Trace element profiles of the sea anemone Anemonia viridis living nearby a natural CO2 vent

Ocean acidification (OA) is not an isolated threat, but acts in concert with other impacts on ecosystems and species. Coastal marine invertebrates will have to face the synergistic interactions of OA with other global and local stressors. One local factor, common in coastal environments, is trace element contamination. CO2 vent sites are extensively studied in the context of OA and are often considered analogous to the oceans in the next few decades. The CO2 vent found at Levante Bay (Vulcano, NE Sicily, Italy) also releases high concentrations of trace elements to its surrounding seawater, and is therefore a unique site to examine the effects of long-term exposure of nearby organisms to high pCO2 and trace element enrichment in situ. The sea anemone Anemonia viridis is prevalent next to the Vulcano vent and does not show signs of trace element poisoning/stress. The aim of our study was to compare A. viridis trace element profiles and compartmentalization between high pCO2 and control environments. Rather than examining whole anemone tissue, we analyzed two different body compartments-the pedal disc and the tentacles, and also examined the distribution of trace elements in the tentacles between the animal and the symbiotic algae. We found dramatic changes in trace element tissue concentrations between the high pCO2/high trace element and control sites, with strong accumulation of iron, lead, copper and cobalt, but decreased concentrations of cadmium, zinc and arsenic proximate to the vent. The pedal disc contained substantially more trace elements than the anemone's tentacles, suggesting the pedal disc may serve as a detoxification/storage site for excess trace elements. Within the tentacles, the various trace elements displayed different partitioning patterns between animal tissue and algal symbionts. At both sites iron was found primarily in the algae, whereas cadmium, zinc and arsenic were primarily found in the animal tissue. Our data suggests that A. viridis regulates its internal trace element concentrations by compartmentalization and excretion and that these features contribute to its resilience and potential success at the trace element-rich high pCO2 vent.

Occurrence (presence-only) of living Lophelia pertusa reefs in the Irish continental margin

The present data set was used as a training set for a Habitat Suitability Model. It contains occurrence (presence-only) of living Lophelia pertusa reefs in the Irish continental margin, which were assembled from databases, cruise reports and publications. A total of 4423 records were inspected and quality assessed to ensure that they (1) represented confirmed living L. pertusa reefs (so excluding 2900 records of dead and isolated coral colony records); (2) were derived from sampling equipment that allows for accurate (<200 m) geo-referencing (so excluding 620 records derived mainly from trawling and dredging activities); and (3) were not duplicated. A total of 245 occurrences were retained for the analysis. Coral observations are highly clustered in regions targeted by research expeditions, which might lead to falsely inflated model evaluation measures (Veloz, 2009). Therefore, we coarsened the distribution data by deleting all but one record within grid cells of 0.02° resolution (Davies & Guinotte 2011). The remaining 53 points were subject to a spatial cross-validation process: a random presence point was chosen, grouped with its 12 closest neighbour presence points based on Euclidean distance and withheld from model training. This process was repeated for all records, resulting in 53 replicates of spatially non-overlapping sets of test (n=13) and training (n=40) data. The final 53 occurrence records were used for model training.

Self-organizing cultured neural networks: Image analysis techniques for longitudinal tracking and modeling of the underlying network structure

Esta tesis estudia la evolución estructural de conjuntos de neuronas como la capacidad de auto-organización desde conjuntos de neuronas separadas hasta que forman una red (clusterizada) compleja. Esta tesis contribuye con el diseño e implementación de un algoritmo no supervisado de segmentación basado en grafos con un coste computacional muy bajo. Este algoritmo proporciona de forma automática la estructura completa de la red a partir de imágenes de cultivos neuronales tomadas con microscopios de fase con una resolución muy alta. La estructura de la red es representada mediante un objeto matemático (matriz) cuyos nodos representan a las neuronas o grupos de neuronas y los enlaces son las conexiones reconstruidas entre ellos. Este algoritmo extrae también otras medidas morfológicas importantes que caracterizan a las neuronas y a las neuritas. A diferencia de otros algoritmos hasta el momento, que necesitan de fluorescencia y técnicas inmunocitoquímicas, el algoritmo propuesto permite el estudio longitudinal de forma no invasiva posibilitando el estudio durante la formación de un cultivo. Además, esta tesis, estudia de forma sistemática un grupo de variables topológicas que garantizan la posibilidad de cuantificar e investigar la progresión de las características principales durante el proceso de auto-organización del cultivo. Nuestros resultados muestran la existencia de un estado concreto correspondiente a redes con configuracin small-world y la emergencia de propiedades a micro- y meso-escala de la estructura de la red. Finalmente, identificamos los procesos físicos principales que guían las transformaciones morfológicas de los cultivos y proponemos un modelo de crecimiento de red que reproduce el comportamiento cuantitativamente de las observaciones experimentales. ABSTRACT The thesis analyzes the morphological evolution of assemblies of living neurons, as they self-organize from collections of separated cells into elaborated, clustered, networks. In particular, it contributes with the design and implementation of a graph-based unsupervised segmentation algorithm, having an associated very low computational cost. The processing automatically retrieves the whole network structure from large scale phase-contrast images taken at high resolution throughout the entire life of a cultured neuronal network. The network structure is represented by a mathematical object (a matrix) in which nodes are identified neurons or neurons clusters, and links are the reconstructed connections between them. The algorithm is also able to extract any other relevant morphological information characterizing neurons and neurites. More importantly, and at variance with other segmentation methods that require fluorescence imaging from immunocyto- chemistry techniques, our measures are non invasive and entitle us to carry out a fully longitudinal analysis during the maturation of a single culture. In turn, a systematic statistical analysis of a group of topological observables grants us the possibility of quantifying and tracking the progression of the main networks characteristics during the self-organization process of the culture. Our results point to the existence of a particular state corresponding to a small-world network configuration, in which several relevant graphs micro- and meso-scale properties emerge. Finally, we identify the main physical processes taking place during the cultures morphological transformations, and embed them into a simplified growth model that quantitatively reproduces the overall set of experimental observations.

Self-organizing cultured neural networks: Image analysis techniques for longitudinal tracking and modeling of the underlying network structure (versión actualizada)

Esta tesis estudia la evolución estructural de conjuntos de neuronas como la capacidad de auto-organización desde conjuntos de neuronas separadas hasta que forman una red (clusterizada) compleja. Esta tesis contribuye con el diseño e implementación de un algoritmo no supervisado de segmentación basado en grafos con un coste computacional muy bajo. Este algoritmo proporciona de forma automática la estructura completa de la red a partir de imágenes de cultivos neuronales tomadas con microscopios de fase con una resolución muy alta. La estructura de la red es representada mediante un objeto matemático (matriz) cuyos nodos representan a las neuronas o grupos de neuronas y los enlaces son las conexiones reconstruidas entre ellos. Este algoritmo extrae también otras medidas morfológicas importantes que caracterizan a las neuronas y a las neuritas. A diferencia de otros algoritmos hasta el momento, que necesitan de fluorescencia y técnicas inmunocitoquímicas, el algoritmo propuesto permite el estudio longitudinal de forma no invasiva posibilitando el estudio durante la formación de un cultivo. Además, esta tesis, estudia de forma sistemática un grupo de variables topológicas que garantizan la posibilidad de cuantificar e investigar la progresión de las características principales durante el proceso de auto-organización del cultivo. Nuestros resultados muestran la existencia de un estado concreto correspondiente a redes con configuracin small-world y la emergencia de propiedades a micro- y meso-escala de la estructura de la red. Finalmente, identificamos los procesos físicos principales que guían las transformaciones morfológicas de los cultivos y proponemos un modelo de crecimiento de red que reproduce el comportamiento cuantitativamente de las observaciones experimentales. ABSTRACT The thesis analyzes the morphological evolution of assemblies of living neurons, as they self-organize from collections of separated cells into elaborated, clustered, networks. In particular, it contributes with the design and implementation of a graph-based unsupervised segmentation algorithm, having an associated very low computational cost. The processing automatically retrieves the whole network structure from large scale phase-contrast images taken at high resolution throughout the entire life of a cultured neuronal network. The network structure is represented by a mathematical object (a matrix) in which nodes are identified neurons or neurons clusters, and links are the reconstructed connections between them. The algorithm is also able to extract any other relevant morphological information characterizing neurons and neurites. More importantly, and at variance with other segmentation methods that require fluorescence imaging from immunocyto- chemistry techniques, our measures are non invasive and entitle us to carry out a fully longitudinal analysis during the maturation of a single culture. In turn, a systematic statistical analysis of a group of topological observables grants us the possibility of quantifying and tracking the progression of the main networks characteristics during the self-organization process of the culture. Our results point to the existence of a particular state corresponding to a small-world network configuration, in which several relevant graphs micro- and meso-scale properties emerge. Finally, we identify the main physical processes taking place during the cultures morphological transformations, and embed them into a simplified growth model that quantitatively reproduces the overall set of experimental observations.

V-substituted In2S3: an intermediate band material with photocatalytic activity in the whole visible light range

We proposed in our previous work V-substituted In2S3 as an intermediate band (IB) material able to enhance the efficiency of photovoltaic cells by combining two photons to achieve a higher energy electron excitation, much like natural photosynthesis. Here this hyper-doped material is tested in a photocatalytic reaction using wavelength-controlled light. The results evidence its ability to use photons with wavelengths of up to 750 nm, i.e. with energy significantly lower than the bandgap (=2.0 eV) of non-substituted In2S3, driving with them the photocatalytic reaction at rates comparable to those of non-substituted In2S3 in its photoactivity range (λ ≤ 650 nm). Photoluminescence spectra evidence that the same bandgap excitation as in V-free In2S3 occurs in V-substituted In2S3 upon illumination with photons in the same sub-bandgap energy range which is effective in photocatalysis, and its linear dependence on light intensity proves that this is not due to a nonlinear optical property. This evidences for the first time that a two-photon process can be active in photocatalysis in a single-phase material. Quantum calculations using GW-type many-body perturbation theory suggest that the new band introduced in the In2S3 gap by V insertion is located closer to the conduction band than to the valence band, so that hot carriers produced by the two-photon process would be of electron type; they also show that the absorption coefficients of both transitions involving the IB are of significant and similar magnitude. The results imply that V-substituted In2S3, besides being photocatalytically active in the whole visible light range (a property which could be used for the production of solar fuels), could make possible photovoltaic cells of improved efficiency.

Design and Simulation of Deep Nanometer SRAM Cells under Energy, Mismatch, and Radiation Constraints

La fiabilidad está pasando a ser el principal problema de los circuitos integrados según la tecnología desciende por debajo de los 22nm. Pequeñas imperfecciones en la fabricación de los dispositivos dan lugar ahora a importantes diferencias aleatorias en sus características eléctricas, que han de ser tenidas en cuenta durante la fase de diseño. Los nuevos procesos y materiales requeridos para la fabricación de dispositivos de dimensiones tan reducidas están dando lugar a diferentes efectos que resultan finalmente en un incremento del consumo estático, o una mayor vulnerabilidad frente a radiación. Las memorias SRAM son ya la parte más vulnerable de un sistema electrónico, no solo por representar más de la mitad del área de los SoCs y microprocesadores actuales, sino también porque las variaciones de proceso les afectan de forma crítica, donde el fallo de una única célula afecta a la memoria entera. Esta tesis aborda los diferentes retos que presenta el diseño de memorias SRAM en las tecnologías más pequeñas. En un escenario de aumento de la variabilidad, se consideran problemas como el consumo de energía, el diseño teniendo en cuenta efectos de la tecnología a bajo nivel o el endurecimiento frente a radiación. En primer lugar, dado el aumento de la variabilidad de los dispositivos pertenecientes a los nodos tecnológicos más pequeños, así como a la aparición de nuevas fuentes de variabilidad por la inclusión de nuevos dispositivos y la reducción de sus dimensiones, la precisión del modelado de dicha variabilidad es crucial. Se propone en la tesis extender el método de inyectores, que modela la variabilidad a nivel de circuito, abstrayendo sus causas físicas, añadiendo dos nuevas fuentes para modelar la pendiente sub-umbral y el DIBL, de creciente importancia en la tecnología FinFET. Los dos nuevos inyectores propuestos incrementan la exactitud de figuras de mérito a diferentes niveles de abstracción del diseño electrónico: a nivel de transistor, de puerta y de circuito. El error cuadrático medio al simular métricas de estabilidad y prestaciones de células SRAM se reduce un mínimo de 1,5 veces y hasta un máximo de 7,5 a la vez que la estimación de la probabilidad de fallo se mejora en varios ordenes de magnitud. El diseño para bajo consumo es una de las principales aplicaciones actuales dada la creciente importancia de los dispositivos móviles dependientes de baterías. Es igualmente necesario debido a las importantes densidades de potencia en los sistemas actuales, con el fin de reducir su disipación térmica y sus consecuencias en cuanto al envejecimiento. El método tradicional de reducir la tensión de alimentación para reducir el consumo es problemático en el caso de las memorias SRAM dado el creciente impacto de la variabilidad a bajas tensiones. Se propone el diseño de una célula que usa valores negativos en la bit-line para reducir los fallos de escritura según se reduce la tensión de alimentación principal. A pesar de usar una segunda fuente de alimentación para la tensión negativa en la bit-line, el diseño propuesto consigue reducir el consumo hasta en un 20 % comparado con una célula convencional. Una nueva métrica, el hold trip point se ha propuesto para prevenir nuevos tipos de fallo debidos al uso de tensiones negativas, así como un método alternativo para estimar la velocidad de lectura, reduciendo el número de simulaciones necesarias. Según continúa la reducción del tamaño de los dispositivos electrónicos, se incluyen nuevos mecanismos que permiten facilitar el proceso de fabricación, o alcanzar las prestaciones requeridas para cada nueva generación tecnológica. Se puede citar como ejemplo el estrés compresivo o extensivo aplicado a los fins en tecnologías FinFET, que altera la movilidad de los transistores fabricados a partir de dichos fins. Los efectos de estos mecanismos dependen mucho del layout, la posición de unos transistores afecta a los transistores colindantes y pudiendo ser el efecto diferente en diferentes tipos de transistores. Se propone el uso de una célula SRAM complementaria que utiliza dispositivos pMOS en los transistores de paso, así reduciendo la longitud de los fins de los transistores nMOS y alargando los de los pMOS, extendiéndolos a las células vecinas y hasta los límites de la matriz de células. Considerando los efectos del STI y estresores de SiGe, el diseño propuesto mejora los dos tipos de transistores, mejorando las prestaciones de la célula SRAM complementaria en más de un 10% para una misma probabilidad de fallo y un mismo consumo estático, sin que se requiera aumentar el área. Finalmente, la radiación ha sido un problema recurrente en la electrónica para aplicaciones espaciales, pero la reducción de las corrientes y tensiones de los dispositivos actuales los está volviendo vulnerables al ruido generado por radiación, incluso a nivel de suelo. Pese a que tecnologías como SOI o FinFET reducen la cantidad de energía colectada por el circuito durante el impacto de una partícula, las importantes variaciones de proceso en los nodos más pequeños va a afectar su inmunidad frente a la radiación. Se demuestra que los errores inducidos por radiación pueden aumentar hasta en un 40 % en el nodo de 7nm cuando se consideran las variaciones de proceso, comparado con el caso nominal. Este incremento es de una magnitud mayor que la mejora obtenida mediante el diseño de células de memoria específicamente endurecidas frente a radiación, sugiriendo que la reducción de la variabilidad representaría una mayor mejora. ABSTRACT Reliability is becoming the main concern on integrated circuit as the technology goes beyond 22nm. Small imperfections in the device manufacturing result now in important random differences of the devices at electrical level which must be dealt with during the design. New processes and materials, required to allow the fabrication of the extremely short devices, are making new effects appear resulting ultimately on increased static power consumption, or higher vulnerability to radiation SRAMs have become the most vulnerable part of electronic systems, not only they account for more than half of the chip area of nowadays SoCs and microprocessors, but they are critical as soon as different variation sources are regarded, with failures in a single cell making the whole memory fail. This thesis addresses the different challenges that SRAM design has in the smallest technologies. In a common scenario of increasing variability, issues like energy consumption, design aware of the technology and radiation hardening are considered. First, given the increasing magnitude of device variability in the smallest nodes, as well as new sources of variability appearing as a consequence of new devices and shortened lengths, an accurate modeling of the variability is crucial. We propose to extend the injectors method that models variability at circuit level, abstracting its physical sources, to better model sub-threshold slope and drain induced barrier lowering that are gaining importance in FinFET technology. The two new proposed injectors bring an increased accuracy of figures of merit at different abstraction levels of electronic design, at transistor, gate and circuit levels. The mean square error estimating performance and stability metrics of SRAM cells is reduced by at least 1.5 and up to 7.5 while the yield estimation is improved by orders of magnitude. Low power design is a major constraint given the high-growing market of mobile devices that run on battery. It is also relevant because of the increased power densities of nowadays systems, in order to reduce the thermal dissipation and its impact on aging. The traditional approach of reducing the voltage to lower the energy consumption if challenging in the case of SRAMs given the increased impact of process variations at low voltage supplies. We propose a cell design that makes use of negative bit-line write-assist to overcome write failures as the main supply voltage is lowered. Despite using a second power source for the negative bit-line, the design achieves an energy reduction up to 20% compared to a conventional cell. A new metric, the hold trip point has been introduced to deal with new sources of failures to cells using a negative bit-line voltage, as well as an alternative method to estimate cell speed, requiring less simulations. With the continuous reduction of device sizes, new mechanisms need to be included to ease the fabrication process and to meet the performance targets of the successive nodes. As example we can consider the compressive or tensile strains included in FinFET technology, that alter the mobility of the transistors made out of the concerned fins. The effects of these mechanisms are very dependent on the layout, with transistor being affected by their neighbors, and different types of transistors being affected in a different way. We propose to use complementary SRAM cells with pMOS pass-gates in order to reduce the fin length of nMOS devices and achieve long uncut fins for the pMOS devices when the cell is included in its corresponding array. Once Shallow Trench isolation and SiGe stressors are considered the proposed design improves both kinds of transistor, boosting the performance of complementary SRAM cells by more than 10% for a same failure probability and static power consumption, with no area overhead. While radiation has been a traditional concern in space electronics, the small currents and voltages used in the latest nodes are making them more vulnerable to radiation-induced transient noise, even at ground level. Even if SOI or FinFET technologies reduce the amount of energy transferred from the striking particle to the circuit, the important process variation that the smallest nodes will present will affect their radiation hardening capabilities. We demonstrate that process variations can increase the radiation-induced error rate by up to 40% in the 7nm node compared to the nominal case. This increase is higher than the improvement achieved by radiation-hardened cells suggesting that the reduction of process variations would bring a higher improvement.

Antisense oligonucleotides against α1E reduce R-type calcium currents in cerebellar granule cells

Many neurons of the central nervous system display multiple high voltage-activated Ca2+ currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the α1E subunit, which yields Ca2+ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against α1E could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6–8 days in vitro. Incubation with α1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from −10 ± 0.6 picoamperes/picofarad (pA/pF) (n = 6) in the untreated controls to −4.8 ± 0.8 pA/pF (n = 11) (P < 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (−11.3 ± 3.2 pA/pF). The specificity of the α1E antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against α1A did not affect R-type current expression (−11.5 ± 1.7 and −11.7 ± 1.7 pA/pF, respectively), whereas the α1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of α1 subunits support the high voltage-activated R-type current in cerebellar granule cells.

Myofibrillogenesis visualized in living embryonic cardiomyocytes

Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green fluorescent protein (GFP) linked to the Z-band protein, α-actinin. The expression of this fluorescent protein provided an in vivo label for structures containing α-actinin. The GFP–α-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed de novo at the spreading edges of cardiomyocytes, contained punctate concentrations of α-actinin, termed Z-bodies. The punctate Z-bodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form de novo near the cell membrane are premyofibrils—i.e., the precursors of mature myofibrils.

Copolymer 1 induces T cells of the T helper type 2 that crossreact with myelin basic protein and suppress experimental autoimmune encephalomyelitis

The synthetic amino acid copolymer copolymer 1 (Cop 1) suppresses experimental autoimmune encephalomyelitis (EAE) and is beneficial in multiple sclerosis. To further understand Cop 1 suppressive activity, we studied the cytokine secretion profile of various Cop 1-induced T cell lines and clones. Unlike T cell lines induced by myelin basic protein (MBP), which secreted either T cell helper type 1 (Th1) or both Th1 and Th2 cytokines, the T cell lines/clones induced by Cop 1 showed a progressively polarized development toward the Th2 pathway, until they completely lost the ability to secrete Th1 cytokines. Our findings indicate that the polarization of the Cop 1-induced lines did not result from the immunization vehicle or the in vitro growing conditions, but rather from the tendency of Cop 1 to preferentially induce a Th2 response. The response of all of the Cop 1 specific lines/clones, which were originated in the (SJL/J×BALB/c)F1 hybrids, was restricted to the BALB/c parental haplotype. Even though the Cop 1-induced T cells had not been exposed to the autoantigen MBP, they crossreacted with MBP by secretion of interleukin (IL)-4, IL-6, and IL-10. Administration of these T cells in vivo resulted in suppression of EAE induced by whole mouse spinal cord homogenate, in which several autoantigens may be involved. Secretion of anti-inflammatory cytokines by Cop 1-induced suppressor cells, in response to either Cop 1 or MBP, may explain the therapeutic effect of Cop 1 in EAE and in multiple sclerosis.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+: poly(adenosine-diphosphate-d-ribosyl)-acceptor ADP-d-ribosyltransferase, EC 2.4.2.30] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP−/− mice either by the alkylating agent N-methyl-N-nitrosourea (MNU) or by γ-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body γ-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP−/− cells displayed a high sensitivity to MNU exposure: a G2/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver

The ability to identify, isolate, and transplant progenitor cells from solid tissues would greatly facilitate the treatment of diseases currently requiring whole organ transplantation. In this study, cell fractions enriched in candidate epithelial progenitor cells from the rat pancreas were isolated and transplanted into the liver of an inbred strain of Fischer rats. Using a dipeptidyl dipeptidase IV genetic marker system to follow the fate of transplanted cells in conjunction with albumin gene expression, we provide conclusive evidence that, after transplantation to the liver, epithelial progenitor cells from the pancreas differentiate into hepatocytes, express liver-specific proteins, and become fully integrated into the liver parenchymal structure. These studies demonstrate the presence of multipotent progenitor cells in the adult pancreas and establish a role for the liver microenvironment in the terminal differentiation of epithelial cells of foregut origin. They further suggest that such progenitor cells might be useful in studies of organ repopulation following acute or chronic liver injury.