VictorJuiz/thesis huggingface dataset:
SCRIBBLE Interactome and Proliferation in Breast Cancer
Keywords: Leucine Rich Repeat
Mammary Gland
Protein-protein interactions
Scribble
Transgenic Mouse Model
Tumourigenesis

Disruption of ductal epithelial organization in the mammary gland during carcinogenesis of the breast is a hallmark and frequently utilized clinical marker of invasive ductal breast cancer. The Scribble (SCRIB) polarity protein is required to regulate organization and distribution of proteins in epithelial cells. Specifically, in the normal mammary gland, SCRIB maintains ductal organization, by acting as a molecular scaffold to assemble regulatory components of signaling pathways. During breast cancer, SCRIB is frequently mislocalized and overexpressed. Mouse models have suggested that overexpressed mislocalized SCRIB is sufficient to cause mammary tumourigenesis, with the development of tumours with basal characteristics and hyperactivation of S6 kinase. With the aim of defining SCRIB function, studies have identified individual interactors for SCRIB, however, the SCRIB interactome in the mammary epithelium remains uncharacterized. My research has validated and characterized a unique interaction between SCRIB and the sub-apical AF6 polarity protein, that arose from our unbiased proteomic screen for SCRIB interactors in mammary epithelial cells. I have shown that this interaction can regulate proliferation/survival of three-dimensional acini under nutrient stress, and loss of this interaction in mammary epithelial cells can compromise p85-S6K phosphorylation and RHEB-GTP hydrolysis. Second, in a mouse model of mammary-specific Tp53 loss, overexpression of mislocalized SCRIB did not accelerate Tp53-loss driven mammary tumourigenesis, and in fact, may increase the latency of mammary tumourigenesis and lung metastases, suggesting that mislocalized SCRIB may retain some tumour suppressive function. Finally, I have screened and identified several cancer-associated mutations in the SCRIB leucine rich repeat region (LRR) that result in mislocalization of a SCRIB-RFP reporter construct, suggesting that acquisition of point mutations in the LRR of SCRIB is a physiologically relevant means of SCRIB mislocalization in tumourigenesis. These studies suggest that novel SCRIB interactions may modulate cell survival under certain conditions, such as nutrient stress and growth factor stimulation, while SCRIB mislocalization is a cancer-associated event and may be attributed to tumourigenesis in specific genetic contexts.
Cellular and Molecular Mechanisms of Transient Receptor Potential Melastatin 7 (TRPM7) Channel in Neuronal Development and Injury
Keywords: Cytoskeleton
Hypoxia
Ion channel
Neuronal development

Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed, calcium-permeable ion channel that regulates intracellular levels of divalent cations, cytoskeletal dynamics, cell adhesion and other processes. As these processes are critical for neurite elongation during development, this thesis investigated the role of TRPM7 in neurite outgrowth and development using a pharmacological approach. It was found that blocking TRPM7 activity preferentially enhanced axonal outgrowth of cultured hippocampal neurons at least in part through TRPM7 interaction with cytoskeletal proteins, actin and α-actinin. TRPM7 activity is potentiated under the conditions of stress that commonly occur in injury or disease, such as increased extracellular acidity and production of reactive oxygen species. Therefore, the effect of hypoxia, a common cause of damage to the developing brain, on TRPM7 activity and TRPM7-mediated neurite outgrowth was investigated next. It was found that prolonged hypoxia caused axonal retraction, while blocking TRPM7 activity attenuated this effect. Finally, the therapeutic potential of TRPM7 blocker has been evaluated in a mouse model of neonatal hypoxic-ischemic brain injury, and blocking TRPM7 pharmacologically had neuroprotective and therapeutic effects (up to 1 hour after injury). Further short and long-term evaluation demonstrated preservation of brain morphology and functional motor and memory outcomes. Proteomic analysis revealed that TRPM7 may mediate neuronal injury at least in part through calcium-dependent cytoskeletal regulation by calcium/calmodulin-dependent kinase and phosphatase. Overall, this thesis establishes the role of TRPM7 in neuronal outgrowth and development under normal and stress conditions. Moreover, it evaluates a specific pharmacological inhibitor of TRPM7 as a candidate for further drug development for hypoxic-ischemic injury to the developing brain.
Building New Senses for Yeast: Responding to Light and Inflammation with S. Cerevisiae
Keywords: G protein-coupled receptors
Protein engineering
Purinergic signaling
Rhodopsin
Synthetic biology

