Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains.

The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin. However, maturation of the N-linked glycan of MHC class I heavy chains was impaired in US3+ cells. The glycoprotein product of US3 (gpUS3) occurs mostly in a high-mannose form and coimmunoprecipitates with beta 2-microglobulin associated class I heavy chains. Mature class I molecules were detected at steady state on the surface of US3+ cells, as in control cells. Substantial perinuclear accumulation of heavy chains was observed in US3+ cells. The data suggest that gpUS3 impairs egress of MHC class I heavy chains from the endoplasmic reticulum.

A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens.

A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.

Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139.

The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.

The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guérin.

The ureABC genes of Mycobacterium tuberculosis were cloned. By using a set of degenerate primers corresponding to a conserved region of the urease enzyme (EC 3.5.1.5), a fragment of the expected size was amplified by PCR and was used to screen a M. tuberculosis cosmid library. Three open reading frames with extensive similarity to the urease genes from other organisms were found. The locus was mapped on the chromosome, using an ordered M. tuberculosis cosmid library. A suicide vector containing a ureC gene disrupted by a kanamycin marker (aph) was used to construct a urease-negative Mycobacterium bovis bacillus Calmette-Guérin mutant by allelic exchange involving replacement of the ureC gene with the aph::ureC construct. To our knowledge, allelic exchange has not been reported previously in the slow-growing mycobacteria. Homologous recombination will be an invaluable genetic tool for deciphering the mechanisms of tuberculosis pathogenesis, a disease that causes 3 x 10(6) deaths a year worldwide.

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the beta subunit of Na+,K(+)-ATPase.

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the beta subunit of Na+,K(+)-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. Our results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the beta subunit of Na+,K(+)-ATPase.

Measurement of wide frequency range structural microvibrations with a pocket digital camera and sub-pixel techniques

Analysis of vibrations and displacements is a hot topic in structural engineering. Although there is a wide variety of methods for vibration analysis, direct measurement of displacements in the mid and high frequency range is not well solved and accurate devices tend to be very expensive. Low-cost systems can be achieved by applying adequate image processing algorithms. In this paper, we propose the use of a commercial pocket digital camera, which is able to register more than 420 frames per second (fps) at low resolution, for accurate measuring of small vibrations and displacements. The method is based on tracking elliptical targets with sub-pixel accuracy. Our proposal is demonstrated at a 10 m distance with a spatial resolution of 0.15 mm. A practical application over a simple structure is given, and the main parameters of an attenuated movement of a steel column after an impulsive impact are determined with a spatial accuracy of 4 µm.

Effects of landscape spatial heterogeneity on dryland restoration success. The combined role of site conditions and reforestation techniques in southeastern Spain

In occidental Europe, Spain is one of countries the most severely affected by desertification (Arnalds & Arsher 2000). Particularly, South-eastern Spain is considered as one of the most threatened areas by desertification in Mediterranean Europe (Vallejo 1997). In 2003, the Valencia Regional Forest Service implemented a restoration demonstration project in this area. The project site is a small catchment (25 ha) located in the Albatera municipality. The catchment is highly heterogeneous, with terraced slopes, south-facing slopes and north-facing slopes. The restoration strategy was based on planting evergreen trees and shrubs which can grow quickly after disturbances, and on field treatments aimed at maximizing water collection (micro-catchments, planting furrows), organic amendment (compost), and conservation (tree shelters, mulching). On south landscape unit, the whole category of restoration treatments was applied: water micro-catchment + Tubex tree shelters + mulching & compost, while on north landscape unit: netting tree shelters + mulching & compost only were applied, while in terrace landscape unit: furrows + netting tree shelters + mulching & compost were applied. Survival and growth of the planted seedlings were used as metrics of restoration success. To assess the effects of the treatments applied for soil conservation, soil loss rates (from 2005 to 2009) were evaluated using the erosion pin method. We conclude that, despite the limiting conditions prevailing on the south unit, this landscape unit showed the highest survival and growth plant rates in the area. The best seedling performances on the south landscape unit were probably due to the highest technical efforts applied, consisting in the water micro-catchment installation and the Tubex plant shelters addition. In addition, soil loss rates followed decreasing trends throughout the assessment period. Soil loss rates were highest on south landscape unit in comparison with the other landscape units, due to the more accentuated relief. North landscape unit and terrace unit showed a net soil mass gain, probably reflecting the trapping of sediments produced by plantation works.

