VictorJuiz/thesis huggingface dataset:
In vitro and In vivo High-throughput Analysis of Protein:DNA Interactions

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

Keywords: transcription factors
protein:DNA interactions
Yeast hybrid assays
Fluorescent proteins
Enterokinase
proteolysis specificity
DNA-binding proteins
bZIP
Affinity tag
Tag removal
Protein purification
Bioprobe
Advisor: Shin, Jumi A.
Investigation of Thermal Hydraulics of a Nuclear Reactor Moderator

A three-dimensional numerical modeling of the thermo hydraulics of Canadian Deuterium Uranium (CANDU) nuclear reactor is conducted. The moderator tank is a Pressurized heavy water reactor which uses heavy water as moderator in a cylindrical tank. The main use of the tank is to bring the fast neutrons to the thermal neutron energy levels. The moderator tank compromises of several bundled tubes containing nuclear rods immersed inside the heavy water.It is important to keep the water temperature in the moderator at sub-cooled conditions, to prevent potential failure due to overheating of the tubes. Because of difficulties in measuring flow characteristics and temperature conditions inside a real reactor moderator, tests are conducted using a scaled moderator in moderator test facility (MTF) by Chalk River Laboratories of Atomic Energy of Canada Limited (CRL, AECL). MTF tests are conducted using heating elements to heat tube surfaces. This is different than the real reactor where nuclear radiation is the source of heating which results in a volumetric heating of the heavy water. The data recorded inside the MTF tank have shown levels of fluctuations in the moderator temperatures and requires in depth investigation of causes and effects. The purpose of the current investigation is to determine the causes for, and the nature of the moderator temperature fluctuations using three-dimensional simulation of MTF with both (surface heating and volumetric heating) modes. In addition, three dimensional simulation of full scale actual moderator tank with volumetric heating is conducted to investigate the effects of scaling on the temperature distribution. The numerical simulations are performed on a 24-processor cluster using parallel version of the FLUENT 12. During the transient simulation, 55 points of interest inside the tank are monitored for their temperature and velocity fluctuations with time.

Keywords: Nuclear
Fluid Mechanics
Advisor: Ashgriz, Nasser
The Role of Inorganic Polyphosphate in the Formation of Bioengineered Cartilage Incorporating a Zone of Calcified Cartilage In Vitro

The development of bioengineered cartilage for replacement of damaged articular cartilage has gained momentum in recent years. One such approach has been developed in the Kandel lab, whereby cartilage is formed by seeding primary articular chondrocytes on the top surface of a porous biodegradable calcium polyphosphate (CPP) bone substitute, permitting anchorage of the tissue within the pores of the substrate; however, the interfacial shear properties of the tissue-substrate interface of these biphasic constructs are 1 to 2 orders of magnitude lower than the native cartilage-subchondral bone interface. To overcome this limitation, a strategy was devised to generate a zone of calcified cartilage (ZCC), thereby mimicking the native architecture of the osteochondral junction; however, the ZCC was located slightly above the cartilage-CPP interface. Thus, it was hypothesized that polyphosphate released from the CPP substrate and accumulating in the tissue inhibits the formation of the ZCC at the tissue-substrate interface. Based on this information, a strategy was devised to generate biphasic constructs incorporating a properly located ZCC. This approach involved the application of a thin calcium phosphate film to the surfaces of porous CPP via a sol-gel procedure, thereby limiting the accumulation of polyphosphate in the cartilaginous tissue. This modification to the substrate surface did not negatively impact the quality of the in vitro-formed cartilage tissue or the ZCC. Interfacial shear testing of biphasic constructs demonstrated significantly improved interfacial shear properties in the presence of a properly located ZCC. These studies also led to the observation that chondrocytes produce endogenous polyphosphate and that its levels in deep zone cartilage appear inversely related to mineral deposition within the tissue. Using an in vitro model of cartilage calcification, it was demonstrated that polyphosphate levels are modulated in part by the inhibitory effects of fibroblast growth factor 18 on exopolyphosphatase activity in the tissue. Polyphosphate also appears to act in a feedback loop to control exopolyphosphatase activity. Interestingly, polyphosphate also exhibits positive effects on cartilage matrix accumulation. The potential implication of polyphosphate in the maintenance of articular cartilage homeostasis is intriguing and must be investigated further.

