Dissertations / Theses: 'Fluorescents probes'

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Ardizzone, Antonio. "New fluorescent nanovesicles, by self-assembly of organic fluorophores, sterols and surfactants, as probes for bioimaging." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/403924.

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El estudio de nuevas nanopartículas orgánicas fluorescentes (FONs) que puedan superar los límites de las comunes sondas fluorescentes como fluoróforos moleculares, proteínas fluorescentes y nanopartículas inorgánicas es un tema de gran interés para los científicos de materiales que desarrollan nuevas sondas para microscopía de fluorescencia y teranóstica. En los últimos años, se han desarrollado nuevas nanovesículas no liposómicas, basadas en el autoensamblaje de tensioactivos y esteroles, denominados Quatsomes (QSs), que constituyen unos prometedores vehículos de fármacos multifuncionales. Dentro de este escenario, el objetivo principal de esta Tesis (realizada en el marco del proyecto Marie Skłodowska-Curie ITN “Nano2Fun”) es explorar la posibilidad de utilizar los Quatsomes como vehículos para la nano-estructuración en medios acuosos de varias moléculas fluorescentes, independientemente de sus propiedades físico-químicas y ópticas, con el fin de obtener nuevas nanopartículas orgánicas fluorescentes (FONs) con elevada estabilidad coloidal y características fluorescentes optimales, especialmente en relación al brillo. El auto-ensamblaje de fluoróforos orgánicos moleculares, esteroles y tensioactivos de amonio cuaternario en Quatsomes fluorescentes se logró mediante el método DELOS-SUSP, un proceso a base de CO2 comprimido, que garantiza una composición de membrana y una disposición supramolecular altamente homogéneas y, como consecuencia, nanovesículas con elevadas propiedades ópticas. Se han explorado diferentes estrategias para la nano-estructuración en medios acuosos, por medio de QSs, de moléculas fluorescentes con diferentes propiedades fisicoquímicas, incluyendo aquellas solubles y no solubles en agua, analizando el impacto de la nano-estructuración sobre las propiedades ópticas de las FONs obtenidas. De esta manera, los fluoroforos aniónicos solubles en agua, como la fluoresceína, fueron nano-estructurados encima de los QSs. Por otra parte, los fluoróforos lipófilos y no solubles en agua, modificados con largas cadenas alquílicas pueden incorporarse de forma estable en la membrana de los QSs, como se muestra en el caso de varias familias de colorantes, por ejemplo, las cianinas, los diketopirrolopirroles (DPP) y los derivados del fluorene. Los QSs fluorescentes mostraron una estabilidad óptica coloidal excepcional (hasta varios meses), un alto grado de homogeneidad estructural y altas eficiencias de fluorescencia, mostrando mejores prestaciones en comparación con otras nanoestructuras de los mismos fluoroforos. Además, con el objetivo de obtener nanopartículas multicolores, los Quatsomes permitieron cargar simultáneamente diferentes fluoroforos dentro de sus membranas, mostrando un mecanismo de transferencia de energía de resonancia de fluorescencia (FRET) altamente eficiente, una interesante herramienta para monitorear la integridad del carrier durante la administración del fármaco y para la adquisición de imágenes multicolores. En conclusión, los Quatsomes fluorescentes se probaron como nano-sondas para la obtención de imágenes de células in vitro. Se ha demostrado que los Quatsomes que incorporan derivados del fluorene (denominados LysoQS) constituyen una sonda lisosómica altamente específica, ideal para la adquisición de imágenes en tiempos largos. Además, los Quatsomes cargados de cianinas se utilizaron como sondas para técnicas de microscopía superresolución (STORM), que permitió la visualización y resolución de Quatsomes individuales tras la internalización en las células. Los resultados de esta tesis muestran que los Quatsomes fluorescentes, gracias a las ventajas ofrecidas en comparación con otros marcadores fluorescentes comúnmente empleados, son unas nanosondas altamente prometedoras, con posibles aplicaciones futuras en bioimagen, teragnostica y, en general, nanomedicina.
Finding new fluorescent organic nanoparticles (FONs) with the potential to overcome the limits of common fluorescent probes as molecular fluorophores, fluorescent proteins and inorganic nanoparticles is a subject of strong interest for materials scientists developing new probes for fluorescence microscopy and theranostics. In the recent years, innovative non-liposomal nanovesicles, based on the self-assembly of quaternary ammonium surfactants and sterols, named Quatsomes (QSs), have been developed as promising candidates for applications as multifunctional drug carriers. Within this scenario, the main objective of this Thesis (conducted in the framework of the Marie Skłodowska-Curie ITN “Nano2Fun”) is to explore the possibility of using Quatsomes as a vehicle for nanostructuring in aqueous media several dye molecules, irrespective of their physicochemical and optical properties, in order to obtain new fluorescent organic nanoparticles (FONs) with superior colloidal stability and enhanced fluorescent features, especially with high brightness, in relation to single molecule flurofores and other type of FONS. The self-assembly of molecular organic fluorophores, sterols and quaternary ammonium surfactants into fluorescent Quatsomes was achieved by the DELOS-SUSP method, a compressed CO2 –based process which guarantees a highly homogeneous membrane composition and supramolecular arrangement, which have impact on the optical properties of the obtained FONs. Different strategies have been explored to nanostructurate in aqueous media, by mean of QSs, molecular dyes with different physicochemical properties, including those water- and non-water soluble, analyzing the impact of their nanostructuration on the optical properties of the obtained FONs. Thus, anionic water-soluble dyes, such as fluorescein, were nanostructured over QSs surface, taking advantage of anionic/cationic interaction among dye and vesicles surface. On the other hand, lipophilic and non-water soluble dyes modified with long alkyl chains can be stably incorporated into QSs membrane, as shown in the case of several dyes families, including cyanine, diketopyroolopyrrole (DPPs) and fluorene derivatives. The fluorescent QSs showed superior colloidal and optical stability (up to several months), a high degree of structural homogeneity and high fluorescence performances, overcoming those of other nanostructures of the same dyes. Furthermore, aiming to obtain multicolor nanoparticles, Quatsomes allowed the simultaneous loading within their membrane of different dyes, which showed a highly efficient fluorescence resonance energy transfer (FRET) mechanism, an interesting tool for monitoring the carrier integrity during the drug delivery and for multiplexed imaging applications. Finally, fluorescent Quatsomes were tested as nanoprobes for in vitro cells imaging. It has been demonstrated that fluorene-based Quatsomes (named LysoQS) constitute a strongly specific lysosomal probe ideal for long-term imaging. Furthermore, cyanines-loaded Quatsomes were used as probes for super-resolution microscopy technique (STORM) which allowed visualizing and resolving single Quatsomes structures upon internalization in cells. The results of this Thesis showed that fluorescent Quatsomes, thanks to the advantages offered in comparison with other commonly employed fluorescent labels, constitute a promising fluorescent nanoprobes with possible future applications in bioimaging, theranostics and, generally, nanomedicine.

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Celeghini, Eneiva Carla Carvalho. "Efeitos da criopreservação do sêmen bovino sobre as membranas plasmática, acrossomal e mitocondrial e estrutura da cromatina dos espermatozóides utilizando sondas fluorescentes." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-21092006-111358/.

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A criopreservação, como já conhecido, causa danos da função celular podendo, com isso, apresentar redução da fertilidade. O desenvolvimento de técnicas para avaliar a viabilidade espermática, que visam aperfeiçoar o processo de avaliação seminal tem sido meta de muitas pesquisas. Este trabalho foi dividido em dois experimentos. O experimento 1 foi realizado para o desenvolvimento e validação de técnicas práticas e de alta repetibilidade para a avaliação simultânea da integridade das membranas plasmática e acrossomal e função mitocondrial de espermatozóides bovinos pela associação de sondas fluorescentes. O experimento 2 foi realizado para avaliar os efeitos que a criopreservação pode causar sobre a motilidade espermática, membranas plasmática, acrossomal e mitocondrial (pela técnica desenvolvida no experimento 1) e estrutura da cromatina de espermatozóides bovinos frente a dois diluidores diferentes, bem como verificar a existência de correlações entre alterações nas membranas e características de motilidade em espermatozóides bovinos criopreservados. No experimento 1 as associações das sondas iodeto de propídio (PI, para avaliar integridade da membrana plasmática) e aglutinina de Pisum sativum conjugada a isotiocionato de fluoresceína (FITC-PSA, para avaliar a integridade acrossomal), foram divididas em quatro protocolos, nos quais foram testadas diferentes sondas para avaliar a função mitocondrial. Rodamina 123 (R123, protocolo 1), MitoTracker Green FM (MITO, protocolo 2), CMXRos ou MitoTracker red (protocolo 3) e no: iodeto de 5,5,6,6-tetracloro-1,1,3,3-tetraetilbenzimidazolilcarbocianina (JC-1, protocolo 4). Estes protocolos foram testados e validados. Os resultados obtidos foram submetidos à análise de variância, teste de Fisher e à análise de regressão linear pelo programa Stat-View® (SAS Institute, Inc. 1998). O protocolo 1 não apresentou resultados satisfatórios, não sendo possível validar, enquanto, os protocolos 2, 3 e 4 foram validados e apresentaram resultados bastante consistentes. O protocolo 4 foi escolhido para a utilização no experimento 2. Para o experimento 2 foram realizadas sete colheitas de sêmen de oito touros puros da raça Simental. Após a colheita, o sêmen foi avaliado quanto ao volume, concentração, motilidade e vigor visualmente, motilidade por sistema computadorizado (CASA), características morfológicas por microscopia de interferência diferencial, integridade das membranas plasmática e acrossomal, função mitocondrial, pela associação de FITC-PSA, PI e JC-1, e integridade da estrutura da cromatina, pela técnica da acridina laranja. Após as análises, o sêmen foi dividido em duas alíquotas, sendo uma das alíquotas diluída com o diluidor Bioxcell® e a outra com o diluidor Botu-Bov®, envasado em palhetas e submetido a congelação com um sistema automatizado. Duas palhetas cada tratamento foram descongeladas e o sêmen foi avaliado quanto a motilidade e vigor visualmente, motilidade pelo CASA, características morfológicas, integridade das membranas plasmática e acrossomal, função mitocondrial e estrutura da cromatina. A análise estatística foi realizada utilizando-se o programa Statistical Analysis System (SAS Institute Inc., 1985), os dados foram submetidos à análise de variância e teste de Tukey. Foram calculadas correlações lineares de Pearson. O nível de significância foi fixado em 5%. Foram observados efeitos da criopreservação e do diluidor sobre motilidade e vigor visual, motilidade pelo CASA, características morfológicas, integridade das membranas plasmática e acrossomal e função mitocondrial. De forma que, após a criopreservação houve aumento dos danos nas estruturas espermática e o diluidor Botu-Bov® apresentou a maioria das características melhores quando comparado ao diluidor Bioxcell®
The process of cryopreservation causes damages in the sperm cellular function which may lead to a reduction in fertility. New techniques to evaluate sperm viability may improve the process of semen evaluation. The present work was divided in two experiments. Experiment 1 aimed the development and validation of practical techniques with high repetibility for simultaneous evaluation of integrity of plasmatic and acrosomal membrane and mitochondrial function of bovine spermatozoa by the strategic association of fluorescent probes. Objective of experiment 2 was to validate the effects of cryopreservation using two differents diluents on sperm motility, plasmatic, acrosomal, mitochondrial membranes integrity and chromatin structure of bovine spermatozoa. A second objective was to verify the correlations between alterations in membranes and motility characteristics in cryopreserved bovine spermatozoa. In experiment 1 propidium iodide (PI, to evaluate plasmatic membrane integrity) and the Pisum sativum agglutinin conjugated with fluorescein isothicyonate- (FITC-PSA, to evaluate acrosomal integrity). Probes were associated to evaluate mitochondrial function in four protocols: Rhodamine 123 (R123, Protocol 1), MitoTracker Green FM (MITO, protocol 2), CMXRos or MitoTracker red (protocol 3), and 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl carbocyanine iodide (JC-1, protocol 4). Results were submitted to analysis of variance, Fishers test and linear regression analysis by the program Stat-View® ( SAS Institute, Inc.1998). It was not possible to validate protocol 1, while the protocols 2, 3 and 4 were validated and showed consistent results. Protocol 4 was used in experiment 2. For experiment 2 seven ejaculates were collected from eight Simental bulls. Volume, concentration, motility and vigour of semen were evaluated subjectively, motility by computer system (CASA), morphological characteristics by differential interference microscopy, plasmatic and acrosomal membranes integrity and, mitochondrial function with FITC-PSA, PI and JC-1 association, and chromatin integrity by acridine orange technique. Then, semen was divided in two aliquots. One was diluted with Bioxcell® and the other with Botu-Bov® diluent, packaged in 0.5 mL straws and frozen using na automated system. Two straws of sêmen from each treatment were thawed and the semen parameter evaluated, as described above. The statistical analysis was performed using the Statistical Analysis System program (SAS Institute Inc., 1985). Data were submitted to analysis of variance and Tukeys test. Pearsons linear correlations were calculated. Cryopreservation increased damages in spermatic structures. Botu-Bov® diluent was better in preserve the motility and membrane integrity (P⁢0,05) when compared to Bioxcell® diluent

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Peyrard, Lisa. "Cyclotrivératrylènes fluorescents pour la détection d’ammoniums d’intérêt biologique." Thesis, Bordeaux 1, 2012. http://www.theses.fr/2012BOR14700/document.

