Development and assessment of traffic-related emission abatement measures for the Madrid city (Spain) through the WRF-SMOKE-CMAQ modelling system

The achievement of the limit values established in the European legislation pose an important handicap for large urban areas with intense road traffic, such as Madrid (Spain). Despite permanent measures included in air quality plans it is important to assess additional measures that may be temporally applied under unfavourable conditions. This paper reports on the simulation of different traffic restriction strategies in Madrid for high-pollution episodes.

Magnetic Attitude Control System for a Small Satellite. Impact on the Thermal Performance

El principal objetivo de la tesis es estudiar el acoplamiento entre los subsistemas de control de actitud y de control térmico de un pequeño satélite, con el fin de buscar la solución a los problemas relacionados con la determinación de los parámetros de diseño. Se considera la evolución de la actitud y de las temperaturas del satélite bajo la influencia de dos estrategias de orientación diferentes: 1) estabilización magnética pasiva de la orientación (PMAS, passive magnetic attitude stabilization), y 2) control de actitud magnético activo (AMAC, active magnetic attitude control). En primer lugar se presenta el modelo matemático del problema, que incluye la dinámica rotacional y el modelo térmico. En el problema térmico se considera un satélite cúbico modelizado por medio de siete nodos (seis externos y uno interno) aplicando la ecuación del balance térmico. Una vez establecido el modelo matemático del problema, se estudia la evolución que corresponde a las dos estrategias mencionadas. La estrategia PMAS se ha seleccionado por su simplicidad, fiabilidad, bajo coste, ahorrando consumo de potencia, masa coste y complejidad, comparado con otras estrategias. Se ha considerado otra estrategia de control que consigue que el satélite gire a una velocidad requerida alrededor de un eje deseado de giro, pudiendo controlar su dirección en un sistema inercial de referencia, ya que frecuentemente el subsistema térmico establece requisitos de giro alrededor de un eje del satélite orientado en una dirección perpendicular a la radiación solar incidente. En relación con el problema térmico, para estudiar la influencia de la velocidad de giro en la evolución de las temperaturas en diversos puntos del satélite, se ha empleado un modelo térmico linealizado, obtenido a partir de la formulación no lineal aplicando un método de perturbaciones. El resultado del estudio muestra que el tiempo de estabilización de la temperatura y la influencia de las cargas periódicas externas disminuye cuando aumenta la velocidad de giro. Los cambios de temperatura se reducen hasta ser muy pequeños para velocidades de rotación altas. En relación con la estrategia PMAC se ha observado que a pesar de su uso extendido entre los micro y nano satélites todavía presenta problemas que resolver. Estos problemas están relacionados con el dimensionamiento de los parámetros del sistema y la predicción del funcionamiento en órbita. Los problemas aparecen debido a la dificultad en la determinación de las características magnéticas de los cuerpos ferromagnéticos (varillas de histéresis) que se utilizan como amortiguadores de oscilaciones en los satélites. Para estudiar este problema se presenta un modelo analítico que permite estimar la eficiencia del amortiguamiento, y que se ha aplicado al estudio del comportamiento en vuelo de varios satélites, y que se ha empleado para comparar los resultados del modelo con los obtenidos en vuelo, observándose que el modelo permite explicar satisfactoriamente el comportamiento registrado. ABSTRACT The main objective of this thesis is to study the coupling between the attitude control and thermal control subsystems of a small satellite, and address the solution to some existing issues concerning the determination of their parameters. Through the thesis the attitude and temperature evolution of the satellite is studied under the influence of two independent attitude stabilization and control strategies: (1) passive magnetic attitude stabilization (PMAS), and (2) active magnetic attitude control (AMAC). In this regard the mathematical model of the problem is explained and presented. The mathematical model includes both the rotational dynamics and the thermal model. The thermal model is derived for a cubic satellite by solving the heat balance equation for 6 external and 1 internal nodes. Once established the mathematical model of the problem, the above mentioned attitude strategies were applied to the system and the temperature evolution of the 7 nodes of the satellite was studied. The PMAS technique has been selected to be studied due to its prevalent use, simplicity, reliability, and cost, as this strategy significantly saves the overall power, weight, cost, and reduces the complexity of the system compared to other attitude control strategies. In addition to that, another control law that provides the satellite with a desired spin rate along a desired axis of the satellite, whose direction can be controlled with respect to the inertial reference frame is considered, as the thermal subsystem of a satellite usually demands a spin requirement around an axis of the satellite which is positioned perpendicular to the direction of the coming solar radiation. Concerning the thermal problem, to study the influence of spin rate on temperature evolution of the satellite a linear approach of the thermal model is used, which is based on perturbation theory applied to the nonlinear differential equations of the thermal model of a spacecraft moving in a closed orbit. The results of this study showed that the temperature stabilization time and the periodic influence of the external thermal loads decreases by increasing the spin rate. However, the changes become insignificant for higher values of spin rate. Concerning the PMAS strategy, it was observed that in spite of its extended application to micro and nano satellites, still there are some issues to be solved regarding this strategy. These issues are related to the sizing of its system parameters and predicting the in-orbit performance. The problems were found to be rooted in the difficulties that exist in determining the magnetic characteristics of the ferromagnetic bodies (hysteresis rods) that are applied as damping devices on-board satellites. To address these issues an analytic model for estimating their damping efficiency is proposed and applied to several existing satellites in order to compare the results with their respective in-flight data. This model can explain the behavior showed by these satellites.

