VictorJuiz/thesis huggingface dataset:
An Ultrasound and Acoustic Study of Turkish Rounded/Unrounded Vowel Pairs

This dissertation grew out of a phonology course paper on the adaption of Turkish words in Serbian. The focus of the paper was on the Turkish vowels /ɯ y oe/. Noticeably, in contrast to vowels /y oe/, the back vowel /ɯ/ showed variable patterns of adaptation. The conclusion drawn from the paper was that the vowel /ɯ/ was different from other vowels. Interestingly, the Turkish vowel inventory is a crowded vowel system with three rounded/unrounded vowel pairs, two front vowel pairs (/i y/ and /e oe/), and one back vowel pair (/ɯ u/). And, while phonetic research has largely focused on front vowel pairs in other languages, I emphasize on the importance of the back vowel pair. The dissertation examines the phonetics of Turkish vowels to determine acoustic and articulatory properties of vowels produced in different contexts. Four experiments were done, two describing vowels in isolation and two describing vowels in four different consonantal contexts, based on place of articulation. First, I discuss the theory behind acoustics and articulation, by outlining theories of coarticulation and describing how the vowel /ɯ/ differs from other vowels. Then, I present methodology used in the dissertation. Chapters 4 and 5 present results of articulation and acoustic experiments on vowels in isolation, while Chapter 6 and 7 present results of the articulation and acoustic experiments on vowels in consonantal contexts. Chapter 8 discusses the main findings. The study discovered that the three rounded/unrounded vowel pairs are true pairs, as they do not articulatorily differ much in tongue height and frontness. Since they differ most consistently in F2, this confirms the non-linear relationship between articulation and acoustics. Both high back vowels /ɯ u/ are more prone to coarticulation in F2 than other vowels. Context variability, short duration, and absence of visual cues can explain why /ɯ/ behaves like no other vowel in loanword adaptations. The dissertation enriches phonetic research by investigating the back vowel pair, by examining front rounded/unrounded vowel pairs in contexts, and by using quantitative ultrasound methodology. Also, it contributes to loanword research by accounting for loanword adaption of the back unrounded vowel /ɯ/.

Keywords: Loanwords
Phonetics
Serbian
Turkish
Vowels
Advisor: Rice, Keren
Activity Induced Plasticity in AMPAR Composition at the developing Calyx of Held / MNTB Synapse

Developing central synapses exhibit robust plasticity and undergo marked experience-dependent remodeling. This is particularly evident in sensory systems such as auditory brainstem circuits where synapses mature rapidly to achieve high-fidelity neurotransmission. This depends on a developmental switch in AMPAR composition from slow-gating GluA1 dominant too fast-gating GluA4 dominant known to occur within the first 2 weeks of postnatal development, but the mechanisms underlying this switch remain unknown. We hypothesize that patterned stimuli mimicking spontaneous or sound evoked activity drives the GluA gating switch. We examined activity-dependent changes in evoked and miniature excitatory postsynaptic currents (eEPSCs and mEPSCs) at the calyx of Held synapse by rupturing the postsynaptic membrane at different time points following 2 minutes of theta burst stimulation (TBS) to afferents in mouse brainstem slices. We found the decay time course of eEPSCs accelerated, manifested as decreases in fast and slow time constants (τf and τs, by exponential fits), but these changes were not apparent until >30 min after TBS. Histogram analyses of the decay time course of mEPSCs for naive and tetanized synapses revealed two τ populations being 0.4 and 0.8 ms, respectively, but the relative weight of the τ0.4 population over the τ0.7 population increased significantly in tetanized synapses. Such changes are blocked by NMDAR or mGluR1/5 antagonists or inhibitors of CaMKII, PKC and protein synthesis, and more importantly precluded in GluA4-/- synapses, suggesting GluA4 is the substrate underlying the acceleration. Interference of an interaction between the immediate early gene product, Neuronal Activity Regulated Pentraxin (Narp), and AMPARs, blocks this switch, substantiating a role for Narp in excitatory synapse remodeling. These results demonstrate a novel form of plasticity working through coincident NMDAR and mGluR activation to trigger a gating switch of synaptic AMPARs with a temporally delayed expression onset, ultimately enhancing development of high-fidelity neurotransmission at an ultra-fast synapse.

