Chloroplast and cytoplasmic ribosomes have been isolated from a number of species of the angiosperm genus Nicotiana. Ribosomal subunit and monosome proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Resultant two-dimensional electrophoretic patterns of chloroplast and cytoplasmic ribosomal proteins were processed by a computer algorithm, developed to formally compare different electrophoretic patterns by the construction of two-dimensional, conformal average electrophoretic mobility maps. The chloroplast ribosomal subunit of N. tabacum contains 22-24 distinct basic polypeptides (pI > 5) and 2-3 acidic proteins (pI < 5). The 50S chloroplast ribosomal subunit possesses at least 1 acidic and 33-35 basic proteins. 40S and 60S cytoplasmic ribosomal subunits of the same species have 26-30 and 47-50 basic polypeptides, respectively. Molecular weights of chloroplast ribosomal proteins (ChRP) and cytoplasmic ribosomal proteins (CyRP) were estimated. There was little similarity between the 2D electrophoretic patterns of ChRP and CyRP of N. tabacum. However, 2D-PAGE patterns of N. tabacum ChRP and CyRP were qualitatively isomorphous with homologous patterns of Chlamydomonas reinhardi, pea and spinach. In terms of molecular weight and electrophoretic pattern N. tabacum ChRP were found to be more closely affiliated with prokaryotic ribosomal proteins than with CyRP from the same species. ChRP were isolated from N. gossei (an Australian species) and its reciprocol interspecies hybrids with N. tabacum (denoted by: T x G and G x T). Interspecies polymorphisms between homologous N. tabacum and N. gossei ChRP were delineated by computerized mobility mapping and co-electrophoresis of radiolabeled N. tabacum ChRP with a large molar excess of N. gossei ChRP. The inheritance mode (Mendelian vs. maternal) of a number of well-defined interspecies ChRP polymorphisms was determined by co-electrophoresis of radioiodinated N. tabacum ChRP with T x G and G x T hybrid ChRP. Results indicate that at least 4 30S ChRP and 3 50S ChRP are encoded by nuclear genes. 30S ChRP from an N. tabacum line carrying a maternally-inherited streptomycin-resistance mutation (SR-1) were compared to N. tabacum 30S ChRP by mobility mapping. Two differences were established between the SR-1 and wild-type 30S ChRP average mobility maps. These findings correlate with the reduced affinity of SR-1 30S chloroplast ribosomal subunits for ('3)H-dihydrostreptomycin, and show that at least one 30S ChRP is encoded by chloroplast DNA. Preparative 2D-PAGE and reverse high performance liquid chromatography (RPHPLC) separation techniques for complex ribosomal protein mixtures were developed. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI

A series of rapid, high sensitivity capillary electrophoresis (CE) separation systems have been developed for the analysis of biological analytes and systems. A majority of the work has focused on development of novel instrumentation, in which new injection and detection strategies were investigated to improve the sensitivity of fast CE. A novel optical injection interface for capillary zone electrophoresis based upon the photophysical activation of caged dye attached to the target analyte was developed. The primary advantage of this approach is the lower background and background-associated noise resulting from reduced caged-fluorescein emission in conjunction with the high quantum yield of the resulting fluorescein. Improved detection limits were obtained compared to those observed in photobleaching-based optical gating. A primary drawback of photolytic optical gating CE is the lack of available caged-dye analogs with sufficiently fast reaction kinetics for online derivatization. To overcome this limitation, we have developed a chemical derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophysical properties of visible, high quantum yield, fluorescent dyes. The feasibility of this approach was evaluated by using an OPA/fluorescent thiol reaction, which was used to monitor neurotransmitter mixtures and proteins. The utilization of a high power ultraviolet light emitting diode for fluorescence detection in CE separations has been introduced to analyze a range of environmentally and biologically important compounds, including polyaromatic hydrocarbons and biogenic amines, such as neurotransmitters, amino acids and proteins, that have been derivatized with UV-excited fluorogenic labels. To understand cellular chemistry, it is imperative that single cells should be studied. This work was focused on developing CE based method to characterize the cellular uptake of TAT-EGFP. We demonstrated TAT mediated delivery of EGFP protein into HeLa cells and TAT-EGFP loaded single cell was analyzed by CE-LIF to determine the intracellular EGFP content. An application of CE-LIF for the determination of biogenic amine levels in the antennal lobes of the Manduca sexta is also explored and methods were developed to analyze a single antennal lobe dissected from moths. The lobe was digested and contents were labeled with the fluorogenic dye prior to CZE analysis.

