VictorJuiz/thesis huggingface dataset:
The Role of the Arabidopsis Auxin Response Factor MONOPTEROS in Local Tissue Patterning

The 23 AUXIN RESPONSE FACTORS (ARFs) are among the best characterized transcription factors in plants, yet their biological roles are still largely unknown. As one of the most studied ARFs, MONOPTEROS(MP)/ARF5, has been functionally characterized based on the phenotypes of loss-of-function mutants and of MP overexpressing plants. The results compiled in this thesis take advantage of a novel genotype, MP∆, which encodes a truncated, irrepressible form of MP and of a newly derived variant of it, MP∆:GR, which enables deliberate posttranslational regulation of MP∆. These new genetic tools allow for the exploration of molecular mechanisms through which MP activity can influence leaf vascular patterning and leaf shape, female gametophyte and ovule development, and enables two independent strategies to identify genes acting downstream of MP. A saturating search for transcripts responsive to posttranslational activation of MP∆ and a genetic screen for extragenic suppressors of the MP∆ phenotype yielded a number of candidate transcripts and suppressor mutations that will provide a more detailed understanding of the mechanisms through which auxin, via MP, controls cell specification in plant organ development. As one example for how these genetic tools can jointly contribute to the dissection of patterning mechanisms, the PIN FORMED1 (PIN1) gene was identified as both an apparent target of MP regulation and as a gene required for a vast shift towards vascular cell specification very early in leaf development. This shift, in turn, provides a plausible explanation for the strong influence of MP∆ activity on the regulation of leaf lamina width.

Advisor: Berleth, Thomas
Investigation of Post-translational Strategies to Enhance Recombinant Human Bone Morphogenetic Protein-2 Production in Mammalian Cell Culture Systems

Bone morphogenetic proteins (BMPs) have been found useful in repairing craniofacial defects, treating non-union long bone fractures, and in spinal fusion procedures. However, the widespread use of recombinant human BMPs (rhBMPs) is hindered by the difficulties in obtaining large quantities of pure and biologically active rhBMPs. Standard approaches to improve recombinant protein yields such as increasing transgene expression by having strong promoters and/or utilizing gene amplification methods still result in low yields when applied to BMPs. The impact of altering the post-translational processing on recombinant BMP-2 yields in mammalian cell expression systems was investigated in this dissertation. We attempted to modify the proteolytic cleavage of BMP-2 by 1) adding a BMP cleavage inhibitor to the cell culture medium, 2) mutating a cleavage site on BMP-2 sequence, and 3) overexpressing BMP-cleaving proprotein convertases in cells producing rhBMP-2. All three methods led to significant increases in the yield of biologically active rhBMP-2. Characterizing the rhBMP-2 produced from these methods, the data revealed that proteolysis of BMP-2 could also influence the biological activity of the mature protein. Taken together, these results suggest that targeting post-translational processing is a useful and practical strategy to improve the yield of BMP-2 and possibly other difficult to express recombinant proteins in mammalian expression systems. As an increasing number of recombinant proteins become extremely valuable therapeutic options today, the findings from this study contribute to the collective understanding of enhancing recombinant protein yield in mammalian cell expression systems.

Keywords: Bone morphogenetic protein
Chinese hamster ovary (CHO) cells
Pro-domain
Proprotein convertase
Proteolytic cleavage
Recombinant protein
Advisor: Clokie, Cameron M.
Peel, Sean A.
Re-Thinking Expert Evidence

The jurisprudence surrounding expert evidence in Canadian law has evolved since the 1994 case, R. v. Mohan, but it is still replete with numerous problems. This thesis challenges the current admissibility test for expert evidence in Canadian law, and suggests a new, more focused test, which places reliability as the predominant factor in admitting expert evidence. This thesis will argue that changing the admissibility test in this direction will make the jurisprudence in this area more coherent, and lead to better admissibility outcomes.

