Contribution of Pannexin1 to Experimental Autoimmune Encephalomyelitis

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.

Time-resolved investigation of low-density plasma channels produced by a kilohertz femtosecond laser in air

By employing pump-probe back longitudinal diffractometry, the electron density and decay dynamics of a weak plasma channel created by a 1-KHz fs laser in air has been investigated. With ultrashort laser pulses of 50 fs and low energy of 0.6 mJ, we observe weak plasma channels with a length similar to 2 cm in air. An analytical reconstruction method of electron density has been analyzed, which is sensitive to the phase shift and channel size. The electron density in the weak plasma channel is extracted to be about 4x10(16) cm(-3). The diameters of the plasma channel and the filament are about 50 and 150 mu m, respectively, and the measurable electron density can be extended to less than 10(15) cm(-3). Moreover, a different time-frequency technique called linearly chirped longitudinal diffractometry is proposed to time-resolved investigate ultrafast ionization dynamics of laser-irradiated gas, laser interaction with cluster beam, etc.

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Chemical-scale studies of G protein-coupled receptors and ligand-gated ion channels

<p>This dissertation describes studies of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) using unnatural amino acid mutagenesis to gain high precision insights into the function of these important membrane proteins.</p> <p>Chapter 2 considers the functional role of highly conserved proline residues within the transmembrane helices of the D2 dopamine GPCR. Through mutagenesis employing unnatural ��-hydroxy acids, proline analogs, and N-methyl amino acids, we find that lack of backbone hydrogen bond donor ability is important to proline function. At one proline site we additionally find that a substituent on the proline backbone N is important to receptor function.</p> <p>In Chapter 3, side chain conformation is probed by mutagenesis of GPCRs and the muscle-type nAChR. Specific side chain rearrangements of highly conserved residues have been proposed to accompany activation of these receptors. These rearrangements were probed using conformationally-biased ��-substituted analogs of Trp and Phe and unnatural stereoisomers of Thr and Ile. We also modeled the conformational bias of the unnatural Trp and Phe analogs employed.</p> <p>Chapters 4 and 5 examine details of ligand binding to nAChRs. Chapter 4 describes a study investigating the importance of hydrogen bonds between ligands and the complementary face of muscle-type and ��4��4 nAChRs. A hydrogen bond involving the agonist appears to be important for ligand binding in the muscle-type receptor but not the ��4��4 receptor.</p> <p>Chapter 5 describes a study characterizing the binding of varenicline, an actively prescribed smoking cessation therapeutic, to the ��7 nAChR. Additionally, binding interactions to the complementary face of the ��7 binding site were examined for a small panel of agonists. We identified side chains important for binding large agonists such as varenicline, but dispensable for binding the small agonist ACh.</p> <p>Chapter 6 describes efforts to image nAChRs site-specifically modified with a fluorophore by unnatural amino acid mutagenesis. While progress was hampered by high levels of fluorescent background, improvements to sample preparation and alternative strategies for fluorophore incorporation are described.</p> <p>Chapter 7 describes efforts toward a fluorescence assay for G protein association with a GPCR, with the ultimate goal of probing key protein-protein interactions along the G protein/receptor interface. A wide range of fluorescent protein fusions were generated, expressed in Xenopus oocytes, and evaluated for their ability to associate with each other.</p>

Theory of ozone isotopic effects and various electron transfer reactions

<p>Three separate topics, each stimulated by experiments, are treated theoretically in this dessertation: isotopic effects of ozone, electron transfer at interfaces, and intramolecular directional electron transfer in a supramolecular system.</p> <p> The strange mass-independent isotope effect for the enrichment of ozone, which has been a puzzle in the literature for some 20 years, and the equally puzzling unconventional strong mass-dependent effect of individual reaction rate constants are studied as different aspects of a symmetry-driven behavior. A statistical (RRKM-based) theory with a hindered-rotor transition state is used. The individual rate constant ratios of recombination reactions at low pressures are calculated using the theory involving (1) small deviation from the statistical density of states for symmetric isotopomers, and (2) weak collisions for deactivation of the vibrationally excited ozone molecules. The weak collision and partitioning among exit channels play major roles in producing the large unconventional isotope effect in "unscrambled" systems. The enrichment studies reflect instead the non-statistical effect in "scrambled" systems. The theoretical results of low-pressure ozone enrichments and individual rate constant ratios obtained from these calculations are consistent with the corresponding experimental results. The isotopic exchange rate constant for the reaction ^(16)O + ^(18)O ^(18)O���+ ^(16)O ^(18)O + ^(18)O provides information on the nature of a variationally determined hindered-rotor transition state using experimental data at 130 K and 300 K. Pressure effects on the recombination rate constant, on the individual rate constant ratios and on the enrichments are also investigated. The theoretical results are consistent with the experimental data. The temperature dependence of the enrichment and rate constant ratios is also discussed, and experimental tests are suggested. The desirability of a more accurate potential energy surface for ozone in the transition state region is also noted.</p> <p>Electron transfer reactions at semiconductor /liquid interfaces are studied using a tight-binding model for the semiconductors. The slab method and a z-transform method are employed in obtaining the tight-binding electronic structures of semiconductors having surfaces. The maximum electron transfer rate constants at Si/viologen^(2-/+) and InP /Me_(2)Fc^(+/O) interfaces are computed using the tight-binding type calculations for the solid and the extended-Huckel for the coupling to the redox agent at the interface. These electron transfer reactions are also studied using a free electron model for the semiconductor and the redox molecule, where Bardeen's method is adapted to calculate the coupling matrix element between the molecular and semiconductor electronic states. The calculated results for maximum rate constant of the electron transfer from the semiconductor bulk states are compared with the experimentally measured values of Lewis and coworkers, and are in reasonable agreement, without adjusting parameters. In the case of InP /liquid interface, the unusual current vs applied potential behavior is additionally interpreted, in part, by the presence of surface states.</p> <p>Photoinduced electron transfer reactions in small supramolecular systems, such as 4-aminonaphthalimide compounds, are interesting in that there are, in principle, two alternative pathways (directions) for the electron transfer. The electron transfer, however, is unidirectional, as deduced from pH-dependent fluorescence quenching studies on different compounds. The role of electronic coupling matrix element and the charges in protonation are considered to explain the directionality of the electron transfer and other various results. A related mechanism is proposed to interpret the fluorescence behavior of similar molecules as fluorescent sensors of metal ions.</p>

Rela����o dos sistemas de reparo de DNA em Escherichia coli K-12 com resist��ncia a antibi��ticos, caracter��sticas adesivas e forma����o de biofilme

As esp��cies reativas de oxig��nio (ERO) s��o geradas durante o metabolismo celular normal e podem produzir v��rios danos oxidativos no DNA, tais como les��es nas bases nitrogenadas ou s��tios apur��nico/apirimid��nico (AP). Essas les��es podem acarretar ac��mulo de s��tios de muta����es, caso esses danos n��o sejam reparados. Entretanto, as bact��rias possuem v��rios mecanismos de defesa contra as ERO que desempenham um importante papel na manuten����o da fisiologia. O objetivo deste trabalho foi o de avaliar se sistemas enzim��ticos, como o reparo por excis��o de bases (BER), sistema SOS e SoxRS, interferem em respostas como a sensibilidade aos antibi��ticos, ader��ncia das c��lulas bacterianas a superf��cies bi��ticas ou abi��ticas e forma����o de biofilme. Os mutantes utilizados no presente estudo s��o todos derivados de Escherichia coli K-12 e os resultados obtidos mostraram que, dos mutantes BER testados, o ��nico que apresentou diferen��a no perfil de sensibilidade aos antimicrobiamos em rela����o �� cepa selvagem (AB1157) foi o mutante xthA- (BW9091), deficiente em exonuclease III. No teste de ader��ncia qualitativo realizado com linhagem de c��lulas HEp-2 (origin��ria de carcinoma de laringe humana) foi observado que onze cepas da nossa cole����o, apresentaram um padr��o denominando like-AA, contrastando com o que era esperado para as cepas de E. coli utilizadas como controle negativo, que apresentam ader��ncia discreta sem padr��o t��pico. A ader��ncia manose-sens��vel via f��mbria do tipo I avaliada nesse estudo mostrou que essa fimbria, possui um papel relevante na intensidade da ader��ncia e filamenta����o nessas cepas estudas. A filamenta����o �� uma resposta SOS importante para que o genoma seja reparado antes de ser partilhado pelas c��lulas filhas. Al��m disso, com rela����o �� forma����o de biofilme, oito cepas apresentaram um biofilme forte sendo que essa resposta n��o foi acompanhada pelo aumento da intensidade de filamenta����o. Nossos resultados em conjunto sugerem o envolvimento de estresse oxidativo na defini����o de par��metros como sensibilidade a antimicrobianos, padr��o e intensidade de ader��ncia, filamenta����o e forma����o de biofilme nas amostras de E. coli K-12 avaliadas neste trabalho. Sugerimos que a ader��ncia gera estresse oxidativo causando danos no DNA, o que leva a indu����o do sistema SOS resultando na resposta de filamenta����o observada.

Controlling the group velocity in a five-level K-type atomic system

The properties of a five-level K-type system are investigated. With the controlling fields, the properties of the dispersion and absorption of the system are changed greatly. The system can produce anomalous dispersion regions with absorption and normal dispersion regions with absorption or transparency. Furthermore, the group velocity can be varied from subluminal to superluminal by varying the intensity of the controlling field and the probe detunings in principle. (C) 2008 Elsevier B.V. All rights reserved.