Cell competition time line: winners kill losers, which are extruded and engulfed by hemocytes

Cell competition is a mechanism that eliminates slow dividing cells from a growing population. It is believed that the genes wasp, psr, and draper are active in the cells that win the competition ("winner cells") and that they are essential in the winner cells for the induction of apoptosis and for the elimination of the "loser cells." Here, we show that lack of those genes in winner cells appears to be dispensable for cell-competition-induced apoptosis and during dmyc-induced supercompetition. Moreover, winner clones do not need those genes in order to preserve their growth advantage. Finally, we find that most of the clearance of the apoptotic debris is not performed by winners but by recruited hemocytes, which are required for the removal of the apoptotic corpses at the very end. Therefore, engulfment is a consequence-not a cause-of loser cells' death.

PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of embryonic blood vascular morphogenesis. However, the molecular mechanisms required for ephrinB2 transduced cellular signaling in vivo have not been characterized. To address this question, we generated two sets of knock-in mice: ephrinB2DeltaV mice expressed ephrinB2 lacking the C-terminal PDZ interaction site, and ephrinB2(5F) mice expressed ephrinB2 in which the five conserved tyrosine residues were replaced by phenylalanine to disrupt phosphotyrosine-dependent signaling events. Our analysis revealed that the homozygous mutant mice survived the requirement of ephrinB2 in embryonic blood vascular remodeling. However, ephrinB2DeltaV/DeltaV mice exhibited major lymphatic defects, including a failure to remodel their primary lymphatic capillary plexus into a hierarchical vessel network, hyperplasia, and lack of luminal valve formation. Unexpectedly, ephrinB2(5F/5F) mice displayed only a mild lymphatic phenotype. Our studies define ephrinB2 as an essential regulator of lymphatic development and indicate that interactions with PDZ domain effectors are required to mediate its functions.

From 'land grabbing' to sustainable investments in land: potential contributions by land change science

This paper assesses possible contributions of land change science to the growing body of knowledge about large-scale land acquisition. Despite obvious commonalities, such as a problem-oriented and interdisciplinary approach to land change, there seems to be little overlap between the two fields thus far. We adopt a sustainability research perspective — an important feature of land change science — to review research questions about large-scale land acquisition that are currently being addressed, and to define questions for further inquiry. Possible contributions of land change science toward more sustainable land investments are based on understanding land use change not only as a consequence, but also as a cause of large-scale land acquisition and as a solution to the problems land acquisition can create.

Are land sparing and land sharing real alternatives for European agricultural landscapes?

There is a lively debate on whether biodiversity conservation and agricultural production could be better reconciled by land sparing (strictly separating production fields and conservation areas) or by land sharing (combining both, agricultural production and biodiversity conservation on the same land). The debate originates from tropical countries, where agricultural land use continues to increase at the expense of natural ecosystems. But is it also relevant for Europe, where agriculture is withdrawing from marginal regions whilst farming of fertile lands continues to be intensified? Based on recent research on farmland biodiversity we conclude that the land sharing – land sparing dichotomy is too simplistic for Europe. Instead we differentiate between productive and marginal farmland. On productive farmland, semi-natural habitats are required to yield ecosystem services relevant for agriculture, to promote endangered farmland species which society wants to conserve even in intensively farmed regions, and to allow migration of non-farmland species through the agricultural matrix. On marginal farmland, high-nature value farming is a traditional way of land sharing, yielding high quality agricultural products and conserving specialized species. To conserve highly disturbance-sensitive species, there is a need for nature reserves. In conclusion, land sparing is not a viable olution for Europe in both productive and marginal farmland but because of different reasons in each type of farmland.

The Caenorhabditis elegans histone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division.

As in all metazoans, the replication-dependent histone genes of Caenorhabditis elegans lack introns and contain a short hairpin structure in the 3' untranslated region. This hairpin structure is a key element for post-transcriptional regulation of histone gene expression and determines mRNA 3' end formation, nuclear export, translation and mRNA decay. All these steps contribute to the S-phase-specific expression of the replication-dependent histone genes. The hairpin structure is the binding site for histone hairpin-binding protein that is required for hairpin-dependent regulation. Here, we demonstrate that the C. elegans histone hairpin-binding protein gene is transcribed in dividing cells during embryogenesis and postembryonic development. Depletion of histone hairpin-binding protein (HBP) function in early embryos using RNA-mediated interference leads to an embryonic-lethal phenotype brought about by defects in chromosome condensation. A similar phenotype was obtained by depleting histones H3 and H4 in early embryos, indicating that the defects in hairpin-binding protein-depleted embryos are caused by reduced histone biosynthesis. We have confirmed this by showing that HBP depletion reduces histone gene expression. Depletion of HBP during postembryonic development also results in defects in cell division during late larval development. In addition, we have observed defects in the specification of vulval cell fate in animals depleted for histone H3 and H4, which indicates that histone proteins are required for cell fate regulation during vulval development.

Cold Microwave-Enabled Protein Detection and Quantification.

Protein screening/detection is an essential tool in many laboratories. Owing to the relatively large time investments that are required by standard protocols, the development of methods with higher throughput while maintaining an at least comparable signal-to-noise ratio is highly beneficial in many research areas. This chapter describes how cold microwave technology can be used to enhance the rate of molecular interactions and provides protocols for dot blots, Western blots, and ELISA procedures permitting a completion of all incubation steps (blocking and antibody steps) within 24-45 min.

Are there age-, strain-, and sex-differences in the prolonged exposure to methylphenidate?

