Combining MAR elements and transposable vectors for more reliable transgene integration and expression

Integration without cytotoxic effects and long-term expression of a transgene constitutes a major challenge in gene therapy and biotechnology applications. In this context, transposons represent an attractive system for gene transfer because of their ability to promote efficient integration of a transgene in a variety of cell lines. However, the transgene integration can lead to insertional mutagenesis and/or unstable transgene expression by epigenetic modifications. These unwanted events may be limited by the use of chromatin control elements called MARs (matrix attachment regions). Indeed, the insertion of these DNA elements next to the transgene usually results in higher and more stable expression by maintaining transgene chromatin in an active configuration and preventing gene silencing. In this study, we tested if the inclusion of the MAR 1-68 in the piggyBac transposon system may lead to efficient and safer transgene integration and ensure reliable stable and long-term expression of a transgene. The MAR-containing transposon construct was tested in CHO cells, for biotechnology applications, and in mesoangioblast cells that can differentiate into muscle cells and are important candidates for potential stem cell therapies of myopathies. We showed that the addition of the MAR 1 -68 in the piggyBac transposon did not interfere with transposition, thereby maintaining high frequency of transgene integrations in these cells. Moreover, the MAR allowed higher transgene expression from fewer transposon integration events. We also found that enriched transgene-expressing cell populations could be obtained without the need of selection pressure. Since antibiotic-enforced selection protocols often result in a higher integrated copy number and mosaic expression patterns, this strategy could benefit many applications in which a low copy number of integrated transgenes and antibiotic-free conditions are desired. In addition, the intramuscular transplantation of mouse tibialis anterior muscles with mesoangioblasts containing the transposon led to widespread and sustained myofiber transgene expression after differentiation of these cells in vivo. These findings indicated that piggyBac vectors may provide a viable approach to achieve stable gene transfer in the context of Duchenne muscular dystrophy therapy. - L'intégration sans effets cytotoxiques et l'expression à long terme d'un transgène constituent un défi majeur en thérapie génique et en biotechnologie. Dans ce contexte, les transposons représentent un système attrayant pour le transfert de gènes en raison de leur capacité à promouvoir l'intégration efficace d'un transgène dans une variété de lignées cellulaires. Toutefois, l'intégration d'un transgène peut conduire à une mutagénèse insertionnelle et/ou à une expression instable due au silençage du transgène suite à des modifications épigénétiques. Ces événements indésirables de silençage génique peuvent être diminués par l'utilisation d'éléments de contrôle de la chromatine appelés MAR (matrix attachment region). En effet, l'insertion de ces éléments d'ADN à proximité du transgène se traduit généralement par une expression plus élevée et plus stable de celui-ci, en permettant le maintien d'une chromatine dans une configuration active autour du transgène et en empêchant l'inactivation du gène. Dans cette étude, nous avons testé si l'inclusion du MAR 1-68 dans le système transposon piggyBac peut améliorer l'efficacité d'intégration de façon sécuritaire et l'expression à long terme d'un transgène. Le transposon contenant l'élément MAR a été testé dans les cellules CHO, couramment utilisées en biotechnologie, et dans des cellules progénitrices appelées mésoangioblastes, qui peuvent se différencier en cellules musculaires, et qui constituent ainsi des candidats prometteurs pour la thérapie à partir de cellules souches de patients souffrant de myopathie. Nous avons montré que l'addition du MAR 1-68 dans le transposon piggyBac n'interfère pas avec la transposition et permet de maintenir une fréquence élevée d'intégration du transgène dans ces deux types cellulaires. De plus, il semble que cette association mène à une meilleure expression du transgène à partir de peu d'événements d'intégration du transposon. En outre, ces populations enrichies en cellules exprimant de façon stable le transgène ont pu être obtenues sans avoir recours à une pression de sélection. Etant donné que les protocoles de sélection basée sur l'utilisation d'antibiotiques conduisent souvent à un nombre plus élevé de copies intégrées et à la variégation de l'expression du transgène et qu'ils impliquent une longue culture in vitro, cette stratégie pourrait profiter à des applications pour lesquelles on souhaite un faible nombre de copies intégrées et/ou l'utilisation d'antibiotiques n'est pas souhaitable. De plus, la transplantation intramusculaire de mésoangioblastes contenant le transposon dans le muscle tibial antérieur de souris a conduit, après la différentiation de ces cellules in vivo, à une expression constante et étendue du transgène dans les myofibres. Ces résultats indiquent que les vecteurs piggyBac pourraient fournir une approche viable pour assurer un transfert de gènes stables dans le contexte d'un traitement de la dystrophic musculaire de Duchenne.

