Aujourd'hui, il n'existe pas d'étalon de référence pour la grandeur physique des pressions dynamiques infrasonores. La demande d'étalonnage d'une telle grandeur est apparue récemment, en réponse à des problématiques du domaine de la géophysique, étudiant la propagation d'ondes acoustiques dans l'atmosphère entre 20 Hz et 0,001 Hz, soutenue par la surveillance du respect du Traité d'Interdiction Complète des Essais Nucléaires (TICE). Dans le but de répondre à cet enjeu, cette thèse a pour objectif la réalisation d'un étalon primaire pour cette grandeur. Le contexte métrologique et quelques bancs d'étalonnage existants, utilisant des générateurs de pression dynamique infrasonore, sont présentés. Afin de concevoir le banc d'étalonnage primaire, les réponses en amplitude et en phase du générateur d'infrasons du CEA sont caractérisées analytiquement et expérimentalement. Par ailleurs, le principe de l'étalon primaire basé sur le pistonphone calculable utilise les mêmes modèles d'admittance acoustique de transfert des cavités cylindriques que ceux préconisés pour l'étalonnage primaire des microphones étalons par la méthode de la réciprocité en pression. Les limites des formulations normalisées sont identifiées pour les fréquences inférieures à 100 Hz. Deux solutions alternatives sont proposées, permettant de généraliser la gamme de fréquences au domaine des infrasons. La validité de ces formulations est démontrée expérimentalement. Enfin, à partir de ces travaux, le développement du banc primaire est détaillé, avec son modèle analytique et les choix mécaniques associés. / Currently, there is no reference standard for the dynamic infrasonic pressures physical quantity. The request for calibration of such a quantity has recently appeared, in response to geophysical issues, studying the propagation of acoustic waves in the atmosphere between 20 Hz and 0.001 Hz, supported by the monitoring of the respect of the Comprehensive Nuclear-Test-Ban Treaty (CTBT). In order to meet this challenge, the aim of this thesis is to realize a primary standard for this quantity. The metrological context and some existing calibration benches, using infrasonic dynamic pressure generators, are presented. To design the primary calibration bench, the amplitude and phase responses of the CEA infrasound generator are characterized analytically and experimentally. In addition, the definition of the standard model is based on the primary method models for pressure calibration of standard microphones in the acoustic pressure range, using cylindrical cavities. The limits of the acoustic transfer admittance standard formulations for these cavities are identified for frequencies below 100 Hz. Two alternative solutions are proposed, allowing the frequency range to be extended to the infrasound domain. The validity of these formulations is demonstrated experimentally. Finally, based on this work, the development of the primary bench is detailed, with its analytical model and the associated mechanical choices.

There is a high demand for quadruplex-specific compounds that not only bind preferentially to quadruplex DNA over duplex DNA, but also bind to one quadruplex motif over other motifs. Quadruplex structures are recognized as common occurrences in cancer cells, and if a compound could stabilize this structure, it may serve as an effective anti-cancer treatment with minimal side effects. In this study, cyanine dyes’ interactions with DNA were analyzed with fluorescence titrations, UV-Vis thermal studies, circular dichroism titrations, and surface plasmon resonance (SPR) analysis. With these techniques, binding affinity, DNA stabilization, and conformational shifts were analyzed to determine if cyanine dyes could act as quadruplex-specific binding compounds for possible cancer treatments.

The development of small-molecules which target nucleic acids, more specifically the minor groove of DNA, in a sequence specific manner and control gene expression are currently being investigated as potential therapeutic compounds for the treatment of various diseases, including cancer, as well as viral and bacterial infections. The naturally occurring compounds netropsin and distamycin have been shown to demonstrate antitumor and antibacterial properties. Currently, there are synthetic efforts to create pyrrole and imidazole-containing polyamide derivatives of netropsin and distamycin that show potential as medicinal agents. Synthetic pyrrole and imidazole-containing polyamides are potentially useful for targeting and modulating the expression of genes, including those associated with cancer cell growth. The key challenges that must be overcome to realize this goal of using synthetic polyamides in the treatment of disease are the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. As part of a systematic study within the authors’ laboratory, our goal is to develop polyamides which can be synthesized readily yet possess excellent sequence specificity, stronger binding affinity, high solubility in biological media and enhanced cell penetration and nuclear localization properties. There is a need to develop a library of modified polyamides which target DNA and exhibit improved biological properties. The present study is a systematic examination of the binding properties of various modified synthetic polyamide compounds. The synthetic polyamide derivatives presented have more potential as therapeutic candidates over other synthetic polyamides because of their increased water solubility, smaller molecular weights, and molecular design, thus, allowing them to penetrate into cells and localize in the nucleus.

