Reconstitution of Membrane-Bound Enzymes for Neutron Scattering Studies: A Case Study of Human Dihydroorotate Dehydrogenase

Membrane proteins are challenging to study due the necessity to maintain a lipid-like environment to preserve their structure and function. Particularly difficult to study are membrane-bound enzymes that interact with lipophilic substrates. The aim of this doctoral thesis was to investigate the mechanism of action and inhibition of membrane-bound enzymes that interact with lipids and lipophilic substrates under non-crystalline, physiologically relevant conditions using neutron scattering methods. Neutron scattering techniques are suitable for the study of biological systems. Neutrons have no net charge and can therefore penetrate deeply into matter. Using neutrons, it is also possible to distinguish between isotopes of the same element, as is the case with the isotopes of hydrogen (protium and deuterium). Selective deuteration can be used to determine the structure and location of biological molecules in complex systems. Neutron reflectometry (NR) is a surface scattering technique capable of determining the structure and composition of thin films in the direction perpendicular to the surface. NR is therefore highly suitable for the study of membrane proteins and lipid bilayers. A relevant example of this type of membrane proteins is human dihydroorotate dehydrogenase (HsDHODH). HsDHODH in an integral protein found in the inner mitochondrial membrane (IMM), where it catalyzes the oxidation of dihydroorotate to orotate with the concomitant reduction of ubiquinone Q10 (coenzyme Q10), thus linking pyrimidine biosynthesis and the respiratory chain. HsDHODH is the target of anti-inflammatory drugs and anti-proliferative compounds, and mutations in the HsDHODH gene have been identified as the cause of Miller syndrome, a rare genetic disorder characterized by malformations of the head, face, and limbs. Firstly, the production of the protein reagents needed for this thesis was established. A full biochemical characterization of HsDHODH and truncated HsDHODH as well as three variants associated with Miller syndrome (G19E, E52G, R135C) was performed. A particular focus was placed on their interaction with lipids including quartz crystal microbalance with dissipation (QCM-D) measurements of their binding to supported lipid bilayers (SLB). Miller syndrome mutants displayed lower activities compared to the wild-type enzymes, but showed also decreased stability, a probably impaired mitochondrial import, as well as differences in interactions with lipids (Papers I – III). As part of this thesis, the interactions between SLBs consisting of either synthetic lipids or a complex lipid mixture extracted from yeast with or without Q10 and a soluble truncated form of HsDHODH, as well as the soluble bacterial analog from E. coli (EcDHODH), were investigated by NR. Q10 was found to be located at the center of all the bilayers studied, between the lipid leaflets. Both enzymes were found to penetrate into the outer lipid leaflet of the bilayer upon interaction. The bacterial enzyme displayed a stronger binding and better retention than the human enzyme. Binding was also found to depend on the lipid composition of the bilayer as both enzymes displayed a stronger binding to complex bilayers consisting of several lipid species as opposed to those consisting of a few synthetic lipids. The interaction between both enzymes and ubiquinone was found to be mediated by protein penetration, as opposed to Q10 migration (Paper IV). The reconstitution of HsDHODH into supported lipid bilayers was attempted using three different methodologies: adsorption of lipid-detergent micelles prepared with dodecyl D-maltoside (DDM), fusion of proteoliposomes, and hybrid approaches combining the adsorption of DDM-protein micelles and lipid vesicle fusion and characterized by NR. Micelle adsorption resulted in membranes with low lipid and protein coverage and with residual detergent. Proteoliposomes yielded a good lipid bilayer coverage but with a low protein content. The hybrid approaches resulted in good protein incorporation but also in the formation of an additional floating layer. The information obtained from these approaches can be used to guide and inform the reconstitution of proteins structurally similar to HsDHODH (Paper V)