Cognitive risk perception system for obstacle avoidance in outdoor mUAV missions

La robótica ha evolucionado exponencialmente en las últimas décadas, permitiendo a los sistemas actuales realizar tareas sumamente complejas con gran precisión, fiabilidad y velocidad. Sin embargo, este desarrollo ha estado asociado a un mayor grado de especialización y particularización de las tecnologías implicadas, siendo estas muy eficientes en situaciones concretas y controladas, pero incapaces en entornos cambiantes, dinámicos y desestructurados. Por eso, el desarrollo de la robótica debe pasar por dotar a los sistemas de capacidad de adaptación a las circunstancias, de entendedimiento sobre los cambios observados y de flexibilidad a la hora de interactuar con el entorno. Estas son las caracteristicas propias de la interacción del ser humano con su entorno, las que le permiten sobrevivir y las que pueden proporcionar a un sistema inteligencia y capacidad suficientes para desenvolverse en un entorno real de forma autónoma e independiente. Esta adaptabilidad es especialmente importante en el manejo de riesgos e incetidumbres, puesto que es el mecanismo que permite contextualizar y evaluar las amenazas para proporcionar una respuesta adecuada. Así, por ejemplo, cuando una persona se mueve e interactua con su entorno, no evalúa los obstáculos en función de su posición, velocidad o dinámica (como hacen los sistemas robóticos tradicionales), sino mediante la estimación del riesgo potencial que estos elementos suponen para la persona. Esta evaluación se consigue combinando dos procesos psicofísicos del ser humano: por un lado, la percepción humana analiza los elementos relevantes del entorno, tratando de entender su naturaleza a partir de patrones de comportamiento, propiedades asociadas u otros rasgos distintivos. Por otro lado, como segundo nivel de evaluación, el entendimiento de esta naturaleza permite al ser humano conocer/estimar la relación de los elementos con él mismo, así como sus implicaciones en cuanto a nivel de riesgo se refiere. El establecimiento de estas relaciones semánticas -llamado cognición- es la única forma de definir el nivel de riesgo de manera absoluta y de generar una respuesta adecuada al mismo. No necesariamente proporcional, sino coherente con el riesgo al que se enfrenta. La investigación que presenta esta tesis describe el trabajo realizado para trasladar esta metodología de análisis y funcionamiento a la robótica. Este se ha centrado especialmente en la nevegación de los robots aéreos, diseñando e implementado procedimientos de inspiración humana para garantizar la seguridad de la misma. Para ello se han estudiado y evaluado los mecanismos de percepción, cognición y reacción humanas en relación al manejo de riesgos. También se ha analizado como los estímulos son capturados, procesados y transformados por condicionantes psicológicos, sociológicos y antropológicos de los seres humanos. Finalmente, también se ha analizado como estos factores motivan y descandenan las reacciones humanas frente a los peligros. Como resultado de este estudio, todos estos procesos, comportamientos y condicionantes de la conducta humana se han reproducido en un framework que se ha estructurado basadandose en factores análogos. Este emplea el conocimiento obtenido experimentalmente en forma de algoritmos, técnicas y estrategias, emulando el comportamiento humano en las mismas circunstancias. Diseñado, implementeado y validado tanto en simulación como con datos reales, este framework propone una manera innovadora -tanto en metodología como en procedimiento- de entender y reaccionar frente a las amenazas potenciales de una misión robótica. ABSTRACT Robotics has undergone a great revolution in the last decades. Nowadays this technology is able to perform really complex tasks with a high degree of accuracy and speed, however this is only true in precisely defined situations with fully controlled variables. Since the real world is dynamic, changing and unstructured, flexible and non context-dependent systems are required. The ability to understand situations, acknowledge changes and balance reactions is required by robots to successfully interact with their surroundings in a fully autonomous fashion. In fact, it is those very processes that define human interactions with the environment. Social relationships, driving or risk/incertitude management... in all these activities and systems, context understanding and adaptability are what allow human beings to survive: contrarily to the traditional robotics, people do not evaluate obstacles according to their position but according to the potential risk their presence imply. In this sense, human perception looks for information which goes beyond location, speed and dynamics (the usual data used in traditional obstacle avoidance systems). Specific features in the behaviour of a particular element allows the understanding of that element’s nature and therefore the comprehension of the risk posed by it. This process defines the second main difference between traditional obstacle avoidance systems and human behaviour: the ability to understand a situation/scenario allows to get to know the implications of the elements and their relationship with the observer. Establishing these semantic relationships -named cognition- is the only way to estimate the actual danger level of an element. Furthermore, only the application of this knowledge allows the generation of coherent, suitable and adjusted responses to deal with any risk faced. The research presented in this thesis summarizes the work done towards translating these human cognitive/reasoning procedures to the field of robotics. More specifically, the work done has been focused on employing human-based methodologies to enable aerial robots to navigate safely. To this effect, human perception, cognition and reaction processes concerning risk management have been experimentally studied; as well as the acquisition and processing of stimuli. How psychological, sociological and anthropological factors modify, balance and give shape to those stimuli has been researched. And finally, the way in which these factors motivate the human behaviour according to different mindsets and priorities has been established. This associative workflow has been reproduced by establishing an equivalent structure and defining similar factors and sources. Besides, all the knowledge obtained experimentally has been applied in the form of algorithms, techniques and strategies which emulate the analogous human behaviours. As a result, a framework capable of understanding and reacting in response to stimuli has been implemented and validated.