The bulk modulus of pure hydraulic oil and its dependency on pressure and temperature has been studied extensively over the past years. A comprehensive review of some of the more common definitions of fluid bulk modulus is conducted and comments on some of the confusion over definitions and different methods of measuring the fluid bulk modulus are presented in this thesis. In practice, it is known that there is always some form of air present in hydraulic systems which substantially decreases the oil bulk modulus. The term effective bulk modulus is used to account for the effect of air and/or the compliance of transmission lines. A summary from the literature of the effective bulk modulus models for a mixture of hydraulic oil and air is presented. Based on the reviews, these models are divided into two groups: “compression only” models and “compression and dissolve” models. A comparison of various “compression only” models, where only the volumetric compression of air is considered, shows that the models do not match each other at the same operating conditions. The reason for this difference is explained and after applying some modifications to the models, a theoretical model of the “compression only” model is suggested. The “compression and dissolve” models, obtained from the literature review, include the effects of the volumetric compression of air and the volumetric reduction of air due to the dissolving of air into the oil. It is found that the existing “compression and dissolve” models have a discontinuity at some critical pressure and as a result do not match the experimental results very well. The reason for the discontinuity is discussed and a new “compression and dissolve” model is proposed by introducing some new parameters to the theoretical model. A new critical pressure (PC) definition is presented based on the saturation limit of oil. In the new definition, the air stops dissolving into the oil after this critical pressure is reached and any remaining air will be only compressed afterwards. An experimental procedure is successfully designed and fabricated to verify the new proposed models and to reproduce the operating conditions that underlie the model assumptions. The pressure range is 0 to 6.9 MPa and the temperature is kept constant at °C. Air is added to the oil in different forms and the amount of air varies from about 1 to 5%. Experiments are conducted in three different phases: baseline (without adding air to the oil), lumped air (air added as a pocket of air to the top of the oil column) and distributed air (air is distributed in the oil in the form of small air bubbles). The effect of different forms and amounts of air and various volume change rates are investigated experimentally and it is shown that the value of PC is strongly affected by the volume change rate, the form, and the amount of air. It is also shown that the new model can represent the experimental data with great accuracy. The new proposed “compression and dissolve” model can be considered as a general model of the effective bulk modulus of a mixture of oil and air where it is applicable to any form of a mixture of hydraulic oil and air. However, it is required to identify model parameters using experimental measurements. A method of identifying the model parameters is introduced and the modeling errors are evaluated. An attempt is also made to verify independently the value of some of the parameters. The new proposed model can be used in analyzing pressure variations and improving the accuracy of the simulations in low pressure hydraulic systems. The new method of modeling the air dissolving into the oil can be also used to improve the modeling of cavitation phenomena in hydraulic systems.

Oil production from EOR and waterflood processes supplies a considerable amount of the world's oil production. Therefore, the screening and selection of the best EOR process becomes important. Numerous steps are involved in evaluating EOR methods for field applications. Binary screening guides in which reservoirs are selected on the basis of reservoir average rock and fluid properties are consulted for initial determination of applicability. However, quick quantitative comparisons and performance predictions of EOR processes are more complicated and important than binary screening that are the objectives of EOR forecasting. Forecasting (predicting) the performance of EOR processes plays an important role in the study, design and selection of the best method for a particular reservoir or a collection of reservoirs. In EOR forecasting, we look for finding ways to get quick quantitative results of the performance of different EOR processes using analytical model/s before detailed numerical simulations of the reservoirs under study. Although numerical simulation of the reservoirs is widely used, there are significant obstacles that restrict its applicability. Lack of necessary reservoir data and time consuming computations and analyses can be barriers even for history matching and/or predicting EOR/waterflood performance of one reservoir. There are different forecasting (predictive) models for evaluation of different secondary/tertiary recovery methods. However, lack of a general purpose EOR/waterflood forecasting model is unsatisfactory because any differences in results can be caused by differences in the model rather than differences in the processes. As the main objective of this study, we address this deficiency by presenting a novel and robust analytical-base general EOR and waterflood forecasting model/tool (UTF) that does not rely on conventional numerical simulation. The UTF conceptual model is based on the fundamental law of material balance, segregated flow and fractional flux theories and is applied for both history matching and forecasting the EOR/waterflood processes. The forecasting model generates the key results of isothermal EOR and waterflooding processes including variations of average oil saturation, recovery efficiency, volumetric sweep efficiency, oil cut and oil rate with real or dimensionless time. The forecasting model was validated against field data and numerical simulation results for isothermal EOR and waterflooding processes. The forecasting model reproduced well (R2> 0.8) all of the field data and reproduced the simulated data even better. To develop the UTF for forecasting when there is no injection/production history data, we used experimental design and numerical simulation and successfully generated the in-situ correlations (response surfaces) of the forecasting model variables. The forecasting model variables were proven to be well correlated to reservoir/recovery process variables and can be reliably used for forecasting. As an extension to the abilities of the forecasting model, these correlations were used for prediction of volumetric sweep efficiency and missing/dynamic pore volume of EOR and waterflooding processes. / text

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative

Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking: Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein). My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions. Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.

