La Casa Domínguez. Alejandro de la Sota: Construir - Habitar

La Tesis Doctoral nace con una intensa vocación pedagógica. La hipótesis de trabajo se establece en torno a una cuestión de interés personal, un tema sobre el que se vertebran, desde el comienzo del doctorado, los diferentes cursos y trabajos de investigación: LA CASA DOMÍNGUEZ como paradigma de la dialéctica en la obra de Alejandro de la Sota. La clasificación de la realidad en categorías antagónicas determina un orden conceptual polarizado, una red de filiaciones excluyentes sobre las que Sota construye su personal protocolo operativo: la arquitectura intelectual o popular, experimental o tradicional, universal o local, ligera o pesada, elevada o enterrada, etc. Se propone el abordaje de una cuestión latente en el conjunto de la obra ‘sotiana’, desde la disección y el análisis de una de sus obras más pequeñas: la casa Domínguez. Se trata de una organización sin precedentes, que eleva la estrategia dialéctica al paroxismo: la vivienda se separa en dos estratos independientes, la zona de día, elevada, y la zona de noche, enterrada; cada uno de los estratos establece su propio orden geométrico y constructivo, su propio lenguaje y carácter, su propia identidad e incluso su propio presupuesto. Las relaciones entre interior y exterior se especializan en función de la actividad o el reposo, estableciéndose una compleja red de relaciones, algunas evidentes y otras celosamente veladas, entre los diferentes niveles. La estancia destinada a las tareas activas se proyecta como un objeto de armazón ligero y piel fría; la precisa geometría del cubo delimita la estancia vigilante sobre el paisaje conquistado. La ladera habitada se destina al reposo y se configura como una topografía verde bajo la que se desarrollan los dormitorios en torno a patios, grietas y lucernarios, generando un paisaje propio: la construcción del objeto frente a la construcción del lugar La casa Domínguez constituye uno de los proyectos menos estudiados, y por lo tanto menos celebrados, de la obra de Don Alejandro. Las publicaciones sucesivas reproducen la documentación gráfica junto a la memoria (epopeya) que el propio Sota compone para la publicación del proyecto. Apenas un par de breves textos críticos de Miguel Ángel Baldellou y, recientemente de Moisés Puente, abordan la vivienda como tema monográfico. Sin embargo, la producción de proyecto y obra ocupó a De la Sota un periodo no inferior a diez años, con casi cien planos dibujados para dos versiones de proyecto, la primera de ellas, inédita. El empeño por determinar hasta el último detalle de la ‘pequeña’ obra, conduce a Sota a controlar incluso el mobiliario interior, como hiciera en otras obras ‘importantes’ como el Gobierno Civil de Tarragona, el colegio mayor César Carlos o el edificio de Correos y Telecomunicaciones de León. La complicidad del cliente, mantenida durante casi cuarenta años, habilita el despliegue de una importante colección de recursos y herramientas de proyecto. La elección de la casa Domínguez como tema central de la tesis persigue por lo tanto un triple objetivo: en primer lugar, el abordaje del proyecto como paradigma de la dialéctica ‘sotiana’, analizando la coherencia entre el discurso de carácter heroico y la obra finalmente construida; en segundo lugar, la investigación rigurosa, de corte científico, desde la disección y progresivo desmontaje del objeto arquitectónico; y por último, la reflexión sobre los temas y dispositivos de proyecto que codifican la identificación entre la acción de construir y el hecho de habitar, registrando los aciertos y valorando con actitud crítica aquellos elementos poco coherentes con el orden interno de la propuesta. This doctoral thesis is the fruit of a profound pedagogical vocation. The central hypothesis was inspired by a question