Direct estimation wave setup as a medium level in swash

The extreme runup is a key parameter for a shore risk analysis in which the accurate and quantitative estimation of the upper limit reached by waves is essential. Runup can be better approximated by splitting the setup and swash semi-amplitude contributions. In an experimental study recording setup becomes difficult due to infragravity motions within the surf zone, hence, it would be desirable to measure the setup with available methodologies and devices. In this research, an analysis is made of evaluated the convenience of direct estimation setup as the medium level in the swash zone for experimental runup analysis through a physical model. A physical mobile bed model was setup in a wave flume at the Laboratory for Maritime Experimentation of CEDEX. The wave flume is 36 metres long, 6.5 metres wide and 1.3 metres high. The physical model was designed to cover a reasonable range of parameters, three different slopes (1/50, 1/30 and 1/20), two sand grain sizes (D50 = 0.12 mm and 0.70 mm) and a range for the Iribarren number in deep water (ξ0) from 0.1 to 0.6. Best formulations were chosen for estimating a theoretical setup in the physical model application. Once theoretical setup had been obtained, a comparison was made with an estimation of the setup directly as a medium level of the oscillation in swash usually considered in extreme runup analyses. A good correlation was noted between both theoretical and time-averaging setup and a relation is proposed. Extreme runup is analysed through the sum of setup and semi-amplitude of swash. An equation is proposed that could be applied in strong foreshore slope-dependent reflective beaches.

Transport of Pru p 3 across gastrointestinal epithelium - an essential step towards the induction of food allergy?

Background Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated. Objective To study the interaction of Pru p 3 with intestinal epithelium, its natural entrance, analyzing transport kinetics and cellular responses that trigger. Methods This was achieved using Pru p 3, the peach LTP, as a model. Enterocytic monolayers were established by culturing Caco 2 cells, as a model of enterocytes, on permeable supports that separate the apical and basal compartments. Pru p 3 was added to the apical compartment, the transepithelial resistance (TEER) was measured, and the transport was quantified. Results The peach allergen that crossed the cell monolayer was detected in the cell fraction and in the basal medium by immunodetection with specific antibodies and the quantity was measured by ELISA assay. Pru p 3 was able to cross the monolayer without disturbing the integrity of the tight junctions. This transport was significantly higher than that of a non-allergenic peach LTP, LTP1, and occurred via lipid raft pathway. The incubation of Caco 2 cells with Pru p 3 and LTP1 produced the expression of epithelial-specific cytokines TSLP, IL33 and IL25. Conclusion These results suggest that Pru p 3 was able to cross the cell monolayer by the transcellular route and then induce the production of Th2 cytokines. The results of the present study represent a step towards clarifying the importance of Pru p 3 as a sensitizer. Clinical relevance The capacity of food allergens to cross the intestinal monolayer could explain their high allergenic capacity and its fast diffusion through the body associating to severe symptoms.

Identificación de áreas prioritarias para la conservación de razas de maíz en la sierra de Ecuador

