3-pyridylcarbene and 3-pyridylnitrene: Ring opening to nitrile ylides

Photolysis of 3-pyridyldiazomethane in an Ar matrix at 7-10 K gives 3-pyridylcarbene. Further photolysis causes ring opening to nitrile ylide 26 (formonitrile pent-2-en-4-ynylide) as the major reaction together with a minor amount of ring expansion to 1-azacyclohepta-1,3,4,6-tetraene, 27. Matrix photolysis of 3-azidopyridine leads to ring opening to formonitrile N-cyanovinylmethylide, 33.

Portugal making a difference with children and families: the GADIF model

Portugal is a culture grounded in strong traditions and family. Yet, social changes like women returning to the workforce and a decreas ed national birth rate are impacting the traditional family structure and care giving environments of children. Female employment has been increasing steadily in P ortugal over the last three decades (Galego & Pereira, 2006) and the total fert ility rate decreasing from 4.1 to 2.8 (INE, 2006). Furthermore, extended family me mbers, like grandparents, no longer reside close by to their children and grandc hildren as in the past, because of a changing labor market. Many of the younger gen eration are leaving their rural communities to flock to urban areas because o f job opportunities, leaving behind older relatives who would have otherwise par ticipated in the daily care of children. Given these social and economic changes, children are spending more time in out-of-home care with non-familial caregive rs. Yet, government regulations and guidelines in early care and educat ion (ECCE) and early intervention (EI) are only just emerging; it contin ues to be a work in progress.

O futurismo russo: Maiakovsky ou a "Nuvem de calças"

Este breve ensaio sobre o poeta e escritor russo Vladimir Mayakovsky é uma modesta tentativa de contribuir para um (re)descobrimento ou para uma simples revisitação da produção literária russa do período soviético, que, por razões muitas vezes apenas de ordem política e não estética, caiu no esquecimento, não sendo traduzida na sua totalidade, como é o caso de Mayakovsky, ou, pura e simplesmente, sendo votada ao “ostracismo”, porque rotulada, a priori ou ab initio, como obras escritas dentro das paredes estreitas do “realismo socialista”, padecendo de falta de criatividade e norteandose, aberta ou dissimuladamente, pelos princípios rígidos e imutáveis de um totalitarismo inflexível. Contudo, nem toda a literatura soviética pode ser enquadrada no realismo socialista, valendo a pena conhecê-la e analisá-la.

Structural investigation of the Bacillus subtilis morphogenic factor RodZ

A thesis to obtain a Master degree in Structural and Functional Biochemistry

Fotoionização de moléculas e átomos

Este trabalho, no âmbito da física atómica e molecular, é essencialmente um estudo experimental, por fotoionização, da estrutura electrónica de moléculas e átomos de elevada instabilidade, relevantes na indústria, atmosfera terrestre e astrofísica. Os estudos de física molecular compreendem a caracterização em fase gasosa, à temperatura ambiente e a decomposição térmica controlada até 1100 K, por fotoionização com radiação de ultravioleta de vácuo usando a fonte de He I (21.22 eV), das seguintes moléculas: azidoformato de metilo, N3COOCH3; azidoformato de etilo, N3COOCH2CH3; azidoacetato de etilo, N3CH2COOCH2CH3; azidoetanol, N3CH2CH2OH;2-azidopropionitrilo, CH3CH(N3)CN; e 3-azidopropionitrilo, N3CH2CH2CN. Os resultados de espectroscopia de fotoelectrões, para cada uma destas moléculas, são comparados com os resultados do estudo de pirólise pela técnica de isolamento em matriz de azoto molecular, à temperatura de 12 K, assistida por espectroscopia de infravermelho. Os compostos intermediários e produtos finais observados permitem propor mecanismos de decomposição térmica para cada um dos sistemas. A interpretação e atribuição das bandas espectrais relativas a cada molécula têm por base os cálculos ab initio (HF e MP2) e de funcional da densidade (BLYP e B3LYP) das propriedades moleculares dos seus confórmeros de equilíbrio. A radiação de sincrotrão de ultravioleta de vácuo é usada nos estudos, por fotoionização, do oxigénio e azoto atómicos e dos radicais OH e OD, os quais são produzidos in situ por descarga de microondas e/ou reacção rápida átomo molécula. Para cada um destes sistemas, apresentam-se medidas da secção eficaz parcial de fotoionização, σ i, e do parâmetro de assimetria, β. Nestes estudos é usada a radiação da estação de trabalho 4.2 do Sincrotrão Elettra, em Itália.