Development and implementation of an air quality integrated assessment modelling system for the Iberian Peninsula

La mejora de la calidad del aire es una tarea eminentemente interdisciplinaria. Dada la gran variedad de ciencias y partes involucradas, dicha mejora requiere de herramientas de evaluación simples y completamente integradas. La modelización para la evaluación integrada (integrated assessment modeling) ha demostrado ser una solución adecuada para la descripción de los sistemas de contaminación atmosférica puesto que considera cada una de las etapas involucradas: emisiones, química y dispersión atmosférica, impactos ambientales asociados y potencial de disminución. Varios modelos de evaluación integrada ya están disponibles a escala continental, cubriendo cada una de las etapas antesmencionadas, siendo el modelo GAINS (Greenhouse Gas and Air Pollution Interactions and Synergies) el más reconocido y usado en el contexto europeo de toma de decisiones medioambientales. Sin embargo, el manejo de la calidad del aire a escala nacional/regional dentro del marco de la evaluación integrada es deseable. Esto sin embargo, no se lleva a cabo de manera satisfactoria con modelos a escala europea debido a la falta de resolución espacial o de detalle en los datos auxiliares, principalmente los inventarios de emisión y los patrones meteorológicos, entre otros. El objetivo de esta tesis es presentar los desarrollos en el diseño y aplicación de un modelo de evaluación integrada especialmente concebido para España y Portugal. El modelo AERIS (Atmospheric Evaluation and Research Integrated system for Spain) es capaz de cuantificar perfiles de concentración para varios contaminantes (NO2, SO2, PM10, PM2,5, NH3 y O3), el depósito atmosférico de especies de azufre y nitrógeno así como sus impactos en cultivos, vegetación, ecosistemas y salud como respuesta a cambios porcentuales en las emisiones de sectores relevantes. La versión actual de AERIS considera 20 sectores de emisión, ya sea equivalentes a sectores individuales SNAP o macrosectores, cuya contribución a los niveles de calidad del aire, depósito e impactos han sido modelados a través de matrices fuentereceptor (SRMs). Estas matrices son constantes de proporcionalidad que relacionan cambios en emisiones con diferentes indicadores de calidad del aire y han sido obtenidas a través de parametrizaciones estadísticas de un modelo de calidad del aire (AQM). Para el caso concreto de AERIS, su modelo de calidad del aire “de origen” consistió en el modelo WRF para la meteorología y en el modelo CMAQ para los procesos químico-atmosféricos. La cuantificación del depósito atmosférico, de los impactos en ecosistemas, cultivos, vegetación y salud humana se ha realizado siguiendo las metodologías estándar establecidas bajo los marcos internacionales de negociación, tales como CLRTAP. La estructura de programación está basada en MATLAB®, permitiendo gran compatibilidad con software típico de escritorio comoMicrosoft Excel® o ArcGIS®. En relación con los niveles de calidad del aire, AERIS es capaz de proveer datos de media anual y media mensual, así como el 19o valor horario más alto paraNO2, el 25o valor horario y el 4o valor diario más altos para SO2, el 36o valor diario más alto para PM10, el 26o valor octohorario más alto, SOMO35 y AOT40 para O3. En relación al depósito atmosférico, el depósito acumulado anual por unidad de area de especies de nitrógeno oxidado y reducido al igual que de azufre pueden ser determinados. Cuando los valores anteriormente mencionados se relacionan con características del dominio modelado tales como uso de suelo, cubiertas vegetales y forestales, censos poblacionales o estudios epidemiológicos, un gran número de impactos puede