"Public on the outside, private on the inside": the organizational hybridization, sense of belonging and identity strategies of the employees of a public unemployment insurance fund in Switzerland

A number of studies show that New Public Management reforms have altered the current identity benchmarks of public officials, particularly by hybridizing values or management practices. However, existing studies have largely glossed over the sense of belonging of officials when their organization straddles the concerns of public service and private enterprise, so that the boundary between public and private sector is blurred. The purpose of this article is precisely to explore this sense of belonging in the context of organizational hybridization. It does so by drawing on the results of research conducted among the employees of a public unemployment insurance fund in Switzerland. On the one hand, the analysis shows how much their markers of belonging are hybrid, multiple and constructed in negative terms (with regard to the State), while indicating that the working practices of the employees point to an identity that is nevertheless closely bound with the public sector. On the other hand, the analysis shows that the organization plays strategically with its State status, by exploiting either its private or public identity in line with the needs related to its external image. The article concludes with a discussion of the results highlighting the strategic functionality of the hybrid identity of the actors.

GLUTX1, a novel mammalian glucose transporter expressed in the central nervous system and insulin-sensitive tissues.

Based on homology with GLUT1-5, we have isolated a cDNA for a novel glucose transporter, GLUTX1. This cDNA encodes a protein of 478 amino acids that shows between 29 and 32% identity with rat GLUT1-5 and 32-36% identity with plant and bacterial hexose transporters. Unlike GLUT1-5, GLUTX1 has a short extracellular loop between transmembrane domain (TM) 1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. When expressed in Xenopus oocytes, GLUTX1 showed strong transport activity only after suppression of a dileucine internalization motif present in the amino-terminal region. Transport activity was inhibited by cytochalasin B and partly competed by D-fructose and D-galactose. The Michaelis-Menten constant for glucose was approximately 2 mM. When translated in reticulocytes lysates, GLUTX1 migrates as a 35-kDa protein that becomes glycosylated in the presence of microsomal membranes. Western blot analysis of GLUTX1 transiently expressed in HEK293T cells revealed a diffuse band with a molecular mass of 37-50 kDa that could be converted to a approximately 35-kDa polypeptide following enzymatic deglycosylation. Immunofluorescence microscopy detection of GLUTX1 transfected into HEK293T cells showed an intracellular staining. Mutation of the dileucine internalization motif induced expression of GLUTX1 at the cell surface. GLUTX1 mRNA was detected in testis, hypothalamus, cerebellum, brainstem, hippocampus, and adrenal gland. We hypothesize that, in a similar fashion to GLUT4, in vivo cell surface expression of GLUTX1 may be inducible by a hormonal or other stimulus.

Ghosts in contemporary African diasporic literature : re-visioning history, memory and identity

Starting from the observation that ghosts are strikingly recurrent and prominent figures in late-twentieth African diasporic literature, this dissertation proposes to account for this presence by exploring its various functions. It argues that, beyond the poetic function the ghost performs as metaphor, it also does cultural, theoretical and political work that is significant to the African diaspora in its dealings with issues of history, memory and identity. Toni Morrison's Beloved (1987) serves as a guide for introducing the many forms, qualities and significations of the ghost, which are then explored and analyzed in four chapters that look at Fred D'Aguiar's Feeding the Ghosts (1998), Gloria Naylor's Mama Day (1988), Paule Marshall's Praisesong for the Widow (1983) and a selection of novels, short stories and poetry by Michelle Cliff. Moving thematically through these texts, the discussion shifts from history through memory to identity as it examines how the ghost trope allows the writers to revisit sites of trauma; revise historical narratives that are constituted and perpetuated by exclusions and invisibilities; creatively and critically repossess a past marked by violence, dislocation and alienation and reclaim the diasporic culture it contributed to shaping; destabilize and deconstruct the hegemonic, normative categories and boundaries that delimit race or sexuality and envision other, less limited and limiting definitions of identity. These diverse and interrelated concerns are identified and theorized as participating in a project of "re-vision," a critical project that constitutes an epistemological as much as a political gesture. The author-based structure allows for a detailed analysis of the texts and highlights the distinctive shapes the ghost takes and the particular concerns it serves to address in each writer's literary and political project. However, using the ghost as a guide into these texts, taken collectively, also throws into relief new connections between them and sheds light on the complex ways in which the interplay of history, memory and identity positions them as products of and contributions to an African diasporic (literary) culture. If it insists on the cultural specificity of African diasporic ghosts, tracing its origins to African cultures and spiritualities, the argument also follows gothic studies' common view that ghosts in literary and cultural productions-like other related figures of the living dead-respond to particular conditions and anxieties. Considering the historical and political context in which the texts under study were produced, the dissertation makes connections between the ghosts in them and African diasporic people's disillusionment with the broken promises of the civil rights movement in the United States and of postcolonial independence in the Caribbean. It reads the texts' theoretical concerns and narrative qualities alongside the contestation of traditional historiography by black and postcolonial studies as well as the broader challenge to conventional notions such as truth, reality, meaning, power or identity by poststructuralism, postcolonialism or queer theory. Drawing on these various theoretical approaches and critical tools to elucidate the ghost's deconstructive power for African diasporic writers' concerns, this work ultimately offers a contribution to "speciality studies," which is currently emerging as a new field of scholarship in cultural theory.