Keywords: Expert Evidence
Mohan
Reliability
White Burgess
Advisor: Stern, Simon
Transrectal Diffuse Optical Tomography Monitoring of Photothermal Therapy for Localized Prostate Cancer

The clinical management for localized, low- to intermediate-risk prostate cancer (PCa) has become more complex as more therapeutic options become available, while it remains very challenging as physicians try to find a treatment that offers both excellent oncological results and minimal morbidity. Photothermal therapy (PTT) is currently undergoing clinical trials and has shown potential to be one such technique. However, the clinical outcome of PTT is suboptimal, mainly due to the lack of direct monitoring of the coagulated lesion that is generated during PTT. Transrectal diffuse optical tomography (TRDOT) has been proposed to localize the photocoagulation boundary based on changes in tissue optical properties upon coagulation, particularly scattering. A baseline study of phantom experiments with corresponding numerical simulations was first conducted using an existing TRDOT system to investigate its signal sensitivity to a growing coagulating lesion. After gaining critical guidance in building an instrumentation, a clinical propotype TRDOT system, which comprises a 32-channel fiberoptic-based, MR-compatible transrectal probe, was designed and fabricated specifically for the clinical application of in vivo monitoring of PTT by localizing the boundary of the coagulated lesion. The performance of the instrument was tested and characterized. Calibration methods were developed for the system and the probe. The accuracy and reliability of calibrated TRDOT measurements were investigated and characterized in homogeneous phantoms. The in vivo feasibility of the system was tested in a canine PCa tumor model. TRDOT’s ability in monitoring PTT and accuracy in localizing the boundary of a coagulated lesion were investigated through a series studies using thermal coagulating phantoms, ex vivo canine prostates, and in vivo canine prostate models. This demonstrates TRDOT’s potential of visualization of the extent of coagulation during PTT, which will contribute improving PTT’s safety and clinical outcome.

Keywords: photothermal therapy
prostate cancer
transrectal diffuse optical tomography
Advisor: Wilson, Brian C
Integrated Circuits for Quantized Analog Signal Processing

This thesis presents the new concept of Quantized Analog Signal Processing, which is a method to expand the dynamic range of an analog system by using multiple units covering different portions of the rail-to-rail input range. This allows amplification of full-swing signals at the input, without saturating the overall output. Another benefit of the proposed system is the ability to linearize the input-output transfer characteristics of a quantized amplifier, irrespective of the linearity properties of a single unit. In the first chapter of the thesis, a theoretical formulation is developed for the thermodynamic minimum power limits of analog circuits by using prior literature. Then it is shown that the quantized analog signal processing can allow expanding this thermodynamic limit beyond what has been theoretically possible with any prior analog architecture. In Chapter 2, theoretical and behavioral simulations are performed on a quantized voltage amplifier, to support the claims of Chapter 1. Chapter 3 presents an RF receiver integrated in a 65nm CMOS, with the quantized-analog RF front-end. Experimental results show that the receiver has an unprecedented power efficiency in the analog signal path for a given dynamic range. The last chapter presents an upgraded version of the receiver, with extra added features, and on-chip delta-sigma ADCs, which is designed in 28nm CMOS.

Keywords: Dynamic Range
Integrated Circuit
Linearity
Quantized Amplifier
RF Receiver
Wireless
Advisor: Liscidini, Antonio
The Regulation of Cerebellar Granule Neuron Development by Suppressor of Fused