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Le développement de sondes moléculaires fluorescentes pour le suivi ex vivo de phénomènes biologiques (communication nerveuse, par exemple) est en plein essor. Dans cette optique, des dérivés fluorescents de cyclotrivératrylènes (CTV), cavitands propices à la complexation de petites molécules organiques, ont été synthétisés. La stratégie utilisée consiste à introduire des groupements électro-donneurs et électro-attracteurs conjugués sur chaque unité aromatique du CTV afin d’avoir des systèmes de type « push-pull ». L’extension de la conjugaison entre les groupements électro-attracteurs et électro-donneurs a été envisagée afin d’améliorer les propriétés spectroscopiques des sondes. Des réactions de couplage organométallique de Sonogashira, ainsi que des cycloadditions alcynes-azotures catalysées par le cuivre (CuAAc), ont été conduites sur le squelette CTV dans ce but. La caractérisation spectroscopique des différentes sondes fluorescentes développées a été réalisée en milieu organique et en conditions physiologiques, pour celles dont la solubilité le permettait. L’étude des propriétés de reconnaissance de ces dernières, pour des ammoniums d’intérêt biologique (tels que les neurotransmetteurs, acétylcholine, dopamine et dérivés) en conditions physiologiques, a également été menée par spectrofluorimétrie, ainsi que par d’autres techniques (RMN, calorimétrie). La formation d’assemblages moléculaires en milieu aqueux a également été mise en évidence (expériences de diffusion dynamique de la lumière, microscopie électronique en transmission), pour certaines sondes lors de ce travail
The development of fluorescent probes for the ex vivo detection of biological phenomena (neuronal communication, for example) presents a growing interest. In this context, fluorescent cyclotriveratrylene (CTV) derivatives have been developed, as these cavitands are known to complex small organic molecules. The strategy used to get interesting spectroscopic properties, was to introduce conjugated electron-donating and electron-withdrawing groups on each aromatic unit of the CTV (leading to “push-pull” systems). To improve the spectroscopic properties of the probes, the conjugation between the electron-donating and the electron-withdrawing groups was extended. Hence, the Sonogashira organometalic coupling reaction and the copper catalyzed cycloaddition directly on CTV skeleton were used. The spectroscopic characterization of the new fluorescent probes synthesized was done, in organic solvent but also in physiological conditions when the solubility permits it. The recognition studies for biological ammoniums, such as neurotransmitters (acetylcholine, dopamine, and derivatives) were performed in physiological conditions by spectrofluorimetry but also other methods (like MNR or micro-calorimetry). The formation of molecular assemblies was also observed (by dynamic light scattering and transmission electron microscopy) during this work with some of the probes

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Oger, Samuel. "Analogues fluorescents de l'epicocconone et sondes pour le piégeage de produits naturels azaphiles." Thesis, Normandie, 2017. http://www.theses.fr/2017NORMR035/document.

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L’étude des phénomènes biologiques et notamment du rôle des protéines au sein d’un mécanisme cellulaire est un défi pour les biologistes. L’avènement de la microscopie de fluorescence à excitation biphotonique et des techniques « super-résolutives » a permis l’amélioration des performances des techniques de microscopie classiques et l’application l’imagerie in vivo pour l’analyse des tissus biologiques. Ces techniques requièrent cependant l’emploi de sondes aux propriétés photophysiques optimisées en complément de la spécificité vis-à-vis de la ou des cible(s) biologique(s). L’epicocconone, une molécule naturelle profluorescente de la famille des azaphilones, est employée en protéomique pour la détection des protéines sur gel d’électrophorèse. Ce composé a la faculté de réagir avec les amines des résidus lysine des protéines pour former un adduit covalent énaminone hautement fluorescent dans le proche infrarouge (610 nm) sous irradiation UV (395 nm) ou visible (520 nm). Différents analogues synthétisés au sein du laboratoire ont permis d’étudier la relation structure-fluorescence de ces composés capables de détecter non spécifiquement les protéines du milieu étudié. Afin d’améliorer la spécificité de ces molécules en vue d’applications en imagerie, la synthèse de sondes polyfonctionnelles, via une réaction de cycloaddition 1,3 dipolaire azoture-alcyne catalysée au cuivre, associant un analogue de l’epicocconone à un agent de reconnaissance possédant une affinité particulière pour une cible biologique a été étudiée. La synthèse d’analogues de l’epicocconone optimisés pour l’absorption biphotonique a également été réalisée lors de cette thèse. Les propriétés optiques linéaires et non linéaires de ces composés ont été étudiées afin de sélectionner le meilleur composé pour des applications en imagerie par microscopie de fluorescence à excitation biphotonique. Enfin, la réactivité particulière des azaphilones a servi de point de départ au développement d’une stratégie d’identification et d’isolement de nouvelles molécules naturelles azaphiles grâce à l’utilisation de sondes à produits naturels capables de cibler spécifiquement ce type de composés
Understanding biological processes that involve proteins is a challenge for biologists. Two-photon excitation and super-resolution microscopy have improved drastically bioimaging techniques allowing in vivo deep tissue analysis. However those techniques require the use of optimized and selective fluorescent probes. Epicocconone is a natural profluorescent azaphilone widely used in proteomics for detecting proteins on electrophoresis gels. This compound can react reversibly with primary amines from lysines forming a covalent enaminone adduct that emits near infrared fluorescence light (610 nm) upon UV (395 nm) or visible (520 nm) excitation. Different analogues, that non-selectively bind to proteins, were previously synthesized in order to understand the structure-fluorescence relationship. The synthesis of polyfunctionnal probes was studied using copper-catalyzed azide-alkyne cycloaddition to connect the epicocconone scaffold to a recognition moiety, which can specifically recognise one biological target. New analogues optimized for two-photon absorption were synthesized. Their linear and non-linear optical properties were determined to select the most suitable molecule for two-photon excitation microscopy.In a last part, the particular reactivity of azaphilones was also regarded as a useful strategy for designing probes which could react specifically with azaphilic natural products in order to identify and isolate new ones

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Nina, Diogo Anthony. "Hétérocycles fluorescents pour la détection des protéines carbonylées associées au vieillissement et à l’inflammation." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS355.

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Les travaux présentés dans ce manuscrit décrivent la synthèse de nouvelles sondes fluorescentes pour le marquage des protéines carbonylées. Dans une première partie, la synthèse de nouvelles molécules fluorescentes à base 1,2,4-triazoles 1,3,5-trisubstitués est décrite à partir des 4H-pyrido[e][1,3]oxazin-4-ones. L’étude photophysique des 1,2,4- triazoles dans plusieurs solvants a permis de mettre en évidence un mécanisme de fluorescence de type ESIPT induisant un large déplacement de Stokes (jusqu’à 250 nm). La deuxième partie de ce manuscrit est consacrée à la synthèse de nouveaux BODIPY avec une fonction hydrazide. Enfin, la dernière partie décrit les applications de ces nouveaux BODIPY en tant que marqueurs des protéines carbonylées, utilisable avec la 2D-Oxi DIGE, la microscopie à fluorescence et la cytométrie en flux
The works presented in this manuscript describe the synthesis of new fluorescent probes for protein labelling. In a first part, the synthesis of new fluorescent molecules 1,2,4-triazoles based 1,3,5-trisubstituted is described from 4H-pyrido[e][1,3]oxazin-4-ones. The photophysical study of 1,2,4-triazoles in several solvents allowed to highlight a fluorescence ESIPT type mechanism leadingto a wide Stokes’shift (close to 250 nm). The second part of this manuscript is dedicated to the synthesis of new BODIPY with a hydrazide function. Finally, the last part describes the applications of these new BODIPY as fluorescent probes for carbonylated proteins labelling, used in 2D - Oxi DIGE, fluorescence microscopy and flow cytometry

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Zhou, Xiaobo. "Design, synthesis and sensing properties of chiral amine-based fluorescent probes." HKBU Institutional Repository, 2012. https://repository.hkbu.edu.hk/etd_ra/1442.

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Brookner, Carrie Kazinoff. "Biological basis of cervical tissue autofluorescence /." Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.

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Karpenko, Iuliia. "Conception, synthesis and evaluation of fluorescent probes and PET radioligands for the oxytocin and vasopressin receptors." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAF045/document.

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Les récepteurs de l’ocytocine (OTR) et de la vasopressine (AVPR) sont connus pour être impliqués dans la modulation d’effets centraux complexes. Récemment l’OTR a été proposé comme une cible thérapeutique pour le traitement des troubles du spectre autistique (TSA).Afin de mieux comprendre le rôle de l’OTR et des AVPR dans les TSA, d’éclaircir des nouveaux traits de sa pharmacologie et d’établir des méthodes du criblage sur les récepteurs sauvages, nous avons développé des traceurs pour la tomographie par émission des positons ainsi que des sondes fluorescentes pour la famille OT/AVP des RCPG. Les ligands fluorescents ont été utilisés pour établir un test de liaison TR-FRET pour l’OTR et pour initier le développement du test alternatif sur les récepteurs sauvages. Les radiotraceurs TEP seront bientôt testés chez la souris et chez le singe pour évaluer leurs performances pour la détection des récepteurs de l’ocytocine centraux avant d’envisager des études chez l’Homme
In order to better understand the role of OTR and AVPR in ASD, to reveal new features in its pharmacology and signaling and to establish high-throughput screening method on wild-type G protein-coupled receptors, we developed imaging probes for the oxytocin-vasopressin receptors family, namely radiotracers for positron emission tomography and optical probes for fluorescence detection and imaging. The fluorescent ligands have been used to establish TR-FRET binding assay for OTR and to initiate the development the screening assay for the wild-type oxytocin receptor. The PET radiotracers will be shortly tested in mice and monkeys to evaluate their potency in detecting the central oxytocin receptors

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Meyer, Yves. "Conception et développement de bras réactifs auto-immolables pour la synthèse de sondes pro-fluorescentes : applications à la détection de peptidases dans un contexte in-vivo." Thesis, Rouen, INSA, 2010. http://www.theses.fr/2010ISAM0018.

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L'objectif de cette thèse est la conception et le développement de bras espaceurs auto-immolables originaux situés entre le substrat peptidique et un fluorophore à phénol. Une première partie de ces travaux porte sur le développement de bras réactifs auto-immolables adaptés à la détection d'exopeptidases et à leur application pour la synthèse de sondes destinées à l'imagerie in-vivo de la caspase 3, une enzime impliquée dans le processus apoptotique. La seconde partie de ce travail relate les efforts effectués afin d'étendre l'utilisation des bras réactifs auto-immolables à la détection des endopeptidases, notamment les MMPs, une famille d'enzimes fortement impliquée dans la progression cancéreuse
The aim of this PhD work is the design and development of novel self-immolative species linking a peptide susbstrate to a phenolic fluorophore. A first part was dedicated to the development of self-immolative linkers for exopeptidases detection and their incorporation in caspase 3 probes to stain the apoptotic process. A second part was devoted to the extension of the strategy to endopeptidases, especially MMPs, an enzyme family mainly involved in cancer progression

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Best, Quinn Adams. "XANTHENE AND SILICON ANALOGS OF XANTHENE FLUOROPHORES AS CHEMICAL SENSORS FOR pH AND HYPOCHLOROUS ACID." OpenSIUC, 2013. https://opensiuc.lib.siu.edu/dissertations/662.

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Chemical sensors capable of detecting a specific atom or molecule under various conditions have been utilized in biological and environmental analyses. Fluorescence based sensors are particularly advantageous in these studies because of their high sensitivity, relative ease in handling, and low technical costs. This dissertation focuses on the detection of two analytes, H+ and hypochlorous acid, which are of interest in biology because the presence of abnormal quantities of these analytes may be indicative of disease. We have established a new platform for which sensitive changes in various regions of pH can be detected using fluorescence. The aminomethylrhodamine (AMR) scaffold is highly versatile, i.e. the pH range in which the sensor is active can be tuned by introducing different substituents on the amine moiety. Overall this systematic approach to the design of pH sensitive fluorophores has allowed for a library of compounds that are responsive over a broad range of pH (pH 3 - 10) by simply changing the substituent on the amino group. We report the synthesis and characterization of a silicon analog of rhodamine for the fluorescence based detection of hypochlorous acid. This fluorophore exhibits a 90 nm bathochromic shift in its absorption and emission, relative to its oxygen counterpart. Hypochlorous acid is a biological agent linked to certain diseases. Therefore, the longer wavelength properties of the this far-red fluorescent sensor will be of significant benefit to imaging experiments of this analyte in biological media and tissue due to its spectral proximity of the so called NIR optical window. Furthermore, the novel synthetic methodology of this sensor possesses a key intermediate, which could potentially lead to future fluorescence based sensors. The characterization of a fluorescent probe designed for the detection of hypochlorous acid (HOCl) using a silicon analog of fluorescein (SiF) was also reported. Over a range of pHs, the probe reacts with a stoichiometric amount of HOCl resulting in a mixture of two pH dependent fluorescent species, a SiF disulfide product and a SiF sulfonate product. The unique colorometric properties of the individual SiF fluorophores were utilized to perform simultaneous detection of HOCl and pH. When an excess of HOCl is present, the SiF fluorophores become chlorinated, via an intermediate halohydrin, resulting in a more pH independent and red-shifted fluorophore. Finally, an attempt was made at developing a pH responsive photodynamic therapy agent. This system was designed to target the relatively low extracellular pH found around tumors. A di-bromohydroxymethylrhodamine system was synthesized and the photophysical properties were characterized. This system absorbs weakly under acidic conditions (ca. pH 3), however was shown to be a moderate photosensitizer under acidic conditions.

