VictorJuiz/thesis huggingface dataset:
Development of a Well Plate-based Multiplexed Organ-on-a-chip Platform with Perfusable Vasculature

Cellular microenvironment forms a multifaceted network that regulates all aspects of tissue organization, specification and function. Animal models have been essential in providing a physiological relevance on drug metabolism and disease model, but they are limited in replicating human physiology and complex disease progression, and in providing high-resolution real time assessment. Traditional in vitro platforms can provide high-throughput studies, but lack the physiological relevance. Organ-on-a-chip technology offers a great promise in building organotypic models of human tissues for drug screening and disease modelling. They are engineered to recapitulate hemodynamic fluid flow in a vascular network and mimic cell-cell crosstalk, organ-organ communication with a high spatiotemporal precision. However, current techniques result with systems mainly in closed-capped format, and with channel networks that limit seeded parenchymal cells from remodeling the cellular environment in three dimension, and organs that are not interconnected together under a single vasculature.Here, we report the design and development of a generic user-friendly, organ-on-a-chip platform for the assembly of micro-vascularized tissues on a multi-well plate footprint. The integrated vasculature for assessing dynamic events (InVADE) platform is constructed by first 3D stamping a UV-cross-linkable polymer into a scaffold that has a built-in central lumen and micro-holes on the side walls, and then situating the scaffold on to a 96-well base plate. The scaffold is mechanically stable to support a perfusable vasculature and a continuously remodeling parenchymal tissue. With this technology, we have successfully engineered (i) a vascular barrier for studying small molecule permeation and cellular trafficking, (ii) a metabolically active liver tissue that processes clinically relevant drugs delivered through the vasculature, (iii) a contracting cardiac tissue that responds to drug treatment and electrical stimulation, (iv) a co-cultured tumor tissue that remodels its microenvironment into desmoplastic like stroma and affects drug bioavailability. Incorporating a cantilever structure to the side wall of the scaffold allows non-invasive, real-time assessment of cardiac contraction. Interconnecting adjacent tissue compartments with a single scaffold initiates modelling of all stages in cancer metastasis cascade, in particular, parenchymal tissue invasion, vascular transmigration, and multi-organ interaction.

Keywords: Organ-on-chip
Tissue Engineering
Vasculature
Advisor: Radisic, Milica
System Wide Views of Proteome Abundance and Dynamic Localization Reveals Novel Saccharomyces cerevisiae Stress Response and Replication Checkpoint Pathways

Cells face a myriad of different environmental stressors and must respond appropriately, as failure to do so can result in disease states. To this end, cells engage intricate signaling pathways that typically involve cellular changes, such as alterations in transcription programs, translation rates, and protein function. Ultimately, protein activity is the final arbiter of function in most cellular pathways. In this work, I investigated the cellular response to stress in the model organism, Saccharomyces cerevisiae, focusing on two of the most important proteomic properties, protein abundance and localization. To begin, I evaluated 21 quantitative analyses of the S. cerevisiae proteome and converted all measurements of protein abundance into the intuitive measurement of absolute number of molecules per cell. I found that the previously described global environmental stress response (ESR) is not detected at the level of protein abundance. In addition, this rich dataset provided insight into proteins that are differentially regulated at the levels of mRNA abundance, mRNA translation, and protein abundance. Next, I developed a quantification method to measure protein localization dynamics in single cells and applied this pipeline to monitor 322 yeast proteins following MMS- and HU-induced replication stress. I discovered that proteins change localization at different rates and with high heterogeneity within a cell population. Moreover, this analysis revealed that Ydr132c/Mrx16 is an unrecognized component of intranuclear quality control (INQ) compartments. Finally, I defined protein factors that govern protein re-localization during conditions of stress, particularly in cells experiencing DNA replication stress. I discovered that Mec1 and Rad53 checkpoint kinases promote the proper subcellular localization of 131 proteins. Importantly, my screening efforts revealed a new checkpoint activation mechanism and gene involved in genome maintenance, RTG3. Together, my data show that a more complete characterization of the proteome, particularly during stressful conditions, provides great insight into cellular functions and reveals novel protein biology.

