Dissertationen zum Thema „Fluorescence coefficients“

La connaissance de la température de la phase gaz au voisinage du front chaud existant lors de l'utilisation de fours solaires à concentration peut s'avérer importante pour la maîtrise des procédés faisant appel à ces technologies. Pour accéder à ce paramètre nous avons utilisé un phénomène inhérent à ces installations: la fluorescence induite par absorption d'une fraction du rayonnement solaire incident servant à chauffer l'échantillon. Cette thèse présente l'étude de la fluorescence de la molécule diatomique YO générée lors de la fusion d'oxyde d'yttrium au foyer d'un four de 1,5 kW dans des atmosphères d'argon ou d'hélium. La température désirée est obtenue en comparant les spectres mesurés par spectroscopie optique des systèmes A2P1/2-X2S+, A2P3/2- X2S+, B2S+- X2S+ de la molécule YO et ceux calculés de ces mêmes bandes. On mesurera aussi ces temperatures par une méthode d'absorption dont le faisceau de référence est obtenu par prélèvement de rayonnement sur le faisceau direct de l'héliostat associé au four solaire. Le modèle de calcul des spectres est un modèle à trois températures (électronique, vibration, rotation). Les résultats obtenus se comparent favorablement à un modèle fluide (Fluent) mais montrent, à basse pression et au voisinage du front chaud, des déséquilibres importants entre ces différentes températures. On explique ces déséquilibres, dans cette situation, par l'existence d'une couche de Knudsen au voisinage de l'échantillon
The knowledge of the gas phase temperature in the vicinity of the hot front in solar processes involving high concentration solar furnaces is an important parameter for the control of these processes. In order to measure this parameter we used a phenomenon inherent in these facilities : fluorescence induced by absorption of a fraction of the incident radiation used to heat the sample. This work concerns the fluorescence of the YO diatomic molecule issuing from an Y2O3 sample melted in argon or helium at the focus of a 1. 5 kW solar furnace. Temperatures are deduced from the comparison between measured and computed spectra of YO bands (A2P1/2-X2S+, A2P3/2-X2S+, B2S+-X2S+). These temperatures are also deduced from spectra obtained with an absorption method, the reference beam being withdrawn from the direct beam from the heliostat associated to the concentrator. The model used to calculate the band spectra is a three temperatures model (electronic, vibrational, rotational). The results are consistent with the calculation from a computational fluid dynamic software (Fluent). However the measurements show large discrepancies at low pressure and near the hot front. These discrepancies can be accounted for by a Knudsen layer in the vicinity of the hot front

The aim of the diploma thesis was to optimize the sequential fractionation method of organic matter to be used for physico-chemical characterization of extracted fractions. Humic acid isolated from oxidized brown coal of Leonardite was used as a source matrix of organic matter. An eluotropic series was assembled and sequential fractionation was performed by extraction on a Soxhlet apparatus. The original humic acid and fractions were characterized by elemental analysis (EA) and thermogravimetric analysis (TGA), followed by Fourier transform infrared spectrometry (FTIR), molecular absorption spectrometry (UV/VIS), fluorescence spectrometry and potentiometric titration. Atomic ratios were determined from the results of the elemental analysis. From the measured UV/Vis and fluorescence excitation and emission spectra, the absorption coefficients, resp. fluorescence coefficients. Used fractionation method proved to be a suitable method for studying HA structure. A total of 62 wt. % of initial materiál was extracted, indiivdual fraction amounted from 0.36–30.92 wt. %. From the results of the structural analysis, it is clear that with increasing polarity of the organic solvent, fractions with long aliphatic chains were first isolated and their aromaticity graddualy increased. Non-polar organic solvents were suitable for the extraction of lipid-like coumpounds, while the most polar organic fractions were rich in polar groups and their structual parameters were close to the original humic acid. The fraction extracted with acetonitrile was the most unique fraction. This fraction was rich on nitrogen and amine groups and was similar to protein-like structures. In the last two fractions, extracted with alcohols, a significant bathochromic shift typical of fluorophore type V was observed. Among other things, they were also characterized by a higher content of plant carbohydrate residues.

