Linear color correction for multiple illumination changes and non-overlapping cameras

Many image processing methods, such as techniques for people re-identification, assume photometric constancy between different images. This study addresses the correction of photometric variations based upon changes in background areas to correct foreground areas. The authors assume a multiple light source model where all light sources can have different colours and will change over time. In training mode, the authors learn per-location relations between foreground and background colour intensities. In correction mode, the authors apply a double linear correction model based on learned relations. This double linear correction includes a dynamic local illumination correction mapping as well as an inter-camera mapping. The authors evaluate their illumination correction by computing the similarity between two images based on the earth mover's distance. The authors compare the results to a representative auto-exposure algorithm found in the recent literature plus a colour correction one based on the inverse-intensity chromaticity. Especially in complex scenarios the authors’ method outperforms these state-of-the-art algorithms.

The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional over-expression

Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β–estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β–estradiol treatment. As a proof of concept for the exploitation of this resource, β–estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl–root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution.

Bayesian scene analysis for multi-camera 3D tracking and camera positioning

Los sistemas de seguimiento mono-cámara han demostrado su notable capacidad para el análisis de trajectorias de objectos móviles y para monitorización de escenas de interés; sin embargo, tanto su robustez como sus posibilidades en cuanto a comprensión semántica de la escena están fuertemente limitadas por su naturaleza local y monocular, lo que los hace insuficientes para aplicaciones realistas de videovigilancia. El objetivo de esta tesis es la extensión de las posibilidades de los sistemas de seguimiento de objetos móviles para lograr un mayor grado de robustez y comprensión de la escena. La extensión propuesta se divide en dos direcciones separadas. La primera puede considerarse local, ya que está orientada a la mejora y enriquecimiento de las posiciones estimadas para los objetos móviles observados directamente por las cámaras del sistema; dicha extensión se logra mediante el desarrollo de un sistema multi-cámara de seguimiento 3D, capaz de proporcionar consistentemente las posiciones 3D de múltiples objetos a partir de las observaciones capturadas por un conjunto de sensores calibrados y con campos de visión solapados. La segunda extensión puede considerarse global, dado que su objetivo consiste en proporcionar un contexto global para relacionar las observaciones locales realizadas por una cámara con una escena de mucho mayor tamaño; para ello se propone un sistema automático de localización de cámaras basado en las trayectorias observadas de varios objetos móviles y en un mapa esquemático de la escena global monitorizada. Ambas líneas de investigación se tratan utilizando, como marco común, técnicas de estimación bayesiana: esta elección está justificada por la versatilidad y flexibilidad proporcionada por dicho marco estadístico, que permite la combinación natural de múltiples fuentes de información sobre los parámetros a estimar, así como un tratamiento riguroso de la incertidumbre asociada a las mismas mediante la inclusión de modelos de observación específicamente diseñados. Además, el marco seleccionado abre grandes posibilidades operacionales, puesto que permite la creación de diferentes métodos numéricos adaptados a las necesidades y características específicas de distintos problemas tratados. El sistema de seguimiento 3D con múltiples cámaras propuesto está específicamente diseñado para permitir descripciones esquemáticas de las medidas realizadas individualmente por cada una de las cámaras del sistema: esta elección de diseño, por tanto, no asume ningún algoritmo específico de detección o seguimiento 2D en ninguno de los sensores de la red, y hace que el sistema propuesto sea aplicable a redes reales de vigilancia con capacidades limitadas tanto en términos de procesamiento como de transmision. La combinación robusta de las observaciones capturadas individualmente por las cámaras, ruidosas, incompletas y probablemente contaminadas por falsas detecciones, se basa en un metodo de asociación bayesiana basado en geometría y color: los resultados de dicha asociación permiten el seguimiento 3D de los objetos de la escena mediante el uso de un filtro de partículas. El sistema de fusión de observaciones propuesto tiene, como principales características, una gran precisión en términos de localización 3D de objetos, y una destacable capacidad de recuperación tras eventuales errores debidos a un número insuficiente de datos de entrada. El sistema automático de localización de cámaras se basa en la observación de múltiples objetos móviles y un mapa esquemático de las áreas transitables del entorno monitorizado para inferir la posición absoluta de dicho sensor. Para este propósito, se propone un novedoso marco bayesiano que combina modelos dinámicos inducidos por el mapa en los objetos móviles presentes en la escena con las trayectorias observadas por la cámara, lo que representa un enfoque