Repeated treatment with psychostimulants produces behavioral sensitization that results in increased locomotor responses so that lower drug doses are required to obtain the same effect and cross-sensitization with other stimulants. Methylphenidate (MPD; Ritalin) is most frequently prescribed to treat children having attention deficit hyperactivity disorder (ADHD), a syndrome with onset in childhood characterized by high levels of inattention, hyperactivity, and impulsivity. Little is known of the consequences involving the long-term use of MPD as treatment for ADHD. This study investigates if there are age, genetic/strain, and sex differences in the prolonged exposure to MPD and cross-sensitization with amphetamine. The objective is to determine whether (a) early exposure to MPD in adolescent rats increases their sensitivity to the drug when they are adult rats, (b) there are strain and sex differences in the response to MPD, and (c) treatment with MPD in adolescent and adult Wistar-Kyoto (WKY), spontaneously hyperactive/hypertensive rat (SHR), and Sprague-Dawley (SD) rat results in cross-sensitization with amphetamine. The hypotheses are that (1) early exposure to MPD in adolescent rats increases their sensitivity to the drug when they reach adulthood, and that this hypersensitivity is dose-, strain-, and sex-dependent and (2) adult rats treated with MPD as adolescents will show a greater cross-sensitization to amphetamine than those adult rats treated with saline as adolescents, and that this cross-sensitization is dose-, strain-, and sex-dependent. The study consists of recording and evaluating locomotor activity of female and male WKY, SHR, and SD rats before and after acute and repeated MPD administration when these rats are young and as adults follows by an amphetamine treatment. Results showed that repeated treatment with MPD elicited behavioral sensitization and cross-sensitization with amphetamine in these animals. The study also found that strain and sex play a crucial role in the differentiated sensitivity to the acute and chronic effects of MPD. The development of behavioral sensitization and cross-sensitization are also dependent on the dose of MPD and the age of the rat. ^

The SasS -SasR two-component signal transduction system required for early Myxococcus xanthus multicellular *development

Initiation of Myxococcus xanthus multicellular development requires both nutrient limitation and high cell density. The extracellular signal, A signal, which consists of a set of amino acids at specific concentrations, serves as a cell density signal in M. xanthus early development. A reporter gene, designated 4521, that requires both starvation and A signal for developmental expression was used to identify mutations in the signal transduction pathways. A group of point mutations located in the chromosomal sasB locus that bypasses both requirements was previously isolated. One of these point mutations, sasB7, was mapped to the sasS gene, which is predicted to encode a transmembrane histidine protein kinase required for normal development. SasS is a positive regulator of 4521 and a candidate A signal sensor. This dissertation continues the characterization of the sasB locus, focusing on the sasR gene and the functional relationship of SasS and SasR. ^ The sasR gene is located 2.2-kb downstream of sasS. It is predicted to encode an NtrC-like response regulator, which belongs to the family of sigma54 transcriptional activators. SasR is a positive regulator of 4521 gene and is required for normal development. The sasR mutant displays phenotypes similar to that of sasS mutant. Both SasS and SasR are required for the A-signal-dependent 4521 expression. Genetic epistasis analysis indicates that SasR functions downstream of SasS. Biochemical studies show that SasS has autokinase activity, and phosphorylated SasS is able to transfer its phosphate to SasR. We propose that SasS and SasR form a two-component signal transduction system in the A signal transduction pathway. ^ To search for the genes regulated by SasS and SasR, expression patterns of a group of developmental genes were compared in wild-type and sasS null mutant backgrounds. SasS and SasR were found to positively regulate sasN and 4521. The sasN gene was previously identified as a negative regulator of 4521, located at about 170-bp downstream of sasR. It is required for normal fruiting body development. Based on the above data, a regulatory network consisting of sasS, sasR, sasN, and 4521 is hypothesized, and the interactions of the components in this network can now be further studied. ^

