Most eukaryotic mRNAs have a sequence of polyadenylic acid [poly(A)] at their 3'-termini. Although it has been almost two decades since the discovery of these poly(A) tracts, their function(s) have yet to be clarified. Earlier results from our laboratory led us to propose that poly(A) has a role in translation. More specifically, we proposed that an interaction of the cytoplasmic poly(A)-binding protein (PABP) with a critical minimum length of poly(A) facilitates the initiation of translation of poly(A)+, but not poly(A)-, mRNAs. The results of several different experimental approaches have provided evidence which indirectly supports this hypothesis. These results include: 1) the correlation of specific changes in mRNA poly(A) tail length with translational efficiency in vivo and in vitro; 2) correlations between the abundance and stability of PABPs and the rate of translational initiation in vivo and in vitro; and 3) the demonstration that exogenous poly(A) is a potent and specific inhibitor of the in vitro translation of poly(A)+, but not poly(A)-mRNAs. To evaluate the hypothesis that the 3'-poly(A) tract of mRNA plays a role in translational initiation, we have constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs are translated, degraded, recruited into polysomes, and assembled into mRNPs or intermediates in the translational initiation pathway. Relative to mRNAs which are polyadenylated, we find that poly(A)- mRNAs have a reduced translational capacity which is not due to an increase in their decay rates, but is attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A)- mRNAs affects a late step in translational initiation, is distinct from the phenotype associated with cap-deficient mRNAs, and results in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibits translation from capped poly(A)+ mRNAs, but stimulates translation from capped poly(A)- mRNAs. We suggest that poly(A) is the formal equivalent of a transcriptional enhancer, i.e., that poly(A)-binding protein (PABP) bound at the 3'-end of mRNA may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5'-end.

Submitted by Natalia de Souza Gonçalves (natalia.goncalves@ufpe.br) on 2016-10-10T12:17:42Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Maralise-081214-VERSÃO corrigida biblioteca ATUALIZADA.pdf: 9951245 bytes, checksum: be34a8b4340d6e8daffcff6422e39f13 (MD5) / Made available in DSpace on 2016-10-10T12:17:42Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Maralise-081214-VERSÃO corrigida biblioteca ATUALIZADA.pdf: 9951245 bytes, checksum: be34a8b4340d6e8daffcff6422e39f13 (MD5) Previous issue date: 2014-12-22 / síndrome de imunodeficiência adquirida (AIDS) é uma doença infecciosa e desde que foi descoberta nos EUA, em 1981, a AIDS se espalhou rapidamente, sendo considerada uma epidemia mundial caracterizada por uma imunodeficiência severa, infecções oportunistas, neoplasia e um resultado fatal. Apesar de já ser empregado a multiterapia anti-HIV, tal tratamento farmacológico traz como inconveniente resistência já relatada aos antivirais e a toxicidade provocada por esses fármacos. Dessa forma, é um desafio para os pesquisadores a busca por fármacos mais eficazes e menos tóxicos. A meta é minimizar o impacto dessa doença no Brasil e no mundo. Para tanto, conhecendo-se as propriedades dos tionucleosídeos, objetivou-se, no presente trabalho, a síntese, a caracterização e a avaliação da atividade antiviral e antitumoral de novos análogos tionucleosídeos da 5-metiluridina. A síntese dos análogos foi efetuada a partir de 2’,2-anidrotimidina. Reação deste composto com 2-(trimetilsilil)etanotiol na presença de carbonato de potássio, seguida da mesilação das hidroxilas de 3’ e 5’ forneceu o derivado dimesilado correspondente que, por reação com benzoato de sódio forneceu o tionucleosídeo 2’, 3’-didesidro correspondente, benzoilado em C-5’. Remoção do grupo benzoíla com hidróxido de potássio/metanol forneceu o nucleosídeo insaturado 2’,3’-didesidro-2’,3’-didesoxi-2’-(2-trimetilsilil)etiltio)timidina. Substituindo-se o 2-(trimetilsilil)etanotiol pelo tiometóxido de sódio o tionucleosídeo insaturado