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The Development of Fluorescent Probes and Slow-Releasing H2S Donors for Studying Biological H2SHammers, Matthew 27 October 2016 (has links)
Hydrogen sulfide (H2S) is an essential small molecule in human physiology. Although quite toxic, H2S is produced endogenously and performs important regulatory functions in the cardiovascular, immune, nervous, and respiratory systems. Varied interactions with intracellular thiols, reactive oxidants, and protein transition-metal centers are highly dynamic and sensitive to fluctuations in redox homeostasis. Furthermore, H2S is implicated in a number of diseases such as cancer, neurodegeneration, and heart disease. Hence, exogenously delivered H2S as a therapeutic agent is an active area of intrigue and research. The complexity and interconnectivity of these processes has stimulated the development of advanced chemical tools with which to study biological H2S, including reaction-based fluorescent probes and slow-releasing H2S donors.
Toward these goals, I present several significant advances in the fields of H2S detection and delivery. An azide reduction-based probe, MeRho-Az, provides a rapid >1,000-fold fluorescence response when treated with H2S. MeRho-Az is sufficiently sensitive to detect endogenous H2S in C6 cells and was used to image H2S in live zebrafish larvae using light sheet fluorescence microscopy, representing the first analyte-responsive experiments with this imaging technology. Using a ratiometric dual-fluorophore fragmentation strategy, NBD-Coum simultaneously detects, differentiates, and measures relative concentration ratios of H2S versus cysteine/homocysteine, two important metabolites in H2S biosynthesis. NBD-Coum was used to monitor changes in redox homeostasis in a simulated sulfur pool and is useful for studying H2S-thiol dynamics. The synthesis and amide-coupling conditions of ADT-NH2, a highly sought dithiolethione H2S donor, allow for hydrolytically stable, H2S-releasing non-steroidal anti-inflammatory drug hybrids. Finally, inspired by polysulfide-containing natural products, functionalized tetrasulfides are a new class of accessible, customizable, and versatile H2S donors with controllable H2S release rates. I hope that by using these investigative tools, chemists and biologists are able to refine our understanding of physiological H2S and exploit H2S activities in disease treatments.
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Development of fluorescent chemosensors based on different signal transduction mechanismsYou, Qihua 09 July 2014 (has links)
A series of fluorescent probes based on different signal transduction mechanisms for the detection of Fe3+, Zn2+, histidine and pH was designed and synthesized. Their photophysical properties, binding abilities and the further application in cell imaging were fully evaluated. Building on the groundwork of our previous study, molecular scaffold 19 has been appended to spirobenzopyran fluorophore to furnish a highly selective and sensitive Zn2+ sensor. To broaden the application scope of this trifunctional receptive molecule, 19 was incorporated onto rhodamine, antipyrine and coumarin moieties to give 20, 21 and 23, respectively. Probe 20 operative on a chelation-enhanced fluorescence mechanism exhibited highly selective response to Fe3+ with 2:1 stoichiometry of 20-Fe3+ complex. However, a possible tendency of probe 20 to hydrolyze induced by Fe3+ and the unsuccessful attempt of cell imaging would limit its application scope. Probe 21 with O-N-N-N-N-ligand showed a highly selective and sensitive detection of Zn2+. The probe displayed suppressed response to Cd2+ which is the most common interference ion in zinc metal detection. The binding of Zn2+ to probe 21 inhibited the photoinduced electron transfer process originating from the lone pair of the nitrogen atom in the antipyrine moiety to quinoline fluorophore. Therefore, a turn-on fluorescent probe was developed. A moderate binding constant with 1:1 stoichiometry of 21-Zn2+ complex was established by fluorescence titration. The binding mechanism was fully explained by 1H NMR titration. To our delight, probe 21 was successfully applied for recognizing Zn2+ in living cells. The preparation of probe 23 was achieved by appendage of 19 to coumarin derived fluorophore and the probe exhibited a good selectivity and fluorescent turn-off property to Cu2+. The 1:1 stoichiometry of 23-Cu2+ ensemble can serve as an efficient probe for the detection of histidine and biothiols. In the presence of NEM, the influence of biothiols could be eliminated. Furthermore, this sensing ensemble was also used in the detection of histidine in hard-to-transfect U87MG cells with very low cytotoxicity. Based on our group’s previous work on the spiropyran platform, a novel ratiometric near-infrared pH probe 27 operating on an excited-state intramolecular electron transfer mechanism was developed. The pKa was calculated to be 5.9 and the ring-opening/ring-closing mechanism triggered by protons was reasonably explained by 1H NMR titration. However, this spiropyran-based probe was found to be unsuitable for cell imaging. To continue the innovation of pH sensing and extend its application in bioimaging, a series of ratiometric pH probes 32 and 38 characterized by their high quantum yield working in the NIR range was developed. The appendage of N,O-disubstituted hemiaminal ether moiety onto coumarin fluorophore with C=C double bond conferred the sensory material with the ability to display a pH-dependent ratiometric output operating on the ring-opening/ring-closing mechanism. The pKa of 32 and 38 were 6.9 and 5.8 – 6.0, respectively, which rendered them suitable for pH measurement in near-neutral and acidic media. A preliminary work of intracellular pH measurement was also conducted and promising results were obtained
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Development of fluorescent chemosensors : mercury sensing and biological molecules sensing probesWang, Hao 01 January 2008 (has links)
No description available.