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Mastrodonato, Cristiano Matteo. "Elaboration of fluorescent molecular probes and molecular-based nanoparticles for bioimaging purposes." Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0652/document.

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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

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12

Sachl, Radek. "Localisation of Fluorescent Probes and the estimation of Lipid Nanodomain sizes by modern fluorescence techniques." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-52619.

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The thesis is divided into two major parts. The first part focuses on the localisation of probes in lipid/polymeric bilayers and in GM1 micelles. Included in this thesis is a new approach based on electronic energy transfer/migration (FRET/DDEM), which efficiently determines transversal positions of fluorescent molecules in lipid bilayers. This approach has been used to locate newly synthesized lipid probes in DOPC bilayers. The label was introduced at the end of sn-2 acyl chains of variable length. Analytical models accounting for FRET exist for a limited number of basic geometries. Here, a combination of FRET and Monte Carlo simulations enables the localisation of probes in bicelles and in bilayers containing pores, i.e. in lipid systems with variable curvature, or in non-homogenous lipid systems. This approach has been used to test whether conical-like fluorescence probes have an increased affinity to highly curved regions, which would enable preferential labelling of membrane pores. A simplified FRET model has been applied to localize 2-pyridones, a class of potential drugs, in GM1 micelles. Since the localisation of drugs within nanoparticles might influence the release kinetics and loading efficiency, knowledge about the drug location is highly relevant. It turned out that all derivatives were localised at the core-shell interface of GM1 micelles. The second part of the thesis focuses mainly on the estimation of lipid nanodomain size by means of FRET, which still remains the most powerful method in this field. Limitations of FRET in the determination of domain size have been explored. We showed that the limitations of FRET are mainly caused by a low probes affinity to either the liquid-ordered or liquid-disordered phase. In the continuing work we provided a detailed dynamic and structural study of crosslinker-triggered formation of nanodomains. Here, two different domains have been revealed, i.e. i) domains whose size grows with increasing amount of added cholera toxin (CTxB), and to which CTxB binds tightly; ii) domains formed in membranes containing a slightly increased amount of sphingomyelin (as compared to i) whose size does not change during titration by additional CTxB and to which CTxB binds less tightly.
Disertace je rozdělena do dvou hlavníchčástí. Prvníčást se zabývá lokalizací značek v lipidových/polymerních dvojvrstvách a v GM1micelách. V práci prezentujeme nový přístup založený na přenosu/migraci elektronické energie (FRET/DDEM), jež umožňuje efektivně určovat vertikální pozici fluorescenčních molekul uvnitř lipidové dvojvrstvy. Tato metoda byla použita k lokalizaci nově syntetizovaných lipidových značek značených na konci sn-2 acylového řetězce s různou délkou v DOPC dvojvrstvách. Analytické modely popisující FRET existují pouze pro limitovaný počet základních geometrií. Kombinace FRETu s Monte Carlo simulacemi nicméně umožňuje lokalizaci značek v bicelách a v dvojvrstvách obsahujících póry, tj. v lipidových systémech s proměnlivým zakřivením a v nehomogenních lipidových útvarech. Tento přístup umožnil např. zjistit, zda kuželovitětvarované značky mají zvýšenou afinitu k vysoce zakřiveným oblastem dvojvrstvy, což by umožnilo preferenční značení pórů. Lokalizovány byly rovněž tři deriváty 2-pyridonů(potencionálních léčiv) v GM1micelách za použití jednoduchého modelu zohledňujícího FRET mezi donory a akceptory nacházejícími se v micelách. Lokalizace léčiv v nanočásticích ovlivňuje kinetiku uvolňování (release kinetics) a množství látky solubilizované v micelách (loading efficiency). Druhá část se především zabývá určováním velikostí lipidových nanodomén pomocí FRETu, který stále zůstává nejvíce výkonnou metodou v této oblasti. Zkoumány byly limitace FRETu v určování lipidových nanodomén. Ukázalo se, že tato omezení jsou především způsobena nízkou afinitou značek buď k Lonebo k Ldfázi. V navazující studii jsme poskytnuli detailní dynamickou a strukturní studii formace nanodomén indukované crosslinkerem. Objevili jsme dva typy domén: a) domény, jejichž velikost se zvětšuje s rostoucím množstvím přidaného cholera toxinu (CTxB) a k nimž se CTxB váže pevně a b) domény vzniklé v membránách se zvýšeným množstvím sfingomyelinu (ve srovnání s a)), jejichž velikost se nemění během titrace dodatečným CTxB a k nimž se CTxB váže méně pevně.
This thesis has been elaborated within the framework of the Agreement on JointSupervision (co-tutelle) of an International Doctoral Degree Programmebetween Charles University in Prague, Czech Republic and the Department of Chemistry at Umeå University, Sweden.

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Sousa, Daniel Bartoli de [UNESP]. "Variabilidade das sub-populações de espermatozóides avaliados pela cinética em sistema computadorizado e combinação de sondas fluorescentes como praâmetro quantitativo do sêmen congelado de ovinos." Universidade Estadual Paulista (UNESP), 2007. http://hdl.handle.net/11449/105992.

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Made available in DSpace on 2014-06-11T19:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-01-17Bitstream added on 2014-06-13T19:24:36Z : No. of bitstreams: 1 sousa_db_dr_botfmvz.pdf: 488884 bytes, checksum: cee00747e11c814a32c18443659e8194 (MD5)
Universidade Estadual Paulista (UNESP)
Várias pesquisas foram desenvolvidas visando a melhoria da congelabilidade do sêmen ovino. Contudo, ainda não houve progressos significativos na fertilidade do sêmen congelado após a inseminação artificial cervical. Diversos sistemas de análise computadorizada do movimento espermático (CASA) têm sido propostos e aplicados na tentativa de quantificar características específicas do movimento espermático, podendo ainda determinar a presença e a cinética das subpopulações de espermatozóides. Muitos testes para avaliar a função espermática foram desenvolvidos, permitindo analisar simultaneamente diferentes aspectos da função espermática. Análise da função mitocondrial oferece uma maneira de acessar a motilidade espermática. Foram objetivos otimizar o sistema CASA na avaliação do sêmen congelado; determinar parâmetros da cinética espermática que expressem analogia com as características da avaliação das membranas plasmática, acrossomal e mitocondrial (IP, FITC-PSA e JC-1; MITO; R-123) e utilizar conjuntamente os parâmetros fornecidos pelo sistema CASA e pelas sondas fluorescentes para agrupar as amostras de maneira qualitativa. Vinte e seis amostras de sêmen congelado de diferentes carneiros foram estudadas pelo CASA obtendo-se para os parâmetros VCL, VAP, VSL, ALH, BCF, LIN, STR, ELONG dados médios e individuais para cada espermatozóide e por sondas fluorescentes para a avaliação simultânea da integridade de membrana plasmática, reação acrossomal e potencial de membrana mitocondrial. Estatisticamente aplicou-se a análise exploratória de técnicas multivariadas obtendo-se três fatoriais sendo o primeiro fator F1 positivo e alto para as variáveis VAP, VSL, STR e LIN, que é interpretado com um fator relacionado à progressividade. Para o segundo fator F2, associam as variáveis VCL, ALH e MT, que representam um fator de deslocamento...
Many researches were developed to improve the ram semen criopreservation. However, no significant advances in the fertility rates with the frozen semen were observed with cervical artificial insemination. Several computer-assisted motility assessments (CASA) systems have been considered and applied in the attempt to quantify specific characteristics of the sperm motion and still them being able to determine the spermatozoa presence and subpopulations kinematics. A large number of sperm functions evaluation had been developed, making it possible to analyze different aspects of the sperm function simultaneously. Analysis of the mitochondrial function offers a way to have access the sperm motility. The aim of this study was to optimize the CASA system in the evaluation of the ram frozen semen; determine parameters of the sperm kinematics that express analogy with the characteristics of the evaluation of plasmatic, acrossomal and mitochondrial membranes (PI, FITC-PSA and JC-1; MITO; R-123) and use CASA parameters together with the fluorescent probes to group samples in a qualitative way. Twenty and six frozen semen samples of different rams were evaluated by CASA system for mean and individual sperm motion parameters VCL, VAP, VSL, ALH, BCF, LIN, STR, ELONG and for fluorescent probes leads for the simultaneous evaluation of the integrity of plasmatic, acrossomal membranes and membrane mitochondrial potential. The statistic applied was multivariate analysis getting to three different factorials. The first factor was positive and high (F1 factor) for variables VAP, VSL, STR and LIN, that were interpreted with the forward displacement. For F2 factor, there were associate variables VCL, ALH and MT that represent the displacement. The F3 factor, whose interpretation has to do with available energy, was associated with variables BCF, MITO and ELONG...(Complete abstract, click electronic address below)

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Sabatini, Carolina Aparecida. "Investigação da hidrólise enzimática de derivados da quinizarina por espectroscopia e microscopia de fluorescência." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/75/75134/tde-08112012-144203/.

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A cinética enzimática dos derivados de quinizarina com cadeias homólogas por lipases imobilizadas foi investigada por espectroscopia de fluorescência. Este estudo foi realizado em nível macroscópico e microscópico. Para o estudo macroscópico, foi utilizada a lipase suportada CALB (Novozyme® 435) e para o estudo microscópico a lipase Rhizopus niveus imobilizada em nanopartículas de sílica. Os derivados de quinizarina são espécies que não apresentam fluorescência, porém, quando são hidrolisados, tornam-se fluorescentes (quinizarina). Com um modelo cinético considerando um mecanismo de dois processos sequenciais do tipo Michaelis-Menten, foi possível fazer uma descrição adequada da evolução temporal da formação da quinizarina. O tempo médio de reação da hidrólise enzimática, em nível macroscópico, foi determinado para os derivados diacetato, dibutirato, dihexanoato e dioctanoato de quinizarina nos solventes hexano, ciclo-hexano e decalina saturados com água. No estudo microscópico, a lipase de Rhizopus niveus foi incorporada em nanopartículas de sílica de 200nm. A hidrólise enzimática foi monitorada por imagens e pela flutuação da intensidade de fluorescência com o tempo, por meio da microscopia de fluorescência confocal. Os resultados mostraram que, após a adição do substrato (derivados da quinizarina), começam a aparecer regiões fluorescentes devido ao trabalho enzimático (formação da quinizarina). As imagens de microscopia de fluorescência confocal não mostraram uma nítida diferença entre os substratos avaliados. Entretanto, o estudo da flutuação da intensidade de fluorescência mostrou que há uma diferença entre os substratos e que é possível estimar constantes de tempo de relaxação da atividade enzimática. Além disso, a atividade da lipase depende da forma em que a mesma está distribuída nas nanopartículas (ligada ou adsorvida) e também do tamanho da cadeia de alquílica que compões os derivados. O decaimento de fluorescência da quinizarina produzida pela hidrólise dos derivados pela lipase foi adquirido por microscopia de fluorescência confocal usando excitação de 2-fótons.
The kinetics of enzymatic hydrolysis of quinizarin diester by supported lipase dispersed beads in organic solvents was investigated by fluorescence spectroscopy. This study was performed on macroscopic and microscopic levels. For the macroscopic study was used CALB immobilized lipase (Novozyme ® 435) on acrylate beads, and for microscopic study Rhizopus niveus lipase immobilized on silica nanoparticles. The quinizarin derivatives (substrates) are non-fluorescent species, and only the end product quinizarin has fluorescence. A kinetic model considering two sequential Michaelis-Menten mechanisms provides a suitable description of the time evolution of the quinizarin formation monitored by emission spectroscopy and photon counting measurements. The average reaction time of the enzymatic hydrolysis was determined for quinizarin diacetate, dibutirate, dihexanoate and dioctanoate in hexane, cyclohexane and decaline water saturated solvents. In the microscopic study, the Rhizopus niveus lipase was dispersed into and bound silica mesoporous 200nm particles. In both systems, dispersed silica nanoparticles and a small fraction of aggregates are found in thin film. The enzyme activity was monitored by images and fluctuations of fluorescence intensity over time using confocal fluorescence microscopy. The results showed that after addition of substrate fluorescent spots due to enzyme activity start to appear. Confocal fluorescence images showed no clear difference among substrates. However, the study of fluorescence intensity fluctuations showed that enzyme activity depends on the type of substrate and enzyme support. In addition, the lipase activity depends on the form in which it is distributed in the nanoparticles (bound or entrapped) and the size of the alkyl diester derivatives. The fluorescence decay of quinizarin produced by lipase hydrolysis of diester was measured by confocal fluorescence microscopy using 2-photon pulse excitation.