La cristallisation est une des opérations élémentaires du génie chimique. Les matières produites sont extraites par cristallisation et purifiées par recristallisation. Mais la nucléation du cristal reste un mystère et la théorie classique de la nucléation est battue en brèche par de nombreuses données expérimentales. Nous avons construit un dispositif microfluidique de précipitation par mélange de solvants pour produire de manière continue et observer la formation d’un grand nombre de cristaux. La molécule étudiée est le DBDCS dont les cristaux sont fluorescents mais pas la molécule. Le germe sera ainsi le premier objet lumineux du mélange.Nous avons calculé la thermodynamique du mélange ternaire DBDCS/1,4-dioxane(diox)/eau à partir de ce qui est connu pour le mélange diox/eau et de la courbe de solubilité du DBDCS dans diox/eau, dans le cadre d’un modèle de solution régulière. Il permet de calculer les conditions de la décomposition spinodale ([DBDCS]= 59 fois la saturation) du mélange ternaire en une phase liquide hypothétique de DBDCS pratiquement pur dans un mélange diox/eau.Nous observons cette phase dans une expérience de précipitation après 6ms de mélange. La mesure du volume de cette phase liquide confirme qu’elle est pratiquement pure. L’apparition de cette phase liquide nécessite une forte sursaturation. Celle-ci fait suite à la diffusion de l’eau qui repousse et concentre le DBDCS au centre du dispositif. L’étude du temps mis à atteindre la concentration critique en fonction de la concentration initiale en DBDCS dans le flux central permet d’obtenir une valeur de 50 à 70 fois la saturation pour la concentration critique d’apparition de la phase liquide DBDCS. Le produit de cette décomposition spinodale est un nuage de gouttelettes sub-micrométriques. Mais le gradient de potentiel chimique peut, dans certaines conditions, regrouper ces nano-gouttes en un chapelet de gouttes micrométriques de même taille.Lorsque l’anti-solvant n’est pas de l’eau pure, mais un mélange diox/eau, la barrière de potentiel ne l’emporte pas sur l’entropie de la diffusion, ce que montre la répartition de la fluorescence résiduelle des molécules (rendement<10-4). Sur des temps de l’ordre de 5s, on observe la formation et la croissance de cristaux dans un mélange localement homogène. La simulation numérique indique que dans ces conditions la sursaturation relative ne dépasse pas 3,5. L’imagerie rapide et la fluorescence permettent d’observer les cristaux un par un. Trois polymorphes différents sont identifiables par leur durée de vie : les phases vertes et bleues déjà observées et une phase de courte durée de vie. Ces cristaux présentent une vitesse de croissance moyenne proportionnelle à la concentration locale.En focalisant un laser sur les nuages de nano-gouttes, on observe un effet de pince optique capable de rassembler ces gouttes. En focalisant ce laser dans la zone de super-saturation maximale dans des conditions de nucléation spontanée, on observe une multiplication du nombre de cristaux formés d’un facteur cinq. Nous sommes en présence d’une nucléation induite par laser. Ces cristaux présentent la même vitesse de croissance, la même distribution en nombre des polymorphes, que les cristaux obtenus spontanément. Cette nucléation induite par laser est donc très douce et induit un changement minimal du mécanisme de la nucléation. Un effet de pince optique qui concentre localement les précurseurs du germe et augment transitoirement la sursaturation pourrait avoir cet effet.Cette nucléation induite par laser permet de localiser la nucléation. Au point focal du laser NPLIN, nous observons l’accumulation d’une phase de durée de vie de fluorescence courte, donc peut être désordonnée. Elle disparaît après le passage dans le laser pendant qu’une phase de grande durée de vie (la phase verte) croit lentement. Ce serait une observation directe d’une nucléation en deux étapes
Crystallization is one of the elementary operations of chemical engineering. The materials produced are extracted by crystallization and purified by recrystallization. But the nucleation of the crystal remains a mystery and the classical theory of nucleation is undermined by numerous experimental data. We have chosen to build a microfluidic precipitation device by mixing solvents to produce and continuously observe the birth of a large number of crystals. The molecule chosen for the study is DBDCS, with fluorescent crystals but not the molecule. The germ will thus be the first luminous object in the mixture.We calculated the thermodynamics of the ternary mixture DBDCS/diox/water from what is known for the mixture diox/water and the solubility curve of DBDCS in diox/water, as part of a regular solution model. We calculate the conditions of the spinodal decomposition ([DBDCS]= 59 times the saturation) of the ternary mixture into a hypothetical liquid phase of DBDCS practically pure in a diox/water mixture.However, this hypothetical phase we observe it as the main initial product of this precipitation experience. The measurement of the volume produced by this liquid phase confirms that it is practically pure. The appearance of this liquid phase requires a strong oversaturation following the diffusion of water. The study of the solubility of DBDCS in diox/water shows that the chemical potential of DBDCS in water is 17 RT higher than its value in diox. The diffusion of water in diox induces the formation of an energy barrier that repels and concentrates DBDCS to the center of the device. The study of the time taken to reach the critical concentration as a function of the initial concentration of DBDCS in the central flow provides a value 50 to 70 times the saturation for the critical concentration of occurrence of the DBDCS liquid phase. This confirms that we observe a spinodal decomposition. The product of this spinodal decomposition is a cloud of sub-micrometric droplets. But the chemical potential gradient can, under certain conditions, group these nanodrops into a string of micrometric drops of the same size.When the anti-solvent is not pure water, but a diox/water mixture, the potential barrier does not outweigh the entropy of the diffusion. This is shown by the distribution of the fluorescence of the molecules (yield<10-4). Over times of the order of 5s, the formation and growth of crystals is observed. The numerical simulation indicates that under the conditions the relative oversaturation does not exceed 3.5. Rapid imaging and fluorescence allow the crystals to be observed one by one. Three different polymorphs are identifiable by their lifetime : the green and blue phases already observed and a short-lived phase. The growth rates are widely dispersed, making it difficult to locate and observe spontaneous nucleation.By focusing a laser on the clouds of nanodrops, we observe an optical tweezer effect capable of collecting these drops. By focusing this laser in the zone of maximum super-saturation under spontaneous nucleation conditions, we observe a multiplication of the number of crystals formed by a factor of five. We are in the presence of laser-induced nucleation. These crystals have the same growth rate, the same distribution in number of polymorphs, as the spontaneously obtained crystals. This laser-induced nucleation is therefore very soft and induces a minimal change in the nucleation mechanism. An optical tweezer effect that locally concentrates the precursors of the germ and increases the over-saturation could have this effect.This laser-induced nucleation makes it possible to locate the nucleation. At the focal point of the NPLIN laser, we observe the accumulation of a phase with a short fluorescence lifetime, so it can be disordered, which disappears after the passage in the laser while the green phase grows slowly. This would be a direct observation of a two-step nucleation