Optimización de áreas funcionales espaciales mediante algoritmos evolutivos multioperador. Aplicación a la delimitación de mercados locales de trabajo

El documento de esta tesis por compendio de publicaciones se divide en dos partes: la síntesis donde se resume la fundamentación, resultados y conclusiones de esta tesis, y las propias publicaciones en su formato original, que se incluyen como apéndices. Dado que existen acuerdo de confidencialidad (véase "Derechos" más adelante) que impiden su publicación en formato electrónico de forma pública y abierta (como es el repositorio de la UA), y acorde con lo que se dictamina en el punto 6 del artículo 14 del RD 99/2011, de 28 de enero, no se incluyen estos apéndices en el documento electrónico que se presenta en cedé, pero se incluyen las referencias completas y sí se incluyen integramente en el ejemplar encuadernado. Si el CEDIP y el RUA así lo decidiesen más adelante, podría modificarse este documento electrónico para incluir los enlaces a los artículos originales.

Measurement of wide frequency range structural microvibrations with a pocket digital camera and sub-pixel techniques

Analysis of vibrations and displacements is a hot topic in structural engineering. Although there is a wide variety of methods for vibration analysis, direct measurement of displacements in the mid and high frequency range is not well solved and accurate devices tend to be very expensive. Low-cost systems can be achieved by applying adequate image processing algorithms. In this paper, we propose the use of a commercial pocket digital camera, which is able to register more than 420 frames per second (fps) at low resolution, for accurate measuring of small vibrations and displacements. The method is based on tracking elliptical targets with sub-pixel accuracy. Our proposal is demonstrated at a 10 m distance with a spatial resolution of 0.15 mm. A practical application over a simple structure is given, and the main parameters of an attenuated movement of a steel column after an impulsive impact are determined with a spatial accuracy of 4 µm.

Fleshing Out and Losing Flesh: The Decomposing Corpse as Narrative Object in American Hardboiled Detective Fiction

This dissertation examines the corpse as an object in and of American hardboiled detective fiction written between 1920 and 1950. I deploy several theoretical frames, including narratology, body-as-text theory, object relations theory, and genre theory, in order to demonstrate the significance of objects, symbols, and things primarily in the clever and crafty work of Dashiell Hammett (1894-1961) and Raymond Chandler (1888-1959), but also touching on the writings of their lesser known accomplices. I construct a literary genealogy of American hardboiled detective fiction originating in the writings of Edgar Allan Poe, compare the contributions of classic or Golden Age detective fiction in England, and describe the socio-economic contexts, particularly the predominance of the “pulps,” that gave birth to the realism of the Hardboiled School. Taking seriously Chandler’s obsession with the art of murder, I engage with how authors pre-empt their readers’ knowledge of the tricks of the trade and manipulate their expectations, as well as discuss the characteristics and effect of the inimitable hardboiled style, its sharpshooting language and deadpan humour. Critical scholarship has rarely addressed the body and figure of the corpse, preferring to focus instead on the machinations of the femme fatale, the performance of masculinity, or the prevalence of violence. I cast new light on the world of hardboiled detective fiction by dissecting the corpse as the object that both motivates and de-composes (or rots away from) the narrative that makes it signify. I treat the corpse as an inanimate object, indifferent to representation, that destabilizes the integrity and self-possession, as well as the ratiocination, of the detective who authors the narrative of how the corpse came to be. The corpse is all deceptive and dangerous surface rather than the container of hidden depths of life and meaning that the detective hopes to uncover and reconstruct. I conclude with a chapter that is both critical denouement and creative writing experiment to reveal the self-reflexive (and at times metafictional) dimensions of hardboiled fiction. My dissertation, too, in the manner of hardboiled fiction, hopes to incriminate my readers as much as enlighten them.

