Coronavírus, incluindo-se o Coronavírus aviário (ACoV), possuem o maior genoma composto por RNA conhecido entre os vírus. Aproximadamente dois terços desse genoma codificam proteínas não estruturais (Nsps), cujas funções parecem estar associadas à replicação e patogênese viral. Até o momento, esses alvos têm sido pouco explorados quanto a sua diversidade em diferentes linhagens de ACoV. O presente estudo teve como objetivo investigar a diversidade dos genes codificadores das proteínas não estruturais Nsp2 e protease Papaína-like (Plpro), utilizando-se linhagens brasileiras de ACoV. Para tanto, 10 linhagens de ACoV, isoladas em ovos embrionados, foram submetidas à RT-PCR direcionada aos genes codificadores de Plpro e Nsp2, seguindo-se o sequenciamento de DNA e a análise filogenética, juntamente com sequências homólogas obtidas no GenBank. Além disso, realizou-se a genotipagem por meio do sequenciamento parcial do gene codificador da proteína de espícula (região S1). Três das amostras virais obtidas e investigadas no presente trabalho apresentaram padrão de segregação discordante para os genes estudados. O isolado CRG I22 agrupou-se com linhagens virais pertencentes ao genótipo Massachusetts para S1 e com o grupamento de ACoVs brasileiros os genes da Nsp2 e Plpro. O isolado CRG I33 agrupou-se com linhagens virais pertencentes ao genótipo brasileiro para s1 e plpro e de maneira divergente para o gene da Nsp2. Para o isolado CRG I38, não foi obtida a genotipagem por s1, entretanto, similarmente ao observado para o isolado CRG I33, esse isolado agrupo-se com linhagens virais brasileiras para o gene plro e de maneira independente para o gene nsp2. As demais linhagens estudadas resultaram na formação de um grupamento especificamente brasileiro de ACoV, para os três genes estudados. Esses achados sugerem a ocorrência de recombinação nessas amostras discrepantes. Quanto às identidades médias entre as sequencias nucleotídicas analisadas, a região de s1 analisada apresentou as menores identidades (73,75% ±16,78), seguido pelo gene plpro (88,06% ±5,7) e do gene nsp2 (92,28% ±4,37), em acordo com a literatura. Assim sendo, os alvos investigados podem constituir ferramentas úteis na epidemiologia molecular do ACoV e na investigação de linhagens recombinantes do vírus. O presente estudo é o primeiro a investigar a diversidade genética de genes codificadores de proteínas não-estruturais em linhagens brasileiras de ACoV. Os resultados aqui apresentados reforçam a existência de um genótipo brasileiro de ACoV, para os 3 genes estudados. Entretanto, discrepâncias pontuais encontradas no padrão genotípico para s1, nsp2 e nsp3 permitem inferir uma diversidade genética maior do que a conhecida até o momento, possivelmente resultante de eventos de recombinação entre ACoVs brasileiros, ACoVs vacinais e outros ainda desconhecidos. Os resultados obtidos auxiliam na compreensão dos padrões e evolução dos ACoVs / Coronaviruses, including Avian coronavirus (ACoV), have the largest known RNA genome. Nearly two thirds of its genome codes for non-structural proteins (Nsps), whose functions appear to be linked to viral replication and pathogenesis. Hitherto these targets have been poorly explored regarding the ACoV lineages diversity. The present study aimed to assess the diversity of non-structural protein 2 (nsp2), papain-like protease (plpro) and spike protein (S1 subunit) coding genes, in Brazilian ACoV strains. To this end, 10 ACoV strains, isolated in embryonated eggs, had its 3rd and 5th passages submitted to RT-PCR targeting nsp2, plpro and s1, followed by DNA sequencing and phylogenetic analysis, herewith homologous sequences obtained from GenBank. Three of