Les interactions plasma surface (PSI) sont considérées comme l'un des défis scientifiques majeurs de la fusion nucléaire magnétique contrôlée. L'interaction entre les isotopes d'hydrogène et les matériaux de l’enceinte à plasma tels que le tungstène revêt une importance particulière. Le coefficient de perte en surface des isotopes atomiques (γ) est un point clé dans les études PSI. Il peut donner des informations sur le recyclage de l'hydrogène atomique à la paroi et constitue ainsi un paramètre clé dans les modélisations des interactions plasma surface. Le but de ce projet est de déterminer les coefficients de perte de surface de l'hydrogène atomique et du deutérium sur échantillons de tungstène (W) et de nitrure de tungstène (WN) en utilisant une technique de fluorescence induite par plasma (PIF) et une technique de fluorescence induite par laser à deux photons (TALIF). Ce projet s'effectue dans le réacteur CAMITER qui est un réacteur plasma radiofréquence à basse pression au laboratoire PIIM de l'Université Aix-Marseille
Plasma surface interaction (PSI) is considered to be one of the key scientific challenges in nuclear fusion. The interaction between hydrogen isotopes and plasma-facing materials such as tungsten is of particular importance. The atomic hydrogen isotope surface loss coefficient (γ) is a key point in PSI studies. It can give information on hydrogen isotope inventory and is an important input for modeling and theoretical work. The aim of this project is to determine atomic hydrogen and deuterium surface loss coefficients on tungsten (W) sample by using two-photon-absorption laser induced fluorescence (TALIF) and pulsed induced fluorescence (PIF) technique. This project is carried out in CAMITER reactor which is a low-pressure radio-frequency ICP reactor at PIIM laboratory in Aix-Marseille University

The main aim of doctoral thesis is the study on physicochemical properties of humic substances (HS) by modern instrumental techniques. The subject of the study were HS isolated from South Moravian lignite, South Bohemian peat, forest soil Humic Podzol and finally extract from brown sea algae Ascophyllum nodosum. With respect on determination of structure and reactivity of these unique “biocolloids”, standard samples (Leonardite HA, Elliott Soil HS and Pahokee Peat HS) were also studied. These samples were obtained from International Humic Substances Society (IHSS). All mentioned substances were characterized by elemental analysis (EA), molecular absorption spectroscopy in ultraviolet and visible region (UV/Vis), infrared spectroscopy with Fourier transformation (FTIR), nuclear magnetic resonance spectroscopy of carbon isotope 13C (LS 13C NMR), steady-state and time resolved fluorescence spectroscopy. Obtained fluorescence, UV/Vis and 13C NMR spectra were used for calculation of fluorescence and absorption indexes, values of specific absorbance and structural parameters respectively, which were used for fundamental characterization of these “biocolloidal” compounds. Infrared spectroscopy with Fourier transformation was utilized for the identification of functional groups and structural units of HS. Evaluation of infrared spectra is quiet complicated by overlapping of absorption bands especially in fingerprint region. This problem was overcome by Fourier self-deconvolution (FSD). Steady-state fluorescence spectroscopy was used for deeper characterization of HS with respect to origin, structural units, amount of substituents with electron-donor and electron-acceptor effects, content of reactive functional groups, “molecular” heterogeneity, the degree of humification, etc. Parameters of complexation of samples Elliott Soil with heavy metal ions (Cu2+, Pb2+ and Hg2+) were obtained by using modified Stern-Volmer equation. These ions were chosen purposefully, because the interaction of HS with these ions is one of the fundamental criteria for the assessment of the reactivity of HS. Key part of the whole doctoral thesis is time-resolved fluorescence spectroscopy. It is able to determine the origin of emission of HS by method Time-Resolved Area Normalized Emission Spectra (TRANES). The viscosity of micro medium about excited fluorophores of HS was determined by Time-Resolved Emission Spectra (TRES).

Monomeric red fluorescent proteins (RFPs) are used extensively for applications in molecular biology research, and are especially suited for whole body imaging applications due to their longer excitation and emission wavelengths, which are less damaging and penetrate deeper into animal tissue. However, these proteins suffer from reduced brightness compared to other fluorescent proteins, and require further engineering, which is often achieved through random methods, incurring large time and resource costs. Here we propose a rational design approach to improve the quantum yield of RFPs by reducing conformational variability of the chromophore. We engineered mRojoA, a mutant containing a π-stack involving Tyr197 and the chromophore phenolate, to include the P63F/H/Y mutations on its other side, by simultaneously mutating neighbouring positions 16, 143, and 163. The brightest mutants that we found in each library, mRojo-VYGV, mRojo-VFAV, and mRojo-VHSV, exhibited 1.8- to 2.4-fold increases in brightness, and quantum yield increases of up to 2.1-fold. In all three mutants, the increases in brightness were predominantly due to improvements in the quantum yield and not the extinction coefficient. Solving the crystal structures of two of these mutants along with a dim variant allowed us to strongly infer a link between rigidity of the chromophore and increased quantum yield. In addition, back-mutating position 63 in the highest quantum yield mutant, mRojo-VYGV, reversed the improvement in quantum yield, indicating that Y63 was the primary residue responsible for the improved brightness of the protein. Unfortunately, the mCherry-VYGV mutant did not achieve a similar increase in quantum yield or brightness. This is likely due to the lack of a second bulky aromatic residue at position 197, which is present in mRojoA. Nevertheless, this rational approach could be applied to some other RFPs whose chromophores exhibit increased conformational variability in order to further improve their brightness.

