Die vorliegende Arbeit untersucht mit bioinformatischen Methoden die biologische Bedeutung von Ähnlichkeiten in Kleinstrukturen und peptidischen Sequenzmotiven sowie lokaler und globaler Sequenzähnlichkeit. Der erste Teil der Arbeit behandelt chemische Ähnlichkeiten. Ausgehend von bekannten Inhibitoren der Fehlfaltung des Prionproteins wurde eine Datenbank pharmakologischer Wirkstoffe nach chemisch und strukturell ähnlichen Substanzen durchsucht und 16 Substanzen als neue potentielle Inhibitoren der Fehlfaltung vorgeschlagen. Der nächste Teil untersucht Ähnlichkeiten in Sequenzmotiven, die eine Interaktion mit Pex19, dem Importrezeptor für peroxisomale Membranproteine, vermitteln. In Zusammenarbeit mit einer experimentellen Arbeitsgruppe konnte die Bindestelle charakterisiert und Präferenzen für bestimmte Aminosäuren herausgearbeitet werden. Das Bindemotiv ist eine vermutlich helikale Region mit verzweigtkettigen aliphatischen und basischen Aminosäuren. Aus experimentellen Daten konnte eine positionsabhängige Vorhersagematrix erstellt und validiert werden. Die Beziehung zwischen lokalen Sequenzähnlichkeiten und der Konformation von Prolylbindungen in Proteinen ist Thema des dritten Teils. Die Aminosäurepräferenzen in der Nachbarschaft von cis- und trans-Prolylresten unterscheiden sich, und beide zeigen unterschiedliche Austauschpräferenzen bei Mutationen. Im Gegensatz zu lokaler Sequenzähnlichkeit ist eine globale Sequenzähnlichkeit von nur 20% ein wesentlich besserer Indikator für das Auftreten von cis-Prolylbindungen. Der letzte Teil befaßt sich mit inverser Sequenzähnlichkeit zwischen Proteinen, die wesentlich öfter auftritt als erwartet. Proteine aus einem nichtredundanten Datensatz wurden gleich- und gegenläufig aligniert und strukturelle Ähnlichkeiten zwischen den aufgefundenen Proteinpaaren untersucht. Es konnte gezeigt werden, daß bis auf kurze Sekundärstruktur-Einheiten eine inverse Sequenzähnlichkeit zwischen Proteinen keine strukturelle Ähnlichkeit impliziert. / This work is dealing with the biological impact of similarities between chemical structures, protein sequence motifs and local sequence surrounding as well as global sequence similarity. All four aspects are analyzed by computational methods. The first part is dealing with chemical similarities. Based on a recently published set of prion protein misfolding inhibitors, a data base of approved drugs has been screened for compounds with chemical and structural similarities to these substances. 16 drugs are proposed as new potential inhibitors of prion protein aggregation. The next part addresses similarities of sequence motifs which mediate the interaction with the peroxisomal membrane protein import receptor Pex19. In cooperation with an experimental group, the binding site could be characterized, and amino acid preferences of the different positions of the motif have been determined. The binding motif is a probably helical region of target proteins bearing branched aliphatic and basic residues. A position specific scoring matrix for the prediction of Pex19 binding sites could be generated and validated. The relation between local sequence similarity and prolyl bond conformation is examined in the third part. Amino acid preferences of neighboring residues differ between cis and trans prolyl residues, and both species show different amino acid exchange patterns upon mutation. In contrast to local sequence similarity, overall sequence similarity between proteins as low as 20% is a much better indicator for the occurrence of cis prolyl bonds. The last part focuses on inverse sequence similarity between proteins which occurs far more often than expected by chance. Proteins from a nonredundant data set have been aligned in parallel and antiparallel, and structural similarities between the detected protein pairs have been examined. It could be shown that, with the exception of short secondary structural elements, inverse sequence similarity does not imply structural similarity.