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Remy, Charlotte. "Synthèse et étude de récepteurs moléculaires fluorescents pour la détection de molécules neutres." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLN070/document.

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La détection de molécules toxiques pour l’Homme et son environnement est d’une importance cruciale et fait partie des préoccupations majeures de la société actuelle. Les résidus de pesticides tels que l’atrazine ainsi que la mélamine font notamment partie de ces molécules dangereuses pour la santé humaine. Ces deux molécules sont principalement dosées par des techniques lourdes et coûteuses comme la spectrométrie de masse, la chromatographie ou l’électrochimie. De même, la détection des amines biogéniques représente un intérêt sociétal. Elles sont produites par des bactéries durant la décarboxylation des acides aminés dans les cellules. Leur détection permet ainsi d’évaluer la contamination microbiologique et la dégradation potentielle d’un aliment. Elles sont aujourd’hui dosées par chromatographie en phase liquide ou en en phase gaz, par électrochromatographie capillaire et par spectroscopie UV-visible. Quelques exemples de détection par fluorescence ont déjà été décrits dans la littérature mais la nécessité de développer de nouveaux récepteurs fluorescents efficaces est bien réelle.La fluorescence est une technique qui offre de multiples avantages tels que la sensibilité, la sélectivité et un faible coût. De nombreuses sondes fluorescentes capables de détecter des métaux lourds ont été développées au laboratoire PPSM. Cependant, la détection de molécules neutres par fluorescence représente un défi supplémentaire en raison de la nature plus faible de l’interaction, comparée à celle entre espèces chargées.La première étape de cette thèse a été de concevoir et de synthétiser un ensemble de sondes moléculaires fluorescentes, aussi bien pour la détection de l’atrazine, de ses produits de dégradation et des dérivés de la mélamine que pour la détection des amines biogéniques. Des fluorophores dérivés de la molécule de maléimide, de naphthalimide et de l’acide barbiturique ont ainsi été développés pour sonder les dérivés de triazine en exploitant leur système de trois liaisons hydrogène pour la reconnaissance moléculaire. De même, un calix[6]arène fluorescent a été conçu pour déceler la présence des amines biogéniques qui provoqueront une réponse fluorescente par encapsulation dans le calixarène.La deuxième étape a consisté à étudier les propriétés photophysiques de ces sondes. La sonde Naphth-AlcyneOMe possède un rendement quantique élevé, s’est révélée fortement solvatochrome. Elle est de plus sensible à la déprotonation de sa fonction imide. Des études RMN et de modélisation moléculaire ont également été menées afin de caractériser les sondes de manière plus approfondie et de comprendre plus précisément leur réactivité. La spectroscopie RMN a confirmé l’interaction par liaison hydrogène entre les sondes maléimide et naphthalimides et la molécule d’atrazine. Elle a aussi mis en évidence l’encapsulation de l’heptylamine dans le calix[6]arène. Pour sa part, la modélisation moléculaire nous a permis de mieux comprendre la photophysique de la sonde Naphth-TriazoleOMe.Enfin, la capacité des sondes à détecter les divers analytes cibles par fluorescence a été évaluée lors de la dernière étape de ce projet. La sonde TPA-BARB a présenté une forte exaltation de fluorescence en présence des dérivés de mélamine alors que le calix[6]arène-quinoléine Calix-Quino est capable de détecter les amines aliphatiques par fluorescence
The detection of molecules toxic for man and his environment is one of the major concerns of our society. Melamine and the pesticide residues such as atrazine are some of these dangerous molecules. These two molecules are usually measured with time-consuming and costly techniques like mass-spectrometry, chromatography or electrochemistry. In the same way, the detection of biogenic amines is of the greatest importance. They are produced by some bacteria during the decarboxylation of amino acids in the cells. So their detection allows to assess the microbiologic contamination and the potential degradation of a food. Today they are measured by chromatography in the liquid or gas phase, capillary electrochromatography and UV-visible spectroscopy. Some examples of detection by fluorescence have been described in scientific literature, but it is really necessary to develop some new efficient fluorescent receptors.Fluorescence is a technique which offers many advantages such as sensitivity, selectivity and a low cost. A lot of fluorescent probes able to detect heavy metals have been developed in PPSM laboratory. However the detection of neutral molecules by fluorescence represents an additional challenge as the interaction is weaker than with charged species.The first step of this thesis was to design and synthesize a set of fluorescent molecular probes designed to detect atrazine, the products of its degradation and melamine derivatives as well as biogenic amines. Some fluorophores based on maleimide, naphtalimide and barbituric acid moieties have been developed for the detection of the triazines derivatives by exploiting their three hydrogen bonds for molecular recognition. In order to detect the presence of biogenic amines, a fluorescent calix[6]arene which lead to a fluorescent change upon encapsulation in the calixarene cavity has been designed.The second step consisted in studying the photophysical properties of these probes. Naphth-AlcyneOMe probe which has a high quantum yield turned out to be highly solvatochromic. Moreover it is sensitive to the deprotonation of its imide function. NMR studies and molecular modeling were conducted in order to deepen the characteristics of the probes and better understand their reactivity. NMR spectroscopy confirmed the interaction through hydrogen bonding between maleimide and naphtalimide probes and the atrazine molecule.It highlighted the encapsulation of heptylamine in the calix[6]arene. Molecular modeling enabled us to better understand the photophysics of Naphth-TriazoleOMe probe.Finally the capacity of probes to detect the various analytes by fluorescence was assessed in our last part. TPA-BARB probe presented a high exaltation of fluorescence in presence of melamine derivatives whereas the calix[6]arène-quinoleine Calix-Quino is able to detect aliphatic amines by fluorescence

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Dilek, Özlem. "Synthesis and spectroscopic characterization of fluorescent boron dipyrromethene (BDP) probes for site specific bioorthogonal labeling of proteins." Diss., Online access via UMI:, 2009.

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Blašková, Martina. "Využití fluorescenčních sond pro sledování aktivity imobilizovaných fotokalyzátorů." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2015. http://www.nusl.cz/ntk/nusl-217128.

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This diploma thesis deals with the use of fluorescent probes for evaluation of photocatalytic activity of immobilized photocatalyst. To the evaluation of photocatalytic activity of TiO2 were used three different fluorescent probes – terephthalic acid, coumarin and benzoic acid, wherein was monitored the increasing intensity of fluorescence of their oxidation products – hydroxyterephthalic acid, 7-hydroxycoumarin and salicylic acid for the photochemical degradation of various fluorescent probes. To the evaluation of photocatalytic activity was used solid phase (photocatalyst) – liquid phase (probe) system and was used three sources of radiation. Fluorescence of oxidation products was monitored by the fiber spectrometer and a conventional cuvette fluorometer.

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Hofmekler, Jonathan. "Investigating the effect of membrane anchoring on photoinduced electron transfer pyrazoline based fluorescent probes." Thesis, Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/42896.

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Fluorescence microscopy is a powerful analytical tool for visualizing biological processes at the subcellular level. In this regard, 1,3,5-triarylpyrazoline based fluorescent probes which act as "turn-on" probes, have been extensively researched. These probes achieve their fluorescence "turn-on" response by inhibition of fluorescence quenching by acceptor-excited photoinduced electron transfer upon binding of an analyte. It has been recently shown that some fluorescent probes used in biological research form colloids composed of nanoparticles, due to their hydrophobic character. This hydrophobic character can also lead to partitioning of the probe into cellular membranes. Colloid formation and membrane partitioning may affect the probes' photophysical properties such as absorption and emission wavelength and quantum yields. Recently, a series of 1,3,5-triarylpyrazolines synthesized in our group by M. T. Morgan, showed no formation of aggregates in aqueous buffer. Surprisingly, these probes increased their fluorescence intensity in the presence of liposomes. The photoinduced electron transfer process is greatly affected by the polarity of the medium in which the probe is used. In this study, the effect of membrane proximity on the photoinduced electron transfer process for pyrazoline based "turn-on" probes has been investigated. A series of water soluble 1,3,5-triarylpyrazolines have been synthesized in which a N,N-dialkylaniline moiety acts as an electron donor and a proton acceptor and an alkylated sulfonamide moiety acts as a molecular anchor for interaction with neutral and anionic liposomes.

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DeArmond, Fredrick Michael. "Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections." PDXScholar, 2017. https://pdxscholar.library.pdx.edu/open_access_etds/4036.

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As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed [1]. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

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Arribat, Mathieu. "Acides aminés phosphole ou silole : vers de nouvelles sondes fluorescentes pour un marquage de peptide innovant." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTS144.

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La première partie de ces travaux de thèse concerne la synthèse d’acides aminés phosphole par formation d’une liaison P-C. Les propriétés de fluorescence (absorption, émission et rendement quantique) sont modulées à la fois par les différents substituants présents sur le phosphore (BH3, O, S…) ainsi que par le squelette aromatique du phosphole. Des couplages peptidiques modèles réalisés en solution et sur support solide démontrent la possibilité d’intégrer ces acides aminés dans des peptides d’intérêts. La deuxième partie concerne la synthèse de nouveaux phospholes fonctionnalisés ainsi que d’une nouvelle méthode d’accrochage pour les introduire sur différents groupes pendants (SH, NH2, OH) d’acide aminés et peptides via la formation de liaisons P-S, P-N ou P-O. La troisième partie de ce travail a consisté en la synthèse d’une nouvelle classe d’acides aminés tétraphénylsilole fluorescents qui présentent des propriétés d’AIE (aggregation-induced emission) et pourront être utilisés pour le marquage de peptides d’intérêts
The first part of this work is focused on phospholyl amino acids synthesis by formation of a P-C bond. The fluorescent properties (absorption, emission and quantum yield) are modulated either by the substituent on the phosphorus atom (BH3, O, S, …) or by the aromatic skeleton of the phosphole. Peptide coupling in solution or on solid support were performed and showed the possibility to introduce such amino acids into peptide of interest. The second part of this work is dedicated to the synthesis of new functionalized phospholes for a chemoselective grafting on amino acid and peptides pendant groups (SH, NH2, OH) via PS, P-N or P-O bonds. The third part consists into the synthesis of a new class of tetraphenylsilole amino acids which exhibit AIE (aggregation-induced emission) fluorescent properties. Those compounds were successfully incorporated into di- an tri- peptides in solution and on solid support

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Preza, Sérgio Leandro Espindola. "Localização e dinâmica de sondas fluorescentes em modelos de membranas: estudos por dinâmica molecular e anisotropia de fluorescência resolvida no tempo." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/59/59135/tde-01042014-112145/.

