Topo IIIalpha and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents

The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.

Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation.

Medulloblastoma, one of the most malignant brain tumors in children, is thought to arise from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer of the cerebellum. However, the mechanism of tumorigenesis remains unknown for the majority of medulloblastomas. In this study, we found that many human medulloblastomas express significantly elevated levels of both myc oncogenes, regulators of neural progenitor proliferation, and REST/NRSF, a transcriptional repressor of neuronal differentiation genes. Previous studies have shown that neither c-Myc nor REST/NRSF alone could cause tumor formation. To determine whether c-Myc and REST/NRSF act together to cause medulloblastomas, we used a previously established cell line derived from external granule layer stem cells transduced with activated c-myc (NSC-M). These immortalized NSCs were able to differentiate into neurons in vitro. In contrast, when the cells were engineered to express a doxycycline-regulated REST/NRSF transgene (NSC-M-R), they no longer underwent terminal neuronal differentiation in vitro. When injected into intracranial locations in mice, the NSC-M cells did not form tumors either in the cerebellum or in the cerebral cortex. In contrast, the NSC-M-R cells did produce tumors in the cerebellum, the site of human medulloblastoma formation, but not when injected into the cerebral cortex. Furthermore, the NSC-M-R tumors were blocked from terminal neuronal differentiation. In addition, countering REST/NRSF function blocked the tumorigenic potential of NSC-M-R cells. To our knowledge, this is the first study in which abnormal expression of a sequence-specific DNA-binding transcriptional repressor has been shown to contribute directly to brain tumor formation. Our findings indicate that abnormal expression of REST/NRSF and Myc in NSCs causes cerebellum-specific tumors by blocking neuronal differentiation and thus maintaining the "stemness" of these cells. Furthermore, these results suggest that such a mechanism plays a role in the formation of human medulloblastoma.

Rainer Werner Fassbinder’s "Garbage, The City, and Death". A Four Act Scandal in Post-war Germany

Rainer Werner Fassbinder’s Garbage, The City, and Death. A Four Act Scandal in Post-war Germany The paper explores the dramaturgy of the scandals around the play Garbage, The City and Death (Der Müll, die Stadt und der Tod) by German playwright, theatre and film maker Rainer Werner Fassbinder. Published in 1976, the play immediately caused a scandal in West Germany, because it was accused of reproducing anti-Semitic stereotypes. The presentation sheds light on the different phases of the scandal and their historical and cultural contexts in post-war Germany – starting as a literary scandal in 1976, being transformed into a theatre scandal in the 1980ies and finally being dissolved by the German premiere in 2009. The paper is structured as follows: Act One: The Literary Scandal. Destroying Fassbinder’s Garbage, Act Two: Preventing the Staging of the Play, Act Three: Blocking the Opening Night, Act Four: Performing the Play in Germany. By analysing the dramaturgical structure of this specific scandal, the paper discusses the following hypotheses: 1. Scandals arise through the circulation of decontextualised information in public. This is due to either a lack of information about the actual object or incident being scandalised or a lack of information about the context of the object or incident. This lack is caused by the logic of the scandal itself: Because the play or the performance is prohibited, it has been withdrawn from the public, making it impossible to form a well-founded opinion on the controversy. 2. The scandal is driven forward by an emotionalising rhetoric built around the decontextualised information. 3. Once the gap of information is filled, the scandalising rhetoric turns into a rhetoric of irrelevance: Reviews of the first performance of Garbage, The City and Death in Germany considered the play hardly a matter of public concern.

The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

Protease-activated receptor-1 signaling plays a major role in melanoma growth and metastasis

Overexpression of the thrombin receptor (Protease-Activated-Receptor-1), PAR-1, in cell lines and tissue specimens correlates with the metastatic potential of human melanoma. Utilizing lentiviral shRNA to stably silence PAR-1 in metastatic melanoma cell lines results in decreased tumor growth and lung metastasis in vivo. Since the use of viral technology is not ideal for clinical therapies, neutral liposomes (DOPC) were utilized as a delivery vehicle for PAR-1 siRNA. Our data suggest that PAR-1 siRNA-DOPC treatment by systemic delivery significantly decreases tumor growth and lung metastasis in nude mice. Concomitant decreases in angiogenic and invasive factors (IL-8, VEGF, MMP-2) were observed in PAR-1 siRNA-DOPC-treated mice. Utilizing a cDNA microarray platform, several novel PAR-1 downstream target genes were identified, including Connexin 43 (Cx-43) and Maspin. Cx-43, known to be involved in tumor cell diapedesis and attachment to endothelial cells, is decreased after PAR-1 silencing. Furthermore, the Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells suggesting transcriptional regulation of Cx-43 by PAR-1. ChIP analysis revealed a reduction in SP-1 and AP-1 binding to the Cx-43 promoter. Moreover, melanoma cell attachment to HUVEC was significantly decreased in PAR-1-silenced cells as well as in Cx-43 shRNA transduced cells. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Maspin, a serine protease inhibitor with tumor-suppressor function, was found to be upregulated after PAR-1 silencing. Our results indicate that PAR-1 transcriptionally regulates Maspin, as the promoter activity was significantly increased after PAR-1 silencing. ChIP analysis revealed that silencing PAR-1 increased binding of Ets and c-Jun to the Maspin promoter. As Maspin was recently found to be a tumor-suppressor in melanoma by reducing the invasive capacity of melanoma cells, invasion assays revealed a decrease in invasion after PAR-1 silencing and in cells transduced with a Maspin expression vector. We propose that PAR-1 is key to the progression and metastasis of melanoma in part by regulating the expression of Cx-43 and Maspin. Taken together, we propose that PAR-1 is an attractive target for the treatment of melanoma.^

