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1
Gowher, Ali. "Characterization of protein factors targeting RNA into human mitochondria." Phd thesis, Université de Strasbourg, 2013. http://tel.archives-ouvertes.fr/tel-01071841.
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The import of yeast tRNALys (tRK1) into human mitochondria in the presence of cytosolic extract suggests that human cell possesses machinery for tRK1 import. Here, we show that precursor of mitochondrial lysyl-tRNA synthetase (preKARS2) interact with tRK1 and its derivatives containing tRK1 import determinants, and facilitates their import into isolated mitochondria and in vivo, when preKARS2 was overexpressed or downregulated. tRK1 import efficiency increased upon addition of glycolytic enzyme enolase, previously found as an actor of RNA import in yeast. We found that tRK1 and its derivatives translocate into mitochondrial matrix in polynucleotide phosphorylase (PNPase) dependent manner. Furthermore, a point mutation preventing trimerization of PNPase affect import of 5S rRNA and MRP RNA into mitochondria and subsequently mitochondrial translation. Overexpression of the wild-type PNPase induced an increase of 5S rRNA import into mitochondria and rescued translation.
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Corsi, Flavia. "Towards the in silico reconstruction of protein interaction networks : identification of DNA- and RNA-protein interfaces, and construction of a database of multiple interactions of proteins." Electronic Thesis or Diss., Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2019SORUS452.pdf.
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Cette thèse porte sur la caractérisation et la prédiction des interfaces protéine-ADN et -ARN, et des comparaisons avec les interfaces protéine-protéine. Nous avons créé un ensemble non-redondant et représentatif de 187 complexes protéine-ADN à haute résolution, comprenant aussi les conformations non liées de 82 protéines. Cette base de données peut servir de référence dans le domaine. Nous avons mené une analyse exhaustive des propriétés de séquence et structurels des interfaces protéine-ADN/ARN et nous les avons comparé avec les propriétés des interfaces protéine-protéine et celles des régions protéiques non-interagissantes. Nous avons développé JET2DNA et JET2RNA, nouvelles méthodes pour la prediction des sites de liaison protéine-ADN/ARN à la surface des protéines. En combinant quatre descripteurs biologiquement pertinents, elles surpassent des méthodes par apprentissage machine. Elles permettent aussi de découvrir des sites de liaison alternatifs avec l'ADN/ARN et de déchiffrer leurs propriétés. Afin de donner un aperçu global de la plasticité des protéines interagissant avec l'ADN, nous avons construit la base de données protéine-(protéine)-ADN (P(P)DNAdb). Elle inclut les 187 complexes protéine-ADN de notre ensemble de référence, les forme libres des protéines et les structures des autres complexes où ces protéines, ou des homologues proches, sont impliqués. L'utilisateur peut accéder aux propriétés des interfaces, visualiser les changements de conformation associés à la liaison avec des partenaires différents et localiser les résidus interagissants avec l'ADN dans les autres structures de la même protéineThis thesis focuses on the characterization and prediction of DNA- and RNA-binding sites on protein structures, with some comparisons with protein-protein ones. We compiled and manually curated a non-redundant and representative set of 187 high resolution protein-DNA complexes, with the available 82 protein unbound conformations, that could be used as a reference benchmark. We conducted a comprehensive analysis of sequence- and structure-based properties of protein-DNA/RNA interfaces and compared them with respect to protein-protein interfaces and to non-interacting protein regions. We developed JET2DNA and JET2RNA, new methods for predicting DNA- and RNA-binding sites on protein surfaces. Combining four biologically meaningful descriptors, they outperform other machine-learning methods, in terms of predictive power and robustness to conformational changes. Our tools demonstrated to be instrumental in discovering alternative DNA/RNA-binding sites and in deciphering their properties. This could be very helpful for drug design and repurposing. To give a comprehensive view of plasticity of DNA-binding proteins and structural information on their multiple interactions, we constructed the Protein-(Protein)-DNA database (P(P)DNAdb). It comprises the 187 protein-DNA complexes in our benchmark, protein unbound forms and structures of other complexes where the proteins, or closed homologs, were in contact with other proteins. The user can access properties of the interfaces, visualize conformational changes associated to the binding of different partners and the location of the DNA-binding residues on the unbound structures and on the complexes with the other protein partners
3
CARUCCI, FEDERICA. "Agronomic strategies for Sustainable Management of Durum Wheat Cultivation in Mediterranean Area." Doctoral thesis, Università di Foggia, 2021. https://hdl.handle.net/11369/425190.
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L’azoto è un elemento nutritivo fondamentale per la produzione di cereali, tu5avia ha un
signi!cativo impa5o ambientale. Nel presente elaborato vengono valutate diverse pratiche
agronomiche al !ne di incrementare la sostenibilità della coltivazione del frumento duro, e
migliorare la sua produ5ività e qualità. Gli studi sono stati tu5i condo5i nell’area del
Mediterraneo, de!nita come una delle regioni maggiormente colpita dai cambiamenti
climatici in corso. In quest’area, quindi, la gestione sostenibile dell’azoto diventa ancora
più importante. L'obie5ivo generale è stato perseguito valutando le risposte di diversi
genotipi di frumento duro alla gestione sostenibile della fertilizzazione azotata sia in
sistemi produ5ivi biologici che a basso input.
De!nire il sistema produ5ivo agricolo che da solo sia in grado di garantire alla
popolazione mondale un'alimentazione sicura e sostenibile è impossibile. Tu5avia, per
garantire contemporaneamente l’accesso al cibo e la sostenibilità ambientale, sarà
necessario ricorrere a sistemi agricoli innovativi, compresi quelli a basso input e biologici.
In conclusione, i risultati descri5i in questo elaborato hanno contribuito a far progredire il
grado di conoscenza sulla sostenibilità, sulla produ5ività e su diversi aspe5i qualitativi del
frumento duro in ambiente mediterraneo. Tu5avia, molte questioni signi!cative restano
da indagare e saranno ogge5o di futuri studi.Nitrogen is the most requested element in cereal systems and has the most signi!cant impact on the environment. This Ph.D. dissertation deals with di$erent agronomic strategies to improve the sustainability of durum wheat cultivation, together with its productivity and quality. Studies were conducted in the Mediterranean area, de!ned as one of the regions most vulnerable to climatic changes. Since, in this area the e'ciently use of nitrogen is more critical. The main objective was pursued evaluating the responses of di$erent durum wheat genotypes to the sustainable management of nitrogen fertilizers in organic and low-input systems. De!ne the best farming system that alone can satisfy the world safe and sustainable feeding is impossible. However, innovative farming systems, including low-input and organic ones, will be necessary for both food access and ecosystem security. In conclusion, the results described in this Ph.D. advance the current state of the knowledge about improving the sustainability, productivity and di$erent quality aspects of durum wheat under Mediterranean conditions. However, several signi!cant issues remain to be investigated in future research.
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Uchikawa, Emiko. "A structural approach of RNA-protein recognition and kinetics of binding in two examples : tRNA aminoacylation by arginyl-tRNA synthetase and 7SK stabilization by LaRP7." Strasbourg, 2011. http://www.theses.fr/2011STRA6052.
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Dans la cellule, les interactions ARN-protéines jouent un rôle fondamental dans divers processus impliqués dans la régulation de l'expression du message génétique. Si l'épissage de l'ARN pré-messager, la polyadénylation, le transport et l'adressage, la stabilité et la traduction sont des régulations de type post-transcriptionnel, les interactions ARN-protéines ont également un rôle-clé dans le domaine de la transcription. Effectivement, en addition aux capacités de codage, qui font de l'ADN et de l'ARN des supports de l'information génétique, la grande variabilité de structures et la flexibilité de l'ARN créent de nombreux sites uniques et le potentiel pour des régulations complexes. Les protéines se liant à l'ARN utilisent une bibliothèque de motifs structuraux pour reconnaître la séquence et la structure de leurs ARN cibles, ce qui conduit à un large éventail d'interactions, de labiles à stables. Ce manuscrit décrit nos travaux consistant à révéler les détails d'interactions RNA-protéines au niveau moléculaires, dans différents exemples concernant deux champs de la biologie cellulaire, la traduction et la transcription du code génétique. Nos cibles ont été choisies afin d'apporter des informations sur des interactions critiques pour la survie cellulaire et représentent différents modes de liaison de protéines à des ARN. Nous avions pour but d'utiliser la cristallographie aux rayons X, qui est une méthode fiable et reconnue pour la finesse des informations à l'échelle atomique que l'on peut en obtenir, et nous avons développé pour chaque cible un protocole de purification conduisant à une préparation homogène et cristallisable. Nous décrivons également les divers tests biophysiques et biochimiques ayant été utilisés pour caractériser nos échantillonsIn the cell, RNA-protein interactions are fundamental to many processes involved in the regulation of gene expression, including pre-mRNA splicing, polyadenylation, editing, transport, cytoplasmic targeting, mRNA turnove and translation. In addition to these post-transcriptional processes, RNA-prote in interactions may also play a key rôle in transcription. Indeed, in addition to its coding capacity, which makes both DNA and RNA recipients of the genetic message, the high variability and conformationnal flexibility of RNA structure creates a number of unique binding sites and the potential for complex regulation by RNA binding proteins. These use a large Iibrary of structural modules in order to recognize RNAs in a combination of sequence- or structure-dependent ways, leading to a wide range of transient to more stable interactions. This manuscript describes our endeavour to reveal the details of RNAprotein interactions at the molecular level in several examples taken in two different fields of cell biology, transcription and translation. Our targets were chosen to better understand the molecular foundation of interactions critical for the cell survival, and represent different binding modes ofproteins to RNA. Aiming to use X-ray crystallography, a well-accepted and reliable mean to analyze recognition details at atomic resolution, we developed for each target a purification protocolleading to homogeneous preparations that were used for crystallization and subjected to various anai}'ses, including functional assays and biophysical characterization
5
Fischer, Tiffany Brink. "A structural and energetic description of protein-protein interactions in atomic detail." Texas A&M University, 2006. http://hdl.handle.net/1969.1/4775.
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Here, we present the program QContacts, which implements Voronoi polyhedra
to determine atomic and residue contacts across the interface of a protein-protein
interaction. While QContacts also describes hydrogen bonds, ionic pair and salt bridge
interactions, we focus on QContactsâ identification of atomic contacts in a protein
interface compared against the current methods. Initially, we investigated in detail the
differences between QContacts, radial cutoff and Change in Solvent Accessible Surface
Area (delta-SASA) methods in identifying pair-wise contacts across the binding interface.
The results were assessed based on a set of 71 double cycle mutants. QContacts
excelled at identifying knob-in-hole contacts. QContacts, closest atom radial cutoff and
the delta-SASA methods performed well at picking out direct contacts; however, QContacts
was the most accurate in excluding false positives. The significance of the differences
identified between QContacts and previous methods was assessed using pair-wise
contact frequencies in a broader set of 592 protein interfaces. The inaccuracies
introduced by commonly used radial cutoff methods were found to produce misleading
bias in the residue frequencies. This bias could compromise pair-wise potentials that are
based on such frequencies. Here we show that QContacts provides a more accurate description of protein interfaces at atomic resolution than other currently available
methods. QContacts is available in a web-based form at http://tsailab.tamu.edu/qcons
(Fischer et al., 2006).
6
Jarrige, Domitille. "Déchiffrer le "code OPR" pour une meilleure compréhension du rôle physiologique des protéines OPR." Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS632.
