VictorJuiz/thesis huggingface dataset:
The Role of Actin Polymerization in the Regulation of the Superficial Zone Chondrocyte Phenotype

The superficial zone (SZ) plays a multifaceted role in maintaining cartilage tissue homeostasis, in part through expression of the chondroprotective molecules proteoglycan 4 (PRG4) and tenascin C (TNC). Although studies have investigated the pathways regulating the primary chondrocyte phenotype, these studies use full-thickness chondrocytes (FTC), which are primarily comprised of chondrocytes exhibiting a round morphology. However, SZ chondrocytes (SZC) have an elongated morphology, accounting for up to 10-20% of the cells of native tissue. Thus, the results obtained using FTC may not be applicable to the SZC population. We observed that the elongated shape of SZC is maintained in vitro at 48 hours and used this feature to facilitate characterization studies and evaluation of signaling pathways that regulate the SZC phenotype. SZC had a lower G-/F-actin ratio, compared to deep zone chondrocytes (DZC), indicating the presence of more polymerized actin. Additionally, SZC expressed the actin-regulated transcription factors myocardin-related transcription factor-A (MRTF-A), yes-associated protein (YAP), and transcriptional co-activator with PDZ-binding motif (TAZ). However, TAZ was more highly expressed in DZC. In this study, we investigated the role of MRTF-A and YAP/TAZ in the regulation of the SZC phenotype downstream of the Rho GTPase CDC42 and actin polymerization. While PRG4 and TNC were regulated by CDC42 and actin polymerization, the pathways downstream of actin diverged. Firstly, treatment with actin depolymerizing agents latrunculin B and cytochalasin D had differential effects on PRG4 and TNC mRNA and protein levels. Secondly, siRNA-mediated knockdown of MRTF-A decreased TNC mRNA and protein levels with no effect on PRG4 accumulation. Thirdly, while PRG4 mRNA and protein levels decreased with siRNA-mediated knockdown of YAP/TAZ, TNC decreased only with TAZ knockdown. YAP/TAZ did not have synergistic effects in regulating expression of PRG4 and TNC in SZC. Importantly, TAZ knockdown did not affect YAP levels and vice versa. However, decreased nuclear TAZ was observed with MRTF-A knockdown (day 5), suggesting crosstalk between these two signaling pathways. While CDC42 may contribute to the SZC phenotype through multiple actin-dependent and independent pathways, this work identifies a novel finding demonstrating the role of MRTF-A and YAP/TAZ in the regulation of the SZC phenotype.

Keywords: Actin Cytoskeleton
Cell division control protein 42 homolog (CDC42)
Myocardin-Related Transcription Factor-A (MRTF-A)
Superficial Zone Chondrocyte Phenotype
Transcriptional Co-Activator with PDZ-Binding Motif (TAZ)
Yes-Associated Protein (YAP)
Advisor: Kandel, Rita
MicroRNA-34a-5p Promotes Joint Destruction During Obesity and Osteoarthritis

Osteoarthritis (OA) is a chronic degenerative joint disease and the most common cause of disability among older individuals. We previously found that levels of microRNA-34a-5p (miR-34a-5p) in synovial fluid increase during late-stage (grade III/IV) compared to grade I/II radiographic knee OA; however, the role of miR-34a-5p in OA pathophysiology and its potential as a therapeutic target is largely unknown. In the current study, I found that miR-34a-5p expression was increased in the plasma, knee articular cartilage and synovial tissue of total knee replacement (TKR) patients compared to healthy controls and early OA synovial tissue. Similarly, miR-34a-5p expression was increased in knee joint tissues of a surgically-induced (destabilization of medial meniscus (DMM)) mouse model of OA. Since obesity is a significant risk factor for OA development, I first showed that plasma miR-34a-5p expression was further elevated in obese versus non obese OA patients. In addition, miR-34a-5p expression was highly elevated in plasma and knee joints of obese mice subjected to high fat diet. My in vitro studies revealed that miR-34a-5p mimic promoted critical mediators involved in catabolic processes in human chondrocytes and increased expression of synovitis-associated markers in human fibroblast-like synoviocytes (FLS). Conversely, miR-34a-5p antisense oligonucleotide (ASO) treatment promoted anabolic effects in chondrocytes and reduced the expression of pro-fibrotic and pro-inflammatory markers in FLS. In conjunction with my in vitro findings, intra-articular injection of miR-34a-5p mimic in mice induced OA pathological changes such as cartilage degeneration and synovitis. Notably, my preclinical studies conclusively demonstrated the therapeutic potential of intra-articular injections of miR-34a-5p ASO, which imparted cartilage-protective effects in both normal weight and high-fat diet-fed mice subjected to OA surgery. To investigate the regulatory mechanisms of miR-34a-5p in articular cartilage, chondrocytes obtained from miR-34a knock out (KO) mice or wild-types (WT) were subjected to RNA Sequencing and putative signaling pathways regulated by miR-34a-5p were determined. This is the first study to propose a link between obesity and OA via dysregulated miR-34a-5p expression and provides first preclinical evidence for OA therapeutic intervention using a miR-34a-5p ASO.