of great personal interest, and this interest has, since the very beginning of the doctorate, been the driving force behind all subsequent lines of research and investigation. The “Casa Domínguez” represents a paradigm of the dialectics found in the work of Alejandro de la Sota. The perception of reality as antagonistic categories determines a polarized conceptual order, a network of mutually excluding associations upon which Sota builds his own personal operative protocol: intellectual or popular architecture, experimental or traditional, universal or local, heavy or light, above or below ground, etc. Through the analysis and dissection of the “Casa Domínguez”, one of Sota’s smallest projects, an attempt is made to approach the underlying question posed in “Sotian” work as a whole. This is about organization without precedent, raising the strategic dialectics to levels of paroxysm. The house is divided into two separate levels, the day-time level above ground, and the lower night-time level beneath the surface of the ground. Each level has its own geometrical and stuctural order, its own language and character, its own identity and even has its own construction budget. The interaction between the two areas is centered on the two functions of rest and activity, and this in turn establishes a complex relationship network between both, which is sometimes self-evident, but at other times jealously guarded. The living area designed for daily activity is presented as an object of light structure and delicate skin; the precise geometry of the cube delimiting the ever watchful living area’s domain over the land it has conquered. A green topography is created on the slope below which lies an area adapted for rest and relaxation. Two bedrooms, built around patios, skylights and light crevices, generate an entirely independent environment: the construction of an object as opposed to the creation of a landscape. The “Casa Domínguez” project has been subject to much less scrutiny and examination than Don Alejandro’s other works, and is consequently less well-known. A succession of journals have printed the blueprint document together with a poetic description (epopee), composed by Sota himself, to mark the project’s publication. There has, however, scarcely been more than two brief critical appraisals, those by Miguel Ángel Baldellou and more recently by Moisés Puente, that have regarded the project as a monographic work. The project and works nevertheless occupied no less than ten years of De La Sota’s life, with over a hundred draft drawings for two separate versions of the project, the first of which remains unpublished. The sheer determination to design this “small” work in the most meticulous detail, drove Sota to manage and select its interior furniture, as indeed he had previously done with more “important” works like the Tarragona Civil Government, César Carlos College, or the Post Office telecommunications building in León. Client collaboration, maintained over a period of almost forty years, has facilitated an impressive array of the project’s tools and resources. The choice of “Casa Domínguez” as the central subject matter of this thesis, was made in pursuance of a triple objective: firstly, to approach the project as a paradigm of the “Sotian” dialectic, the analysis of the discourse between the heroic character and the finished building; secondly, a rigorous scientific investigation, and progressive disassembling and dissecting of the architectonic object; and finally, a reflection on aspects of the project and its technology which codify the identification between the action of construction and the reality of living, thus marking its achievements, whilst at the same time subjecting incoherent elements of the proposal’s established order to a critical evaluation.

