Submitted by Suethene Souza (suethene.souza@ufpe.br) on 2015-03-09T19:15:35Z No. of bitstreams: 2 DISSERTAÇÃO Anderson Diego Farias da Silva.pdf: 1954053 bytes, checksum: a236bf641f60387745d491e1418d6598 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-09T19:15:35Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Anderson Diego Farias da Silva.pdf: 1954053 bytes, checksum: a236bf641f60387745d491e1418d6598 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-09-30 / Este estudo teve por objetivo desvelar como ocorreu o processo de regulação cultural em meio à [re]conversão do Arranjo Produtivo Local (APL) monotemático de Tecnologia da Informação e Comunicação (TIC), denominado Porto Digital, em um polo de Economia Criativa. Criado em 2001, o Porto Digital é um Arranjo Institucional constituído por uma hélice tríplice, compreendida pela atuação do poder público e privado por meio de uma Organização Social (OS), que busca promover uma rede colaborativa de produção cientifica e tecnológica extremamente complexa, na Cidade do Recife. Em 2011, o Porto Digital, por meio da lei municipal nº 17.762, expandiu o seu território de atuação e incorporou setores da economia criativa, consolidados por meio da criação do Portomídia - Centro de Empreendedorismo e Tecnologia da Economia Criativa. Para tanto, esta pesquisa qualitativa buscou compreender, mediante realização de um estudo de caso, como ocorreu o processo de expansão do escopo de atuação do Porto Digital, sob o prisma dos Estudos Culturais (EC), especificamente no que tange a teoria da Regulação Cultural. Baseado nas mudanças que ocorrem nas relações tempo e espaço no mundo contemporâneo, e que sugerem a urgência em se revisar e descortinar outras formas de se compreender, decodificar e analisar o ambiente simbólico das organizações, onde certas perspectivas epistemometodológicas vigentes reduzem o papel da cultura a uma espécie de construto manipulável acrescentado ao desempenho empresarial, que a escolha pela abordagem dos EC se materializou, por possibilitar uma versatilidade teórica e o despertar crítico e reflexivo mais profundo, considerando as relações de poder e a força política do indivíduo. Dessa forma, buscou-se averiguar como a compreensão de cultura nos EC poderá contribuir para o entendimento da dimensão cultural e o avanço das pesquisas apresentadas pela literatura dos estudos organizacionais sobre os APL brasileiros. Os resultados revelam que outros aspectos estão associados à regulação das aglomerações produtivas, não compreendidos pelas teorias vigentes nos estudos organizacionais e que estão estabelecidos por meio do poder institucional (regras e regulação), do poder econômico (relações de poder) e do poder subjetivo (o ato empreendedor, a força política do indivíduo).

Pesquisa descritivo-explicativa e exploratória sobre a relação entre Turismo e desenvolvimento regional que apresenta o modelo de análise APL Tur, elaborado com base em elementos de referenciais teóricos de economia, geografia, sociologia, administração, comunicação, antropologia e Turismo. O objetivo é mostrar se é possível realizar o desenvolvimento regional por meio do Turismo. Uma região é um contexto territorial delimitado por critérios geográficos, econômicos e políticos. Um dos modelos da análise e gestão do desenvolvimento regional é o Arranjo Produtivo Local APL, uma categoria especial de cluster. Em razão das limitações do APL, elabora-se o modelo particular de análise APL Tur Arranjo Produtivo Local de Turismo. O modelo APL Tur estrutura-se nas dimensões econômica, cultural e organizacional. Os elementos do desenvolvimento regional na dimensão econômica são: delimitação espacial; disparidades intra-regionais; externalidades; sustentabilidade ambiental; e inclusão social. Os elementos do Turismo circunscritos na dimensão econômica são: oferta e demanda; desempenho; priorização; exportação; circuito produtivo; interatividade extra-regional; e acessibilidade. Na dimensão cultural, destacam-se: aspectos históricos; acervos e incentivos; estética; produtos e atrativos; animação; e motivação e satisfação da comunidade. Na dimensão organizacional, têm-se os elementos: poder e capital social; gestão sistêmica; divulgação e imagem; mercadologia e comercialização; planejamento; empreendedorismo e inovação; e conhecimento. Realizou-se o teste do modelo APL Tur, aplicando-o à Região das Hortênsias (Rio Grande do Sul, Brasil), formada pelos municípios de Gramado, Canela, Nova Petrópolis e São Francisco de Paula. Com base no quadro de indicadores dos elementos das dimensões do modelo APL Tur, verifica-se que o Turismo contribui para o desenvolvimento regional, pois proporciona o ingresso, produção e distribuição de riquezas. Para que uma região se desenvolva economicamente por meio do Turismo, é preciso, entretanto, uma gestão adequada das dimensões e elementos do APL Tur, visando à realização dos seus indicadores. O modelo APL Tur é um instrumento adequado para diagnosticar e para identificar oportunidades de melhoria da atividade turística como polarizadora ou como alternativa do desenvolvimento regional. / This is a descriptive-explanatory and exploratory research about the relationship between Tourism and regional development that presents the LPS Tour analysis model and that was worked out taking its basis elements of theoretical references from economics, geography, sociology, administration, communication, anthropology and Tourism. The objective is to show if it is possible to accomplish regional development through Tourism. A region is a territorial context delimitated by geographical, economic, and political criteria. It is taken into consideration that the general model of Local Productive System of Tourism LPS, a especific cathegory of cluster, has limitations to the analysis and management of Tourism. For this reason, the LPS Tour Local Productive System of Tourism - particular model of analysis - is developed. The LPS model is structured in the economic, cultural, and organizational dimensions. The regional development elements in the economic dimension are: spatial delimitation; intra-regional dissimilarities; externalities; environmental sustainability; and social inclusion. The Tourism elements circumscribed in the economic dimension are: supply and demand; performance; prioritization; exportation; productive circuit; extra-regional interactivity; and accessibility. In the cultural dimension, the elements that stand out are: historical aspects; collections and incentives; esthetics; products and attractions; animation; and motivation and satisfaction of the community. In the organizational dimension, we have the following elements: power and stock capital; systemic management; divulgation and image; marketing and commercialization; planning; entrepreneurship and innovation; and knowledge. The test of the LPS Tour model was performed applying it to the Região das Hortênsias (Rio Grande do Sul, Brasil) (The Hydrangeas Region), that is formed by the municipal districts of Gramado, Canela, Nova Petrópolis, and São Francisco de Paula. Based on the panel of indicators of the dimensions elements of the LPS Tour model, it is possible to verify that Tourism contributes to the regional development because it accomplishes the ingression, production and distribution of richness. However, for a region to develop economically through Tourism, an adequate management of its dimensions and elements is necessary, aiming at the accomplishment of the LPS Tour indicators. The LPS Tour model is an adequate instrument to diagnose and to identify opportunities of improvement of the tourist activity, as a polarizer or as an alternative to regional development.

