This work presents a design methodology and analyss of the selfoscillating electronic ballast used in fluorescent lamps. The self-oscillating electronic ballast is represented as a control system with a nonlinear behavior. In this representation the describing function method and extended Nyquist stability criterion are used to determine the circuit parameters. Besides, the time domain analysis is performed by the differential equations that describe the circuit behaviour. From the analysis carried out it is possible to get some interesting simple alternatives for the self-oscillating electronic ballast applications, such as: dimming capability, power factor correction, and lamp current creast factor correction. It allows increasing the circuit applications whithout compromising the simplicity, reliability, and low cost that characterizes the self-oscillating electronic ballast. Experimental and simulation results are presented to confirm the performance and to validate the analysis, design, and applications presented using the self-oscillating electronic ballast / Neste trabalho são apresentados a análise e o projeto do circuito de comando auto-oscilante empregado em reatores eletrônicos para lâmpadas fluorescentes. O reator eletrônico auto-oscilante é representado através de um sistema de controle com comportamento não-linear. Sua análise e projeto são realizados através do método da função descritiva e o critério estendido de estabilidade de Nyquist. Realiza-se também uma análise no domínio do tempo em que se solucionam as equações diferenciais que descrevem seu comportamento. Como resultado das análises realizadas, apresentam-se aplicações possíveis deste circuito, tais como: técnica de controle de intensidade luminosa de lâmpadas fluorescentes, correção de fator de potência e controle do fator de crista da corrente da lâmpada fluorescente, permitindo estender as aplicações do circuito de comando auto-oscilante sem comprometer suas características de simplicidade, confiabilidade e baixo custo. Os resultados experimentais e de simulação são apresentados no intuito de demonstrar e validar o projeto, análise e aplicações realizadas empregando o reator eletrônico auto-oscilante

L’objet de cette thèse a été la réalisation de sondes moléculaires fluorescentes pour de la détection de césium et de potassium en milieu aqueux. Deux problématiques ont été abordées : la détection de traces de césium en vue d’applications environnementales, et la mesure de variations de potassium en milieu biologique en vue d’applications biologiques. La première partie de cette thèse concerne la détection du césium. Dans un premier temps, différentes entités complexantes du césium ont été étudiées dans le but de mesurer des concentrations de césium comprises entre 1.10-3 et 5 ppm. Certaines de ces sondes ont ensuite été utilisées au sein d’un système de mesures basé sur un circuit micro-fluidique destiné à mesurer le césium de façon continue. La seconde partie de cette thèse s’intéresse à la détection du potassium. Dont le but a été de mettre au point des sondes pour mesurer le potassium extracellulaire par imagerie de fluorescence. Une cage complexante sélective du potassium a tout d’abord été identifiée. Différentes stratégies ont ensuite été développées pour remplacer la coumarine par un fluorophore excitable à de plus hautes longueurs d’ondes. Parmi les sondes étudiées, le Calix-COU-Alcyne-Sulf a permis d’effectuer des mesures in vitro préliminaires qui ont montré que ce type de sondes ne perturbe pas l’activité neuronale et permet de détecter le potassium dans la gamme de concentration visée. / The aim of this PhD was to study fluorescent molecular sensors in order to detect cesium and potassium in aqueous media. Two different issues have been addressed: the detection of cesium traces for environmental applications, and the measure of potassium fluctuations for biological applications. The first part concerns the detection of cesium. Several complexing units were first studied, to measure cesium concentration between 1.10-3 and 5 ppm. Some of the molecules made were then used in a measuring system based on a micro-fluidic chip to measure cesium in a continuous way. The second part concerns the detection of potassium. The aim was to design sensors to measure extracellular potassium fluctuations by fluorescence imaging. A selective complexing unit was first found. Several strategies were then explored to replace a coumarin by a fluorophore excitable at higher wavelengths. Among the molecules made, the Calix-COU-Alcyne-Sulf enabled preliminary in vitro measurements and showed that this type of molecules does not affect the neuronal activity and enables to measure potassium in the range of concentration targeted.