Surface InGaAs nanostructures for sensing applications

El desarrollo de sensores está ganando cada vez mayor importancia debido a la concienciación ciudadana sobre el medio ambiente haciendo que su desarrollo sea muy elevado en todas las disciplinas, entre las que cabe destacar, la medicina, la biología y la química. A pesar de la existencia de estos dispositivos, este área está aún por mejorar, ya que muchos de los materiales propuestos hasta el momento e incluso los ya comercializados muestran importantes carencias de funcionamiento, eficiencia e integrabilidad entre otros. Para la mejora de estos dispositivos, se han propuesto diversas aproximaciones basadas en nanosistemas. Quizá, uno de las más prometedoras son las nanoestructuras de punto cuántico, y en particular los semiconductores III-V basados en la consolidada tecnología de los arseniuros, las cuáles ofrecen excelentes propiedades para su uso como sensores. Además, estudios recientes demuestran su gran carácter sensitivo al medio ambiente, la posibilidad de funcionalizar la superficie para la fabricación de sensores interdisciplinares y posibilididad de mejorar notablemente su eficiencia. A lo largo de esta tesis, nos centramos en la investigación de SQD de In0.5Ga0.5As sobre substratos de GaAs(001) para el desarrollo de sensores de humedad. La tesis abarca desde el diseño, crecimiento y caracterización de las muestras hasta la el posterior procesado y caracterización de los dispositivos finales. La optimización de los parámetros de crecimiento es fundamental para conseguir una nanoestructura con las propiedades operacionales idóneas para un fin determinado. Como es bien sabido en la literatura, los parámetros de crecimiento (temperatura de crecimiento, relación de flujos del elemento del grupo V y del grupo I II (V/III), velocidad de crecimiento y tratamiento térmico después de la formación de la capa activa) afectan directamente a las propiedades estructurales, y por tanto, operacionales de los puntos cuánticos (QD). En esta tesis, se realiza un estudio de las condiciones de crecimiento para el uso de In0.5Ga0.5As SQDs como sensores. Para los parámetros relacionados con la temperatura de crecimiento de los QDs y la relación de flujos V / I I I se utilizan los estudios previamente realizados por el grupo. Mientras que este estudio se centrará en la importancia de la velocidad de crecimiento y en el tratamiento térmico justo después de la nucleación de los QDs. Para ello, se establece la temperatura de creciemiento de los QDs en 430°C y la relación de flujos V/III en 20. Como resultado, los valores más adecuados que se obtienen para la velocidad de crecimiento y el tratamiento térmico posterior a la formación de los puntos son, respectivamente, 0.07ML/s y la realización de una bajada y subida brusca de la temperatura del substrato de 100°C con respecto a la temperatura de crecimiento de los QDs. El crecimiento a una velocidad lo suficientemente alta que permita la migración de los átomos por la superficie, pero a su vez lo suficientemente baja para que se lleve a cabo la nucleación de los QDs; en combinación con el tratamiento brusco de temperatura que hace que se conserve la forma y composición de los QDs, da lugar a unos SQDs con un alto grado de homogeneidad y alta densidad superficial. Además, la caracterización posterior indica que estas nanoestructuras de gran calidad cristalina presentan unas propiedades ópticas excelentes incluso a temperatura ambiente. Una de las características por la cual los SQD de Ino.5Gao.5As se consideran candidatos prometedores para el desarrollo de sensores es el papel decisivo que juega la superficie por el mero hecho de estar en contacto directo con las partículas del ambiente y, por tanto, por ser capaces de interactuar con sus moléculas. Así pues, con el fin de demostrar la idoneidad de este sistema para dicha finalidad, se evalúa el impacto ambiental en las propiedades ópticas y eléctricas de las muestras. En un primer lugar, se analiza el efecto que tiene el medio en las propiedades ópticas. Para dicha evaluación se compara la variación de las propiedades de emisión de una capa de puntos enterrada y una superficial en distintas condiciones externas. El resultado que se obtiene es muy claro, los puntos enterrados no experimentan un cambio óptico apreciable cuando se varían las condiciones del entorno; mientras que, la emisión de los SQDs se modifica significativamente con las condiciones del medio. Por una parte, la intensidad de emisión de los puntos superficiales desaparece en condiciones de vacío y decrece notablemente en atmósferas secas de gases puros (N2, O2). Por otra parte, la fotoluminiscencia se conserva en ambientes húmedos. Adicionalmente, se observa que la anchura a media altura y la longitud de onda de emisión no se ven afectadas por los cambios en el medio, lo que indica, que las propiedades estructurales de los puntos se conservan al variar la atmósfera. Estos resultados apuntan directamente a los procesos que tienen lugar en la superficie entre estados confinados y superficiales como responsables principales de este comportamiento. Así mismo, se ha llevado a cabo un análisis más detallado de la influencia de la calidad y composición de la atmósfera en las propiedades ópticas de los puntos cuánticos superficiales. Para ello, se utilizan distintas sustancias con diferente polaridad, composición atómica y masa molecular. Como resultado se observa que las moléculas de menor polaridad y más pesadas causan una mayor variación en la intensidad de emisión. Además, se demuestra que el oxígeno juega un papel decisivo en las propiedades ópticas. En presencia de moléculas que contienen oxígeno, la intensidad de fotoluminiscencia disminuye menos que en atmósferas constituidas por especies que no contienen oxígeno. Las emisión que se observa respecto a la señal en aire es del 90% y del 77%, respectivamente, en atmósferas con presencia o ausencia de moléculas de oxígeno. El deterioro de la señal de emisión se atribuye a la presencia de defectos, enlaces insaturados y, en general, estados localizados en la superficie. Estos estados actúan como centros de recombinación no radiativa y, consecuentemente, se produce un empeoramiento de las propiedades ópticas de los SQDs. Por tanto, la eliminación o reducción de la densidad de estos estados superficiales haría posible una mejora de la intensidad de emisión. De estos experimentos de fotoluminiscencia, se deduce que las interacciones entre las moléculas presentes en la atmósfera y la superficie de la muestra modifican la superficie. Esta alteración superficial se traduce en un cambio significativo en las propiedades de emisión. Este comportamiento se atribuye a la posible adsorción de moléculas sobre la superficie pasivando los centros no radiativos, y como consecuencia, mejorando las