Teses / dissertações sobre o tema "Xist RNA"

X inactivation is the process of silencing one of the two X chromosomes in mammalian female cells in order to equalise the dosage of X-linked genes with males. The process is initiated by the long noncoding RNA XIST, which is transcribed from the future inactive X and localises to it in cis. How XIST RNA is able to localise to the X chromosome is not well defined. The aim of the current study was to deduce mechanisms of XIST RNA localisation. This was addressed in various ways, including 1) testing the ability of an XIST transgene integrated into a variety of autosomes to localise to those autosomes, as opposed to the X chromosome; 2) assessing the ability of XIST transgenes with different regions deleted to localise, in order to identify sequences required for localisation; and 3) knocking down various proteins implicated in X inactivation in order to assess any effect on the ability of XIST to localise. We find that the XIST transgene is able to localise to a wide variety of different autosomes and furthermore, is able to direct the enrichment of the histone variant macroH2A on an autosome and the deposition of a repressive histone modification, H3K27me3, onto an autosome. We also find that a region of XIST encompassing repeats B and C, and sequences downstream of exon 1 are involved in localising XIST RNA, and that they do so in a redundant fashion. Lastly, we show that the knockdown of five proteins - YY1, hnRNP-U, SPOP, CUL3 and ASH2L - prevent the formation of an intact XIST focus. The results presented here add to the limited knowledge of how XIST RNA is able to localise, an essential step in the process of X chromosome inactivation.

X-chromosome inactivation ensures equal expression of mammalian male and female X-linked genes by transcriptionally silencing one X chromosome in each female cell. The pivotal molecule responsible for the silencing is a long non-coding RNA XIST; however, an all-encompassing model explaining how XIST induces silencing of the whole X chromosome is yet to emerge. This thesis aims to broaden our understanding of XIST action in humans by leveraging an inducible XIST transgene capable of silencing downstream reporters to identify sequences within XIST and XIST-interacting proteins critical for gene silencing. First, we demonstrate that the repeat A region of XIST is necessary and sufficient to induce gene silencing, at least locally, irrespective of the makeup of the surrounding chromatin, and that XIST induces silencing of a distal gene in one of the HT1080 cell lines. Second, we show that individual repeats of a consensus repeat A sequence contribute additively to silencing. Mutations within a construct consisting of two repeat A units both demonstrate that the two palindromic sequences within the repeat A units spanning ‘ATCG’ and ‘ATAC’ tetranucleotides are critical for repeat A function and add to the evidence that the first palindrome forms a hairpin, rather than engaging in pairing between repeat A units. Third, we explore which proteins are critical for XIST-induced silencing. We show that histone deacetylation, an early mark of an X-chromosome inactivation, is likely a consequence, and not the cause of XIST-induced silencing. We next demonstrate that in the transgenic HT1080 system, gene silencing is not accompanied by recruitment of the H3K27me3 repressive histone mark and XIST induces silencing independently of its previously reported associations with the polycomb repressive complex 2 (PRC2). Finally, we performed siRNA-mediated knock-down of 31 proteins previously implicated to play a role in X-chromosome inactivation. Our results show that proteins involved in XIST RNA localization (YY1), chromatin organization (SATB2, HNRNPU), and cell cycle (ATM), as well as an E3 ubiquitin ligase (SPOP) contribute to XIST-induced gene silencing in the HT1080 system. Thus, we demonstrate that the repeat A alone induces gene silencing and identify candidate pathways critical for its function.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.
Includes bibliographical references.
