935 resultados para Three-hybrid system
Resumo:
An efficient high-resolution (HR) three-dimensional (3D) seismic reflection system for small-scale targets in lacustrine settings was developed. In Lake Geneva, near the city of Lausanne, Switzerland, the offshore extension of a complex fault zone well mapped on land was chosen for testing our system. A preliminary two-dimensional seismic survey indicated structures that include a thin (<40 m) layer of subhorizontal Quaternary sediments that unconformably overlie south-east-dipping Tertiary Molasse beds and a major fault zone (Paudeze Fault Zone) that separates Plateau and Subalpine Molasse (SM) units. A 3D survey was conducted over this test site using a newly developed three-streamer system. It provided high-quality data with a penetration to depths of 300 m below the water bottom of non-aliased signal for dips up to 30degrees and with a maximum vertical resolution of 1.1 m. The data were subjected to a conventional 3D processing sequence that included post-stack time migration. Tests with 3D pre-stack depth migration showed that such techniques can be applied to HR seismic surveys. Delineation of several horizons and fault surfaces reveals the potential for small-scale geologic and tectonic interpretation in three dimensions. Five major seismic facies and their detailed 3D geometries can be distinguished. Three fault surfaces and the top of a molasse surface were mapped in 3D. Analysis of the geometry of these surfaces and their relative orientation suggests that pre-existing structures within the Plateau Molasse (PM) unit influenced later faulting between the Plateau and SM. In particular, a change in strike of the PM bed dip may indicate a fold formed by a regional stress regime, the orientation of which was different from the one responsible for the creation of the Paudeze Fault Zone. This structure might have later influenced the local stress regime and caused the curved shape of the Paudeze Fault in our surveyed area.
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When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.
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The phytochrome family of red/far-red (R/FR)-responsive photoreceptors plays a key role throughout the life cycle of plants . Arabidopsis has five phytochromes, phyA-phyE, among which phyA and phyB play the most predominant functions . Light-regulated nuclear accumulation of the phytochromes is an important regulatory step of this pathway, but to this date no factor specifically required for this event has been identified . Among all phyA signaling mutants, fhy1 and fhy3 (far-red elongated hypocotyl 1 and 3) have the most severe hyposensitive phenotype, indicating that they play particularly important roles . FHY1 is a small plant-specific protein of unknown function localized both in the nucleus and the cytoplasm . Here we show that FHY1 is specifically required for the light-regulated nuclear accumulation of phyA but not phyB. Moreover, phyA accumulation is only slightly affected in fhy3, indicating that the diminished nuclear accumulation of phyA observed in fhy1 seedlings is not simply a general consequence of reduced phyA signaling. By in vitro pull-down and yeast two-hybrid analyses, we demonstrate that FHY1 physically interacts with phyA, preferentially in its active Pfr form. Furthermore, FHY1 and phyA colocalize in planta. We therefore identify the first component required for light-regulated phytochrome nuclear accumulation.
Resumo:
Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the alpha1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The alpha1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected alpha1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the alpha1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired alpha1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (DeltaR8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G protein-coupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.
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Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.
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hShroom1 (hShrm1) is a member of the Apx/Shroom (Shrm) protein family and was identified from a yeast two-hybrid screen as a protein that interacts with the cytoplasmic domain of melanoma cell adhesion molecule (MCAM). The characteristic signature of the Shrm family is the presence of a unique domain, ASD2 (Apx/Shroom domain 2). mRNA analysis suggests that hShrm1 is expressed in brain, heart, skeletal muscle, colon, small intestine, kidney, placenta and lung tissue, as well a variety of melanoma and other cell lines. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments indicate that hShrm1 and MCAM interact in vivo and by immunofluorescence microscopy some co-localization of these proteins is observed. hShrm1 partly co-localises with beta-actin and is found in the Triton X-100 insoluble fraction of melanoma cell extracts. We propose that hShrm1 is involved in linking MCAM to the cytoskeleton.