Keywords: Antisense oligonucleotide
MicroRNA
Obesity
Osteoarthritis
Preclinical study
Therapeutic
Advisor: Kapoor, Mohit
Postprandial Dyslipidemia and Intestinal Dysfunction in Adolescents with Obesity and Insulin Resistance

Obesity and the metabolic syndrome are becoming increasingly prevalent in adolescents. Dyslipidemia, a key metabolic syndrome component, contributes to increased cardiovascular disease (CVD) risk. While traditionally measured in the fasting state, postprandial dyslipidemia independently predicts CVD risk. Postprandial gut peptides, glucagon-like peptide (GLP)-1 and GLP-2, as well as bile acids are secreted in response to fat ingestion and modulate intestinal dietary fat absorption and triglyceride-rich lipoprotein (TRL) output. Postprandial dyslipidemia and metabolic regulators have been seldom examined in adolescents, despite the early genesis of atherosclerosis. I hypothesized that postprandial GLP-1, GLP-2, and bile acids are impaired in adolescents with insulin resistance and obesity and associate with postprandial dyslipidemia. Normal weight adolescents and adolescents with obesity and varying degrees of insulin resistance underwent an oral fat tolerance test with blood collected up to 6 hours. The postprandial lipoprotein phenotype profile was determined using various assays, including nuclear magnetic resonance spectroscopy, to characterize lipoprotein particle number, size, lipid content, and apolipoprotiens. Adolescents with obesity and insulin resistance exhibited dyslipidemia, particularly reduced high-density lipoprotein particle size and exaggerated postprandial intestinally-derived large TRLs. Both total and active GLPs were quantified to estimate their secretion and biological activity, respectively. Postprandial secretion and activity of GLPs were blunted in adolescents with obesity and insulin resistance and inversely correlated with postprandial dyslipidemia. Conversely, fasting total GLPs positively correlated with postprandial dyslipidemia, suggesting a compensatory increase in fasting secretion. Postprandial total bile acids were diminished in adolescents with obesity. Further quantifying the postprandial bile acid pool composition by mass spectrometry revealed a specific reduction in postprandial lithocholic acid, a potent stimulator of GLP-1 secretion. In summary, postprandial GLP-1, GLP-2, and bile acids were blunted in response to a high-fat meal in adolescents with obesity and insulin resistance and may contribute to the development of postprandial dyslipidemia.

Keywords: Adolescents
Dyslipidemia
GLP-1
Insulin Resistance
Obesity
Postprandial
Advisor: Adeli, Khosrow
Delivery of Drug Combination Therapy in Polymeric Nanoparticles for Treatment of Breast Cancer

