Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.

Quantification of nanoparticles at the single-cell level: an overview about state-of-the-art techniques and their limitations.

With the increasing production and use of engineered nanoparticles it is crucial that their interaction with biological systems is understood. Due to the small size of nanoparticles, their identification and localization within single cells is extremely challenging. Therefore, various cutting-edge techniques are required to detect and to quantify metals, metal oxides, magnetic, fluorescent, as well as electron-dense nanoparticles. Several techniques will be discussed in detail, such as inductively coupled plasma atomic emission spectroscopy, flow cytometry, laser scanning microscopy combined with digital image restoration, as well as quantitative analysis by means of stereology on transmission electron microscopy images. An overview will be given regarding the advantages of those visualization/quantification systems, including a thorough discussion about limitations and pitfalls.

Autonomic angiotensinergic fibres in the human heart with an efferent sympathetic cophenotype

AIM The autonomic innervation of the heart consists of sympathetic and parasympathetic nerve fibres, and fibres of the intrinsic ganglionated plexus with noradrenaline and acytylcholine as principal neurotransmitters. The fibres co-release neuropeptides to modulate intracardiac neurotransmission by specific presynaptic and postsynaptic receptors. The coexpression of angiotensin II in sympathetic fibres of the human heart and its role are not known so far. METHODS Autopsy specimens of human hearts were studied (n=3; ventricles). Using immunocytological methods, cryostat sections were stained by a murine monoclonal antibody (4B3) directed against angiotensin II and co-stained by polyclonal antibodies against tyrosine hydroxylase, a catecholaminergic marker. Visualisation of the antibodies was by confocal light microscopy or laser scanning microscopy. RESULTS Angiotensin II-positive autonomic fibres with and without a catecholaminergic cophenotype (hydroxylase-positive) were found in all parts of the human ventricles. In the epicardium, the fibres were grouped in larger bundles of up to 100 and more fibres. They followed the preformed anatomic septa and epicardial vessels towards the myocardium and endocardium where the bundles dissolved and the individual fibres spread between myocytes and within the endocardium. Generally, angiotensinergic fibres showed no synaptic enlargements or only a few if they were also catecholaminergic. The exclusively catechalominergic fibres were characterised by multiple beaded synapses. CONCLUSION The autonomic innervation of the human heart contains angiotensinergic fibres with a sympathetic efferent phenotype and exclusively angiotensinergic fibers representing probably afferents. Angiotensinergic neurotransmission may modulate intracardiac sympathetic and parasympathetic activity and thereby influence cardiac and circulatory function.

Design, synthesis and cellular and molecular pharmacology of 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401): A novel non-cross-resistant anthracycline antibiotic with the intrinsic ability to circumvent P -glycoprotein-mediated drug resistance

We designed and synthesized a novel daunorubicin (DNR) analogue that effectively circumvents P-glycoprotein (P-gp)-mediated drug resistance. The fully protected carbohydrate intermediate 1,2-dibromoacosamine was prepared from acosamine and effectively coupled to daunomycinone in high yield. Deprotection under alkaline conditions yielded 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401). The in vitro cytotoxicity and cellular and molecular pharmacology of WP401 were compared with those of DNR in a panel of wild-type cell lines (KB-3-1, P388S, and HL60S) and their multidrug-resistant (MDR) counterparts (KB-V1, P388/DOX, and HL60/DOX). Fluorescent spectrophotometry, flow cytometry, and confocal laser scanning microscopy were used to measure intracellular accumulation, retention, and subcellular distribution of these agents. All MDR cell lines exhibited reduced DNR uptake that was restored, upon incubation with either verapamil (VER) or cyclosporin A (CSA), to the level found in sensitive cell lines. In contrast, the uptake of WP401 was essentially the same in the absence or presence of VER or CSA in all tested cell lines. The in vitro cytotoxicity of WP401 was similar to that of DNR in the sensitive cell lines but significantly higher in resistant cell lines (resistance index (RI) of 2-6 for WP401 vs 75-85 for DNR). To ascertain whether drug-mediated cytotoxicity and retention were accompanied by DNA strand breaks, DNA single- and double-strand breaks were assessed by alkaline elution. High levels of such breaks were obtained using 0.1-2 $\mu$g/mL of WP401 in both sensitive and resistant cells. In contrast, DNR caused strand breaks only in sensitive cells and not much in resistant cells. We also compared drug-induced DNA fragmentation similar to that induced by DNR. However, in P-gp-positive cells, WP401 induced 2- to 5-fold more DNA fragmentation than DNR. This increased DNA strand breakage by WP401 was correlated with its increased uptake and cytotoxicity in these cell lines. Overall these results indicate that WP401 is more cytotoxic than DNR in MDR cells and that this phenomenon might be related to the reduced basicity of the amino group and increased lipophilicity of WP401. ^

