Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to evaluate the in vitro germination and multiplication of grapia for plantlet production and conservation of selected genotypes. For in vitro establishment, three concentrations (0; 2.5 and 5.0%) of sodium hypochlorite (NaOCl) and seed immersion times of 5, 10 and 15 min were tested. Seeds were than inoculated in glass flasks with 5 mL of distilled water, sucrose (30 g L-1) and agar (7g L-1) medium. The evaluations were done at 30 days of cultivation for the percentage of germination and desinfestation, mean germination time (TMG) and germination speed index (IVG). Disinfected seeds were also inoculated in WPM (Wood Plant Medium) medium supplemented with 4, 5 or 6 g L-1 of agar combined with 10, 20 or 30 g L-1 of sucrose, and maintained under dark condition of the first seven days or light during the whole period of cultivation. The evaluations were done at 15 days for the percentage of germination, height of aerial part, number of leaves and internodes, total root length, TMG and IVG. Aseptic seedlings were transplanted to WPM, MS or ½ MS culture medium. After 15 days, they were evaluated for height of aerial part, total root length and number of internodes and leaves. The treatment of breaking seed dormancy associated with ethanol 70% promotes efficient desinfestation of grapia seeds, even without NaOCl immersion. Seeds maintained in dark showed the lowest TMG, the greatest IVG and the highest aerial part. The WPM medium supplemented with 4 g L-1 of agar and 10 g L-1 of sucrose is indicated for maintenance of grapia seedlings. For multiplication, segments of different positions (basal, medium and apical) were inoculated in WPM medium supplemented with 0; 2.2; 4.4; 6.6 or 8.8 μM of 6-benzylaminopurine (BAP). Nodal segments were inoculated in WPM medium supplemented with 0; 2.2; 4.4; 6.6 or 8.8 μM of BAP and 0 and 1.5 g L-1of activated charcoal. Nodal segments were also inoculated in WPM medium supplemented with 0; 2.3; 4.6; 6.9 and 9.2 μM of kinetin (KIN) and 0 and 1.5 g L-1of activated charcoal. The three experiments were evaluated at 30 days for the presence of callus, number and total length of sprouts, number of leaves, rooting percentage, and number and total length of roots. Microstumps were maintained in WPM medium with 0; 2.2; 4.4; 6.6 and 8.8 μM of BAP and 0 and 1.5 g L-1of activated charcoal. After 30 days, microstumps were subcultured in WPM medium with 1.5 g L-1 of activated charcoal. After 30 days of cultivation in BAP and subcultivated in activated charcoal, microstumps were evaluated for survival, percentage of sprouting, number and total length of sprouts and number of internodes and leaves. Basal segments showed the greatest number and length of sprouts. The WPM medium supplemented with 6.6 μM of BAP resulted in the greatest number of sprouts. The activated charcoal reduced callus formation and favored root formation. KIN didnot favor sprout formation. The WPM medium supplemented with 8.8 μM of BAP increases the number of sprouts and leaves in microstumps subcultivated in WPM medium with 1.5 g L-1 of activated charcoal. / O objetivo deste estudo foi avaliar a germinação e multiplicação in vitro de grápia, visando a produção de mudas e a conservação de genótipos selecionados. Para a desinfestação, as sementes passaram primeiramente pelo tratamento de quebra de dormência tegumentar com ácido sulfúrico (H2SO4) concentrado por 20 min. Posteriormente foram avaliadas as concentrações de 0; 2,5 e 5,0% de hipoclorito de sódio (NaOCl) nos tempos de imersão de 5, 10 e 15 min. na desifestação das sementes. As sementes foram inoculadas em frascos de vidro contendo 5 mL de meio de cultura composto por água destilada, sacarose (30 g L-1) e ágar (7g L-1). Aos 30 dias foram avaliadas as porcentagens de germinação e desinfestação, o tempo médio de germinação (TMG) e o índice de velocidade de germinação (IVG). Sementes desinfestadas também foram inoculadas em meio de cultura WPM (Wood Plant Medium) suplementado com 4, 5 e 6 g L-1 de ágar, combinado com 10, 20 ou 30 g L-1 de sacarose e cultivadas no escuro durante os sete primeiros dias ou na luz durante todo o período. Aos 15 dias foram avaliadas a porcentagem de germinação, a altura da parte aérea (cm), o número de folhas e entrenós, o comprimento total das raízes (cm), o TMG e o IVG. As plântulas assépticas foram transplantadas para meio de cultura WPM, MS ou ½ MS. Aos 15 dias foram avaliadas quanto à altura da parte aérea (cm), o comprimento total das raízes (cm) e o número de segmentos nodais e de folhas. O tratamento de superação de dormência das sementes de grápia com (H2SO4) associado à pré-desinfestação com etanol 70% promoveram eficiente desinfestação das sementes, sem a necessidade do uso de NaOCl. Sementes mantidas no escuro apresentaram menor TMG, maior IVG e altura da parte aérea. O meio de cultura WPM suplementado com 4 g L-1 de ágar e 10 g L-1 de sacarose é indicado para a conservação in vitro das plântulas de grápia. Para a multiplicação, segmentos de diferentes posições da parte aérea da plântula (basal, mediana e apical) foram inoculados em meio de cultura WPM acrescido de 0; 2,2; 4,4; 6,6 ou 8,8 μM de 6-benzilaminopurina (BAP). Segmentos nodais foram inoculados em meio de cultura WPM suplementado com 0; 2,2; 4,4; 6,6 ou 8,8 μM de BAP e com 0 e 1,5 g L-1 de carvão ativado. Segmentos nodais também foram inoculados em meio de cultura WPM acrescido