Co-creación de semiconductores

El mercado de los semiconductores está saturado de productos similares y de distribuidores con una propuesta de servicios similar. Los procesos de Co-Creación en los que el cliente colabora en la definición y desarrollo del producto y proporciona información sobre su utilidad, prestaciones y valor percibido, con el resultado de un producto que soluciona sus necesidades reales, se están convirtiendo en un paso adelante en la diferenciación y expansión de la cadena de valor. El proceso de diseño y fabricación de semiconductores es bastante complejo, requiere inversiones cada vez mayores y demanda soluciones completas. Se requiere un ecosistema que soporte el desarrollo de los equipos electrónicos basados en dichos semiconductores. La facilidad para el diálogo y compartir información que proporciona internet, las herramientas basadas en web 2.0 y los servicios y aplicaciones en la nube; favorecen la generación de ideas, el desarrollo y evaluación de productos y posibilita la interacción entre diversos co-creadores. Para iniciar un proceso de co-creación se requiere métodos y herramientas adecuados para interactuar con los participantes e intercambiar experiencias, procesos para integrar la co-creación dentro de la operativa de la empresa, y desarrollar una organización y cultura que soporten y fomenten dicho proceso. Entre los métodos más efectivos están la Netnografía que estudia las conversaciones de las comunidades en internet; colaboración con usuarios pioneros que van por delante del Mercado y esperan un gran beneficio de la satisfacción de sus necesidades o deseos; los estudios de innovación que permiten al usuario definir y a menudo crear su propia solución y la externalización a la multitud, que mediante una convocatoria abierta plantea a la comunidad retos a resolver a cambio de algún tipo de recompensa. La especialización de empresas subcontratistas en el desarrollo y fabricación de semiconductores; facilita la innovación abierta colaborando con diversas entidades en las diversas fases del desarrollo del semiconductor y su ecosistema. La co-creación se emplea actualmente en el sector de los semiconductores para detectar ideas de diseños y aplicaciones, a menudo mediante concursos de innovación. El servicio de soporte técnico y la evaluación de los semiconductores con frecuencia es fruto de la colaboración entre los miembros de la comunidad fomentada y soportada por los fabricantes del producto. Con el programa EBVchips se posibilita el acceso a empresas pequeñas y medianas a la co-creación de semiconductores con los fabricantes en un proceso coordinado y patrocinado por el distribuidor EBV. Los semiconductores configurables como las FPGAs constituyen otro ejemplo de co-creación mediante el cual el fabricante proporciona el circuito integrado y el entorno de desarrollo y los clientes crean el producto final definiendo sus características y funcionalidades. Este proceso se enriquece con bloques funcionales de diseño, IP-cores, que a menudo son creados por la comunidad de usuarios. ABSTRACT. The semiconductor market is saturated of similar products and distributors with a similar proposal for services. The processes of co-creation in which the customer collaborates in the definition and development of the product and provides information about its utility, performance and perceived value, resulting in a product that solves their real needs, are becoming a step forward in the differentiation and expansion of the value chain. The design and semiconductor manufacturing process is quite complex, requires increasingly higher investments and demands complete solutions. It requires an ecosystem that supports the development of electronic equipments based on such semiconductors. The ease of dialogue and sharing information that provides internet, web 2.0-based tools and services and applications in the cloud; favor the generation of ideas, the development and evaluation of products and allows the interaction between various co-creators. To start a process of co-creation adequate methods and tools are required to interact with the participants and exchange experiences, processes to integrate the co-creation within the operations of the company, and developing an organization and culture that support and promote such process. Among the most effective methods are the Netnography that studies the conversations of the communities on the internet; collaboration with Lead Users who are ahead of the market and expect a great benefit from the satisfaction of their needs or desires; Innovation studies that allow the user to define and often create their own solution and Crowdsourcing, an open call to the community to solve challenges in exchange for some kind of reward. The specialization of subcontractors in the development and manufacture of semiconductors; facilitates open innovation in the context of collaboration with different entities working in the different phases of the development of the semiconductor and its ecosystem. Co-creation is used currently in the semiconductor sector to detect ideas of designs and applications, often through innovation’s contests. Technical support and evaluation of semiconductors frequently is the result of collaboration between members of the community fostered and supported by the manufacturers of the product. The EBVchips program provides access to small and medium-sized companies to the co-creation of semiconductors with manufacturers in a process coordinated and sponsored by the Distributor EBV. Configurable semiconductors like FPGAs are another example of co-creation whereby the manufacturer provides the integrated circuit and the development environment and customers create the final product by defining their features and functionality. This process is enriched with IP-cores, designs blocks that are often created by the user community.

Evaluación de las Consecuencias de la Nueva Regulación de la OMI sobre Combustibles Marinos