Cerebellar granule cell (GC) development relies on precise regulation of the Sonic Hedgehog (Shh) signaling pathway. Mutations that impair the pathway cause reduced GCP proliferation and cerebellar hypoplasia, underlying multiple motor dysfunction disorders. In contrast, aberrantly activated signalling is associated with medulloblastoma, a debilitating pediatric malignant brain tumour. However, intracellular mechanisms that establish the correct level of pathway activity within GCPs have not been defined. The pathway regulator, Suppressor of Fused (Sufu) controls early cerebellar development and is mutated in human and mouse medulloblastomas. As it interacts with both Gli activator and repressor (Gli-A/R) proteins, its precise function in Shh pathway regulation is highly context-dependent and remains enigmatic. Here, Math1-Cre-mediated deletion of Sufu from GCPs demonstrates that Sufu is required to restrain postnatal GCP proliferation and promote cell cycle exit, together restricting the GCP proliferative window. This function relies on maintenance of Gli3R levels and, surprisingly, suppression of Gli2, together allowing Sufu to dynamically modulate Shh activity. Genetic overexpression of Gli3R and reduction of Gli2 not only rescued the Sufu-deficient phenotype, but revealed intriguing spatial differences in Sufu function. GCPs in the central cerebellar lobules were more deleteriously impacted by Sufu deficiency, and more strongly dependent on suppression of Gli2. RNA-Sequencing further identified novel Sufu gene targets, including Fgf8. The overproliferation of Sufu-deficient GCPs was suppressed by Fgf8 neutralising antibodies, while exogenous Fgf8 was sufficient to stimulate GCP proliferation, demonstrating a previously unknown mitogenic function of Fgf8. Finally, interrogation of non-Cre targeted cells identified non-cell autonomous effects of GC-localised Sufu on BG differentiation. Collectively, these findings implicate Sufu in the spatial and temporal modulation of Shh signalling, and in mediating cross-talk with other signalling partners, to control GCP proliferation and cerebellar development. Moreover, they provide a platform to interrogate the dysregulation of Shh signalling underlying congenital and pathogenic disorders.

Keywords: cerebellum development
granule cell proliferation
medulloblastoma
sonic hedgehog
spatial patterning
sufu-gli
Advisor: Rosenblum, Norman D
Phosphoinositide metabolism and signaling during late phagosome maturation and phagolysosome resolution

Phagocytosis, the regulated uptake of particulate matter by cells, is essential to immunity and homeostasis. Phagocytes are tasked with the immune surveillance that prevents infection by invading pathogens. Additionally, these cells clear accumulated apoptotic debris that results from the daily turnover of billions of cells. In order to fulfill these paramount functions, phagocytes recognize targets and internalize them into membrane-bound compartments termed phagosomes. Following internalization, cells degrade the phagosomal contents and dispose of them resuming the immune response. Phagocytosis is therefore divided into three stages: formation, maturation and resolution. Formation refers to the recognition and engulfment of prey. The transformation that enables degradation of the target is termed maturation. Finally, the elimination and often re-utilization of degraded contents is known as resolution. Most of the phenomena that govern each of these stages are orchestrated by acute signaling events. Specialized signaling phospholipids known as phosphoinositides mediate the recruitment of effector proteins to phagosomes. While the roles of diverse phosphoinositides during formation and the early maturation have been studied and partially elucidated, little is known about them during the late maturation and resolution stages. These late stages are essential for degradation of the phagocytic targets, processing of phagosomal contents and return to homeostasis by phagocytes in order to resume their responses. The work described in this dissertation was conducted with the aim of characterizing phosphatidylinositol 4-phosphate (PtdIns4P)—a phosphoinositide present in the plasma membrane, Golgi apparatus and late compartments—during phagocytosis. Additionally, bacterial species such as Legionella, have been shown to commandeer PtdIns4P metabolism during infection, emphasizing the potential importance of this phosphoinositide during the innate immune response. For this work, I specifically focused on elucidating the dynamics, metabolism and functions of PtdIns4P during each stage of phagocytosis. Chapter I, consists of relevant background on the cell biology of phagocytosis and the involvement of phosphoinositides during the process. The aims and hypothesis of my work are described in Chapter 2. Chapter 3 describes the methodology used throughout this work. In Chapter 4, I detail PtdIns4P dynamics and metabolic mechanisms during phagosome formation and maturation. Additionally, I define the functional implications of PtdIns4P in maturing phagosomes. In Chapter 5 I detail how the endoplasmic reticulum regulates both phagosomal PtdIns4P levels and recruitment of its effectors to the compartment. Finally, I explore the relevance of these events during resolution. This work establishes the importance of PtdIns4P during phagocytosis emphasizing the necessity of an exquisite coordination of lipid metabolism during the process.

