Characterization of tomato Cycling Dof Factors reveals conserved and new functions in the control of flowering time and abiotic stress responses.

DNA binding with One Finger (DOF) transcription factors are involved in multiple aspects of plant growth and development but their precise roles in abiotic stress tolerance are largely unknown. Here we report a group of five tomato DOF genes, homologous to Arabidopsis Cycling DOF Factors (CDFs), that function as transcriptional regulators involved in responses to drought and salt stress and flowering-time control in a gene-specific manner. SlCDF1?5 are nuclear proteins that display specific binding with different affinities to canonical DNA target sequences and present diverse transcriptional activation capacities in vivo. SlCDF1?5 genes exhibited distinct diurnal expression patterns and were differentially induced in response to osmotic, salt, heat, and low-temperature stresses. Arabidopsis plants overexpressing SlCDF1 or SlCDF3 showed increased drought and salt tolerance. In addition, the expression of various stress-responsive genes, such as COR15, RD29A, and RD10, were differentially activated in the overexpressing lines. Interestingly, overexpression in Arabidopsis of SlCDF3 but not SlCDF1 promotes late flowering through modulation of the expression of flowering control genes such as CO and FT. Overall, our data connect SlCDFs to undescribed functions related to abiotic stress tolerance and flowering time through the regulation of specific target genes and an increase in particular metabolites

Asplund versus Lewerentz

La tesis Asplund versus Lewerentz analiza, por el método comparado, los invariantes, las convergencias y las divergencias entre las obras de los arquitectos suecos Erik Gunnar Asplund y Sigurd Lewerentz. Autores del Cementerio del Bosque de Estocolmo, que desarrollaron en colaboración durante más de veinte años, supone una de las obras maestras de la arquitectura escandinava, y uno de los ejemplos de referencia para entender la cultura arquitectónica nórdica moderna en relación a su tradición. Del estudio contrastado de los distintos proyectos y versiones del cementerio, ejecutados conjuntamente o por separado, se obtienen los valores compartidos entre ambos arquitectos, así como sus afinidades o diferencias respecto a un mismo tema. Estos aspectos, extrapolados al resto de sus trabajos, permiten entender sus actuaciones según las referencias e influencias mutuas, y la definición de las variables que configuran sus respectivos espacios arquitectónicos. La investigación se ha estructurado en ocho capítulos: La Escuela Klara, que trata la relación de Asplund y Lewerentz en su época de estudiantes, así como la influencia de sus maestros respectivos; El Grand Tour, que analiza sus viajes y las miradas de cada uno de ellos en sus visitas a los mismos lugares; Los Estudios, que profundiza en sus caracteres, reflejados a través de la comparación de los distintos lugares de trabajo; Las Tumbas, donde se concretan a pequeña escala los temas desarrollados ampliamente en el Cementerio del Bosque, constituyendo así un laboratorio de ideas; La Exposición de Estocolmo de 1930, que vuelve a aclarar las posturas divergentes de ambos, en este caso, en relación a un tema lúdico, y que supuso un giro de su arquitectura y, con ello, un importante apoyo a los cambios sociales del momento; Los Concursos, realizados por separado, donde se observan con precisión las afinidades y las diferencias respecto a un mismo planteamiento inicial; Los Accesos del Cementerio del Bosque, que supone un trabajo conjunto con alternancia de decisiones, en el que se perfila con claridad los intereses específicos de ambos; y por último, las Capillas del Cementerio del Bosque, especialmente la Capilla del Bosque y de la Resurrección, que concretan, en los dos lugares más íntimos del cementerio, las ideas y personalidades opuestas de Asplund y Lewerentz en relación al tema común de la muerte. Las conclusiones de este estudio comparado y centrado en varios temas, determinan una confluencia de intereses en la conexión de sus obras con la topografía como medio de contextualización, en las acciones sobre la vegetación como un aspecto fundamental y no sólo como un acompañamiento a la arquitectura, y en la manipulación de la luz como hecho diferencial del carácter de los distintos espacios. Se reúnen así en esta tesis unas constantes del trabajo de Asplund y Lewerentz, que planteadas muchas veces de forma distinta, apuntan directamente a los sentimientos del Hombre en el momento de la muerte y ante una despedida en relación a la Arquitectura. ABSTRACT The thesis Asplund versus Lewerentz analyses, by means of comparative methodology, the constants, the convergences and the divergences between the works of the Swedish architects Erik Gunnar Asplund and Sigurd Lewerentz. They are the authors of Stockholm’s Woodland Cemetery, which they developed in collaboration over a period of more than twenty years, one of the masterpieces of Swedish architecture and one of the key examples in terms of understanding modern Nordic architectural culture in relation to its tradition. From the contrasted study of the various projects and versions of the cemetery, executed jointly or separately, one obtains the values shared by both architects as well as their affinities or differences with respect to one and the same theme. These aspects, extrapolated from the rest of their works, permit an understanding of their actions according to mutual references and influences, and the definition of the variables that shape their respective architectural spaces. The research is divided into eight chapters: The Klara School, dealing with Asplund and Lewerentz’s relationship during their student days, as well as the influence of their respective teachers; The Grand Tour, which analyses their travels and the viewpoints of each of them on their visits to the same places; The Studies, looks closely at their personalities, reflected by a comparison of their different workplaces; The Tombs, where the themes extensively developed in the Woodland Cemetery are set out on a small scale, thus constituting one of their laboratories of ideas; The Stockholm exhibition of 1930, which again clarifies the diverging postures of the two, in this case, in relation to a recreational topic which represented a turnabout in their architecture and, moreover an important support for the social changes of the age; The Competitions, which they carried out separately, where one closely observes affinities and differences with respect to an initial approach; The Accesses to the Woodland Cemetery, which represents a joint work with alternating decision-making, in which the interests of both are clearly outlined; and finally, the Chapels of the Woodland Cemetery, especially the Woodland Chapel and the Chapel of the Resurrection, which, in the two most intimate spots in the cemetery, embody the opposing ideas and personalities of Asplund and Lewerentz in relation to the common theme of death. The conclusions of this comparative study centred on various topics, determine a confluence of interests in the connection of their works with the topography as a means of contextualization, in actions relating to vegetation as a fundamental aspect and not just an accompaniment to architecture, and in the manipulation of light as a differential factor in the character of the different spaces. Thus this thesis brings together some of the constants of the work of Asplund and Lewerentz which, frequently dealt with in a different manner, point directly to Man’s feelings at the point of death and in the face of a farewell in relation to Architecture.