Report of Recommendations to the Iowa Department of Cultural Affairs, June 30, 2002

State Agency Audit Report

Conditional and syllogistic deductive tasks dissociate functionally during premise integration.

Deduction allows us to draw consequences from previous knowledge. Deductive reasoning can be applied to several types of problem, for example, conditional, syllogistic, and relational. It has been assumed that the same cognitive operations underlie solutions to them all; however, this hypothesis remains to be tested empirically. We used event-related fMRI, in the same group of subjects, to compare reasoning-related activity associated with conditional and syllogistic deductive problems. Furthermore, we assessed reasoning-related activity for the two main stages of deduction, namely encoding of premises and their integration. Encoding syllogistic premises for reasoning was associated with activation of BA 44/45 more than encoding them for literal recall. During integration, left fronto-lateral cortex (BA 44/45, 6) and basal ganglia activated with both conditional and syllogistic reasoning. Besides that, integration of syllogistic problems additionally was associated with activation of left parietal (BA 7) and left ventro-lateral frontal cortex (BA 47). This difference suggests a dissociation between conditional and syllogistic reasoning at the integration stage. Our finding indicates that the integration of conditional and syllogistic reasoning is carried out by means of different, but partly overlapping, sets of anatomical regions and by inference, cognitive processes. The involvement of BA 44/45 during both encoding (syllogisms) and premise integration (syllogisms and conditionals) suggests a central role in deductive reasoning for syntactic manipulations and formal/linguistic representations.

Report of Recommendations to the Iowa Department of Cultural Affairs, June 30, 2003

State Agency Audit Report

El reto del paisaje urbano en China, del Mall Comercial al espacio público

Las categorías del espacio introvertido y extrovertido definen por excelencia la estructura espacial, que ordena el ámbito de lo privado y lo público en la ciudad. La dualidad del espacio abierto y cerrado, vinculado a la estructura espacial de la ciudad clásica China, quedaba ordenado, justificado y vinculado mediante una estructura espacial muy definida. Sin embargo en el modelo de ciudad contemporánea, aflora la cuestión de la integración de estas categorías espaciales y su articulación dentro de una estructura urbana heterogénea y fuertemente alterada. ¿Cuáles serán los factores de cambio de la transición de un espacio comunitario introvertido a un espacio cívico abierto? Este proceso es un proceso que debe discurrir a un doble nivel espacial y político. Debe discurrir en paralelo a la transición de las categorías espaciales que ordenan identitariamente el ámbito urbano y la progresiva transición socio-cultural y política de una sociedad que debe gradualmente identificar y adoptar como propias las instituciones de gobierno local y su escenario, la ciudad.