En Ecuador el maíz es el cultivo más importante en superficie y es base de la alimentación para la población rural que vive en los Andes. A diferencia de lo que sucede en la Costa, en la región Sierra todavía se cultivan numerosas variedades tradicionales que se agrupan en veinticuatro razas. Mantener esta diversidad es, pues, de gran importancia no solo para la seguridad alimentaria, sino también como fuente de genes para tolerancia a factores abióticos que podrían ser incorporados a las variedades modernas. Si bien parte de esta diversidad fue recolectada a mediados del siglo pasado y está siendo conservada en distintos bancos de germoplasma, es deseable que su conservación in situ también esté asegurada, entre otras razones, porque de esta manera el cultivo puede seguir evolucionando. Para poder implementar un plan de conservación en finca que contribuya a preservar este patrimonio, resulta imprescindible identificar áreas idóneas donde concentrar los recursos y conocer las características y tipologías de los agricultores que manejan la diversidad actual. Generar esta información es el objetivo principal de esta investigación y para lograrlo se han llevado a cabo cuatro estudios: (1) Análisis de la diversidad a nivel de razas e identificación de áreas de alta riqueza de razas, alta diversidad morfológica y/o alta diversidad ecogeográfica en la Sierra de Ecuador, (2) Identificación del perfil y las características de los agricultores que conservan y manejan las variedades tradicionales de maíz en la Sierra de Ecuador, (3) Análisis del conocimiento local, manejo y usos de variedades tradicionales de maíz en la Sierra de Ecuador, y (4) Identificación de áreas de alta diversidad y bajo riesgo de pérdida para la conservación en finca de maíz en la Sierra de Ecuador. Para el primer estudio se visitaron 303 fincas distribuidas a lo largo de la Sierra y se recolectaron 636 muestras que fueron caracterizadas morfológicamente mediante 14 variables: 8 relacionadas con la mazorca (forma, longitud y diámetro de la mazorca, color y diámetro de olote y número y disposición de hileras) y 7 referidas el grano (número total de granos, color, forma, longitud, anchura y grosor de grano y tipo de endospermo). Adicionalmente, las fincas donde se tomaron las muestras fueron caracterizadas ecogeográficamente mediante 5 variables climáticas (temperatura media estacional, rango de temperatura media anual, temperatura mínima de diciembre, precipitación estacional y precipitación de octubre), 2 geofísicas (altitud y pendiente) y 5 edáficas (textura principal del suelo, profundidad a roca, pH, contenido en materia orgánica y fertilidad). A partir de esta información y mediante técnicas de sistemas de información geográfica (SIG), se generaron mapas de distribución por raza en formato vectorial y un mapa de riqueza de razas, un mapa de diversidad morfológica y un mapa de diversidad ecogeográfica en formato ráster con celdas de 10 km x 10 km. Los resultados permitieron constatar que, en los últimos 60 años, no se ha perdido ninguna raza. Sin embargo, Canguil, Chaucho y Clavito han dejado de cultivarse en algunas provincias con la consiguiente erosión genética del cultivo. La caracterización morfológica detectó diferencias en el grado de variabilidad intra-raza, siendo Patillo Ecuatoriano, Racimo de Uva y Uchima las razas más heterogéneas tanto para los caracteres cualitativos como cuantitativos. A nivel climático y geofísico, también se detectaron diferencias en el grado de variación intra-raza; Cuzco Ecuatoriano, Kcello Ecuatoriano y Montaña Ecuatoriana fueron las razas que en promedio presentaron mayores rangos y coeficientes de variación para estas variables ecogeográficas. En cuanto a las condiciones edáficas todas las razas, excepto Cónico Dentado, presentaron una gran heterogeneidad, pudiendo crecer tanto en suelos ricos como pobres, con valores de pH entre ácido y moderadamente alcalino. La comparación entre razas reveló diferencias significativas en los rangos ambientales de algunas razas como Cónico Dentado, que tiende a cultivarse a menor altitud y, por tanto, en ambientes menos fríos y de mayor precipitación que Blanco Blandito, Patillo Ecuatoriano, Sabanero Ecuatoriano, Uchima y Zhima. Para la mayoría de las razas se encontraron materiales potencialmente adaptados a condiciones de estrés (precipitación estacional inferior a 500 mm y suelos con pH entre 4.5 y 5.5). Finalmente, los mapas de riqueza, de diversidad morfológica y de diversidad ecogeográfica mostraron 36 celdas de alta diversidad repartidas en las 10 provincias de la Sierra: 11 celdas en las provincias del norte, 11 en las provincias del centro y 14 en las provincias del sur. Para la caracterización e identificación de las tipologías de los agricultores que cultivan maíz en la Sierra de Ecuador y el análisis de los posibles factores de riesgo de pérdida de diversidad, se realizaron entrevistas individuales y semiestructuradas a los agricultores dueños de las fincas donde se recolectaron las muestras para el estudio de diversidad (254 en total). Las preguntas que se formularon (11 abiertas y 5 cerradas) estuvieron organizadas en seis bloques: datos del agricultor, características de la finca, diversidad y conocimiento del cultivo, manejo del cultivo, usos y flujo de semillas. Los resultados indicaron que la diversidad de maíz que hay en la Sierra de Ecuador es manejada mayoritariamente por agricultores mestizos, de entre 30 y 55 años, que cultivan una o dos variedades tradicionales para autoconsumo, en parcelas de menos de 0.5 ha y en asocio con fréjol. El análisis de segmentación mediante el algoritmo Chi-square automatic interaction detection (CHAID) permitió identificar un pequeño grupo de agricultores indígenas con parcelas medianas (entre 0.5 ha y 1.5 ha) que conservan un mayor número de variedades tradicionales por finca que el agricultor promedio. Los análisis estadísticos no detectaron diferencias significativas entre etnias (mestizo vs. indígena), géneros (hombre vs. mujer) y grupos de edad (jóvenes menores de 30 años, adultos entre 30 y 55 años y adultos mayores de 55 años) en lo que respecta al conocimiento del cultivo (criterios de reconocimiento y razones de preferencia) y manejo (tipo de cultivo), pero sí detectaron diferencias entre regiones, principalmente en el modo de cultivar el maíz; mientras que en el norte y sur tienden a sembrarlo en asocio y con un mayor número de especies, en el centro acostumbran a cultivarlo preferentemente solo. En cuanto a los usos, se recopilaron hasta 39 modos diferentes de consumir maíz, siendo Kcello Ecuatoriano y Zhima las razas para las que se registró un mayor número de usos. La comparación del número medio de usos por variedad entre etnias evidenció que los agricultores mestizos utilizan sus variedades tradicionales de forma más variada que los indígenas. Entre los factores de riesgo que se analizaron, el bajo porcentaje de jóvenes agricultores que se ocupan de las fincas podría suponer una amenaza a medio plazo por falta de relevo generacional. Adicionalmente, las numerosas sinonimias y homonimias que se detectaron y el bajo intercambio de semillas también podrían ser causa de pérdida de diversidad, bien por reemplazo o por envejecimiento de la semilla. Finalmente, se concluyó que las razas Chaucho, Complejo Chillo-Huandango, Complejo Mishca-Huandango, Cónico Dentado, Montaña Ecuatoriana y Sabanero Ecuatoriano son particularmente vulnerables, no solo por su baja presencia, sino también por el color de grano que tienen (los mismos que la mayoría de las razas más comunes) y carecer de nombres y usos específicos. Finalmente, para la priorización de áreas de conservación en finca para maíz en la Sierra de Ecuador, se utilizaron 13 criterios de diferente naturaleza: 2 ecogeográficos (precipitación, diversidad ecogeográfica), 6 biológicos (grado de presencia del cultivo, riqueza de razas, diversidad morfológica, presencia de mezclas, presencia de razas locales y riesgo de erosión genética), 3 culturales (abundancia de variedades por finca, diversidad de usos y frecuencia de intercambio) y 2 demográficos (tamaño de la población y distancia a núcleos urbanos). Mediante técnicas SIG y de evaluación multicriterio, los valores originales de las capas-criterio fueron transformados a una escala de 0 a 100. Posteriormente, las capas-criterio normalizadas fueron sumadas utilizando tres métodos de ponderación: (1) mismo peso, (2) diferente peso según la puntuación otorgada por 72 expertos, y (3) diferente peso según el método de comparación entre pares de criterios. Los resultados permitieron identificar ocho celdas de 10 km x 10 km con alta puntuación (> 65): tres celdas en el norte (una en cada una de las provincias), una celda en el centro (en la provincia de Cotopaxi), y cuatro celdas en la región sur (dos en Azuay y otras dos en Loja). ABSTRACT In Ecuador, the maize is the most important cultivation in surface and it is a base of the feeding for the rural population who lives in the Andes. In contrast to what it happens on the Coast, in the Sierra region still there are cultivated numerous traditional varieties that are grouped into twenty-four races. Maintaining this diversity is, therefore, of great importance not only for food security, but also as a source of genes for tolerance to abiotic factors could be incorporated into modern varieties. Although part of this diversity was collected in the middle of the last century and is still preserved in various germplasm banks, it is desirable for the in situ conservation also is assured, among other reasons, because in this way the crop can continue to evolve. To be able to implement a conservation plan on farm that contribute to preserving this heritage, it is essential to identify suitable areas where to concentrate resources and know the characteristics and typology of farmer who managed the current diversity. To generate this information is the main target of this investigation and to achieve this, four studies have been carried out: (1) Analysis of the diversity at races and identification of areas of high richness of races, high morphological diversity and / or ecogeographical high diversity in the Sierra of Ecuador, (2) Identification of the profile and characteristics of farmers who conserve and manage traditional varieties of maize in the Sierra of Ecuador, (3) Analysis of local knowledge, management and use of traditional varieties of maize in the Sierra of Ecuador, and (4) Identification of areas of high diversity and low risk of loss for the conservation of maize in the Sierra of Ecuador. For the first study were visited 303 farms distributed along the Sierra and collected 636 samples that were characterized morphologically by 14 variables: 8 related to the ear (shape, length and diameter of the cob, colour, and diameter of cob and number and arrangement of rows) and 7 referred to the grain (total number of grain, colour, shape, length, width, and thickness and type of grain endosperm). In addition, the farms where the samples were taken were characterized ecogeographically through 5 climatic variables (seasonal average temperature, range of average annual temperature, minimum temperature for December, seasonal precipitation and precipitation of October), 2 geophysical (altitude and slope) and edaphic 5 (main texture of the soil, deep rock, pH, content of organic matter and fertility). From this information and techniques of geographic information systems (GIS), maps were generated for distribution by race in vector format and a map of richness of races, a map of morphological diversity and a map of ecogeographical diversity in raster format with cells of 10 km x 10 km. The results allowed observing that, over the past 60 years, it has not lost any race. Nevertheless, Canguil, Chaucho and Clavito have stopped being cultivated in some provinces with the consequent genetic erosion of the cultivation. The morphological characterization detected differences in the degree of variability intra-race, being Patillo Ecuatoriano, Racimo de Uva and Uchima races more heterogeneous both for the qualitative and quantitative characters. At climate and geophysical level, also detected differences in the degree of variation intra-race; Cuzco Ecuatoriano, Kcello Ecuatoriano and Montaña Ecuatoriana were races that, on average, showed higher ranges and coefficients of variation for these geographical characters. In terms of the edaphic conditions, all races, except Cónico Dentado, showed a great heterogeneity, and can grow both in rich and poor soils, with pH values between acid and moderately alkaline. The comparison between races revealed significant differences in the environmental ranges in some races as Cónico Dentado, which tends to be grown at lower elevations and, therefore, in environments less cold and greater precipitation than Blanco Blandito, Patillo Ecuatoriano, Sabanero Ecuatoriano, Uchima and Zhima. For most of the races were found materials potentially adapted to stress conditions (seasonal precipitation less than 500 mm and soil with a pH between 4.5 and 5.5). Finally, the maps of richness, morphologic diversity and ecogeographical diversity showed 36 cells high diversity distributed in 10 provinces of the Sierra: 11 cells in the northern provinces, 11 in the central provinces and 14 in the southern provinces. For the characterization and identification of the typology of the farmers who cultivate corn in the Sierra of Ecuador and the analysis of the possible factors of risk of loss of diversity, there were realized interviews individual and semistructured to the farmers’ owners of the farms where the samples were gathered for the study of diversity (254 in whole). The questions that were formulated (11 opened ones and 5 closed ones) were organized in six blocks: data of the farmer, characteristics of the farm, diversity and knowledge of the crop, crop management, uses and seed flow. The results indicated that the maize diversity that exist in the Sierra of Ecuador is managed mainly by mestizo farmers, aged between 30 and 55, who cultivate one or two traditional varieties for self-consumption, on plots of less than 0.5 has and in associated with beans. The segmentation analysis algorithm using the Chi-square automatic interaction detection (CHAID technique), allowed to identify a small group of indigenous farmers with medium-sized plots (between 0.5 there is and 1.5 it is) that a major number of traditional varieties preserves for farm that the average farmer. The statistical analysis did not detect significant differences between ethnic groups (mestizos vs. indigenous), genres (man vs. women) and age groups (young people under 30 years of age, adults between 30 and 55 years and adults over 55 years old) in regards to the knowledge of the cultivation (recognition criteria and reasons of preference) and management (type of crop), but if detected differences between regions, mainly on the mode of cultivating the maize; while in the north and south they tend to sow in associate and with a greater number of species, in the center accustomed to cultivate it preferably only. In regards to the uses, they were compiled up to 39 different ways of consuming maize, being Kcello Ecuatoriano and Zhima the races for which a major number of uses registered. The comparison of the average number of uses per variety between ethnic groups showed that the mestizo farmers used their traditional varieties of form more varied than the indigenous people. Between the factors of risk that were analyzed, the low percentage of young farmers who deal with the farms might suppose a medium-term threat for lack of generational relief. In addition, the numerous synonyms and homonyms that were detected and the low seed exchange could also be a cause of loss of diversity, either by replacement or by aging of the seed. Finally, it was concluded that the races Chaucho, Complex Chillo-Huandango, Complex Mishca-Huandango, Cónico Dentado, Montaña Ecuatoriana and Sabanero Ecuatoriano are particularly vulnerable, not only because of their low presence, but also by the grain color they have (the same as the majority of races more common) and lack of names and specific uses. Finally, for the prioritization of maize conservation areas on farm in the Sierra of Ecuador, used 13 criteria of different nature: 2 ecogeographic (precipitation, diversity ecogeographical), 6 biological (degree of presence of the crop, races richness, morphological diversity, the presence of mixtures, presence of local races and risk of genetic erosion), 3 cultural (abundance of varieties per farm, diversity of uses and frequency of exchange) and 2 demographic (population size and distance to urban centers). Using GIS techniques and multicriteria evaluation, the original values of the layers-criterion were transformed to a scale of 0 to 100. Later, the normalized layers - criteria were added using three weighting methods: (1) the same weight, (2) different weight according to the score given by 72 experts, and (3) different weight according to the method of comparison between pairs of criteria. The results allowed to identify eight 10 km cells x 10 km with high punctuation (> 65): three cells in the north (one in each of the provinces), a cell in the center (in the Cotopaxi province), and four cells in the south region (two in Azuay and other two in Loja).