A new repetitive DNA sequence from Trypanosoma cruzi

Tandemly repeated DNA sequences are found in the genome of higher eukaryotes, and have also been demonstrated in Trypanosoma cruzi. Repeated DNA sequences are potentially useful for the diagnostic detection of T. cruzi (A. Gonzales et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 3356-3360). We have isoleted two clones from a genomic library of T. cruzi (Y strain) that contain, in one clone a family of at least seven copies of a repetitive sequence of approximately 600 base pairs, and in the other an independent copy of the same sequence. One copy of the repetition (HSP) and the independent clone (HCR) were sequenced by the Sanger procedure (Fig.). This sequence hybridized to four strains of T. cruzi tested and did not hybridize to eleven species of trypanosotids from five different Genera, being a good candidate for diagnostic assays. GenBank accession numbers: HSP#m31919, HCR#31920.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

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.

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.

CTCF-T, a paralogue of CTCF, directs sequence specific DNA methylation of the imprinting control region of Igf2/H19

SUMMARY Genomic imprinting is an epigenetic mechanism of transcriptional regulation that ensures restriction of expression of a subset of mammalian genes to a single parental allele. The best studied example of imprinted gene regulation is the Igf2/H19 locus, which is also the most commonly altered by loss of imprinting (LOT) in cancer. LOT is associated with numerous hereditary diseases and several childhood, and adult cancers. Differential expression of reciprocal H19 and 1gf2 alleles in somatic cells depends on the methylation status of the imprinting control region (ICR) which regulates binding of CTCF, an ubiquitously expressed 11-zinc finger protein that binds specifically to non-methylated maternal ICR and thereby attenuates expression of Igf2, while it does not bind to methylated paternal ICR, which enables Igf2 expression. Initial ICR methylation occurs during gametogenesis by an as yet unknown mechanism. The accepted hypothesis is that the event of differential maternal and paternal DNA methylation depends on germ-line specific proteins. Our Laboratory identified a novel 11-zinc-finger protein CTCF-T (also known as CTCFL and BORIS) that is uniquely expressed in the male germ-line and is highly homologous within its zinc-finger region with CTCF. The amino-acid sequences flanking the zinc-finger regions of CTCF and CTCF-T have widely diverged, suggesting that though they could bind to the same DNA targets (ICRs) they are likely to have different functions. Interestingly, expression of CTCF-T and CTCF is mutually exclusive; CTCF-T-positive (CTCF-negative) cells occur in the stage of spermatogenesis that coincides with epigenetic reprogramming, including de novo DNA methylation. In our study we demonstrate the role that CTCF-T plays in genomic imprinting. Here we show that CTCF-T binds in vivo to the ICRs of Igf2/H19 and Dlk/Gt12 imprinted genes. In addition, we identified two novel proteins interacting with CTCF-T: a protein arginine methyltransferase PRMT7 and an arginine-rich histone H2A variant that we named trH2A. These interactions were confirmed and show that the two proteins interact with the amino-teiminal region of CTCF-T. Additionally, we show interaction of the amino- terminal region of CTCF-T with histones H1, H2A and H3. These results suggest that CTCF-T is a sequence-specific DNA (ICR) binding protein that associates with histones and recruits PRMT7. Interestingly, PRMT7 has a histone-methyltransferase activity. It has been shown that histone methylation can mark chromatin regions thereby directing DNA-methylation; thus, our hypothesis is that the CTCF-T protein-scaffold directs PRMT7 to methylate histone(s) assembled on ICRs, which marks chromatin for the recruitment of the de novo DNA methyltransferases to methylate DNA. To test this hypothesis, we developed an in vivo DNA-methylation assay using Xenopus laevis' oocytes, where H19 ICR and different expression cDNAs, including CTCF-T, PRMT7 and the de novo DNA methyltransferases (Dnmt3a, Dnmt3b and Dnmt3L) are microinjected into the nucleus. The methylation status of CpGs within the H19 ICR was analysed 48 or 72 hours after injection. Here we demonstrate that CpGs in the ICR are methylated in the presence of both CTCF-T and PRMT7, while control oocytes injected only with ICR did not show any methylation. Additionally, we showed for the first time that Dnmt3L is crucial for the establishment of the imprinting marks on H19 ICR. Moreover, we confirmed that Dnmt3a and Dnmt3b activities are complementary. Our data indicate that all three Dnmt3s are important for efficient de novo DNA methylation. In conclusion, we