No description available.

No description available.

Les microtubules sont des polymères cylindriques de tubuline-αβ, membres du cytosquelette eucaryote. Ils possèdent une dynamique intrinsèque nécessaire à de nombreuses fonctions cellulaires telle que la mitose. L’hydrolyse du nucléotide GTP dans les polymères de tubuline-αβ ainsi que les interactions entre la tubuline et les protéines partenaires ou les molécules à visées pharmacologiques, jouent un rôle critique sur la dynamique des microtubules. Durant cette thèse, des approches de modélisation moléculaire ont été utilisées pour mieux appréhender les interactions tubuline-ligand à l’échelle atomique et contribuer au développement de nouvelles molécules actives. Des simulations de dynamiques moléculaires ont été réalisées pour étudier l’effet de différents nucléotides dans la tubuline-β sur la structure et la dynamique du protofilament de tubuline. Nous proposons un rôle du résidu αE254 dans la coordination du magnésium catalytique. Nous observons également des changements conformationnels aux interfaces latérales et un réarrangement de structure aux interfaces longitudinales qui peuvent affecter la stabilisation du microtubule. Des travaux menés au laboratoire ont montré que la colchicine et le carbendazime se fixent dans des poches voisines dans la sous-unité tubuline-β et inhibent la prolifération cellulaire. Nous avons proposé un site de fixation du carbendazime dans les complexes tubuline-colchicine à l’aide de l’amarrage moléculaire et de simulations de dynamiques moléculaires. Ces expériences ont mené au design de molécules hybrides composées des noyaux colchicine et carbendazime reliés par un linker. Une de ces molécules hybrides a été synthétisée et testée avec succès sur des lignées de cellules HeLa. Enfin, nous avons construit des peptides cycliques dérivées d’I19L, un peptide anti-microtubule identifié au laboratoire. Des simulations de dynamique moléculaire et des calculs d’énergie libre de liaisons ont permis d’évaluer ces peptides. Enfin, des mutations ont été proposées afin d’optimiser l’interaction entre le meilleur peptide et la tubuline. / Microtubules are cylindrical polymers of αβ-tubulin heterodimers, members of the eukaryotic cytoskeleton. They possess an intrinsic dynamics which is necessary to any cellular functions such as the mitosis. It has long been recognized that GTP hydrolysis in αβ-tubulin polymers plays a critical role in this dynamics as well as the interactions between tubulin and the protein partners or the drugs. In this thesis, molecular modeling approaches are applied to three theoretical studies to gain insight at the atomic scale about tubulin-ligand interactions and to contribute to the development of new active compounds. Molecular dynamics simulations were used to study the effect of the different nucleotide states at β-tubulin on the protofilament structure and dynamics. We propose a role for residue αE254 in catalytic magnesium coordination. We also observe conformational changes and structure rearrangement at lateral and longitudinal interfaces that can affect the microtubule stabilization. Previous work carried out in the laboratory showed that colchicine and carbendazime bind neighboring pockets in the β-tubulin subunit and inhibit cell proliferation. We proposed a binding site of carbendazime on the tubulin-colchicine complex, using docking and molecular dynamics simulation, which lead to the design of hybrid molecules composed of both colchicines and carbendazime moieties attached with a linker. One of these hybrid molecules has been synthesized and successfully tested on HeLa cells. Finally, we designed four cyclic peptides based on I19L, an anti-microtubule peptide identified at the laboratory. Molecular dynamic simulations and binding free energy calculations were used to evaluate these peptides. Mutations were then proposed on the best peptide to increase its interactions with tubulin.

No description available.

L'amélioration de la technique, permettant d'induire in situ la gynogenèse afin de fiabiliser la production de lignées haploïdes doublées (HD) de maïs, a été entreprise. L'intérêt de son intégration dans les schémas de sélection, par rapport aux méthodes conventionnelles a été évalué. Le pourcentage moyen de plantes haploïdes obtenues, est passé d'environs 2,5 à environs 5 , puis 7. Dans une très large variabilité génétique il n'a généralement pas été observé de génotypes ne répondant pas à l'induction de la gynogenèse. Les lignées HD ont des phénotypes très semblables à ceux des lignées obtenues par autofécondation. Pour le rendement, la variance génétique des lignées HD est presque deux fois supérieure à celle des familles S1 dont elles sont issues. Par cycle de sélection récurrente, les lignées HD sont plus efficaces que les familles S1. L'haplométhode, mise au point au cours de ce travail, peut donc être considéré comme efficace et intéressante pour la selection d'hybrides de maïs.

No description available.

The abundance of microtubules in receptive dendrites suggests they may function in sensory transduction. Responses of frog muscle spindle receptors and joint receptors is inhibited within 25 minutes by 50 mM colchicine, a microtubuledisrupting agent. The inhibition is reversible upon removal of colchicine, and the time course of recovery is comparable to that of inhibition. Frog olfactory responses are briefly inhibited by washing the olfactory mucosa with perfusion fluid. Colchicine accentuates the inhibition and substantially retards the rate of recovery in a dose-dependent fashion. Colchicine does not affect axonal conduction, nor the oxygen uptake of isolated crab or frog leg nerves. The inhibitory action of colchicine is therefore an effect on the electrical excitability of the receptive dendrites or soma, and not an effect on axonal conduction.

