Calcium (Ca2+), a crucial effector for many biological systems, has been associated with diseases such as cardiovascular disease, Alzheimer’s, Parkinson’s, cancer, and osteoporosis. It is important to develop calcium sensors to measure intracellular Ca2+ dynamics at various biological and pathological states. Our lab has engineered such probes by designing a Ca2+ binding site into fluorescent proteins such as Enhanced Green Fluorescent Protein (EGFP) and mCherry. In this thesis, we aim to improve optical properties and metal binding properties of green EGFP-based sensor CatchER and mCherry based red sensors by site-directed mutagenesis and protein engineering, various spectroscopic methods and cell imaging. The green EGFP-based sensor CatchER, with a Ca2+ binding pocket charge of -5, displays the greatest affinity for Ca2+ and has the greatest fluorescence intensity change with Ca2+ when compared to its variants with a less negative binding pocket charge. In addition, we have also designed several SR/ER targeting CatchER variants using Ryanodine receptor and Calnexin transmembrane domains. These constructs were shown to display a strong presence in the SR/ER lumen and further designed for a new luminal orientation. Further, we have shown that the optical properties of two red calcium sensors can be significantly improved by modifying the local environment of the chromophore.

acase@tulane.edu / Current microscopy-based tissue diagnostics, particularly hematoxylin and eosin (H&E) histology, requires multiple complex tissue processing steps: fixation, paraffin embedding, microtome sectioning, dying the tissue, and imaging individual slides through a bright field microscope. The time and labor-intensive result of this process makes it unsuitable for patient point-of-care evaluation. Therefore, many bedside procedures are completed without efficient real-time analysis of tissue adequacy and diagnostic results are unnecessarily delayed. Additionally, research experiments that require information regarding changes to tissue morphology or function before proceeding to the next experimental phase are severely interrupted by histology processing in their workflow. Fluorescence histology, which relies on rapid fluorescent staining of tissue, optical sectioning microscopy, and image processing for digital viewing, can provide an inexpensive, non-destructive, 3-dimensional, and fast alternative to traditional histology and point-of-care screening protocols. The objective of this work is to further advance the concept of “fluorescence histology,” in which traditional histopathology preparation methods are replaced by optical-sectioning (in lieu of physical sectioning), sensitive and flexible fluorescence-based contrast (in lieu of chromophore-based contrast), and computational strategies to replicate traditional color-schemes. In this work, we demonstrate the development and use of a fluorescent analogue to H&E on fixed and frozen tissue sections and fresh human biopsies. This fluorescent analogue, DRAQ5 & eosin, is compared against the current single-agent, monochrome fluorescence histology system, and their effects on diagnostic downstream molecular analyses, including quantitative-PCR and immunohistochemistry, is evaluated. We create a methodology to develop and characterize fluorescent analogues for any histological stain, with demonstration using Masson’s Trichrome and Periodic Acid-Schiff, enabling the expansion of fluorescence histology for multiple applications. This work demonstrates the ability to improve point-of-care pathology and research by replacing destructive, incomplete, and time-consuming histology with fluorescence histology, which preserves the tissue for later analysis or experiments while providing accurate and rapid histology assessment. / 1 / Katherine Elfer

Thèse de doctorat : Sciences de la Vie et de la Santé. Biophysique : Strasbourg 1 : 2007. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 16 p.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 89-94).

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Rebecca Richards-Kortum. Vita. Includes bibliographical references. Also available from UMI.

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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Digitized by Kansas Correctional Industries

ITC/USA 2015 Conference Proceedings / The Fifty-First Annual International Telemetering Conference and Technical Exhibition / October 26-29, 2015 / Bally's Hotel & Convention Center, Las Vegas, NV / Research, design, construction, and operation of a portable mixed Raman and Fluorescence type spectrometer will be presented. The spectrometer was used on a wheeled vehicle which competed in the University Rover Challenge sponsored by the Mars Society. It uses a 50 mW, 532 nm continuous wave laser to probe a sample of soil for bacteria or biomarkers. The device costs 2,000 USD, weighs 1.4 kg and is 23 cm x 23 cm x 10 cm in size. Results from the competition, complications of analyzing mixed Raman-Fluorescence spectra via digital signal processing, and future ideas and improvements will also be discussed.

No description available.