Estudio Cinético de Reacciones de Transferencia de átomo de cloro de haloalcanos con radicales asimétricos: Análisis comparativo con cálculos semiempíricos

El objetivo es aportar parámetros cinéticos de interés en la Fotoquímica de la Atmósferica, relacionada con la comprensión de los procesos de abstracción de átomo de cloro de halometanos, con radicales asimétricos del tipo C2FxCly (donde x + y = 5). La fuente de radicales (ICF2CFECl Y ICF2CFC12) son ioduros asimétricos sintetizados y purificados con cromatografía gaseosa y caracterizado por espectroscopía IR, UV y espectrometría de masa. Se utiliza un sistema de medición para reacciones competitivas en la determinación de las constantes de velocidad, con análisis de los productos mediante cromatografía gaseosa. Se calcularán además algunas funciones termodinámicas de los radicales involucrados y de los procesos de transferencia de átomo de cloro de las reacciones directas e inversas. Los factores experimentales son reproducidos por el método de Benson. Para explicar la correlación entre las energías de activación, estructura electrónica y geometría del estado de transición se utilizarán métodos ab-initio y semiempíricos que pueden dar una explicación satisfactoria de los valores experimentales.

Parámetros magnéticos y estructura molecular

Los estudios realizados a través de este proyecto pretenden abarcar dos aspectos fundamentales en la Física Experimental y Teórica, los cuales son: a) Realizar estudios en investigación básica, orientados a acrecentar el conocimiento en ramas de la Física Molecular, utilizando la técnica de Resonancia Magnética Nuclear (RMN). Para ello se obtienen e interpretan los parámetros magnéticos moleculares característicos y se desarrollan e implementan métodos de cálculo de gran potencia y tiempo de cómputo a fin de correlacionar los estudios de tales parámetros con la estructura geométrica y electrónica molecular y/o con las diversas interacciones moleculares que se puedan presentar. b) Presentar y desarrollar tecnología adicional que permita aplicaciones de la RMN a otras líneas de estudio y también la consolidación de la infraestructura experimental existente en la Universidad Nacional de Río Cuarto. Objetivos generales y específicos: Se plantea como objetivo global, el acrecentar el conocimiento de un conjunto de propiedades moleculares, estudiando una familia de compuestos orgánicos mediante la técnica de la RMN de alta resolución y el empleo de métodos de cálculo tipo ab-initio. (...) Se mencionan a continuación los objetivos específicos más importantes a alcanzar: * Proponer modelos y leyes empíricas que describan el comportamiento de los parámetros magnéticos pertenecientes a familias de compuestos orgánicos heterocíclicos en relación a su estructura electrónica y conformacional. * Compatibilizar los estudios empíricos con resultados teóricos. * Desarrollar nuevos métodos de cálculo a fin de obtener una función de onda que mejor aproxime lo anterior. * Orientar parte del trabajo experimental a diversas aplicaciones de la RMN, adaptando el equipamiento existente a otras técnicas y/o adquiriendo equipamiento para tal fin.

Química de l'espín prohibit: la importància dels intermedis a capa oberta en l'activació d'enllaços C-H