Sensitization profiles and cross-reactivity among plant allergens. Molecular mechanisms of food allergy development and the role of enhancers

La prevalencia de las alergias está aumentando desde mediados del siglo XX, y se estima que actualmente afectan a alrededor del 2-8 % de la población, pero las causas de este aumento aún no están claras. Encontrar el origen del mecanismo por el cual una proteína inofensiva se convierte en capaz de inducir una respuesta alérgica es de vital importancia para prevenir y tratar estas enfermedades. Aunque la caracterización de alérgenos relevantes ha ayudado a mejorar el manejo clínico y a aclarar los mecanismos básicos de las reacciones alérgicas, todavía queda un largo camino para establecer el origen de la alergenicidad y reactividad cruzada. El objetivo de esta tesis ha sido caracterizar las bases moleculares de la alergenicidad tomando como modelo dos familias de panalergenos (proteínas de transferencia de lípidos –LTPs- y taumatinas –TLPs-) y estudiando los mecanismos que median la sensibilización y la reactividad cruzada para mejorar tanto el diagnóstico como el tratamiento de la alergia. Para ello, se llevaron a cabo dos estrategias: estudiar la reactividad cruzada de miembros de familias de panalérgenos; y estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas. Para estudiar la reactividad cruzada entre miembros de la misma familia de proteínas, se seleccionaron LTPs y TLPs, descritas como alergenos, tomando como modelo la alergia a frutas. Por otra parte, se estudiaron los perfiles de sensibilización a alérgenos de trigo relacionados con el asma del panadero, la enfermedad ocupacional más relevante de origen alérgico. Estos estudios se llevaron a cabo estandarizando ensayos tipo microarrays con alérgenos y analizando los resultados por la teoría de grafos. En relación al estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas, se llevaron a cabo estudios sobre la interacción de los alérgenos alimentarios con células del sistema inmune humano y murino y el epitelio de las mucosas, analizando la importancia de moléculas co-transportadas con los alérgenos en el desarrollo de una respuesta Th2. Para ello, Pru p 3(LTP y alérgeno principal del melocotón) se selección como modelo para llevarlo a cabo. Por otra parte, se analizó el papel de moléculas activadoras del sistema inmune producidas por patógenos en la inducción de alergias alimentarias seleccionando el modelo kiwi-alternaria, y el papel de Alt a 1, alérgeno mayor de dicho hongo, en la sensibilización a Act d 2, alérgeno mayor de kiwi. En resumen, el presente trabajo presenta una investigación innovadora aportando resultados de gran utilidad tanto para la mejora del diagnóstico como para nuevas investigaciones sobre la alergia y el esclarecimiento final de los mecanismos que caracterizan esta enfermedad. ABSTRACT Allergies are increasing their prevalence from mid twentieth century, and they are currently estimated to affect around 2-8% of the population but the underlying causes of this increase remain still elusive. The understanding of the mechanism by which a harmless protein becomes capable of inducing an allergic response provides us the basis to prevent and treat these diseases. Although the characterization of relevant allergens has led to improved clinical management and has helped to clarify the basic mechanisms of allergic reactions, it seems justified in aspiring to molecularly dissecting these allergens to establish the structural basis of their allergenicity and cross-reactivity. The aim of this thesis was to characterize the molecular basis of the allergenicity of model proteins belonging to different families (Lipid Transfer Proteins –LTPs-, and Thaumatin-like Proteins –TLPs-) in order to identify mechanisms that mediate sensitization and cross reactivity for developing new strategies in the management of allergy, both diagnosis and treatment, in the near future. With this purpose, two strategies have been conducted: studies of cross-reactivity among panallergen families and molecular studies of the contribution of cofactors in the induction of the allergic response by these panallergens. Following the first strategy, we studied the cross-reactivity among members of two plant panallergens (LTPs , Lipid Transfer Proteins , and TLPs , Thaumatin-like Proteins) using the peach allergy as a model. Similarly, we characterized the sensitization profiles to wheat allergens in baker's asthma development, the most relevant occupational disease. These studies were performed using allergen microarrays and the graph theory for analyzing the results. Regarding the second approach, we analyzed the interaction of plant allergens with immune and epithelial cells. To perform these studies , we examined the importance of ligands and co-transported molecules of plant allergens in the development of Th2 responses. To this end, Pru p 3, nsLTP (non-specific Lipid Transfer Protein) and peach major allergen, was selected as a model to investigate its interaction with cells of the human and murine immune systems as well as with the intestinal epithelium and the contribution of its ligand in inducing an allergic response was studied. Moreover, we analyzed the role of pathogen associated molecules in the induction of food allergy. For that, we selected the kiwi- alternaria system as a model and the role of Alt a 1 , major allergen of the fungus, in the development of Act d 2-sensitization was studied. In summary, this work presents an innovative research providing useful results for improving diagnosis and leading to further research on allergy and the final clarification of the mechanisms that characterize this disease.