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À la suite de l’endosymbiose, le génome chloroplastique a rétréci et dépend maintenant du génome nucléaire pour son expression. Chez Chlamydomonas reinhardtii, les protéines Octotricopeptide repeat (OPR), codées dans le noyau, contrôlent l’expression d’ARNm chloroplastiques spécifiques. La répétition OPR est un motif dégénéré de 38 acides aminés, qui forme un tandem d’hélices α antiparallèles qui lient l’ARN. Une répétition OPR est prédite pour interagir avec un nucléotide spécifique grâce à des résidus variables à des positions précises. La succession de répétitions permet aux protéines OPR de se lier à une séquence donnée. En partant d’un « code OPR » théorique, j’ai cherché à étudier cette spécificité de reconnaissance. J’ai mute in vivo les cibles chloroplastiques de facteurs OPR pour empêcher l’interaction OPR/ARN, puis j’ai tenté de la restaurer en mutant les résidus conférant la spécificité dans les répétitions correspondantes. Étonnamment, les interactions OPR/ARN sont très résilientes, ce qui a complétement changé notre vision de ces interactions in vivo. Des études fonctionnelles complémentaires que j’ai réalisées sur les facteurs OPR MDB1 and MTHI1 ont révélé que l’expression des gènes chloroplastiques dépend probablement de systèmes de facteurs nucléaires. En coopérant ces facteurs auraient une affinité combinée plus forte et seraient ainsi plus résilientsFollowing endosymbiosis, the chloroplast genome shrunk and became reliant on the host genome for its expression. In Chlamydomonas reinhardtii, Octotricopeptide repeat proteins (OPR), encoded in the nucleus, control the expression of a specific organellar mRNA. The OPR repeat is a degenerate motif of 38 amino-acids, folding into a tandem of antiparallel α-helices which can bind to RNA. An individual OPR repeat is predicted to interact with one given nucleotide thanks to specificity-conferring residues at defined positions within the repeat. OPR proteins contain tracks of successive OPR motifs, thus they can bind to a specific RNA “target” sequence and act on it. I aimed to study this specificity, called the “OPR code”, starting with a draft code based on known OPR protein/mRNA couples. I mutated in vivo the chloroplast targets of some OPR factors to disrupt the OPR/RNA interaction, and then tried to restore it by mutating the specificity-conferring residues in the corresponding repeats. Surprisingly, OPR/RNA interactions seem very resilient, challenging our view of how the specificity is established in vivo. Complementary functional studies that I performed on the OPR factors MDB1 and MTHI1 revealed that chloroplast gene expression might rely on complex networks of nuclear factors. By cooperating those putative systems would be both more specific and more resilient
7
Li, Yi. "Study of Arnt-interacting proteins on Arnt-dependent signaling pathways." Scholarly Commons, 2006. https://scholarlycommons.pacific.edu/uop_etds/2786.
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In an effort to better understand the Ah receptor nuclear translocator (Arnt)-dependent signaling mechanisms, we employed a phage display system to identify Arnt-interacting peptides. Human liver cDNA library was utilized to screen for Arnt-interacting peptides using an Arnt construct fused to thioredoxin (TH-ArntCΔ418). Two clones, namely Ainp1 and Ainp2 (Arnt-interacting peptide), were identified and subsequently characterized. Ainp2 interacted with TH-ArntCΔ418 in the GST pull-down, TALON co-precipitation, and mammalian two-hybrid assays. Northern blot results revealed that Ainp2 is predominantly expressed in human liver. The putative full-length Ainp2 cDNA sequence was subsequently cloned using RACE PCR. Endogenous expression of Ainp2 was found in Jurkat cells and human fetal/adult liver medleys. Results from the transient transfection studies using a DRE- or ERE-driven reporter plasmid and the real-time QPCR experiments examining the endogenous CYP1A1 or GREB-1 expression demonstrated that Ainp2 enhances the 3MC-induced AhR signaling pathway in HepG2 cells, while suppresses the E2-induced ER signaling pathway in MCF-7 cells. These results suggested that Ainp2 plays a role in the Arnt-dependent signaling pathways. The suppressive effect of Ainp2 in the ER signaling pathway was not observed in Arnt-knockdown cells. Additionally, co-precipitation data showed that Ainp2 did not interact with ER α and ER β, suggesting that Ainp2 suppresses the ER signaling via an Arnt-mediated mechanism. The phage display technique also revealed another potential Arnt-interacting peptide Ainp1, which contains an open reading frame of 58 amino acids. The GST pull-down and mammalian two-hybrid assays showed that Ainp1 interacts with TH-ArntCΔ418. Northern blot results demonstrated that Ainp1 is ubiquitously present in all the tested tissues, including brain, placenta, skeletal muscle, heart, kidney, pancreas, liver, lung, spleen, and colon.
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Takeuchi, Akiko Krol Alain Allmang-Cura Christine. "RNA-protein interaction in the selenoprotein synthesis machinery." Strasbourg : Université de Strasbourg, 2009. http://eprints-scd-ulp.u-strasbg.fr:8080/1133/01/TAKEUCHI_Akiko_2009.pdf.
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Thèse de doctorat : Sciences du Vivant. Aspects moléculaire et cellulaire de la Biologie : Strasbourg : 2009.Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 11 p.
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Takeuchi, Akiko. "RNA-protein interaction in the selenoprotein synthesis machinery." Strasbourg, 2009. http://www.theses.fr/2009STRA6054.
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La sélénocystéine est incorporée co-traductionnellement dans les sélénoprotéines en réponse à un codon UGA habituellement l’un des 3 codons stop. La protéine SBP2 joue un rôle majeur dans ce mécanisme de recodage en se liant à une structure en tige-boucle (SECIS) située dans la région 3’UTR de l’ARNm des sélénoprotéines. Nous avons isolé et caractérisé fonctionnellement SBP2 de Drosophila melanogaster. Par comparison avec SBP2 humaine, nous avons identifié un domaine de liaison à l’ARN additionnel essentiel à la liaison au SECIS et à la sous-unité ribosomique 60S et permettant une sélectivité structurale du SECIS. Des prédictions structurales et des analyses biophysiques ont établi que SBP2 était une protéine globalement désordonnée ou “Intrinsically Disordered Protein” qui ne se replie qu’en présence de partenaires. Enfin, nous avons établi que l’assemblage des mRNP de sélénoprotéines faisait appel à des facteurs communs et présentait de multiples similarités avec celui des sn/snoRNPThe 21st amino acid selenocysteine is encoded by a UGA codon that usually signifies translational termination. Selenoprotein synthesis therefore requires specialized factors. Among these is SBP2 that binds the SECIS, a stem-loop structure in the 3’UTR of selenoprotein mRNAs. In structural analyses of SBP2, we isolated and functionally characterized Drosophila melanogaster SBP2. By comparing it with human SBP2, we identified an additional RNA binding domain that is essential for SECIS and 60S ribosomal subunit binding, and also enables SECIS structure selectivity. In addition, computational and biophysical analyses established that SBP2 is globally unfolded, supporting our hypothesis that SBP2 is an Intrinsically Disordered Protein and becomes folded in the presence of partners yet to be identified. Finally, we searched for potential partners of SBP2 and our results showed that the molecular assembly of selenoprotein mRNPs has many similarities with that of sn/snoRNPs
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Mendez, Giraldez Raul. "Critical assessment of predicted interactions at atomic resolution." Doctoral thesis, Universite Libre de Bruxelles, 2007. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210664.
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Molecular Biology has allowed the characterization and manipulation of the molecules of life in the wet lab. Also the structures of those macromolecules are being continuously elucidated. During the last decades of the past century, there was an increasing interest to study how the different genes are organized into different organisms (‘genomes’) and how those genes are expressed into proteins to achieve their functions. Currently the sequences for many genes over several genomes have been determined. In parallel, the efforts to have the structure of the proteins coded by those genes go on. However it is experimentally much harder to obtain the structure of a protein, rather than just its sequence. For this reason, the number of protein structures available in databases is an order of magnitude or so lower than protein sequences. Furthermore, in order to understand how living organisms work at molecular level we need the information about the interaction of those proteins. Elucidating the structure of protein macromolecular assemblies is still more difficult. To that end, the use of computers to predict the structure of these complexes has gained interest over the last decades.The main subject of this thesis is the evaluation of current available computational methods to predict protein – protein interactions and build an atomic model of the complex. The core of the thesis is the evaluation protocol I have developed at Service de Conformation des Macromolécules Biologiques et de Bioinformatique, Université Libre de Bruxelles, and its computer implementation. This method has been massively used to evaluate the results on blind protein – protein interaction prediction in the context of the world-wide experiment CAPRI, which have been thoroughly reviewed in several publications [1-3]. In this experiment the structure of a protein complex (‘the target’) had to be modeled starting from the coordinates of the isolated molecules, prior to the release of the structure of the complex (this is commonly referred as ‘docking’).
The assessment protocol let us compute some parameters to rank docking models according to their quality, into 3 main categories: ‘Highly Accurate’, ‘Medium Accurate’, ‘Acceptable’ and ‘Incorrect’. The efficiency of our evaluation and ranking is clearly shown, even for borderline cases between categories. The correlation of the ranking parameters is analyzed further. In the same section where the evaluation protocol is presented, the ranking participants give to their predictions is also studied, since often, good solutions are not easily recognized among the pool of computer generated decoys.
An overview of the CAPRI results made per target structure and per participant regarding the computational method they used and the difficulty of the complex. Also in CAPRI there is a new ongoing experiment about scoring previously and anonymously generated models by other participants (the ‘Scoring’ experiment). Its promising results are also analyzed, in respect of the original CAPRI experiment. The Scoring experiment was a step towards the use of combine methods to predict the structure of protein – protein complexes. We discuss here its possible application to predict the structure of protein complexes, from a clustering study on the different results.
In the last chapter of the thesis, I present the preliminary results of an ongoing study on the conformational changes in protein structures upon complexation, as those rearrangements pose serious limitations to current computational methods predicting the structure protein complexes. Protein structures are classified according to the magnitude of its conformational re-arrangement and the involvement of interfaces and particular secondary structure elements is discussed. At the end of the chapter, some guidelines and future work is proposed to complete the survey.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Armaos, Alexandros 1989. "Computational characterization of protein-RNA interactions and implications for phase separation." Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/668546.
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Despite what was previously considered, the role of RNA is not only to carry the genetic
information from DNA to proteins. Indeed, RNA has proven to be implicated in more
complex cellular processes. Recent evidence suggests that transcripts have a regulatory
role on gene expression and contribute to the spatial and temporal organization of the
intracellular environment. They do so by interacting with RNA-binding proteins (RBPs)
to form complex ribonucleoprotein (RNP) networks, however the key determinants that
govern the formation of these complexes are still not well understood. In this work, I will
describe algorithms that I developed to estimate the ability of RNAs to interact with
proteins. Additionally, I will illustrate applications of computational methods to propose
an alternative model for the function of Xist lncRNA and its protein network.
Finally, I will show how computational predictions can be integrated with high
throughput approaches to elucidate the relationship between the structure of the RNA and
its ability to interact with proteins. I conclude by discussing open questions and future
opportunities for computational analysis of cell’s regulatory network.
Overall, the underlying goal of my work is to provide biologists with new insights into
the functional association between RNAs and proteins as well as with sophisticated tools
that will facilitate their investigation on the formation of RNP complexesA pesar de lo que se consideraba anteriormente, el papel del ARN no es solo transportar la información genética del ADN a las proteínas. De hecho, el ARN ha demostrado estar implicado en muchos procesos celulares más complejos. La evidencia reciente sugiere que los transcriptos tienen un papel regulador en la expresión génica y contribuyen a la organización espacial y temporal del entorno intracelular. Lo hacen interactuando con proteínas de unión a ARN (RBP) para formar redes complejas de ribonucleoproteína (RNP), sin embargo, los determinantes clave que rigen la formación de estos complejos aún no se conocen bien. En este trabajo, describiré algoritmos que he desarrollado para estimar la capacidad de los ARN de interactuar con las proteínas. Además, ilustraré aplicaciones de métodos computacionales para proponer una maquinaria alternativa para el Xist lncRNA y su red de interacciones. Finalmente, mostraré cómo las predicciones computacionales pueden integrarse con enfoques de alto rendimiento para dilucidar la relación entre la estructura del ARN y su capacidad para interactuar con las proteínas. Concluyo discutiendo preguntas abiertas y oportunidades futuras para el análisis computacional de la red reguladora de la célula. En general, el objetivo subyacente de mi trabajo es proporcionar a los biólogos nuevas ideas sobre la asociación funcional entre ARN y proteínas, así como herramientas sofisticadas que facilitarán su investigación sobre la formación de complejos RNP.
