VictorJuiz/thesis huggingface dataset:
A Biological Investigation of the Proteins Required for Nickel Insertion into Escherichia coli [NiFe] Hydrogenase

[NiFe] hydrogenases are found in a variety of microorganisms and catalyze the reversible oxidation of hydrogen gas to protons and electrons. This enzyme has generated intense interest due to its contribution to pathogenicity in certain organisms as well as its application in bioremediation and the production of hydrogen as an alternative fuel source. The biosynthesis of the dinuclear active site requires a number of accessory proteins to chaperone and insert the metal cofactors to the awaiting large subunit of hydrogenase. The proteins responsible for nickel delivery to Escherichia coli hydrogenase 3 are HypA, HypB, and SlyD, however the mechanism by which this is accomplished is unclear. The goal of this work was to analyze the metal-binding abilities and protein interactions of these nickel insertion proteins to enhance our understanding of their roles. Isolated N-terminal peptide of HypB has similar high-affinity metal-binding to the full-length protein. This peptide binds nickel in a square planar site with three cysteinyl and a fourth N-terminal amine ligand. Additionally, studies with SlyD and HypA reveal protein interactions that occur during hydrogenase maturation. Pull-down experiments of a tagged variant of hydrogenase revealed multi-protein complexes with HypA, HypB, and SlyD. A complex between SlyD and hydrogenase forms prior to both nickel and iron insertion, supporting chaperone activity of SlyD during hydrogenase maturation. HypA can interact with hydrogenase in the absence of HypB and SlyD, and a possible role as the bridging protein during the nickel insertion event is proposed. In addition, fluorescent imaging of E. coli cells using a fluorescently labeled streptavidin conjugate revealed localization of both Strep-tagged II hydrogenase and HypA at or near the cell membrane, suggesting that enzyme maturation occurs proximal to metal transporters. This work provided a deeper understanding of the role that each of these proteins play in [NiFe] hydrogenase assembly and is helpful for any future applications of this enzyme.

Keywords: Metalloproteins
Bacterial nickel homeostasis
[NiFe] Hydrogenase
Advisor: Zamble, Deborah
Role of the Actin Cytoskeleton in Pro-fibrotic Signaling

The development of fibrosis involves disruption of connective tissue homeostasis that may include inhibition of collagen remodeling pathways such as phagocytosis, as well as the differentiation of myofibroblasts, pro-fibrotic cells. Myofibroblast differentiation is dependent on actin assembly, which can alter cell shape and is required for collagen phagocytosis and remodeling. Cyclosporin A (CsA) is a commonly used drug for prevention of organ transplant rejection that causes marked fibrosis in periodontal tissues by inhibiting collagen phagocytosis. As gelsolin is a Ca2+-dependent actin severing protein that mediates collagen phagocytosis, I determined whether gelsolin is a CsA target. Compared to vehicle-treated controls, CsA-treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. From a series of in vitro experiments, I conclude that CsA-induced accumulation of collagen in the periodontal ECM involves disruption of the actin severing properties of gelsolin. This disruption inhibits the binding step of collagen phagocytosis and promotes fibrosis. During the development of pressure-induced cardiac hypertrophy, collagen accumulates in the interstitium, due to myofibroblasts which express alpha-smooth muscle actin (SMA). As focal adhesion complexes are putative mechanosensing organelles, I examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin, in the regulation of SMA expression. After application of mechanical force to cultured fibroblasts through collagen-coated magnetite beads attached to beta1 integrins, FAK and gelsolin were recruited to beads and there was increased nuclear translocation of MRTF-A, a transcriptional co-activator of SMA. These data suggested a novel pathway in which mechanosensing by FAK regulates actin assembly through gelsolin; actin assembly in turn controls SMA expression through MRTF-A. I also examined a potential role for the actin nucleators, mammalian Diaphanous-related formins (mDia), in the mechanosensing pathway that leads to force-induced expression of SMA. siRNA knockdown of mDia inhibited actin assembly at force-induced focal adhesions. In anchored collagen gels to model myofibroblast-mediated contraction of the matrix, mDia knockdown reduced contraction by 50%. Collectively, these experiments indicate that the regulation of actin assembly plays an important role in the development of force-induced transcriptional activation of SMA, myofibroblast differentiation and collagen phagocytosis.

