Cette thèse est dédiée à la conception de machines moléculaires pH-sensibles inédites de type muscles et nœuds moléculaires. Le premier muscle moléculaire pH-sensible a été synthétisé de manière très directe, et publié en 2008, en utilisant une stratégie en deux étapes: 1) fermeture des axes encapsulés par cycloaddition 1,3-dipolaire de type Huisgen catalysée par le cuivre (I), 2) méthylation des triazoles formés en triazoliums, capables d'être reconnus par les macrocycles DB24C8. Deux états étirés ou contractés, déclenchés par simple variation de pH, permettent le contrôle de l'orientation et de la distance entre les deux « stoppeurs » glucidiques non reliés de manière covalente. Des stations pyridiniums amides mono ou disubstitués inédites ont également été utilisées pour la synthèse de muscles moléculaires de plus large amplitude à effets co-conformationnels induits. Lors de la contraction du muscle par carbamoylation des ammoniums, la différence de localisation des macrocycles autour des stations pyridiniums amides, dépendante de la substitution des amides, engendre deux effets très différents : rôle de frein moléculaire de la DB24C8 ou basculement conformationnel impressionnant des chaises des mannopyranoses. Une étude méthodologique a été menée afin de classer les stations moléculaires rencontrées dans ce manuscrit, selon leurs affinités respectives pour la DB24C8, et a conduit à la conception d'un muscle moléculaire oscillant dont l'état varie continuellement entre contracté et semi-contracté en fonction de la température et de la nature du solvant. Enfin, différentes stratégies de synthèse ont été explorées pour obtenir des nœuds moléculaires inédits en forme de double lasso par cyclisation de synthons dimères de rotaxanes. Un double lasso dont la vitesse de rotation et la taille de la cavité peuvent être modulées en fonction du pH a ainsi été obtenu. / This thesis is devoted to the synthesis of pH-sensitive molecular muscles and knots. The first molecular muscle has been readily synthesized and published in 2008, using a two-step strategy: 1) end-capping of the interlocked axles by copper(I)-catalyzed Huisgen alkyne-azide 1,3-dipolar cycloaddition, 2) methylation of triazoles to triazoliums, which are able to interact with the macrocycle DB24C8. Two stretched and contracted states, triggered by variation of pH, allow the control of the distance and of the orientation of the two glucidic ends, which are not covalently linked. Novel mono- and disubstituted pyridinium amide stations have been used for the synthesis of large-amplitude molecular muscles, whose translation of the macrocycles trigger a second co-conformational induced effect. In fact, upon contraction of the molecular muscle, using carbamoylation of the ammoniums, the slight different localizations of the macrocycles around the pyridinium amides (depending on their mono- or disubstitution) trigger two very different effects. The first one is a molecular break played by the DB24C8, whereas the second one is a flipping of the chair-like conformation of the mannopyranosyl ends. A methodologic study was then carried out with the aim to determine the relative affinity of the new described molecular stations for the DB24C8, and led to the synthesis of a molecular muscle which oscillates from the contracted to the semi-contracted co-conformation, depending on solvent and temperature. Eventually, different routes to very new double-lasso molecular knots were investigated from a molecular muscle building-block. One molecular knotted machine has been obtained, and has a double-lasso structure, whose rotation and size of its cavity can both been modulated by variation of pH.

We present the bioconjugation of avidin as a central and/or bridging building block with mono-, bi- and tetravalent biotinylated glycodendrimers to fabricate defined supramolecular nanostructures for future (bio)medical applications. For this purpose mono-, bi- and tetravalent biotinylated glycodendrimers, decorated with short alkyl-linked or long PEG-linked biotin ligands, were synthesized and characterized by NMR, IR and mass spectrometry and HABA displacement assay. Various techniques (UV/Vis, DLS, TEM, LILBID-MS and AF4) were used in order to obtain information about the structural properties of different conjugates of avidin and mono-, bi- and tetravalent biotinylated glycodendrimers. The biotin ligand’s spacer length, its chemical structure and the degree of biotin functionalization are essential parameters in the formation of nanostructures with avidin having a controlled composition and size dimension up to 100 nm. Biohybrid structures with avidin as a central unit require monovalent glycodendrimers with PEG-linked biotin, while bi- and tetravalent glycodendrimers with short alkyl-linked biotin ligands are more efficient than their counterparts with longer PEG–biotin ligands in the fabrication of defined biohybrid structures (∅ up to 100 nm) with avidin as a bridging unit. The most dominating key issue, combined with other conjugation issues, is the optimal ligand–receptor stoichiometry to fabricate biohybrid structures with diameter of <20, <30 or up to 100 nm.

