difference are unknown. We hypothesized that the bovine casein translational efficiency is influenced by characteristics of the untranslated regions (UTRs) and coding regions. The main objective of this study was to identify molecular mechanisms that explain differential translational regulation between bovine β- and αs2-casein by assessing the role of each putative translational regulatory factor found throughout full-length sequences in both in cellular and cell-free translation systems. This dissertation begins with the cloning and initial characterization of bovine β- and αs2-casein. Transcript analysis indicates that the two genes share similar characteristics of nucleotide sequence around the coding region and secondary structure. It is confirmed that αs2-casein mRNA has a lower translational efficiency compared to that of β-casein in a cell-free system. The latter portion of this thesis investigates further the UTRs and codon usage effect on difference in translational efficiency between β- and αs2-casein. Overall, our data suggest that β-casein 3’ UTR and αs2-casein 5’ UTR exert stimulatory effects on translation yet their effectiveness depends on the upstream and downstream sequences with which they are associated. Replacement of the UTRs of αs2-casein mRNA with those of β-casein did not stimulate translation. A stronger effect on translational efficiency was found in the coding region of αs2-casein which displays unfavourable codons at the 3’ terminus. Deletion of a 28-codon fragment from the 3’ terminus of the αs2-casein coding region increased translation to a par with β-casein. We suggest that the last 28 codons of αs2-casein is the main regulatory sequence that attenuates its expression and is responsible for the different translational expression of β- and αs2-casein mRNAs. Identification of regulatory factors that are responsible for translation efficiency improves our understanding of the molecular mechanisms of control of milk protein prodiction in secretory cells of the bovine mammary glands. / NSERC canada

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.

No description available.

No description available.

The E2A locus is involved in chromosomal translocations associated with acute lymphoblastic leukemia. The most common of these involves a translocation between chromosomes 1 and 19 (t1;19), resulting in expression of the chimeric oncoprotein E2A-PBX1. A direct interaction between transcriptional activation domain 1 (AD1) of E2A and KIX domain of the histone acetyltransferase (HAT) /co-activator CBP is required for E2A-PBX1-mediated leukemia induction in mice. This thesis examines the functional consequences of the direct, physical interaction between E2A and CBP, for both proteins. We demonstrate that the interaction between E2A and CBP/p300, as well as another HAT/co-activator, p/CAF, results in acetylation of E2A. Mutagenesis-based mapping studies identify several lysine residues as substrates for acetylation. Of particular interest, a conserved lysine (K34) located within AD1 is acetylated in vitro and in vivo. Substitution of this residue to arginine impairs transcriptional activation of a luciferase reporter while substitution to glutamine, mimicking the acetylation, restores E2A-mediated transcriptional activation. Recent studies have shown that several transcription factors can modulate the intrinsic HAT activity of CBP/p300. We were surprised to find that E2A proteins enhance acetylation of histones by CBP, in vitro and in vivo, in a KIX domain-independent manner. Acetylation of E2A is also not required for stimulation of CBP/p300 histone acetylation. It appears that E2A interacts with the other CBP domains to mediate this effect, presumably through allosteric effects. In summary, we demonstrate that acetylation of E2A plays a role in mediating the transcriptional activation activity of E2A. Furthermore, acetylation of E2A enhances its interaction with CBP/p300, at least in the presence of additional nuclear factors. We show evidence that p/CAF may mediate this effect. Enhancement of CBP/p300 HAT activity by oncogenic E2A-PBX1 proteins in vivo, suggests that some of its leukemia-promoting effects may be due to E2A-induced gain of function effects on CBP/p300. The enhanced interaction between acetylated E2A and CBP/p300, as well as the E2A-mediated stimulation of histone acetyltransferase activity might play a role in the DNA-binding-independent induction of proliferation. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2007-09-26 13:37:21.905