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Lindenbaum, Pierre. "Roxan, une nouvelle proteine cellulaire interagissant avec la proteine non-structurale nsp3 du rotavirus : clonelt* : un programme en ligne trouvant des strategies de clonage (doctorat : microbiologie)." Paris 11, 2000. http://www.theses.fr/2000PA114811.
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Chernov, Konstantin Grigorievich. "Interplay of YB-1 between tubulin and mRNA." Thesis, Evry-Val d'Essonne, 2008. http://www.theses.fr/2008EVRY0040/document.
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YB-1 est un régulateur important de l’expression des gènes dans les cellules eucaryotes. En plus de son rôle dans la transcription, YB-1 joue un rôle clé dans la traduction et la stabilisation des ARN messagers. Nous avons identifié plusieurs nouveaux partenaires de la protéine YB-1 par chromatographie d’affinité à partir de différents extraits tissulaires. Parmi ces partenaires, nous avons démontré que YB-1 interagit avec la tubuline et les microtubules et stimule fortement l'assemblage des microtubules in vitro. Les microtubules assemblés en présence de YB-1 ont une ultrastructure normale, et les données montrent que YB-1 recouvre probablement la surface extérieure des microtubules. De la même façon YB-1 stimule aussi l'assemblage de la tubuline-MAP qui est plus proche des complexes protéiques qui existent dans la cellule, et de la tubuline clivée par subtilisine ce qui suggère que son interaction avec la tubuline ne relève pas seulement d’effets électrostatiques. Nous avons enfin découvert que la tubuline interfère avec la formation des complexes ARNm:YB-1. Ces résultats suggèrent que YB-1 peut réguler l'assemblage des microtubules in vivo et que son interaction avec la tubuline peut contribuer à la régulation de la traduction des ARN messagers. En effet, in vivo, la traduction des mRNPs dépend de l’état de saturation de l’ARN messager par YB-1. Nous avons montré ici que lorsque le rapport YB-1:ARNm est faible, les complexes mRNPs possèdent des structures non-compactes, alors que les mRNPs saturés sont compacts. Ce changement structural est observé de façon parallèle à l'inhibition de la traduction des ARN messagers lorsqu’ils passent des polysomes (traduits) aux mRNPs libres (non traduits). De façon intéressante, nous avons découvert que les mRNPs saturés se lient aux microtubules via des interactions protéine:protéine et ont tendance à former des agrégats sur la surface des microtubules. Cette dernière propriété pourrait contribuer à la formation de granules de stress et à la localisation des mRNPs dans le cytoplasme. Finalement, un modèle de diffusion facilité a été développé pour expliquer l'assemblage des microtubules orchestré par les polyamines naturelles (telles que YB-1 qui sont positivement chargées dans la cellules). L’ensemble de ces données contribuent à une meilleure compréhension de processus biologiques fondamentaux concernant l’assemblage de la tubuline en microtubules et le trafic des ARN dans la cellule. Ils pourraient avoir un intérêt pour développer de nouveaux médicaments qui ciblent les microtubulesYB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. We identify several novels YB-1 protein partners by affinity chromatography of different tissue extracts. We observed that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. Microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure where YB-1 probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Additionally, we demonstrated that tubulin interferes with mRNA:YB-1 complexes. These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation. The translational status of mRNPs in vivo depends on amount of YB-1 associated with mRNA. We show here that at low YB-1:mRNA ratios mRNP complexes possess an incompact structures, whereas saturated mRNPs are compact. This structural change corresponds to translation inhibition when mRNA moves from polysomal (translatable) to free (untranslatable) mRNPs. Saturated mRNPs bind to microtubules via protein:protein interactions and tend to self-aggregate on microtubule surface. This property could contribute to stress granule formation, mRNPs traffic and localization of translation apparatus within cytoplasm. Finally, the facilitated diffusion model was developed to explain enhancement of microtubule assembly by positively charged natural polyamines in living cells. Altogether our data contribute to the understanding of fundamental biological processes
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PARK, YOUNG CHUL. "Stabilite d'une proteine dimerique complexe : la tyrosyl-arnt synthetase." Paris 7, 1998. http://www.theses.fr/1998PA077266.
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La tyrosyl-arnt synthetase (tyrrs) de bacillus stearothermophilus comprend un domaine n-terminal (residus 1-319), qui est dimerique, forme le tyrosyl-adenylate et possede le repliement caracteristique de rossmann. Le domaine c-terminal (320-419) fixe l'anticodon de l'arnt. J'ai etudie et modelise le depliement du domaine n-terminal par l'uree a 25\c a l'equilibre dans des experiences de spectrofluorimetrie, dichroisme circulaire et chromatographie d'exclusion de taille. Les resultats ont montre l'existence d'un equilibre entre l'etat natif dimerique du domaine n-terminal, un etat monomerique intermediaire et l'etat deplie. L'intermediaire etait replie et n'etait pas en globule fondu. La variation d'energie libre deltag(h 2o) et son coefficient m de dependance vis-a-vis de la concentration en uree ont ete determines avec precision pour la dissociation du dimere natif et pour le depliement de l'intermediaire monomerique. J'ai identifie un groupement dense de 8 residus dans la tyrrs du thermophile b. Stearothermophilus dont 4 residus ne sont pas conserves dans la tyrrs du mesophile escherichia coli. J'ai construit les mutations correspondantes, t51p, i52l, m55l et l105v, et certaines mutations multiples dans la tyrrs de b. Stearothermophilus, pour etudier le role et le mode devolution de ce groupement dense. Les mutations n'affectaient pas l'activite de la tyrrs ou l'augmentaient. I52l destabilisait l'association entre les deux sous-unites du dimere de tyrrs bien que le residus ile52 soit a plus de 20 angstroms de leur interface. Au contraire, l105v destabilisait principalement l'intermediaire monomerique de depliement. Les effets des 2 mutations etaient antagonistes avec une forte compensation de la mutation l105v par i52l pour la stabilite de l'intermediaire monomerique. Le gain d'activite du a t51p se faisait au depend d'une destabilisation. Ce travail ouvre la voie a l'etude quantitative de la stabilite et du repliement des proteines dimeriques complexes.
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Le, Borgne Maïlys. "Étude in vivo de la fonction biologique de la protéine de liaison aux ARN Mex-3B." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10141.
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La protéine de liaison aux ARN MEX-3 est un régulateur essentiel du développement embryonnaire chez le nématode Caenorhabditis elegans. Une famille de quatre gènes homologues à hMex-3 (dénommés hMex-3A, 3B, 3C et 3D) a été identifiée chez les mammifères par notre équipe. Afin de mieux comprendre la fonction physiologique in vivo des protéines Mex-3, nous avons invalidé le gène Mex-3B chez la souris. Cette approche expérimentale a révélé que Mex-3B est un acteur majeur de la spermatogenèse. Les souris mâles nullizygotes présentent une obstruction des tubes séminifères conduisant à une réduction importante du nombre des spermatozoïdes produits. L’ablation de Mex-3B ciblée à la cellule de Sertoli, cellule somatique essentielle à la fonction de l’épithélium séminifère, a permis d’établir que le phénotype testiculaire a pour origine une perturbation des propriétés biologiques de cette cellule. En effet, les cellules de Sertoli déficientes pour Mex-3B présentent des défauts de la phagocytose qui conduisent à une élimination défectueuse des corps résiduels au cours de la spemiogenèse. L’exploration des mécanismes moléculaires impliqués a montré que Mex-3B contrôle la phagocytose via la régulation de l’activité et de la localisation membranaire de Rap1GAP, une protéine qui régule négativement la petite protéine G Rap1. En accord avec ces données, l’absence de Mex-3B provoque une déstabilisation de la barrière hémato-testiculaire due à un défaut de localisation à la membrane plasmique des molécules de jonction connexine 43 et N-Cadhérine, protéines dont la translocation et la stabilité dépendent de Rap1. En conclusion, mes travaux de thèse ont permis de mettre en évidence un rôle clé de Mex-3B dans le contrôle spatial de la voie de signalisation Rap1 au cours de la spermatogenèseThe RNA binding-protein MEX-3 is a post-transcriptional regulator involved in early embryogenesis of the nematode Caenorhabditis elegans. We have recently reported the characterization of a novel family of four mammalian genes homologous to hMex-3 (called hMex-3A, 3B, 3C and 3D). To gain insight into the biological functions of these proteins in vivo, we disrupted the Mex-3B gene in mice. Using this experimental approach, we found that Mex-3B is as a major regulator of spermatogenesis. We observed that male Mex-3B null mice hypofertile and present an obstruction of seminiferous epithelium. Phagocytic properties of Sertoli cells were impaired, thus impeding the clearance of residual bodies released during spermiogenesis. Exploration of the underlying molecular mechanisms revealed that Mex-3B regulates phagocytosis through the activation and the transport at the peripheral membrane of Rap1GAP, a protein that downregulates the small G protein Rap1. Consistently, the Rap1-dependent recruitment of the junction proteins, connexin 43 and N-Cadherin at the cell surface was compromised in Mex-3B deficient mice. In conclusion, my work highlights a key role gor Mex-3B in the spatial control of Rap1 signaling during spermatogenesis
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Karlikow, Margot. "Drosophila CG4572 protein and the spread of the RNAi antiviral immune signal." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066713/document.
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Au cours d’une infection virale, la survie des cellules dépend d’informations adéquatement distribuées, reçues et traitées, permettant l’établissement d’une réponse antivirale performante. La communication cellulaire est donc essentielle pour permettre la propagation de signaux immuns protecteurs à tout l’organisme.Chez les insectes, la principale réponse antivirale est l’ARN interférent (ARNi), activé lors de la détection d’ARN double brin (ARNdb) d’origine virale. Le mécanisme antiviral de l’ARNi peut être cellulaire ou systémique. Dans la première catégorie, la régulation de l’expression génique est limitée à la cellule dans laquelle l’ARNdb est produit, alors que dans la seconde, cette même régulation s’effectue dans des cellules distinctes de celles produisant l’ARNdb. Chez les insectes, l’ARNi systémique reste très peu décrit.Ma thèse explore le rôle de la protéine de drosophile CG4572/DORA, dans les mécanismes permettant l’établissement de l’ARNi systémique. J’ai également cherché la nature des signaux déclencheurs de cette réponse antivirale. Nous montrons l’existence de deux mécanismes de communication cellulaire permettant la propagation de signaux antiviraux: des vésicules extracellulaires et des nanotubes. Nous mettons en évidence que des vésicules contenant DORA et des fragments d’ARN viraux peuvent se propager dans les mouches en leur conférant une protection antivirale spécifique. Nous montrons également pour la première fois la présence de nanotubes membranaires qui contiennent des protéines de la machinerie ARNi ainsi que DORA.Les mécanismes que nous proposons sont pour la première fois associés à la réponse antivirale chez Drosophila melanogasterDuring viral infection, cell survival will depend on adequately giving, receiving and processing information to establish an efficient antiviral immune response. Cellular communication is therefore essential to allow the propagation of immune signals that will confer protection to the entire organism.The major antiviral defense in insects is the RNA interference (RNAi) mechanism that is activated by detection of viral double-stranded RNA (dsRNA). The antiviral RNAi mechanism can be divided in cell- and non-cell- autonomous. In cell-autonomous RNAi, the silencing process is limited to the cell in which the viral dsRNA is produced. In non-cell-autonomous (systemic) RNAi, the interfering effect occurs in cells different from where the viral dsRNA was produced. In insects the systemic RNAi response remains poorly characterized. My PhD explores the role of the Drosophila CG4572/DORA protein in the establishment of systemic antiviral RNAi. It also investigates the nature of immune signals that trigger the antiviral response. I provide evidence for the existence of two different mechanisms of cell-cell communication that allow the spread of the immune signal: extracellular vesicles and tunneling nanotubes. I describe that DORA-positive extracellular vesicles carry fragments of viral RNAs that can spread and confer specific antiviral protection in flies. I also present the characterization of tunneling nanotubes (TNTs) containing components of the RNAi machinery, DORA and dsRNA and I hypothesize on the use of TNTs in the spread of the immune signal.Both mechanisms of cell-to-cell communication are coupled for the first time to the antiviral response in Drosophila melanogaster
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Dodds, Anna Louise. "Functional analysis of a major nitrogen regulatory protein : AREA of Aspergillus nidulans." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311142.