Keywords: DNA Replication Stress
Protein Abundance
Protein Localization
Advisor: Brown, Grant W
The Regulation of C-type Natriuretic Peptide Signalling in Aortic Valves: Sex Differences and Shear Stress-dependency

Aortic valve disease initiates on the fibrosa (aorta-facing) side of the valve leaflets, whereas the ventricularis side is disease-protected. Spatially predictable disease correlates with side-specific hemodynamics and gene expression, as fibrosa valve endothelial cells (VECs) experience low, oscillatory shear stress and have lower expression of pathological inhibitors, whereas ventricularis VECs experience high shear stress and have a more protective phenotype. Among the genes expressed in a side-dependent manner, C-type natriuretic peptide (CNP) stands out as a factor that suppresses fibrotic differentiation of aortic valve interstitial cells (VICs). However, the regulation of CNP in aortic valves has not been characterized. In this thesis, shear stress regulation of CNP was studied in vitro using porcine VECs. Steady, high magnitude shear stress and ventricular side-specific shear stress waveforms upregulated NPPC (encoding CNP) in a KLF2-dependent manner. KLF2 and NPPC were upregulated by shear stress in VECs isolated from both the fibrosa and ventricularis sides, but with greater sensitivity in fibrosa-side cells. Notably, while male VECs exhibited side-specific shear stress regulation of KLF2 and NPPC, female VECs did not. Immunohistochemical staining of human valves validated side- and sex-specific CNP expression in normal endothelium. Given the putative role of CNP in valve homeostasis and emerging clinical evidence that stenotic female aortic valves present with more fibrosis than male valves, we hypothesized that the predominance of valvular fibrosis in women would associate with deficiencies in CNP signalling. Here, for the first time, sex differences in CNP expression were demonstrated for human aortic valves: female valves, on average, had lower CNP and higher fibrosis upon disease, but the amount of disease (collagen) was not correlated with levels of CNP. In contrast, in diseased regions of male valves, collagen deposition was inversely correlated with CNP, suggesting an anti-fibrotic relationship not present in female valves. Further, treatment of male VICs with CNP suppressed myofibrogenic differentiation in vitro, but female VICs were unresponsive to CNP treatment, indicating deficient CNP signaling. These studies reveal new insights into the hemodynamic regulation of CNP signalling and its role in sex differences of valvular fibrosis.

Keywords: Aortic valve disease
C-type natriuretic peptide
Sex Differences
Shear stress
Valve endothelial cell
Valve interstitial cell
Advisor: Simmons, Craig A
Mechanisms Contributing to Animal Cell Size Homeostasis

While molecules that promote the growth of animal cells have been identified, it remains unclear how such signals are orchestrated to determine and maintain a characteristic target size for the different cell types. More recent studies have established that cell size is determined by size checkpoints – mechanisms that restrict the cell cycle progression of cells that are smaller than their target size. However, what determines such a target size remains unclear. Through a chemical screen and subsequent validation studies, I have identified CDK4 activity to set the critical size referenced to by homeostatic mechanisms in proliferating animal cells (Chapter 2). In other words, CDK4 activity shifts the size threshold at which checkpoint mechanisms consider a cell to be ‘too small’. While these findings shed light on the molecular processes involved with target size determination, the cell size checkpoint remains limited in rectifying cells that are too large. To that end, I followed up on previous reports highlighting mechanisms of growth rate regulation (i.e., how fast cells accumulate mass) that function in parallel with size checkpoints to maintain cell size homeostasis. It has been shown that large cells slow down rates of growth; however, mechanistic studies on such a compensatory response are lacking. I have demonstrated that large cells inhibit growth not by slowing down protein production but rather by upregulating rates of protein degradation (Chapter 3). Together, these findings contribute insights into the mechanisms that control cell size in animal cells.

Keywords: CDK4
cell growth
cell size
cell size checkpoint
size homeostasis
target size
Advisor: Kafri, Ran
EVALUATION OF ROBOTIC SURGERY AND PUBLIC PREFERENCE

Introduction:Robotic surgery has rapidly integrated into our Canadian healthcare system for the management of surgical diseases. However, the benefits in patient-important outcomes from robotic surgery compared with laparoscopic or open procedures remain unclear and controversial. The objective of this thesis is to evaluate robotic surgery in Ontario and the public’s perception of this new technology. Methods: Using linked health administrative data, we identified adults who received the most common robotic surgeries in Ontario, which include radical prostatectomy, total hysterectomy, thoracic lobectomy, and partial nephrectomy, since the beginning of documenting robotic surgery in administrative databases the province, 2008-2018. We first determined the trend in the utilization of the robotic approach compared to the non-robotic approach. Subsequently, we examined the patient and system-level characteristics that could determine the receipt of robotic compared to laparoscopic and open approaches. Then we compared 90-day perioperative adverse events across the 3 surgical approaches for all procedures. Finally, we examined the public’s perception of and preference for robotic surgery. Results: We demonstrated that the use of robotic surgery has increased. Lower comorbidity burden, earlier disease stage (among cancer cases), higher surgeon case volume, early career surgeons, and surgery at a teaching hospital, were consistently associated with receipt of robotic surgery. The robotic approach was associated with fewer 90-day adverse events compared to the open approach. However, the robotic approach was not associated with 90-day adverse events compared to the laparoscopic approach. More participants feared outcomes of robotic surgery more than laparoscopic surgery, while participants preferred laparoscopic over robotic surgery. Conclusion: The trend of robotic surgery has increased and is offered to a selected group of patients by selected surgeons. The benefit of robotic surgery is related to the minimally invasive approach rather than the platform itself. The public prefers laparoscopic surgery over robotic surgery and is more fearful of undergoing surgery using the robotic approach. The rapid implementation of robotic surgery is less likely to be due to the public’s preference and is more likely related to other factors, such as marketing or system-level factors. The study of additional real-world clinical outcomes and associated costs is needed before further expanding use among additional providers and hospitals.