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As moléculas AHBA (2-Amino-N-hexadecil-benzamida) e DPH (1,6-Difenil-1,3,5- hexatrieno) são sondas fluorescentes com características particulares, comumente utilizadas para monitorar diferentes regiões das bicamadas lipídicas, no entanto, pouco se sabe sobre a mobilidade e dinâmica destas sondas em membranas e quais os principais fatores que influenciam as suas interações com solventes polares e apolares. Esta tese teve por objetivo estudar essas sondas em diferentes ambientes, para ampliar o entendimento de suas estruturas, mobilidade e dinâmicas rotacionais em diferentes solventes e em bicamadas lipídicas. Utilizou-se a técnica de Dinâmica Molecular (DM) para obter as trajetórias das sondas em caixas com diferentes proporções de água e 1,4-dioxano e também nas membranas de POPC (1-palmitoil-2-oleoil-sn-glicerol-3-fosfocolina) e DMPC (1,2-dimiristoil-sn-glicerol-3-fosfocolina). Com as trajetórias geradas, foram analisadas a estrutura, a solvatação e a dinâmica rotacional das sondas em misturas de solventes e membranas modelo. Para as DM em solventes, os resultados indicaram um comportamento atípico das duas moléculas, com a diminuição da interação com a água a medida que diminuía-se a proporção de 1,4-dioxano na caixa. Em membranas, a localização e mobilidade da sonda AHBA apresentaram comportamento semelhante em POPC e DMPC, com os tempos obtidos a partir da curva de autocorrelação rotacional do seu dipolo comparáveis aos medidos pelo experimento de anisotropia de fluorescência resolvida no tempo. Já para o DPH, os resultados em POPC indicaram que a sonda alinha-se paralelamente à superfície da membrana e apresenta muito mais liberdade para se movimentar quando comparada às aos resultados de DM em DMPC, onde a sonda se alinhou paralelamente às caudas dos fosfolipídios e teve uma restrição bem maior para seus movimentos. Os tempos de correlação rotacional do seu dipolo em POPC apresentaram boa concordância com os obtidos experimentalmente. Em contrapartida, os resultados em DMPC mostraram que é preciso mais tempo de DM para comparação entre a correlação rotacional teórica e a experimental, por ser um sistema mais compactado. De qualquer forma, os resultados indicam que a DM é uma técnica promissora para modelagem da dinâmica rotacional de moléculas em membranas.
AHBA (2-Amino-N-hexadecyl benzamide) and DPH (1,6-diphenyl-1,3,5-hexatriene) molecules are fluorescent probes with particular characteristics commonly used to monitor different regions of the lipid bilayers, however, little is known about the mobility and dynamics of these probes in membranes and the main factors that influence their interactions with polar and non-polar solvents. This thesis aimed to study these probes in different environments, to extend the understanding of their structures, mobility and rotational dynamics in different solvents and in lipid bilayers. It was used the Molecular Dynamic (MD) technique to obtain the trajectories of the probes in boxes with different proportions of water and 1,4-dioxane, and also in membranes of POPC (1-palmitoyl-2- oleoy l-sn-glycerol-3 -phosphocholine) and DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphocholine). With the trajectories generated, the structure, solvation and rotational dynamics of the probes were analyzed in solvent mixtures and model membranes. For simulations in solvents, the results indicate an atypical behavior of the two molecules with the decrease of the interaction with water, when decreased the proportion of 1,4-dioxane in the box. In membranes, the location and mobility of AHBA showed similar behavior for on DMPC and POPC, with the decay times obtained from the dipole rotational autocorrelation curve comparable to experimental time-resolved fluorescence anisotropy data. For the DPH in POPC, the results indicated that the probe is aligned parallel to the membrane surface and is much more free to move when compared to simulations in DMPC, where the probe is aligned parallel to the tails of the phospholipids, and had a greater restriction for their movement. The rotational correlation times of their dipole in POPC showed good agreement with those obtained experimentally. On the other hand, the results in DMPC, showed that it needs more time of simulation for comparison between the theoretical and experimental rotational correlation, because it a more compressed system. In any way, the results indicate that MD is a promising technique for modeling the rotational dynamics of molecules in membranes.

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Rai, Prabin. "Design and synthesis of fluorescent probes." Thesis, Kent State University, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3618852.

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The fundamental objective of this project is to design, synthesize, and characterize fluorescent dyes, which may be utilized in super resolution imaging techniques. In Chapters 1, 2 and 3, we concentrated on photoswitchable rhodamine dyes. We synthesized several rhodamine dyes and increased their water solubility, installed a bioconjugation unit and, more importantly, we optimized the absorption properties (close to 400 nm) of the rhodamine spirolactams in their closed state and studied their basic photophysical properties as well. In Chapter 4, we synthesized azido-DCDHF fluorogens that can be converted to the bright state after a 1,3-dipolar cycloaddition reaction between an azide-Ph-DCDHF and a strained alkene. We synthesized some strained alkenes, which may speed up the kinetics in 1,3-dipolar cycloaddition. This chemical method of turning the dyes from dark to bright state is a new dimension in the bioconjugation arena. In Chapter 5, we synthesized Nile red derivatives which can switch to a bright state from a dark state by collision on the cell surface utilizing PAINT methodology. We expected that the design of new Nile red derivatives may have better properties than the parent Nile red. Besides the PAINT technique, we worked on some active control of emission by enzymatic cleavage of fluorescent dyes in a dark state to the bright state, which can be utilized in super resolution imaging. Related to the 1,3-dipolar cycloaddition reaction between azido-DCDHF and norbornene, we have examined recently popularized tetrazine chemistry. We linked pyridyl tetrazines to DCDHF with short spacer. In Chapter 6, we describe the preparation of co-crystals between perfluorophenazine and several polynuclear aromatic compounds/polynuclear heteroaromatic compounds. In Chapter 7 we describe the preparation of some partially fluorinated heteropolynuclear aromatic compounds such phenzaine and acridine class of compounds for possible use in organic semiconductors.

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Rai, Prabin. "Design and Synthesis of Fluorescent Probes." Kent State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=kent1375091914.

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Banerjee, Anusuya. "Novel, Targettable Bioimaging Probes Using Conjugates of Quantum Dots and DNA." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066376/document.

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Les boîtes quantiques (ou Quantum Dots en anglais - QD) sont une nouvelle génération de sondes polyvalentes pour la biologie, en particulier pour l’imagerie. Pour des applications de marquage des voies intra-cellulaires, les QDs peuvent être conjugués à des biomolécules telles que des acides nucléiques ou des protéines. En partant des travaux du LPEM portant sur le développement de ligands permettant la dispersion des QDs dans l’eau et leur fonctionnalisation, une nouvelle méthode de conjugaison de l'ADN sur les QDs a été développée dans cette thèse. Cette méthode utilise les motifs présents sur les polymères des QDs pour le greffage d'ADN. Les paramètres affectant cette réaction ont été étudiés et cette stratégie de couplage a été étendue à d'autres nanoparticules et biomolécules. En partant de ces QDs-ADN, des protéines modifiées ADN ont pu être attachées aux QDs en utilisant le principe d’hybridation de l’ADN. Les propriétés des conjugués ainsi générés ont été mises en évidence en utilisant la Transferrine (QD-ADN-Tf) et ces complexes ont été étudiés in vitro et in cellulo. Ces conjugués ont ensuite été utilisés pour le suivi de la dynamique des endosomes, exploitant ainsi pleinement le potentiel des QDs pour l’imagerie directe. Dans la dernière partie, des études supplémentaires sur les facteurs influençant la «performance biologique» des QDs ont été réalisées. Pour cela, une large gamme de ligands polymères développée par le groupe a été utilisée pour sonder l'interaction de la surface des QDs avec l'interface biologique. Des expériences biochimiques et cellulaires ont permis de démontrer que les QDs revêtus de divers polymères ont des comportements différents
Quantum dots (QD) are new generation of versatile probes for biology, particularly for bioimaging. For specific applications, QDs are conjugated to biomolecules such as nucleic acid or proteins and subsequently targeted to unique intra-cellular pathways. Building upon the state-of-the-art ligands for water-dispersible QDs developed by the lab, a novel and highly generalizable method to conjugate DNA to QD is developed in this thesis. This method employs thiols present on polymers on QDs for conjugation to maleimide-functionalized DNA. Extensive characterization of parameters affecting this reaction is carried out and the strategy is extended to other nanoparticles and biomolecules. Following this, a novel method to conjugate proteins to QD via DNA hybridization is discussed. Using a model protein Transferrin (Tf), the unique properties of thus generated QD-DNA-Tf conjugates are studied in-vitro and in-cellulo. These conjugates are subsequently used for tracking endosomal dynamics for up-to 20 minutes, exploiting the fullest potential of QDs for live imaging. In the last part, additional studies on factors affecting the ‘biological performance’ of QDs are carried out. Using a range of highly adaptable polymeric ligands developed by the group, interactions of surface-modified QDs with the biological interface are probed. Systematic biochemical and cellular experiments demonstrate that QDs coated with zwitterionic polymers have superior antifouling properties compared to poly(ethylene glycol)-based polymers and stability in diverse biological contexts

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Carter, Ramirez Daniel Marcelo. "Fluorescent and Photocaged Lipids to Probe the Ceramide-mediated Reorganization of Biological Membranes." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23713.

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This thesis describes the development of novel fluorescent and photocaged lipids, and their application as tools to probe the morphological effects of ceramide (Cer)-mediated membrane reorganization in supported lipid bilayers. Cer is a sphingolipid found in eukaryotic cells that plays a key role in regulating biological processes such as apoptosis, cell-to-cell communication, differentiation and some types of pathogenesis. Sphingolipid and cholesterol-rich lipid rafts in the plasma membrane are thought to be the point of origin for many of this lipid second messenger’s effects. Cer is formed in the exoplasmic leaflet of the plasma membrane via the enzymatic hydrolysis of sphingomyelin. The compositional complexity of biological membranes has prompted the adoption of simpler model systems to study the effects of Cer generation. When it is directly incorporated into model membranes, Cer segregates into highly ordered domains with physical properties that are distinct from those of the surrounding fluid environments. However, enzymatic generation of Cer induces complex and dynamic membrane heterogeneity that is difficult to interpret and reconcile with its direct incorporation. Here I describe the synthesis of 4-nitrobenzo-2-oxa-1,3-diazol-7-yl (NBD)-labelled cholesterol (Chol) and Cer analogs, and their use as probes in model membranes exhibiting liquid-disordered (Ld) and liquid-ordered (Lo) phase coexistence. The Chol probes reproduce the modest enrichment of Chol in Lo membrane domains as well as the Cer-induced displacement of cholesterol. One of the NBD Chol probes is used to provide direct visualization of Chol redistribution during enzymatic Cer generation, and assists in identifying new features as Cer-rich regions. The NBD-labelled Cer quantifies membrane order using orientational order parameter measurements derived from polarized total internal reflection fluorescence microscopy (pTIRFM) images. The probe reports on changes in membrane order upon enzymatic generation of Cer, and indicates a significant increase in the molecular order of Ld membrane regions that is consistent with the redistribution of Chol into these areas. The probe also identifies de novo Cer-rich domains as areas of particularly high molecular order. In the final project area, 6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc)-caged Cers are shown to release Cer rapidly and efficiently upon irradiation with near-visible UV light. The caged lipids are then incorporated into supported membranes and photolyzed to release Cer with a high degree of spatial and temporal control. Controlled Cer generation is then used to drive protein-ganglioside clustering in lipid bilayers.

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Cavalcante, Victor Fernandes. "Síntese de seleno- e teluro-cumarinas para estudos de emissão e supressão de fluorescência e aplicações analíticas e/ou biológicas." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/46/46136/tde-27112017-163750/.

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Nos últimos anos, o desenvolvimento e a aplicação de sondas contendo átomos de calcogênio, expandiu significativamente, devido principalmente à reatividade dos elementos dessa família que são facilmente oxidados aos seus correspondentes calcogenóxidos e calcogenonas, permitindo diversas aplicações, especialmente em sistemas biológicos. A inserção de átomos pesados como os calcogênios, ao núcleo fluorofórico, leva à supressão de fluorescência, processo conhecido por \"efeito do átomo pesado\" também atribuída por Transferência Eletrônica Fotoinduzida (Photoinduced Electron Transfer). A oxidação do calcogênio ao correspondente calcogenóxido ou calcogenona inibe esse processo reestabelecendo a fluorescência. Todavia, moléculas com núcleo fluorofórico contendo, principalmente, os átomos de selênio e telúrio tem suas propriedades fotofísicas pouco investigadas, se comparado com moléculas contendo o átomo de enxofre. Neste trabalho foi tratado do desenvolvimento de metodologias de preparação de sondas contendo os átomos de selênio (II) e telúrio (II), mais especificamente, através da funcionalização da 7-hidróxi-4-metil-cumarina. Foram preparadas 6 calcogeno-cumarinas inéditas em rendimentos que variaram de 27% a 69%. Esses compostos apresentaram comportamento fluorescente condizente com o que havia sido idealizado: suas propriedades fotofísicas foram determinadas em acetonitrila, a 298 K, observando-se máximos de absorção em 290 nm e em 320 nm e máximo de emissão de fluorescência em 380 nm. Demais propriedades fotofísicas como rendimento quântico e tempo de vida do estado excitado também foram obtidas. Também foram realizados estudos com os compostos sintetizados frente a espécies oxidantes endógenas (ClO- e H2O2) permitindo inicializar estudos em sistemas celulares, observando-se que as cumarinas contendo o átomo de telúrio (II) demonstraram resultados promissores para seu uso como sondas fluorescentes.
In the last years, the development and application of chalcogen-containing dyes has expanded significantly, mainly due to the chalcogen elements reactivity that are are easily oxidized to their correspondent chalcogenides and chalcogenones, allowing several applications, especially in biological systems. The insertion of heavy atoms such as chalcogens to the fluorophoric core of the molecule leads to a fluorescence suppression, process known as \"heavy atom effect\", also attributed as Photoinduced Electron Transfer (PeT). The chalcogen oxidation to its correspondent chalcogenoxide or chalcogenone inhibts this process reestablishing the fluorescence of the molecule. However, fluorophoric molecules containing selenium and tellurium are not very investigated towards its photophysical properties if compared to their sulfur analogues. It is discussed in this this work, the development of methodologies for the preparation of probes containing selenium (II) and tellurium (II), more specifically, through the functionalization of the 7-methyl-4-hydroxi-coumarin. Six novel chalcogen-coumarins were prepared presenting yields varying from 27% to 69%. These compounds presented consistent fluorescent behavior for what it was predicted: their photophysical properties were determined observing absorption maxima at 290 nm and 320 nm and fluorescence maxima at 380 nm. Other photophysical properties such as quantum yields and excited state lifetime were also obtained. Studies with the synthetized compounds related to their behavior against endogenous oxidant species (ClO- and H2O2) were also conducted, allowing initial studies in cell systems, which demonstrated that the tellurium (II) derived coumarins presented promising results as fluorescent probes

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Valente, John Vic. "Synthetic studies towards potential lead(II) specific fluorescent probes /." Title page, contents and abstract only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phv154.pdf.