Keywords: activity
AMPAR
development
ion channel
MNTB
Advisor: Wang, Lu-Yang
A Ternary Brain-computer Interface based on Single-trial Readiness Potentials of Self-initiated Fine Movements

This thesis investigates the feasibility of a ternary brain-computer interface (BCI) using the single-trial readiness potential (RP) of self-initiated fine movements. The BCI classified among an idle state and fine motor movements in the left and right hands. Three studies were conducted, each investigating a step towards this ternary BCI. The first study presented a novel technique of feature fusion from an optimal number of electrodes, for the prediction of a self-initiated fine movement from an idle state in single-trials. This technique achieved significantly lower classification error than the best single classifier and some conventional classifier fusion methods. The first study also confirmed the detection of the self-initiated fine movements on a single-trial basis, at above-chance levels, starting 396 ms before their motoric realization. The highest detection accuracy was 82.21% at 250 ms before movement. The second study formulated a novel pipeline of spatio-temporal filtering (PSTF) feature extraction method for laterality classification of self-initiated fine movements. The PSTF achieved significantly higher average classification accuracy (74.99%) than two conventional methods. This second study achieved a significant improvement in single-trial fine movement laterality prediction from RP features alone. The final study investigated a ternary BCI based on a single-trial RP of self-initiated fine movements. The study extended the PSTF for feature extraction in the ternary case, and proposed a diversified classification algorithm for multiclass binary decomposition. The proposed approach achieved significantly higher average classification accuracy (96.24% for idle class; 77.79% for left class; 72.37% for right class) than the conventional multiclass classifier and popular binary decomposition methods. This thesis demonstrates the potential of a ternary BCI based exclusively on RP features. It supports further study of RP-based BCI for intuitive asynchronous control of the environment, such as in augmentative communication or wheelchair navigation.

Keywords: brain-computer interface
EEG
human machine inteface
readiness potential
spatio-temporal filtering
voluntary movement
Advisor: Chau, Tom
Bouffet, Eric
Genome Evolution of Smittium and Allies (Harpellales)

The Harpellales (Kickxellomycotina) is an early-diverging fungal lineage associated with the digestive tracts of aquatic arthropod hosts (lower Diptera). Smittium, with 94 reported species, is the largest genus of the Harpellales and has served as a model to help advance our understanding of insect gut-associated fungi, from aspects of biodiversity, immunology, physiology, and symbiosis. Comparative genomics is shedding new light in all fields of biology. However, little is known about the genomics and natural history of Smittium and allies. The goals of this dissertation are: 1) to place Smittium and allies in a temporal dimension in the Tree of Life and test whether or not these fungi have co-evolved with their lower Diptera hosts; 2) to produce the first whole genome sequences of Smittium and allies and characterize fundamental aspects of their genome features; and 3) to conduct comparative analyses to infer their genomic diversity and reveal unique genetic elements that could be responsible for insect-fungus symbiosis. Chapter 2 shows that the divergence time of the Smittium clade was around 243 Ma, which corresponds to that of their Diptera hosts. However, no strong co-phylogenetic congruence was detected between the symbiotic pairs. Chapter 3 describes the genome features of four Harpellales taxa (three Smittium and one ally) and reports a horizontally transferred ubiquitin gene from a mosquito host to its gut fungal symbiontâ Zancudomyces culisetae. Chapter 4 reveals that Smittium and allies are diploid; suggests that ancient genome duplications have occurred more than once among Smittium and allies; and identifies a ten-gene toolbox that could be responsible for the fungus-insect symbiosis. This work offers insights into the genome evolution of a group of early-diverging fungi and places Smittium and allies into the broader context of fields of molecular biology and comparative genomics.

Keywords: DNA
Fungi
Genomics
Insect
Mycology
Symbiosis
Advisor: Moncalvo, Jean-Marc
Vitamin D Receptor (VDR) and Cholesterol Homeostasis: Interplay of VDR Enzyme Targets and Vitamin D Deficiency

Vitamin D deficiency is speculated to play a role in hypercholesterolemia. However, there has been little molecular evidence to link the two until recent evidence identified the vitamin D receptor (VDR) and its natural ligand, 1Îą,25-dihydroxyvitamin D3 [1,25(OH)2D3], as key regulators of cholesterol metabolism. In the liver, 1,25(OH)2D3-liganded VDR directly inhibited the small heterodimer partner (Shp) to increase expression of cholesterol 7Îą-hydroxylase (Cyp7a1), the rate-limiting enzyme for cholesterol metabolism to bile acids, a mechanism independent of the farnesoid X receptor. Vitamin D deficiency was established in mice after 8 weeks of the D-deficient diet, which resulted in decreased levels of plasma and liver 1,25(OH)2D3, downregulation of hepatic Vdr and Cyp7a1, and elevation of Shp. Consequently, higher plasma and liver cholesterol levels were observed. Intervention with 1,25(OH)2D3 or vitamin D3 reversed the altered expression of these cholesterol-regulating genes and lowered cholesterol levels back to baseline levels. The correlations between liver cholesterol vs. liver 1,25(OH)2D3 and Cyp7a1 expression in mice were also found in human liver tissue, suggesting that the VDR could be a potential therapeutic target for cholesterol lowering. However, the therapeutic utility of 1,25(OH)2D3 is limited by hypercalcemia and by its feedback control mechanisms. When given exogenously, 1,25(OH)2D3 triggered inhibition of its synthesis enzyme (Cyp27b1) but induction of its degradation enzyme (Cyp24a1) in a dose-dependent manner, events that are predicted by compartmental and physiologically-based pharmacokinetic/pharmacodynamic modeling. These models provide a foundation for predicting 1,25(OH)2D3 disposition for inter-species scaling and for exploration of alternative dosing schemes and routes of administration to describe the dynamics of 1,25(OH)2D3 in its new therapeutic roles. Overall, these findings highlight the importance of vitamin D status and the VDR as regulators of hypercholesterolemia.