propose a mechanism for the establishment of de novo imprinting marks during spermatogenesis: the CTCF-T/PRMT7 protein complex directs histone methylation leading to sequence-specific de novo DNA methylation of H19 ICR. RESUME L'empreinte génomique parentale est un mécanisme épigénétique de régulation transcriptionelle qui se traduit par une expression différentielle des deux allèles de certains gènes, en fonction de leur origine parentale. L'exemple le mieux caractérisé de gènes soumis à l'empreinte génomique parentale est le locus Igf2/H19, qui est aussi le plus fréquemment altéré par relaxation d'empreinte (en anglais: loss of imprinting, LOI) dans les cancers. Cette relaxation d'empreinte est aussi associée à de nombreuses maladies héréditaires, ainsi qu'à de nombreux cancers chez l'enfant et l'adulte. Dans les cellules somatiques, les différences d'expression des allèles réciproques H19 et Ig12 est sous le contrôle d'une région ICR (Imprinting Control Region). La méthylation de cette région ICR régule l'ancrage de la protéine à douze doigts de zinc CTCF, qui se lie spécifiquement à l'ICR maternel non-méthylé, atténuant ainsi l'expression de Igf2, alors qu'elle ne s'ancre pas à l'ICR paternel méthyle. Le mécanisme qui accompagne la méthylation initiale de la région ICR durant la gamétogenèse n'a toujours pas été élucidé. L'hypothèse actuelle propose que la différence de méthylation entre l'ADN maternel et paternel résulte de l'expression de protéines propres aux zones germinales. Notre laboratoire a récemment identifié une nouvelle protéine à douze doigts de zinc, CTCF-T (aussi dénommée CTCFL et BORRIS), qui est exprimée uniquement dans les cellules germinales mâles, dont la partie à douze doigts de zinc est fortement homologue à la protéine CTCF. La séquence d'acides aminés de part et d'autre de cette région est quant à elle très divergente, ce qui implique que CTCF-T se lie sans doute au même ADN cible que CTCF, mais possède des fonctions différentes. De plus, l'expression de CTCF-T et de CTCF s'oppose mutuellement; l'expression de la protéine CTCF-T (cellules CTCF-T positives, CTCF negatives) qui a lieu pendant la spermatogenèse coïncide avec la reprogrammation épigénétique, notamment la méthylation de novo de l'ADN. La présente étude démontre le rôle essentiel joué par la protéine CTCF-T dans l'acquisition de l'empreinte génomique parentale. Nous montrons ici que CTCF-T s'associe in vivo avec les régions ICR des loci Igf2/H19 et Dlk/Gt12. Nous avons également identifié deux nouvelles protéines qui interagissent avec CTCF-T : une protéine arginine méthyl transférase PRMT7, et un variant de l'histone H2A, riche en arginine, que nous avons dénommé trH2A. Ces interactions ont été analysées plus en détail, et confinnent que ces deux protéines s'associent avec la région N-terminale de CTCF-T. Aussi, nous présentons une interaction de la région N-terminale de CTCF-T avec les histones H1, H2, et H3. Ces résultats suggèrent que CTCF-T est une protéine qui se lie spécifiquement aux régions ICR, qui s'associe avec différents histones et qui recrute PRMT7. PRMT7 possède une activité méthyl-tansférase envers les histones. Il a été montré que la méthylation des histones marque certains endroits de la chromatine, dirigeant ainsi la méthylation de l'ADN. Notre hypothèse est donc la suivante : la protéine CTCF-T sert de base qui dirige la méthylation des histones par PRMT7 dans les régions ICR, ce qui contribue à marquer la chromatine pour le recrutement de nouvelles méthyl transférases pour méthyler l'ADN. Afin de valider cette hypothèse, nous avons développé un système de méthylation de l'ADN in vivo, dans des oeufs de Xenopus laevis, dans le noyau desquels nous avons mico-injecté la région ICR du locus H19, ainsi que différents vecteurs d'expression pour CTCF-T, PRMT7, et les de novo méthyl transférases (Dnmt3a, Dnmt3b et Dnmt3L). Les CpGs méthyles de la région ICR du locus H19 ont été analysé 48 et 72 heures après l'injection. Cette technique nous a permis de démontrer que les CpGs de la région ICR sont méthyles en présence de CTCF-T et de PRMT7, tandis que les contrôles injectés seulement avec la région ICR ne présentent aucun signe de méthylation. De plus, nous démontrons pour la première fois que la protéine méthyl transférase Dnmt3L est déterminant pour l'établissement de l'empreinte génomique parentale au niveau de la région ICR du locus H19. Aussi, nous confirmons que les activités méthyl transférases de Dnmt3a et Dnmt3b sont complémentaires. Nos données indiquent que les trois protéines Dnmt3 sont impliquées dans la méthylation de l'ADN. En conclusion, nous proposons un mécanisme responsable de la mise en place de nouvelles empreintes génomiques pendant la spermatogenèse : le complexe protéique CTCF-T/PRMT7 dirige la méthylation des histones aboutissant à la méthylation de novo de l'ADN au locus H19.