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Mendler, Claudia Theresa [Verfasser], Arne [Akademischer Betreuer] [Gutachter] Skerra, and Markus [Gutachter] Schwaiger. "Protein-Engineering für die In-Vivo-Bildgebung / Claudia Theresa Mendler ; Gutachter: Markus Schwaiger, Arne Skerra ; Betreuer: Arne Skerra." München : Universitätsbibliothek der TU München, 2017. http://d-nb.info/1129874621/34.
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Chant, Alan. "Characterisation of the domain structure of the gene regulatory protein AreA from Aspergillus nidulans." Thesis, University of Portsmouth, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369475.
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AreA, a 96 kDa gene regulatory protein involved in nitrogen metabolite repression in Aspergillus nidulans, is a member of the GATA family of zinc finger DNA binding proteins, and regulates the expression of around 100 genes. This project was designed to examine the domain structure of AreA in this region of the protein, and to characterise the DNA binding domain Limited proteolysis has been employed to identify structural domains in the Cterminal region of AreA, which has been cloned and over-produced in E.coli. A variety of proteases have been used, and each reveals a dominant stable fragment of approximately 17-22 kDa. N-terminal sequencing and mass spectroscopy have been used to identify a number of these fragments. The major product following limited proteolysis by Glu-C is composed of two closely related species, a 164 residue fragment (17,489 Da) and a 157 residue fragment (16,857 Da). Both fragments encompass the Zn-finger motif, and share the same Cterminus, differing at the N-terminus by only 7 amino acids. The DNA sequence coding for the 157 residue fragment (16,857 Da) has been cloned and over-produced as a His-tag fusion protein. Further studies on this domain have shown that this putative domain has a relatively strong DNA binding constant with values in the nanomolar range. Structural analysis using Circular Dichroism, NMR and fluorescence suggests that the domain contains some irregular or unstructured regions. The regions that are structured are likely to be from the zinc-finger region, since DNA binding is maintained.
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Manival, Xavier. "Etude fonctionnelle et structurale de l'interaction de la protéine antiterminatrice SacY avec son ARN cible chez Bacillus subtilis : mise en évidence d'un nouveau type de domaine protéique de liaison à l'ARN." Montpellier 1, 1997. http://www.theses.fr/1997MON1T010.
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Nguyen, Chi Mai. "Post-transcriptional regulation during spermatogenesis : Role of the RNA-binding protein hu." Toulouse 3, 2008. http://thesesups.ups-tlse.fr/365/.
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La spermatogenèse est un processus élaboré permettant d'une part le maintien de cellules souches par divisions mitotiques et d'autre part la production de spermatozoïdes par différenciation. Au cours des dernières étapes de la différenciation, la chromatine se compacte, ne laissant plus à la cellule la possibilité de transcrire ses gènes. Du fait de l'arrêt brutal de la transcription, bien avant la fin du processus de différenciation, la cellule germinale utilise le stock d'ARN messagers (ARNm) préexistants pour finaliser sa différenciation. Ce phénomène repose sur la régulation fine du stockage et de la traduction des ARNm au cours du temps, deux régulations post-transcriptionnelles encore très peu documentées dans les cellules germinales. Au cours de ma thèse je me suis intéressée au rôle potentiel de deux protéines de liaison à l'ARN exprimées dans le testicule de souris: HuR/ELAVL1 et AUF1/hnRNP D. Dans les cellules somatiques, ces protéines lient les séquences riches en adénines et uridines (AU-rich element ou ARE) localisées dans la région 3' non codante de certains ARNm (ARN à ARE). HuR protége de la dégradation ses ARN à ARE cibles et favorise leur traduction, alors qu'AUF1 induit leur dégradation. Afin d'étudier la contribution d'HuR et d'AUF1 aux mécanismes post-transcriptionnels indispensables au bon déroulement de la spermatogénèse, nous avons dans un premier temps examiné leur patron d'expression. Nous avons montré que l'expression d'HuR est étroitement régulée au cours de la spermatogénèse, alors que celle d'AUF1 est ubiquitaire. Dans un second temps, nous avons utilisé des lignées de souris transgéniques surexprimant HuR (HuRtg) ou AUF1 (AUF1tg) établies au laboratoire et montré que la surexpression d'HuR et non celle d'AUF1 altère la spermatogenèse, entraînant leur stérilité dans 25% des cas (Sertoli Cells Only syndrome). Par la suite, nous avons mis évidence que de nombreux ARN à ARE, naturellement abondamment exprimés dans le testicule, sont dérégulés dans les cellules germinales HuRtg et AUF1tg. Une étude approfondie des ARN cibles d'HuR et d'AUF1, a révélé que ces deux protéines ont une activité différente car elles s'associent à des ARN différents dans les cellules germinales. .Spermatogenesis, the elaborate process by which sperm are produced, is marked by dramatic proliferation and differentiation. During the late steps of spermatogenesis, transcription suddenly ceases prior the end of differentiation, because of drastic epigenetic modifications that result in chromatin compaction. Thus, haploid germ cells make use of extensive temporal mRNA storage and translation regulation to ensure stage-specific protein synthesis. Factors and cellular compartments involved in these post-transcriptional controls are still poorly understood. During my PhD, I hypothesized that the two RNA binding proteins HuR/ELAVL1 and AUF1/hnRNP D, might play a role in these controls. They bind AU-rich element-containing mRNAs (ARE-mRNAs) in somatic cells and regulate their stability and translation: HuR protects ARE-mRNAs from degradation and favours their translation, whereas AUF1 usually induces their degradation. First, to investigate the contribution of HuR and AUF1 to the post-transcriptional mechanisms occurring in germ cells, I used transgenic mice derived in our laboratory overexpressing HuR (HuRtg) and AUF1 (AUF1tg) in their testes. Strikingly, whereas spermatogenesis proceeded normally in AUF1tg mice, HuR overexpression impaired spermatogenesis, revealing the importance of a regulated expression of HuR to fulfill male germ cell differentiation. The comparative analysis of AU-transcriptome of pre-pubertal wild type testes with that of HuRtg and AUF1tg testes, combined with computational analyses and RNA/Protein immunoprecipitation experiments, revealed that these two proteins regulate different targets mRNAs and thus exhibit different activities. .
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Ribeiro, Diogo. "Discovery of the role of protein-RNA interactions in protein multifunctionality and cellular complexity." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0449/document.
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Au fil du temps, la vie a évolué pour produire des organismes remarquablement complexes. Pour faire face à cette complexité, les organismes ont développé une pléthore de mécanismes régulateurs. Par exemple, les mammifères transcrivent des milliers d'ARN longs non codants (ARNlnc), accroissant ainsi la capacité régulatrice de leurs cellules. Un concept émergent est que les ARNlnc peuvent servir d'échafaudages aux complexes protéiques, mais la prévalence de ce mécanisme n'a pas encore été démontrée. De plus, pour chaque ARN messager, plusieurs régions 3’ non traduites (3’UTRs) sont souvent présentes. Ces 3’UTRs pourraient réguler la fonction de la protéine en cours de traduction, en participant à la formation des complexes protéiques dans lesquels elle est impliquée. Néanmoins, la fréquence et l’importance ce mécanisme reste à aborder.Cette thèse a pour objectif de découvrir et comprendre systématiquement ces deux mécanismes de régulation méconnus. Concrètement, l'assemblage de complexes protéiques promus par les ARNlnc et les 3'UTRs est étudié avec des données d’interactions protéines-protéines et protéines-ARN à grande échelle. Ceci a permis (i) de prédire le rôle de plusieurs centaines d'ARNlnc comme molécules d'échafaudage pour plus de la moitié des complexes protéiques connus, ainsi que (ii) d’inférer plus d’un millier de complexes 3'UTR-protéines, dont certains cas pourraient réguler post-traductionnellement des protéines moonlighting aux fonctions multiples et distinctes. Ces résultats indiquent qu'une proportion élevée d'ARNlnc et de 3'UTRs pourrait réguler la fonction des protéines en augmentant ainsi la complexité du vivantOver time, life has evolved to produce remarkably complex organisms. To cope with this complexity, organisms have evolved a plethora of regulatory mechanisms. For instance, thousands of long non-coding RNAs (lncRNAs) are transcribed by mammalian genomes, presumably expanding their regulatory capacity. An emerging concept is that lncRNAs can serve as protein scaffolds, bringing proteins in proximity, but the prevalence of this mechanism is yet to be demonstrated. In addition, for every messenger RNA encoding a protein, regulatory 3’ untranslated regions (3’UTRs) are also present. Recently, 3’UTRs were shown to form protein complexes during translation, affecting the function of the protein under synthesis. However, the extent and importance of these 3’UTR-protein complexes in cells remains to be assessed.This thesis aims to systematically discover and provide insights into two ill-known regulatory mechanisms involving the non-coding portion of the human transcriptome. Concretely, the assembly of protein complexes promoted by lncRNAs and 3’UTRs is investigated using large-scale datasets of protein-protein and protein-RNA interactions. This enabled to (i) predict hundreds of lncRNAs as possible scaffolding molecules for more than half of the known protein complexes, as well as (ii) infer more than a thousand distinct 3’UTR-protein complexes, including cases likely to post-translationally regulate moonlighting proteins, proteins that perform multiple unrelated functions. These results indicate that a high proportion of lncRNAs and 3’UTRs may be employed in regulating protein function, potentially playing a role both as regulators and as components of complexity
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Falcon, de Longevialle Alexis. "Identification des protéines PPR impliquées dans l'épissage des ARN messagers dans les chloroplastes et les mitochondries chez Arabidopsis Thaliana." Thesis, Evry-Val d'Essonne, 2010. http://www.theses.fr/2010EVRY0015.
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Le mécanisme d’épissage dans les organites est décrit comme étant l’ancêtre du spliceosome nucléaire. Cependant même si les protéines composant ce dernier sont bien connues, seulement quelques facteurs d’épissage ont été identifiés et caractérisés dans les chloroplastes et les mitochondries. Beaucoup de protéines ayant la faculté de se lier à l’ARN ont acquis des fonctions dans l’épissage, en effet un certain nombre de protéines sans véritable lien ont un rôle essentiel, avec différents degrés de spécificité dans l’épissage de la plupart des introns chloroplastiques chez les plantes. La plus grande famille de protéines se liant à l’ARN est la famille des protéines à domaines « pentatricopetide repeat » (PPR). Ces protéines sont impliquées dans la plupart des processus post-transcriptionnels dans les organites. En 2006, parmi les centaines de protéines PPR décrites chez les plantes, seulement une PPR avait été décrite comme nécessaire à l’épissage d’un intron. Ainsi, PPR4 est absolument et spécifiquement nécessaire pour l’épissage en trans de l’intron 1 de rps12 dans les plastes (Schmitz-Linneweber et al., 2006), suggérant que d’autres protéines PPR pourraient être impliquées dans l’épissage des ARN des organites. Le sujet de cette thèse porte sur la caractérisation d’autres protéines PPR impliquées dans ce processus. En utilisant des approches de génétique inverse et des outils mis en place dans le cadre de la thèse afin de détecter des défauts d’épissage par PCR quantitative, sept nouvelles PPRs impliquées dans l’épissage d’un certain nombre d’introns dans les plastes et les mitochondries ont pu être caractérisées. Dans l’optique de rechercher si des protéines PPR, impliquées dans l’épissage mais aussi dans l’édition des ARN, interagissent avec d’autres protéines, des approches de TAP-TAG ont été réalisées et sont également présentées dans ce manuscrit. L’identification de partenaires protéiques pour 3 PPRs impliquées, nous a ainsi permis de redessiner nos modèles et d’émettre de nouvelles hypothèses. Enfin, une dernière partie est consacrée à la découverte d’isoformes d’épissage pour des gènes PPR sans introns. Phénomène qui permettrait de réguler l’expression des gènes PPR, et/ou d’augmenter la diversité des protéines PPRThe RNA splicing mechanism in organelles is described to be ancestral to that of the nuclear spliceosome. However, whereas this last complex is well known, only very few splicing factors have been identified and characterized in chloroplasts and mitochondria. Many RNA binding proteins have acquired roles in RNA splicing, and indeed a variety of often unrelated RNA binding proteins have essential functions in splicing of many plastid introns in plants, with varying degrees of specificity. The largest family of RNA binding proteins in plant organelles is the pentatricopeptide repeat (PPR) family. PPR proteins are involved in diverse post-transcriptional processes in organelles. In 2006, among hundreds of higher plant proteins of this family, only one was described as being required for a splicing event - PPR4 was shown to be absolutely and specifically required for the trans-splicing of the rps12 intron 1 in plastids (Schmitz-Linneweber et al., 2006). The main purpose of this PhD thesis was to characterize other PPR proteins involved in this process. By using a reverse genetics approach and by developing tools for the detection of splicing defects, seven new PPR proteins involved in RNA splicing of a subset of chloroplast or mitochondria introns have been characterized. In parallel, in order to characterize proteins involved in PPR-containing complexes, a TAP-TAG approach has been carried out on a few PPR proteins involved in splicing or editing of organellar RNA. The identification of partner proteins of 3 PPR proteins allows us to draw new mechanistic models and new hypotheses. Finally, the final part of the manuscript describes the discovery of splicing isoforms of PPR-encoding mRNAs. Alternative splicing may be involved in regulation of PPR gene expression and/or in increasing the diversity of the PPR protein family
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Cirillo, Davide. "Prediction of protein and nucleic acid interactions." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/403537.