Quantitative analysis of morphological changes in yeast colonies growing on solid medium: the eccentricity and Fourier indices

The colony shape of four yeast species growing on agar medium wasmeasured for 116 days by image analysis. Initially, all the colonies are circular, with regular edges. The loss of circularity can be quantitatively estimated by the eccentricity index, Ei, calculated as the ratio between their orthogonal vertical and horizontal diameters. Ei can increase from 1 (complete circularity) to a maximum of 1.17–1.30, depending on the species. One colony inhibits its neighbour only when it has reached a threshold area. Then, Ei of the inhibited colony increases proportionally to the area of the inhibitory colony. The initial distance between colonies affects those threshold values but not the proportionality, Ei/area; this inhibition affects the shape but not the total surface of the colony. The appearance of irregularities in the edges is associated, in all the species, not with age but with nutrient exhaustion. The edge irregularity can be quantified by the Fourier index, Fi, calculated by the minimum number of Fourier coefficients that are needed to describe the colony contour with 99% fitness. An ad hoc function has been developed in Matlab v. 7.0 to automate the computation of the Fourier coefficients. In young colonies, Fi has a value between 2 (circumference) and 3 (ellipse). These values are maintained in mature colonies of Debaryomyces, but can reach values up to 14 in Saccharomyces.All the species studied showed the inhibition of growth in facing colony edges, but only three species showed edge irregularities associated with substrate exhaustion. Copyright © 2014 John Wiley & Sons, Ltd.