The development of resistance and the serious side effects associated with the commercial preparation of paclitaxel (PTX) necessitate the development of new PTX formulations that have a better safety profile. The use of synergistic drug combination therapies has emerged as one promising avenue towards reducing the toxicities associated with high doses of single agents while maintaining therapeutic efficacy. The molar ratios at which the drugs are administered have significant impact on their synergistic effects. In this thesis, for the first time, we provide a systematic assessment of the synergistic effects of PTX and everolimus (EVER) combination and define the optimal synergistic ratio. Polymeric nanoparticles represent an effective nanocarrier platform for the delivery of drug combinations to the tumor site at the optimal synergistic ratio, allowing for selective delivery and sparing normal tissues from exposure to their cargo and the toxicities that result. This thesis describes the design and characterization of a dual-targeted polymeric nanoparticle formulation based on poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-b-PLGA) co-encapsulating PTX and EVER at the optimal synergistic molar ratio (Dual-NPs). HER2 and EGFR-targeted antibody Fab fragments were conjugated to the surface of the NPs to enhance specific delivery and facilitate NP internalization in breast cancer co-overexpressing HER2 and EGFR. Dual-targeting of HER2 and EGFR resulted in a significant increase in the cellular uptake and cytotoxicity of the Dual-NPs in breast cancer cells relative to untargeted and HER2 mono-targeted NPs. Cellular uptake was dependent on the level of expression of HER2 and EGFR, confirming receptor-mediated internalization of the NPs. Limited in vivo stability is one of the main practical challenges in the application of polymeric NPs for the delivery of PTX to the tumor site. Importantly, Dual-NPs maintained their stability in mouse models which is a essential to exploit the EPR effect, leading to increased tumor accumulation and reduced toxicities. As a result, the levels of body weight loss were significantly reduced and neurotoxicity was overall reduced with Dual-NP treatment relative to the free-drug combination. In conclusion, Dual-NPs represent a promising tool for the delivery of PTX and EVER drug combination therapy to BC while reducing the toxicities of the drugs.

Keywords: Active Targeting
Breast Cancer
Combination Therapy
Dual Targeting
Nanoparticles
Paclitaxel
Advisor: Allen, Christine
Clinical Performance of Lithium Disilicate Glass-Ceramic CAD/CAM Crowns Provided by Predoctoral Students at the University of Toronto

The clinical success of monolithic lithium disilicate glass-ceramic (LDGC) crowns manufactured with computer-aided design (CAD) / computer-aided manufacturing (CAM) technology provided by predoctoral students has not yet been investigated. The aims of this retrospective clinical study were to evaluate the clinical performance of monolithic posterior LDGC CAD/CAM (IPS e.max CAD, Ivoclar Vivadent) crowns fabricated with conventional and digital workflows provided by predoctoral students at the Faculty of Dentistry, University of Toronto. In addition, it assessed the effect of different patient and operator-related factors on their longevity. Furthermore, their clinical performance was compared to the metal-ceramic (MC) posterior crowns. Clinical assessment of the crowns and supporting periodontal structures was performed following the modified California Dental Association criteria. Intra-oral photographs, periapical and bitewing radiographs were taken for further assessment by two evaluators. In part 1 of this study, the clinical performance of 210 LDGC-CAD/CAM crowns fabricated with polyvinyl siloxane impressions for 6 years was evaluated. Crowns were milled in-house with CEREC Bluecam system using IPS e.max CAD blocks. Twenty-eight complications were observed (12-technical, 11-biological, and 5-esthetic). The 6-year survival and success rates were 93% and 86.4%, respectively. In part 2, the clinical performance of 40 chairside LDGC-CAD/CAM posterior crowns fabricated with digital impressions was evaluated. Impressions were taken with the Omnicam system and crowns were milled using a CEREC MC-XL machine. Three complications were observed (1-biological and 2-technical). The 4-year survival and success rates were 92.6% and 89.7%, respectively. In part 3, the clinical performance of 112 MC crowns for up to 10 years was evaluated. The 10-year survival and success rates were 87.1% and 81.0%%, respectively. Esthetic problems accounted for the majority of complications. In part 4, the clinical performance of LDGC-CAD/CAM was compared to MC crowns using split-mouth design for 6 years. Twelve complications were observed in the MC crown group (9-esthetic, 2-technical and 1-biological) in comparison to 2 complications in the LDGC-CAD/CAM crown group (1-technical and 1-esthetic). The 6-year survival rates for MC crowns and LDGC-CAD/CAM crowns were 90.8% and 96%, respectively, whereas, the success rates were 83.4% and 96%, respectively. LDGC-CAD/CAM crowns proved to be a predictable, reliable and viable alternative to the “gold standard” MC crowns.