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

Interconexión monolítica de dispositivos fotovoltaicos basados en a-Si mediante ablación directa con fuentes láser ultravioleta

Resumen En la última década la tecnología láser se ha convertido en una herramienta imprescindible en la fabricación de dispositivos fotovoltaicos, muy especial¬mente en aquellos basados en tecnología de lámina delgada. Independiente¬mente de crisis coyunturales en el sector, la evolución en los próximos años de estas tecnologías seguirá aprovechándose de la flexibilidad y calidad de proceso de la herramienta láser para la consecución de los dos objetivos básicos que harán de la fotovoltaica una opción energética económicamente viable: la reducción de costes de fabricación y el aumento de eficiencia de los dispositivos. Dentro de las tecnologías fotovoltaicas de lámina delgada, la tecnología de dispositivos basados en silicio amorfo ha tenido un gran desarrollo en sistemas estándar en configuración de superestrato, pero su limitada efi¬ciencia hace que su supervivencia futura pase por el desarrollo de formatos en configuración de substrato sobre materiales flexibles de bajo coste. En esta aproximación, las soluciones industriales basadas en láser actualmente disponibles para la interconexión monolítica de dispositivos no son aplica¬bles, y desde hace años se viene investigando en la búsqueda de soluciones apropiadas para el desarrollo de dichos procesos de interconexión de forma que sean transferibles a la industria. En este contexto, esta Tesis propone una aproximación completamente orig¬inal, demostrando la posibilidad de ejecutar una interconexión completa de estos dispositivos irradiando por el lado de la lámina (es decir de forma com¬patible con la opción de configuración de substrato y, valga la redundancia, con el substrato del dispositivo opaco), y con fuentes láser emitiendo en UV. Este resultado, obtenido por primera vez a nivel internacional con este trabajo, aporta un conocimiento revelador del verdadero potencial de estas fuentes en el desarrollo industrial futuro de estas tecnologías. Si bien muy posiblemente la solución industrial final requiera de una solución mixta con el empleo de fuentes en UV y, posiblemente, en otras longitudes de onda, esta Tesis y su planteamiento novedoso aportan un conocimiento de gran valor a la comunidad internacional por la originalidad del planteamiento seguido, los resultados parciales encontrados en su desarrollo (un número importante de los cuales han aparecido en revistas del JCR que recogen en la actualidad un número muy significativo de citas) y porque saca además a la luz, con las consideraciones físicas pertinentes, las limitaciones intrínsecas que el desarrollo de procesos de ablación directa selectiva con láseres UV en parte de los materiales utilizados presenta en el rango temporal de in¬teracción de ns y ps. En este trabajo se han desarrollado y optimizado los tres pasos estándar de interconexión (los habitualmente denominados Pl, P2 y P3 en la industria fotovoltaica) demostrando las ventajas y limitaciones del uso de fuentes en UV tanto con ancho temporal de ns como de ps. En particular destaca, por el éxito en los resultados obtenidos, el estudio de procesos de ablación selectiva de óxidos conductores transparentes (en este trabajo utilizados tanto como contacto frontal así como posterior en los módulos) que ha generado resultados, de excelente acogida científica a nivel internacional, cuya aplicación trasciende el ámbito de las tecnologías de silicio amorfo en lámina delgada. Además en este trabajo de Tesis, en el desarrollo del objetivo citado, se han puesto a punto técnicas de análisis de los procesos láser, basadas en métodos avanzados de caracterización de materiales (como el uso combi¬nado de la espectroscopia dispersiva de rayos X y la microscopía confocal de barrido) que se presentan como auténticos avances en el desarrollo de técnicas específicas de caracterización para el estudio de los procesos con láser de ablación selectiva de materiales en lámina delgada, procesos que no solo tienen impacto en el ámbito de la fotovoltaica, sino también en la microelectrónica, la biotecnología, la microfabricación, etc. Como resultado adicional, parte de los resultados de este trabajo, han sido aplicados exi¬tosamente por el grupo de investigaci´on en la que la autora desarrolla su labor para conseguir desarrollar procesos de enorme inter´es en otras tec-nolog´ıas fotovoltaicas, como las tecnolog´ıas est´andar de silicio amorfo sobre vidrio en configuraci´on de superestrato o el procesado de capas delgadas en tecnolog´ıas convencionales de silicio cristalino. Por u´ltimo decir que este trabajo ha sido posible por una colaboraci´on muy estrecha entre el Centro L´aser de la UPM, en el que la autora de¬sarrolla su labor, y el Grupo de Silicio Depositado del Centro de Inves¬tigaciones Energ´eticas, Medioambientales y Tecnol´ogicas, CIEMAT, que, junto al Grupo de Energ´ıa Fotovoltaica de la Universidad de Barcelona, han preparado la mayor parte de las muestras utilizadas en este estudio. Dichas colaboraciones se han desarrollado en el marco de varios proyectos de investigaci´on aplicada con subvenci´on pu´blica, tales como el proyecto singular estrat´egico PSE-MICROSIL08 (PSE-120000-2006-6), el proyecto INNDISOL (IPT-420000-2010-6), ambos