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The purpose of my doctoral studies has been the development of bioinformatics methods to quantitatively evaluate associations between proteins and nucleic acids (NAs). This thesis aims at providing insights into molecular features and still relatively unknown mechanisms of protein-NAs associations, such as RNA-binding proteins and long noncoding RNAs as well as transcription factors and regulatory DNA elements. In this work, I present two algorithms, catRAPID omics express and PAnDA, for the prediction of RNA- and DNA-protein interaction respectively. Those computational methods offer the possibility to address experimental problems and guide new approaches largely facilitating experimental design and proceduresMis estudios de doctorado han tenido como propósito principal el desarrollo de herramientas bioinformáticas para la evaluación de interacciones entre proteínas y ácidos nucleicos (ANs) de forma cuantitativa. Por consiguiente, esta tesis apunta a proporcionar conocimientos sobre características moleculares y mecanismos de asociación proteína-AN aún relativamente desconocidos; concretamente, la asociación de proteínas a ARNs y ARNs no codificantes, a la vez que factores de transcripción y elementos de regulación del ADN. En este proyecto presento dos algoritmos: catRAPIDomics express y PAnDA, cuyas finalidades son las de predecir interacciones proteína-ARN y proteína-ADN respectivamente. Dichos métodos computacionales ofrecen la posibilidad de abordar problemas experimentales, así como de guiar el diseño y procedimiento de nuevas estrategias para su resolución.
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Mason, Aaron Charles. "Functional analysis of an alternative Replication Protein A complex containing RPA4." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/1020.
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Replication Protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism including, but not limited to, DNA replication, DNA repair and recombination. The `canonical' RPA is composed of three subunits (RPA1, RPA2, and RPA3). In addition to the three canonical subunits, there is a human homolog to the RPA2 subunit, termed RPA4, which can substitute for RPA2 in complex formation. The resulting RPA complex has been termed `alternative' RPA (aRPA). The normal function of aRPA is not known; however, previous studies have shown that it does not support S-phase progression in vivo. The goal of this thesis was to characterize the function of aRPA in DNA replication, DNA repair and recombination and profile its expression in human tissues.
The studies presented in this thesis show that the aRPA complex has solution and DNA binding properties indistinguishable from the canonical RPA complex as determined by gel mobility shift assays. However, aRPA was unable to support DNA replication and inhibited canonical RPA function in a cell-free simian virus 40 system. aRPA inhibited both initiation and elongation of DNA synthesis in the SV40 system. Two regions of RPA4, the putative L34 loop and the C-terminal winged helix domain, were responsible for inhibiting SV40 DNA replication.
The mechanism of SV40 DNA replication inhibition during initiation and elongation was characterized using assays for DNA polymerase α and DNA polymerase δ. aRPA was shown to have reduced interaction with DNA polymerase α and was not able to efficiently stimulate DNA synthesis by DNA polymerase α on aRPA coated single-stranded DNA. However, aRPA stimulated DNA synthesis by DNA polymerase δ in the presence of PCNA and RFC even though a reduced interaction was observed between aRPA and polymerase δ.
The role of aRPA in DNA repair was also investigated. aRPA interacted with both Rad52 and Rad51 but had a reduced interaction with Rad51. However, aRPA was still able to stimulate Rad51-dependent strand exchange. aRPA also supported the dual incision/excision reaction of nucleotide excision repair. aRPA was less efficient in nucleotide excision repair than canonical RPA and this reduction was attributed to reduced interactions with the repair factor XPA. In contrast, aRPA exhibited higher affinity for damaged DNA than canonical RPA.
The expression of RPA4 and RPA2 was determined by quantitative PCR in established cell lines, human normal tissues and human tumor tissue. RPA4 was shown to be expressed in all normal tissues examined but the level of expression was tissue specific. Additionally, RPA4 expression was decreased in all tumor tissues examined and was at the limit of detection in established cell lines. Taken together, the results presented in this thesis suggest that aRPA is a `non-proliferative' form of RPA that functions to maintain the genomic stability of non-dividing cells.
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Yavrom, Sheena. "Evidence that ARNT plays a role in the regulation of the immunoglobulin heavy chain enhancer and identification of a putative ARNT ligand." Scholarly Commons, 1998. https://scholarlycommons.pacific.edu/uop_etds/516.
Full textAbstract:
Basic helix-loop-helix (bHLH) proteins are involved in the regulation of a multitude of developmental processes including cellular differentiation, cellular proliferation and xenobiotic metabolism. Among the members of the bHLH protein family are the products of the Pan gene Pan-1, Pan-2 and ITF -1. Pan proteins have been demonstrated to be required for proper B cell development, suggesting a unique role for Pan proteins during B cell formation. In our study we tested the function of ARNT (Ah receptor nuclear translocator) at the IgH (immunoglobulin heavy chain) enhancer. We were able to determine that ARNT appears to partially down-regulate activation at the IgH enhancer by Pan-1 in transient transfection assays by cotransfection of the multimerized murine form of the IgH enhancer elements 1-1E2, !-LE3 , and 1-1ES upstream of a luciferase reporter gene, a rodent Pan-1 (human homolog E47) expression vector, and an ARNT expression vector. Furthermore, during our investigation we discovered a putative ARNT -binding ligand that increases DNA-binding activity of the ARNT homodimer. This ligand was partially characterized by UV crosslinking studies and a variety of biochemical studies using electrophoretic mobility-shift assays. Preliminary data suggests that it is hydrophilic, heat-stable, small, and non-protein.
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Holla, Andrea Dorothee [Verfasser], Arne [Akademischer Betreuer] Skerra, and Dirk [Akademischer Betreuer] Haller. "Protein-Engineering eines Anticalins mit Bindungsspezifität für DC-SIGN / Andrea Dorothee Holla. Gutachter: Arne Skerra ; Dirk Haller. Betreuer: Arne Skerra." München : Universitätsbibliothek der TU München, 2012. http://d-nb.info/1031075763/34.
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Serfiotis-Mitsa, Dimitra. "Biophysical and structural studies of the antirestriction proteins ArdA and KlcA." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4358.
Full textAbstract:
Gene orf18, which is situated in the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA) protein. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli and the recombinant ArdA protein was purified to homogeneity. Biophysical characterisation of ArdA demonstrated tight association between ArdA and the M.EcoKI. Also, ArdA was shown to efficiently inhibit restriction and modification by all four major classes of Type I R/M enzymes in vivo. Thus, ArdA can overcome the restriction barrier following conjugation and so helps to increase the spread of antibiotic resistance genes by horizontal gene transfer. The amino acid sequence of KlcA, from the incompatibility plasmid pBP136 from Bordetella pertussis, showed a high degree of similarity with the antirestriction protein ArdB from the IncN plasmid pKM101. In this study the solution structure of KlcA was solved with high-resolution NMR and its antirestriction function demonstrated. The structure of KlcA showed a rigid globular molecule with a novel fold. No antimodification function was observed for KlcA in vivo and the antirestriction function of KlcA has been successfully shown in vivo but not in vitro. Because no direct binding of KlcA to EcoKI was observed in vitro, the mechanism of the endonuclease blocking was assumed to be different from that of ArdA. Preliminary experiments including coimmunoprecipitation assays were conducted in order to elucidate the antirestriction mechanism of KlcA.
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Cid, Samper Fernando 1991. "Computational approaches to characterize RNP granules." Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/668449.
Full textAbstract:
Ribonucleoprotein granules (RNP granules) are liquid-liquid phase separated
complexes composed mainly by proteins and RNA. They are responsible of many
processes involved in RNA regulation. Alterations in the dynamics of these proteinRNA complexes are associated with the appearance of several neurodegenerative
disorders such as Amyotrophic Lateral Sclerosis ALS or Fragile X Tremor Ataxia
Syndrome FXTAS. Yet, many aspects of their organization as well as the specific
roles of the RNA on the formation and function of these complexes are still unknown.
In order to study RNP granules structure and formation, we integrated several state of
the art high-throughput datasets. This includes protein and RNA composition obtained
from RNP pull-downs, protein-RNA interaction data from eCLIP experiments and
transcriptome-wide secondary structure information (produced by PARS). We used
network analysis and clustering algorithms to understand the fundamental properties
of granule RNAs. By integrating these properties, we produced a model to identify
scaffolding RNA. Scaffolding RNAs are able to recruit many protein components into
RNP granules. We found that the main protein components of stress granules (a kind
of RNP granules) are connected through protein-RNA interactions. We also analyzed
the contribution of RNA-RNA interactions and RNA post-transcriptional
modifications on the granule internal organization.
We applied these findings to understand the biochemical pathophysiology of FXTAS
disease, employing as well some novel experimental data. In FXTAS, a mutation on
the FMR1 gene produces a 5´microsatellite repetition that enhances its scaffolding
ability. This mutated mRNA is able to sequester some important proteins into nuclear
RNP granules, such as TRA2A (i.e. a splicing factor), impeding their normal function
and therefore producing some symptoms associated with the progress of the disease.
