Neste trabalho, utilizamos a técnica de espalhamento de raios-X a baixos Ângulos (SAXS) para estudar a influência de dois derivados fenotiazínicos na estrutura de sistemas micelares, assim como suas propriedades de auto-associação, além de investigar a influência da variação de pH e de concentração nas interações entre proteínas em solução. Para tanto, utilizamos dois fármacos fenotiazínicos, (Trifluoperazina, TFP e a Clorpromazina, CPZ), em presença de L--fosfatidilcolina (LPC), um surfactante zwiteriônico (30 mM), a pH 4.0 e 7.0. Os resultados de SAXS indicam que a micela de LPC, em ausência de fenotiazina, pode ser representada por uma micela com forma elipsoidal (com razão axial 1.6 0.1). No entanto, em presença de TFP e de CPZ a forma da micela se altera, passando para um cilindro (com razão axial 2.5 0.1). Este efeito é acompanhado por uma diminuição do raio parafínico da micela (22.5 0.3 Å), em ausência de fármaco, para 20.0 0.5 em presença de 10 mM de fármaco. Em paralelo, realizamos medidas de EPR (Ressonância Paramagnética eletrônica) destes sistemas. Combinando os resultados de SAXS e de EPR, propusemos um sítio para a localização destes compostos nas micelas de LPC, que seria na interface polar/apolar da mesma. Em um segundo momento, utilizamos as técnicas de SAXS e de EPR para investigar as características estruturais dos agregados formados por TFP e CPZ (a 20 e 60 mM, a pH 4.0 e 7.0). As curvas de SAXS são compatíveis com o espalhamento de agregados pequenos com diferentes geometrias: elipsoidal, cilíndrico e tipo-paralelepípedo. Devido à resolução da técnica, dentro do intervalo de vetores de espalhamento utilizada (até cerca de 0.3 Å-1), não é possível determinar, de forma absoluta, a correta geometria dos agregados, ou seja, todas as geometrias citadas acima ajustam de forma satisfatória as curvas de SAXS. As análises dessas curvas também não excluem a possibilidade de que estes fármacos mantenham-se como nano-cristais em solução (compostos por cerca de 10 celas unitárias, empilhadas na direção-z), seguindo sua estrutura cristalográfica. Medidas de EPR indicam que os auto-agregados a pH 4.0 possuem características semelhantes às micelas, mas a pH 6.5 este efeito não foi evidenciado, uma vez que ocorre uma forte interação entre a sonda e os agregados. Este fato indica que os agregados, a pH 6.5, têm um maior empacotamento, em comparação aos sistemas a pH 4.0. Por fim, utilizamos a Albumina de Soro Bovina (BSA, a 10 50 mg/ml), em diferentes pHs (2.0 9.0), para investigar os efeitos de concentração e de pH nos potenciais de interação das macromoléculas em solução. O fator de forma da proteína foi obtido através da estrutura cristalográfica da HSA (Human Serum Albumine, proteína humana homóloga a BSA), enquanto que as interações proteína-proteína foram calculadas através da relação de fechamento RPA (Random Phase Approximation). Nossos dados indicam que a BSA mantém sua estrutura terciária inalterada de pH 4.0 a 9.0, independente de sua concentração. No entanto, a pH 2.0 a proteína sofre um processo de desenovelamento, indicado pelo aumento da dimensão máxima da mesma. Nossos dados dão suporte para concluir que as interações entre as proteínas, a 10 mg/ml, são praticamente desprezíveis, exceto para os sistemas compostos a pH 2.0 (onde a proteína está desenovelada) e a pH 4.0 (onde evidenciamos a presença de interferência atrativa entre as proteínas). Entretanto, a medida em que aumentamos a concentração proteica, uma função de interferência do tipo repulsiva aparece na curvas de SAXS (para os sistemas de pH 4.0 a 9.0). Além disso, no sistema composto por BSA, pH 5.4 e 50 mg/ml, evidenciamos a existência de monômeros e dímeros em solução, provavelmente devido a proximidade do ponto isoelétrico da proteína (entre 4.8 5.6). Este efeito não foi evidenciado para os outros pHs, nesta mesma concentração. A pH 2.0 (25 e 50 mg/ml) evidenciamos uma compactação da proteína, sendo que sua forma é diferente da forma nativa da BSA. Nestas condições, é possível que a proteína tenha alcançado um estado molten globule, como evidenciado em outros trabalhos. Acreditamos que os efeitos de volume excluído são de grande importância para a estabilidade da proteína in vivo. / In this work we study, mainly by means of small angle X-ray scattering (SAXS), the influence of two phenothiazine derivatives on biomimetic systems as well as the self-assembly features. At the same time, the conformational stability of proteins in the presence of denaturant agents (pH and concentration) was evaluated. First of all, the phenothiazine compounds trifluoperazine (TFP) and chlorpromazine (CPZ) with micelles of the zwitterionic surfactant L--lysophosphatidylcholine (LPC), at pHs 4.0 and 7.0, are reported. The SAXS results demonstrate that, upon addition of both phenothiazines, the LPC micelle of prolate ellipsoidal shape changes into a cylindrically shaped micelle, increasing its axial ratio from 1.6 0.1 (in the absence of drug) to 2.5 0.1 (for 5 and 10 mM of phenothiazine). Such an effect is accompanied by a shrinking of the paraffinic shortest semiaxis from 22.5 0.3 to 20.0 0.5 Å. Besides, EPR (Electronic Paramagnetic Resonance) evidenced a bigger motion immobilization of the nitroxe probe, in the presence of phenothiazines. Our results provide evidence that the positively charged phenothiazine molecule must be accommodated near the hydrophobic/hydrophilic inner micellar interface. Furthermore, SAXS and EPR experiments were carried out to investigate the structure of the self-aggregates of CPZ and TFP, in aqueous solution. SAXS studies (drug solutions of 20 and 60 mM, at pH 4.0 and 7.0) evidenced that several different particle form factors with a homogeneous electron density distribution, in respect to the water environment, could reproduce the scattering curves. Due to the limitation of scattering intensity in the q range above 0.15 Å-1, precise determination of the aggregate shape was not possible and all of the tested models for ellipsoids, cylinders, or parallelepipeds fitted the experimental data equally well. The SAXS data allows inferring, however, that CPZ molecules might self-assemble in a basis set of an orthorhombic