Triquentrinas A são produtos naturais marinhos com atividade biológica e alta complexidade estrutural. Estes fatores tornam estes alcaloides e compostos análogos, como os herbindóis, alvos para a síntese total e plataforma para o desenvolvimento de novas metodologias sintéticas. Nesta Dissertação de Mestrado, procurou-se completar a síntese estereosseletiva da (+)-cis-triquentrina A empregando-se intermediários sintéticos protegidos com o grupo benzila que poderão ser usados para a futura prospecção de novos compostos com atividade biológica. A rota proposta baseia-se na obtenção do ácido (S)-3-(1-benzil-4-etil-1H-indol-7-il)butanóico, um intermediário-chave já descrito por Silva e colaboradores no percurso da síntese total da (+)-trans-triquentrina A, e da finalização da síntese de acordo com a abordagem proposta pelo grupo de RajanBabu para conversão do análogo protegido com grupo tosila à (+)-cis-triquentrina A. A resolução enzimática do intermediário-chave com lipase de Pseudomonas cepacia imobilizada em terra diatomácea foi otimizada, resultando em rendimentos de 32% e 99% ee na metade do tempo descrito anteriormente. Na etapa-chave da síntese, o (S)-ácido foi submetido a uma acilação de Friedel-Crafts intramolecular na presença de anidrido trifluoroacético que produziu o produto de ciclização desejado com 40% de rendimento. Na etapa final da síntese, o intermediário sintético protegido com grupo benzila não pode ser convertido à (+)-cis-triquentrina A, empregando-se a metodologia desenvolvida para a redução do composto análogo tosilado, dada a diferença de reatividade imposta pela troca do grupo protetor. As etapas realizadas até o penúltimo intermediário sintético (S)-8, consta com 10 etapas e rendimento global de 1,3%. viii As diferenças eletrônicas e estruturais relacionadas a diferentes grupos protetores poderão ser refletidas em variações na atividade antiproliferativa de indóis relacionados a triquentrinas. Ainda no interesse de preparar moléculas para envio à análises de atividade antiproliferativa preparou-se um composto relacionado à síntese da trans-triquentrina A tendo como etapa-chave uma contração de anel mediada por I(III) em 21% de rendimento, cujo trabalho foi incluído nos anexos. / Trikentrins A are marine natural products with biological activity and high structural complexity. These factors make these alkaloids and analogous compounds, such as herbidoles, targets for total synthesis and platform for the development of new synthetic methodologies. In this Master\'s Dissertation, we attempted to complete the stereoselective synthesis of (+)-cis-trikentrin A using synthetic intermediates protected with the benzyl group that could be used for the future prospection of new compounds with biological activity. The proposed route is based on the preparation of (S)-3-(1-benzyl-4-ethyl-1H-indol-7-yl) butanoic acid, a key intermediate already described by Silva et al., in the course of total synthesis (+)-trans-triquentrin A, and the final part of the synthesis according to the approach proposed by the RajanBabus group for conversion of the protected analogue with tosyl group to (+)-cis-trikentrin A. The enzymatic resolution of the key intermediate with Pseudomonas cepacia lipase immobilized on diatomaceous earth was optimized, resulting in 32% yield and 99% ee in half the time described above. In the key step of the synthesis, the (S)-acid was subjected to an intramolecular Friedel-Crafts acylation in the presence of trifluoroacetic anhydride which yielded the desired cyclization product in 40% yield. In the final step of the synthesis, the synthetic intermediate protected with benzyl group couldnt be converted to (+)-cis-trikentrin A, employing the methodology developed for the reduction of the tosylated analogous compound, given the difference of reactivity imposed by exchange of the protective group. The steps carried out up to the synthetic intermediate (S)-8, consists of 10 steps and overall yield of 1.3%. Electronic and structural differences related to different protective groups may be reflected in variations in the antiproliferative activity of indoles related to trikentrins. A compound related to the synthesis of trans-triquentrin A having as its key step an I(III) mediated ring contraction in 21% yield was also prepared in the interest of preparing molecules for antiproliferative activity analysis, whose work was included in the appendix.

Estudos funcionais anteriores identificaram um módulo cis-regulador (MCR) de 67 pb (-253/- 187) na região promotora do gene de pufe de DNA BhC4-1 que dirige a expressão do gene repórter na glândula anelar de Drosophila melanogaster. Uma análise bioinformática identificou 67 sequências de D. melanogaster que são similares a sequências contidas no MCR de glândula anelar. Uma das sequências identificadas reside em um fragmento genômico de 657 pb localizado aproximadamente 2500 pb à montante do CG13711, 400 pb à montante do CG12493, em uma região genômica que constitui um dos íntrons do CG32239 (Gef64C). A caracterização preliminar de três linhagens transformadas com a construção 657 pb-lacZ mostrou expressão do gene repórter no sistema traqueal de larvas e prépupas e no tórax de adultos. Baseado padrão de expressão promovido por este MCR, o mesmo foi denominado Traqueia-Tórax (TT). O principal objetivo do presente trabalho constituiu estender a caracterização molecular das linhagens da série TT-lacZ. Inicialmente embriões, larvas de primeiro, segundo e terceiro estádio, prépupas 0 h, 1 h e 2 h, pupas 24 h e adultos com 1, 3 e 5 dias foram investigados quanto ao padrão de expressão do repórter utilizando ensaio histoquímico que detecta atividade de ?