We investigated the elastic scattering and fluorescence from laser dye solutions inside 5000, 1100, and 96.5 micron inner-diameter hollow-core capillaries. Incident 4416 A laser illumination of Coumarin 7 dye dissolved in ethanol caused fluorescence from approximately 4600 to 6000 A. This was studied over an angular range from 0° to 360°. A light scattering nephelometer coupled with a spectrometer gave intensity measurements as functions of wavelength (at fixed detection angles) and angle (at fixed wavelengths), while the illumination source, dye-filled capillary, and detector remained stationary. We saw capillary size and detection-angle dependence of the fluorescence and elastic scattering. Results show that angular variations of the elastic scattering and emitted fluorescence can be used to determine an optimum detection angle from the capillary with respect to the incident illumination direction. This work is important in the design and execution of Capillary Zone Electrophoresis (CZE) experiments.

Single enzymes of E. coli sourced B-galactosidase were analysed in effort to expand the wealth of knowledge in the area of heterogeneity. Static and dynamic heterogeneity was studied with respect to catalytic rate, electrophoretic mobility, and heat shock protein chaperone systems. Temperature was found to be a contributing factor to the observed range of dynamic heterogeneity, with the range increasing with temperature. The inhibitor dissociation constant was determined to be a heterogeneous property of B-galactosidase. A novel assay was developed in which a single enzyme molecule was subjected to three separate solutions while the enzyme itself remained free in solution. / October 2016

Photodynamic therapy (PDT) is a cancer treatment method in which a photosensitizer, light of a particular wavelength, and also oxygen are used to destroy cancerous cells. Cancer cells absorb the photosensitizing agent which is injected into the body, and it is triggered to cause cell destruction upon absorption of light. This occurs because of the excitation of the photosensitizer produces reactive oxygen species that induce a cascade of cellular and molecular events in the body. Photosensitizing agents that can photo-cleave DNA at long wavelengths are highly demanded in PDT, because the long wavelengths of light can penetrate through tissue deeply compared to visible light. While most of the photosensitizers are activated at wavelengths less than 690 nm, penetration of light continues to increase at increasing wavelengths. In this thesis, photosensitizers that can be activated to oxidize DNA with long wavelengths of light will be discussed. Using quinoline cyanine dyes, here we report the first example of DNA photocleavage at a wavelength of light above 800 nm.

Mixed-function oxidases play a major role in the metabolism of xenobiotics. The main component of this system is the cytochrome P450, it oxidizes substrates coming into our body to more polar products. Another component of mixed-function system - the cytochrome b5 (cyt b5) is able to modulate the function of cytochrome P450, the mechanism of this modulation is yet unknown. However, it is believed that it could be mediated via transfer of electron or allosteric modulation of cytochrome P450 caused by interaction with cyt b5. The aim of this thesis was to find and prepare analogs of cyt b5, which are unable to transfer electrons to cytochrome P450 and simultaneously are structurally very similar to native cyt b5. The conformational stability of cyt b5 and its analogs was monitored using pulse proteolysis. This method employs proteases to cleave the evaluated protein at varying concentration of a denaturant. For soluble proteins, urea is typically used as denaturant in combination with thermolysin as protease. While for membrane proteins, sodium dodecyl sulfate (SDS) is usually used as denaturant together with subtilisin as protease. The aim of this thesis was to use these methods to compare a conformational stability of the native human cyt b5 with apo-cyt b5 and analogs of the cyt b5 reconstituted...

La nanofluidique est l’étude du transport de molécules au travers de nanostructures filtrantes dont la taille avoisine l'épaisseur de la double-couche diffuse à la surface du verre. A cette échelle de la centaine de nanomètres, la charge de surface qui induit une exclusion des ions négatifs à l'extérieur du "nanofiltre" produit un effet de rétention des biomolécules. Des études menées sur le transport électrocinétique au sein d’un nanocanal unique ont permis de montrer qu’il était théoriquement possible de concentrer des solutions, même fortement diluées, avec des taux élevés (jusqu’à 103) grâce à un effet de concentration de polarisation. Ce phénomène pourrait être exploité dans de nombreuses applications de diagnostic médical (analyses rapides et précoces d’échantillons bruts), de contrôle qualité (agroalimentaire) ou encore de défense (suivi continu de zones à risques pour la menace terroriste biologique).La modélisation de la dynamique des phénomènes d’électropréconcentration (sous champ électrique) et de rétention (sous gradient de pression) d’un nanocanal unique s’avère extrêmement ardue. Une multitude d’observations, souvent contradictoires quant au profil de préconcentration obtenu, ont été par ailleurs rapportées dans la littérature, avec des points focaux de préconcentration observés parfois du côté anodique ou du coté cathodique pour une même protéine. Certaines expériences observent ces points focaux soit très loin en amont dans le microcanal réservoir soit directement à proximité de l’entrée du nanocanal. C’est dans ce contexte qu’a été développé précédemment un modèle unidimensionnel permettant de prédire le profil de concentration de l’analyte en tout point de la structure MNM(Micro/Nano/Microcanal) proposée. Ce travail de modélisation a démontré l’existence de quatre régimes distincts: deux régimes coté anodique et deux régimes coté cathodique, plus ou moins éloignés du nanocanal. Ce modèle a mis en avant la sélectivité de ce processus vis-à-vis de la mobilité électrophorétique et de la valence des analytes préconcentrés, et il a permis d’appréhender un peu mieux la diversité des expériences rapportées. Cependant, le régime de préconcentration obtenu dépend du bioanalyte étudié. Il serait pourtant intéressant de ne plus être tributaire des caractéristiques intrinsèques de la solution analysée et de ne plus subir le régime obtenu mais, au contraire, d’effectuer ce que l’on pourrait appeler une électropréconcentration sélective du dit analyte. Ceci pourrait permettre d’effectuer deux étapes primordiales de tout diagnostic que sont la séparation et la préconcentration d’un mélange. Pour se faire, nous avons introduit un paramètre expérimental, une composante hydrodynamique (ou surpression), en sus du champ électrique, pour moduler la localisation de la préconcentration.A l’aide d’une technologie "tout verre" récemment brevetée, nous élaborons aujourd’hui des puces intégrant une nanofente dans un long microcanal. Ces puces sont parfaitement isolantes, biocompatibles et présentent une tenue exceptionnelle au cours du temps. Elles sont combinées à un banc expérimental "fait maison" complètement automatisé (interfacé avec Matlab), qui permet de contrôler les différents paramètres imposés. Les données recueillies sont ensuite prétraitées par d’autres codes Matlab que nous avons développés. Grâce à ces divers outils, de nombreuses expériences d’électropréconcentration "classique" (champ électrique seul) et assistée en pression ont été réalisées pour deux bioanalytes modèles : la fluorescéine et la BSA (Bovin Serum Albumin). Elles ont permis de déterminer les différents paramètres influant sur la préconcentration de ces deux analytes et de prouver la sélectivité et l’efficacité de la méthode proposée ici. Des régimes de préconcentration inattendus, stables et présentant des taux élevés ont en effet été obtenus au cours de cette thèse. / Nanofluidics is the study of the transport of molecules through filtering nanostructures whose size approximates the thickness of the diffuse double layer at the surface of the glass. At this scale of hundreds of nanometers, the surface charge induces an exclusion of negative species outside the "nanofilter" and a retention effect of biomolecule. Studies on the electrokinetic transport in a single nanochannel have shown that it was theoretically possible to concentrate solutions, even highly diluted, with high rates (up to 103) thanks to a concentration polarization effect. This phenomenon can be exploited in many medical diagnostic applications (early and fast sample analysis), quality control (food, water) or defense (continuous monitoring of risky areas for biological terrorist threat). Modeling the dynamics of electropreconcentration phenomena (under an electric field) and retention phenomena (under a pressure gradient) of a single nanochannel is extremely difficult. A multitude of observations, often contradictory regarding the obtained preconcentration profile, were also reported with focal points observed sometimes in the anodic side and other times in the cathodic side for the same protein . Some experiments observe these focal points either upstream in the microchannel reservoir or directly at the entrance of the nanochannel. In this context, a one-dimensional model was previously developed to predict the concentration profile of the analyte at any point of the proposed MNM (Micro/Nano/Microchannel) structure. This modeling work has demonstrated the existence of four distinct regimes: two regimes in the anodic side and two regimes in the cathodic side, more or less distant from the nanochannel. This model highlighted the selectivity of the process regarding the electrophoretic mobility and the valence of the preconcentrated analytes and allowed to understand a little better the diversity of reported experiments. However, the obtained regime depends on the bioanalyte. Though it would be interesting not to be dependent of the characteristics of the analyzed solution and, on the contrary, to realize a selective electropreconcentration of the analyte. This could allow to perform two important steps in any diagnosis: the separation and the preconcentration of a mixture. To do so, we introduced an experimental setting, a hydrodynamic component (or pressure) in addition to the electric field to modulate the localization of the preconcentration .Using an "all glass" technology patented in LPN, chips perfectly insulating, biocompatible and with an exceptional resistance over time are now manufactured. These chips presenting a nanoslit in a straight microchannel are combined with a "homemade" experimental set-up fully automated (interfaced with Matlab®) which allow a perfect control of the various experimental parameters. The data obtained during experiments are then preprocessed by other Matlab codes that we have developed. Thanks to these various tools, many electropreconcentration experiments were performed for two bioanalytes: fluorescein and BSA (Bovin Serum Albumin). They have identified different parameters affecting the preconcentration of these analytes and they have demonstrated the selectivity and the efficiency of the method proposed in this thesis. Unexpected and stable preconcentration regime have been obtained with high rates of preconcentration.