the ACoV strains sequenced showed a discordant segregation pattern for target genes. CRG I22 strain clustered with Massachusetts genotipe strains for S1, and with Brazilian cluster for nsp3 and plpro genes. CRG I33 strain, clustered with Brazilian strains for S1 and plpro genes, and was divergent for nsp2 gene. For CRG I38 strain, the S1 sequence was not obtained, however, similarly to what was observed for CRG I33, this strain grouped with the Brazilian lineage for plpro gene and was divergent for nsp2 gene. All the other ACoV here sequenced resulted in a specific Brazilian cluster for the three studied genes. Regarding the mean nucleotide identities measured, s1 gene showed the lowest identity (73.75% ±16.78), followed by plpro gene (88.06% ±5.7) and nsp2 gene (92.28% ±4.37), in accordance with previous reported data. Therefore, the targets of the present study are useful tools for ACoV molecular epidemiology studies and for the survey of recombinant ACoV strains. The presented study is the first one investigating the molecular diversity of non-structural proteins coding genes in Brazilian strains of ACoV. Results achieved herein reinforce the data over the circulation of ACoV Brazilian strains in this country, for the three investigated genes. However, divergences found between S1, nsp2 and plpro genetic patters allow inferring a higher molecular diversity than previously known. It is possible that this divergence is due to recombination events between ACoV from vaccines, Brazilian field strains and others still unknown. These results contribute on the comprehension over genetic patters and evolution of ACoV

A beta-tubulin gene (benA) is widely used in taxonomy and identification of Aspergillus spp. and other Fungi.Across Aspergillus spp. There is either one (benA) or two beta-tubulin paralogs (benA and tubC). The risk ofcontemporary use of sequences of paralogous genes with non-homologous function in the same phylogeneticanalysis is well known. It is evident that it had happened repeatedly in Aspergillus section Nigri. It is alarmingthat conventional primers for amplification of partial benA sequence can specifically amplify tubC paralog insome species. In this work, both paralogs were characterised in a set of species. The beta-tubulin primers in usewere revised and new, more benA specific primers were designed. Applicability of some markers such as basecomposition, codon usage and length of introns for distinguishing -tubulin paralogs benA and tubC is tested. Alarge study on molecular diversity of 349 isolates of Aspergillus (PCR-fingerprint, sequence data - ITS, benA,rpb2, caM) originating from Czech culture collections and from clinical material is also included. 82 specieswere identified, togetherwith nine tentative new taxa belonging to sections with high economic impact - Nigri,Fumigati or Aspergillus (Eurotium spp.). Five species from Section Aspergillus could be synonymised withexisting taxa. A study...

<p>U&nbsp; ovom&nbsp; radu&nbsp; sagledan&nbsp; je&nbsp; molekularni&nbsp; diverzitet,&nbsp; filogeografska&nbsp; struktura,<br />prostorna&nbsp; distribucija&nbsp; molekularnog&nbsp; diverziteta,&nbsp; kao&nbsp; i&nbsp; prisustvo&nbsp; selekcionih<br />signala i genetičkih signala lokalnih adaptacija kod 251 jedinke&nbsp; vrste&nbsp; Lepus<br />europaeus (Pallas,&nbsp; 1778)&nbsp; sa&nbsp; teritorije&nbsp; Evrope&nbsp; i&nbsp; Bliskog&nbsp; Istoka,&nbsp; a&nbsp; na&nbsp; osnovu<br />analize&nbsp; varijabilnosti&nbsp; sekvenci&nbsp; D&nbsp; petlje&nbsp; mtDNK, MT-ND2,&nbsp; MT-ND6,&nbsp; MHCDQA, MHC-DQB i TLR2 gena. Uočen je visok nivo parametara molekularnog<br />diverziteta&nbsp; za&nbsp; sve&nbsp; ispitivane&nbsp; molekularne&nbsp; markere.