VictorJuiz/thesis huggingface dataset:
The Role of PIDD in Apoptosis and Innate Antiviral Immunity

PIDD has previously been described as a death domain (DD)-containing protein that is inducible upon p53 activation and plays a role in programmed cell death. It has previously been shown that PIDD interacts with RAIDD (RIP-associated ICE/CED3 homologous protein with a death domain) in a cytoplasmic complex known as the PIDDosome, which results in the activation of capsase-2 and ultimately in cell death in response to DNA damage. Despite earlier studies on PIDD, however, the physiological role of PIDD has not been elucidated. Thus, we have generated PIDD-deficient mice and examined its in vivo functions particularly in cell death and in antiviral innate immunity. The first major aim of the thesis is to determine whether or not PIDD is required in cell death. PIDD mice are developmentally normal and do not display a pronounced phenotype. Surprisingly, PIDD deficiency perturbed neither DNA damage-induced nor stress-induced cell death in a variety of cell types, suggesting that PIDD may not play a critical role in cell death. In addition, caspase-2 processing occurred normally in the absence of PIDD in response to ionizing irradiation or etoposide treatment, indicating that PIDD is dispensable in the cleavage of caspase-2. The second major aim is to examine the role of PIDD and RAIDD in LCMV-induced innate immunity. To study the role of PIDD and RAIDD in antiviral immune responses, I have generated PIDD/RAIDD double-deficient mice and challenged them with lymphocytic choriomeningitis virus (LCMV). Interestingly, I observed that ablation of both PIDD and RAIDD together resulted in defective viral clearance in the spleen, but not in other organs including the lung, liver, and kidney. In addition, the production of type I IFN was also decreased in the mice deficient in both PIDD and RAIDD. However, the cytotoxicity of the T lymphocytes was largely intact in the absence of both PIDD and RAIDD. Collectively, our results suggest that PIDD is dispensable in cell death, yet PIDD and RAIDD together have a synergistic effect in LCMV-induced antiviral innate immunity. The findings presented in this thesis provide a better understanding of the physiological role of PIDD and may ultimately contribute to the novel therapeutic strategies for the proper control of viral infection.

Keywords: PIDD
apoptosis
Innate antiviral immunity
caspase-2
death domain
leucine-rich repeats
Advisor: Ohashi, Pamela
High-Throughput Screening for Novel Anti-cancer Radiosensitizers for Head and Neck Cancer

Despite advances in therapeutic options for head and neck cancer (HNC), treatment-associated toxicities and overall clinical outcomes have remained disappointing. Even with radiation therapy (RT), which remains the primary curative modality for HNC, the most effective regimens achieve local control rates of 45-55%, with disease-free survival rates of only 30-40%. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to the surrounding normal tissues, is critical for improving cure rates with RT. Accordingly, we sought to identify novel radiosensitizing therapies for HNC, exploiting a high-throughput screening (HTS) approach. Initially, a cell-based phenotype-driven HTS of ~2,000 commercially available natural products was conducted, utilizing the short-term MTS cell viability assay. Cetrimonium bromide (CTAB) was identified as a novel anti-cancer agent, exhibiting in vitro and in vivo efficacy against several HNC models, with minimal effects on normal fibroblasts. Two major limitations of our findings, however, were that CTAB did not synergize with radiation, nor was its precise cellular target(s) elucidated. Consequently, an alternative strategy was proposed involving a target-driven RNAi-based HTS. Since the colony formation assay (CFA) is the gold standard for measuring cellular effects of radiation in vitro, an automated high-throughput colony-formation read-out was developed as a more appropriate end-point for radiosensitivity. Although successful as a tool for the discovery of potent anti-cancer cytotoxics, a technical drawback was its limited dynamic range. Thus, the BrdU incorporation assay, which measures replicative DNA synthesis and is a viable CFA alternative, was employed. From an RNAi-based screen of ~7000 human genes, uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified as a novel tumor-selective radiosensitizing target against HNC in vitro and in vivo. Radiosensitization appeared to be mediated via tumor-selective enhancement of oxidative stress from perturbation of iron homeostasis and increased ROS production. UROD was significantly over-expressed in HNC patient biopsies, wherein lower pre-RT UROD levels correlated with improved disease-free survival, suggesting that UROD expression could also be a potential predictor for radiation response. Thus, employing a HTS approach, this thesis identified two novel therapeutic strategies with clinical potential in the management of HNC.

