877 resultados para Segmentation, Targeting and Positioning
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The endothelial nitric oxide synthase (ec-NOS) plays a key role in the transduction of signals from the bloodstream to the underlying smooth muscle. ecNOS undergoes a complex series of covalent modifications, including myristoylation and palmitoylation, which appear to play a role in ecNOS membrane association. Mutagenesis of the myristoylation site, which prevents both myristoylation and palmitoylation, blocks ecNOS targeting to cell membranes. Further, as described for some G-protein alpha subunits, both membrane association and palmitoylation of ecNOS are dynamically regulated: in response to agonists, the enzyme undergoes partial redistribution to the cell cytosol concomitant with depalmitoylation. To clarify the role of palmitoylation in determining ecNOS subcellular localization, we have constructed palmitoylation-deficient mutants of ecNOS. Serine was substituted for cysteine at two potential palmitoylation sites (Cys-15 and Cys-26) by site-directed mutagenesis. Immunoprecipitation of ecNOS mutants following cDNA transfection and biosynthetic labeling with [3H]palmitate revealed that mutagenesis of either cysteine residue attenuated palmitoylation, whereas replacement of both residues completely eliminated palmitoylation. Analysis of N-terminal deletion mutations of ecNOS demonstrated that the region containing these two cysteine residues is both necessary and sufficient for enzyme palmitoylation. The cysteines thus identified as the palmitoylation sites for ecNOS are separated by an unusual (Gly-Leu)5 sequence and appear to define a sequence motif for dual acylation. We analyzed the subcellular distribution of ecNOS mutants by differential ultracentrifugation and found that mutagenesis of the ecNOS palmitoylation sites markedly reduced membrane association of the enzyme. These results document that ecNOS palmitoylation is an important determinant for the subcellular distribution of ecNOS and identify a new motif for the reversible palmitoylation of signaling proteins.
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Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. The resulting "knockout" mice have confirmed some hypotheses, have upset others, but have rarely been uninformative. Models of several human genetic diseases have been produced by targeting--including Gaucher disease, cystic fibrosis, and the fragile X syndrome. These diseases are primarily determined by defects in single genes, and their modes of inheritance are well understood. When the disease under study has a complex etiology with multiple genetic and environmental components, the generation of animal models becomes more difficult but no less valuable. The problems associated with dissecting out the individual genetic factors also increases substantially and the distinction between causation and correlation is often difficult. To prove causation in a complex system requires rigorous adherence to the principle that the experiments must allow detection of the effects of changing only a single variable at one time. Gene targeting experiments, when properly designed, can test the effects of a precise genetic change completely free from the effects of differences in any other genes (linked or unlinked to the test gene). They therefore allow proofs of causation.
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A fundamental catalytic principle for protein enzymes in the use of binding interactions away from the site of chemical transformation for catalysis. We have compared the binding and reactivity of a series of oligonucleotide substrates and products of the Tetrahymena ribozyme, which catalyzes a site-specific phosphodiester cleavage reaction: CCCUCUpA+G<-->CCCUCU-OH+GpA. The results suggest that this RNA enzyme, like protein enzymes, can utilize binding interactions to achieve substantial catalysis via entropic fixation and substrate destabilization. The stronger binding of the all-ribose oligonucleotide product compared to an analog with a terminal 3' deoxyribose residue gives an effective concentration of 2200 M for the 3' hydroxyl group, a value approaching those obtained with protein enzymes and suggesting the presence of a structurally well defined active site capable of precise positioning. The stabilization from tertiary binding interactions is 40-fold less for the oligonucleotide substrate than the oligonucleotide product, despite the presence of the reactive phosphoryl group in the substrate. This destabilization is accounted for by a model in which tertiary interactions away from the site of bond cleavage position the electron-deficient 3' bridging phosphoryl oxygen of the oligonucleotide substrate next to an electropositive Mg ion. As the phosphodiester bond breaks and this 3' oxygen atom develops a negative charge in the transition state, the weak interaction of the substrate with Mg2+ becomes strong. These strategies of "substrate destabilization" and "transition state stabilization" provide estimated rate enhancements of approximately 280- and approximately 60-fold, respectively. Analogous substrate destabilization by a metal ion or hydrogen bond donor may be used more generally by RNA and protein enzymes catalyzing reactions of phosphate esters.