Les travaux présentés dans cette thèse concernent la paramétrisation de la distribution verticale de la biomasse et de la structure des communautés phytoplanctoniques dans l’océan global. Nous avons d’abord développé une méthode neuronale de calibration de la fluorescence en concentration en chlorophylle a ([Chl]) associée à la biomasse phytoplanctonique totale et à trois classes de taille de phytoplancton. Cette méthode, FLAVOR, a été entrainée et validée à l’aide une base de données de ~900 profils de fluorescence et de pigments mesurés pat HPLC. Une base de données globale de ~49000 profils de fluorescence a ensuite été assemblée et calibrée en termes de biomasse chlorophyllienne et composition du phytoplancton. Ce travail représente une première étape vers une vision tridimensionnelle de la biomasse phytoplanctonique. Nous avons ensuite développé deux réseaux de neurones (SOCA) pour estimer la distribution verticale de deux paramètres bio-optiques, [Chl] et le coefficient de rétrodiffusion. Ces réseaux de neurones requièrent comme données d’entrée des données satellites de couleur de l’eau co-localisées avec un profil hydrologique collecté par un flotteur Argo. Ils ont été entrainés et validés avec une base de données globale composée de ~5 000 profils de propriétés bio-optiques et hydrologiques acquises par des flotteurs Bio-Argo. Les bases de données utilisées pour développer les méthodes FLAVOR et SOCA proviennent de régions océaniques représentatives de l’océan global, permettant ainsi l’application de ces méthodes à la majorité des eaux océaniques. Finalement, nous avons mené une étude focalisée sur l’Atlantique Nord qui exploite les outils développés. Les champs tridimensionnels de biomasse obtenus, couplés à un modèle bio-optique de production primaire, permettent d’étudier les cycles saisonniers de la distribution verticale de la biomasse phytoplanctonique et de la production primaire dans différentes bio-régions de l’Atlantique Nord
This PhD work focuses on the parameterization of the vertical distribution of phytoplankton biomass and community structure in the global open ocean. First we have developed a neural network-based method for the calibration of the fluorescence in chlorophyll a concentration [Chl] associated with the total phytoplankton biomass and with three phytoplankton size classes. This method, (FLAVOR for Fluorescence to Algal communities Vertical distribution in the Oceanic Realm), was trained and validated using a database of ~900 concomitant fluorescence and HPLC-determined pigment profiles. A global database comprising ~49 000 fluorescence profiles was assembled and calibrated with FLAVOR. The resulting database represents a first step towards a global three-dimensional view of phytoplankton biomass and community composition. Second, two neural networks (SOCA for Satellite Ocean Color and Argo data to infer vertical distribution of bio-optical properties) were developed to infer the vertical distribution of two bio-optical proxies of the phytoplankton biomass, [Chl] and the particulate backscattering coefficient, using as input satellite-derived products matched up with a hydrological Argo profile. The SOCA methods were trained and validated using a global database of ~5 000 profiles of bio-optical and hydrological properties collected from Bio-Argo floats with concomitant satellite products. The database used to develop FLAVOR and SOCA originates from various oceanic regions largely representative of the global ocean, making the methods applicable to most oceanic waters. Finally, we proposed a study dedicated to the North Atlantic where the tools developed in this thesis are used in conjunction with a bio-optical primary production model. This allows us to characterize the seasonal cycle of the vertical distribution of the phytoplankton biomass and primary production in various bio-regions of the North Atlantic

Lubricin, or proteoglycan 4 (PRG4), is a secreted, glycosylated protein that binds to cartilage surfaces, which functions as a boundary lubricant. The loss of lubricin from cartilage is identified as a major pathogenic factor in post-traumatic osteoarthritis (PTOA) that has now been the aim of therapeutic intervention. Intra-articular injection of PRG4 protein provides short-term benefits that might be extended using sustained delivery methods such as in gene therapy. Here we describe the development and testing of such therapy using adeno-associated virus (AAV) as a vector for the transfer of PRG4-green fluorescent protein (GFP) fusion gene. Our recombinant PRG4 gene produces a PRG4-GFP fusion protein to facilitate tracking of its expression and distribution on joint surfaces. We hypothesized that PRG4-GFP is fully functional as a cartilage lubricant and that PRG4-GFP produced in vivo is expressed by synoviocytes and other joint cells, and cartilage surfaces remained coated for several weeks up to months after intra-articular injection of the virus. PRG4-GFP showed lubricin-like cartilage binding in vitro, and lubrication immunoblot analysis confirmed that purified PRG4-GFP from cultured media conditioned by PRG4-GFP-transduced synoviocytes was heavily glycosylated, while confocal microscopy revealed binding of the fluorescent fusion protein to cartilage surfaces. Metal-on-cartilage friction tests showed that PRG4-GFP reduced friction coefficients to a degree comparable to that of synovial fluid and had strong chondro-protective effects in explanted cartilage exposed to shear loading. The chondrocyte viability after shear loading showed that PRG4-GFP had a strong chondro-protective effect on par with that of the synovial fluid. Confocal microscopy and immunohistology confirmed that cartilage surfaces in the stifle joints of mice injected with viruses were coated with PRG4-GFP for up to 2 or 4 weeks after the treatment. The overexpression of PRG4-GFP and coating of cartilage surfaces in the stifle joints of mice injected with Adeno-Associated Virus for the transfer of PRG4-GFP fusion gene (AAV-PRG4-GFP) was confirmed by confocal microscopy and immunohistology for up to 2 or 4 weeks post-injection. The μCT imaging and immunohistology in AAV-PRG4-GFP injected rabbit knees showed stronger inhibition in degeneration of damaged tissues than in AAV-GFP injected control group. Collectively these findings indicate that AAV-PRG4-GFP transduction is a valuable new tool for evaluating the effects of long-term lubricant supplementation on PTOA in animal models.

