961 resultados para siRNA interference


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Report for the scientific sojourn at the Department of Information Technology (INTEC) at the Ghent University, Belgium, from january to june 2007. All-Optical Label Swapping (AOLS) forms a key technology towards the implementation of All-Optical Packet Switching nodes (AOPS) for the future optical Internet. The capital expenditures of the deployment of AOLS increases with the size of the label spaces (i.e. the number of used labels), since a special optical device is needed for each recognized label on every node. Label space sizes are affected by the wayin which demands are routed. For instance, while shortest-path routing leads to the usage of fewer labels but high link utilization, minimum interference routing leads to the opposite. This project studies and proposes All-Optical Label Stacking (AOLStack), which is an extension of the AOLS architecture. AOLStack aims at reducing label spaces while easing the compromise with link utilization. In this project, an Integer Lineal Program is proposed with the objective of analyzing the softening of the aforementioned trade-off due to AOLStack. Furthermore, a heuristic aiming at finding good solutions in polynomial-time is proposed as well. Simulation results show that AOLStack either a) reduces the label spaces with a low increase in the link utilization or, similarly, b) uses better the residual bandwidth to decrease the number of labels even more.

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In the course of their coevolution with insects, plants have learnt to protect themselves by chemical means. Semiochemical act as antifeedants or deterrents, others by disrupting growth and development. By use of the Epilachna varivestis bioassay we isolated from Azadirachta indica seed a group of triterpenoids which interfee with larval growth and development in ppm range. Main components are the azadirachtins A and B with identical biological activity. Various other azadirachtins were obtained, either as minor seed components or by chemical modification of the naturally occuring compounds. Structure vs. activity relation studies enabled us to postulate a basic structural element that should still be biologically active and with much simpler chemical structure than natural compounds. What underlies the biological activity of these insect growth inhibitors? Their interference with the hormonal regulation of development and reproduction has been studied in Locusta migratoria and Rhodnius prolixus. In addition, tritiated dihydroazadirachtin A was used. With this approach, a precise correlation between administered dose, resulting effects, and retention of the compound was established. The azadirachtins either interrupt, delay, or deviate whole developmental programs. Results from these studies provide another chemical probe for studies in insect endocrinology and physiology.

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In Egypt the "national schistosomiasis control program" was formulated to control transmission by reduction of prevalence and intensity of current infections, and thereby achieve an acceptable level of schistosomiasis disease control. The program was implemented foremost in Middle Egypt (1977) and Upper Egypt (1980), collectively extending 800 km alongside of the River Nile and accommodate about 10.5 million people. Schistosoma haematobium has been essentially the prevailing species infection in both areas. The strategy of control entailed both area-wide mollusciciding with niclosamide, and selective population chemotherapy with metrifonate. Evaluation in 1986 showed that prevalence dropped from pre-control 29.4% in Middle Egypt and 26.3% in Upper Egypt to 6% and 7.8% respectively, together with a remarkable drop of infections among children. Also mean intensity attained low levels consistent of low grade infections. It is evident therefore that in these areas where an enhancement of schistosomiasis infections had been anticipated the employment of the twofold strategy effected a state of low-prevalence/low-intensity signifying a lowered reservoir of infection and a substantial interference with the potentials of transmission.