El 10 de octubre de 2008 la Organización Marítima Internacional (OMI) firmó una modificación al Anexo VI del convenio MARPOL 73/78, por la que estableció una reducción progresiva de las emisiones de óxidos de azufre (SOx) procedentes de los buques, una reducción adicional de las emisiones de óxidos de nitrógeno (NOx), así como límites en las emisiones de dióxido de Carbono (CO2) procedentes de los motores marinos y causantes de problemas medioambientales como la lluvia ácida y efecto invernadero. Centrándonos en los límites sobre las emisiones de azufre, a partir del 1 de enero de 2015 esta normativa obliga a todos los buques que naveguen por zonas controladas, llamadas Emission Control Area (ECA), a consumir combustibles con un contenido de azufre menor al 0,1%. A partir del 1 de enero del año 2020, o bien del año 2025, si la OMI decide retrasar su inicio, los buques deberán consumir combustibles con un contenido de azufre menor al 0,5%. De igual forma que antes, el contenido deberá ser rebajado al 0,1%S, si navegan por el interior de zonas ECA. Por su parte, la Unión Europea ha ido más allá que la OMI, adelantando al año 2020 la aplicación de los límites más estrictos de la ley MARPOL sobre las aguas de su zona económica exclusiva. Para ello, el 21 de noviembre de 2013 firmó la Directiva 2012 / 33 / EU como adenda a la Directiva de 1999. Tengamos presente que la finalidad de estas nuevas leyes es la mejora de la salud pública y el medioambiente, produciendo beneficios sociales, en forma de reducción de enfermedades, sobre todo de tipo respiratorio, a la vez que se reduce la lluvia ácida y sus nefastas consecuencias. La primera pregunta que surge es ¿cuál es el combustible actual de los buques y cuál será el que tengan que consumir para cumplir con esta Regulación? Pues bien, los grandes buques de navegación internacional consumen hoy en día fuel oil con un nivel de azufre de 3,5%. ¿Existen fueles con un nivel de azufre de 0,5%S? Como hemos concluido en el capítulo 4, para las empresas petroleras, la producción de fuel oil como combustible marino es tratada como un subproducto en su cesta de productos refinados por cada barril de Brent, ya que la demanda de fuel respecto a otros productos está bajando y además, el margen de beneficio que obtienen por la venta de otros productos petrolíferos es mayor que con el fuel. Así, podemos decir que las empresas petroleras no están interesadas en invertir en sus refinerías para producir estos fueles con menor contenido de azufre. Es más, en el caso de que alguna compañía decidiese invertir en producir un fuel de 0,5%S, su precio debería ser muy similar al del gasóleo para poder recuperar las inversiones empleadas. Por lo tanto, el único combustible que actualmente cumple con los nuevos niveles impuestos por la OMI es el gasóleo, con un precio que durante el año 2014 estuvo a una media de 307 USD/ton más alto que el actual fuel oil. Este mayor precio de compra de combustible impactará directamente sobre el coste del trasporte marítimo. La entrada en vigor de las anteriores normativas está suponiendo un reto para todo el sector marítimo. Ante esta realidad, se plantean diferentes alternativas con diferentes implicaciones técnicas, operativas y financieras. En la actualidad, son tres las alternativas con mayor aceptación en el sector. La primera alternativa consiste en “no hacer nada” y simplemente cambiar el tipo de combustible de los grandes buques de fuel oil a gasóleo. Las segunda alternativa es la instalación de un equipo scrubber, que permitiría continuar con el consumo de fuel oil, limpiando sus gases de combustión antes de salir a la atmósfera. Y, por último, la tercera alternativa consiste en el uso de Gas Natural Licuado (GNL) como combustible, con un precio inferior al del gasóleo. Sin embargo, aún existen importantes incertidumbres sobre la evolución futura de precios, operación y mantenimiento de las nuevas tecnologías, inversiones necesarias, disponibilidad de infraestructura portuaria e incluso el desarrollo futuro de la propia normativa internacional. Estas dudas hacen que ninguna de estas tres alternativas sea unánime en el sector. En esta tesis, tras exponer en el capítulo 3 la regulación aplicable al sector, hemos investigado sus consecuencias. Para ello, hemos examinado en el capítulo 4 si existen en la actualidad combustibles marinos que cumplan con los nuevos límites de azufre o en su defecto, cuál sería el precio de los nuevos combustibles. Partimos en el capítulo 5 de la hipótesis de que todos los buques cambian su consumo de fuel oil a gasóleo para cumplir con dicha normativa, calculamos el incremento de demanda de gasóleo que se produciría y analizamos las consecuencias que este hecho tendría sobre la producción de gasóleos en el Mediterráneo. Adicionalmente, calculamos el impacto económico que dicho incremento de coste producirá sobre sector exterior de España. Para ello, empleamos como base de datos el sistema de control de tráfico marítimo Authomatic Identification System (AIS) para luego analizar los datos de todos los buques que han hecho escala en algún puerto español, para así calcular el extra coste anual por el consumo de gasóleo que sufrirá el transporte marítimo para mover todas las importaciones y exportaciones de España. Por último, en el capítulo 6, examinamos y comparamos las otras dos alternativas al consumo de gasóleo -scrubbers y propulsión con GNL como combustible- y, finalmente, analizamos en el capítulo 7, la viabilidad de las inversiones en estas dos tecnologías para cumplir con la regulación. En el capítulo 5 explicamos los numerosos métodos que existen para calcular la demanda de combustible de un buque. La metodología seguida para su cálculo será del tipo bottom-up, que está basada en la agregación de la actividad y las características de cada tipo de buque. El resultado está basado en la potencia instalada de cada buque, porcentaje de carga del motor y su consumo específico. Para ello, analizamos el número de buques que navegan por el Mediterráneo a lo largo de un año mediante el sistema AIS, realizando “fotos” del tráfico marítimo en el Mediterráneo y reportando todos los buques en navegación en días aleatorios a lo largo de todo el año 2014. Por último, y con los datos anteriores, calculamos la demanda potencial de gasóleo en el Mediterráneo. Si no se hace nada y los buques comienzan a consumir gasóleo como combustible principal, en vez del actual fuel oil para cumplir con la regulación, la demanda de gasoil en el Mediterráneo aumentará en 12,12 MTA (Millones de Toneladas Anuales) a partir del año 2020. Esto supone alrededor de 3.720 millones de dólares anuales por el incremento del gasto de combustible tomando como referencia el precio medio de los combustibles marinos durante el año 2014. El anterior incremento de demanda en el Mediterráneo supondría el 43% del total de la demanda de gasóleos en España en el año 2013, incluyendo gasóleos de automoción, biodiesel y gasóleos marinos y el 3,2% del consumo europeo de destilados medios durante el año 2014. ¿Podrá la oferta del mercado europeo asumir este incremento de demanda de gasóleos? Europa siempre ha sido excedentaria en gasolina y deficitaria en destilados medios. En el año 2009, Europa tuvo que importar 4,8 MTA de Norte América y 22,1 MTA de Asia. Por lo que, este aumento de demanda sobre la ya limitada capacidad de refino de destilados medios en Europa incrementará las importaciones y producirá también aumentos en los precios, sobre todo del mercado del gasóleo. El sector sobre el que más impactará el incremento de demanda de gasóleo será el de los cruceros que navegan por el Mediterráneo, pues consumirán un 30,4% de la demanda de combustible de toda flota mundial de cruceros, lo que supone un aumento en su gasto de combustible de 386 millones de USD anuales. En el caso de los RoRos, consumirían un 23,6% de la demanda de la flota mundial de este tipo de buque, con un aumento anual de 171 millones de USD sobre su gasto de