110 research outputs found

    Identification and characterization of TNFalpha responsive genes in human breast cancer cells

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    One of the hallmarks of cancer is the escape of the transformed cells from apoptosis. Therefore, the identification of survival genes, allowing cancer cells to circumvent programmed cell death, could provide new diagnostic markers as well as targets for therapeutic intervention. A well known transcription factor regulating the balance between pro- and anti- apoptotic factors is NF-kappaB, which is strongly induced by tumor necrosis factor alpha (TNFalpha). When cells are stimulated by TNFalpha their response is biphasic with an initial NF-kappaB induction of survival genes which is overridden by the subsequent activation of initiator caspases triggering apoptosis. By combining gene trap mutagenesis with site specific recombination a strategy was developed, which enriches for genes induced by TNFalpha in the human breast cancer cell line MCF-7. The strategy relies on a one way gene expression switch based on Cre/loxP mediated recombination, which uncouples the expression of a marker gene from the trapped cellular promoter thereby enabling the recovery of genes that are only transiently induced by TNFalpha. The marker gene used in these experiments was a dominant negative variant of the TNFalpha-receptor associated protein FADD (dnFADD), which blocks the apoptotic branch of the TNFalpha induced signaling pathway. Initial experiments indicated that MCF-7 cells expressing high levels of dnFADD were insensitive to TNFalpha induced apoptosis and therefore suitable for the installment of a one way gene expression switch susceptible to Cre/loxP mediated recombination. A MCF-7 reporter clone harboring the recombinase dependent gene expression switch was infected with the gene trap retrovirus U3Cre, which inserts the Cre recombinase gene into a large collection of chromosomal sites. Insertion of Cre downstream of an active cellular promoter induces dnFADD expression from the gene expression switch enabling the cells to block TNFalpha triggered apoptosis. From a gene trap integration library containing approximately 2000000 unique proviral integrations, 69 unique TNFalpha inducible gene trap insertion sites were recovered in a two step selection procedure. Sequencing of the genomic regions adjacent to the insertion sites, which were obtained by inverse PCR (gene trap sequence tags, GTSTs), and data base analysis revealed that 42% of the GTSTs belonged to annotated genes, 13% to known cDNAs with open reading frames, 17% to Genscan predicted genes, 9% to ESTs, 9% to repetitive sequences and 10% to unannotated genomic sequence. Overall, 44% of the annotated genes recovered in this screen were directly or indirectly related to cancer, indicating that the gene trap strategy developed here is suitable for the identification of cancer relevant genes. Analysis of the expression patterns of the trapped and annotated genes in wild type cells revealed that 19 out of 24 genes were either up- or down- regulated by a factor of at least 1.45 by TNFalpha. A large fraction of the gene trap insertions were located upstream, in introns or in opposite orientation to annotated transcripts, indicating that the strategy efficiently recovers non-coding RNAs (ncRNAs). While the biological significance of these transcripts still needs to be elucidated, they fall into two main categories. The first category includes gene trap insertions upstream of genes, which could either represent regulatory RNAs interacting with promoter elements or transcripts driven by bidirectional promoters. The second includes inverse orientation gene trap insertions in introns of annotated genes suggesting the presence of natural antisense transcripts (NATs). Interestingly, more than 50% of all antisense integrations are located downstream of transcription start sites predicted by different algorithms supporting the existence of RNAs transcribed from the corresponding genomic regions. Intronic integrations on the coding strand could be derived from cryptic splicing, alternative promoter usage or additional, so far uncharacterized transcripts. Preliminary functional analysis of two genes recovered in this screen encoding the transcription factor ZFP67 and the FLJ14451 protein revealed that FLJ14451 but not ZFP67 inhibited anchorage independent growth in soft agar, suggesting that FLJ14451 might have some tumor suppressor functions. In summary, besides identifying a putative tumor suppressor protein, the present experiments have shown that gene trapping is useful in identifying non-coding transcripts in living cells and may turn out to be the method of choice in characterizing these transcripts whose functions are still largely unknown.Ein zentrales Charakteristikum von Krebs ist die Unterdrückung des zellulären Apoptoseprogramms in den transformierten