Keywords: CAD/CAM
Clinical survival
Glass ceramic
Lithium disilicate
Longevity
Advisor: El-Mowafy, Omar
Extended Intelligence and Mediated Reality Veillametrics in the Space, Time, Frequency, and Amplitude Domains

Sensing is a key part of human life, and also plays a key role in technological systems. Technology and humans share a need to sense the physical world accurately and precisely, preferably with low error rates, low noise, high dynamic range, and high bandwidth. With the rise of surveillance (institutional sensing) and sousveillance (individual grassroots sensing) --- or more generally, veillance --- in the world around us, it is desired to enhance, control, measure, and understand the capacity-to-sense, in the context of extended intelligence, in order that this capacity-to-sense can benefit humanity. This thesis proposes new algorithms, circuits, and devices to extend the sensory capability of machines and humans. This includes sensory enhancement systems, user-interface devices that embody humanistic intelligence (HI) and a proposed new field of veillametrics. Veillametrics --- the measurement of sensing --- is a quantified approach to the sensing of sensing itself, introducing the new concept of “veillance flux.” Veillance flux is the quantified capacity-to-sense “emitted” from sensors, as that capacity-to-sense propagates through space, reflects, refracts and scatters. The mathematics and physics of veillance flux are introduced, along with measurement systems and visualizations of it. Sensory aid systems are further proposed, to expand sensory capability for machines and humans, in the spatial, temporal, frequency, and amplitude domains. A first application is cameras, where the capability to sense extreme-dynamic-range signals is improved by compositing together dynamic ranges from multiple sensors. A new concept called “coupled dynamic dynamic range” is introduced, where sensors themselves are controlled in a feedback loop, to expand their range of sensing. This work is then expanded to a wider class of sensor signals, including audio, high voltages, and seismic vibrations. This has led to newly-published concepts such as the amplitude-frequency transform, and harmonic range density functions. Novel user-interfaces and algorithms are introduced, to sense human input continuously over spatial-temporal-range domains, and provide tactile, audible and visual feedback to the user, for a tightly coupled interaction. Finally, space-time-frequency-amplitude domain signal processing is used to characterize sensing itself, using veillance flux, to help us understand, mediate, and augment the sensory capability of machines and humans.

Keywords: augmented reality
mediated reality
sensing
sensors
sensory enhancement
veillametrics
Advisor: Mann, Steve
Methods and Probes towards Time-resolved Multiparametric Imaging with Mass Cytometry

Mass cytometry (MC) provides highly-multiparametric analysis of biological systems, with up to 45 parameters measurable per cell. Imaging mass cytometry (IMC) extends MC to account for spatial heterogeneity in biological samples by measuring 2-dimensional images of tissues with the same parametricity. Our lab previously developed tellurophene probes for cellular biochemistries of interest to be used with MC and IMC. Tellurium has seven commercially-available isotopes, allowing for synthesis of probe ‘isotopologues’: isotopically-enriched versions of a probe that are chemically identical but distinguishable by MC and IMC. Sequential labelling with isotopologous probe (SLIP) experiments analyzed by IMC are able to provide time-resolved images of a biochemical process of interest, and so represent a powerful method of quantifying biology with spatiotemporal resolution. This thesis details work towards new methods and new probes compatible with SLIP experiments, MC, and IMC. First, we use SLIP experiments to quantify the action of the drug metformin on tumour hypoxia. We found unexpectedly that metformin only transiently reduces the hypoxia found in solid tumours, and after 6~hours its effect disappears. This brings into question the mechanism by which metformin potentiates radiotherapy. Second, we detail the development of our next-generation hypoxia probe, Telox~3. The probe clears rapidly, making it compatible with SLIP experiments to study cycling tumour hypoxia over time: a complication in cancer known to promote metastasis and aggressive disease. Third, we present our tellurophene probe for protein synthesis, TePhe. The tellurophene ring is a faithful bioisostere of a phenyl ring, allowing TePhe to be incorporated into proteins in vitro and in vivo. Quantification of tellurium incorporation by MC and IMC reports on the rate of protein synthesis in the cells or tissue of interest. Finally, we discuss data processing strategies arising from the idiosyncrasies of tellurium. We remove xenon background signal, quantify transmission efficiency biases across the tellurium isotopes, and present a strategy for combining images from natural abundance tellurium experiments to improve signal-to-noise ratio. In conclusion, the work in this thesis has made progress towards a more complete toolkit for studying complex biology with spatiotemporal resolution.