financiados porel Fondo Europeo de Desarrollo Regional FEDER (UE) ”Una manera de hacer Europa y el MICINN, y los proyectos de Plan Nacional AMIC (ENE2010-21384-C04-´ 02) y CLASICO (ENE2007-6772-C04-04), cuya financiaci´on ha permitido en gran parte llevar a t´ermino este trabajo Abstract In the last decade, the laser technology has turned into an indispensable tool in the production of photovoltaic devices, especially of those based on thin film technology. Regardless the current crisis in the sector, the evolution of these technologies in the upcoming years will keep taking advantage of the flexibility and process quality of the laser tool for the accomplishment of the two basic goals that will convert the photovoltaic energy into economically viable: the manufacture cost reduction and the increase in the efficiency of the devices. Amongst the thin film laser technologies, the technology of devices based on amorphous silicon has had a great development in standard systems of superstrate configuration, but its limited efficiency makes its survival de¬pendant on the development of formats in substrate configuration with low cost flexible materials. In this approach, the laser industrial solutions cur¬rently available for the monolithic interconnection are not applicable, and in the last few years the investigations have been focused on the search of appropriate solutions for the development of such interconnection processes in a way that the same are transferable to the industry. In this context, this Thesis proposes a totally original approach, proving the possibility of executing a full interconnection of these devices by means of irradiation from the film side, i.e., compatible with the substrate con¬figuration, and with UV laser sources. This result, obtained for the first time at international level in this work, provides a revealing knowledge of the true potential of these sources in the future industrial development of these technologies. Even though very probably the final industrial solution will require a combination of the use of UV sources along with other wave¬lengths, this Thesis and its novel approach contribute with a high value to the international community because of the originality of the approach, the partial results found throughout its development (out of which, a large number has appeared in JCR journals that currently accumulate a signifi¬cant number of citations) and brings to light, with the pertinent scientific considerations, the intrinsic limitations that the selective direct ablation processes with UV laser present in the temporal range of interaction of ns and ps for part of the materials used in this study. More particularly, the three standard steps of interconnection (usually de¬nominated P1, P2 and P3 in the photovoltaic industry) have been developed and optimized, showing the advantages as well as the limitations of the use of UV sources in both the ns and ps pulse-width ranges. It is highly remark¬able, because of the success in the obtained results, the study of selective ablation processes in transparent conductive oxide (in this work used as a front and back contact), that has generated results, of excellent interna¬tional scientific reception, whose applications go beyond the scope of thin film photovoltaic technologies based on amorphous silicon. Moreover, in this Thesis, with the development of the mentioned goal, differ¬ent techniques of analysis of laser processes have been fine-tuned, basing the same in advanced methods for material characterization (like the combined use of EDX Analysis and Confocal Laser Scanning Microscopy) that can be presented as true breakthroughs in the development of specific techniques for characterization in the study of laser processes of selective ablation of materials in thin film technologies, processes that not only have impact in the photovoltaic field, but also in those of microelectronics, biotechnology, micro-fabrication, etc. As an additional outcome, part of the results of this work has been suc¬cessfully applied, by the investigation group to which the author belongs, to the development of processes of enormous interest within other photo¬voltaic technologies, such as the standard technologies on amorphous silicon over glass in superstrate configuration or the processing of thin layers in conventional technologies using crystalline silicon. Lastly, it is important to mention that this work has been possible thanks to the close cooperation between the Centro L´aser of the UPM, in which the author develops her work, and the Grupo de Silicio Depositado of Centro de Investigaciones Energ´eticas, Medioambientales y Tecnol´ogicas, CIEMAT, which, along with the Grupo de Energ´ıa Fotovoltaica of Univer¬sidad de Barcelona, has prepared the largest part of the samples utilized in this study. Such collaborations have been carried out in the context of several projects of applied investigation with public funding, like Proyecto Singular Estrat´egico PSE-MICROSIL08 (PSE-120000-2006-6), Proyecto IN-NDISOL (IPT-420000-2010-6), both funded by the European Regional De¬velopment Fund (ERDF), ”Una manera de hacer Europa” and MICINN, and the projects of Plan Nacional AMIC (ENE2010-21384-C04-02) and ´ CLASICO (ENE2007-6772-C04-04), whose funds have enabled the devel-opment of large part of this work.