Resumo:
Tant en la meva etapa d’estudiant com en la meva curta vida laboral, he participat en el desenvolupament de varies aplicacions de gestió, tant d’escriptori com web, que majoritàriament sempre estaven dividides en tres capes però en dos nivells: la base de dades i l’aplicació, que contenia tant la lògica de negoci com la vista. Des de fa temps he tingut la curiositat i la necessitat d’intentar desenvolupar una aplicació de gestió que estigui distribuïda en tres o més nivells, separant el model de dades i la lògica de negoci de la vista. Crec que el treball de fi de carrera és idoni per desenvolupar-la. L’aplicació a desenvolupar anirà destinada a la gestió d’un servei d’informàtica, que s’encarrega de resoldre les incidències, inventariar i realitzar el manteniment preventiu dels equips informàtics d’una o de varies empreses. Es crearà una aplicació que contindrà el model de dades i la lògica de negoci. També es crearan dues aplicacions clients: una aplicació d’escriptori que donarà accés a totes i cadascuna de les funcionalitats del sistema i una aplicació web que oferirà un accés lleuger i des de qualsevol ubicació a les funcionalitats d’explotació del sistema. Per últim, també es crearà una altre aplicació web que s’encarregarà de generar els llistats sol•licitats per les dues aplicacions clients. D’aquesta manera també es centralitzarà la gestió dels llistats de les aplicacions del sistema. Per desenvolupar el sistema, s’utilitzarà la plataforma J2EE de Java, i es comptarà amb l’ajuda de marcs de treball com, per exemple, Oracle ADF o JasperReports. Com a conclusió general, puc assegurar que si es distribueixen les tres capes d’un sistema en tres nivells s’obtenen beneficis en l’administració, en el manteniment, en l’escalabilitat, en la flexibilitat i en la reutilització de les aplicacions. El principal inconvenient és el possible augment de lentitud en les aplicacions clients.
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Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.
Resumo:
This line of research of my group intends to establish a Silicon technological platform in the field of photonics allowing the development of a wide set of applications. Particularly, what is still lacking in Silicon Photonics is an efficient and integrable light source such an LED or laser. Nanocrystals in silicon oxide or nitride matrices have been recently demonstrated as competitive materials for both active components (electrically and optically driven light emitters and optical amplifiers) and passive ones (waveguides and modulators). The final goal is the achievement of a complete integration of electronic and optical functions in the same CMOS chip. The first part of this paper will introduce the structural and optical properties of LEDs fabricated from silicon nanostructures. The second will treat the interaction of such nanocrystals with rare-earth elements (Er), which lead to an efficient hybrid system emitting in the third window of optical fibers. I will present the fabrication and assessment of optical waveguide amplifiers at 1.54 ¿m for which we have been able to demonstrate recently optical gain in waveguides made from sputtered silicon suboxide materials.
Resumo:
La liberalización del transporte aéreo que se llevó a término en la Unión Europea a principios de los años noventa ha tenido efectos positivos sobre el bienestar del viajero. No obstante, existe un consenso en la literatura académica que estos efectos dependen de la existencia de una competencia efectiva en el nivel de la ruta. En este sentido, se plantea el problema que puede llegar a suponer las ventajas de escalera de las compañías dominantes en cada mercado interior. Además, se pretende capturar la diferenciación de productos como característica esencial de la industria del transporte aéreo. El análisis de estas cuestiones se realiza de la forma siguiente. En primer lugar, se hace referencia a los principales aspectos económicos que condicionan la competencia en el transporte aéreo. Y en segundo lugar, se implementa un modelo empírico basado en un sistema de tres ecuaciones, que se estima mediante la técnica de las variables instrumentales. La muestra utilizada hace referencia al año 2001 para la mayoría de las rutas del mercado interior español de vuelos regulares en dónde hay competencia. Los resultados de la estimación muestran la existencia de unas condiciones de competencia diferentes según el segmento del mercado al cual se dirigen las compañías aéreas. Efectivamente, la competencia en precios (calidad) parece ser predominante en el segmento de viajeros por motivos personales (negocios). Adicionalmente, el dominio que la compañía dominante tiene sobre la mayoría de las rutas parece descansar en las ventajas competitivas, tanto en términos de costes como en términos de demanda, que le proporciona el control de la red aeroportuaria nacional. De todo esto se puede inferir que el mantenimiento y/o aumento de los beneficios de la liberalización de los servicios de transporte aéreo exige extender la liberalización al uso del aeropuertos así como descentralizar su gestión.