Zellen. Daher könnte die Identifizierung von Überlebensgenen, welche den Zellen die Umgehung des programmierten Zelltods erlauben, neue diagnostische Marker oder Zielmoleküle für therapeutische Intervention liefern. Die Balance zwischen pro- und anti-apoptotischen Signalen wird durch NF-kappaB, einen gut charakterisierter Transkriptionsfaktor reguliert, welcher wiederum durch Tumornekrosefaktor alpha (TNFalpha) induziert wird. Zellen, die mit TNFalpha stimuliert werden zeigen eine biphasische Antwort, während derer es zu einer initialen Induktion von Überlebensgenen kommt, bevor im folgenden Initiator-Caspasen aktiviert werden, die dann die Apoptose einleiten. Zur Identifizierung TNFalpha induzierbarer Gene in der menschlichen Brustkrebszellinie MCF-7 wurde eine Strategie benutzt, welche auf einer Kombination von Genfallen-Mutagenese und sequenzspezifischer Rekombination beruht. Diese Strategie macht sich einen irreversiblen molekularen Schalter zunutze, der die Expression eines Markergens von dem, die Genfalle aktivierenden, zellulären Promotor entkoppelt. Als Markergen wurde eine dominant negative Variante des TNFalpha-Rezeptor-assozierten Proteins FADD ("Fas-associated death domain protein"; dnFADD) benutzt, die den pro-apoptotische Zweig des TNFalpha-Rezeptor-Signalwegs blockiert. Ein MCF-7 Zellklon mit diesem Rekombinase-aktivierbaren, molekularen Schalter wurde mit dem Genfallen-Retrovirus U3Cre transduziert, welcher das Cre-Rekombinase-Gen in eine große Anzahl unterschiedlicher chromosomaler Loci inserieren kann. Kommt Cre dabei unter die Kontrolle eines aktiven zellulären Promoters, wird die Expression von dnFADD induziert und somit die Apoptose-Induktion durch TNFalpha verhindert. Aus einer Genfallen-Integrationsbank mit ca. 2000000 unabhängigen proviralen Integrationen wurden nach einer zweistufigen Selektion 69 Zellklone mit Genfallen-Integrationen in TNFalpha induzierten Genen erhalten. Die Sequenzierung der benachbart zu den Genfallen-Proviren liegenden, über inverse PCRs amplifizierten, genomischen Regionen und anschließende Datenbankanalysen ergaben folgende Verteilung der Genfallen-Integrationen: 42% lagen in annotierten Genen, 13% in Genen mit offenen Leserahmen unbekannter Funktion, 17% in hypothetischen Genen, 9% in ESTs ("expressed sequence tags"), 9% in repetitiven Elementen und 10% in nicht annotierten genomischen Regionen. 44% der aus diesem Screening-Verfahren erhaltenen, annotierten Gene ließen sich direkt oder indirekt mit Krebserkrankungen korrelieren. Dies ist ein Indiz dafür, dass der hier entwickelte, experimentelle Ansatz zur Identifizierung Krebs-relevanter Gene geeignet ist. Die Expressionanalyse ausgewählter, annotierter Gene in wildtypischen MCF-7 Zellen ergab eine Herauf- bzw. Herunterregulation um einen Faktor von mindestens 1.45 bei 19 von 24 untersuchten Genen. Ein großer Teil der Genfallen-Integrationen befand sich 5'-oberhalb von Genen, in Introns oder in umgekehrter Orientierung zu annotierten Transkripten, was darauf hindeutet, dass mit Hilfe der gewählten Strategie nicht-kodierende RNAs identifiziert werden können. Obwohl der Nachweis dieser Transkripte und ihrer biologischen Relevanz noch aussteht, können sie in zwei Kategorien eingeteilt werden. Die erste umfasst Integrationen 5' zu Genen, die entweder regulatorische RNAs repräsentieren, die mit Promotorelementen interagieren, oder Transkripte, welche unter der Kontrolle von bidirektionalen Promotoren stehen. Die zweite Kategorie, Insertionen auf dem nicht-kodierenden Strang innerhalb von Introns, legen das Vorkommen natürlicher antisense Transkripte (NATs) nahe. Interessanterweise liegen mehr als 50% aller antisense-Integrationen 3' zu potentiellen Transkriptionsstartstellen, die mit verschiedenen Algorithmen vorausgesagt wurden. Dies kann als Hinweis darauf gewertet werden, dass höchstwahrscheinlich RNAs existieren, die Transkripte der entsprechenden genomischen Regionen darstellen. Intronische Integrationen auf dem kodierenden Strang können als Resultat kryptischer Spleißvorgänge oder durch alternative Promotoren transkriptionell aktiviert werden; alternativ könnten es sich bei den durch die Genfalle "abgefangenen" RNAs um zusätzliche, bisher nicht charakterisierte Transkripte handeln. Vorläufige funktionelle Analysen zweier Gene, die für den Tanskriptionsfaktor ZFP67 und das Protein FLJ14451 kodieren, ergaben, dass FLJ14451, aber nicht ZFP67 das Substrat-unabhängige Wachstum von MCF-7 Zellen in Weichagar inhibieren kann. Dies weist auf eine mögliche Tumorsuppresorfunktion des Proteins hin. Die im Rahmen dieser Dissertation durchgeführten Experimente führten nicht nur zur Identifizierung eines potentiellen, neuen Tumorsuppressorgens, sondern zeigten auch, dass Genfallen ein nützliches Werkzeug bei der Suche nach nicht-kodierenden RNAs in lebenden Zellen sein können und ihr Einsatz möglicherweise die Methode der Wahl für die Identifizierung derartiger Transkripte darstellt