Keywords: Articular cartilage
Chondrocyte
Tissue engineering
Biphasic construct
Inorganic polyphosphate
Mineralization
Extracellular matrix
Fibroblast growth factor 18
Advisor: Kandel, Rita A.
Pilliar, Robert M.
Revisiting Cognitive and Neuropsychological Novelty Effects

Recent proposals have attributed a key role to novelty in the formation of new episodic memories. These proposals are based on evidence of enhanced memory and greater metabolic activity in the hippocampus in response to novel relative to familiar materials. However, such novelty effects are incongruous with long-standing observations that familiar items and lists are associated with better memory than novel ones. In four experiments, I explored possible reasons for this apparent discrepancy. In Experiment 1, I directly tested whether previously observed novelty effects were the result of novelty, discrimination demands, or both. I used linguistic materials (proverbs) to replicate the novelty effect but found it occurred only when familiar items were subject to source confusion. In Experiment 2, to examine better how novelty influences episodic memory, I used experimentally familiar, pre-experimentally familiar, and novel proverbs in a paradigm designed to overcome discrimination demand confounds. Memory was better for both types of familiar proverbs. These cognitive results indicate that familiarity, not novelty, leads to better episodic memory for studied items, regardless of whether familiarity is experimentally induced or based on prior knowledge. I also conducted two fMRI experiments to evaluate the neural correlates of the encoding of novel and familiar forms of information. In Experiment 3, I compared the neural encoding correlates of source memory for novel and familiar visual scenes using fMRI. Replicating previous neuroimaging studies, I observed an anterior novelty-sensitive region of the hippocampus specialized in novelty encoding. Unlike past studies, I also probed for familiarity-encoding regions and identified such regions in the posterior hippocampus. I replicated this pattern in Experiment 4 using proverbs as stimuli. As in Experiment 2, I found the effect held whether familiarity was based on prior knowledge or experimental induction. In both fMRI experiments, anterior and posterior hippocampal regions were functionally connected with different large-scale networks, helping to explain local variation in hippocampal functional specialization in terms of different neural contexts. Together, these experiments show that stimulus familiarity enhances episodic memory for materials, and that novelty is processed differently, not preferentially, in the hippocampus. A new model of hippocampal novelty processing is proposed.

Keywords: novelty
familiarity
long-term memory
episodic memory
memory encoding
medial temporal lobe
hippocampus
subsequent memory effect
functional connectivity
fMRI
Advisor: Moscovitch, Morris
McIntosh, Anthony Randal
Coordinating Cell Cycle Exit and Differentiation in the Mammalian Retina and its Dependence on Rb