combustible anterior. El mayor incremento de coste lo sufrirán los portacontenedores, con 1.168 millones de USD anuales sobre su gasto actual. Sin embargo, su consumo en el Mediterráneo representa sólo el 5,3% del consumo mundial de combustible de este tipo de buques. Estos números plantean la incertidumbre de si semejante aumento de gasto en buques RoRo hará que el transporte marítimo de corta distancia en general pierda competitividad sobre otros medios de transporte alternativos en determinadas rutas. De manera que, parte del volumen de mercancías que actualmente transportan los buques se podría trasladar a la carretera, con los inconvenientes medioambientales y operativos, que esto produciría. En el caso particular de España, el extra coste por el consumo de gasóleo de todos los buques con escala en algún puerto español en el año 2013 se cifra en 1.717 millones de EUR anuales, según demostramos en la última parte del capítulo 5. Para realizar este cálculo hemos analizado con el sistema AIS a todos los buques que han tenido escala en algún puerto español y los hemos clasificado por distancia navegada, tipo de buque y potencia. Este encarecimiento del transporte marítimo será trasladado al sector exterior español, lo cual producirá un aumento del coste de las importaciones y exportaciones por mar en un país muy expuesto, pues el 75,61% del total de las importaciones y el 53,64% del total de las exportaciones se han hecho por vía marítima. Las tres industrias que se verán más afectadas son aquellas cuyo valor de mercancía es inferior respecto a su coste de transporte. Para ellas los aumentos del coste sobre el total del valor de cada mercancía serán de un 2,94% para la madera y corcho, un 2,14% para los productos minerales y un 1,93% para las manufacturas de piedra, cemento, cerámica y vidrio. Las mercancías que entren o salgan por los dos archipiélagos españoles de Canarias y Baleares serán las que se verán más impactadas por el extra coste del transporte marítimo, ya que son los puertos más alejados de otros puertos principales y, por tanto, con más distancia de navegación. Sin embargo, esta no es la única alternativa al cumplimiento de la nueva regulación. De la lectura del capítulo 6 concluimos que las tecnologías de equipos scrubbers y de propulsión con GNL permitirán al buque consumir combustibles más baratos al gasoil, a cambio de una inversión en estas tecnologías. ¿Serán los ahorros producidos por estas nuevas tecnologías suficientes para justificar su inversión? Para contestar la anterior pregunta, en el capítulo 7 hemos comparado las tres alternativas y hemos calculado tanto los costes de inversión como los gastos operativos correspondientes a equipos scrubbers o propulsión con GNL para una selección de 53 categorías de buques. La inversión en equipos scrubbers es más conveniente para buques grandes, con navegación no regular. Sin embargo, para buques de tamaño menor y navegación regular por puertos con buena infraestructura de suministro de GNL, la inversión en una propulsión con GNL como combustible será la más adecuada. En el caso de un tiempo de navegación del 100% dentro de zonas ECA y bajo el escenario de precios visto durante el año 2014, los proyectos con mejor plazo de recuperación de la inversión en equipos scrubbers son para los cruceros de gran tamaño (100.000 tons. GT), para los que se recupera la inversión en 0,62 años, los grandes portacontenedores de más de 8.000 TEUs con 0,64 años de recuperación y entre 5.000-8.000 TEUs con 0,71 años de recuperación y, por último, los grandes petroleros de más de 200.000 tons. de peso muerto donde tenemos un plazo de recuperación de 0,82 años. La inversión en scrubbers para buques pequeños, por el contrario, tarda más tiempo en recuperarse llegando a más de 5 años en petroleros y quimiqueros de menos de 5.000 toneladas de peso muerto. En el caso de una posible inversión en propulsión con GNL, las categorías de buques donde la inversión en GNL es más favorable y recuperable en menor tiempo son las más pequeñas, como ferris, cruceros o RoRos. Tomamos ahora el caso particular de un buque de productos limpios de 38.500 toneladas de peso muerto ya construido y nos planteamos la viabilidad de la inversión en la instalación de un equipo scrubber o bien, el cambio a una propulsión por GNL a partir del año 2015. Se comprueba que las dos variables que más impactan sobre la conveniencia de la inversión son el tiempo de navegación del buque dentro de zonas de emisiones controladas (ECA) y el escenario futuro de precios del MGO, HSFO y GNL. Para realizar este análisis hemos estudiado cada inversión, calculando una batería de condiciones de mérito como el payback, TIR, VAN y la evolución de la tesorería del inversor. Posteriormente, hemos calculado las condiciones de contorno mínimas de este buque en concreto para asegurar una inversión no sólo aceptable, sino además conveniente para el naviero inversor. En el entorno de precios del 2014 -con un diferencial entre fuel y gasóleo de 264,35 USD/ton- si el buque pasa más de un 56% de su tiempo de navegación en zonas ECA, conseguirá una rentabilidad de la inversión para inversores (TIR) en el equipo scrubber que será igual o superior al 9,6%, valor tomado como coste de oportunidad. Para el caso de inversión en GNL, en el entorno de precios del año 2014 -con un diferencial entre GNL y gasóleo de 353,8 USD/ton FOE- si el buque pasa más de un 64,8 % de su tiempo de navegación en zonas ECA, conseguirá una rentabilidad de la inversión para inversores (TIR) que será igual o superior al 9,6%, valor del coste de oportunidad. Para un tiempo en zona ECA estimado de un 60%, la rentabilidad de la inversión (TIR) en scrubbers para los inversores será igual o superior al 9,6%, el coste de oportunidad requerido por el inversor, para valores del diferencial de precio entre los dos combustibles alternativos, gasóleo (MGO) y fuel oil (HSFO) a partir de 244,73 USD/ton. En el caso de una inversión en propulsión GNL se requeriría un diferencial de precio entre MGO y GNL de 382,3 USD/ton FOE o superior. Así, para un buque de productos limpios de 38.500 DWT, la inversión en una reconversión para instalar un equipo scrubber es más conveniente que la de GNL, pues alcanza rentabilidades de la inversión (TIR) para inversores del 12,77%, frente a un 6,81% en el caso de invertir en GNL. Para ambos cálculos se ha tomado un buque que navegue un 60% de su tiempo por zona ECA y un escenario de precios medios del año 2014 para el combustible. Po otro lado, las inversiones en estas tecnologías a partir del año 2025 para nuevas construcciones son en ambos casos convenientes. El naviero deberá prestar especial atención aquí a las características propias de su buque y tipo de navegación, así como a la infraestructura de suministros y vertidos en los puertos donde vaya a operar usualmente. Si bien, no se ha estudiado en profundidad en esta tesis, no olvidemos que el sector marítimo debe cumplir además con las otras dos limitaciones que la regulación de la OMI establece sobre las emisiones de óxidos de Nitrógeno (NOx) y Carbono (CO2) y que sin duda, requerirán adicionales inversiones en diversos equipos. De manera que, si bien las consecuencias del consumo de gasóleo como alternativa al cumplimiento de la Regulación MARPOL son ciertamente preocupantes, existen alternativas al uso del gasóleo, con un aumento sobre el coste del transporte marítimo menor y manteniendo los beneficios sociales que pretende dicha ley. En efecto, como hemos demostrado, las opciones que se plantean como más rentables desde el punto de vista financiero son el consumo de GNL en los buques pequeños y de línea regular (cruceros, ferries, RoRos), y la instalación de scrubbers para el resto de buques de grandes dimensiones. Pero, por desgracia, estas inversiones no llegan a hacerse realidad por el elevado grado de incertidumbre asociado a estos dos mercados, que aumenta el riesgo empresarial, tanto de navieros como de suministradores de estas