VictorJuiz/thesis huggingface dataset:
Developing Methods to Enable Multiplexed Signal Transduction Measurements in Single Cells

Signalling states of cells are heterogeneous even within clonally derived populations due to cell cycle status and their local microenvironment. As a result multiplexed single-cell signal transduction measurements represent a powerful tool which could potentiate a much greater understanding of subcellular processes. However, multiplexed single-cell analysis remains challenging due to several factors, most notably the low copy number of analytes present, difficulties in cellular manipulation and the availability of well characterized and stable probes for use in intact cells. In order to address these issues, a capillary electrophoresis system with laser induced fluorescence (CE-LIF) suitable for screening methods to facilitate single cell analysis was designed and assembled. This system has the requisite sensitivity for single-cell analysis, with the capability of detecting down to 10000 molecules of fluorescein, and has been designed to reduce the time required for analyte separations compared to similar systems by integrating a compact detection module which allows for shorter electrophoretic separation distances. This system has been employed to develop a method to determine the sampling efficiency of laser-based cell lysis of single cells allowing more accurate quantitative measurements of fluorescent peptides from single cells. Furthermore, a fluorescent probe based on amyloid precursor protein (β-APP peptide) has been designed and conditions were found which allowed resolution of enzyme-modified versions within single cells. To identify the enzymatic conversion products produced, a novel method was developed employing bulk cell samples in conjunction with LC-MS. As a testament to the resolution afforded by this technique, the peptide fragments identified from single cells represented peptides which differed by single uncharged amino acids. Together, the methods here developed are able to provide higher quality quantitative data and more informative analysis of fluorescent signal transduction reporters in single cells and represents progress towards being able to obtain highly multiplexed data needed for accurate cellular models.

Keywords: Single-cell
Capillary Electrophoresis
Advisor: Audet, Julie
Biofilm-derived Planktonic Cell Yield: A Mechanism for Bacterial Proliferation

The development of biofilms at solid-liquid interfaces has been investigated extensively, whereas the yield of planktonic cells from biofilms has received comparatively little attention. The detachment of single cells from biofilms has been attributed mainly to the erosive action of flowing liquid or the dispersal of cells from within biofilm microcolonies. The result has been an underestimation of the active role that biofilms can play in microbial proliferation through the production and release of planktonic cells to the environment. In this study, the cultivation of Pseudomonas sp. strain CT07 biofilms in conventional flowcells, glass tubes and a novel CO2 evolution measurement system was utilized to show that biofilm-derived planktonic cell yield was initiated within 6 hours of initial surface colonization and increased in conjunction with biofilm development. The magnitude of the yield was influenced by the metabolic activity of the biofilm, which was in turn dependent on environmental conditions, such as carbon availability. The physiologically active region of the biofilm was responsible for the yield of significant numbers of planktonic cells (~107 CFU.cm-2.h-1), whereas a less active biofilm zone was optimized for survival during unfavourable conditions and shown to be responsible for the subsequent re-establishment of biofilm structure, activity and cell yield. Despite the yield of numerically considerable numbers of planktonic cells (~1010 CFU), a carbon balance revealed that the carbon investment required to maintain this yield was insignificant (~1%) compared to the amount of carbon channelled into CO2 production (~54%). Together, these results indicate that biofilm-derived planktonic cell yield represents an efficient proliferation mechanism and support the view that the biofilm lifestyle affords microbes a dual survival-proliferation strategy, where the dominant strategy depends on the prevailing environmental conditions. An alternative model of biofilm development is presented to account for planktonic cell yield during all stages of biofilm development.

Keywords: biofilm
planktonic
proliferation
bacteria
Advisor: Wolfaardt, Gideon
Edwards, Elizabeth A.
Systems and Synthetic Biology in E. coli Cells Quantitative System Characterization, Programming and Engineering Novel Cellular Functions