La fertilidad masculina puede ser medida mediante un espermatograma convencional, sin embargo este examen no incluye la valoración de la integridad del ADN espermático. Esta variable ha sido correlacionada con las tasas de fertilización, viabilidad y desarrollo del embrión, convirtiéndose en una herramienta de importancia clínica tanto para los programas de reproducción animal como los tratamientos de fertilidad asistida. El bioensayo Cometa es capaz de determinar de una manera exacta el valor de la integridad del ADN espermático, lamentablemente este examen no es de uso rutinario por su elevado costo de implementación ya que utiliza microscopia especializada y tinciones fluorescentes para evidenciar la migración del ADN. El objetivo de esta investigación fue evaluar la compatibilidad de las tinciones no fluorescentes Diffquik, Giemsa, de Feulgen y FastBlast en el bioensayo Cometa usando un método visual y automatizado. Se utilizaron 15 eyaculados previamente seleccionados de acuerdo al manual OMS 2010, para luego ser capacitados en búsqueda de homogeneidad adecuada para la experimentación. Cada muestra fue expuesta a una gradiente de Peróxido de hidrogeno (0, 10, 30,60 y 100 mM) por 1 hora a 4°C para luego evaluar el coeficiente de daño mediante el método visual y porcentaje de ADN en la cola mediante el método automatizado. Las pendientes de la regresión lineal en el método visual indican que los valores obtenidos por la tinción control SybrGreen (m=3,69) difieren con Giemsa (m=3,45) y Diffquik (m=2,57). En el método automatizado de igual manera SybrGreen (m=0.83), Giemsa (m=0,79) y Diffquik (m=0,77). Sin embargo SybrGreen es 1,06 veces más efectivo que Giemsa en el visual y 1,05 veces en el automatizado, sugiriendo una compatibilidad con el bioensayo cometa. De igual manera SybrGreen es 1,07 veces más efectivo que Diffquik en el visual y 1,44 veces en el automatizado, concluyendo una compatibilidad solo en el método visual.Male fertility can be measured by a conventional semen analysis, however, this examination does not include the assessment of sperm DNA integrity. This variable has been correlated with fertilization rates, embryo viability and development, becoming a tool of clinical importance for both animal breeding programs and assisted fertility treatments. Comet bioassay is able to determine an exact way the value of sperm DNA integrity, unfortunately this test is not routinely used because of its high cost of implementation because it uses specialized microscopy and fluorescent dyes to demonstrate DNA migration. The objective of this research was to evaluate the compatibility of non-fluorescent dyes Diffquik, Giemsa, Feulgen and Comet FastBlast in the bioassay using a visual and automated method. 15 ejaculates were used previously manually selected according to WHO 2010 and then be trained in finding adequate homogeneity for experimentation. Each sample was exposed to a hydrogen peroxide gradient (0, 10, 30,60 and 100 mM) for 1 hour at 4 ° C and then assess the damage coefficient by visual method and percentage of DNA in the tail by automated method. The slopes of the linear regressions on the visual method indicate that the values obtained by the SybrGreen Control staining (m = 3.69) differ with Giemsa (m = 3.45) and Diffquik (m = 2.57). In the same way automated method SybrGreen (m = 0.83), Giemsa (m = 0.79) and Diffquik (m = 0.77). However SybrGreen is 1.06 times more effective than Giemsa visual and 1.05 times in the automated, suggesting a comet support bioassay. Similarly SybrGreen is 1.07 times more effective than Diffquik visual and 1.44 times in the automated, concluding compatibility only in the visual method. Keywords:

Dans ce travail, nous avons etudie la reaction en phase gazeuse des atomes de cuivre dans l'etat fondamental cu(#2s#1#/#2, #2d#5#/#2) avec un compose halogene rx (x: f, cl, br, i) pour former la molecule cux a l'etat fondamental ou dans des etats electroniques excites. La spectroscopie d'emission (chimiluminescence) et la molecule cux excitee fournit beaucoup d'informations sur la repartition de l'energie disponible dans les differents etats de la molecule (electroniques, vibrationnels, rotationnels). Un pompage optique au moyen d'un laser a cuivre permet d'agir sur la concentration de chaque metastable #2d#3#/#2 et #2d#5#/#2 et de departager leur role dans la reaction (coefficient de reaction totale, repartition des populations). Les durees de vie des etats excites ont ete egalement mesurees. Une etude comparative entre les reactions des atomes de cuivres avec differents reactants rx montre que la repartition de l'energie des produits formes est differente dans chaque cas. Il semble qu'une distribution statistique soit favorisee en allant vers les halogenes plus lourds. Pour rendre compte des differents mecanismes de formation, nous proposons deux modeles de reaction: modele de harponnage et modele d'insertion. L'ensemble de cette etude a ete realise en utilisant la technique de post-decharge en ecoulement ou les produits de la reaction sont immediatement evacues. Cette technique permet de separer spatialement la zone de decharge de la zone d'injection du reactant de sorte que seuls les atomes de cuivre (#2d#1#/#2, #2d#3#/#2, #2d#3#/#2) interviennent dans la reaction etudiee

L’objectif de la recherche est la mise au point d'un capteur photophysique pour l'étude des phénomènes de transfert de matière en milieu biphasique gaz-liquide. Le principe repose sur l'inhibition de fluorescence de molécules greffées sur le capteur, par l'oxygène dissous dans l'eau. La variation d'intensité de fluorescence est une fonction linéaire de la concentration en oxygène, celle-ci étant elle-même reliée au coefficient de transfert volumique, kLa. Dans ce but, deux actions principales ont été développées: - la réalisation et l'étude de la fixation chimique de la molécule fluorescente sur une surface plane en silice: l'acide 4-(1-pyrenyl)butyrique a été choisi comme fluorophore pour sa forte réactivité vis-à-vis de l'oxygène. Différentes conditions de greffage ont été testées pour déterminer l'influence de divers paramètres (quantité d'eau, présence de catalyseur, type de surface) et leurs actions analysées de manière qualitative (utilisations de techniques d'analyse de surface: microscopie par fluorescence, microscopie à force atomique (de cisaillement ou de friction), ) et quantitative (fluorimétrie). L’utilisation d'un post-traitement, jouant sur la désorption, a permis de dégager un modèle cinétique. - La mise au point du capteur: la détermination de la droite de Stern-Vollmer (If=f(O2)) en milieu statique et dynamique et les essais dans le milieu biphasique gaz-liquide ont permis de déterminer le coefficient kLa

