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Anthraquinone (AQ) has been used in electron transfer studies in DNA. The focus of this dissertation is the synthesis of conjugates between AQ derivatives and 2’-deoxyadenosine (dA), which can be used to induce adenine oxidation in DNA. Different AQ derivatives were attached to dA via ethynyl or ethanyl linkers. If incorporated into DNA, these short linkers should enable regiocontrol for electron transfer from adenine within the DNA duplex structure. The challenge in working with anthraquinone-2’-deoxynucleosides conjugates is that they and their intermediates are insoluble in water and only sparingly soluble in most organic solvents. A strategy used to overcome this problem was the use of either tert-butyldiphenylsilyl (TBDPS) or 4’,4-dimethoxytrityl (DMTr) 5’-protected deoxynucleosides as starting materials. A water-soluble, ethynyl-linked AQ-dA conjugate with a 3’-benzyl hydrogen phosphate was synthesized using DMTr protection. The DMTr group was not stable to the hydrogenation required to make the ethanyl-linked AQ-dA conjugate with 3’-benzyl hydrogen phosphate. Hence the latter was synthesized starting with the TBDPS protecting group. Both of these syntheses were based on the Pd coupling between ethynylanthraquinone and 8-bromodeoxyadenosine derivatives. New conjugates between AQ and A, in which the AQ moieties have been modified with formyl, trifluoroacetyl and methyl ester groups as electron withdrawing substituents were also synthesized. The synthesis of these AQ-dA conjugates was based on Pd coupling between bromoanthraquinone and 8-ethynyldeoxyadenosine derivatives. This route avoided the use of ethynylanthraquinone derivatives that had extremely low solubility and photoinstability. Other anthraquinones with electron withdrawing groups (which should provide enhanced driving force to enable respective AQ derivative to oxidize adenine) were synthesized as models. Cyclic voltammetry showed that the conjugate with the two ester groups and ethynyl linker was the most easily reduced of the derivatives synthesized. Conjugates between AQ and dU were also synthesized. Those conjugates can potentially be used to oxidize guanine or adenine or they can be used as a deep trap for an electron in reduced DNA.

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Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 3 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Made available in DSpace on 2016-12-23T14:37:36Z (GMT). No. of bitstreams: 1 dissertacao.pdf: 478233 bytes, checksum: 59f53a87f76279d2bcf9fe36ad7f2a18 (MD5) Previous issue date: 2006-02-23 / Experiments were accomplished in establishment and multiplication medium with the objective of evaluating the micropropagation of the papaya tree (Carica papaya L.) Hybrid 'Tainung 01', generation F1, being used apical sections and apical sections of lateral buds of juvenile plants, cultivated in greenhouse. In the MS establishment medium, being used apical sections, different combinations of growth regulators were tested NAA (0.093; 0.931 and 1.862 mg L-1) and kinetin (5.38; 10.76 and 21.52 mg L-1) for identification of the best relationship for the survival, reactivity and callus mass. With the use of 0.093 mg L-1 of NAA combined with 5.38 mg L-1 of kinetin was obtained the best rosette of leaves formation with 100% of cultures reactivate, indicating there to be adaptation of the explants in vitro, besides to smallest callus formation, could be proven with the good reaction of these explants in MS multiplication medium with NAA to 0.093 mg L-1 and BAP to 0.45 mg L-1 during five subculture, with rate of constant multiplication of 5.288:1. In a second stage, with the use of apical sections of lateral buds, the adenine sulfate was evaluated in the multiplication medium. For the preparation of the establishment medium the best combination of the growth regulators was used NAA and kinetin obtained previously, while for the multiplication medium NAA was used to 0.093 mg L-1 and BAP to 0.45 mg L-1, and different levels of adenine sulfate (0; 30; 60; 90 and 120 mg L-1), associated with removal or not of the leaves of the rosette of leaves formed in the establishment medium. The tufts of shoots were subculture every 30 days in smaller tuft and, at the end of the fourth subculture they were made the evaluations of the fresh mass of the aerial part, number of capable shoots (&#8805; 5 mm) and no capable (< 5 mm) to the promoting root formation, and the visual aspect of the tufts of shoots. The best answers for the number of capable shoots to the promoting root formation (&#8805; 5 mm) they were found with the presence of the leaves, for the treatments 0, 30, 60 and 90 mg L-1 of adenine sulfate. It is recommended to use 30 mg L-1 of adenine sulfate in the multiplication medium for the quality and homogeneity of the produced aerial parts, with prolonged shoots and long petiole, with good pattern for subsequent promoting