The emerging field of synthetic biology aims to use artificially designed genetic circuits to program living cells, much as engineers program a computer or control electronic or mechanical systems. This thesis focuses on the design and implementation of synthetic gene circuits in the bacterium Escherichia coli to create new cellular functions, and on the quantitative characterization and modelling of these circuits. Though important in any engineering discipline, quantitative system characterization has been poorly explored in synthetic biology. We have performed a quantitative system characterization by implementing simple gene circuits in Escherichia coli. The work showed that the level and variability of gene expression varied across different cell strains, and we investigated how these effects manifested through the coupled effects of cell division, cellular growth rate, and plasmid copy number regulation. The work suggests that gene circuit modules from a standard library cannot be used universally; the cellular context and the time dependent dynamics must be considered when implementing gene circuits. In order to work precisely as engineering devices, synthetic gene circuits must be appropriately tuned. One standard method of tuning genetic circuits requires altering the DNA sequences by extensive molecular biology work. Part of this thesis focuses on developing easily tunable gene circuits. A set of circuits were developed in E. coli where the shape of the chemically induced signal response curves can be tuned from a band structure to a sigmoidal structure simply by altering the temperature in a single system. Another set of circuits was developed which demonstrate a range of chemically tunable signal response curves along with multiple functions in a single device. One of the ultimate goals of synthetic biology is to program living cell in a human-controlled way. To this end, I developed a set of genetic devices that could work as ‘in cell disease prevention devices,’ preventing an otherwise fatal viral infection in E. coli. The device displays a number of ‘device’ properties: being dormant under normal conditions, detecting the onset of the disease state, turning on automatically to prevent a lethal outcome, and being subject to external deactivation when desired. The combination of design, characterization, and mathematical understanding explored in this work represents a contribution in the direction of developing synthetic biology as a well-founded engineering discipline.

Keywords: Synthetic Biology, Systems Biology
Physical Chemistry
Advisor: McMillen, David
Platonic Craft and Medical Ethics

Platonic Craft and Medical Ethics examines the Platonic theory of craft and shows its application to different ethical problems in medicine, both ancient and modern. I begin by elucidating the Platonic use of the term “craft” or “technē”, using especially the paradigmatic craft of medicine, and explicate a number of important principles inherent in his use of the term. I then show how Plato’s framework of crafts can be applied to two ancient debates. First, I show how Plato’s understanding of crafts is used in discussing the definition of medicine, and how he deals with the issue of “bivalence”, that medicine seems to be capable of generating disease as well as curing it. I follow this discussion into Aristotle, who, though he has a different interpretation of bivalence, has a solution in many ways similar to Plato’s. Second, I discuss the relevance of knowledge to persuasion and freedom. Rhetors like Gorgias challenge the traditional connections of persuasion to freedom and force to slavery by characterizing persuasion as a type of force. Plato addresses this be dividing persuasion between sorcerous and didactic persuasion, and sets knowledge as the new criterion for freedom. Finally, I discuss three modern issues in medical ethics using a Platonic understanding of crafts: paternalism, conclusions in meta-analyses and therapeutic misconceptions in research ethics. In discussing paternalism, I argue that tools with multiple excellences, like the body, should not be evaluated independently of the uses to which the patient intends to put them. In discussing meta-analyses, I show how the division of crafts into goal-oriented and causal parts in the Phaedrus exposes the confusion inherent in saying that practical conclusions can follow directly from statistical results. Finally, I argue that authors like Franklin G. Miller and Howard Brody fail to recognize the hierarchical relationship between medical research and medicine when they argue that medical research ethics should be autonomous from medical ethics per se.

Keywords: Plato
Medical Ethics
Aristotle
Techne
Craft
Rhetoric
Contraries
Predication
Autonomy
Paternalism
Research Ethics
Therapeutic Misconception
Meta-Analysis
Gorgias
Laws
Euthydemus
Eleutheria
Episteme
Hippocrates
Persuasion
Bia
Peitho
Health
Disease
Measurement
Metaphysics
Phaedrus
Privation
Franklin G. Miller
Education
Informed Consent
Freedom
Lysias
Hippias Minor
Philebus
Statesman
Sophist
Charmides
Republic
Protagoras
Kolakeia
Flattery
Arche
Essence
Definition
Suppliant Maidens
Heraclidae
Philoctetes
Slavery
Competition
Advisor: Barney, Rachel
Vitamin D and Breast Cancer Risk

It has long been known that vitamin D is important for calcium absorption and bone health. More recently, vitamin D has been found to modulate breast cancer cell growth and increasingly epidemiologic studies suggest vitamin D may be associated with reduced breast cancer risk. The primary objective of this thesis was to evaluate the associations between vitamin D from all sources (food, supplements and sunlight exposure) and breast cancer. Secondary objectives were focused on methodological issues including the development of a solar vitamin D score and adapting the measurement of vitamin D from foods for use among Canadians. The data source for this study was the “Ontario Women’s Diet and Health Study”, a population-based case-control study of women in Ontario. Cases (n = 3,101) diagnosed between 2002 and 2003 were identified through the Ontario Cancer Registry and controls (n = 3,471) were identified through random digit dialing of Ontario households. Study participants completed mailed risk factor and food frequency questionnaires. Vitamin D intake from supplements (>400 IU/day compared to none) was found to be associated with reduced breast cancer risk (OR = 0.76; 95% CI: 0.59, 0.98). However, total vitamin D intake (from food and supplements) and intake from food alone were not associated with breast cancer risk. Time spent outdoors during 4 periods of life (including adolescence) was associated with reduced breast cancer (e.g., highest versus lowest categories of exposure at age 40 to 59: OR = 0.74; 95% CI: 0.61, 0.88). The novel solar vitamin D score, derived from time spent outdoors, skin color, sun protection practices, and ultraviolet radiation of residence, was also associated with reduced breast cancer risk. In summary, there is some evidence to suggest that vitamin D intake from supplements and determinants of cutaneous vitamin D production are associated with reduced breast cancer risk.

Keywords: breast cancer
vitamin D
sunlight
Advisor: Cotterchio, Michelle
Producing Small Droplets of Aqueous Solutions and Molten Metals using a Pneumatic Droplet Generator

A pneumatic droplet generator is described, which consists of a T-junction with a nozzle fit into one opening, the second opening is connected to a gas cylinder through a solenoid valve and the third connected to a length of steel tubing. The droplet generator is filled with liquid. Opening the valve for a preset time creates a pulse of alternating negative and positive pressure in the gas above the surface of the liquid. A jet of liquid issues far enough out of the nozzle that its tip becomes unstable, detaches and forms a droplet. Experiments were conducted using water/glycerin mixtures and molten metals including tin, zinc and zinc alloy. Droplet formation was photographed and the pressure variation inside the droplet generator recorded. The effect of various experimental parameters such as nozzle size, pressure pulse width, secondary gas flow pressure, liquid viscosity and temperature on droplet formation were investigated. An analytic model of incompressible liquid motion in the nozzle is used to explain the behavior of water/glycerin solutions inside the nozzle and droplet formation. The model demonstrates that the motion of the surface is out of phase with the exciting pressure oscillation. Experiments showed the oscillation of the liquid surface prior to droplet ejection and the time lag between the pressure oscillation and droplet ejection. The model predicts that maximum liquid velocity is attained at an intermediate value of viscosity, and experiments confirmed that the largest liquid motion was achieved with this intermediate value, which eventually leads to droplet formation. Similarly, with molten metals, a simple analytical method was used to estimate the diameter of droplets. The size of tin droplets measured from experiments was in good agreement with that obtained from the model.

Keywords: drop-on-demand
Advisor: Chandra, Sanjeev
Becoming a Family Physician – Exploring the Experiences of Residents During the First Six Months of PostgraduateTraining

Becoming a Family Physician – Exploring the Experiences of Residents During the First Six Months of Postgraduate Training describes the early training experience of residents from their perspective as they begin a postgraduate program in Family Medicine. A case study approach using focus groups and individual interviews was used to gain insight into the resident-participant’s experiences of the first six months of training. Resident-participants were asked to describe their concerns, changes that occurred and the influences they attributed to those changes as a way to explore their early training experiences from their perspective. This study found resident-trainees do not begin a Family Medicine postgraduate training program knowing what it means to be a Family Physician, but must learn what it means to fulfill this role. From the participants’ perspective, this process involves adjusting to significant shifts in responsibility in the areas of Knowledge, Practice Management and Relationships that occur when they make the transition from being medical students in undergraduate training to doctors responsible for the outcome of care during postgraduate training. As the participants began postgraduate training they were eager to accept the responsibility of being the doctor, but were uncertain they had the necessary medical experience and expertise for someone calling themselves the doctor. The experience of practice, which included developing relationships with different patients over time (continuity of care) was particularly influential in helping the participants gain confidence in fulfilling the role of doctor and learning that the role of Family Physician is complex, multifaceted and not limited to their initial concept of doctoring. As the participants adjusted to their new responsibilities, they gained confidence in their new role as doctor, which subsequently led to a more comprehensive understanding of what it meant to be a Family Physician. This study was able to contribute to what little is know about the transition into a postgraduate Family Medicine program by illuminating from the resident-participant’s perspective how the transition is experienced. In doing so, medical educators have a better understanding of the early training experience of resident-trainees and how these experiences contribute to consolidating their new professional identity.

Keywords: transitions
medical education
postgraduate
Advisor: Thiessen, Dennis
Young Women's Provisioning: A Study of the Social Organization of Youth Employment

This study uses institutional ethnography (IE) to address the question of how young women, considered to be “at risk” youth, make decisions about their working lives. Based on interviews with young women and program workers in housing, employment, young mothers’ and girls’ programs, field observations, and document analysis at Gen-Y (pseudonym for a women’s community-based social services agency), young women’s provisioning experiences are used to critique current program and policy models that feature notions of choice and risk. Provisioning is a concept that captures a wide range of work and work-related activities that young women perform for themselves and people they feel responsible for. IE is applied to understand how institutional processes and practices give rise to the conditions under which young women participants at Gen-Y make career and life decisions.