Keywords: fibrosis
actin cytoskeleton
Advisor: McCulloch, Christopher
An Investigation of Educators’ Data Habit of Mind

Educators are increasingly being asked to interact with data to facilitate students’ learning in the classroom. However, as an educational measurement community, we have little understanding of the factors and/or contexts that facilitate educators’ successful use of data. Educators’ use of score reports and the relationship to the intended use is integral to the concept of validity. A conceptual model, “Data Habit of Mind,” is proposed to study educators’ understanding, interpretation and potential applications of results from large-scale assessments. The metaphor, “Habit of Mind,” was originally coined by Robert Sternberg and Dan Keating, and has been applied in the education sector to describe educators’ habits of inquiry when interacting with assessments. Based on an extensive review of the literature, Data Habit of Mind is defined as a combination of statistical literacy and score report interpretation. Statistical literacy is the extent to which an individual is able to describe, organize and reduce, represent, and analyze and interpret data. Score report interpretation is the extent to which an individual is able to describe, summarize, question, and propose an application for a given set of elements on a score report. The combination of these two makes up an individual’s Data Habit of Mind. Twenty educators were interviewed to assess their level of statistical literacy and their score report interpretation skills. A cognitive interview approach was used to capture the educators’ cognitive processes as they solved performance-based tasks, and protocol analysis procedures were used to encode the responses into the conceptual model. Descriptions of educators’ Data Habit of Mind were then generated through qualitative matrix analysis. Four groups of educators were identified based on the patterns of relationship between their statistical literacy and score report interpretation scores. Demographic factors, including teaching experience, gender and educational background were not meaningful predictors of educators’ Data Habit of Mind. These results add to our understanding of how educators interpret and use test results and have implications for test validation processes.

Keywords: Data Habit of Mind
Score report interpretation
Statistical LIteracy
Assessment
Data-Use
testing
Advisor: Childs, Ruth A.
The Nucleolus and Nucleolar Proteins of Dictyostelium

Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.

Keywords: nucleolus
Dictyostelium
nuclear localization signal
nucleolar localization signal
nucleomorphin
subcompartment
Advisor: O'Day, Danton H.
Teaching Toward Equity in Mathematics

This research is a qualitative case study examining changes in urban Canadian elementary teachers’ conceptualizations of equity and approaches to pedagogy in their mathematics teaching in relation to their involvement in multiple professional learning contexts. The study focuses on four major professional development (PD) efforts in which five focal teachers participated over a school year. Data sources include researcher observations, field notes, video-recordings of PD sessions and classroom mathematics teaching, as well as a series of one-on-one interviews. Data analysis revealed three main ideas related to equity that were adopted by focal teachers: 1) the importance of developing awareness of students and their communities; 2) teaching strategies to scaffold students’ development of mathematical proficiency; and 3) strategies for structuring student-driven, inquiry-based learning for mathematics. The multiple contexts of professional learning presented contradictory messages. Thus, teachers took up some ideas and left others behind and sometimes took up ideas that served conflicting goals of education. Future studies of teacher PD should focus on the teacher’s perspective and the role of any individual PD within the multiple contexts of professional learning in which teachers participate.

Keywords: Mathematics Education
equity
professional development
teacher change
Advisor: Esmonde, Indigo
Sedimentology of the Squantum ‘Tillite’, Boston Basin, USA: Modern Analogues and Implications for the Paleoclimate during the Gaskiers Glaciation (c. 580 Ma)

