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Développement de sondes activables à deux photons pour une utilisation en neurosciences / Development of two photon activable molecular probes for applications in neuroscienceTran, Christine 19 November 2015 (has links)
Des sondes photoactivables (composés “cagés”) dérivées de la 2-hydroxyméthylène-diméthylaminoquinoléine, ont été préparées et testées pour une application en neurophysiologie. Ces sondes montrent une stabilité hydrolytique élevée et une faible fluorescence, avec des cinétiques de photofragmentation rapides sous irradiation UV (365 nm). Il en est également de même dans des conditions biphotoniques en IR proche (730 nm). Une optimisation de cette plateforme a été réalisée en modifiant la nature et la position des substituants du chromophore, en augmentant la conjugaison et en incorporant des éléments de symétrie C2 et S3, conduisant aux sondes dipolaires, quadrupolaires (dimériques) et octupolaires (trimériques) à haute sensibilité biphotonique ( < 2,50 GM). Les dérivés les plus efficaces ont été testés dans des expériences en neurophysiologie. Tandis que les dérivés de kaïnate sont suffisamment stables en solution aqueuse à pH 7,4 pour les expériences en conditions physiologiques, les dérivés de L-glutamate et GABA ont nécessité une connexion de type carbamate avec la plateforme photoactivable. Sans irradiation, les solutions « stock » de ces composés « cagés » (c = 200-300 µM) n’ont pas produit d’effets majeurs sur l'excitabilité des neurones à en juger par le manque d'effet sur l'activité neuronale ou de potentiels d’actions synaptiques spontanés provoqués par une dépolarisation. La photolyse en lumière blanche par pulses courts d’irradiation a permis la libération de substances actives en quantité suffisante pour produire de grands courants (jusqu’à 5 nA) dans les neurones de Purkinje. / Photosensitive molecular probes (‘caged’ compounds) derived from 2-hydroxymethylenedimethylaminoquinoline were prepared and tested for applications in neurophysiology. These compounds show high hydrolytic stability and low fluorescence, with fast fragmentation kinetics upon UV irradiation (365 nm), and under two photon photolysis conditions (730 nm). This platform was optimized by modifying the substitution pattern, increasing the conjugation length and incorporating C2 or S3 symmetry elements. Dipolar, quadrupolar (dimer) and octupolar (trimer) derivatives were thus synthesized and were found exhibiting high two-photon sensitivity ( < 2,50 GM). The most efficient probes were tested in neurophysiological experiments. While kaïnate derivatives are stable in aqueous solution at pH 7.4 in physiological conditions, L-glutamate and GABA derivatives required the use of a carbamate linker. Without irradiation, any major changes were observed on neuron excitability with “stock” solutions of these caged compounds (c = 200-300 µM), according to the lack of effects on neuron activity or action potentials evoked by depolarization. White light photolysis by short pulses generated sufficient active substances to induce large inward currents (up to 5 nA) in Purkinje neurons.
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Copper at the Interface of Chemistry and Biology: New Insights into hCtr1 Function and the Role of Histidine in Human Cellular Copper AcquisitionHaas, Kathryn Louise January 2010 (has links)
<p>Mechanisms of copper homeostasis are of great interest partly due to their connection to debilitating genetic and neurological disorders. The family of high-affinity copper transporters (Ctr) is responsible for extracellular copper acquisition and internalization in yeast, plants, and mammals, including human. The extracellular domain of the human high-affinity copper transporter (hCtr1) contains essential Cu-binding methionine-rich MXXM and MXM (Mets) motifs that are important for copper acquisition and transport. The hCtr1 extracellular domain also contains potential copper binding histidine (His) clusters, including a high-affinity Cu(II) ATCUN site. As of yet, extracellular His clusters have no established significance for hCtr1 function. We have made model peptides based on the extracellular copper acquisition domain of hCtr1 that is rich in His residues and Mets motifs. The peptides' Cu(I) and Cu(II) binding properties have been characterized by UV-Vis and mass spectrometry. Our findings have been extended to a mouse cell model and we show that His residues are important for hCtr1 function likely because of their contribution to strong copper-binding sites in the hCtr1 extracellular domain responsible for copper acquisition. </p>
<p>Copper's pro-oxidant property is also medicinally promising if it can be harnessed to induce oxidative stress as a cancer chemotherapy strategy. Our lab has designed a photocleavable caged copper complex that can selectively release redox-active copper in response to light. The thermodynamic copper binding properties of these potential chemotherapeutics have been characterized</p> / Dissertation
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THE DEVELOPMENT OF INTRACELLULAR NANOSENSORS: ACID-DEGRADABLE POLYMERIZED PHOSPHOLIPID VESICLES AND FLUORESCENT LABELSRoberts, David January 2010 (has links)