Advisor: Pang, K. Sandy
Tet1 Deficiency Leads to Aberrant Maintenance of Stemness in Normal and Neoplastic Brain Cells

Glioblastoma is the deadliest adult malignant brain tumour, having a dismal prognosis even with the best therapeutic options available. Glioblastoma has been shown to be hierarchically organized with a stem-like cell at the apex, known as the cancer stem cell, having the ability to self-renew and give rise to more differentiated progeny that make up the bulk of the tumour tissue. Although its behavior atop a hierarchically structured tissue is reminiscent of the role of a neural stem cell in brain development, the functional processes that the cancer stem cell undergoes to produce tumour bulk are poorly understood. In this thesis, I strive to understand what the necessary genetic factors are for a cancer stem cell to switch from a self-renewing state to a more differentiated state. First, I use an unbiased screening approach in a collection of >1300 mouse embryonic stem cells to comprehensively investigate the effect of mono-allelic genetic loss across the genome, on neurogenesis in vitro. Second, I follow up on a screen hit region of mouse chromosome 10 by using CRISRP-Cas9 targeted mutant embryonic stem cells to confirm that the epigenetic modifier Tet1 is necessary for the consolidation of neuronal differentiation from neural stem cells. Lastly, I use this data as a model and apply it to glioblastoma biology in which TET1 is found hemizygous in >85% of patient tumours. Using CRISPR-Cas9 technology to create mono-allelic or bi-allelic loss of function of TET1, this time in patient-derived glioblastoma cells, I show that mono-allelic loss of TET1 is sufficient to bias cells towards self-renewal instead of differentiation, and decrease the survival of mice bearing tumours derived from these cells. Further, I suggest that treatment with methylase inhibitors may be an effective therapeutic option for tumours with elevated DNA methylation due to low TET1 expression.

Keywords: glioblastoma
glioma
neural development
neurogenesis
Tet1
Advisor: Dirks, Peter B
Improved Approaches to Protein-Protein Coupling and the Efficient Formation of Hemoglobin bis-Tetramers

My research focused on developing new methods for coupling proteins, with an emphasis on producing hemoglobin (Hb) bis-tetramers. These are predicted to be safe and effective alternatives to red cells in transfusions. Based on previous work, it was known that stabilized tetrameric Hb causes increases in blood pressure in circulation, presumably as the result of their extravasation, which leads to scavenging of nitric oxide, the signal for relaxation of the muscles that surround blood vessels. Our initial approach involved optimizing solubility-directed click chemistry based on the copper-catalyzed azide-alkyne cycloaddition (CuAAC). To increase coupling specificity, we developed a double cross-linking approach in which reactive sites on the α-subunits are blocked with a fumaryl cross-linker and with azide installed on a linker specifically bridging the β-subunits. While this approach gives substantial success, the inefficiencies of the CuAAC process with our protein of interest motivated us to improve the process. Thus, strain-promoted alkyne-azide cycloaddition (SPAAC) was successfully adapted for coupling proteins. Using SPAAC, we also developed extended coupling to produce ‘Hb clusters’ with albumin. The resultant Hb-albumin cluster is a breakthrough product that can be scaled with minimal manipulation. We took the project in a new direction using biotin-avidin affinity as the basis for protein-protein coupling. The approach adds a biotin derivative to a cysteine thiol on each β-subunit of Hb. Avidin then scavenges the cysteine biotinylated Hbs by the high affinity biotin-avidin interaction to give the fully functional triple protein HbAvHb. We evaluated the physical and circulatory properties of the products developed here. These approaches not only apply to creating Hb assemblies but also provide a general route to coupling like proteins.