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L'ubiquitination est une modification des protéines conservée, consistant en l'addition de résidus « ubiquitine » et régulant le destin cellulaire des protéines. La protéine « TRAF-interacting protein » TRAIP (ou TRIP) est une ligase E3 qui catalyse l'étape finale de l'ubiquitination. TRAIP est conservé dans l'évolution et est nécessaire au développement des organismes puisque l'ablation de TRAIP conduit à la mort embryonnaire aussi bien de la drosophile que de la souris. De plus, la réduction de l'expression de TRAIP dans des kératinocytes épidermiques humains réprime la prolifération cellulaire et induit un arrêt du cycle cellulaire en phase Gl, soulignant le lien étroit entre TRAIP et la prolifération cellulaire. Comme les mécanismes de régulation de la prolifération jouent un rôle majeur dans l'homéostasie de la peau, il est important de caractériser la fonction de TRAIP dans ces mécanismes. En utilisant des approches in vitro, nous avons déterminé que la protéine TRAIP est instable, modifiée par l'addition d'ubiquitine et ayant une demi-vie d'environ 4 heures. Nos analyses ont également révélé que l'expression de TRAIP est dépendante du cycle cellulaire, atteignant un pic d'expression en phase G2/M et que l'induction de son expression s'effectue principalement au cours de la transition Gl/S. Nous avons identifié le facteur de transcription E2F1 comme en étant le responsable, en régulant directement le promoteur de TRAIP. Aussi, TRAIP endogène ou surexprimée est surtout localisée au niveau du nucléole, une organelle nucléaire qui est désassemblée pendant la division cellulaire. Pour examiner la localisation subcellulaire de TRAIP pendant la mitose, nous avons imagé la protéine TRAIP fusionnée à une protéine fluorescente, à l'intérieur de cellules vivantes nommées HeLa, à l'aide d'un microscope confocal. Dans ces conditions, TRAIP est majoritairement localisée autour des chromosomes en début de mitose, puis est arrangée au niveau de l'ADN chromosomique en fin de mitose. La détection de TRAIP endogène à l'aide d'un anticorps spécifique a confirmé cette localisation. Enfin, l'inactivation de TRAIP dans les cellules HeLa par interférence ARN a inhibé leur capacité à s'arrêter en milieu de mitose. Nos résultats suggèrent que le mécanisme sous-jacent peut être lié au point de contrôle de l'assemblage du fuseau mitotique. - Ubiquitination of proteins is a post-translational modification which decides the cellular fate of the protein. The TRAF-interacting protein (TRAIP, TRIP) functions as an E3 ubiquitin ligase mediating addition of ubiquitin moieties to proteins. TRAIP interacts with the deubiquitinase CYLD, a tumor suppressor whose functional inactivation leads to skin appendage tumors. TRAIP is required for early embryonic development since removal of TRAIP either in Drosophila or mice by mutations or knock¬out is lethal due to aberrant regulation of cell proliferation and apoptosis. Furthermore, shRNA- mediated knock-down of TRAIP in human epidermal keratinocytes (HEK) repressed cell proliferation and induced a Gl/S phase block in the cell cycle. Additionally, TRAIP expression is strongly down- regulated during keratinocyte differentiation supporting the notion of a tight link between TRAIP and cell proliferation. We thus examined the biological functions of TRAIP in epithelial cell proliferation. Using an in vitro approach, we could determine that the TRAIP protein is unstable, modified by addition of ubiquitin moieties after translation and exhibits a half-life of 3.7+/-1-6 hours. Our analysis revealed that the TRAIP expression is modulated in a cell-cycle dependent manner, reaching a maximum expression level in G2/M phases. In addition, the expression of TRAIP was particularly activated during Gl/S phase transition and we could identify the transcription factor E2F1 as an activator of the TRAIP gene promoter. Both endogenous and over-expressed TRAIP mainly localized to the nucleolus, a nuclear organelle which is disassembled during cell division. To examine the subcellular localization of TRAIP during M phase, we performed confocal live-cell imaging of a functional fluorescent protein TRAIP-GFP in HeLa cells. TRAIP was distributed in the cytoplasm and accumulated around mitotic chromosomes in pro- and meta-phasic cells. TRAIP was then confined to chromosomal DNA location in anaphase and later phases of mitosis. Immune-detection of endogenous TRAIP protein confirmed its particular localization in mitosis. Finally, inactivating TRAIP expression in HeLa cells using RNA interference abrogated the cells ability to stop or delay mitosis progression. Our results suggested that TRAIP may involve the spindle assembly checkpoint.

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Throughout the animal kingdom, steroid hormones have been implicated in the defense against microbial infection, but how these systemic signals control immunity is unclear. Here, we show that the steroid hormone ecdysone controls the expression of the pattern recognition receptor PGRP-LC in Drosophila, thereby tightly regulating innate immune recognition and defense against bacterial infection. We identify a group of steroid-regulated transcription factors as well as two GATA transcription factors that act as repressors and activators of the immune response and are required for the proper hormonal control of PGRP-LC expression. Together, our results demonstrate that Drosophila use complex mechanisms to modulate innate immune responses, and identify a transcriptional hierarchy that integrates steroid signalling and immunity in animals.

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Accurate chromosome segregation during mitosis is temporally and spatially coordinated by fidelity-monitoring checkpoint systems. Deficiencies in these checkpoint systems can lead to chromosome segregation errors and aneuploidy, and promote tumorigenesis. Here, we report that the TRAF-interacting protein (TRAIP), a ubiquitously expressed nucleolar E3 ubiquitin ligase important for cellular proliferation, is localized close to mitotic chromosomes. Its knockdown in HeLa cells by RNA interference (RNAi) decreased the time of early mitosis progression from nuclear envelope breakdown (NEB) to anaphase onset and increased the percentages of chromosome alignment defects in metaphase and lagging chromosomes in anaphase compared with those of control cells. The decrease in progression time was corrected by the expression of wild-type but not a ubiquitin-ligase-deficient form of TRAIP. TRAIP-depleted cells bypassed taxol-induced mitotic arrest and displayed significantly reduced kinetochore levels of MAD2 (also known as MAD2L1) but not of other spindle checkpoint proteins in the presence of nocodazole. These results imply that TRAIP regulates the spindle assembly checkpoint, MAD2 abundance at kinetochores and the accurate cellular distribution of chromosomes. The TRAIP ubiquitin ligase activity is functionally required for the spindle assembly checkpoint control.