Le mercure est universellement reconnu comme un contaminant largement distribué dans l'environnement en raison d'activités naturelles et anthropiques. Comme certaines de ses espèces peuvent se propager et s'accumuler à travers la chaine alimentaire, ce polluant peut poser un risque élevé pour la santé humaine. Heureusement, ces dangers sont atténués par la mise en place de politiques et par le suivi de la qualité de l'eau et des aliments dans des laboratoires d'analyse équipés d'instruments de pointe conçus pour fournir une réponse analytique précise et sensible. Malgré cela, cette approche est plus ou moins adéquate pour répondre aux besoins spécifiques des communautés de régions éloignées, où l'accès limité aux installations d'analyse et les coûts d'expédition élevés entravent le suivi exhaustif des aliments. Pourtant, les peuples autochtones habitant l'Arctique, comme les Inuits, courent un risque plus élevé d'exposition au mercure en raison de l'accumulation préférentielle du contaminant aux pôles et d'un mode de vie reposant fortement sur la chasse et la pêche traditionnelles. Considérant que l'alimentation traditionnelle est une partie vitale de la culture et qu'elle procure d'autres nutriments essentiels à la santé, limiter la consommation de ces aliments n'est pas une option viable et l'amélioration du suivi de la qualité alimentaire est donc capitale. Dès lors, le développement d'une plateforme de détection qui serait facile à utiliser, peu coûteuse à produire et qui présenterait une faible empreinte spatiale permettrait d'accomplir le suivi de la qualité directement sur le terrain, favorisant alors l'autonomie de ces communautés. Compte tenu de ce qui précède, l'objectif principal encadrant les travaux de cette thèse était de développer une sonde moléculaire fluorescente pour la détection sélective et sensible du mercure en bénéficiant de la simplicité instrumentale associée à la récolte de signaux optiques. La mise au point de cette stratégie a d'abord exigé la synthèse d'un fluorophore sensible au Hg et l'étude de ses propriétés de détection. Dans un deuxième temps, la photostabilité et la sensibilité au Hg de la molécule ont été améliorées par son assemblage sur des nanoparticules d'argent ayant des propriétés plasmoniques propices à l'exaltation de la fluorescence par le métal (MEF). Troisièmement, le capteur hybride argent-fluorophore développé a été intégré dans un dispositif microfluidique facilitant l'utilisation de la sonde en éliminant la manipulation des solutions typiquement requise par l'utilisateur pour faire l'analyse du mercure. De plus, comme une préparation d'échantillon précède généralement l'analyse en laboratoire, le potentiel de la méthode de détection dans un dispositif portatif qui intégrerait des étapes de préparation d'échantillon a aussi été évalué et discuté. / Mercury is universally established as a contaminant widely distributed in the environment due to natural and anthropogenic activities. As some of its species can distribute and accumulate through the food chain, this pollutant can cause a significant threat to human health. Fortunately, these hazards are alleviated by establishing policies and monitoring this contaminant in analytical laboratories equipped with state-of-the-art instruments designed to provide a precise and sensitive analytical response. Regardless, this approach is sometimes ill-suited to meet the specific needs of populations living in remote regions where access to facilities is limited and high shipping costs impede timely and reasonably priced food monitoring. Yet, the indigenous peoples inhabiting the Artic, such as the Inuits, are at a higher risk of mercury exposure due to the latter's preferential accumulation at the poles combined with a livelihood that strongly relies on traditional hunting and fishing. Since country food is an integral part of their culture and provides other essential nutrients, limiting the consumption of these foods is not a sustainable route and improving the efficiency of quality monitoring is therefore imperative. This could be achieved through the development of a field-deployable detection platform that would be easy to operate, inexpensive to produce and would exhibit a small spatial footprint. This strategy would eventually enable northern communities to have a better autonomy with regards to food monitoring and the ensuing decision-making. The main objective of the project presented in this thesis was to design a fluorescent molecular probe for the sensitive and selective sensing of mercury by benefitting from the instrumental simplicity associated with the detection of optical signals. Developing this strategy first required the synthesis of a Hg-sensitive fluorophore and the study of its detection properties. In a second stage, the molecule's photostability and Hg-sensitivity was enhanced by its assembly onto silver nanoparticles which exhibit plasmonic properties that are conducive to fluorescence enhancement through a phenomenon called metal-enhanced fluorescence (MEF). Thirdly, the MEF assembly was integrated into a microfluidic cartridge that facilitates the usage of the developed sensor without requiring the complex handling of solutions to perform mercury analysis. Moreover, as a sample preparation is usually carried out beforehand in a laboratory set-up, the compatibility of the detection method in a portable device that would also integrate sample preparation steps was evaluated and discussed.

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2011-2012 / Pour bénéficier de la grande sensibilité de la spectroscopic de fluorescence, l'emploi de marqueurs luminescents stables et brillants est nécessaire. Cependant, les fluorophores organiques les plus couramment utilisés souffrent de certaines limitations importantes (hydrophobicité, extinction collisionnelle en milieu aqueux, faible rendement quantique de fluorescence, faible résistance à la photodégradation). À cet égard, le phénomène d'exaltation plasmonique de la fluorescence, soit, le couplage entre les plasmons de surface de nanostructures métalliques et les fluorophores à proximité, représente une technologie puissante pour surmonter ces limitations et augmenter la sensibilité de détection des différentes applications biologiques de ses fluorophores. Dans cette thèse, nous avons développé des nanosondes composées d'un noyau métallique recouvert d'une matrice de silice contenant le(s) fluorophore(s) d'intérêt(s) (Figure 1.13). Dans de tels systèmes, le noyau métallique manifeste une forte résonance plasmonique dont l'interaction avec les molécules fluorescentes environnantes améliore considérablement l'efficacité d'excitation et le taux d'émission radiatif. Par ailleurs, la réduction de la durée de vie de l'état excité du fluorophore qui s'ensuit est responsable de l'augmentation de la photostabilité et de la détectabilité. En outre, la coquille de silice protège les fluorophores contre la désactivation collisionnelle et peut être aisément fonctionnalisée avec des biomolécules cibles ou des fluorophores et former des complexes électrostatiques avec des molécules chargées. Enfin, l'exaltation plasmonique permet d'améliorer l'efficacité, le taux de transfert et la portée du transfert d'énergie par résonance de type Fôrster (FRET) au sein de la nanoparticule en augmentant la force des interactions donneur-accepteur. Tout cela peut conduire à l'excitation par un seul donneur de plusieurs accepteurs sur des distances dépassant la portée normale du FRET. Ces nanoparticules multicouches de type coeur-coquille présentent de nombreuses caractéristiques requises pour l'obtention d'une sonde fluorescente idéale, ce qui a été démontré avec succès avec l'utilisation de ces nanosondes pour la détection ultrasensible et spécifique de faibles quantités d'ADN en solution, sans aucun marquage ou amplification enzymatique des acides nucléiques.