Arquitectura antidesertización. Fluidez, biodiversidad, hidrofilia y transpirabilidad

La escasez de recursos, la desertización creciente y el previsible calentamiento global forman un escenario físico sin precedentes que urge la revisión de las relaciones entre arquitectura y agua en la urbanización de los paisajes secos, donde la aridez es el factor limitante principal. Cuestionar la idea de escasez, la de residuo o la de confort es el punto de partida para analizar la hidrología urbana. La condición ubicua y dinámica del agua, sus continuos cambios de estado y las implicaciones energéticas y ambientales involucradas argumentan a favor de un entendimiento integral, holístico, que aborda el diseño en relación a lo paisajístico, lo infraestructural y lo ambiental como un único ámbito de investigación, planteado en términos no solo de materia o de escasez, sino de energía. Este trabajo se interesa por el rol de la arquitectura en este proyecto conjunto, integral, del espacio físico con el ciclo hidrológico donde lo orgánico, lo geológico y lo atmosférico están ligados, y donde las categorías de lo hidrológico y lo hidráulico se confunden. Las profundas implicaciones culturales en la construcción de la naturaleza urbana y en la relación con el cuerpo humano adquieren especial notoriedad en los modelos de paisaje y de espacio público adoptados, importados desde las geografías húmedas, y en la estigmatización del agua desde los presupuestos del higienismo decimonónico, que conduce al hidrofugado general de una ciudad aséptica y estéril, que se extiende hasta las envolventes de fachadas estancas y los interiores blancos y satinados. La búsqueda de referentes salta la modernidad hacia contextos de baja energía como el oasis tradicional de las tierras áridas, que concilia la formación de un ecosistema productivo con la climatización de un espacio público exterior, o los ambientes aislados de la investigación aeroespacial, con ciclos cerrados de materia. Las condiciones del ciclo del agua, como la vinculación con el suelo y el territorio, la capacidad de disolver, de mezclarse y de lavar, su volumen variable y su papel como termorregulador señalan el interés que características físicas como la fluidez, la biodiversidad, la hidrofilia y la transpirabilidad tienen para una arquitectura con competencias hidráulicas y un espacio urbano transparente a los procesos del agua. La morfología y localización, la elección de escalas, jerarquías y relaciones entre espacios y la reformulación de los sistemas constructivos aparecen como herramientas y categorías propias desde las que proponer mejores respuestas a problemas como la deshidratación, la erosión y la contaminación. Una urbanización permeable e hidrófila que absorba el agua como un recurso valioso, nuevos ensamblajes para la bioquímica urbana (que introduzcan una idea de limpieza más próxima a fertilidad que a la desinfección), y el diseño de las condiciones atmosféricas a través de una arquitectura transpirable, que se empapa y rezuma frescor, son las claves de este nuevo proyecto. La ciudad se piensa como una síntesis multiescalar de espacios hidráulicos que aporta mayor resiliencia frente a la desertificación y las condiciones climáticas extremas, y mayor visibilidad en la escena pública al agua y a las inevitables conexiones entre ecología y economía. Pero también es una oportunidad para revisar las categorías disciplinares, para renovar las consistencias materiales, las calidades ambientales y las relaciones entre el cuerpo y el espacio. ABSTRACT The shortage of resources, foreseeable global warming and increasing desertification create an unprecedented prospect that question the existing relationships between water and architecture, in the urbanization of the arid lands. The awareness of the huge volumes of water that pierce unnoticed through urban space, their impact on the landscape as well as on environmental qualities, promote a design field where cultural, social and political considerations intersect, related to the body and the physical experience of space within the built environment. Fluidity and ubiquity, solution ability, variability and cyclical processes are characteristic of water as material, directly related with the fields of potential, chemical and thermal energy and the reality of its mass, as it occupies a changing volume in space. These are also the imposing cross sections that water introduces into the project, that argue in favor of a comprehensive and holistic understanding, of addressing design in relationship to landscape, infrastructure and environmental issues as a one single area of research. This work attempts to investigate how architecture, with its specific tools, can partake in the design of water cycle in the space, linking the organic, geological, and atmospheric, blurring the lines between hydrology and hydraulic. It aims to identify issues, within the continuous query associated with water, that deal with the architectural project and may have here better results. The deep cultural implications in the construction of urban nature and the relationship with the body, acquire special notoriety in the models of landscape and public space adopted, imported from humid geographies. Also in the stigmatization of water from the premises of nineteenth- century hygienics, which lead to the entire waterproofing of an aseptic and sterile city, to the sealed facades and white and polished interiors. The search for alternative references goes beyond modernity towards a mindset of low energy, as the traditional oasis of arid lands, which aims to reconcile the formation of a productive ecosystem with the conditioning of an outdoor public space, or the controlled environments of aerospace research, with closed cycles of matter. Fluidity, biodiversity, hydrophilicity and breathability are characteristic of an architecture with hydraulic competences. The distributing phenomenon of water, its necessary connectivity to the ground and to small cycles in the ecosystems, shows strong affinities with an infrastructural architecture, as an alternative to large-scale centralized networks. Its volume has approximated to the dimensions of the built space, promoting a new found condition of coexistence. A permeable and hydrophilic urbanization absorbs water as a valuable resource; new assemblies for urban biochemistry introduce an idea of sanitation closer to fertility than to disinfection; a breathable architecture that soaks and exudes freshness design the atmospheric conditions: these are the essential components of this new project. The city is understood as a synthesis of multi-scale hydraulic spaces that provides greater resilience against desertification and increases the visibility of water and the linkages between ecology and economy in the public scene. It is also an opportunity to review the disciplinary categories of architecture, the material consistencies, the environmental qualities and the relationship between body and space.

