VictorJuiz/thesis huggingface dataset:
Transcriptome-wide Measurements of mRNA Stability

Numerous regulatory processes work at the level of RNA to ensure proper control of gene expression. Of these, transcription and decay are particularly important as together they determine the abundance of a given transcript. Several methods have been developed over the last two decades to interrogate the stability of transcripts genome-wide. Despite inherent limitations, methods based on transcription inhibition remain popular. I have developed two redundant normalization methods for in vivo metabolic labeling time course data generated by high-throughput RNA sequencing (RNA-seq) that will make such experiments more accessible. I demonstrate that the use of exogenous whole organism spike-ins can help overcome transcript specific biases by averaging the behavior of thousands of exogenous transcripts. Additionally, I demonstrate that internal short-lived RNA species, e.g. introns, can be used to normalize the same time course data, often yielding more reproducible results than those obtained by normalizing to exogenous spike-ins.

Keywords: 4-thiouridine
DRUID
introns
metabolic labeling
RNA degradation
transcription inhibition
Advisor: Rissland, Olivia S
Evolution and epigenetic control of acute inflammation in mammalian endothelial cells

Vascular endothelial cells (ECs) line the interior of blood vessels and respond to inflammation through a series of well-characterized chemokines and receptors. Less is known about the cis-regulatory elements (CREs) that control the endothelial inflammatory response in humans and other mammals. The NF-κB complex, in particular its subunit RELA (p65), is an essential inflammatory mediator in ECs. To study the evolution and function of endothelial acute inflammatory response, we characterized RELA binding sites and epigenetic changes that occur after TNF-α treatment in primary aortic ECs isolated from human, mouse, and cow. We discovered ~5000 RELA binding sites at orthologous regions in all three species near genes involved in vascular development and pro-inflammatory responses. The conserved RELA binding sites were enriched for known regulatory disease mutations. We established a CRISPR/Cas9 gene editing approach in an immortalized human aortic EC line and used this platform to study the function of TNF-α-induced CREs.

Keywords: cis-regulatory elements
CRISPR/Cas9
endothelial cells
evolution
inflammation
NF-kB
Advisor: Wilson, Michael D
The Role of an Autoimmune Disease-Associated Phosphatase in Dendritic Cell Biology

PTPN22 encodes the human lymphoid tyrosine phosphatase, Lyp, a protein exclusively expressed in hematopoietic cells and involved in numerous signaling pathways. Lyp has been of great interest since the discovery of a variant affiliated with multiple autoimmune diseases. The variant, PTPN22-C1858T, has been investigated in human and animal models with conflicting results. Some groups claim the variant causes a gain-of-function effect while others claim a loss- of-function effect. This study sheds light on how the variant influences the dendritic cell (DC) network, a network comprised of multiple subpopulations, each with specific and overlapping functions. Here we show mice expressing the orthologous variant have abnormal splenic DC frequencies and distribution, with an increase in the CD4+ conventional type 2 DC population. Furthermore, all DC subpopulations investigated consistently displayed increased expression of CD40, indicating a hyperactive phenotype. Additionally, bone marrow-derived DCs produced more IL-12 after TLR9 stimulation and collectively these factors may contribute to autoimmune disease.

Keywords: Autoimmune Disease
Dendritic Cells
Genetics
Mouse model
Phosphatase
Advisor: Siminovitch, Katherine
Sequencing Effects in Memories of Online Video Technical Quality

While researchers have shown that memories of experience diverge from actual experience, little is known about how variations in online video service quality impact overall ratings of Technical Quality (TQ), satisfaction, and frustration after a sequence of videos is viewed. Additionally, it is yet to be determined how accumulated experiences of TQ may impact customer loyalty measures like Likelihood-to-Recommend (L2R) and Likelihood-to-Churn (L2C). This thesis addresses these gaps in the literature by conducting two experiments that vary the sequencing of videos with good, mediocre, and bad TQ. In the first experiment, a negative end effect was found. The second experiment found the negative end effect as well as an effect of linear trend (both increasing and decreasing). Overall ratings of L2R and L2C at the end of a one-hour viewing session suggest that satisfaction is the closest predictor to significance of L2R while frustration is the best predictor of L2C.