Hacia un análisis cuantitativo de la Ciudad Informal: una aproximación desde la Habitabilidad Básica y la experiencia en Makeni (Sierra Leona)

La importantísima expansión urbana que sufren las principales ciudades de los países en vías de desarrollo, es el mayor reto que afronta la habitabilidad a nivel mundial. Dentro de la teoría general de la Habitabilidad Básica (HaB-ETSAM-UPM, 1995) la ordenación del territorio y el planeamiento urbanístico son las herramientas determinantes para orientar coherentemente los procesos de urbanización, como se reconoce también desde las principales esferas técnicas a nivel internacional. Pero tales herramientas deben enfocarse a una construcción eficiente del territorio, actuando desde una aproximación multidisciplinar, flexible y directa, que incida en las prioridades específicas de cada contexto. Para ello, resulta fundamental comprender a fondo las realidades específicas de estos ámbitos. La ciudad es un fenómeno complejo en esencia. El tejido construido, en constante proceso de cambio, es el caparazón visible que alberga una maravillosa mezcla entrelazada de espacios, funciones, flujos, personas.... Cada ciudad, diferente y única, se integra en su medio, se adapta a geografías, contextos y climas distintos, evoluciona según dinámicas propias, en incomprensibles (o casi) procesos evolutivos. El estudio de la ciudad, supone siempre una simplificación de la misma. La realidad urbana, por detallado que sea nuestro análisis, siempre contendrá indescifrables relaciones que se nos escapan. En cambio, necesitamos de métodos analíticos que nos ayuden a comprender algo esa complejidad. Acercarnos en ese análisis, es un paso previo fundamental para la formulación de respuestas. En este plano, de avance en la comprensión del hecho urbano, se sitúa este trabajo. Se pone el acento en el enfoque cuantitativo, profundizando en datos básicos concretos, siempre aceptando de partida que esta información es una componente mínima, pero esperamos que sustantiva, de un fenómeno inabordable. Y es esta búsqueda de comprensión material y cuantitativa de la ciudad, el objetivo esencial de la investigación. Se pretende proporcionar una base detallada de aquéllos aspectos fundamentales, que pueden ser medidos en los entornos urbanos y que nos proporcionan información útil para el diagnóstico y las propuestas. Para ello, se aportan rangos y referencias deseables, a través de una herramienta para la comprensión y la valoración de cada contexto, la Matriz de Indicadores. Esta herramienta se concibe desde la reflexión a la aplicación práctica, a la utilidad directa, al aporte concreto para quien pueda servir. Ésta es la voluntad decidida con la que se aborda este trabajo, centrado en los entornos urbanos donde el aporte técnico es prioritario: la Ciudad Informal. La Ciudad Informal, entendida aquí como aquélla que se desarrolla sin los medios suficientes (técnicos, económicos e institucionales) que proporciona la planificación, aquélla por donde la habitabilidad precaria se extiende. Es la ciudad que predomina en los países en vías de desarrollo, en los contextos de bajos recursos, allí donde, precisamente, se concentran los principales déficits y necesidades a nivel global. El enfoque nace de la teoría de la Habitabilidad Básica, de la definición de mínimos posibles para, desde allí, construir el espacio necesario para el desarrollo humano. Éste es el ámbito genérico objeto del trabajo que, a su vez, se nutre, de forma muy importante, de la experiencia directa en la ciudad de Makeni, en Sierra Leona. Esta ciudad nos sirve de prototipo experimental en un doble sentido. Por un lado, sirve como espacio empírico en el que chequear la metodología de valoración cuantitativa; y, por otro, el conocimiento de esta ciudad de tamaño medio africana, que se ha ido adquiriendo en los últimos cinco años, es una base directa para el desarrollo teórico de la propia metodología, que ayuda a atisbar lo esencial en contextos similares. El encaje de todo este recorrido se ha articulado desde una experiencia académica que, como docente, he coordinado directa e intensamente. Experiencia muy enriquecedora, que ha sumado muchas manos y mucho aprendizaje en este tiempo. Teoría y práctica en planeamiento urbano se alternan en el trabajo, nutriéndose la una de la otra y a la inversa. Trabajo que nace desde la pasión por la ciudad y el urbanismo. Desde la búsqueda por comprender y desde la vocación de actuar, de intentar mejorar y hacer más habitables los entornos urbanos. Especialmente allí donde las dificultades se agolpan y el camino se alarga, se llena de polvo. Acumular preguntas a cada paso. Cada vez más preguntas. Las respuestas, si existen, aparecen entrelazadas en dinámicas indescifrables de las que queremos formar parte. Fundirnos por momentos en la misma búsqueda, acompañarla. Sentirnos cerca de quiénes comienzan de cero casi cada día. Y otra vez, arrancar. Y compartir, desde el conocimiento, si acaso es que se puede. Y la ciudad. Brutal, imponente, asfixiante, maravillosa, imposible. Creación colectiva insuperable, de energías sumadas que se cosen sin patrón aparente. O sin más razón que la del propio pulso de la vida. Así se siente Makeni. ABSTRACT The important urban growth suffering major cities of developing countries, is the biggest challenge facing the global habitability. Within the general theory of Basic Habitability (HAB-ETSAM-UPM, 1995) spatial planning and urban planning are the crucial tools to consistently guide the urbanization process, as also recognized from the main technical areas worldwide. But such tools should focus on an efficient construction of the territory, working from a multidisciplinary, flexible and direct approach, that affects the specific priorities of each context. To do this, it is essential to thoroughly understand the specific realities of these areas. The city is essentially a complex phenomenon. The urban fabric in constant flux, is the visible shell that houses a wonderful mixture interlocking spaces, functions, flows, people.... Every city, different and unique, is integrated into its environment, adapted to geographies, contexts and climates, it evolves according to its own dynamics, along (almost) incomprehensible evolutionary processes. The study of the city, is always a simplification of it. The urban reality, even studied from a detailed analysis, always contain undecipherable relationships that escape us. Instead, we need analytical methods that help us understand something that complexity. Moving forward in this analysis is an essential first step in formulating responses. At this level, progressing in understand the urban reality, is where this work is located. The emphasis on the quantitative approach is placed, delving into specific basic data, starting always accepting that this information is just a minimal component, but we hope that substantive, of an intractable phenomenon. And it is this search for materials and quantitative understanding of the city, the main objective of the research. It is intended to provide a detailed basis of those fundamental aspects that can be measured in urban environments that provide us useful information for the diagnosis and proposals. To do this, desirable ranges and references are provided, through a tool for understanding and appreciation of each context, the Indicator Matrix. This tool is conceived from reflection to practical application, to a direct utility, concrete contribution to who can serve. This is the firm resolve with which this work is addressed, centered in urban environments where the technical contribution is a priority: the Informal City. The Informal City, understood here as the urban areas developed without the sufficient resources (technical, economic and institutional) which planning provides, where it is extended the precarious habitability. It is the city that prevails in developing countries, in low-resource settings, where, precisely, the main gaps and needs in the global context are concentrated. The approach stems from the theory of Basic Habitability, the definition of possible minimum, to build the necessary space for human development. This is the generic scope of the work object, that is also based in the direct experience in the town of Makeni, Sierra Leone. This city serves as an experimental prototype in two ways. On the one hand, it serves as a space where empirically check the quantitative assessment methodology; and, secondly, the knowledge of this African city of medium size, which has been acquired in the last five years, is a direct basis for the theoretical development of the methodology itself, which helps to glimpse the essence in similar contexts. The engagement of this whole journey has been articulated from an academic experience, directly and intensely coordinated as teacher. Enriching experience that has added many hands and much learning at this time. Theory and practice in urban planning are mixed at work, enriching the one of the other and vice versa. Work is born from the passion for the city and urbanism. From the search for understanding and from the vocation of acting, trying to improve and make more livable urban environments. Especially where the difficulties are crowded and the road is so long, full of dust. To accumulate questions at every turn. More and more questions. The answers, if do exist, appears inside indecipherable dynamics in which we want to be involved. Merge at times in the same search. Feel close to those who start from scratch almost every day. And again, move forward. And share, from knowledge, if possible. And the city. Brutal, impressive, suffocating, wonderful, impossible. Unsurpassed collective creation, combined energy mix that are sewn with no apparent pattern. Or for no reason other than the pulse of life itself. As it feels the city of Makeni.