Keywords: Head and neck cancer
Radiation therapy
High-throughput screens
Experimental therapeutics
Advisor: Liu, Fei-Fei
Molecular Mechanisms Involved in the Regulation of Circadian Clock Gene and Neuropeptide Transcription: Influence of Palmitate

Canadians live in a society where the sun does not dictate the workday. Our lifestyles must shift to cater to the 24-hour demands of a fast paced global community. As a result our circadian rhythms are altered, leading to dysregulation of key physiological processes responsible for the maintenance of essential functions like energy homeostasis. Energy homeostasis is controlled by neuropeptide-expressing neurons within the hypothalamus. These neurons are affected by circulating hormone and nutrient levels in addition to their endogenous molecular clock machinery that controls cellular processes. Therefore, hypotheses were generated that non-SCN hypothalamic neurons express orexigenic neuropeptides in a rhythmic fashion without external influence from the SCN as a result of internal rhythmicity; and that elevated concentrations of palmitate, a ubiquitous saturated FFA common in a high fat diet, have direct influence on the mRNA levels of circadian clock components Bmal1, Clock, Per2, Rev-erbα and the potent orexigenic neuropeptides NPY, AgRP and ppGhrelin through mechanisms related to HAT, SIRT1 and AMPK. Using the mHypoE-44 neurons, a well characterized cell line that expresses the molecular clock and specific neuropeptides these hypotheses were explored in four studies. Neuropeptide expression within the mHypoE-44 neurons was determined to be rhythmic. NPY and NT demonstrate significant 24-hour rhythms. CRH and ppGhrelin mRNA cycled significantly in an ultradian fashion, oscillating approximately every 18 h. AgRP mRNA did not show a significant rhythm. We identified rhythmic binding of BMAL1 to the NPY promoter, suggesting clock-mediated control of neuropeptide expression. Bmal1 and Clock mRNA levels were elevated with palmitate, whereas Per2 and Rev-erbα mRNA showed significant decreases following palmitate treatment. Palmitate increased the acetylation of both BMAL1 and PER2 proteins. Alteration of AMPK activity altered the mRNA levels of all clock genes assayed and AMPK activation diminished the palmitate-induced changes in Bmal1 mRNA. Palmitate significantly elevated both NPY and ppGhrelin mRNA levels. Chemical modifiers that decrease acetylation altered these systems. AMPK activation reduced the palmitate-induced changes in NPY mRNA levels. These findings demonstrate that non-SCN neurons have rhythmic neuropeptide transcript levels. This thesis elucidates a direct effect of palmitate on the molecular clock and neuropeptide expression at the level of the hypothalamic neuron; and these findings highlight a role for HAT/SIRT1 activation and AMPK in these important processes, which ultimately contribute to the understanding of circadian dysregulation and energy balance.

Keywords: neuroendocrinology
circadian rhythms
hypothalamus
neuropeptides
Advisor: Belsham, Denise
The Molecular Ecology of Hyporheic Zones: Characterization of Dissolved Organic Matter and Bacterial Communities in Contrasting Stream Ecosystems