G protein-coupled receptors (GPCRs) detect a diverse range of biologically relevant signals, including light which enables us to see, and molecules relevant to disease which enables our immune system to respond accordingly. These crucial functions can be disrupted by mutations in GPCRs, often contributing to disease. The function of GPCRs can also be harnessed in synthetic gene circuits, to sense and respond in a programmed manner. In this thesis, I apply synthetic biology methods to the study of GPCRs, to determine how mutations contribute to disease, how GPCRs can be engineered to grant new function, and designing synthetic gene circuits harnessing GPCR function. In Chapter 2, I present novel strains of S. cerevisiae that functionally couple human rhodopsin to the yeast mating pathway. This provided a high-throughput method to rapidly characterize mutations implicated in inherited retinal disease. In Chapter 3, I apply directed evolution methods to engineer the human purinergic GPCR P2Y2, to improve its natural ability to sense and respond to extracellular ATP. A synthetic gene circuit was devised in yeast, utilizing these novel engineered P2Y2 variants, so that pro-inflammatory concentrations of extracellular ATP elicited secretion of anti-inflammatory proteins (apyrase and IL-10). In Chapter 4, I again focus on rhodopsin, and engineer additional strains of yeast to enable high-throughput drug screening to discover novel drugs which may help to rescue misfolded mutants of rhodopsin. I developed screening methodology, and performed an initial screen of 4280 compounds, characterizing one lead compound in detail. In Chapter 5, I expanded the use of light-activated rhodopsin in yeast to perform deep mutational scanning. Large libraries of mutant human rhodopsin were generated and sorted for function, assigning functional relevance to >1200 mutations. Regions and residues in rhodopsin known to be required for function were confirmed in my analysis, in addition to many novel mutations that have not previously been studied. Synthetic biology methods and concepts enabled a diverse range of yeast-based studies of human GPCRs, which are presented in this thesis. More broadly, this thesis combines studies of human mutations implicated in disease, with the engineering of cellular receptors and pathways to treat disease.
Functional and Structural Characterization of human Rvb1 and Rvb2 and Screening for Inhibitors of their ATPase Activity
Keywords: ATPase
Rvb1
Rvb2

Rvb1 and Rvb2 are highly conserved and essential eukaryotic AAA+ proteins linked to a wide range of cellular processes. AAA+ proteins are ATPases associated with diverse cellular activities. Both proteins have been found to be part of critical cellular complexes and processes in many different species: the chromatin remodeling complexes Ino80 and SWR-C, the telomerase complex, mitotic spindle assembly, as well as phosphatidylinsitol 3-kinase-related protein kinase (PIKK) signaling. Rvb1 and Rvb2 are linked to DNA damage sensing and repair, transcriptional regulation and cell growth, therefore, implicating them in carcinogenesis. Due to wide range of Rvb1 and Rvb2 involvement, their pivotal roles and overexpression in several cancers, investigating their molecular structure and activity will aid in understanding their physiological function and explain their multifaceted role. This project sheds light on the structural and functional characteristics of human Rvb1 and Rvb2 that have been highly controversial as per several independent studies. Rvb1 and Rvb2 were shown to form a hetero-hexameric ring complex that exhibits enhanced ATPase activity in comparison to the individual proteins, with WB motif of both subunits being essential for activity. Our investigation of human Rvbs’ oligomerization using multiple techniques has shown that an N-terminal tag induced dodecamerization of human Rvb1/Rvb2 complex and affected the oligomerization of human Rvb2 alone. A mutation in the DLLDR motif was found to cause a significant enhancement in the ATPase activity of human Rvb2. In addition, our high-throughput screen for inhibitors of the ATPase activity of human Rvbs led to the discovery of sorafenib as a weak inhibitor of human Rvb2 and human Rvb1/2 complex. Sorafenib was characterized to be a mixed-non competitive inhibitor of human Rvb2 with an IC50 of 3.1 µM and Kd of ~ 22 µM. Our work suggests that sorafenib binds to human Rvb2 through the insertion domain (DII) as shown by SPR and ATPase assays, and that it possibly has a physiological role in the SHQ1-DKC1 disassembly. This work advances our understanding of human Rvb1 and Rvb2 and lays out a strong foundation for targeting their ATPase activity as a strategy for the identification of novel cancer therapeutics.
Canonizing Le Corbusier: The Making of an Architectural Icon as Colonial Hegemony
Keywords: Colonialism
Hegemony
Le Corbusier
Modern Architecture
Pedagogy