Experimental and numerical study of cw green laser crystallization of a-Si:H thin films

Crystallization and grain growth technique of thin film silicon are among the most promising methods for improving efficiency and lowering cost of solar cells. A major advantage of laser crystallization and annealing over conventional heating methods is its ability to limit rapid heating and cooling to thin surface layers. Laser energy is used to heat the amorphous silicon thin film, melting it and changing the microstructure to polycrystalline silicon (poly-Si) as it cools. Depending on the laser density, the vaporization temperature can be reached at the center of the irradiated area. In these cases ablation effects are expected and the annealing process becomes ineffective. The heating process in the a-Si thin film is governed by the general heat transfer equation. The two dimensional non-linear heat transfer equation with a moving heat source is solve numerically using the finite element method (FEM), particularly COMSOL Multiphysics. The numerical model help to establish the density and the process speed range needed to assure the melting and crystallization without damage or ablation of the silicon surface. The samples of a-Si obtained by physical vapour deposition were irradiated with a cw-green laser source (Millennia Prime from Newport-Spectra) that delivers up to 15 W of average power. The morphology of the irradiated area was characterized by confocal laser scanning microscopy (Leica DCM3D) and Scanning Electron Microscopy (SEM Hitachi 3000N). The structural properties were studied by micro-Raman spectroscopy (Renishaw, inVia Raman microscope).

Estudio de los materiales que componen el sistema constructivo de las viviendas del casco histórico de Maracaibo (Venezuela)