Cell cycle exit (“birth”) of retinal progenitor cells (RPCs) is considered a watershed that is preceded by changing levels of cell cycle regulators, and followed rapidly by induction of a post M-phase differentiation cascade. Yet the actual dynamics of these events are largely unclear, thus whether mitosis separates pre- and post- birth differentiation cascades is unproven. We characterized the regulation of many division and differentiation markers relative to each other and final mitosis. Unexpectedly, classic “cell cycle” markers were present well beyond exit (e.g. Ki67, Pcna), early embryonic RPCs expressed “differentiation” markers that later labeled post-mitotic neurons exclusively (e.g. Brn3b, Tubb3, Ptf1a), and factors detected just after cell birth in the embryo were induced well beyond M-phase post-natally (e.g. Nrl, Crx). Thus, the dynamics of birth-associated events shift dramatically during development, even to either side of mitosis. Instead of mitosis behaving as a cog that activates post-exit differentation events we suggest that a common trigger induces both the exit and differentiation programs in RPCs, precisely coordinating their startpoints, but that each subsequent cascade unfolds independently. This model explains the convergence of birth and differentiation but also their temporal maliability. This view fits with our observation that in the absence of the Rb tumor suppressor, differentiation still initiates even without cell cycle exit. Finally, neoplastic transformation in the mouse retina requires loss of Rb and its relative p107, and emerging tumor features suggest an amacrine cell-of-origin. We studied Rb/p107 null clones, and noted two striking features. First, despite initial expansion of aberrantly dividing differentiating cells, apoptosis pruned clones precisely to wild type sizes. “Cell competition” maintains tissue size by selecting fitter over weaker progenitors; our data provide a unique example of competition among differentiating cells. Second, despite normal numbers of amacrine cells per Rb/p107 null clone, more clones contained amacrine cells and fewer had bipolar cells. Both this effect and ectopic division were E2f1-dependent. Thus, the oncogenic initiation event in mouse retinoblastoma triggers a very early fate switch, even before neoplastic transformation, broadening the possibilities for the cell-of-origin of retinoblastoma, and arguing that even very early stage tumors cannot be used to define cancer origin.

Keywords: Retina
Retinoblastoma cell of origin
Cell cycle exit control
Advisor: Bremner, Rod
Development of Novel Antiangiogenic Biologics

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

Keywords: angiogenesis
VEGF
biologics
cancer
age-related macular degeneration
diabetic retinopathy
Advisor: Nagy, Andras
Design and Practical Implementation of Advanced Reconfigurable Digital Controllers for Low-power Multi-phase DC-DC Converters

The main goal of this thesis is to develop practical digital controller architectures for multi-phase dc-dc converters utilized in low power (up to few hundred watts) and cost-sensitive applications. The proposed controllers are suitable for on-chip integration while being capable of providing advanced features, such as dynamic efficiency optimization, inductor current estimation, converter component identification, as well as combined dynamic current sharing and fast transient response. The first part of this thesis addresses challenges related to the practical implementation of digital controllers for low-power multi-phase dc-dc converters. As a possible solution, a multi-use high-frequency digital PWM controller IC that can regulate up to four switching converters (either interleaved or standalone) is presented. Due to its configurability, low current consumption (90.25 μA/MHz per phase), fault-tolerant work, and ability to operate at high switching frequencies (programmable, up to 10 MHz), the IC is suitable to control various dc-dc converters. The applications range from dc-dc converters used in miniature battery-powered electronic devices consuming a fraction of watt to multi-phase dedicated supplies for communication systems, consuming hundreds of watts. A controller for multi-phase converters with unequal current sharing is introduced and an efficiency optimization method based on logarithmic current sharing is proposed in the second part. By forcing converters to operate at their peak efficiencies and dynamically adjusting the number of active converter phases based on the output load current, a significant improvement in efficiency over the full range of operation is obtained (up to 25%). The stability and inductor current transition problems related to this mode of operation are also resolved. At last, two reconfigurable digital controller architectures with multi-parameter estimation are introduced. Both controllers eliminate the need for external analog current/temperature sensing circuits by accurately estimating phase inductor currents and identifying critical phase parameters such as equivalent resistances, inductances and output capacitance. A sensorless non-linear, average current-mode controller is introduced to provide fast transient response (under 5 μs), small voltage deviation and dynamic current sharing with multi-phase converters. To equalize the thermal stress of phase components, a conduction loss-based current sharing scheme is proposed and implemented.