The role of Xist RNA in silencing the inactive X of female somatic cells was investigated by generating a conditional allele of the Xist gene. A system was set up in which reactivation of two X-linked genes, the endogenous Hprt gene and an X-linked GFP transgene, can be quantitatively assessed. Mouse embryonic fibroblasts derived from mice carrying the conditional Xist mutation were cultured and infected with an adenovirus vector carrying the gene for Cre recombinase. After Cre mediated deletion of Xist, the inactive X remained transcriptionally silent, late replicating, and hypoacetylated on histone H4, confirming that X-inactivation can be maintained in the absence of Xist RNA. However, the Xist mutant inactive X was no longer enriched in histone macroH2A 1. Furthermore, the reactivation rate of GFP and Hprt increased, indicating Xist RNA does contribute to gene repression on the inactive X. DNA methylation, histone hypoacetylation and Xist RNA were found to act synergistically in X chromosome silencing. To investigate whether Xist RNA can also silence the active X chromosome of male somatic cells, Xist expression on the active X was induced by demethylation. Demethylation was achieved by Cre mediated deletion of a conditional mutant allele of DNA methyltransferase-l (Dnmtl) in male fibroblasts. In these cells, Xist RNA coated the active X chromosome, in a pattern indistinguishable from coating of the inactive X of female cells. Although many Xist expressing chromosomes also transcribed X-linked genes Pgk-i and Hprt, in a small percent of cells Xist expression led to X chromosome inactivation. The proportion of chromosome expressing X-linked genes declined, and occasionally the X became late replicating, indicating that X-inactivation can be initiated in male somatic cells.
by Györgyi Csankovszki.
Ph.D.

L’inactivation du chromosome X (XCI) est un mécanisme qui permet l’extinction transcriptionelle d’un des deux chromosomes X chez la femelle. XCI est régulé par une région spécifique nommée centre de l’inactivation du chromosome (Xic), contenant plusieurs gènes produisant de longs ARNs non codants (lncRNAs). Parmi ces lncRNAs, le transcrit Xist est l’effecteur principal pour l’XCI. Xist peut s’accumuler en cis sur le chromosome et recruter la machinerie qui permettra l’initiation et la propagation de l’extinction transcriptionnelle à l’échelle du chromosome.Le laboratoire d’accueil a identifié un nouveau gène du Xic qui produit le lncRNA Ftx. Dans cette étude, on a pu montrer que l’inactivation du chromosome X est fortement perturbée dans les cellules Ftx-/- et s’accompagne par une forte baisse du niveau d’expression et d’accumulation de Xist. Dans ce contexte, certaines cellules parviennent à maintenir l’expression de Xist mais le profil de couverture du chromosome X par Xist est anormal, présentant un profil diffus ; ceci est associé à une extinction transcriptionnelle déficiente des gènes liés à l’X. Dans les lignées hétérozygotes Ftx+/-, l’expression et l’accumulation de Xist est aussi affectée mais dans une moindre mesure, si bien qu’il apparaît que le nombre de copies de Ftx soit important pour sa fonction. Par ailleurs, l’inactivation du chromosome X dans les cellules Ftx+/- est biaisée de telle sorte que le chromosome X portant une copie fonctionnelle de Ftx est préférentiellement inactivé, suggérant un rôle en cis de Ftx. Ces résultats montrent que Ftx est un activateur de Xist et qu’il est essentiel pour la mise en place de l’inactivation
X-chromosome inactivation (XCI) is a female-specific, chromosome-wide regulatory process that, in eutherians, ensures dosage compensation for X-linked genes between sexes. XCI is controlled by a cis-acting locus on the X-chromosome, the X-inactivation center (Xic), enriched in genes producing long non-coding RNAs (lncRNAs). The Xic-linked gene Xist is the master player of XCI, and produces a lncRNA that accumulates in cis on the X-chromosome and recruits the machinery responsible for initiation and propagation of silencing.The laboratory has identified an additional Xic-linked non-coding gene, Ftx. In this study, we could find that, in female Ftx-/- lines, XCI is strongly impaired, with a significant decrease in the levels of Xist expression and in the percentage of cells showing normal Xist accumulation patterns. Importantly, a high proportion of the cells that still retain Xist expression show abnormal X-chromosome coating and a decreased ability to silence X-linked genes. These data reveal that Ftx is a positive Xist regulator and it is required for proper XCI establishment. In female Ftx+/- lines, the levels of Xist expression and the percentage of cells showing normal Xist accumulation patterns are also decreased, albeit to a lower extent compared to Ftx-/- lines, suggesting that Ftx works in a copy-dependent manner. In addition, a high proportion of Ftx+/- cells display skewed X-inactivation, with preferential inactivation of the wild-type X chromosome. This suggests that Ftx role on Xist accumulation is mostly restricted in cis. Taken together, these results demonstrate that Ftx is required for XCI establishment, where it functions as a strong Xist activator

The XIST gene is implicated in X-chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST RNA does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2-D and 3-D space; hence the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its poly-adenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of non-chromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging.

La récidive et la progression métastatique du cancer du sein ne sont toujours pas curables. Le concept des cellules souches cancéreuses (CSC) pourrait apporter une explication à ces échecs. Les CSC résisteraient aux thérapies conventionnelles (chimiothérapies, radiothérapie) et seraient responsables de la rechute et de la progression du cancer. L'élimination des CSC semble être un pré-requis indispensable pour le traitement des patientes. L'identité et le destin des cellules souches sont finement régulés par des acteurs épigénétiques. Les travaux de cette thèse se sont intéressés aux conséquences de la dérégulation de deux acteurs épigénétiques en particulier : les enzymes HDAC et le long ARN non-codant Xist. Nous avons montré que la modulation épigénétique via l'inhibition des HDAC (HDACi) permet d'éliminer les CSC en induisant leur différenciation. Nous présentons une nouvelle stratégie thérapeutique pour le cancer du sein : la thérapie différenciante. Nous avons déterminé Xist comme étant le biomarqueur prédictif de la réponse aux HDACi. Xist étant un partenaire clé de la plasticité cellulaire, les travaux de cette thèse se sont ensuite intéressés aux conséquences de la dérégulation de Xist dans l'initiation tumorale. Nous avons observé que l'inhibition de Xist favorise la division des cellules souches mammaires normales. Nous proposons un nouveau modèle de l'initiation tumorale où la dérégulation épigénétique est une modification précoce sans conséquence sur l'homéostasie tissulaire mais pourrait être la première étape de la transformation cancéreuse
These last decades have allowed deciphering the biology of breast cancer and improving the therapeutic management. However, recurrence and metastatic progression of the disease are still not curable. The concept of cancer stem cells (CSC) could provide an explanation for these failures. CSC would resist conventional therapies (chemotherapy, radiotherapy) and would be responsible for both relapse and progression of cancer. The elimination of CSC seems to be an essential prerequisite for the treatment of patients. The identity and fate of stem cells are tightly regulated by epigenetic mechanisms. The work of this thesis investigated the consequences of deregulation of two epigenetic players: HDAC enzymes and long non-coding RNA Xist. We have shown that epigenetic modulation via HDAC inhibitor (HDACi) eliminates the CSC by inducing their differentiation. We present a new therapeutic strategy for breast cancer: differentiation therapy. We determined Xist as the predictive biomarker of response to HDACi. Xist is a key partner of cell plasticity, the work of this thesis therefore interested in the consequences of Xist deregulation in tumor initiation. We observed that Xist inhibition promotes division of normal breast stem cells. We propose a new model of tumor initiation: epigenetic deregulation is an early change without consequence on tissue homeostasis but could be the first step of the cancerous transformation

XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.

XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.

A inativação do cromossomo X (ICX) é o fenômeno através do qual um dos cromossomos X das fêmeas de mamíferos é silenciado para atingir compensação de dose em relação aos machos. Ela envolve a expressão do gene XIST exclusivamente no X inativo, e a associação em cis de seu RNA nesse cromossomo. Isso inicia a imposição de várias marcas epigenéticas no cromossomo X inativo, que garantem a manutenção deste estado de silenciamento transcricional de maneira estável durante todas as mitoses num organismo. Uma dessas modificações epigenéticas é a metilação do DNA, desempenhada principalmente pela enzima DNMT1. Os papéis de XIST e DNMT1 na manutenção da inativação do cromossomo X ainda são controversos em humanos, e nesse sentido foi objetivo desse trabalho analisar a possível função desses genes nesse processo em células humanas não transformadas. Foi otimizado um sistema experimental para o estudo de possíveis perturbações na manutenção da inativação do cromossomo X, onde a re-expressão de genes submetidos a esse processo pode ser monitorada. Nesse sistema foram identificados dois genes, MAOA e GYG2, cujo padrão de expressão no X inativo difere do previamente descrito. Demonstrou-se que baixos níveis de expressão do gene XIST foram suficientes para manter seu RNA associado ao X inativo, conservando o estado silenciado desse cromossomo. Além disso, foram obtidos indicativos de que a inibição de XIST em fibroblastos humanos gera uma diminuição da viabilidade celular. Foi possível demonstrar que DNMT1 é necessária para a manutenção da metilação global do genoma em células humanas não transformadas, e que eXISTe um mecanismo de compensação da inibição desse gene que leva ao aumento da expressão de DNMT3B. Ainda se observou que a repressão de DNMT1 não é suficiente para levar à reativação de genes no cromossomo X inativo. Além disso, a desmetilação encontrada nos promotores de MAOA e XIST não foi suficiente para levar à expressão destes genes nos cromossomo X inativo e ativo, respectivamente. Estes resultados enfatizam a necessidade de se estudar os mecanismos moleculares da ICX em humanos utilizando sistemas experimentais adequados para a análise de herança epigenética.
X chromosome inactivation (XCI) is the phenomenon through which one of the X chromosomes in female mammals is silenced to achieve dosage compensation related to males. It involves the expression of XIST gene exclusively from the inactive X, and the association of its RNA in cis in this chromosome. This leads to a series of epigenetic modifications in the chromatin of the inactive X (Xi) that guarantee a stable maintenance of the transcriptional silence through all the mitoses in the organism. One of these epigenetic modifications is DNA methylation, achieved mainly by the maintenance DNA methylase DNMT1. The roles of XIST and DNMT1 in the maintenance phase of XCI are controversial in humans. Therefore, the main goal of this present work was to analyze some of the possible functions of these genes in this process in untransformed human cells. An experimental system was optimized to study possible disturbances in maintenance of XCI, where the re-expression of genes submitted to this process could be monitored. In this system we identified two genes, MAOA and GYG2, whose pattern of expression on the Xi, differed from what had been previously described. It was demonstrated that low levels of XIST expression were sufficient to keep its RNA associated to the Xi, assuring the silenced state of this chromosome. Besides, evidences have been found that XIST inhibition in human fibroblasts reduces cellular viability. It was possible to demonstrate that DNMT1 is necessary to the maintenance of global genome methylation in untransformed human cells, and the eXISTence of a compensation mechanism involving DNMT3B upregulation. It was also observed that repression of DNMT1 was not sufficient to reactivate genes of the Xi chromosome. Additionally, demethylation of MAOA and XIST promoters was not enough to cause expression of these genes on the inactive and active Xs, respectively. All these results emphasize the requirement of studying the molecular mechanisms of XCI in humans using experimental systems appropriate for the analysis of epigenetic inheritance.

L’inactivation d’un des deux chromosomes X dans les cellules d’organismes femelles permet d’assurer un taux similaire des transcrits des gènes liés aux chromosomes X entre les deux sexes. L’ARN non codant Xist d’environ 17000 nts joue un rôle central dans ce processus. Il habille le futur chromosome X inactivé et induit la mise en place de modifications épigénétiques qui permettent d’éteindre l’expression des gènes. Une région d’approximativement 500 nts située à l’extrémité 5’ de l’ARN Xist est nécessaire à l’initiation de l’inactivation. Cette région appelée region des A-repeats contient 8 répétitions d’une séquence de 24 nucléotides. La délétion de cette région provoque un défaut d’inactivation, ce qui souligne son importance dans le processus. Etant donné que la fonction d’un ARN est bien souvent conditionnée par sa structure 2D, mon travail de thèse a consisté à réaliser l’étude expérimentale de la structure 2D de la région des A-repeats, ceci en utilisant des sondes de la structure secondaire des ARN en solution et une méthode de FRET. Nous avons montré que la région des A-repeats se structure selon 2 grandes structures tige-boucle irrégulières formées par l’appariement 2 à 2 des éléments répétés. Par purification des RNP et identification de leurs protéines, nous avons démontré que le complexe PRC2, impliqué dans la mise en place des marques épigénétiques du Xi, se lie à la région des A-repeats. Nous avons également identifié un grand nombre d’autres protéines pouvant avoir un rôle dans l’activité de la région des A-repeats (PTB, KSRP, Sam68, Vigiline, RHA, TIAR, DEK, H1, BRML1, Rod1, Lin28). Leurs implications dans l’inactivation du chromosome X est en cours de vérification
Silencing of one X chromosome (XCI) in cells of mammalian female ensures sex chromosome dosage compensation between male and female. The 17kb Xist ncRNA plays an essential role in XCI. Its spread along the future inactivated X chromosome is associated with major modifications of the epigenetic status of this chromosome, including histone H3K27 methylations mediated by PRC2 complex. One key part of Xist necessary for XCI initiation is the phylogenetically conserved A region. It lies at the 5’ end of the Xist molecule and contains 8 of a 24-nucleotides motif. Female mouse embryos carrying a mutated Xist deleted for the A region are selectively lost during embryogenesis, which underlines the importance of this element. We performed the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we established a 2D structure for the A region that contains two long stem-loop structures each including 4 repeats which interact together two by two. By immunoprecipitation assays and mass spectrometry analysis, we identified the protein partners of the A region. We demonstrated that the A region associate with PRC2 components which is responsible for the apposition of epigenetic modifications of X inactive chromosome. Others proteins which would have a role in A region function were also identified (PTB, KSRP, Sam68, Vigiline, RHA, TIAR, DEK, H1, BRML1, Rod1, Lin28)

Submitted by PPG Comunica??o Social (famecos-pg@pucrs.br) on 2018-04-27T18:10:26Z No. of bitstreams: 1 RENATA_ANDREONI_TES.pdf: 4557075 bytes, checksum: 0e51179b7449adc96aa68d424586c772 (MD5)
Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-05-10T14:39:26Z (GMT) No. of bitstreams: 1 RENATA_ANDREONI_TES.pdf: 4557075 bytes, checksum: 0e51179b7449adc96aa68d424586c772 (MD5)
Made available in DSpace on 2018-05-10T15:07:46Z (GMT). No. of bitstreams: 1 RENATA_ANDREONI_TES.pdf: 4557075 bytes, checksum: 0e51179b7449adc96aa68d424586c772 (MD5) Previous issue date: 2018-03-27
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
(Re) Thinking about the memory and communication interface, based on the assumptions of the New Theory of Communication - NTC (MARCONDES FILHO, 2013a; 2013b; 2010a; 2010b; 2008; 2004) and the Philosophy of Henri Bergson (2006; 2005a; 2005b; 1999); 1988) is the journey we propose in this research. To accomplish this, in the light of complex thinking (MORIN, 2015a, 2015b, 2013, 2008), we set three objectives: To critically discuss the proposed interfaces between memory and communication, under the organizational memory configuration; to show possible dimensions that reveal how the organizational memory is configured, considering the academic propositions and their practices in the companies; (re) dimensioning the memory in the Communication, in addition to an instrumental approach. These objectives are designed to help us understand the landscapes that unveil at each step. The first landscape of this course is constituted by a critical and complex perspective of the praxis of academic productions and actions of different organizations on possible interfaces between memory and communication. For this, we consider the configuration of organizational memory, under which we show the predominance of the functional communication approach (SODR?, 2014a). In the second landscape we intertwine the NTC with the Bergsonian philosophical thought, constituting an ontological path for understanding memory in Communication. It is a theoretical study, in which we defend the thesis that the interface memory and communication can be established beyond a transmissive and instrumental relationship. Our efforts are aimed at developing an awareness to (re) think the communicational approach, having memory as a ubiquitous dimension in the relationships we establish in the continuous and indeterminate movement of life (I / Other).
(Re) penser l?interface m?moire et communication, ? partir des hypoth?ses de la Nouvelle Th?orie de la Communication - NTC (MARCONDES FILHO, 2013e; 2013b; 2010a; 2010b; 2008; 2004) et de la Philosophie de Henri Bergson (2006, 2005a; 2005b; 1999 1988) est le parcours que nous proposons dans cette recherche. Pour ce faire, ? la lumi?re de la pens?e complexe (Morin, 2015e; 2015b; 2013; 2008), nous avons ?tabli trois objectifs: discuter de fa?on critique les interfaces propos?es entre la m?moire et la communication, dans la configuration de la m?moire organisationnelle; mettre en ?vidence les dimensions possibles qui r?v?lent la constituition de la m?moire organisationnelle, en consid?rant les propositions acad?miques et leurs pratiques dans les entreprises; (re) dimensionner la m?moire dans la Communication, en plus d'une approche instrumentale. Ces objectifs sont con?us pour nous aider ? comprendre les paysages qui se d?roulent ? chaque ?tape. Le premier paysage de ce parcours se compose d'une perspective critique et complexe de la praxis des productions acad?miques et les actions des diff?rentes organisations sur les possibles interfaces entre la m?moire et de la communication. Pour cela, nous consid?rons la configuration de la m?moire organisationnelle, sous laquelle nous montrons la pr?dominance de l'approche de la communication fonctionnelle (SODR?, 2014a). Le second paysage parcourut d?veloppe une (re) tessiture entre la NTC et la pens?e bergsonienne, ce qui constitue un chemin ontologique ? la compr?hension de la m?moire en Communication. Il s?agit d?une ?tude th?orique dans laquelle nous soutenons la th?se, selon laquelle, l?interface m?moire et communication peut s'?tablir au-del? d'une relation transmissible et instrumentale. Nos efforts visent ? d?velopper une prise de conscience pour (re) penser l'approche communicative, ayant la m?moire comme dimension omnipr?sente dans les relations ?tablies dans le mouvement continu et ind?termin? de la vie (Moi/ Autre).
(Re)Pensar a interface mem?ria e comunica??o, partindo dos pressupostos da Nova Teoria da Comunica??o ? NTC (MARCONDES FILHO, 2013a; 2013b; 2010a; 2010b; 2008; 2004) e da Filosofia de Henri Bergson (2006; 2005a; 2005b; 1999; 1988), ? a jornada que propomos nesta pesquisa. Para realiz?-la, ? luz do pensamento complexo (MORIN, 2015a; 2015b; 2013; 2008), estabelecemos tr?s objetivos: discutir criticamente as interfaces propostas entre mem?ria e comunica??o, sob a configura??o da mem?ria organizacional; evidenciar poss?veis dimens?es que revelam como se configura a mem?ria organizacional, considerando as proposi??es acad?micas e suas pr?ticas nas empresas; e (re)dimensionar a mem?ria na Comunica??o para al?m de uma abordagem instrumental. Esses objetivos s?o tra?ados para nos auxiliarem a compreender as paisagens que se descortinam a cada passo. A primeira paisagem deste percurso ? constitu?da por uma perspectiva cr?tica e complexa da pr?xis de produ??es acad?micas e de a??es de diferentes organiza??es sobre poss?veis interfaces entre mem?ria e comunica??o. Para tanto, consideramos a configura??o da mem?ria organizacional, sob a qual evidenciamos o predom?nio da abordagem da comunica??o funcional (SODR?, 2014a). Na segunda paisagem percorrida, desenvolvemos uma (re)tecitura entre a NTC e o pensamento bergsoniano, constituindo um caminho ontol?gico para a compreens?o da mem?ria na Comunica??o. Trata-se de um estudo te?rico, no qual defendemos a tese que a interface mem?ria e comunica??o pode se estabelecer para al?m de uma rela??o transmissiva e instrumental. Nossos esfor?os s?o destinados ao desenvolvimento de uma consci?ncia para (re)pensarmos a abordagem comunicacional, tendo a mem?ria como dimens?o onipresente nas rela??es que estabelecemos no movimento cont?nuo e indeterminado da vida (Eu/Outro).

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-07T11:25:20Z No. of bitstreams: 1 2011 - Jo?o Antonio de Lima Vilela.pdf: 5449457 bytes, checksum: 8ea1eef4359358286afd4bf39023ac77 (MD5)
Made available in DSpace on 2016-10-07T11:25:20Z (GMT). No. of bitstreams: 1 2011 - Jo?o Antonio de Lima Vilela.pdf: 5449457 bytes, checksum: 8ea1eef4359358286afd4bf39023ac77 (MD5) Previous issue date: 2011-10-10
The research concerning Environmental Education in association with the Pedagogy of Projects originated from the necessity of developing a pedagogical work which valued the participation of the student and teacher in the teaching-learning process. This work was aimed to study the contribution of the application of Pedagogy of Projects in the praxis of Environmental Education for the formation in Farming Technicians at IFTM ? Campus Uberl?ndia under the focus of sustainable dimension; verify in which way the proposal of works with projects of Environmental Education determines a transforming character in the practice of students, in relation to the scholar community. Action-research was used in order to develop the Environmental Education projects, because this methodoly has strong relations with an action or with the resolution of problems in a cooperative or participative way. Participated in the research 120 students ? 60 from 2nd high school and 60 from 3rd high school of the Farming course of IFTM, who developed 6 projects: Water and Sewage treatment, PEPA (Permanent Environmental Protection Area) Reforesting project, the Environmental problems at IFTM, Sustainability at IFTM, Disposal at IFTM, and of proposal of Solid residue Management at IFTM. The knowledge gained with the application of Pedagogy of Projects in the praxis of Environmental Education at IFTM contributed to develop in the scholar community an awareness process about the importance of sustainability in the use of natural resources. This research has prompted a new way of teaching, enabling the construction of a new look on the way of working the subjects, breaking up the paradigms of traditional pedagogy, through creativity and autonomy of students.
A pesquisa com a tem?tica Educa??o Ambiental aliada ? pedagogia de projetos surgiu da necessidade de desenvolver um trabalho pedag?gico que valorizasse a participa??o de educando e do educador no processo de ensino-aprendizagem. Este trabalho teve como objetivos estudar a contribui??o da pedagogia de projetos na pr?xis da Educa??o Ambiental para a forma??o de T?cnicos em Agropecu?ria do IFTM - Campus Uberl?ndia; verificar de que forma a proposta de trabalhos com projetos de Educa??o Ambiental determina um car?ter transformador nas pr?ticas dos estudantes, em rela??o ? comunidade escolar. Utilizamos a pesquisa-a??o para desenvolver os projetos de Educa??o Ambiental, pois essa metodologia estabelece estreita associa??o com uma a??o ou com a resolu??o de problemas de modo cooperativo ou participativo. Participaram da pesquisa 120 estudantes - 60 das 2? s?ries e 60 das 3? s?ries do curso de agropecu?ria do IFTM, os quais desenvolveram seis projetos, sendo eles: Tratamento de ?gua e esgoto, Reflorestamento de APP (?rea de Prote??o Permanente), Problemas Ambientais no IFTM, Sustentabilidade, Desperd?cio, e Proposta de Implanta??o de Gerenciamento dos Res?duos S?lidos. Os conhecimentos adquiridos com a aplica??o da pedagogia de projetos na pr?xis da Educa??o Ambiental no IFTM contribu?ram para desenvolver na comunidade escolar uma tomada de consci?ncia sobre a import?ncia da sustentabilidade na utiliza??o dos recursos naturais. Esta pesquisa proporcionou uma nova forma de ensinar, possibilitando a constru??o de um novo olhar sobre a maneira de trabalhar os conte?dos, rompendo os paradigmas da pedagogia tradicional, por meio da criatividade e autonomia dos estudantes.

The typical female mammal has two X chromosomes. However, only one copy remains active, while the other copy is silenced. The Xist gene has been found to remain active on the silenced X chromosome. Other lab experiments have determined that the Xist RNA plays a significant role in the process of X - chromosome inactivation. Our goal in this thesis is to predict a possible consensus structure for the Xist RNA using comparative sequence analysis. Since the Xist RNA structure is not known, we evaluate our approaches on sequences with known structures. There are three main phases for this approach: (i) aligning multiple sequences, (ii) finding stems, and (iii) building the structure. Phases (ii) and (iii) are the main focus of this research. We used the concept of mutual information content to detect potential stems containing mutations. We found potential conserved stems by searching for sequences of complementary paired bases. The stems were then used to find the consensus structure. We investigated two general approaches for building the consensus structure, greedy and randomized methods. We tested these methods on data comprising the following types of RNA: tRNA, 16S rRNA, 23S rRNA, and Xist RNA. We found that our methods accurately predicted the consensus structure for tRNA. However, when we analyzed data sets with longer sequences, the accuracy of our predictions decreased. Of the methods we tested, we found that one of our greedy approaches performed better than the others on all the data sets. We predicted several possible structures for Xist from different alignments. While the overall accuracy of our predicted structures is likely to be low, some of the substructures predicted may give us clues about the true consensus structure.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019
Introduction: Due to the discrepancy in the number of X chromosomes between the sexes in many animal species, dosage compensation mechanisms have evolved to equalize X-linked gene dosage between males and females. In female mammals, dosage compensation is achieved by a remarkable process known as X-chromosome inactivation (XCI), that leads to the epigenetic silencing of an entire X chromosome. XCI is master-regulated by a long non-coding RNA (lncRNA) called X-inactive specific transcript (Xist in mouse/XIST in human), which is exclusively expressed from the future inactive X-chromosome (Xi). Xist gene is peculiar, since it encodes for a 17 kb capped, spliced and polyadenylated RNA that is never translated. This lncRNA is monoallelically upregulated randomly from only one of the X chromosomes chosen for inactivation, coating the entire chromosome in cis and inducing transcriptional silencing and heterochromatin formation that is stably inherited through cell divisions. Xist lncRNA is poorly conserved at sequence level, with the exception of six tandem-repeated regions, known as the A to F repeats. These regions are believed to act as regulatory modules that exert their function through the interaction with specific RNA-binding proteins. However, the relative contribution of each of these RNA modules to XCI is not fully understood. Methods: Using a well-described system whereby Xist is expressed from a tetracycline-inducible promoter in its endogenous position in J1 male mouse embryonic stem cells, we performed deletions for Xist D and E tandem-repeated regions by CRISPR/Cas9. Then, we validated and characterized these mutants alongside another mutant type previously generated in the lab (Xist ΔF). Three independent clones per mutant type were selected and their potential impact on XCI was addressed. We specifically explored several features related to XCI, namely: Xist coating of the X chromosome, through RNA Fluorescence In Situ Hybridization (FISH) for Xist; Xist capacity to silence the genes on the X chromosome, by RT-qPCR and RNA-FISH for X-linked genes; recruitment of typical heterochromatin marks of the Xi, using RNA FISH for Xist combined with Immunofluorescence for the histone marks H3K27me3, H2AK119ub and H4K20me1. Results: Xist-TetOP mutants for D- and E-repeats were generated, and, jointly with the previous deletion for the F-repeat, successfully validated. All the characterized mutants (Xist ΔF, ΔD and ΔE) were able to form Xist domains (or clouds) around the Xi, as observed by RNA-FISH for Xist. Xist ΔF clouds visually seemed smaller, but this was not quantified. By measuring Xist expression levels by RT-qPCR analysis, we suspected a possible instability in Xist ΔD transcripts that should be further explored. The X-linked gene silencing of some candidate genes seemed not to be affected in all the Xist mutants, but further characterization at X chromosome global level would give a definitive answer. Finally, typical heterochromatin marks of the Xi were explored for two clones of Xist ΔF mutants and no major defects were observed, although the two clones (F2 and F3) exhibited some variability. Conclusion: As a result of this work, a new series of inducible Xist mutants was generated, which will be a valuable set of experimental tools for further investigation in the field of XCI. Our initial characterization gave the first hints on the functional role of some unexplored conserved repeats of Xist during XCI. In the future, these Xist-inducible mutants can be used in high-throughput approaches to enquiry about gene silencing (RNA-seq), chromatin status (ChIP-seq) or to fish protein interactors of the different repeats (ChIRP-MS), to get mechanistic insights in this puzzled process of XCI.