Multimode laser emission from dye doped polymer optical fiber

Multimode laser emission is observed in a polymer optical fiber doped with a mixture of Rhodamine 6G (Rh 6G) and Rhodamine B (Rh B) dyes. Tuning of laser emission is achieved by using the mixture of dyes due to the energy transfer occurring from donor molecule (Rh 6G) to acceptor molecule (Rh B). The dye doped poly(methyl methacrylate)-based polymer optical fiber is pumped axially at one end of the fiber using a 532 nm pulsed laser beam from a Nd:YAG laser and the fluorescence emission is collected from the other end. At low pump energy levels, fluorescence emission is observed. When the energy is increased beyond a threshold value, laser emission occurs with a multimode structure. The optical feedback for the gain medium is provided by the cylindrical surface of the optical fiber, which acts as a cavity. This fact is confirmed by the mode spacing dependence on the diameter of the fiber.

Development of Electrochemical and Fluorescent Sensors

Chemical sensors have growing interest in the determination of food additives, which are creating toxicity and may cause serious health concern, drugs and metal ions. A chemical sensor can be defined as a device that transforms chemical information, ranging from the concentration of a specific sample component to total composition analysis, into an analytically useful signal. The chemical information may be generated from a chemical reaction of the analyte or from a physical property of the system investigated. Two main steps involved in the functioning of a chemical sensor are recognition and transduction. Chemical sensors employ specific transduction techniques to yield analyte information. The most widely used techniques employed in chemical sensors are optical absorption, luminescence, redox potential etc. According to the operating principle of the transducer, chemical sensors may be classified as electrochemical sensors, optical sensors, mass sensitive sensors, heat sensitive sensors etc. Electrochemical sensors are devices that transform the effect of the electrochemical interaction between analyte and electrode into a useful signal. They are very widespread as they use simple instrumentation, very good sensitivity with wide linear concentration ranges, rapid analysis time and simultaneous determination of several analytes. These include voltammetric, potentiometric and amperometric sensors. Fluorescence sensing of chemical and biochemical analytes is an active area of research. Any phenomenon that results in a change of fluorescence intensity, anisotropy or lifetime can be used for sensing. The fluorophores are mixed with the analyte solution and excited at its corresponding wavelength. The change in fluorescence intensity (enhancement or quenching) is directly related to the concentration of the analyte. Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular rearrangements, energy transfer, ground-state complex formation and collisional quenching. Generally, fluorescence quenching can occur by two different mechanisms, dynamic quenching and static quenching. The thesis presents the development of voltammetric and fluorescent sensors for the analysis of pharmaceuticals, food additives metal ions. The developed sensors were successfully applied for the determination of analytes in real samples. Chemical sensors have multidisciplinary applications. The development and application of voltammetric and optical sensors continue to be an exciting and expanding area of research in analytical chemistry. The synthesis of biocompatible fluorophores and their use in clinical analysis, and the development of disposable sensors for clinical analysis is still a challenging task. The ability to make sensitive and selective measurements and the requirement of less expensive equipment make electrochemical and fluorescence based sensors attractive.

Facilitating folding of outer membrane proteins, roles of the periplasmic chaperone Skp and the outer membrane lipoprotein BamD

This thesis describes several important advancements in the understanding of the assembly of outer membrane proteins of Gram-negative bacteria like Escherichia coli. A first study was performed to identify binding regions in the trimeric chaperone Skp for outer membrane proteins. Skp is known to facilitate the passage of unfolded outer membrane proteins (OMPs) through the periplasm to the outer membrane (OM). A gene construct named “synthetic chaperone protein (scp)” gene was used to express a fusion protein (Scp) into the cytoplasm of E. coli. The scp gene was used as a template to design mutants of Scp suitable for structural and functional studies using site-directed spectroscopy. Fluorescence resonance energy transfer (FRET) was used to identify distances in Skp-OmpA complexes that separate regions in Scp and in outer membrane protein A (OmpA) from E. coli. For this study, single cysteine (Cys) mutants and single Cys - single tryptophan (Trp) double mutants of Scp were prepared. For FRET experiments, the cysteines were labeled with the tryptophan fluorescence energy acceptor IAEDANS. Single Trp mutants of OmpA were used as fluorescence energy donors. In the second part of this thesis, the function of BamD and the structure of BamD-Scp complexes were examined. BamD is an essential component of the β-barrel assembly machinery (BAM) complex of the OM of Gram-negative bacteria. Fluorescence spectroscopy was used to probe the interactions of BamD with lipid membranes and to investigate the interactions of BamD with possible partner proteins from the periplasm and from the OM. A range of single cysteine (Cys) and single tryptophan (Trp) mutants of BamD were prepared. A very important conclusion from the extensive FRET study is that the essential lipoprotein BamD interacts and binds to the periplasmic chaperone Skp. BamD contains tetratrico peptide repeat (TPR) motifs that are suggested to serve as docking sites for periplasmic chaperones such as Skp.

