Assembly and Activation of the Plasmodial Pyridoxal 5’-Phosphate Synthase Complex. Understanding the Structural Mechanism of PLP Biosynthesis

Biosynthesis of vitamin B6 is essential for all living cells. Most organisms use the pyridoxal 5’-phosphate (PLP) synthase complex to synthesize the cofactor form, PLP, from the three substrates ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P) and ammonia. PLP synthase complex is a glutamine amidotransferase (GATase) class I, consisting of 12 Pdx1 and 12 Pdx2 subunits. Pdx1 is responsible for the PLP synthesis and Pdx2 is the glutaminase that hydrolyses glutamine to produce ammonia, which is transfered to the Pdx1 active site. In this PhD Thesis, studying Pdx1 and Pdx2 proteins from the human parasite Plasmodium falciparum and from the rodent parasite Plasmodium berghei gave important insights into the assembly, activation and substrate tunneling of the PLP synthase complex. Electron microscopy analyses showed that association of the PLP synthase and glutaminase subunits was random, suggesting a non cooperative mechanism independent of neighboring Pdx1 binding sites to be occupied by Pdx2. Complex assembly is critical for glutamine hydrolysis by Pdx2, although the presence of an ammonia acceptor in the Pdx1 active site did neither enhance the Pdx1/Pdx2 interaction nor the catalytic rate of Pdx2 in vitro, as tested by biophysical and kinetic experiments. In particular, the PLP synthase complex does not show allosteric activation by R5P or glutamine binding that would result in synchronization of the glutaminase and PLP synthesis reactions. Therefore, the Pdx1/Pdx2 interaction is enough to stimulate glutamine hydrolysis. A particular motivation of this Thesis was to crystallize the PLP synthase complex from the malaria causing parasite, P. falciparum, for the potential use of the 3D structure in drug design. However, the complex assembled into fibers in vitro, induced by the Pdx1 subunit, making the crystallization trials of this enzyme impossible. A chimeric complex formed by Pdx1 from P. berghei and Pdx2 from P. falciparum proved to be a catalytically active system, suitable for structural studies of the plasmodial complex. Crystal structure of this enzyme complex gave two major advances in the understanding how prokaryotic and eukaryotic PLP synthase complexes resemble each other or differ in protein interaction and activation. Variations at the Pdx1/Pdx2 interface occur through insertion sequences in eukaryotic systems, notably in the plasmodial PLP synthase complex by the loop 95-111 in Pdx2. Activation of the glutaminase is highly conserved in both systems. The process entails reorganization of structural regions at the Pdx1/Pdx2 interface through stabilization of alpha-N and the oxyanion hole region. Activation of the PLP synthase requires a helical segment, named alpha-2’, for sugar binding. The helix is observed in two alternative positions in the Pdx1/Pdx2 and Pdx1-R5P structures: an open conformation to allow the entrance of the substrate and a closed conformation oriented towards R5P, sequestering the substrate in the catalytic center. The pentose substrate is bound in the P1 site to the catalytic Lys84 via a Schiff base with the ribose C1 atom. GATases are characterized by two separate active sites for glutamine hydrolysis and enzyme-specific metabolite syntheses. Previously, ammonia transfer between two catalytic centers was proposed to occur by flexible methionine residues within a transient tunnel in Pdx1. The plasmodial proteins show an ammonia tunnel distict from bacterial orthologs as some of the residues lining the passage are exchanged. Biochemical analysis confirmed that the (beta/alpha)8 -barrel of Pdx1 passes the reactive ammonia produced in Pdx2 to Pdx1 active site, assigning function of key residues for the ammonia channeling. The differences between eukaryotic and prokaryotic systems provide insight into PLP synthase complex regulation, which may be exploitable in drug design for the treatment of malaria

Mapping the interaction site and effect of the Siglec-9 inflammatory biomarker on human primary amine oxidase