nunca utilizado en la literatura existente. El sistema de localización se divide en dos sub-tareas diferenciadas, debido a que cada una de estas tareas requiere del diseño de algoritmos específicos de muestreo para explotar en profundidad las características del marco desarrollado: por un lado, análisis de la ambigüedad del caso específicamente tratado y estimación aproximada de la localización de la cámara, y por otro, refinado de la localización de la cámara. El sistema completo, diseñado y probado para el caso específico de localización de cámaras en entornos de tráfico urbano, podría tener aplicación también en otros entornos y sensores de diferentes modalidades tras ciertas adaptaciones. ABSTRACT Mono-camera tracking systems have proved their capabilities for moving object trajectory analysis and scene monitoring, but their robustness and semantic possibilities are strongly limited by their local and monocular nature and are often insufficient for realistic surveillance applications. This thesis is aimed at extending the possibilities of moving object tracking systems to a higher level of scene understanding. The proposed extension comprises two separate directions. The first one is local, since is aimed at enriching the inferred positions of the moving objects within the area of the monitored scene directly covered by the cameras of the system; this task is achieved through the development of a multi-camera system for robust 3D tracking, able to provide 3D tracking information of multiple simultaneous moving objects from the observations reported by a set of calibrated cameras with semi-overlapping fields of view. The second extension is global, as is aimed at providing local observations performed within the field of view of one camera with a global context relating them to a much larger scene; to this end, an automatic camera positioning system relying only on observed object trajectories and a scene map is designed. The two lines of research in this thesis are addressed using Bayesian estimation as a general unifying framework. Its suitability for these two applications is justified by the flexibility and versatility of that stochastic framework, which allows the combination of multiple sources of information about the parameters to estimate in a natural and elegant way, addressing at the same time the uncertainty associated to those sources through the inclusion of models designed to this end. In addition, it opens multiple possibilities for the creation of different numerical methods for achieving satisfactory and efficient practical solutions to each addressed application. The proposed multi-camera 3D tracking method is specifically designed to work on schematic descriptions of the observations performed by each camera of the system: this choice allows the use of unspecific off-the-shelf 2D detection and/or tracking subsystems running independently at each sensor, and makes the proposal suitable for real surveillance networks with moderate computational and transmission capabilities. The robust combination of such noisy, incomplete and possibly unreliable schematic descriptors relies on a Bayesian association method, based on geometry and color, whose results allow the tracking of the targets in the scene with a particle filter. The main features exhibited by the proposal are, first, a remarkable accuracy in terms of target 3D positioning, and second, a great recovery ability after tracking losses due to insufficient input data. The proposed system for visual-based camera self-positioning uses the observations of moving objects and a schematic map of the passable areas of the environment to infer the absolute sensor position. To this end, a new Bayesian framework combining trajectory observations and map-induced dynamic models for moving objects is designed, which represents an approach to camera positioning never addressed before in the literature. This task is divided into two different sub-tasks, setting ambiguity analysis and approximate position estimation, on the one hand, and position refining, on the other, since they require the design of specific sampling algorithms to correctly exploit the discriminative features of the developed framework. This system, designed for camera positioning and demonstrated in urban traffic environments, can also be applied to different environments and sensors of other modalities after certain required adaptations.

Laser induced forward transfer of silver pastes for printing of fingers in c-si cells.

The main objective of this work is to adapt the Laser Induced Forward Techniques (LIFT), a well- known laser direct writing technique for material transfer, to define metallic contacts (fingers and busbars) onto c-Si cells. The silver paste (with viscosity around 30-50 kcPs) is applied over a glass substrate using a coater. The thickness of the paste can be control changing the deposit parameters. The glass with the silver paste is set at a controlled gap over the c-Si cell. A solid state pulsed laser (532 nm) is focused at the glass/silver interface producing a droplet of silver that it is transferred to the c-Si cell. A scanner is used to print lines. The process parameters (silver paste thickness, gap and laser parameters -spot size, pulse energy and overlapping of pulses) are modified and the morphology of the lines is studied using confocal microscopy. Long lines are printed and the uniformity (in thickness and height) is studied. Some examples of metallization of larger areas (up to 10 cm x 10 cm) are presented.

