Improved ore processing assessed by digital image análisis of the ores: a key to the sustainable development of the Iberian Pyrite Belt

Mining in the Iberian Pyrite Belt (IPB), the biggest VMS metallogenetic province known in the world to date, has to face a deep crisis in spite of the huge reserves still known after ≈5 000 years of production. This is due to several factors, as the difficult processing of complex Cu-Pb-Zn-Ag- Au ores, the exhaustion of the oxidation zone orebodies (the richest for gold, in gossan), the scarce demand for sulphuric acid in the world market, and harder environmental regulations. Of these factors, only the first and the last mentioned can be addressed by local ore geologists. A reactivation of mining can therefore only be achieved by an improved and more efficient ore processing, under the constraint of strict environmental controls. Digital image analysis of the ores, coupled to reflected light microscopy, provides a quantified and reliable mineralogical and textural characterization of the ores. The automation of the procedure for the first time furnishes the process engineers with real-time information, to improve the process and to preclude or control pollution; it can be applied to metallurgical tailings as well. This is shown by some examples of the IPB.

Research lines in Hyperthermia at the Bioinstrumentation Laboratory of the Centre for Biomedical Technology

The Bioinstrumentation Laboratory belongs to the Centre for Biomedical Technology (CTB) of the Technical University of Madrid and its main objective is to provide the scientific community with devices and techniques for the characterization of micro and nanostructures and consequently finding their best biomedical applications. Hyperthermia (greek word for “overheating”) is defined as the phenomenon that occurs when a body is exposed to an energy generating source that can produce a rise in temperature (42-45��C) for a given time [1]. Specifically, the aim of the hyperthermia methods used in The Bioinstrumentation Laboratory is the development of thermal therapies, some of these using different kinds of nanoparticles, to kill cancer cells and reduce the damage on healthy tissues. The optical hyperthermia is based on noble metal nanoparticles and laser irradiation. This kind of nanoparticles has an immense potential associated to the development of therapies for cancer on account of their Surface Plasmon Resonance (SPR) enhanced light scattering and absorption. In a short period of time, the absorbed light is converted into localized heat, so we can take advantage of these characteristics to heat up tumor cells in order to obtain the cellular death [2]. In this case, the laboratory has an optical hyperthermia device based on a continuous wave laser used to kill glioblastoma cell lines (1321N1) in the presence of gold nanorods (Figure 1a). The wavelength of the laser light is 808 nm because the penetration of the light in the tissue is deeper in the Near Infrared Region. The first optical hyperthermia results show that the laser irradiation produces cellular death in the experimental samples of glioblastoma cell lines using gold nanorods but is not able to decrease the cellular viability of cancer cells in samples without the suitable nanorods (Figure 1b) [3]. The generation of magnetic hyperthermia is performed through changes of the magnetic induction in magnetic nanoparticles (MNPs) that are embedded in viscous medium. The Figure 2 shows a schematic design of the AC induction hyperthermia device in magnetic fluids. The equipment has been manufactured at The Bioinstrumentation Laboratory. The first block implies two steps: the signal selection with frequency manipulation option from 9 KHz to 2MHz, and a linear output up to 1500W. The second block is where magnetic field is generated ( 5mm, 10 turns). Finally, the third block is a software control where the user can establish initial parameters, and also shows the temperature response of MNPs due to the magnetic field applied [4-8]. The Bioinstrumentation Laboratory in collaboration with the Mexican company MRI-DT have recently implemented a new research line on Nuclear Magnetic Resonance Hyperthermia, which is sustained on the patent US 7,423,429B2 owned by this company. This investigation is based on the use of clinical MRI equipment not only for diagnosis but for therapy [9]. This idea consists of two main facts: Magnetic Resonance Imaging can cause focal heating [10], and the differentiation in resonant frequency between healthy and cancer cells [11]. To produce only heating in cancer cells when the whole body is irradiated, it is necessary to determine the specific resonant frequency of the target, using the information contained in the spectra of the area of interest. Then, special RF pulse sequence is applied to produce fast excitation and relaxation mechanism that generates temperature increase of the tumor, causing cellular death or metabolism malfunction that stops cellular division