Asplund versus Lewerentz

La tesis Asplund versus Lewerentz analiza, por el método comparado, los invariantes, las convergencias y las divergencias entre las obras de los arquitectos suecos Erik Gunnar Asplund y Sigurd Lewerentz. Autores del Cementerio del Bosque de Estocolmo, que desarrollaron en colaboración durante más de veinte años, supone una de las obras maestras de la arquitectura escandinava, y uno de los ejemplos de referencia para entender la cultura arquitectónica nórdica moderna en relación a su tradición. Del estudio contrastado de los distintos proyectos y versiones del cementerio, ejecutados conjuntamente o por separado, se obtienen los valores compartidos entre ambos arquitectos, así como sus afinidades o diferencias respecto a un mismo tema. Estos aspectos, extrapolados al resto de sus trabajos, permiten entender sus actuaciones según las referencias e influencias mutuas, y la definición de las variables que configuran sus respectivos espacios arquitectónicos. La investigación se ha estructurado en ocho capítulos: La Escuela Klara, que trata la relación de Asplund y Lewerentz en su época de estudiantes, así como la influencia de sus maestros respectivos; El Grand Tour, que analiza sus viajes y las miradas de cada uno de ellos en sus visitas a los mismos lugares; Los Estudios, que profundiza en sus caracteres, reflejados a través de la comparación de los distintos lugares de trabajo; Las Tumbas, donde se concretan a pequeña escala los temas desarrollados ampliamente en el Cementerio del Bosque, constituyendo así un laboratorio de ideas; La Exposición de Estocolmo de 1930, que vuelve a aclarar las posturas divergentes de ambos, en este caso, en relación a un tema lúdico, y que supuso un giro de su arquitectura y, con ello, un importante apoyo a los cambios sociales del momento; Los Concursos, realizados por separado, donde se observan con precisión las afinidades y las diferencias respecto a un mismo planteamiento inicial; Los Accesos del Cementerio del Bosque, que supone un trabajo conjunto con alternancia de decisiones, en el que se perfila con claridad los intereses específicos de ambos; y por último, las Capillas del Cementerio del Bosque, especialmente la Capilla del Bosque y de la Resurrección, que concretan, en los dos lugares más íntimos del cementerio, las ideas y personalidades opuestas de Asplund y Lewerentz en relación al tema común de la muerte. Las conclusiones de este estudio comparado y centrado en varios temas, determinan una confluencia de intereses en la conexión de sus obras con la topografía como medio de contextualización, en las acciones sobre la vegetación como un aspecto fundamental y no sólo como un acompañamiento a la arquitectura, y en la manipulación de la luz como hecho diferencial del carácter de los distintos espacios. Se reúnen así en esta tesis unas constantes del trabajo de Asplund y Lewerentz, que planteadas muchas veces de forma distinta, apuntan directamente a los sentimientos del Hombre en el momento de la muerte y ante una despedida en relación a la Arquitectura. ABSTRACT The thesis Asplund versus Lewerentz analyses, by means of comparative methodology, the constants, the convergences and the divergences between the works of the Swedish architects Erik Gunnar Asplund and Sigurd Lewerentz. They are the authors of Stockholm’s Woodland Cemetery, which they developed in collaboration over a period of more than twenty years, one of the masterpieces of Swedish architecture and one of the key examples in terms of understanding modern Nordic architectural culture in relation to its tradition. From the contrasted study of the various projects and versions of the cemetery, executed jointly or separately, one obtains the values shared by both architects as well as their affinities or differences with respect to one and the same theme. These aspects, extrapolated from the rest of their works, permit an understanding of their actions according to mutual references and influences, and the definition of the variables that shape their respective architectural spaces. The research is divided into eight chapters: The Klara School, dealing with Asplund and Lewerentz’s relationship during their student days, as well as the influence of their respective teachers; The Grand Tour, which analyses their travels and the viewpoints of each of them on their visits to the same places; The Studies, looks closely at their personalities, reflected by a comparison of their different workplaces; The Tombs, where the themes extensively developed in the Woodland Cemetery are set out on a small scale, thus constituting one of their laboratories of ideas; The Stockholm exhibition of 1930, which again clarifies the diverging postures of the two, in this case, in relation to a recreational topic which represented a turnabout in their architecture and, moreover an important support for the social changes of the age; The Competitions, which they carried out separately, where one closely observes affinities and differences with respect to an initial approach; The Accesses to the Woodland Cemetery, which represents a joint work with alternating decision-making, in which the interests of both are clearly outlined; and finally, the Chapels of the Woodland Cemetery, especially the Woodland Chapel and the Chapel of the Resurrection, which, in the two most intimate spots in the cemetery, embody the opposing ideas and personalities of Asplund and Lewerentz in relation to the common theme of death. The conclusions of this comparative study centred on various topics, determine a confluence of interests in the connection of their works with the topography as a means of contextualization, in actions relating to vegetation as a fundamental aspect and not just an accompaniment to architecture, and in the manipulation of light as a differential factor in the character of the different spaces. Thus this thesis brings together some of the constants of the work of Asplund and Lewerentz which, frequently dealt with in a different manner, point directly to Man’s feelings at the point of death and in the face of a farewell in relation to Architecture.