Computational and Experimental Modelling of Mooring Lines Dynamics for Offshore Floating Wind Turbines

El cálculo de cargas de aerogeneradores flotantes requiere herramientas de simulación en el dominio del tiempo que consideren todos los fenómenos que afectan al sistema, como la aerodinámica, la dinámica estructural, la hidrodinámica, las estrategias de control y la dinámica de las líneas de fondeo. Todos estos efectos están acoplados entre sí y se influyen mutuamente. Las herramientas integradas se utilizan para calcular las cargas extremas y de fatiga que son empleadas para dimensionar estructuralmente los diferentes componentes del aerogenerador. Por esta razón, un cálculo preciso de las cargas influye de manera importante en la optimización de los componentes y en el coste final del aerogenerador flotante. En particular, el sistema de fondeo tiene gran impacto en la dinámica global del sistema. Muchos códigos integrados para la simulación de aerogeneradores flotantes utilizan modelos simplificados que no consideran los efectos dinámicos de las líneas de fondeo. Una simulación precisa de las líneas de fondeo dentro de los modelos integrados puede resultar fundamental para obtener resultados fiables de la dinámica del sistema y de los niveles de cargas en los diferentes componentes. Sin embargo, el impacto que incluir la dinámica de los fondeos tiene en la simulación integrada y en las cargas todavía no ha sido cuantificada rigurosamente. El objetivo principal de esta investigación es el desarrollo de un modelo dinámico para la simulación de líneas de fondeo con precisión, validarlo con medidas en un tanque de ensayos e integrarlo en un código de simulación para aerogeneradores flotantes. Finalmente, esta herramienta, experimentalmente validada, es utilizada para cuantificar el impacto que un modelos dinámicos de líneas de fondeo tienen en la computación de las cargas de fatiga y extremas de aerogeneradores flotantes en comparación con un modelo cuasi-estático. Esta es una información muy útil para los futuros diseñadores a la hora de decidir qué modelo de líneas de fondeo es el adecuado, dependiendo del tipo de plataforma y de los resultados esperados. El código dinámico de líneas de fondeo desarrollado en esta investigación se basa en el método de los Elementos Finitos, utilizando en concreto un modelo ”Lumped Mass” para aumentar su eficiencia de computación. Los experimentos realizados para la validación del código se realizaron en el tanque del École Céntrale de Nantes (ECN), en Francia, y consistieron en sumergir una cadena con uno de sus extremos anclados en el fondo del tanque y excitar el extremo suspendido con movimientos armónicos de diferentes periodos. El código demostró su capacidad para predecir la tensión y los movimientos en diferentes posiciones a lo largo de la longitud de la línea con gran precisión. Los resultados indicaron la importancia de capturar la dinámica de las líneas de fondeo para la predicción de la tensión especialmente en movimientos de alta frecuencia. Finalmente, el código se utilizó en una exhaustiva evaluación del efecto que la dinámica de las líneas de fondeo tiene sobre las cargas extremas y de fatiga de diferentes conceptos de aerogeneradores flotantes. Las cargas se calcularon para tres tipologías de aerogenerador flotante (semisumergible, ”spar-buoy” y ”tension leg platform”) y se compararon con las cargas obtenidas utilizando un modelo cuasi-estático de líneas de fondeo. Se lanzaron y postprocesaron más de 20.000 casos de carga definidos por la norma IEC 61400-3 siguiendo todos los requerimientos que una entidad certificadora requeriría a un diseñador industrial de aerogeneradores flotantes. Los resultados mostraron que el impacto de la dinámica de las líneas de fondeo, tanto en las cargas de fatiga como en las extremas, se incrementa conforme se consideran elementos situados más cerca de la plataforma: las cargas en la pala y en el eje sólo son ligeramente modificadas por la dinámica