Contribution to the quality improvement of the antenna measurement using post-processing techniques = Contribución a la mejora de la calidad de medida de antena usando técnicas de post-procesado

One important task in the design of an antenna is to carry out an analysis to find out the characteristics of the antenna that best fulfills the specifications fixed by the application. After that, a prototype is manufactured and the next stage in design process is to check if the radiation pattern differs from the designed one. Besides the radiation pattern, other radiation parameters like directivity, gain, impedance, beamwidth, efficiency, polarization, etc. must be also evaluated. For this purpose, accurate antenna measurement techniques are needed in order to know exactly the actual electromagnetic behavior of the antenna under test. Due to this fact, most of the measurements are performed in anechoic chambers, which are closed areas, normally shielded, covered by electromagnetic absorbing material, that simulate free space propagation conditions, due to the absorption of the radiation absorbing material. Moreover, these facilities can be employed independently of the weather conditions and allow measurements free from interferences. Despite all the advantages of the anechoic chambers, the results obtained both from far-field measurements and near-field measurements are inevitably affected by errors. Thus, the main objective of this Thesis is to propose algorithms to improve the quality of the results obtained in antenna measurements by using post-processing techniques and without requiring additional measurements. First, a deep revision work of the state of the art has been made in order to give a general vision of the possibilities to characterize or to reduce the effects of errors in antenna measurements. Later, new methods to reduce the unwanted effects of four of the most commons errors in antenna measurements are described and theoretical and numerically validated. The basis of all them is the same, to perform a transformation from the measurement surface to another domain where there is enough information to easily remove the contribution of the errors. The four errors analyzed are noise, reflections, truncation errors and leakage and the tools used to suppress them are mainly source reconstruction techniques, spatial and modal filtering and iterative algorithms to extrapolate functions. Therefore, the main idea of all the methods is to modify the classical near-field-to-far-field transformations by including additional steps with which errors can be greatly suppressed. Moreover, the proposed methods are not computationally complex and, because they are applied in post-processing, additional measurements are not required. The noise is the most widely studied error in this Thesis, proposing a total of three alternatives to filter out an important noise contribution before obtaining the far-field pattern. The first one is based on a modal filtering. The second alternative uses a source reconstruction technique to obtain the extreme near-field where it is possible to apply a spatial filtering. The last one is to back-propagate the measured field to a surface with the same geometry than the measurement surface but closer to the AUT and then to apply also a spatial filtering. All the alternatives are analyzed in the three most common near-field systems, including comprehensive noise statistical analyses in order to deduce the signal-to-noise ratio improvement achieved in each case. The method to suppress reflections in antenna measurements is also based on a source reconstruction technique and the main idea is to reconstruct the field over a surface larger than the antenna aperture in order to be able to identify and later suppress the virtual sources related to the reflective waves. The truncation error presents in the results obtained from planar, cylindrical and partial spherical near-field measurements is the third error analyzed in this Thesis. The method to reduce this error is based on an iterative algorithm to extrapolate the reliable region of the far-field pattern from the knowledge of the field distribution on the AUT plane. The proper termination point of this iterative algorithm as well as other critical aspects of the method are also studied. The last part of this work is dedicated to the detection and suppression of the two most common leakage sources in antenna measurements. A first method tries to estimate the leakage bias constant added by the receiver’s quadrature detector to every near-field data and then suppress its effect on the far-field pattern. The second method can be divided into two parts; the first one to find the position of the faulty component that radiates or receives unwanted radiation, making easier its identification within the measurement environment and its later substitution; and the second part of this method is able to computationally remove the leakage effect without requiring the substitution of the faulty component. Resumen Una tarea