Temporalización de la historia y perspectivismo metodológico

This essay aims to explore the issue of methodologic perspectivism in Kosselleck’s thought considering as cornerstone the concept of temporalization. The first section links the concepts of temporalization and secularization introducing beforehand philosophies of the history from Sattelzeit time. Then the text focuses on reconstructing the notion of temporalization based on an emerging tension between the language and the reality it describes. This article concludes bringing up the notion of ficcionality as a key element in Koselleck’s theory of history making up for the methodological deficits after this tension. The unifying thread of this essay is that the theoretical project of a conceptual history it is not only an analysis method but mainly a theory of modernity.Este ensayo quiere explorar la problemática del perspectivismo metodológico en el pensamiento de Koselleck tomando como eje central el concepto de temporalización. Tras un primer apartado en el que se pone en relación los conceptos de temporalización y secularización haciendo referencia previamente a las filosofías de la historia correspondientes al periodo de la Sattelzeit, el texto se centra en reconstruir la noción de temporalización sobre la base de una tensión creciente entre el lenguaje y la realidad que éste describe. El artículo se cierra planteando la noción de ficcionalidad como un elemento central en la teoría de la historia de Koselleck que viene a subsanar los déficits metodológicos que se derivan de esa tensión. El hilo conductor que vertebra el ensayo es que el proyecto teórico de una historia conceptual no es únicamente un método de análisis, sino también, y sobre todo, una teoría de la modernidad

The problem of objectivity in history: A dialogue between Max Weber and Ankersmit

El estatuto epistemológico de la historia en particular y las ciencias humanas en general depende fundamentalmente de su grado de objetividad. El problema radica en qué se entiende por objetividad y hasta qué punto o en qué sentido puede ser objetivo el conocimiento histórico. ¿Es el conocimiento histórico meramente subjetivo? Si es así, ¿cómo es posible que grupos sociales consideren que tienen un pasado común? En el presente artículo se quiere dar respuestas a estas preguntas estableciendo un diálogo entre Max Weber, un autor clásico en la epistemología de las ciencias humanas y sociales, y un autor contemporáneo como es el caso de AnkersmitThe epistemological status of history in particular and the human sciences in general depends on objectivity. The problem stems from how the concept of objectivity is understood, and to what extent or in which sense historical knowledge may be considered objective. Is knowledge of history merely subjective? If so, why do some social groups consider that they have a common past? The present article attempts to answer these questions by establishing a dialogue between Max Weber, a classic author on the epistemology of human and social sciences, and the contemporary author Ankersmi

The Role and Impact of Remittances on Small Business Development during Cuba’s Current Economic Reforms

This paper explores the extent to which Cuban remittance recipients are responding to the Cuban government’s current economic reforms which seek to incentivize entrepreneurial activities as an economic growth strategy and state liberalization policy. In so doing we hope to make some preliminary observations and recommendations about the potential role and impact of remittances in Cuba’s socio-economic development. It is based on an original survey conducted in Cuba in 2012

Presentación

Publicamos ahora el volumen XIV de esta obra colectiva iniciada en los umbrales del siglo XXI. Sin afanes de autocomplacencia, nos congratulamos de haber arribado hasta esta colina, en el largo e interminable camino de la creación intelectual y la investigación científica

Diagnóstico de situación actual del proceso de producción de pan bizcotela en la panadería Linda Vista Nº 2 para la implementación de las BPM, en la ciudad de Matagalpa, durante el segundo semestre 2014

El pan ha estado unido a la evolución del hombre hasta nuestros días, donde la oferta de este producto es extensa. En la panadería Linda Vista #2 se ha llevado a cabo un estudio que muestra las condiciones en las que se encuentra operando actualmente, en función del tema: diagnóstico de situación actual de procesos productivos en empresas, para la implementación de técnicas orientadas al mejoramiento del proceso (BPM, HCCP, otros), con el propósito de diagnosticar la situación actual del proceso de producción de pan en la panadería Linda Vista #2 en la ciudad de Matagalpa durante el segundo semestre 2014. Entre los principales temas abordados están: 1. Materias primas en la elaboración de pan. 2. Proceso de panificación. 3. Las Buenas Prácticas de Manufactura (BPM). 4. Análisis de procesos industriales. Para el proceso investigativo se utilizaron herramientas para recopilar información tales como observaciones directas y entrevista. La información recopilada se utilizó para diagnosticar la situación actual de la empresa, además de brindar recomendaciones a dificultades encontradas en torno al proceso productivo orientadas a un mejor control del mismo. Al finalizar la investigación las principales conclusiones son las siguientes: 1. Las fases actuales del proceso de producción de pan bizcotela en la panadería son: Compra de materia prima, mezclado, pasteado, troceado, figurado, fermentado, horneado, preparación del dulce, bañado, secado, empacado y almacenado. 2. La distribución actual de la planta no es adecuada para el proceso. 3. No se cumplen con todas las normas del BPM. 4. Los cuellos de botella se encuentran en los subprocesos de mezclado, pasteado y hornead