-galactosidase. A expressão do gene repórter é inicialmente detectada no sistema traqueal durante o terceiro estádio larval e continua a ser detectada neste tecido em prépupas 0 h, 1 h e 2 h e pupas 24 h. Em adultos, a expressão do gene repórter é verificada nos músculos longitudinais dorsais em adultos de 3 e 5 dias. Uma vez que o MCR TT reside em uma região intergênica e a informação disponível sobre os CGs próximos ainda é escassa, não foi possível inferir qual dos CGs é regulado pelo MCR TT. Neste contexto, o padrão de expressão do RNAm do gene repórter lacZ e do CG13711, CG12493 e CG32239 foi investigado no sistema traqueal de larvas e prépupas e no tórax de adultos de uma das linhagens da série TT-lacZ utilizando RT-qPCR. Os níveis de expressão do RNAm lacZ aumentam cerca de 3 vezes em prépupas 0 horas, quando comparados com os níveis de expressão do RNAm lacZ presentes no sistema traqueal de larvas de terceiro estádio. Um padrão de expressão similar foi observado no caso do CG32239 e do CG13711. Nos tóraxes de adultos de 3 e 5 dias de idade os níveis de expressão do RNAm lacZ aumentam cerca de 37 vezes e 11 vezes, respectivamente, quando comparados aos níveis de expressão iv do RNAm lacZ presentes nos tóraxes de adultos de 1 dia. No tórax de adultos, o único CG que apresenta um padrão de expressão similar ao padrão de expressão de lacZ constitui o CG12493. Em conjunto, nós concluímos que o MCR TT promove um padrão dinâmico de expressão durante o desenvolvimento. Além disso, com base nos resultados de RT-qPCR, nós sugerimos que o MCR TT regula a expressão do RNAm do CG32239 no sistema traqueal durante a transição larva-prépupa e também a expressão do RNAm do CG12493 no tórax de adultos de 3 e 5 dias de idade. Além de estender a caracterização funcional de um novo MCR, nossos resultados também contribuem com novas informações acerca dos padrões de expressão no desenvolvimento de três CGs de D. melanogaster. / Previous functional studies identified in the DNA puff BhC4-1 promoter region a 67 bp (- 253/-187) cis-regulatory module (CRM) that drives reporter gene expression in the ring gland of D. melanogaster. A bioinformatics analysis identified 67 Drosophila melanogaster sequences that are similar to sequences contained in the ring gland CRM. One of the identified sequences resides in a 657 bp genomic fragment located about 2500 bp upstream CG13711, about 400 bp upstream CG12493, in a genomic region that constitutes one of the introns of CG32239 (Gef64C). The preliminary characterization of three transgenic lines transformed with a 657 bp-lacZ construct revealed reporter gene expression in the larval/prepupal tracheal system and in adult thorax. Based on the pattern of expression driven by this CRM we named it Trachea-Thorax (TT). The main goal of this work was to extend the molecular characterization of the lines of the TT-lacZ series. Initially ?-galactosidase histochemical assays were performed in embryos, first, second and third instar larvae, 0h, 1h and 2h prepupae, 24 h pupae and 1, 3 and 5 days old adults. Reporter gene expression is initially detected during the third larval instar in the tracheal system and continues to be detected in this tissue at 0 h, 1h and 2 h prepupa and, 24 h pupa. During the adult stage, reporter gene expression is verified in the dorsal longitudinal muscles of 3 and 5 days old adults. Since the TT CRM lies in an intergenic region and the available information about the nearby CGs is still scarce it was not possible to infer which of the CGs is regulated by the TT CRM. In this context, the mRNA pattern of expression of the lacZ reporter gene and of CG13711, CG12493 and CG32239 was investigated in the tracheal system of both larvae and prepupae and in adult thoraxes of one of the transgenic lines of the TT-lacZ series using RTqPCR. The lacZ mRNA expression levels increase about 3 times in 0 h prepupae when compared to the lacZ mRNA expression levels present in the tracheal system of third instar larvae. A similar pattern of expression was observed for both CG32239 and CG13711. In three and five days old adult thoraxes lacZ mRNA expression levels increase about 37 times and 11 times, respectively, when compared to lacZ mRNA expression levels present in one day old thoraxes. In the adult thorax, the only CG that presents a similar pattern of expression constitutes CG12493. Overall, we conclude that the TT CRM drives a dynamic pattern of ii expression throughout development. Additionally, based on RT-qPCR results, we suggest that the TT CRM regulates the expression of CG32239 mRNA in the tracheal system during the larvae to prepupae transition, as well as the expression of CG12493 mRNA in the thorax of 3 and 5 days old adults. Besides extending the functional characterization of a novel CRM our results also contribute new information about the developmental patterns of expression of three Drosophila melanogaster CGs.