In 2016, it was announced that iconic Franco-Swiss modern architect, Le Corbusier, and his nearly complete oeuvre of seventeen buildings, would be enshrined on the United Nations Educational, Scientific and Cultural Organization (UNESCO)’s World Heritage List. With this notable decision, Le Corbusier, the figure, moved from beatification as a “modern master” to achieving the architectural status of canonical sainthood. No other architect had been so completely enshrined on UNESCO’s list. The discussions and debates centered on this landmark decision provide evidence regarding how and why Le Corbusier’s figuration persists and is sustained. The Corbusiern figure remains foundational in architectural pedagogy, modern architectural history text writing and in the imaginaries formed in aspiring students of architecture as a global project that is both colonial and hegemonic. How this decision to reify Le Corbusier came about after a decade of debate and deliberation provides the basis for this project. The argument suggests ways to explain how we continue to tell and perform the history of modern architecture today, using Le Corbusier as a figure, as well as the legacy of the Bauhaus and International Style modernism, as pedagogical tools to perpetuate an ongoing hegemonic colonial regime situated in architectural knowledge production and pedagogy.
Increasing Plant Reliability of Offshore Gas Installations
Keywords: Fuzzy Logic Theory
Human Error
Maintennace Improvement Factor
Preventive Maintenance
Simulation Model
Survey

The maintenance improvement factor (IF), which provides a mathematical description of maintenance efficiency, can be used to determine the probability of component failure, the foundational block for calculating the optimal preventive maintenance interval. The IF can be estimated via probabilistic analysis of failure data. However, this body of data does not usually incorporate subjective information such as procedure compliance, the quality of spare parts, or the time available for completing an activity. Subjective information provides vital insight into the quality of maintenance. Since poor quality maintenance is assumed to promote component failure in the short term, an IF incorporating subjective information can improve estimates of the probability of component failure. The required information can be provided by maintenance experts. Unfortunately, the literature has shown that their judgment is inherently biased, and this diminishes the accuracy of the IF estimate. This thesis provides a framework within which to estimate a bias-free IF, referred to here as the expert improvement factor (EIF). The EIF is obtained from a combination of an expert interview and a survey administered to maintenance personnel; the survey incorporates questions regarding factors that promote human error in maintenance. The optimal maintenance interval, of a system consisting of two components, based on the EIF was estimated using a simulation model. The model suggests that considerable savings in the total maintenance cost can be realized by estimating the optimal preventive maintenance interval based on the EIF. The current thesis also proposes a second framework that can be applied to determine the effect of reducing the factors considered in the survey, providing a managerial tool for reducing the total maintenance cost over the long term. As an application of the first framework, a specific framework was developed for estimating the optimal maintenance interval for multiple components in a real-world offshore oil and gas installation.
Analyzing Heterogeneity in Cell Images Using Machine Learning
Keywords: 3D Image Analysis
Cell Heterogeneity
Cell Imaging
High Content Screening
Machine Learning
Microscopy