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Fluorescent chemosensor development based on multifunctional spirobenzopyransZhu, Jianfa 01 January 2011 (has links)
No description available.
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Development and analysis of recombinant fluorescent probes for use in live cell imaging of filamentous fungiAltenbach, Kirsten January 2010 (has links)
The molecular cloning and subsequent engineering of the green fluorescent protein (GFP) of the jellyfish Aequoria victoria allowed a novel approach to the investigation of cell signalling. GFP and its mutants can now not only be used to target specific organelles in living cells but also function as a basis for a variety of sensors for biologically important ions and molecular interactions. GFP-based Ca2+- sensors have been successfully used for studies in mammalian and plant cells. In filamentous fungi, however, they have not yet been reported to work. Since only little is known about calcium signalling in filamentous fungi, this project aimed to improve existing GFP-based Ca2+- sensors by exchanging the original fluorophores for improved versions and expressing those in the filamentous fungus Aspergillus niger. During this project, the donor and acceptor fluorophores of 3 existing Ca2+-FRETprobes based on cameleons and troponin C-sensors, have been changed, 2 novel positive FRET controls have been designed and these , as well as donor and acceptor fluorophores alone, have been expressed in the filamentous fungus Aspergillus niger. The probes were assessed using different imaging techniques, such as conventional confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM) and spectral imaging using a Leica TSC SP5 confocal and IRIS, a novel spectral imaging device designed at Heriot Watt University. Problems were encountered that prevented FRET analysis using CLSM and IRIS. These were due mainly to the difference in expression level of the constructs and the distribution of the emission bandpasses of the IRIS system. Analysis of the spectral data obtained on the Leica confocal system and analysis of the FLIM results, however, revealed significant differences between the donor only and the positive FRET controls. Spectra of the positive FRET controls and the Ca2+-sensitive probes showed emission peaks of both the donor and the acceptor fluorophores upon excitation of the donor fluorophore alone while analysis of the FLIM results revealed an additional decay component in the positive FRET controls. Both results are very strong indicators that we can detect FRET in living hyphae of Aspergillus niger transformed with the probes designed during this project.
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Synthesis and applications of trifluoromethyl aryldiazirine photophoreValles-Miret, Mariona January 2011 (has links)
Photoreactive groups have been used in photoaffinity labelling of chemical macromolecules via the generation of highly reactive species upon short wave light irradiation. One of the most efficient photoreactive functional groups is trifluoromethyl aryldiazirine (TFMAD). This compound was synthesised as part of the work discussed in this thesis, making use of microwave irradiation to shorten reaction times (Chapter I). An investigation of properties allowed the development of three different applications for conjugation to biomolecules. The first application consisted of the development of an approach for generation of small-molecule microarrays, where a 2,000 compound library was immobilised onto the glass surface through carbene insertion. The microarray was then used to screen for potential binders to beta-transducin repeat containing protein (b-TrCP1) allowing the reduction of possible candidates to less than 25 compounds (Chapter II). The second application was the synthesis of two probes to allow the selective delivery of active compounds inside specific organelles or cells. The diazirine moiety was used as a rapid way to covalently capture a number of cargos. The approach allowed a peptoid and an anticancer drug to be conjugated to the two probes and their cell penetrability properties and therapeutic effect were studied, respectively (Chapter III). Finally, the insertion properties of TFMAD were used to develop approaches to attach DNA onto microspheres and the efficiency of this delivery system was evaluated (Chapter IV).