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Cheng, Hok Yan. "Near infrared fluorescence probes : towards applications in fluorescence guided surgery." Thesis, University of Hull, 2017. http://hydra.hull.ac.uk/resources/hull:16529.

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Surgery has been a popular method for the treatment of cancers, in particular solid tumours; but the surgical margins for cancerous tissues are often indistinct and in most cases, the poor identification of residual cancer tissues can result in re-excision. Therefore, near infrared (NIR) fluorescence-guided surgery (FGS) is being developed as a real time intra-operative imaging technique to assist surgeons by improving the accuracy and precision of the removal of tumours. However, current FDA approved fluorophores suffer from poor chemical stability, limited water-solubility, and lack selectivity toward neoplastic tissue, limiting their clinical application. These current challenges have led to the development of new and improved fluorophores capable of absorbing and emitting light at NIR wavelengths, negating autofluorescence and improving deeper light transmission. Throughout this project, a series of BODIPYs, aza-BODIPYs and bacteriochlorins were synthesised and developed for bioimaging applications. Despite many of them showing interesting fluorescence properties, the investigation suggested aza-BODIPYs were the most promising red / NIR fluorophores (λem 600-700 nm) due to their excellent photostability. Methods have been developed to incorporate functionalities suitable for bioconjugation. Different bioconjugation strategies have been explored to covalently conjugate the NIR fluorophores to a clinically relevant protein, peptide and antibody under mild conditions. The viability of aza-BODIPY conjugates against biological targets were investigated and a range of other novel targeted NIR fluorophores were successfully developed. In vitro fluorescence imaging was subsequently carried out to demonstrate the enhanced selectivity of the targeting NIR fluorophores toward overexpressed receptors on various cancer cells lines. This project has demonstrated the potential of aza-BODIPY in biological imaging and developed targeted NIR fluorophores. Further biological evaluation is progressing with the eventual aim of developing a pre-clinical model for NIR FGS in oncology.

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Davenport, Eric Parker. "Fluorescent Probes to Investigate Homologous Recombination Dynamics." DigitalCommons@USU, 2016. https://digitalcommons.usu.edu/etd/5007.

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There are multiple mechanisms by which DNA can become damaged. Such damage must be repaired for the cell to avoid ill-health consequences. Homologous recombination (HR) is a means of repairing one specific type of damage, a double-strand break (DSB). This complex pathway includes the Rad51-DNA nucleoprotein filament as its primary machinery. Current methodology for studying HR proteins includes the use of fluorescently labeled DNA to probe for HR dynamics. This technique limits the number of proteins that can be involved in experimentation, and often only works as an end reporter. The work here aims at improving upon standard techniques by creating two fluorescent protein probes. The first probe was developed by directly attaching a fluorophore to Saccharomyces cerevisiae Rad51 with the use of click chemistry and the incorporation of unnatural amino acids. This probe could function as a primary reporter on the formation and dissociation of the Rad51-DNA filament in the presence of pro- and anti- HR mediator proteins. The second probe was created by labeling the exterior cysteine residues of Plasmodium falciparum single strand DNA binding protein (SSB) with a fluorophore via maleimide chemistry. This probe acts as a secondary reporter for HR dynamics by signaling for when free single stranded DNA (ssDNA) is available.

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Cassell, Ryan T. "Synthesis of a PbTx-2 photoaffinity and fluorescent probe and an alternative synthetic route to photoaffinity probes." FIU Digital Commons, 2014. http://digitalcommons.fiu.edu/etd/1612.

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A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom. K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence. Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein. Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached. A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds. The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described. An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.

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Barros, Ana Eliza Barbosa. "Estudos espectroscópicos da hemoglobina extracelular de Glossoscolex paulistus (HbGp)." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/75/75134/tde-29012015-092108/.

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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.

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Lecellier, Daphné. "Fluorescence probes: towards automatic coagulant dosing." Thesis, KTH, Hållbar utveckling, miljövetenskap och teknik, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-227995.

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There is a current lack of accurate tools to determine the concentration of cyanobacteria in situ. Besides, cyanobacterial blooms have to be carefully monitored in reservoirs as they are more frequent because of climate change and can lead to potential released of toxins, along with other components. This project investigates the possible use of fluorescent probes to measure the concentration of different types of organic matter released by the algae. Three different species of toxic cyanobacteria were chosen to carry out this research as they are representative of the local harmful blooms found across Australia. Furthermore, the efficiency of two different chemicals (powdered activated carbon, also known as PAC, and alum) used in drinking water treatment plants were investigated, in order to determine a method for automatic dosage adjustment in water treatment plant. The organic matter was characterized by LC-OCD and fluorescence spectroscopy and statistical analysis such as principal component analysis was performed on the generated data. General characterization of the different species was firstly performed and globally, similar comportments were observed among the three cyanobacteria species. There is indeed a general increase in the release of organic material throughout the cell’s growth phase. Results from the jar tests showed that PAC mainly targeted humic-like substances and building blocks, which are middle size particles. The average removal rate obtained was 40µg/L per mg/L of PAC added into the water. Therefore, there is indication that the decreased efficiency for the removal of the taste and odor compounds observed in certain plants can be partially attributed to direct competition of organic matter adsorption onto PAC instead of a blockage of the PAC pores by larger particles. On the other hand, alum was able to remove large particles, particularly biopolymers and also humic-substances. However, a great increase of the low molecular weight molecules at very high doses of alum was seen, which suggest that a too high dose of alum is toxic for the algae cells. Based on the bench scale testing the recommended dose of 50 mg/L seems to be optimum for the studied water treatment plant. Specific ultraviolet absorbance and dissolved organic carbon measurements were also investigated and good correlations were found between the concentration of humic-like substances and absorbance, confirming that they are good measure to assess the content of organic matter in the water. However, as the slope coefficient of the linear trend varied between the species, it was not possible to obtain a common conversion factor for all the species. Finally, one fluorophore was found in common to all the samples and is characterized by the excitation-emission wavelength: 240/440 nm. Correlations with the chromatography’s results were investigated and this component seems to match the biopolymers and humic-like substances concentrations. Furthermore, its intensity decreases continuously with the addition of PAC whereas a drop was observed at the lower doses of alum. In regards on these findings, a method for automatic chemicals dosing from the fluorescence measures was proposed.
Detta examensarbete handlar om hur dricksvattenkvalitet kan kontrolleras och övervakas i vattenreningsverk. Nu existerar inte någon exakt metod för att bestämma koncentrationen av cyanobakterier i vatten då det finns många olika arter. Men det är viktigt att övervaka algers tillväxt in i vattenreningsverk för de kan släppa ut skadliga ämnen till dricksvattnet. De tre arter som studerats i detta projekt är giftiga. Det organiska materialet i råvattnet kan också blockera membranporer eller leda till n biprodukter, som är cancerframkallande. Till sist konkurrerar några organiska substansen med smak- och luktföreningar för adsorptionsställena hos det pulverformiga aktiverade kolet. Därför är smaken och luktföreningarna inte väl borttagna, vilket leder till kundernas klagomål. Cyanobakterier måste övervakas noggrant. För att bestämma biologisk och kemisk egenskap hos vatten används flera tekniker för närvarande. I examensarbetet har undersökningar med vätskekromatografi och fluorescensteknik företagits. Kromatografi användes för att klassificera den organiska substansen i mindre grupper: biopolymerer, humus substanser, byggstenar och neutralmolekyler med låg molekylvikt (LMVN). Statistisk analys med R, inklusive huvudkomponentanalys företogs på insamlade data. Fluorescensdata registrerades också och visas i en excitationsutsläppsmatris. Experimenten reproducerade en behandlingsprocess och undersökte effektiviteten hos två kemikalier: pulveriserat aktivt kol (PAK) och alun. Resultaten visade att humusämnen och dess byggstenar var väl borttagna av PAK medan även biopolymerer och humusämnen var väl bortagna av alun. Emellertid var en för hög dos av alun skadlig eftersom det ledde till en ökad frisättning av LMVN. I synnerhert kunde PAK ta bort 40µg/L av både humusämnen och dess byggstenar per mg/L av PAK tillagd. Det föreslår att de är de viktigaste konkurrenterna och att endast direkt konkurrens för adsorptionsställena sker. Om det fanns blockeringsfenomen, skulle det också finnas en minskning för biopolymererna. Den optimala doseringen av alun som bestämdes för det undersökta vattenreningsverket var 50 mg/L. Det kunde ta bort 60-70% av biopolymerer och 40-50% av humusämnen. Specifik ultraviolett absorbans och fluorescens registrerades. Båda visade riktigt bra korrelationer med humusämnen, vilket gör de till bra verktyg för att bedöma vattenkvaliteten. Men det kräver fortfarande att arten av cyanobakterie urskiljs eftersom koefficientens lutningar var olika. De kan därför vara ett verktyg för att mäta koncentrationen av organisk material, men arten måste vara känd. Fluorescencedata visade en topp vid 440 nm. En parallellfaktoranalys utfördes på data och endast en komponent hittades gemensam i alla prover. Därför studerades den maximala fluorescensintensiteten hos denna komponent. Å ena sidan kunde vi observera en kontinuerlig minskning av intensiteten när PAK tillsattes. Det är därför möjligt att veta hur man justerar den kemiska doseringen från fluorescensintensiteterna. I slutet av examensarbetet föreslås en metod för automatisk kemisk dosering. Fluorescensprober kan ännu inte exakt indikera cellkoncentrationen. Men med flera sonder som riktar sig till olika våglängder kan de redan vara till stor hjälp för styrning vid vattenverk.

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33

Sánchez, Cid Antonio Alberto. "Organophosphorus compounds as fluorescent probes for cell imaging." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/4043.

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Small molecules containing fluorescent moieties can be used as a means of studying cell structure and function, as a result of the high sensitivity of fluorescence microscopy. This technique allows one to obtain specific information about the cell and has recently attracted considerable interest by many research groups. This work presents three projects in which the main aim was to develop multi-modal imaging agents. They will possess a fluorescent group and also another moiety which provides predictable biological properties. Our interest is centred on two types of fluorophore: Polycyclic Aromatic Hydrocarbons (PAHs) and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). The first project describes the synthesis of the phosphorus analogues of the biologically-active indazole core which remain rare in the literature. The synthesis presented here shows the versatility of our approach and allows for substitution on the phenyl ring of the newly formed phosphindole core simply by changing the nitrile used. Position 3 of the phosphindoles was also varied to bear different aromatic groups; the chosen aromatic systems were phenyl, naphthyl and anthracyl. These were chosen in order to prepare a fluorescent and biologically-active core. In practice, the phosphindoles showed near zero quantum yields. Photoinduced Electron Transfer and Dexter Energy Transfer seem to be the plausible responsible phenomena behind this lack of fluorescence. Temperature was found to be a key variable in the synthesis of phosphindoles since a temperature below 110 °C in the last step led to the formation of two chlorothiophosphonates. One of these unexpected chlorothiophosphonates showed strong activity against Bacillus subtilis and Streptococcus pyogenes. The second project describes the synthesis of the pyrene-based ligand 109, which is significant as it was based on an air-stable alkyl primary phosphine. This remarkable stability is provided by the electronic properties that both the pyrene and the butyl linker ii confer on the corresponding primary phosphine. The tridentate ligand 109 was obtained following a double hydrophosphination reaction of the primary phosphine, and 109 was subsequently used to create complexes with the transition metals from groups 9 and 10. These demonstrated demonstrated weak fluorescence despite the presence of a metallic core. The presence of the DNA intercalating pyrene unit and the presence of the square-planar Pt centre in complex 116 required an assessment of the cytotoxicity of the complex. In assays, 116 was shown to exert similar cytotoxicity towards bone osteosarcoma (U2OS) and transformed mammary cancer (HMLER) cell lines as the anticancer drug Cisplatin. The advantage of complex 116 is that it contains an intercalating function, a potential cytotoxic platinum centre and moderate/mild loss of fluorescence for cell imaging by optical microscopy. The final project discussed in this thesis is the synthesis of a phosphonium salt containing BODIPY as fluorophore, bound to a macrocycle which is able to undergo complexation reactions with d-block metals. This is another example of a molecule capable of multi-modal functionality, since phosphonium salts have been shown to target mitochondria. Positively charged compounds freely diffuse across the negatively charged mitochondrial membrane and the BODIPY moiety allows for imaging of the compound’s fate by optical microscopy. Finally, the tetraamine macrocycle of the molecule allowed ligand 154 to be reacted with [Cu(OAc)2] which gave the fluorescent Cu(II) complex 155. This complex is interesting because it proves that coorduination to Cu is possible. The next step in this research would be to prepare the 64Cu analogue, which would be a candidate for Positron Emission Tomography (PET) imaging. In this manner, 64Cu-154 would be a fluorescent organelle-specific PET imaging agent.

