Winter wheat has several advantages over spring varieties, higher (25 % more) yield, efficient use of spring moisture, reduction of soil erosion by providing ground cover during the fall and early spring, rapid initial spring growth to out - compete weeds and circumvent the peak of Fusarium head blight infections by flowering early. Winter wheat is planted in early autumn when it germinates and developing seedlings acclimate to cold. The crown survives under snow cover and in spring rapidly grows into a vigorously growing plant for grain to be harvested in summer. However, the harsh Canadian prairie winters require that winter wheat has increased cold hardiness and improved winter survival to reduce losses from sudden cold snaps during winter and spring. Low temperature (LT) tolerance is one of the major components of cold hardiness. Genetic mapping studies have revealed a major quantitative trait locus (Fr-A2) at wheat chromosome 5A which can explain at least 50 % of LT tolerance in wheat. Physical mapping of 5A LT QTL in a hardy winter wheat cv Norstar revealed a cluster of at least 23 C - repeat binding factors (CBF) coinciding with peak of Fr-A2 QTL. The objective of this study is biochemical, and molecular characterization of CBF co - located at Fr-A2 to identify key CBF participating in conferring LT tolerance in winter wheat. A comparative analysis of CBF gene cluster at the Fr-A2 collinear region among Poaceae members showed an expansion in the number of CBF genes with increased LT tolerance. Rice, a cold sensitive member, had only three CBF genes, whereas cold hardy winter wheat cv Norstar has 23 CBF genes. Amino acid sequence - based cluster analysis of complete CBF genes, or their major functional components such as the AP2 - DNA binding domain and C - terminal trans - activation domain, divide Norstar CBF into Pooideae specific clades. However, analyses of Norstar CBF amino acid sequences of different functional groups revealed a shift in clade members. These results suggest divergence of CBF functions which could lead to possible differences / similarity in the regulon activated by a CBF in a specific group. The 15 CBF genes from winter wheat cv Norstar were expressed in E. coli to produce recombinant TrxHisS - CBF fusion proteins in adequate quantities for structural and functional assays. All CBF fusion proteins could be recovered in the E. coli soluble phase of cell extract, except that the CBF17.0 fusion protein could only be recovered with 6 M urea extraction. Eleven of the 15 CBF fusion proteins were very stable in heat (98 oC), 10 % SDS and 6 M urea treatment. The five other CBF members were very labile under native conditions, but were stable in E. coli cell extracts or when extracted under denaturing conditions. Most of the CBF recombinant proteins in denaturing gel electrophoresis migrated slower than expected from their predicted molecular mass, based on amino acid sequence. The slow migration could be associated to their elongated protein structure as determined by dynamic light scattering (DLS). CBF 12.2 and CBF 17.0 were highly resistant to denaturation and retained their secondary structure in these conditions as determined by circular dichroism (CD) spectra. The high stability of these two CBF proteins may be important for cold acclimation or maintenance of cold hardiness in wheat. CBF proteins are transcription factors that bind to the dehydration-responsive element / C-repeat element (DRE / CRT) motif (CCGAC). Ten of the 15 Norstar recombinant CBFs whether purified under native or denaturing conditions showed in vitro binding to the CRT motif. Within hours of cold exposure (4 oC) the native CBF increased their affinity to CRT interaction which could be due to changes in the CBF secondary structures. Some of the CBF for binding preferred the core GGCCGAC motif while others preferred TGCCGAC. Similarly binding assays with truncated CBF revealed that for some CBF proteins, the second signature motif (DSAWR) and remaining C - terminal were not needed, while for others a considerable portion of the C -terminal region was needed for binding. Norstar CBF 12.1 has a memory of cold experience, and upon exposure to cold, has a high and immediate affinity to CRT elements. A homolog CBF12.2 in less cold - hardy winter wheat cv Cappelle - Desprez had a non - functional protein due to a R → Q substitution in a highly conserved residue within the AP2 domain. Several of the cv Norstar CBFs showed increased activity under LT and denaturing conditions, which may be the reason for the greater cold hardiness in Norstar. In conclusion, detailed and extensive analyses of CBF in this study characterized their structure and function relationships, which are important for understanding and improving LT tolerance in plants. The identification of specific CRT binding motifs and two CBFs which were very stable under adverse conditions may be prime candidates for further study to improve LT tolerance in plants.