Les techniques de fluorescence sont des outils de choix pour l’étude et la compréhension fine des processus biologiques. Ceci requiert toutefois l’utilisation de sondes fluorescentes parfaitement adaptées au but visé et répondant aux différentes exigences requises pour l’application visée. Dans ce cadre, nous nous sommes plus particulièrement intéressés à l’élaboration de sondes biphotoniques de pH adaptées à une mesure très sensible de faibles variations de pH autour du pH neutre. Les variations et gradients de pH sont en effet impliqués dans un certain nombre de processus biologiques importants et peuvent être associées à des dysfonctionnements liés à certaines maladies. Dans ce cadre, nous avons développé de nouvelles sondes fluorescentes de pH fluorescentes présentant à la fois un comportement ratiométrique, une forte sensibilité autour du pH neutre et facilement excitables dans le proche IR par absorption à deux photons. Ces sondes de structure quadrupolaire et bolamamphiphile permettent ainsi la détection ratiométrique du pH dans des environnements biologiques au moyen d'une excitation biphotonique dans le proche IR. En parallèle, nous nous sommes intéressés à l’élaboration de nanoparticules hyperbrillantes dédiées à l’imagerie biologique par microscopie de fluorescence induite par excitation à deux photons. Nous nous sommes plus particulièrement attachées au design de nanoparticules organiques fluorescentes constituées de molécules organiques de bas poids moléculaire (FONs). Cette approche offre en effet une grande flexibilité et la possibilité d’accéder à des nanosondes ayant des brillances comparables aux très populaires quantum dots mais moins toxiques et plus facilement dégradables. L’ingénierie moléculaire des fluorophores utilisés pour la préparation des FON est cruciale puisqu’elle influence fortement à la fois les propriétés photophysiques (brillance, couleur…) et leur propriétés physico-chimiques (stabilité chimique et structurale, stabilité colloïdale). Dans ce contexte, une librairie de nouveaux chromophores dipolaires a été synthétisée et utilisées pour la préparation de FON par la méthode de nano-précipitation. Leurs propriétés ont été étudiées afin de déterminer la relation entre la structure du chromophore et les propriétés globales des nanoparticules constituées de ces colorants. Ce travail a permis d’identifier les paramètres structuraux permettant d’accéder à des nanoparticules présentant à la fois une brillance exceptionnelle, une émission modulable du vert au rouge et proche IR et une remarquable stabilité colloïdale. Ces nanoparticules présentent des potentialités majeures pour l’imagerie in vivo par excitation et détection dans le proche IR. / Fluorescence-based techniques are popular tools for the study and understanding of biological processes. This has prompted continuous research aimed at the development of a wide range of fluorescent probes specifically designed for specific applications. Among them, fluorescent pH probes are of much interest as pH variations or gradients are involved in many biological events and anomalous alterations are often related to the onset of dysfunctions and diseases. In this framework we have developed a series of promising two-photon pH fluorescent molecular probes. These quadrupolar bolaamphiphilic probes are of great interest, as they combine a steep pH dependence of their optical properties close to neutral pH, ratiometric behavior and large response to two-photon (2P) excitation in the NIR region. As such they offer much promise for ratiometric detection of the pH in biological environments and in situ monitoring of acidification. In parallel, we have been interest in the design of ultrabright nanoparticles for bioimaging purpose (in particular highly sensitive optical imaging). We chose to focus on Fluorescent Organic Nanoparticles made of organic molecules with low molecular weight (FONs) as they offer a flexible route and promising alternatives to toxic quantum dots. In this case the design of the dye used as building blocks of the FONs is of crucial importance and strongly influence the chemical and physical properties of the nanoparticles generated, such as their one and two-photon brightness and both their structural and colloidal stability. In that context a library of novel dipolar chromophores have been synthesized and used to prepare FONs using the nanoprecipitation method. Their properties were thoroughly investigated in order to determine the relationship between the molecular design of the isolated dye and the overall properties of the nanoparticles made of these dyes. As a result, Hyperbright FONs emitting in the green to NIR region and combining giant brightness and remarkable stability have been achieved. They offer major promise for bioimaging based on both excitation and detection in the NIR region.