Keywords: Chemical biology
imaging mass cytometry
mass cytometry
Advisor: Nitz, Mark
Notch Signaling in Pre-thymic T-cell Differentiation and Intrathymic γδ T-cell Development

T-cell development in the postnatal thymus is contingent on the successful recruitment of bone marrow (BM)-derived thymus-seeding progenitors (TSPs). Upon entry into the thymus, TSPs are termed early T-cell progenitors (ETPs) and subsequently engage Notch ligands on thymic epithelial cells (TECs) to specify and commit to the T-cell lineage. Specification occurs at the CD4-CD8- double negative (DN)2 stage of T-cell development while commitment occurs at the DN3 stage. At the DN3 stage, T-cell progenitors also bifurcate into either the - or the -lineage. -lineage committed cells develop into CD4+CD8+ double positives (DPs) before maturing into CD4+ or CD8+ single positive (SP) T-cells. -lineage committed cells remain DN and mature through strong TCR signaling. In contrast to T-cells, T-cells are able to be programmed to specific effector fates during their thymic development. The role of Notch signaling throughout intrathymic T-cell development from T-lineage specification/commitment, to /-lineage bifurcation, and CD8+ SP differentiation is well understood. However, whether Notch plays a role in a pre-thymic setting to generate T-lineage competent TSPs remain unclear. In addition, the role of Notch signaling, along with other factors such as TCR signaling and temporal regulation, in intrathymic effector programming of T-cells remain to be further elucidated. Using an RBPJ-inducible (RBPJind) mouse model in which Notch responsiveness can be regulated in hematopoietic cells, I revealed that Notch signaling plays a critical pre-thymic role to generate T-cell competent TSPs and thus thymic appearance of ETPs. With the RBPJind system, I also revealed the influence of different temporal periods in the generation of specifc T-cell V subsets and effector subsets. Using a TCR transgenic mouse model (termed KN6tg), I revealed that strong TCR signaling promotes the generation of IL-4 producers, while weak TCR signaling promotes the generation of IL-17 producers. The combination of KN6tg and RBPJind approaches revealed that Notch signaling plays an inhibitory role in -lineage commitment and thus optimal IFN production in the presence of weak TCR signals. However, Notch signaling plays a positive role in promoting optimal generation of IL-4 producers in the presence of strong TCR signals. Taken together, this work highlights an important role for Notch in T-cell differentiation in a pre-thymic setting, and establishes the precise requirements for temporal timing, TCR, and Notch in the thymic programming of specific T-cell effector fates.

Advisor: Zuniga-Pflucker, Juan Carlos
The Socio-economic Organization of Votive Pottery Production at Abydos, Egypt in the New Kingdom: A Metric Analysis Study

The present study examines the metric variability of beer jars used for the official temple cult and private votive activities at the Thutmosis III chapel in the North Abydos Votive Zone (NAVZ), and beer jars and votive dishes deposited at Umm el-Qa’ab as private offerings for the Abydos Festival. An understanding of the socio-economic organization of pottery connected to private cultic activities and the official temple cult helps address broader debates regarding the pervasiveness of state/administrative control over the ancient Egyptian economy and the degree to which individuals had autonomy to participate in economic activities. The socio-economic organization of pottery production and procurement are examined in the present studying using metric analysis, or the statistical analysis of measurements of vessel attributes (e.g., rim diameter, height, etc.) of an assemblage that inform on its relative degree of standardization. Several issues persist with the theoretical foundation, methodology, and research design of metric analysis. In an effort to contribute to scholarship that seeks to address these issues, the present study additionally conducts metric analysis of modern Egyptian pottery. To contextualize the results of the Abydos metric analysis, this study also examines the extant archaeological and textual evidence related to the socio-economic organization of pottery production in the New Kingdom. The results of the Abydos metric analysis study and the examination of textual and arcaheological evidence of New Kingdom pottery production suggests that rather than directly administered state-controlled pottery production, the demand for pottery used in temple and private ritual activities was met by networks of dispersed pottery workshops that were connected through social, economic, and work obligation relationships. Utlimately, this study shows that the organization of pottery production was distributed in ancient Egypt with both individuals and larger institutions (in this case temples) employing diverse strategies to produce and obtain pottery.

Keywords: Ancient Egyptian Economy
Ceramic Metric Analysis
Craft Production
Egyptian Archaeology
Pottery Production
Advisor: Pouls Wegner, Mary-Ann