Vivienda y fondo edificatorio: análisis de las consecuencias del parámetro fondo en el edificio de vivienda colectiva

Este estudio ofrece una herramienta de aproximación al espacio morfológico-métrico en el que se formula la ciudad de alta densidad desde la vivienda colectiva. La vivienda colectiva es la célula básica de la ciudad. El estudio configurativo y dimensional del tejido urbano muestra la importancia del fondo edificatorio como parámetro clave a mitad de camino entre la vivienda y la ciudad. El fondo edificatorio traza el margen de la arquitectura en la ciudad y desde él se equipa y cuantifica el territorio urbano. Sus dinámicas van caracterizando los distintos entornos, mientras en su interior se formula el tipo en un ajuste de continua verificación y adaptación. La forma de la ciudad y sus distintas posibilidades configurativas —en cuanto masa construida y espacio público, pero sin perder de vista la relación entre ambos— depende en gran medida del fondo edificatorio. Se trata, por tanto, de un parámetro importante de relación entre las distintas configuraciones del espacio exterior e interior. Al proyectar, una vez establecido un fondo, algunas propiedades se adaptan con facilidad mientras que otras requieren un cierto grado de interpretación o deben ser descartadas. Dada una superficie, la especificación del fondo fuerza la dimensión del frente en las configuraciones posibles. Ambas dimensiones son vitales en el valor del factor de forma del continuo edificado y en su relación se produce el complejo rango de posibilidades. Partiendo de la ciudad, un gran fondo encierra y mezcla en su interior todo tipo de usos sin distinción, repercute un menor coste por unidad de superficie edificada y comparte su frente reduciendo los intercambios térmicos y lumínicos. Sin embargo la ciudad de fondo reducido ajusta la forma al uso y se desarrolla linealmente con repetitividad a lo largo de sus frentes exteriores. En ella, el fuerte intercambio energético se opone a las grandes posibilidades del espacio libre. En cambio desde la casa las distintas medidas del fondo se producen bajo determinados condicionantes: clima, compacidad, ocupación, hibridación, tamaño de casa, etc., mientras que el tipo se desarrolla en base a una métrica afín. Este trabajo parte de esta dialéctica. Estudia la relación de dependencia entre las condiciones del edificio de viviendas y su métrica. Jerarquiza edificios en base al parámetro “fondo” para constituir una herramienta que como un ábaco sea capaz de visibilizar las dinámicas relacionales entre configuración y métrica bajo la condición de alta densidad. Para ello en una primera fase se gestiona una extensa muestra de edificios representativos de vivienda colectiva principalmente europea, extraída de tres prestigiosos libros en forma de repertorio. Se ordenan y categorizan extrayendo datos conmensurables y temas principales que ligan la profundidad de la huella a la morfología y posteriormente, esta información se estudia en diagramas que ponen de manifiesto convergencias y divergencias, acumulaciones y vacíos, límites, intervalos característicos, márgenes y ejes, parámetros y atributos... cuya relación trata de factorizar el lugar morfológico y métrico de la casa como metavivienda y ciudad. La herramienta se establece así como un complejo marco relacional en el que posicionar casos concretos y trazar nexos transversales, tanto de tipo morfológico como cultural, climático o técnico, normativo o tecnológico. Cada nuevo caso o traza añadida produce consonancias y disonancias en el marco que requieren interpretación y verificación. De este modo este instrumento de análisis comparativo se tempera, se especializa, se completa y se perfecciona con su uso. La forma de la residencia en la ciudad densa se muestra así sobre un subsistema morfológico unitario y su entendimiento se hace más fácilmente alcanzable y acumulable tanto para investigaciones posteriores como para el aprendizaje o el ejercicio profesional. ABSTRACT This research study offers a tool to approach the morphometric space in which (multi-family) housing defines high-density cities. Multi-family housing is the basic cell of the city. The configuration and dimension studies of the urban fabric render the importance of building depth as a key parameter half way between the dwelling and the city. The building depth traces de limit of architecture in the city. It qualifies and quantifies the urban territory. Its dynamics characterize the different environments while in its essence, an adjustment process of continuous verification and adaption defines type. The shape of the city and its different configuration possibilities —in terms of built fabric and public space, always keeping an eye on the relationship between them— depend majorly on the building depth. Therefore, it is a relevant parameter that relates the diverse configurations between interior and exterior space. When designing, once the depth is established, some properties are easily adpated. However, others require a certain degree of interpretation or have to be left out of the study. Given a ceratin surface, the establishment of the depth forces the dimensions of the facade in the different configurations. Both depth and facade dimensions are crucial for the form factor of the built mass. Its relationship produces a complex range of possibilities. From an urban point of view, great depth means multiple uses (making no distinction whatsoever,) it presents a lower cost per unit of built area and shares its facade optimizing temperature and light exchange. On the contrary, the city of reduced depth adjusts its shape to the use, and develops linearly and repetitively along its facades. The strong energy exchange opposes to the great possibilities of free space. From the perspective of the dwelling, the different dimensions of depth are produced under certain determinants: climate, compactness, occupancy, hybridization, dwelling size, etc. Meanwhile, the type is developed based on a related meter (as in poetry). This work starts from the previous premise. It studies the dependency relation bewteen the conditions of the dwellings and their meter (dimensions). It organizes buildings hierarchically based on the parameter “depth” to create a tool that, as an abacus, is able to visibilise the relational dynamics between configuration and dimension in high density conditions. For this, in the first stage a large group of representative multi-family housing buildings is managed, mostly from Europe, picked from three prestigious books as a repertoir. They are categorized and ordered drawing commensurable data and key issues that link the depth of the fooprint to its morphology. Later, this information is studied deeply with diagrams that bring out connections and discrepancies, voids and accumulations, limits, charasteristic intervals, margins and axii, parameters, attributes, etc. These relationships try to create factors from a morphological and metrical point of view of the house as a metadwelling. This tool is established as a complex relation frame in which case studies are postitioned and cross-cutting nexii are traced. These can deal with morphology, climate, technique, law or technology. Each new case or nexus produces affinities and discrepancies that require interpretation and verification. Thus, this instrument of comparative analysis is fine-tuned, especialized and completed as its use is improved. The way housing is understood in high density cities is shown as a unitary metric subsystem and its understanding is easy to reach and accumulate for future researchers, students or practicing architects.

1950 En torno al Museo Louisiana 1970

La tesis “1950 En torno al Museo Louisiana 1970” analiza varias obras relacionadas con el espacio doméstico, que se realizaron entre 1950 y 1970 en Dinamarca, un periodo de esplendor de la Arquitectura Moderna. Tras el aislamiento y restricciones del conflicto bélico que asoló Europa, los jóvenes arquitectos daneses, estaban deseosos por experimentar nuevas ideas de procedencia internacional, favorecidos por diferentes circunstancias, encuentran el mejor campo de ensayo en el espacio doméstico. La mejor arquitectura doméstica en Dinamarca, de aquel periodo, debe entenderse como un sistema compuesto por diferentes autores, que tienen en común muchas más similitudes que diferencias, se complementan unos a otros. Para la comprensión y el entendimiento de ello se hace necesario el estudio de varias figuras y edificios, que completen este sistema cuya investigación está escasamente desarrollada. La tesis propone un viaje para conocer los nombres de algunos de sus protagonistas, que mostraron con su trabajo, que tradición y vanguardia no estarán reñidas. El objetivo es desvelar las claves de la Modernidad Danesa, reconocer, descubrir y recuperar el legado de algunos de sus protagonistas en el ámbito doméstico, cuya lección se considera de total actualidad. Una arquitectura que asume las aportaciones extranjeras con moderación y crítica, cuya íntima relación con la tradición arquitectónica y la artesanía propias, será una de sus notas especiales. Del estudio contrastado de varios proyectos y versiones, se obtienen valores comunes entre sus autores, al igual que se descubren sus afinidades o diferencias respecto a los mismos asuntos; que permitirán comprender sus actuaciones según las referencias e influencias, y definir las variables que configuran sus espacios arquitectónicos. La línea de conexión entre los edificios elegidos será su particular relación con la naturaleza y el lugar en que se integran. La fachada, lugar donde se negociará la relación entre el interior y el paisaje, será un elemento entendido de un modo diferente en cada uno de ellos, una relación que se extenderá en todas ellas, más allá de su perímetro. La investigación se ha estructurado en seis capítulos, que van precedidos de una Introducción. En el capítulo primero, se estudian y se señalan los antecedentes, las figuras y edificios más relevantes de la Tradición Danesa, para la comprensión y el esclarecimiento de algunas de las claves de su Modernidad en el campo de la Arquitectura, que se produce con una clara intención de encontrar su propia identidad y expresión. Esta Modernidad floreciente se caracteriza por la asimilación de otras culturas extranjeras desde la moderación, con un punto de vista crítico, y encuentra sus raíces ancladas a la tradición arquitectónica y la artesanía propia, que fragua en la aparición de un ideal común con enorme personalidad y que hoy se valora como una auténtica aportación de una cultura considerada entonces periférica. Se mostrará el debate y el camino seguido por las generaciones anteriores, a las obras análizadas. Las sensibilidades por lo vernáculo y lo clásico, que aparentemente son contradictorias, dominaran el debate con la misma veracidad y respetabilidad. La llamada tercera generación por Sigfried Giedion reanudará la práctica entre lo clásico y lo vernáculo, apoyados en el espíritu del trabajo artesanal y de la tradición, con el objetivo de conocer del acto arquitectónico su “la verdad” y “la esencia original”. El capítulo segundo, analiza la casa Varming, de 1953, situada en un área residencial de Gentofte, por Eva y Nils Koppel, que reinterpreta la visión de Asplund de un paisaje interior continuación del exterior, donde rompen la caja de ladrillo macizo convencional propia de los años 30. Es el ejemplo más poderoso de la unión de tradición e innovación en su obra residencial. Sus formas sobrias entre el Funcionalismo Danés y la Modernidad se singularizarán por su abstracción y volúmenes limpios que acentúan el efecto de su geometría, prismática y brutalista. El desplazamiento de los cuerpos que lo componen, unos sobre otros, generan un ritmo, que se producirá a otras escalas, ello unido a las variaciones de sus formas y a la elección de sus materiales, ladrillo y madera, le confieren a la casa un carácter orgánico. El edificio se ancla a la tierra resolviéndose en diferentes niveles tras el estudio del lugar y su topografía. El resultado es una versión construida del paisaje, en la cual el edificio da forma al lugar y ensalza la experiencia del escenario natural. La unidad de las estructuras primitivas, parece estar presente. Constituye un ejemplo de la “La idea de Promenade de Asplund”, el proyecto ofrece diferentes recorridos, permitiendo su propia vivencia de la casa, que ofrece la posibilidad vital de decidir. El capítulo tercero trata sobre el pabellón de invitados de Niels Bohr de 1957, situado un área boscosa, en Tisvilde Hegn, fue el primer edificio del arquitecto danés Vilhelm Wohlert. Arraigado a la Tradición Danesa, representa una renovación basada en la absorción de influencias extranjeras: la Arquitectura Americana y la Tradición Japonesa. La caja de madera, posada sobre un terreno horizontal, tiene el carácter sensible de un organismo vivo, siempre cambiante según las variaciones de luz del día o temperatura. Cuando se abre, crea una prolongación del espacio interior, que se extiende a la naturaleza circundante, y se expande hacia el espacio exterior, permitiendo su movilización. Se establece una arquitectura de flujos. Hay un interés por la materia, su textura y el efecto emocional que emana. Las proporciones y dimensiones del edificio están reguladas por un módulo, que se ajusta a la medida del hombre, destacando la gran unidad del edificio. La llave se su efecto estético está en su armonía y equilibrio, que transmiten serenidad y belleza. El encuentro con la naturaleza es la lección más básica del proyecto, donde un mundo de relaciones es amable al ser humano. El capítulo cuarto, analiza el proyecto del Museo Louisiana de 1958, en Humlebæk, primer proyecto de la pareja de arquitectos daneses Jørgen Bo y Vilhelm Wohlert. La experiencia en California de Wohlert donde será visitado por Bo, será trascendental para el desarrollo de Louisiana, donde la identidad Danesa se fusiona con la asimilación de otras culturas, la arquitectura de Frank Lloyd Wright, la del área de la Bahía y la Tradición Japonesa principalmente. La idea del proyecto es la de una obra de arte integral: arquitectura, arte y paisaje, coexistirían en un mismo lugar. Diferentes recursos realzarán su carácter residencial, como el uso de los materiales propios de un entorno doméstico, la realización a la escala del hombre, el modo de usar la iluminación. Cubiertas planas que muestran su artificialidad, parecen flotar sobre galerías acristaladas, acentuarán la fuerza del plano horizontal y establecerán un recorrido en zig-zag de marcado ritmo acompasado. Ritmo que tiene que ver con la encarnación del pulso de la naturaleza, que se acompaña de juegos de luz, y de otras vibraciones materiales a diferentes escalas, imagen, que encuentra una analogía semejante en la cultura japonesa. Todo se coordina con la trama estructural, que conlleva a una construcción