propiedades ópticas. Además, los resultados demuestran que las moléculas que contienen oxígeno con mayor polaridad y más ligeras son adsorbidas con mayor facilidad, lo que hace que la intensidad óptica sufra variaciones despreciables con respecto a la emisión en aire. Con el fin de desarrollar sensores, las muestras se procesan y los dispositivos se caracterizan eléctricamente. El procesado consiste en dos contactos cuadrados de una aleación de Ti/Au. Durante el procesado, lo más importante a tener en cuenta es no realizar ningún ataque o limpieza que pueda dañar la superficie y deteriorar las propiedades de las nanostructuras. En este apartado, se realiza un análisis completo de una serie de tres muestras: GaAs (bulk), un pozo cuántico superficial (SQW) de Ino.5Gao.5As y SQDs de Ino.5Gao.5As. Para ello, a cada una de las muestras se le realizan medidas de I-V en distintas condiciones ambientales. En primer lugar, siguiendo los resultados obtenidos ópticamente, se lleva a cabo una comparación de la respuesta eléctrica en vacío y aire. A pesar de que todas las muestras presentan un carácter más resistivo en vacío que en aire, se observa una mayor influencia sobre la muestra de SQD. En vacío, la resistencia de los SQDs decrece un 99% respecto de su valor en aire, mientras que la variación de la muestras de GaAs e Ino.5Gao.5As SQW muestran una reducción, respectivamente, del 31% y del 20%. En segundo lugar, se realiza una evaluación aproximada del posible efecto de la humedad en la resistencia superficial de las muestras mediante la exhalación humana. Como resultado se obtiene, que tras la exhalación, la resistencia disminuye bruscamente y recupera su valor inicial cuando dicho proceso concluye. Este resultado preliminar indica que la humedad es un factor crítico en las propiedades eléctricas de los puntos cuánticos superficiales. Para la determinación del papel de la humedad en la respuesta eléctrica, se somete a las muestras de SQD y SQW a ambientes con humedad relativa (RH, de la siglas del inglés) controlada y se analiza el efecto sobre la conductividad superficial. Tras la variación de la RH desde 0% hasta el 70%, se observa que la muestra SQW no cambia su comportamiento eléctrico al variar la humedad del ambiente. Sin embargo, la respuesta de la muestra SQD define dos regiones bien diferenciadas, una de alta sensibilidad para valores por debajo del 50% de RH, en la que la resistencia disminuye hasta en un orden de magnitud y otra, de baja sensibilidad (>50%), donde el cambio de la resistencia es menor. Este resultado resalta la especial relevancia no sólo de la composición sino también de la morfología de la nanostructura superficial en el carácter sensitivo de la muestra. Por último, se analiza la influencia de la iluminación en la sensibilidad de la muestra. Nuevamente, se somete a las muestras SQD y SQW a una irradiación de luz de distinta energía y potencia a la vez que se varía controladamente la humedad ambiental. Una vez más, se observa que la muestra SQW no presenta ninguna variación apreciable con las alteraciones del entorno. Su resistencia superficial permanece prácticamente inalterable tanto al modificar la potencia de la luz incidente como al variar la energía de la irradiación. Por el contrario, en la muestra de SQD se obtiene una reducción la resistencia superficial de un orden de magnitud al pasar de condiciones de oscuridad a iluminación. Con respecto a la potencia y energía de la luz incidente, se observa que a pesar de que la muestra no experimenta variaciones notables con la potencia de la irradiación, esta sufre cambios significativos con la energía de la luz incidente. Cuando se ilumina con energías por encima de la energía de la banda prohibida (gap) del GaAs (Eg ~1.42 eV ) se produce una reducción de la resistencia de un orden de magnitud en atmósferas húmedas, mientras que en atmósferas secas la conductividad superficial permanece prácticamente constante. Sin embargo, al inicidir con luz de energía menor que Eg, el efecto que se produce en la respuesta eléctrica es despreciable. Esto se atribuye principalmente a la densidad de portadores fotoactivados durante la irradiación. El volumen de portadores excita dos depende de la energía de la luz incidente. De este modo, cuando la luz que incide tiene energía menor que el gap, el volumen de portadores generados es pequeño y no contribuye a la conductividad superficial. Por el contrario, cuando la energía de la luz incidente es alta (Eg), el volumen de portadores activados es elevado y éstos contribuyen significantemente a la conductividad superficial. La combinación de ambos agentes, luz y humedad, favorece el proceso de adsorción de moléculas y, por tanto, contribuye a la reducción de la densidad de estados superficiales, dando lugar a una modificación de la estructura electrónica y consecuentemente favoreciendo o dificultando el transporte de portadores. ABSTRACT Uncapped three-dimensional (3D) nanostructures have been generally grown to assess their structural quality. However, the tremendous growing importance of the impact of the environment on life has become such nanosystems in very promising candidates for the development of sensing devices. Their direct exposure to changes in the local surrounding may influence their physical properties being a perfect sign of the atmosphere quality. The goal of this thesis is the research of Ino.5Gao.5As surface quantum dots (SQDs) on GaAs(001), covering from their growth to device fabrication, for sensing applications. The achievement of this goal relies on the design, growth and sample characterization, along with device fabrication and characterization. The first issue of the thesis is devoted to analyze the main growth parameters affecting the physical properties of the Ino.5Gao.5As SQDs. It is well known that the growing conditions (growth temperature , deposition rate, V/III flux ratio and treatment after active layer growth) directly affect the physical properties of the epilayer. In this part, taking advantage of the previous results in the group regarding Ino.5Gao.5As QD growth temperature and V/III ratio, the effect of the growth rate and the temperature treatment after QDs growth nucleation is evaluated. Setting the QDs growth temperature at 430°C and the V/III flux ratio to ~20, it is found that the most appropriate conditions rely on growing the QDs at 0.07ML/s and just after QD nucleation, rapidly dropping and again raising 100°C the substrate temperature with respect to the temperature of QD growth. The combination of growing at a fast enough growth rate to promote molecule migration but sufficiently slow to allow QD nucleation, together with the sharp variation of the temperature preserving their shape and composition yield to high density, homogeneous Ino.5Gao.5As SQDs. Besides, it is also