correspondente foi obtido. Quando o derivado anidro foi submetido a reação com hidreto de sódio em DMF, seguida da reação do epóxido formado com 2-(trimetilsilil)etanotiol, obteve-se um intermediário de configuração D-xilo, contendo a cadeia sulfurada em C-3’. Reação desse composto com carbonato de difenila em DMF, em presença de bicarbonato de sódio forneceu o derivado 2’,3’-insaturado correspondente, contendo o grupo fenoxicarbonila em C-5’. Esse intermediário foi convertido no tionucleosídeo insaturado correspondente de forma semelhante à descrita para a obtenção de 2’,3’-didesidro-2’,3’-didesoxi-2’-(2-trimetilsilil)etiltio)timidina. Também nesse caso, a substituição de 2-(trimetilsilil)etanotiol pelo tiometóxido de sódio levou, ao final da síntese, ao tionucleosídeo insaturado contendo o grupo metiltio em C-3’. Os produtos finais e alguns intermediários foram avaliados em testes de atividade citotóxica contra linhagem de células tumorais A549 (linhagem humana de câncer de pulmão). O intermediário 2’, 3’-didesidro benzoilado em C-5’ e derivado 2’,3’-insaturado contendo o grupo fenoxicarbonila em C-5’ destacaram-se entre os mais ativos da série, apresentando, respectivamente CC50 32,8 μM e 85,5 μM. Os testes de atividade contra o herpes vírus simplex humano tipo I (HSV-I) estão em andamento. Os testes contra o HIV serão realizados oportunamente. / Acquired immune deficiency syndrome (AIDS) is an infectious disease and since its discovery in the USA in 1981, AIDS has spread rapidly and is considered a global epidemic characterized by severe immunodeficiency, opportunistic infections, cancer and a fatal outcome. Despite being already employed an anti-HIV multi-therapy, pharmacotherapy has drawbacks like viral resistance to antiviral drugs and drug toxicity. Thus, it is a challenge for researchers the search for more effective and less toxic drugs.The goal is to minimize the impact of this disease in Brazil and worldwide. To that end, knowing the properties of thionucleosides, the aim of the present work was the synthesis, characterization and evaluation of antiviral and antitumor activity of novel thionucleosides analogues of 5-methyluridine. Analogues synthesis was performed from 2', 2-anhydrothymidine. Reaction of this compound with 2- (trimethylsilyl)ethanethiol in the presence of potassium carbonate, followed by mesylation of hydroxyl groups at 3' and 5' provided the corresponding mesylated derivative, which, by reaction with sodium benzoate, provided the corresponding 2',3'-didehydro, benzoylated at C-5'. Removal of benzoyl group with potassium hydroxide/methanol gave the unsaturated nucleoside 2',3'- didehydro-2',3'-dideoxy-2'-(2-trimethylsilyl)ethylthio)thymidine. Replacing 2- (trimethylsilyl)ethanethiol by sodium thiomethoxide, the corresponding unsaturated thionucleoside was obtained. When the anhydro derivative was subjected to reaction with sodium hydride in DMF, followed by reaction of the epoxide formed with 2-(trimethylsilyl)ethanethiol, a D-xylo intermediate, with the side chain at C-3' was obtained. Reaction of this compound with diphenyl carbonate in DMF in the presence of sodium bicarbonate provided the corresponding 2',3'-unsaturated derivative containing phenoxycarbonyl group at C-5'. This intermediate was converted to the corresponding unsaturated thionucleoside in a similar way to that described to obtain 2', 3'-didehydro-2',3'- dideoxy-2'- (2-trimethylsilyl)ethylthio)thymidine. Also in this case, replacing 2- (trimethylsilyl)ethanethiol by sodium thiomethoxide led to the unsaturated thionucleoside containing methylthio group at C-3'. Final and some intermediate compounds were evaluated for cytotoxicity activity against tumor cell line A549 (human lung cancer strain). 2',3'-didehydro thionucleoside benzoylated at C-5' and 2',3'-unsaturated thionucleoside containing phenoxycarbonile group at C-5' derivative were the most active compounds in this series, presenting CC50 values of 32.8 μM and 85.5 μM, respectively. Tests for antiviral activity against human herpes simplex virus type I (HSV-I) are in progress. Anti-HIV screening will be conducted in the near future.