y proporción disciplinada. Louisiana atiende al principio de crecimiento de la naturaleza, con la que su conexión es profunda. Hay un dinamismo expresado por el despliegue del edificio, que evoca a algunos proyectos de la Tradición Japonesa. Los blancos muros tienen su propia identidad como formas en sí mismas, avanzan prolongándose fuera de la línea del vidrio, se mueven libremente siguiendo el orden estructural, acompañando al espacio que fluye, en contacto directo con la naturaleza que está en un continuo estado de flujos. Se da todo un mundo de relaciones, donde existe un dialogo entre el paisaje, arte y arquitectura. El capítulo quinto, se dedica a analizar la segunda casa del arquitecto danés Halldor Gunnløgsson, de 1959. Evoca a la Arquitectura Japonesa y Americana, pero es principalmente resultado de una fuerte voluntad y disciplina artística personal. La cubierta, plana, suspendida sobre una gran plataforma pavimentada, que continúa la sección del terreno construyendo de lugar, tiene una gran presencia y arroja una profunda sombra bajo ella. En el interior un espacio único, que se puede dividir eventualmente, discurre en torno a un cuerpo central. El espacio libre fluye, extendiéndose a través de la transparencia de sus ventanales a dos espacios contrapuestos: un patio ajardinado íntimo, que inspira calma y sosiego; y la naturaleza salvaje del mar que proyecta el color del cielo, ambos en constante estado de cambio. El proyecto se elabora de un modo rigurosamente formal, existiendo al mismo tiempo un perfecto equilibrio entre la abstracción de su estructura y su programa. La estructura de madera cuyo orden se extiende más allá de los límites de su perímetro, está formada por pórticos completos como elementos libres, queda expuesta, guardando una estrecha relación con el concepto de modernidad de Mies, equivalente a la arquitectura clásica. La preocupación por el efecto estético es máxima, nada es improvisado. Pero además la combinación de materiales y el juego de las texturas hay una cualidad táctil, cierto erotismo, que flota alrededor de ella. La precisión constructiva y su refinamiento se acercan a Mies. La experiencia del espacio arquitectónico es una vivencia global. La influencia de la arquitectura japonesa, es más conceptual que formal, revelada en un respeto por la naturaleza, la búsqueda del refinamiento a través de la moderación, la eliminación de los objetos innecesarios que distraen de la experiencia del lugar y la preocupación por la luz y la sombra, donde se establece cierto paralelismo con el oscuro mundo del invierno nórdico. Hay un entendimiento de que el espacio, en lugar de ser un objeto inmaterial definido por superficies materiales se entiende como interacciones dinámicas. El capítulo sexto. Propone un viaje para conocer algunas de las viviendas unifamiliares más interesantes que se construyeron en el periodo, que forman parte del sistema investigado. Del estudio comparado y orientado en varios temas, se obtienen diversa conclusiones propias del sistema estudiado. La maestría de la sustancia y la forma será una característica distintiva en Dinamarca, se demuestra que hay un acercamiento a la cultura de Oriente, conceptual y formal, y unos intereses comunes con cierta arquitectura Americana. Su lección nos sensibiliza hacia un sentido fortalecido de proporción, escala, materialidad, textura y peso, densidad del espacio, se valora lo táctil y lo visual, hay una sensibilidad hacia la naturaleza, hacia lo humano, hacia el paisaje, la integridad de la obra. ABSTRACT The thesis “1950 around the Louisiana Museum 1970” analyses several works related to domestic space, which were carried out between 1950 and 1970 in Denmark, a golden age of modern architecture. After the isolation and limitations brought about by the war that blighted Europe, young Danish architects were keen to experiment with ideas of an international origin, encouraged by different circumstances. They find the best field of rehearsal to be the domestic space. The best architecture of that period in Denmark should be understood as a system composed of different authors, who have in common with each other many more similarities than differences, thus complimenting each other. In the interests of understanding, the study of a range of figures and buildings is necessary so that this system, the research of which is still in its infancy, can be completed. The thesis proposes a journey of discovery through the names of some of those protagonists who were showcased through their work so that tradition and avant- garde could go hand in hand. The objective is to unveil the keys to Danish Modernity; to recognise, discover and revive the legacy of some of its protagonists in the domestic field whose lessons are seen as entirely of the present. For an architect, the taking on of modern contributions with both moderation and caution, with its intimate relationship with architectural tradition and its own craft, will be one of his hallmarks. With the study set against several projects and versions, one can derive common values among their authors. In the same way their affinities and differences in respect of the same issue emerge. This will allow an understanding of their measures in line with references and influences and enable the defining of the variables of their architectural spaces. The common line between the buildings selected will be their particular relationship with nature and the space with which they integrate. The façade, the place where the relationship between the interior and the landscape would be negotiated, wouldl be the discriminating element in a distinct way for each one of them. It is through each of these facades that this relationship would extend, and far beyond their physical perimeter. The investigation has been structured into six chapters, preceded by an introduction. The first chapter outlines and analyses the backgrounds, figures and buildings most relevant to the Danish Tradition. This is to facilitate the understanding and elucidation of some of the keys to its modernity in the field of architecture, which came about with the clear intention to discover its own identity and expression. This thriving modernity is characterized by its moderate assimilation with foreign cultures with a critical eye, and finds its roots anchored in architectural tradition and its own handcraft. It is forged in the emergence of a common ideal of enormous personality which today has come to be valued as an authentic contribution to the sphere from a culture that was formerly seen as on the peripheries. What will be demonstrated is the path taken by previous generations to these works and the debate that surrounds them. The sensibilities for both the vernacular and the classic, which at first glance may seem contradictory, will dominate the debate with the same veracity and respectability. The so-called third generation of Sigfried Giedion will revive the process between the classic and the vernacular, supported in spirit by the handcraft work and by tradition, with the objective of discovering the “truth” and the “original essence” of the architectural act. The second chapter analyzes the Varming house, built by Eva and Nils Koppel 1953, which is situated in a residential area of Gentofte. This