Suite au projet de séquençage du génome d’Arabidopsis thaliana, plus de 400 récepteurs de types serine/thréonine kinases (Protein Receptor Kinase ou PRK) ont été prédits. Par contre, seulement sept paires de récepteurs/ligands ont été caractérisées jusqu’à présent par des techniques de biochimie et d’analyse, de mutants. Parmi ceux-ci figurent les PRK : BRI1, CLV1, SRK, SR160, Haesa-IDA et PEPR1 qui jouent un rôle important dans le développement, l’auto-incompatibilité sporophytique et les mécanismes de défense. Le but de mon projet de maîtrise était de développer un bioessai à haut débit qui permettra la découverte de ligands peptidiques. Le bioessai utilisera des PRK chimériques composés du domaine extracellulaire (l’ectodomaine) de la PRK à l’étude fusionnée au domaine intracellulaire d’une PRK qui agira comme rapporteur. Deux stratégies sont présentement développées dans notre laboratoire : la première consiste à fusionner la PRK à l’étude avec le domaine intracellulaire (l’endodomaine) du récepteur tyrosine kinase animal EGFR (Epidermal Growth Factor Receptor). Suite à l’interaction avec une fraction protéique contenant un ligand correspondant à la PRK étudiée, une transphosphorylation de l’endodomaine (le domaine kinase) serait détectable. La seconde stratégie utilise l’endodomaine du récepteur BRI1, un récepteur répondant aux brassinostéroïdes. Suite à l’interaction avec une fraction protéique contenant un ligand correspondant à la PRK étudiée, cette fois-ci nous devrions être en mesure de mesurer l’activation d’un gène rapporteur répondant normalement à une activation par les brassinostéroïdes. / The complete sequence of the genome of Arabidopsis thaliana was achieved in year 2000 and has resulted in the prediction of more than 400 receptor serine/threonine kinase or Plant Receptor Kinase (PRK). Despite this tremendous work, only seven pairs of ligand/receptor have been characterized through conventional techniques such as mutant analysis and biochemical characterization. These receptors have been found to play an important role in plant defense (SP160), development (BRI1, CLV1) and sporophytic autoincompatibility (SRK). The aim of the project was to develop a high throughput bioassay in order to find new ligands for known receptors. In order to do so, the bioassay will use chimeric protein technology, by fusing the ectodomain of a receptor to a known endodomaine. The latter will play the role of a reporter. Two strategies were developed in our laboratory and are being tested. The first strategy is to fuse the ectodomain of an unknown PRK to the phylogeneticaly unrelated kinase domain of the animal Epidermal Grown Factor Receptor (EGFR). When tested with a crude protein extract containing the specific ligand of the unknown PRK, a transphosphorylation should occur and be detected. The second strategy will use the endodomain of BRI1 as a reporter, a receptor responding to the brassinosteroid phytohormone, which will relay the message to a second construct used as a reporter gene once the ligand has bound the PRK ectodomain fused to the BRI1 endodomain.