The Discursive double game of EMU reform: the clash of titans between French White Knight and German Iron Lady. Paper presented at the 9th Biennial Conference European Community Studies Association–Canada (ECSA-C), Ottawa, Ontario, April 27-28, 2012

From the Introduction. In the aftermath of the EU’s enlargement towards Central and Eastern Europe, many scholars and observers of European integration were proclaiming that the French-German “engine” of Europe had come to an end. The political legitimacy of French-German initiatives was contested by coalitions of smaller member states and the ‘new Europe’ was calling for new leadership dynamics. However, the experience of the Eurozone debt crisis provided dramatic evidence that no alternative to the Franco-German partnership has yet to emerge in the enlarged EU. In a time of existential crisis, Franco-German initiatives appear to have remained the basic dynamic of integration. However, unlike in the past, agreements on steps forward have proven to be particularly difficult. This is largely due to these countries’ contrasting political economic policy ideas, cultures, and practices....the paper analyses the ideational ‘frames’ of the two leaders while tracing their discursive interactions against changing background conditions since the European debt crisis was triggered by Greece in October 2009 until the last measures taken in 2012 before the French Presidential elections. The empirical analysis is based on a systematic corpus of press conferences and media interviews by Nicolas Sarkozy and Angela Merkel after European summits. It is complemented by a number of press interviews including some given by their respective Finance Ministers) and important speeches in that same period of time.

EU Social Policy: Developments in the field and insights for the study of social policy. ACES Cases No. 2012.1

This paper focuses on the key features of EU social policy and the way it has been interpreted and seeks to identify new directions for study. EU social policy is considered along two main dimensions: its content and hallmark features and the main approaches to conceptualizing and theorizing it. Rather than the classic negative depiction of EU social policy, this piece suggests that it is more significant than usually allowed, not least because the empirical and theoretical lenses which have been applied to it were developed for other purposes. The implication is that developments in EU social policy are often overlooked, not least in how the EU has carved out a role for itself by constantly framing and reframing discourses relevant to social policy and social problems in an attempt to both influence how social actors at all levels of governance approach policy and secure their acceptance of its role in social policy. Therefore analyzing EU social policy outside of the traditional frames reveals interesting and significant developments especially around innovation in social policy and the attempt to legitimate the EU as a social policy actor.

Identification of differentially expressed genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metronidazole.

OBJECTIVES The characterization of differential gene expression in Giardia lamblia WB C6 strain C4 resistant to metronidazole and nitazoxanide using microarray technology and quantitative real-time PCR. METHODS In a previous study, we created and characterized the G. lamblia WB C6 clone C4 resistant to nitazoxanide and metronidazole. In this study, using a microarray-based approach, we have identified open-reading frames (ORFs) that were differentially expressed in C4 when compared with its wild-type WB C6. Using quantitative real-time PCR, we have validated the expression patterns of some of those ORFs, focusing on chaperones such as heat-shock proteins in wild-type and C4 trophozoites. In order to induce an antigenic shift, trophozoites of both strains were subjected to a cycle of en- and excystation. Expression of selected genes and resistance to nitazoxanide and metronidazole were investigated after this cycle. RESULTS Forty of a total of 9115 ORFs were found to be up-regulated and 46 to be down-regulated in C4 when compared with wild-type. After a cycle of en- and excystation, resistance of C4 to nitazoxanide and metronidazole was lost. Resistance formation and en-/excystation were correlated with changes in expression of ORFs encoding for major surface antigens such as the variant surface protein TSA417 or AS7 ('antigenic shift'). Moreover, expression patterns of the cytosolic heat-shock protein HSP70 B2, HSP40, and of the previously identified nitazoxanide-binding proteins nitroreductase and protein disulphide isomerase PDI4 were correlated with resistance and loss of resistance after en-/excystation. C4 trophozoites had a higher thermotolerance level than wild-type trophozoites. After en-/excystation, this tolerance was lost. CONCLUSIONS These results suggest that resistance formation in Giardia to nitazoxanide and metronidazole is correlated with altered expression of genes involved in stress response such as heat-shock proteins.

Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact

Nonsense-mediated mRNA decay (NMD) was originally coined to define a quality control mechanism that targets mRNAs with truncated open reading frames due to the presence of a premature termination codon. Meanwhile, it became clear that NMD has a much broader impact on gene expression and additional biological functions beyond quality control are continuously being discovered. We review here the current views regarding the molecular mechanisms of NMD, according to which NMD ensues on mRNAs that fail to terminate translation properly, and point out the gaps in our understanding. We further summarize the recent literature on an ever-rising spectrum of biological processes in which NMD appears to be involved, including homeostatic control of gene expression, development and differentiation, as well as viral defense.