cell, remaining as nanocrystallites in solution. Such nanocrystals are composed of a small number of unit cells (up to 10, in c-direction), with CPZ aggregation numbers of 60-80. EPR spectra of 5- and 16-doxyl stearic acids bound to the aggregates were also performed, indicating a micelle-like aggregate at pH 4.0, and a significant motional restriction of the nitroxide was observed at pH 6.5. This implies that the aggregate is densely packed at this pH and that the nitroxide is tightly bound to it producing a strongly immobilized EPR spectrum. Finally, the effect of concentration and pH on the protein-protein interactions of BSA (Bovine Serum Albumin, from 10 up to 50 mg/ml) was evaluated by SAXS. Our results give support to infer that BSA keeps its native shape (similar to the Human Serum Albumin, HSA, crystallographic structure) unaltered at middle-acid (pH 4.0) up to basic pHs (9.0). At pH 2.0, however, BSA undergoes an unfolding process, indicated by a non globular shape. The protein-protein interactions were analysed into the Random Phase Approximation. The results show that at smaller amounts of BSA (10 mg/ml) the interference effects are not significative over the SAXS curve for pH 5.4 up to 9.0. At pH 4.0 and 10 mg/ml, however, an attractive potential takes place over the SAXS curves, that becomes repulsive with increasing BSA concentration. Besides, at pH 5.4 and 50 mg/ml, we evidenced a dimer-monomer co-existence in the solution. At pH 2.0 and 25 and 50 mg/ml, BSA undergoes to a compact conformation. Probably, BSA is in a molten globule state. Our results give also support to infer that probably, the exclude volume effect plays an important role on the protein stability in vivo.

O Vírus Respiratório Sincicial Humano, do inglês human Respiratory Syncytial Virus (hRSV), é uma das maiores causas de doenças respiratórias agudas, principalmente em crianças e bebês entre seis meses e dois anos de idade. Não há drogas eficazes ou vacina aprovada até o momento para esse vírus, apesar das décadas de intensa pesquisa e grande quantidade de dados sobre ele acumulados. O genoma do hRSV codifica onze proteínas e a compreensão das interações entre essas proteínas virais e as proteínas do hospedeiro é essencial para que possíveis alvos terapêuticos contra o hRSV sejam identificados. No laboratório, anteriormente, foi dado enfoque às interações entre as proteínas celulares e as proteínas virais de matriz (M), nucleoproteína (N) e fosfoproteína (P). Neste trabalho, analisamos as interações da proteína viral M2-1 (cofator essencial para a transcrição) através da mesma estratégia utilizada naqueles experimentos, de fusão a FLAG (gerando FLAG-M2-1) e imunoprecipitação com anticorpos contra esse peptídeo. As proteínas co-imunoprecipitadas, identificadas por espectrometria de massas, foram: poly(A)-binding protein cytoplasmic 1 (PABPC1), Y-box binding protein 3 (YBX3), e Nuclease-sensitive element-binding protein 1 (YBX1). M2-1 é capaz de integrar-se ao complexo chamado de semelhante a corpúsculos de inclusão (IB like, do inglês), formado por N e P, que é similar estruturalmente aos corpúsculos de inclusão encontrados em células infectadas (IBs). Essa propriedade foi usada para analisar que proteínas celulares seriam recrutadas para esse outro nível de organização dessas três proteínas virais, envolvidas na transcrição. O complexo FLAG-N/P/M2-1 co-imunoprecipitou as proteínas celulares: Hsp70, Hsp90 (Heat shock proteins 70 e 90), Npm (Nucleophosmin), que podemos agrupar como chaperonas; PABPC1, YBX1, YBX3, ligantes de RNA; e sub-unidade pICIn do metilossomo, associada a modificação pós-tradução. Detalhamos a análise para YBX3, obtendo evidências adicionais de sua interação com M2-1 em ensaios de complementação de proteína fragmentada (Split-NanoLuc), e de co-localização por imunofluorescência indireta. Finalmente, utilizamos a metodologia de expressão em bactérias para demonstrar a interação entre M2-1 e os domínios funcionais de PABPC1, porém esses ensaios não foram conclusivos. / Human Respiratory Syncytial Virus (hRSV) is one of the leading causes of acute respiratory diseases, especially in children and infants between six months and two years of age. There is no effective drug or vaccine approved so far for this virus, despite decades of intensive research and large amount of data on it. The genome of hRSV encodes 11 proteins and the understanding of the interactions between these viral proteins and host proteins is essential to identify possible therapeutic targets against hRSV. In the lab, previously, was given focus to the interactions between cellular proteins and viral proteins matrix (M), nucleoprotein (N) and phosphoprotein (P). In this paper, we analyze the viral M2-1 (cofactor essential for transcription) protein interactions through the same strategy used in those experiments: fusion with FLAG (generating FLAG-M2-1) and immunoprecipitation with antibodies against this peptide. The co-immunoprecipitated proteins, identified by mass spectrometry, were: Poly (A)-binding protein cytoplasmic 1 (PABPC1), Y-box binding protein 3 (YBX3), and Nuclease-sensitive element-binding protein 1 (YBX1). M2-1 is able to integrate the complex called similar to inclusion bodies (IB like), formed by N and P, which is similar structurally to the inclusion bodies found in infected cells (IBs). This property has been used to analyze which cellular proteins would be recruited for this new level of organization of these three viral proteins involved in transcription. The cellular proteins co-immunoprecipitated with the complex FLAG-N/P/M2-1, were: Hsp70, Hsp90 (Heat shock proteins 70 and 90), Npm (Nucleophosmin), that we can group as chaperones; PABPC1, YBX1, YBX3, RNA ligands; and the methylosome sub-unit pICIn, post-translational modification-associated. We detailed the analysis for YBX3, obtaining additional evidence of its interaction with M2-1 in fragmented protein complementation tests (Split-NanoLuc), and co-localization by indirect immunofluorescence. Finally, we used the methodology of expression in bacteria to demonstrate the interaction between M2-1 and functional domains of PABPC1, but these tests were not conclusive.

In dieser Arbeit werden Strategien zur Entwicklung von biohybriden Photoelektroden, die Licht in elektrische Energie umwandeln, demonstriert und diskutiert. Der natürliche Photonen-transformierende Superkomplex der oxygenen Photosynthese aus Thermosynechococcus elongatus, das Photosystem I (PSI), kann durch die nicht-native Interaktion zum Redoxprotein Cytochrom c (Cyt c), erfolgreich funktional in Elektroden integriert werden. Hierfür wurden unterschiedliche Strategien entwickelt, z. B. bilden beide Biokomponenten unspezifische Komplexe in Lösung und assemblieren gemeinsam auf modifizierten Goldoberflächen. Aus der Kontaktierung des PSI mit einer thiol-modifizierten Goldelektrode via Cyt c ergeben sich unidirektionale kathodische Photoströme. DNA, als ein Polyelektrolytmatrixelement, kann zum Aufbau von 3D-Protein-Mehrschichtarchitekturen höherer Stabilität und Leistungsfähigkeit verwendet werden. Der Einsatz von mesoporösen Indium-Zinnoxid-Elektroden vergrößert die Photostromgenerierung um mehr als eine Größenordnung, wodurch sich hieraus skalierbare transparente Photobioelektroden mit hohen Quanteneffizienzen (bis zu 30%) erzeugen lassen. / In this thesis, strategies are demonstrated and discussed for the development of biohybrid photoelectrodes transforming light into electrical energy. The natural photon-to-charge carrier converting super-complex from oxygenic photosynthesis of Thermosynechococcus elongatus, photosystem I (PSI), can be functionally implemented into such electrodes, due to the non-native interaction with the small redox protein cytochrome c (cyt c). Different strategies have been developed, e. g. both biocomponents form complexes in solution and self-assemble on modified gold-surfaces. The electrical connection of PSI to thiol-modified gold electrodes via cyt c results in unidirectional cathodic photocurrents of high efficiency. DNA, as a polyelectrolyte matrix element, can be used to build up 3D protein multilayer architectures of higher stability and performance. The use of mesoporous indium tin oxide electrodes further enhances the photocurrent generation more than one order of magnitude, thus resulting in scalable transparent photobioelectrodes of high quantum efficiencies (up to 30 %).

Made available in DSpace on 2015-03-05T13:56:59Z (GMT). No. of bitstreams: 0 Previous issue date: 6 / Hewlett-Packard Brasil Ltda / Um dos mais importantes desafios para a biologia pós-genômica é atender a estrutura e o comportamento das interações moleculares complexas que controlam o comportamento celular. Para tanto é essencial à integração dos dados biológicos referentes a estas interações armazenadas em diversos banco de dados. Este é um problema difícil, pois estes dados estão disponíveis em banco de dados públicos espalhados geograficamente na rede mundial de computadores e cada um destes possui um sistema diferente de gerenciamento, formato ou visão de como representar os dados. Os principais problemas para a realização desta tarefa são:a necessidade de se desenvolver e aplicar parsers para cada banco de dados sem ausência de um vocabulário unificado. Como uma alternativa para facilitar estes problemas, este trabalho propõe a ontologia MONET (Molecular Network Ontology) que tem como objetivo ser um modelo integrado para a rede de redes que existe dentro da celula. Tal visão integrada ajuda a entender as interações de larga escala / One of the most important challenges for biology in the post-genomic is to understand the structure and behavior of the molecular interactions that controls cell behavior. Therefore is essential to integrate biological data concerning these interactions, which are stored in different databases. The integration task is dificult because these data are distributed in public databases on the world wide web and each database has diferent management systems, formats and views of how to represent biological data. The two main problems involved here are the dificulty in parsing the data when dealing with heterogeneous at file formats and the inconsistencies due to the absence of an united vocabulary. As an alternative to facilitate these problems this work proposes MONET (the Molecular Network) ontology, an integration model for the unifying of diferent molecular networks that exist inside the cell. Such integrated view facilitates the understanding of the large-scale interactions responsible for the behavior of

Les immunoglobulines ne sont pas les seules protéines capables de reconnaissance spécifique. D’autres systèmes d’immunité adaptative existent et beaucoup d’autres protéines peuvent aussi générer des interactions spécifiques de hautes affinités. Ce sont des ossatures/squelettes protéiques intéressants pour concevoir de nouveaux interacteurs.Une nouvelle famille de protéines synthétiques, appelées AlphaReps, basée sur la famille des protéines à HEAT repeat contenant un motif structural répété en double hélice alpha, a été construite au laboratoire. Le motif répété, d’abord identifié chez une archée thermostable, a été idéalisé en concevant une séquence consensus, grâce à l’alignement de séquences de motifs naturels. Une banque de protéines a alors été construite à partir de ce motif. Toutes les protéines de la banque ont une structure générale similaire mais elles diffèrent par le nombre de motifs insérés et par les cinq résidus hautement diversifiés situés sur la face externe de la seconde hélice de chacun des motifs. Ces nouvelles protéines sont exprimées très efficacement chez E. Coli, solubles, sans pont disulfure et très stables (50-100 mg. L-1 de culture, Tm > 70°C).Le travail de thèse présenté dans ce manuscrit s’intéresse à l’utilisation de ces nouvelles protéines synthétiques comme outils de reconnaissance moléculaire. Pour cela, plusieurs applications ont été développées. Dans la première partie, à l’aide de la technique du phage display, des interacteurs de hautes affinités pour la Green Fluorescent Protein ont pu être isolés au sein de la banque. Les interactions des protéines partenaires ont été caractérisées par la détermination des constantes d’affinité, ainsi que la résolution des structures cristallographiques de deux complexes contenant une AlphaRep spécifique et la GFP. Avec cette cible modèle, la possibilité d’utiliser les AlphaReps sélectionnées à l’intérieur des cellules eucaryotes vivantes pour reconnaître spécifiquement une cible protéique dans un milieu complexe a aussi été démontrée. L’utilisation des AlphaReps comme outils de diagnostique a été développée pour la détection de la cible membranaire FSHr (récepteur de l’hormone folliculo-stimulante), protéine surexprimée dans de nombreuses tumeurs. Ce projet a permis d’expérimenter des approches de sélections sur cellules entières, soulignant les progrès restant encore à accomplir pour la sélection contre des cibles plus complexes.La seconde partie de ce travail s’est intéressée à l’ingénierie et à l’évolution des AlphaReps. Ainsi, l’insertion de résidus variables sur le dernier motif (C-cap) de protéines de la banque a pu être validé. Une approche innovante de shuffling modulaire, adaptée à l’ossature AlphaRep a permis de cerner les limites de cette méthode et les améliorations à apporter pour être en mesure d’augmenter l’affinité d’interacteurs présélectionnés. La banque d’AlphaReps de phage display a également été transférée dans un vecteur de PCA (Protein Fragment Complementation Assay) utilisant la protéine scindée DHFR (Dihydrofolate Reductase) comme protéine rapportrice. Cela a permis de sélectionner des AlphaReps spécifiques pour des cibles non exploitables en phage display. L’utilisation de la cis-fusion entre une AlphaRep et sa cible, combinée à la technique de PCA, s’est révélée très efficace pour la sélection et la cristallisation de protéines réfractaires telles que la protéine ComD, ici présentée comme preuve de la réussite de cette approche.Les AlphaReps sont donc des protéines artificielles, parmi lesquelles des interacteurs spécifiques peuvent être isolés pour des cibles variées. Un large panel d’applications peut être envisagé comme le développement d’outils d’aide à la cristallogenèse ou celui d’outils de reconnaissance moléculaire in vivo. / Immunoglobulin fold is not the only basis for specific recognition proteins. Other adaptive immunity systems exist and many other proteins are also able to mediate specific high-affinity interactions. These are interesting scaffolds to generate alternative binding molecules.A new family of artificial proteins, named AlphaRep, based on HEAT repeat proteins containing an alpha-helical repeated motif, was designed in the laboratory. The repeated motif, first identified in a thermostable archae protein of unknown function was refined and idealized using a consensus design strategy. A library of artificial proteins based on this design was then constructed. All proteins from this library share the same general fold but differ both in the number of repeats and in a set of five highly randomized positions per repeat. The randomized side chains are located on the outside surface of the second helix. Sequences from this library are efficiently expressed as soluble, folded and very stable proteins (50-100 mg. L-1 of culture, Tm > 70°C).The work presented in this manuscript is focused on the use of those new synthetic proteins as molecular recognition tools. Then, different applications have been developed.In a first part, binders with high affinity for the green fluorescent protein were selected by phage display. Complexes were characterized. Affinity between partners was measured and structures of two of those complexes containing a specific AlphaRep and the protein target were solved by X-ray crystallography. Thanks to this model target, it was demonstrated that AlphaReps could be used in living cells for the specific recognition of the protein they have been selected for. AlphaReps have also been developed as a diagnostic tool to detect the membrane protein FSHr (Follicle stimulating Hormone receptor), shown to be overexpressed in various tumors. In this project, selections on entire cells have been performed, showing the limit of the selections approaches with complex targets.The second part of this work focused on engineering and evolutions of AlphaRep proteins. The insertion of randomized residues at specific positions in the last motif (C-cap) was validated. An innovative approach of modular shuffling, adjusted to the AlphaRep scaffold, was assessed. Limitations of this approach to perform affinity maturation of AlphaReps could then be understood. Finally, the AlphaRep Library was transferred to a PCA (Protein Fragment Complementation Assay) vector using the split DHFR (Dihydrofolate Reductase) as reporter protein. With this new selection system, specific Alphareps could be selected for protein targets not suitable for phage display selection. A cis-fusion strategy was employed to express the AlphaRep fused to its partner in order to increase the stability and solubility of the target as well as helping for its crystallogenesis. This approach, combined with the PCA selection, was successful to obtain crystals of the ComD protein (unstable protein), shown here as an example of success for this new method.AlphaReps are thus artificial proteins, among which specific binders can be isolated for various targets, showing a strong potential for a large range of applications from crystallogenesis helpers to in vivo molecular recognition tools.

Les travaux décrits dans ce mémoire ont pour objectif d’une part le développementd’une nouvelle famille de protéines artificielles et d’autre part la création de nouveaux sitesde fixation spécifiques dans ces protéines. L’objectif général était de développer une approchegénérale permettant d’obtenir rapidement des protéines reconnaissant toute macromoléculecible choisie. On peut voir ces protéines artificielles spécifiques comme des sortes d’anticorpsartificiels pour leur spécificité et leur affinité mais dont les propriétés physiques : stabilité,solubilité, efficacité d’expression, insensibilité à l’agrégation sont nettement plus favorablesque celles des anticorps et de leur dérivés.Le premier chapitre, présente la conception et la construction d’une bibliothèque deprotéines artificielles dite de première génération où les protéines sont formées par larépétition d’un motif idéalisé à partir d’une famille de motifs naturels appelés HEAT repeats.Toutes les protéines de la bibliothèque, dénommées αRep, sont conçues pour avoir la mêmearchitecture générale mais diffèrent les unes des autres par le nombre de motifs et par laséquence dans certaines positions rendues variables au sein de chaque motif. Cette banquenous a permis de valider l’architecture αRep choisie : Les protéines s’expriment sous formesoluble, sont très stables et adoptent la structure secondaire et tertiaire attendue quel que soitla séquence des positions hypervariables. Le second chapitre présente alors les approchessuivies pour l’amélioration de la qualité et de la diversité de la bibliothèque et a conduit à laconstruction d’une bibliothèque d’αRep de deuxième génération. Cette dernière bibliothèque(2 .1) repose sur le même schéma général mais contient une diversité ayant été optimiséelors de la conception puis améliorée expérimentalement par une procédure dite deFiltration/shuffling. Cette bibliothèque très diverse (1.7*109 clones indépendants) a été alorsexploitée pour y rechercher, par des méthodes d’exposition sur phages, de nouvelles αRepreconnaissant des protéines cibles préalablement choisies. L’ensemble des résultats montretrès clairement que des αRep reconnaissant spécifiquement, avec une affinité élevée, desprotéines cibles choisies arbitrairement peuvent être effectivement obtenues. Les structurestridimensionnelles de plusieurs complexes formés entre les αRep et leur cible a été résoluepermet de comprendre la nature et l’organisation précise de ces capacités de reconnaissancemoléculaire nouvellement créées. / The main objective of this work was to design, produce and characterize a new familyof artificial proteins and to introduce new tailored specific binding sites within this structuralframework. Our general goal was to develop method allowing to rapidly generate newprotein binding specifically to any predefined target macromolecule. Binders based onartificial proteins can be viewed as antibody-like molecules but due to their different structurehave more favorable physical properties (expression, solubility, folding efficiency, stability)than antibodies and derivatives.The design and experimental assembly of a first generation artificial protein library isdescribed in part I. Proteins of this library are made by a repetition of a motif idealized from afamily of natural protein repeats (HEAT repeat). These artificial proteins, named αRep, havethe same general fold but the number of the repeated motif vary from protein to protein.Furthermore, a set of positions of each motif is highly variable within the library. Proteinisolated from this first generation library are well expressed, soluble, extremely stable andwere shown to have the designed secondary and tertiary structure.The methods used to improve the diversity and the experimental quality of the protein libraryare described in the second part of this thesis and have allowed us to create a secondgeneration αRep library. This library is based on the same general scheme but its diversitywas optimized by an improved design and experimental procedures known as filtration/shuffling.This highly diverse second generation library (1.7*109 independent clones) was usedto select variants with tailored binding specificities using phage display method. The resultsclearly show that news αReps binding tightly and specifically a range of arbitrarily definedprotein targets can be efficiently selected. The tertiary structure of complexes between αRepand their cognate target molecule were solved and allow to analyze the nature and detailedorganization of this newly engineered molecular recognition capacities.

Made available in DSpace on 2016-01-26T12:51:42Z (GMT). No. of bitstreams: 1 arielifernandagavioligomes_dissert.pdf: 1557143 bytes, checksum: 898e0714fcf73f6bc8ba53f5719efc71 (MD5) Previous issue date: 2011-12-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Introduction: The Yellow Fever is characterized by severe hepatitis, renal failure, hemorrhage, and rapid terminal events that lead to shock and death. This disease is caused by the infection with the Yellow Fever Virus (YFV), considered the prototype of the Flavivirus genus. Its mechanism of replication is not well known but includes interactions of viral RNA with cellular and viral proteins. The nonstructural protein 5 (NS5) is the largest and most conserved protein of the Flavivirus genus; it encodes RNA-dependent RNA polymerase (RdRp) domains, besides possessing many important functions during viral replication, such as genic regulation of host cells. The protein hSlu7 is a homologous human protein, which was isolated interacting with the U5 that is involved in the second the step of alternative splicing. The hSlu7 is a predominantly nuclear protein and participates at the alternative splicing, influencing the correct choice of the alternative AGs of exon 3' so that the spliceossome is able to bind and start alternative splicing. Objective: To characterize the interaction of the hSlu7 protein with the YFV-NS5 protein and its cellular localization during viral infection. Material and Method: We confirmed the interaction of various NS5 with human proteins by two-hybrid assays. Deletion mutants were constructed and co-transformed with hSlu7 in yeast to determine the minimal domain of NS5 required for interaction. The cellular localization of the hSlu7 fused with GFP during the response of vaccine strain 17D of YFV in cells Vero E6, marked with anti-NS4AB and anti-NS5 for detection of the infection was also tested. Results: hSlu7 interacts with initial and final portions of the RdRp and the cytoplasmic sublocalization of hSlu7 occurs in the cells infected with YFV. Conclusions: Our results suggest that hSlu7 interacts with the YFV by two-hybrid system and the cellular sublocalization occurs due to the presence of viral infection. Further studies using RNA interference should be addressed to confirm the cellular function of hSlu7 , and to evaluate which alterations that infected and uninfected cells will suffer with low levels of hSlu7. / Introdução: A Febre Amarela é uma doença decorrente da infecção pelo Vírus da Febre Amarela (YFV) que é um protótipo do gênero Flavivirus que provoca uma severa hepatite, falência renal, hemorragia, e eventos que rapidamente levam ao choque e morte do indivíduo. Os mecanismos de replicação genômico do YFV não são bem conhecidos. A proteína não estrutural 5 (NS5) é a maior proteína e a mais conservada dos Flavivirus, ela codifica a RNA polimerase dependente de RNA (RdRp). A hSlu7 é uma proteína celular, predominantemente nuclear, e foi isolada interagindo com a U5 no segundo passo do splicing alternativo. A hSlu7 auxilia na correta seleção dos AGs alternativos do exon 3 para a realização da reação de splicing. Objetivo: Caracterizar a interação de hSlu7 com a proteína NS5 de YFV, quanto a sua localização celular durante a infecção. Material e Método: Pelo sistema duplo-híbrido em leveduras utilizando plasmid-linkage avaliamos a interação de hSlu7 com deleções mutantes da RdRp de YFV, e a localização celular de GFP-hSlu7 durante a replicação da cepa vacinal 17D de YFV em cultura de células Vero E6, marcadas com anticorpos NS4AB e NS5 para detecção da infecção. Resultados: A hSlu7 interage com as porções inicial e final da RdRp, e a sublocalização citoplasmática de hSlu7, ocorre nas células infectadas com YFV. Conclusão: Nossos resultados sugerem que a hSlu7 possui uma sublocalização celular durante a replicação do YFV, além de interagir com a RdRp viral. Ainda será necessária a confirmação da função celular de hSlu7 utilizando RNA de interferência para avaliar quais as alterações que a célula não infectada e infectada sofrerá diante dos níveis baixos de hSlu7.

Made available in DSpace on 2016-01-26T12:51:47Z (GMT). No. of bitstreams: 1 anacarolinabterzian_tese.pdf: 3328386 bytes, checksum: 0bae9bd7c5453a781cfbba32fac30196 (MD5) Previous issue date: 2013-11-08 / Fundação de Amparo a Pesquisa do Estado de São Paulo / Introduction. Yellow Fever Virus (YFV) causes a hemorrhagic fever and it is the prototype of genus Flavivirus. Kunjin virus (KUNV) is naturally attenuated and is used to develop vaccine candidates against more pathogenic WNV strains. Flavivirus replication is a complex mechanism that involves interaction between viral RNA and cellular and viral proteins. The NS5 protein is the largest and highly conserved viral protein and it is critical for many functions, including replication, RNA capping and virus-host interactions. Once protein-protein interactions present basic importance for the activation, the regulation and the control of diverse enzymatic functions related to these interactions, the identification and the characterization of them are essential for a better comprehension of the pathogenesis and for the rational design of drugs for YFV. Previously, it was identified that the cellular protein p54nrb/NonO interacts with the RNA dependent RNA polymerase domain of YFV NS5. The p54nrb/NonO protein is a nuclear transcription factor associated with nuclear membrane and exhibits multifunction characteristics in nuclear processes in eukaryotic cells, in frequent association with the U1A and PSF proteins. Interaction between NS5 and p54nrb/NonO may influence localization and transport of proteins and viral RNA within the cell. Objective. The purpose of this study was to confirm the interaction between p54nrb/NonO and YFV and KUNV NS5 and determine the role of p54nrb/NonO on viral replication. Material and Method. Co-immunoprecipitation, mass spectrometry and indirect immunofluorescence assays were realized to confirm the interaction and co-localization between the proteins. To determine the effect on viral replication, the p54nrb/NonO and PSF were overexpressed in cellular culture, as well, the silencing of p54nrb/NonO. After, the replication level was determined by Tempo Real PCR, plaque assay, measuring of β-galactosidase and luciferase activity assays. Results. Immunofluorescence assays showed co-localization of p54nrb/NonO with YFV NS4 in the perinuclar region and with NS5 in the nucleus. In contrast, KUNV NS5 co-localized with p54nrb/NonO in the perinuclear region and co-precipitated with p54nrb/NonO. The co-precipitation between p54nrb/NonO and NS5 YFV was not identified. Again, it was identified by mass spectrometry analysis the co-precipitation of p54nrb/NonO by monoclonal antibodies to KUNV NS5 protein. The p54nrb/NonO overexpression did not affect the YFV and KUNV replication, however, PSF overexpression showed inhibitory effect on viral replication. The RNA interference assays were inconclusive about the role of p54nrb/NonO silencing on YFV replication. Conclusion. p54nrb/NonO and KUNV NS5 interact physically and co-localize in the cytoplasm, while, the co-localization with YFV NS5 occcurs in the nucleus, although, there is no physical interaction between them. However, the overexpression of p54nrb/NonO does not affect the viral replication. PSF was confirmed as an interactive partern of p54nrb/NonO and, when it is overexpressed, it inhibits YFV and KUNV replication. / Introdução. O vírus da Febre Amarela (YFV) causa febre hemorrágica e é o protótipo do gênero Flavivirus. O vírus Kunjin (KUNV) é naturalmente atenuado e usado para o desenvolvimento de candidatos vacinais contra linhagens mais patogênicas do WNV. A replicação do Flavivirus é um mecanismo complexo que envolve interações entre o RNA viral e proteínas virais e celulares. A NS5 é a maior e mais conservada proteína viral e é crítica para muitas funções, incluindo replicação, capeamento do RNA e interação vírus-hospedeiro. Como interações proteicas são de fundamental importância para ativação, regulação e controle de diversas funções enzimáticas a elas relacionadas, fica clara a relevância da identificação e caracterização das interações participantes desse processo para uma melhor compreensão da patogênese e para o desenho racional de drogas para a febre amarela. Foi identificado previamente que a proteína celular p54nrb/NonO interage com o domínio RNA polimerase RNA dependente de NS5 de YFV. p54nrb/NonO é um fator de transcrição nuclear associada a membrana nuclear e apresenta características multifuncionais nos processos celulares em células eucariotas, ocorrendo frequentemente em associação com as proteínas U1A e PSF. Dessa forma, a interação entre p54nrb/NonO e NS5 pode influenciar a localização, transporte das proteínas e do RNA viral dentro da célula. Objetivo. O objetivo deste estudo foi confirmar a interação entre p54nrb/NonO e NS5 de YFV e KUNV, e determinar o efeito da interação sobre a replicação viral. Materiais e Métodos. Para tanto, experimentos de co-imunoprecipitação, espectrometria de massa e imunofluorescência indireta foram realizados para confirmar a interação e a co-localização entre as proteínas. Para determinar o efeito sobre a replicação viral, foi realizado, em cultura celular, a superexpressão p54nrb/NonO e PSF, bem como, o silenciamento de p54nrb/NonO. Posteriormente, a taxa de replicação viral foi determinada por técnicas de qPCR, ensaio de placa, mensuração da atividade de β-galactosidase e luciferase. Resultados. O ensaio de imunofluorescência mostrou co-localização entre p54nrb/NonO e NS4 de YFV na região perinuclear e com NS5 no núcleo. Em contraste, NS5 de KUN co-localizou com p54nrb/NonO na região perinuclear, e da mesma forma, NS5 de KUNV foi identificado co-precipitando p54nrb/NonO. Não foi identificada a co-precipitação entre p54nrb/NonO e NS5 de YFV. Novamente, p54nrb/NonO foi identificada co-precipitando com NS5 de KUNV pela análise por espectrometria de massa com o uso de anticorpo monoclonal para a proteína NS5 de KUNV. A superexpressão de p54nrb/NonO não mostrou afetar a replicação de YFV e KUNV, entretanto, a superexpressão de PSF mostrou efeito inibitório sobre a replicação viral. Os estudos com interferência de RNA, contudo, foram inconclusivos sobre o efeito do silenciamento de p54nrb/NonO sobre a replicação de YFV. Conclusão. p54nrb/NonO e NS5 de KUNV interagem fisicamente e co-localizam no citoplasma, enquanto que, a co-localização com NS5 de YFV ocorre no núcleo, embora não ocorra interação física. Entretanto, a superexpressão de p54nrb/NonO não afeta a replicação viral. PSF foi confirmada como parceira interativa de p54nrb/NonO e, quando superexpressa, inibe a replicação de YFV e KUNV.

A proteína fosfatase do tipo-1 (PP1) é uma das principais proteínas serina/treonina fosfatases (PSTPs) e desempenha papeis fisiológicos tão diversos quanto importantes, tais como a regulação do metabolismo de carboidratos e do ciclo celular. A holoenzima PP1 é constituída por uma subunidade catalítica conservada (PP1c) que está associada a subunidades não-catalíticas que modulam sua localização subcelular, especificidade de substrato e atividade enzimática. Mais de 100 proteínas que interagem com a PP1c já foram identificadas em distintos organismos eucarióticos. Proteínas que interagem com a PP1c são, portanto, a chave para compreender os diferentes papéis biológicos da PP1. A subunidade catalítica da PP1 da ameba social Dictyostelium discoideum (DdPP1c) é codificada por um gene em cópia única o qual é expresso ao longo de todo o ciclo de vida desse organismo. Algumas proteínas que interagem e possivelmente modulam a atividade da PP1 de D. discoideum já foram identificadas, utilizando-se tanto buscas por similaridades na sequência genômica deste microorganismo como ensaios utilizando o sistema de duplo-híbrido em leveduras, utilizando-se a PP1c como isca. Com esta última abordagem, foram selecionados mais de 25 clones distintos de cDNA que codificam proteínas que potencialmente interagem com a DdPP1c, após varreduras de bibliotecas de cDNA de diferentes estágios de desenvolvimento de D. discoideum. Neste trabalho, nós confirmamos que o produto protéico de 11 destes clones interagem com a isca DdPP1c com base em novos ensaios de duplo-híbrido. Os demais clones codificam proteínas que não interagem com DdPP1c ou promovem auto-ativação do gene repórter. Selecionamos para estudos adicionais um clone do gene DDB_G0269300 cujo produto protéico predito de 423 de aminoácidos não tem função ainda conhecida. A sequência codificadora completa de DDB_G0269300 foi clonada para realização de novos ensaios de duplo-híbrido em leveduras, os quais confirmaram a especificidade de sua interação com DdPP1c. A proteína recombinante rDDB_G0269300 foi obtida com sucesso em bactérias, possibilitando a obtenção de anticorpos policlonais em camundongos. O anti-soro anti-rDDB_G0269300 é aparentemente específico no reconhecimento da proteína correspondente em extratos celulares de D. discoideum coletados em 12h e 16 da fase de desenvolvimento. Estes resultados coincidem com dados obtidos através de RT-qPCR que mostram aumento nos níveis dos transcritos de DDB_G0269300 entre 8h e 12h da fase de desenvolvimento, o que é indicativo da sua importância desta proteína durante esta fase do ciclo de vida de Dictyostelium como uma potencial parceira molecular da DdPP1c / Protein phosphatase type-1 (PP1) is a major protein serine/threonine phosphatase (PSTP) which plays as diverse as important physiological roles, such as regulation of carbohydrate metabolism and of cell cycle. The PP1 holoenzyme comprises a conserved catalytic subunit (PP1c) associated with non-catalytic subunits that modulate its subcellular localization, substrate specificity and enzymatic activity. More than 100 proteins that interact with PP1c have been identified in different eukaryotic organisms. Therefore proteins that interact with PP1c are key to the understanding of PP1 different biological roles. The catalytic subunit of PP1 the social amoeba Dictyostelium discoideum (DdPP1c) is encoded by a single copy gene which is expressed throughout the life cycle of this organism. Some proteins that interact with and possibly modulate the activity of D. discoideum PP1 have been identified, using both similarity searches in the genome sequence of this microorganism as yeast two-hybrid screenings using PP1c as bait. With the latter approach, we have selected more than 25 distinct cDNA clones encoding proteins that potentially interact with DdPP1c after screening D. discoideum cDNA libraries from different developmental stages. In this study, we confirmed that the protein product from 11 of these clones interact with the bait DdPP1c based on two-hybrid assays. The other clones encode proteins that either does not interact or promote self-activation of the reporter gene. The clone related to DDB_G0269300 gene that encodes a predicted protein of 423 amino acids with unknown function was selected for further studies. DDB_G0269300 full-length coding sequence was cloned and new yeast two-hybrid assays were performed confirming the specificity of the interaction with DdPP1c. The recombinant protein rDDB_G0269300 was successfully obtained in bacteria and further used for polyclonal antibodies production in mice. The antiserum anti-rDDB_G0269300 is apparently specific for recognition of the corresponding protein in D. discoideum cell extracts collected after 12h and 16h of development. These results agree with RT-qPCR data showing that the levels of DDB_G0269300 transcripts are increased between 8 h and 12 h during the development, which is indicative of its importance during this phase in Dictyostelium life cycle as a DdPP1c potential molecular partner.

Le réseau de signalisation qui répond à l’insuline et aux nutriments est conservé chez les métazoaires, où il joue un rôle central dans le contrôle du métabolisme et de la croissance. Les nutriments assimilés sont soit directement utilisés pour la croissance tissulaire, soit stockés principalement sous forme de triglycérides. Chez la drosophile, l’activation de ce réseau de signalisation dans le corps gras, un organe qui remplit à la fois les fonctions hépatiques et destockage, induit une augmentation du stockage de lipides sous forme de nombreuses gouttelettes lipidiques (LDs). A l’inverse, la carence alimentaire se traduit par une augmentation de la taille des LDs et une diminution de lipides stockés. La kinase TOR (TargetOf Rapamycine) et son substrat S6 Kinase (S6K) jouent un rôle central dans cette régulation.Chez la drosophile, ces 2 kinases (dTOR et dS6K) contrôlent les aspects autonome-cellulaireset hormonaux de la croissance. En dépit de nombreuses études sur divers organismes modèles,destinées à comprendre les mécanismes régulateurs de S6K, rien n’est connu à ce jour sur lecontrôle de sa dégradation.Nous avons utilisé une banque de lignées exprimant des ARN interférant (RNAi) contre unegrande quantité de gènes de la drosophile, pour réaliser 3 des cribles génétiques destinés à identifier de nouveaux régulateurs du métabolisme et de la croissance. Dans le premier crible,les RNAi ont été induits dans la glande prothoracique, siège de la production de l’hormonestéroïde ecdysone connue pour réguler la croissance et les étapes du développement, souscontrôle de la nutrition et de la signalisation dTOR. Sur 7000 gènes criblés, 620 ont étéidentifiés comme nécessaire à la production d’ecdysone. Dans le second crible, nous avonsexprimé les RNAi de 4000 gènes dans le corps gras pour rechercher ceux qui induisaient uneaugmentation de la taille des LDs. L’objectif était d’identifier des gènes impliqués dans la réponse à la carence alimentaire, et nous avons ainsi retenu 24 candidats intéressants. Le troisième crible représente la majeure partie du travail de thèse, où nous avons criblé les RNAi susceptibles de modifier un phénotype de croissance induit par dS6K. Sur 7000 gènes testés,nous en avons retenu 45 qui ont ensuite été utilisés pour générer un diagramme d’interaction en utilisant les informations disponibles dans les banques de données. Les candidats les plus intéressants ont ensuite été analysés en culture de cellules pour identifier ceux qui régulent l’activité de dS6K et ceux qui régulent sont niveau d’expression. Parmi ces derniers, nousavons identifié le gène codant pour Archipelago (Ago), connue pour contrôler la dégradationrégulée des protéines-cibles au niveau du protéasome. Nous avons réalisé de nombreusesexpériences qui montrent que ago et dS6K interagissent génétiquement. En outre, il est indiquédans les banques de données que ces protéines interagissent entre elles par la technique des 2-hybrides en levure. Tous ces résultats révèlent que Ago régule la dégradation de dS6K, etposent les premières pierres de ce niveau de régulation. / The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.