Una leve Arqueología de la escala

Esta es una tesis con dos partes bien diferenciadas; en la primera se habla de conceptos como la escala, el tamaño y la medida en el mundo de la antigua Roma. Para ello se eligen edificios con características específicas que pueden explicar estos términos, como la” Villa Adriana” en la que se habla del tamaño desde la acumulación de dimensiones medias, el “Ara Pacis”, en la que se describe la ambigüedad dimensional y el tamaño, el Panteón en el que habla de la escala, y el Santuario de Baalbek a través del cual se reflexiona sobre la condición de monumentalidad. Dichos edificios se comparan con piezas de arte contemporáneo que ayudan a entender el contenido histórico. En ese sentido la tesis propone un viaje desde lo moderno a lo antiguo, instalándose en la certeza de que el conocimiento ampliado de los conceptos contemporáneos otorga nueva vida a los edificios históricos, en un paseo inverso al clásico de estudiar lo antiguo para entender el presente. La tesis defiende que el presente se lee desde el presente y el pasado también desde el presente, elaborando nuevas narrativas que permiten superponer realidades distintas para alumbrar lo real. La segunda parte de la tesis compara la medida de lo público y lo privado así como la medida del paisaje. Habla de la medida del Poder ejemplificada en el proyecto de los Foros Imperiales desde el Foro de César hasta el Foro de Trajano y elabora una comparación dimensional desde la disección de sesenta casas pompeyanas, que son sometidas a un cuidadoso análisis métrico y relacional. Se estudian así mismo sus distintas proporciones desde el tratado de Vitrubio y se analizan sus sistemas de proyecto, encontrando en la ampliación acumulativa un mecanismo que relaciona lo público y lo privado. Por último se estudia la medida del paisaje, encontrando un apoyo en la pintura mural romana y también en la relación entre los cuatro Santuarios Republicanos de Lacio. ABSTRACT This is a thesis with two different parts; the first one deals with concepts from the Ancient Rome, such as scale, size and measure. Some buildings with specific characteristics have been chosen in order to explain these terms, such as "Villa Adriana" where size is formed by the accumulation of medium sizes, the "Ara Pacis",where the dimensional ambiguity is described, the Pantheon where the scale is explained and the Sanctuary of Baalbek where the condition of monumentality is understood. These buildings are compared with contemporary artworks that help to understand the historical contents. In that sense, the thesis proposes a journey from the modern to the old, settling in the certainty that the extended knowledge of contemporary concepts gives new life to historic buildings, instead of the usual walk to the old to understand the present . The thesis argues that both the present and the past can be read through the present, developing new narratives that are overlayed to light reality. The second part of the thesis compares the measures in public and private worlds as well as the measure in the landscape. It is also about the measure of the power, that is exemplified in the Fori Imperiali as the Forum of Caesar and Trajan's Forum. Besides a dimensional comparison is made by dissecting sixty Pompeian houses, which are subjected to careful and relational analysis. Furthermore, their different proportions are studied, since the Treaty of Vitruvius and their design systems are analyzed, finding the mechanism that links the public and private spheres in the cumulative expansion. Finally , the measure of the landscape is studied supported by Roman wall painting and in the relationship between the four Sanctuaries Republicans of Lazio.

Post-embryonic organogenesis and plant regeneration from tissues: two sides of the same coin?

Plants have extraordinary developmental plasticity as they continuously form organs during post-embryonic development. In addition they may regenerate organs upon in vitro hormonal induction. Advances in the field of plant regeneration show that the first steps of de novo organogenesis through in vitro culture in hormone containing media (via formation of a proliferating mass of cells or callus) require root post-embryonic developmental programs as well as regulators of auxin and cytokinin signaling pathways. We review how hormonal regulation is delivered during lateral root initiation and callus formation. Implications in reprograming, cell fate and pluripotency acquisition are discussed. Finally, we analyze the function of cell cycle regulators and connections with epigenetic regulation. Future work dissecting plant organogenesis driven by both endogenous and exogenous cues (upon hormonal induction) may reveal new paradigms of common regulation.

Descripción de parámetros glóticos basados en el modelado de los pliegues vocales para la detección de patología de la voz