importante en el diseño de una antena es llevar a cabo un análisis para averiguar las características de la antena que mejor cumple las especificaciones fijadas por la aplicación. Después de esto, se fabrica un prototipo de la antena y el siguiente paso en el proceso de diseño es comprobar si el patrón de radiación difiere del diseñado. Además del patrón de radiación, otros parámetros de radiación como la directividad, la ganancia, impedancia, ancho de haz, eficiencia, polarización, etc. deben ser también evaluados. Para lograr este propósito, se necesitan técnicas de medida de antenas muy precisas con el fin de saber exactamente el comportamiento electromagnético real de la antena bajo prueba. Debido a esto, la mayoría de las medidas se realizan en cámaras anecoicas, que son áreas cerradas, normalmente revestidas, cubiertas con material absorbente electromagnético. Además, estas instalaciones se pueden emplear independientemente de las condiciones climatológicas y permiten realizar medidas libres de interferencias. A pesar de todas las ventajas de las cámaras anecoicas, los resultados obtenidos tanto en medidas en campo lejano como en medidas en campo próximo están inevitablemente afectados por errores. Así, el principal objetivo de esta Tesis es proponer algoritmos para mejorar la calidad de los resultados obtenidos en medida de antenas mediante el uso de técnicas de post-procesado. Primeramente, se ha realizado un profundo trabajo de revisión del estado del arte con el fin de dar una visión general de las posibilidades para caracterizar o reducir los efectos de errores en medida de antenas. Después, se han descrito y validado tanto teórica como numéricamente nuevos métodos para reducir el efecto indeseado de cuatro de los errores más comunes en medida de antenas. La base de todos ellos es la misma, realizar una transformación de la superficie de medida a otro dominio donde hay suficiente información para eliminar fácilmente la contribución de los errores. Los cuatro errores analizados son ruido, reflexiones, errores de truncamiento y leakage y las herramientas usadas para suprimirlos son principalmente técnicas de reconstrucción de fuentes, filtrado espacial y modal y algoritmos iterativos para extrapolar funciones. Por lo tanto, la principal idea de todos los métodos es modificar las transformaciones clásicas de campo cercano a campo lejano incluyendo pasos adicionales con los que los errores pueden ser enormemente suprimidos. Además, los métodos propuestos no son computacionalmente complejos y dado que se aplican en post-procesado, no se necesitan medidas adicionales. El ruido es el error más ampliamente estudiado en esta Tesis, proponiéndose un total de tres alternativas para filtrar una importante contribución de ruido antes de obtener el patrón de campo lejano. La primera está basada en un filtrado modal. La segunda alternativa usa una técnica de reconstrucción de fuentes para obtener el campo sobre el plano de la antena donde es posible aplicar un filtrado espacial. La última es propagar el campo medido a una superficie con la misma geometría que la superficie de medida pero más próxima a la antena y luego aplicar también un filtrado espacial. Todas las alternativas han sido analizadas en los sistemas de campo próximos más comunes, incluyendo detallados análisis estadísticos del ruido con el fin de deducir la mejora de la relación señal a ruido lograda en cada caso. El método para suprimir reflexiones en medida de antenas está también basado en una técnica de reconstrucción de fuentes y la principal idea es reconstruir el campo sobre una superficie mayor que la apertura de la antena con el fin de ser capaces de identificar y después suprimir fuentes virtuales relacionadas con las ondas reflejadas. El error de truncamiento que aparece en los resultados obtenidos a partir de medidas en un plano, cilindro o en la porción de una esfera es el tercer error analizado en esta Tesis. El método para reducir este error está basado en un algoritmo iterativo para extrapolar la región fiable del patrón de campo lejano a partir de información de la distribución del campo sobre el plano de la antena. Además, se ha estudiado el punto apropiado de terminación de este algoritmo iterativo así como otros aspectos críticos del método. La última parte de este trabajo está dedicado a la detección y supresión de dos de las fuentes de leakage más comunes en medida de antenas. El primer método intenta realizar una estimación de la constante de fuga del leakage añadido por el detector en cuadratura del receptor a todos los datos en campo próximo y después suprimir su efecto en el patrón de campo lejano. El segundo método se puede dividir en dos partes; la primera de ellas para encontrar la posición de elementos defectuosos que radian o reciben radiación indeseada, haciendo más fácil su identificación dentro del entorno de medida y su posterior substitución. La segunda parte del método es capaz de eliminar computacionalmente el efector del leakage sin necesidad de la substitución del elemento defectuoso.

