Studies of the serotonin type 3A receptor and the chemical preparation of tRNA

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT₃AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.

In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT₃A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-π interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT₃AR, which establishes a binding orientation for these drugs.

In Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT₃A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-π interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.

In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes.

Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, K_B, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT₃A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.

Group velocity of the light pulse in an open V-type system

We investigate the group velocity of the probe light pulse in an open V-type system with spontaneously generated coherence. We find that, not only varying the relative phase between the probe and driving pulses can but varying the atomic exit rate or incoherent pumping rate also can manipulate dramatically the group velocity, even make the pulse propagation switching from subluminal to superluminal; the subliminal propagation can be companied with gain or absorption, but the superluminal propagation is always companied with absorption. (c) 2006 Elsevier GmbH. All rights reserved.

Electromagnetically induced negative refractive index in a V-type four-level atomic system

We propose a scheme for realizing negative refractive index in a V-type four-level atomic system. It is shown that the negative refractive index can be achieved in a wide frequency band based on the effect of quantum coherence. It is also found that the frequency band of negative refractive index and the absorption property of left-handed material are manipulated by the pump and control fields. Furthermore, left-handed material with reduced absorption is possible by choosing appropriate parameters. (c) 2006 Elsevier B.V. All rights reserved.

Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors

This dissertation primarily describes studies of serotonin type 3 (5-HT₃) receptors of the Cys-loop super-family of ligand gated ion channels. The first chapter provides a general introduction to these important proteins and the methods used to interrogate their structure and function. The second chapter details the delineation of a structural unit of the ligand binding site of homomeric 5-HT₃A receptors on which the ligands serotonin (5-HT) and m-chlorophenyl biguanide (mCPBG) are reliant for effective receptor activation. Unnatural amino acid mutagenesis results show that the details of each ligand’s interaction with this organizing feature of the binding site differ, providing insights into general principles of receptor activation.

The third chapter describes a study in which florescent protein fusions of the A and B subunits of the heteromeric 5-HT₃AB receptor are employed to determine the subunit stoichiometry and order within functional receptors. Strong evidence is found for an A₃B₂ stoichiometry with A-A-B-A-B order. The fourth chapter investigates the potential for ligand binding across heteromeric binding sites in the 5-HT₃AB receptor. Unlike serotonin, mCPBG is found to bind the receptor at heteromeric binding sites. Further mCPBG is capable of allosterically modulating the response of serotonin on the 5-HT₃AB receptor from these heteromeric sites.

Finally, the fifth chapter describes progress towards the application of unnatural amino acid mutagenesis to an important new class of proteins, transcription factors. Experiments optimizing novel methods for the detection of function are described, using RARα of the nuclear receptor family of transcription factors.

Radio frequency studies of surface resistance and critical magnetic field of type I and type II superconductors

The surface resistance and the critical magnetic field of lead electroplated on copper were studied at 205 MHz in a half-wave coaxial resonator. The observed surface resistance at a low field level below 4.2°K could be well described by the BCS surface resistance with the addition of a temperature independent residual resistance. The available experimental data suggest that the major fraction of the residual resistance in the present experiment was due to the presence of an oxide layer on the surface. At higher magnetic field levels the surface resistance was found to be enhanced due to surface imperfections.

The attainable rf critical magnetic field between 2.2°K and T_c of lead was found to be limited not by the thermodynamic critical field but rather by the superheating field predicted by the one-dimensional Ginzburg-Landau theory. The observed rf critical field was very close to the expected superheating field, particularly in the higher reduced temperature range, but showed somewhat stronger temperature dependence than the expected superheating field in the lower reduced temperature range.