En este trabajo de investigación que se presenta para optar al grado de Doctor, se analizan y estudian los materiales que conforman las viviendas de mampostería de arenisca “Piedra de Ojo” del casco histórico de Maracaibo, Venezuela, construidas en el siglo XIX. No existe una bibliografía descriptiva técnico-constructiva del sistema constructivo, por lo que esta tesis complementa la escasa descripción estilística existente donde apenas se mencionan algunos materiales de construcción. Definido el marco histórico y las manzanas del área en donde se encontraban las viviendas preseleccionadas a estudiar, en la visita de campo se seleccionaron 12 que se encontraban en estado de deterioro, y que permitieron recolectar las muestras más fácilmente. Para realizar la caracterización y comportamientos de los diferentes materiales utilizados: piedra y morteros en los cerramientos, maderas en armaduras de tejados, techos y carpintería de puertas y ventanas, cerámicos en muros y acabados, etc. Para complementar lo antes dicho se ha dividido esta tesis en seis capítulos: En el capítulo I se desarrolla el estado del arte a nivel nacional e internacional de trabajos de investigación, similares. Se aborda la memoria histórica, que es una reseña de la evolución de la vivienda en la ciudad de Maracaibo. En el capítulo II se describe la metodología empleada en la tesis, de acuerdo a los objetivos, tanto generales como específicos de la investigación. Que ha cubierto diferentes frentes: consulta bibliográfica, levantamiento planimétrico, toma de muestras, análisis de visu, caracterización físico-química y correlación de resultados. Se ha desarrollado el trabajo tanto in situ como en laboratorio y despacho. El capítulo III presenta la caracterización de la arenisca “Piedra de Ojo”, se desarrolla: la descripción geológica y caracterización petrológica. Se reseñan los ensayos realizados en laboratorio como: caracterización de visu, caracterización petrográfica, estudio petrográfico por microcopia óptica de trasmisión, estudio petrográfico por microcopia electrónica de barrido, microscopia electrónica de barrido en modo electrones secundarios (SSE) y microscopia electrónica de barrido en modo electrones retrodispersados. También las propiedades escalares de los mampuestos y los siguientes valores: densidades, porosidades y resistencia mecánicas, entre otros. En el capítulo IV se analizan las características de los morteros aplicados en las viviendas, y la patología o lesiones que presentan. Se clasifican en tres tipos: mortero de junta o asiento, de enfoscado y revoco. Se documenta la realización de los ensayos físicos y químicos, resistencia mecánica y de granulometría; se explican sus componentes principales: conglomerante de cal, áridos y aditivos y la tecnología de fabricación, así como las características físicas, hídricas, químicas y granulométricas. El capítulo V, contiene las aplicaciones constructivas de los materiales de albañilería, Se describen otros elementos de la vivienda como; cimentaciones, muros mixtos, molduras, apliques y pinturas y finalmente pavimentos. Y en el capítulo VI se analizan las especies de madera más representativas usadas en las armaduras de las cubiertas, así como los elementos de cubrición. De igual forma se describe la carpintería de puertas y ventanas, así como sus dinteles o cargaderos de madera y se realiza la identificación anatómica, las propiedades físicas y mecánicas de las utilizadas. Entre los resultados y conclusiones se determinó que el 90% de los materiales utilizados en su construcción proceden de zonas cercanas a la construcción de la vivienda, como la formación El Milagro convertida en cantera de piedra y que el resto de los materiales provenían de la Isla de Toas y de la exportación de las islas del Caribe y de Europa como el cemento. El principal aporte de esta investigación es el análisis técnico constructivo y la caracterización física, mecánica y química de los materiales de la vivienda, con el fin de que dicha información sea usada para definir los materiales nuevos a utilizar en las restauraciones de las viviendas y en futuras líneas de investigación. ABSTRACT In this research paper submitted to opt to the degree of Doctor, the materials that make the “Piedra de Ojo” sandstone masonry houses of the historical center of Maracaibo, Venezuela, built in the XIX century, are analyzed and studied. There exists no technical-constructive descriptive literature of the constructive system, so this thesis complements the very limited existing stylistic description, where barely some construction materials are mentioned. With the historical context and the blocks of the area where the preselected houses to be studied being defined, 12 of these houses that were in a state of decay (deterioration) were selected and this condition allowed to collect samples more easily, in order to carry out the characterization and behavior of the different materials used: stone and mortars in the walls, wood trusses in roofs, ceilings and woodwork of doors and windows, walls and ceramic finishes, etc. To complement the foregoing, this thesis has been divided in six chapters: In Chapter I, the state of art at national and international levels of similar research is developed, which is a review of the evolution of housing in the city of Maracaibo. In Chapter II, the methodology used in the thesis is described, according to the research’s objectives, general and specific ones, which have covered several fronts: literature survey, planimetric survey, sampling, visu analysis, physical-chemical characterization and correlation of results. Chapter III presents the characterization of the “Piedra de Ojo” sandstone; geological description and petrologic characterization are developed. Essays performed in the laboratory are reviewed, such as: visu characterization, petrographic characaterization, petrographic study by optical microscopy of transmission, petrographic study by electronic scanning microscopy in secondary electron mode (SSE) and electron microscopy scaning by backscattered electron mode. Also scalar properties of the masonry and the following: density, porosity and mechanical resistance, among others. In Chapter IV, characteristics of the mortars used in the houses are analyzed and pathology or damages are presented. They are classified into three types: grout, cement render and plaster. Physical and chemical testing, mechanical strength and grain size (granulometric) is documented; its main components are explained: lime binder, aggregates and additives and manufacturing technology as well as the physical, water, chemical and granulometric characteristics. Chapter V contains the constructive applications of masonry materials. Other housing elements are described, such as; foundations, mixed walls, moldings, wall paintings and finally floorings (pavements). And in chapter VI the most representative species of wood used in the overhead fixtures and cover elements are analyzed. Likewise, woodwork of doors and windows and their wooden lintels or landings are described; anatomical identification and physical and mechanical properties of the ones used is made. The results and conclusions determined that 90% of the materials used in its construction came from areas near the construction of housing, such as El Milagro formation, converted into stone quarry and other materials came from the Toas Island and from the export of the Caribbean islands and Europe, such as cement. The main contribution of this research is the constructive technical analysis and physical, mechanical and chemical characterization of the materials of the houses, in order that such information is used to define the new materials to be used in the housing restoration and future research lines.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.

Neuronal calcium sparks and intracellular calcium “noise”

Intracellular calcium ions are involved in many forms of cellular function. To accommodate so many control functions, a complex spatiotemporal organization of calcium signaling has developed. In both excitable and nonexcitable cells, calcium signaling was found to fluctuate. Sudden localized increases in the intracellular calcium concentration—or calcium sparks—were found in heart, striated and smooth muscle, Xenopus Laevis oocytes, and HeLa and P12 cells. In the nervous system, intracellular calcium ions were found important in key processes such as transmitter release, repetitive firing, and gene expression. Hence, we examined whether calcium sparks also exist in neurons. Using confocal laser-scanning microscopy and fluorescent probes, we found that calcium sparks exist in two types of neuronal preparations: the presynaptic boutons of the lizard neuromuscular junction and rat hippocampal neurons in cell culture. Control experiments exclude the possibility that these calcium sparks originate from instrumental or biological artifacts. Calcium sparks seem to be just the tip of the iceberg of a more general phenomenon of intracellular calcium “noise.” We speculate that calcium sparks and calcium noise may be of key importance in calcium signaling in the nervous system.

Localization and Recycling of gp27 (hp24γ3): Complex Formation with Other p24 Family Members

We report here the characterization of gp27 (hp24γ3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15°C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24α2), p24 (hp24β1), and p23 (hp24δ1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24γ4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.

Glutathione biosynthesis in Arabidopsis trichome cells

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode γ-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238 ± 25, 80 ± 2, and 144 ± 19 μM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH]cyt is reduced to 47 ± 5 μM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.

Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex

Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human–hamster hybrid (AL) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5′,6′-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in AL cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors α- and γ-mediated gene expression via liver fatty acid binding protein: A signaling path to the nucleus

Peroxisome proliferator-activated receptor α (PPARα) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPARα colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPARα. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPARα and PPARγ but not with PPARβ and retinoid X receptor-α by protein–protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. Final and quantitative proof for L-FABP mediation was obtained in transactivation assays upon incubation of transiently and stably transfected HepG2 cells with saturated, monounsaturated, and polyunsaturated fatty acids as well as with hypolipidemic drugs. With all ligands applied, we observed strict correlation of PPARα and PPARγ transactivation with intracellular concentrations of L-FABP. This correlation constitutes a nucleus-directed signaling by fatty acids and hypolipidemic drugs where L-FABP acts as a cytosolic gateway for these PPARα and PPARγ agonists. Thus, L-FABP and the respective PPARs could serve as targets for nutrients and drugs to affect expression of PPAR-sensitive genes.