Keywords: Robotic surgerySurgical innovation
Advisor: Karanicolas, Paul MD PhD
The Relationship between Food Environments and Nutritional Status of School-aged Children and Adolescents in Low- and Middle- income Countries: Evidence from Pakistan

Food environments in low- and middle- income countries (LMICs) have undergone unprecedented changes over recent decades with the emergence of modern supply chains, coupled with shifts in consumer food choice. The accessibility and availability of cheap, ultra- processed foods have led to suboptimal diets, and a rise in the double burden of malnutrition particularly in school-aged children and adolescents (SACA). The aim of this dissertation is to improve the understanding of the food environment and its relationship with diet-related health outcomes in SACA in LMICs and advance the methodological approaches to assess and monitor food environments in such settings and population. The first study led to a comprehensive understanding of methodologies for conceptualizing and analyzing food environments in LMICs and presented a systematic, inclusive portfolio of methods and metrics for SACA, which can be used to study the relationship between the food environment, diet and nutritional outcomes. To pilot test these indicators, an empirical assessment of retail food environments in Pakistan and its relationship to adolescent nutrition outcomes was conducted. Findings from the second study suggest that in the peri-urban district of Sindh, modernization is underway with the increasing accessibility and availability of unhealthy foods as compared to healthy foods. When examining the association between food environment characteristics and nutrition outcomes in adolescents, the total number of food outlets were significantly associated to hemoglobin in both adolescent girls and boys, whereas for BMI z-score, mean total number of healthy food items was significantly associated in adolescent boys, but not girls. Finally, a systematic review and meta-analysis on the effectiveness of food environment interventions on diet-related health outcomes in SACA in LMICs was conducted. Seven randomized controlled trials (RCTs), and six quasi-experimental studies were included. A pooled analysis of RCTs indicated no significant effect of the intervention on BMI (kg/m2), (mean difference: -0.08, 95% confidence interval of -0.18, 0.02). Taken together, this body of work provides novel and necessary evidence to inform the design and development of standardized tools and future food environment interventions related to improving diet-related health outcomes in SACA in LMICs.

Keywords: Adolescents
Children
Developing Countries
Double Burden of Malnutrition
Food Environments
Global Health
Advisor: Bhutta, Zulfiqar A
Learning Scalable Physics-based Motion Skills with Reinforcement Learning

The fast development of deep neural networks and reinforcement learning (RL) has enabled a large number of applications that generate motions in robotics, video games and movies.However, current motion controllers are task and agent dependent, and are sensitive to agents' small dynamical changes. Their performance also degenerates with high dimensional agents such as humanoids. Therefore existing research methods cannot scale to real-life applications. In this thesis, we present a series of research works that improve the scalability of physics-based motion controllers. First, we identify one of the potential causes of the scalability gap as the task-dependent objective modeling and reward design.We propose UniCon, which instead is a two level framework that disentangles high-level behaviors and low-level motion skills. By developing multi-task objective functions and constraints, UniCon enables learning of thousands of motion skills with a single neural controller, and can be used for various interactive applications in a plug-and-play fashion. The second line of research uses structured prior in RL, which aims to improve the scalability of modeling agents with different topologies and structures.We propose NerveNet, which models the agent as a graph and learns a structured policy with graph neural networks. NerveNet greatly increases the scalability and transferability of the learnt features between different agents, demonstrating zero-shot performance across agents with similar structures. Following NerveNet, we further propose neural graph evolution (NGE).NGE improves the graph modeling which scales and shares the graph controllers to agents with arbitrary structures. NGE empowers the search optimizer to find the optimal graph structures and policies jointly. We demonstrate the effectiveness of NGE with the task of robot search, where NGE successfully evolves effective agents much faster and more efficiently. Our final contribution is GraphCon, where the insights from the aforementioned research are combined.We utilize the RL formulation from UniCon, and the graph policy modeling from NerveNet and NGE. We show that GraphCon generates realistic physics-based control with the scalability across a large variety of motions and characters, and models object interactions naturally with the graphs.