ser calculado. Centrándose en los impactos a ecosistemas y suelos, AERIS es capaz de estimar las superaciones de cargas críticas y las superaciones medias acumuladas para especies de nitrógeno y azufre. Los daños a bosques se calculan como una superación de los niveles críticos de NO2 y SO2 establecidos. Además, AERIS es capaz de cuantificar daños causados por O3 y SO2 en vid, maíz, patata, arroz, girasol, tabaco, tomate, sandía y trigo. Los impactos en salud humana han sido modelados como consecuencia de la exposición a PM2,5 y O3 y cuantificados como pérdidas en la esperanza de vida estadística e indicadores de mortalidad prematura. La exactitud del modelo de evaluación integrada ha sido contrastada estadísticamente con los resultados obtenidos por el modelo de calidad del aire convencional, exhibiendo en la mayoría de los casos un buen nivel de correspondencia. Debido a que la cuantificación de los impactos no es llevada a cabo directamente por el modelo de calidad del aire, un análisis de credibilidad ha sido realizado mediante la comparación de los resultados de AERIS con los de GAINS para un escenario de emisiones determinado. El análisis reveló un buen nivel de correspondencia en las medias y en las distribuciones probabilísticas de los conjuntos de datos. Las pruebas de verificación que fueron aplicadas a AERIS sugieren que los resultados son suficientemente consistentes para ser considerados como razonables y realistas. En conclusión, la principal motivación para la creación del modelo fue el producir una herramienta confiable y a la vez simple para el soporte de las partes involucradas en la toma de decisiones, de cara a analizar diferentes escenarios “y si” con un bajo coste computacional. La interacción con políticos y otros actores dictó encontrar un compromiso entre la complejidad del modeladomedioambiental con el carácter conciso de las políticas, siendo esto algo que AERIS refleja en sus estructuras conceptual y computacional. Finalmente, cabe decir que AERIS ha sido creado para su uso exclusivo dentro de un marco de evaluación y de ninguna manera debe ser considerado como un sustituto de los modelos de calidad del aire ordinarios. ABSTRACT Improving air quality is an eminently inter-disciplinary task. The wide variety of sciences and stakeholders that are involved call for having simple yet fully-integrated and reliable evaluation tools available. Integrated AssessmentModeling has proved to be a suitable solution for the description of air pollution systems due to the fact that it considers each of the involved stages: emissions, atmospheric chemistry, dispersion, environmental impacts and abatement potentials. Some integrated assessment models are available at European scale that cover each of the before mentioned stages, being the Greenhouse Gas and Air Pollution Interactions and Synergies (GAINS) model the most recognized and widely-used within a European policy-making context. However, addressing air quality at the national/regional scale under an integrated assessment framework is desirable. To do so, European-scale models do not provide enough spatial resolution or detail in their ancillary data sources, mainly emission inventories and local meteorology patterns as well as associated results. The objective of this dissertation is to present the developments in the design and application of an Integrated Assessment Model especially conceived for Spain and Portugal. The Atmospheric Evaluation and Research Integrated system for Spain (AERIS) is able to quantify concentration profiles for several pollutants (NO2, SO2, PM10, PM2.5, NH3 and O3), the atmospheric deposition of sulfur and nitrogen species and their related impacts on crops, vegetation, ecosystems and health