VictorJuiz/thesis huggingface dataset:
Predicting Treatment Response and the Role of the ISG15/USP18 Ubiquitin-like Signaling Pathway in Hepatitis C Viral Infection
Keywords: Hepatitis C Virus
ISG15/USP18
gene expression profiling
treatment response prediction

Hepatitis C Virus (HCV) infects 170 million people worldwide. The current treatment regimen, which is combination therapy with pegylated interferon (PegIFN) and Ribavirin (Rib), cures only 50% of the patients infected with the most prevalent HCV genotype. Therefore, there is a pressing need to understand the molecular mechanism of interferon resistance and to develop a prognostic tool to predict who will respond to treatment before initiation of therapy. It has been firmly established that the virus-host interaction plays an important role in determining treatment outcomes. My thesis investigated the host factors that are involved in interferon resistance with an aim to provide insights into the molecular mechanism of IFN resistance. cDNA microarray analysis identified 18 differentially expressed hepatic genes from pretreatment liver tissues of responders (Rs) and non-responders (NRs). Based on the differential expression levels of these 18 genes, a prognostic tool was developed to predict who will respond to therapy, with a positive predicting value (PPV) of 96%. Most of these 18 genes are interferon stimulated genes (ISGs) and they are more highly expressed in NR livers, indicating that preactivation of interferon signaling in the pre-treatment liver tissues contributes to NR. 3 out of the 18 genes are involved in an ubiquitin-like ISG15/USP18 signaling pathway that plays an important role in interferon response. Over-expression of USP18 and ISG15 in the pretreatment liver tissues of NR promotes HCV production and blunts interferon anti-HCV activity. There exists a distinct cell-type specific ISG activation in the pretreatment liver tissues of Rs and NRs. Up-regulation of the two ISGs that I tested (ISG15 and MxA) was found mainly in hepatocytes in NRs while ISG activation was preferentially observed in macrophages in Rs. Taking all these data together, pre-activation of interferon signaling and cell-type specific gene activation in the pretreatment liver tissues of patients infected with HCV are associated with treatment non-response. HCV exploits the host interferon system to favour its persistence by enhanced replication /secretion stimulated by a few ISGs (ISG15, USP18) in response to IFN. The developed prognostic tool can be used to stratify patients for treatment and the novel insights of the molecular mechanism of IFN resistance in HCV patients offer potential drug targets for future development.
Hypoxia Suppresses DNA Repair: Implications for Cancer Progression and Treatment
Keywords: Hypoxia
DNA Repair

Acute and chronic hypoxia exists within the microenvironment of solid tumours and drives therapy resistance, genetic instability and metastasis. Despite its importance in solid tumour progression, very little is known regarding the functional consequences of hypoxia-mediated changes in the expression of DNA repair proteins. I studied the relationship between hypoxia and DNA repair using a prolonged chronic hypoxic gas treatment model in a variety of human tumour cell lines to mimic the dynamic state of proliferation and DNA repair in cells distant from the tumour blood vasculature. I observed decreased expression of homologous recombination (HR) and base excision repair (BER) proteins due to a novel mechanism involving decreased protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O2 as measured by the DR-GFP reporter system and functional BER was impaired as assessed with a functional glycosylase assay. This decrease in protein expression and function resulted in increased sensitivity to the DNA damaging agents MMC, cisplatin, H2O2 and MMS. Additionally, chronically hypoxic cells were relatively radiosensitive (OER = 1.37) when compared to acutely hypoxic or anoxic cells (OER = 1.96 - 2.61). As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, I evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. I observed increased clonogenic killing in HR-deficient hypoxic cells following inhibition or depletion of PARP1. PARP-inhibited hypoxic cells accumulated γH2AX foci consistent with an accumulation of collapsed replication forks. Additionally, tumour xenografts exposed to PARP1 inhibition showed increased γH2AX and cleaved caspase-3 expression in hypoxic subregions with suppressed RAD51 protein expression and decreased ex vivo clonogenic survival. I conclude that persistent down-regulation of DNA repair components by the microenvironment could result in faulty DNA repair with significant implications for therapeutic response and genetic instability in human cancers. Specifically, hypoxic cells may be sensitized to PARP inhibitors and other agents targeting repair pathways down-regulated by hypoxia as a consequence of microenvironment-mediated “contextual synthetic lethality”.
Genetic Determinants of Serum Ascorbic Acid Concentrations
Keywords: Ascorbic acid
Nutrigenomics
Vitamin C
Chronic disease
Genotypes
Diet-gene interaction
GSTT1 polymorphism
GSTM1 polymorphism
Hp polymorphism
Vitamin C transporters 1 and 2