Keywords: Customer loyalty
Peak-end effect
Quality of Experience
Sequencing
Technical quality
Video streaming
Advisor: Chignell, Mark
Measuring Cerebrovascular Pulsatility Using Cardiac Cycle Fluctuations of fMRI BOLD Data

Arterial pulsatility is linked to cerebral small vessel damage and neurodegeneration, but measuring cerebrovascular pulsatility in humans has largely been impeded by the skull. This thesis describes a method that generates cerebrovascular pulsatility maps based on resorting blood-oxygenation level dependent (BOLD) volumes according to their cardiac cycle position. Sensitivity of this method was tested using 20 minutes of moderate-intensity exercise as an acute physiological stressor in 45 healthy adolescents. Further examinations evaluated the influence of repetition time (TR) and echo time (TE) via simulation and multi-echo data, respectively. There were global, tissue-specific, and region-specific decreases in cerebrovascular pulsatility 20 minutes following exercise cessation. Cardiac-related pulsatility detection was comparable over a range of TR and TE values, with highest detection during rapid TRs (â ¤300ms) or shorter TE (~14ms). These results suggest that cardiac-related fMRI may represent a potent and easily adoptable method of mapping cerebrovascular pulsatility influences with voxel-wise specificity.

Keywords: aerobic exercise
BOLD fMRI
cardiac cycle
cerebrovascular pulsatility
physiological fluctuations
Advisor: MacIntosh, Bradley J
Cardiofaciocutaneous Syndrome-Associated BRAF Mutants in Development and Cancer

Cardiofaciocutaneous syndrome (CFCS) is an autosomal-dominant disorder caused by germ-line mutations in members of the RAS/ERK signaling pathway, most commonly in BRAF. Selected mutations that either activate or impair BRAF kinase activity and that are known to cause CFCS have also been identified in melanoma. I found that knock-in mouse mutants expressing BrafK499E (kinase-activating) and BrafG469E (kinase-impaired) alleles have many features of CFCS, including short stature, craniofacial dysmorphia, and an increased heart weight to body weight ratio. Studies of the phenotype of BrafK499E mice show that the effects of this mutation might be affected by genetic modifiers on the 129/B6 mouse background. Endogenous expression of BrafG469E in mouse embryonic fibroblasts (MEFs) caused increased MEK and ERK activation compared to wild-type MEFs . These effects correlated with increased heterodimerization of mutant BRAF with RAF1, as assessed by co-immunoprecipitation. Finally, I found that BrafK499E expression induced post-natally in vivo in melanocytes is not sufficient to cause hyperpigmentation or nevus formation, unlike the highly activated melanoma-associated allele BrafV600E .

Advisor: Neel, Benjamin G
Investigating the Role of the Vascular Endothelium in Insulin Delivery

Insulin delivery is an integrated process that first requires perfusion of the capillary bed supplying the tissue, followed by the transit of insulin across the endothelium that lines each vessel. Methodological limitations have hindered the ability to study transendothelial insulin transfer in isolation of hemodynamic forces that also promote the delivery of the hormone. We aimed to determine whether or not the aortic endothelium represented a rate-limiting barrier to insulin transit towards the underlying smooth muscle. Using an ex vivo approach, we determined that the aortic endothelium may not be rate-limiting to insulin action. Two permeability-inducing agents (histamine or platelet activating factor) induced inter-endothelial gap formation, but had no effect on insulin action in the aorta. Together, these data demonstrated that the aortic endothelium may not be a rate-limiting barrier to insulin delivery.

Keywords: diabetes
endothelium
insulin
macrovasculature
microvasculature
Advisor: Lee, Warren L
Klip, Amira
A Population-Based Study of the Impact of Pregnancy on Breast Cancer Mortality in Women in Ontario.