    Study of the turbocharger shaft motion by means of infrared sensors

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    This work describes a technique for measuring the precession movement of the shaft of small automotive turbochargers. The main novelty is that the technique is based on infrared light diode sensors. With presented technique it is possible to perform secure mounting of electronics and also to measure, with good accuracy, far enough from the turbocharger shaft. Both advantages allow applying it even in critical lubrication conditions and when blade contact occurs. The technique's main difficulties arise from the small size of the turbocharger shaft and the high precession movement in critical conditions. In order to generate the optimum albedo reflection for infrared measurement, a special cylindrical nut with larger diameter than the original one is assembled at the shaft tip in the compressor side. Following, shaft balancing, the calibration of the sensors and the compensation of errors from different sources are needed steps before the method is able to identify the main frequencies of shaft motion. Once synchronous and sub-synchronous frequencies have been obtained it is possible to reconstruct the instantaneous position of the shaft to determine its precession movement.This research has also been partially supported by the Programa de Desarrollo del Talento Humano de la Secretaria Nacional de Educacion Superior, Ciencia, Tecnologia e Innovacion del Gobierno Ecuatoriano No. 20100289.Serrano Cruz, JR.; Guardiola, C.; Dolz García, VM.; López, M.; Bouffaud, F. (2015). Study of the turbocharger shaft motion by means of infrared sensors. Mechanical Systems and Signal Processing. 56-57:246-258. https://doi.org/10.1016/j.ymssp.2014.11.006S24625856-5

    Clustering Groundwater Level Time Series of the Exploited Almonte-Marismas Aquifer in Southwest Spain

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    Groundwater resources are regularly the principal water supply in semiarid and arid climate areas. However, groundwater levels (GWL) in semiarid aquifers are suffering a general decrease because of anthropic exploitation of aquifers and the repercussions of climate change. Effective groundwater management strategies require a deep characterization of GWL fluctuations, in order to identify individual behaviors and triggering factors. In September 2019, the Guadalquivir River Basin Authority (CHG) declared that there was over-exploitation in three of the five groundwater bodies of the Almonte-Marismas aquifer, Southwest Spain. For that reason, it is critical to understand GWL dynamics in this aquifer before the new Spanish Water Resources Management Plans (2021–2027) are developed. The application of GWL series clustering in hydrogeology has grown over the past few years, as it is an extraordinary tool that promptly provides a GWL classification; each group can be related to different responses of a complex aquifer under any external change. In this work, GWL time series from 160 piezometers were analyzed for the period 1975 to 2016 and, after data pre-processing, 24 piezometers were selected for clustering with k-means (static) and time series (dynamic) clustering techniques. Six and seven groups (k) were chosen to apply k-means. Six characterized types of hydrodynamic behaviors were obtained with time series clustering (TSC). Number of clusters were related to diverse affections of water exploitation depending on soil uses and hydrogeological spatial distribution parameters. TSC enabled us to distinguish local areas with high hydrodynamic disturbance and to highlight a quantitative drop of GWL during the studied period

    Olive Pomace and Soybean-Sunflower Acid Oils as Alternative Fat Sources in European Seabass ( Dicentrarchus labrax) Diets: Effects on Performance, Digestibility and Flesh Fatty Acid Composition and Quality Parameters