Elementos peatonales de las ciudades medias españolas : tipos, orígenes, relaciones y articulaciones

A través de esta tesis se busca aportar un análisis de los elementos peatonales que cubren las necesidades y las infraestructuras de apoyo al peatón, siendo un campo poco desarrollado en nuestro país a diferencia de otros como Dinamarca, Reino Unido, Alemania y Holanda, donde las actuaciones peatonales ejecutadas son tradicionales y muy exitosas. Se trata por tanto de un tema poco estudiado pero que adquiere singular importancia en estos años de búsqueda de nuevos modelos urbanos que eviten el derroche de recursos, y avancen hacia formas de movilidad más adecuadas a un mundo limitado y vulnerable. La investigación tiene un carácter eminentemente descriptivo cuyo principal valor es documentar y analizar sistemáticamente una realidad poco conocida, aportándose datos para entender el origen y desarrollo de las actuaciones peatonales. El propósito es ayudar a cimentar este campo en nuestro país, definiendo con precisión el estado de la cuestión geográficamente y dejando para un futuro una cartografía temática completa. El objeto general es estudiar y comprender los elementos peatonales de un número limitado de ciudades medias españolas, marcándose como objetivos específicos los siguientes: - Identificar y describir los principales elementos que conforman la red peatonal de las ciudades. ‐ Estudiar las tipologías espaciales dominantes, sus características y orígenes, pudiendo distinguir de un lado, las desarrolladas en la ciudad consolidada, como las actuaciones en los cascos antiguos; y por otro, las ejecutadas en la ciudad nueva a través de fórmulas arquitectónicas generalmente residenciales con espacios peatonales adjuntos. ‐ Analizar los referidos elementos para comprobar si han conformado redes de uso peatonal, si se han constituido articulando la ciudad como en el caso de los ejes lineales en las ciudades con río o mar, o si han influenciado el carácter del entorno en el que se han realizado, centrando el interés en los cascos históricos peatonalizados. ‐ Comprobar si hay actuaciones complementarias a los procesos de urbanización y mejora urbanística, así como la aprobación de ayudas y subvenciones para afrontar las mismas. ‐ Finalmente, describir y evaluar los procesos y procedimientos mediante los que se produce la peatonalización de determinadas áreas urbanas, los orígenes, los instrumentos y fechas de su puesta en marcha, aportando una cronología peatonal. La metodología comienza con la acotación de 22 ciudades españolas, capitales de provincia con una población entre 100.000 y 500.000 habitantes según los datos del INE del año 2009. La distribución es equilibrada con la selección de ciudades de costa, de interior, con río/ría, con carácter patrimonial, ciudades en islas, etc. El análisis de los elementos peatonales de estas urbes se realiza gracias a la gran resolución de las bases cartográficas públicas y privadas que hay en la actualidad en internet, especialmente con el uso de las herramientas de Google Earth y Google Maps en su versión “street view”, softwares que permiten un paseo virtual de cada ciudad detallándose con gran precisión las actuaciones peatonales. Las ciudades elegidas son: ‐ 11 ciudades costeras: Alicante, Almería, Bilbao, Cádiz, Castellón de la Plana, Huelva, Las Palmas de Gran Canaria, Palma de Mallorca, Santander, San Sebastián y Tarragona. ‐ 11 ciudades de interior: Albacete, Burgos, Córdoba, León, Lérida, Logroño, Oviedo, Murcia, Pamplona, Valladolid y Vitoria. Finalmente, por tener los porcentajes mas elevados de las áreas peatonales de sus casco histórico, se eligen 3 casos de ciudades representativas para un desarrollo peatonal en profundidad, seleccionándose Burgos, San Sebastián y Pamplona. Otros aspectos metodológicos interesantes a destacar, es la extensa documentación adquirida a través de las fuentes de Internet, sobre todo a la hora de desarrollar el último capítulo “Procesos de peatonalización de las ciudades españolas” centrado en las 3 ciudades elegidas. Se consulta las hemerotecas de periódicos locales, los boletines oficiales, las actas de los plenos de los Ayuntamientos y los documentos de planificación (Planes Generales, Planes Especiales, Planes y Proyectos Municipales, Planes de Movilidad), adquiriéndose una precisa información en cuanto a la cronología de las fechas y extensión del trabajo. Se complementa la metodología con las entrevistas a actores claves en el proceso peatonal, como el personal técnico de los Ayuntamiento y los técnicos redactores de los proyectos; y con la colaboración directa de los técnicos de los consistorios de las 3 ciudades seleccionadas, aportando todo tipo de información demandada. En la tesis, se introduce los elementos peatonales mediante el estudio de los espacios peatonales y los procesos de peatonalización existentes, a través del análisis de la literatura consultada aportándose información valiosa en cuanto a la definición de los modelos peatonales, exponiéndose una breve historia de las peatonalizaciones. Destacan las publicaciones de los expertos europeos en la materia como los autores Carmen Hass-Klau, Rolf Monheim, Rob Krier y Collin Buchanan. En España, las actuaciones peatonales fueron tardías en el tiempo en comparación con el resto de Europa, con casi 40 años de retraso. Las referencias bibliográficas españolas en esta materia son escasas siendo las mas significativas las realizadas por Alfonso Sanz y por el profesor Julio Pozueta destacando una de sus últimas publicaciones “La ciudad paseable” describiéndose los elementos peatonales existentes en las ciudades, sirviendo como modelo y ejemplo para esta investigación. En base a lo anterior, se definen los elementos peatonales como aquellos espacios públicos libres de edificación que, por sus características y diseño, garantizan un confortable uso estancial, de tránsito peatonal y están expresamente reservados para ello. En la tesis se detectan y se especifican los siguientes elementos peatonales: las calles y plazas peatonales, las aceras y bulevares de una anchura superior a 8 metros, los paseos peatonales, los elementos de paso (tradicionales y mecánicos), espacios peatonales ligados a nuevas áreas residenciales, los soportales y pasajes peatonales con una anchura mínima de 4 metros, recintos feriales, espacios libres y zonas verdes y parques. Para cada una de las 22 ciudades seleccionadas se realiza un exhaustivo inventario de los anteriores elementos a través de fichas, aportando un gran número de proyectos individuales ejecutados en cada urbe. Esta información se recoge gráficamente en 2 fotoplanos, conformándose como herramientas fundamentales para esta investigación. El Fotoplano 1 se hace sobre la base de la ciudad del Google Earth diferenciándose las áreas peatonales y las zonas verdes. En el Fotoplano 2, y con el soporte anterior, se distinguen las tipologías persistentes y dominantes como son los grandes ejes peatonales, las redes peatonales locales y los elementos peatonales singulares. Finalmente, a partir de los 2 fotoplanos, se realiza la denominada “Huella peatonal” que destaca por su atractivo y pregnancia, algo de lo que el campo de lo peatonal es muy útil. A través de la huella se obtiene una rápida información al tener el plano dos colores, color naranja para lo peatonal (formado por las áreas peatonales y zonas verdes) y color negro para el resto el resto no peatonal. Toda esta documentación gráfica y descriptiva de los elementos peatonales de cada ciudad, queda ampliamente recogida en el Anexo de la tesis. La “Huella peatonal” se considera como el plano básico en la investigación obteniendo una percepción rápida, clara y visual del espacio peatonal de cada ciudad. Se arroja información morfológica con respecto a la distribución espacial, articulación, equilibrio, modulación, cohesión, concentración peatonal, etc. de cada una de las ciudades seleccionadas. A través de la huella se resuelve muchos de las cuestiones enmarcadas como objetivos de la tesis, comprobándose de forma general, que los elementos peatonales dentro de la trama urbana están desarticulados ya que no hay una estructura que module la ciudad como un conjunto cohesionado. Se localizan pequeñas redes conectadas entre sí, que de forma heterogénea, están dispersas por la ciudad. Se verifica que los elementos peatonales con mayor concentración y presencia en las ciudades analizadas son los ejes peatonales, los cascos históricos y los elementos peatonales en las nuevas áreas residenciales. En la investigación, además de estos 3 elementos mencionados, se analiza con mayor profundidad otros tipos que destacan por su persistencia y repetición en la “Huella peatonal” de cada una de las 22 ciudades, como son las plazas peatonales, los soportales y los elementos singulares peatonales: espacios tradicionales (recintos feriales, pasajes, puentes históricos), nuevos elementos peatonales (pasarelas peatonales, elementos mecánicos de paso), áreas peatonales entorno a edificios culturales y otros casos como los consistentes en la recuperación y transformación de edificaciones y construcciones urbanas de diferente uso y tipología, en nuevas áreas peatonales. Se aporta luz sobre su definición, localización, cronología, proporción, presencia y génesis, facilitando resultados de cada uno de ellos. De forma paralela, se obtiene información de índole cuantitativa a través de las mediciones de la huella, obteniéndose datos y porcentajes de extensión de la ciudad, de las zonas peatonales y de las zonas verdes, de los cascos históricos y de sus superficies peatonales, de los metros lineales de dichos cascos históricos y de sus calles peatonales. Se proporcionan ratios e índices que se consideran fundamentales para una mejor comprensión peatonal de cada urbe. Se establece una clasificación de las ciudades en cada uno de los ámbitos descritos, destacando las ciudades de Pamplona, San Sebastián y Burgos por arrojar las cifras peatonales más positivas encabezando la mayoría de las tablas clasificatorias. La peor ciudad valorada es Alicante, seguida según criterio, de Almería, Palma de Mallorca y Las Palmas de GC. En el capítulo final de la investigación, se eligen las ciudades de Burgos, San Sebastián y Pamplona como casos de estudio representativos. El objetivo es obtener un conocimiento más preciso en cuanto a su origen, cronología y procesos de las peatonalizaciones. Cada ciudad se compone de un cuadro cronológicos por etapas, desarrollándose de forma exhaustiva cada una de ellas, enunciadas normalmente, como instrumentos de planeamiento o proyectos de urbanización, acompañadas de los planos de las actuaciones peatonales. Al final de cada etapa se aportan datos de las cifras de las peatonalizaciones que se van realizando, con un cuadro numérico de localizaciones, superficie peatonal, superficie reurbanizada, longitud peatonal y el grado de peatonalización del casco histórico, además de los datos acumulados por etapa. De las conclusiones cruzadas de estas 3 ciudades, destaca la comprobación de la tendencia de ir peatonalizando los centros antiguos llegando incluso al 100% de calles peatonales en el caso de San Sebastián. No obstante, es difícil confirmar si el objetivo es una peatonalización global de los cascos, ya que son muchas las variables que afectan e influyen en el proceso peatonal de cada ciudad. Se coteja adicionalmente cómo se extiende la cultura peatonal desde el interior al exterior, mas allá de los cascos históricos hacia los ensanches próximos. En cuanto al origen de las peatonalizaciones, se concluye que no hay una concepción inicial y teórica marcada, a partir de la cual se va desarrollando la trama peatonal de cada ciudad. Se puede afirmar que los procesos peatonales ejecutados son variados y de diversa índole, sin poder precisar un hecho como causa principal. Según el momento y las circunstancias, las peatonalizaciones responden a distintas iniciativas promovidas por los comerciantes, por las administraciones locales o por los técnicos de planeamiento, con sus expertas aportaciones en los planes. Estos tres agentes actúan como impulsores de proyectos individuales peatonales que se van solapando en el tiempo. Como punto final de la tesis, se propone una serie de nuevas líneas de investigación que pueden servir como estudio adicional y complementario, respondiendo a aspectos tan relevantes como cuestiones de índole económica, social y de movilidad, fuertemente ligadas a las