de 0; 2,3; 4,6; 6,9 e 9,2 μM de cinetina (KIN) e de 0 e 1,5 g L-1 de carvão ativado. Os três experimentos foram avaliados aos 30 dias quanto à presença de calo, o número e comprimento total dos brotos (cm), o número de folhas, a porcentagem de enraizamento e o número e comprimento total das raízes (cm). Microcepas foram mantidas em meio de cultura WPM com 0; 2,2; 4,4; 6,6 e 8,8 μM de BAP e 0 e 1,5 g L-1 de carvão ativado. Após 30 dias de cultivo as microcepas foram subcultivadas em meio de cultura WPM suplementado com 1,5 g L-1 de carvão ativado. Aos 30 dias de cultivo ou de subcultivo as microcepas foram avaliadas quanto à sobrevivência, a porcentagem de brotação, o número e comprimento total dos brotos (cm) e o número de segmentos nodais e de folhas. Segmentos basais apresentaram o maior número e comprimento dos brotos. A adição de 6,6 μM de BAP proporcionou o maior número de brotos. O carvão ativado reduziu a formação de calo e favoreceu o enraizamento. A KIN não favoreceu a formação de brotos. A adição de 8,8 μM de BAP ao meio WPM resulta no maior número de brotos em microcepas subcultivadas em meio WPM com 1,5 g L-1 de carvão ativado.

The present thesis investigates the electronic and structural properties of adenine, cytosine, and guanine layers on hydrogen passivated silicon (111)(7x7). The (7x7) reconstruction of the silicon surface was achieved by direct current heating of the samples in UHV conditions. After in situ hydrogen passivation layers of the DNA bases were prepared in different thicknesses by means of organic molecular beam deposition, all samples were characterized employing valence band and core level photoemission spectroscopy. Additionally the near edge x-ray absorption fine structure of the DNA base layers was investigated. A detailed and consistent picture of structural and electronic properties of the nucleotide bases in the solid state could be developed by comparison of measurements and DFT/B3LYP calculations.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Computer modeling of enzymes is a valuable complement to experiments. Quantum chemical studies of enzymatic reactions can provide a detailed description of the reaction mechanism and elucidate the roles of various residues in the active site. Different reaction pathways can be analyzed, and their feasibility be established based on calculated energy barriers. In the present thesis, density functional theory has been used to study the active sites and reaction mechanisms of three different enzymes, cytosine deaminase (CDA) from Escherichia coli, ω-transaminase from Chromobacterium violaceum (Cv-ωTA) and dinitrogenase reductase-activating glycohydrolase (DraG) from Rhodospirillum rubrum. The cluster approach has been employed to design models of the active sites based on available crystal structures. The geometries and energies of transition states and intermediates along various reaction pathways have been calculated, and used to construct the energy graphs of the reactions. In the study of CDA (Paper I), two different tautomers of a histidine residue were considered. The obtained reaction mechanism was found to support the main features of the previously proposed mechanism. The sequence of the events was established, and the residues needed for the proton transfer steps were elucidated. In the study of Cv-ωTA (Paper II and Paper III), two active site models were employed to study the conversion of two different substrates, a hydrophobic amine and an amino acid. Differences and similarities in the reaction mechanisms of the two substrates were established, and the role of an arginine residue in the dual substrate recognition was confirmed. In the study of DraG (Paper IV), two different substrate-binding modes and two different protonation states of an aspartate residue were considered. The coordination of the first-shell ligands and the substrate to the two manganese ions in the active site was characterized, and a possible proton donor in the first step of the proposed reaction mechanism was identified. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Dans ce travail, trois types de lésions des bases de l'ADN ont été étudiés d'un point de vue théorique à l'aide de méthodes de chimie quantique basées sur la théorie de la fonctionnelle de la densité : la désamination spontanée de la cytosine et de ses dérivés, la formation de lésions tandem induites par des radicaux hydroxyles en milieu anaérobie et la formation de dimères de bases pyrimidiques suite à une exposition à un rayonnement ultraviolet. L'utilisation complémentaire de méthodes quantitatives statiques permettant d'explorer en partie les surfaces d'énergie potentielle associées à une réaction chimique et de la « DFT conceptuelle » ont permis d'obtenir des renseignements quant aux mécanismes réactionnels mis en jeu et de rationaliser des différences de réactivité entre bases nucléiques vis-à-vis de la formation d'un même type de lésion.<br /> Parallèlement à ces études, une réflexion a été menée concernant le concept de mécanisme concerté asynchrone, en particulier en termes de sens physique de l'état de transition, de respect du principe de dureté maximum, et de détermination du nombre de processus primitifs impliqués. Enfin, un nouvel indice de réactivité locale a été développé, pertinent pour décrire la réactivité de systèmes moléculaires dans un état excité.

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