Keywords: bis-tetramer
hemoglobin
hemoglobin-based oxygen carrier
protein coupling
red blood cell substitute
Advisor: Kluger, Ronald
Structural and Functional Insights into the Molecular Mechanisms of Viral-cell and Cell-cell Fusion

Membrane fusion is an essential step in various developmental, physiological, and pathological processes in eukaryotes. Specific fusion proteins catalyze the merger of two lipid bilayers. The molecular mechanisms of many membrane fusion reactions still remain unclear. Here, we characterized the molecular details of various adhesion and fusion proteins involved in viral-cell, placental trophoblast, and sperm-egg fusion events to better understand their mechanisms. In total, nine crystal structures of these human and viral glycoproteins are presented. Furthermore, structural comparisons combined with mutagenesis, biochemical, biophysical, and cell culture experiments allowed us to investigate the implications of specific residues of these proteins in mediating membrane fusion. Structures of class I viral fusion proteins revealed that electrostatic interactions are critical for post-fusion stability in viruses that fuse at neutral pH, whereas the salt bridges do not play a stabilizing role in fusion proteins that proceed through low pH. Instead, hydrophobic residues stabilize the fusion core, and histidine/arginine residues in the chain reversal region stabilize a helix-dipole moment. Moreover, the structure of human syncytin-1 fusion protein unveiled a striking overall structural similarity to class I viral fusion proteins, and the presence of salt bridges within the fusion subunit zipper the inner and outer helices. These electrostatic interactions stabilize the post-fusion structure and provide the energetics for trophoblast cell fusion in placentation. While class I fusion proteins share similar structural and functional features, the mechanism of sperm-egg fusion requires the involvement of sperm Izumo1 and egg Juno proteins. Atomic resolution structures of Izumo1 and Juno display an interface stabilized through extensive interactions and a major conformational change within Izumo1 upon Juno binding. Moreover, mutational studies at the Izumo1-Juno interface revealed the structural determinants required for binding. We provide a comprehensive analysis that now identifies general trends and signatures important in viral-cell and cell-cell fusion events.

Keywords: fertilization
Fusion
glycoprotein
Membrane
structural biology
Virus
Advisor: Lee, Jeffrey` E.
High-throughput Analysis of Alternative Splicing Regulatory Networks that Control Cell Fate

Advances in high-throughput profiling technologies have revolutionized our view of transcriptome complexity and regulation. Alternative splicing (AS) is increasingly recognized as a widespread mechanism for gene regulation and is responsible for greatly expanding transcriptome and proteome diversity in metazoans. It is often controlled in a precise spatiotemporal manner by an intricate RNA-protein network. Given the extensive roles of AS in development and disease, understanding the molecular mechanisms underlying AS regulation is of critical importance. The main focus of my thesis is to develop and apply high-throughput strategies to systematically discover AS networks that control cell fate, as well as the factors and mechanisms that regulate these AS networks. By combining high-throughput RNA sequencing and splicing code analyses, I describe the identification of the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved negative regulators of a large program of alternative exons that are differentially spliced between embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) and diverse non-ESCs/tissues. My results from this study provided the first evidence that AS regulatory networks play a central role in the core circuitry required for ESC pluripotency and iPSC reprogramming. Next, I present the development of a new multiplexed functional genomics screen whereby thousands of gene perturbations can be simultaneously assayed for effects on dozens of endogenous AS and gene expression events of interest. Using this system, I revealed a large network of novel positive and negative factors from multiple gene layers that regulate distinct subsets of AS events linked to ESC pluripotency, neural differentiation, and transitions in reprogramming. In summary, these studies have significantly broadened our knowledge of the global regulatory networks that determine cell fate decisions, and provided powerful tools for large-scale investigation of the regulation of AS and other RNA metabolic pathways in diverse cell types.

Keywords: alternative splicing
cell fate control
gene regulation
high-throughput screening
RNA biology
Advisor: Blencowe, Benjamin J
Moffat, Jason
Determinants of Disability and Disablement in Ontario Long-term Care Residents

Purpose: Disability is difficulty with or dependence on others to conduct activities of daily living, such as bathing, eating and dressing; disablement is worsening disability measured over time. Among long-term care residents, disability and disablement lower quality of life and increase health care costs. Understanding the determinants of disability and disablement in this population is critical to guide clinical practice and accountability policies in long-term care homes. Methods: This thesis is theoretically grounded in the Disablement Process Model. It consists of a literature review and two retrospective studies done using Ontario health administrative data. Study 1 features a critical literature review and analytic framework of the determinants of disability and disablement in older adults. Study 2 examines the relative effect of long-term care home versus resident characteristics in explaining residentsâ disability. Study 3 focuses on the association between disability and geriatric syndromes present at admission and disablement experienced by long-term care residents over time. Hierarchical linear regression models were used in both Studies 2 and 3. Implications: The conceptually-grounded, evidence-based analytic framework from Study 1 can be used to advance future research on disability and disablement in older adults, whether or not they live in the community or long-term care. Study 2 demonstrates that the majority of variation in disability among Ontario long-term care home residents is explained by residentsâ geriatric syndromes, not characteristics of the homes in which they live. Study 3 shows that residents with lower disability at admission become disabled more rapidly over the course of their stay; our exploration of possible mechanisms for this finding is relevant to frontline providers and researchers alike.

Keywords: activities of daily living
disability
Disablement Process
long-term care
nursing homes
Advisor: Wodchis, Walter P