The rf critical magnetic field was also studied at 90 MHz for pure tin and indium, and for a series of SnIn and InBi alloys spanning both type I and type II superconductivity. The samples were spherical with typical diameters of 1-2 mm and a helical resonator was used to generate the rf magnetic field in the measurement. The results of pure samples of tin and indium showed that a vortex-like nucleation of the normal phase was responsible for the superconducting-to-normal phase transition in the rf field at temperatures up to about 0.98-0.99 T_c' where the ideal superheating limit was being reached. The results of the alloy samples showed that the attainable rf critical fields near T_c were well described by the superheating field predicted by the one-dimensional GL theory in both the type I and type II regimes. The measurement was also made at 300 MHz resulting in no significant change in the rf critical field. Thus it was inferred that the nucleation time of the normal phase, once the critical field was reached, was small compared with the rf period in this frequency range.

Phase effects on group velocity propagation in a V-type system with spontaneously generated coherence

The effects of the relative phase between two laser beams on the propagation of a weak electromagnetic pulse are investigated in a V-type system with spontaneously generated coherence (SGC). Due to the relative phase, the subluminal and superluminal group velocity can be unified. Meanwhile, SGC can be regarded as a knob to manipulate light propagation between subluminal and superluminal.

Upper level and phase controlling group velocity in V-type system

With the external field coupling the two upper levels, we investigate the light pulse propagation properties with weak probe field in a V-type system. Due to the external upper level (UL) coupling field, the dispersion of the system has been influenced by the relative phase. It is shown that the UL field and the relative phase can be regarded as switches to manipulate light propagation between subluminal and superluminal. (c) 2007 Elsevier B.V. All rights reserved.

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.

Alteración de lípidos en la enfermedad de Alzheimer. Estudio en tejido cerebral humano postmortem mediante MALDI-IMS.

En el presente estudio, se analizó la distribución lipídica de 3 áreas de hipocampo (CA1, subiculum y giro dentado) en muestras cerebrales de pacientes con enfermedad de Alzheimer y se comparó con muestras de sujetos sin patología neurológica. Las muestras de tejido de cerebro humano postmortem de la región de hipocampo, fueron procesadas mediante la técnica MALDI-IMS.

Part I. Coenzyme B12 as a hydroformylation-type catalyst. Part II. Mechanisms of hydrogen transfer in the methylmalonyl coenzyme a mutase reaction

Part I

The mechanism of the hydroformylation reaction was studied. Using cobalt deuterotetracarbonyl and 1-pentene as substrates, the first step in the reaction, addition of cobalt tetracarbonyl to an olefin, was shown to be reversible.

Part II

The role of coenzyme B₁₂ in the isomerization of methylmalonyl coenzyme A to succinyl coenzyme A by methylmalonyl coenzyme A mutase was studied. The reaction was allowed to proceed to partial completion using a mixture of methylmalonyl coenzyme A and 4, 4, 4-tri-²H-methylmalonyl coenzyme A as substrate. The deuterium distribution in the product, succinyl coenzyme A, was shown to best fit a model in which hydrogen is transferred from C-4 of methylmalonyl coenzyme A to C-5’ of the adenosyl moiety of coenzyme B₁₂ in the rate determining step. The three hydrogens at the 5’-adenosyl position of the coenzyme B₁₂ intermediate are then able to become enzymatically equivalent before hydrogen is transferred from the coenzyme B₁₂ intermediate to form succinyl coenzyme A.

Theoretical research on noncollinear match conditions of the type I optical parametric process

Broad bandwidth group match conditions are reported for a noncollinear type I optical parametric process. The theoretical calculations corresponding to two special situations in practice were made, respectively, which are in accordance with the published experimental results. Furthermore, we provide a method to not only achieve maximal parametric bandwidth output but also match the group velocities between three waves. (c) 2006 Optical Society of America.

Optical setup and analysis of disk-type photopolymer high-density holographic storage

A relatively simple scheme for disk-type photopolymer high-density holographic storage based on angular and spatial multiplexing is described. The effects of the optical setup on the recording capacity and density are studied. Calculations and analysis show that this scheme is more effective than a scheme based on the spatioangular multiplexing for disk-type photopolymer high-density holographic storage, which has a limited medium thickness. Also an optimal beam recording angle exists to achieve maximum recording capacity and density. (C) 2002 Society of Photo-Optical Instrumentation Engineers.