The better understanding of the principles governing granules formation and structure
will enable to develop novel therapies (e.g. aptamers) to mitigate the development of
several neurodegenerative diseases.Los gránulos ribonucleoproteicos (gránulos RNP, por sus siglas en inglés) son complejos producidos mediante separación líquido-líquido y están constituidos principalmente por proteínas y ARN. Son responsables de numerosos procesos involucrados con la regulación del ARN. Alteraciones en la dinámica de estos complejos de proteínas y ARN están asociadas con la aparición de diversas enfermedades neurodegenerativas como el ELA o FXTAS. Sin embargo, todavía se desconocen muchos aspectos relativos a su organización interna así como las contribuciones específicas del RNA en la formación y funcionamiento de estos complejos. A fin de estudiar la estructura y formación de los gránulos RNP, hemos integrado varias bases de datos de alto rendimiento de reciente aparición. Esto incluye datos sobre la composición proteica y en ARN de los RNP, sobre la interacción de proteínas y ARN extraída de experimentos de eCLIP y sobre la estructura secundaria del transcriptoma (producida mediante PARS). Todos estos datos han sido procesados para comprender las propiedades fundamentales de los ARNs que integran los gránulos, mediante el empleo de métodos computacionales como el análisis de redes o algoritmos de agrupamiento. De esta manera, hemos producido un modelo que integra varias de estas propiedades e identifica candidatos denominados ARNs de andamiaje. Definimos ARNs de andamiaje como moléculas de ARN con una alta propensión a formar gránulos y reclutar un gran número de componentes proteicos a los gránulos RNP. También hemos encontrado que las interacciones proteína-ARN conectan los principales componentes proteicos de consenso de los gránulos de estrés (un tipo específico de gránulos RNP). También hemos estudiado la contribución de las interacciones ARN-ARN y las modificaciones post-transcriptionales del RNA en la organización interna del gránulo. Hemos aplicado estos resultados para la comprensión de la fisiopatología molecular de FXTAS, empleando también algunos datos experimentales originales. En FXTAS, una mutación en el gen FMR1 produce una repetición de microsatélite en 5´ que incrementa su capacidad como ARN de andamiaje. Este mARN mutado es capaz de secuestrar algunas proteínas importantes como TRA2A (un factor de ayuste alternativo) en gránulos RNP nucleares, impidiendo su normal funcionamiento y por consiguiente produciendo algunos síntomas asociados con el progreso de la enfermedad. Una mejor comprensión de los principios que gobiernan la formación y estructura de los gránulos puede permitir desarrollar nuevas terapias (ej: aptámeros) para mitigar el desarrollo de diversas enfermedades neurodegenerativas.
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Christians, Arne [Verfasser], and Stefan [Akademischer Betreuer] Wiemann. "Funktionelle Charakterisierung des putativen Tumorsuppressors "Epithelial Membrane Protein 3" / Arne Christians ; Betreuer: Stefan Wiemann." Heidelberg : Universitätsbibliothek Heidelberg, 2014. http://d-nb.info/1180300955/34.
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Rall, Nils Arne [Verfasser], Heinz [Akademischer Betreuer] Neumann, and Henning [Akademischer Betreuer] Urlaub. "A Method for the Quantitative Analysis of Protein-Protein Interactions In Vivo / Nils Arne Rall. Betreuer: Heinz Neumann. Gutachter: Heinz Neumann ; Henning Urlaub." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://d-nb.info/1096752069/34.
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Stefani, Arno Gert [Verfasser], and Johannes [Gutachter] Huber. "Nonparametric and Nonasymptotic Confidence Intervals for Estimation of Mutual Information with Applications in Protein-Protein Docking Analysis / Arno Gert Stefani ; Gutachter: Johannes Huber." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2018. http://d-nb.info/115500616X/34.
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Mayer, Jan-Peter Andreas [Verfasser], Arne [Akademischer Betreuer] Skerra, Wolfgang [Akademischer Betreuer] Liebl, and Bernhard [Akademischer Betreuer] Küster. "Rationales und kombinatorisches Protein-Engineering mit Lipocalinen / Jan-Peter Andreas Mayer. Gutachter: Wolfgang Liebl ; Arne Skerra ; Bernhard Küster. Betreuer: Arne Skerra." München : Universitätsbibliothek der TU München, 2013. http://d-nb.info/1036262170/34.
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Fichera, Claudia Annunziata. "Building a ¦Â-structured artificial pore-forming protein by PA83 Bacillus anthracis toxin." Doctoral thesis, Università di Catania, 2014. http://hdl.handle.net/10761/1573.
Full textAbstract:
Le porine naturali sono delle proteine che si trovano principalmente nella membrana esterna dei batteri Gram negativi, come Escherichia coli, ma che sono state ritrovate anche nelle cellule vegetali e nelle membrane di organuli cellulari eucariotici come mitocondri e cloroplasti. Le porine generano dei canali acquosi poco o nulla selettivi, a volte voltaggio dipendenti, e alcune rivestono molta importanza in determinati pathway cellulari. L¡¯utilizzo sempre pi¨´ frequente di peptidi strutturalmente definiti con funzioni chimiche specifiche ha portato alla realizzazione di questo lavoro. Attraverso una preliminare analisi bioinformatica ¨¨ stato individuato un pattern proteico di base con una struttura a forcina (¦Â-strand/loop/ ¦Â-strand) partendo dalla proteina PA83 di Bacillus anthracis. Mediante la realizzazione di un protocollo sperimentale semplice e molto rapido ¨¨ stato possibile costruire multimeri del modulo di base, ovvero costrutti contenenti varie ripetizioni dello stesso. I dati dell¡¯analisi bioinformatica suggerivano che il giusto numero di ripetizioni del modulo di base necessario alla produzione di una proteina formante poro fosse sette, che tra l¡¯altro ¨¨ il numero di oligomeri che si assemblano nella proteina naturale da cui siamo partiti. Il piano sperimentale messo a punto ci ha permesso di ottenere in pochi giorni una proteina chimerica completamente artificiale, la quale ¨¨ stata clonata in un vettore di espressione batterico. L¡¯espressione della proteina ¨¨ stata ottenuta difficilmente e inoltre le colture batteriche in cui veniva indotta l¡¯espressione della proteina chimerica risultavano meno torbide, indice di una ridotta crescita cellulare. In accordo con la recente letteratura siamo propensi a credere che la proteina agisca in qualche modo da antibiotico, inserendosi massivamente in membrana e determinando la lisi cellulare dell¡¯ospite batterico. Studi futuri saranno volti a valutare le strutture secondaria e terziaria della porina artificiale prodotta e le sue propriet¨¤ elettrofisiologiche.
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Belfiore, Ramona. "Protein Misfolding and Aggregation in Neurodegeneration: In Vitro And In Vivo Study Cases." Doctoral thesis, Università di Catania, 2018. http://hdl.handle.net/10761/4178.
Full textAbstract:
Neurodegenerative diseases are nowadays increasing in incidence and widely distributed around the world. Despite those disorders show very different symptoms and morbidity, intracellular and extracellular protein misfolding and accumulation appears as a common pathological pathway. In the present thesis work I analyzed two cases of toxic protein deposition involved in ALS and AD. First, I looked at SOD1-G93A mutant protein, whose neuronal deposit is associated to familial and sporadic ALS. The mitochondrial porin VDAC1 has been proposed as a binding target of SOD1 mutant forms to mitochondria. By affinity studies we found that VDAC1 protein specifically binds SOD1-G93A but not wild type SOD1. Notably, it is known that the N-Terminal end of Hexokinase 1 (N-HK1) interacts with VDAC1: thus, we produced a synthetic peptide corresponding to the first 11 aa of human HK1 and tested its action as a potential interfering molecule between VDAC1/SOD1-G93A bond. Both in a protein-protein interaction and in a protein mitochondrial interaction we obtained a decrease of VDAC1/SOD1-G93A binding with respect of the increased N-HK1 peptide concentration. Summarizing, SOD1-G93A binds VDAC1 and impairs HK1 binding and our results suggest for N-HK1 peptide a neuroprotective potential in ALS patients.
The second part of my thesis work was focused on Amyloid and tau protein accumulation in 3xTg-AD mice. In order to study neuropathology and cognitive deficits in (AD), several transgenic models of AD have been identified. Accumulation of Abeta and fibrillary tangles as well as impairments in working and learning memory are age-related hallmark of AD pathology. To produce a progressive characterization of Abeta and tau pathology in 3xTg-AD mice we aged female mice at 2, 6, 12 and 20 months of age. We tested mice in a behavioral assay named Morris Water Maze (MWM) and we used in vitro biochemical assays, to observe Abeta soluble and insoluble fraction as well as tau phosphorylation in both cortex and hippocampus of 3xTg-AD mice. Our data on MWM demonstrate a progressive impairment in learning with a strongly significant difference between 3xTg-AD mice and controls, from 6 months of age. Notably, we also found a progressive increase in both soluble and insoluble Abeta40 and Abeta42, an age dependent tau hyperphosphorylation at specific AD linked phospho-sites, and an intense glial reactivity. Overall, our data confirm that female 3xTg-AD mice consistently show AD-like pathology, therefore this transgenic mouse model can be used as an extremely powerful tool to investigate pathogenic mechanisms underlying Alzheimer s disease.
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Bourdeau-Julien, Isabelle. "ALS-associated RNA-binding protein FUS and mRNA translation regulation." Master's thesis, Université Laval, 2020. http://hdl.handle.net/20.500.11794/68742.
Full textAbstract:
Des mutations dans plusieurs gènes ont été liés à la sclérose latérale amyotrophique (SLA),en particulier dans celui codant pour la protéine Fused in Sarcoma (FUS). Les mutations sont retrouvées dans la partie codant pour le signal de localisation nucléaire, rendant la protéine anormalement abondante dans le cytoplasme. Combiné à d’autres observations, ça suggère qu’un gain de fonction toxique de FUS dans le cytoplasme serait à l’origine de la neurodégénérescence. La SLA est une maladie neurodégénérative qui affecte les neurones moteurs et cause une paralysie progressive. Les mécanismes moléculaires causant la maladies ont toujours inconnus. Une des pistes serait la perturbation de la traduction locale desARNm, qui permet aux synapses de répondre rapidement et indépendamment du corps cellulaire. Une traduction locale insuffisante pour soutenir l’activité synaptique à long terme mènerait à la perte des synapses et à la neurodégénérescence. Mon objectif est donc de déterminer le rôle de FUS dans régulation de la traduction des ARNm en caractérisant son interaction avec les composantes traductionnelles et d’évaluer sa fonction dans une condition reproduisant les caractéristiques de la SLA. J’ai montré que FUS s’associe aux polyribosomes inactifs, ce qui suggère que FUS jouerait un rôle dans la régulation de la traduction des ARNm en interagissant avec le cœur de la traduction. Il est également possible d’observer une augmentation de la présence de FUS dans le cytoplasme et de son interaction avec les polyribosomes suite à une inhibition de la traduction par mTOR, suggérant son rôle de régulateur négatif. De plus, les mutations liées à la SLA amplifient la fonction inhibitrice de FUS en rendant FUS cytoplasmique et en réduisant la synthèse des protéines. Mes résultats montrent que la protéine FUS aurait un rôle d’inhibiteur de la traduction quand celle-ci est cytoplasmique. Par conséquent, l’augmentation de la présence de FUS dans le cytoplasme dans la SLA entrainerait une inhibition de la traduction importante, à un niveau insuffisant pour soutenir l’activité synaptique.Mutations in several genes have been linked to amyotrophic lateral sclerosis (ALS),particularly in the gene coding for the Fused in Sarcoma protein (FUS). Those mutations are found in the part encoding for the nuclear localization signal, making the protein abnormallyabundant in the cytoplasm. Combined with other observations, it suggests that a toxic gainof function of FUS in the cytoplasm would be the cause of the neurodegeneration. ALS is a neurodegenerative disease that affects motor neurons and causes progressive paralysis. The molecular mechanisms causing the disease are still unknown. One of the hypotheses is the disruption of local translation of mRNAs, which allows synapses to respond quickly and independently from the cell body. Insufficient local translation to support long-term synapticactivity would lead to synaptic loss and neurodegeneration. Thereby, the objective of mystudy is to determine the role of FUS in the regulation of mRNA translation by characterizing its interaction with translational components and evaluate its function in an ALS-linked condition. I have shown that FUS is associated with stalled polyribosomes, which suggests that it plays a role in regulating mRNA translation by interacting with the core of translation.There is also an increase in the presence of FUS in the cytoplasm and in its interaction with polyribosomes following inhibition of translation through mTOR, suggesting its role as anegative regulator. In addition, ALS-related mutations amplify FUS inhibitory function bymaking FUS cytoplasmic and reducing protein synthesis. My results show that the FUSprotein would have a role as a translation inhibitor when it is cytoplasmic. There fore, increasing the presence of FUS in the cytoplasm in ALS would result in significant translation inhibition, at a level insufficient to support synaptic activity.
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Konji, Sandra. "Gestational and Postnatal Exposure to a Contaminant Mixture: Effects on Estrogen Receptor Protein Expression In the Postpartum Maternal Brain." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/38792.