nuevas tecnologías. Observamos así una gran reticencia del sector privado a decidirse por estas dos alternativas. Este elevado nivel de riesgo sólo puede reducirse fomentando el esfuerzo conjunto del sector público y privado para superar estas barreras de entrada del mercado de scrubbers y GNL, que lograrían reducir las externalidades medioambientales de las emisiones sin restar competitividad al transporte marítimo. Creemos así, que los mismos organismos que aprobaron dicha ley deben ayudar al sector naviero a afrontar las inversiones en dichas tecnologías, así como a impulsar su investigación y promover la creación de una infraestructura portuaria adaptada a suministros de GNL y a descargas de vertidos procedentes de los equipos scrubber. Deberían además, prestar especial atención sobre las ayudas al sector de corta distancia para evitar que pierda competitividad frente a otros medios de transporte por el cumplimiento de esta normativa. Actualmente existen varios programas europeos de incentivos, como TEN-T o Marco Polo, pero no los consideramos suficientes. Por otro lado, la Organización Marítima Internacional debe confirmar cuanto antes si retrasa o no al 2025 la nueva bajada del nivel de azufre en combustibles. De esta manera, se eliminaría la gran incertidumbre temporal que actualmente tienen tanto navieros, como empresas petroleras y puertos para iniciar sus futuras inversiones y poder estudiar la viabilidad de cada alternativa de forma individual. ABSTRACT On 10 October 2008 the International Maritime Organization (IMO) signed an amendment to Annex VI of the MARPOL 73/78 convention establishing a gradual reduction in sulphur oxide (SOx) emissions from ships, and an additional reduction in nitrogen oxide (NOx) emissions and carbon dioxide (CO2) emissions from marine engines which cause environmental problems such as acid rain and the greenhouse effect. According to this regulation, from 1 January 2015, ships travelling in an Emission Control Area (ECA) must use fuels with a sulphur content of less than 0.1%. From 1 January 2020, or alternatively from 2025 if the IMO should decide to delay its introduction, all ships must use fuels with a sulphur content of less than 0.5%. As before, this content will be 0.1%S for voyages within ECAs. Meanwhile, the European Union has gone further than the IMO, and will apply the strictest limits of the MARPOL directives in the waters of its exclusive economic zone from 2020. To this end, Directive 2012/33/EU was issued on 21 November 2013 as an addendum to the 1999 Directive. These laws are intended to improve public health and the environment, benefiting society by reducing disease, particularly respiratory problems. The first question which arises is: what fuel do ships currently use, and what fuel will they have to use to comply with the Convention? Today, large international shipping vessels consume fuel oil with a sulphur level of 3.5%. Do fuel oils exist with a sulphur level of 0.5%S? As we conclude in Chapter 4, oil companies regard marine fuel oil as a by-product of refining Brent to produce their basket of products, as the demand for fuel oil is declining in comparison to other products, and the profit margin on the sale of other petroleum products is higher. Thus, oil companies are not interested in investing in their refineries to produce low-sulphur fuel oils, and if a company should decide to invest in producing a 0.5%S fuel oil, its price would have to be very similar to that of marine gas oil in order to recoup the investment. Therefore, the only fuel which presently complies with the new levels required by the IMO is marine gas oil, which was priced on average 307 USD/tonne higher than current fuel oils during 2014. This higher purchasing price for fuel will have a direct impact on the cost of maritime transport. The entry into force of the above directive presents a challenge for the entire maritime sector. There are various alternative approaches to this situation, with different technical, operational and financial implications. At present three options are the most widespread in the sector. The first option consists of “doing nothing” and simply switching from fuel oil to marine gas oil in large ships. The second option is installing a scrubber system, which would enable ships to continue consuming fuel oil, cleaning the combustion gases before they are released to the atmosphere. And finally, the third option is using Liquefied Natural Gas (LNG), which is priced lower than marine gas oil, as a fuel. However, there is still significant uncertainty on future variations in prices, the operation and maintenance of the new technologies, the investments required, the availability of port infrastructure and even future developments in the international regulations themselves. These uncertainties mean that none of these three alternatives has been unanimously accepted by the sector. In this Thesis, after discussing all the regulations applicable to the sector in Chapter 3, we investigate their consequences. In Chapter 4 we examine whether there are currently any marine fuels on the market which meet the new sulphur limits, and if not, how much new fuels would cost. In Chapter 5, based on the hypothesis that all ships will switch from fuel oil to marine gas oil to comply with the regulations, we calculate the increase in demand for marine gas oil this would lead to, and analyse the consequences this would have on marine gas oil production in the Mediterranean. We also calculate the economic impact such a cost increase would have on Spain's external sector. To do this, we also use the Automatic Identification System (AIS) system to analyse the data of every ship stopping in any Spanish port, in order to calculate the extra cost of using marine gas oil in maritime transport for all Spain's imports and exports. Finally, in Chapter 6, we examine and compare the other two alternatives to marine gas oil, scrubbers and LNG, and in Chapter 7 we analyse the viability of investing in these two technologies in order to comply with the regulations. In Chapter 5 we explain the many existing methods for calculating a ship's fuel consumption. We use a bottom-up calculation method, based on aggregating the activity and characteristics of each type of vessel. The result is based on the installed engine power of each ship, the engine load percentage and its specific consumption. To do this, we analyse the number of ships travelling in the Mediterranean in the course of one year, using the AIS, a marine traffic monitoring system, to take “snapshots” of marine traffic in the Mediterranean and report all ships at sea on random days throughout 2014. Finally, with the above data, we calculate the potential demand for marine gas oil in the Mediterranean. If nothing else is done and ships begin to use marine gas oil instead of fuel oil in order to comply with the regulation, the demand for marine gas oil in the Mediterranean will increase by 12.12 MTA (Millions Tonnes per Annum) from 2020. This means an increase of around 3.72 billion dollars a year in fuel costs, taking as reference the average price of marine fuels in 2014. Such an increase in demand in the Mediterranean would be equivalent to 43% of the total demand for diesel in Spain in 2013, including automotive diesel fuels, biodiesel and marine gas oils, and 3.2% of European consumption of middle distillates in 2014. Would the European market be able to supply enough to meet this greater demand for diesel? Europe has always had a surplus of gasoline and a deficit of middle distillates. In 2009, Europe had to import 4.8 MTA from North America and 22.1 MTA from Asia. Therefore, this increased demand on Europe's already limited capacity for refining middle distillates would lead to increased imports and higher prices, especially in the diesel market. The sector which would suffer the greatest impact of increased demand for marine gas oil would be Mediterranean cruise ships, which