Neste trabalho, estudou-se o efeito da adição de acetonitrila nas propriedades de micelas do detergente aniônico, dodecil sulfato de sódio (SDS), e do detergente catiônico, cloreto de hexadeciltrimetilamônio (CTACl). Medidas de condutividade foram utilizadas para determinar a concentração micelar crítica, cmc, e o grau de dissociação, α, das micelas em função da fração molar de acetonitrila, XAc. Medidas de supressão de fluorescência, resolvida no tempo, com pireno como sonda, foram utilizadas para determinar a influência de acetonitrila no número de agregação das micelas, N, e na dinâmica de migração de solutos entre as fases aquosa e micelar. Em baixas frações molares XAc < 0,2), a acetonitrila insere-se nas cavidades da água, quebrando parcialmente as pontes de hidrogênio da água com a formação de novas pontes de hidrogênio entre as moléculas de acetonitrila e as moléculas de água. Nesta faixa de concentração, ocorre um aumento da cmc e do α, acompanhada de uma diminuição de N. Observa-se também alterações na dinâmica da interação de contra-íons e co-íons supressores na micela. Assim, as micelas de SDS e CTACl formadas em misturas acetonitrila-água são menores, mais dissociadas e apresentam maior fluidez interna. Ao redor de XAc = 0,2, as misturas de água-acetonitrila tornam-se microheterogêneos com o aparecimento de microdomínios ricos em acetonitrila e microdomínios ricos em água. A proporção das regiões ricas em acetonitrila aumenta com o aumento da fração molar de acetonitrila, com apenas pequenas modificações das propriedades dos dois tipos de microdomínios. Em XAc > 0,2 a variação de cmc e de α com a XAc passa a ser menos acentuada, sugerindo que o detergente forma agregados preferencialmente nas regiões mais aquosas; a sonda fluorescente pireno começa sair das micelas durante o tempo de vida do estado excitado; e há claras mudanças na dinâmica de incorporação de íons nos agregados.
This work presents a study of the effect of added acetonitrile on the properties of the micelles of the anionic detergent sodium dodecylsulfate (SDS) and the cationic detergent hexadecyltrimethylammonium chloride (CTACl). Conductimetric measurements were employed to determine the critical micelle concentration, cmc, and the degree of counterion dissociation, α, of the micelles as a function of the mole fraction of added acetonitrile, XAc. Time resolved fluorescence quenching measurements with pyrene as probe were employed to determine the effect of acetonitrile on the micellar aggregation number, N, and the dynamics of solute migration between the micellar and aqueous phases. At low mole fractions (XAc < 0.2), acetonitrile inserts into the cavities present in liquid water, partially disrupting the hydrogen bonding of water, with formation of new hydrogen bonds between water and acetonitrile. In this range, both the cmc and α increase, while N decreases. The dynamics of incorporation of counterionic and coionic quenchers into the micelles is also altered. Thus, the SDS and CTACl micelles formed in these acetonitrile-water mixtures are smaller, more highly dissociated and internally more fluid than those in aqueous solution. Above XAc of ca. 0,2, acetonitrile-water mixtures become microheterogeneous, the solution containing microdomains rich in acetonitrile and microdomains rich in water. The proportion of acetonitrile-rich microdomians increases with increasing XAc, with only small changes in the properties of the two types of microdomains. Correspondingly, at XAc > > ca. 0.2: the variation of the cmc and α with XAc is much less pronounced, suggesting that the detergent forms aggregates preferentially in the aqueous-rich domains; the fluorescence probe pyrene begins to exit the micelles during its excited state lifetime; and there are distinct changes in the rate constants for the incorporation of ions into the micelles.

Tato práce se zabývá studiem fluorescenčně značeného hyaluronanu, konkrétně rhodaminylamino hyaluronátu sodného (Hya-Rh, 40 kDa), pomocí dvouohniskové fluorescenční korelační spektroskopie (2f-FCS). Nejdříve byla prostudována literatura týkající se využití FCS techniky v koloidní chemii a při studiu polymerů, přičemž následně byly shrnuty veškeré poznatky o využití 2f-FCS metody. Na základě prvotních měření byl zjištěn vhodný postup přípravy a způsob uchovávání vzorku Hya-Rh používaném pro následující experimenty. Záhy byly prostudovány možné vlivy koncentrace Hya-Rh na jeho difúzní charakteristiky jak ve vodě, tak ve fyziologickém roztoku. Následně bylo studováno chování Hya-Rh a vliv koncentrace solí alkalických kovů ve vodných roztocích těchto solí a fluorescenčně značeného hyaluronanu. Poté bylo sledováno chování Hya-Rh v závislosti na koncentraci velmi nízkomolekulárního hyaluronanu (VLMW HA, 404 kDa) v čisté vodě i ve fyziologickém roztoku, přičemž získané výsledky byly mezi sebou porovnány. Nakonec bylo využití 2f-FCS metody celkově zhodnoceno a popsáno z hlediska studia chování fluorescenčně značeného hyaluronanu v roztocích.