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    The effects of dietary inclusion of soybean-sunflower and olive pomace acid oils on growth, digestibility and flesh composition were studied in European seabass. Eight diets were fed for 100 days (101.37 ± 0.33 g initial weight, mean ± SD), differing in the added fat source (25% fish oil, 75% experimental oil): S (crude soybean oil), SA (soybean-sunflower acid oil), O (crude olive pomace oil) or OA (olive pomace acid oil); 3 blends: S-O, S-OA, SA-OA at a 1:1 ratio; and a diet containing only fish oil (F) as a control. Animals fed OA showed the worst performance among dietary treatments, with the lowest weight, specific growth ratio, average daily gain and the highest feed conversion ratio (p 0.05), but a lower digestibility of lipids and saturated fatty acids was observed (p 0.05). Hence the results suggest that the studied acid oils may potentially be used in fish diets although further studies are needed. Keywords: acid oil; alternative energy source; dietary fat; fat by-product; fish nutrition; flesh quality; free fatty acid

    Olive Pomace and Soybean-Sunflower Acid Oils as Alternative Fat Sources in European Seabass (Dicentrarchus labrax) Diets : effects on Performance, Digestibility and Flesh Fatty Acid Composition and Quality Parameters

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    Altres ajuts: ERDFThe effects of dietary inclusion of soybean-sunflower and olive pomace acid oils on growth, digestibility and flesh composition were studied in European seabass. Eight diets were fed for 100 days (101.37 ± 0.33 g initial weight, mean ± SD), differing in the added fat source (25% fish oil, 75% experimental oil): S (crude soybean oil), SA (soybean-sunflower acid oil), O (crude olive pomace oil) or OA (olive pomace acid oil); 3 blends: S-O, S-OA, SA-OA at a 1:1 ratio; and a diet containing only fish oil (F) as a control. Animals fed OA showed the worst performance among dietary treatments, with the lowest weight, specific growth ratio, average daily gain and the highest feed conversion ratio (p 0.05), but a lower digestibility of lipids and saturated fatty acids was observed (p 0.05). Hence the results suggest that the studied acid oils may potentially be used in fish diets although further studies are needed

    Selecting Suitable MODFLOW Packages to Model Pond–Groundwater Relations Using a Regional Model

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    In large-scale regional models, used for the management of underground resources, it is quite common to find that relationships between the regional aquifer and small wetlands are not included. These models do not consider this connection because of the small amount of water involved, but they should consider the potential for significant ecological impacts if the groundwater resources in the ecosystems associated with these wetlands are mismanaged. The main objective of this work is to investigate the possibilities offered by MODFLOW LGR-V2 to represent (at small scale) the Santa Olalla pond, located in the Doñana Natural Park (South of Spain), and its relationship with the Almonte-Marismas regional aquifer. As a secondary objective, we propose to investigate the advantages and disadvantages that DRAIN, RIVER and LAKE MODFLOW packages offer within the MODFLOW LGR-V2 discretizations. The drain boundary condition with a coarse discretization implemented through ModelMuse allows the most adequate performance of the groundwater levels in the environment of the pond. However, when using lake boundary condition, the use of the MODFLOW LGR-V2 version is particularly useful. The present work also gives some guidelines to employ these packages with the MODFLOW graphical user’s interface, ModelMuse 4.2

    Creating a safe operating space for wetlands in a changing climate

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    Many of the world’s wetlands may be profoundly affected by climate change over the coming decades. Although wetland managers may have little control over the causes of climate change, they can help to counteract its effects through local measures. This is because direct anthropogenic impacts, such as water extraction and nutrient loading, work in concert with climate change to damage wetlands. Control of these local stressors may therefore ameliorate undesired effects of climate change, such as a shift towards dominance by invasive floating plants, increasingly frequent cyanobacteria blooms, or extinction of key species. Using the iconic Doñana wetlands in Spain as a case study, we illustrate how the concept of creating a “safe operating space” may be implemented to better ensure that ecosystems do not surpass thresholds for collapse during an era of global change.WIMEK grant for a research stay at WUR. JAE DOCTORES. European Union. European Social Fund. Grant Number: ESF2007‐2013. Spanish Ministry for Economy and Competitiveness. EU FP7 project FAST. Grant Number: 607131. European Union's Horizon 2020 research and innovation program. Grant Number: 641762Peer reviewe