The aims of this thesis were to characterize the molecular ecology of the hyporheic zone – between dissolved organic matter (DOM) and microbes – and to test whether seasonal and spatial patterns existed in correlation with seasonal ecosystem processes. The hyporheic zone is an area of vertical integration between groundwater and surface water, and lateral integration between terrestrial and stream ecosystems. Colonization corers were used to collect in situ DOM and bacterial communities from the hyporheic sediments of two streams that varied in hydroperiod (i.e., permanent vs. intermittent). DOM was collected using passive samplers and analyzed using 1H NMR and fluorescence spectroscopy; bacteria were characterized using terminal-restriction fragment length polymorphism. At the permanent site, bacteria correlated significantly with seasonal environmental factors including: fall communities with DOM concentration; spring and winter communities with nitrate concentrations; and summer communities with temperature. Bacterial communities at the intermittent site were significantly correlated with flooding as a function of hydrologic connectivity. Sediment communities were discriminated between hyporheic sediments and interstitial porewaters, and shared several operational taxonomic units (OTUs). Sediment communities were more distinct when hydrologic connectivity was low, and porewater communities changed dramatically upon flooding. Fifteen out of 259 OTUs were shared across aquatic sediments, interstitial porewater and watershed soil samples. DOM was spatially and seasonally dynamic in both sites. Five key DOM groups described using 1H NMR spectroscopy revealed spatial differences between the permanent and intermittent sites. EEM-PARAFAC models confirmed that despite significantly different molecular components, the relative sources of DOM at both sites were similar, including humic-like terrestrial sources and tyrosine (microbial) sources. This study provides new knowledge on both organic matter dynamics and bacterial communities in a dynamic aquatic ecotone, and also confirmed the hypothesis that bacterial communities correlated significantly with ecosystem processes within a watershed.

Keywords: 0329
0425
0793
0307
Advisor: Williams, D. Dudley
Modeling Dust Formation in Lime Kilns

Dusting is one of the major problems in the operation of lime kilns because dust particles interfere with kiln operation and reduce its efficiency. A numerical model is developed to predict the rate of dust formation in rotary lime kilns. The model consists of four major components: 1) a 3D model for the kiln gas, solving fluid flow, heat transfer, and combustion in the gas region; 2) a 1D model for the kiln bed, solving for variation of the solids composition, including moisture content, along the kiln; 3) a 3D model to predict the motion of the solids in the bed, and to estimate the reaction rates; 4) a mathematical model to predict the rate of particle pickup from the bed. Additionally, motion of dust particles was modeled, for the first time, using Stochastic Separated Flow model (a Lagrangian approach). The developed model of particle tracking enables the user to predict distribution of dust particles in the gas section of the kiln. Different components of the model were validated using experimental data published in the literature. The developed model was used to simulate operation of a full-scale lime kiln at typical operating conditions, i.e. at different fuel and air flow-rates. Dusting signatures were also estimated for each setting to determine the effect each operating condition has on dusting. The results presented in this thesis indicate that dust formation is mainly affected by the kiln gas velocity. Effect of other operating conditions was found to be negligible within the ranges studied. The results presented here suggest that dust formation can be controlled by minimizing the input gas flow rate.

Keywords: Lime Kiln
Dust
Advisor: Sullivan, Pierre E.
Chip-based Sensors for Disease Diagnosis

Nucleic acid analysis is one of the most important disease diagnostic approaches in medical practice, and has been commonly used in cancer biomarker detection, bacterial speciation and many other fields in laboratory. Currently, the application of powerful research methods for genetic analysis, including the polymerase chain reaction (PCR), DNA sequencing, and gene expression profiling using fluorescence microarrays, are not widely used in hospitals and extended-care units due to high-cost, long detection times, and extensive sample preparation. Bioassays, especially chip-based electrochemical sensors, may be suitable for the next generation of rapid, sensitive, and multiplexed detection tools. Herein, we report three different microelectrode platforms with capabilities enabled by nano- and microtechnology: nanoelectrode ensembles (NEEs), nanostructured microelectrodes (NMEs), and hierarchical nanostructured microelectrodes (HNMEs), all of which are able to directly detect unpurified RNA in clinical samples without enzymatic amplification. Biomarkers that are cancer and infectious disease relevant to clinical medicine were chosen to be the targets. Markers were successfully detected with clinically-relevant sensitivity. Using peptide nucleic acids (PNAs) as probes and an electrocatalytic reporter system, NEEs were able to detect prostate cancer-related gene fusions in tumor tissue samples with 100 ng of RNA. The development of NMEs improved the sensitivity of the assay further to 10 aM of DNA target, and multiplexed detection of RNA sequences of different prostate cancer-related gene fusion types was achieved on the chip-based NMEs platform. An HNMEs chip integrated with a bacterial lysis device was able to detect as few as 25 cfu bacteria in 30 minutes and monitor the detection in real time. Bacterial detection could also be performed in neat urine samples. The development of these versatile clinical diagnostic tools could be extended to the detection of various cancers, genetic, and infectious diseases.

Keywords: Sensors
Diagnosis
Eletrochemistry
Nanotechnology
Advisor: Kelley, Shana O.
Conductive Anodic Filament (CAF) Formation

Conductive anodic filament (CAF) is a failure mode in printed wiring boards (PWBs) which occurs under high humidity and high voltage gradient conditions. The filament, a copper salt, grows from anode to cathode along the epoxy-glass interface. Ready and Turbini (2000) identified this copper salt as the Cu2(OH)3Cl, atacamite compound. This work has investigated the influence of polyethylene glycol (PEG) and polyethylene propylene glycol (PEPG) fluxing agents on the chemical nature of CAF. For coupons processed with PEPG flux, with and without chloride, a copper-chloride containing compound was formed in the polymer matrix. This compound was characterized using x-ray photoelectron spectroscopy (XPS) as CuCl and an electrochemical mechanism for the formation of the chloride-containing CAF has been proposed. For PEG flux, with and without chloride, it has been shown that CAF only formed, but no copper containing compound formed in the matrix. It appears for PEG fluxed coupons, a PEG-Cu-Cl complex forms, binds the available Cu and acts as a barrier to the formation of CuCl in the polymer matrix. Meeker and Lu Valle (1995) have previously proposed that CAF failure is best represented by two competing reactions – the formation of a copper chloride corrosion compound (now identified as Cu2(OH)3Cl) and the formation of innocuous trapped chlorine compounds. Since no evidence of any trapped chloride compounds has been found, we propose that the formation of CAF is best represented by a single non-reversible reaction. For coupons processed with a high bromide-containing flux, bromide containing CAF was created and characterized using transmission electron microscopy (TEM) to be Cu2(OH)3Br. In addition, a copper-containing compound was formed in the polymer matrix and characterized using XPS as CuBr. An electrochemical mechanism for the formation of bromide-containing CAF has been proposed based on the XPS data. .

Keywords: solder flux
printed wiring boards
conductive anodic filament
Advisor: Perovic, Doug
Turbini, Laura
New Icelandic Ethnoscapes: Material, Visual, and Oral Terrains of Cultural Expression in Icelandic-Canadian history, 1875 - Present

This dissertation uses the Icelandic-Canadian community to discuss alternate media and the production of “ethnoscapes,” or landscapes of ethnic identity, on the prairies from 1875 to the present. Drawing from larger historiographies of food, gender, material culture, oral history, and commemoration, it offers an investigation into power, acculturation, and representation using often-marginalized terrains of Canadian ethnic expression. Each of the project’s five chapters examines the cultural history of the community through a different medium. The first chapter uses clothing, one of the most intimate and immediate ways that migrants experienced transition in North America, to explore the impact of poverty, marginalization, disease, climate, and eventual access to Anglo commercial goods on migrant culture. Chapter two analyses the role of food and drink, specifically coffee, alcohol, and vínarterta (a festive layered torte) in everyday life and the development of migrant identity. The third chapter analyses the growth of conservatism and depictions of women in the Icelandic-Canadian community in the twentieth century, with a focus on the decline of radical Icelandic language publications and the rise of ethnic spectacles. Chapter four analyses the impact of centennial and multicultural heritage campaigns on Icelandic-Canadian life, popular narrative, and domestic space by tracing the emergence of the koffort (immigrant trunk) in intergenerational family commemorative practices. Chapter five continues the discussion of popular memory with an examination of the compelling hjátru (superstitious) narrative tradition in the community. It illustrates that Icelandic migrants imported and adapted this tradition to the North American context in a way that also reflected their understanding of colonial violence as an unresolved, disruptive, and damaging intergenerational inheritance. Providing an alternate view of the community beyond either cultural endurance or assimilation, this dissertation argues that the multiple material, visual, and oral conduits through which members have experienced life in the New World have been crucial to the construction of Icelandic-Canadian identity. It is through these terrains that community members have continually engaged with public expectations and demands for both ethnic performance and suppression. The fluidity of these forms and forums and their facilitation of members’ engagement with, adaptations to, and contestation of images of ethnicity and history have enabled the continual construction of Icelandic identities in North America 135 years after departure.