demonstrated that this high quality SQDs show excellent optical properties even at room temperature (RT). One of the characteristics by which In0.5Ga0.5As/GaAs SQDs are considered promising candidates for sensing applications is the crucial role that surface plays when interacting with the gases constituting the atmosphere. Therefore, in an attempt to develop sensing devices, the influence of the environment on the physical properties of the samples is evaluated. By comparing the resulting photoluminescence (PL) of SQDs with buried QDs (BQDs), it is found that BQDs do not exhibit any significant variation when changing the environmental conditions whereas, the external conditions greatly act on the SQDs optical properties. On one hand, it is evidenced that PL intensity of SQDs sharply quenches under vacuum and clearly decreases under dry-pure gases atmospheres (N2, O2). On the other hand, it is shown that, in water containing atmospheres, the SQDs PL intensity is maintained with respect to that in air. Moreover, it is found that neither the full width at half maximun nor the emission wavelength manifest any noticeable change indicating that the QDs are not structurally altered by the external atmosphere. These results decisively point to the processes taking place at the surface such as coupling between confined and surface states, to be responsible of this extraordinary behavior. A further analysis of the impact of the atmosphere composition on the optical characteristics is conducted. A sample containing one uncapped In0.5Ga0.5As QDs layer is exposed to different environments. Several solvents presenting different polarity, atomic composition and molecular mass, are used to change the atmosphere composition. It is revealed that low polarity and heavy molecules cause a greater variation on the PL intensity. Besides, oxygen is demonstrated to play a decisive role on the PL response. Results indicate that in presence of oxygen-containing molecules, the PL intensity experiments a less reduction than that suffered in presence of nonoxygen-containing molecules, 90% compared to 77% signal respect to the emission in air. In agreement with these results, it is demonstrated that high polarity and lighter molecules containing oxygen are more easily adsorbed, and consequently, PL intensity is less affected. The presence of defects, unsaturated bonds and in general localized states in the surface are proposed to act as nonradiative recombination centers deteriorating the PL emission of the sample. Therefore, suppression or reduction of the density of such states may lead to an increase or, at least, conservation of the PL signal. This research denotes that the interaction between sample surface and molecules in the atmosphere modifies the surface characteristics altering thus the optical properties. This is attributed to the likely adsoption of some molecules onto the surface passivating the nonradiative recombination centers, and consequently, not deteriorating the PL emission. Aiming for sensors development, samples are processed and electrically characterized under different external conditions. Samples are processed with two square (Ti/Au) contacts. During the processing, especial attention must be paid to the surface treatment. Any process that may damage the surface such as plasma etching or annealing must be avoided to preserve the features of the surface nanostructures. A set of three samples: a GaAs (bulk), In0.5Ga0.5As SQDs and In0.5Ga0.5As surface quantum well (SQW) are subjected to a throughout evaluation. I-V characteristics are measured following the results from the optical characterization. Firstly, the three samples are exposed to vacuum and air. Despite the three samples exhibit a more resistive character in vacuum than in air, it is revealed a much more clear influence of the pressure atmosphere in the SQDs sample. The sheet resistance (Rsh) of SQDs decreases a 99% from its response value under vacuum to its value in air, whereas Rsh of GaAs and In0.5Ga0.5As SQW reduces its value a 31% and a 20%, respectively. Secondly, a rough analysis of the effect of the human breath on the electrical response evidences the enormous influence of moisture (human breath is composed by several components but the one that overwhelms all the rest is the high concentration of water vapor) on the I-V characteristics. Following this result, In0.5Ga0.5As SQDs and In0.5Ga0.5As SQW are subjected to different controlled relative humidity (RH) environments (from 0% to 70%) and electrically characterized. It is found that SQW shows a nearly negligible Rsh variation when increasing the RH in the surroundings. However, the response of SQDs to changes in the RH defines two regions. Below 50%, high sensitive zone, Rsh of SQD decreases by more than one order of magnitude, while above 50% the dependence of Rsh on the RH becomes weaker. These results remark the role of the surface and denote the existence of a finite number of surface states. Nevertheless, most significantly, they highlight the importance not only of the material but also of the morphology. Finally, the impact of the illumination is determined by means of irradiating the In0.5Ga0.5As SQDs and In0.5Ga0.5As SQW samples with different energy and power sources. Once again, SQW does not exhibit any correlation between the surface conductivity and the external conditions. Rsh remains nearly unalterable independently of the energy and power of the incident light. Conversely, Rsh of SQD experiences a decay of one order of magnitude from dark-to-photo conditions. This is attributed to the less density of surface states of SQW compared to that of SQDs. Additionally, a different response of Rsh of SQD with the energy of the impinging light is found. Illuminating with high energy light results in a Rsh reduction of one order of mag nitude under humid atmospheres, whereas it remains nearly unchanged under dry environments. On the contrary, light with energy below the bulk energy bandgap (Eg), shows a negligible effect on the electrical properties regardless the local moisture. This is related to the density of photocarriers generated while lighting up. Illuminating with excitation energy below Eg affects a small absorption volume and thus, a low density of photocarriers may be activated leading to an insignificant contribution to the conductivity. Nonetheless, irradiating with energy above the Eg can excite a high density of photocarriers and greatly improve the surface conductivity. These results demonstrate that both illumination and humidity are therefore needed for sensing. The combination of these two agents improves the surface passivation by means of molecule adsorption reducing the density of surface states, thus modifying the electronic structures, and consequently, promoting the carrier motion.