Enquête sur le fer(II) noyau transition de spin centrée métal coordonner avec des ligands photo-actif (isomérisation tran / cis) et l'étude de la lumière induite par le changement de spin conduit ligand (LD-LISC) propriétés en fer(II) fonctionnalisé complexes sous la forme de nanoparticules dispersées dans des films minces transparents. Pour cette étude, nous avons choisi un ensemble de complexes mononucléaires incorporant un ligand bipyridine substitué soit par un groupe méthyle (de la fonction électronique et de la structure) ou un groupe styryle (fonction photoactivable). L'introduction dans la sphère de trois de coordination (forte cas ligand-domaine) ou deux (intermédiaire force ligand-domaine) des ligands bipyridine (avec NCSe- co-ligands) a permis à la modulation de la résistance au ligand champ subi par l'ion métallique dans des systèmes différents. Propriétés de commutation optiques et thermiques des solides ont été étudiés dans les poudres polycristallines et de la dispersion des objets de taille réduite (micro, nanoparticules). Nous avons présenté l'élaboration et l'étude des micro- et nano-particules, complexes Fe(II) mononuclées non-ioniques, y compris ligand bidenté (4,4-méthyl-2,2 'bipyridine, Me₂-bpy) ont été sélectionnés. La synthèse de nano-et micro-objets a nécessité l'adaptation de la méthode (précipitation rapide anti-solvant). Il a été réalisé avec ionique [Fe(L)₃](NCSe)₂ précurseurs en jouant avec l'équilibre chimique pour induire la précipitation de connexes neutre Fe(L)₂(NCSe)₂ espèces. Nano et micro-cristaux de Fe(Me₂-bpy)₂(NCSe)₂ avec des tailles de 56 à 1200 nm ont été caractérisés et très dilués dans des films minces polymères pour optimiser l'absorption de la lumière et de l'étude des processus photocommutation. Fe (Me₂-bpy)₂(NCSe)₂ sous la forme de nano-et micro-particules, traitées dans des films polymères minces ont montré à spin-croisé thermique centrée à des températures supérieures à la température ambiante; pas d'effet significatif de l'environnement a été détectée. Nous avons étudié la réactivité photochimique de la precusors ioniques [Fe(msbpy)₃](NCSe)₂ (msbpy = trans et cis-isomères de 4-méthyl-4'-styryl-2,2 '-bipyridine), a été d'abord sondé en solution. Il a été établi que les cis isomérisation trans de 4-méthyl-4'-styryle 2,2 bipyidine a lieu d'une manière quantitative lorsque le complexe Fe(II) diamagnétique a été exposé à une lumière bleue (405 nm); cette réaction peut être partiellement inversée par une excitation UV. Nous avons élaboré des nanoparticules dérivées croisées sur la base du Fe(II) sont décrits; la commutation de ces particules (60nm) embarqués dans des films minces de PMMA et l'effet LD-LISC sont étudiés. Dans l'environnement très confiné de nanoparticules, la Feᴵᴵ(t-msbpy)₂(NCSe)₂ complexe réagit aussi lors de l'irradiation. La combinaison des UV-Vis, Raman et mesures photomagnétiques des matériaux hautement diluées nous a permis d'analyser l'effet de l'irradiation de lumière et le comportement de commutation état de spin. L'étude de Feᴵᴵ(t-msbpy)₂(NCSe)₂a montré la présence à la température ambiante des différents processus en fonction de la longueur d'onde d'excitation: un procédé de ligand centrée (trans à cis isomérisation, 365 nm) et un métal un centré (entraînant un changement MLCT, 405 nm). / Investigation of the iron(II) metal-centered spin-crossover core coordinate with photo-active ligands (tran / cis isomerization) and study of the ligand-driven light-induced spin change (LD-LISC) properties with functionalized iron(II) complexes in the form of nanoparticles dispersed in transparent thin films. For this investigation, we have selected a set of mononuclear complexes incorporating a bipyridine ligand substituted either with a methyl group (electronic and structural function) or a styryl group (photoactivable function). The introduction in the coordination sphere of three (strong ligand-field case) or two (intermediate ligand-field strength) bipyridine ligands (with NCSe- co-ligands) has allowed the modulation of the ligand-field strength experienced by the metal ion in different systems. Optical and thermal switching properties of solids have been investigated in polycrystalline powders and in dispersion of size-reduced objects (micro-, nano-particles).We have presented elaboration and study of micro- and nanoparticles, non-ionic mononuclear FeII complexes including bidentate ligand (4,4’-methyl-2,2’-bipyridine, Me2-bpy) have been selected. The synthesis of nano- and micro-objects has