Evidence for a disaggregation of the universe.

Combining the kinematical definitions of the two dimensionless parameters, the deceleration q(x) and the Hubble t 0 H(x), we get a differential equation (where x=t/t 0 is the age of the universe relative to its present value t 0). First integration gives the function H(x). The present values of the Hubble parameter H(1) [approximately t 0 H(1)≈1], and the deceleration parameter [approximately q(1)≈−0.5], determine the function H(x). A second integration gives the cosmological scale factor a(x). Differentiation of a(x) gives the speed of expansion of the universe. The evolution of the universe that results from our approach is: an initial extremely fast exponential expansion (inflation), followed by an almost linear expansion (first decelerated, and later accelerated). For the future, at approximately t≈3t 0 there is a final exponential expansion, a second inflation that produces a disaggregation of the universe to infinity. We find the necessary and sufficient conditions for this disaggregation to occur. The precise value of the final age is given only with one parameter: the present value of the deceleration parameter [q(1)≈−0.5]. This emerging picture of the history of the universe represents an important challenge, an opportunity for the immediate research on the Universe. These conclusions have been elaborated without the use of any particular cosmological model of the universe

Characterisation of the operation & maintenance phase in PV rural electrification programmes

Con 1.300 millones de personas en el mundo sin acceso a la electricidad (la mayoría en entornos rurales de países empobrecidos), la energía solar fotovoltaica constituye una solución viable técnica y económicamente para electrificar las zonas más remotas del planeta donde las redes eléctricas convencionales no llegan. Casi todos los países en el mundo han desarrollado algún tipo de programa de electrificación fotovoltaica rural durante los últimos 40 años, principalmente los países más pobres, donde a través de diferentes modelos de financiación, se han instalado millones de sistemas solares domiciliarios (pequeños sistemas fotovoltaicos para uso doméstico). Durante este largo período, se han ido superando muchas barreras, como la mejora de la calidad de los sistemas fotovoltaicos, la reducción de costes, la optimización del diseño y del dimensionado de los sistemas, la disponibilidad financiera para implantar programas de electrificación rural, etc. Gracias a esto, la electrificación rural descentralizada ha experimentado recientemente un salto de escala caracterizada por la implantación de grandes programas con miles de sistemas solares domiciliarios e integrando largos períodos de mantenimiento. Muchos de estos grandes programas se están llevando a cabo con limitado éxito, ya que generalmente parten de supuestos e hipótesis poco contrastadas con la realidad, comprometiendo así un retorno económico que permita el desarrollo de esta actividad a largo plazo. En este escenario surge un nuevo reto: el de cómo garantizar la sostenibilidad de los grandes programas de electrificación rural fotovoltaica. Se argumenta que la principal causa de esta falta de rentabilidad es el imprevisto alto coste de la fase de operación y mantenimiento. Cuestiones clave tales como la estructura de costes de operación y mantenimiento o la fiabilidad de los componentes del sistema fotovoltaico no están bien caracterizados hoy en día. Esta situación limita la capacidad de diseñar estructuras de mantenimiento capaces de asegurar la sostenibilidad y la rentabilidad del servicio de operación y mantenimiento en estos programas. Esta tesis doctoral tiene como objetivo responder a estas cuestiones. Se ha realizado varios estudios sobre la base de un gran programa de electrificación rural fotovoltaica real llevado a cabo en Marruecos con más de 13.000 sistemas solares domiciliarios instalados. Sobre la base de este programa se ha hecho una evaluación en profundidad de la fiabilidad de los sistemas solares a partir de los datos de mantenimiento recogidos durante 5 años con más de 80.000 inputs. Los resultados han permitido establecer las funciones de fiabilidad de los equipos tal y como se comportan en condiciones reales de operación, las tasas de fallos y los tiempos medios hasta el fallo para los principales componentes del sistema, siendo este el primer caso de divulgación de resultados de este tipo en el campo de la electrificación rural fotovoltaica. Los dos principales componentes del sistema solar domiciliario, la batería y el módulo fotovoltaico, han sido analizados en campo a través de una muestra de 41 sistemas trabajando en condiciones reales pertenecientes al programa solar marroquí. Por un lado se ha estudiado la degradación