Made available in DSpace on 2017-07-10T18:34:01Z (GMT). No. of bitstreams: 1 Marlowa Zachow.pdf: 1273004 bytes, checksum: 081277ea29c172abe5d0122c9c704ac4 (MD5) Previous issue date: 2012-12-04 / This research aims to analyze the impact that the association of companies of Information Technology of Paraná has on their competitiveness. The process of globalization has brought the need for companies of all sizes move to compete with multinational giants, especially in this line of business. An alternative was the creation of a local productive arrangement, encouraged by governmental and nongovernmental organizations. Through the results of the survey found that companies know that the joint actions could enhance or improve competitiveness. It was noticed that there was a considerable growth in terms of turnover and number of employees after the association. Entrepreneurs realize the advantages it has for being part of the arrangement, noting among these advantages the issue of personnel training and training. It was also noticed that the arrangement researched is very young, because it had its foundation in 2008. There is plenty to evolve, realizing the need to attract more businesses to the arrangement, as well as other priority action plans that address all activities of information technology and communication. / Esta pesquisa tem por objetivo analisar o impacto que a associação das empresas de Tecnologia da Informação do Oeste do Paraná tem sobre a competitividade das mesmas. O processo de globalização trouxe a necessidade das empresas de qualquer porte em passar a competir com gigantes multinacionais, especialmente nesse ramo de atuação. Uma alternativa encontrada foi a criação de um arranjo produtivo local, incentivado por instituições governamentais e não governamentais. Através dos resultados da pesquisa constatou-se que as empresas sabem que as ações conjuntas melhoram ou poderiam melhorar a competitividade. Percebeu-se que houve um crescimento considerável em termos de faturamento e número de funcionários após a associação. Os empresários percebem as vantagens que tem por fazerem parte do arranjo, destacando entre essas vantagens a questão da capacitação de pessoal e treinamento. Percebeu-se ainda que o arranjo pesquisado é muito jovem, pois teve sua fundação em 2008. Há muito para evoluir, percebendo-se a necessidade de atrair mais empresas para o arranjo, bem como priorizar outros planos de ação que contemplem todas as atividades de tecnologia da informação e comunicação.

This study aims to analyze ways of creating and managing knowledge in Petrogas Sergipe Network. Accordingly, we analyze various forms of knowledge generated in the network, as well as its management. It presents significant content based on discussion Enterprises Networks, Local Production - APLs based on normative concepts and experiences in Oil and Gas Networks. Emphasis is also important concepts on Knowledge Management and organizational knowledge. The context presented by Petrogas Sergipe Network provides understand that organizations acquire competitiveness and market innovative, admittedly competitive process required by the economy, in which companies increasingly need to improve their skills for business continuity. Strategy that includes work in interorganizational network, presents itself as appropriate, because that constitutes a privileged space for interaction and cooperation between different actors that are part of the network. At the same time, you can see that the process of knowledge organizational management comprises an important condition in the development of interorganizational business networks in order to add value to the companies that comprise it. Therefore, Petrogas Sergipe Network has important significance for the economy in Sergipe, in terms of income generation, employment and business opportunities, especially as an arrangement also fundamental for the consolidation of System Innovation and knowledge generation in State of Sergipe. / O presente trabalho tem como objetivo, analisar formas de criação e gestão do conhecimento, na Rede PETROGAS Sergipe. Nesse sentido, são analisadas diversas formas do conhecimento gerado nessa rede, bem como sua gestão. Apresenta-se significativo conteúdo com base na discussão sobre Redes de Empresas, Arranjos Produtivos Locais - APLs com base em conceitos normativos e experiências em Redes de Petróleo e Gas. Enfatiza-se também, importantes conceitos sobre o Conhecimento Organizacional e a Gestão do conhecimento organizacional. O contexto apresentado pela Rede PETROGÁS Sergipe, proporciona entender que as organizações adquirem competitividade inovativa e mercadológica, reconhecidamente exigida pelo processo concorrencial da economia, no qual as empresas necessitam cada vez mais aprimorar suas habilidades para continuidade dos seus negócios. Desta forma, a estratégia que compreende o trabalho em rede interorganizacional, apresenta-se como adequada, em virtude de que se constitui como espaço privilegiado de cooperação e interação entre diversos atores que fazem parte da rede. Ao mesmo tempo, é possível perceber que o processo de gestão do conhecimento organizacional, compreende uma condição importante no desenvolvimento de redes empresariais interorganizacionais, no sentido de agregar valor às empresas que a compõem. Portanto, a Rede PETROGÁS Sergipe apresenta importante significado para a economia sergipana, do ponto de vista da geração de renda, emprego e oportunidades de negócios, destacando- se também como um arranjo fundamental para a consolidação do Sistema de Inovação e de geração de conhecimento no Estado de Sergipe.

Trois protéines de la famille TRIM (Motif TRIpartite), TIF1α, β (Transcriptional Intermediary Factor 1) et PML (ProMyelocytic Leukaemia¬), font l’objet de cette étude. TIF1α est connu comme un coactivateur des récepteurs nucléaires et TIF1β comme le corépresseur universel des protéines KRAB-multidoigt de zinc dont le prototype étudié ici est ZNF74. PML possède divers rôles dont le plus caractérisé est celui d’être l’organisateur principal et essentiel des PML-NBs (PML-Nuclear Bodies), des macrostructures nucléaires très dynamiques regroupant et coordonnant plus de 40 protéines. Il est à noter que la fonction de TIF1α, β et PML est régulée par une modification post-traductionnelle, la sumoylation, qui implique le couplage covalent de la petite protéine SUMO (Small Ubiquitin like MOdifier) à des lysines de ces trois protéines cibles. Cette thèse propose de développer des méthodes utilisant le BRET (Bioluminescence Resonance Energy Transfert) afin de détecter dans des cellules vivantes et en temps réel des interactions non-covalentes de protéines nucléaires mais aussi leur couplage covalent à SUMO. En effet, le BRET n’a jamais été exploré jusqu’alors pour étudier les interactions non-covalentes et covalentes de protéines nucléaires. L’étude de l’interaction de protéines transcriptionnellement actives est parfois difficile par des méthodes classiques du fait de leur grande propension à agréger (famille TRIM) ou de leur association à la matrice nucléaire (ZNF74). L’homo et l’hétérodimérisation de TIF1α, β ainsi que leur interaction avec ZNF74 sont ici testées sur des protéines entières dans des cellules vivantes de mammifères répondant aux résultats conflictuels de la littérature et démontrant que le BRET peut être avantageusement utilisé comme alternative aux essais plus classiques basés sur la transcription. Du fait de l’hétérodimérisation confirmée de TIF1α et β, le premier article présenté ouvre la possibilité d’une relation étroite entre les récepteurs nucléaires et les protéines KRAB- multidoigt de zinc. Des études précédentes ont démontré que la sumoylation de PML est impliquée dans sa dégradation induite par l’As2O3 et dépendante de RNF4, une E3 ubiquitine ligase ayant pour substrat des chaînes de SUMO (polySUMO). Dans le second article, grâce au développement d’une nouvelle application du BRET pour la détection d’interactions covalentes et non-covalentes avec SUMO (BRETSUMO), nous établissons un nouveau lien entre la sumoylation de PML et sa dégradation. Nous confirmons que le recrutement de RNF4 dépend de SUMO mais démontrons également l’implication du SBD (Sumo Binding Domain) de PML dans sa dégradation induite par l’As2O3 et/ou RNF4. De plus, nous démontrons que des sérines, au sein du SBD de PML, qui sont connues comme des cibles de phosphorylation par la voie de la kinase CK2, régulent les interactions non-covalentes de ce SBD mettant en évidence, pour la première fois, que les interactions avec un SBD peuvent dépendre d’un évènement de phosphorylation (“SBD phospho-switch”). Nos résultats nous amènent à proposer l’hypothèse que le recrutement de PML sumoylé au niveau des PML-NBs via son SBD, favorise le recrutement d’une autre activité E3 ubiquitine ligase, outre celle de RNF4, PML étant lui-même un potentiel candidat. Ceci suggère l’existence d’une nouvelle relation dynamique entre phosphorylation, sumoylation et ubiquitination de PML. Finalement, il est suggéré que PML est dégradé par deux voies différentes dépendantes de l’ubiquitine et du protéasome; la voie de CK2 et la voie de RNF4. Enfin une étude sur la sumoylation de TIF1β est également présentée en annexe. Cette étude caractérise les 6 lysines cibles de SUMO sur TIF1β et démontre que la sumoylation est nécessaire à l’activité répressive de TIF1β mais n’est pas impliquée dans son homodimérisation ou son interaction avec la boîte KRAB. La sumoylation est cependant nécessaire au recrutement d’histones déacétylases, dépendante de son homodimérisation et de l’intégrité du domaine PHD. Alors que l’on ne connaît pas de régulateur physiologique de la sumoylation outre les enzymes directement impliquées dans la machinerie de sumoylation, nous mettons en évidence que la sumoylation de TIF1β est positivement régulée par son interaction avec le domaine KRAB et suggérons que ces facteurs transcriptionnels recrutent TIF1β à l’ADN au niveau de promoteur et augmentent son activité répressive en favorisant sa sumoylation. / Three TRIM proteins (TRIpartite Motif), TIF1α, β (Transcriptional Intermediary Factor 1) and PML (ProMyelocytic Leukaemia¬), were studied in this thesis. TIF1α is a nuclear receptor coactivator and TIF1β is the universal corepressor of the KRAB-zinc finger repressor family of which, ZNF74 is studied here as a prototypic member. PML functions as a tumor suppressor and is the essential organiser of PML-NBs (PML-Nuclear Bodies) which are very dynamic nuclear macrostructures containing more than 40 proteins. The function of these three TRIM proteins is regulated by sumoylation, a post-translational modification involving the covalent linkage of SUMO (Small Ubiquitin like MOdifier) to specific targets lysine. In this thesis, we propose to develop new methods based on BRET (Bioluminescence Resonance Energy Transfer) to detect non-covalent nuclear protein interactions but also covalent linkage to SUMO in real time in living cells. To date, BRET was never used to assess non-covalent or covalent nuclear protein interactions. Studying transcriptionally active protein interactions represents a challenge by classical methods in particular when proteins have a tendency to aggregate (TRIM family) or when characterizing nuclear matrix proteins (ZNF74). In the first article, homo- and heterodimerisation of TIF1 α and β as well as their interaction with ZNF74 was assessed by BRET using full length proteins in living mammalian cells. We ascertained the heterodimerisation of TIF1α and β. Whereas ZNF74 interacts strongly with TIF1β, no interaction was detected with TIF1α. However, we unravelled the existence of ternary complexes involving ZNF74, TIF1α and TIF1β. This suggested that a mechanisms for cross-talk between nuclear receptors and KRAB-zinc finger proteins. Thus, we showed that BRET can be advantageously used as a non-transcription-based interaction system for studying transcriptionally active proteins, including nuclear matrix proteins, in living cells. Previous studies have shown that the sumoylation of PML (a tumour suppressor) is involved in its proteasome degradation that is As2O3-inducible and dependent on the polySUMO E3 ubiquitin ligase, RNF4. In the second article, we describe the development of a new application of the BRET method for the detection of covalent and non-covalent interactions with SUMO. Owing to this SUMO BRET assay, we established that the As2O3 / RNF4-mediated degradation of PML, not only depends on PML sumoylation as previously demonstrated, but also on the integrity of its SUMO binding domain. We also demonstrated that As2O3 which increases PML sumoylation, also enhances PML / RNF4 interaction. Our study revealed that most PML SBD non covalent interactions with sumoylated proteins required the phosphorylation of serines within PML SBD that were previously described as target sites for CK2 kinase and involved in PML degradation. Despites the involvement of PML SBD in RNF4-mediated degradation, these serines which function as an SBD phospho-switch, were not required for RNF4-mediated degradation. This suggested that CK2- and RNF4-mediated PML degradation represents two distinct pathways triggering PML ubiquitin / proteasome-dependent degradation. At last, our study led to the hypothesis that the recruitment of sumoylated PML at PML-Nuclear Bodies subnuclear structures via the PML SBD and / or possibly an E3 ubiquitin ligase activity other than RNF4 (PML itself being candidate) may favour PML degradation. Our study also stresses the dynamic involvement of three PML post-translational modifications, phosphorylation, sumoylation and ubiquitination in its degradation. A third article addressing the role of TIF1β sumoylation is presented in the Appendix. We characterized the 6 SUMO targets lysine of TIF1β and demonstrated that sumoylation is required for TIF1β transcriptional repressive activity. This is in part explained by the fact that TIF1β sumoylation is a pre-requisite for histone deacetylases recruitment since TIF1β repressive activity is partly dependent on histone deacetylases. We found that TIF1β sumoylation does not influence its homodimerisation or interaction with the KRAB box of KRAB zinc finger proteins recruiting TIF1β to promoters. TIF1β sumoylation is however relying on the integrity of TIF1β PHD finger and on its self-oligomerisation. Interestingly, we demonstrated that TIF1β sumoylation is positively regulated by its interaction with KRAB domain. It is thus suggested that KRAB-zinc finger proteins recruit TIF1β at DNA promoters where they trigger increase of TIF1β sumoylation and thus enhance its repressive activity.

Nous avons étudié la famille des composés isostructuraux du bore alpha (BxC, B1202, "B12As2", "B12P2") grâce à la spectrométrie infrarouge et leurs propriétés notamment électriques et mécanique (microdureté). No us avons préparé du bore pur sous forme alpha par réduction de BBr3 par l'hydrogène, car la dissociation de BI3 donne de mauvais résultats. La meilleure valorisation industrielle de ces céramiques ne pouvant se faire que sur des échantillons massifs, nous avons comprimé à chaud du bore et du sous oxyde de bore. Nous avons comparé les modèles de frittage et les cinétiques calculées à partir des compressions, avant de développer une méthode informatique graphique originale, pouvant avoir des appliquations tant au niveau de la qualité, qu'au niveau informatique lui même. Nous avons été conduits à mettre au point des barrières de diffusion chimique qui ont fait l'objet d'un brevet. Différentes propriétés physiques du bore et des borures ont été envisagées : pour les diverses phases : métallographies, pour le bore : diffraction X, microdureté, résistivité électrique, coefficient Seebeck, diffusivité thermique, vitesse du son. pour le sous oxyde de bore : diffraction X, stabilité de la poudre, oxydation d'échantillons massifs, diffusivité thermique.

The aim of this essay is to examine the supervisor’s view on the structure and content of the tasks given to students during their vocational training. The investigation could be of help for teachers when devising new vocational training tasks. The study is based upon qualitative interviews with five supervisors, all of whom work at preschools. The supervisors were asked to grade all the vocational training tasks on a scale from one (easy) to five (difficult). The results were then used as a starting point for a discussion with the supervisors about their grading of the tasks. When devising vocational training tasks, one of the foundations is the knowledge requirements as expressed in the syllabus, and how they are reflected in the vocational training. An important aspect of the student’s learning is the supervisor’s ability to clarify the goals and criteria of vocational training. The supervisor plays an important role and is responsible for the student’s vocational training period. This study has given me a better understanding of the supervisor’s view on the structure of the vocational training tasks and I have been given many useful suggestions as to how it can be improved to strengthen the quality of the education. The Swedish Schools Inspectorate carried out an investigation in 2011 and noticed shortcomings in the grading of the vocational training, which is interesting and relevant for my study. The supervisor assesses the student’s performance during the vocational training according to a certain scale of grades. The findings suggest that we need to revise the structure and assessment of the vocational training tasks. At the upper secondary school used in this study, the tasks are devised in a way that makes it difficult for the supervisor to support the student in his/her learning process, because the tasks are too numerous and the instructions contain many ill-defined terms. The supervisors also want closer contact with, and visits from, the upper secondary school teachers, so that both the former and the students can receive more feedback and better support. / Syftet med denna uppsats är att undersöka handledares syn på praktikuppgifternas utformning och innehåll. Undersökningen kan vara till hjälp för lärare när nya praktikuppgifter ska utarbetas. Arbetet bygger på fem kvalitativa intervjuer som har genomförts med handledare som är verksamma inom förskoleverksamheten. Handledarna fick göra en bedömning av praktikuppgifterna utifrån en svårhetsskala från ett till fem, där fem är mycket svår och ett är mycket lätt. Utifrån skalans bedömning samtalade vi kring varför de gjord den bedömningen. Vid utformning av praktikuppgifter är en av byggstenarna kunskapsmålen som kopplas till praktiken utifrån ämnesplanerna. En viktig del i elevens lärande är att handledaren kan tydliggöra mål och kriterier i det arbetsplatsförlagda lärandet. Handledaren har en viktig roll och ansvar för eleven under praktikperioden. Under undersökningens gång har jag fått inblick i hur handledarna ser på praktikuppgifternas utformning och fått bra förslag på hur de kan förbättras för att stärka kvalitén på utbildningen. Då skolinspektionen genomfört en kvalitets undersökning 2011 och sett brister i betygsättningen kopplat till praktiken är detta intressant och relevant att koppla till mitt arbete. Handledarna sätter omdöme på eleven under praktikperioden utifrån en betygsskala. Resultatet tyder på att vi behöver se över praktikuppgifternas utformning och bedömning innan eleverna går ut på praktik i höst. Som praktikuppgifterna ser ut idag i åk 1 på den för studien aktuella gymnasieskolan har handledarna svårt att stödja elevens lärande, uppgifterna består av många svåra begrepp samt att de är för många till antalet. Det finns också önskemål om tätare kontakt med skolan samt praktikbesök för att stötta och ge återkoppling till både handledare och elev.

En mamíferos, así como en plantas, mutaciones en AND helicasas/ATPasas del la família SNF2, no solo afectan a la estructura de la cromatina, sino que también afectan al patrón global de la metilación del ADN. Sugiriendo una relación funcional entre la estructura de la cromatina y la epigenética. El complejo NuRD, el cual posee una ATPasa de la familía SNF2, está relacionado con la represión de la transcripción y en el remodelamiento de la cromatina. Nuestro laboratorio demostró que la proteína leucémica PML-RARα reprime la transcripción de sus genes diana por el reclutamiento de DNMTs y el complejo PRC2. En esta tesis, demostramos una relación directa del complejo NuRD en la represión génica y en los cambios epigenéticos en la leucemia promielocítica aguda (APL). Mostramos que PML-RARα se une y recluta NuRD a sus genes diana, incluyendo el gen supresor de tumores RAR2, facilitando que el complejo de Polycomb se reclute y metile la lisina 27 de la histona H3. Tratamiento con Acido Retinóico (RA), el qual se utiliza en pacientes, reduce la ocupación de NuRD en células leucémicas. Eliminando NuRD no solo provoca que las histonas no se deacetilen y que la cromatina no se compacte, sino que también provoca que tanto la metilación del ADN y de las histonas no se produzca, así como la represión génica del gen RAR2, favoreciendo la diferenciación celular. Nuestros resultados caracterizan un nuevo papel del complejo NuRD en el establecimiento de los patrones epigenéticos en APL, demostrando una relación esencial entre la estructura de la cromatina y epigenética durante el desarrollo de la leucemia, pudiéndose aplicar a la terapia de esta enfermedad. / In mammals, as in plants, mutations in SNF2-like DNA helicases/ATPases were shown to affect not only chromatin structure but also global methylation patterns, suggesting a potential functional link between chromatin structure and epigentic marks. The SNF2-like containing NuRD complex is involved in gene transcriptional repression and chromatin remodeling. We have previously shown that the leukemogenic protein PMLRARα represses target genes through recruitment of DNMTs and Polycomb complex. In this thesis, we demonstrate a direct role of the NuRD complex in aberrant gene repression and transmission of epigenetic repressive marks in acute promyelocytic leucemia (APL). We show that PML-RARα binds and recruits NuRD to target genes, including to the tumor-suppressor gene RAR2. In turn, the NuRD complex facilitates Polycomb binding and histone methylation at lysine 27. Retinoic acid treatment reduced the promoter occupancy of the NuRD complex. Knock-down of the NuRD complex in leukemic cells not only prevented histone deacetylation and chromatin compaction, but also impaired DNA and histone methylation as well as stable silencing, thus favoring cellular differentiation. These results unveil an important role for NuRD in the establishment of altered epigenetic marks in APL, demonstrating an essential link between chromatin structure and epigenetics in leukemogenesis that could be exploited for therapeutic intervention.

Trois protéines de la famille TRIM (Motif TRIpartite), TIF1α, β (Transcriptional Intermediary Factor 1) et PML (ProMyelocytic Leukaemia¬), font l’objet de cette étude. TIF1α est connu comme un coactivateur des récepteurs nucléaires et TIF1β comme le corépresseur universel des protéines KRAB-multidoigt de zinc dont le prototype étudié ici est ZNF74. PML possède divers rôles dont le plus caractérisé est celui d’être l’organisateur principal et essentiel des PML-NBs (PML-Nuclear Bodies), des macrostructures nucléaires très dynamiques regroupant et coordonnant plus de 40 protéines. Il est à noter que la fonction de TIF1α, β et PML est régulée par une modification post-traductionnelle, la sumoylation, qui implique le couplage covalent de la petite protéine SUMO (Small Ubiquitin like MOdifier) à des lysines de ces trois protéines cibles. Cette thèse propose de développer des méthodes utilisant le BRET (Bioluminescence Resonance Energy Transfert) afin de détecter dans des cellules vivantes et en temps réel des interactions non-covalentes de protéines nucléaires mais aussi leur couplage covalent à SUMO. En effet, le BRET n’a jamais été exploré jusqu’alors pour étudier les interactions non-covalentes et covalentes de protéines nucléaires. L’étude de l’interaction de protéines transcriptionnellement actives est parfois difficile par des méthodes classiques du fait de leur grande propension à agréger (famille TRIM) ou de leur association à la matrice nucléaire (ZNF74). L’homo et l’hétérodimérisation de TIF1α, β ainsi que leur interaction avec ZNF74 sont ici testées sur des protéines entières dans des cellules vivantes de mammifères répondant aux résultats conflictuels de la littérature et démontrant que le BRET peut être avantageusement utilisé comme alternative aux essais plus classiques basés sur la transcription. Du fait de l’hétérodimérisation confirmée de TIF1α et β, le premier article présenté ouvre la possibilité d’une relation étroite entre les récepteurs nucléaires et les protéines KRAB- multidoigt de zinc. Des études précédentes ont démontré que la sumoylation de PML est impliquée dans sa dégradation induite par l’As2O3 et dépendante de RNF4, une E3 ubiquitine ligase ayant pour substrat des chaînes de SUMO (polySUMO). Dans le second article, grâce au développement d’une nouvelle application du BRET pour la détection d’interactions covalentes et non-covalentes avec SUMO (BRETSUMO), nous établissons un nouveau lien entre la sumoylation de PML et sa dégradation. Nous confirmons que le recrutement de RNF4 dépend de SUMO mais démontrons également l’implication du SBD (Sumo Binding Domain) de PML dans sa dégradation induite par l’As2O3 et/ou RNF4. De plus, nous démontrons que des sérines, au sein du SBD de PML, qui sont connues comme des cibles de phosphorylation par la voie de la kinase CK2, régulent les interactions non-covalentes de ce SBD mettant en évidence, pour la première fois, que les interactions avec un SBD peuvent dépendre d’un évènement de phosphorylation (“SBD phospho-switch”). Nos résultats nous amènent à proposer l’hypothèse que le recrutement de PML sumoylé au niveau des PML-NBs via son SBD, favorise le recrutement d’une autre activité E3 ubiquitine ligase, outre celle de RNF4, PML étant lui-même un potentiel candidat. Ceci suggère l’existence d’une nouvelle relation dynamique entre phosphorylation, sumoylation et ubiquitination de PML. Finalement, il est suggéré que PML est dégradé par deux voies différentes dépendantes de l’ubiquitine et du protéasome; la voie de CK2 et la voie de RNF4. Enfin une étude sur la sumoylation de TIF1β est également présentée en annexe. Cette étude caractérise les 6 lysines cibles de SUMO sur TIF1β et démontre que la sumoylation est nécessaire à l’activité répressive de TIF1β mais n’est pas impliquée dans son homodimérisation ou son interaction avec la boîte KRAB. La sumoylation est cependant nécessaire au recrutement d’histones déacétylases, dépendante de son homodimérisation et de l’intégrité du domaine PHD. Alors que l’on ne connaît pas de régulateur physiologique de la sumoylation outre les enzymes directement impliquées dans la machinerie de sumoylation, nous mettons en évidence que la sumoylation de TIF1β est positivement régulée par son interaction avec le domaine KRAB et suggérons que ces facteurs transcriptionnels recrutent TIF1β à l’ADN au niveau de promoteur et augmentent son activité répressive en favorisant sa sumoylation. / Three TRIM proteins (TRIpartite Motif), TIF1α, β (Transcriptional Intermediary Factor 1) and PML (ProMyelocytic Leukaemia¬), were studied in this thesis. TIF1α is a nuclear receptor coactivator and TIF1β is the universal corepressor of the KRAB-zinc finger repressor family of which, ZNF74 is studied here as a prototypic member. PML functions as a tumor suppressor and is the essential organiser of PML-NBs (PML-Nuclear Bodies) which are very dynamic nuclear macrostructures containing more than 40 proteins. The function of these three TRIM proteins is regulated by sumoylation, a post-translational modification involving the covalent linkage of SUMO (Small Ubiquitin like MOdifier) to specific targets lysine. In this thesis, we propose to develop new methods based on BRET (Bioluminescence Resonance Energy Transfer) to detect non-covalent nuclear protein interactions but also covalent linkage to SUMO in real time in living cells. To date, BRET was never used to assess non-covalent or covalent nuclear protein interactions. Studying transcriptionally active protein interactions represents a challenge by classical methods in particular when proteins have a tendency to aggregate (TRIM family) or when characterizing nuclear matrix proteins (ZNF74). In the first article, homo- and heterodimerisation of TIF1 α and β as well as their interaction with ZNF74 was assessed by BRET using full length proteins in living mammalian cells. We ascertained the heterodimerisation of TIF1α and β. Whereas ZNF74 interacts strongly with TIF1β, no interaction was detected with TIF1α. However, we unravelled the existence of ternary complexes involving ZNF74, TIF1α and TIF1β. This suggested that a mechanisms for cross-talk between nuclear receptors and KRAB-zinc finger proteins. Thus, we showed that BRET can be advantageously used as a non-transcription-based interaction system for studying transcriptionally active proteins, including nuclear matrix proteins, in living cells. Previous studies have shown that the sumoylation of PML (a tumour suppressor) is involved in its proteasome degradation that is As2O3-inducible and dependent on the polySUMO E3 ubiquitin ligase, RNF4. In the second article, we describe the development of a new application of the BRET method for the detection of covalent and non-covalent interactions with SUMO. Owing to this SUMO BRET assay, we established that the As2O3 / RNF4-mediated degradation of PML, not only depends on PML sumoylation as previously demonstrated, but also on the integrity of its SUMO binding domain. We also demonstrated that As2O3 which increases PML sumoylation, also enhances PML / RNF4 interaction. Our study revealed that most PML SBD non covalent interactions with sumoylated proteins required the phosphorylation of serines within PML SBD that were previously described as target sites for CK2 kinase and involved in PML degradation. Despites the involvement of PML SBD in RNF4-mediated degradation, these serines which function as an SBD phospho-switch, were not required for RNF4-mediated degradation. This suggested that CK2- and RNF4-mediated PML degradation represents two distinct pathways triggering PML ubiquitin / proteasome-dependent degradation. At last, our study led to the hypothesis that the recruitment of sumoylated PML at PML-Nuclear Bodies subnuclear structures via the PML SBD and / or possibly an E3 ubiquitin ligase activity other than RNF4 (PML itself being candidate) may favour PML degradation. Our study also stresses the dynamic involvement of three PML post-translational modifications, phosphorylation, sumoylation and ubiquitination in its degradation. A third article addressing the role of TIF1β sumoylation is presented in the Appendix. We characterized the 6 SUMO targets lysine of TIF1β and demonstrated that sumoylation is required for TIF1β transcriptional repressive activity. This is in part explained by the fact that TIF1β sumoylation is a pre-requisite for histone deacetylases recruitment since TIF1β repressive activity is partly dependent on histone deacetylases. We found that TIF1β sumoylation does not influence its homodimerisation or interaction with the KRAB box of KRAB zinc finger proteins recruiting TIF1β to promoters. TIF1β sumoylation is however relying on the integrity of TIF1β PHD finger and on its self-oligomerisation. Interestingly, we demonstrated that TIF1β sumoylation is positively regulated by its interaction with KRAB domain. It is thus suggested that KRAB-zinc finger proteins recruit TIF1β at DNA promoters where they trigger increase of TIF1β sumoylation and thus enhance its repressive activity.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A indÃstria nacional calÃadista tem enfrentado o desafio de uma globalizaÃÃo inevitÃvel que acarreta uma concorrÃncia desleal com os paÃses asiÃticos. Esse cenÃrio sugere como uma ferramenta eficaz, a integraÃÃo entre as empresas para o seu fortalecimento e assim, responder a esta globalizaÃÃo atravÃs do aumento da sua capacidade competitiva, por meio do uso eficiente de inovaÃÃes tecnolÃgicas que tÃm impulsionado mundialmente o setor, como a tecnologia da informaÃÃo aplicada ao gerenciamento do fluxo logÃstico. No entanto, alÃm do associativismo essencial, as empresas necessitam rever sua tradicional cultura organizacional e alinhar sua visÃo estratÃgica para o cenÃrio descrito anteriormente e, disseminar, fazer com que essa visÃo alcance de maneira satisfatÃria o setor produtivo, atingindo principalmente a quem interessa: os consumidores. Com isso, o presente trabalho propÃe um mÃtodo de anÃlise da eficiÃncia do fluxo logÃstico em uma empresa do ramo calÃadista, fundamentado no mapeamento do processo e no desenvolvimento de um modelo de gestÃo de indicadores estratÃgicos, apresentando uma metodologia que auxiliarà na avaliaÃÃo das suas operaÃÃes logÃsticas, mapeando seu fluxo logÃstico e auxiliando na tomada de decisÃo para tornar a cadeia de suprimentos mais eficiente e flexÃvel. Portanto, contribuindo para a capacidade de absorÃÃo das oportunidades tecnolÃgicas de integraÃÃo dos elos da cadeia, como a tecnologia da informaÃÃo. O estudo se dà atravÃs do mapeamento do fluxo logÃstico e na gestÃo da utilizaÃÃo dos indicadores