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Micro-particles as cellular delivery devicesAlexander, Lois Meryl January 2009 (has links)
Narrowly dispersed amino-functionalised polystyrene microspheres, with a range of diameters, were successfully synthesised via emulsion and dispersion polymerisation. Fluorescent labelling allowed cellular translocation to be assessed in a variety of cell lines and was found to be very high, but controllable, whilst exhibiting no detrimental effect on cellular viability. In order to fully determine the mode of microsphere uptake, “beadfected” melanoma (B16F10) cells were studied using both chemical and microscopic methods. Uptake was found to be wholly unreliant upon energetic processes, with microspheres located cytoplasmically and not encapsulated within endosomes, an important characteristic for delivery devices. In order to demonstrate the effective delivery of exogenous cargo mediated by microspheres, short interfering (si)-RNAs were conjugated to beads and investigated for the gene silencing of enhanced green fluorescent protein (EGFP) in cervical cancer (HeLa) and embryonic (E14) stem cells. EGFP knockdown was found to be highly efficient after 48 – 72 hours. Dual-functionalised microspheres displaying a fluorophore (Cy5) and siRNA allowed only those cells beadfected with the delivery vehicle (and thus containing siRNA) to be assessed for EGFP expression, yielding an accurate assessment of microsphere-mediated gene silencing. In addition, by manipulation of the microsphere preparation conditions, micro-doughnuts and paramagnetic microspheres were produced and their cellular uptake assessed. Paramagnetic microspheres were found to enter cells efficiently and were subsequently used to bias the movement of beadfected cells in response to an externally applied magnet, while micro-doughnuts were found to exhibit cell selective properties and were noted to traffic specifically to the liver in vivo.
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Development of Fluorescent Probes for Imaging Synaptic Activity at Individual Presynaptic TerminalsMerchant, Paolomi January 2014 (has links)
This thesis describes the design, synthesis and development of fluorescent probes to monitor synaptic transmission at individual presynaptic terminals in the mouse brain. Two distinct approaches to accomplish this are discussed. The first approach seeks to monitor synaptic activity by using pH-sensitive endocytic membrane probes to label active presynaptic terminals. The second approach seeks to monitor synaptic activity by loading small fluorescent molecules into presynaptic vesicles and studying their evoked release upon stimulation.
The first chapter of this thesis describes currently available techniques that are used to study synaptic transmission in the brain. The use of electrochemical techniques is discussed and the use of fluorescent reporters is introduced as a means to image single synapses with high resolution.
Chapter II of this thesis describes the rational design of pH-sensitive membrane probes for labeling recycling vesicles. The synthesis, photophysical properties and biological characterization of these probes are described. Although these probes proved to be too lipophilic to work well in the brain tissue and neuronal culture, their use on the cell surface is demonstrated. Furthermore, the structure activity relationship established by this library of probes can be used to direct the future development of pH-sensing endocytic dyes.
Chapter III and IV of this thesis describe the development of new generations of Fluorescent False Neurotransmitters (FFNs) for imaging vesicular content release from individual presynaptic terminals in the brain. Chapter III introduces a novel imaging agent, FFN200, for monitoring and quantifying dopamine release from individual synaptic terminals in the mouse brain. Chapter IV describes the exploration and screening of small fluorescent molecules in the mouse brain for the purpose of developing FFNs at synaptic terminals other than dopamine. FFN7122 is introduced as the first FFN to be developed for terminals outside of dopamine. FFN7122 is shown to be a marker for glutamatergic terminals in the hippocampus, dorsal striatum, and motor cortex of the mouse brain. The evoked release of this probe from presynaptic vesicles is demonstrated and two hypotheses for its uptake mechanism are proposed.
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Localização e dinâmica de sondas fluorescentes em modelos de membranas: estudos por dinâmica molecular e anisotropia de fluorescência resolvida no tempo / Location and dynamics of fluorescent probes in model membranes: study by Molecular Dynamics and Time-resolved Fluorescence Anisotropy.Preza, Sérgio Leandro Espindola 27 August 2013 (has links)
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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Síntese de seleno- e teluro-cumarinas para estudos de emissão e supressão de fluorescência e aplicações analíticas e/ou biológicas / Synthesis of selenium- and tellurium-coumarins for fluorescence emission and supression studies and analytical and/or biological applicationsCavalcante, Victor Fernandes 17 July 2017 (has links)
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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