Population-based, retrospective cohort study, linking health administrative databases of Ontario, Canada, comprising 7,553 women aged 20 to 45 years with invasive breast cancer, from January 1, 2003, to December 31, 2014. Women were classified into four exposure groups: no pregnancy (n = 5,832; the referent), pregnancy before breast cancer (n = 1,108), pregnancy-associated breast cancer (n = 501), and pregnancy following breast cancer (n = 112). The 5-year overall survival rate was 87.5% for women with no pregnancy (the referent); 85.3% (age-adjusted HR, 1.03; 95%CI, 0.85-1.27; P = .73) for women with pregnancy before breast cancer; 82.1% (age-adjusted HR, 1.18; 95%CI, 0.91-1.53; P = .20) for women with pregnancy-associated breast cancer; and 96.7% (age-adjusted HR, 0.22; 95%CI, 0.10-0.49; P

Keywords: Breast cancer
Pregnancy
Survival
Women's Health
Advisor: Narod, Steven A
Rochon, Paula A
Assessing the Therapeutic Potential of CRISPR/Cas9-Mediated Gene Modulation in Merosin-Deficient Congenital Muscular Dystrophy Type 1A

Merosin-deficient congenital muscular dystrophy type 1A is an autosomal recessive disease caused by mutations in the LAMA2 gene, which codes for the laminin ⍺2 chain of the laminin-211 cell adhesion molecule. MDC1A is the most common type of congenital muscular dystrophy. MDC1A patients exhibit severe hypotonia, progressive muscle weakness and wasting, delayed developmental milestones and often die prematurely. Lama1 is a potential disease modifier gene for MDC1A that could be modulated to treat or prevent the disease phenotype. The Lama1 gene encodes the laminin ⍺1 chain protein, which is structurally similar to laminin ⍺2 chain. We hypothesize that using the S.aureus CRISPR/Cas9 system, we will be able to upregulate expression of Lama1 and compensate for the deficiency of laminin ⍺2 chain in mice to re-establish the integrity of muscle fibers. We have evaluated the effectiveness of local intramuscular and systemic delivery of CRISPR/Cas9 components to modulate Lama1 expression in vivo to decrease disease severity in laminin alpha 2 deficient dy2j/dy2j mice. Successful results from this project will provide evidence to develop CRISPR/Cas9-mediated gene modulation as a therapeutic option for patients with MDC1A.

Advisor: Cohn, Ronald D
Identifying interacting ligands of human RORγ using transgenic zebrafish

The retinoic acid receptor-related orphan receptor gamma, or RORγ, is a member of the nuclear receptor ROR family and its activity can be regulated by the binding of ligands. Our lab has developed an in vivo GFP reporter system, the ‘Ligand Trap’ system, that can be incorporated into the zebrafish genome to identify ligands that target RORγ. I have performed a drug screen, using human RORγ Ligand Trap zebrafish, against a compound library that includes around 4000 natural compounds and approved drugs. 20 hits were identified in the screen, with an antagonist chosen for further characterization: echinocystic acid (EA). Studies were performed to show that EA acts as an antagonist of RORγ by binding to RORγ to inhibit transcription of RORγ target genes. In the future, EA can be used to identify ligand specific cofactors and target genes that are associated with RORγ.

Advisor: Krause, Henry
Development of Site-Specific CFTR Gene Integration Tools for Testing Gene Editing in Pig Cells

Development of Site-Specific CFTR Gene Integration Tools for Testing Gene Editing in Pig Cells Liang Yang Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2017 Abstract Cystic Fibrosis (CF) is a genetic disorder caused by autosomal recessive mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. In this study, we proposed a novel gene therapy strategy to integrate a transgene expression cassette into GGTA1 locus utilizing the precise genome cleavage capability of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) and the large packaging capacity of Helper Dependent Adenoviral vector (HD-Ad). Using a LacZ reporter gene expression cassette, we determined this novel strategy can achieve stable and sufficient integration in the pig IPEC-J2 cell line. In addition, the CFTR transgene mRNA and protein can be successfully detected post HD-Ad delivery. Future experiments include investigating the CFTR functional correction and the impact of enhancing homology directed repair (HDR), which is the major pathway we rely on for transgene integration, on integration efficiency and assess CFTR transgene functional corrections.