de las líneas, las cargas en la base de la torre pueden cambiar significativamente dependiendo del tipo de plataforma y, finalmente, la tensión en las líneas de fondeo depende fuertemente de la dinámica de las líneas, tanto en fatiga como en extremas, en todos los conceptos de plataforma que se han evaluado. ABSTRACT The load calculation of floating offshore wind turbine requires time-domain simulation tools taking into account all the phenomena that affect the system such as aerodynamics, structural dynamics, hydrodynamics, control actions and the mooring lines dynamics. These effects present couplings and are mutually influenced. The results provided by integrated simulation tools are used to compute the fatigue and ultimate loads needed for the structural design of the different components of the wind turbine. For this reason, their accuracy has an important influence on the optimization of the components and the final cost of the floating wind turbine. In particular, the mooring system greatly affects the global dynamics of the floater. Many integrated codes for the simulation of floating wind turbines use simplified approaches that do not consider the mooring line dynamics. An accurate simulation of the mooring system within the integrated codes can be fundamental to obtain reliable results of the system dynamics and the loads. The impact of taking into account the mooring line dynamics in the integrated simulation still has not been thoroughly quantified. The main objective of this research consists on the development of an accurate dynamic model for the simulation of mooring lines, validate it against wave tank tests and then integrate it in a simulation code for floating wind turbines. This experimentally validated tool is finally used to quantify the impact that dynamic mooring models have on the computation of fatigue and ultimate loads of floating wind turbines in comparison with quasi-static tools. This information will be very useful for future designers to decide which mooring model is adequate depending on the platform type and the expected results. The dynamic mooring lines code developed in this research is based in the Finite Element Method and is oriented to the achievement of a computationally efficient code, selecting a Lumped Mass approach. The experimental tests performed for the validation of the code were carried out at the `Ecole Centrale de Nantes (ECN) wave tank in France, consisting of a chain submerged into a water basin, anchored at the bottom of the basin, where the suspension point of the chain was excited with harmonic motions of different periods. The code showed its ability to predict the tension and the motions at several positions along the length of the line with high accuracy. The results demonstrated the importance of capturing the evolution of the mooring dynamics for the prediction of the line tension, especially for the high frequency motions. Finally, the code was used for an extensive assessment of the effect of mooring dynamics on the computation of fatigue and ultimate loads for different floating wind turbines. The loads were computed for three platforms topologies (semisubmersible, spar-buoy and tension leg platform) and compared with the loads provided using a quasi-static mooring model. More than 20,000 load cases were launched and postprocessed following the IEC 61400-3 guideline and fulfilling the conditions that a certification entity would require to an offshore wind turbine designer. The results showed that the impact of mooring dynamics in both fatigue and ultimate loads increases as elements located closer to the platform are evaluated; the blade and the shaft loads are only slightly modified by the mooring dynamics in all the platform designs, the tower base loads can be significantly affected depending on the platform concept and the mooring lines tension strongly depends on the lines dynamics both in fatigue and extreme loads in all the platform concepts evaluated.