Human primary amine oxidase (hAOC3), also known as vascular adhesion protein 1, mediates leukocyte rolling and trafficking to sites of inflammation by a multistep adhesion cascade. hAOC3 is absent on the endothelium of normal tissues and is kept upregulated during inflammatory conditions, which is an applicable advantage for imaging inflammatory diseases. Sialic acid binding immunoglobulin like-lectin 9 (Siglec-9) is a leukocyte ligand for hAOC3. The peptide (CARLSLSWRGLTLCPSK) based on the region of inflammation using Positron Emission Tomography (PET). In the present study, we show that the Siglec-9 peptide binds to hAOC3 and triggers its amine oxidase activity towards benzylamine. Furthermore, the hAOC3 inhibitors semicarbazide and imidazole reduce the binding of wild type and Arg/Ala mutated Siglec-9 peptides to hAOC3. Molecular docking of the Siglec-9 peptide is in accordance with the experimental results and predicts that the R3 residue in the peptide interacts in the catalytic site of hAOC3 when the topaquinone cofactor is in the non-catalytic on-copper conformation. The predicted binding mode of Siglec-9 peptide to hAOC3 is supported by the PET studies using rodent, rabbit and pig AOC3 proteins

Functional measurement in the field of ethics in politics

We present, in a synthetic way, some of the main findings from ten studies that were conducted in the field of ethics in politics, using the Functional Measurement framework. These studies were about (a) Angolan and Mozambican people’s views about the legitimacy of military-humanitarian interventions, (b) French people’s perspectives regarding the government’s responsibility for the health of consumers of illicit substances, (c) Togolese people’s views about the acceptability of political amnesties in a time of political transition, (d) the perspective of victims of the genocide of the Tutsis in Rwanda regarding the attribution of guilt by association to offspring of perpetrators, (e) slave descendants’ views about the acceptability of national policies on reparations for slavery, (f) Colombian people’s willingness to forgive perpetrators of violence who harmed family members during the civil war, (g) the attitudes of French and Colombian people about national drug control policies, (h) Indian students’ views about the appropriateness of the death penalty for murder or rape, (i) Colombian people’s perspectives regarding corruption, and finally (j) Venezuelan people’s conceptualization of human rights. The main findings are discussed in reference to six of the foundations of Moral Foundations Theory.Este texto presenta, de forma resumida, algunos de los principales resultados de diez estudios que se realizaron en el campo de la ética y la política, en el marco de la Medición Funcional. Estos estudios trataron de: (a) los puntos de vista de la gente de Angola y Mozambique sobre la legitimidad de las intervenciones militares-humanitaria; (b) las perspectivas de los franceses con respecto a la responsabilidad del gobierno con la salud de los consumidores de sustancias ilícitas; (c) las perspectivas de las personas de Togo acerca de la aceptabilidad de amnistías políticas en un momento de transición política; (d) las perspectivas de las víctimas del genocidio de los Tutsis en Ruanda en cuanto a la atribución de culpabilidad a los descendientes de los perpetradores de violencia; (e) los puntos de vista de los descendientes de esclavos acerca de la aceptabilidad de las políticas nacionales de reparaciones por la esclavitud; (f) la disposición de los colombianos a perdonar autores de la violencia que causaron daño a miembros de la familia durante el conflicto armado interno; (g) las actitudes de los franceses y colombianos acerca de las políticas nacionales de control de drogas; (h) los puntos de vista de los estudiantes indios acerca de la idoneidad de la pena de muerte por asesinato o violación; (i) las perspectivas de los colombianos con respecto a la corrupción, y, finalmente, (j) la conceptualización de los derechos humanos en la población venezolana. Los principales resultados se discuten en relación con seis de los fundamentos de la Teoría de los Fundamentos Morales

Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase

Most organisms synthesise the B(6) vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I(320), and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex

La percepción de riesgo social por laicos que viven en Venezuela. Una comparación entre países Latinos