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Maternal behaviours are those that increase offspring survival. Estrogens affect maternal behaviour by activating Estrogen Receptors (ER) in the brain. Maternal brain plasticity was explored by characterizing the effects of exposure to a mixture of environmental pollutants on number of ERs. Following exposure to the toxicants during pregnancy and lactation, brains of female rats were collected, sectioned at 30 μm and immunohistochemistry for ERα performed. Immuno-positive cells in the mPOA, VTA and NAc were counted. A two way ANOVA revealed no main effect of Treatment on the number of immunopositive cells for all three brain regions. However, a significant difference between the High and Low Doses with the high dose reducing the number of ERα+ cells in the mPOA and VTA. Our work showcases the importance of studying the effects of multiple chemical co-exposures on the mother's brain, as maternal brain changes impact maternal behaviour consequently affecting offspring neurodevelopment.
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Sharma, Kamal Kant. "Molecular mechanism of the hepatitis C virus core protein chaperone properties : Physicochemical investigation by fluorescence and surface plasmon resonance." Strasbourg, 2011. https://publication-theses.unistra.fr/public/theses_doctorat/2011/SHARMA_Kamal_Kant_2011.pdf.
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La protéine core de virus de l’hépatite C (HCV) est l’une des dix protéines codées par l’ARN génomique de 9. 6kb du virus. C’est une protéine chaperonne multifonctionnelle impliquée dans plusieurs processus viraux comme la prolifération cellulaire, la différentiation, l’encapsidation de l’ARN, la formation de la nucléocapside, et la variabilité génétique. Grâce à ses propriétés de chaperonne, le domaine D1 dimérise la région 3’ non traduite (3’UTR) de l’ARN génomique. Cependant le mécanisme de cette activité chaperonne ainsi que l’interaction entre la protéine Core et différents oligonucléotides de la partie 3’ de l’ARN restent inconnus. Dans ce but, nous avons utilisé différentes approches de fluorescence et la résonance plasmonique de surface. Ainsi, en utilisant le peptide D1, correspondant à un fragment de la protéine core, ainsi que plusieurs dérivés de ce peptide nous avons caractérisé les paramètres de l’interaction et montré que la protéine core se lie spécifiquement aux oligonucléotides en tige-boucle de la partie 3’. Ensuite, nous avons suivi la conformation de ces oligonucléotides et montré que la protéine core n’est pas capable de déstabiliser leur structure secondaire. Enfin, nous avons décrit au niveau moléculaire le mécanisme permettant à la protéine core d’hybrider des oligonucléotides complémentaires et montré que la cinétique d’hybridation repose sur une réaction à deux étapes impliquant un contact boucle-boucle. Ce travail devrait nous permettre de mieux comprendre le rôle de la protéine core lors de l’encapsidation et la transcription de l’ARN et notamment dans les mécanismes de recombinaison expliquant les variations génétiques du virusOpen reading frame (ORF) of 9. 6kb HCV genomic RNA encodes at least 10 proteins, 4 structural and 6 non-structural, during translation process. The core is one of those 4 structural proteins and considered as a multifunctional chaperone involving in several viral processes like cell proliferation, differentiation, RNA packaging, nucleocapsid formation and recombinant genetic variability. With the virtue of its chaperone properties, Domain D1 dimerises the 3’ untranslated region (3’ UTR) of the genomic RNA. However, the mechanism of the core chaperone activity in the dimerisation of the genomic RNA and in binding with its target nucleic acids are still unknown and were investigated in this present project. To reach this objective, we used fluorescence and surface plasmon resonance (SPR) techniques. By using the native D1 domain and peptides derived from this domain, we first characterized the binding parameters and the conformational changes associated with the binding of these peptides to the native and mutated sequences from HCV 3’ UTR sequences. Next, we investigated the destabilization of model and HCV ODNs secondary structure by the D1 domain and its mutants and found that core peptides only marginally destabilise the secondary structures of ODNs. In a last step, we described the molecular mechanisms of the core chaperone properties based on the hybridization kinetics of various HCV and model oligonucleotides. These chaperone properties of core are thought to intervene in processes like the encapsidation, the synthesis of the complementary strand of the genomic RNA and the recombination mechanisms participating to the genetic variability of the virus
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Pintard, Lionel. "Spb1p est une méthylase de levure impliquée dans la maturation des ARNr." Montpellier 1, 2000. http://www.theses.fr/2000MON1T019.
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Orelle, Béatrice. "Pancreatitis associated protein (PAP) : clonage, séquençage et expression de l'ARN messager chez le rat et l'homme." Lyon 1, 1991. http://www.theses.fr/1991LYO10069.
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La pancreatitis associated protein ou pap est une proteine secretoire mise en evidence au cours de la pancreatite aigue experimentale chez le rat. Le clonage et le sequencage de son adnc ont permis de mieux caracteriser sa structure et d'etudier l'expression de son gene. Cette expression est tres faible dans le pancreas sain et augmente considerablement (environ 300 fois) au cours de l'inflammation, alors que celle des enzymes digestives diminue. L'adnc de la pap de rat nous a permis de cloner l'adnc de la pap humaine. La sequence en acide amine deduite de la proteine presente 69% d'homologie avec la pap de rat. L'expression du gene de la pap humaine est tres importante lors d'une pancreatite necro-hemorrhagique severe. Son expression est beaucoup plus faible dans les pancreatites obstructives et chroniques, et reste indetectable chez les individus sains, dans les adenocarcinomes du pancreas et dans les tumeurs endocrines. L'expression de la pap semble correlee avec la reponse a l'inflammation. Son induction possede les caracteristiques de l'expression des proteines de stress dans d'autres tissus: elle est rapide, intense et transitoire. La pap pourrait donc etre un marqueur specifique de l'inflammation pancreatique. Son role est pour l'instant inconnu. L'analogie structurale de la pap avec les lectines de types c, calcium dependantes, suggere cependant que la proteine agirait en se fixant a un sucre specifique
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Kirchner, Christian Oliver [Verfasser], Arne [Akademischer Betreuer] [Gutachter] Skerra, and Wolfgang [Gutachter] Liebl. "Protein-Engineering eines Anticalins mit Bindungsspezifität für das Anthrazyklin-Zytostatikum Doxorubicin / Christian Oliver Kirchner. Betreuer: Arne Skerra. Gutachter: Wolfgang Liebl ; Arne Skerra." München : Universitätsbibliothek der TU München, 2016. http://d-nb.info/1113181958/34.
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Sessa, Gaetana. "Role of the Interaction of BRCA2 and DDX5 in the DNA Damage Response BRCA2 promotes DNA-RNA hybrid resolution by DDX5 at DNA double strand breaks to facilitate homologous recombination Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS116.
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Un nombre croissant d’études soutiennent le fait que les protéines majeures du métabolisme des ARN, telles que les hélicases ARN, sont impliquées dans la réponse aux dommages à l’ADN. Cette activité est généralement accomplie par leur interaction avec des facteurs de réparation de l’ADN. BRCA2, une protéine suppressive de tumeurs, joue un rôle crucial dans la réparation des cassures double-brin (CDB) de l'ADN par recombinaison homologue (RH) et donc, est un facteur essentiel pour l’intégrité du génome. Les cellules déficientes pour BRCA2 accumulent des hybrides ADN-ARN ou R-loops, une source de dommage à l'ADN, suggérant ainsi l’importance de cette protéine dans la prévention ou la suppression de ces structures. Toutefois, le rôle spécifique de BRCA2 dans la résolution des hybrides ADN-ARN reste inconnu.Afin de connaître des potentiels partenaires de BRCA2, une analyse par spectrométrie de masse réalisée dans notre laboratoire a révélé un enrichissement en protéines impliquées dans le métabolisme de l'ARN, comme les hélicases ARN. Ces résultats nous ont menés à examiner la coopération entre BRCA2 et les hélicases ARN dans la séparation des structures ADN-ARN. Nous avons d’abord confirmé l'interaction entre l'hélicase ARN DDX5 et BRCA2, qui est améliorée dans les cellules exposées à γ-irradiation. Ensuite, nous avons réduit l’interaction aux premiers 250 aa de BRCA2 (BRCA2T1) et avons constaté que celle-ci est directe en utilisant des protéines purifiées. En collaboration avec le laboratoire du docteur A. Aguilera (Cabimer, SP), nous avons montré que la déplétion de DDX5 conduit à une accumulation des hybrides ADN-ARN dans l’entièreté du génome, particulièrement aux sites de dommages à l’ADN. De plus, nos résultats indiquent que DDX5 localise aussi aux hybrides ARN-ADN qui se forment à proximité de CDB.De manière intéressante, nous avons constaté que BRCA2 est important pour la rétention de DDX5 aux sites de dommage à l’ADN induit par l’irradiation laser. Notamment, des tests de déroulement de brins in vitro en utilisant les protéines purifiées DDX5 et BRCA2 ont révélé que BRCA2 stimule l’activité de déroulement des R-loops de DDX5.Un variant de signification inconnue (VSI) trouvé dans de patients atteints de cancer du sein situé dans la région BRCA2T1 (T207A) réduit l’interaction de BRCA2 avec DDX5 et conduit à l’accumulation des hybrides ADN-ARN. Les cellules exprimant stablement BRCA2-T207A montrent également une diminution de l’association de DDX5 avec les hybrides ARN-ADN, en particulier lors d’une exposition de cellules à l’irradiation. L’analyse de l’efficacité de la réparation des CDB par RH dans les cellules déficientes en DDX5 ou exprimant BRCA2-T207A, montre une cinétique retardée de l’apparition des foyers de réparation RAD51 lors de l’irradiation, ce qui suggère un rôle actif de l’interaction BRCA2-DDX5 pour assurer la réparation par RH efficacement. En accord avec cette hypothèse, la ribonucléase RNAseH1, qui dégrade spécifiquement la fraction d’ARN dans les structures d’ADN-ARN, restaure partiellement le phénotype de cinétique des foyers RAD51 dans les cellules BRCA2 T207A. De plus, les cellules portant le variant BRCA2-T207A ont également montré un nombre réduit de foyers RPA par rapport aux cellules qui expriment BRCA2 sauvage, témoins d’un défaut dans l’étape qui précède le chargement de RAD51 aux CDB.Ensemble, nos résultats suggèrent que les hybrides ADN-ARN représentent un obstacle à la réparation des CDB par RH et révèlent BRCA2 et DDX5 en tant que facteurs actifs dans leur suppressionIncreasing evidence support the idea that proteins involved in RNA metabolism such as RNA binding proteins (RBPs) and RNA helicases are directly implicated in the DNA damage response (DDR). This activity is generally achieved through their interaction with DNA repair factors.BRCA2 is a tumor suppressor protein that plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) as well as protecting stalled replication forks from unscheduled degradation; therefore, it is essential to maintain genome integrity. Interestingly, BRCA2 deficient cells accumulate DNA-RNA hybrids or R-loops, a known source of DNA damage and genome instability, providing evidence for its role in either R-loop prevention or processing. However, the specific role of BRCA2 on these structures remains poorly understood.A mass spectrometry screen to identify partners of BRCA2 performed in our laboratory revealed an enrichment of proteins involved in RNA metabolism such as RNA helicases. These findings led us to investigate whether BRCA2 could cooperate with these candidate interacting RNA helicases in processing DNA-RNA structures. First, we confirmed the interaction of BRCA2 and the DEAD-box RNA helicase DDX5, which we found is enhanced in cells exposed to -irradiation. Then, we narrowed down the interaction to the first 250 aa of BRCA2 (BRCA2T1) and found that it is direct using purified proteins. In collaboration with A. Aguilera lab (Cabimer, SP), we could show that depletion of DDX5 leads to a genome-wide accumulation of DNA-RNA hybrids that is particularly enriched at DNA damage sites. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs. Interestingly, we found that BRCA2 is important for the retention of DDX5 at laser irradiation-induced DNA damage. Notably, in vitro R-loop unwinding assays using purified DDX5 and BRCA2 proteins revealed that BRCA2 stimulates the R-loop helicase activity of DDX5.A breast cancer variant of unknown clinical significance (VUS) located in BRCA2T1 (T207A) reduced the interaction between BRCA2 and DDX5 and led to the accumulation of DNA-RNA hybrids. Cells stably expressing BRCA2-T207A also showed a decreased association of DDX5 with DNA-RNA hybrids, especially upon irradiation. Notably, monitoring RAD51 foci to evaluate HR-mediated DSBs repair efficiency in either DDX5-depleted cells or in BRCA2-T207A cells resulted in a delayed kinetics of appearance of RAD51 foci upon irradiation suggesting an active role of BRCA2-DDX5 interaction in ensuring timely HR repair. In agreement with this, overexpression of the RNAseH1 ribonuclease, that specifically degrades the RNA moiety in DNA-RNA structures, partially restored RAD51 kinetics phenotype of BRCA2-T207A cells. Moreover, cells bearing BRCA2-T207A variant also showed a reduced number of RPA foci compared to BRCA2 WT expressing cells, a step that precedes RAD51 loading at DSBs.Taken together, our results are consistent with DNA-RNA hybrids being an impediment for the repair of DSBs by HR and reveal BRCA2 and DDX5 as active players in their removal
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Soicke, Arne [Verfasser]. "Stereoselektive Synthese neuartiger Prolin- und Pseudoprolin-Dipeptidmimetika zur Modulation von Peptid-Protein-Wechselwirkungen / Arne Soicke." München : Verlag Dr. Hut, 2014. http://d-nb.info/1067707913/34.
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Reynolds, Lindsey. "DNA binding and structural studies of truncated forms of the AreA protein from Aspergillus nidulans." Thesis, University of Portsmouth, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264456.
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AreA is a transcription activation protein regulating over 100 genes in Aspergillus nidulans. It is a member of the 4 cysteine zinc finger family, with significant sequence homology in the zinc finger domain with related proteins. As with similar proteins, the zinc finger domain has been identified as the DNA binding motif. Other regions of the protein do not appear to playa role in directly binding DNA. A truncated form of AreA, known as the minimal zinc finger protein, containing the zinc finger domain alone, has been cloned and over-expressed in this study. This domain is sufficient to bind DNA specifically, but weakly. However, the addition of a further 30 amino acids, C-terminal to the minimal zinc finger, containing a highly basic tail is shown to increase the specific binding affinity. Other forms of AreA have been characterised and do not significantly increase the affinity of DNA binding. Identification of a consensus binding sequence by SELEX has also demonstrated that the minimal zinc finger is sufficient to specifically recognise the core sequence GA T A but suggests a degree of tolerance for TAT A. Addition of further regions of the protein do not extend the limits of the recognition sequence or change the consensus sequence. The SELEX experiments did not give any evidence of AreA functioning as a dimer, through proteinprotein interactions. Structural studies of the truncated forms of AreA, by circular dichroism, have suggested the formation of secondary structures for the zinc finger motif similar to those of other proteins and in agreement with the published NMR structure of the AreA zinc finger bound to DNA (Starich et ai., 1998a).
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Fournier, Cécile. "Caractérisation de la fixation de la protéine NEF du virus HIV-1 à l'ARN et mise en évidence de son rôle dans la rétrotranscription." Montpellier 1, 2000. http://www.theses.fr/2000MON1T015.
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Di, Battista Maria Elena. "Dementia in Parkinson s Disease: relationship between clinical and neurobiological aspects." Doctoral thesis, Università di Catania, 2015. http://hdl.handle.net/10761/3836.
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Il deficit cognitivo è uno degli aspetti più comuni e importanti della malattia di Parkinson (PD), influisce notevolmente la qualità e l aspettativa di vita del paziente. Lo spettro delle disfunzioni cognitive nei PD è stato recentemente codificato in termini di Mild Cognitive Impairment (PD-MCI) e Demenza (PDD) con criteri clinici e diagnostici forniti da specialisti del Movement Disorders Society (MDS). La nostra attenzione è stata inizialmente concentrata sulla affidabilità dei criteri diagnostici proposti per PDD; in seguito i pazienti sono stati seguiti per cinque anni per intercettare un possibile profilo malattia, includendo il profilo di disabilità motoria, i deficit neuropsicologici e la compromissione neuropsichiatrica che potevano essere associati al fenomeno di involuzione cognitiva. Nel setting laboratoristico, abbiamo condotto uno studio di correlazione genotipo-fenotipo per valutare il possibile ruolo degli aplotipi MAPT nel determinare l'espressione del profilo motorio e non-motorio nei pazienti con PD, prima dello sviluppo di demenza. I risultati di questo studio suggeriscono un ruolo dell aplotipo H1 MAPT in tutto il corso della malattia, agendo come cuneo di pressione degenerativa nel corso degli anni: un fattore di rischio per lo sviluppo di PD con un OR di ~ 1,5, un fattore genetico di co-determinazione con un OR > 2 del fenotipo motorio durante le fasi intermedie e, infine, un fattore di rischio per la demenza con un OR> 10. I primi risultati sul profilo dei sintomi non-motori suggeriscono un possibile ruolo degli aplotipi MAPT e della proteina tau anche per quanto riguarda la disfunzione cardiaca disautonomica, anche prima dello sviluppo di demenza e in assenza di evidente associazione con altri sintomi non motori. I risultati dello studio di associazione genotipo-fenotipo sugli aspetti non-motori rappresentano un dato preliminare condotto in una porzione della nostra coorte, devono quindi essere considerati con cautela. I dati, nel loro insieme, indicherebbero il gene MAPT e la proteina tau come un legame biologico tra le manifestazione fenotipiche della malattia ed il successivo sviluppo di demenza aumentando la suscettibilità agli insulti biochimici che sono propri del processo della Malattia di Parkinson.
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BESSE, LAURENT. "Mise au point d'un reactif chimique permettant l'etude des interactions proteine-proteine. Application aux arn polymerases de levure." Paris 11, 1996. http://www.theses.fr/1996PA112232.
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Les methodes classiques d'etude de la structure quaternaire de proteines permettent difficilement, a l'heure actuelle, d'obtenir des informations structurales sur les complexes multimeriques de haut poids moleculaire. Nous avons donc concu et synthetise un reactif bifonctionnel, permettant l'etude des interactions entre les differentes sous-unites constituant ces proteines oligomeriques. Ce reactif comporte, a l'une de ses extremites, un complexe organique du ruthenium(ii) capable de se lier de maniere specifique a une sequence poly-histidines introduite par genie genetique sur une sous-unite donnee et, a l'autre extremite, un groupement photoactivable radioactif. Ces deux fonctionnalites sont reliees par une chaine carbonee hydrolysable. En premier lieu, nous avons montre a travers l'etude d'un compose modele - le carbonatobis(2,2'-bipyridine)ruthenium(ii) - qu'une fonction carbonate porte par le ruthenium permet au metal de se fixer sur des histidines en milieu aqueux tamponne et a temperature ambiante, conditions requises par l'emploi du reactif sur des proteines. Nous avons ensuite toujours grace a ce complexe modele, demontre la complexation des histidines sur des peptides synthetiques. Cette etude a de plus ete realisee par rmn du proton et par modelisation moleculaire. Dans un deuxieme temps, la synthese des complexes bifonctionnels comportant les proprietes fonctionnelles requises pour l'etude des interactions entre proteines a ete mise au point. Les systemes biologiques auxquels nous nous sommes interesses sont les arn polymerases i, ii et iii de la levure saccharomyces cerevisiae. Les premiers resultats ont ete obtenus sur ces macromolecules biologiques. En conclusion, les resultats encourageants nous permettent d'envisager l'etude de l'agencement des sous-unites les unes par rapport aux autres afin d'obtenir des informations structurales sur les proteines multimeriques
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Collet, Axelle. "Caractérisation des enzymes de formation de la coiffe du virus du Nil Occidental et du métapneumovirus humain." Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4087.
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Ma thèse a porté sur l’étude des activités enzymatiques impliquées dans la formation de la coiffe de deux virus à ARN: le virus du Nil Occidental (WNV) et le métapneumovirus humain (hMPV). Ces virus codent pour des enzymes assurant l’ajout de la coiffe de type-1 (m7GpppN2’Om) à l’extrémité 5’ de leur ARNm.Le domaine N-terminal de la protéine NS5 (NS5MTase) du WNV porte les activités N7- et 2’O-méthyltransférases (N7- et 2’O-MTases) et il a été proposé que NS5MTase puisse également porter l’activité guanylyltransférase (GTase). J’ai identifié in vitro des résidus clés impliqués dans l’interaction entre NS5MTase et des ARN substrats de chaque activité MTase. Nos résultats démontrent que le site de fixation de la coiffe est nécessaire lors de la 2’O-méthylation et ne l’est pas pour la N7-méthylation. En parallèle, j’ai recherché des résidus catalytiques de la GTase par la méthode de génétique inverse. Des résultats préliminaires indiquent que la mutation K29A induit un défaut de réplication. Ce résidu pourrait donc être impliqué dans l’activité GTase de NS5MTase.Concernant hMPV, j’ai effectué une analyse fonctionnelle du domaine CR-VI+ de la protéine L. J’ai démontré que CR-VI+ possède les activités N7- et 2’O-MTases et j’ai identifié les résidus impliqués dans le recrutement de l’ARNm. L’ordre de méthylation est non canonique avec la 2’O-méthylation qui précède la N7-méthylation. Enfin, j’ai également démontré que CR-VI+ possède une activité d’hydrolyse du GTP.Ce travail démontre que ces MTases possèdent 2 voire 3 des activités enzymatiques nécessaires à la formation de la coiffe, et représentent donc une cible de choix pour le développement d’inhibiteursMy PhD project is focus on the study of the enzymatic activities involved in the RNA capping pathway of two RNA viruses: the West Nile Virus (WNV) and the human metapneumovirus (hMPV). These viruses encode for enzymes allowing the addition of a cap-1 structure (m7GpppN2’Om) to their mRNA 5’ ends. The NS5 N-terminal domain (NS5MTase) of WNV harbours the N7- and 2’O-methyltransferase activities (N7- and 2’O-MTase); and it has been proposed that NS5MTase also bears a guanylyltransferase activity (GTase). I have identified residues involved in the NS5MTase interaction sites with their RNAs substrate. My assays demonstrate the importance of the cap-binding site for the 2’O-methylation but not for the N7-methylation. In parallel, I have tried to identify putative catalytic residues of the GTase activity by reverse genetics. Preliminary results suggest that NS5MTase K29 could be a catalytic residue.Concerning hMPV, I performed a functional analysis of CR-VI+ domain of the protein L. I demonstrated that the CR-VI+ domain harbours the N7- and 2’O-MTase activities and identified the residues involved in the mRNA recruitment. I showed that the methylation order is not canonical with the 2’O-methylation preceding the N7-methylation. Finally, I showed that the domain harbours an additional GTP hydrolysis activity, representing the first step of RNA cap formation for Mononegavirales.This work demonstrates that this MTase domains harbour 2 or 3 of the enzymatic activities required for viral RNA cap synthesis and represent attractive targets for the development of antivirals
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Barkovskiy, Mikhail [Verfasser], Arne [Akademischer Betreuer] Skerra, Arne [Gutachter] Skerra, and Kay H. [Gutachter] Schneitz. "Protein design of hapten-specific Anticalins as therapeutic antidotes and biochemical reagents / Mikhail Barkovskiy ; Gutachter: Arne Skerra, Kay H. Schneitz ; Betreuer: Arne Skerra." München : Universitätsbibliothek der TU München, 2020. http://d-nb.info/1216626367/34.
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Carlton, Morgan. "Saliva biomarkers for physical healing and psychological wellbeing in paediatric small area thermal burns." Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/228564/8/Morgan%20Carlton%20Thesis.pdf.
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This thesis is the first study to catalogue the proteins in the saliva of children with small area thermal burns and investigate changes in saliva protein and cytokine levels in relation to different burn characteristics and clinical outcomes. It identifies proteins that may be influenced by burn injury and discusses the future studies that are required before saliva can be used as a diagnostic tool in paediatric burn assessment.