Keywords: Immunity
Macrophages
Phagocytosis
Phosphoinositides
Phospholipids
Advisor: Grinstein, Sergio
Structural and functional characterization of dual kinase-endoribonucleases, IRE1 and RNase L

IRE1 and RNase L are a unique set of protein kinases found in the human kinome. They are unique in that both possess a protein kinase domain fused to an endoribonuclease domain. Although IRE1 and RNase L share similar structural domain architectures, they are involved in regulating different cellular pathways. IRE1 is involved in the unfolded protein response (UPR) signaling pathway while RNase L is involved in the interferon antiviral response signaling pathway. In the first data chapter, I describe purified and crystallized IRE1 bound to three hydroxy-aryl-aldehyde (HAA) small molecule analogues that inhibit IRE1’s ribonuclease activity but not its protein kinase activity. The IRE1-HAA structures revealed that the HAA analogues engaged a preformed pocket in the ribonuclease active site of IRE1. Inhibitors binding to the pocket is supported by an essential covalent yet reversible Schiff base interaction between the lysine 907 and the aldehyde moiety of the inhibitor. Further mutational studies, modeling, SAR, and enzymology characterizations were performed to validate the binding mode and mechanism of action of the HAA inhibitors. These IRE1-HAA structures provided the first view of an inhibitor engaging the ribonuclease active site of IRE1. In the second data chapter, I discovered two potent polyphenol compounds that potently and selectively inhibited RNase L’s ribonuclease activity relative to its closest relative IRE1. I performed further SAR and enzymology characterization to gain insight into the binding mode and mechanism of action of these small molecules. Additionally, in collaboration with the Silverman and Weiss labs, we performed in cell studies that demonstrated that our newly discovered inhibitors are functional against RNase L enzyme and superior to the only other RNase L inhibitor characterized to date, namely sunitinib. These polyphenol compounds represent the first small molecules to inhibit RNase L’s ribonuclease activity by binding specifically to the enzyme- RNA substrate complex.

Keywords: Biochemistry
Small molecule
Structural biology
Advisor: Sicheri, Frank
VITAMIN D AND HEALTH SERVICE UTILIZATION FOR UPPER RESPIRATORY TRACT INFECTIONS AND ASTHMA IN YOUNG CHILDREN

Upper respiratory tract infections (URTI) and asthma are major causes of childhood morbidity. They are two of the most common reasons for children seeking health care in the outpatient and inpatient settings, posing a large burden on society and the health care system. It has been suggested that vitamin D may decrease airway inflammation and have an immune protecting effect, which may lead to a reduced risk of health service utilization (HSU) for URTI and asthma in childhood. If vitamin D has an impact on HSU for these common health issues, measures to increase vitamin D status could potentially reduce the burden of these diseases. This doctoral thesis uses observational methods to examine the relationship between 25-hydroxyvitamin D concentration in children ages 0-6 years, vitamin D supplementation during pregnancy and childhood and HSU over 2 years for URTI and asthma in childhood. Healthy children ages 0-6 years were recruited through the TARGet Kids! practice based research network from 7 primary care clinics in Toronto ON, Canada. Data from Ontario’s publicly funded health-care system was linked to TARGet Kids! data at the individual level and used to evaluate the relationship between vitamin D and HSU for URTI and asthma in childhood. I did not find statistically significant evidence to support an association between 25-hydroxyvitamin D concentration, vitamin D supplementation in pregnancy and childhood and HSU for URTI or asthma in a sample (n=2926) of healthy children ages 0-6 years that generally had adequate vitamin D concentrations. However, due to the relatively low frequency of asthma outcomes, future studies could be conducted in an asthma prevalent cohort with low 25-hydroxyvitamin D levels. Measuring asthma prevalence in young children is challenging, thus I conducted a measurement study to evaluate the agreement between the Ontario Asthma Surveillance Information System (OASIS) algorithm (a validated health services algorithm) and parent-reported asthma in children ages 1-5 years. The OASIS algorithm performed similarly to parent-reported asthma and may be a useful tool for identifying children 1-5 years of age to develop a cohort of children with a high likelihood of having asthma for inclusion into future studies.

Keywords: Asthma
Child
Health service utilization
Nutrition
Upper respiratory tract infections
Vitamin D
Advisor: Maguire, Jonathon L
O'Connor, Deborah L
Clinical Burden of Antimicrobial Resistance Following Implementation of an Antimicrobial Stewardship Team Prospective Audit and Feedback Initiative at a Tertiary Care Centre: A Controlled Interrupted Time Series Over 14 Years

Background: Reducing antimicrobial resistance (AMR) is a major incentive for institutional antimicrobial stewardship programs (ASPs). However, there remains a paucity of high-quality data evaluating the impact of these programs on nosocomial AMR. The Sunnybrook Health Sciences Centre (SHSC) Bayview Campus is a shared site, home to a 627-bed acute care hospital and a 530-bed long-term-care facility (LTCF). A prospective audit-and-feedback (PAF) ASP was implemented in the acute care facility in October 2009. No specific intervention was initiated in LTCF, but there was potential for antimicrobial prescribing in the LTCF to be positively influenced by policy and practice at the adjoining acute care facility. This research evaluated the impact of the SHSC PAF-ASP on the burden of antibiotic-resistant organisms (ARO) and multidrug-resistant organisms (MDRO) and on inpatient antimicrobial use (AMU) in the 7 years following program implementation. Methods: Patient-level microbiologic and AMU data were retrospectively obtained over a 14-year study period (October 2002–September 2016). Interrupted time series Poisson regression models were used to detect PAF-ASP associated changes in the incidence and trend of hospital-acquired (HA-) ARO, HA-MDRO, and targeted (TGD) AMU and infer program impact. Changes in community-acquired (CA-) and long-term-care facility-acquired (LTCF-) ARO and MDRO were assessed for comparison. Results: PAF-ASP implementation was associated with improvements in HA-ARO incidence (9.3% reduction/post-intervention period, p

Advisor: Walker, Sandra A.N.
Walker, Scott
Assessment of Potassium Focused Dietary Strategies in Individuals with Cardiovascular Disease

Despite the wealth of clinical data investigating the benefits of a high K+ intake, there is a lack of knowledge regarding the safety of a high K+ diet in individuals that may have disturbances in K+ homeostasis. This thesis will focus on both HTN and heart failure (HF) populations as there are several factors common to both that can predispose to hyperkalemia. In addition, the final piece of work will report on the postprandial impact of K+ intake in augmenting endothelial function and its subsequent role as a possible mechanism in driving changes in blood pressure. The overall objectives of this work were to investigate the safety of a high K+ diet in individuals that may be at risk for hyperkalemia due to medical management; and to investigate the acute changes in endothelial function after ingestion of a high and low K+ meal. We determined that: (1) high K+ diet did not cause hyperkalemia or any statistically significant or clinically relevant changes in serum K⁺ in hypertensive individuals receiving angiotensin converting enzyme inhibitors or/and angiotensin receptor blocker therapy; (2) in heart failure patients, despite increases in reported K+ consumption and accompanying changes in urinary K+ between groups, there were no overall significant changes in serum K+ concentrations. However, hyperkalemia occurred in 3 patients: 2 in the HKD group and 1 in the UD group. Based on these results, we cannot confirm the safety of consuming any amount of dietary K+ without monitoring serum K+ in patients with HF; (3) A single meal high in K+ did not improve endothelial function. This suggests that the beneficial effects of dietary K+ may not occur acutely or that a change in one nutrient, like K +, was not sufficient to improve FMD, a marker of endothelial function. In summary, these experiments provide novel information related to the safety of increasing K+ intake through K+ rich foods in populations at risk for hyperkalemia. This work also gives insight into the postprandial effects of high K+ intake on vascular function in a population likely to have endothelial dysfunction due to disease.

Keywords: Cardiovascular Disease
Heart Failure
Hypertension
Potassium
Advisor: Allard, Johane P
Newton, Gary E