This thesis makes two significant contributions to the advancement of knowledge within crime prevention. The first of these is to evaluate the success of repeat victimisation prevention interventions. Interventions across four crime types are assessed herein, and the context-mechanisms-outcome configurations examined. The second contribution of this thesis is to assess two techniques of meta-evaluation: systematic reviews and realist syntheses. Each of these techniques is used in turn to assess the repeat victimisation prevention interventions. The contribution of each technique to the knowledge pool is then discussed, and the question of whether they are complementary or contradictory approaches answered. The thesis is framed in the context of evolutionary epistemology, which is the philosophy underpinning both approaches to meta-evaluation addressed herein. The thesis starts, with an examination of: firstly, how the evaluation methods in question have evolved, and the background to their scientific worth; and secondly, how situational crime prevention measures have evolved over time. The thesis then examines the two competing approaches for their contribution to the evaluation ecosystem by using both to assess repeat victimisation prevention interventions. Finally, the last section poses the question of whether it is survival of the fittest, or whether co-existence or adaptation could be the key to survival for these two meta-evaluative methodologies. Repeat victimisation prevention is revealed as an effective way of reducing crime, with a need for further research to apply the principle across further crime types. A requirement is identified for a greater breadth and depth of information to be included in future crime prevention evaluations. The systematic review is shown to be a useful way of assessing the overall effectiveness of the interventions, whilst the realist synthesis fills in the detail of why some interventions work and others fail. It is concluded that both approaches to meta-evaluation have useful contributions to make, and that a third way incorporating the best elements from each method should be developed.

Les subtélomères forment la transition entre les séquences spécifiques des chromosomes et les répétitions télomériques terminales. Ils semblent capables d’influencer les fonctions télomériques mais les connaissances sur les mécanismes mis en jeu sont encore limitées. Les subtélomères sont pourtant associés à de nombreuses pathologies comme la myopathie facio-scapulo-humérale (FSHD), une dystrophie musculaire secondaire à la contraction de répétitions macrosatellites D4Z4 dans la région subtélomérique 4q35. Afin d’étudier les propriétés de la séquence subtélomérique D4Z4, nous avons créé des constructions reproduisant l’organisation génomique au locus 4q35. Nous avons montré que D4Z4 est capable d’adresser un télomère à la périphérie du noyau. Cette activité est couplée à une activité insulatrice au niveau d’une séquence proximale de 80 pb et est dépendante de CTCF et des Lamines A. De plus, la relocalisation périphérique d’un télomère par D4Z4 s’accompagne d’une réplication plus tardive de celui-ci. Par ailleurs, la recherche de séquences capables de s’opposer à l’effet de position télomérique (TPE) a identifié un élément de 30 pb contenant un site CTCF dans la séquence insulatrice proximale de D4Z4. De même,   l’introduction d’un signal de poly-adénylation entre un gène rapporteur et les répétitions télomériques interfère avec le TPE et est accompagnée d’une diminution d’un transcrit hybride contenant le gène rapporteur et des répétitions télomériques, suggérant un rôle des transcrits télomériques TERRAs dans la régulation du TPE. En conclusion, ce travail a permis de caractériser l’implication de séquences subtélomériques, et notamment D4Z4, dans la régulation des télomères, leur compartimentalisation nucléaire, la réplication ou l’effet de position télomérique. De plus, il apporte un éclairage nouveau sur la physiopathologie de la FSHD et ouvre des perspectives dans la compréhension d’autres pathologies liées aux subtélomères. / Subtelomeres form the transition between chromosome specific sequences and terminal telomeric repeats. They might influence telomeric functions but underlying mechanisms are still unclear. Nevertheless, subtelomeres are associated with a number of human pathologies such as facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant disease secondary to the contraction of an array of D4Z4 macrosatellite repeats in the subtelomeric region 4q35. In order to study the biological function of the D4Z4 sequence, we created contructs that mimic the genomic organization of the 4q35 locus. We showed that D4Z4 is able to localize a telomere at the nuclear periphery. This perinuclear activity was dependant on interactions with CTCF and A type lamins and lied within a 80 bp proximal sequence that harbors an insulator activity. Moreover, the peripheral positionning of a telomere by D4Z4 is accompanied by a late replication timing of the telomere. We also searched for sequences able to counteract telomeric position effect (TPE) and identified a 30 bp element containing a CTCF binding site in the proximal region of D4Z4. In another construct, the introduction of a poly-adenylation signal between a reporter gene and telomeric repeats counteracted TPE. This effect is accompanied by the production of a hybrid transcript encompassing the reporter gene and telomeric repeats, suggesting a role for the TERRAs telomeric transcripts in TPE regulation. This work contibuted to characterize the role of subtelomeric sequences, especially the D4Z4 macrosatellite, in telomere regulation, their nuclear compartimentalization, their replication or the telomeric position effect. We will discuss the implications in the understanding of the pathophysiology of FSHD and other subtelomeric diseases.