Multiwall carbon nanotubes (MWNTs) have extraordinary mechanical and thermal transport properties. They significantly improve the static and dynamic mechanical properties of acrylic orthopaedic bone cement when added to the dry cement polymer powder. Understanding the role MWNTs play on bone cement polymerization temperatures will lead to improved mechanical integrity of the cement-bone interface in joint arthroplasties. It was determined through thermal testing that MWNTs increased the polymerization time of the methylmethacrylate by 45-460% and decreased the peak exothermic temperature of bone cement with and without antibiotics. The flow of heat produced during polymerizing cement was reduced 25-85% with the addition of MWNTs to the cement powder. This decreases the probability of thermal necrosis and “hot” spots caused by high exothermic polymerization temperatures that can destroy the bone adjacent to the cement. These high temperatures also affect the potency and range of antibiotics used in arthroplasty. Isothermal and elution studies determined that MWNTs altered the heat flow and amount of antibiotic release from bone cement during polymerization. Antibiotic elution from bone cement containing MWNTs could match the elution seen in pure cement. The alteration of the flow of heat from bone cement leads to new options for heat-labile antibiotics in total joint arthroplasty.

Colloidal particles serve as useful building blocks for materials applications ranging from controlled band-gap materials to rationally designed drug delivery systems. Thus, developing approaches to direct the assembly and disassembly of sub-micron sized particles will be paramount to further advances in materials science engineering. This project focuses on using programmable and reversible binding between oligonucleotide strands to assemble and then disassemble polystyrene colloidal particles. It is shown that DNA-mediated assembly can be reversed at a fixed temperature using secondary oligonucleotide strands to competitively displace the primary strands linking particles together. It was found that 1) titrating the surface density of hybridizing probe strands and 2) adjusting the base length difference between primary and secondary target strands was key to successful isothermal disassembly. In order to titrate the surface density of primary probe-target duplexes, colloidal particles were conjugated with mixtures of probe strands and "diluent" strands in order to minimize the number of DNA linkages between particles. To reduce the steric interference of the diluent strands to hybridization events, diluent strands were clipped with a restriction enzyme in select cases. Kinetics studies revealed that a four to six base-length difference between primary and secondary target strands resulted in extensive competitive hybridization at secondary oligonucleotide concentrations as low as 10 nM. Importantly, it was found that the timing for release of either DNA alone or DNA-conjugated nanoparticles could be tuned through choices in the DNA sequences and concentration. Lastly, competitive hybridization was explored in select studies to drive the "shedding" of PEGylated DNA targets from microspheres to reveal underlying adhesive groups or ligands on the particle surface. Unlike prior work relying on elevated temperatures to melt DNA-linkages, this work presents an important first step towards extending DNA as a reversible assembly tool for physiological applications such as multifunctional drug delivery vehicles programmed to disassemble at targeted tissue sites such as malignant tumors.

abstract: HIV/AIDS is the sixth leading cause of death worldwide and the leading cause of death among women of reproductive age living in low-income countries. Clinicians in industrialized nations monitor the efficacy of antiretroviral drugs and HIV disease progression with the HIV-1 viral load assay, which measures the copy number of HIV-1 RNA in blood. However, viral load assays are not widely available in sub-Saharan Africa and cost between 50-$139 USD per test on average where available. To address this problem, a mixed-methods approach was undertaken to design a novel and inexpensive viral load diagnostic for HIV-1 and to evaluate barriers to its adoption in a developing country. The assay was produced based on loop-mediated isothermal amplification (LAMP). Blood samples from twenty-one individuals were spiked with varying concentrations of HIV-1 RNA to evaluate the sensitivity and specificity of LAMP. Under isothermal conditions, LAMP was performed with an initial reverse-transcription step (RT-LAMP) and primers designed for HIV-1 subtype C. Each reaction generated up to a few billion copies of target DNA within an hour. Presence of target was detected through naked-eye observation of a fluorescent indicator and verified by DNA gel electrophoresis and real-time fluorescence. The assay successfully detected the presence of HIV in samples with a broad range of HIV RNA concentration, from over 120,000 copies/reaction to 120 copies/reaction. In order to better understand barriers to adoption of LAMP in developing countries, a feasibility study was undertaken in Tanzania, a low-income country facing significant problems in healthcare. Medical professionals in Northern Tanzania were surveyed for feedback regarding perspectives of current HIV assays, patient treatment strategies, availability of treatment, treatment priorities, HIV transmission, and barriers to adoption of the HIV-1 LAMP assay. The majority of medical providers surveyed indicated that the proposed LAMP assay is too expensive for their patient populations. Significant gender differences were observed in response to some survey questions. Female medical providers were more likely to cite stigma as a source problem of the HIV epidemic than male medical providers while males were more likely to cite lack of education as a source problem than female medical providers. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011