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34

Moshiri, Houta. "Fluorescence-based reporter substrate for monitoring RNA editing in Trypanosomatid pathogens." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116117.

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Mitochondrial gene expression in trypanosomatid pathogens requires extensive post transcriptional modification called RNA editing. This unique molecular mechanism, catalyzed by a multiprotein complex (the editosome), generates translatable transcripts for essential components of parasite respiratory complex. How editosome proteins are assembled and perform RNA editing is not fully understood. Moreover, previous studies have shown that editosome proteins are essential for parasite survival, which makes editosome as a suitable target for drug discovery. Currently, researchers use radio-labeled based assays to monitor RNA editing process. However, these assays are not suitable for high throughput screening of editosome inhibitors, have low detection limits, and cannot monitor RNA editing in real time.
Therefore, to develop a sensitive high throughput RNA editing assay, we have designed a sensitive hammerhead ribozyme-based fluorescence assay. Ribozyme structure was remodeled by adding or removing uridylate in its conserved catalytic core to make an inactive ribozyme. In the presence of the editosome, inactive ribozyme is edited to an active ribozyme. Consequently, hammerhead ribozyme activity can be measured by cleaving its fluorescently labeled substrate. We have shown that higher sensitivity is achieved using fluorescent based assay than conventional radio-labeled assay. Moreover, we can use this assay for rapid identification and characterization of the editosome inhibitors against RNA editing activities in trypanosomatids.

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Prado, Willian Andrade. "Obtenção de materiais orgânicos conjugados com corantes fluorescentes para a marcação de nanocápsulas poliméricas com potencial aplicação em diagnóstico e terapêutica." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2014. http://hdl.handle.net/10183/98998.

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Sistemas nanocarreadores de moléculas orgânicas têm emergido como promissores sistemas para aplicação na terapêutica e/ou diagnóstico. No presente trabalho foram desenvolvidas nanocápsulas poliméricas contendo, em seus diferentes domínios, sondas fluorescentes capazes de fornecer imagem quando analisadas em microscópio de fluorescência confocal, mostrando as potencialidades do sistema como agente para imagem ótica. Uma importante característica de moléculas propostas como marcadores fluorescentes, é que não haja sobreposição dos espectros de absorção e emissão de fluorescência. Para tanto, foi utilizado um corante sintético do grupo dos benzoxazólicos (HBO-C8) para marcação do núcleo lipofílico, e os polímeros PCL e quitosana foram ligados covalentemente aos corantes rodamina B e alaranjado de acridina, respectivamente. As formulações desenvolvidas e caracterizadas com dois ou três marcadores apresentaram caráter nanotecnológico e as fotomicrografias de transmissão revelaram que as nanopartículas contendo HBO-C8 e PCL-Rho (sem quitosana), preservou a morfologia da nanopartícula. Adicionalmente, o HBO-C8 foi quantificado na formulação contendo HBO-C8 e PCL-Rho, onde verificou-se que 98% do corante está internalizado na nanopartícula, alcançando assim, o objetivo proposto de usá-lo como sonda para marcação do núcleo lipofílico. Por meio da microscopia de fluorescência confocal, foram obtidas imagens dos marcadores fluorescentes colocalizados, para as formulações contendo dois ou três domínios fluorescentes, demonstrando que essas nanocápsulas podem ser propostas como uma plataforma promissora para serem empregadas como sondas para imagem ótica.
Nanocarriers systems of organic molecules have emerged as promising systems for application in therapy and diagnosis. In the present study polymeric nanocapsules containing fluorescent probes in their different domains, capable of providing image when analyzed in confocal fluorescence microscope, showing the potential of the system as an agent for optical imaging. An important feature of proposed molecules as fluorescent probes, there is no overlap of the spectra of absorption and fluorescence emission. Thus, a group of synthetic dye benzoxazólicos (HBO-C8) to mark the lipophilic core, and PCL and chitosan polymers were covalently linked to dye rhodamine B and acridine orange , respectively. The formulations developed and characterized with two or three markers showed nanotechnological character and transmission photomicrographs showed that nanoparticles containing PCL-Rho and HBO-C8 (without chitosan) preserved morphology of the nanoparticle. Additionally, the HBO-C8 was was quantified on formulation contained HBO-C8 and PCL-Rho, where it was found that 98% of the dye is internalized in the nanoparticle thus reaching the purpose proposed to use as a probe for marking the lipophilic core . By images of confocal fluorescence microscopy were obtained the colocalized of fluorescent markers to formulations containing two or three fluorescent domains, demonstrating that these nanocapsules can be proposed as a promising platform to be used as probes for optical image.

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36

Wakelin, Stuart. "Fluorescence and transient kinetic analysis of the dictyostelium myosin-II motor domain using green fluorescent protein (GFP) and its variants as probes." Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/29706.

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The determination of the structure of the myosin motor domain has made it possible to introduce fluorescent probes at defined sites, thereby allowing the resolution of the mechanochemical steps in greater detail. Here, modern genetic cloning techniques were utilised to create and express within Dictyostelium discoideum novel myosin-II motor fusion proteins containing various fluorescent probes, in an attempt to investigate conformational changes within the motor domain during the actin-bound stages of the crossbridge cycle. Stopped-flow analysis showed that the myosin ATPase of the single tryptophan myosin-II motor W501 was unaffected by N- and C-terminal YFP and CFP probes, whereas ATP-induced actomyosin dissociation was disrupted (potentially by the probes' propensity to dimerise in close proximity), thus rendering the system unsuitable for investigation of the actin-bound stages of the actomyosin ATPase. By combining total internal reflection microscopy with flash photolysis of an inert caged-ATP precursor, it was shown that kinetic information for the ATP-induced dissociation of fluorescently-labelled myosin motor domains may be achieved using only nanogram quantities, while simultaneously avoiding bundling artefacts common to solution kinetics. A slight adaptation of this process could yield a highly sensitive assay for the processivity of non-classical myosin types, yielding information on their rotational and lateral movement concurrently. Lever arm movement was assessed via the analysis of fluorescence resonance energy transfer (FRET) changes between the YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein) probes. A FRET efficiency increase was observed upon nucleotide occupancy of the active site, in direct contrast to previous FRET studies. Anisotropy studies showed no change upon nucleotide binding, suggesting the FRET increase was due to a distance change, rather than a variation of relative dipole orientations. However, due to the high anisotropy (i.e. slow rotation in solution) of the protein, it was also shown that results from this type of FRET system are qualitative rather than quantitative.

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Liu, Chuangjun. "MODIFICATION OF XANTHENE AND SILICON ANALOGUES OF XANTHENE FLUOROPHORES FOR CHEMICAL PROBES DEVELOPMENT." OpenSIUC, 2016. https://opensiuc.lib.siu.edu/dissertations/1158.

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Fluorescence techniques have been widely used in chemistry and many areas of biology due to its high sensitivity, simplicity, fast response, and capability of spatial imaging. Currently there are six major classes of small-molecular fluorophores-coumarins, boron dipyrromethene (BODIPY) dyes, fluoresceins, rhodamines, oxazines, and cyanines. This dissertation mainly focuses on modifications of fluoresceins and development of pH probes based on rhodamines. We have modified the xanthene core of fluorescein by replacing the xanthene oxygen with a dimethylsilyl group which shows 90 nm red-shift relative to fluorescein (λabs/λem =582 nm/598 nm). These fluorophores have advantages over their oxygen counterparts because longer-wavelength allows deeper penetration of the light, causing less damage to the cells and less interference from the biological medium. Based on this scaffold, we have tried to develop copper, mercury, and hydrogen peroxide probes by functionalizing the hydroxyl group of the fluorophore with different groups. Although the results weren’t satisfactory, we have gained some new insights about this scaffold. The hydroxyl group of the fluorophore is not very reactive and this scaffold is not stable under strong basic conditions and its electronic configuration is very different with the regular fluorescein. We have synthesized and characterized Si-rhodol fluorophore, the hybrid structure of Si-fluorescein and Si-rhodamine. The preliminary investigation of Si-rhodol, UV-vis and fluorescence, has been performed. It shows that the absorption and fluorescence emission of Si-rhodol are around 30 nm red-shift compared to Si-fluorescein (λabs/λem =617 nm/630 nm). And Si-rhodol is more fluorescent than Si-fluorescein (pKa = 6.8) at pH 7.4 (biological condition) due to its lower pKa (5.3). Despite the excellent photophysical properties of Si-rhodol, there are actually limited examples about its application. Among these examples, none of them gave the detailed information about functionalization of the hydroxyl group of Si-rhodol. We have found a method to functionalize the hydroxyl group of Si-rhodol. We have developed a series of pH probes through modification of rhodamines which possess either an aniline-methyl or cyclic ring moiety. These analogues maintain a coloress, non-fluorescent spirocyclic structure at high pH and the fluorescence goes up while the pH decreases. The substituent effects on anilinomethylrhodamines (AnMR) system follows Hammett’s equation. The effect of cycloalkane ring size on the acid/base properties of the AMR system can be predicted by Baeyer’s ring strain theory. The pKa of these rhodamine analogues can be tuned from around 3 to 8. We have also investigated aminomethylfluoroscein (AMF) system. In this system, we found that once the phenolic hydroxyl group was functionalized at one side, the fluorescence was off at pH>3. In addition, variation of the substituents on aminomethyl moiety didn't affect the pKa of this system significantly. Therefore, we were set out to develop mitochondria-targetable hydrogen peroxide probe. However, in the presence of triphenylphosphine cation, the conversion from the OTf group to the boronate group didn't work.

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Lai, Yau-tsz, and 黎佑芷. "Lighting-up metalloproteins in living cells : seeing is believing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/206989.

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One third of proteins in nature have been revealed as metalloproteins, whereas most of them remain uncharacterized, probably due to the lack of robust methods especially for tracking metalloproteins within the living context. Fluorescent labeling is capable to detect biomolecules with molecular resolution in living cells. Tracking metal-binding proteins in living cells by fluorescence could provide invaluable information in understanding their localization and potential functions in the native environment. A synthetic molecular probe NTA-AC was designed and synthesized to track metal-associated proteins in living cells upon chelation with metal ions. The fluorescent probe consists of a small molecular fluorophores, a metal-chelating moiety to direct the metal-chelated probe to the protein targets, and a photo-active crosslinker. Metal being chelated could help further explore potential binding targets and direct the fluorescent agent to the appropriate region, then subsequently covalent linkage to targets could be generated through photo-activation. NTA-AC was therefore chelated with different metals to examine its binding preference to different proteins. The Ni2+-chelating probe was applied to track Ni2+-binding proteins as an example to validate its applicability. Ni2+-NTA-AC preferentially binds to histidine-rich peptides and proteins thus verified its binding specificity. The Ni2+-chelated probe was further exploited to light up over-expressed histidine-rich proteins in Escherichia coli cells to validate its membrane permeability and binding specificity. In addition, the probe was applied to label His-tagged proteins expressed in tobacco plant cells to further evaluate its applicability in detecting and localizing the protein targets in eukaryotic cells. Afterwards, Ni2+-NTA-AC was exploited to track Ni2+-binding proteins in living Helicobacter pylori cells and incorporated with gel electrophoresis and mass spectrometry for protein identification. Many proteins identified are correlated to Ni2+-association and thus validating the applicability of the probe. Bi3+-chelated NTA-AC was therefore used to mine potential targets in H. pylori. Intense fluorescence was observed within H. pylori cells thus indicating the effectiveness of the fluorescent labeling. Protein separation and identification was therefore initiated to trace potential targets, while finding that some of the Bi 3+-coordinated proteins participate in various functioning pathways of the pathogens. The effects of colloidal bismuth subcitrate (CBS) on pH buffering and redox defense systems were therefore determined and verified, confirming that respective proteins could be potential therapeutic targets of the drug. Cr3+-NTA-AC was further applied to human Hep G2 cell line to determine Cr3+-binding targets in mammalian cells. Their localization on mitochondria was revealed, implying the potential effects of Cr3+ on mitochondria. Further confirmation of protein targets was performed through protein separation and identification. Proteins identified could be positively correlated to mitochondrial functions and thus revealing that Cr3+ might exert its effect at mitochondria. Addition of Cr3+ to Hep G2 could prevent mitochondrial fragmentation induced by hyperglycemia, which thus suggests the possible therapeutic function of Cr3+. The extensive application of NTA-AC in tracking Ni2+-, Bi3+- and Cr3+-associated proteins has validated the effectiveness of such strategy in detecting and localizing metalloproteins within the living context and thus could be extended to investigate other metalloproteomes.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

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39

Carson, Travis D. "Development of a DNA probe and anisotropic films with an emphasis on self-assembly and fluorescence /." abstract and full text PDF (free order & download UNR users only), 2005. http://0-wwwlib.umi.com.innopac.library.unr.edu/dissertations/fullcit/3198195.