as a response to percentual changes in the emissions of relevant sectors. The current version of AERIS considers 20 emission sectors, either corresponding to individual SNAP sectors or macrosectors, whose contribution to air quality levels, deposition and impacts have been modeled through the use of source-receptor matrices (SRMs). Thesematrices are proportionality constants that relate emission changes with different air quality indicators and have been derived through statistical parameterizations of an air qualitymodeling system (AQM). For the concrete case of AERIS, its parent AQM relied on the WRF model for meteorology and on the CMAQ model for atmospheric chemical processes. The quantification of atmospheric deposition, impacts on ecosystems, crops, vegetation and human health has been carried out following the standard methodologies established under international negotiation frameworks such as CLRTAP. The programming structure isMATLAB ® -based, allowing great compatibility with typical software such as Microsoft Excel ® or ArcGIS ® Regarding air quality levels, AERIS is able to provide mean annual andmean monthly concentration values, as well as the indicators established in Directive 2008/50/EC, namely the 19th highest hourly value for NO2, the 25th highest daily value and the 4th highest hourly value for SO2, the 36th highest daily value of PM10, the 26th highest maximum 8-hour daily value, SOMO35 and AOT40 for O3. Regarding atmospheric deposition, the annual accumulated deposition per unit of area of species of oxidized and reduced nitrogen as well as sulfur can be estimated. When relating the before mentioned values with specific characteristics of the modeling domain such as land use, forest and crops covers, population counts and epidemiological studies, a wide array of impacts can be calculated. When focusing on impacts on ecosystems and soils, AERIS is able to estimate critical load exceedances and accumulated average exceedances for nitrogen and sulfur species. Damage on forests is estimated as an exceedance of established critical levels of NO2 and SO2. Additionally, AERIS is able to quantify damage caused by O3 and SO2 on grapes, maize, potato, rice, sunflower, tobacco, tomato, watermelon and wheat. Impacts on human health aremodeled as a consequence of exposure to PM2.5 and O3 and quantified as losses in statistical life expectancy and premature mortality indicators. The accuracy of the IAM has been tested by statistically contrasting the obtained results with those yielded by the conventional AQM, exhibiting in most cases a good agreement level. Due to the fact that impacts cannot be directly produced by the AQM, a credibility analysis was carried out for the outputs of AERIS for a given emission scenario by comparing them through probability tests against the performance of GAINS for the same scenario. This analysis revealed a good correspondence in the mean behavior and the probabilistic distributions of the datasets. The verification tests that were applied to AERIS suggest that results are consistent enough to be credited as reasonable and realistic. In conclusion, the main reason thatmotivated the creation of this model was to produce a reliable yet simple screening tool that would provide decision and policy making support for different “what-if” scenarios at a low computing cost. The interaction with politicians and other stakeholders dictated that reconciling the complexity of modeling with the conciseness of policies should be reflected by AERIS in both, its conceptual and computational structures. It should be noted however, that AERIS has been created under a policy-driven framework and by no means should be considered as a substitute of the ordinary AQM.