Afin d’améliorer l’efficacité des traitements des pathologies articulaires, en tenant compte de leur complexité et de leur ampleur, des études récentes ont mis en évidence le rôle des assemblages lipidiques associés à la structure discontinue du fluide synovial dans le contrôle du fonctionnement tribologique articulaire. Ceci à conduit à la mise au point d’un modèle tribologique ex vivo (thèse AM Sfarghiu, 2006), proposant un « motif élémentaire » de la biolubrification articulaire, constitué de l’empilement d’interfaces phospholipidiques et de couches aqueuses. En utilisant ce modèle, l’objectif de ce travail a été d’étudier l’évolution des interfaces phospholipidiques du fluide synovial en présence de pathologies. Pour ce faire, une méthodologie nano-bio-tribologique alliant des analyses biochimiques, physicochimiques, nano-mécaniques et tribologiques a été utilisée. Les résultats de ces analyses montrent : l’influence de la faible rugosité des surfaces frottantes caractérisant les stades précoces des pathologies et celle des propriétés des interfaces phospholipidiques (liées à la variation de leur composition) sur la résistance mécanique, l’évolution au cours du frottement et la dégradation in situ des assemblages lipidiques des fluides synoviaux pathologiques. Le comportement des assemblages lipidiques est accentué par l’action des enzymes associées aux pathologies. Par conséquent, le fonctionnement articulaire dépend de la résistance mécanique des interfaces phospholipidiques et pour obtenir des coefficients de frottement très bas, l’accommodation de vitesse doit s’effectuer au niveau des couches d’hydratation qui entourent les ions présents dans la couche aqueuse. Ces résultats permettront de comprendre à court terme l’évolution des interfaces phospholipidiques dans les pathologies articulaires et, à plus long terme le bon enchaînement cause/conséquence responsable d’une pathologie articulaire afin de développer des traitements plus efficaces, ciblés et non prothétiques
In order to improve the effectiveness of joint diseases’ treatments, given their complexity and magnitude, recent studies have highlighted the role of lipid assemblies associated with the discontinuous structure of the synovial fluid (SF) in the tribological performance of joint operation. Thus, an ex vivo tribological model (AM Sfarghiu, PhD thesis, 2006) providing a "basic pattern" for joint biolubrification was developed. It consists of the stack of phospholipidic interfaces and aqueous layers. Using this model, the objective of this work was to study the evolution of phospholipidic interfaces of SF within pathological state. Therefore, a nano-bio-tribological methodology combining biochemical, physicochemical, nano-mechanical and tribological analysis was used. The results of these analyses show: the influence of even small rubbing surfaces’ roughness characteristics of early stage illness and that of phospholipidic interfaces’ properties (related to their composition change) on the mechanical strength, changes in friction and in situ degradation of lipidic assemblies of pathological SF. The tribological operation is highlighted by enzymes’ associated with diseases. Thus, joint operation depends on the mechanical strength of phospholipidic interfaces and to obtain very low friction coefficients, velocity accommodation must be done at the level of hydration layers surrounding ions in the aqueous solution. These results would therefore allow better understanding of the evolution of phospholipidic interfaces in joint diseases and of the proper cause/consequence sequence responsible for a joint disease in order to develop more effective, targeted and non prosthetic treatments

An implantable closed-loop insulin delivery device (INsmart device) containing a glucose responsive gel has been developed within the INsmart research group, over a period of 10 years, to mimic pancreas. In this thesis, the reliability and performance capability of the INsmart device was studied for future clinical use. Investigations into the device material compatibility with insulin solution, assessed by monitoring insulin loss and degradant formation over a period of 31 days using RP-HPLC have shown that stainless steel and titanium are the most compatible materials. Polycarbonate contributes to insulin loss after 11 days, resin might not be the best material and polyurethane is the least compatible for future device designs. To study insulin delivery mechanism and kinetics from the device, fluorescently labelled human insulin (FITC-insulin) was synthesised and characterised using RP-HPLC and MS, to produce a product with predominantly di-labelled conjugate (>75%) with no unreacted FITC or native insulin. Clinically used insulin analogues were also fluorescently labelled to produce predominantly di-labelled FITC-insulin conjugate with potential future biological and in vitro applications. The drug release mechanism from the glucose sensitive gel held in the INsmart device, studied using fluorescein sodium was determined as a Fickian diffusion controlled release mechanism. The diffusion coefficient (D) for FITC-insulin in the non-polymerised dex2M-conA gel (NP gel) determined using mathematical models, QSS and TL slope methods was 1.05 ± 0.02 x 10-11 m2/s and in the cross-linked dex500MA-conAMA gel (CL gel) was 0.75 ± 0.06 x 10-11 m2/s. In response to physiologically relevant glucose triggers in the NP gel, the diffusivity of FITC-insulin increases with increasing glucose concentrations, showing a second order polynomial fit, device thus showing glucose sensitivity and graded response, mimicking pancreas. Rheological measurements further confirmed the gel glucose responsiveness demonstrated by a third order polynomial fit between FITC-insulin D and the NP complex viscosity in response to increasing glucose concentration. The knowledge of FITC-insulin diffusion kinetics in the gel has aided in making some theoretical predictions for the capability and performance of the INsmart device. Alternate device geometry and design optimisation is also explored.

Afin d'améliorer l'efficacité des traitements des pathologies articulaires, en tenant compte de leur complexité et de leur ampleur, des études récentes ont mis en évidence le rôle des assemblages lipidiques associés à la structure discontinue du fluide synovial dans le contrôle du fonctionnement tribologique articulaire. Ceci à conduit à la mise au point d'un modèle tribologique ex vivo (thèse AM Sfarghiu, 2006), proposant un " motif élémentaire " de la biolubrification articulaire, constitué de l'empilement d'interfaces phospholipidiques et de couches aqueuses. En utilisant ce modèle, l'objectif de ce travail a été d'étudier l'évolution des interfaces phospholipidiques du fluide synovial en présence de pathologies. Pour ce faire, une méthodologie nano-bio-tribologique alliant des analyses biochimiques, physicochimiques, nano-mécaniques et tribologiques a été utilisée. Les résultats de ces analyses montrent : l'influence de la faible rugosité des surfaces frottantes caractérisant les stades précoces des pathologies et celle des propriétés des interfaces phospholipidiques (liées à la variation de leur composition) sur la résistance mécanique, l'évolution au cours du frottement et la dégradation in situ des assemblages lipidiques des fluides synoviaux pathologiques. Le comportement des assemblages lipidiques est accentué par l'action des enzymes associées aux pathologies. Par conséquent, le fonctionnement articulaire dépend de la résistance mécanique des interfaces phospholipidiques et pour obtenir des coefficients de frottement très bas, l'accommodation de vitesse doit s'effectuer au niveau des couches d'hydratation qui entourent les ions présents dans la couche aqueuse. Ces résultats permettront de comprendre à court terme l'évolution des interfaces phospholipidiques dans les pathologies articulaires et, à plus long terme le bon enchaînement cause/conséquence responsable d'une pathologie articulaire afin de développer des traitements plus efficaces, ciblés et non prothétiques.