A hemoglobina de Glossoscolex paulistus (HbGp) é caracterizada por uma massa molecular de 3.600 kDa, alta estabilidade oligomérica, resistência a auto-oxidação, e alta afinidade em ligar oxigênio. A estrutura quaternária desta proteína apresenta 144 cadeias com grupo heme (globinas) e 36 cadeias sem grupo heme (linkers), dispostos em duas camadas hexagonais. No presente trabalho, foi realizado o estudo da estabilidade da oxi-HbGp frente aos processos de dissociação oligomérica e desnaturação, utilizando duas classes de desnaturantes, ou seja, o surfactante brometo de dodeciltrimetilamônio (DTAB), e os agentes caotrópicos cloridrato de guanidina (GuHCl) e ureia. Convém mencionar ainda, que este estudo foi desenvolvido através do uso de duas sondas fluorescentes, 1-Anilino-8-naftaleno-sulfonato (1,8-ANS) e fluoresceína isotiocianato (FITC), usando as técnicas de absorção óptica, fluorescência estática, espalhamento de luz dinâmico (DLS) e fluorescência resolvida no tempo. Os resultados de fluorescência estática mostram que o DTAB induz um aumento na intensidade de emissão de fluorescência da sonda ANS, com o deslocamento do máximo de emissão para o azul de 517 para 493 nm. Duas transições são observadas, em 2,5 e 9,5 mmol/L de DTAB, e estão associadas à interação da sonda ANS com agregados pré-micelares e micelas, respectivamente. Na oxi-HbGp, ANS liga a sítios menos expostos ao solvente, quando comparado às micelas de DTAB, caracterizados pela emissão em 467-472 nm. A adição de DTAB ao sistema oxi-HbGp-ANS, no pH 7,0, induz a agregação da proteína, a dissociação oligomérica e desenovelamento da oxi-HbGp. No pH 5,0, a formação de agregados não foi observada. Além disso, o processo de desenovelamento induzido pelo DTAB apresenta duas transições, a primeira em virtude da dissociação oligomérica, e a segunda, provavelmente, devido à desnaturação das subunidades dissociadas. Por outro lado, GuHCl e ureia com concentrações acima de 1,5 e 4,0 mol/L respectivamente, induzem a desnaturação completa da oxi-HbGp, com redução dos grupos hidrofóbicos na superfície da proteína, e o deslocamento do ANS para o meio aquoso, detectado pela redução de intensidade de fluorescência. A técnica de fluorescência resolvida no tempo permitiu avaliar os valores dos tempos de vida para a sonda 1,8-ANS, bem como, para oxi-HbGp. Por último, a oxi-HbGp foi marcada com a sonda covalente fluoresceína isotiocianato (FITC) em dois valores de pH 7,0 e 9,0, e nas proporções sonda:heme de 1:5 e 2:1. A quantidade de FITC efetivamente ligada a oxi-HbGp por heme foi estimada a partir dos dados de absorção óptica. Supondo que o rendimento quântico de FITC no tampão é 100%, os rendimentos quânticos de FITC ligada a oxi-HbGp também foram encontrados. Além disso, estudos preliminares de dissociação e desnaturação da oxi-HbGp marcada com FITC, na presença do surfactante DTAB, foram realizados. / Glossoscolex paulistus hemoglobin (HbGp) is characterized by a molecular mass of 3,600 kDa, a high oligomeric stability, resistance to oxidation and a high affinity to oxygen. The quaternary structure of this macromolecule consists of 144 globin chains, and 36 additional chains lacking the heme group, named linkers, organized in a double-layered hexagonal structure. In this study, the oxy-HbGp stability, as well as, the oligomeric dissociation and unfolding processes were studied, using two types of denaturants,the surfactant Dodecyl-trimethylammonium bromide (DTAB), and chaotropic agents guanidine hydrochloride (GuHCl) and urea. Moreover, this study was developed based on 8-anilino-1-naphtalene-sulfonic acid (ANS) and fluorescein isothiocyanate (FITC) fluorescence probes, using the techniques of optical absorption, static fluorescence, dynamic light scattering (DLS) and time resolved fluorescence. The results of static fluorescence show that dodecyl-trimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles, characterized by emission at 467 - 472 nm. At pH 7.0, the addition of DTAB to the oxy-HbGp-ANS system induced the protein aggregation, oligomeric dissociation and unfolding of oxy-HbGp. At pH 5.0, no formation of aggregates was observed. Moreover, DTAB-induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, due to the denaturation of dissociated subunits. On the other hand, guanidine hydrochloride (GuHCl) and urea, at concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches on the surface of the proteins. The shift of ANS to the aqueous medium was detected by the reduction in the fluorescence intensity. Time resolved fluorescence technique allowed to evaluate the lifetimes of ANS, as well as, for oxy-HbGp. Finally, oxy-HbGp was labeled with covalent probe fluorescein isothiocyanate (FITC), at pH values 7.0 and 9.0, and at probe: heme ratios of 1:5 and 2:1. The quantity of FITC effectively bound to oxy-HbGp, on the heme basis was estimated from the optical absorption data. Assuming a 100% quantum yield for FITC in buffer, the quantum yield of FITC bound to oxy-HpGp was also estimated. In addition, preliminary studies of dissociation and denaturation of oxy-HbGp labeled with FITC, in the presence of DTAB surfactant, were accomplished.