At very fine resolution the stochastic nature of gene expression and penetrance leading to phenotypic heterogeneity among individual cells. This heterogeneity results in differences in cellular responses to environmental and metabolic cues leading to morphological heterogeneity. Thus, to understand the fundamental biological processes, it is necessary to identify and quantify such morphological heterogeneity. This is important especially in diseases such as cancer where tumor heterogeneity plays a significant role in cancer therapy. The goal of this research is to develop image analysis methods to quantify heterogeneity in cell images from monolayer as well as three-dimensional (3D) culture systems. A large database of cell images displaying localization of EGFP fused marker proteins to different organelles (landmarks) was used as a biological model for heterogeneity in monolayer cultures. The landmark images were used to develop techniques to deal with focus, segmentation and expression artifacts as well as to assign subcellular localization to 995 tail anchor proteins with unknown localization and deduce sequence motif required for tail anchor proteins to localize to different organelles. The large set of images were further used to quantify heterogeneity within landmarks and construct a map of protein trafficking that can be used to quantify localization dynamics. For 3D cultures, clonally derived premalignant human epithelial cell (MCF10A) organoids expressing different oncogenes were used as a model system. Typically, images of 3D organoids are analyzed by projecting them on a single plane using maximum intensity projection and using conventional algorithms that were developed for monolayer cultures resulting in the loss of information from the third dimension. To overcome the limitations, I have developed a novel 3D segmentation free image analysis algorithm and applied it to quantify heterogeneity in MCF10A derived organoids expressing different oncogenes. I have further applied this algorithm to quantify response of primary cortical neuronal cultures to different anti-apoptotic inhibitor compounds. The techniques developed in this thesis can be broadly applied to quantify heterogeneity in other biological systems.
Neurobiology of Predation Risk and the Inheritance of Stress in Snowshoe Hares (Lepus americanus)
Keywords: Boreal
HPA axis
Maternal effects
Neurobiology
Predator-prey
Stress

The population dynamics of snowshoe hares (Lepus americanus) are fundamental to the ecosystem dynamics of Canada’s boreal forest. During their 8-11 year population cycle, hare densities can fluctuate up to 40-fold. Predators in this system (lynx, coyotes, great-horned owls) affect hare demography not only through direct mortality but also through non-consumptive effects on reproduction that are mediated by chronic stress. There is strong evidence for the acute physiological sensitivity of snowshoe hares to fluctuating predator pressure, characterized by major shifts in glucocorticoid profiles, energy mobilization, immunity and body condition under situations of extreme risk. This thesis aimed first to determine whether the predator-induced variation in hare stress physiology seen across the cycle is associated with predictable neurobiological changes in transcriptional activity and the expression of corticosteroid receptors in key limbic regulatory regions of the hypothalamic-pituitary-adrenal (HPA) axis, and second, whether predation risk has intergenerational impacts on offspring phenotype that may act to prolong the lag in population recovery that follows the decline phase of the cycle, even though food resources are plentiful and predators have all but disappeared. I found changes in the expression of gene networks that mediate hormone responses and hematological condition, and in the expression of glucocorticoid-sensitive receptors that regulate the HPA axis as a function of increasing predation risk in the natural population. During the increase and peak phases of the cycle, predator-induced maternal stress programmed for a neuroresilient offspring phenotype characterized by key transcriptional changes that allow for sensitive physiological responses to environmental stress through activation of the amygdala, balanced by gene activity that confers neuroendocrine resilience and maintains basal HPA function in the hippocampus. Offspring stress physiology was associated with a number of anti-predator behaviours that could confer an initial survival advantage in risky environments, but become maladaptive when conditions change. Taken together, these results provide support for predator-induced maternal stress effects on offspring phenotype that could become important mediators of the lag in population recovery under conditions of chronic maternal stress.
Feeling Real: Affective Dimensions of Reality in Contemporary Canadian Performance
Keywords: affect
audience
Canada
reality
spectatorship
theatre of the real