Keywords: Icelandic Canadians
Material culture
Immigration and ethnicity
Scandinavian migration
Manitoba history
Advisor: Iacovetta, Franca
Identifying Targets of ERA1 Involved in Plant Development and Abiotic Stress Signaling

In Arabidopsis thaliana (Arabidopsis), by screening for the inability to germinate on low concentrations of exogenous abscisic acid (ABA), loss-of-function mutations in the β-subunit of a protein farnesyltransferase (FTase) were identified (Cutler et al., 1996). Designated era1-2, these mutants are pleiotropic and show a hypersensitive ABA response at the level of germination and stomatal closure, thereby conferring drought resistance, besides having particular developmental phenotypes (Pei et al., 1998; Bonetta et al., 2000). Although a number of proteins have been shown to be farnesylated in plants, which has provided some insight into how farnesylation regulates various processes, there is still no clear understanding of how loss of farnesylation can confer ABA hypersensitivity, for example. The simplest interpretation is that farnesylation acts as a negative regulator of ABA signal transduction. The primary goal of this thesis is to carry out several reverse genetic screens using a Arabidopsis homozygous T-DNA knockout collection to discover potential targets of farnesylation as well as to determine the overall function of these farnesylated targets in plant growth and development. This included screening for morphological changes related to era1-2, altered responses to ABA at the level of germination, and altered drought responses. In total, 15 unique mutants were identified from the aforementioned reverse genetic screens. A knockout in the gene At3g30180 became particularly interesting for further study since it exhibited several phenotypes that resemble era1-2, including ABA hypersensitivity in germination, drought resistance, protruding carpels, reduced fertility, and round and broadened leaves. At3g30180, otherwise known as CYP85A2, is a cytochrome P450 that mediates the final step in the biosynthesis of brassinolide (BL), a brassinosteroid (Kim et al., 2005). At3g30180 was also identified through a bioinformatic screen (Brady and Provart, 2009; Usadel et al., 2009). Overall, ERA1 positively regulates CYP85A2 function through farnesylation, and therefore BL production, which negatively regulates ABA signaling.

Keywords: farnesylation
Arabidopsis
Advisor: McCourt, Peter
The Effect of Lactobacillus rhamnosus GR-1 Supernatant on Cytokine Production and Prostaglandins in Gestational Tissues

Preterm birth remains a major challenge in obstetrics. It complicates up to 13% of all pregnancies and accounts for approximately 80% of neonatal mortality and morbidity. Bacterial Vaginosis (BV) is associated with a 1.4-fold increased risk of preterm birth. Due to ineffectiveness of antibiotics in preventing preterm labour, probiotics have been proposed to serve as an alternative for treatment of BV and prevention of preterm birth. The objectives of this thesis were to determine 1) the effect of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) supernatant on cytokine profile and prostaglandin (PG)-regulating enzyme expression in lipopolysaccharide (LPS)-stimulated human chorion and placental trophoblast cells from human placentae, 2) the potential signaling pathways through which lactobacilli act and 3) the potential role of immune and placental trophoblast cells in initiating a response to LPS and L. rhamnosus GR-1 treatments. Primary cultures of human placental trophoblast cells were pre-treated with lactobacilli supernatant and then with LPS. In addition, immune cells were removed from cell suspensions using a magnetic purification technique to determine their role in modulating cytokine levels. The expression of pro- and anti-inflammatory cytokines and prostaglandin-regulating enzymes was then determined. We found sex-specific differences in the ability of LPS to increase the output of TNF-α, IL-10, and PTGS2. We also showed that L. rhamnosus GR-1 is able to act through the JAK/STAT and MAPK pathways to increase IL-10 and G-CSF, and independently down-regulates PTGS2 and TNF-α and up-regulates PGDH. The increase in G-CSF and PGDH were only observed in women carrying a female fetus. L. rhamnosus GR-1 may serve as an alternative to antibiotics in preventing some infection/inflammation-mediated cases of preterm birth.

Keywords: Preterm Birth
L. rhamnosus GR-1
Cytokines
Prostaglandins
Bacterial Vaginosis
Fetal Sex
Advisor: Bocking, Alan
Challis, John R. G.