required the adaptation of the method (sudden precipitation in antisolvent). It was achieved with ionic [Fe(L)3](NCSe)2 precursors by playing with chemical equilibrium for inducing the precipitation of related neutral Fe(L)2(NCSe)2 species. Nano- and micro-crystals of Fe(Me2-bpy)2(NCSe)2 with sizes 56-1200 nm were characterized and then highly diluted in polymeric thin films for optimizing light absorption and investigating photoswitching processes. Fe(Me2-bpy)2(NCSe)2) in the form of nano-and micro-particles, processed in thin polymeric films were shown to exhibit thermal spin-crossover centered at temperatures higher than room temperature; no significant effect of environment was detected.We have studied the photochemical reactivity of the ionic precusors [Fe(msbpy)3](NCSe)2 (msbpy = trans- and cis-isomers of 4-methyl-4’-styryl-2,2’-bipyridine,) was first probed in solution. It has been established that the cis to trans isomerization of 4-methyl-4’-styryl-2,2’-bipyidine takes place in a quantitative way when the diamagnetic FeII complex was exposed to a blue light (405 nm); this reaction can be partly reversed with a UV excitation.We have elaborated spin-crossover nanoparticles based on the Fe(II) complexes are described; the switching of these particles (60nm) embedded in PMMA thin films and the LD-LISC effect are investigated. In the highly confined environment of nanoparticles, the FeII(t-msbpy)2(NCSe)2 complex also reacts upon irradiation. The combination of UV-Vis, Raman and photomagnetic measurements of the highly diluted materials has allowed us to analyze the effect of light irradiation and the spin-state switching behavior. The study of FeII(t-msbpy)2(NCSe)2 has shown the occurrence at room temperature of different processes depending on the excitation wavelength: a ligand-centered process (trans-to-cis isomerization, 365 nm) and a metal-centered one (resulting in a MLCT change, 405 nm).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nesta Dissertação foi utilizado um sistema catalítico Zieger-Natta à base de neodímio para avaliar a influência do agente de halogenação e da razão molar halogênio:Nd sobre a atividade catalítica, a constante de velocidade de propagação, a conversão da polimerização, a microestrutura, a massa molecular e a polidispersão do polibutadieno 1,4-cis. O sistema utilizado era constituído por versatato de neodímio (NdV), hidreto de diisobutilalumínio (DIBAH) e um agente de halogenação. Os agentes halogenantes estudados foram: cloreto de t-butila (t-BuCl), sesquicloreto de etilalumínio (EASC) e cloreto de dietilalumínio (DEAC), em valores de razão molar Cl:Nd que variaram entre 0,5:1 e 5:1 e o dietil-eterato de trifluoreto de boro (BF3.Et2O), na razão molar F:Nd = 3:1. Os polímeros foram caracterizados por espectroscopia na região do infravermelho para determinação da microestrutura e por cromatografia de exclusão por tamanho para determinação das massas moleculares. O teor de unidades 1,4-cis variou de 90 a 98%, a massa molecular numérica média ( ) permaneceu na faixa entre 0,2 e 2x105, e a massa molecular ponderal média ( ) variou de 1,4 a 4x105 / In this Dissertation a catalyst system based on neodymium was employed to evaluate the influence of halogen source and the halogen:Nd molar ratio on the catalyst activity, polymerization constant rate, polymerization conversion, molecular weight characteristics and microstructure of polybutadiene. The catalyst systems were formed by diisobutylaluminium hydride (DIBAH), neodymium versatate (NdV) and a halogen source. The halogenating agents studied were t-butyl chloride (t-BuCl), ethylaluminium sesquichloride (EASC) and diethylaluminium chloride (DEAC) in a nCl:nNd molar ratio in the range from 0,5 to 5, and boron trifluoride diethyl ether (BF3.Et2O). The polymers were characterized by infrared espectroscopy for determining the microstructure and by size exclusion chromatography to obtain the molecular mass characteristics. The cis-1,4 units contents varied from 90 to 98%, the number-average molecular mass ( ) varied from 0,2 to 2x105 and the weight-average molecular mass ( ) varied from 1,4 to 4x105