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Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

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Biocorrosion means any process of corrosion in wich microorganisms are somehow involved. As far as the petroleum industry is concerned, the anaerobic type is the more important, with Sulphate-Reducing Bacteria (SRB) accouting for half of the described processes. SRB are obligate anaerobs that use sulphur, sulphate or other oxidized sulphur compounds as oxidizing agents when decomposing organic material. A typical product of SRB metabolism, hydrogen sulphide -H2S-, is extremely toxic. In the present work we review the literature on mechanisms underlying biocorrosive process in wich SRB are involved and summarize some of the ultrastructural and eletrochemical work developed using SRB obtained from water injection flow in wells located on PETROBRAS offshore marine plataforms, sampled directly in the field over metallic probes, or cultured under laboratory conditions. Biofilms develop when SRB adhere to inert surfaces. A high diversity of morphological types is found inside these biofilms. Their extracellular matrix is highly hydrated and mainly anionic, as shown by its avid reaction with cationic compounds like ruthenium red. We have noted that variations in iron contet lead to interesting changes in the ultrastructure of the bacterial cell coat and also in the rate of corrosion induced in metallic test cupons. Since routine methods to prevent and treat SRB contamination and biodeterioration involve the use of biocides that are toxic and always have some environmental impact, an accurate diagnosis of biocorrosion is always required prior to a treatment decision. We developed a method that detects and semi-quantifies the presence of living or dead SRB by using free silver potentials as an indicator of corrosive action by SRB-associated sulphides. We found a correlation between sulphide levels (determined either by spectrophotometry, or using a silver electrode -E(Ag)- that measured changes in free potentials induced by the presence of exogeneously added sulphide) and SRB concentration (enumerated by a culturing method). E (Ag) was characterized under a variety of conditions andwas found to be relatively immune to possible interference resulting from aeration of media or from the psence of iron corrosion products. The method offers a simple, rapid, and effective means of diagnosing biocorrosive processes prior to their control.

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Dendritic cells (DCs) are professional APCs that have a role in the initiation of adaptive immune responses and tolerance. Among the tolerogenic mechanisms, the expression of the enzyme IDO1 represents an effective tool to generate T regulatory cells. In humans, different DC subsets express IDO1, but less is known about the IDO1-related enzyme IDO2. In this study, we found a different pattern of expression and regulation between IDO1 and IDO2 in human circulating DCs. At the protein level, IDO1 is expressed only in circulating myeloid DCs (mDCs) and is modulated by PGE2, whereas IDO2 is expressed in both mDCs and plasmacytoid DCs and is not modulated by PGE2. In healthy subjects, IDO1 expression requires the presence of PGE2 and needs continuous transcription and translation, whereas IDO2 expression is constitutive, independent from suppressor of cytokine signaling 3 activity. Conversely, in patients suffering from inflammatory arthritis, circulating DCs express both IDO1 and IDO2. At the functional level, both mDCs and plasmacytoid DCs generate T regulatory cells through an IDO1/IDO2-dependent mechanism. We conclude that, in humans, whereas IDO1 provides an additional mechanism of tolerance induced by proinflammatory mediators, IDO2 is stably expressed in steady-state conditions and may contribute to the homeostatic tolerogenic capacity of DCs.

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Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.