Keywords: digital controller
high-frequency dc-dc converters
switch-mode power supply
digital pulse-width modulator
efficiency optimization
phase shedding
current estimation
thermal management
identification
sensorless average current-mode controller
current sensing
overload protection
fault protection
temperature protection
fast transient response
current-mode control
fault detection
power management
integrated circuit
low-power consumption
low-power applications
dynamic current sharing
Advisor: Prodic, Aleksandar
Growth and Characterization of ZnSe and ZnTe Alloy Nanowires

The objective of this thesis is to explore the synthesis and characterization of high quality binary ZnTe nanowires with great potential for development of optoelectronic devices including high efficiency photovoltaic cells for energy conversion and high sensitivity photodetectors for green fluorescent protein bioimaging at single molecule level. To systematically explore the fabrication process for high quality nanowires, a chemical vapour deposition system was built for nanowire growth. Computational fluid dynamics simulations were used to optimize the reactor and growth parameters. The simulations were validated by experimental measurements. Room temperature photoluminescence measurements showed that high crystal quality with very low defects by single step growth was achieved. This single step growth technique makes a great improvement compared to the reported growth followed by annealing, which achieved equivalent crystal quality. This simplification could be of use in large scale synthesis of nanowires. The simulation results also showed that reactant species concentration is a key factor influencing the growth. A metal-organic chemical vapour deposition system was thus built to independently control reactant concentrations for ZnTe nanowire growth. Temperature-dependent photoluminescence measurements of as-grown ZnTe nanowires showed a strong near band-edge emission. In addition, a deep level oxygen-related band was observed for the first time. From the detailed analysis of thermal quenching of the photoluminescence, it was shown that the deep level emission was partially from the intermediate band of the material. This is of great importance due to the theoretical absorption efficiency that is as high as 63% for intermediate band materials, which is more than two times of that of current single junction concentrators, and few materials possessing this property. Individual ZnTe nanowires, grown after optimization, were patterned and contacted, and their conductivity and photoconductivity were measured at room temperature. A single ZnTe nanowire serving as a photodetector was shown to have the highest reported visible responsivity of 360 A/W (at 530 nm), and a gain of 8,640 (at 3 V bias). The responsivity is roughly 18 times higher than that of silicon avalanche photodiodes. This demonstrates that ZnTe nanowires are strong candidates for single photon detection.

Keywords: Nanowires
II-VI semiconductors
Synthesis
Characterization
Advisor: Ruda, Harry E.
“Desire” Viewed through Ethical Optics: A Comparative Study of Dai Zhen and Levinas

This research project investigates Confucian thinker Dai Zhen (1724-1777) and Jewish thinker Emmanuel Levinas’s (1906-1995) philosophical discourses on desire from a comparative perspective. First, I look at Dai Zhen and Levinas individually each in their own philosophical contexts, while framing my readings with parallel structure that pivots on a hermeneutic strategy to examine their ideas of desire within the larger prospect of the human relation with transcendence. Then, my inquiry leads to a critical analysis of several interesting issues yielded in my interpretive readings of the two thinkers as regards transcendence and immanence and the self-other relationship. Methodologically, my study combines careful textual analysis, philosophical reflection, and historical sensitivity. We might want to say that there is in fact no correlative of the Levinasian desire in Dai Zhen’s philosophy. Dai Zhen’s notion of desire perhaps comes closer to Levinas’s concept of need. However, the disparity of their conceptual formulations does not keep us from discerning their shared ethical concern for the other, the weak, marginalized, and underprivileged group of society, which provides me the very ground for a dialogical comparison between the two thinkers. Henceforth, my writing is hinged on a comprehension of their conception of desire as an articulation of human striving for what is lying beyond themselves, as a redefinition of the being or essence of humankind in relation to the transcendent which in both philosophers’ ethical thinking is translated into a sympathetic understanding of and care for the other, particularly the stranger, the widow, the orphan, the young, the weak and the like. Through the comparative study of the two thinkers’ ideas of desire, I want to argue that “desire,” which is most readily directed to human egoism and instinctive propensity in both Confucian and Western philosophical traditions, can be at once the very driving force to open us to the other beyond ourselves and an actual moral creativity to produce ethical being out of material existence.

Keywords: Philosophy of religion
Religious thought
Ethics
Comparative religious ethics
Chinese philosophy
Jewish philosophy
Jewish thought
Confucianism
Confucian thought
Confucian ethics
Neo-Confucianism
Dai Zhen
Emmanuel Levinas
the Other
Desire
Emotion
Human relationship
Advisor: Shen, Vincent