Estudi realitzat a partir d’una estada a la School of Chemistry de la University of Bristol, Gran Bretanya, entre 2006 i 2008. La reacció d’activació del pre-catalitzador CH3-Fe(P(iPr)2CH2)3B-Ph, que es dóna en presència de H2 i P(CH3)3, és una reacció spin-prohibida ja que els reactius i els productes tenen diferents estat d’espín en el seu estat fonamental: el pre-catalitzador és quintet, mentre que el producte format després de l’eliminació de metà, PMe3(H3)-Fe[(P(iPr)2CH2)3B-Ph, és singlet. Un dels intermedis d’aquesta reacció és d’especial interès ja que catalitza la hidrogenació d’olefines. Intermedi que experimentalment s’ha proposat com a H-Fe[(P(iPr)2CH2)3B-Ph] o el Hx-Fe[(P(iPr)2CH2)3B-Ph]. Aquestes espècies han estat estudiades computacionalment, mitjançant la combinació de mètodes del funcional de la densitat calibrats mitjançant càlculs ab initio. Els resultats obtinguts mostren que les superfícies d’energia potencial singlet, triplet i quintet es creuen al llarg de les reaccions descrites. La reacció d’activació del pre-catalitzador és una reacció multi spin-prohibida (més d’un canvi d’espín), en la que el mecanisme preferit addiciona primer hidrogen i després la fosfina, el pas determinant de la velocitat és el creuament de la superfície d’energia potencial quintet a la triplet a la geometria del reactiu, que es produeix amb una barrera d’aproximadament 18 kcal/mol. La reacció d’hidrogenació d’olefines en canvi pot ser o no una reacció espín prohibida depenent de les condicions en que es dugui a terme. En cas de que la reacció es dugui a terme en un excés d’hidrogen, en el mecanisme principal l’espècie activa seria el (H2)H-Fe[(P(iPr)2CH2)3B-Ph] singlet en el seu estat fonamental que hidrogenaria l’olefina sense cap pas espín prohibit. Aquest mecanisme és el de barrera més baixa però s’espera que estigui més desafavorit entropicament. Si les condicions no són d’un important excés d’hidrogen, llavors l’espècie activa s’espera que sigui H-Fe[(P(iPr)2CH2)3B-Ph], la reacció llavors seria multi espín prohibida amb una barrera de unes 13 kcal/mol corresponents al creuament entre la superfície quintet i triplet a la geometria del H-Fe[(P(iPr)2CH2)3B-Ph].

Structural prediction of peptides bound to MHC class I.

An ab initio structure prediction approach adapted to the peptide-major histocompatibility complex (MHC) class I system is presented. Based on structure comparisons of a large set of peptide-MHC class I complexes, a molecular dynamics protocol is proposed using simulated annealing (SA) cycles to sample the conformational space of the peptide in its fixed MHC environment. A set of 14 peptide-human leukocyte antigen (HLA) A0201 and 27 peptide-non-HLA A0201 complexes for which X-ray structures are available is used to test the accuracy of the prediction method. For each complex, 1000 peptide conformers are obtained from the SA sampling. A graph theory clustering algorithm based on heavy atom root-mean-square deviation (RMSD) values is applied to the sampled conformers. The clusters are ranked using cluster size, mean effective or conformational free energies, with solvation free energies computed using Generalized Born MV 2 (GB-MV2) and Poisson-Boltzmann (PB) continuum models. The final conformation is chosen as the center of the best-ranked cluster. With conformational free energies, the overall prediction success is 83% using a 1.00 Angstroms crystal RMSD criterion for main-chain atoms, and 76% using a 1.50 Angstroms RMSD criterion for heavy atoms. The prediction success is even higher for the set of 14 peptide-HLA A0201 complexes: 100% of the peptides have main-chain RMSD values < or =1.00 Angstroms and 93% of the peptides have heavy atom RMSD values < or =1.50 Angstroms. This structure prediction method can be applied to complexes of natural or modified antigenic peptides in their MHC environment with the aim to perform rational structure-based optimizations of tumor vaccines.

Exploring chromium (VI) dioxodihalides chemistry: is density functional theory the most suitable tool?

A comparative systematic study of the CrO2F2 compound has been performed using different conventional ab initio methodologies and density functional procedures. Two points have been analyzed: first, the accuracy of results yielded by each method under study, and second, the computational cost required to reach such results. Weighing up both aspects, density functional theory has been found to be more appropriate than the Hartree-Fock (HF) and the analyzed post-HF methods. Hence, the structural characterization and spectroscopic elucidation of the full CrO2X2 series (X=F,Cl,Br,I) has been done at this level of theory. Emphasis has been given to the unknown CrO2I2 species, and specially to the UV/visible spectra of all four compounds. Furthermore, a topological analysis in terms of charge density distributions has revealed why the valence shell electron pair repulsion model fails in predicting the molecular shape of such CrO2X2 complexes

An assessment of gene prediction accuracy in large DNA sequences

One of the first useful products from the human genome will be a set of predicted genes. Besides its intrinsic scientific interest, the accuracy and completeness of this data set is of considerable importance for human health and medicine. Though progress has been made on computational gene identification in terms of both methods and accuracy evaluation measures, most of the sequence sets in which the programs are tested are short genomic sequences, and there is concern that these accuracy measures may not extrapolate well to larger, more challenging data sets. Given the absence of experimentally verified large genomic data sets, we constructed a semiartificial test set comprising a number of short single-gene genomic sequences with randomly generated intergenic regions. This test set, which should still present an easier problem than real human genomic sequence, mimics the approximately 200kb long BACs being sequenced. In our experiments with these longer genomic sequences, the accuracy of GENSCAN, one of the most accurate ab initio gene prediction programs, dropped significantly, although its sensitivity remained high. Conversely, the accuracy of similarity-based programs, such as GENEWISE, PROCRUSTES, and BLASTX was not affected significantly by the presence of random intergenic sequence, but depended on the strength of the similarity to the protein homolog. As expected, the accuracy dropped if the models were built using more distant homologs, and we were able to quantitatively estimate this decline. However, the specificities of these techniques are still rather good even when the similarity is weak, which is a desirable characteristic for driving expensive follow-up experiments. Our experiments suggest that though gene prediction will improve with every new protein that is discovered and through improvements in the current set of tools, we still have a long way to go before we can decipher the precise exonic structure of every gene in the human genome using purely computational methodology.

Comparative gene prediction in human and mouse

The completion of the sequencing of the mouse genome promises to help predict human genes with greater accuracy. While current ab initio gene prediction programs are remarkably sensitive (i.e., they predict at least a fragment of most genes), their specificity is often low, predicting a large number of false-positive genes in the human genome. Sequence conservation at the protein level with the mouse genome can help eliminate some of those false positives. Here we describe SGP2, a gene prediction program that combines ab initio gene prediction with TBLASTX searches between two genome sequences to provide both sensitive and specific gene predictions. The accuracy of SGP2 when used to predict genes by comparing the human and mouse genomes is assessed on a number of data sets, including single-gene data sets, the highly curated human chromosome 22 predictions, and entire genome predictions from ENSEMBL. Results indicate that SGP2 outperforms purely ab initio gene prediction methods. Results also indicate that SGP2 works about as well with 3x shotgun data as it does with fully assembled genomes. SGP2 provides a high enough specificity that its predictions can be experimentally verified at a reasonable cost. SGP2 was used to generate a complete set of gene predictions on both the human and mouse by comparing the genomes of these two species. Our results suggest that another few thousand human and mouse genes currently not in ENSEMBL are worth verifying experimentally.

Modeling of the TCR-MHC-peptide complex.

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.

Molecular modeling study of the binding and signaling properties of the TCRpMHC complex