peatonalizaciones, tal y como se demuestra en la literatura consultada. ABSTRACT This thesis aims to analyse the pedestrian elements dealing with the needs and infrastructures that support pedestrians, as this is a field that is little developed in our country, in comparison to other regions such as Denmark, the United Kingdom, Germany and the Netherlands, where the pedestrian actions implemented are traditional and highly successful. Therefore, even though little research has been done about this topic, this field is increasingly important throughout these years of search for urban models intended to prevent the waste of resources and to develop new mobility ways, more adapted to a limited, vulnerable world. This research is essentially descriptive and it mainly aims to document and systematically analysed a scarcely known reality by providing data in order to understand the origin and development of pedestrian actions. This thesis intends to lay the foundations of this field in Spain, accurately defining the state of the art from a geographical point of view and preparing a full thematic map that may be used in the future. The overall aim is to study and understand the pedestrian elements of a limited number of Spanish medium-size cities, establishing the following specific goals: - To identify and describe the main elements comprising cities' pedestrian networks. - To study of special predominant typologies, their features and origins, with the possibility to, on the one hand, make a difference between the typologies developed in well-established cities, such as the actions in historic quarters, and, on the other hand, those implemented in new cities through generally residential architectural formulae with adjoining pedestrian areas. - To analyse the said elements in order to check whether they have resulted in pedestrian-use networks, whether they have been established by organising the city, such as the linear axes in coastal and riverside cities, or whether they have had an impact on the character of the areas where these elements have been implemented, focusing on pedestrian historic quarters. - Check whether there are actions supplementary to the urban development and urban improvement processes, as well as the approval of financial support and subventions to deal with these actions. - Finally, to describe and assess the processes and procedures by which the pedestrianisation of certain urban areas is carried out, the origins, the instruments and the date of their implementation, providing a pedestrian timeline. The methodology starts by defining 22 Spanish cities, province capitals with a population ranging from 100,000 to 500,000 inhabitants, according to the data recorded by the Spanish Statistics Institute (INE) in 2009. The distribution of coastal, riverside and interior cities, as well as of patrimonial cities and cities in islands, etc. is well balanced. The analysis of the pedestrian elements of these cities is made with the great resolution of the public and private map databases that can be accessed on the Internet, especially using the "street-view" version of tools such as Google Earth and Google Maps, software applications that allow to go for a virtual walk in each city, providing highly precise details about the pedestrian actions. The following cities have been chosen: ‐ 11 coastal cities: Alicante, Almería, Bilbao, Cádiz, Castellón de la Plana, Huelva, Las Palmas de Gran Canaria, Palma de Mallorca, Santander, San Sebastián and Tarragona. ‐ 11 interior cities: Albacete, Burgos, Córdoba, León, Lérida, Logroño, Oviedo, Murcia, Pamplona, Valladolid and Vitoria. Finally, as they have the highest percentages regarding the pedestrian areas found in the historic quarters, 3 cities representing a deep pedestrian development have been chosen: Burgos, San Sebastián and Pamplona. Other significant methodological aspects are the many documents found from online sources, especially when preparing the last chapter: “Processes for the pedestrianisation of Spanish cities”, which focuses on the 3 cities chosen. Local newspaper and periodical libraries, official gazettes, the minutes of plenary sessions of councils and the zoning regulation documents (General Zoning Plans, Special Zoning Plans, Municipal Plans and Projects, Mobility Plans, etc.) have been consulted, obtaining accurate information regarding the timeline and the extension of the works carried out. The methodology is supplemented by interviews with key players in the pedestrianisation process, such as the technical staff in councils and the officers drafting and designing the projects, as well as with the direct collaboration by the officers of the councils of the 3 cities chosen, who provided all the information requested. The thesis introduces the pedestrian elements by studying pedestrian areas and existing pedestrianisation processes through the analysis of the literature consulted, providing valuable information for the definition of pedestrian models and showing a brief history and background of the pedestrianisation process. Remarkable papers published by some European experts in the field, such as Carmen Hass-Klau, Rolf Monheim and Collin Buchanan, are covered in the thesis. In Spain, the pedestrianisation actions were late in time in comparison to the rest of Europe, with at least 40 years of delay. The Spanish literature references in this field are limited. The most significant papers are those published by Alfonso Sanz and by Professor Julio Pozueta, with a special mention of one of his last works "La ciudad paseable" (The Walkable City), which describes the pedestrian elements found in the cities and is used a model and an example for this research. Based on the elements above, pedestrian elements are defined as those construction-free public areas that, due to their features and design, ensure the comfortable, convenient use of the spaces, characterised by pedestrian traffic and specifically reserved for this purpose. The thesis detects and specifies the following pedestrian elements: pedestrian streets and squares, pavements (sidewalks) and boulevards with a width exceeding 8 metres, pedestrian promenades, crossing elements (traditional and mechanical), pedestrian spaces linked to new residential areas, colonnades and pedestrian passages or narrow streets with a minimum width of 4 metres, exhibition sites, free spaces and green areas and parks. For each of the 22 cities chosen, a thorough inventory of the elements mentioned above has been made by using worksheets, providing a significant number of individual projects developed in each city. This information is graphically collected and displayed on 2 photomaps, resulting in tools essential for this research. Photomap 1 is made based on the city displayed by Google Earth, making a difference between pedestrian areas and green areas. On Photomap 2, using the tool mentioned above, a difference can be made between persistent and predominant typologies, such as the big pedestrian axes, the local pedestrian networks and singular pedestrian elements. Finally, the 2 photomaps are used in order to establish the so-called "pedestrian footprint", which is highlighted by its attractiveness and appeal, concepts for which the pedestrian field is very useful. With the pedestrian footprint, quick information can be obtained, since the map shows two colours: orange for pedestrian elements (made up of pedestrian areas and green areas) and black for the other non-pedestrian elements. A significant part of these graphic, descriptive documents about each city's pedestrian elements can be found in the thesis appendix. The "Pedestrian Footprint" is regarded in the research as the basic map, obtaining a quick, clear and visual perception of each city's pedestrian space. This footprint provides morphological information regarding the space distribution, the organisation, the balance, the modulation, the cohesion, the pedestrian concentration, etc. in each of the cities chosen. The pedestrian footprint helps solve many of the questions established as the thesis goals, proving that, in general, the pedestrian elements are not organised in the urban plot, as there is no structure modulating the city as a properly linked set of elements. Small networks linked to each other and heterogeneously scattered all over the city are found. It has been verified that the pedestrian elements with the highest concentration and presence in the cities analysed are the pedestrian axes, the historic quarters and the pedestrian elements found in the new residential areas. Besides these 3 elements mentioned, the research analyses in depth other types that are remarkable due to their persistence and repetition in the "Pedestrian Footprint" of each of the 22 cities, such as the pedestrian squares, the colonnades and the singular pedestrian elements: traditional spaces (exhibition sites, passages, historic bridges), new pedestrian elements (pedestrian footbridges, mechanical crossing elements), pedestrian areas around cultural buildings and other cases such as those consisting of recovering and transforming building and urban constructions, intended for a wide range of purposes and of different types, into new pedestrian areas. This work puts light on the definition, location, timeline, proportion, presence and origin, providing results for each of these concepts. At the same time, quantitative information is obtained by measuring the footprint, getting data and percentages on the size of the city, the pedestrian areas and the green areas, the historic quarters and the pedestrian zones, the linear metres of such historic quarters and pedestrian streets. The footprint also provides ratios and rates that are considered as essential in order to better understand the pedestrian elements of each city. A classification of cities is established for each of the areas described, highlighting the cities of Pamplona, San Sebastián and Burgos, as they provide the most positive pedestrian figures and lead most of the classification tables or rankings. According to the criteria considered, the city with the worst values is Alicante, followed by Almería, Palma de Mallorca and Las Palmas de Gran Canaria. In the final chapter in the thesis, the cities of Burgos, San Sebastián and Pamplona are chosen as representative study cases. The aim is to gain more accurate knowledge regarding the pedestrianisation origin, timeline and processes. Each city comprises a chronological sequence made of stages, each of which is thoroughly developed and usually announced as zoning plans or urban development projects, accompanied by the plans of the pedestrian actions. At the end of each stage, data with the figures of the pedestrianisation projects are provided, including a numerical chart with locations, pedestrian area, redeveloped urban areas, pedestrian length and pedestrianisation degree in the historic quarter, as well as the data cumulated throughout each stage. In the crossed conclusions on the three cities, the trend of gradually pedestrianising the historic quarters (even reaching the 100% of the pedestrian streets as in San Sebastián) is verified. However, it is difficult to confirm whether the purpose is to reach an overall pedestrianisation of the historic quarters, since there are many variables that affect and influence each city's pedestrianisation process. In addition, the spread of the pedestrian culture from the internal areas to the external areas, beyond the historic quarters to the nearby expansion areas, is compared. Regarding the origin of pedestrianisations, the thesis comes to the conclusion that there is no initial or theoretical conception from which each city's pedestrian plot is developed. The pedestrian processes implemented are varied and diverse but no fact can be specified as the main cause. Based on the moment and circumstances, the pedestrianisation processes are due to different initiatives promoted by shopkeepers, by the local administrations or by the zoning officers, with their expert contributions to the plans. These three players promote and drive individual pedestrianisation projects that overlap in the course of time. Finally, new lines of research are put forwards, as they can be taken as an additional study and as a supplement to this research, dealing with aspects as significant as the economic, social and mobility factors, closely linked to the pedestrianisation processes, as proven in the literature consulted.