    An on-engine method for dynamic characterisation of NOx concentration sensors

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    An on-engine method for dynamic characterisation of automotive NOx concentration sensors is presented. Steps in start of injection on a diesel engine are employed to achieve step-like NOx concentration variations on exhaust flow. On the basis of the sensor response, delay and dynamic response can be easily identified; the paper shows a simple least squares procedure although other models and identification techniques could be used. Application data is presented for three NOx sensors: a research-grade chemiluminescence exhaust gas analyser, and two different commercial ZrO2-based sensors. © 2010 Elsevier Inc.The authors thanks R. Lujan and G. Couture for their valuable contribution in the experimental part of the present work. This work has been partially supported by Ministerio de Ciencia y Tecnologia through Project PLANUCO No. TRA2006-15620-C02-02.Galindo, J.; Serrano Cruz, JR.; Guardiola, C.; Blanco-Rodriguez, D.; Cuadrado, I. (2011). An on-engine method for dynamic characterisation of NOx concentration sensors. Experimental Thermal and Fluid Science. 35(3):470-476. https://doi.org/10.1016/j.expthermflusci.2010.11.010S47047635

    Semiempirical in-cylinder pressure based model for NOx prediction oriented to control applications

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    This work describes the development of a fast NO X predictive model oriented to engine control in diesel engines. The in-cylinder pressure is the only instantaneous input signal required, along with several mean variables that are available in the ECU during normal engine operation. The proposed model is based on the instantaneous evolution of the heat release rate and the adiabatic flame temperature (both obtained among other parameters from the in-cylinder pressure evolution). Corrections for considering the NO X reduction due to the re-burning mechanism are also included. Finally, the model is used for providing a model-based correction of tabulated values for the NO X emission at the reference conditions. The model exhibits a good behaviour when varying exhaust gas recirculation rate, boost pressure and intake temperature, while changes in the engine speed and injection settings are considered in the tabulated values. Concerning the calculation time, the model is optimised by proposing simplified sub-models to calculate the heat release and the adiabatic flame temperature. The final result is suitable for real time applications since it takes less than a cycle to complete the NO X prediction.Guardiola García, C.; López Sánchez, JJ.; Martín Díaz, J.; García Sarmiento, D. (2011). Semiempirical in-cylinder pressure based model for NOx prediction oriented to control applications. Applied Thermal Engineering. 31(16):3275-3286. doi:10.1016/j.applthermaleng.2011.05.048S32753286311

    Análisis de la actividad laboral de los profesionales sanitarios en formación de la Región de Murcia ante la pandemia por COVID-19

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    The present study intends to analyze the situation of professionals in training in the Murcian Health Service (SMS) during the state of sanitary emergency in Spain (BOE Order SND/232/2020) due to the pandemic of the new coronavirus 2019-nCoV. To achieve this objective, a descriptive study has been carried out through the collection of data via direct form, channeled through the network that makes up the Comité de Empresa. A response from at least one resident of each of the services that make up the SMS has been obtained. This has allowed a sample size that represents a large amount of the group under study. This is a study that stands out for the rapid acquisition and analysis of the sample, which serves as an important tool to condition future policies and decisions in the field of professionals in training. The lack of access to protection material for more than half of the population under study, as well as difficulties in accessing updated information from the administration are some of the results obtained after analyzing the responses. Overall, there is a majority of residents that continue with their work assistance and which maintain the same number of 24-hour shifts. It is also a main opinion that there is an appropriate health training and contingency plan against COVID-19.El presente estudio pretende analizar, ante el estado de emergencia sanitaria española (BOE Orden SND/232/2020) decretado por la pandemia del nuevo coronavirus 2019-nCoV, la situación en la que se encuentran los profesionales en formación del Servicio Murciano de Salud (SMS). Para ello se ha realizado un estudio descriptivo llevado a cabo a través de la recogida de datos mediante formulario directo, canalizado a través de la red que conforma el Comité de Empresa, habiéndose obtenido respuesta por parte de al menos un residente de cada uno de los servicios que conforman el SMS y alcanzando un tamaño de muestra que permite la total representatividad del colectivo a estudio. Se trata de un estudio que destaca por la rapidez de reclutamiento de la muestra y la rapidez en su redacción, lo que sirve como una importante herramienta para condicionar futuras políticas y decisiones en el ámbito de los profesionales en formación. La falta de acceso a material de protección por más de la mitad de la población a estudio junto con dificultades para el acceso a información actualizada por parte de la administración, son algunos de los resultados obtenidos tras el análisis de las respuestas. En general, existe una mayoría de residentes que continúan con su labor asistencial, que mantienen el número de guardias mensuales, que piensa que recibe formación oficial adecuada y que se está realizando un plan de contingencia apropiado
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