碩士
國立交通大學
應用化學系
87
The absolute rate coefficient for the reactions of Cl radicals with CF3CH2Cl (HCFC-133a) have been measured using laser photolysis — resonance fluorescence technique between 297-550 K and at pressure 10-100 torr。Cl radicals were produced by photolyzing Cl2 molecules with a Nd-YAG laser output at 355nm。 Cl + CF3CH2Cl → HCl + CF3CHCl We obtained k(T)=(6.3±3.1)×10-12exp[-(1940±186)/ T] cm3 molecule-1 s-1，with k(297K)= (1.07±0.08)×10-14 cm3 molecule-1 s-1。No pressure and laser power effect were observed。

碩士
國立交通大學
應用化學系
87
The absolute rate coefficient for the reaction of Cl atoms with CH3CFCl2 (HCFC-l41b) has been determined using laser photolysis-resonance fluorescence technique at 298 K and 45 torr. Cl + CH3CFCl2 → HCl + CH2CFCl2 (1-19) Two Cl radicals source reactions were tested, (1)CCl4 + hν(l93 nm) → Cl(2P) + CCl3 CCl4 + hν(l93 nm) → 2Cl(2P) + CCl2 (1-20) (2)Cl2 + hν(355 nm) → 2Cl(2P3/2) (1-21) Cl2 was found to be a better precursor for the rate coefficient measurements. We obtained a second-order rate coefficient : kⅡ=(5.12─6.07) ╳lO-14 cm3 molecule-1 s-1.The energy of photolysis laser (1.1─18.6 mJ/cm2) did not affect the measured rate coefficient. When [Cl] was higher than 1 ╳1013 molecule cm-3, Cl regenerations were observed .

abstract: The cell is a dense environment composes of proteins, nucleic acids, as well as other small molecules, which are constantly bombarding each other and interacting. These interactions and the diffusive motions are driven by internal thermal fluctuations. Upon collision, molecules can interact and form complexes. It is of interest to learn kinetic parameters such as reaction rates of one molecule converting to different species or two molecules colliding and form a new species as well as to learn diffusion coefficients. Several experimental measurements can probe diffusion coefficients at the single-molecule and bulk level. The target of this thesis is on single-molecule methods, which can assess diffusion coefficients at the individual molecular level. For instance, super resolution methods like stochastic optical reconstruction microscopy (STORM) and photo activated localization microscopy (PALM), have a high spatial resolution with the cost of lower temporal resolution. Also, there is a different group of methods, such as MINFLUX, multi-detector tracking, which can track a single molecule with high spatio-temporal resolution. The problem with these methods is that they are only applicable to very diluted samples since they need to ensure existence of a single molecule in the region of interest (ROI). In this thesis, the goal is to have the best of both worlds by achieving high spatio-temporal resolutions without being limited to a few molecules. To do so, one needs to refocus on fluorescence correlation spectroscopy (FCS) as a method that applies to both in vivo and in vitro systems with a high temporal resolution and relies on multiple molecules traversing a confocal volume for an extended period of time. The difficulty here is that the interpretation of the signal leads to different estimates for the kinetic parameters such as diffusion coefficients based on a different number of molecules we consider in the model. It is for this reason that the focus of this thesis is now on using Bayesian nonparametrics (BNPs) as a way to solve this model selection problem and extract kinetic parameters such as diffusion coefficients at the single-molecule level from a few photons, and thus with the highest temporal resolution as possible.
Dissertation/Thesis
Source code related to chapter 3
Source code related to chapter 4
Doctoral Dissertation Physics 2020

Ziel der vorgestellten Arbeit war die Synthese von Nucleolipiden zur Lipophilisierung von Oligonucleotiden sowie deren Untersuchung im Hinblick auf ihre Wechselwirkung und Duplex-Bildung an horizontalen Lipidmembranen und verschiedenen Phasengrenzen zur Entwicklung eines neuartigen Bio-Chips für die RNA/DNA-Analyse. Mit der Synthese N(3)-prenylierter und 2’,3’-O-ketalisierter Pyrimidinbasen Uridin und Methyluridin wurden Nucleolipid-Bausteine dargestellt, die auch als terminale Kopfgruppen eines Oligonucleotid-Dodecamers den lipophilen Charakter dieser Oligonucleotid-Sequenz erhöhten. Für den Einsatz solcher LONs (Lipo-Oligonucleotide) in einer vereinfachten RNA/DNA-Analytik wurde eine Vielzahl von Lipo-Oligonucleotiden mit diversen Nucleolipid-Kopfgruppen synthetisiert und auf ihr Einlagerungsverhalten in künstliche Lipid-Bilayer untersucht. Fluoreszenz-spektroskopische Untersuchungen zeigten, dass alle Lipo-Oligonucleotide in der Lage sind, sich in künstliche Lipid-Bilayer einzulagern. Abhängig von der Struktur, der Länge und der Anzahl der C-Atom-Ketten dieser lipophilen Anker-Bausteine wurden die Geschwindigkeit und die Festigkeit der Verankerung im Lipid-Bilayer beeinflusst. Des Weiteren wurde die Hybridisierung von LONs mit komplementären Oligomeren an Lipidmembranen untersucht. Es konnte gezeigt werden, dass die im Bilayer verankerten Lipo-Oligonucleotide mit komplementären Oligomeren DNA-Duplexe bilden. Die hybridisierte DNA wurde nicht nur über einen kovalent gebundenen Cy5-Fluorophor am Gegenstrang nachgewiesen, sondern auch über den DNA-Interkalator SYBR Green I (SG). Am Beispiel von zwei Lipo-Oligonucleotiden (LON 20 und 23), die sich schnell und fest in der Bilayermembran verankern, konnte eine spontane Akkumulation dieser LONs an CHCl3/H2O sowie H2O/n-Decan Grenzflächen direkt nach der Probenzugabe beobachtet werden. Diese und andere Ergebnisse stützen den Einsatz von Lipo-Oligonucleotiden als Ziel-Oligomere in einem neuartigen RNA/DNA-Nachweisverfahren an Phasengrenzen.