reinterprets Asplund’s vision of an interior landscape extending to the exterior, where we see a break with the conventional sturdy brick shell of the 1930s. It is the most powerful example of the union of tradition and innovation in his their residential work. Their sober forms caught between Danish Functionalism and modernity are characterized by their abstraction and clean shapes which accentuate their prismatic and brutal geometry, The displacement of the parts of which they are composed, one over the other, generate a rhythm. This is produced to varying scales and is closely linked to its forms and the selection of materials – brick and wood – that confer an organic character to the house. The building is anchored to the earth, finding solution at different levels through the study of place and topography. The result is an adaption constructed out of the landscape, in which the building gives form to the place and celebrates the experience of the natural setting. The unity of primitive structures appears to be present. It constitutes an example of “Asplund’s Promenade Idea”. Different routes of exploration within are available to the visitor, allowing for one’s own personal experience of the house, allowing in turn for the vital chance to decide. The third chapter deals with Niels Bohr’s guest pavilion. Built in 1957, it is situated in a wooded area of Tisvilde Hegn and was the architect Vilhelm Wohlert’s first building. Rooted in the Danish Tradition, it represents a renewal based on the absorption of foreign influences: American architecture and the Japanese tradition. The wooden box, perched atop a horizontal terrain, possesses the sensitive character of the living organism, ever-changing in accordance with the variations in daylight and temperature. When opened up, it creates an elongation of the interior space which extends into the surrounding nature and it expands towards the exterior space, allowing for its mobilisation. It establishes an architecture of flux. There is interest in the material, its texture and the emotional effect it inspires. The building’s proportions and dimensions are regulated by a module, which is adjusted by hand, bringing out the great unity of the building. The key to its aesthetic effect is its harmony and equilibrium, which convey serenity and beauty. The meeting with nature is the most fundamental lesson of the project, where a world of relationships softens the personality of the human being. The fourth chapter analyzes the Louisiana Museum project of 1958 in 1958. It was the first project of the Danish architects Jørgen Bo and Vilhelm Wohlert. Wohlert’s experience in California where he was visited by Bo would be essential to the development of Louisiana, where the Danish identity is fused in assimilation with other cultures, the architecture of Frank Lloyd Wright, that of the Bahía area and principally the Japanese tradition. The idea of the project was for an integrated work of art: architecture, art and landscape, which would coexist in the same space. A range of different resources would realize the residential character, such as the use of materials taken from a domestic environment, the attainment of human scale and the manner in which light was used. Flat roof plans that show their artificiality and appear to float over glassed galleries. They accentuate the strength of the horizontal plan and establish a zigzag route of marked and measured rhythm. It is a rhythm that has to do with the incarnation of nature’s pulse, which is accompanied with plays of light, as well as material vibrations of different scales, imagery which uncovers a parallel analogy with Japanese culture. Everything is coordinated along a structural frame, which involves a disciplined construction and proportion. Louisiana cherishes nature’s principle of growth, to which its connection is profound. Here is a dynamism expressed through the disposition of the building, which evokes in some projects the Japanese tradition. The white walls possess their own identity as forms in their own right. They advance, extending beyond the line of glass, moving freely along the structural line, accompanying a space that flows and in direct contact with nature that is itself in a constant state of flux. It creates a world of relationships, where dialogue exists between the landscape, art and architecture. The fifth chapter is dedicated to analyzing the Danish architect Halldor Gunnløgsson’s second house, built in 1959. It evokes both Japanese and American architecture but is principally the result of a strong will and personal artistic discipline. The flat roof suspended above a large paved platform – itself continuing the constructed terrain of the place – has great presence and casts a heavy shadow beneath. In the interior, a single space, which can at length be divided and which flows around a central space. The space flows freely, extending through the transparency of its windows which give out onto two contrasting locations: an intimate garden patio, inspiring calm and tranquillity, and the wild nature of the sea which projects the colour of sky, both in a constant state of change. The project is realized in a rigorously formal manner. A perfect balance exists between the abstraction of his structure and his project. The wooden structure, whose order extends beyond the limits of its perimeter, is formed of complete porticos of free elements. It remains exposed, maintaining a close relationship with Mies’ concept of modernity, analogous to classical architecture. The preoccupation with the aesthetic effect is paramount and nothing is improvised. But in addition to this - the combination of materials and the play of textures - there is a tactile quality, a certain eroticism, which lingers all about. The constructive precision and its refinement are close to Mies. The experience of the architectural space is universal. The influence of Japanese architecture, more conceptual than formal, is revealed in a respect for nature. It can be seen in the search for refinement through moderation, the elimination of the superfluous object that distract from the experience of place and the preoccupation with light and shade, where a certain parallel with the dark world of the Nordic winter is established. There is an understanding that space, rather than being an immaterial object defined by material surfaces, extends instead as dynamic interactions. The sixth chapter. This proposes a journey to discover some of the unfamiliar residences of most interest which were constructed in the period, and which form part of the system being investigated. Through the study of comparison and one which is geared towards various themes, diverse conclusions are drawn regarding the system being researched. The expertise in substance and form will be a distinctive characteristic in Denmark, demonstrating an approach to the culture of the Orient, both conceptual and formal, and some common interests in certain American architecture. Its teachings sensitize us to a strengthened sense of proportion, scale, materiality, texture and weight and density of space. It values both the tactile and the visual. There is a sensitivity to nature, to the human, to the landscape and to the integrity of the work.

RECONHECENDO-SE SEM-RELIGIÃO NAS PERIFERIAS DA CIDADE: LIBERDADE COMPARTILHADA COMO RESISTÊNCIA

Reconhecendo, a partir da constatação empírica, a multiplicidade de escolhas de crenças no Mundo e em particular na periferia urbana paulistana, reconhecemos, também, a emergência criativa de novas possibilidades de crer e não crer. Tal amplitude não apenas aponta para o crer (segundo as ofertas de um sem número de religiões) e o não crer (ateu e agnóstico), mas para uma escolha que poderia vir a ser silenciada e esquecida, neste binômio arcaico e obsoleto, quando alguém se dá à liberdade crer sem ter religião. Reconhecer interessadamente os sem-religião nas periferias urbanas paulistanas é dar-se conta das violências a que estes indivíduos estão submetidos: violência econômica, violência da cidadania (vulnerabilidade) e proveniente da armas (grupos x Estado). Tanto quanto a violência do esquecimento e silenciamento. A concomitância espaço-temporal dos sem-religião nas periferias, levou-nos buscar referências em teorias de secularização e de laicidade, e, a partir destas, traçar uma história do poder violento, cuja pretensão é a inelutabilidade, enquanto suas fissuras são abertas em espaços de resistências. A história da legitimação do poder que se quer único, soberano, de caráter universal, enquanto fragmenta a sociedade em indivíduos atomizados, fragilizando vínculos horizontais, e a dos surgimentos de resistências não violentas questionadoras da totalidade trágica, ao reconhecer a liberdade de ser com autonomia, enquanto se volta para a produção de partilha de bens comuns. Propomos reconhecer a igual liberdade de ser (expressa na crença da filiação divina) e de partilhar o bem comum em reconhecimentos mútuos (expressa pela ação social), uma expressão de resistência não violenta ao poder que requer a igual abdicação da liberdade pela via da fragmentação individualizante e submissão inquestionável à ordem totalizante. Os sem-religião nas periferias urbanas, nossos contemporâneos, partilhariam uma tal resistência, ao longo da história, com as melissas gregas, os profetas messiânicos hebreus, os hereges cristãos e os ateus modernos, cuja pretensão não é o poder, mas a partilha igual da liberdade e dos bens comuns. Estes laicos, de fato, seriam agentes de resistências de reconhecimento mútuos, em espaços de multiplicidade crescente, ao poder violento real na história.

T cell positive selection by a high density, low affinity ligand

Interaction of the αβ T cell receptor (TCR) with major histocompatibility (MHC) molecules occupied with any of a large collection of peptides derived from self proteins is a critical step in driving T cell “positive” selection in the thymus. Interaction with this same pool of self-peptide/MHC ligands deletes T cells with potential self-reactivity. To examine how T cells survive both of these processes to form a self-tolerant mature repertoire, mice were constructed whose entire class II MHC IEk specific repertoire was positively selected on a single peptide covalently attached to the IEk molecule. In these mice T cells were identified that could respond to a variant of the positively selecting peptide bound to IEk. The affinities of the TCRs from these T cells for the positively selecting ligand were extremely low and at least 10-fold less than those for the activating ligand. These results support the theory that positive selection is driven by TCR affinities lower than those involved in T cell deletion or activation and that, if present at high concentration, even very low affinity ligands can positively select.

The design, computer modeling, solution structure, and biological evaluation of synthetic analogs of bryostatin 1

The bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with Ki = 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like bryostatin, acetal 7c exhibited significant levels of in vitro growth inhibitory activity (1.8–170 ng/ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the bryostatins.

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell–collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (Kd ≈ 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence α1(I)87–92, KGHRGF, with intermediate affinities (Kd ≈ 2 μM); and THPs including other collagenous sequences, or single-stranded sequences, negligibly (Kd ≫ 10 μM). Thus, heparin–type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen–heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.

The role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter

The human and shark Na–K–Cl cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding. We have utilized shark–human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3.1 and its reverse sh3.1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive 86Rb influx with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role. Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-Cl cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect Km(Cl), demonstrating that the affinity difference for Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, bumetanide binding was strongly affected by shark–human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the Cl-binding site.

The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis δ-endotoxin are consistent with an “umbrella-like” structure of the pore

The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by Bacillus thuringiensis δ-endotoxin. We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal. In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state. The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations. Our results are consistent with a situation in which helices α4 and α5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the “umbrella model”). Our results also support the suggestion that α7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain.

Three metal ions at the active site of the Tetrahymena group I ribozyme

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3′-oxygen of the oligonucleotide substrate and the 3′- and 2′-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing d amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.

Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype

Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALB/c mice with rabbit polyclonal anti-idiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an “internal image” monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 × 109 M−1 and 2.4 × 107 M−1, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 × 106 M−1 and 3.8 × 106 M−1). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen.