Kinome-wide CRISPR-Cas9 screen to identify kinases involved in Taxol resistance in breast cancer

Breast cancer is the most frequently diagnosed cancer in women, accounting for over 25% of cancer diagnoses and 13% of cancer-related deaths in Canadian women. There are many types of therapies for treatment or management of breast cancer, with chemotherapy being one of the most widely used. Taxol (paclitaxel) is one of the most extensively used chemotherapeutic agents for treating cancers of the breast and numerous other sites. Taxol stabilizes microtubules during mitosis, causing the cell cycle to arrest until eventually the cell undergoes apoptosis. Although Taxol has had significant benefits in many patients, response rates range from only 25-69%, and over half of Taxol-treated patients eventually acquire resistance to the drug. Drug resistance remains one of the greatest barriers to effective cancer treatment, yet little has been discerned regarding resistance to Taxol, despite its widespread clinical use. Kinases are known to be heavily involved in cancer development and progression, and several kinases have been linked to resistance of Taxol and other chemotherapeutic agents. However, a systematic screen for kinases regulating Taxol resistance is lacking. Thus, in this study, a set of kinome-wide screens was conducted to interrogate the involvement of kinases in the Taxol response. Positive-selection and negative-selection CRISPR-Cas9 screens were conducted, whereby a pooled library of 5070 sgRNAs targeted 507 kinase-encoding genes in MCF-7 breast cancer cells that were Taxol-sensitive (WT) or Taxol-resistant (TxR) which were then treated with Taxol. Next generation sequencing (NGS) was performed on cells that survived Taxol treatment, allowing identification and quantitation of sgRNAs. STK38, Blk, FASTK and Nek3 stand out as potentially critical kinases for Taxol-induced apoptosis to occur. Furthermore, kinases CDKL1 and FRK may have a role in Taxol resistance. Further validation of these candidate kinases will provide novel pre-clinical data about potential predictive biomarkers or therapeutic targets for breast cancer patients in the future.

DEVELOPMENT OF A CONDENSED PHASE VELOCITY MAP IMAGING APPARATUS: DISSOCIATION PHOTODYNAMICS OF NITROGEN DIOXIDE AT 355 NM

The photochemistry of the polar regions of Earth, as well as the interstellar medium, is driven by the effect of ultraviolet radiation on ice surfaces and on the materials trapped within them. While the area of ice photochemistry is vast and much research has been completed, it has only recently been possible to study the dynamics of these processes on a microscopic level. One of the leading techniques for studying photoreaction dynamics is Velocity Map Imaging (VMI). This technique has been used extensively to study several types of reaction dynamics processes. Although the majority of these studies have utilized molecular beams as the main medium for reactants, new studies showed the versatility of the technique when applied to molecular dynamics of molecules adsorbed on metal surfaces. Herein the development of a velocity map imaging apparatus capable of studying the photochemistry of condensed phase materials is described. The apparatus is used to study of the photo-reactivity of NO2 condensed within argon matrices to illustrate its capabilities. A doped ice surface is formed by condensing Ar and NO2 gas onto a sapphire rod which is cooled using a helium compressor to 20 K. The matrix is irradiated using an Nd:YAG laser at 355 nm, and the resulting NO fragment is state-selectively ionized using an excimer-pumped dye laser. In all, we are able to detect transient photochemically generated species and can collect information on their quantum state and kinetic energy distribution. It is found that the REMPI spectra changes as different sections of the dissociating cloud are probed. The rotational and translational energy populations are found to be bimodal with a low temperature component roughly at the temperature of the matrix, and a second component with much higher temperature, the rotational temperature showing a possible population inversion, and the translational temperature of 100-200 K. The low temperature translational component is found to dominate at long delay times between dissociation and ionization, while at short time delays the high temperature component plays a larger role. The velocity map imaging technique allows for the detection of both the axial and radial components of the translational energy. The distribution of excess energy over the rotational, electronic and translational states of the NO photofragments provides evidence for collisional quenching of the fragments in the Ar-matrix prior to their desorption.

The Psychometric Properties of the Developmental Reading Assessment

ABSTRACT The purpose of this study was to examine the technical adequacy of the Developmental Reading Assessment (Beaver & Carter, 2004). Internal consistency analysis, factor analysis, and linear regression analyses were used to test whether the DRA is a statistically reliable measuring of reading comprehension for Grades 7 and 8 students. Correlational analyses, decision consistency analyses, and a focus group of experienced Intermediate (Grades 7 and 8) teachers examined whether there is evidence that the results from the DRA provide valid interpretations regarding students’ reading skills and comprehension. Results indicated that, as currently scored, internal consistency is low and skewness of distribution is high. Factor analyses did not replicate those cited by the DRA developers to prove construct validity. Two-way contingency analyses determined that decision consistency did not vary greatly between the DRA, EQAO, scores and report card marks. Views expressed during the focus group echoed many of the challenges to validity found in the statistical analysis. The teachers found that the DRA was somewhat useful, as there were limited alternative reading assessments available for the classroom, but did not endorse it strongly. The study found little evidence that the DRA provides valid interpretations regarding Intermediate students’ reading skills. Indicated changes to the structure and administration procedures of the DRA may ameliorate some of these issues.

The Most Complete Photometric Analysis of 548 CALIFA Galaxies

We present an extensive photometric catalog for 548 CALIFA galaxies observed as of the summer of 2015. CALIFA is currently lacking photometry matching the scale and diversity of its spectroscopy; this work is intended to meet all photometric needs for CALIFA galaxies while also identifying best photometric practices for upcoming integral field spectroscopy surveys such as SAMI and MaNGA. This catalog comprises gri surface brightness profiles derived from Sloan Digital Sky Survey (SDSS) imaging, a variety of non-parametric quantities extracted from these pro files, and parametric models fitted to the i-band pro files (1D) and original galaxy images (2D). To compliment our photometric analysis, we contrast the relative performance of our 1D and 2D modelling approaches. The ability of each measurement to characterize the global properties of galaxies is quantitatively assessed, in the context of constructing the tightest scaling relations. Where possible, we compare our photometry with existing photometrically or spectroscopically obtained measurements from the literature. Close agreement is found with Walcher et al. (2014), the current source of basic photometry and classifications of CALIFA galaxies, while comparisons with spectroscopically derived quantities reveals the effect of CALIFA's limited field of view compared to broadband imaging surveys such as the SDSS. The colour-magnitude diagram, star formation main sequence, and Tully-Fisher relation of CALIFA galaxies are studied, to give a small example of the investigations possible with this rich catalog. We conclude with a discussion of points of concern for ongoing integral field spectroscopy surveys and directions for future expansion and exploitation of this work.

Genomic consequences of mating system evolution in the Pacific coastal dune endemic, Abronia umbellata (Nyctaginaceae)

The advent of next-generation sequencing has significantly reduced the cost of obtaining large-scale genetic resources, opening the door for genomic studies of non-model but ecologically interesting species. The shift in mating system, from outcrossing to selfing, has occurred thousands of times in angiosperms and is accompanied by profound changes in the population genetics and ecology of a species. A large body of work has been devoted to understanding why the shift occurs and the impact of the shift on the genetics of the resulting selfing populations, however, the causes and consequences of the transition to selfing involve a complicated interaction of genetic and demographic factors which are difficult to untangle. Abronia umbellata is a Pacific coastal dune endemic which displays a striking shift in mating system across its geographic range, with large-flowered outcrossing populations south of San Francisco and small-flowered selfing populations to the north. Abronia umbellata is an attractive model system for the study of mating system transitions because the shift appears to be recent and therefore less obscured by post-shift processes, it has a near one-dimensional geographic range which simplifies analysis and interpretation, and demographic data has been collected for many of the populations. In this study, we generated transcriptome-level data for 12 plants including individuals from both subspecies, along with a resequencing study of 48 individuals from populations across the range. The genetic analysis revealed a recent transition to selfing involving a drastic reduction in genetic diversity in the selfing lineage, potentially indicative of a recent population bottleneck and a transition to selfing due to reproductive assurance. Interestingly, the genetic structure of the populations was not coincident with the current subspecies demarcation, and two large-flowered populations were classified with the selfing subspecies, suggesting a potential need for re-evaluation of the current subspecies classification. Our finding of low diversity in selfing populations may also have implications for the conservation value of the threatened selfing subspecies.

β-Arrestin-mediated Regulation of the Human Ether-a-go-go-Related Gene (hERG) Potassium Channel

The human ether-a-go-go-related gene (hERG) encodes the voltage-gated K+ channel, hERG (Kv11.1). This channel passes the rapidly-activating delayed rectifier K+ current (IKr), which is important for cardiac repolarization. A reduction in IKr due to loss-of-function mutations or drug interactions causes long QT syndrome (LQTS), which can lead to cardiac arrhythmias and sudden cardiac death. The density of hERG channels in the plasma membrane is a key determinant of normal physiological function, and is balanced by trafficking to and from the cell surface. Many LQTS-associated hERG mutations result in a trafficking deficiency of otherwise functional channels. Thus, elucidating mechanisms of hERG regulation at the plasma membrane is useful for the prevention and treatment of LQTS. We previously demonstrated that M3 muscarinic receptor activation increases mature hERG expression through a Gq protein-dependent protein kinase C (PKC) pathway. In addition to conventional Gq protein-coupling, M3 receptors recruit β-arrestins upon agonist binding. Traditionally known for their role in receptor desensitization and internalization, β-arrestins also act as adaptor proteins to facilitate G protein-independent signaling. In the present work, I investigated the exclusive effect of β-arrestin signaling on hERG expression by utilizing an arrestin-biased M3 designer receptor (M3D-arr) exclusively activated by clozapine-N-oxide (CNO). By expressing M3D-arr in hERG-HEK cells and treating with CNO under various conditions, I found that M3D-arr activation increased mature hERG expression and current. Within this paradigm, M3D-arr recruited β-arrestin to the plasma membrane, and promoted the PI3K-dependent activation of Akt. I further found that the activated Akt acted through phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) and Rab11 to facilitate endosomal recycling of hERG channels to the plasma membrane.

An investigation of lower limb joint powers as a predictive measure of countermovement vertical jump height.

Measuring and tracking athletic performance is crucial to an athlete’s development and the countermovement vertical jump is often used to measure athletic performance, particularly lower limb power. The linear power developed in the lower limb is estimated through jump height. However, the relationship between angular power, produced by the joints of the lower limb, and jump height is not well understood. This study examined the contributions of the kinetic value of angular power, and its kinematic component, angular velocity, of the lower limb joints to jump height in the countermovement vertical jump. Kinematic and kinetic data were gathered from twenty varsity-level basketball and volleyball athletes as they performed six maximal effort jumps in four arm swing conditions: no-arm involvement, single-non-dominant arm swing, single-dominant arm swing, and two-arm swing. The displacement of the whole body centre of mass, peak joint powers, peak angular velocity, and locations of the peaks as a percentage of the jump’s takeoff period, were computed. Linear regressions assessed the relationship of the variables to jump height. Results demonstrated that knee peak power (p = 0.001, ß = 0.363, r = 0.363), its location within takeoff period (p = 0.023, ß = -0.256, r = 0.256), and peak knee peak angular velocity (p = 0.005, ß = 0.310, r = 0.310) were moderately linked to increased jump height. Additionally, the location, within the takeoff period, of the peak angular velocities of the hip (p = 0.003, ß = -0.318, r = 0.419) and ankle (p = 0.011, ß = 0.270, r = 0.419) were positively linked to jump height. These results highlight the importance of training the velocity and timing of joint motion beyond traditional power training protocols as well as the importance of further investigation into appropriate testing protocol that is sensitive to the contributions by individual joints in maximal effort jumping.

Modeling the impact of large-scale solid mass transfers on the gravity field

The focus of this thesis is to explore and quantify the response of large-scale solid mass transfer events on satellite-based gravity observations. The gravity signature of large-scale solid mass transfers has not been deeply explored yet; mainly due to the lack of significant events during dedicated satellite gravity missions‘ lifespans. In light of the next generation of gravity missions, the feasibility of employing satellite gravity observations to detect submarine and surface mass transfers is of importance for geoscience (improves the understanding of geodynamic processes) and for geodesy (improves the understanding of the dynamic gravity field). The aim of this thesis is twofold and focuses on assessing the feasibility of using satellite gravity observations for detecting large-scale solid mass transfers and on modeling the impact on the gravity field caused by these events. A methodology that employs 3D forward modeling simulations and 2D wavelet multiresolution analysis is suggested to estimate the impact of solid mass transfers on satellite gravity observations. The gravity signature of various submarine and subaerial events that occurred in the past was estimated. Case studies were conducted to assess the sensitivity and resolvability required in order to observe gravity differences caused by solid mass transfers. Simulation studies were also employed in order to assess the expected contribution of the Next Generation of Gravity Missions for this application.