Dynamics of contacts between lamellae of fibroblasts: Essential role of the actin cytoskeleton

We investigated actin cytoskeletal and adhesion molecule dynamics during collisions of leading lamellae of nontransformed and oncogene-transformed fibroblasts. By using real-time video microscopy, it was found that during lamellar collision there was considerable overlapping of leading lamellae followed by subsequent retraction. Overlapping of nontransformed fibroblasts was accompanied by formation of β-catenin-positive contact structures organized into strands oriented parallel to the long axis of the cell that were associated with bundles of actin filaments. Maintenance of such cell–cell contact structures critically depended on the contractility of actin cytoskeleton, as inhibition of contractility with serum-free medium or 2,3-butanedione 2-monoxime (BDM) resulted in loss of strand formation. Strand formation was recovered when cells in serum-free medium were incubated with the microtubule inhibitor nocodazole, which is known to increase contractility. Oncogene-transformed fibroblasts reacted to collisions with responses similar to nontransformed fibroblasts but did not develop well-organized cell–cell contacts. A model is presented to describe how differences in the organization of the actin cytoskeleton could account for the structurally distinct responses to cell–cell contact by polarized fibroblastic cells versus nonpolarized epithelial cells.

Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II

It is shown that restoration of photoinduced electron flow and O2 evolution with Mn2+ in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0–8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H2CO3 dissociation (6.4), which indicates that HCO3− is the essential species for the restoration effect. The addition of just 2 Mn2+ atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant (Kd) of ≈2.5 μM and 20–34 μM when 2,6-dichloro-p-benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O2 evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the Kd of 20–34 μM is associated with the donor side of PSII while the location of the lower Kd binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.

MCD4 Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p’s lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4–174) harbors a single amino acid change in motif 2. The mcd4–174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4–174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4–174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.

Glutathione synthesis is essential for mouse development but not for cell growth in culture

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of γ-glutamylcysteine synthetase (γGCS-HStm1), an essential enzyme in GSH synthesis. Embryos homozygous for γGCS-HStm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

Brownian flocculation of polymer colloids in the presence of a secondary minimum

Most analyses of Brownian flocculation apply to conditions where London–van der Waals attractive forces cause particles to be strongly bound in a deep interparticle potential well. In this paper, results are reported that show the interaction between primary- and secondary-minimum flocculation when the interparticle potential curve reflects both attractive and electrostatic repulsive forces. The process is highly time-dependent because of transfer of particles from secondary- to primary-minimum flocculation. Essential features of the analysis are corroborated by experiments with 0.80-μm polystyrene spheres suspended in aqueous solutions of NaCl over a range of ionic strengths. In all cases, experiments were restricted to the initial stage of coagulation, where singlets and doublets predominate.

cemA homologue essential to CO2 transport in the cyanobacterium Synechocystis PCC6803.

We have isolated mutants of Synechocystis PCC6803 that grew very slowly in a low-sodium medium, which is unfavorable for HCO3(-) transport, and examined two of these mutants (SC1 and SC2) for their ability to take up CO2 and HCO3(-) in the light. The CO2 transport activity of SC1 and SC2 was much lower than that of the wild type (WT), whereas there was no difference between the mutants and the WT in their activity of HCO3(-) transport. A clone containing a 3.9-kilobase-pair insert DNA that transforms both mutants to the WT phenotype was isolated from a genomic library of WT Synechocystis. Sequencing of the insert DNA in the region of mutations in SC1 and SC2 revealed an open reading frame (designated cotA), which showed significant amino-acid sequence homology to cemA encoding a protein found in the inner envelope membrane of chloroplasts. The cotA gene is present in a single copy and was not cotranscribed with any other gene(s). cotA encodes a protein of 247 amino acids containing four transmembrane domains. There was substitution of a single base in SC1 and two bases in SC2 in their cotA genes. A possible role of the cotA gene product in CO2 transport is discussed.

Ascorbic acid is essential for the release of insulin from scorbutic guinea pig pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with elevated D-glucose. Islets from normal guinea pigs released insulin in a D-glucose-dependent manner showing a rapid initial secretion phase and three secondary secretion waves during a 120-min period. Islets from scorbutic guinea pigs failed to release insulin during the immediate period, and only delayed and decreased responses were observed over the 40-60 min after D-glucose elevation. Insulin release from scorbutic islets was greatly elevated if 5 mM L-ascorbic acid 2-phosphate was supplemented in the perifusion medium during the last 60 min of perifusion. When 5 mM L-ascorbic acid 2-phosphate was added to the perifusion medium concurrently with elevation of medium D-glucose, islets from scorbutic guinea pigs released insulin as rapidly as control guinea pig islets and to a somewhat greater extent. L-Ascorbic acid 2-phosphate without elevated D-glucose had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, suggesting that the decreased insulin release from the scorbutic islets was not due to decreased insulin synthesis but due to abnormal insulin secretion.

The ABC transporter gene in the sirodesmin biosynthetic gene cluster of Leptosphaeria maculans is not essential for sirodesmin production but facilitates self-protection

Epipolythiodioxopiperazine toxins are secreted by a range of fungi, including Leptosphaeria maculans, which produces sirodesmin, and Aspergillus fumigatus, which produces gliotoxin. The L. maculans biosynthetic gene cluster for sirodesmin includes an ABC transporter gene, sirA. Disruption of this gene led to increased secretion of sirodesmin into the medium and an altered ratio of sirodesmin to its immediate precursor. The transcription pattern of a peptide synthetase that catalyses an early step in sirodesmin biosynthesis was elevated in the sirA mutant by 47% over a 7-day period. This was consistent with the finding that the transporter mutant had elevated sirodesmin levels. Despite increased production of sirodesmin, the sit-A mutant was more sensitive to both sirodesmin and gliotoxin. The putative gliotoxin transporter gene, gliA, (a major facilitator superfamily transporter) from A.fumigatus complemented the tolerance of the L. maculans sirA mutant to gliotoxin, but not to sirodesmin. The results indicate that SirA contributes to self-protection against sirodesmin in L. maculans and suggest a transporter other than SirA is primarily responsible for efflux of endogenously produced sirodesmin. (C) 2004 Elsevier Inc. All rights reserved.