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Thesis (Ph. D.)--University of Nevada, Reno, 2005.
"May, 2005." Includes bibliographical references. Online version available on the World Wide Web. Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2005]. 1 microfilm reel ; 35 mm.

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40

Recalde, Elena Carolina Serrano. "Influência da qualidade do sêmen criopreservado equino sobre a taxa de prenhez, hemodinâmica uterina e endometrite pós-cobertura." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-08012015-131457/.

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As condições do trato reprodutivo da fêmea, assim como a qualidade do sêmen são fatores que interferem na fertilidade. Os objetivos deste trabalho foram avaliar a resposta inflamatória uterina e a taxa de prenhez em éguas, considerando hemodinâmica uterina e citologia endometrial após a inseminação artificial (IA) com sêmen congelado de alta e baixa qualidade. Para o presente estudo foram realizados dois experimentos. No Experimento 1 foram utilizadas 15 éguas distribuídas de forma aleatória em quatro grupos: CT - controle: mimetização do procedimento de IA (n=7), DIL: infusão intrauterina de diluidor a base de leite desnatado (n=7), ALTA: IA com sêmen de alta qualidade (n=7), e BAIXA: IA com sêmen de baixa qualidade (n=7). A avaliação uterina foi realizada por ultrassonografia transretal com Doppler nos modos Espectral e Color-flow em sete momentos: prévio à indução da ovulação (TIO), imediatamente antes da IA (TIA), 2 (T2), 6 (T6), 12 (T12), 24 (T24) e 48 horas (T48) após a IA. Foram considerados os valores de índice de resistência (RI) da artéria uterina e de escore de vascularização (EV) uterino. A citologia uterina foi realizada 6 h após a IA. No Experimento 2 foram utilizadas 12 éguas, e seus ciclos foram distribuídos nos tratamentos: CT - controle: mimetização do procedimento de IA (n=8), DIL: infusão intrauterina de diluidor (n=8), ALTA: IA com sêmen de alta qualidade (n=8), e BAIXA: IA com sêmen de baixa qualidade (n=8). O delineamento experimental foi em Quadrado Latino 4X4. A avaliação uterina foi realizada por ultrassonografia transretal com Doppler modos Espectral e Color-flow em três momentos: prévio à indução da ovulação (TIO), imediatamente antes da IA (TIA) e 6 horas (T6) após a IA. A citologia uterina foi realizada 6 h após a IA. Neste experimento foi considerada a taxa de prenhez comparando-se os ciclos utilizados para os grupos ALTA e BAIXA. O diagnóstico de gestação foi realizado 14 dias após a ovulação. Foi utilizado o procedimento misto (PROC MIXED) do SAS (Versão 9.3) para a análise estatística e foi considerada diferença significativa quando p ≤0,05. No Experimento 1, não foi encontrada diferença estatística entre os grupos para os valores de hemodinâmica uterina RI e EV. Houve aumento significativo de células inflamatórias no endométrio de éguas inseminadas com sêmen de baixa qualidade, porém não diferiu do grupo de éguas inseminadas com sêmen de alta qualidade. No Experimento 2, não houve diferença estatística entre os grupos para os valores de RI, mas se observou maior EV uterino nos grupos inseminados com sêmen de alta e de baixa qualidade quando comparados com o grupo controle. Na citologia uterina não foi encontrada diferença estatística. A taxa de prenhez para os grupos ALTA (81,82%) e BAIXA (54,55%) não foi estatisticamente diferente. Conclui-se que a deposição de sêmen no útero, leva a um processo inflamatório do endométrio, e esta pode alterar a hemodinâmica uterina detectável por ultrassonografia Doppler em éguas. Não existe diferença significativa na resposta inflamatória entre as qualidades de sêmen. A inseminação com sêmen congelado de menor qualidade não altera a taxa de prenhez, mas são necessários mais estudos para verificar esta diferença.
The female reproductive tract soundness, as well as semen quality are factors that interfere in fertility. The aims of this study were to evaluate inflammatory uterine response and pregnancy rate in mares, considering uterine hemodynamics and endometrial cytology after artificial insemination (IA) with frozen semen of high and low quality. Two experiments were executed for the present study. For Experiment 1, fifteen mares were randomly distributed between four groups: CT - control: mimic of the procedure of IA (n=7), DIL: intrauterine infusion of skim milk semen extender (n=7), ALTA: IA with high quality semen (n=7), and BAIXA: IA with low quality semen (n=7). Uterine evaluation was done using transrectal Doppler ultrasonography by Spectral and Color-flow modes on seven moments: immediately before ovulation induction (TIO), immediately before AI (TIA), 2 (T2), 6 (T6), 12 (T12), 24 (T24) and 48 (T48) hours after IA. There were considered the numerical values of resistance index (RI) of uterine arteries and vascularity scores (EV) of uterine horns. Endometrial cytology was done at 6 h after IA. On Experiment 2, were used 12 mares which their cycles were distributed between the treatments: CT - control: only mimic of IA procedure (n=8), DIL: intrauterine infusion of semen extender (n=8), ALTA: IA with high quality semen (n=8), and BAIXA: IA with low quality semen (n=8). The experimental design was a Latin Square. Uterine evaluation was done using transrectal Doppler ultrasonography by Spectral and Color-flow modes on three moments: before ovulation induction (TIO), immediately before AI (TIA) and 6 hours (T6) after IA. Endometrial cytology was done at 6 h after IA. In this experiment it was considered the pregnancy rate comparing the cycles for groups ALTA e BAIXA. The pregnancy diagnosis was done 14 days after ovulation. It was used the Mixed Procedure (PROC MIXED) from SAS (Version 9.3) for statistical analysis and significant difference was considered when p≤0.05. On Experiment 1, no statistical difference was found between the groups for uterine hemodynamic values RI and EV. There was a significant increase of inflammatory cells on the mares inseminated with low quality semen, however it wasnt different from the group if mares inseminated with high quality semen. On Experiment 2, there was no statistical difference between the groups for RI values, but the uterine EV was higher for the groups inseminated with high and low quality when compared with the control group. There was no statistical difference on endometrial cytology. There was no statistical difference on pregnancy rate for the groups ALTA (81,82%) and BAIXA (54,55%). As a conclusion, the deposition of semen in the uterus causes an inflammatory process on the endometrium, and it could alter uterine dynamics detected by Doppler ultrasonography in mares. It does not exist significant difference on the inflammatory response between semen qualities. Low quality frozen-semen insemination does not alter pregnancy rate, but more studies are necessary in order to verify this difference.

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41

Lançoni, Renata. "Uso da melatonina e do ácido ferúlico como promotores da função do espermatozoide equino criopreservado." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-17092015-103200/.

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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.

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Alves, Maíra Bianchi Rodrigues. "Tratamento da degeneração testicular em carneiros com suplementação de vitamina A ou laserterapia de baixa intensidade." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-18092014-112108/.

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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.

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43

Ghorbanian, Shohreh. "Studies of potentially useful thiol-reactive fluorescent probes and novel ion-responsive fluorescent quinoxalinone derivatives." Thesis, Brunel University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360815.

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44

Rami, H. "A study of potential fluorescent probes for hypoxic cells." Thesis, Brunel University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377946.

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45

Dempsey, Graham Thomas. "Photoswitchable Fluorescent Probes for Localization-Based Super-Resolution Imaging." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10376.

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In recent years, localization-based super-resolution imaging has been developed to overcome the diffraction limit of far-field fluorescence microscopy. Photoswitchable probes are a hallmark of this technique. Their fluorescence can be modulated between an emissive and dark state whereby the sequential, nanoscale measurement of individual fluorophore positions can be used to reconstruct an image at higher spatial resolution. Despite the importance of photoswitchable probes for localization-based super-resolution imaging, both a mechanistic and quantitative understanding of the essential photoswitching properties is lacking for most fluorophores. In this thesis, we begin to address this need. Furthermore, we demonstrate the development of new probes and methodologies for both multicolor and live-cell super-resolution imaging. Chapter 2 describes our mechanistic insights into the photoswitching of a common class of dyes called carbocyanines. Red carbocyanines, such as Cy5, enter a long-lived dark state upon illumination with red light in the presence of a primary thiol. We show that the dark state is a covalent conjugate between the thiol and dye and that this dark state recovers by illumination with ultraviolet light. We also speculate on possible reactivation mechanisms. Our mechanistic studies may ultimately lead to the creation of new probes with improved photoswitching properties. Chapter 3 details our quantitative characterization of the photoswitching properties of 26 organic dyes, including carbocyanines and several other structural classes. We define the essential properties of photoswitchable probes, including photons per switching event, on/off duty cycle, photostability, and number of switching cycles, and demonstrate how these properties dictate super-resolution image quality. This rigorous evaluation will enable more effective use of probes. In Chapters 4 and 5, we focus on expanding the super-resolution toolbox with novel strategies for multicolor and live-cell imaging. Chapter 4 discusses two approaches we have developed for multicolor super-resolution imaging, which distinguish probes based on either the color of activation or emission light. These tools allow multiple cellular targets to be resolved with high spatial resolution. Lastly, Chapter 5 introduces a method for targeted cellular labeling with photoswitchable probes using a small peptide tag, as well as a new sulfonate-protection strategy for intracellular delivery of high performing photoswitchable dyes.

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Lister, Francis George Alexander. "Fluorescent probes of conformational signal relay in membrane environments." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/fluorescent-probes-of-conformational-signal-relay-in-membrane-environments(baa2cddd-94a1-4505-bf45-13403e0a6548).html.

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G-Protein Coupled Receptors (GPCRs) are a class of membrane-bound receptor proteins capable of relaying a biological signal across a cell membrane through a solely conformational change in their transmembrane domain. Previous work has shown that helical foldamers composed of achiral monomeric units can be used in an analogous manner to relay stereochemical information on the nano-scale through the conformational control of screw-sense preference. While this work has produced some highly successful examples of signal relay, mimicking the function of GPCRs, its reliance on screw-sense responsive NMR probes has restricted further development into membrane environments. This thesis describes the successful development of a pyrene based screw-sense responsive fluorescence probe and its subsequent use in the development of a series of membrane-based GPCR mimics. This thesis has also details the preliminary steps towards the development of light-responsive controllers of screw-sense preference for nano-scale signal relay devices.

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47

Burckel, Hélène. "Synthèse et évaluation de molécules bifonctionnelles alkylantes de l’ADN et inhibitrices de la PARP pour la radiochimiothérapie concomitante." Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAJ092.

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Cette étude a consisté en le développement et l’évaluation de nouvelles molécules pour la radiochimiothérapie concomitante. Ce travail a abouti à la conception de nouveaux agents chimiothérapeutiques duaux basés sur la combinaison covalente de deux radiosensibilisateurs: un inhibiteur de la PARP d’une part, et un alkylant de l’ADN (complexe de platine ou témozolomide) d’autre part. Les évaluations biologiques ont permis de mettre en évidence l’intérêt d’une molécule duale inhibiteur de la PARP/platine. Parallèlement à ce projet, le développement d’outils moléculaires pour l’étude d’inhibiteurs de la PARP a été entrepris. Ainsi, plusieurs sondes d’affinité ont été conçues pour une étude d’inhibiteurs de la PARP par protéomique chimique. Ce travail a permis de valider la spécificité d’une sonde d’affinité pour la PARP1 et la PARP2. Finalement, des molécules fluorescentes inhibitrices de la PARP ont été développées dans l’objectif d’un criblage d’inhibiteurs de PARP3 par anisotropie de fluorescence
The main topic of this work was the development and biological evaluation of dual molecules for concomitant chemoradiotherapy. To this end, new dual chemotherapeutic agents were designed by linking covalently two radiosensitizers: a PARP inhibitor and an alkylating agent (platinum complex or temozolomide). This study led to an efficient PARP inhibitor/platinum dual molecule. A complementary approach was to develop affinity probes to study PARP inhibitors by a chemical proteomic method. This study permitted to validate the selectivity of an affinity probe for PARP1 and PARP2. Finally, fluorescent PARP inhibitor probes were synthesised and evaluated for a PARP3 screening by fluorescence anisotropy

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Zhu, Jianfa. "Fluorescent chemosensor development based on multifunctional spirobenzopyrans." HKBU Institutional Repository, 2011. https://repository.hkbu.edu.hk/etd_ra/1299.

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49

Hester, Jeffrey D. "Nitroxide-labeled oligonucleotides as hybridization probes a comparative study between nitroxide- and fluorescent-labeled probes /." Cincinnati, Ohio : University of Cincinnati, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=ucin.

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Hester, Jeffery Dean. "Nitroxide-Labeled Oligonucleotides as Hybridization Probes: A Comparative Study Between Nitroxide- and Fluorescent-Labeled Probes." University of Cincinnati / OhioLINK, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1069433831.

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Dissertations / Theses on the topic 'Fluorescents probes'

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