Digging Deeper than Platinum: South Africa's Participation in an Emergent Fuel Cell Industry

South’s Africa’s position as global platinum supplier provides a unique opportunity for an emergent fuel cell industry. The innovative technology’s reliance on platinum has sparked interest in the mining sector, promoting the clean energy-producing devices in their own operations. This research focuses upon contemporary structures of racial oppression within the industry, to analyse how these dynamics influence the development and implementation of innovative technology. It also challenges the sustainability discourse associated with fuel cell technology in South Africa. The study follows a qualitative research approach, incorporating a political ecology focus to highlight the politicized nature of these interactions. The methodology incorporates a literature review, key informant interviews, fieldwork observations and document analysis. Findings indicate that the implementation of fuel cell technology in South Africa’s platinum mines will disproportionately burden historically disadvantaged South Africans, with the lack in technical knowledge-base considered a major challenge. Additionally, it was found that sustainability claims surrounding fuel cell technology are largely based on environmental characteristics. This has resulted in an oversimplification and a depoliticised account of the impacts of the technology. This study looked critically at the convergence of history and innovation, placing emphasis on context, power relations and knowledge to provide a more holistic account of the research problem. Opportunities exist for making a meaningful and viable contribution towards development and sustainability by means of investing in a South African fuel cell industry. The challenge will be in deliberately seeking pathways which address the more complex components of sustainability, benefitting all stakeholders and paying particular attention to the historical, political and social contexts from which the technology emerges. It is this particular context which allows for a questioning and perhaps even a re-evaluation of the sustainability narratives broadly applied to fuel cell technology.

Irreducibility of Random Hilbert Schemes

We prove that a random Hilbert scheme that parametrizes the closed subschemes with a fixed Hilbert polynomial in some projective space is irreducible and nonsingular with probability greater than $0.5$. To consider the set of nonempty Hilbert schemes as a probability space, we transform this set into a disjoint union of infinite binary trees, reinterpreting Macaulay's classification of admissible Hilbert polynomials. Choosing discrete probability distributions with infinite support on the trees establishes our notion of random Hilbert schemes. To bound the probability that random Hilbert schemes are irreducible and nonsingular, we show that at least half of the vertices in the binary trees correspond to Hilbert schemes with unique Borel-fixed points.

An Ergonomic Evaluation of a Wrist Brace for Canadian Tree Planters

Tree planting is one of the most physically demanding occupations in Canada and as a result, tree planters are at an elevated risk of injury, specifically at the wrist. Wrist injuries develop on account of the highly repetitive nature of the job, as well as other musculoskeletal risk factors including non-neutral wrist postures and high impact forces sustained at the wrist during shovel-ground impact. As a result, wrist brace use has become common among planters, in an effort to limit deviated wrist postures while also providing enhanced stability at the wrist. The external stability provided by a wrist brace is thought to reduce the muscular effort required to provide stiffness at the wrist during shovel-ground impact. Since these prospective benefits have not been formally investigated, the purpose of this study was to determine the effect of a wrist brace on wrist posture, muscle activity, and joint rotational stiffness about the wrist joint (for two degrees of freedom: flexion/extension and ulnar/radial deviation). We hypothesized that the brace would promote more neutrally aligned wrist angles, and that muscle activity and joint rotational stiffness would also decrease when participants wore the brace. Fourteen tree planters with at least one season of experience were recruited to complete two planting conditions in a laboratory setting: one condition while wearing the brace (with brace, WB) and one condition without the brace (no brace, NB). The results from this study showed that at shovel-ground impact muscle activity trended towards increasing in three muscles when participants wore the brace. Additionally, wrist angles improved about the flexion/extension axis of rotation while increasing in deviation about the ulnar/radial axis of rotation when participants wore the brace. Joint rotational stiffness increased when participants wore the wrist brace. Participants from this study indicated difficulty gripping the shovel due to the bulk of the wrist brace, and this feature is discussed with possible suggestions for future iterations of design. In addition to grip diameter this analysis also prompts the suggestion that hand length and experience should also be considered in the design of tree planting tools, specifically an ergonomic aid such as a wrist brace.

Polymer Process Partitioning: Extruding Plastic Pipe

When plastic pipe is solidified, it proceeds through a long cooling chamber. Inside this chamber, inside the hollow extrudate, the plastic is molten, and this inner surface solidifies last. Sag, the flow due to the self-weight of the molten plastic, then happens in this cooling chamber, and sometimes, thickened regions (called knuckles) arise in the lower quadrants, especially of large diameter thickwalled pipes. To compensate for sag, engineers normally shift the die centerpiece downward. This thesis focuses on the consequences of this decentering. Specifically, when the molten polymer is viscoelastic, as is normally the case, a downward lateral force is exerted on the mandrel. Die eccentricity also affects the downstream axial force on the mandrel. These forces govern how rigidly the mandrel must be attached (normally, on a spider die). We attack this flow problem in eccentric cylindrical coordinates, using the Oldroyd 8-constant constitutive model framework. Specifically, we revise the method of Jones (1964), called polymer process partitioning. We estimate both axial and lateral forces. We develop a corresponding map to help plastics engineers predict the extrudate shape, including extrudate knuckles. From the mass balance over the postdie region, we then predict the shape of the extrudate entering the cooling chamber. We further include expressions for the stresses in the extruded polymer melt. We include detailed dimensional worked examples to show process engineers how to use our results to design pipe dies, and especially to suppress extrudate knuckling.