Resumo:
AKAP-Lbc is a member of the A-kinase anchoring protein (AKAP) family that has been recently associated with the development of pathologies, such as cardiac hypertrophy and cancer. We have previously demonstrated that, at the molecular level, AKAP-Lbc functions as a guanine nucleotide exchange factor (GEF) that promotes the specific activation of RhoA. In the present study, we identified the ubiquitin-like protein LC3 as a novel regulatory protein interacting with AKAP-Lbc. Mutagenesis studies revealed that LC3, through its NH(2)-terminal alpha-helical domain, interacts with two binding sites located within the NH(2)-terminal regulatory region of AKAP-Lbc. Interestingly, LC3 overexpression strongly reduced the ability of AKAP-Lbc to interact with RhoA, profoundly impairing the Rho-GEF activity of the anchoring protein and, as a consequence, its ability to promote cytoskeletal rearrangements associated with the formation of actin stress fibers. Moreover, AKAP-Lbc mutants that fail to interact with LC3 show a higher basal Rho-GEF activity as compared with the wild type protein and become refractory to the inhibitory effect of LC3. This suggests that LC3 binding maintains AKAP-Lbc in an inactive state that displays a reduced ability to promote downstream signaling. Collectively, these findings provide evidence for a previously uncharacterized role of LC3 in the regulation of Rho signaling and in the reorganization of the actin cytoskeleton.
Resumo:
Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor alpha. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARgamma but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARgamma in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.
Resumo:
La liberalización del transporte aéreo que se llevó a término en la Unión Europea a principios de los años noventa ha tenido efectos positivos sobre el bienestar del viajero. No obstante, existe un consenso en la literatura académica que estos efectos dependen de la existencia de una competencia efectiva en el nivel de la ruta. En este sentido, se plantea el problema que puede llegar a suponer las ventajas de escalera de las compañías dominantes en cada mercado interior. Además, se pretende capturar la diferenciación de productos como característica esencial de la industria del transporte aéreo. El análisis de estas cuestiones se realiza de la forma siguiente. En primer lugar, se hace referencia a los principales aspectos económicos que condicionan la competencia en el transporte aéreo. Y en segundo lugar, se implementa un modelo empírico basado en un sistema de tres ecuaciones, que se estima mediante la técnica de las variables instrumentales. La muestra utilizada hace referencia al año 2001 para la mayoría de las rutas del mercado interior español de vuelos regulares en dónde hay competencia. Los resultados de la estimación muestran la existencia de unas condiciones de competencia diferentes según el segmento del mercado al cual se dirigen las compañías aéreas. Efectivamente, la competencia en precios (calidad) parece ser predominante en el segmento de viajeros por motivos personales (negocios). Adicionalmente, el dominio que la compañía dominante tiene sobre la mayoría de las rutas parece descansar en las ventajas competitivas, tanto en términos de costes como en términos de demanda, que le proporciona el control de la red aeroportuaria nacional. De todo esto se puede inferir que el mantenimiento y/o aumento de los beneficios de la liberalización de los servicios de transporte aéreo exige extender la liberalización al uso del aeropuertos así como descentralizar su gestión.
Resumo:
Polynomial constraint solving plays a prominent role in several areas of hardware and software analysis and verification, e.g., termination proving, program invariant generation and hybrid system verification, to name a few. In this paper we propose a new method for solving non-linear constraints based on encoding the problem into an SMT problem considering only linear arithmetic. Unlike other existing methods, our method focuses on proving satisfiability of the constraints rather than on proving unsatisfiability, which is more relevant in several applications as we illustrate with several examples. Nevertheless, we also present new techniques based on the analysis of unsatisfiable cores that allow one to efficiently prove unsatisfiability too for a broad class of problems. The power of our approach is demonstrated by means of extensive experiments comparing our prototype with state-of-the-art tools on benchmarks taken both from the academic and the industrial world.
Resumo:
This paper addresses the application of a PCA analysis on categorical data prior to diagnose a patients data set using a Case-Based Reasoning (CBR) system. The particularity is that the standard PCA techniques are designed to deal with numerical attributes, but our medical data set contains many categorical data and alternative methods as RS-PCA are required. Thus, we propose to hybridize RS-PCA (Regular Simplex PCA) and a simple CBR. Results show how the hybrid system produces similar results when diagnosing a medical data set, that the ones obtained when using the original attributes. These results are quite promising since they allow to diagnose with less computation effort and memory storage