root formation. / Foram realizados experimentos em meio de estabelecimento e de multiplicação com o objetivo de avaliar a micropropagação do mamoeiro (Carica papaya L.) Híbrido Tainung 01 , geração F1, utilizando-se segmentos apicais e segmentos apicais de brotações laterais provenientes das plantas juvenis cultivadas em casa de vegetação. No meio de estabelecimento MS, utilizando-se segmentos apicais, foram testadas diferentes combinações dos reguladores de crescimento ANA (0,093; 0,931 e 1,862 mg L-1) e Cinetina (5,38; 10,76 e 21,52 mg L-1) para identificação da melhor relação para a sobrevivência, a reatividade e a massa de calo. Com a utilização de 0,093 mg L-1 de ANA combinado com 5,38 mg L-1 de Cinetina, obteve-se a melhor formação de roseta foliar com 100% de culturas reativas, indicando haver adaptação dos explantes in vitro, além da menor formação de calo, podendo ser comprovado com a boa reação destes explantes em meio de multiplicação MS com ANA a 0,093 mg L-1 e BAP a 0,45 mg L-1, durante cinco subcultivos, com taxa de multiplicação constante de 5,288:1. Numa segunda etapa, com a utilização de segmentos apicais de brotações laterais, avaliou-se a multiplicação em meio MS com diferentes níveis de sulfato de adenina. Para o preparo do meio de estabelecimento, utilizou-se a melhor combinação dos reguladores de crescimento ANA e Cinetina obtidos anteriormente, enquanto que para o meio de multiplicação, usou-se ANA a 0,093 mg L-1, BAP a 0,45 mg L-1 e diferentes níveis de sulfato de adenina (0; 30; 60; 90 e 120 mg L-1), associados com remoção ou não das folhas da roseta foliar formada no meio de estabelecimento. Os tufos de ramos foram subcultivados a cada 30 dias em tufos menores, e ao final do quarto subcultivo, foram feitas as avaliações da massa fresca da parte aérea, número de ramos aptos (&#8805; 5 mm) e não aptos (< 5 mm) ao enraizamento e o aspecto visual dos tufos de ramos. As melhores respostas para o número de ramos aptos ao enraizamento (&#8805; 5 mm) foram encontradas com a presença das folhas para os tratamentos 0, 30, 60 e 90 mg L-1 de sulfato de adenina. Recomenda-se utilizar 30 mg L-1 de sulfato de adenina no meio de multiplicação pela qualidade e homogeneidade das partes aéreas produzidas, com ramos alongados e pecíolos longos, com bom padrão para posterior enraizamento.

A oxidação do resíduo de cisteína 56 (ANT-cys56) do transportador de nucleotídeos de adenina (ANT) é descrita como evento crítico da Transição de Permeabilidade Mitocondrial (TPM), fenômeno caracterizado pela sensibilidade ao fármaco imunossupressor ciclosporina A (CsA), responsável pela ligação e inibição do componente promotor da abertura do Poro de Transição de Permeabilidade (PTP), a enzima peptidil-prolil-cis trans isomerase (cyp D). Aspectos bioquímico-estruturais do ANT, das cardiolipinas (CDL) que envolvem o transportador e da cyp D na TPM foram avaliados por meio de ensaios turbidimétricos de inchamento mitocondrial e estado conformacional do ANT em mitocôndrias isoladas de fígado de rato, associados a abordagens de química computacional para análises de campos de interação molecular (MIF) e dinâmica molecular (MD), visando a predição de eventos envolvidos na abertura do PTP. As análises computacionais revelaram aumento da mobilidade relativa do ANT-cys56, como resultado da interação preferencial do Ca2+ com a molécula de CDL ligada à hélice 4 do transportador, enquanto que a inversão da configuração do resíduo de prolina do ANT (ANT-pro61) potencializou o efeito induzido por Ca2+. A presença de ADP no interior do ANT preveniu o aumento da mobilidade relativa do ANT-cys56 promovida pelo Ca2+, enquanto que a inversão da configuração do ANT-pro61, de trans para cis, potencializou o efeito promovido pelo Ca2+ na mobilidade relativa do ANT-cys56, de forma insensível ao nucleotídeo. Os ensaios com mitocôndrias isoladas demonstraram que o Ca2+ induz a conformação c do ANT e promove abertura do PTP, de forma sensível à CsA e ADP. A presença de cyp D estabilizou a conformação c do ANT induzida por Ca2+, sendo que Atractilosídeo (ATR) tornou o efeito parcialmente insensível aos inibidores da TPM. Os resultados sugerem que a abertura do PTP induzida por Ca2+ envolve a mudança conformacional do ANT para o estado c, cuja estabilização é obtida pela cyp D na função de inversão do ANT-pro61, com base na avaliação da mobilidade relativa do ANT-cys56 parcialmente sensível ao ADP. / Oxidation of the Adenine Nucleotide Translocase (ANT) cysteine residue 56 (ANT-cys56) is potentially involved in Ca2+-induced Mitochondrial Permeability Transition (MPT), a process which is prevented by cyclosporine A (CsA), due to its inhibition of Permeability Transition Pore (PTP) opener component, the peptidyl-prolyl cis-trans isomerase cyclophylin D (cyp D). The main aspects of ANT, cardiolipins (CDL) and cyp D on Ca2+-induced PTP opening were addressed by employing light scattering techniques in isolated rat liver mitochondria to assess