As espécies reativas de oxigênio (ROS) podem ser responsáveis por causar danos às membranas dos espermatozoides, fragmentação de DNA, entre outros fatores, influenciando assim na fertilidade principalmente no processo de criopreservação do sêmen. A melatonina (MEL) e o ácido ferúlico (AF) são potentes agentes antioxidantes que poderiam atuar no controle da produção de ROS no sêmen equino. Este estudo teve como objetivo avaliar o efeito dos antioxidantes AF e MEL na criopreservação do sêmen equino. Foram utilizados 5 ejaculados de 4 garanhões. Dentre os tratamentos aplicados, foram utilizadas duas concentrações de cada antioxidante (AF 0,5mM, AF 1,2mM, MEL 2mM e MEL 1µM) além do controle (diluidor de congelação convencional BotuCrio®), totalizando 5 tratamentos. As variáveis analisadas foram cinética espermática pelo sistema CASA (programa SCA - Sperm Class Analyser), morfologia, integridade de membranas plasmática, acrossomal e potencial de membrana mitocondrial, com o uso das sondas fluorescentes PI, Hoescht 33342, FITC-PSA e JC-1 além da produção de (ROS) pelo espermatozoide com a sonda fluorescente CellRox Deep Red®. Comparações entre os tratamentos foram realizadas pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3) e as diferenças entre eles foram localizadas através do teste de Duncan. A probabilidade de P0,05 foi considerada como diferença significativa. Os resultados para características da motilidade tiveram diferença significativa em alguns aspectos, porém nenhum tratamento foi superior ao controle. Houve diminuição no percentual de defeitos maiores nas amostras tratadas com AF 1,2mM, MEL 2mM e MEL 1µM comparadas ao grupo controle. No que diz respeito à integridade de membranas, o tratamento MEL 1µM apresentou porcentagens significativamente melhores nas células com membrana plasmática intacta, acrossomo intacto e alto potencial de membrana mitocondrial, quando comparadas ao grupo controle. As células em estresse oxidativo não se diferenciaram entre os tratamentos. O uso da sonda fluorescente CellRox Deep Red® foi validado para espermatozoides de equinos. Foram utilizados 4 ejaculados de 4 garanhões aos quais eram submetidos aos tratamentos T0 (fração do ejaculado não submetida à indução do estresse oxidativo), T50 (50% da amostra não induzida e 50% induzida ao estresse oxidativo) e T100 (amostra induzida ao estresse oxidativo). Os dados de porcentagem de células positivas (com estresse oxidativo) foram submetidos à análise de regressão polinomial pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3). O valor do coeficiente de determinação (R2) foi igual a 0,88 e a probabilidade de P0,05 foi considerada significativa. Pode-se concluir que o tratamento MEL 1µM contribui para a preservação da integridade de membranas espermáticas durante o processo de criopreservação do sêmen equino e que a sonda fluorescente CellRox Deep Red® é eficiente na detecção de espécies reativas de oxigênio no espermatozoide de garanhões. / Reactive oxygen species (ROS) can be responsible for causing damage to the membranes of sperm, DNA fragmentation, among other factors influencing fertility especially in cryopreservation. Melatonin (MEL) and ferulic acid (FA) are potent antioxidants that could act in the control of ROS production in equine semen. This study aimed to evaluate the effect of antioxidants AF and MEL in cryopreservation of equine semen. Five ejaculates from four stallions were used. Among the treatments, we used two concentrations of each antioxidant (AF 0.5mM, AF 1.2mM, MEL 2 mM and MEL 1µM) beyond the control (conventional freezing extender BotuCrio®), totaling five treatments. The parameters analyzed were sperm kinetics with the CASA system (SCA program - Sperm Class Analyzer), morphology, plasma and acrossomal membrane integrity mitochondrial membrane potential, using fluorescent probes PI, Hoechst 33342, FITC-PSA and JC- 1 over production ROS by the sperm with the fluorescent probe CellRox Deep Red®. Comparisons between treatments were performed by generalized linear model (PROC GLM) of SAS (version 9.3) and the differences between them were located with the Duncan test. The probability of P0.05 was considered significant. The results for the motility characteristics were significant differences in some aspects, but no treatment was superior to the control. There was a decrease in the percentage of major defects in the samples treated with AF 1.2mM, MEL 2 mM and MEL 1µM compared to the control group. Regarding to membrane integrity, treatment MEL 1µM showed significantly better in percentages of cells with intact plasma membrane, intact acrosome and high mitochondrial membrane potential compared to the control group. Cells with oxidative stress not differ between treatments. The fluorescent probe CellRox Deep Red® was validated for equine sperm previously. Was used ejaculates of 4 stallion which were subjected to the treatments T0 (fraction of the ejaculate not subjected to induction of oxidative stress), T50 (50% sample uninduced and 50% induced to oxidative stress) and T100 (sample induced to oxidative stress). The data of percentage of positive cells (with oxidative stress) were submitted to polynomial regression analysis based on generalized linear model (GLM PROC) of SAS (version 9.3). The value of the coefficient of determination (R2) was 0.88 and a probability of P0.05 was considered significant. It can be conclude that the treatment MEL 1µM contributes to the preservation of the integrity of sperm membranes during the equine sperm cryopreservation process and the fluorescent probe CellRox Deep Red® is efficient in the detection of reactive oxygen species in stallions sperm.