Keywords: animation
control methods
graph neural network
machine learning
reinforcement learning
robotics
Advisor: Filder, Sanja
Ba, Jimmy
Disentangling the Roles of Pollination and Seed Dispersal on the Demography of a Tropical Understory Herb (Heliconia Tortuosa) in a Fragmented Landscape

Forest loss and fragmentation can affect plant populations directly by altering their demographic vital rates or indirectly by altering the movement behavior, abundance, and diversity of animals, which can affect pollen and seed flow. Most studies assess how forest loss and fragmentation affects plant population dynamics, pollination or seed dispersal independently, but little is known about how these processes are jointly affected for the same plant species. My thesis assesses the relative importance of forest loss and fragmentation for pollination, seed dispersal and plant demographic vital rates of the tropical understory herb Heliconia tortuosa Griggs. In Chapter 2, I found that, contrary to previous research on pollinators of H. tortuosa, the community of seed dispersers and Turdus grayi (one of the main seed dispersers of H. tortuosa) were not significantly affected by forest loss and fragmentation. Furthermore, I found high intraspecific variation in T. grayi habitat preference that was independent of patch size and proportion of forest in a 1 km radius. In Chapter 3, using a movement behavior model developed from radio-tracked individuals and an experimental assessment of seed passage time of H. tortuosa seeds by T. grayi individuals, I found that the probability of seeds landing in the same forest patch, in the matrix or in a new forest patch were mainly driven by habitat preference and patch size, while seed dispersal distance depended on step length. Lastly, in Chapter 4, demographic models obtained by following H. tortuosa individuals during 2013-2015 and 2015-2017 showed that pollination and seed dispersal had a low effect on population growth rate (λ), while survival, probability of reproduction and growth had larger effects. I found that the high mortality during 2013 - 2015 coincided with an unusually dry and warm wet season of 2015. Such unfavorable conditions are predicted to occur more frequently due to global warming. Overall, the results from my thesis suggest that, at least in the short term, inter-annual variation in meteorological conditions may have a stronger effect on H. tortuosa populations in the remaining forest habitat than direct and indirect effects of forest loss and fragmentation.

Keywords: Climate change
Forest fragmentation
Forest loss
Heliconia tortuosa
Plant demography
Seed dispersal
Advisor: Wagner, Helene H
Hadley, Adam S
Medicinal Chemistry of Cannabis and Cannabinoids

As societal attitudes towards cannabis evolve along with our understanding of the endocannabinoid system, medical cannabis is poised to re-enter the mainstream pharmacopeia, especially in North America. Presently, significant gaps in knowledge exist regarding the true chemical composition of medical cannabis as well as the pharmacodynamics of cannabinoids and cannabis extracts. As such, deconvoluting the chemical complexity of cannabis is essential for maximizing its therapeutic benefits and reducing adverse effects in the clinic. Accordingly, this thesis presents a comprehensive investigation of cannabis and cannabinoids at the pre-clinical level, drawing on core aspects of medicinal chemistry and applying developed techniques under large study settings. Through pilot experiments, extraction protocols for the optimal recovery of cannabinoids from different types of cannabis plant material were investigated and the diverse phytochemistry of cannabis was revealed through extensive chemical profiling, reiterating the need for standardization and consistency in therapeutic-grade cannabis extracts. Meanwhile, receptor profiling at CB1 and CB2 receptors confirmed the importance of decarboxylation for the optimal biological activity of cannabinoids and cannabis extracts, demonstrating the suitability of microwave-assisted extraction for producing pharmacologically active cannabis extracts. Following this, high-level chemoinformatic analyses of receptor responses versus cannabinoid concentrations of cannabis extracts generated meaningful prediction models for CB1R and CB2R agonism, predicting the cannabinoid receptor activities of individual phytocannabinoids with reasonable accuracy and revealing differences in cannabinoid receptor responses between pure cannabinoids and cannabis extracts. Finally, the total synthesis and characterization of two pharmaceutically novel cis stereoisomers of CBD was undertaken, resulting in the first unambiguous identification of cis-CBD species and the isolation of a hitherto undescribed cis-Δ9-THC species. Through this thesis, we report significant real-world evidence for the case that cannabis and cannabis extracts behave as complex mixtures of physiologically active molecules, whereby their effects on receptors vary significantly with chemotype and in turn reflects their chemical compositions. Several first-in-kind studies detailed in this thesis pave the way for further investigations of cannabinoids into their chemical structures, mechanism(s) of receptor binding, as well as synergistic and antagonistic effects during co-administration that together bring about the unique and medically important effects of cannabis.

Keywords: Analytical chemistry
Cannabinoids
Medical cannabis
Medicinal chemistry
Natural products
Synthetic chemistry
Advisor: Kotra, Lakshmi P