Análisis del gen adra2a receptor alfa 2a adrenergico en pacientes con trastorno de hiperactividad y déficit de atención

El trastorno de hiperactividad y déficit de atención (THDA), es definido clínicamente como una alteración en el comportamiento, caracterizada por inatención, hiperactividad e impulsividad. Estos aspectos son clasificados en tres subtipos, que son: Inatento, hiperactivo impulsivo y mixto. Clínicamente se describe un espectro amplio que incluye desordenes académicos, trastornos de aprendizaje, déficit cognitivo, trastornos de conducta, personalidad antisocial, pobres relaciones interpersonales y aumento de la ansiedad, que pueden continuar hasta la adultez. A nivel global se ha estimado una prevalencia entre el 1% y el 22%, con amplias variaciones, dadas por la edad, procedencia y características sociales. En Colombia, se han realizado estudios en Bogotá y Antioquia, que han permitido establecer una prevalencia del 5% y 15%, respectivamente. La causa específica no ha sido totalmente esclarecida, sin embargo se ha calculado una heredabilidad cercana al 80% en algunas poblaciones, demostrando el papel fundamental de la genética en la etiología de la enfermedad. Los factores genéticos involucrados se relacionan con cambios neuroquímicos de los sistemas dopaminérgicos, serotoninérgicos y noradrenérgicos, particularmente en los sistemas frontales subcorticales, corteza cerebral prefrontal, en las regiones ventral, medial, dorsolateral y la porción anterior del cíngulo. Basados en los datos de estudios previos que sugieren una herencia poligénica multifactorial, se han realizado esfuerzos continuos en la búsqueda de genes candidatos, a través de diferentes estrategias. Particularmente los receptores Alfa 2 adrenérgicos, se encuentran en la corteza cerebral, cumpliendo funciones de asociación, memoria y es el sitio de acción de fármacos utilizados comúnmente en el tratamiento de este trastorno, siendo esta la principal evidencia de la asociación de este receptor con el desarrollo del THDA. Hasta la fecha se han descrito más de 80 polimorfismos en el gen (ADRA2A), algunos de los cuales se han asociado con la entidad. Sin embargo, los resultados son controversiales y varían según la metodología diagnóstica empleada y la población estudiada, antecedentes y comorbilidades. Este trabajo pretende establecer si las variaciones en la secuencia codificante del gen ADRA2A, podrían relacionarse con el fenotipo del Trastorno de Hiperactividad y el Déficit de Atención.

Domain swapping in the human histamine H-1 receptor

G-protein-coupled receptors (GPCRs) represent the largest family of receptors involved in transmembrane signaling. Although these receptors were generally believed to be monomeric entities, accumulating evidence supports the presence of GPCRs in multimeric forms. Here, using immunoprecipitation as well as time-resolved fluorescence resonance energy transfer to assess protein-protein interactions in living cells, we unambiguously demonstrate the occurrence of dimerization of the human histamine H-1 receptor. We also show the presence of domain-swapped H-1 receptor dimers in which there is the reciprocal exchange of transmembrane domain TM domains 6 and 7 between the receptors present in the dimer. Mutation of aspartate(107) in transmembrane (TM) 3 or phenylalanine(432) in TM6 to alanine results in two radioligand-binding-deficient mutant H-1 receptors. Coexpression of H-1 D(107)A and H-1 F(432)A, however, results in a reconstituted radioligand binding site that exhibits a pharmacological profile that corresponds to the wildtype H-1 receptor. Interestingly, the H-1 receptor radioligands [H-3] mepyramine and [H-3]-(-)- trans-1-phenyl-3-N, N-dimethylamino-1,2,3,4-tetrahydronaphthalene show differential saturation binding values (B-max) for wild-type H-1 receptors but not for the radioligand binding site that is formed upon coexpression of H-1 D(107)A and H-1 F(432)A receptors, suggesting the presence of different H-1 receptor populations.