O conflito como realidade e desafio cultural no exercício da gerência do enfermeiro

O exercício da gerência do enfermeiro é permeado de conflitos que podem ser interpretados pelos referenciais de cultura. O presente estudo tem como objetivo denotar especificidades culturais, analisadas segundo a Teoria Interpretativa da Cultura de Geertz, que se expressam como conflitos no âmbito da gerência do enfermeiro num Hospital Universitário. Os resultados denotam a incorporação de elementos ideológicos e de mecanismos de controle e de poder, que manifestam sua origem na forma de organização do trabalho. Percebe-se a efetivação de políticas alicerçadas em valores da própria profissão. As práticas exercidas destacam uma construção cultural que elucida algumas compreensões dos processos cognitivos, sociais e comportamentais, pois estes organizam as interpretações e as respostas para os eventos da prática gerencial do enfermeiro. Os resultados deste estudo apontam para a importância da cultura organizacional no exercício da gerência na enfermagem diante das incertezas laborais, na complexidade do ambiente hospitalar

Cross-cultural adaptations of the Oxford-12 HIP score to the French speaking population.

BACKGROUND: An objective measurement of surgical procedures outcomes is inherent to professional practices quality control; this especially applies in orthopaedics to joint replacement outcomes. A self-administered questionnaire offers an attractive alternative to surgeon's judgement but is infrequently used in France for these purposes. The British questionnaire, the 12-item Oxford Hip Score (OHS) was selected for this study because of its ease of use. HYPOTHESIS: The objective of this study was to validate the French translation of the self-assessment 12-item Oxford Hip Score and compare its results with those of the reference functional scores: the Harris Hip Score (HHS) and the Postel-Merle d'Aubigné (PMA) score. MATERIALS AND METHODS: Based on a clinical series of 242 patients who were candidates for total hip arthroplasty, the French translation of this questionnaire was validated. Its coherence was also validated by comparing the preoperative data with the data obtained from the two other reference clinical scores. RESULTS: The translation was validated using the forward-backward translation procedure from French to English, with correction of all differences or mistranslations after systematized comparison with the original questionnaire in English. The mean overall OHS score was 43.8 points (range, 22-60 points) with similarly good distribution of the overall value of the three scores compared. The correlation was excellent between the OHS and the HHS, but an identical correlation between the OHS and the PMA was only obtained for the association of the pain and function parameters, after excluding the mobility criterion, relatively over-represented in the PMA score. DISCUSSION AND CONCLUSION: Subjective questionnaires that contribute a personal appreciation of the results of arthroplasty by the patient can easily be applied on a large scale. This study made a translated and validated version of an internationally recognized, reliable self-assessment score available to French orthopaedic surgeons. The results obtained encourage us to use this questionnaire as a complement to the classical evaluation scores and methods.

Conhecimento e significado cultural da menopausa para um grupo de mulheres

Este estudo etnográfico, realizado junto a um grupo de mulheres, teve como objetivo verificar o nível de conhecimento sobre a menopausa e compreender como ocorre a vivência deste estágio biológico próprio do sexo feminino. Para a coleta dos dados, usou-se a observação participante, a entrevista, mediante uso de gravador, além de desenhos previamente reproduzidos. A análise foi efetuada com base nos pressupostos teóricos, de método etnográfico e de alguns pressupostos do interpretativismo biográfico. Das narrativas coletadas, extraíram-se as categorias e os temas culturais. Assim, foi possível verificar que o processo da menopausa era entendido como resultante de uma construção singular, estando integrado a uma rede de significados, instituídos pelo grupo, os quais condicionam o conhecimento e a vivência dentro de determinados padrões culturais, entre eles, o fato de que a menopausa significa deixar de ser mulher.

Diagnòstic en profunditat sobre la integració dels i les joves de 14 i 18 anys migrats a Catalunya