Nearly half of all pregnancies in the United States are unintended. In 2002, the direct medical costs of unintended pregnancies were estimated to be almost five billion dollars. Moreover, women with unintended pregnancies tend to delay seeking prenatal care and making the necessary life style changes for the fetus. Subsequently, unintended pregnancies have the potential to lead to low birth weight infants and potentially poorer long-term child development. Although there are negative consequences with unintended pregnancies, repeat unintended pregnancies impose even greater health risks for both mothers and infants. To prevent unintended and repeat unintended pregnancies, family planning methods must be utilized effectively. Despite the potential risk associated with negative health outcomes of mothers and infants, repeated unintended pregnancies have not been studied extensively. Therefore, the purpose of this study was to examine the relationship between the number of unintended pregnancies and effective use of family planning methods. A secondary analysis of The National Statistics of Family Growth, cycle 6 (2002) was done. The study showed a statistically significant relationship association between the number of unintended pregnancies and effective contraceptive use after adjusting for confounders which include age, race, annual household income, marital status and types of insurance. The recommendation of this study include creating better strategies for family planning practices and the need to improving contraceptive education and service delivery for those with higher risk.

Doctor of Philosophy / Department of Geography / Kevin Blake / The American West is a land of great diversity and stark contrast. It is also a landscape marked by rapid change as a result of such forces as globalization, population growth, and heightened interest in natural resources (either for recreation or extraction). This dissertation investigates these changes to the region through a repeat photography analysis. Between 1982 and 1984, Thomas and Geraldine Vale traveled along U.S. Highway 89 from Glacier National Park, Montana to Nogales, Arizona. Their subsequent work, Western Images, Western Landscapes: Travels Along U.S. 89 (University of Arizona Press, 1989), contained fifty-three photographs from this journey, representing a cross section of the West from border to border. Nearly every facet of the region was represented, from the remote prairie landscapes of Montana to the bustling Phoenix downtown, and from the largest open pit mine in the world to seldom visited corners of Yellowstone National Park. Between March 2009 and August 2010, I retraced the steps taken by the Vales and successfully rephotographed all of the locations contained within their book. The observed continuity or change is examined thematically in order to address the landscapes and cultures of the West in greater detail. Specifically, chapters within this dissertation visually and textually describe changes that have occurred along national borders, within Native American reservations, throughout the rural landscapes and national parks of the region, within the many resource extraction industries, and within towns and cities of every size. Significant findings, which are well depicted in the photographic pairings, include heightened national security along the borders, problems of overuse in many parks and protected areas, the transition of traditional small towns into communities increasingly dependent upon tourism for survival, and both beautification and revitalization efforts taking place in the urban cores of Phoenix and Salt Lake City. By painting a vivid picture of recent Western geography, this research provides for greater ability for residents and scholars of the region to understand the forces at work within their communities and surroundings.

Pannexin 1 (Panx1) is a metabolite channel enriched in the brain and known to localize to the cell surface, where it is involved in a variety of neuronal processes including cell proliferation and differentiation. The mechanisms through which Panx1 is trafficked or stabilized at the surface, however, are not fully understood. The proximal Panx1 C-terminus (Panx1CT), upstream of a caspase-cleavage site has been demonstrated to be required for Panx1 cell-surface expression. We discovered a previously unreported putative leucine-rich repeat (LRR) motif within the proximal Panx1CT. I investigated the involvement of this putative LRR motif on Panx1 localization and oligomerization. Deletion of the putative LRR motif or uniquely the highly conserved segment of the putative LRR motif resulted in a significant loss of Panx1 cell surface expression. Finally, ectopic expression of Panx1-EGFP in HEK293T cells increased cell proliferation, which was not recapitulated by a Panx1 deletion mutant lacking the putative LRR motif. Overall the findings presented in this thesis provide new insights into the molecular determinants of Panx1 trafficking and oligomerization. / Graduate / 2020-02-14