The Gaskiers glaciation (c. 580 Ma) has been classically traced along the Neoproterozoic Avalonian-Cadomian Terranes, which are now found scattered around the North Atlantic Ocean. Around 625 Ma these terranes were composed of volcanoes and arc-type basins. ‘Till-like’ diamictite horizons identified within these basins have been used as evidence for a ‘Snowball Earth-type’ glaciation at 580 Ma. However, others argue that these deposits are non-glacial debris flow deposits. To test the non-glacial interpretation of these deposits, a detailed sedimentological and basin analysis was conducted on the Neoproterozoic Squantum Member that occurs conformably with the volcanic-sedimentary rocks of the Boston Bay Group (eastern Massachusetts); this deposit is one of the most referenced ‘tillite’ deposits for the Gaskiers glaciation. This thesis shows that the ‘tillites’ of this succession are volcanically-influenced non-glacial debrites. Using the Lesser Antilles Arc and the adjacent Grenada Basin in the Caribbean Sea as a modern depositional analogue for the Avalonian-Cadomian Terranes, this study further reveals that debris flow facies types (diamicts) comparable to those of the Avalonian-Cadomian Terranes are produced at this modern arc and are recorded in the fill of the Grenada Basin. A similar study was conducted on the modern diamicts produced at the heavily glaciated Mount Rainier volcano (Washington, USA), revealing that despite the presence of local glaciers, debris flow is the dominant process depositing diamicts due to eruptions and flood events. The major thrust of this thesis is that it highlights the key role of tectonics and volcanism, not glaciation, in producing the diamictites of the Avalonian-Cadomian Terranes, and the importance of examining Neoproterozoic diamictite facies in their wider sedimentary, stratigraphic and tectonic context.

Keywords: Neoproterozoic glaciation
Diamictite
Snowball Earth
Debrite
Advisor: Eyles, Nick
Bitter Sweet Morality: An Investigation of the Role of Ethical Orientation on Workplace Necessary Evils

In almost all types of work it is sometimes necessary to do harm as a means of doing good; as such, necessary evils are a wide spread phenomenon in work life. The present research furthers our understanding of necessary evils through exploring how one’s ethical orientation (measured in terms of idealism and relativism) affects one’s experience of performing such tasks. Molinsky and Margolis (2005) theorized that nine dimensions of necessary evil affect a person’s judgments, beliefs, and attitudes about the task they are required to perform, the self, and the impact of the necessary evil. When performing a necessary evil, individuals evaluate its dimensions -- this evaluation involves assessing the alternative actions one might take to carry out the necessary evil, and the possible consequences of each alternative course of action. Workers are thus faced with an ethical judgment as they realize that if the necessary evil is to be carried out, someone will be harmed. Two studies were conducted to develop a better understanding of workers’ experience of necessary evils in general, and to explore whether people’s idealistic and relativistic values are related to how they experience performing necessary evils. In an effort to gain rich and contextualized information about peoples’ experiences, 30 individuals with experience performing necessary evils were interviewed for Study 1. Respondents were asked to comment on their experiences before, during and after they performed necessary evils, as well as whether they felt that such tasks were ethical or moral in nature. Study 2 presented necessary evil vignettes to 150 university students and used structural equation modeling to test hypotheses about the relationship between ethical orientation and the necessary evil experience. Two models of how necessary evils are performed fit the data. Theoretical and practical implications are discussed.

Keywords: Ethics
Necessary evils
Advisor: Gunz, Hugh P.
Theorizing Praxis in Citizenship Learning: Civic Engagement and the Democratic Management of Inequality in AmeriCorps

Over the last twenty years, the academic work on citizenship education and democracy promotion has grown exponentially. This research investigates the United States federal government’s cultivation of a ‘politics of citizenship’ through the Corporation for National and Community Service and the AmeriCorps program. Drawing on Marxist-feminist theory and institutional ethnography, this research examines the ways in which democratic learning is organized within the AmeriCorps program through the category of ‘civic engagement’ and under the auspices of federal regulations that coordinate the practice of AmeriCorps programs trans-locally. The findings from this research demonstrate that the federal regulations of the AmeriCorps program mandate a practice and create an environment in which ‘politics,’ understood broadly as having both partisan and non-partisan dimensions, are actively avoided in formalized learning activities within the program. The effect of these regulations is to create an ideological environment in which learning is separated from experience and social problems are disconnected from the political and material relations in which they are constituted. Further, the AmeriCorps program cultivates an institutional discourse in which good citizenship is equated with participation at the local scale, which pivots on a notion of community service that is actively disengaged from the State. Through its reliance on these forms of democratic consciousness, the AmeriCorps program engages in reproductive praxis, ultimately reproducing already existing inequalities within U.S. society. The primary elements of this reproductive praxis have been identified as ‘a local fetish’ and the ‘democratic management of inequality.’ The local fetish refers to the solidification of the local as the preferential terrain of democratic engagement and is characterized by an emphasis on face-to-face moral relationships, local community building, and small-scale politics. The democratic management of inequality refers to the development of discursive practices and the organization of volunteer labor in the service of poverty amelioration, which is in turn labeled ‘good citizenship.’ This research directs our attention to a more complicated notion of praxis and its relationship to the reproduction of social relations. Also, this research brings into focus the problem of the conceptualization of civil society and its relationship to democracy and capitalism.