La finalitat d'aquest projecte és aprofundir en el procés d'integració socioeducativa de la joventut migrada a Catalunya, específicament, dels i les joves entre 14 i 18 anys. Entre els resultats destaquem que els ioves tenen un concepte força tancat i excloent del concepte de ciutadania com a estatus , i un baix grau, de coneixement de la diversitat cultural, especialment els joves autòctons, tot i que tenen una actitud de reconeixement i acceptació, favorable a la convivència intercultural, tant al centre educatiu com al seu barri. No obstant, tenen una visió de la diversitat cultural i del fet migratori en clau de coexistència, sense el reconeixement que suposaria un pas més cap a la convivència. La immigració és vista com un col•lectiu vulnerable, amb un baix nivell econòmic i cultural i que també és percebut com un problema i, fins i tot, com a causant de les dificultutats que actualment pateix i travessa el conjunt de la societat. Els joves tendeixen a relacionar-se amb el seu grup cultural, i responen de forma passiva en la comprensió i actuació davant dels problemes i assumptes públics. Entre els elements que valoren per sentir-se ciutadà té una especial importància la llengua. Eis joves que fa més de 10 anys que viuen a Catalunya són els que tendeixen a tenir un sentiment de pertinença cívica amb el lloc de residència. De fet, com mes temps porten els joves estrangers vivint al lloc d’acollida, perden importància elements d’identificació cultural del país d’origen, però la religió, les celebracions populars, l’art i la tendència a formar parella són elements identitaris del lloc d’origen, més estables. Es demana una intervenció urgent en diversos nivells i destinades a diferents agents. Destaquem: la importància d’una acollida afectiva i efectiva; potenciar espais de trobada; treballar els prejudicis; transversalitzar l’educació intercultural; potenciació de l’aprenentatge de la llengua catalana; formació del professorat en competències interculturals.

Hours worked - Productivity puzzle: identification in fractional integration settings

A recent finding of the structural VAR literature is that the response of hours worked to a technology shock depends on the assumption on the order of integration of the hours. In this work we relax this assumption, allowing for fractional integration and long memory in the process for hours and productivity. We find that the sign and magnitude of the estimated impulse responses of hours to a positive technology shock depend crucially on the assumptions applied to identify them. Responses estimated with short-run identification are positive and statistically significant in all datasets analyzed. Long-run identification results in negative often not statistically significant responses. We check validity of these assumptions with the Sims (1989) procedure, concluding that both types of assumptions are appropriate to recover the impulse responses of hours in a fractionally integrated VAR. However, the application of longrun identification results in a substantial increase of the sampling uncertainty. JEL Classification numbers: C22, E32. Keywords: technology shock, fractional integration, hours worked, structural VAR, identification

Ser mulher portadora do HPV: uma abordagem cultural

Este trabalho foi realizado com o objetivo de investigar o nível de conhecimento das mulheres sobre o HPV, para, a partir daí, identificar crenças, mitos e tabus sobre a doença e analisar a influência destes elementos culturais no comportamento da mulher. Realizamos um estudo baseado nos pressupostos da teoria do cuidado transcultural. A pesquisa foi desenvolvida a partir do depoimento de quinze mulheres que realizavam tratamento para HPV. Através da investigação, foi possível perceber que, apesar das inúmeras fontes de informação sobre DST, o HPV ainda é uma doença desconhecida e cercada de mistério. Este desconhecimento, interagindo com fatores culturais, favorece o desenvolvimento de conceitos equivocados, tais como crenças e mitos.

Adaptação cultural e validação do instrumento The Bowel Function in the Community para o Brasil

Estudos sobre hábito intestinal, considerando cultura, hábitos alimentares e de vida entre outros, não existem no Brasil. O objetivo deste artigo é apresentar o The Bowel Function in the Community, ferramenta específica para avaliação do hábito intestinal das populações, adaptado e validado para o Brasil. O processo de adaptação cultural incluiu tradução, retrotradução e avaliação por comitê de especialistas, obtendo-se uma versão traduzida do instrumento, posteriormente submetida a análises que atestaram a validade de conteúdo do mesmo. A confiabilidade inter-observadores e estabilidade (teste-reteste) foram confirmadas por níveis de concordância de boa a excelente e de moderada a excelente para a maioria das questões e agrupamentos do instrumento. Concluiu-se que a versão adaptada do instrumento pode ser aplicada em nosso meio para dar continuidade ao processo de validação, bem como para ampliar o conhecimento do hábito intestinal na população brasileira.