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The future of antimalarial chemotherapy is particulary alarming in view of the spread of parasite cross-resistances to drugs that are not even structurally related. Only the availability of new pharmacological models will make it possible to select molecules with novel mechanisms of action, thus delaving resistance and allowing the development of new chemotherapeutic strategies. We reached this objective in mice. Our approach is hunged on fundamental and applied research begun in 1980 to investigate to phospholipid (PL) metabolism of intraerythrocytic Plasmodium. This metabolism is abundant, specific and indispensable for the production of Plasmodium membranes. Any drug to interfere with this metabolism blocks parasitic development. The most effective interference yet found involves blockage of the choline transporter, which supplies Plasmodium with choline for the synthesis of phosphatidylcholine, its major PL, this is a limiting step in the pathway. The drug sensitivity thereshold is much lower for the parasite, which is more dependent on this metabolism than host cells. The compounds show in vitro activity against P. falciparum at 1 to 10 nM. They show a very low toxicity against a lymphblastoid cell line, demonstrating a total abscence of correlation between growth inhibition of parasites and lymphoblastoid cells. They show antimalarial activity in vivo, in the P. berghei or P. chabaudi/mouse system, at doses 20-to 100-fold lower than their in acute toxicity limit. The bioavailability of a radiolabeled form of the product seemed to be advantageous (slow blood clearance and no significant concentration in tissues). Lastly, the compounds are inexpensive to produce. They are stable and water-soluble.

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Los filtros de microondas son uno de los elementos clave en el diseño de la mayoría de sistemas de RF que soportan servicios de telecomunicaciones modernos. Algunas de las aplicaciones actuales son, por ejemplo, los nuevos sistemas radar de banda ultra ancha (UWB – Ultra Wide Band) y sistemas de transmisión de servicios multimedia, donde se requieren dimensiones pequeñas del circuito, bajas pérdidas de inserción y una alta selectividad. Una de las tendencias que ha surgido como alternativa a las teorías clásicas de diseño de filtros, es el diseño de filtros basados en técnicas de señales interferentes, conocidos como filtros transversales. En esta memoria es muestra como diseñar un filtro transversal mediante el Híbrido de 90º y mediante el Híbrido de 90º con líneas acopladas, planteando 3 posibles soluciones de la ubicación de las líneas acopladas.

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There are far-reaching conceptual similarities between bi-static surface georadar and post-stack, "zero-offset" seismic reflection data, which is expressed in largely identical processing flows. One important difference is, however, that standard deconvolution algorithms routinely used to enhance the vertical resolution of seismic data are notoriously problematic or even detrimental to the overall signal quality when applied to surface georadar data. We have explored various options for alleviating this problem and have tested them on a geologically well-constrained surface georadar dataset. Standard stochastic and direct deterministic deconvolution approaches proved to be largely unsatisfactory. While least-squares-type deterministic deconvolution showed some promise, the inherent uncertainties involved in estimating the source wavelet introduced some artificial "ringiness". In contrast, we found spectral balancing approaches to be effective, practical and robust means for enhancing the vertical resolution of surface georadar data, particularly, but not exclusively, in the uppermost part of the georadar section, which is notoriously plagued by the interference of the direct air- and groundwaves. For the data considered in this study, it can be argued that band-limited spectral blueing may provide somewhat better results than standard band-limited spectral whitening, particularly in the uppermost part of the section affected by the interference of the air- and groundwaves. Interestingly, this finding is consistent with the fact that the amplitude spectrum resulting from least-squares-type deterministic deconvolution is characterized by a systematic enhancement of higher frequencies at the expense of lower frequencies and hence is blue rather than white. It is also consistent with increasing evidence that spectral "blueness" is a seemingly universal, albeit enigmatic, property of the distribution of reflection coefficients in the Earth. Our results therefore indicate that spectral balancing techniques in general and spectral blueing in particular represent simple, yet effective means of enhancing the vertical resolution of surface georadar data and, in many cases, could turn out to be a preferable alternative to standard deconvolution approaches.

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Animals communicate with conspecifics to resolve conflicts over how resources are shared. Since signals reflect individuals' resource-holding potential and motivation to compete, it is crucial that opponents efficiently transmit and receive information to adjust investment optimally in competitive interactions. Acoustic communication is particularly flexible as it can be quickly modulated according to background noise and audience. Diverse mechanisms have evolved to minimize acoustic signal interference, one being the avoidance of signal overlap by adjusting the timing of call production to alternate calls with those of competitors. However, the occurrence and function of overlap avoidance in the resolution of competition among relatives have barely been studied. Using young barn owl siblings, Tyto alba, which vocally negotiate over who will have priority access to food provided by parents, we investigated the extent to which nestlings avoid calling simultaneously and the function of this behaviour. We found that nestlings overlapped both their live siblings' calls and experimentally broadcast calls at least five times less often than expected at random. Furthermore, a focal nestling engaged more intensely in vocal negotiation when competing with nestmates that called simultaneously compared to those that did not overlap their respective calls. This suggests that barn owl nestlings avoid calling simultaneously, as overlapped calls are less efficient at deterring siblings from competing. Overlap avoidance reduces signal interference and, as a consequence, would improve the efficiency of communication among kin.

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A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity