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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
331

Une nouvelle approche computationnelle pour la découverte des sites de fixation de facteurs de transcription à l’ADN, adaptée aux données de ChIP-chip et de ChIP-séquençage

Aid, Malika 09 1900 (has links)
Les facteurs de transcription sont des protéines spécialisées qui jouent un rôle important dans différents processus biologiques tel que la différenciation, le cycle cellulaire et la tumorigenèse. Ils régulent la transcription des gènes en se fixant sur des séquences d’ADN spécifiques (éléments cis-régulateurs). L’identification de ces éléments est une étape cruciale dans la compréhension des réseaux de régulation des gènes. Avec l’avènement des technologies de séquençage à haut débit, l’identification de tout les éléments fonctionnels dans les génomes, incluant gènes et éléments cis-régulateurs a connu une avancée considérable. Alors qu’on est arrivé à estimer le nombre de gènes chez différentes espèces, l’information sur les éléments qui contrôlent et orchestrent la régulation de ces gènes est encore mal définie. Grace aux techniques de ChIP-chip et de ChIP-séquençage il est possible d’identifier toutes les régions du génome qui sont liées par un facteur de transcription d’intérêt. Plusieurs approches computationnelles ont été développées pour prédire les sites fixés par les facteurs de transcription. Ces approches sont classées en deux catégories principales: les algorithmes énumératifs et probabilistes. Toutefois, plusieurs études ont montré que ces approches génèrent des taux élevés de faux négatifs et de faux positifs ce qui rend difficile l’interprétation des résultats et par conséquent leur validation expérimentale. Dans cette thèse, nous avons ciblé deux objectifs. Le premier objectif a été de développer une nouvelle approche pour la découverte des sites de fixation des facteurs de transcription à l’ADN (SAMD-ChIP) adaptée aux données de ChIP-chip et de ChIP-séquençage. Notre approche implémente un algorithme hybride qui combine les deux stratégies énumérative et probabiliste, afin d’exploiter les performances de chacune d’entre elles. Notre approche a montré ses performances, comparée aux outils de découvertes de motifs existants sur des jeux de données simulées et des jeux de données de ChIP-chip et de ChIP-séquençage. SAMD-ChIP présente aussi l’avantage d’exploiter les propriétés de distributions des sites liés par les facteurs de transcription autour du centre des régions liées afin de limiter la prédiction aux motifs qui sont enrichis dans une fenêtre de longueur fixe autour du centre de ces régions. Les facteurs de transcription agissent rarement seuls. Ils forment souvent des complexes pour interagir avec l’ADN pour réguler leurs gènes cibles. Ces interactions impliquent des facteurs de transcription dont les sites de fixation à l’ADN sont localisés proches les uns des autres ou bien médier par des boucles de chromatine. Notre deuxième objectif a été d’exploiter la proximité spatiale des sites liés par les facteurs de transcription dans les régions de ChIP-chip et de ChIP-séquençage pour développer une approche pour la prédiction des motifs composites (motifs composés par deux sites et séparés par un espacement de taille fixe). Nous avons testé ce module pour prédire la co-localisation entre les deux demi-sites ERE qui forment le site ERE, lié par le récepteur des œstrogènes ERα. Ce module a été incorporé à notre outil de découverte de motifs SAMD-ChIP. / Transcription factors (TF) play important roles in various biological processes such as differentiation, cell cycle progression and tumorigenesis. They regulate gene expression by binding to specific DNA sequences (TFBS). Identifying these cis-regulatory elements is a crucial step to understand gene regulatory networks. Technological developments have enhanced DNA sequencing at genomic scale. On the basis of the resulting sequences, computational biologists now attempt to localize the most important functional regions, starting with genes, but also importantly the whole genome characterization of transcription factor binding sites and allow the development of several computational DNA motif discovery tools. Although these various tools are widely used and have been successful at discovering novel motifs, they are not adapted to ChIP-chip and ChIP-sequencing data. The main drawback of these approaches is that most of the predicted motifs represent artifacts due to an inefficient assessment of their enrichment. This thesis is about transcription factor proteins and statistical analysis of their binding sites in ChIP-chip and ChIP-sequencing data. The first objective was to develop a new do novo DNA motif discovery tool adapted to ChIP-chip and ChIP-sequencing data. SAMD-ChIP combines enumerative and stochastic strategies to predict enriched motifs in the vicinity of the ChIP peak summits. Our approach is an automated pipeline that includes motif discovery, motif clustering, motif optimization and finally motif identification using transcription factor (TF) databases. SAMD-ChIP outperforms state-of-the-art motif discovery tools in term of the number of predicted motifs and the prediction of rare and degenerate motifs. In particular, SAMD-ChIP efficiently identifies gapped motifs such as inverted or direct repeats bound by nuclear receptors and composite motifs resulting from the association of different single TF binding sites. The underlying assumption of the second objective is that in regulatory regions, binding sites of interacting transcription factors co-occur more often than expected by chance in the vicinity of the ChIP-peak summits. We proposed an approach to predict transcription factor binding sites co-localization based on the prediction of single motifs by do novo motif discovery tools or by using TFBS models from TF data bases.
332

Agentury a klientský systém zprostředkovávající práci migrantům z SNS a Ukrajiny v Česku (pozitivní a negativní aspekty jejích činnosti) / Agencies and the client system of mediating work for migrants from SNS and Ukraine in Czechia (pros and cons)

Kniazeva, Anastasiia January 2019 (has links)
1 Abstract International migration has an impact on the economy in both the receiving and the sending countries due to the fact that foreign workers will 'reinvest' the financial remittances in their home country and support the domestic economy in the source state while fulling the demand for work in the receiving country and improving the financial climate in the host state. Work agencies and the client system are some of the most important details of this system that allows migrants to get a job and often facilitates participation and provides additional services to organize their stay in the host country. The theoretical basis of this thesis is the migration network theory and also the neoinstitutional theory with its concept of limited human rationality as well as a concept of institutional "limitations" in the context of created institutional rules. In the theoretical part of this work the agency and the so-called client system, which performs the same duties on an illegal level, - were explored as institutional units in their historical, real, social dimensions. Basic qualitative research examines the concept of the migration network theory and the concept of limited choice of institutional theory on the example of nine migrants from the Commonwealth of Independent States (CIS) and Ukraine with...
333

Estudos por modelagem e dinâmica molecular integradas a técnicas físicas para biomoléculas em solução - interação de receptores nucleares a elementos responsivos no DNA e dinâmica inter-domínios da celobiohidrolase I / Integrated experimental biophysics and molecular dynamics simulations of biomolecules in solution - the interaction of nuclear receptors with DNA response elements and the inter-domain dynamics of Cellobiohydrolase I

Lima, Leonardo Henrique França de 26 September 2011 (has links)
Movimentos coletivos prestam um papel fundamental na dinâmica e energética de biomoléculas em solução. Estes movimentos permitem o acoplamento de regiões significativamente distantes, apresentando considerável influência, por exemplo, no alosterismo para a formação de complexos macromoleculares e no funcionamento integrado de proteínas multidomínios como \"máquinas moleculares\". Neste trabalho de doutoramento, serão apresentados os resultados referentes à aplicação conjunta de técnicas experimentais biofísicas, de modelagem estrutural e de dinâmica molecular no estudo de dois sistemas para os quais estes movimentos coletivos demonstram considerável importância funcional. Para a interação do receptor nuclear do ácido 9-cis-retinóico com seu elemento responsivo específico no DNA (HRE), a comparação de estudos de dinâmica molecular com ensaios de afinidade por anisotropia de fluorescência sugere que a resistência inicial para a associação do monômero, seguida da acentuada colaboratividade na associação do dímero é regida por um impedimento da associação do domínio de ligação ao DNA (DBD) para o primeiro à sequência responsiva devido, em última análise, a uma não complementaridade dos modos coletivos mútuos. Este impedimento para a associação monomérica inicial é mais acentuado para o monômero 5\' (para o qual a menor especificidade de ligação à seqüência específica já é bem documentada), devido aos efeitos conjuntos de um \"defeito\" natural no empacotamento de bases da seqüência responsiva, que se manifesta mais significativamente na interface entre o meio-sítio 5\' e a seqüência espaçadora, e dos modos vibracionais entre os dois sítios decorrentes de seu faseamento relativo na topologia do DNA na seqüência responsiva, caracterizando um mecanismo \"chave e fechadura\" para a interação obrigatoriamente simultânea dos dois monômeros ao DNA. No segundo caso, um estudo integrado utilizando a técnica experimental de espalhamento de raios X a baixos ângulos e uma abordagem de modelagem estrutural baseada em dinâmica molecular foi realizado para a celobiohidrolase I de Trichoderma harziannum. Este estudo permitiu tanto a elaboração de um modelo estrutural de maior resolução para esta enzima de alto potencial biotecnológico como a constatação dos possíveis mecanismos moleculares a partir dos quais as glicosilações no peptídeo conector impõem restrições à orientação e modos vibracionais entre seus dois domínios de forma condizente com sua ação concertada na interação e no deslize da enzima sobre a superfície celulósica, ambos de fundamental importância para a processividade da enzima na hidrólise do substrato microcristalino. / Collective motions play a fundamental role in solution biomolecule dynamics and energetics. These movements can couple very distant regions in the protein structures affection, for instance, allosteric mechanisms, the establishment of macromolecular complexes, and on the integrated function of multidomain proteins as molecullar machines. In this thesis, we present results concerning to the joint use of experimental biophysical techniques, structural modeling and molecular dynamics simulations on the study of two systems for which these collective motions have substantial importance. First, we study the interaction of the nuclear retinoid X receptor with its specific DNA hormone response element (HRE) using a combination of molecular dynamics simulations and affinity assays performed by using fluorescence anisotropy. We find out that collective motions mediate the low binding affinity of monomers and the high cooperative binding of HRE dimers. The lower binding affinity of the monomer is more prominent for 5´ monomers. This occur due to an natural ineffective stacking of the last base pair step at the 5´-half-site and to the phasing of the two binding half-sites in the DNA topology, that impose a collective motions that tends to occlude the 5´ binding site. This behavior, in turn, is concurrent with the well known 3´ polarity and the decreased binding specificity to the 5´ half site for the hRXRα monomer. This same pattern impose a lock-and-key mechanisms dependent on the binding of the full dimer. Second, an integrated Small angle X ray scattering and molecular dynamics based structural modeling was used to comprehend the interdomain motions of cellobiohydrolase I of Trichoderma harziannum. We manage to build a refined model for this enzime, with important biotechnological potential. We also provide insights into molecular mechanisms of linker and glycosylation imposed restraints on the orientation and vibrational modes of the full-length enzyme, supporting a mechanism of sliding of on the cellulose surface. This mechanism is fundamental for the high processivity on the hydrolysis of microcrystalline cellulose.
334

Expression et évolution de gènes de la famille des ARN hélicases à boîte DEAD chez Arabidopsis thaliana (L.) Heynh

Mingam, Annaïck 08 June 2004 (has links) (PDF)
L'évolution de la régulation de l'expression des gènes participe à l'évolution de leur fonction. L'existence de profils d'expression spécifiques évitant la redondance fonctionnelle expliquerait le maintien des gènes dupliqués dans les génomes. La PCR quantitative en temps réel se révèle adaptée à l'étude de l'expression des familles de gènes présentant des niveaux très variés et des séquences proches. Le génome modèle d'Arabidopsis thaliana contient une famille d'ARN hélicases à boîte DEAD (RH) de 58 membres, i.e. environ deux fois plus que n'en comptent les génomes d'animaux ou celui de la levure. L'expression transcriptionnelle de 20 AtRH a été obtenue par RT-PCR quantitative dans neuf organes différents. Dans cette famille de gènes " de ménage ", deux AtRH présentent des profils spécifiques alors que les 18 autres AtRH présentent le même profil spatial, mais des niveaux d'expression transcriptionnelle très différents. L'élément régulateur principal du niveau transcriptionnel est la présence simultanée d'une boîte TATA caractéristique et d'un intron en 5' UTR. Dès lors que la boîte TATA est présente, il y a une corrélation positive significative entre la taille de l'intron en 5' UTR et le niveau d'expression. Notre travail sur l'expression des AtRH permet d'introduire un scénario sur la dynamique évolutive des gènes dupliqués qui forment une même branche terminale d'un arbre phylogénétique et dont le niveau d'expression diffère fréquemment. Après la duplication d'un gène fortement transcrit, l'altération de l'activité transcriptionnelle des copies se produirait par des événements successifs de suppression de la boîte TATA et/ou de l'intron en 5' UTR.
335

Macromolecular Interactions in West Nile Virus RNA-TIAR Protein Complexes and of Membrane Associated Kv Channel Peptides

Zhang, Jin 01 July 2013 (has links)
Macromolecular interactions play very important roles in regulation of all levels of biological processes. Aberrant macromolecular interactions often result in diseases. By applying a combination of spectroscopy, calorimetry, computation and other techniques, the protein-protein interactions in the system of the Shaw2 Kv channel and the protein-RNA interactions in West Nile virus RNA-cellular protein TIAR complex were explored. In the former system, the results shed light on the local structures of the key channel components and their potential interaction mediated by butanol, a general anesthetic. In the later studies, the binding modes of TIAR RRM2 to oligoU RNAs and West Nile virus RNAs were investigated. These findings provided insights into the basis of the specific cellular protein–viral RNA interaction and preliminary data for the development of strategies on how to interfere with virus replication
336

Θέματα στην εφαρμογή προτύπων ποιότητας στην ασφάλεια των πληροφοριακών συστημάτων : Η περίπτωση της Εθνικής Τράπεζας της Ελλάδος

Παναγόπουλος, Αιμίλιος-Χρήστος 13 January 2015 (has links)
Η χρήση των Πληροφοριακών Συστημάτων συνεχώς αυξάνεται. Πλέον οι περισσότεροι οργανισμοί βασίζονται στην λειτουργία τους. Αχίλλειος πτέρνα αυτών είναι η ασφάλεια τους. Στη παρούσα μελέτη παρουσιάζονται τα βασικά θέματα που αφορούν την διαχείριση προσωπικών δεδομένων αναλύοντας την πολιτική ασφαλείας μιας εταιρείας του ελληνικού τραπεζικού τομέα . Αρχικά εντάσσεται η έννοια των Πληροφοριακών Συστημάτων. Ακολουθεί η έννοια της Πολιτικής Ασφάλειας στον ευρύτερο τομέα της Διαχείρισης της Ασφάλειας των Πληροφοριακών Συστημάτων καθώς και οι κατηγοριοποιήσεις των κινδύνων και των ζημιογόνων γεγονότων. Έπειτα προσδιορίζονται οι βασικές αρχές για την ανάπτυξη Πολιτικών Ασφάλειας των Πληροφοριακών Συστημάτων, διευκρινίζοντας το νομικό πλαίσιο προστασίας τραπεζικών δεδομένων και το απόρρητο τους. Η επόμενη ενότητα αφορά την εφαρμογή των Πολιτικών Ασφάλειας στο πλαίσιο της εταιρείας και καταγράφει τα απαραίτητα μέτρα για την επιτυχή και αποτελεσματική εφαρμογή τους. Ακολουθούν τα αποτελέσματα της μελέτης και οι προτάσεις για την βελτιστοποίηση της παρούσας κατάστασης και την αποφυγή μελλοντικών κινδύνων. / The use of Information Systems is constantly increasing. Now most of the organizations rely on them for their operation. Their vulnerable spot is their security. This study presents the main issues related to the management of personal data by analyzing the security policy of a company of Greek banking sector. Firstly, the concept of Information Systems is presented.Then a part of the concept of security policy in the broader field of Safety Management Information Systems and classifications of risks and loss events is presented. Afterwards identifying the key principles for the development of Rules of Security of Information Systems, specifying the legal framework for the protection of bank data and their privacy. The next section involves the implementation of security policies within the company and record the necessary steps for the successful and effective implementation. Then are the results of the study presented and recommendations for optimization of this situation and avoiding future risks.
337

Functional Genetic Analysis Reveals Intricate Roles of Conserved X-box Elements in Yeast Transcriptional Regulation

Voll, Sarah 13 November 2013 (has links)
Understanding the functional impact of physical interactions between proteins and DNA on gene expression is important for developing approaches to correct disease-associated gene dysregulation. I conducted a systematic, functional genetic analysis of protein-DNA interactions in the promoter region of the yeast ribonucleotide reductase subunit gene RNR3. I measured the transcriptional impact of systematically perturbing the major transcriptional regulator, Crt1, and three X-box sites on the DNA known to physically bind Crt1. This analysis revealed interactions between two of the three X-boxes in the presence of Crt1, and unexpectedly, a significant functional role of the X-boxes in the absence of Crt1. Further analysis revealed Crt1- independent regulators of RNR3 that were impacted by X-box perturbation. Taken together, these results support the notion that higher-order X-box-mediated interactions are important for RNR3 transcription, and that the X-boxes have unexpected roles in the regulation of RNR3 transcription that extend beyond their interaction with Crt1.
338

Caracterização química e genética da interação Capsicum spp. (Solanacea), pulgão Aphis gossypii Glover (Hemiptera: Aphididae) e o parasitóide Aphidius colemani Viereck (Hymenoptera, Braconidae, Aphidiinae) / Chemical and genetic characterization of the interaction Capsicum ssp. (Solanacea), Aphid Aphis gossypii Glover (Hemiptera: Aphididae) and the paraitoid Aphidius colemani Viereck (Hymenoptera, Braconidae, Aphidiinae)

Costa, João Gomes da 23 August 2010 (has links)
Pest control of cultivated plant species has been usually performed by insecticides, which is undesirable because of economical and environmental concerns, since successive applications affect natural enemies and increase the possibility of development of resistant population toward insecticides. These problems can be minimized with alternative control methods as the use of resistant varieties, use of substances that induce resistance and biological control. Those studies involving the interaction of plant, pest and natural enemies are of fundamental importance. Thus, this study aimed: a) to study the effect of volatile organic compounds in tritrophic interactions between pepper Capsicum spp., the aphid Aphis gossypii and its parasitoid Aphidius colemani; b) to study the role of cis-jasmone in the tritrophic interaction between the pepper, the aphid A. gossypii and the parasitoid A. colemani and its role in activating the defense mechanism of the plant. Pepper varieties were evaluated for resistance to the aphid A. gossypii and their volatiles were collected before and after infestation. Volatiles compounds were tentatively identified by gas chromatography/mass spectrometry. Olfactometry bioassays were performed with volatile regarding the behavior of A. gossypii and A. colemani. The main conclusions obtained in this work were: a) there is genetic variability among genotypes of Capsicumin relation to the release of volatile compounds and in the susceptibility toward A. gossypii; b) genotype Cambuci can be used in breeding programs aiming Capsicum cultivars more resistant to A. gossypii; c) there were significant differences between the effects of volatiles from the two cultivars on behavior of A. gossypii and A. colemani; d) the volatiles emitted by Cambuci cultivar after infestation produced repellent effect on A. gossypii and were attractive to A. colemani; e) the cis-jasmone applied to pepper plants provided emission of volatiles that had repellent action on the A. gossypii and attractive one to A. colemani; f) the genetic variability between genotypes, after infestation indicates that volatile organic compounds present as variables can be used for selection and development of bell pepper cultivars resistant to the aphid A. gossypii. / O controle de pragas das espécies vegetais cultivadas tem sido normalmente realizado por meio de inseticidas, o que é indesejável tanto por motivos econômicos quanto ambientais, já que as aplicações sucessivas afetam os inimigos naturais e aumentam a possibilidade de desenvolvimento de populações da praga resistentes aos inseticidas. Esses problemas podem ser minimizados com métodos alternativos de controle como o emprego de variedades resistentes, o uso de substâncias indutoras e o controle biológico. Para isso, estudos envolvendo a interação planta, praga e inimigo natural são de fundamental importância. Dessa forma, este trabalho teve como objetivos: a) Estudar a ação dos compostos orgânicos voláteis na interação tritrófica entre o pimentão Capsicum spp., o pulgão Aphis gossypiie seu parasitóide Aphidiuscolemani; b) Estudar a ação da cis-jasmona na interação tritrófica entre o pimentão, o pulgão A. gossypii e o parasitóide A. colemani e seu papel na ativação do mecanismo de defesa do vegetal. Variedades de pimentão foram avaliadas em relação à resistência ao pulgão A. gossypii e os seus compostos voláteis foram coletados antes e após a infestação. Os compostos voláteis foram tentativamente identificados por cromatografia gasosa/espectrometria de massas. Bioensaios de olfatometria foram realizados com os compostos voláteis em relação ao comportamento de A. gossypii e A. colemani. As principais conclusões obtidas neste trabalho foram: a) Existe variabilidade genética entre os genótipos de Capsicum em relação à emissão de compostos voláteis e em relação à susceptibilidade ao A. gossypii; b) O genótipo Cambuci poderá ser utilizado em programas de melhoramento genético visando cultivares de Capsicum mais resistentes ao A. gossypii; c) Houve diferenças significativas entre os efeitos dos compostos voláteis das duas cultivares sobre os comportamentos de A. gossypii e de A. colemani; d) Os compostos voláteis emitidos pela cultivar Cambuci após a infestação proporcionaram efeito repelente a A. gossypii e atrativo a A. colemani; f) A cis-jasmona aplicada sobre plantas de pimentão induziu a emissão e/ou produção de compostos voláteis que teve ação de repelência a A. gossypii e ação atraente para A. colemani; i) A variabilidade genética entre os genótipos, após a infestação, indica que os compostos orgânicos voláteis apresentam-se como variáveis que podem ser utilizadas para seleção e desenvolvimento de cultivares de pimentão resistente ao pulgão A. gossypii.
339

O Partido Comunista (1922-1962): lugar de mem?ria, espa?o de disputa

Souza, Vital Nogueira de 27 March 2007 (has links)
Made available in DSpace on 2014-12-17T15:25:09Z (GMT). No. of bitstreams: 1 VitalNS.pdf: 421939 bytes, checksum: dfb24bac7ed8f9c2b6f1c9cb48e4b676 (MD5) Previous issue date: 2007-03-27 / This work studies the dispute by Party Communist s memory in Brazil during and after division which it resulted in the appearance of two parties entitled communists, in the brazilian political context, in the final of the fifties and beginning of the sixties: the Brazilian Communist Party (in Portuguese, Partido Comunista Brasileiro) and the Communist Party of Brazil (in portuguese, Partido Comunista do Brasil). Along of yours three chapters, the author tries to answer some questions as: what were the elements which constituted the Communist Party s memory, in the period from 1922 to 1956? At which moment did this memory pass to be fiercely contested? Who and which documents reveal this? How did it happen the dispute by this memory? The approach to topic consider which the production of memory by Communist Party and the dispute for your possession by communists of PCB and of PCdoB happened in scope of national reality and of party, spaces notions understand from concept of memory developed for authors as Halbwachs, Le Goff, Pollack and Pierre Nora. To explain the objectives of historical research it has used as methodology the exam and the interpretation of documentary and bibliographical sources, with priority for the party s documents then it was depicted a collective interpretation about the events considered more importants. The sites of Partido Comunista do Brasil (www.pcdob.org.br or vermelho.org.br) and of Partido Popular Socialista (www.pps.org.br) also went consulted / Este trabalho estuda a disputa pela mem?ria do Partido Comunista no Brasil durante e depois da cis?o que resultou no aparecimento de duas agremia??es intituladas comunistas, no cen?rio pol?tico brasileiro, no final da d?cada de 1950 e in?cio dos anos de 1960: o Partido Comunista Brasileiro (PCB) e Partido Comunista do Brasil (PCdoB). Ao longo de seus tr?s cap?tulos, o autor tenta responder quest?es como: quais os elementos que constitu?ram a mem?ria do Partido Comunista, no per?odo de 1922 a 1956? Em que momento essa mem?ria passou a ser disputada, por quem e quais documentos revelam isso? Como aconteceu essa disputa pela mem?ria? A abordagem do tema considera que a produ??o da mem?ria pelo Partido Comunista e a disputa por sua apropria??o pelos comunistas do PCB e do PCdoB aconteceram no ?mbito da realidade nacional e partid?ria, no??es espaciais compreens?veis a partir do conceito de mem?ria desenvolvido por autores como Halbwachs, Le Goff, Pollack, e Pierre Nora. Para dar conta dos objetivos da pesquisa hist?rica foi utilizado como metodologia o exame e a interpreta??o de fontes documentais e bibliogr?ficas, com prioridade para os documentos partid?rios que representavam uma interpreta??o coletiva sobre os acontecimentos considerados mais relevantes. Os sites do Partido Comunista do Brasil (www.pcdob.org.br ou vermelho.org.br) e do Partido Popular Socialista (www.pps.org.br) tamb?m foram consultados
340

Estudos por modelagem e dinâmica molecular integradas a técnicas físicas para biomoléculas em solução - interação de receptores nucleares a elementos responsivos no DNA e dinâmica inter-domínios da celobiohidrolase I / Integrated experimental biophysics and molecular dynamics simulations of biomolecules in solution - the interaction of nuclear receptors with DNA response elements and the inter-domain dynamics of Cellobiohydrolase I