The present study presents the mean risk magnitude judgments on 91 activities, substances, and technologies expressed by Venezuelan adults living in the two main cities of this country: Caracas and Maracaibo. These judgments were compared methodically with findings on other samples of previous studies, namely four other Latin countries: France, Spain, Brazil, and Portugal. The aim of this study was to structure the cross-country differences in risk perception between the aforementioned countries and Venezuela using cluster analysis. A 91-hazard x 5 country matrix was created. Two main clusters were found. The Economically and Socially Challenging group (Venezuela and Portugal) and the Western Europe group (France and Spain). Brazil was situated closer to the Venezuelan and Portugal cluster than was the Western Europe group. The common denominator in the Economically and Socially Challenging group can be the economic and social problems that both of these countries struggle against. It was reasonable that Brazil was closer to this cluster, given its similarities to both countries (in geographical and cultural terms). More explanations for these clusters were presented in the discussion. Finally, some recommendations and limitations are also presented and more research in this field is suggested as well.El presente estudio presenta la media de los juicios de magnitud del riesgo expresadas por los adultos venezolanos que viven en las dos principales ciudades de este país: Caracas y Maracaibo, en 91 actividades, sustancias y tecnologías. Estos juicios se compararon metódicamente con los resultados de otras muestras de estudios anteriores, específicamente con otros cuatro países de raíz Latina: Francia, España, Brasil y Portugal. El objetivo del presente estudio fue estructurar las diferencias en la percepción de riesgo entre los países antes mencionados y Venezuela utilizando análisis de conglomerados. Se creó una matriz de 5 países x 91 afirmaciones. Se encontraron dos clusters principales: El grupo económica y socialmente desafiante (Venezuela y Portugal) y el grupo de Europa Occidental (Francia y España). Brasil se encuentra más cerca de la agrupación venezolana, Portugal se asignó al grupo de Europa Occidental. El común denominador en el grupo económica y socialmente desafiante pueden ser los problemas económicos y sociales de estos dos países en la coyuntura. Es razonable que Brasil esté más cerca a este grupo debido a sus similitudes de ambos países (geográfica y culturalmente). Más explicaciones para estos clusters se presentan en la discusión. Por último, se presentan algunas recomendaciones y limitaciones, y se sugieren campos de investigación

La percepción de riesgo social por laicos que viven en Venezuela. Una comparación entre países Latinos

The present study presents the mean risk magnitude judgments on 91 activities, substances, and technologies expressed by Venezuelan adults living in the two main cities of this country: Caracas and Maracaibo. These judgments were compared methodically with findings on other samples of previous studies, namely four other Latin countries: France, Spain, Brazil, and Portugal. The aim of this study was to structure the cross-country differences in risk perception between the aforementioned countries and Venezuela using cluster analysis. A 91-hazard x 5 country matrix was created. Two main clusters were found. The Economically and Socially Challenging group (Venezuela and Portugal) and the Western Europe group (France and Spain). Brazil was situated closer to the Venezuelan and Portugal cluster than was the Western Europe group. The common denominator in the Economically and Socially Challenging group can be the economic and social problems that both of these countries struggle against. It was reasonable that Brazil was closer to this cluster, given its similarities to both countries (in geographical and cultural terms). More explanations for these clusters were presented in the discussion. Finally, some recommendations and limitations are also presented and more research in this field is suggested as well.El presente estudio presenta la media de los juicios de magnitud del riesgo expresadas por los adultos venezolanos que viven en las dos principales ciudades de este país: Caracas y Maracaibo, en 91 actividades, sustancias y tecnologías. Estos juicios se compararon metódicamente con los resultados de otras muestras de estudios anteriores, específicamente con otros cuatro países de raíz Latina: Francia, España, Brasil y Portugal. El objetivo del presente estudio fue estructurar las diferencias en la percepción de riesgo entre los países antes mencionados y Venezuela utilizando análisis de conglomerados. Se creó una matriz de 5 países x 91 afirmaciones. Se encontraron dos clusters principales: El grupo económica y socialmente desafiante (Venezuela y Portugal) y el grupo de Europa Occidental (Francia y España). Brasil se encuentra más cerca de la agrupación venezolana, Portugal se asignó al grupo de Europa Occidental. El común denominador en el grupo económica y socialmente desafiante pueden ser los problemas económicos y sociales de estos dos países en la coyuntura. Es razonable que Brasil esté más cerca a este grupo debido a sus similitudes de ambos países (geográfica y culturalmente). Más explicaciones para estos clusters se presentan en la discusión. Por último, se presentan algunas recomendaciones y limitaciones, y se sugieren campos de investigación