La diversité morphologique des muscles squelettiques permet la précision et la coordination des mouvements propres à chaque espèce animale. L'établissement du patron musculaire a lieu au cours du développement embryonnaire durant le processus de myogenèse. Il a été décomposé en quatre étapes chez la drosophile : la spécification de groupes de myoblastes équivalents (groupes promusculaires) à des positions précises du mésoderme, la sélection d'une ou plusieurs cellules progéniteurs à partir de chaque groupe, la division asymétrique des progéniteurs en cellules fondatrices des muscles, et enfin, la fusion d'une cellule fondatrice avec un nombre défini de myoblastes compétents pour la fusion qui forme une myofibre syncytiale. Ce processus aboutit à la mise en place d'un patron stéréotypé de muscles morphologiquement distincts par leur taille, orientation, forme, et sites d'attachement au squelette ; ces caractères définissant l'identité du muscle. Chez la drosophile, chacun des 30 muscles par hémisegment de la larve est constitué d'une seule myofibre. Il a été proposé que l'identité morphologique de cette fibre soit contrôlée par une combinatoire de facteurs de transcription identitaires (FTi) exprimés par la cellule fondatrice. Mon projet de thèse a porté sur le contrôle transcriptionnel de l'identité musculaire, avec comme modèle d'étude, un muscle dorso-latéral de la larve de drosophile, le muscle DA3 dont un FTi est Collier/EBF (Col). La transcription de col est activée dans un groupe promusculaire, puis transitoirement dans les quatre progéniteurs issus de ce groupe, avant d'être maintenue spécifiquement dans la myofibre DA3. Dans des embryons mutants pour col, le DA3 est transformé en muscle plus dorsal, DA2. Les travaux précédents de l'équipe ont montré que la transcription de col dans le lignage DA3 est contrôlée par deux modules cis-régulateurs, EarlyCRM et LateCRM, séparés physiquement sur le chromosome et agissant séquentiellement. Leur chevauchement temporel d'activité restreint au progéniteur DA3 et l'autorégulation directe du LateCRM ont mené à l'hypothèse d'un mécanisme de " passage de témoin " entre ces deux CRM, spécifique au progéniteur DA3. L'objectif de ma thèse était de tester cette hypothèse et de comprendre comment une information temporelle et spatiale intégrée par un CRM est transmise à un autre CRM, pour définir une identité cellulaire, une question fondamentale au-delà du cas d'espèce que constitue le muscle DA3.[...] / The morphological diversity of skeletal muscles allows the precision and coordination of movements specific to each animal species. Establishment of a stereotypic pattern of muscles takes places during the process of myogenesis. Studies in Drosophila, an insect model, have identified four steps in this process: the specification of equivalence groups of myoblasts (promuscular clusters) at defined positions within the somatic mesoderm, the selection of progenitor(s) from each group, asymmetric division of each progenitor into post-mitotic muscle founder cells, and finally the fusion of each founder cell with a given number of fusion competent cells to form a syncytial myofiber. This dynamic, integrated process leads to establishing a stereotyped pattern of morphologically distinct muscles which can each be distinguished, based on size, orientation, shape, sites of attachment to the skeleton, all properties defining muscle identity. In the Drosophila larva, each of the about 30 different muscles per hemisegment is made of a single myofiber. It has been proposed that final morphology of a myofiber reflects the combinatorial code of identity Transcription Factors (iTF) expressed by its founder cell, although many questions remain unanswered. My thesis project aimed at better understanding the mechanism of specification of muscle identity, using as model a dorso-lateral muscle of the Drosophila larva, the DA3 muscle whose identity is controlled by the Collier/EBF (Col) iTF. col transcription is activated in one promuscular cluster, transient in the 4 progenitors issued from this cluster and stably maintained in the DA3 myofiber. In col mutant embryos, the DA3 muscle is transformed into a more dorsal, DA2-like muscle. Previous work has shown that col transcription in the DA3 lineage is controlled by two cis-regulatory modules (EarlyCRM and LateCRM), physically distant on the chromosome and acting sequentially. The temporal overlap of EarlyCRM and LateCRM in the DA3 progenitor and direct col autoregulation via the LateCRM led to hypothesize a handover between the two CRM in the DA3 progenitor. One goal of my thesis project was to challenge this hypothesis and understand how positional and temporal information integrated by EarlyCRM could be memorized via LateCRM, in order to specify cell identity, a fundamental question of developmental biology beyond the specific case of the Drosophila DA3 muscle. [...]