Leonardo Henrique França de Lima 26 September 2011 (has links)
Movimentos coletivos prestam um papel fundamental na dinâmica e energética de biomoléculas em solução. Estes movimentos permitem o acoplamento de regiões significativamente distantes, apresentando considerável influência, por exemplo, no alosterismo para a formação de complexos macromoleculares e no funcionamento integrado de proteínas multidomínios como \"máquinas moleculares\". Neste trabalho de doutoramento, serão apresentados os resultados referentes à aplicação conjunta de técnicas experimentais biofísicas, de modelagem estrutural e de dinâmica molecular no estudo de dois sistemas para os quais estes movimentos coletivos demonstram considerável importância funcional. Para a interação do receptor nuclear do ácido 9-cis-retinóico com seu elemento responsivo específico no DNA (HRE), a comparação de estudos de dinâmica molecular com ensaios de afinidade por anisotropia de fluorescência sugere que a resistência inicial para a associação do monômero, seguida da acentuada colaboratividade na associação do dímero é regida por um impedimento da associação do domínio de ligação ao DNA (DBD) para o primeiro à sequência responsiva devido, em última análise, a uma não complementaridade dos modos coletivos mútuos. Este impedimento para a associação monomérica inicial é mais acentuado para o monômero 5\' (para o qual a menor especificidade de ligação à seqüência específica já é bem documentada), devido aos efeitos conjuntos de um \"defeito\" natural no empacotamento de bases da seqüência responsiva, que se manifesta mais significativamente na interface entre o meio-sítio 5\' e a seqüência espaçadora, e dos modos vibracionais entre os dois sítios decorrentes de seu faseamento relativo na topologia do DNA na seqüência responsiva, caracterizando um mecanismo \"chave e fechadura\" para a interação obrigatoriamente simultânea dos dois monômeros ao DNA. No segundo caso, um estudo integrado utilizando a técnica experimental de espalhamento de raios X a baixos ângulos e uma abordagem de modelagem estrutural baseada em dinâmica molecular foi realizado para a celobiohidrolase I de Trichoderma harziannum. Este estudo permitiu tanto a elaboração de um modelo estrutural de maior resolução para esta enzima de alto potencial biotecnológico como a constatação dos possíveis mecanismos moleculares a partir dos quais as glicosilações no peptídeo conector impõem restrições à orientação e modos vibracionais entre seus dois domínios de forma condizente com sua ação concertada na interação e no deslize da enzima sobre a superfície celulósica, ambos de fundamental importância para a processividade da enzima na hidrólise do substrato microcristalino. / Collective motions play a fundamental role in solution biomolecule dynamics and energetics. These movements can couple very distant regions in the protein structures affection, for instance, allosteric mechanisms, the establishment of macromolecular complexes, and on the integrated function of multidomain proteins as molecullar machines. In this thesis, we present results concerning to the joint use of experimental biophysical techniques, structural modeling and molecular dynamics simulations on the study of two systems for which these collective motions have substantial importance. First, we study the interaction of the nuclear retinoid X receptor with its specific DNA hormone response element (HRE) using a combination of molecular dynamics simulations and affinity assays performed by using fluorescence anisotropy. We find out that collective motions mediate the low binding affinity of monomers and the high cooperative binding of HRE dimers. The lower binding affinity of the monomer is more prominent for 5´ monomers. This occur due to an natural ineffective stacking of the last base pair step at the 5´-half-site and to the phasing of the two binding half-sites in the DNA topology, that impose a collective motions that tends to occlude the 5´ binding site. This behavior, in turn, is concurrent with the well known 3´ polarity and the decreased binding specificity to the 5´ half site for the hRXRα monomer. This same pattern impose a lock-and-key mechanisms dependent on the binding of the full dimer. Second, an integrated Small angle X ray scattering and molecular dynamics based structural modeling was used to comprehend the interdomain motions of cellobiohydrolase I of Trichoderma harziannum. We manage to build a refined model for this enzime, with important biotechnological potential. We also provide insights into molecular mechanisms of linker and glycosylation imposed restraints on the orientation and vibrational modes of the full-length enzyme, supporting a mechanism of sliding of on the cellulose surface. This mechanism is fundamental for the high processivity on the hydrolysis of microcrystalline cellulose.

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