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita.

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.

Immunogenicity and antigenicity of the N-term repeat amino acid sequence of the Plasmodium falciparum P126 antigen

The P126 protein, a parasitosphorus vacuole antigen of Plasmodium falciparum has beenshoen to induce protective immunity in Saimiri and Aotus monkeys. In the present work we investigated its immunogenicity. Our results suggest that the N-term of P126 is poorly immunogenic and antibody response against the P126 could be under a MHC restricted control in C57BL/6(H-2b) mice, which could be problematic in ternms of a use of the P126 in a vaccine program. However, we observed that a synthetic peptide, copying the 6 octapeptide repeat corresponding to the N-term of the P126, induces an antibody response to the native molecule in C57BL/6 non-responder mice. Moreover, the vaccine-P126 recombinant induced anmtibodies against the N-term of the molecule in rabbits while the unprocessed P126 did not.

Sequence conservation in Plasmodium falciparum alpha-helical coiled coil domains proposed for vaccine development.

BACKGROUND: The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to alpha-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative alpha-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB. METHODOLOGY/PRINCIPAL FINDINGS: 14 alpha-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 alpha-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative alpha-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for alpha-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved alpha-helical coiled coil motifs. CONCLUSION/SIGNIFICANCE: We conclude that the selection of alpha-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.