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Au cours de mes travaux de thèse, je me suis penché sur le pendant génétique de l'adaptation. Certes, les mutations sont générées au hasard; mais quelles sont celles que l'évolution retient et pourquoi? Peut-on définir certains principes qui déterminent les chemins génétiques favoris qu'emprunte et ré-emprunte l'évolution ? Ces principes génétiques bornent-ils parfois le jeu des adaptations possibles ? Le substrat génétique qui nourrit la sélection est-il le même lorsqu'il s'agit de l'émergence d'un caractère nouveau ou bien lorsqu'il s'agit de la diversification ou le raffinement d'un caractère existant ?Pour répondre à ces questions, je me suis penché sur des drosophiles à pois noirs. En particulier, des drosophiles du groupe melanogaster, dont un ancêtre s'est paré d'une tache noire sur le bout de l'aile, il y a environ 20 millions d'années. Cette tache s'est transmise de génération en génération, en se modifiant de façon spectaculaire dans certaines lignées.J'ai d'abord tenté de mettre à jour la logique génétique du bricolage qui a fondé l'émergence puis la diversification de cette tache. Ces travaux suggèrent un modèle général dans lequel les deux versants de l'adaptation, émergence et diversification, correspondent à deux bricolages génétiques distincts.Dans un deuxième temps, je me suis concentré sur l'émergence de cette tache. Ce travail en cours a permis de dégager certaines caractéristiques génétiques du module de régulation qui sous-tend le développement de cette tache. Mon travail de thèse contribue, dans son ensemble, à une meilleure compréhension des ressorts génétiques sur lesquels repose l'adaptation. / In my thesis, I studied the genetics of adaptation. Certainly, mutations are randomly generated, yet evolutionary relevant mutations are not randomly distributed; why? Can we define some principles that determine the most favorable genetic paths, in the light of evolution? Would these principles sometimes limit the range of possible adaptations? Are mutations that feed emergence of a new character and diversification or refinement of an existing character the same? To answer these questions, I studied fruit flies with black spots. In particular, fruit flies from the melanogaster group, whose ancestor evolved a black spot on the tip of its wing, about 20 million years ago. This spot has been inherited through generations, and diversified dramatically in some lineages.I first tried to uncover the logic of genetic tinkering underlying the emergence and diversification of the spot. Our results suggest a general model in which the two sides of adaptation, emergence and diversification, correspond to two distinct genetic tinkering.Second, I focused strictly on the emergence of this spot. This ongoing work has identified some general genetic features of the regulatory module underlying the development of this spot.Overall, my thesis contributes to a better understanding of the genetics of adaptation.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nesta Dissertação foi utilizado um sistema catalítico Zieger-Natta à base de neodímio para avaliar a influência do agente de halogenação e da razão molar halogênio:Nd sobre a atividade catalítica, a constante de velocidade de propagação, a conversão da polimerização, a microestrutura, a massa molecular e a polidispersão do polibutadieno 1,4-cis. O sistema utilizado era constituído por versatato de neodímio (NdV), hidreto de diisobutilalumínio (DIBAH) e um agente de halogenação. Os agentes halogenantes estudados foram: cloreto de t-butila (t-BuCl), sesquicloreto de etilalumínio (EASC) e cloreto de dietilalumínio (DEAC), em valores de razão molar Cl:Nd que variaram entre 0,5:1 e 5:1 e o dietil-eterato de trifluoreto de boro (BF3.Et2O), na razão molar F:Nd = 3:1. Os polímeros foram caracterizados por espectroscopia na região do infravermelho para determinação da microestrutura e por cromatografia de exclusão por tamanho para determinação das massas moleculares. O teor de unidades 1,4-cis variou de 90 a 98%, a massa molecular numérica média ( ) permaneceu na faixa entre 0,2 e 2x105, e a massa molecular ponderal média ( ) variou de 1,4 a 4x105 / In this Dissertation a catalyst system based on neodymium was employed to evaluate the influence of halogen source and the halogen:Nd molar ratio on the catalyst activity, polymerization constant rate, polymerization conversion, molecular weight characteristics and microstructure of polybutadiene. The catalyst systems were formed by diisobutylaluminium hydride (DIBAH), neodymium versatate (NdV) and a halogen source. The halogenating agents studied were t-butyl chloride (t-BuCl), ethylaluminium sesquichloride (EASC) and diethylaluminium chloride (DEAC) in a nCl:nNd molar ratio in the range from 0,5 to 5, and boron trifluoride diethyl ether (BF3.Et2O). The polymers were characterized by infrared espectroscopy for determining the microstructure and by size exclusion chromatography to obtain the molecular mass characteristics. The cis-1,4 units contents varied from 90 to 98%, the number-average molecular mass ( ) varied from 0,2 to 2x105 and the weight-average molecular mass ( ) varied from 1,4 to 4x105

Estudos funcionais anteriores identificaram um módulo cis-regulador (MCR) de 67 pb (-253/- 187) na região promotora do gene de pufe de DNA BhC4-1 que dirige a expressão do gene repórter na glândula anelar de Drosophila melanogaster. Uma análise bioinformática identificou 67 sequências de D. melanogaster que são similares a sequências contidas no MCR de glândula anelar. Uma das sequências identificadas reside em um fragmento genômico de 657 pb localizado aproximadamente 2500 pb à montante do CG13711, 400 pb à montante do CG12493, em uma região genômica que constitui um dos íntrons do CG32239 (Gef64C). A caracterização preliminar de três linhagens transformadas com a construção 657 pb-lacZ mostrou expressão do gene repórter no sistema traqueal de larvas e prépupas e no tórax de adultos. Baseado padrão de expressão promovido por este MCR, o mesmo foi denominado Traqueia-Tórax (TT). O principal objetivo do presente trabalho constituiu estender a caracterização molecular das linhagens da série TT-lacZ. Inicialmente embriões, larvas de primeiro, segundo e terceiro estádio, prépupas 0 h, 1 h e 2 h, pupas 24 h e adultos com 1, 3 e 5 dias foram investigados quanto ao padrão de expressão do repórter utilizando ensaio histoquímico que detecta atividade de ?-galactosidase. A expressão do gene repórter é inicialmente detectada no sistema traqueal durante o terceiro estádio larval e continua a ser detectada neste tecido em prépupas 0 h, 1 h e 2 h e pupas 24 h. Em adultos, a expressão do gene repórter é verificada nos músculos longitudinais dorsais em adultos de 3 e 5 dias. Uma vez que o MCR TT reside em uma região intergênica e a informação disponível sobre os CGs próximos ainda é escassa, não foi possível inferir qual dos CGs é regulado pelo MCR TT. Neste contexto, o padrão de expressão do RNAm do gene repórter lacZ e do CG13711, CG12493 e CG32239 foi investigado no sistema traqueal de larvas e prépupas e no tórax de adultos de uma das linhagens da série TT-lacZ utilizando RT-qPCR. Os níveis de expressão do RNAm lacZ aumentam cerca de 3 vezes em prépupas 0 horas, quando comparados com os níveis de expressão do RNAm lacZ presentes no sistema traqueal de larvas de terceiro estádio. Um padrão de expressão similar foi observado no caso do CG32239 e do CG13711. Nos tóraxes de adultos de 3 e 5 dias de idade os níveis de expressão do RNAm lacZ aumentam cerca de 37 vezes e 11 vezes, respectivamente, quando comparados aos níveis de expressão iv do RNAm lacZ presentes nos tóraxes de adultos de 1 dia. No tórax de adultos, o único CG que apresenta um padrão de expressão similar ao padrão de expressão de lacZ constitui o CG12493. Em conjunto, nós concluímos que o MCR TT promove um padrão dinâmico de expressão durante o desenvolvimento. Além disso, com base nos resultados de RT-qPCR, nós sugerimos que o MCR TT regula a expressão do RNAm do CG32239 no sistema traqueal durante a transição larva-prépupa e também a expressão do RNAm do CG12493 no tórax de adultos de 3 e 5 dias de idade. Além de estender a caracterização funcional de um novo MCR, nossos resultados também contribuem com novas informações acerca dos padrões de expressão no desenvolvimento de três CGs de D. melanogaster. / Previous functional studies identified in the DNA puff BhC4-1 promoter region a 67 bp (- 253/-187) cis-regulatory module (CRM) that drives reporter gene expression in the ring gland of D. melanogaster. A bioinformatics analysis identified 67 Drosophila melanogaster sequences that are similar to sequences contained in the ring gland CRM. One of the identified sequences resides in a 657 bp genomic fragment located about 2500 bp upstream CG13711, about 400 bp upstream CG12493, in a genomic region that constitutes one of the introns of CG32239 (Gef64C). The preliminary characterization of three transgenic lines transformed with a 657 bp-lacZ construct revealed reporter gene expression in the larval/prepupal tracheal system and in adult thorax. Based on the pattern of expression driven by this CRM we named it Trachea-Thorax (TT). The main goal of this work was to extend the molecular characterization of the lines of the TT-lacZ series. Initially ?-galactosidase histochemical assays were performed in embryos, first, second and third instar larvae, 0h, 1h and 2h prepupae, 24 h pupae and 1, 3 and 5 days old adults. Reporter gene expression is initially detected during the third larval instar in the tracheal system and continues to be detected in this tissue at 0 h, 1h and 2 h prepupa and, 24 h pupa. During the adult stage, reporter gene expression is verified in the dorsal longitudinal muscles of 3 and 5 days old adults. Since the TT CRM lies in an intergenic region and the available information about the nearby CGs is still scarce it was not possible to infer which of the CGs is regulated by the TT CRM. In this context, the mRNA pattern of expression of the lacZ reporter gene and of CG13711, CG12493 and CG32239 was investigated in the tracheal system of both larvae and prepupae and in adult thoraxes of one of the transgenic lines of the TT-lacZ series using RTqPCR. The lacZ mRNA expression levels increase about 3 times in 0 h prepupae when compared to the lacZ mRNA expression levels present in the tracheal system of third instar larvae. A similar pattern of expression was observed for both CG32239 and CG13711. In three and five days old adult thoraxes lacZ mRNA expression levels increase about 37 times and 11 times, respectively, when compared to lacZ mRNA expression levels present in one day old thoraxes. In the adult thorax, the only CG that presents a similar pattern of expression constitutes CG12493. Overall, we conclude that the TT CRM drives a dynamic pattern of ii expression throughout development. Additionally, based on RT-qPCR results, we suggest that the TT CRM regulates the expression of CG32239 mRNA in the tracheal system during the larvae to prepupae transition, as well as the expression of CG12493 mRNA in the thorax of 3 and 5 days old adults. Besides extending the functional characterization of a novel CRM our results also contribute new information about the developmental patterns of expression of three Drosophila melanogaster CGs.