Induction of potent antitumor CTL responses by recombinant vaccinia encoding a melan-A peptide analogue.

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

Identification and quantification techniques in mass spectrometry-based proteomics

Résumé La protéomique basée sur la spectrométrie de masse est l'étude du proteome l'ensemble des protéines exprimées au sein d'une cellule, d'un tissu ou d'un organisme - par cette technique. Les protéines sont coupées à l'aide d'enzymes en plus petits morceaux -les peptides -, et, séparées par différentes techniques. Les différentes fractions contenant quelques centaines de peptides sont ensuite analysées dans un spectromètre de masse. La masse des peptides est enregistrée et chaque peptide est séquentiellement fragmenté pour en obtenir sa séquence. L'information de masse et séquence est ensuite comparée à une base de données de protéines afin d'identifier la protéine d'origine. Dans une première partie, la thèse décrit le développement de méthodes d'identification. Elle montre l'importance de l'enrichissement de protéines comme moyen d'accès à des protéines de moyenne à faible abondance dans le lait humain. Elle utilise des injections répétées pour augmenter la couverture en protéines et la confiance dans l'identification. L'impacte de nouvelle version de base de données sur la liste des protéines identifiées est aussi démontré. De plus, elle utilise avec succès la spectrométrie de masse comme alternative aux anticorps, pour valider la présence de 34 constructions de protéines pathogéniques du staphylocoque doré exprimées dans une souche de lactocoque. Dans une deuxième partie, la thèse décrit le développement de méthodes de quantification. Elle expose de nouvelles approches de marquage des terminus des protéines aux isotopes stables et décrit la première méthode de marquage des groupements carboxyliques au niveau protéine à l'aide de réactifs composé de carbone 13. De plus, une nouvelle méthode, appelée ANIBAL, marquant tous les groupements amines et carboxyliques au niveau de la protéine, est exposée. Summary Mass spectrometry-based proteomics is the study of the proteome -the set of all expressed proteins in a cell, tissue or organism -using mass spectrometry. Proteins are cut into smaller pieces - peptides - using proteolytic enzymes and separated using different separation techniques. The different fractions containing several hundreds of peptides are than analyzed by mass spectrometry. The mass of the peptides entering the instrument are recorded and each peptide is sequentially fragmented to obtain its amino acid sequence. Each peptide sequence with its corresponding mass is then searched against a protein database to identify the protein to which it belongs. This thesis presents new method developments in this field. In a first part, the thesis describes development of identification methods. It shows the importance of protein enrichment methods to gain access to medium-to-low abundant proteins in a human milk sample. It uses repeated injection to increase protein coverage and confidence in identification and demonstrates the impact of new database releases on protein identification lists. In addition, it successfully uses mass spectrometry as an alternative to antibody-based assays to validate the presence of 34 different recombinant constructs of Staphylococcus aureus pathogenic proteins expressed in a Lactococcus lactis strain. In a second part, development of quantification methods is described. It shows new stable isotope labeling approaches based on N- and C-terminus labeling of proteins and describes the first method of labeling of carboxylic groups at the protein level using 13C stable isotopes. In addition, a new quantitative approach called ANIBAL is explained that labels all amino and carboxylic groups at the protein level.

Per una comprensió històrica de les activitats aquàtiques : una mirada educativa

Una atenta observació de les activitats aquàtiques de caire educatiu mostra que aquestes pràctiques es fonamenten, principalment, en constructes que no són propis de la pedagogia, ans provenen de l’antropologia i/o de les cultures establertes a l’entorn de l’aigua. Així, el present article es pregunta pels sentits que acompanyen aquests imaginaris que sorgeixen de la relació que s’estableix entre l’home i el medi aquàtic. Per a fer-ho, s’utilitza el recurs hermenèutic de comprendre quines han estat les pràctiques aquàtiques que han anat sorgint al llarg de la història, ja que la idea principal del treball sosté que l’activitat educativa en el medi aquàtic s’ha construït a partir dels imaginaris culturals, sorgits de les mateixes pràctiques aquàtiques que l’han precedit.