Keywords: CFTR
CRISPR/Cas9
Cystic Fibrosis
Gene therapy
Helper-Dependent Adenoviral vector
Advisor: Hu, Jim
A Starvation-Induced Heritable Stress-Resistant State in Saccharomyces cerevisiae

Transient exposures to diverse stimuli induce altered physiological properties in exposed cells that persist after the stimulus has been removed. This state, referred to as cellular memory, can be passed on to daughter cells. In the budding yeast Saccharomyces cerevisiae, I found that meiotically produced yeast spores possess a state of heightened stress resistance that, following their germination, is propagated through their descendants. As yeast meiotic development is essentially a starvation response that diploid cells engage, I modelled this phenomenon by subjecting haploid cells to starvation conditions. Haploid cells exposed to glucose withdrawal acquire elevated hydrogen peroxide resistance following the reintroduction of the cells to glucose-replete-media. In both haploid starved cells and diploid produced meiotic spores I show that their cellular memories are not attributable to trehalose, a proposed stress protectant. My findings identify nutritionally induced states of cellular memory in yeast that exhibit striking degrees of persistence and inheritance.

Keywords: Heritable
Starvation
Stress Resistance
Advisor: Meneghini, Marc D
Computational Simulations of Fenestrated Endovascular Aneurysm Repair

Fenestrated endovascular aneurysm repair is a minimally invasive method to treat abdominal aortic aneurysms. Stent grafts used in this process contain fenestrations that need to be aligned with the visceral arteries; however, fenestration misalignment (rotation) is a common occurrence and can result in severe complications. The objective of this work is to develop a finite element model of the deployment of these devices to predict potential rotation. As a first step, a model for simulating the response of iliac arteries to stiff guidewire insertion is presented and validated against clinical data. This model is then extended into a model of the entire stent graft deployment process and validated against six clinical cases, with an average error of 17%. Finally, a parameterization study is undertaken as a means of potentially minimizing or eliminating device rotation. Simulated rotation values are more than halved by modifying various coefficients of friction.

Advisor: Amon, Cristina H
Forbes, Thomas L
Optimization of Polymerase Chain Reaction Machine

In this thesis, a numerical investigation was carried out on Polymerase Chain Reaction (PCR) machine. PCR is used in a wide variety of industrial applications. One of its main applications is in biomedical field where it is used to multiply DNA molecules. To duplicate DNA samples with a high quality and precision, the temperature uniformity on DNA samples plays a significant role. To study the temperature field, a three-dimensional Computational Fluid Dynamics (CFD) model of PCR device was developed in ANSYS CFX. In this study, initially, both the denaturation and post elongation steps were simulated in the complete PCR device, where temperature uniformities of 0.36â and 0.06â were obtained for these steps, respectively. Subsequently, only the air duct and heat sink sections of the PCR device were considered in the simulations to investigate the effect of both the fin length and the gap between the heat sink and air duct on heat removal performance of the device. It was found that by removing the gap between the heat sink and the air duct and cutting the heat sink in half, the efficiency of the heat sink will increase. Finally, an experimental investigation was carried out to compare the performance of aluminum metal foams with regular aluminum heat sinks in terms of heat removal, temperature uniformity, and the outlet temperature of the heat exchanger. It was observed that due to the high surface area to volume ratio of metal foams they can remove heat at a faster rate which leads to a shorter cycling time and a better temperature uniformity in PCR devices.

Advisor: Mostaghimi, Javad
Thompson Sampling for the Control of a Queue with Demand Uncertainty

We study an admission control problem in which the customer arrival rate is unknown and needs to be learned from data using Bayesian inference. Two key defining features of this model are that: (1) when the arrival rate is known, the DP equations can be solved explicitly to obtain the optimal policy over the infinite horizon, and (2) uninformative actions are unavoidable and occur infinitely often. We extend the standard proof techniques for Thompson sampling to admission control, in which uninformative actions occur infinitely often, and show that asymptotically optimal convergence rates of the posterior error and worst-case average regret are achieved. Finally, we show that under simple assumptions, our techniques generalize to a broader class of policies, which we call Generalized Thompson sampling. We show that this class of policies achieves asymptotically optimal convergence rates and can outperform standard Thompson sampling in numerical simulation.