Keywords: adult education
citizenship learning
demoxcracy
Advisor: Mojab, Shahrzad
Global Analyses of Alternative Splicing in Evolution and Nervous System Development

Technological advancements have sparked discovery in biology, enabling important questions to be addressed experimentally at unprecedented depth and scale. One such advance, the development of large-scale approaches to study gene expression, has transformed the way we view the transcriptome. In recent years, these approaches have been applied to studies of alternative RNA splicing, a process where multiple distinct messenger RNAs can be generated from precursor transcripts to produce extensive transcriptomic diversity from a limited repertoire of genes. Global analyses have not only reinforced models initially based on single gene studies, they have also led to numerous insights into general principles governing the regulation and evolution of alternative splicing. In this thesis, I describe how I have combined both large-scale and focused approaches to study alternative splicing regulation during development and in an evolutionary context. Using microarray profiling and comparative genomics approaches, I describe the first large-scale comparative analysis of alternative splicing patterns between humans and chimpanzees. Next, I describe the discovery of a novel neural-specific RS domain splicing factor and the network of alternative exons it regulates to promote nervous system development in vertebrates. Finally, I describe the profiling of alternative splicing patterns during C. elegans development using splicing microarrays and high-throughput sequencing. In this latter study, I also describe two resources that facilitate the analysis of tissue- or cell type-specific splicing events, and enable the function of isoforms to be assessed in vivo. Collectively, these studies have shed light on how differential regulation of alternative splicing has contributed to the evolution of complexity and diversity in biological systems.

Keywords: Alternative splicing
genomics
neurobiology
biochemistry
Advisor: Blencowe, Benjamin
Zhen, Mei
Mapping Futures, Making Selves: Subjectivity, Schooling and Rural Youth

This dissertation explores how rural young people imagine their futures in neo-liberal times. The analysis is based upon three months of ethnographic research with grade 7/8 students in ‘Fieldsville,’ a predominantly white and working-class rural community in Southeastern Ontario. I examine students' participation in a widely-used career-education program called The Real Game, in which they are encouraged to become entrepreneurial subjects capable of crafting productive futures in an uncertain world. My study asks: How do these young people produce and perform their imagined future selves, and what does this suggest about the opportunities and constraints that shape their current identities? Integrating insights from feminist poststructural theory and cultural geography, the project extends and challenges studies of the neo-liberal subject by integrating an analysis of place. The thesis builds upon, and contributes to, critical scholarship theorizing young lives as socially, spatially and temporally situated by exploring processes of location within subjectivity formation. Integrating classroom and playground observations with focus groups and interviews, the analysis reveals that young people draw upon diverse discourses in order to envision the person they hope to become. In addition to the subject positions on offer in The Real Game, popular culture provides a key resource in practices of self-making, as students invest in middle-class ideals of the “good life,” and distinguish their own rural location from racialized mappings of urban and global others. Although Fieldsville students are deeply invested in their rural community, tensions emerge where local attachments meet dominant narratives of mobility that encourage them to locate their futures elsewhere. These place-based tensions present particular challenges for girls, who must negotiate the gendered dynamics of rural social space alongside popular discourses of “girl power” that proffer unlimited possibilities for today's young women. Teasing apart the intersections of gender, race, class and space within students' narratives, I argue that studies of neo-liberal subjectivity must examine how dominant discourses are negotiated from particular social and geographical locations. Methodologically, the analysis demonstrates how school-based ethnography can shed light on broader socio-historical processes as they are lived in specific geographical and cultural spaces.

Keywords: subjectivity
rural youth
schooling
space
Advisor: Dehli, Kari