STRAP (Stress responsive activator of p300) is a 440 amino acid protein, predicted to have 6 TPR (Tetra-Tri-Co-Peptide Repeats) motifs, known to mediate protein-protein interactions. STRAP has been shown to form a complex with proteins p300 and JMY (Junctional Mediatory Protein), and is implicated in the DNA damage, heat shock response pathway, regulation of the Glucocorticoid receptor and in the function of p53.The aims of this project were to clone, express and purify full length and truncated human STRAP (hSTRAP) variants in high quantities. Full length and shorter hSTRAP fragments, which contain different combinations of the predicted TPR motifs and hence cover different regions, would be then structurally characterised by various structural and biophysical experiments. Another important aim was to identify interacting partners of hSTRAP in breast cancer and to map the position of their interaction sites to different parts of the protein. To this direction GST- and His- tagged full length hSTRAP, as well as His- tagged truncated hSTRAP protein variants have been successfully cloned, expressed and purified. Independent and reproducible biochemical pull-down assays have been carried out in MCF7 breast cancer cells, followed by mass spectrometry-based proteomics analysis which identified 25 hSTRAP-interacting partners from various signaling pathways such as regulation of the actin cytoskeleton and translation. In addition, crystallization trials were carried out using pure His-hSTRAP(1-440) protein, which were unfortunately un-successful. Various hSTRAP protein variants have been characterized by CD, showing that hSTRAP(1-150), His-hSTRAP(1-440), hSTRAP(1-219), hSTRAP(151-284) and hSTRAP(285-440) comprise of alpha and β structures, but the hSTRAP protein variants show no clear cooperative unfolding transitions, suggestive of molten globule states. NMR on hSTRAP(1-219), hSTRAP(1-150) and hSTRAP(151-284) have shown these proteins are not folded at a tertiary structure level. We conclude that a protocol has been established to clone, express and purify various hSTRAP variants and the thermal and secondary structure characteristics of each have been determined, although the 3D structure could not be solved. Pull-down assays followed by proteomic analysis have shown that hSTRAP is implicated in many aspects of cellular regulation.

A trinucleotide repeat (TNR) is a 3 base pair (bp) DNA sequence tandemly repeated in an array. In humans, TNR sequences have been found to be associated with at least 14 severe neurological diseases including Huntington disease, myotonic dystrophy and several of the spinocerebellar ataxias. Such diseases are caused by an expansion of the repeat sequence beyond a threshold length and are characterized by non-Mendelian patterns of inheritance which lead to genetic anticipation. Although the mechanism of the genetic instability in these arrays is not yet fully understood, various models have been suggested based on the in vitro observation that TNR sequences can form secondary structures such as pseudo-hairpins. In order to investigate the mechanisms responsible for instability of TNR sequences, a study was carried out on Escherichia coli cells in which TNR arrays had been integrated into the chromosomal lacZ gene. This genetic assay was used to identify proteins and pathways involved in deletion and/or expansion instability. Deletion instability was clearly dependent on orientation of the TNR sequence relative to the origin of replication. Interestingly, it was found that expansion instability is not dependent on the orientation of the repeat array relative to the origin of replication. The replication fork reversal pathway and the RecFOR mediated gap repair pathway were found to have no statistically significant influence on the instability of TNR arrays. However, the protein UvrD was found to affect the deletion instability of TNR sequences. The roles of key helicase genes were investigated for their effects on instability of chromosomal CTG•CAG repeats. Mutation of the rep gene increased deletion in the CTG leading-strand orientation of the repeat array, and expansion in both orientations - destabilizing the TNR array. RecQ helicase was found to have a significant effect on TNR instability in the orientation in which CAG repeats were present on the leading-strand relative to the origin of replication. Mutation of the recQ gene severely limited the number of expansion events in this orientation, whilst having no effect on deletions. This dependence of expansions on RecQ was lost in a rep mutant strain. In a rep mutant expansions were shown to be partially dependent on the DinG helicase. All together, these results suggest a model of TNR instability in which expansions are due to events occurring at either the leading or lagging strand of an arrested replication fork, facilitated by helicase action. The identity of the helicase implicated is determined by the nature of the arrest.