1950 En torno al Museo Louisiana 1970

La tesis “1950 En torno al Museo Louisiana 1970” analiza varias obras relacionadas con el espacio doméstico, que se realizaron entre 1950 y 1970 en Dinamarca, un periodo de esplendor de la Arquitectura Moderna. Tras el aislamiento y restricciones del conflicto bélico que asoló Europa, los jóvenes arquitectos daneses, estaban deseosos por experimentar nuevas ideas de procedencia internacional, favorecidos por diferentes circunstancias, encuentran el mejor campo de ensayo en el espacio doméstico. La mejor arquitectura doméstica en Dinamarca, de aquel periodo, debe entenderse como un sistema compuesto por diferentes autores, que tienen en común muchas más similitudes que diferencias, se complementan unos a otros. Para la comprensión y el entendimiento de ello se hace necesario el estudio de varias figuras y edificios, que completen este sistema cuya investigación está escasamente desarrollada. La tesis propone un viaje para conocer los nombres de algunos de sus protagonistas, que mostraron con su trabajo, que tradición y vanguardia no estarán reñidas. El objetivo es desvelar las claves de la Modernidad Danesa, reconocer, descubrir y recuperar el legado de algunos de sus protagonistas en el ámbito doméstico, cuya lección se considera de total actualidad. Una arquitectura que asume las aportaciones extranjeras con moderación y crítica, cuya íntima relación con la tradición arquitectónica y la artesanía propias, será una de sus notas especiales. Del estudio contrastado de varios proyectos y versiones, se obtienen valores comunes entre sus autores, al igual que se descubren sus afinidades o diferencias respecto a los mismos asuntos; que permitirán comprender sus actuaciones según las referencias e influencias, y definir las variables que configuran sus espacios arquitectónicos. La línea de conexión entre los edificios elegidos será su particular relación con la naturaleza y el lugar en que se integran. La fachada, lugar donde se negociará la relación entre el interior y el paisaje, será un elemento entendido de un modo diferente en cada uno de ellos, una relación que se extenderá en todas ellas, más allá de su perímetro. La investigación se ha estructurado en seis capítulos, que van precedidos de una Introducción. En el capítulo primero, se estudian y se señalan los antecedentes, las figuras y edificios más relevantes de la Tradición Danesa, para la comprensión y el esclarecimiento de algunas de las claves de su Modernidad en el campo de la Arquitectura, que se produce con una clara intención de encontrar su propia identidad y expresión. Esta Modernidad floreciente se caracteriza por la asimilación de otras culturas extranjeras desde la moderación, con un punto de vista crítico, y encuentra sus raíces ancladas a la tradición arquitectónica y la artesanía propia, que fragua en la aparición de un ideal común con enorme personalidad y que hoy se valora como una auténtica aportación de una cultura considerada entonces periférica. Se mostrará el debate y el camino seguido por las generaciones anteriores, a las obras análizadas. Las sensibilidades por lo vernáculo y lo clásico, que aparentemente son contradictorias, dominaran el debate con la misma veracidad y respetabilidad. La llamada tercera generación por Sigfried Giedion reanudará la práctica entre lo clásico y lo vernáculo, apoyados en el espíritu del trabajo artesanal y de la tradición, con el objetivo de conocer del acto arquitectónico su “la verdad” y “la esencia original”. El capítulo segundo, analiza la casa Varming, de 1953, situada en un área residencial de Gentofte, por Eva y Nils Koppel, que reinterpreta la visión de Asplund de un paisaje interior continuación del exterior, donde rompen la caja de ladrillo macizo convencional propia de los años 30. Es el ejemplo más poderoso de la unión de tradición e innovación en su obra residencial. Sus formas sobrias entre el Funcionalismo Danés y la Modernidad se singularizarán por su abstracción y volúmenes limpios que acentúan el efecto de su geometría, prismática y brutalista. El desplazamiento de los cuerpos que lo componen, unos sobre otros, generan un ritmo, que se producirá a otras escalas, ello unido a las variaciones de sus formas y a la elección de sus materiales, ladrillo y madera, le confieren a la casa un carácter orgánico. El edificio se ancla a la tierra resolviéndose en diferentes niveles tras el estudio del lugar y su topografía. El resultado es una versión construida del paisaje, en la cual el edificio da forma al lugar y ensalza la experiencia del escenario natural. La unidad de las estructuras primitivas, parece estar presente. Constituye un ejemplo de la “La idea de Promenade de Asplund”, el proyecto ofrece diferentes recorridos, permitiendo su propia vivencia de la casa, que ofrece la posibilidad vital de decidir. El capítulo tercero trata sobre el pabellón de invitados de Niels Bohr de 1957, situado un área boscosa, en Tisvilde Hegn, fue el primer edificio del arquitecto danés Vilhelm Wohlert. Arraigado a la Tradición Danesa, representa una renovación basada en la absorción de influencias extranjeras: la Arquitectura Americana y la Tradición Japonesa. La caja de madera, posada sobre un terreno horizontal, tiene el carácter sensible de un organismo vivo, siempre cambiante según las variaciones de luz del día o temperatura. Cuando se abre, crea una prolongación del espacio interior, que se extiende a la naturaleza circundante, y se expande hacia el espacio exterior, permitiendo su movilización. Se establece una arquitectura de flujos. Hay un interés por la materia, su textura y el efecto emocional que emana. Las proporciones y dimensiones del edificio están reguladas por un módulo, que se ajusta a la medida del hombre, destacando la gran unidad del edificio. La llave se su efecto estético está en su armonía y equilibrio, que transmiten serenidad y belleza. El encuentro con la naturaleza es la lección más básica del proyecto, donde un mundo de relaciones es amable al ser humano. El capítulo cuarto, analiza el proyecto del Museo Louisiana de 1958, en Humlebæk, primer proyecto de la pareja de arquitectos daneses Jørgen Bo y Vilhelm Wohlert. La experiencia en California de Wohlert donde será visitado por Bo, será trascendental para el desarrollo de Louisiana, donde la identidad Danesa se fusiona con la asimilación de otras culturas, la arquitectura de Frank Lloyd Wright, la del área de la Bahía y la Tradición Japonesa principalmente. La idea del proyecto es la de una obra de arte integral: arquitectura, arte y paisaje, coexistirían en un mismo lugar. Diferentes recursos realzarán su carácter residencial, como el uso de los materiales propios de un entorno doméstico, la realización a la escala del hombre, el modo de usar la iluminación. Cubiertas planas que muestran su artificialidad, parecen flotar sobre galerías acristaladas, acentuarán la fuerza del plano horizontal y establecerán un recorrido en zig-zag de marcado ritmo acompasado. Ritmo que tiene que ver con la encarnación del pulso de la naturaleza, que se acompaña de juegos de luz, y de otras vibraciones materiales a diferentes escalas, imagen, que encuentra una analogía semejante en la cultura japonesa. Todo se coordina con la trama estructural, que conlleva a una construcción y proporción disciplinada. Louisiana atiende al principio de crecimiento de la naturaleza, con la que su conexión es profunda. Hay un dinamismo expresado por el despliegue del edificio, que evoca a algunos proyectos de la Tradición Japonesa. Los blancos muros tienen su propia identidad como formas en sí mismas, avanzan prolongándose fuera de la línea del vidrio, se mueven libremente siguiendo el orden estructural, acompañando al espacio que fluye, en contacto directo con la naturaleza que está en un continuo estado de flujos. Se da todo un mundo de relaciones, donde existe un dialogo entre el paisaje, arte y arquitectura. El capítulo quinto, se dedica a analizar la segunda casa del arquitecto danés Halldor Gunnløgsson, de 1959. Evoca a la Arquitectura Japonesa y Americana, pero es principalmente resultado de una fuerte voluntad y disciplina artística personal. La cubierta, plana, suspendida sobre una gran plataforma pavimentada, que continúa la sección del terreno construyendo de lugar, tiene una gran presencia y arroja una profunda sombra bajo ella. En el interior un espacio único, que se puede dividir eventualmente, discurre en torno a un cuerpo central. El espacio libre fluye, extendiéndose a través de la transparencia de sus ventanales a dos espacios contrapuestos: un patio ajardinado íntimo, que inspira calma y sosiego; y la naturaleza salvaje del mar que proyecta el color del cielo, ambos en constante estado de cambio. El proyecto se elabora de un modo rigurosamente formal, existiendo al mismo tiempo un perfecto equilibrio entre la abstracción de su estructura y su programa. La estructura de madera cuyo orden se extiende más allá de los límites de su perímetro, está formada por pórticos completos como elementos libres, queda expuesta, guardando una estrecha relación con el concepto de modernidad de Mies, equivalente a la arquitectura clásica. La preocupación por el efecto estético es máxima, nada es improvisado. Pero además la combinación de materiales y el juego de las texturas hay una cualidad táctil, cierto erotismo, que flota alrededor de ella. La precisión constructiva y su refinamiento se acercan a Mies. La experiencia del espacio arquitectónico es una vivencia global. La influencia de la arquitectura japonesa, es más conceptual que formal, revelada en un respeto por la naturaleza, la búsqueda del refinamiento a través de la moderación, la eliminación de los objetos innecesarios que distraen de la experiencia del lugar y la preocupación por la luz y la sombra, donde se establece cierto paralelismo con el oscuro mundo del invierno nórdico. Hay un entendimiento de que el espacio, en lugar de ser un objeto inmaterial definido por superficies materiales se entiende como interacciones dinámicas. El capítulo sexto. Propone un viaje para conocer algunas de las viviendas unifamiliares más interesantes que se construyeron en el periodo, que forman parte del sistema investigado. Del estudio comparado y orientado en varios temas, se obtienen diversa conclusiones propias del sistema estudiado. La maestría de la sustancia y la forma será una característica distintiva en Dinamarca, se demuestra que hay un acercamiento a la cultura de Oriente, conceptual y formal, y unos intereses comunes con cierta arquitectura Americana. Su lección nos sensibiliza hacia un sentido fortalecido de proporción, escala, materialidad, textura y peso, densidad del espacio, se valora lo táctil y lo visual, hay una sensibilidad hacia la naturaleza, hacia lo humano, hacia el paisaje, la integridad de la obra. ABSTRACT The thesis “1950 around the Louisiana Museum 1970” analyses several works related to domestic space, which were carried out between 1950 and 1970 in Denmark, a golden age of modern architecture. After the isolation and limitations brought about by the war that blighted Europe, young Danish architects were keen to experiment with ideas of an international origin, encouraged by different circumstances. They find the best field of rehearsal to be the domestic space. The best architecture of that period in Denmark should be understood as a system composed of different authors, who have in common with each other many more similarities than differences, thus complimenting each other. In the interests of understanding, the study of a range of figures and buildings is necessary so that this system, the research of which is still in its infancy, can be completed. The thesis proposes a journey of discovery through the names of some of those protagonists who were showcased through their work so that tradition and avant- garde could go hand in hand. The objective is to unveil the keys to Danish Modernity; to recognise, discover and revive the legacy of some of its protagonists in the domestic field whose lessons are seen as entirely of the present. For an architect, the taking on of modern contributions with both moderation and caution, with its intimate relationship with architectural tradition and its own craft, will be one of his hallmarks. With the study set against several projects and versions, one can derive common values among their authors. In the same way their affinities and differences in respect of the same issue emerge. This will allow an understanding of their measures in line with references and influences and enable the defining of the variables of their architectural spaces. The common line between the buildings selected will be their particular relationship with nature and the space with which they integrate. The façade, the place where the relationship between the interior and the landscape would be negotiated, wouldl be the discriminating element in a distinct way for each one of them. It is through each of these facades that this relationship would extend, and far beyond their physical perimeter. The investigation has been structured into six chapters, preceded by an introduction. The first chapter outlines and analyses the backgrounds, figures and buildings most relevant to the Danish Tradition. This is to facilitate the understanding and elucidation of some of the keys to its modernity in the field of architecture, which came about with the clear intention to discover its own identity and expression. This thriving modernity is characterized by its moderate assimilation with foreign cultures with a critical eye, and finds its roots anchored in architectural tradition and its own handcraft. It is forged in the emergence of a common ideal of enormous personality which today has come to be valued as an authentic contribution to the sphere from a culture that was formerly seen as on the peripheries. What will be demonstrated is the path taken by previous generations to these works and the debate that surrounds them. The sensibilities for both the vernacular and the classic, which at first glance may seem contradictory, will dominate the debate with the same veracity and respectability. The so-called third generation of Sigfried Giedion will revive the process between the classic and the vernacular, supported in spirit by the handcraft work and by tradition, with the objective of discovering the “truth” and the “original essence” of the architectural act. The second chapter analyzes the Varming house, built by Eva and Nils Koppel 1953, which is situated in a residential area of Gentofte. This reinterprets Asplund’s vision of an interior landscape extending to the exterior, where we see a break with the conventional sturdy brick shell of the 1930s. It is the most powerful example of the union of tradition and innovation in his their residential work. Their sober forms caught between Danish Functionalism and modernity are characterized by their abstraction and clean shapes which accentuate their prismatic and brutal geometry, The displacement of the parts of which they are composed, one over the other, generate a rhythm. This is produced to varying scales and is closely linked to its forms and the selection of materials – brick and wood – that confer an organic character to the house. The building is anchored to the earth, finding solution at different levels through the study of place and topography. The result is an adaption constructed out of the landscape, in which the building gives form to the place and celebrates the experience of the natural setting. The unity of primitive structures appears to be present. It constitutes an example of “Asplund’s Promenade Idea”. Different routes of exploration within are available to the visitor, allowing for one’s own personal experience of the house, allowing in turn for the vital chance to decide. The third chapter deals with Niels Bohr’s guest pavilion. Built in 1957, it is situated in a wooded area of Tisvilde Hegn and was the architect Vilhelm Wohlert’s first building. Rooted in the Danish Tradition, it represents a renewal based on the absorption of foreign influences: American architecture and the Japanese tradition. The wooden box, perched atop a horizontal terrain, possesses the sensitive character of the living organism, ever-changing in accordance with the variations in daylight and temperature. When opened up, it creates an elongation of the interior space which extends into the surrounding nature and it expands towards the exterior space, allowing for its mobilisation. It establishes an architecture of flux. There is interest in the material, its texture and the emotional effect it inspires. The building’s proportions and dimensions are regulated by a module, which is adjusted by hand, bringing out the great unity of the building. The key to its aesthetic effect is its harmony and equilibrium, which convey serenity and beauty. The meeting with nature is the most fundamental lesson of the project, where a world of relationships softens the personality of the human being. The fourth chapter analyzes the Louisiana Museum project of 1958 in 1958. It was the first project of the Danish architects Jørgen Bo and Vilhelm Wohlert. Wohlert’s experience in California where he was visited by Bo would be essential to the development of Louisiana, where the Danish identity is fused in assimilation with other cultures, the architecture of Frank Lloyd Wright, that of the Bahía area and principally the Japanese tradition. The idea of the project was for an integrated work of art: architecture, art and landscape, which would coexist in the same space. A range of different resources would realize the residential character, such as the use of materials taken from a domestic environment, the attainment of human scale and the manner in which light was used. Flat roof plans that show their artificiality and appear to float over glassed galleries. They accentuate the strength of the horizontal plan and establish a zigzag route of marked and measured rhythm. It is a rhythm that has to do with the incarnation of nature’s pulse, which is accompanied with plays of light, as well as material vibrations of different scales, imagery which uncovers a parallel analogy with Japanese culture. Everything is coordinated along a structural frame, which involves a disciplined construction and proportion. Louisiana cherishes nature’s principle of growth, to which its connection is profound. Here is a dynamism expressed through the disposition of the building, which evokes in some projects the Japanese tradition. The white walls possess their own identity as forms in their own right. They advance, extending beyond the line of glass, moving freely along the structural line, accompanying a space that flows and in direct contact with nature that is itself in a constant state of flux. It creates a world of relationships, where dialogue exists between the landscape, art and architecture. The fifth chapter is dedicated to analyzing the Danish architect Halldor Gunnløgsson’s second house, built in 1959. It evokes both Japanese and American architecture but is principally the result of a strong will and personal artistic discipline. The flat roof suspended above a large paved platform – itself continuing the constructed terrain of the place – has great presence and casts a heavy shadow beneath. In the interior, a single space, which can at length be divided and which flows around a central space. The space flows freely, extending through the transparency of its windows which give out onto two contrasting locations: an intimate garden patio, inspiring calm and tranquillity, and the wild nature of the sea which projects the colour of sky, both in a constant state of change. The project is realized in a rigorously formal manner. A perfect balance exists between the abstraction of his structure and his project. The wooden structure, whose order extends beyond the limits of its perimeter, is formed of complete porticos of free elements. It remains exposed, maintaining a close relationship with Mies’ concept of modernity, analogous to classical architecture. The preoccupation with the aesthetic effect is paramount and nothing is improvised. But in addition to this - the combination of materials and the play of textures - there is a tactile quality, a certain eroticism, which lingers all about. The constructive precision and its refinement are close to Mies. The experience of the architectural space is universal. The influence of Japanese architecture, more conceptual than formal, is revealed in a respect for nature. It can be seen in the search for refinement through moderation, the elimination of the superfluous object that distract from the experience of place and the preoccupation with light and shade, where a certain parallel with the dark world of the Nordic winter is established. There is an understanding that space, rather than being an immaterial object defined by material surfaces, extends instead as dynamic interactions. The sixth chapter. This proposes a journey to discover some of the unfamiliar residences of most interest which were constructed in the period, and which form part of the system being investigated. Through the study of comparison and one which is geared towards various themes, diverse conclusions are drawn regarding the system being researched. The expertise in substance and form will be a distinctive characteristic in Denmark, demonstrating an approach to the culture of the Orient, both conceptual and formal, and some common interests in certain American architecture. Its teachings sensitize us to a strengthened sense of proportion, scale, materiality, texture and weight and density of space. It values both the tactile and the visual. There is a sensitivity to nature, to the human, to the landscape and to the integrity of the work.