de la capacidad de las baterías y por otro la degradación de potencia de los módulos fotovoltaicos. En el caso de las baterías, los resultados nos han permitido caracterizar la curva de degradación en capacidad llegando a obtener una propuesta de nueva definición del umbral de vida útil de las baterías en electrificación rural. También sobre la base del programa solar de Marruecos se ha llevado a cabo un estudio de caracterización de los costes reales de operación y mantenimiento a partir de la base de datos de contabilidad del programa registrados durante 5 años. Los resultados del estudio han permitido definir cuáles son costes que más incidencia tienen en el coste global. Se han obtenido los costes unitarios por sistema instalado y se han calculado los montantes de las cuotas de mantenimiento de los usuarios para garantizar la rentabilidad de la operación y mantenimiento. Finalmente, se propone un modelo de optimización matemática para diseñar estructuras de mantenimiento basado en los resultados de los estudios anteriores. La herramienta, elaborada mediante programación lineal entera mixta, se ha aplicado al programa marroquí con el fin de validar el modelo propuesto. ABSTRACT With 1,300 million people worldwide deprived of access to electricity (mostly in rural environments), photovoltaic solar energy has proven to be a cost‐effective solution and the only hope for electrifying the most remote inhabitants of the planet, where conventional electric grids do not reach because they are unaffordable. Almost all countries in the world have had some kind of rural photovoltaic electrification programme during the past 40 years, mainly the poorer countries, where through different organizational models, millions of solar home systems (small photovoltaic systems for domestic use) have been installed. During this long period, many barriers have been overcome, such as quality enhancement, cost reduction, the optimization of designing and sizing, financial availability, etc. Thanks to this, decentralized rural electrification has recently experienced a change of scale characterized by new programmes with thousands of solar home systems and long maintenance periods. Many of these large programmes are being developed with limited success, as they have generally been based on assumptions that do not correspond to reality, compromising the economic return that allows long term activity. In this scenario a new challenge emerges, which approaches the sustainability of large programmes. It is argued that the main cause of unprofitability is the unexpected high cost of the operation and maintenance of the solar systems. In fact, the lack of a paradigm in decentralized rural services has led to many private companies to carry out decentralized electrification programmes blindly. Issues such as the operation and maintenance cost structure or the reliability of the solar home system components have still not been characterized. This situation does not allow optimized maintenance structure to be designed to assure the sustainability and profitability of the operation and maintenance service. This PhD thesis aims to respond to these needs. Several studies have been carried out based on a real and large photovoltaic rural electrification programme carried out in Morocco with more than 13,000 solar home systems. An in‐depth reliability assessment has been made from a 5���year maintenance database with more than 80,000 maintenance inputs. The results have allowed us to establish the real reliability functions, the failure rate and the main time to failure of the main components of the system, reporting these findings for the first time in the field of rural electrification. Both in‐field experiments on the capacity degradation of batteries and power degradation of photovoltaic modules have been carried out. During the experiments both samples of batteries and modules were operating under real conditions integrated into the solar home systems of the Moroccan programme. In the case of the batteries, the results have enabled us to obtain a proposal of definition of death of batteries in rural electrification. A cost assessment of the Moroccan experience based on a 5���year accounting database has been carried out to characterize the cost structure of the programme. The results have allowed the major costs of the photovoltaic electrification to be defined. The overall cost ratio per installed system has been calculated together with the necessary fees that users would have to pay to make the operation and maintenance affordable. Finally, a mathematical optimization model has been proposed to design maintenance structures based on the previous study results. The tool has been applied to the Moroccan programme with the aim of validating the model.