Esse trabalho é sobre a agregação da droga leishmanicida miltefosina e um análogo fluorescente e sua interação com vesículas fosfolipídicas. A leishmaniose é uma doença tropical causada por diferentes espécies do gênero Leishmania que atinge boa parte do mundo e, nas duas últimas décadas, sua manifestação visceral reapareceu de forma preocupante, uma vez que sua letalidade vem aumentando de forma gradativa. Vários medicamentos estão sendo testados, incluindo o análogo lipídico sintético hexadecilfosfocolina (miltefosina), que é um agente antitumoral e antileishmania administrado oralmente que age nas membranas celulares e pode induzir apoptose. O primeiro local de interação dos análogos de fosfolipídios é a membrana celular e eles apresentam atividade citotóxica não específica em concentrações acima da sua concentração micelar crítica, sendo importante o conhecimento de suas propriedades de agregação em meio aquoso e sua forma de interação com outros agregados presentes no meio. Além disso, derivados fluorescentes da miltefosina permitem o uso de técnicas de fluorescência para a caracterização de sua atividade leishmanicida. Neste trabalho examinamos propriedades de agregação da miltefosina (MT) e de seu análogo fluorescente MT-BODIPY em meio aquoso, utilizando técnicas baseadas em medidas de tensão superficial e de espectroscopia de fluorescência, tanto estática como com resolução temporal. Os resultados de cmc da miltefosina, a 25oC utilizando diferentes métodos, foram 60M em meio aquoso puro (água Milli-Q), 50M em tampão fosfato 10mM (pH 7,4), e 35M em tampão fosfato com NaCl 150mM. Através do estudo de vários parâmetros de fluorescência, verificamos que para a MT-BODIPY, um limite superior para sua cmc é de 10M. Utilizando a sonda fluorescente amino-hexadecil-benzamida (Ahba) estudamos a interação entre a miltefosina e vesículas fosfolipídicas de DMPC e DPPC, analisando os espectros de absorção e emissão fluorescente, a anisotropia estática e os decaimentos da intensidade fluorescente e da anisotropia. O conjunto de resultados mostrou que os efeitos do acréscimo de miltefosina às vesículas, no intervalo de razões molares entre 1:100 e 5:100, não são monitorados pela sonda Ahba, uma sonda localizada na região das cabeças polares. Por outro lado, a análise da fluorescência intrínseca da MT-BODIPY mostrou que seu acréscimo, no mesmo intervalo de razões molares, promove desorganização da bicamada lipídica. Como nesse caso o grupo fluorescente localiza-se no final da cadeia alifática, concluimos que os maiores efeitos da miltefosina sobre as bicamadas ocorrem na região interna das cadeias apolares. / This work is about the aggregation of the leishmanicidal drug miltefosine and a fluorescent analogue and their interaction with phospholipid vesicles. Leishmaniasis is a complex of tropical diseases caused by different species of the genus Leishmania which reaches almost the whole world, including Brazil, and, on the last decades, its visceral form reappeared with hundreds of million people at risk of infection, and the mortality rate increases every year. Several drugs have been used to treat the disease, and miltefosine, a synthetic phospholipid analogue, has been tested and it is already used in some countries. This drug has a confirmed antitumor and orally antileishmanial action on the cell membranes, which is the first local of interaction of a phospholipid analogue. The cytotoxic activity is not specific on concentrations above its critical micelle concentration (cmc), and the knowledge of the aggregation properties of the drug in aqueous medium becomes important, as well as its interaction with other aggregates presents in the environment. Fluorescent analogues of miltefosine allow the use of fluorescent techniques to characterize the antileishmanial activity of miltefosine. In this work we have investigated the aggregation properties of the drug miltefosine (MT), and the fluorescent analogue MT-BODIPY in aqueous medium, by using techniques of surface tension measurements and both, steady-state and time-resolved fluorescence spectroscopy. The values of miltefosine cmc at 25°C from different methods were about 60M in pure aqueous medium (Milli-Q water), 50M in phosphate buffer 10mM (pH 7,4), and 35M in phosphate buffer with the addition of NaCl 150mM. The fluorescent probe amino-hexadecyl-benzamide (Ahba) was used to study the interaction of miltefosine with phospholipid vesicles of DMPC and DPPC, from absorption and fluorescent emission spectra, steady-state anisotropy and fluorescence and anisotropy decays. The results have shown that the effects of miltefosine addition in the vesicles, with molar ratios between 1:100 and 5:100, are not monittored by the probe Ahba, which is located on the polar head groups region on the bilayer. On the other hand, analysing the intrinsic fluorescence of the analogue MT-BODIPY, we concluded that when the molecule is added, in the same molar ratio intervals, there is a disorder in the lipidic bilayer. The fluorescent group BODIPY is located on the aliphatic chain of MT, therefore, the more accentuated effects of miltefosine in the bilayers occur in the region of the apolar tails.

A degeneração testicular (DT) possui grande relevância dentre os distúrbios da reprodução e pode ser causada pelo aumento da temperatura testicular. Este provoca aumento do metabolismo celular, levando ao estresse oxidativo (EO) e apoptose. O tratamento usual consiste na retirada do agente causador e administração de antioxidantes; entretanto, pode não ser eficiente. Dessa forma, o presente estudo preconizou o tratamento da DT por meio de agentes com poder proliferativo: vitamina A e laserterapia de baixa intensidade (LTBI). Foram realizados três experimentos; o experimento 1 objetivou definir a dose de energia da LTBI necessária para a bioestimulação testicular. Foram utilizados seis carneiros distribuídos em três grupos: GC) insulação escrotal (IE) e sem tratamento (n=2); G28) IE e tratado com LTBI com 808 nm de comprimento de onda, 30 mW de potência e 28 J/cm² de densidade de energia por 15 dias a cada 48 horas (n=2); G56) IE e tratado com LTBI com 808 nm, 30 mW e 56 J/cm² por 15 dias a cada 48 horas (n=2). Foram feitas análises clínicas, reprodutivas e histopatológicas. Os dados foram submetidos à análise de variância (ANOVA) e teste de Tukey. Apesar da LTBI diminuir as taxas de espermatozoides com membrana acrossomal íntegra, esta foi eficiente em aumentar a população celular dos túbulos seminíferos no G28. Portanto, a LBTI provocou efeito bioestimulatório em testículos degenerados de carneiros. O experimento 2 objetivou validar a técnica de avaliação do EO espermático por meio da sonda fluorescente CellROX Deep Red®. Foram realizados dois experimentos; o primeiro utilizou ejaculados de três carneiros tratados em T0 (ejaculado não submetido à indução de EO), T50 (50% não induzido e 50% induzido ao EO) e T100 (submetido ao EO). Os dados foram submetidos à regressão linear. No segundo experimento foram utilizados 16 carneiros submetidos à IE. Foram feitas avaliações do EO antes e após a IE. Os dados foram submetidos à ANOVA e teste LSD de Fisher. O coeficiente de determinação foi de 0,728 e houve aumento do EO após a IE. Assim, a sonda CellROX® foi capaz de detectar o EO espermático. No experimento 3 foi proposto tratamento para a DT baseado na suplementação vitamínica ou LTBI. Foram utilizados 33 carneiros distribuídos em seis grupos: CC) sem IE e sem tratamento (n=5); CA) sem IE e tratado com vitamina A IM 120.000 UI/animal, duas vezes por semana durante três semanas (n=6); CL) sem IE e tratado com LTBI protocolo G28 (experimento 1) (n=5); IC) IE e sem tratamento (n = 5); IA) IE e tratado com vitamina A IM 120.000 UI/animal, duas vezes por semana durante três semanas (n=6); IL) IE e tratado com LTBI protocolo G28 (n=6). Foram realizadas análises clínicas, reprodutivas, hormonais e histopatológicas. Os dados foram analisados usando o procedimento de modelos mistos e os efeitos dos tratamentos foram avaliados utilizando contrastes ortogonais. Não houve efeito benéfico dos tratamentos para as características ultrassonográficas, qualidade espermática, concentração de testosterona e aspectos histopatológicos. Assim, os tratamentos não foram eficientes para melhorar a qualidade espermática nem promover a proliferação celular. / The testicular degeneration (TD) has great significance among the reproductive disorders and one of the main causes is the increase in testicular temperature. High testicular temperature results in increase cellular metabolism, leading to oxidative stress and apoptosis. The treatment consists in removing the causative agent and administration of antioxidants; however, it could be not efficient. The objective of this study is to recommend the treatment of TD by administering agents with proliferative action: vitamin A and low level laser therapy (LLLT). For this, three experiments were conducted. In experiment 1 the objective was to define the dose of energy for LLLT testicular biostimulation; it was used six rams distributed in three groups: GC) scrotal insulation (SI) and untreated (n=2); G28) SI and treated with LLLT with 808 nm, 30 mW and 28 J/cm ² of power density for 15 days every 48 hours (n=2); G56) SI and treated with LLLT with 808 nm, 30 mW and 56 J/cm² for 15 days every 48 hours (n=2). Clinics, reproductive and histopathological analyzes were done. Data were subjected to analysis of variance and Tukey test. The rates of sperm with intact acrosome membrane were decreased by LLLT, but the LLLT was effective in increasing the cell population of the seminiferous tubules in the G28. Thus, LLLT was able of causing stimulatory effect in degenerate testis of rams. The objective of experiment 2 was to assess the technique of evaluation of sperm oxidative stress (OS). This study was divided in two experiments; in experiment 1 was used ejaculates of three rams treated in T0 (ejaculate that was not submitted to OS induction), T50 (50% without OS and 50% inducted to OS) and T100 (entire submitted to OS induction). Data obtained were evaluated by linear regression analysis. In experiment 2, sixteen rams were submitted to SI. Analyses of OS were done before and after the SI. Data obtained were evaluated by analysis of variance and Fisher\'s LSD test. The determination coefficient was of 0.728 and there were increase in sperm showing OS after SI period. Thus, CellROX® fluorescent probe was able to detect sperm OS. The objective of experiment 3 was establish a treatment for TD based on vitamin A supplementation or LLLT; 33 rams were distributed in six groups: CC) no SI and non-treated (n=5); CA) no SI and treated with 120,000 IU/animal of IM vitamin A, twice a week for three weeks (n=6); CL) no SI and treated with LLLT G28 protocol (experiment 1) (n=5); IC) SI and untreated (n=5); IA) SI and treated with 120,000 IU/animal of IM vitamin A, twice a week for three weeks (n=6); IL) SI and treated with LLLT G28 protocol (n=6). Clinics, reproductive, hormonal and histopathological analyzes were performed. Data were analyzed using the mixed models procedure and treatments effects were evaluated using orthogonal contrasts. There was no beneficial effect of treatments for ultrasonographic characteristics, sperm quality, testosterone concentration and histopathological aspects. Thus, the treatments were not effective for improving sperm quality or promoting cell proliferation.

A criopreservação do sêmen resulta em danos à estrutura espermática, sendo nítida a importância da avaliação das partidas de sêmen antes de serem submetidas à inseminação artificial em tempo fixo (IATF). Todavia, nem sempre as avaliações convencionais do sêmen são suficientes para identificar partidas que possam resultar em baixa taxa de prenhez no campo, sendo necessária uma investigação mais profunda e acurada. Neste sentido, este experimento foi realizado com o objetivo de identificar partidas de sêmen que apresentam falhas na fertilidade, mesmo sendo aprovadas pelas avaliações convencionais. Foram realizadas análises convencionais (motilidade, vigor, concentração e morfologia espermática) de 72 partidas de sêmen de 22 touros antes da estação de monta. Destas, 55 partidas de 18 touros foram aprovadas para o uso na IATF, mas somente 28 partidas de 10 touros foram utilizadas. As partidas de sêmen utilizadas na IATF foram submetidas a outras avaliações: análise computadorizada da motilidade espermática (CASA), integridade das membranas plasmática e acrossomal e potencial de membrana mitocondrial (por sondas fluorescentes em microscopia de epifluorescência). Os dados foram analisados pelo Proc Mixed do SAS usando o Test T. As taxas de prenhez das diferentes partidas de sêmen variaram de 71 a 37%, sendo a média e desvio padrão das partidas de 55,57±7,57%. Os dados de fertilidade a campo permitiram a separação das partidas de sêmen como de Alta (>50% de prenhez) e Baixa (50% de prenhez) fertilidade, sendo comparadas quatro partidas de Alta e quatro de Baixa fertilidade. Os dados das características seminais de todas as partidas de sêmen foram submetidos à análise por boxplot e separados em quartis superior e inferior. Quando se comparou as partidas de Alta e Baixa fertilidade notou-se diferença na taxa de prenhez (p<0,01), mas não foi notada diferença para motilidade (p=0,91), vigor (p=0,63), concentração (p=0,27), número de espermatozoides por palheta (NEP, p=0,27), número de espermatozoides móveis e normais por palheta (p=0,18), defeitos maiores (p=0,17), defeitos totais (p=0,43), integridade de membrana plasmática (MPI, p=0,07), alto potencial de membrana mitocondrial (AP, p=0,94), motilidade total (MT, p=0,10), VCL (p=0,80), VSL (p=0,75), VAP (p=0,88), LIN (p=0,78), STR (p=0,71) e BCF (p=0,13). No entanto, foram encontradas diferenças entre os grupos (Alta e Baixa) para defeitos menores (p=0,05), espermatozoides com integridade das membranas plasmática e acrossomal e função mitocondrial (PIAIA)/Palheta (p=0,01), integridade de acrossomo (AI, p=0,03), motilidade progressiva (MP, p<0.01) e rápidos (p<0,01). Quando comparados os quartis superior e inferior das características seminais foram encontradas diferenças para concentração espermática (p<0,01), NEP (p<0,01), número de espermatozoides móveis por palheta (p<0,01), espermatozoides móveis e normais (p<0,01), defeitos maiores (p<0,01), defeitos menores (p=0,01), defeitos totais (p=0,05), MPI (p<0,01), AI (p<0,01), AP (p=0,03), PIAIA (p<0,01), PIAIA/palheta (p<0,01), mas esta divisão de grupos por quartis superior e inferior não apresentaram diferença sobre a fertilidade (p>0,05); sendo que somente. Entretanto, para MT (p<0,01) e MP (p<0,01) além da diferença entre os quartis foi notado efeito da fertilidade (MT, p=0,05 e MP, p=0,01), sendo maior para os de baixa fertilidade. Pode-se concluir que os padrões de qualidade de partidas de sêmen que apresentam fertilidade distinta podem ser semelhantes, além disso, que pode haver diferença na qualidade espermática entre as partidas que não influenciam a fertilidade. Desta forma, são necessárias outras análises mais acuradas para investigar as causas de falha na fertilidade de algumas partidas de sêmen. / Semen cryopreservation results in damage to sperm structure, and it is clear the importance of evaluating the semen batches before submitted it to timed artificial insemination (TAI). However, conventional semen evaluations are not always sufficient to identify batch that may result in reduced pregnancy rate in the field, requiring a more thorough and accurate investigation. Thus, this experiment was conducted in order to identify semen batches that have gaps in fertility, even being adopted by conventional assessments. Conventional analysis (motility, vigor, concentration and morphology) were performed of 72 semen batches from 22 bulls before the breeding season. Of these, 55 batches from 18 bulls were approved for use in TAI, but only 28 batches from 10 bulls were used. Semen batches used in TAI were subjected to further assessment: computer-assisted sperm analysis (CASA), integrity of plasma and acrosomal membranes and mitochondrial membrane potential (by fluorescent probes under epifluorescence microscopy). Data were analyzed by Proc Mixed of the SAS using Test \"T\". Pregnancy rates of different semen batches ranged 71-37%, and the mean and standard deviation of the batches was 55.57 ± 7.57%. Field fertility data allowed the separation of the semen batches as \"High\" (>50% pregnancy rate) and \"Low\" (50% pregnancy rate) fertility. It was compared four batches of \"High\" and four batches of \"Low\" fertility. Data of the seminal characteristics of all the semen batches were analyzed by boxplot and separated into upper and lower quartiles. When comparing the batches of \"High\" and \"Low\" fertility was noticed a difference in the pregnancy rate (p<0.01), but was not noticeable difference in motility (p=0.91), vigour (p=0.63), concentration (p=0.27), number of spermatozoa per straw (NEP, p=0.27), number of motile and normal sperm per straw (p=0.18), major defects (p=0.17), total defects (p=0.43) plasma membrane integrity (MPI, p=0.07), high mitochondrial membrane potential (AP, p=0.94), total motility (TM, p=0.10), VCL (p=0.80), VSL (p=0.75), VAP (p=0.88), LIN (p=0.78), STR (p=0.71) and BCF (p=0.13). However, differences were found between the groups (\"High\" and \"Low\") for minor defects (p=0.05), sperm with plasma and acrosomal membranes integrity and high mitochondrial membrane potential (PIAIA)/straw (p=0.01), acrosomal integrity (Al, p=0.03) progressive motility (PM, p <0.01) and rapid (p<0.01). When comparing the upper and lower quartiles of the seminal characteristics differences were found for sperm concentration (p <0.01), NEP (p <0.01), number of motile sperm per straw (p<0.01), motile and normal sperm (p<0.01), major defects (p<0.01), minor defects (p=0.01), total defects (p=0.05), MPI (p<0.01), AI (p<0.01), AP (p=0.03), PIAIA (p<0.01), PIAIA/straw (p<0.01), but this division groups by upper and lower quartiles showed no difference in fertility rate (p>0.05). However, to MT (p<0.01) and MP (p<0.01) the difference between quartiles of fertility effect was noted (MT, p=0.05 and MP, p=0.01), been higher for low fertility. It can be concluded that patterns of semen quality of batches that have distinct fertility may be similar, furthermore, there may be differences in sperm quality among batches that do not affect fertility. Thus, it takes other more accurate analysis to investigate the causes of failure on fertility of some semen batches.