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We present new tools for the segmentation and analysis of musical scores in the OpenMusic computer-aided composition environment. A modular object-oriented framework enables the creation of segmentations on score objects and the implementation of automatic or semi-automatic analysis processes. The analyses can be performed and displayed thanks to customizable classes and callbacks. Concrete examples are given, in particular with the implementation of a semi-automatic harmonic analysis system and a framework for rhythmic transcription.
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After advocating flexibilization of non-standard work contracts for many years, some European and international institutions and several policy makers now indicate the standard employment relationship and its regulation as a cause of segmentation between the labour market of "guaranteed" insiders, employed under permanent contracts with effective protection against unfair dismissal, and the market of the “not-guaranteed” outsiders, working with non-standard contracts. Reforms of employment legislation are therefore being promoted and approved in different countries, allegedly aiming to balance the legal protection afforded to standard and non-standard workers. This article firstly argues that this approach is flawed as it oversimplifies reasons of segmentation as it concentrates on an “insiders-outsiders” discourse that cannot easily be transplanted in continental Europe. After reviewing current legislative changes in Italy, Spain and Portugal, it is then argued that lawmakers are focused on “deregulation” rather than “balancing protection” when approving recent reforms. Finally, the mainstream approach to segmentation and some of its derivative proposals, such as calls to introduce a “single permanent contract”, are called into question, as they seem to neglect the essential role of job protection in underpinning the effectiveness of fundamental and constitutional rights at the workplace.
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Has inflation targeting (IT) conferred benefits in terms of economic growth on countries that followed this particular monetary policy strategy during the crisis period 2007-12? This paper answers this question in the affirmative. Countries with an IT monetary regime with flexible exchange rates weathered the crisis much better than countries with other monetary regimes, predominantly countries with fixed exchange rates. Part of this difference in growth performance reflects differences in export performance during the initial years of the crisis, which in turn can be explained by real exchange rate depreciations. However, IT seems also to confer other benefits on the countries above and beyond the effects from currency depreciation.
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As stated in the opening sentence of the proposal submitted for the ACES grant in 2009, the research that this seed grant is supporting is ambitious and large in scale. The primary goal is to produce a book-length study that assesses the priorities and impact of European and American foreign aid targeting youth in the Middle East and North Africa (MENA). To date, the research undertaken with the support of the grant has helped in providing some preliminary data for a) testing few hypotheses, b) fine-tuning the research design; and c) pointing to the direction where more conceptual and ethnographic research should be undertaken.
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This study compares monetary and multidimensional poverty measures for the Lao People’s Democratic Republic. Using household data of 2007/2008, we compare the empirical outcomes of the country’s current official monetary poverty measure with those of a multidimensional poverty measure. We analyze which population subgroups are identified as poor by both measures and thus belong to the category of the poorest of the poor; and we look at which subgroups are identified as poor by only one of the measures and belong either to the category of the income-poor (identified as poor only by the monetary measure) or to that of the overlooked poor (identified as poor only by the multidimensional poverty measure). Furthermore, we examined drivers of these differences using a multinomial regression model and found that monetary poverty does not capture the multiple deprivations of ethnic minorities, who are only identified as poor when using a multidimensional poverty measure. We conclude that complementing the monetary poverty measure with a multidimensional poverty index would enable more effective targeting of poverty reduction efforts.
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Mode of access: Internet.
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Mode of access: Internet.
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National Highway Traffic Safety Administration, Office of Vehicle Crashworthiness, Washington, D.C.
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Mode of access: Internet.
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"Serial no. 105-7."