Keywords: young women
employment
provisioning
social policy
Advisor: Neysmith, Sheila
Mechanomyography as an Access Pathway for Binary and Multifunction Control

Individuals with severe physical disabilities and minimal motor behaviour may benefit from access technologies that harness the volitional activity of muscles. In this thesis, we investigate the use of muscle activity, indicated by the mechanomyogram (MMG), as a binary and multifunction control signal for access devices. A challenge in the design of a binary MMG switch is to reliably recognize the timing of voluntary muscle contractions, and to subsequently translate the MMG signal into a switch-activation signal. A continuous wavelet transform (CWT) algorithm based on a simplified MMG generation model of recurring impulsive morphological patterns was proposed for automatic detection of muscle activity from MMG signals. CWT coefficients of the MMG signal were compared to scale-specific thresholds derived from the baseline signal to estimate the timings of muscle activity. The automatic detection algorithm was implemented as a binary switch controlled by a single muscle site at the forehead, shoulders or forearm. The binary control algorithm was verified on able-bodied participants, and the switch was tested on individuals with neuromuscular and neurological disabilities. The switch showed very high sensitivity and specificity when the muscle and its control were minimally affected by spasticity, involuntary movement, or involuntary muscle activity. We further investigate the potential of improving the functionality of the MMG-controlled switching interface. We demonstrate the practical use of the vibratory artefact measured at the forehead during vocalization to control a second switch. The proposed integrated MMG-vocalization access solution may augment access alternatives for individuals with physical disabilities using a single access site. Further, we show that multi-site MMG signals exhibit distinctive patterns of forearm muscle activity, and 7+/-1 hand movements can be identified with an accuracy of 90+/-4%. This suggests that MMG may have applications in multi-function body-machine interfaces when multiple muscle sites are available. However, MMG signal features vary with sensor location. We show that sensor displacements significantly diminish classification accuracy, emphasizing the importance of consistent sensor placement between MMG classifier training and deployment for accurate control of switching interfaces. Collectively, the findings and developments of this thesis lay the foundation for future research on wearable, MMG-driven access technologies.

Keywords: access technology
muscles
mechanomyography
rehabilitation
biomedical engineering
Advisor: Chau, Tom
Molecular Modeling of Immobilized Single and Double Stranded Oligonucleotides in Mixture with Oligomers

Interactions between single and double stranded oligonucleotides with SiO2 surfaces and the interactions between oligonucleotides and immobilized oligomers have been studied computationally. The oligonucleotide is the 18-base-pair sequence for the survival motor neuron gene SMN1. The oligomer consisted of a 50 unit 2-hydroxyethyl methacrylate (PHEMA) molecule. A linker used to tether the oligonucleotide was either a 10 Å or a 30 Å long succinimdyl 4-[N-maleimidomethyl]cyclohexane-1-caroxylate (sulfo-SMCC-Cn). The surface consisted of a SiO2 crystal that was 50 Å long and 50 Å wide, one unit thick and covered with modified-(3-aminopropyl)trimethoxysilane (m-APTMS) molecules. It was determined that explicit water, sodium counterions and excess salt were necessary to produce computationally stable oligonucleotide structures on surfaces. Artificial partial charges were introduced to the surface, and linkers, oligomers and oligonucleotides were immobilized and studied. The linkers collapsed onto a positive but not onto a negative surface. Oligomers moved closer to the SiO2 surface regardless of the surface charge. Immobilized oligonucleotides tilted significantly from an initial upright position but did not collapse completely onto the surfaces. The interactions between immobilized oligonucleotides and oligomers were examined. The number of oligomers surrounding the oligonucleotide was varied between two and four. Single stranded oligonucleotides were prevented from interacting with the surface as they were inhibited by the presence of oligomers. Double stranded oligonucleotides collapsed onto the surface when only two oligomers were present but remained upright when four oligomers were present. This was due to the four oligomers interacting with one another and effectively shielding the surface. The oligomers interacted with the bases in the single stranded oligonucleotides, making them energetically accessible. Presence of a high density of oligomers prevented the dsDNA from collapsing onto the surface. These results suggest design criteria for preparation of mixed oligonucleotide and oligomer films for use in biosensors.

Keywords: Molecular Modelling
Biosensors
AMBER
DNA
Silica surfaces
computational Chemistry
Advisor: Krull, Ulrich Jorg