Dual role of the beta(2)-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization

Homologous desensitization of beta(2)-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta(2)-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta(2)-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.

beta-arrestin binding to the beta(2)-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta(2)-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

The troposphere-to-stratosphere transition in kinetic energy spectra and nonlinear spectral fluxes as seen in ECMWF analyses

Global horizontal wavenumber kinetic energy spectra and spectral fluxes of rotational kinetic energy and enstrophy are computed for a range of vertical levels using a T799 ECMWF operational analysis. Above 250 hPa, the kinetic energy spectra exhibit a distinct break between steep and shallow spectral ranges, reminiscent of dual power-law spectra seen in aircraft data and high-resolution general circulation models. The break separates a large-scale ‘‘balanced’’ regime in which rotational flow strongly dominates divergent flow and a mesoscale ‘‘unbalanced’’ regime where divergent energy is comparable to or larger than rotational energy. Between 230 and 100 hPa, the spectral break shifts to larger scales (from n 5 60 to n 5 20, where n is spherical harmonic index) as the balanced component of the flow preferentially decays. The location of the break remains fairly stable throughout the stratosphere. The spectral break in the analysis occurs at somewhat larger scales than the break seen in aircraft data. Nonlinear spectral fluxes defined for the rotational component of the flow maximize between about 300 and 200 hPa. Large-scale turbulence thus centers on the extratropical tropopause region, within which there are two distinct mechanisms of upscale energy transfer: eddy–eddy interactions sourcing the transient energy peak in synoptic scales, and zonal mean–eddy interactions forcing the zonal flow. A well-defined downscale enstrophy flux is clearly evident at these altitudes. In the stratosphere, the transient energy peak moves to planetary scales and zonal mean–eddy interactions become dominant.

Dehydrodipeptide hydrogelators containing naproxen N‑Capped tryptophan: self-assembly, hydrogel characterization, and evaluation as potential drug nanocarriers

In this work, we introduce dipeptides containing tryptophan N-capped with the nonsteroidal anti-inflammatory drug naproxen and C-terminal dehydroamino acids, dehydrophenylalanine (ΔPhe), dehydroaminobutyric acid (ΔAbu), and dehydroalanine (ΔAla) as efficacious protease resistant hydrogelators. Optimized conditions for gel formation are reported. Transmission electron microscopy experiments revealed that the hydrogels consist of networks of micro/nanosized fibers formed by peptide self-assembly. Fluorescence and circular dichroism spectroscopy indicate that the self-assembly process is driven by stacking interactions of the aromatic groups. The naphthalene groups of the naproxen moieties are highly organized in the fibers through chiral stacking. Rheological experiments demonstrated that the most hydrophobic peptide (containing C-terminal ΔPhe) formed more elastic gels at lower critical gelation concentrations. This gel revealed irreversible breakup, while the C-terminal ΔAbu and ΔAla gels, although less elastic, exhibited structural recovery and partial healing of the elastic properties. A potential antitumor thieno[3,2-b]pyridine derivative was incorporated (noncovalently) into the gel formed by the hydrogelator containing C-terminal ΔPhe residue. Fluorescence and Förster resonance energy transfer measurements indicate that the drug is located in a hydrophobic environment, near/associated with the peptide fibers, establishing this type of hydrogel as a good drug-nanocarrier candidate.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

De-aggregation of polyfluorene derivative by blending with a series of poly(alkyl methacrylate)s with varying sidegroup sizes