de desempenho estratÃgicos, teve como foco uma empresa do ramo calÃadista pertencente ao arranjo produtivo local (APL) do cariri cearense que, por sua vez, se destaca como um dos principais polos produtores calÃadistas no cenÃrio nacional. / A indÃstria nacional calÃadista tem enfrentado o desafio de uma globalizaÃÃo inevitÃvel que acarreta uma concorrÃncia desleal com os paÃses asiÃticos. Esse cenÃrio sugere como uma ferramenta eficaz, a integraÃÃo entre as empresas para o seu fortalecimento e assim, responder a esta globalizaÃÃo atravÃs do aumento da sua capacidade competitiva, por meio do uso eficiente de inovaÃÃes tecnolÃgicas que tÃm impulsionado mundialmente o setor, como a tecnologia da informaÃÃo aplicada ao gerenciamento do fluxo logÃstico. No entanto, alÃm do associativismo essencial, as empresas necessitam rever sua tradicional cultura organizacional e alinhar sua visÃo estratÃgica para o cenÃrio descrito anteriormente e, disseminar, fazer com que essa visÃo alcance de maneira satisfatÃria o setor produtivo, atingindo principalmente a quem interessa: os consumidores. Com isso, o presente trabalho propÃe um mÃtodo de anÃlise da eficiÃncia do fluxo logÃstico em uma empresa do ramo calÃadista, fundamentado no mapeamento do processo e no desenvolvimento de um modelo de gestÃo de indicadores estratÃgicos, apresentando uma metodologia que auxiliarà na avaliaÃÃo das suas operaÃÃes logÃsticas, mapeando seu fluxo logÃstico e auxiliando na tomada de decisÃo para tornar a cadeia de suprimentos mais eficiente e flexÃvel. Portanto, contribuindo para a capacidade de absorÃÃo das oportunidades tecnolÃgicas de integraÃÃo dos elos da cadeia, como a tecnologia da informaÃÃo. O estudo se dà atravÃs do mapeamento do fluxo logÃstico e na gestÃo da utilizaÃÃo dos indicadores de desempenho estratÃgicos, teve como foco uma empresa do ramo calÃadista pertencente ao arranjo produtivo local (APL) do cariri cearense que, por sua vez, se destaca como um dos principais polos produtores calÃadistas no cenÃrio nacional. / The domestic footwear industry has faced the challenge of an inevitable globalization that leads to unfair competition with Asian countries. This scenario suggests as an effective tool, the integration between the companies for their strengthening and thus respond to this globalization by increasing their competitiveness through the efficient use of technological innovations that have driven the industry globally, as technology information applied to the management of logistics flow. However, beyond the essential associations, companies need to review their traditional organizational culture and align its strategic vision for the scenario described above, and disseminate, make that vision satisfactorily reach the productive sector, affecting mostly those who matter: the consumers. Thus, this paper proposes a method of analyzing the efficiency of logistics flow in a company in the footwear business, based on the process mapping and the development of a management model of strategic indicators, presenting a methodology that will assist in the evaluation of its operations logistics, mapping its logistics flow and aiding in the decision to make the supply chain more efficient and flexible. Thus contributing to the absorption capacity of the technological opportunities of integrating parts of the chain, such as information technology. The study is done by mapping the logistics flow and managing the use of strategic performance indicators, a company focused on the footwear branch belonging to the local productive arrangement (APL) in Cearà cariri which, in turn, stands out as a major footwear producers poles on the national scene. / The domestic footwear industry has faced the challenge of an inevitable globalization that leads to unfair competition with Asian countries. This scenario suggests as an effective tool, the integration between the companies for their strengthening and thus respond to this globalization by increasing their competitiveness through the efficient use of technological innovations that have driven the industry globally, as technology information applied to the management of logistics flow. However, beyond the essential associations, companies need to review their traditional organizational culture and align its strategic vision for the scenario described above, and disseminate, make that vision satisfactorily reach the productive sector, affecting mostly those who matter: the consumers. Thus, this paper proposes a method of analyzing the efficiency of logistics flow in a company in the footwear business, based on the process mapping and the development of a management model of strategic indicators, presenting a methodology that will assist in the evaluation of its operations logistics, mapping its logistics flow and aiding in the decision to make the supply chain more efficient and flexible. Thus contributing to the absorption capacity of the technological opportunities of integrating parts of the chain, such as information technology. The study is done by mapping the logistics flow and managing the use of strategic performance indicators, a company focused on the footwear branch belonging to the local productive arrangement (APL) in Cearà cariri which, in turn, stands out as a major footwear producers poles on the national scene.