Suite au projet de séquençage du génome d’Arabidopsis thaliana, plus de 400 récepteurs de types serine/thréonine kinases (Protein Receptor Kinase ou PRK) ont été prédits. Par contre, seulement sept paires de récepteurs/ligands ont été caractérisées jusqu’à présent par des techniques de biochimie et d’analyse, de mutants. Parmi ceux-ci figurent les PRK : BRI1, CLV1, SRK, SR160, Haesa-IDA et PEPR1 qui jouent un rôle important dans le développement, l’auto-incompatibilité sporophytique et les mécanismes de défense. Le but de mon projet de maîtrise était de développer un bioessai à haut débit qui permettra la découverte de ligands peptidiques. Le bioessai utilisera des PRK chimériques composés du domaine extracellulaire (l’ectodomaine) de la PRK à l’étude fusionnée au domaine intracellulaire d’une PRK qui agira comme rapporteur. Deux stratégies sont présentement développées dans notre laboratoire : la première consiste à fusionner la PRK à l’étude avec le domaine intracellulaire (l’endodomaine) du récepteur tyrosine kinase animal EGFR (Epidermal Growth Factor Receptor). Suite à l’interaction avec une fraction protéique contenant un ligand correspondant à la PRK étudiée, une transphosphorylation de l’endodomaine (le domaine kinase) serait détectable. La seconde stratégie utilise l’endodomaine du récepteur BRI1, un récepteur répondant aux brassinostéroïdes. Suite à l’interaction avec une fraction protéique contenant un ligand correspondant à la PRK étudiée, cette fois-ci nous devrions être en mesure de mesurer l’activation d’un gène rapporteur répondant normalement à une activation par les brassinostéroïdes. / The complete sequence of the genome of Arabidopsis thaliana was achieved in year 2000 and has resulted in the prediction of more than 400 receptor serine/threonine kinase or Plant Receptor Kinase (PRK). Despite this tremendous work, only seven pairs of ligand/receptor have been characterized through conventional techniques such as mutant analysis and biochemical characterization. These receptors have been found to play an important role in plant defense (SP160), development (BRI1, CLV1) and sporophytic autoincompatibility (SRK). The aim of the project was to develop a high throughput bioassay in order to find new ligands for known receptors. In order to do so, the bioassay will use chimeric protein technology, by fusing the ectodomain of a receptor to a known endodomaine. The latter will play the role of a reporter. Two strategies were developed in our laboratory and are being tested. The first strategy is to fuse the ectodomain of an unknown PRK to the phylogeneticaly unrelated kinase domain of the animal Epidermal Grown Factor Receptor (EGFR). When tested with a crude protein extract containing the specific ligand of the unknown PRK, a transphosphorylation should occur and be detected. The second strategy will use the endodomain of BRI1 as a reporter, a receptor responding to the brassinosteroid phytohormone, which will relay the message to a second construct used as a reporter gene once the ligand has bound the PRK ectodomain fused to the BRI1 endodomain.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-04-13T12:21:40Z No. of bitstreams: 1 viniciusschmitzpereiranunes.pdf: 31716642 bytes, checksum: 9cfc92a2078f124a8a9e3fa1c6509c7e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-04-24T03:28:04Z (GMT) No. of bitstreams: 1 viniciusschmitzpereiranunes.pdf: 31716642 bytes, checksum: 9cfc92a2078f124a8a9e3fa1c6509c7e (MD5) / Made available in DSpace on 2016-04-24T03:28:04Z (GMT). No. of bitstreams: 1 viniciusschmitzpereiranunes.pdf: 31716642 bytes, checksum: 9cfc92a2078f124a8a9e3fa1c6509c7e (MD5) Previous issue date: 2015-12-07 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Esquistossomose é uma doença negligenciada causada por parasitas do gênero Schistosoma. Segundo a Oganização Mundial da Saúde, mais de 200 milhões de pessoas no mundo estão infectadas, sendo de 4 a 6 milhões de pessoas somente no Brasil. A principal forma de tratamento da doença é o uso do medicamento praziquantel, porém, há relatos na literatura de resistência do parasita ao medicamento. Tal situação levantou a necessidade pela busca de novos alvos moleculares e novos medicamentos para o tratamento da doença. Um grupo de proteínas apresentadas como potenciais alvos moleculares de esquistomicidas é o das NTPDases. Estas enzimas hidrolisam nucleotídeos di e trifosfatados (e.g. ADP e ATP) em presença de cátions bivalentes, e estão descritas em diferentes organismos. No parasita Schistosoma mansoni foi descrita uma isoforma de NTPDase ancorada na membrana plasmática das células do tegumento do verme adulto e conhecida como SmATPDase1 ou Sm1. Segundo a literatura, esta isoforma é a segunda proteína mais expressa no tegumento dos vermes adultos. Devido à localização e a importância dos nucleotídeos di e trifosfatos na ativação hemostática e de células do sistema imunológico, foi sugerido que a Sm1 estaria envolvida na regulação das concentrações de nucleotídeos em torno do parasita, o que contribuiria com sua evasão. Baseado nos pressupostos acima, tem sido proposto o uso da Sm1 como possível alvo molecular de novos esquistomicidas. Nos mamíferos foram descritas oito isoformas de NTPDases. A isoforma 1 de humanos (HsNTPDase1), mais conhecida como CD39, está presente nas células do endotélio dos vasos sanguíneos, sendo responsável pela regulação das concentrações extracelulares de ATP e ADP. No presente trabalho, apresentamos os modelos 3D da Sm1 e CD39, construídos por meio da técnica de modelagem por homologia. Devido a semelhança da estrutura 3D de ambos os modelos, foi necessário realizar uma análise estrutural comparativa entre as duas enzimas, por meio de simulações de dinâmica molecular e estudos de docking receptor-ligante. Predição de cavidades foram feitas no modelo da Sm1 a fim de detectar cavidades diferentes do sítio-ativo que pudessem ser usadas em estudos de docking. Dessa etapa resultou a seleção de duas cavidades, o sítio-ativo e um sítio alternativo. Em seguida foram realizadas simulações de dinâmica molecular envolvendo os modelos da Sm1 e CD39, e os moldes PDB3ZX3 e PDB3CJA. Para cada modelo foram feitas duas simulações, uma com a presença do ANP (análogo do ATP) no sítio-ativo e uma na ausência do ANP. Em todas as quatro simulações os modelos estão acoplados a uma membrana do tipo POPC, e o tempo de simulação foi de 32ns. Para os moldes o tempo de simulação foi 40ns. As simulações mostraram que a região do sítio alternativo apresentou, no modelo da Sm1 sem o ligante, mudanças na conformação que não foram observadas na Sm1 com ligante e nas duas simulações envolvendo o modelo da CD39. Isso sugere que o ligante contribui na estabilização da estrutura da Sm1 e CD39. Também foram feitas análises de drogabilidade e de agrupamento das conformações geradas ao longo das simulações envolvendo os modelos. Para as análises de agrupamento foi proposto o uso da metodologia GLCM, juntamente com o algoritmo K-means. Essas análises tinham como objetivo selecionar conformações das trajetórias que pudessem ser usadas nos estudos de docking. Para os estudos de docking foram analisadas a afinidade de três ligantes (ANP, ARL67153 e praziquantel) no sítio ativo e no sítio alternativo, de ambas as enzimas. Para o sítio ativo, os melhores resultados foram obtidos com os ligantes ANP e ARL67153, em todas as simulações. Foi observado que o ARL67153 apresentou resultados similares com os do ANP. No sítio alternativo os melhores resultados foram obtidos com as conformações da simulação da Sm1 sem o ANP no sítio ativo, sendo que na CD39 nenhum dos ligantes foram atracados dentro do sítio. Com relação ao praziquantel, os melhores resultados foram observados no sítio ativo da Sm1 e CD39. É possível concluir que o sítio alternativo da Sm1 é uma região que pode ser usada para o atracamento de possíveis inibidores dessa enzima. / Schistosomiasis is a neglected disease caused by parasites of the genus Schistosoma. According to the World Health Organization, over 200 million people worldwide were infected, and 4 to 6 million people only in Brazil. The main treatment is the use of the drug praziquantel, but there are reports in the literature suggesting the parasite’s resistance to praziquantel. This situation raised the need for search new molecular targets and new drugs for treating this disease. A group of proteins as potential targets is NTPDase. These enzymes hydrolyse nucleotides (e.g., ADP and ATP) in the presence of bivalent cations, and they are described in different organisms. In the parasite Schistosoma mansoni, an isoform of NTPDase„ named SmATPDase1 or Sm1, was described as a protein anchored in the plasma membrane of cells in the seed coat of the adult worm. According to the literature, this isoform is the most expressed enzyme in the tegument of adult worms. Due to the location and importance of nucleotide triphosphates in the hemostatic and activation of immune cells, Sm1 has been suggested to be involved in the regulation of nucleotide concentrations around the parasite, which could be a mechanism of immune evasion in schistosomiasis. Based on the above assumptions, it has been proposed the use of Sm1 as a possible target for new schistosomicide drugs. In mammals have been described eight isoforms. Isoform 1 of human NTPDase (HsNTPDase1), also known as CD39, is present in the endothelial cells of blood vessels, and it is responsible for the regulation of extracellular concentrations of ATP and ADP. In this paper, we present 3D models of Sm1 and CD39, constructed by homology modeling technique. Due to the similarity of 3D structures of both models, it was necessary to perform a comparative structural analysis of both enzymes by molecular dynamics simulations and and receptorligand docking. Cavities prediction were made in Sm1’s model and two cavities were selected, the active site and an alternative binding site proposed in this work. After, it was performed molecular dynamics simulations involving models of Sm1 and CD39, and PDB3ZX3 and PDB3CJA templates. Two simulations were performed for each model, one in the presence of ANP (ATP analog) in the active-site and other in the absence. In models’ simulations, structures were coupled to a POPC membrane, and the simulation time was 32ns. Simulation time for templates was 40ns. Simulations showed that the alternative site in Sm1 without ANP presented conformational changes which were not observed in simulations of CD39 and Sm1 with ANP. This suggests that the presence of ligand in the active site contributes in stabilizing the structure of Sm1 and CD39. Druggability and clustering analysis were performed with conformations generated through simulations. In this work, we proposed the use of GLCM methodology with Kmeans algorithm for clustering analysis, aiming to select conformations from trajectories that could be used in receptor-ligand docking. We analyzed the affinity of three ligands (ANP, ARL67153 and praziquantel) in the active site and in the alternative site of Sm1 and CD39 enzymes. The best results for active site were obtained with ANP and ARL67153 ligands, in all simulations. For alternative site, the best results were obtained with conformations from the simulation of Sm1 without ANP in the active site. For CD39’s simulations, none of the ligands were docked within the cavity of alternative site. With regard to praziquantel, the best results were observed in the active site of Sm1 and CD39. It is possible to conclude that the alternative site of Sm1 is a region which can be used for docking of possible inhibitors of this enzyme.

No description available.

La dynamique structurelle des biomolécules est intimement liée à leur fonction, mais très coûteuse à étudier expériementalement. Pour cette raison, de nombreuses méthodologies computationnelles ont été développées afin de simuler la dynamique structurelle biomoléculaire. Toutefois, lorsque l'on s'intéresse à la modélisation des effects de milliers de mutations, les méthodes de simulations classiques comme la dynamique moléculaire, que ce soit à l'échelle atomique ou gros-grain, sont trop coûteuses pour la majorité des applications. D'autre part, les méthodes d'analyse de modes normaux de modèles de réseaux élastiques gros-grain (ENM pour "elastic network model") sont très rapides et procurent des solutions analytiques comprenant toutes les échelles de temps. Par contre, la majorité des ENMs considèrent seulement la géométrie du squelette biomoléculaire, ce qui en fait de mauvais choix pour étudier les effets de mutations qui ne changeraient pas cette géométrie. Le "Elastic Network Contact Model" (ENCoM) est le premier ENM sensible à la séquence de la biomolécule à l'étude, ce qui rend possible son utilisation pour l'exploration efficace d'espaces conformationnels complets de variants de séquence. La présente thèse introduit le pipeline computationel ENCoM-DynaSig-ML, qui réduit les espaces conformationnels prédits par ENCoM à des Signatures Dynamiques qui sont ensuite utilisées pour entraîner des modèles d'apprentissage machine simples. ENCoM-DynaSig-ML est capable de prédire la fonction de variants de séquence avec une précision significative, est complémentaire à toutes les méthodes existantes, et peut générer de nouvelles hypothèses à propos des éléments importants de dynamique structurelle pour une fonction moléculaire donnée. Nous présentons trois exemples d'étude de relations séquence-dynamique-fonction: la maturation des microARN, le potentiel d'activation de ligands du récepteur mu-opioïde et l'efficacité enzymatique de l'enzyme VIM-2 lactamase. Cette application novatrice de l'analyse des modes normaux est rapide, demandant seulement quelques secondes de temps de calcul par variant de séquence, et est généralisable à toute biomolécule pour laquelle des données expérimentale de mutagénèse sont disponibles. / The dynamics of biomolecules are intimately tied to their functions but experimentally elusive, making their computational study attractive. When modelling the effects of thousands of mutations, time-stepping methods such as classical or enhanced sampling molecular dynamics are too costly for most applications. On the other hand, normal mode analysis of coarse-grained elastic network models (ENMs) provides fast analytical dynamics spanning all timescales. However, the vast majority of ENMs consider backbone geometry alone, making them a poor choice to study point mutations which do not affect the equilibrium structure. The Elastic Network Contact Model (ENCoM) is the first sequence-sensitive ENM, enabling its use for the efficient exploration of full conformational spaces from sequence variants. The present work introduces the ENCoM-DynaSig-ML computational pipeline, in which the ENCoM conformational spaces are reduced to Dynamical Signatures and coupled to simple machine learning algorithms. ENCoM-DynaSig-ML predicts the function of sequence variants with significant accuracy, is complementary to all existing methods, and can generate new hypotheses about which dynamical features are important for the studied biomolecule's function. Examples given are the maturation efficiency of microRNA variants, the activation potential of mu-opioid receptor ligands and the effect of point mutations on VIM-2 lactamase's enzymatic efficiency. This novel application of normal mode analysis is very fast, taking a few seconds CPU time per variant, and is generalizable to any biomolecule on which experimental mutagenesis data exist.