Las patologías de la voz se han transformado en los últimos tiempos en una problemática social con cierto calado. La contaminación de las ciudades, hábitos como el de fumar, el uso de aparatos de aire acondicionado, etcétera, contribuyen a ello. Esto alcanza más relevancia en profesionales que utilizan su voz de manera frecuente, como, por ejemplo, locutores, cantantes, profesores o teleoperadores. Por todo ello resultan de especial interés las técnicas de ayuda al diagnóstico que son capaces de extraer conclusiones clínicas a partir de una muestra de la voz grabada con un micrófono, frente a otras invasivas que implican la exploración utilizando laringoscopios, fibroscopios o videoendoscopios, técnicas en cualquier caso mucho más molestas para los pacientes al exigir la introducción parcial del instrumental citado por la garganta, en actuaciones consideradas de tipo quirúrgico. Dentro de aquellas técnicas se ha avanzado mucho en un período de tiempo relativamente corto. En lo que se refiere al diagnóstico de patologías, hemos pasado en los últimos quince años de trabajar principalmente con parámetros extraídos de la señal de voz –tanto en el dominio del tiempo como en el de la frecuencia– y con escalas elaboradas con valoraciones subjetivas realizadas por expertos a hacerlo también con parámetros procedentes de estimaciones de la fuente glótica. La importancia de utilizar la fuente glótica reside, a grandes rasgos, en que se trata de una señal vinculada directamente al estado de la estructura laríngea del locutor y también en que está generalmente menos influida por el tracto vocal que la señal de voz. Es conocido que el tracto vocal guarda más relación con el mensaje hablado, y su presencia dificulta el proceso de detección de patología vocal. Estas estimaciones de la fuente glótica han sido obtenidas a través de técnicas de filtrado inverso desarrolladas por nuestro grupo de investigación. Hemos conseguido, además, profundizar en la naturaleza de la señal glótica: somos capaces de descomponerla y relacionarla con parámetros biomecánicos de los propios pliegues vocales, obteniendo estimaciones de elementos como la masa, la pérdida de energía o la elasticidad del cuerpo y de la cubierta del pliegue, entre otros. De las componentes de la fuente glótica surgen también los denominados parámetros biométricos, relacionados con la forma de la señal, que constituyen por sí mismos una firma biométrica del individuo. También trabajaremos con parámetros temporales, relacionados con las diferentes etapas que se observan dentro de la señal glótica durante un ciclo de fonación. Por último, consideraremos parámetros clásicos de perturbación y energía de la señal. En definitiva, contamos ahora con una considerable cantidad de parámetros glóticos que conforman una base estadística multidimensional, destinada a ser capaz de discriminar personas con voces patológicas o disfónicas de aquellas que no presentan patología en la voz o con voces sanas o normofónicas. Esta tesis doctoral se ocupa de varias cuestiones: en primer lugar, es necesario analizar cuidadosamente estos nuevos parámetros, por lo que ofreceremos una completa descripción estadística de los mismos. También estudiaremos cuestiones como la distribución de los parámetros atendiendo a criterios como el de normalidad estadística de los mismos, ocupándonos especialmente de la diferencia entre las distribuciones que presentan sujetos sanos y sujetos con patología vocal. Para todo ello emplearemos diferentes técnicas estadísticas: generación de elementos y diagramas descriptivos, pruebas de normalidad y diversos contrastes de hipótesis, tanto paramétricos como no paramétricos, que considerarán la diferencia entre los grupos de personas sanas y los grupos de personas con alguna patología relacionada con la voz. Además, nos interesa encontrar relaciones estadísticas entre los parámetros, de cara a eliminar posibles redundancias presentes en el modelo, a reducir la dimensionalidad del problema y a establecer un criterio de importancia relativa en los parámetros en cuanto a su capacidad discriminante para el criterio patológico/sano. Para ello se aplicarán técnicas estadísticas como la Correlación Lineal Bivariada y el Análisis Factorial basado en Componentes Principales. Por último, utilizaremos la conocida técnica de clasificación Análisis Discriminante, aplicada a diferentes combinaciones de parámetros y de factores, para determinar cuáles de ellas son las que ofrecen tasas de acierto más prometedoras. Para llevar a cabo la experimentación se ha utilizado una base de datos equilibrada y robusta formada por doscientos sujetos, cien de ellos pertenecientes al género femenino y los restantes cien al género masculino, con una proporción también equilibrada entre los sujetos que presentan patología vocal y aquellos que no la presentan. Una de las aplicaciones informáticas diseñada para llevar a cabo la recogida de muestras también es presentada en esta tesis. Los distintos estudios estadísticos realizados nos permitirán identificar aquellos parámetros que tienen una mayor contribución a la hora de detectar la presencia de patología vocal. Alguno de los estudios, además, nos permitirá presentar una ordenación de los parámetros en base a su importancia para realizar la detección. Por otra parte, también concluiremos que en ocasiones es conveniente realizar una reducción de la dimensionalidad de los parámetros para mejorar las tasas de detección. Por fin, las propias tasas de detección constituyen quizá la conclusión más importante del trabajo. Todos los análisis presentes en el trabajo serán realizados para cada uno de los dos géneros, de acuerdo con diversos estudios previos que demuestran que los géneros masculino y femenino deben tratarse de forma independiente debido a las diferencias orgánicas observadas entre ambos. Sin embargo, en lo referente a la detección de patología vocal contemplaremos también la posibilidad de trabajar con la base de datos unificada, comprobando que las tasas de acierto son también elevadas. Abstract Voice pathologies have become recently in a social problem that has reached a certain concern. Pollution in cities, smoking habits, air conditioning, etc. contributes to it. This problem is more relevant for professionals who use their voice frequently: speakers, singers, teachers, actors, telemarketers, etc. Therefore techniques that are capable of drawing conclusions from a sample of the recorded voice are of particular interest for the diagnosis as opposed to other invasive ones, involving exploration by laryngoscopes, fiber scopes or video endoscopes, which are techniques much less comfortable for patients. Voice quality analysis has come a long way in a relatively short period of time. In regard to the diagnosis of diseases, we have gone in the last fifteen years from working primarily with parameters extracted from the voice signal (both in time and frequency domains) and with scales drawn from subjective assessments by experts to produce more accurate evaluations with estimates derived from the glottal source. The importance of using the glottal source resides broadly in that this signal is linked to the state of the speaker's laryngeal structure. Unlike the voice signal (phonated speech) the glottal source, if conveniently reconstructed using adaptive lattices, may be less influenced by the vocal tract. As it is well known the vocal tract is related to the articulation of the spoken message and its influence complicates the process of voice pathology detection, unlike when using the reconstructed glottal source, where vocal tract influence has been almost completely removed. The estimates of the glottal source have been obtained through inverse filtering techniques developed by our research group. We have also deepened into the nature of the glottal signal, dissecting it and relating it to the biomechanical parameters of the vocal folds, obtaining several estimates of items such as mass, loss or elasticity of cover and body of the vocal fold, among others. From the components of the glottal source also arise the so-called biometric parameters, related to the shape of the signal, which are themselves a biometric signature of the individual. We will also work with temporal parameters related to the different stages that are observed in the glottal signal during a cycle of phonation. Finally, we will take into consideration classical perturbation and energy parameters. In short, we have now a considerable amount of glottal parameters in a multidimensional statistical basis, designed to be able to discriminate people with pathologic or dysphonic voices from those who do not show pathology. This thesis addresses several issues: first, a careful analysis of these new parameters is required, so we will offer a complete statistical description of them. We will also discuss issues such as distribution of the parameters, considering criteria such as their statistical normality. We will take special care in the analysis of the difference between distributions from healthy subjects and the distributions from pathological subjects. To reach these goals we will use different statistical techniques such as: generation of descriptive items and diagramas, tests for normality and hypothesis testing, both parametric and nonparametric. These latter techniques consider the difference between the groups of healthy subjects and groups of people with an illness related to voice. In addition, we are interested in finding statistical relationships between parameters. There are various reasons behind that: eliminate possible redundancies in the model, reduce the dimensionality of the problem and establish a criterion of relative importance in the parameters. The latter reason will be done in terms of discriminatory power for the criterion pathological/healthy. To this end, statistical techniques such as Bivariate Linear Correlation and Factor Analysis based on Principal Components will be applied. Finally, we will use the well-known technique of Discriminant Analysis classification applied to different combinations of parameters and factors to determine which of these combinations offers more promising success rates. To perform the experiments we have used a balanced and robust database, consisting of two hundred speakers, one hundred of them males and one hundred females. We have also used a well-balanced proportion where subjects with vocal pathology as well as subjects who don´t have a vocal pathology are equally represented. A computer application designed to carry out the collection of samples is also presented in this thesis. The different statistical analyses performed will allow us to determine which parameters contribute in a more decisive way in the detection of vocal pathology. Therefore, some of the analyses will even allow us to present a ranking of the parameters based on their importance for the detection of vocal pathology. On the other hand, we will also conclude that it is sometimes desirable to perform a dimensionality reduction in order to improve the detection rates. Finally, detection rates themselves are perhaps the most important conclusion of the work. All the analyses presented in this work have been performed for each of the two genders in agreement with previous studies showing that male and female genders should be treated independently, due to the observed functional differences between them. However, with regard to the detection of vocal pathology we will consider the possibility of working with the unified database, ensuring that the success rates obtained are also high.

Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1

Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory–fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.

Mechanism-based inhibition of the melatonin rhythm enzyme: Pharmacologic exploitation of active site functional plasticity

Serotonin N-acetyltransferase is the enzyme responsible for the diurnal rhythm of melatonin production in the pineal gland of animals and humans. Inhibitors of this enzyme active in cell culture have not been reported previously. The compound N-bromoacetyltryptamine was shown to be a potent inhibitor of this enzyme in vitro and in a pineal cell culture assay (IC50 ≈ 500 nM). The mechanism of inhibition is suggested to involve a serotonin N-acetyltransferase-catalyzed alkylation reaction between N-bromoacetyltryptamine and reduced CoA, resulting in the production of a tight-binding bisubstrate analog inhibitor. This alkyltransferase activity is apparently catalyzed at a functionally distinct site compared with the acetyltransferase activity active site on serotonin N-acetyltransferase. Such active site plasticity is suggested to result from a subtle conformational alteration in the protein. This plasticity allows for an unusual form of mechanism-based inhibition with multiple turnovers, resulting in “molecular fratricide.” N-bromoacetyltryptamine should serve as a useful tool for dissecting the role of melatonin in circadian rhythm as well as a potential lead compound for therapeutic use in mood and sleep disorders.

Sequential and coordinated action of phytochromes A and B during Arabidopsis stem growth revealed by kinetic analysis

Photoreceptor proteins of the phytochrome family mediate light-induced inhibition of stem (hypocotyl) elongation during the development of photoautotrophy in seedlings. Analyses of overt mutant phenotypes have established the importance of phytochromes A and B (phyA and phyB) in this developmental process, but kinetic information that would augment emerging molecular models of phytochrome signal transduction is absent. We have addressed this deficiency by genetically dissecting phytochrome-response kinetics, after having solved the technical issues that previously limited growth studies of small Arabidopsis seedlings. We show here, with resolution on the order of minutes, that phyA initiated hypocotyl growth inhibition upon the onset of continuous red light. This primary contribution of phyA began to decrease after 3 hr of irradiation, the same time at which immunochemically detectable phyA disappeared and an exclusively phyB-dependent phase of inhibition began. The sequential and coordinated actions of phyA and phyB in red light were not observed in far-red light, which inhibited growth persistently through an exclusively phyA-mediated pathway.

Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

Analysis of homeodomain function by structure-based design of a transcription factor.

The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits.

Signal transduction in T lymphocytes using a conditional allele of Sos.

While Ras activation has been shown to play an important role in signal transduction by the T-lymphocyte antigen receptor, the mechanism of its activation in T cells is unclear. Membrane localization of the guanine nucleotide exchange factor Sos, but not Vav or Dbl, was sufficient for Ras-mediated signaling in T lymphocytes. Activation of Sos appears to involve membrane recruitment and not allosteric changes, because interaction of Sos with the linking molecule Grb-2 was not required for Ras activation. To extend this analysis, we constructed a modified Sos that could be localized to the membrane inducibly by using a rationally designed chemical inducer of dimerization, FK1012. The role of Grb-2 in signaling was mimicked with this technique, which induced the association of a modified Sos with the membrane, resulting in rapid activation of Ras-induced signaling. In contrast, inducible localization of Grb-2 to the membrane did not activate signaling and suggests that the interaction of Grb-2 with Sos in T cells is subject to regulation. This conditional allele of Sos demonstrates that membrane localization of Sos is sufficient for Ras activation in T cells and indicates that the role of Grb-2 is to realize the biologic advantages of linker-mediated dimerization: enhanced specificity and favorable kinetics for signaling. This method of generating conditional alleles may also be useful in dissecting other signal transduction pathways regulated by protein localization or protein-protein interactions.

Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase.

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free system activators. NADPH oxidase activation by PA plus DG is reduced approximately 70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by approximately 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-phox precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.

The inositol 1,4,5-trisphosphate receptor is essential for T-cell receptor signaling.