Dendrites of cockroach tibial spine mechanoreceptors contain hundreds of free microtubules, which may have some relation to the generation of electrical activity. Deflection of a spine produces a train of action potentials. Continuous perfusion over a period of 4 hours results in no response decrement. Perfusion with 10mM colchicine reversibly inhibits the response within 5-7 minutes. Irreversible inhibition is produced by perfusion with 1mM vinblastine sulfate in perfusion solution containing 1% dimethyl sulfoxide. Deuterium oxide does not inhibit at concentrations less than 50%, nor does it counteract inhibition by 10mM colchicine. Colchicine may be affecting (1) intracellular microtubules, (2) membraneous tubulin, (3) other membrane components, or (4) axoplasmic transport of essential materials to the sensory dendrites.

abstract / Medicine / Master / Master of Philosophy

A poliploidia induzida é uma ferramenta valiosa para o melhoramento genético de plantas, resultando em genótipos com características superiores aos diploides correspondentes. Considerando a importância da cultura de eucalipto para o setor florestal e para a economia brasileira, o presente trabalho teve como objetivo estabelecer um protocolo de poliploidização in vitro para eucalipto, utilizando a espécie Eucalyptus urophylla ST Blake. Segmentos nodais contendo gemas laterais de plântulas desenvolvidas in vitro foram utilizados como explantes para os testes de indução. Os tratamentos avaliados consistiram na imersão e agitação dos explantes em soluções de colchicina nas concentrações: 0; 0,125% e 0,250% em tempos de exposição de 12 e 24 horas e posterior inoculação in vitro em meio de cultura para multiplicação. Após 60 dias, as brotações emitidas foram analisadas por citometria de fluxo e constatou-se indivíduos diploides, tetraploides e mixoploides. Estas foram micropropagadas para a obtenção de mudas clonais ao final do processo. As mudas obtidas foram avaliadas por meio de análises morfológicas organográficas e anatômicas para verificar possíveis alterações resultantes da poliploidização in vitro. Os tratamentos promoveram a indução de 16 poliploides, sendo 11 mixoploides, quatro tetraploides e um octaplóide. A eficência máxima de indução obtida foi de 28% de poliploides no tratamento com 0,125% de colchicina por 12 horas sob agitação, com a obtenção tanto de mixoploides como de tetraploides puros. O protocolo geral de micropropagação utilizado, apesar de algumas perdas durante as etapas, garantiu a obtenção de altos percentuais de mudas clonais inteiramente tetraploides, comprovando a eficiência da técnica no isolamento de setores poliploides. A análise morfo-anatômica das mudas mostrou diferenças significativas em relação às características estomáticas, apontando este tipo de análise como indicador de poliploidização na separação preliminar dos indivíduos. A estrutura anatômica das folhas, no entanto, não sofreu modificações significativas, com exceção da espessura e área foliar da epiderme adaxial. A análise de componentes principais realizada para algumas variáveis morfo-anatômicas demonstrou que, apesar da análise estatística não ter apontado diferenças significativas, existe uma tendência em agrupar os genótipos diploides e tetraploides, diferenciando-os principalmente em relação às características estomáticas, espessura foliar, área do mesofilo, parênquimas paliçádico e lacunoso e espaço intercelular. Os resultados obtidos neste trabalho instigam a continuidade do estudo em diversas possibilidades de abordagens, como o estudo do desenvolvimento das mudas em condições de campos, anatomia da madeira e avaliação do comportamento meiótico. / The induced polyploidy is a valuable tool for genetic improvement of plants, resulting in genotypes with superior characteristics to their corresponding diploid. Considering the Eucalyptus culture importance to the forest sector and Brazilian economy, the aim of this study was establish an in vitro polyploidy protocol for eucalyptus, using the species Eucalyptus urophylla ST Blake. Nodal segments containing lateral buds developed from in vitro seedlings were used as explants for induction tests. The treatments tested consisted of soak and shake the explants in colchicine solutions at the concentrations: 0; 0.125% and 0.250% in soaking by periods of 12 or 24 hours and subsequent in vitro inoculation in culture medium for multiplication. After 60 days, the emitted shoots were classified by flow cytometry in levels of ploidy (diploid, tetraploid and mixoploids) and were subsequently micropropagated to obtain clonal seedlings at the end of the process. The clonal seedlings were evaluated using morphological and anatomical analysis to check the possible changes resulting from the in vitro polyploidy. The treatments promoted the induction of sixteen polyploids, being eleven mixoploids, four tetraploid and one octaploid. The maximum induction efficiency was obtained in 28% of polyploidy in the treatment with 0.125% of colchicine by soaking period of 12 hours, having obtained both mixoploids and pure tetraploids. The general protocol of micropropagation used, although presenting some losses during the steps, guaranted the inducing of high percentages of enterelly tetraploid, proving the efficiency of micropropagation in the isolation of polyploid sectors. The morpho-anatomical analysis of the poliploidy seedlings showed significant differences in relation to stomatal characteristics, indicating this type of analysis as polyploidy indicator in the preliminary separation of individuals. The anatomical structure of the leaves, however, did not change significantly, except for the thickness and leaf area of the adaxial epidermis. The principal component analysis (PCA) performed for some morphological and anatomical variables demonstrated that despite the statistical analysis did not have pointed out significant differences, there is a tendency to group the diploid and tetraploid genotypes, differentiating them mainly in relation to stomatal characteristics, leaf thickness, mesophyll area, palisade and spongy parenchyma areas and intercellular space. The results of this work instigate to continue the study in several possibilities of approaches, such as the study of the development of seedlings in fields conditions, wood anatomy and study of meiotic behavior.