Este trabalho descreve a construção e avaliação de um detector condutométrico sem contato (oscilométrico) para sua aplicação em eletroforese capilar de zona e cromatografia micelar eletrocinética (MEKC). A construção do detector contou com a avaliação de diversos materiais e métodos para a confecção dos eletrodos, tão bem como o aperfeiçoamento do seu circuito eletrônico. O seu comportamento e desempenho foram verificados através do estudo dos diversos parâmetros que influenciam sua resposta, como freqüência e amplitude do sinal aplicado, temperatura e condutividade do meio. Além disso, a simulação do circuito equivalente da cela de detecção auxiliou no entendimento das propriedades do detector frente a alterações na condutividade do meio, na freqüência de operação e nas dimensões da cela. A otimização dos parâmetros operacionais foi racionalizada pela formulação de equações analíticas que descrevem o fator de resposta do detector a partir de parâmetros obtidos experimentalmente. Para o desenvolvimento do sistema de detecção, dois equipamentos completos de eletroforese capilar foram construídos. Sistemas de injeção de amostra por pressão, por gravidade, e eletrocinética foram desenvolvidos. Um dos equipamentos permite que a injeção da amostra seja feita do lado aterrado da fonte de alta tensão. Conseqüentemente, o detector é posicionado próximo do ponto de aplicação da alta tensão. Este é, de que se tem notícia, o primeiro sistema de detecção eletroquímico para eletroforese capilar com essa característica. A termostatização dos capilares foi efetuada por convecção forçada de ar. De maneira a aumentar o grau de automação e facilitar o seu manuseio, os dois equipamentos foram interfaceados a microcomputador. Os equipamentos de eletroforese com detecção oscilométrica construídos permitiram a análise de diversas substâncias em matrizes variadas, como por exemplo íons orgânicos e inorgânicos em água de chuva e material particulado, ácidos graxos de cadeia linear em gordura de coco, formaldeído na fase gasosa da atmosfera, e, pela primeira vez, espécies neutras, como álcoois alifáticos, separadas por MEKC. / This work describes the construction and evaluation of a contactless conductometric (or oscillometric) detector, for its application in capillary zone electrophoresis and micellar electrokinetic chromatography (MEKC). The materials and methods used for the construction of the electrodes, as well as the improvements on the electronic circuit, were evaluated. The behavior and performance of the detector were studied by changing the parameters that affect its response, e. g., the frequency and amplitude of the input signal, temperature and solution conductivity. The simulation of the cell equivalent electric circuit helped to understand the detector properties related to the variations in the solution conductivity, frequency, and cell dimensions. The optimization of the parameters was conducted by the formulation of analytical equations that describe the response factor using experimental data. For the detection development, two complete capillary electrophoresis equipments were constructed. Pressure, gravity, and electrokinetic sample injection systems were developed. One of the equipments allows the sample injection on the grounded side of the capillary. Consequently, the detector is placed near the high voltage application point. This is the first electrochemical detector described for capillary electrophoresis with this characteristic. The capillary thermostating was made by passing a forced air stream. Both equipments were interfaced to microcomputers. The capillary electrophoresis equipments with oscillometric detection were applied to the analysis of many types of compounds in different matrices, such as organic and inorganic ions in rainwater and particulate material, fatty acids in cocoa oil, formaldehyde in the atmosphere and, for the first time, neutral species, like aliphatic alcohols, separated by MEKC.

Nos últimos anos a eletroforese capilar tem substituído a eletroforese em gel e está sendo usada para uma ampla variedade de aplicações forenses, incluindo tipagem de DNA. A fim de superar as desvantagens com relação à análise simultânea de amostras que a eletroforese em gel oferece, equipamentos com arranjos de capilares foram idealizados, assim como a possibilidade de análises multiplexadas em um único capilar por meio da utilização de corantes intercaladores. Neste trabalho foi otimizada a metodologia para amplificação de DNA pela reação em cadeia da polimerase para sete primers correspondentes a sete regiões padronizadas e legalmente aceitas para testes de paternidade. Três casos foram avaliados por eletroforese em microchip, indicando que um método mais reprodutível e de maior resolução deveria ser utilizado, fato que levou ao desenvolvimento de um método para separação de um padrão de tamanho de DNA de 25 pares de base por eletroforese capilar em soluções poliméricas em um equipamento comercial. Este método foi aplicado à separação do mesmo padrão intercalado a um corante dimérico em um equipamento de eletroforese capilar lab-made miniaturizado, com detecção espectrofotométrica na região visível, sugerindo a possibilidade de o equipamento desenvolvido ser utilizado para análises genéticas multiplexadas com custo e tempo minimizados. / In recent years capillary electrophoresis has substituted slab gel electrophoresis and has been used in a variety of forensic applications, such as DNA typing. In order to overcome the disavantages regarding the simultaneous samples analysis that slab gel offers, equipments with capillary arrays were developed, as well as the possibility of multiplex analysis in a single capillary by using intercalating dyes. In this work the metodology to amplify DNA by polimerase chain reaction was studied to seven primers corresponding to seven standardized and legaly accepted regions in paternity tests. Three cases were evaluated by microchip electrophoresis, indicating the need for a more reproductive and with better resolution method has to be used. This fact lead to the development of a method to separate a 25 base pairs DNA ladder by gel capillary electrophoresis in a comercial equipment. In the sequence, this method was apllied to the separation of the same ladder intercalated to a dimeric dye in a lab-made miniaturized capillary electrophoresis system with spectrophotometric detection at visible region, suggesting that the developed equipment can be used for multiplexed genetic analysis with reduced cost and time.

O hiperadrenocorticismo é uma das endocrinopatias mais frequentes em cães. As manifestações clínicas e as lesões associadas com o hiperadrenocorticismo resultam primariamente da hipercortisolemia crônica. Várias são as alterações clínicas observadas em diversos sistemas orgânicos, como também as laboratoriais que ocorrem como consequência dos efeitos gliconeogênico, lipolítico, catabólico, protéico e anti-inflamatório dos glicocorticóides. No que concerne aos rins, a hipercortisolemia crônica pode causar lesão do glomérulo, como também esta estrutura pode sér comprometida secundariamente pela hipertensão arterial sistêmica e, assim, evoluir para doença renal crônica. Neste estudo foram avaliados 10 cães normotensos com hiperadrenocorticismo hipófise dependente, antes e após a terapia com trilostano, com o intuito de verificar a existência ou não de proteinúria patológica pelos métodos quantitativos (razão proteína-creatinina urinária) e qualitativos (eletroforese de proteínas urinárias), como também de acompanhar a intensidade da mesma ao longo da evolução da doença e do curso da terapia. A principal lesão renal detectada nos cães com HAC foi no segmento tubular, constatada pelo predomínio de bandas de proteínas urinárias de baixo peso molecular, indicando o comprometimento na absorção dessas proteínas no segmento proximal do néfron, sendo que a presença dessas bandas perduraram ao longo da terapia, mesmo quando as concentrações séricas de cortisol diminuíram gradativamente após a terapia com trilostano. Ainda, o bom controle do hiperadrenocorticismo pela terapia e a pressão arterial sistêmcia dentro dos valores de normalidade podem ter contribuído para a prevenção do desenvolvimento de lesão glomerular. / Hyperadrenocorticism is one of the most common endocrine disorders in dogs. Clinical sings and organs lesions associated with hyperadrenocorticism result primarily from chronic hypercortisolemia. There are several clinical changes observed in different organ systems, as well as laboratory alterations that occur as a result of the effects of gluconeogenic, lipolytic, catabolic, protein and anti-inflammatory glucocorticoids. Regarding to the kidneys, chronic hypercortisolemia can cause damage to the glomerulus, but also that structure may be compromised by secondary hypertension and, thus, evolve into chronic kidney disease. This study evaluated 10 normotensive dogs with pituitary dependent hyperadrenocorticism, before and after therapy with trilostane, in order to verify the existence of pathological proteinuria by quantitative (urinary protein-to-creatipine) and qualitative (urinary protein electrophoresis) methods, and also to monitor its intensity over the course of the disease and therapy. The main renal lesion detected in dogs with hyperadrenocorticism was in the tubular segment, evidenced by the prevalence of urinary protein bands of lower molecular weight, indicating the lack absorption of these proteins in the proximal segment of the nephron. Low molecular weight proteins persisted throughout therapy, even when serum cortisol concentrations gradually decreased after treatment with trilostane. Moreover, good control of hyperadrenocorticism and blood pressure within the normal range may have contributed to the prevention ofthe development of glomerular injury.