3D Maize Plant Reconstruction Based on Georeferenced Overlapping LiDAR Point Clouds

3D crop reconstruction with a high temporal resolution and by the use of non-destructive measuring technologies can support the automation of plant phenotyping processes. Thereby, the availability of such 3D data can give valuable information about the plant development and the interaction of the plant genotype with the environment. This article presents a new methodology for georeferenced 3D reconstruction of maize plant structure. For this purpose a total station, an IMU, and several 2D LiDARs with different orientations were mounted on an autonomous vehicle. By the multistep methodology presented, based on the application of the ICP algorithm for point cloud fusion, it was possible to perform the georeferenced point clouds overlapping. The overlapping point cloud algorithm showed that the aerial points (corresponding mainly to plant parts) were reduced to 1.5%–9% of the total registered data. The remaining were redundant or ground points. Through the inclusion of different LiDAR point of views of the scene, a more realistic representation of the surrounding is obtained by the incorporation of new useful information but also of noise. The use of georeferenced 3D maize plant reconstruction at different growth stages, combined with the total station accuracy could be highly useful when performing precision agriculture at the crop plant level.

PÓS-GRADUAÇÃO, PRODUÇÃO ACADÊMICA, INTERDISCIPLINARIDADE E SOCIALIZAÇÃO DO CONHECIMENTO: A PRODUÇÃO CIENTÍFICA DO GRANDE ABC À LUZ DO PNPG 2011-2020

O objetivo desta investigação foi verificar se, diante da autonomia e vocação que possuem, bem como da possibilidade de receber amparo e incentivos governamentais, as universidades pertencentes à região do Grande ABC atenderam as recomendações feitas pelo PNPG vigente. Para tanto, foram analisadas cinquenta e sete dissertações e duas teses da área de ciências sociais aplicadas, publicadas no período entre 2011 e 2014, pelas seguintes instituições: Universidade Metodista de São Paulo (UMESP); Universidade Municipal de São Caetano do Sul (USCS); Universidade Federal do ABC (UFABC) e Faculdade de Engenharia Industrial (FEI). Essa averiguação se deu ao redor de dois eixos organizadores do PNPG 2011-2014: o terceiro eixo – o aperfeiçoamento da avaliação e sua expansão para outros segmentos do sistema de CT&I (formação de pós-graduados voltados para atividades extra-acadêmicas/setor empresarial) e o quarto eixo – a multi e a interdisciplinaridade entre as principais características da pós-graduação e importantes temas da pesquisa (promover, por meio de programas, áreas de concentração e linhas de pesquisa, a convergência de temas e compartilhamento de problemas em oposição à sua mera associação ou sobreposição). Este estudo – qualitativo, bibliográfico, documental, exploratório, descritivo, tipo estado do conhecimento – se desenvolveu por meio de pesquisa documental e de análise de conteúdo temático categorial. A coleta de dados foi feita por meio dos repositórios digitais de teses e dissertações mantidos na internet pelas Universidades, cuja produção científica foi investigada. Após a análise dos dados ficou demonstrado que aproximadamente 68,96% da produção científica da área de ciências sociais aplicadas, publicada pelas universidades do Grande ABC, no período entre 2011 e 2014, corresponde às expectativas do PNPG atual no que diz respeito às recomendações constantes no terceiro eixo. Em relação às recomendações feitas no texto do quarto eixo, vemos que aproximadamente 31,03% dos trabalhos selecionados atendem às expectativas do Plano, apresentando em sua estrutura, segundo a fundamentação teórica utilizada neste trabalho, características de interdisciplinaridade.

PÓS-GRADUAÇÃO, PRODUÇÃO ACADÊMICA, INTERDISCIPLINARIDADE E SOCIALIZAÇÃO DO CONHECIMENTO: A PRODUÇÃO CIENTÍFICA DO GRANDE ABC À LUZ DO PNPG 2011-2020

O objetivo desta investigação foi verificar se, diante da autonomia e vocação que possuem, bem como da possibilidade de receber amparo e incentivos governamentais, as universidades pertencentes à região do Grande ABC atenderam as recomendações feitas pelo PNPG vigente. Para tanto, foram analisadas cinquenta e sete dissertações e duas teses da área de ciências sociais aplicadas, publicadas no período entre 2011 e 2014, pelas seguintes instituições: Universidade Metodista de São Paulo (UMESP); Universidade Municipal de São Caetano do Sul (USCS); Universidade Federal do ABC (UFABC) e Faculdade de Engenharia Industrial (FEI). Essa averiguação se deu ao redor de dois eixos organizadores do PNPG 2011-2014: o terceiro eixo – o aperfeiçoamento da avaliação e sua expansão para outros segmentos do sistema de CT&I (formação de pós-graduados voltados para atividades extra-acadêmicas/setor empresarial) e o quarto eixo – a multi e a interdisciplinaridade entre as principais características da pós-graduação e importantes temas da pesquisa (promover, por meio de programas, áreas de concentração e linhas de pesquisa, a convergência de temas e compartilhamento de problemas em oposição à sua mera associação ou sobreposição). Este estudo – qualitativo, bibliográfico, documental, exploratório, descritivo, tipo estado do conhecimento – se desenvolveu por meio de pesquisa documental e de análise de conteúdo temático categorial. A coleta de dados foi feita por meio dos repositórios digitais de teses e dissertações mantidos na internet pelas Universidades, cuja produção científica foi investigada. Após a análise dos dados ficou demonstrado que aproximadamente 68,96% da produção científica da área de ciências sociais aplicadas, publicada pelas universidades do Grande ABC, no período entre 2011 e 2014, corresponde às expectativas do PNPG atual no que diz respeito às recomendações constantes no terceiro eixo. Em relação às recomendações feitas no texto do quarto eixo, vemos que aproximadamente 31,03% dos trabalhos selecionados atendem às expectativas do Plano, apresentando em sua estrutura, segundo a fundamentação teórica utilizada neste trabalho, características de interdisciplinaridade.

Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of ≈100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5–10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth.

A link density clustering algorithm based on automatically selecting density peaks for overlapping community detection

Peer reviewed

β-Catenin mutations in cell lines established from human colorectal cancers

β-catenin has functions as both an adhesion and a signaling molecule. Disruption of these functions through mutations of the β-catenin gene (CTNNB1) may be important in the development of colorectal tumors. We examined the entire coding sequence of β-catenin by reverse transcriptase–PCR (RT-PCR) and direct sequencing of 23 human colorectal cancer cell lines from 21 patients. In two cell lines, there was apparent instability of the β-catenin mRNA. Five different mutations (26%) were found in the remaining 21cell lines (from 19 patients). A three-base deletion (codon 45) was identified in the cell line HCT 116, whereas cell lines SW 48, HCA 46, CACO 2, and Colo 201 each contained single-base missense mutations (codons 33, 183, 245, and 287, respectively). All 23 cell lines had full-length β-catenin protein that was detectable by Western blotting and that coprecipitated with E-cadherin. In three of the cell lines with CTNNB1 mutations, complexes of β-catenin with α-catenin and APC were detectable. In SW48 and HCA 46, however, we did not detect complexes of β-catenin protein with α-catenin and APC, respectively. These results show that selection of CTNNB1 mutations occurs in up to 26% of colorectal cancers from which cell lines are derived. In these cases, mutation selection is probably for altered β-catenin function, which may significantly alter intracellular signaling and intercellular adhesion and may serve as a complement to APC mutations in the early stages of tumorigenesis.

Increased sensitivity to apoptotic stimuli in c-abl-deficient progenitor B-cell lines

The protooncogene c-abl encodes a nonreceptor tyrosine kinase whose cellular function is unknown. To study the possible involvement of c-Abl in proliferation, differentiation, and cell cycle regulation of early B cells, long-term lymphoid bone marrow cultures were established from c-abl-deficient mice and their wild-type littermates. Interleukin 7-dependent progenitor B-cell clones and lines expressing B220 and CD43 could be generated from both mutant and wild-type mice. The mutant and wild-type lines displayed no difference in their proliferative capacity as measured by thymidine incorporation in response to various concentrations of interleukin 7. Similarly, c-abl deficiency did not interfere with the ability of mutant clones to differentiate into surface IgM-positive cells in vitro. Analysis of cultures after growth factor deprivation, however, revealed a strikingly accelerated rate of cell death in c-abl mutant cells, due to apoptosis as confirmed by terminal deoxynucleotidyltransferase-mediated UTP nick end labeling analysis. Furthermore, a greater susceptibility to apoptotic cell death in c-abl mutant cells was also observed after glucocorticoid treatment. These results suggest that mutant c-Abl renders the B-cell progenitors more sensitive to apoptosis, and may account for some of the phenotypes observed in c-abl-deficient animals.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

Natural killer cell lines kill autologous β2-microglobulin-deficient melanoma cells: Implications for cancer immunotherapy

Cancer vaccines used to generate specific cytotoxic T lymphocytes are not effective against tumor cells that have lost or suppressed expression of their class I major histocompatibility complex proteins. This loss is common in some cancers and particularly in metastatic lesions. We show that β2-microglobulin-deficient class I-negative melanoma variants derived from patients undergoing specific T cell therapy are lysed by heterologous as well as autologous natural killer (NK) lines and clones, but not by specific T cells. Moreover, the minor NK cell fraction but not the major T cell fraction derived from heterologous lymphokine activated killer cells kills those tumor cell lines. ICAM-1 expression by the different class I protein deficient tumors was correlated with their sensitivity to lysis by NK cells. Adoptive autologous NK therapy may be an important supplement to consider in the design of new cancer immunotherapies.