Consumo y convergencia mediática de la prensa local en Cataluña

Los nuevos hábitos de consumo de medios en el marco de la convergencia digital están propiciando un cambio en las estrategias de los medios de comunicación. Mientras unos usuarios siguen prefiriendo la radio, la televisión y la prensa tradicional para informarse otros participan de la experiencia digital en internet (blogs, redes sociales...) y dispositivos móviles, etc. En este panorama es complicado precisar dónde nace y termina el contenido o hasta qué punto es la participación del usuario quien (re) construye la información; así los contenidos mutan y se diluyen en varios soportes. En este contexto, la prensa local también vive las consecuencias del nuevo paradigma y asume los retos del reacomodo del sector ante la nueva realidad. Esta comunicación presenta las conclusiones del estudio cualitativo con entrevistas en profundidad a los responsables de medios locales en Cataluña sobre sus usuarios, contenidos y soportes en el marco de la convergencia digital.

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

Enhanced EGFP Fluorescence Emission in Presence of PEG Aqueous Solutions and PIB1000-PEG6000-PIB1000 Copolymer Vesicles.

An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1 : 10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

Algunes reflexions sobre la construcció d'indicadors indirectes pel seguiment de l'activitat industrial regional

En un gran nombre d'economies, l'evolució de la producció industrial s'analitza a partir de la informació sobre el Producte Industrial Brut i/o el Valor Afegit Brut que proporcionen les Comptabilitats Nacionals. A Espanya, la utilització d'aquestes dades presenta el problema que no estan disponibles tan ràpidament com seria desitjable. En conseqüència, no és possible realitzar un seguiment a curt termini de l'activitat industrial a partir dels mateixos. Per a solucionar aquest problema, l'Institut Nacional d'Estadística elabora un Índex de Producció Industrial mensual a partir de la informació obtinguda a través d'una enquesta dirigida a una mostra representativa de les empreses espanyoles. No obstant això, a nivell regional, les dificultats per a realitzar un seguiment de l'activitat industrial són majors a causa de l'escassesa d'informació estadística. Durant els últims anys, diferents institucions públiques i privades han començat a elaborar indicadors d'activitat per a algunes regions espanyoles, encara que a partir de metodologies no homogènies, de manera que aquests índexs no són directament comparables. Per a corregir aquesta situació, en diferents fòrums s'ha proposat emprar la metodologia utilitzada per l'Institut d'Estadística de Catalunya (IEC) per a la comunitat catalana com alternativa per a aquelles comunitats espanyoles que no disposen d'un indicador de l'activitat industrial, atès que per a Catalunya resulta una metodologia adequada. En aquest treball s'estudia la idoneïtat d'estendre aquesta metodologia a la resta de regions espanyoles. Per a això, es construeixen uns indicadors d'acord amb la metodologia del IEC i es comparen amb els índexs regionals obtinguts per mètodes directes per a tres de les quatre regions que existeixen: Andalusia, Astúries i Euskadi

Growth, convergence and public investment : a bayesian model averaging approach

The aim of this paper is twofold. First, we study the determinants of economic growth among a wide set of potential variables for the Spanish provinces (NUTS3). Among others, we include various types of private, public and human capital in the group of growth factors. Also,we analyse whether Spanish provinces have converged in economic terms in recent decades. Thesecond objective is to obtain cross-section and panel data parameter estimates that are robustto model speci¯cation. For this purpose, we use a Bayesian Model Averaging (BMA) approach.Bayesian methodology constructs parameter estimates as a weighted average of linear regression estimates for every possible combination of included variables. The weight of each regression estimate is given by the posterior probability of each model.

Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

Personality and Culture Cross-Cultural Psychology at the Next Crossroads

A review of nearly three decades of cross-cultural research shows that this domain still has to address several issues regarding the biases of data collection and sampling methods, the lack of clear and consensual definitions of constructs and variables, and measurement invariance issues that seriously limit the comparability of results across cultures. Indeed, a large majority of the existing studies are still based on the anthropological model, which compares two cultures and mainly uses convenience samples of university students. This paper stresses the need to incorporate a larger variety of regions and cultures in the research designs, the necessity to theorize and identify a larger set of variables in order to describe a human environment, and the importance of overcoming methodological weaknesses to improve the comparability of measurement results. Cross-cultural psychology is at the next crossroads in it's development, and researchers can certainly make major contributions to this domain if they can address these weaknesses and challenges.

Growth, convergence and public investment : a bayesian model averaging approach

The aim of this paper is twofold. First, we study the determinants of economic growth among a wide set of potential variables for the Spanish provinces (NUTS3). Among others, we include various types of private, public and human capital in the group of growth factors. Also,we analyse whether Spanish provinces have converged in economic terms in recent decades. Thesecond objective is to obtain cross-section and panel data parameter estimates that are robustto model speci¯cation. For this purpose, we use a Bayesian Model Averaging (BMA) approach.Bayesian methodology constructs parameter estimates as a weighted average of linear regression estimates for every possible combination of included variables. The weight of each regression estimate is given by the posterior probability of each model.

Algunes reflexions sobre la construcció d'indicadors indirectes pel seguiment de l'activitat industrial regional

En un gran nombre d'economies, l'evolució de la producció industrial s'analitza a partir de la informació sobre el Producte Industrial Brut i/o el Valor Afegit Brut que proporcionen les Comptabilitats Nacionals. A Espanya, la utilització d'aquestes dades presenta el problema que no estan disponibles tan ràpidament com seria desitjable. En conseqüència, no és possible realitzar un seguiment a curt termini de l'activitat industrial a partir dels mateixos. Per a solucionar aquest problema, l'Institut Nacional d'Estadística elabora un Índex de Producció Industrial mensual a partir de la informació obtinguda a través d'una enquesta dirigida a una mostra representativa de les empreses espanyoles. No obstant això, a nivell regional, les dificultats per a realitzar un seguiment de l'activitat industrial són majors a causa de l'escassesa d'informació estadística. Durant els últims anys, diferents institucions públiques i privades han començat a elaborar indicadors d'activitat per a algunes regions espanyoles, encara que a partir de metodologies no homogènies, de manera que aquests índexs no són directament comparables. Per a corregir aquesta situació, en diferents fòrums s'ha proposat emprar la metodologia utilitzada per l'Institut d'Estadística de Catalunya (IEC) per a la comunitat catalana com alternativa per a aquelles comunitats espanyoles que no disposen d'un indicador de l'activitat industrial, atès que per a Catalunya resulta una metodologia adequada. En aquest treball s'estudia la idoneïtat d'estendre aquesta metodologia a la resta de regions espanyoles. Per a això, es construeixen uns indicadors d'acord amb la metodologia del IEC i es comparen amb els índexs regionals obtinguts per mètodes directes per a tres de les quatre regions que existeixen: Andalusia, Astúries i Euskadi

Objetivo: cuantificar la reproducción

Se estudian en este trabajo algunas magnitudes relacionadas con el enfoque de la reproducción social. Ante todo se hace hincapié en tres ideas fundamentales, las nociones de: a) Salidas menos entradas; b) Salidas dividido por entradas; c) Subsistemas. A continuación se subrayan los obstáculos para la cuantificación directa de estos conceptos, y se repasan las vías sugeridas para sortear las dificultades (por medio de constructos teóricos propuestos por Leontief, von Neumann y Sraffa). Luego se examinan dos nuevos indicadores: la ¿tasaespecífica de excedente¿, que se refiere a los bienes autorreproducibles, y el ¿coeficiente neto de reproducción¿, que se predica de todos los bienes básicos. De pasada se apuntan algunas pistas para establecer indicadores del mismogénero en campos como la economía ecológica y la economía feminista. Por último, se anotan algunas conjeturas relacionadas con la dirección adecuada del cambio técnico

Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology.

PURPOSE: Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS: Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS: Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS: Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.