Taking as its central focus the question, “What feels real to contemporary audiences?” this project investigates the use, production, and perception of realness in current Canadian performance practices. By considering the tools for and effects of realness present in three case studies, this thesis examines how audience members locate realness within a show’s unique temporal and spatial dimensions. Such an examination of the “real” destabilizes the arguably foundational binary between reality and fiction and illustrates the complexity of determining what constitutes realness in contemporary culture. The first chapter contains an overview of the concept of realness in performance, establishes the utility of feeling and affect as modes for investigating realness, and concludes with an argument for audience-led research into perceptions of realness. The second chapter focuses on Downstage Theatre Company’s Good Fences to argue that the show’s use of multivalent spaces and times results in the presentation of multiple truths; making space for audience members to debate, discuss, and compare perceptions of what is real. The third chapter considers Rising Tide Theatre’s Trinity Pageant and its use of site-specificity and historical re-enactment to outline how the presentation of a blended temporality ultimately inspires audiences to act to preserve Newfoundland culture. Through comparison with other immersive theatre projects, the final chapter considers how Outside the March’s TomorrowLoveÔ’s unique composition requires audience labour that goes beyond the necessary physical navigation of an immersive space and the immediacy of sensory exploration. The chapter concludes that the show’s provocation of real feelings in audience members, as a form of affect-labour, lends to the perception of the show itself as real. The case studies illustrate the ways in which performances cultivate “real-ish-ness”: a term that attempts to capture the fragmentary, partial, complex feeling that is generated by and through the act of engaging elements of reality. This thesis thus provokes a consideration of the ways in which performances of theatre of the real impact and complicate reality in ways pressingly important for a contemporary culture both excited by and anxious about advancements that bring us closer to a blurred line between the real and fictive.
Discovery-Based Transcriptome and Translatome Profiling Reveals Molecular Pathways in the Pathogenesis of Alzheimer’s Disease
Keywords: Alzheimer's disease
cerebrospinal fluid
exosomes
microRNA
transcriptome
translatome

Alzheimer’s disease (AD) is a complex, multifactorial neurodegenerative disease of aging that results in a cascade of spatiotemporally heterogeneous gene expression changes. Limbic lobe regions such as the amygdala, entorhinal cortex, hippocampus, and posterior cingulate cortex (PCC), along with cholinergic neurons of the basal forebrain are particularly susceptible early in AD. Comprehensively characterizing these early changes is a critical area of pre-clinical and clinical study. In this thesis, we hypothesized that discovery-based transcriptional and translational profiling approaches would uncover previously unidentified pathways causing neurodegeneration in Alzheimer’s disease, as well as the potential contribution of the aging brain to the disease process. We combined novel RNA extraction techniques, mouse models, young-onset AD (YOAD) patient biofluids, published transcriptome data, and bioinformatics to investigate limbic brain region and cholinergic neuron vulnerability in AD. We first demonstrated that mRNA and microRNA (miRNA) expression is altered during young adulthood in the amygdala, entorhinal cortex, and hippocampus of wild-type mice. While minute changes were observed in the mRNA transcriptome across these regions with age, the miRNA expression profile converged on FoxO signaling and long-term potentiation pathways. The amygdala demonstrated the most robust age-related miRNA expression changes, enriching for GABAergic synapses and axon guidance pathways. Next, we sequenced the actively translating mRNAs in anterior forebrain cholinergic neurons captured by translating ribosome affinity purification (TRAP) in a rapid onset mouse model of AD (TgCRND8). The cholinergic translatome showed long-term potentiation, axon guidance, and neurotrophin pathway changes which distinguished the immune, complement, and microglial response evident in the anterior forebrain transcriptome, a frequent observation in post-mortem AD brain and AD mouse model transcriptomes. Third, we uncovered four miRNAs altered in the cerebrospinal fluid-derived exosomes from sporadic YOAD patients versus healthy controls (miR-16-5p, miR-125b-5p, miR-451a, and miR-605-5p). These miRNAs were predicted to target many pathways reflected in the sporadic YOAD post-mortem PCC and also overlapped with the cholinergic translatome and anterior forebrain transcriptome of TgCRND8 mice, including long-term potentiation, axon guidance, and immune system pathways. These collective findings provide insight into the shared and divergent molecular alterations affecting limbic regions and neuron subtypes throughout aging and early stage AD pathogenesis.