represent 30.4% of the fuel demand of the entire world cruise fleet, meaning their fuel costs would rise by 386 million USD per year. ROROs in the Mediterranean, which represent 23.6% of the demand of the world fleet of this type of ship, would see their fuel costs increase by 171 million USD a year. The greatest cost increase would be among container ships, with an increase on current costs of 1.168 billion USD per year. However, their consumption in the Mediterranean represents only 5.3% of worldwide fuel consumption by container ships. These figures raise the question of whether a cost increase of this size for RORO ships would lead to short-distance marine transport in general becoming less competitive compared to other transport options on certain routes. For example, some of the goods that ships now carry could switch to road transport, with the undesirable effects on the environment and on operations that this would produce. In the particular case of Spain, the extra cost of switching to marine gas oil in all ships stopping at any Spanish port in 2013 would be 1.717 billion EUR per year, as we demonstrate in the last part of Chapter 5. For this calculation, we used the AIS system to analyse all ships which stopped at any Spanish port, classifying them by distance travelled, type of ship and engine power. This rising cost of marine transport would be passed on to the Spanish external sector, increasing the cost of imports and exports by sea in a country which relies heavily on maritime transport, which accounts for 75.61% of Spain's total imports and 53.64% of its total exports. The three industries which would be worst affected are those with goods of lower value relative to transport costs. The increased costs over the total value of each good would be 2.94% for wood and cork, 2.14% for mineral products and 1.93% for manufactured stone, cement, ceramic and glass products. Goods entering via the two Spanish archipelagos, the Canary Islands and the Balearic Islands, would suffer the greatest impact from the extra cost of marine transport, as these ports are further away from other major ports and thus the distance travelled is greater. However, this is not the only option for compliance with the new regulations. From our readings in Chapter 6 we conclude that scrubbers and LNG propulsion would enable ships to use cheaper fuels than marine gas oil, in exchange for investing in these technologies. Would the savings gained by these new technologies be enough to justify the investment? To answer this question, in Chapter 7 we compare the three alternatives and calculate both the cost of investment and the operating costs associated with scrubbers or LNG propulsion for a selection of 53 categories of ships. Investing in scrubbers is more advisable for large ships with no fixed runs. However, for smaller ships with regular runs to ports with good LNG supply infrastructure, investing in LNG propulsion would be the best choice. In the case of total transit time within an ECA and the pricing scenario seen in 2014, the best payback periods on investments in scrubbers are for large cruise ships (100,000 gross tonnage), which would recoup their investment in 0.62 years; large container ships, with a 0.64 year payback period for those over 8,000 TEUs and 0.71 years for the 5,000-8,000 TEU category; and finally, large oil tankers over 200,000 deadweight tonnage, which would recoup their investment in 0.82 years. However, investing in scrubbers would have a longer payback period for smaller ships, up to 5 years or more for oil tankers and chemical tankers under 5,000 deadweight tonnage. In the case of LNG propulsion, a possible investment is more favourable and the payback period is shorter for smaller ship classes, such as ferries, cruise ships and ROROs. We now take the case of a ship transporting clean products, already built, with a deadweight tonnage of 38,500, and consider the viability of investing in installing a scrubber or changing to LNG propulsion, starting in 2015. The two variables with the greatest impact on the advisability of the investment are how long the ship is at sea within emission control areas (ECA) and the future price scenario of MGO, HSFO and LNG. For this analysis, we studied each investment, calculating a battery of merit conditions such as the payback period, IRR, NPV and variations in the investors' liquid assets. We then calculated the minimum boundary conditions to ensure the investment was not only acceptable but advisable for the investor shipowner. Thus, for the average price differential of 264.35 USD/tonne between HSFO and MGO during 2014, investors' return on investment (IRR) in scrubbers would be the same as the required opportunity cost of 9.6%, for values of over 56% ship transit time in ECAs. For the case of investing in LNG and the average price differential between MGO and LNG of 353.8 USD/tonne FOE in 2014, the ship must spend 64.8% of its time in ECAs for the investment to be advisable. For an estimated 60% of time in an ECA, the internal rate of return (IRR) for investors equals the required opportunity cost of 9.6%, based on a price difference of 244.73 USD/tonne between the two alternative fuels, marine gas oil (MGO) and fuel oil (HSFO). An investment in LNG propulsion would require a price differential between MGO and LNG of 382.3 USD/tonne FOE. Thus, for a 38,500 DWT ship carrying clean products, investing in retrofitting to install a scrubber is more advisable than converting to LNG, with an internal rate of return (IRR) for investors of 12.77%, compared to 6.81% for investing in LNG. Both calculations were based on a ship which spends 60% of its time at sea in an ECA and a scenario of average 2014 prices. However, for newly-built ships, investments in either of these technologies from 2025 would be advisable. Here, the shipowner must pay particular attention to the specific characteristics of their ship, the type of operation, and the infrastructure for supplying fuel and handling discharges in the ports where it will usually operate. Thus, while the consequences of switching to marine gas oil in order to comply with the MARPOL regulations are certainly alarming, there are alternatives to marine gas oil, with smaller increases in the costs of maritime transport, while maintaining the benefits to society this law is intended to provide. Indeed, as we have demonstrated, the options which appear most favourable from a financial viewpoint are conversion to LNG for small ships and regular runs (cruise ships, ferries, ROROs), and installing scrubbers for large ships. Unfortunately, however, these investments are not being made, due to the high uncertainty associated with these two markets, which increases business risk, both for shipowners and for the providers of these new technologies. This means we are seeing considerable reluctance regarding these two options among the private sector. This high level of risk can be lowered only by encouraging joint efforts by the public and private sectors to overcome these barriers to entry into the market for scrubbers and LNG, which could reduce the environmental externalities of emissions without affecting the competitiveness of marine transport. Our opinion is that the same bodies which approved this law must help the shipping industry invest in these technologies, drive research on them, and promote the creation of a port infrastructure which is adapted to supply LNG and handle the discharges from scrubber systems. At present there are several European incentive programmes, such as TEN-T and Marco Polo, but we do not consider these to be sufficient. For its part, the International Maritime Organization should confirm as soon as possible whether the new lower sulphur levels in fuels will be postponed until 2025. This would eliminate the great uncertainty among shipowners, oil companies and ports regarding the timeline for beginning their future investments and for studying their viability.