On the actions that one nerve cell can have on another: Distinguishing “drivers” from “modulators”

When one nerve cell acts on another, its postsynaptic effect can vary greatly. In sensory systems, inputs from “drivers” can be differentiated from those of “modulators.” The driver can be identified as the transmitter of receptive field properties; the modulator can be identified as altering the probability of certain aspects of that transmission. Where receptive fields are not available, the distinction is more difficult and currently is undefined. We use the visual pathways, particularly the thalamic geniculate relay for which much relevant evidence is available, to explore ways in which drivers can be distinguished from modulators. The extent to which the distinction may apply first to other parts of the thalamus and then, possibly, to other parts of the brain is considered. We suggest the following distinctions: Cross-correlograms from driver inputs have sharper peaks than those from modulators; there are likely to be few drivers but many modulators for any one cell; and drivers are likely to act only through ionotropic receptors having a fast postsynaptic effect whereas modulators also are likely to activate metabotropic receptors having a slow and prolonged postsynaptic effect.

The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake

The CHL1 (NRT1) gene of Arabidopsis encodes a nitrate-inducible nitrate transporter that is thought to be a component of the low-affinity (mechanism II) nitrate-uptake system in plants. A search was performed to find high-affinity (mechanism I) uptake mutants by using chlorate selections on plants containing Tag1 transposable elements. Chlorate-resistant mutants defective in high-affinity nitrate uptake were identified, and one had a Tag1 insertion in chl1, which was responsible for the phenotype. Further analysis showed that chl1 mutants have reduced high-affinity uptake in induced plants and are missing a saturable component of the constitutive, high-affinity uptake system in addition to reduced low-affinity uptake. The contribution of CHL1 to constitutive high-affinity uptake is higher when plants are grown at more acidic pH, conditions that increase the level of CHL1 mRNA. chl1 mutants show reduced membrane depolarization in root epidermal cells in response to low (250 μM) and high (10 mM) concentrations of nitrate. Low levels of nitrate (100 μM) induce a rapid increase in CHL1 mRNA. These results show that CHL1 is an important component of both the high-affinity and the low-affinity nitrate-uptake systems and indicate that CHL1 may be a dual-affinity nitrate transporter.

The Nucleoporin Nup153 Plays a Critical Role in Multiple Types of Nuclear Export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin β to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin α/β or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.

Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

We report here the isolation and functional analysis of the rfc3+ gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3+ gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3+ is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

The LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.

Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression

The yeast transcriptional repressor Tup1, tethered to DNA, represses to strikingly different degrees transcription elicited by members of two classes of activators. Repression in both cases is virtually eliminated by mutation of either member of the cyclin-kinase pair Srb10/11. In contrast, telomeric chromatin affects both classes of activators equally, and in neither case is that repression affected by mutation of Srb10/11. In vitro, Tup1 interacts with RNA polymerase II holoenzyme bearing Srb10 as well as with the separated Srb10. These and other findings indicate that at least one aspect of Tup1's action involves interaction with the RNA polymerase II holoenzyme.

One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases

Carotenoids in the photosynthetic membranes of plants typically contain two β-rings (e.g., β-carotene and zeaxanthin) or one ɛ- and one β-ring (e.g., lutein). Carotenoids with two ɛ-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene ɛ-cyclase (LCYe) adds one ɛ-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene β-cyclase (LCYb) adds two β-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two ɛ-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic ɛ-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis ɛ-cyclases involved in the determination of ring number were mapped by analysis of chimeric ɛ-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one ɛ-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two ɛ-rings. An R residue in this position also yields a bi-ɛ-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

Yeast Rab GTPase-activating Protein Gyp1p Localizes to the Golgi Apparatus and Is a Negative Regulator of Ypt1p

A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Δ strain displays a growth defect on synthetic medium at 37°C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Δ cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Δ cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.