A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete–scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.

In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 102-fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation in vivo was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm3 s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.

Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

We have previously shown that the G protein of vesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracycline-dependent and can be modulated by beta-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.

Subcutaneous vaccination with irradiated, cytokine-producing tumor cells stimulates CD8+ cell-mediated immunity against tumors located in the "immunologically privileged" central nervous system.

Vaccination with cytokine-producing tumor cells generates potent immune responses against tumors outside the central nervous system (CNS). The CNS, however, is a barrier to allograft and xenograft rejection, and established tumors within the CNS have failed to respond to other forms of systemic immunotherapy. To determine what barriers the "immunologically privileged" CNS would pose to cytokine-assisted tumor vaccines and what cytokines would be most efficacious against tumors within the CNS, we irradiated B16 murine melanoma cells producing murine interleukin 2 (IL-2), IL-3, IL-4, IL-6, gamma-interferon, or granulocyte-macrophage colony stimulating factor (GM-CSF) and used these cells as subcutaneous vaccines against tumors within the brain. Under conditions where untransfected B16 cells had no effect, cells producing IL-3, IL-6, or GM-CSF increased the survival of mice challenged with viable B16 cells in the brain. Vaccination with B16 cells producing IL-4 or gamma-interferon had no effect, and vaccination with B16 cells producing IL-2 decreased survival time. GM-CSF-producing vaccines were also able to increase survival in mice with pre-established tumors. The response elicited by GM-CSF-producing vaccines was found to be specific to tumor type and to be abrogated by depletion of CD8+ cells. Unlike the immunity generated against subcutaneous tumors by GM-CSF, however, the effector responses generated against tumors in the CNS were not dependent on CD4+ cells. These data suggest that cytokine-producing tumor cells are very potent stimulators of immunity against tumors within the CNS, but effector responses in the CNS may be different from those obtained against subcutaneous tumors.

Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo.

A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that microinjection of wild-type embryonic stem (ES) cells into ak/ak blastocysts produces chimeras with normal ES-cell-derived lenses and that microinjection of Rb-/- ES cells generates an aberrant lens phenotype identical to that obtained through conventional gene targeting methodology. Our determination that a cell autonomous defect underlies the aphakia condition assures that lenses generated through LCS are necessarily ES-cell-derived. LCS provides for the rapid phenotypic analysis of loss-of-function mutations, circumvents the need for germ-line transmission of null alleles, and, most significantly, facilitates the study of essential genes whose inactivation is associated with early lethal phenotypes.

Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.

An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p-hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides.

Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease.

Activated components of the complement system are potent mediators of inflammation that may play an important role in numerous disease states. For example, they have been implicated in the pathogenesis of inflammatory joint diseases including rheumatoid arthritis (RA). To target complement activation in immune-mediated joint inflammation, we have utilized monoclonal antibodies (mAbs) that inhibit the complement cascade at C5, blocking the generation of the major chemotactic and proinflammatory factors C5a and C5b-9. In this study, we demonstrate the efficacy of a mAb specific for murine C5 in the treatment of collagen-induced arthritis, an animal model for RA. We show that systemic administration of the anti-C5 mAb effectively inhibits terminal complement activation in vivo and prevents the onset of arthritis in immunized animals. Most important, anti-C5 mAb treatment is also highly effective in ameliorating established disease. These results demonstrate a critical role for activated terminal complement components not only in the induction but also in the progression of collagen-induced arthritis and suggest that C5 may be an attractive therapeutic target in RA.

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the beta subunit of Na+,K(+)-ATPase.

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the beta subunit of Na+,K(+)-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. Our results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the beta subunit of Na+,K(+)-ATPase.