Conceptualización de franceses y venezolanos de los derechos humanos: Problemas de indivisibilidad y universalidad

The present study aimed to examine French unpaid participants’ (N = 50) perceptions about Human Rights (HR). The material was a series of scenarios depicting a socio-political situation of a fictitious state and a response scale. Four critical items of information were provided: (a) the degree in which the State offers social protection to the citizens (not at all, intermediate or complete);(b) the level of respect for Civil Liberties in the country (no respect, intermediate, full respect);(c) the level of Equality between citizens (inequality of rights vs. equality of right);and (d) the level of Respect for the private life of the citizens (no respect for private life vs. full respect for private life). The 36 stories were obtained by the orthogonal crossing of the four factors: 3 x 3 x 2 x 2 = 36. Results from the French sample were compared with previous results from a Venezuelan sample (Guédez & Mullet, 2014). Results showed no difference in the importance given to Social Protection between French and Venezuelan participants. Also, the crucial four-way interaction was significant at a very stringent level, and the five-way interaction involving Country was not. Thus, it can be safely considered that the way HR are conceptualized is correctly expressed by the equation: Judged Respect for HR = Privacy x Civil Liberties x Equality x Social Protection.El objetivo del presente estudio fue examinar las percepciones acerca de los derechos humanos de participantes franceses no pagados (N = 50). Los materiales fueron una serie de escenarios que representaban la situación sociopolítica de un estado ficticio, y una escala de respuesta. Se proporcionaron cuatro ítems críticos de información: a) el grado en el que el estado ofrece protección social a los ciudadanos (ninguno, intermedio, o completo); b) el nivel de respeto por las libertades civiles en el país (no hay respeto, intermedio, o respeto total); c) el nivel de equidad entre ciudadanos (inequidad de derechos vs. equidad de derechos); y d) el nivel de respeto por la vida privada de los ciudadanos (no hay respeto por la vida privada vs. respeto total por la vida privada). Las 36 historias fueron obtenidas por el cruce ortogonal de los 4 factores: 3 x 3 x 2 x 2 = 36. Los resultados de la muestra francesa fueron comparados con resultados previos de una muestra venezolana (Guédez & Mulllet, 2014). Los resultados no mostraron diferencias en la importancia dada a la protección social entre los participantes franceses y venezolanos. Además, la interacción crucial de cuatro formas fue significativa en un nivel muy riguroso, mientras que la interacción de cinco formas, incluyendo país, no lo fue. Entonces, se puede considerar con seguridad que la manera en que los derechos humanos son conceptualizados es correctamente expresada por la ecuación: Respeto por los derechos humanos juzgado = Privacidad x Libertades civiles x Equidad x Protección Social

Factorización LU de matrices dispersas sobre una arquitectura MIMD mediante la obtención de conjuntos compatibles: Trabajo especial de grado

En este trabajo se paralelizó de manera novedosa la factorización LU de matrices dispersas, mediante la obtención de un conjunto de pivotes que cumplan ciertas propiedades para que puedan procesarse simultánemente, lográndose muy buenos resultados en tiempo de ejecución en comparación con el conocido algoritmo Y12M