Digital geometric-sequence control technique for bidirectional dual active bridge DC-DC converters used in future electric vehicles

Bidirectional DC-DC converters are widely used in different applications such as energy storage systems, Electric Vehicles (EVs), UPS, etc. In particular, future EVs require bidirectional power flow in order to integrate energy storage units into smart grids. These bidirectional power converters provide Grid to Vehicle (V2G)/ Vehicle to Grid (G2V) power flow capability for future EVs. Generally, there are two control loops used for bidirectional DC-DC converters: The inner current loop and The outer loop. The control of DAB converters used in EVs are proved to be challenging due to the wide range of operating conditions and non-linear behavior of the converter. In this thesis, the precise mathematical model of the converter is derived and non-linear control schemes are proposed for the control system of bidirectional DC-DC converters based on the derived model. The proposed inner current control technique is developed based on a novel Geometric-Sequence Control (GSC) approach. The proposed control technique offers significantly improved performance as compared to one for conventional control approaches. The proposed technique utilizes a simple control algorithm which saves on the computational resources. Therefore, it has higher reliability, which is essential in this application. Although, the proposed control technique is based on the mathematical model of the converter, its robustness against parameter uncertainties is proven. Three different control modes for charging the traction batteries in EVs are investigated in this thesis: the voltage mode control, the current mode control, and the power mode control. The outer loop control is determined by each of the three control modes. The structure of the outer control loop provides the current reference for the inner current loop. Comprehensive computer simulations have been conducted in order to evaluate the performance of the proposed control methods. In addition, the proposed control have been verified on a 3.3 kW experimental prototype. Simulation and experimental results show the superior performance of the proposed control techniques over the conventional ones.

Using phase-space localized basis functions to obtain vibrational energies of molecules

For decades scientists have attempted to use ideas of classical mechanics to choose basis functions for calculating spectra. The hope is that a classically-motivated basis set will be small because it covers only the dynamically important part of phase space. One popular idea is to use phase space localized (PSL) basis functions. This thesis improves on previous efforts to use PSL functions and examines the usefulness of these improvements. Because the overlap matrix, in the matrix eigenvalue problem obtained by using PSL functions with the variational method, is not an identity, it is costly to use iterative methods to solve the matrix eigenvalue problem. We show that it is possible to circumvent the orthogonality (overlap) problem and use iterative eigensolvers. We also present an altered method of calculating the matrix elements that improves the performance of the PSL basis functions, and also a new method which more efficiently chooses which PSL functions to include. These improvements are applied to a variety of single well molecules. We conclude that for single minimum molecules, the PSL functions are inferior to other basis functions. However, the ideas developed here can be applied to other types of basis functions, and PSL functions may be useful for multi-well systems.

Applying life cycle assessment to analyze the environmental sustainability of public transit modes for the City of Toronto

One challenge related to transit planning is selecting the appropriate mode: bus, light rail transit (LRT), regional express rail (RER), or subway. This project uses data from life cycle assessment to develop a tool to measure energy requirements for different modes of transit, on a per passenger-kilometer basis. For each of the four transit modes listed, a range of energy requirements associated with different vehicle models and manufacturers was developed. The tool demonstrated that there are distinct ranges where specific transit modes are the best choice. Diesel buses are the clear best choice from 7-51 passengers, LRTs make the most sense from 201-427 passengers, and subways are the best choice above 918 passengers. There are a number of other passenger loading ranges where more than one transit mode makes sense; in particular, LRT and RER represent very energy-efficient options for ridership ranging from 200 to 900 passengers. The tool developed in the thesis was used to analyze the Bloor-Danforth subway line in Toronto using estimated ridership for weekday morning peak hours. It was found that ridership across the line is for the most part actually insufficient to justify subways over LRTs or RER. This suggests that extensions to the existing Bloor-Danforth line should consider LRT options, which could service the passenger loads at the ends of the line with far greater energy efficiency. It was also clear that additional destinations along the entire transit line are necessary to increase the per passenger-kilometer energy efficiency, as the current pattern of commuting to downtown leaves much of the system underutilized. It is hoped that the tool developed in this thesis can be used as an additional resource in the transit mode decision-making process for many developing transportation systems, including the transit systems across the GTHA.