A polyfluorene derivative of the PPV, poly(9,9`-n-dihexyl-2,7-fluorenedilvinylene-alt-1,4-phenylenevinylene), with a strong tendency to aggregation was blended with several members of a series composed by poly(alkyl methacrylate)s with the following substituents in the ester position: methyl, ethyl, isopropyl, isobutyl, n-butyl, and cyclohexyl. The de-aggregation effect in blends was studied by steady-state photoluminescence spectroscopy using several blend compositions. The efficiency of each dispersing phase was discussed in terms of the polymer miscibility, controlled by interaction parameters between the polyfluorene and every poly(alkyl methacrylate)s, here described using Hilde-brand solubility parameters. (c) 2009 Elsevier Ltd. All rights reserved.

Mapping the Intramolecular Vibrational Energy Flow in Proteins Reveals Functionally Important Residues

Unveiling the mechanisms of energy relaxation in biomolecules is key to our understanding of protein stability, allostery, intramolecular signaling, and long-lasting quantum coherence phenomena at ambient temperatures. Yet, the relationship between the pathways of energy transfer and the functional role of the residues involved remains largely unknown. Here, we develop a simulation method of mapping out residues that are highly efficient in relaxing an initially localized excess vibrational energy and perform site-directed mutagenesis functional assays to assess the relevance of these residues to protein function. We use the ligand binding domains of thyroid hormone receptor (TR) subtypes as a test case and find that conserved arginines, which are critical to TR transactivation function, are the most effective heat diffusers across the protein structure. These results suggest a hitherto unsuspected connection between a residue`s ability to mediate intramolecular vibrational energy redistribution and its functional relevance.

Study on the observation of Eu(2+) and Eu(3+) valence states in low silica calcium aluminosilicate glasses

The optical, magnetic and structural properties of Eu doped low silica calcium aluminosilicate glasses were investigated. The optical absorption coefficient presented two bands at 39 246 and 29 416 cm(-1), which were assigned respectively to the 4f(7) ((8)S(7/2)) -> 4f(6) (4F(J)) 5d (T(2g)), and 4f(7) ((8)S(7/2)) -> 4f(6) (4F(J)) 5d (E(g)) transitions of Eu(2+). The fluorescence measured at 300 K on a sample doped with 0.5 wt% of Eu(2)O(3) exhibited a broad band centered at 17 350 cm(-1), which is attributed to the 4f(6)5d -> 4f(7) transition of Eu(2+), whereas the additional peaks are due to the (5)D(0) -> (7)F(J) (J = 1, 2, 4) transitions of Eu(3+). From magnetization and XANES data it was possible to evaluate the fractions of Eu(2+) and Eu(3+) for the sample doped with 0.5 and 5.0 wt% of Eu(2)O(3), the values of which were approximately 30 and 70%, respectively.

Covalent attachment of 3,4,9,10-perylenediimides onto the walls of mesoporous molecular sieves MCM-41 and SBA-15

This work describes the covalent grafting of 3,4,9,10-perylenediimides (PDI), which are fluorescent dyes with very interesting optical properties, onto the walls of mesoporous molecular sieves MCM-41 and SBA-15. The mesoporous materials were first treated with 3-aminopropyltriethoxysilane (APTES) in anhydrous toluene, generating amine-containing surfaces. The amine-containing materials were then reacted with 3,4,9,10-perylenetetracarboxylic dianhydride (PTCA), generating surface-grafted PDI. Infrared spectra of the materials showed that the reaction with amino groups took place at both anhydride ends of the PTCA molecule, resulting in surface attached diimides. No sign of unreacted anhydride groups were found. The new materials, designated as MCMN2PDI and SBAN(2)PDI, presented absorption and emission spectra corresponding to weakly coupled PDI chromophores, in contrast to the strongly coupled rings usually found in solid PDI samples. The materials showed a red fluorescence, which could be observed by the naked eye under UV irradiation or with a fluorescence microscope. The PDI-modified mesoporous materials showed electrical conductivity when pressed into a pellet. The results presented here show that the new materials are potentially useful in the design of nanowires. (C) 2007 Elsevier Inc. All rights reserved.