A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana

Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind SC10-RNase in SI N. alata SC10SC10 and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia × SI N. alata SC10SC10) hybrids with reduced levels of HT-protein continued to express SC10-RNase but failed to reject SC10-pollen. Control hybrids expressing both SC10-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

tRNA binding to the ribosomal P site is dependent not only on correct codon–anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitution–interference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNAPhe from Escherichia coli which are required for P-site binding. Stereo-specific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp- or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNAPhe and E. coli tRNAfMet indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification–interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNAPhe, we propose that specific Rp-phosphate oxygens are required for anticodon loop (“U-turn”) stabilization or are involved in interactions with the ribosome on correct tRNA–mRNA complex formation.

Schizosaccharomyces pombe cdc20+ encodes DNA polymerase ɛ and is required for chromosomal replication but not for the S phase checkpoint

In fission yeast both DNA polymerase alpha (pol α) and delta (pol δ) are required for DNA chromosomal replication. Here we demonstrate that Schizosaccharomyces pombe cdc20+ encodes the catalytic subunit of DNA polymerase epsilon (pol ɛ) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20+ is required early in S phase very near to the initiation step. In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol ɛ (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995) Cell 80, 29–39]. We have investigated whether cdc20+ is required for the checkpoint control operating in fission yeast, and our data indicate that pol ɛ does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol α rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol α, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.

Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation

Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation. Clostridium botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCK are required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP.

Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1 Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.