The activation of the specific immune response against tumor cells is based on the recognition by the CD8+ Cytotoxic Τ Lymphocytes (CTL), of antigenic peptides (p) presented at the surface of the cell by the class I major histocompatibility complex (MHC). The ability of the so-called T-Cell Receptors (TCR) to discriminate between self and non-self peptides constitutes the most important specific control mechanism against infected cells. The TCR/pMHC interaction has been the subject of much attention in cancer therapy since the design of the adoptive transfer approach, in which Τ lymphocytes presenting an interesting response against tumor cells are extracted from the patient, expanded in vitro, and reinfused after immunodepletion, possibly leading to cancer regression. In the last decade, major progress has been achieved by the introduction of engineered lypmhocytes. In the meantime, the understanding of the molecular aspects of the TCRpMHC interaction has become essential to guide in vitro and in vivo studies. In 1996, the determination of the first structure of a TCRpMHC complex by X-ray crystallography revealed the molecular basis of the interaction. Since then, molecular modeling techniques have taken advantage of crystal structures to study the conformational space of the complex, and understand the specificity of the recognition of the pMHC by the TCR. In the meantime, experimental techniques used to determine the sequences of TCR that bind to a pMHC complex have been used intensively, leading to the collection of large repertoires of TCR sequences that are specific for a given pMHC. There is a growing need for computational approaches capable of predicting the molecular interactions that occur upon TCR/pMHC binding without relying on the time consuming resolution of a crystal structure. This work presents new approaches to analyze the molecular principles that govern the recognition of the pMHC by the TCR and the subsequent activation of the T-cell. We first introduce TCRep 3D, a new method to model and study the structural properties of TCR repertoires, based on homology and ab initio modeling. We discuss the methodology in details, and demonstrate that it outperforms state of the art modeling methods in predicting relevant TCR conformations. Two successful applications of TCRep 3D that supported experimental studies on TCR repertoires are presented. Second, we present a rigid body study of TCRpMHC complexes that gives a fair insight on the TCR approach towards pMHC. We show that the binding mode of the TCR is correctly described by long-distance interactions. Finally, the last section is dedicated to a detailed analysis of an experimental hydrogen exchange study, which suggests that some regions of the constant domain of the TCR are subject to conformational changes upon binding to the pMHC. We propose a hypothesis of the structural signaling of TCR molecules leading to the activation of the T-cell. It is based on the analysis of correlated motions in the TCRpMHC structure. - L'activation de la réponse immunitaire spécifique dirigée contre les cellules tumorales est basée sur la reconnaissance par les Lymphocytes Τ Cytotoxiques (CTL), d'un peptide antigénique (p) présenté à la suface de la cellule par le complexe majeur d'histocompatibilité de classe I (MHC). La capacité des récepteurs des lymphocytes (TCR) à distinguer les peptides endogènes des peptides étrangers constitue le mécanisme de contrôle le plus important dirigé contre les cellules infectées. L'interaction entre le TCR et le pMHC est le sujet de beaucoup d'attention dans la thérapie du cancer, depuis la conception de la méthode de transfer adoptif: les lymphocytes capables d'une réponse importante contre les cellules tumorales sont extraits du patient, amplifiés in vitro, et réintroduits après immunosuppression. Il peut en résulter une régression du cancer. Ces dix dernières années, d'importants progrès ont été réalisés grâce à l'introduction de lymphocytes modifiés par génie génétique. En parallèle, la compréhension du TCRpMHC au niveau moléculaire est donc devenue essentielle pour soutenir les études in vitro et in vivo. En 1996, l'obtention de la première structure du complexe TCRpMHC à l'aide de la cristallographie par rayons X a révélé les bases moléculaires de l'interaction. Depuis lors, les techniques de modélisation moléculaire ont exploité les structures expérimentales pour comprendre la spécificité de la reconnaissance du pMHC par le TCR. Dans le même temps, de nouvelles techniques expérimentales permettant de déterminer la séquence de TCR spécifiques envers un pMHC donné, ont été largement exploitées. Ainsi, d'importants répertoires de TCR sont devenus disponibles, et il est plus que jamais nécessaire de développer des approches informatiques capables de prédire les interactions moléculaires qui ont lieu lors de la liaison du TCR au pMHC, et ce sans dépendre systématiquement de la résolution d'une structure cristalline. Ce mémoire présente une nouvelle approche pour analyser les principes moléculaires régissant la reconnaissance du pMHC par le TCR, et l'activation du lymphocyte qui en résulte. Dans un premier temps, nous présentons TCRep 3D, une nouvelle méthode basée sur les modélisations par homologie et ab initio, pour l'étude de propriétés structurales des répertoires de TCR. Le procédé est discuté en détails et comparé à des approches standard. Nous démontrons ainsi que TCRep 3D est le plus performant pour prédire des conformations pertinentes du TCR. Deux applications à des études expérimentales des répertoires TCR sont ensuite présentées. Dans la seconde partie de ce travail nous présentons une étude de complexes TCRpMHC qui donne un aperçu intéressant du mécanisme d'approche du pMHC par le TCR. Finalement, la dernière section se concentre sur l'analyse détaillée d'une étude expérimentale basée sur les échanges deuterium/hydrogène, dont les résultats révèlent que certaines régions clés du domaine constant du TCR sont sujettes à un changement conformationnel lors de la liaison au pMHC. Nous proposons une hypothèse pour la signalisation structurelle des TCR, menant à l'activation du lymphocyte. Celle-ci est basée sur l'analyse des mouvements corrélés observés dans la structure du TCRpMHC.

Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling.

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.