The MIS 5 palaeoenvironmental record in the SE Mediterranean coast of the Iberian Peninsula (Río Antas, Almería, Spain)

Se realiza un estudio detallado del episodio cálido MIS 5 en la zona sureste de la Península Ibérica. Se realiza la reconstrucción paleoambiental a partir del estudio polínico y biomarcadores de un sondeo perforado en la costa de Almería. La cronología se estableció a partir del método de racemizaciónd e aminoácidos.Landwards of a MIS5 bar, a borehole core (SRA) was analyzed to establish the relationship between the lagoonal record and the raised beach deposits in the surroundings of the Antas river mouth and to reconstruct the Pleistocene palaeoenvironmental evolution 5 of the southern Mediterranean coast of the Iberian Peninsula. 63 samples were recovered for amino acid racemization dating, 86 samples for sedimentological and paleontological determination, 37 samples for pollen identification and 54 for biomarker analysis. AAR revealed that the borehole record contains MIS11, MIS6 and MIS5 deposits, the latter extensively represented. During the end of MIS6 and MIS5, a sand 10 barrier developed and created a shallow lagoon with alternating terrestrial inputs this process being common in other Mediterranean realms. Litho- and biofacies allowed the identification of distinct paleoenvironments through time, with the presence of a lagoonal environment alternating with alluvial fan progradation. Biomarkers indicated constant input from terrestrial plants, together with variable development of aquatic 15 macrophytes. The palynological content allowed the reconstruction of the paleoclimatological conditions during MIS6 and 5, with evidence of seven scenarios characterized by alternating arid and relatively humid conditions

Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na+ and K+ and urinary Na+ and K+ excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO2−/NO3− (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-d,l-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60–70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to ≈70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus

The α C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from the bca gene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: Possible role in antigen-driven reactions in the absence of germinal centers

The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark and Xenopus Ig. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses.

A first-generation X-inactivation profile of the human X chromosome

In females, most genes on the X chromosome are generally assumed to be transcriptionally silenced on the inactive X as a result of X inactivation. However, particularly in humans, an increasing number of genes are known to “escape” X inactivation and are expressed from both the active (Xa) and inactive (Xi) X chromosomes; such genes reflect different molecular and epigenetic responses to X inactivation and are candidates for phenotypes associated with X aneuploidy. To identify genes that escape X inactivation and to generate a first-generation X-inactivation profile of the X, we have evaluated the expression of 224 X-linked genes and expressed sequence tags by reverse-transcription–PCR analysis of a panel of multiple independent mouse/human somatic cell hybrids containing a normal human Xi but no Xa. The resulting survey yields an initial X-inactivation profile that is estimated to represent ≈10% of all X-linked transcripts. Of the 224 transcripts tested here, 34 (three of which are pseudoautosomal) were expressed in as many as nine Xi hybrids and thus appear to escape inactivation. The genes that escape inactivation are distributed nonrandomly along the X; 31 of 34 such transcripts map to Xp, implying that the two arms of the X are epigenetically and/or evolutionarily distinct and suggesting that genetic imbalance of Xp may be more severe clinically than imbalance of Xq. A complete X-inactivation profile will provide information relevant to clinical genetics and genetic counseling and should yield insight into the genomic and epigenetic organization of the X chromosome.

Delayed Activation of the Store-operated Calcium Current Induced by Calreticulin Overexpression in RBL-1 Cells

Calreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation.

In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone

Large sections of the 3′ untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3′ terminal conserved region or the poly(U–UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem–loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3′ UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U–UC) region and the conserved region, but not the variable region, of the 3′ UTR seem to be critical for in vivo infectivity of HCV.

Mutation of Residue Threonine-2 of the D2 Polypeptide and Its Effect on Photosystem II Function in Chlamydomonas reinhardtii1

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by 32P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [14C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5���-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.