&nbsp; Utvrđeno&nbsp; je&nbsp; postojanje<br />filogeografske&nbsp; strukturiranosti&nbsp; vrste&nbsp; na&nbsp; osnovu&nbsp; mtDNK,&nbsp; kao&nbsp; i&nbsp; asimetričan<br />protok gena jedinki sa teritorije Anadolije na teritoriju Balkana&nbsp; na osnovu D<br />petlje mtDNK, MT-ND2 i MT-ND6 gena, dok je na osnovu sekvenci D petlje<br />mtDNK uočena gotovo tri puta veća stopa protoka gena sa Balkana u centralnu<br />i&nbsp; zapadnu&nbsp; Evropu.&nbsp; Utvrđeno&nbsp; je&nbsp; prisustvo&nbsp; signala&nbsp; poizivne&nbsp; selekcije&nbsp; u&nbsp; okviru<br />MT-ND6&nbsp; gena,&nbsp; kao&nbsp; i&nbsp; efekat&nbsp; klimatskih&nbsp; parametara&nbsp; (precipitacije)&nbsp; na<br />distribuciju&nbsp; proteinskih&nbsp; varijanti&nbsp; ND6C&nbsp; i&nbsp; ND6F,&nbsp; kao&nbsp; moguća&nbsp; posledica<br />regionalnih adaptacija na razlike u sredinskim uslovima. Pokazano je odsustvo<br />signala&nbsp; filogeografske&nbsp; strukturiranosti&nbsp; na&nbsp; osnovu MHC-DQA, MHC-DQB i<br />TLR2 gena.&nbsp; Uočeno&nbsp; je&nbsp; postojanje&nbsp; prostorne&nbsp; strukturiranosti&nbsp; na&nbsp; osnovu&nbsp; gena<br />imunskog&nbsp; sistema,&nbsp; i&nbsp; definisane&nbsp; su&nbsp; dve&nbsp; prostorne&nbsp; grupe,&nbsp; jedna&nbsp; koja&nbsp; je<br />obuhvatala&nbsp; jedinke&nbsp; sa&nbsp; teritorije&nbsp; Bliskog&nbsp; Istoka,&nbsp; i&nbsp; druga&nbsp; koja&nbsp; je&nbsp; obuhvatala<br />jedinke&nbsp; sa&nbsp; teritorije&nbsp; Evropi.&nbsp; Vi&scaron;e&nbsp; vrednosti&nbsp; parametara&nbsp; molekularnog<br />diverziteta uočene su u anadolijskoj grupi, u poređenju sa evropskom grupom.<br />Uočen je signal delovanja pozitivne i negativne selekcije u MHC-DQA i MHCDQB genima, kao i signal negativne selekcije u TLR2 genu. Pokazan je efekat<br />klimatskih&nbsp; parametara&nbsp; na&nbsp; distribuciju&nbsp; najzastupljenijih&nbsp; proteinskih&nbsp; varijanti<br />MHC-DQA&nbsp; i&nbsp; MHC-DQB&nbsp; gena kao&nbsp; indirektni&nbsp; pokazatelj&nbsp; imunogenetičkih<br />adaptacija&nbsp; na&nbsp; sredinski&nbsp; uslovljene&nbsp; pretpostavljene&nbsp; razlike&nbsp; u&nbsp; distribuciji<br />patogena.&nbsp; Mehanizam&nbsp; oblikovanja&nbsp; varijabilnosti&nbsp; MHC&nbsp; gena&nbsp; rezultat&nbsp; je<br />uzajamnog delovanja mutacija, rekombinacija i selekcije.</p> / <p>In&nbsp; this&nbsp; doctoral&nbsp; dissertation,&nbsp; molecular&nbsp; diversity,&nbsp; phylogeographic&nbsp; structure,<br />spatial&nbsp; distribution&nbsp; of&nbsp; molecular&nbsp; diversity,&nbsp; detection&nbsp; of&nbsp; possible&nbsp; selection<br />signals&nbsp; shaping&nbsp; the&nbsp; evolution of&nbsp; these&nbsp; genes,&nbsp; as&nbsp; well&nbsp; as&nbsp; the&nbsp; presence&nbsp; of<br />local/regional&nbsp; adaptations&nbsp; in&nbsp; correlation&nbsp; was&nbsp; examined&nbsp; in&nbsp; 251&nbsp; brown&nbsp; hares<br />from&nbsp; Europe&nbsp; and&nbsp; the&nbsp; Middle East&nbsp; based&nbsp; on&nbsp; the&nbsp; analyses&nbsp; of&nbsp; mitochondrial&nbsp; D<br />loop,&nbsp; mitochondrially&nbsp; Encoded&nbsp; NADH&nbsp; Dehydrogenase&nbsp; 2&nbsp; (MT-ND2),<br />mitochondrially&nbsp; Encoded&nbsp; NADH&nbsp; Dehydrogenase&nbsp; 6&nbsp; (MT-ND6),&nbsp; exon&nbsp; 2&nbsp; of<br />MHC Class II genes MHC-DQA,MHC-DQB and Toll Like Receptor 2 (TLR2)<br />gene sequences. A high level of molecular diversity was found based on the all<br />applied&nbsp; markers.&nbsp; Strong&nbsp; signal&nbsp; of&nbsp; phylogeographical&nbsp; and&nbsp; spatial&nbsp; structuring<br />was&nbsp; observed&nbsp; for&nbsp; mtDNA,&nbsp; most&nbsp; likely&nbsp; as&nbsp; a&nbsp; consequence&nbsp; of&nbsp; climatic<br />perturbations&nbsp; during&nbsp; the&nbsp; Pleistocene.