Keywords: admission control
Bayesian inference
parameter uncertainty
queueing
Regret bounds
Thompson sampling
Advisor: Kim, Michael J
The Impact of Catalytically Inactive Sirt1 on Age-Associated Cardio-Renal Dysfunction

Aging is one key risk factor that leads to kidney and cardiovascular disease; however, little is known about the pathogenesis of these age-related disorders. One potential target is the Sirtuin 1 (Sirt1) enzyme, which has been shown to mediate health span extension. While Sirt1 activation may attenuate the aging process, we hypothesized that a reduction in Sirt1 activity will accelerate age-related cardio-renal disease. Wild type mice were compared with Sirt1Y/Y mice, known to have undetectable Sirt1 catalytic activity, at 4 weeks and 14 months of age. Compared to their wild type counterparts, Sirt1Y/Y mice had lower glomerular filtration rates, fewer glomeruli, and abnormal measures of cardiac contractility. Interestingly, diminished Sirt1 activity during development plays a role in glomerular endowment. These catalytically inactive Sirt1 mice also provide a model of cardiac aging. Therefore, strategies that increase Sirt1 activity may provide a new approach to treating cardio-renal disease.

Keywords: Aging
Cardio-renal function
Glomerular number
Kidney disease
Sirt1
Advisor: Gilbert, Richard E
INTERACTOME REWIRING THROUGH CANCER-ASSOCIATED MUTATION IN GROWTH FACTOR SIGNALING PATHWAYS

Many somatic mutations acquired in cancer have been identified in upstream regulators that normally relay growth factor signals to downstream effectors, notably kinases (PI3 kinase, AKT, RAF), phosphatases (PTEN) and G-proteins (K-Ras). This work seeks to apply both affinity purification coupled to MS (AP-MS) and BioID to quantitatively characterize changes in protein interactions between wild-type (wt) and cancer-associated mutations. In this study, 26 wt proteins and 27 cancer-associated mutants were profiled using both AP-MS and BioID. Several novel interactions and multiple differential associations were revealed. I validated that mutant Raf-1 and A-Raf display increased interactions with subunits of protein phosphatase 6, which may impact the activation of the classical Raf-ERK pathway and the Hippo pathway. The results presented in this thesis show that interaction profiles are often altered in cancer-associated mutants in growth factor pathways, identifying many routes of potential crosstalk with the Hippo pathway, HER2 signaling, and translation initiation.

Keywords: AP-MS
BioID
Growth factor signaling
Protein phosphatase 6
Proteomics
Raf
Advisor: Gingras, Anne-Claude
ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-tozygotic transition

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryoâ s own genome in a process referred to as the maternal-to-zygotic transition (MZT). Here, I show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B undergoes a switch in its regulatory role throughout the MZT mediated by its protein binding partners. In the early embryo, ME31B represses translation, while after MZT, it promotes mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a global change in mRNP composition and, thus, the mode of repression. I propose that these changes in the ME31B mRNP complex provide an explanation for the shift in post-transcriptional regulatory environment in the early embryo.

Keywords: Development
Drosophila
Maternal-to-zygotic transition
mRNPs
Post-transcriptional regulation
RNA-sequencing
Advisor: Rissland, Olivia
Loss of L3mbtl2 in Cardiac Progenitor Cells Causes Subtle Defects in the Developing Heart Leading to Cardiac Dysfunction in Adulthood

Congenital heart defects are caused by abnormalities in heart development, which can result from defects in regulation of cardiogenic gene expression by chromatin modifiers. Lethal (3) Malignant Brain Tumour-Like Protein 2 (L3mbtl2), a component of the chromatin-modifying complex PRC1.6, interacts with regulators of heart development and disease; however, the function of L3mbtl2 and PRC1.6 in heart development and disease has yet to be determined. To uncover the function of L3mbtl2 in the heart, its gene was inactivated in cardiac progenitor cells by Cre-mediated recombination. I found that at E15.5 L3mbtl2-deficient hearts are hypoplastic with thinner ventricular walls. Surprisingly, L3mbtl2 mutants survive to adulthood but have defective heart function with reduced systolic function and left ventricular dilation. Additionally, expression of heart disease markers was not observed in the adult hearts. These results suggest that expression of L3mbtl2 in embryonic cardiac progenitors is required for proper heart development and adult cardiac homeostasis.

Advisor: Delgado-Olguin, Paul