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Ademais de estudar o fenómeno da cortesía, o presente traballo tamén presta atención a cuestións relacionadas coa adquisición lingüística no desenvolvemento ontoxenético e, máis concretamente, coa adquisición da competencia pragmática. Así, tentarase analizar o proceso polo cal os nenos e nenas galegofalantes adquiren as estratexias de cortesía empregadas nas peticións. Os principais obxectivos radican no estudo das estruturas lingüísticas preferidas polos nenos galegos para levaren a cabo unha petición, na análise do peso de certas variables sociais e contextuais (familiaridade, poder relativo e custo da petición) á hora da escolla dunhas ou outras estratexias e, finalmente, na comprobación da relevancia dos factores da idade e incluso do sexo, tendo en conta sempre a importancia das diferenzas individuais

L’objet de notre travail est justement l’analyse d’un type de proposition subordonnée : la relative. Comme toutes les subordonnées, il s’agit de propositions qui fonctionnent comme un constituant d’une principale. Cependant, il faut dire que ces subordonnées présentent une caractéristique particulière : le subordonnant n’est pas une simple conjonction – comme, par exemple, dans les complétives – mais un pronom qui réalise une fonction à l’intérieur de la subordonnée. Justement, les propositions relatives sont décrites comme un type de subordonnées dont la caractéristique fondamentale est d’être introduites par un pronom relatif, qui joue en même temps le rôle de subordonnant et de constituant de la subordonnée. Comme on le verra, les propositions subordonnées relatives peuvent occuper la fonction d’un constituant adjectival (elles ont un antécédent qu’elles complètent) ou nominal (elles n’ont pas d’antécédent)

There are currently limited treatment options for AAA when surgery is inapplicable. Therefore, insights into molecular mechanisms underlying AAA pathogenesis may reveal therapeutic targets that could be manipulated pharmacologically or biologically to halt disease progression. Using an elastase-induced AAA mouse model, we previously established that the complement alternative pathway (AP) plays a critical role in the development of AAA. However, the mechanism by which complement AP is initiated remains undefined. The complement protein properdin, traditionally viewed as a positive regulator of the AP, may also initiate complement activation by binding directly to target surfaces. In this study, we sought to determine whether properdin serves as a focal point for the initiation of the AP complement activation in AAA. Using a properdin loss of function mutation in mice and a mutant form of the complement factor B protein that produces a stable, properdin-free AP C3 convertase, we show that properdin is required for the development of elastase-induced AAA in its primary role as a convertase stabilizer. Unexpectedly, we find that, in AAA, natural IgG antibodies direct AP-mediated complement activation. The absence of IgG abrogates C3 deposition in elastase-perfused aortic wall and protects animals from AAA development. We also determine that blockade of properdin activity prevents aneurysm formation. These results indicate that an innate immune response to self-antigens activates the complement system and initiates the inflammatory cascade in AAA. Moreover, the study suggests that properdin-targeting strategies may halt aneurysmal growth., Abdominal aortic aneurysm (AAA) is among the top 20 leading causes of mortality in the United States. It accounts for ∼15,000 deaths/y. AAA is a complex disease associated with male sex, advanced age, hypertension, hypercholesterolemia, coronary artery disease, atherosclerosis, and cigarette smoking (1⇓–3). Presently, open surgical repair of large AAA (greater than 5.5 cm in diameter) is an effective option in preventing death from rupture. However, surgical treatment is associated with high postoperative mortality (up to 6%) (4). Endovascular repair is associated with lower postoperative mortality, but it offers no significant difference in overall long-term survival rate and has a higher incidence of reintervention (5⇓⇓–8). Given the high complication rate and morbidities associated with surgical treatment, medical therapy designed to slow progression of AAA would be desirable. Although the causes of AAA are not completely understood, it is apparent that the immune system is a major contributor to pathogenesis. Therefore, a better understanding of the underlying immune-mediated pathways involved in AAA may identify new targets that could be manipulated pharmacologically or biologically to halt disease progression. The complement system is a branch of immunity that can rapidly respond to foreign intruders or pathogens without prior sensitization or exposure. It plays a key role in the identification and removal of injured tissue and debris (9), modulates the antibody repertoire (10), and participates in the activation of T-cell populations (11). Complement activation is mediated by the classical pathway (CP), which is commonly initiated by antigen–antibody complexes, the lectin pathway (LP), which is initiated by LP-specific carbohydrate recognition complexes, and the alternative pathway (AP), which is constitutively active at low level because of continuous hydrolysis of the complement protein C3. The three pathways converge on a central enzyme, the C3 convertase., The AP C3 convertase is formed from three components: fB, a zymogen that carries the convertase catalytic site, C3b, which anchors the convertase to the target surface, and properdin, which stabilizes the complex by 5- to 10-fold (14). More recently, evidence has emerged that properdin may act as an initiator of the AP, binding directly to target cell surfaces and providing a platform for the AP C3 convertase assembly (15, 16). We previously obtained compelling histochemical evidence for the presence of properdin on the luminal surface of human AAA specimens (12), and this finding led us to hypothesize that properdin may serve as a focal point for the initiation of the AP in AAA. Here, we show that properdin plays a critical role in the pathogenesis of elastase-induced AAA, suggesting that properdin-targeting strategies merit additional investigation. We assessed the role of properdin in AAA as a stabilizer vs. initiator of AP convertase by using a mutant form of the fB protein that produces a stable properdin-free AP C3 convertase. Our findings indicate that the primary role of properdin in elastase-induced AAA is as a convertase stabilizer. However, we found that complement activation in AAA is dependent on the presence of natural antibodies, lending support to previous reports suggesting that recognition of self-antigen(s) by antibodies initiates the inflammatory cascade in AAA (17)

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%).

Complement factor B (fB) circulates in plasma as a proenzyme that, upon binding to C3b in the presence of Mg2+, is cleaved by factor D to produce Ba and Bb fragments. Activated Bb remains bound to C3b organizing the alternative pathway C3 convertase (C3bBb). Recently, we have visualized the stable C3bB(Ni2+) proconvertase using electron microscopy, revealing a large conformational change of the C3b-bound fB likely exposing the fD-cleavage site. In contrast, the crystal structure of the proconvertase formed by human fB and the cobra venom factor reveals fB in the closed conformation of the proenzyme. In this study, we have used single-particle electron microscopy and image processing to examine the C3bB(Mg2+) proconvertase. We describe two C3bB(Mg2+) conformations, one resembling cobra venom factor, likely representing the loading state of fB to C3b, and another identical with C3bB(Ni2+). These data illustrate the coexistence of C3b-bound fB in closed and open conformations that either exist in equilibrium or represent structural transitions during the assembly of the C3bB proconvertase

Penicillin G acylase (PGA) is an enzyme that hardly interacts with polycationic polymers (e.g., polyethyleneimine, PEI) and thus the enzyme cannot be stabilized against the action of organic solvents by its co-immobilization with the polymer in the same support, neither covalently attached to the support nor adsorbed on the already immobilized enzyme. However, a new mutant PGA bearing eight additional Glu residues homogenously distributed throughout the enzyme surface may interact with the polymer. The co-immobilization of the enzyme and PEI on glyoxyl-agarose allows one to fully take advantage of the stabilization produced by the multipoint covalent attachment and by the protective hydrophilic micro-environment generated by the polycationic polymer, enabling a significant stabilization of the immobilized PGA in the presence of organic solvents.

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.