The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and diseases, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding RNAs, called trans-acting factors. Biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made important discoveries in characterizing how these factors regulate gene expression and determining the RNA sequence to which they bind. However, the spatiotemporal information regarding these interactions in subcellular compartments have been difficult to determine or to quantify accurately. To image and quantify native RNA and RNA–protein interactions simultaneously in situ, we developed a proximity ligation assay that combines peptide-modified RNA imaging probes. It can detect the RNAs in live cells and the interactions at a single-interaction level. Lastly, it can produce results that are easily quantifiable. We tested the specificity and sensitivity of this technique using two models: interactions between the genomic RNA and the N protein of human respiratory syncytial virus as well as those between exogenous transcripts with or without the Human antigen R (HuR) binding site and HuR. To validate this method, its accuracy and utility have been demonstrated in three models: poly(A)+ or β-actin mRNAs binding to different cytoskeleton for localization, poly(A)+ or β-actin mRNAs interacting with HuR for stabilization, and programmed cell death 4 (PDCD4) mRNA binding to HuR or T-cell intracellular antigen (TIA1) for translational regulation.

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Call number: LD2668 .T4 1979 C52 / Master of Science

Call number: LD2668 .T4 1979 J32 / Master of Science

Cellular transformation is accomplished in vitro through the concerted action of growth factors and oncogenes. This association has demonstrated that malignant growth results from aberrations in pathways that normally operate to control proliferation. Activation of genes that code for growth factors, their receptors, and/or molecules essential in the transduction of signals from the cell surface to the nucleus are all potential mechanisms by which tumor cells could establish a selective growth advantage over normal cells. This dissertation addresses the question of what oncogenic mechanisms are important in the development and progression of human melanoma. These studies show that melanoma growth is regulated by endogenous substances produced by the melanoma cells themselves (autocrine stimulation), as well as by exogenous substances supplied by neighboring cells and platelets (paracrine stimulation). These factors work to drive the expression of the transformed phenotype for melanoma as evidenced by induction of serum-free soft agar growth. Human platelets were found to the the richest source of paracrine growth promoters. The factor from human platelets was characterized and partially purified. Melanoma cells respond to this 60,000 molecular weight, disulfide-bond-containing protein in colony formation assays. In addition, the protein has endothelial cell growth factor activity. Purified fractions which promoted optimal colony formation for human melanoma cells also maximally stimulated monolayer growth of bovine aortic endothelial cells, while melanocytes were nonresponsive. This implies that melanoma cells are expressing receptors for a protein which plays no known or apparent role in the normal growth of melanocytes. Melanoma cells are sensitive to growth regulatory molecules of autocrine and paracrine nature. This dissertation provides clues to the genetic lesions which have occurred in these melanoma cells to influence their proliferation. The aberrations appear to reside in those genes important in growth factor pathways at the level of endogenous production and misguided response to exogenous factors through receptor expression. We can not hope to fully inhibit the proliferation of tumor cells until we identify and understand those forces which drive their growth. These studies have increased our knowledge of those signals which stimulate melanoma cellular proliferation, and thus provide insight into important therapeutic targets.

SRL has been posited to explain student-athletes concurrent success in sport and academics. The purpose of this dissertation was to empirically explore student-athletes’ self-regulated learning (SRL) in and across their academic and sport learning. Three manuscripts addressed two overarching goals: (a) explore the relation between SRL in sports and academics, and (b) explore methods of measuring SRL. First, in McCardle, Jonker, Elferink-Gemser, and Visscher’s (2014) study, competitive youth athletes (N = 215) self-reported on self-regulatory and motivational engagement in sport and academics. Findings revealed a positive relation between SRL in these contexts and more reported engagement of SRL in sports than in school. Second, McCardle (2014) conducted a case study of one student-athlete’s SRL in sport and school. Based on interviews, journals, and video-stimulated recall, the student-athlete demonstrated clear similarities in how he engaged SRL in both contexts. Some differences between sport and academic learning emerged, suggesting potential differences in support for SRL in the two contexts. This paper explored potential of qualitative measures of SRL in by combining multiple qualitative measures of SRL to create SRL profiles in sport and academics. Third, McCardle and Hadwin (2015) explored use of two types of self-reports considered event measures of SRL as they focused on single learning episodes (N = 263): (a) a quantitative questionnaire measure of SRL related to one study episode for an exam, and (b) a qualitative diary related to setting and attainment of one study goal. Contrasting these two methods revealed varying degrees of similarities in students’ self-reports. Together, this research highlights the potential of transfer of SRL across sport and academic domains and the importance of appropriate measures to capture event- and aptitude-based SRL and suggests several avenues for future research. To conclude, I suggest Winne and Hadwin’s (1998) model of SRL serve as a framework for researching SRL transfer with a focus on conditions. New research in transfer has potential for contributing to SRL research on how learners draw on previous regulatory experiences to adapt to new challenges. / Graduate / mccardle@uvic.ca

In this paper we propose an approach to implement environmental standards into Data Envelopment Analysis (DEA) and in this way to measure their regulatory impact on eco-efficiency of firms. As one standard feature of basic DEA models (as e.g. CCR from Charnes et al. (1978)) lies in the exogeneity of inputs, desirable and undesirable outputs, it is not possible to introduce environmental constraints for these parameters directly into basic DEA models. Therefore, we use a bounded-variable way, which allows constraints on the efficiency frontier. The regulatory impact is assessed as difference in eco-efficiency scores before and after fictive introduction of an environmental standard. Furthermore, we distinguish between weak and strong disposability of undesirable outputs and develop corresponding models. Assessing the regulatory impact of environmental standards in advance provides support for environmental policy makers in choosing appropriate instruments and in adjusting the intensity of regulation. (authors' abstract)

Ways to reduce the impact of worry in generalized anxiety disorder (GAD) have received little experimental research attention. Previous research has found that those with GAD are vulnerable to negative emotionality immediately following periods of worry; emotion regulation strategies could be useful to mitigate reactivity following worry. One promising strategy is mindfulness, defined as sustained attention toward the present moment with an attitude of curiosity and acceptance. Experimental research has found that mindfulness reduces negative affect and improves emotion regulation. This strategy is likely more effective than thought suppression, a common strategy used in GAD. This online study recruited 300 individuals with analogue GAD who completed several self-report measures of worry severity, emotion dysregulation, mindfulness, and experiential avoidance and underwent experimental inductions of worry (versus no-worry control) and regulation strategy (mindfulness versus thought suppression versus no-strategy control) before watching a sad film clip and reporting state affect and emotion dysregulation. Contrary to hypotheses, the mindfulness manipulation did not have a buffering effect on the relation between worry and negative affect or emotion dysregulation. The only predicted significant finding indicated that the mindfulness manipulation had a main effect on negative affect, with visual trends indicating that this effect was driven by those who did not worry. An exploratory analysis indicated that a mindfulness manipulation increased positive affect following worry, however. Clinical implications and future directions are discussed.

AreA, a 96 kDa gene regulatory protein involved in nitrogen metabolite repression in Aspergillus nidulans, is a member of the GATA family of zinc finger DNA binding proteins, and regulates the expression of around 100 genes. This project was designed to examine the domain structure of AreA in this region of the protein, and to characterise the DNA binding domain Limited proteolysis has been employed to identify structural domains in the Cterminal region of AreA, which has been cloned and over-produced in E.coli. A variety of proteases have been used, and each reveals a dominant stable fragment of approximately 17-22 kDa. N-terminal sequencing and mass spectroscopy have been used to identify a number of these fragments. The major product following limited proteolysis by Glu-C is composed of two closely related species, a 164 residue fragment (17,489 Da) and a 157 residue fragment (16,857 Da). Both fragments encompass the Zn-finger motif, and share the same Cterminus, differing at the N-terminus by only 7 amino acids. The DNA sequence coding for the 157 residue fragment (16,857 Da) has been cloned and over-produced as a His-tag fusion protein. Further studies on this domain have shown that this putative domain has a relatively strong DNA binding constant with values in the nanomolar range. Structural analysis using Circular Dichroism, NMR and fluorescence suggests that the domain contains some irregular or unstructured regions. The regions that are structured are likely to be from the zinc-finger region, since DNA binding is maintained.

Chromatin structure and epigenetic mechanisms play an important role in initiating and maintaining the intricate patterns of gene expression required for embryonic development. One such mechanism, DNA methylation (5mC), involves the chemical modification of cytosine bases in DNA and is implicated in maintaining patterns of transcription. However, many fundamental aspects of DNA methylation are not fully understood, including the mechanisms by which it influences transcriptional states. Recent data suggest functional links between DNA methylation and a second epigenetic mechanism that has important roles in transcriptional repression, the polycomb group (PcG) repressor system. Here, I suggest that an intact DNA methylation system is required for the repression of many PcG target genes by influencing the genomic targeting of the polycomb repressor 2 complex (PRC2) and its signature histone modification, H3K27me3 (K27me3). I demonstrate differential genomic localisation of K27me3 at gene promoter regions in hypomethylated mouse embryonic fibroblast (MEF) cells deficient for the major maintenance DNA methyltransferase, Dnmt1. Globally, Dnmt1-/- MEFs have a higher level of the K27me3 mark than controls, as assessed by western blot and immunofluorescence. I observe increased K27me3 at a relatively small number of gene promoters in Dnmt1-/- MEFs that often are associated with high levels of DNA methylation in wildtype MEFs, consistent with the notion that DNA methylation is capable of antagonising PRC2 binding at certain loci. Conversely, I show that a large number of developmentally important genes that are normally repressed and highly bound by K27me3, including classic polycomb targets, the Hox genes, display dramatically reduced association with K27me3 in Dnmt1-/- MEFs. Many of these genes, but not all, show reciprocal increases in promoter H3K4me3 modification and are transcriptionally de-repressed in Dnmt1-/- MEFs. I suggest that these genes are mostly associated with CpG-rich promoters with low levels of DNA methylation in wildtype cells, implying that their silencing is not dependent on the canonical role of DNA methylation. Consistent with the findings of recently published work, I suggest a working model where PRC2 binding in wildtype cells is restricted by CpG methylation. According to this model, the differential genomic location of K27me3 in hypomethylated Dnmt1-/- MEFs is explained by a redistribution of PRC2 to normally DNA methylated, unbound loci, resulting in a titration effect and coincident loss of K27me3 from normal targets. It was also apparent that certain PRC2-target genes, including the developmentally important Hox gene clusters, are strongly affected in Dnmt1-/- MEFs, displaying striking loss of K27me3. As intergenic transcription has been implicated in relief from polycomb silencing and abundant intergenic transcription has been reported within Hox clusters, I measured RNA expression at Hox clusters and a small number of other PcG target genes in Dnmt1-/- MEFs using highdensity tiling arrays. In Dnmt1-deficient MEFs, widespread increases in intergenic transcription were observed within Hox clusters. In addition, mapping of the elongatingpolymerase- associated H3K36me3 histone modification showed widespread increases in this mark at intergenic and promoter regions in Dnmt1-/- MEFs. Increased local intergenic RNA and H3K36me3 were found to correlate with K27me3 loss for this cohort of genes. I suggest a working model where increased intergenic transcription and H3K36me3 in Dnmt1-/- MEFs leads to accelerated loss of K27me3 at certain loci, including Hox clusters. Taken together with recently published data, this work suggests that a major role of DNA methylation is in shaping the PRC2/K27me3 landscape. The potential implications of this putative role for DNA methylation are widespread, including our knowledge of how DNA methylation influences transcriptional regulation, and the consequence of rearranged DNA methylation patterns that are observed in many diseases including cancers.