Background: The adequacy of serum ascorbic acid (vitamin C) concentrations in young Canadian adults is unknown. Individuals have varied serum ascorbic acid response to dietary vitamin C, possibly due to genetic variation. Objective: To investigate the prevalence of serum ascorbic acid deficiency in young Canadians and to determine whether common genotypes modify the association between dietary vitamin C and serum ascorbic acid. Methods: Subjects were 1277 men and women aged 20-29 years from the Toronto Nutrigenomics and Health study. Vitamin C intakes were estimated by a 196-item FFQ. Fasting blood was collected to measure serum ascorbic acid by HPLC and to genotype for common polymorphisms in genes that code for glutathione S-transferase (GST) (GSTM1, GSTT1 and GSTP1), haptoglobin (Hp), and vitamin C transporters (SLC23A1 and SLC23A2). Results: 53% of subjects had adequate, 33% had suboptimal and 14% had deficient serum ascorbic acid. Subjects with deficiency had higher mean C-reactive protein, waist circumference, BMI and blood pressure than subjects with adequate serum ascorbic acid. The odds ratio (95% confidence interval) for serum ascorbic acid deficiency was 3.43 (2.14, 5.50) for subjects who did not meet the vitamin C recommendation compared to those who did. The corresponding odds ratios were 2.17 (1.10, 4.28) and 12.28 (4.26, 33.42) for individuals with the GSTT1 functional and null genotypes respectively (interaction p=0.01), and 2.29 (0.96, 5.45) and 4.03 (2.01, 8.09) for the GSTM1 functional and null genotypes (interaction p=0.04). These odds ratios were 4.77 (2.36, 9.65) for the Hp2-2 genotype, but 1.69 (0.80, 3.63) for carriers of the Hp1 allele (interaction p=0.02). Serum ascorbic acid concentrations (mean +/- SE) differed among SLC23A1 rs4257763 genotypes (GG: 24.4 +/- 1.3, GA: 26.8 +/- 1.1, AA: 29.7 +/- 1.4, p=0.002). Conclusions: Serum ascorbic acid deficiency is prevalent and associated with markers of chronic disease. Individuals with GST null or Hp2-2 genotypes had an increased risk of deficiency if they did not meet the recommendation for vitamin C, suggesting that GSTs and haptoglobin may spare ascorbic acid when dietary vitamin C is insufficient, thus protecting against serum ascorbic acid deficiency.
Synthesis of Aromatic Heterocycles and Carbocycles Through Tandem Palladium-catalyzed Cross-couplings of gem-dihaloolefins
Keywords: Cross-Coupling
Palladium

Our group has developed a strategy for the synthesis of benzofused carbocycles and heterocycles through tandem palladium-catalyzed reactions of gem-dibromoolefins. In these syntheses, one bromide undergoes a Pd-catalyzed cyclization reaction, and the other participates in an orthogonal inter- or intramolecular Pd-catalyzed reaction to functionalize or annulate that ring, respectively. Chapter 1 describes the pairing of an intramolecular C–N bond forming reaction (the Buchwald-Hartwig coupling) with an intramolecular direct arylation for the synthesis of fused indole derivatives. A range of previously unknown heterocycles were synthesized through this method. Chapter 2 describes the synthesis of benzothiophenes through the combination of a Pd-catalyzed C–S coupling with an orthogonal Suzuki, Heck, or Sonogashira reaction. This represents the first example of the incorporation of Pd-catalyzed C–S coupling into a tandem reaction. In Chapter 3, a tandem intramolecular Heck / intermolecular Suzuki reaction is described for the synthesis of methyleneindenes. Studies on this reaction have provided advanced understanding of the mechanism, including how variation of the ligand controls the regioselectivity of the reaction.
Structural Color and Odors: Towards a Photonic Crystal Nose Platform
Keywords: Photonic Crystals
Electronic Noses

The present thesis describes a novel photonic crystal platform dubbed the photonic nose, a color-based analogue of the human olfactory system. The platform is founded on a one dimensional photonic crystal architecture known as Bragg stacks, which are fabricated using bottom-up self-assembly approaches. Structural and compositional aspects of this novel class of photonic crystals are established that provide them with functionality and utility. Silicon dioxide, titanium dioxide, tin oxide, clays and zeolites are among the materials incorporated into one-dimensional photonic structures. Retention of materials functionality is demonstrated by vapor and liquid sensing experiments. This class of Bragg stacks displays well defined optical properties that have been thoroughly investigated by use of spectroscopic ellipsometry, as we demonstrate in a chapter dedicated to the technique. Utilizing conventional building blocks comprised of nanostructured silicon and titanium dioxide we discuss various aspects of technique pertaining single layered as well as multilayered films. In terms of practical applications these kinds of Bragg stacks show significant potential in areas such as display and sensors that exploit their vibrant and tunable colors. These colors are an important attribute of photonic crystals with bandgaps in the visible range and in this thesis work we present new approaches for characterizing photonic crystal color using well established methods from the field of color imagery. With this knowhow we have been able to assemble a pixilated array of chemically functionalized Bragg stacks in which each pixel responds differently to vapor phase analytes. The combinatorial response of the entire array enables a unique diagnostic fingerprint of a given analyte vapor as determined from color imagery and multivariate statistical methods of analysis. It was possible to discriminate between ethanol, butanol, hexanol, hexane, octane and decane. We also demonstrate the power of the photonic nose platform by distinguishing different bacteria from a photonic nose color analysis of the complex mixture of vapors in the bacteria culture headspace. Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa could be discriminated based on this technique.
Developing Methods to Enable Multiplexed Signal Transduction Measurements in Single Cells
Keywords: Single-cell
Capillary Electrophoresis

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.
Biofilm-derived Planktonic Cell Yield: A Mechanism for Bacterial Proliferation
Keywords: biofilm
planktonic
proliferation
bacteria

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.
Systems and Synthetic Biology in E. coli Cells Quantitative System Characterization, Programming and Engineering Novel Cellular Functions
Keywords: Synthetic Biology, Systems Biology
Physical Chemistry

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.
Platonic Craft and Medical Ethics
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

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.
Vitamin D and Breast Cancer Risk
Keywords: breast cancer
vitamin D
sunlight

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.