both ANT conformational change and mitochondrial swelling in association with computational chemistry analysis of Molecular Interaction Fields (MIF) and Molecular Dynamics (MD) for PTP events predictions. Computational analysis revealed that Ca2+ interacts preferentially with the ANT surrounding CDL bound to the H4 helix of the carrier and weakens the CDL/ANT interactions accounting for the ADP-sensitive increase of ANT-cys56 relative mobility while ANT-pro61 cis to trans configuration inversion intensified the Ca2+ effect in a ADP-insensitive way. The ANT conformation and mitochondrial swelling analyses demonstrated that Ca2+ induces conformation c of ANT and opens PTP in a CsA- and ADP-sensitive way. Cyp D stabilizes Ca2+-induced ANT conformation c, whereas ATR renders a PTP opening less sensitive to the inhibition by CsA or ADP. The results suggest that Ca2+-induced PTP opening involves ANT conformation c change supported by a cyp D-induced trans to cys ANT-pro61 configuration inversion based on the relative mobility of ANT-cys56, in a ADP-sensitive manner.

O excesso de adenina na dieta (ADE) promove precipitação intrabular de cristais, levando a uma nefrite intersticial progressiva com perda de função renal. Estudo recente demonstrou que esse processo requer ativação do sistema NF-kB. No presente estudo investigamos o possível envolvimento de outros componentes da imunidade inata, além do NF-kB. Verificamos também a hipótese de que a nefropatia associada aos cristais continua a progredir mesmo depois de cessada a sobrecarga de adenina. Foram estudados ratos Munich-Wistar machos e adultos sem tratamento (C) ou recebendo 0.5% de ADE na dieta. Após 1 semana, a ADE foi removida da dieta e os animais foram seguidos por 4 ou 24 semanas. A administração de ADE por 1 semana promoveu uma inflamação intersticial aguda, com perda de função renal, alteração da pressão caudal, sem alterações glomerulares. Os mediadores da imunidade inata, como TLR2, TLR4, inflamassoma NLRP3, IL1beta e IL-6, apresentaram-se ativados sem, no entanto, ativar o sistema NF-kB. Após cessada a sobrecarga de ADE, a inflamação persistiu, com infiltração por macrófagos, expressão elevada de AngII, deposição progressiva de colágeno e, na fase mais tardia, glomeruloesclerose, caracterizando um processo inflamatório crônico, autônomo, que não contou com a participação do eixo NLR/IL1beta. Em contraste, o sistema NF-kB foi ativado, sendo um dos possíveis estímulos a produção intra-renal de AngII. Dois mecanismos patogênicos podem ser identificados neste estudo: 1) agudo, associado à ativação do eixo NLR-IL1beta; 2) crônico, associado à produção de AngII renal e à ativação do sistema NF-kB / Excess adenine in the diet (ADE) promotes intratubular crystal precipitation, leading to progressive interstitial nephritis and loss of renal function. A recent study has shown that this process requires activation of the NF-kB system. In the present study we investigated the possible involvement of other components of innate immunity, in addition to NF-kB, as well as whether nephropathy associated with excess adenine continues to progress even after dietary cessation. Male Munich-Wistar rats without treatment (C) or receiving 0.5% of ADE in the diet were studied. After 1 week, ADE was removed from the diet and the animals were followed for 4 or 24 weeks. Administration of ADE for 1 week promote acute interstitial inflammation, with loss of renal function, alteration of caudal pressure, without glomerular changes. Mediators of innate immunity, such as TLR2, TLR4, NLRP3 inflamassome, IL1beta and IL-6 , were shown to be activated, with no apparent activation of the NF-kB system. In the late phases of the model, the inflammation persisted, with significant infiltration by macrophages, high expression of AngII, progressive collagen deposition and glomerulosclerosis, characterizing a chronic, autonomic inflammatory process that did not involve the participation of the NLR/IL1beta axis. By contrast, the NF-kB system was activated, with intra-renal AngII production as a possible stimulus. Two mechanisms operated this study: 1) an acute one, associated with activation of the NLR-IL1beta axis; 2) a chronic one, associated with intrarenal AngII production and NF-kB activation