The polyglutamine diseases are a clinically heterogeneous group of inherited neurodegenerative disorders caused by expansion of polyglutamine-encoding (CAG)n trinucleotide repeats within the disease genes. It is increasingly clear that the amino acid sequences flanking the polyglutamine expansion in each disease protein, i.e. the specific protein context, contribute to selective neuronal toxicity by influencing the behavior of the disease protein within selectively vulnerable neuronal populations. In the studies described here, I explore the role that protein context plays in the polyglutamine disease, Spinocerebellar ataxia type 3 (SCA3). Toward this end, I utilize biochemical, cell-based, and animal models to gain a broader understanding of the SCA3 disease protein, ataxin-3, and generate tools for further exploration of the molecular properties of ataxin-3 that modulate its toxicity during disease. In Chapter 1, I provide an overview of the recognized polyglutamine diseases, emphasizing the elements of protein context that are distinct among the polyglutamine disease proteins and may contribute to the neuropathological and clinical heterogeneity within this family of diseases. Alternative splicing of the polyglutamine disease gene products adds an additional level of complexity to the tissue-specific protein context of expanded polyglutamine, yet this phenomenon has been underinvestigated. In Chapter 2, I examine the significance of ataxin-3 splice variation. Several minor 5' variants and both known 3' splice variants of ataxin-3, a deubiquitinating enzyme, are expressed at the mRNA level in brain. At the protein level, however, the C-terminal splice isoform with three ubiquitin interacting motifs (3UIM ataxin-3) is the predominant isoform in brain, independent of age or (CAG)n expansion. Although both C-terminal ataxin-3 splice isoforms display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and is more rapidly degraded by the proteasome. These observations demonstrate how alternative splicing of sequences distinct from the polyglutamine-encoding (CAG)n repeat can alter disease-related components of protein context. Knock-in models of polyglutamine diseases utilize pathogenic (CAG)n expansions within the endogenous genomic, transcript, and protein context to recreate key features of individual polyglutamine diseases. In chapter 3, I describe the creation of the first knock-in mouse model of SCA3. Hemizygous knock-in mice transmit the knock-in allele in Mendelian ratios and broadly express both the expanded Atxn3(Q3KQ82) protein and the wildtype murine Atxn3(Q6) protein. In this chapter, I also compare the gene targeting efficiencies and rates of chromosomal instability of a novel C57BL/6J ES cell line (UMB6JD7) and two well established ES cell lines (W4 and Bruce4.G9). Of these, Bruce4.G9 ES cells proved superior based on lower rates of aneuploidy and the production of germline transmitting chimeras. Finally, in Chapter 4 I discuss questions and concepts raised during the course of these studies, and suggest avenues of future research aimed at broadening our understanding of ataxin-3 physiology and of protein context-dependent elements in polyglutamine disease pathogenesis.

The literature suggests that a correlation exists between visitor satisfaction and a visitor's choice to re-experience a product. The higher one's satisfaction level, the more likely he/she is to experience the product again and/or provide positive word-of-mouth advertising to friends and family. The Tian-Cole and Crompton model was chosen because of its acknowledgment and explanation of the differences between Visitor Satisfaction (VS) and Service Quality (SQ) and how their relationship influences satisfaction and contributes to Future Destination Selection. In addition to identifying a theoretical framework that explains why visitors return, it was necessary to identify a survey methodology to be used in developing the Mountain Pine Ridge Forest Reserve (MPRFR) visitor satisfaction surveys. It was determined that a combination of the Relative Performance Assessment (RPA) and Customer Service Questionnaire (CSQ) was an appropriate survey framework for a visitor satisfaction survey. As a result of the research, two visitor satisfaction surveys were developed: Intercept Survey and Mail-back Survey. The Intercept Survey will be administered at a MPRFR site. It was designed to be concise and not take much of the visitor's time. This survey will be used to gather general information about visitor characteristics and level of satisfaction while at a site. The Mail-back Survey will be sent to the visitor after their trip so more time can be devoted to answering a more detailed visitor satisfaction survey. This survey asks visitors to indicate what site characteristics are important to them and then rate their satisfaction with each item. Instruments from the report entitled "A Front Country Visitor Study for Grand Staircase-Escalante National Monument" were used as an example of how to format a visitor satisfaction survey and how to use the collected data as a managerial tool. The two main objectives of this research were accomplished and have established a foundation upon which subsequent research efforts will begin. This work serves as a catalyst to improving Belizean site planning, design, and management by better understanding what site characteristics contribute to visitor satisfaction.