Study of interaction between Si(O,C) nanowires and the biological system

Nanomedicine is a new branch of medicine, based on the potentiality and intrinsic properties of nanomaterials. Indeed, the nanomaterials ( i.e. the materials with nano and under micron size) can be suitable to different applications in biomedicine. The nanostructures can be used by taking advantage of their properties (for example superparamagnetic nanoparticles) or functionalized to deliver the drug in a specific target, thanks the ability to cross biological barriers. The size and the shape of 1D-nanostructures (nanotubes and nanowires) have an important role on the cell fate: their morphology plays a key role on the interaction between nanostructure and the biological system. For this reason the 1D nanostructure are interesting for their ability to mime the biological system. An implantable material or device must therefore integrate with the surrounding extracellular matrix (ECM), a complex network of proteins with structural and signaling properties. Innovative techniques allow the generation of complex surface patterns that can resemble the structure of the ECM, such as 1D nanostructures. NWs based on cubic silicon carbide (3C-SiC), either bare (3C-SiC NWs) or surrounded by an amorphous shell (3C-SiC/SiO2 core/shell NWs), and silicon oxycarbide nanowires (SiOxCy NWs) can meet the chemical, mechanical and electrical requirements for tissue engineering and have a strong potential to pave the way for the development of a novel generation of implantable nano-devices. Silicon oxycarbide shows promising physical and chemical properties as elastic modulus, bending strength and hardness, chemical durability superior to conventional silicate glasses in aggressive environments and high temperature stability up to 1300 °C. Moreover, it can easily be engineered through functionalization and decoration with macro-molecules and nanoparticles. Silicon carbide has been extensively studied for applications in harsh conditions, as chemical environment, high electric field and high and low temperature, owing to its high hardness, high thermal conductivity, chemical inertness and high electron mobility. Also, its cubic polytype (3C) is highly biocompatible and hemocompatible, and some prototypes of biomedical applications and biomedical devices have been already realized starting from 3C-SiC thin films. Cubic SiC-based NWs can be used as a biomimetic biomaterial, providing a robust and novel biocompatible biological interface . We cultured in vitro A549 human lung adenocarcinoma epithelial cells and L929 murine fibroblast cells over core/shell SiC/SiO2, SiOxCy and bare 3C-SiC nanowire platforms, and analysed the cytotoxicity, by indirect and direct contact tests, the cell adhesion, and the cell proliferation. These studies showed that all the nanowires are biocompatible according to ISO 10993 standards. We evaluated the blood compatibility through the interaction of the nanowires with platelet rich plasma. The adhesion and activation of platelets on the nanowire bundles, assessed via SEM imaging and soluble P-selectin quantification, indicated that a higher platelet activation is induced by the core/shell structures compared to the bare ones. Further, platelet activation is higher with 3C-SiC/SiO2 NWs and SiOxCyNWs, which therefore appear suitable in view of possible tissue regeneration. On the contrary, bare 3C-SiC NWs show a lower platelet activation and are therefore promising in view of implantable bioelectronics devices, as cardiovascular implantable devices. The NWs properties are suitable to allow the design of a novel subretinal Micro Device (MD). This devices is based on Si NWs and PEDOT:PSS, though the well know principle of the hybrid ordered bulk heterojunction (OBHJ). The aim is to develop a device based on a well-established photovoltaic technology and to adapt this know-how to the prosthetic field. The hybrid OBHJ allows to form a radial p–n junction on a nanowire/organic structure. In addition, the nanowires increase the light absorption by means of light scattering effects: a nanowires based p-n junction increases the light absorption up to the 80%, as previously demonstrated, overcoming the Shockley-Queisser limit of 30 % of a bulk p-n junction. Another interesting employment of these NWs is to design of a SiC based epicardial-interacting patch based on teflon that include SiC nanowires. . Such contact patch can bridge the electric conduction across the cardiac infarct as nanowires can ‘sense’ the direction of the wavefront propagation on the survival cardiac tissue and transmit it to the downstream surivived regions without discontinuity. The SiC NWs are tested in terms of toxicology, biocompatibility and conductance among cardiomyocytes and myofibroblasts.

1,2-Functionalized Imidazoles as Palladium Ligands: An Efficient and Robust Catalytic System for the Fluorine-Free Hiyama Reaction

A variety of hydroxy- and amino-functionalized imidazoles were prepared from 1-methyl- and 1-(diethoxymethyl)imidazole by means of isoprene-mediated lithiation followed by reaction with an electrophile. These compounds in combination with palladium acetate were screened as catalyst systems for the Hiyama reaction under fluorine-free conditions using microwave irradiation. The systematic study of the catalytic system showed 1-methyl-2-aminoalkylimidazole derivative L1 to be the best ligand, which was employed under solvent-free conditions with a 1:2 Pd/ligand ratio and TBAB (20 mol-%) as additive. The study has revealed an interaction between the Pd/ligand ratio and the amount of TBAB. The established catalytic system presented a certain degree of robustness, and it has been successfully employed in the coupling of a range of aryl bromides and chlorides with different aryl siloxanes. Furthermore, both reagents were employed in an equimolecular amount, without an excess of organosilane.