Phospholipid vesicles are biocompatible, and have potential for intracellular applications, but minimal membrane integrity limits their use in membrane-rich environments. Stabilized membranes overcome this limitation while maintaining biocompatible surface structures. Additionally, the modularity of phospholipid bilayer makes them ideal components when designing biologically inspired sensors. Membrane composition can be tailored to specific applications, transmembrane proteins can provide added functionalities, and the isolated interior can prevent cytotoxic and interfering detection chemistries from altering the cellular environment. This work has focused on expanding the capabilities of stabilized phospholipid membranes, and determining which formulations hold promise in developing stabilized phospholipid vesicle nanosensors.Current membrane stabilization methods suffer from either incomplete stabilization, or irreversible stabilization limiting the applications of vesicle nanosensors. Therefore, a facile method to prepare robust phospholipid vesicles using commonly available phospholipids stabilized via the formation of an interpenetrating, acid-labile, cross-linked polymer network that imparts controlled polymer destabilization and subsequent vesicle degradation was developed. Upon exposure to acidic conditions, the highly cross-linked polymer network was converted to linear polymers, substantially reducing vesicle stability upon exposure to chemical and physical insults. The resultant transiently stabilized vesicles have potential for enhanced drug delivery and chemical sensing applications requiring minimal membrane defects, and allow for improved physiological clearance.Some vesicle nanosensor schemes may require the passive diffusion of low molecular weight species across the membrane in addition to controllable degradation. Therefore, the acid-degradable, polymer-stabilized, phospholipid vesicle production method was extended to bis-SorbPC membranes by simultaneously polymerizing the vesicle with an acetal-containing cross-linker. The vesicles display prolonged stability under physiological conditions, and significant additional stability compared to vesicles composed of naturally occurring phospholipids. The vesicles demonstrated potential utility for sensing and therapeutic applications.Phospholipid vesicles can also serve as labels to observe movement in macromolecular biological assemblies, but a dearth of caged fluorescent labels limits design and function. Therefore, the first caged fluorescent thiol was synthesized, shown to label amines rapidly, and demonstrated the required photolytic properties. The caged fluorescent thiol has potential as a label in observing the movement of macromolecular biological assemblies and as a fluorescent probe for observing endosomal trafficking and release.
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Design and development of a two dimensional scanning molecular tagging velocimetry (MTV) systemAhmad, Farhan Unknown Date
No description available.
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Self-immolative spacers in profluorophore strategies : determination of kinetic parameters for release of single and multiple substrates. / Espaceurs auto-immolables dans des stratégies profluorophores : détermination de paramètres cinétiques pour la libération de substrats simples et multiplesAlouane, Ahmed 30 September 2014 (has links)
Les espaceurs auto-immolables sont des assemblages moléculaires inactifs permettant de découpler deux entités chimiques A (un groupe protecteur subissant une activation) et B (un composé d'intérêt, tel qu'un principe actif, un rapporteur ou un autre espaceur) par des liaisons covalentes. Sous l'effet d'un stimulus externe (activation), A est clivé spontanément lors d'un processus irréversible appelé auto-immolation, qui provoque la libération de l'espèce B. Cette dernière est généralement inactive lorsqu'elle est intégrée dans l'espaceur ; en revanche, une fois libérée, elle retrouve son activité intrinsèque.La cinétique du processus d'auto-immolation contrôle la corrélation spatio-temporelle existant entre l'activation et la libération de la substance active. L'objectif principal de ce travail a été précisément de fournir un ensemble complet de données permettant d'établir des relations structure/propriétés cinétiques fiables dans une série d'espaceur auto-immolable contenant une structure aromatique portant un substituant hydroxyle et s'appuyant sur un mécanisme impliquant une cascade électronique. Ainsi, nous avons utilisé un groupe photactivable et la lumière pour déclencher le processus d'auto-immolation d'une grande banque d'espaceurs auto-immolables, qui présentent un temps caractéristique d'auto-immolation s'étalant sur un large éventail d'échelle de temps (10-4-104s).Au cours de ce travail, nous avons également développé des espaceurs auto-immolables, qui libérent deux composés après activation. Ces systèmes ont été mis en ¿uvre pour répondre à l'analyse quantitative d'un substrat libéré par photodéprotection dans les systèmes biologiques. / Self-immolative spacers are inactive molecular assemblies decoupling two chemical entities A (a protecting group undergoing activation) and B (a compound of interest such as a drug, a reporter or another spacer) via covalent bonds. Under the effect of an external stimulus (activation), A is cleaved thus spontaneously initiating an irreversible process called self-immolation, which ultimately causes the release of the species B. The latter is usually inactive by binding on the spacer but, once released, it recovers its intrinsic activity. The kinetics of the self-immolation process controls the spatiotemporal correlation existing between activation and release of the active compound. Although many kinetic studies have been already reported on various series of self-immolative spacers, literature still lacked homogeneous information permitting comparative analysis of the kinetics of self-immolation. The major aim of this work was precisely to provide an extensive set of data drawing reliable structure property relationships in a series of self-immolative spacers containing an aromatic structure bearing a hydroxyl substituent and relying on a mechanism involving an electronic cascade. Thus we used a photactivable group and light to trigger the disassembly process of a large collection of self-immolative spacers, which exhibit self-immolation time varying over a wide range of time scale (10-4-104s). During this work, we also developed self-immolative spacers, which could release two compounds after a single activation. These systems have been implemented to address the quantitative analysis of a substrate released by uncaging in biological systems.