&nbsp; The&nbsp; evolutionary&nbsp; development&nbsp; of&nbsp; hares<br />from&nbsp; Anatolia/Israel&nbsp; to&nbsp; the&nbsp; Balkans,&nbsp; and&nbsp; furthermore&nbsp; to&nbsp; central&nbsp; and&nbsp; western<br />Europe was suggested by several lines of evidences, which include dating the<br />population&nbsp; demography&nbsp; based&nbsp; on&nbsp; D-loop&nbsp; sequences,&nbsp; the&nbsp; observed&nbsp; migration<br />patterns,&nbsp; results&nbsp; of&nbsp; demographic&nbsp; tests,&nbsp; and&nbsp; apparent&nbsp; reduction&nbsp; in&nbsp; molecular<br />diversity&nbsp; indices&nbsp; along&nbsp; this&nbsp; trajectory.&nbsp; Positive&nbsp; selection&nbsp; acting&nbsp; on MT-ND6<br />gene&nbsp; was&nbsp; detected,&nbsp; together&nbsp; with&nbsp; significant&nbsp; climatic&nbsp; effect&nbsp; shaping&nbsp; the<br />distribution&nbsp; of&nbsp; the&nbsp; most&nbsp; prevalent&nbsp; protein&nbsp; variants&nbsp; found&nbsp; in&nbsp; this&nbsp; gene,<br />supposedly&nbsp; as&nbsp; a&nbsp; consequence&nbsp; to&nbsp; local/regional&nbsp; adaptations&nbsp; due&nbsp; to&nbsp; the<br />environmentally induced different energetic requirements and optimization of<br />OXPHOS&nbsp; genes.&nbsp; On&nbsp; the&nbsp; other&nbsp; side,&nbsp; less&nbsp; evident&nbsp; phylogeographic&nbsp; signal&nbsp; and<br />absence&nbsp; of&nbsp; strong&nbsp; structuring&nbsp; was&nbsp; revealed&nbsp; in&nbsp; MHC&nbsp; genes.&nbsp; High&nbsp; diversity&nbsp; at<br />MHC genes seems to be shaped by the interplay of recombination, selection<br />mechanisms&nbsp; and&nbsp; adaptations.&nbsp; Balancing&nbsp; selection&nbsp; seems&nbsp; to&nbsp; maintain&nbsp; a&nbsp; high<br />molecular&nbsp; diversity&nbsp; within&nbsp; these&nbsp; genes,&nbsp; while&nbsp; directional selection&nbsp; promotes<br />local/regional adaptations to pathogenic landscapes, as indirectly suggested by<br />a&nbsp; significant&nbsp; effect&nbsp; of&nbsp; climatic&nbsp; parameters&nbsp; on&nbsp; the&nbsp; distribution&nbsp; of&nbsp; protein<br />variants in both examined MHC genes.</p>

The plant genus Tolpis (Asteraceae) is a predominantly insular plant lineage. It inhabits four of the five archipelagoes that comprise the Atlantic region of Macaronesia and also occurs in Mediterranean Europe and North Africa. Twelve species are currently recognized in Tolpis, of which ten are insular and two continental. The majority of the insular species inhabit the five western Canarian islands, where they constitute endemics to specific ecological habitats. A comprehensive molecular phylogeny of Tolpis is generated via DNA sequences of one nuclear ribosomal and two low-copy nuclear DNA markers. Considerable phylogenetic uncertainty among inferred tree topologies is detected, and incongruence between these topologies is resolved via statistical hypotheses testing. The extant diversity of the genus is identified to be the result of two independent colonization pathways and adaptive radiations on several islands. Moreover, potential hybridization is detected between species that inhabit different islands and archipelagoes, indicating a more widespread historical distribution of the genus. Details of the biogeographic history of Tolpis are inferred via ancestral area reconstructions under parsimony and likelihood optimality criteria. The hypothesis that Tolpis may have undergone a back-dispersal from an island to a continental habitat is also tested. Uncertainty in taxon cladograms owing to the presence of hybrid or allopolyploid taxa is characterized and a potential adjustment strategy evaluated. Averaging reconstruction results over all optimal phylogenetic trees and the manual pruning of cloned DNA sequences are found potential adjustment strategies against the impact of topological uncertainty owning to hybrid or allopolyploid taxa. Adjusted ancestral area reconstructions in Tolpis do not support the scenario that the genus has undergone a reverse colonization of the continent. In addition to the phylogenetic and biogeographic history of the genus, the diversity of symbiotic mycorrhizal fungi associated with Tolpis is characterized. A molecular survey using two nuclear ribosomal DNA markers and 454 pyrosequencing is performed. Particular emphasis is placed on the quality filtering of resulting fungal DNA sequences, the generation of operational taxonomic units, and their taxonomic assignment via similarity searches against DNA sequence databases. Numerous potentially novel fungal genotypes are identified. / text

Les microorganismes occupent une place centrale dans la diversité du vivant et les processus écosystémiques, notamment dans le sol où ils sont en interaction avec les plantes. Cette thèse vise à caractériser l'influence respective des plantes et du contexte abiotique dans la distribution spatiale des microorganismes. Le travail s'appuie sur un modèle simplifié de la biologie des sols, les plantes en coussins des falaises de haute montagne. Nous avons suivi une seule espèce aux morphotypes variés, Silene acaulis, une espèce ingénieure de l'écosystème dont la croissance mène à la création d'un sol de novo. L'échantillonnage comprend le prélèvement de sol de plante et de sol extérieur comme témoin, pour des coussins distribués le long de gradients altitudinaux et géologiques. Des méthodes moléculaires ont été utilisées pour décrire la diversité microbienne et le génotypage des coussins.Les coussins structurent la beta diversité bactérienne et fongique à l'échelle régionale en agissant comme un tampon à échelle locale sur les effets de la roche mère et de l'altitude en homogénéisant le pH et par un apport de nutriments. Cet effet ingénieur est d'autant plus fort que la contrainte abiotique augmente et varie selon le génotype des coussins. La beta diversité bactérienne diffère de la beta diversité fongique. Alors que les communautés bactériennes sont sensibles au pH du sol et convergent sous les coussins, les communautés fongiques sont corrélées à la génétique des coussins, particulièrement les clades aux modes de vie biotrophes/pathogènes. Ce travail montre que les plantes sont un filtre biotique majeur de la biogéographie microbienne. / Microorganisms are key component of Hearth biodiversity and ecosystem processes, especially in soils where they interact with plants. The objectives of the PhD was to caracterize the plant and abiotic respective influence on microbial spatial distribution. The work was based on a simplified soil biology model, the alpine cushion plants. We choose one species composed of variable morphotype, Silene acaulis,can ecosystem engineer species that creates de novo soil through growth. Sampling design includes soil within cushions and outside, spanning altitudinal and geological gradients. Molecular approachs were used to describe diversity and to genotype cushions.Cushions structures bacterial and fungal regional beta diversity through a local buffering of the influence of abiotic context, homogeneizing soil pH and by nutrient supply. This engineering effect increased in stressful conditions and varied according to plant genotype. Betadiversity differed between bacteria and fungi. Bacterial communities are mainly influenced by pH and converge within cushions while fungal communities correlate to cushion genetic, especially plant-associated biotrophs fungal clades. This work shows that plants act as a major biotic filter on microbial biogeography.

Chez la tomate, l'amélioration pour la qualité du fruit est rendue difficile par la multiplicité et la complexité des caractères. La cartographie de QTL a permis la caractérisation génétique de ces caractères. L'objectif est maintenant d'identifier les gènes sous-jacents aux QTL. Nous avons utilisé la cartographie par déséquilibre de liaison (DL) dans ce but. Pour éviter les fausses associations entre caractères et polymorphismes moléculaires, la structure génétique a été prise en compte dans l'analyse. La tomate cultivée montre un faible niveau de diversité génétique, ce qui réduit la résolution de cartographie. Le génome de la tomate de type cerise est décrit comme une mosaïque entre celui de la tomate cultivée et de l'ancêtre sauvage. Ce mélange devrait augmenter la résolution des études d'association. Nous avons utilisé une « core collection » focalisée sur des accessions de type cerise pour valider la région génomique contenant un QTL pour le nombre de loges. Deux mutations sont associées avec le caractère. Ces deux SNP ont évolué différemment du reste du chromosome 2, en subissant une sélection balancée qui témoigne de l'augmentation de la diversité morphologique lors de la domestication. L'étude, focalisée sur le chromosome 2, a permis d'analyser l'étendue du DL en fonction de la distance génétique et physique. Des associations, entre polymorphismes et phénotypes étudiés, ont été détectés avec des méthodes prenant en compte la structure génétique. Nous avons montré l'intérêt d'utiliser la structure en mosaïque du génome des accessions de type cerise pour surmonter les limitations de résolution dans les analyses d'associations chez une espèce cultivée autogame. / In Tomato (Solanum lycopersicum), breeding for fruit quality is difficult due to the multiplicity and complexity of the traits. QTL mapping has allowed the genetic characterization of these traits. One of the challenges is now to identify the genes underlying these QTLs. Following this aim, we used linkage-disequilibrium (LD) mapping. To avoid hazardous associations between traits and polymorphisms, the genetic structure has to be taken into account for LD mapping. Cultivated tomato showed low genetic diversity reducing mapping resolution. Cherry type tomato genome is described to be admixture between cultivated tomato and its wild ancestor. Such admixture may increase resolution of association mapping. We used a core collection focused on cherry type accessions to validate a candidate gene for a fruit locule-number QTL. We found that two single nucleotide polymorphisms (SNP) were highly associated with the trait. These two SNP evolved differently from the rest of the chromosome 2. They underwent a balanced selection which testifies a selection for fruit morphology diversity by human. Association mapping, focused on whole chromosome 2, allowed us to assess the extent of linkage disequilibrium over genetic and physical distances. Associations of polymorphisms with phenotypes were detected with structured association methods. We thus showed efficiency of genome admixture to overcome the low-resolution limitation of association mapping for an inbred crop. We validated previously identified QTLs and found associations with new QTLs and new candidate genes. An evolutionary model including bottleneck and gene flow between wild and domesticated forms is also presented.

Les anticorps catalytiques (ou abzyme) ont fait l’objet de nombreuses recherches et ont été produits pour réaliser de nombreuses réactions. Ces protéines ont été ensuite découvertes dans le sérum d’individus sains ou atteints de pathologies, dont les pathologies autoimmunes. Les études suggèrent que ces abzymes peuvent avoir des effets bénéfiques ou délétères sur la santé des individus. L’origine des anticorps catalytiques et leur rôle restent ambigus et doivent être approfondis. Nous avons développé une nouvelle stratégie visant à étudier les abzymes, basée sur la technologie du phage display. Nous avons construit 4 banques de fragments d’anticorps, chacune présentant un répertoire immun différent (fond génétique et état d’immunitaire) : saine et naïve, saine et immunisée, autoimmune et naïve, et autoimmune et immunisée. Les stratégies d’amplification et de clonage des régions variables des immunoglobulines ont été conçues afin d’optimiser la taille et la diversité des banques. Nous avons rassemblé les 4 banques en une banque unique élargie contenant 2.7×109 séquences. L’analyse des séquences a mis en évidence des différences dans les profils d’expression des sous-groupes de gènes selon la banque. Nous avons ensuite procédé à la sélection d’abzymes à activité β-lactamase en utilisant deux cibles : un peptide cyclique, et un dérivé de sulfone pénam, inhibiteurs de l’enzyme. Nous avons sélectionné 5 abzymes. Chacun de ces immunoglobulines ont des séquences protéiques propres, incluant un potentiel site actif. Ces résultats montrent que différents motifs peuvent assurer la fonction catalytique de la β-lactamase, confirmant la flexibilité moléculaire de cette enzyme. / Catalytic antibodies (or abzymes) have been the focus numerous studies for some decades and have been produced with the ability to catalyze a wide range of reactions. They have also been discovered naturally in normal physiological and pathological conditions, notably on the background of autoimmune disease. Some have beneficial effects and others are detrimental to individual’s health. Hence, the origin of abzymes and their role in the immune response are ambiguous and must be enhanced. We have developed a novel strategy for the study of abzymes based on the phage display technology. We have constructed 4 libraries representing 4 murine immune repertoires with different genetic backgrounds and immunological states : healthy and naïve, healthy and immunized, autoimmune and naïve , and autoimmune and immunized. The strategies for the amplification and cloning of the immunoglobulin (lg) variable regions have been designed to optimize the size and diversity of the libraries. We have been able to pool the four libraries to create a large repertoire of size 2.7x109. After sequence analysis, we have found a number of statistically significant differences between the libraries. We have then used two strategically chosen targets to select for antibodies endowed with β lactamase activity : a cycle peptide and a penam sulfone, both inhibitors of the enzyme. We have selected for a total of 5 lgs endowed with β lactamase activity. The selected abzymes have different amino acid sequences. 3D modeling has provides insights on potential active sites demonstrating the ability of different structures to maintain the β lactamase activity and confirming the flexibility of the active site.