Antigen-specific activation of T lymphocytes, via stimulation of the T-cell antigen receptor (TCR) complex, is marked by a rapid and sustained increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). It has been suggested that the second messenger inositol 1,4,5-trisphosphate (IP3) produced after TCR stimulation binds to the IP3 receptor (IP3R), an intracellular Ca(2+)-release channel, and triggers the increase in [Ca2+]i that activates transcription of the gene for T-cell growth factor interleukin 2 (IL-2). However, the role of the IP3R in T-cell signaling and possibly in plasma membrane Ca2+ influx in T cells remains unproven. Stable transfection of T cells (Jurkat) with antisense type 1 IP3R cDNA prevented type 1 IP3R expression, providing a tool for dissecting the role of IP3 signaling during T-cell activation. T cells lacking type 1 IP3R failed to increase [Ca2+]i or produce IL-2 after TCR stimulation. Moreover, depletion of intracellular Ca2+ stores without TCR activation stimulated Ca2+ influx in cells lacking the type 1 IP3R. These results establish that the type 1 IP3R is required for intracellular Ca2+ release that triggers antigen-specific T-cell proliferation but not for plasma membrane Ca2+ influx.

Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension.

Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. The resulting "knockout" mice have confirmed some hypotheses, have upset others, but have rarely been uninformative. Models of several human genetic diseases have been produced by targeting--including Gaucher disease, cystic fibrosis, and the fragile X syndrome. These diseases are primarily determined by defects in single genes, and their modes of inheritance are well understood. When the disease under study has a complex etiology with multiple genetic and environmental components, the generation of animal models becomes more difficult but no less valuable. The problems associated with dissecting out the individual genetic factors also increases substantially and the distinction between causation and correlation is often difficult. To prove causation in a complex system requires rigorous adherence to the principle that the experiments must allow detection of the effects of changing only a single variable at one time. Gene targeting experiments, when properly designed, can test the effects of a precise genetic change completely free from the effects of differences in any other genes (linked or unlinked to the test gene). They therefore allow proofs of causation.

Expression profiling and sequence diversity of novel DREB genes from common bean (Phaseolus vulgaris L.) and their association with drought-related traits

Common bean is a major dietary component in several countries, but its productivity is negatively affected by abiotic stresses. Dissecting candidate genes involved in abiotic stress tolerance is a paramount step toward the improvement of common bean performance under such constraints. Thereby, this thesis presents a systematic analysis of the DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) gene subfamily, which encompasses genes that regulate several processes during stress responses, but with limited information for common bean. First, a series of in silico analyses with sequences retrieved from the P. vulgaris genome on Phytozome supported the categorization of 54 putative PvDREB genes distributed within six phylogenetic subgroups (A-1 to A-6), along the 11 chromosomes. Second, we cloned four novel PvDREB genes and determined their inducibility-factors, including the dehydration-, salinity- and cold-inducible genes PvDREB1F and PvDREB5A, and the dehydration- and cold-inducible genes PvDREB2A and PvDREB6B. Afterwards, nucleotide polymorphisms were searched through Sanger sequencing along those genes, revealing a high number of single nucleotide polymorphisms within PvDREB6B by the comparison of Mesoamerican and Andean genotypes. The nomenclature of PvDREB6B is discussed in details. Furthermore, we used the BARCBean6K_3 SNP platform to identify and genotype the closest SNP to each one of the 54 PvDREB genes. We selected PvDREB6B for a broader study encompassing a collection of wild common bean accessions of Mesoamerican origin. The population structure of the wild beans was accessed using sequence polymorphisms of PvDREB6B. The genetic clusters were partially associated with variation in latitude, altitude, precipitation and temperature throughout the areas such beans are distributed. With an emphasis on drought stress, an adapted tube-screening method in greenhouse conditions enabled the phenotyping of several drought-related traits in the wild collection. Interestingly, our data revealed a correlation between root depth, plant height and biomass and the environmental data of the location of the accessions. Correlation was also observed between the population structure determined through PvDREB6B and the environmental data. An association study combining data from the SNP array and DREB polymorphisms enabled the detection of SNP associated with drought-related traits through a compressed mixed linear model (CMLM) analysis. This thesis highlighted important features of DREB genes in common bean, revealing candidates for further strategies aimed at improvement of abiotic stress tolerance, with emphasis on drought tolerance