Le vieillissement musculaire est caractérisé par des pertes progressives de masse et de fonction. Des altérations de l’énergétique mitochondriale pourraient être impliquées dans ce processus. Dans cette thèse, l’analyse modulaire du contrôle métabolique a été appliquée chez le rat à différents niveaux d’intégration pour caractériser les effets du vieillissement sur la fonction mitochondriale. Combinée à la spectroscopie RMN du 31P, cette approche a permis de montrer in vivo dans le muscle gastrocnemius une diminution de la réponse de phosphorylation oxydative mitochondriale face à des variations de concentration des intermédiaires énergétiques chez les rats âgés (21 mois vs. 6 mois). Suivant les principes de l’analyse Top-Down, les propriétés de la phosphorylation oxydative ont été étudiées sur des mitochondries isolées à partir du muscle gastrocnemius. La capacité maximale de production d’ATP est réduite chez les rats âgés, alors que le rendement maximal (rapport ATP/O) de la phosphorylation oxydative reste inchangé. L’application de l’analyse modulaire in vitro a révélé chez les rats âgés une augmentation de la réponse (élasticité) du module phosphorylation face à des variations du potentiel de membrane. Cette élasticité plus élevée explique la modification du schéma de contrôle de la phosphorylation oxydative pour des activités de phosphorylation compatibles avec celles étudiées in vivo. Elle explique également le plus faible potentiel de membrane généré par les mitochondries de rats âgés pour un même niveau d’activité de phosphorylation. De nombreux processus pourraient de fait être affectés : production de radicaux libres, homéostasie calcique, voies de signalisation impliquées dans le contrôle de la masse musculaire. Les modifications des propriétés fonctionnelles de l’ANT démontrées dans cette thèse sont en mesure d’expliquer, au moins en partie, les modifications de l’énergétique mitochondriale révélées à la fois in vitro et in vivo chez les rats âgés. / Skeletal muscle aging is characterized by a progressive loss of muscle mass and function. Involvement in this process of an impaired mitochondrial bioenergetics was proposed but is still extensively debated. The aim of this thesis was to take adventage of the capabilities of modular control analysis approach to get better insights in the effects of aging on mitochondrial function. We first studied the integrated muscle energetics in adult (6 months) and aged (21 months) rats using the modular control analysis approach combined with non-invasive 31P NMR spectroscopy measurements of energetic intermediates. The in vivo activation of mitochondrial oxidative phosphorylation in response to an increase in ATP demand was markedly decreased in the gastrocnemius muscle of aged rats. To further define the effects of aging on mitochondrial energetics, we thus studied the oxidative phosphorylation in mitochondria isolated from the gastrocnemius muscle. Maximal oxidative phosphorylation capacity is clearly reduced in aged rats, while mitochondrial efficiency is unaffected. Application of modular control analysis to the study of oxidative phosphorylation revealed an increased sensitivity (elasticity) of the phosphorylation module in response to changes in membrane potential in aged rats. This increased elasticity is responsible for a modified control pattern of oxidative phosphorylation under low phosphorylation activities. Interestingly these low activities certainly correspond to those we studied in vivo. This increased elasticity of the phosphorylation module is responsible for a modified mitochondrial response toward changes in cellular ATP demand, leading to a decreased membrane potential, which may in turn affect many cellular processes such as ROS production, calcium homeostasis and some signaling pathways involved in the control of muscle mass. The modified ANT properties evidenced in this thesis certainly explain, at least in part, the modified mitochondrial energetics reaveled both in vitro and in vivo in aged rats.

Nucleoside 5'-Diphosphate Kinase (NdPK EC 2.7.4.6) is an enzyme (phosphotransferase) with extraordinary characteristics due to its unique ability to transfer phosphor groups to interconvert all nucleoside di- and triphosphates as a part of the DNA synthesis. Due to Biovica International AB's use of signaling and complementing enzymes in their in vitro diagnostic (IVD) test assays for Thymidine Kinase activity, an investigation was proposed to evaluate NdPK, which is a complementing enzyme in the assay. The aim of the study was to evaluate the enzymatic turnover of the enzyme NdPK with a spectrophotometric assay to obtain the specific activity (Units/mg solid protein). To determine the specific activity, enzyme kinetic methodology was applied, including the Michaelis-Menten model. In this study, the method is proposed as a general internal control procedure for the company, as a tool for function control of the different purchased enzymes used in their products in development. Results from the study reflects the different methods used to gain the specific activity for NdPK, where they were compared with the already specified specific activity from the manufacturing company. The results were auspicious, but before the method's authorization as an internal quality procedure, a few amendments are in mind. For instance, determining a method for the graphical readings, validating the method for quality control, and investigating if the method is applicable to other complementing enzymes. In conclusion, the method for determining the specific activity of the enzyme NdPK can be done, by executing the procedure of colorimetric enzyme assay.