El objetivo de la presente tesis es el de aportar conocimiento sobre la bioquímica y la farmacología de los receptores de adenosina, así como entender las relaciones entre estructura química y actividad farmacológica de los ligandos existentes para estos receptores. Con este objetivo se han empleado distintas técnicas y metodologías del diseño de fármacos asistido por ordenador. Los resultados presentados en este trabajo incluyen:· El desarrollo de una estrategia original para la selección de una muestra que cubra adecuadamente la diversidad molecular existente en una base de datos de compuestos químicos· La construcción de un modelo de la región transmembrana del receptor A1 humano de adenosina, en el que se ha localizado y caracterizado un sitio de unión de agonistas compatible con los datos experimentales.· Predicciones teóricas de las energías de unión de ligandos, realizadas a partir de los complejos agonista-receptor predichos sobre el modelo mencionado, obteniendo un grado de acuerdo con los datos experimentales que resulta esperanzador / The goal of the present thesis is to gain knowledge about the biochemistry and pharmacology of adenosine receptors, as well as to understand structure-activity relationships for the existing ligands for this receptors. In order to achieve this goal, we have used several techniques and methodologies from the computer-aided drug design field. Results presented in this work include:· The development of an original strategy of selection of a maximum diversity sample that adequately covers the original molecular diversity contained in a compound database· The building of the transmembrane region of a human A1 adenosine receptor model. In such a model, an agonists binding site has been located and characterized, showing agreement with experimental data.· The resulting ligand-receptor complexes have been studied with computational approaches for the prediction of ligand-binding free energies. A nice correlation with experimental results was observed

No description available.

La nécessité grandissante de disposer d’une large librairie de diverses petites molécules pour le screening biologique constitue une puissante force motrice pour les chimistes organiciens et requiert en amont le développement de méthodologies rapides et efficaces. Dans ce cadre, nous nous sommes plus particulièrement intéressés à la fonctionnalisation d’énamides qui représentent des blocs moléculaires intéressants permettant d’introduire des fonctionnalités aminées dans des systèmes variés. Notre objectif a été de synthétiser des petites bibliothèques de molécules azotées à partir de substrats communs tout en mettant en oeuvre les différentes stratégies de la synthèse orientée vers la diversité et en s’attachant à respecter les règles suivantes : économie d’atomes, processus catalysés, synthèses rapides en peu d’étapes et contrôle de la stéréoselectivité. Dans un premier temps, nous avons principalement synthétisé divers énamides, nous permettant par la suite de développer des méthodologies innovantes et d’accéder à des « structures privilégiées » ou des fragments clés présents dans des produits naturels ou dans des substances potentiellement biologiquement actives en mettant en jeu des processus variés telles que des réactions d’aza-Michael, d’oxyamidation ou en cascade et la chimie du palladium avec de la CH insertion, des dioxoazoborocanes ou encore l’utilisation de l’auxiliaire chiral SAMP. / The continuing demand to synthesize new and original collections of small molecules for the biological screening is an attractive subject for organic chemists and requires upstream the development of fast and easy synthetic methods. In this context, we decided to focus particularly on the functionalization of enamides which represent valuable building blocks in order to introduce nitrogen based functionality into various organic systems. Our objective was to synthesize new nitrogen containing compound libraries starting from common substrates by applying Diversity-Oriented Synthesis strategy and following these rules: atom economy, catalyzed reactions, fast synthesis in few steps and control of stereoselectivity. Firstly we mainly synthesized enamides. Thereafter, we developped efficient methodologies giving access to motifs frequently found in “privileged structures” or key scaffolds present in natural products or potential bioactive compounds thanks to various processes like aza-Michael, oxyamidation or cascade reactions, palladium chemistry with CH activation, dioxoazoborocanes or chiral auxiliary SAMP.