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Photochemistry of Masked Pyrene-4,5-DioneKarabaeva, Kanykey E. 23 July 2013 (has links)
No description available.
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An Experimental Study of Power Losses of Full-complement Needle Bearings of Planetary Gear SetsStilwell, Alex William 22 June 2012 (has links)
No description available.
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Synthesis of unnatural amino acids for genetic encoding by the pyrrolysyl-tRNA/RNA synthetase systemKnight, William A 01 January 2015 (has links)
The complexity of all biomolecules in existence today can be attributed to the variation of the amino acid repertoire. In nature, 20 canonical amino acids are translated to form these biomolecules, however, many of these amino acids have revealed posttranslational modifications (i.e. acetylation, methylation) after incorporation. Amino acids that exhibit PTM are known for their involvement in cellular processes such as DNA repair and DNA replication; these PTMs are commonly found on histones within the chromatin complex. Utilization of in vivo site-specific incorporation has recently reported functionality of post-translationally modified amino acids.1 xii Here we report the synthesis and in vivo site-specific incorporation of the histone PTM, 2-hydroxyisobutyrl lysine (Khib), with the pyrrolysyl tRNA/ RNA synthetase system. This translational machine can better serve to probe Khib for functional benefits. Additionally, this thesis focuses much of its attention on the development of unnatural amino acids (UAA) with optogenetic characteristics. These UAAs, if site-specifically incorporated, can be used to control enzymes and proteins through rapid light perturbation (365nm UV light). Furthermore, discussed is the synthesis of photo-caged threonine and photo-caged serine as potential substrates for the pyrrolysyl translational machinery.
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Synthesis and Analysis of Modified SNARE Proteins with Respect to Assembly and Disassembly of the SNARE ComplexJunius, Meike Pauline Wilhelmine 26 August 2016 (has links)
No description available.
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The choreography of protein vibrations : Improved methods of observing and simulating the infrared absorption of proteinsKarjalainen, Eeva-Liisa January 2011 (has links)
The work presented in this thesis has striven toward improving the capability to study proteins using infrared (IR) spectroscopy. This includes development of new and improved experimental and theoretical methods to selectively observe and simulate protein vibrations. A new experimental method of utilising adenylate kinase and apyrase as helper enzymes to alter the nucleotide composition and to perform isotope exchange in IR samples was developed. This method enhances the capability of IR spectroscopy by enabling increased duration of measurement time, making experiments more repeatable and allowing investigation of partial reactions and selected frequencies otherwise difficult to observe. The helper enzyme mediated isotope exchange allowed selective observation of the vibrations of the catalytically important phosphate group in a nucleotide dependent protein such as the sarcoplasmic reticulum Ca2+-ATPase. This important and representative member of P-type ATPases was further investigated in a different study, where a pathway for the protons countertransported in the Ca2+-ATPase reaction cycle was proposed based on theoretical considerations. The transport mechanism was suggested to involve separate pathways for the ions and the protons. Simulation of the IR amide I band of proteins enables and supports structure-spectra correlations. The characteristic stacking of beta-sheets observed in amyloid structures was shown to induce a band shift in IR spectra based on simulations of the amide I band. The challenge of simulating protein spectra in aqueous medium was also addressed in a novel approach where optimisation of simulated spectra of a large set of protein structures to their corresponding experimental spectra was performed. Thereby, parameters describing the most important effects on the amide I band for proteins could be determined. The protein spectra predicted using the optimised parameters were found to be well in agreement with experiment. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>
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