The strain promoted azide alkyne cycloaddition (SPAAC) of azido nucleobase modified nucleosides and nucleotides with cyclooctynes to give fluorescent triazoles has been relatively unexplored. Thus, SPAAC between azido-nucleobases and various cyclooctynes in aqueous solution at ambient temperature resulted in the efficient formation (3 min - 2 h) of triazole products with inherent fluorescent properties. The 2- and 8-azidoadenine nucleosides reacted with fused cyclopropyl cyclooctyne, dibenzylcyclooctyne or monofluorocyclooctyne to produce click products functionalized with hydroxyl, amino, N-hydroxysuccinimide, or biotin moieties. The previously unexplored 5-azidouridine and labile 5-azido-2'-deoxyuridine were similarly converted to the analogous triazole products in quantitative yields in less than 5 minutes. The 8-azido-ATP quantitatively afforded the triazole product with fused cyclopropyl cyclooctyne (3 h). Addition of a triazole ring at the 2 or 8 position of adenine or 5-position of uracil induces fluorescent properties which were used for direct imaging with fluorescent microscopy in MCF-7 cancer cells without the need for traditional fluorogenic reporters. Fluorescent lifetime imaging microscopy of the click adducts in live cells were used to determine the lifetime of each fluorophore in the cellular nuclei demonstrating the potential utility of the synthesized triazole adducts for dynamic measuring and tracking of events inside single living cancer cells. The SPAAC methodology developed has also been applied to study the cellular targets in protozoal parasite, Trichomonas vaginalis and bacteria, Pseudomonas aeruginosa. The 9-(2-deoxy-2-fluoro-β,D-arabino-furanosyl)adenine (arabino-F-Ado) was modified with an azido moiety at the C8 position for use in click chemistry. Tagging and subcellular localization studies using azido modified arabino-F-Ado could provide insight into the mechanism of action of arabino-F-Ado. An activated analogue of S-adenosyl-L-methionine (SAM) with an EnYn group on the sulfur instead of a methyl group was prepared to study the transfer of the methyl group from SAM. I found that the EnYn group was transferred from SAM to a guanosine on tRNA by methytransferase Trm1. Thus, AdoEnYn is a competitive inhibitor of SAM and can be incorporated into tRNA in place of SAM.

Orientadores: Yuji Takahata, Rogerio Custodio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-14T14:03:54Z (GMT). No. of bitstreams: 1 Oliveira_KesleyMoraesGodinhode_D.pdf: 5766531 bytes, checksum: 5d60e17b9a5062a054d9bb7de66054f4 (MD5) Previous issue date: 2009 / Resumo: O objeto de estudo desta tese é a forma mais severa e letal de Leishmaniose: a Leishmaniose Visceral, LV, cujo principal agente etiológico é a espécie Leishmania donovani. O objetivo deste trabalho é o desenvolvimento de modelos Quantitativos das Relações Estrutura-Atividade para duas séries de compostos com atividade antileishmaniose contra formas amastigotas de Leishmania donovani. A primeira série envolve vinte e um análogos de nucleosídeos pirazolo-pirimidínicos e a segunda série compreende oito antifúngicos. Para garantir a robustez dos modelos, além dos compostos que compõem as respectivas séries, foram selecionadas moléculas com conhecida atividade contra LV para compor um conjunto de teste e avaliar a capacidade de previsão dos respectivos modelos. Para a série dos nucleosídeos foram desenvolvidos modelos de regressão logística e árvore de classificação. Em ambas as abordagens os descritores Mor26v e o GAP(HOMO, HOMO-1) se mostraram relevantes para a explicação da atividade leishmanicida destes compostos. O modelo de regressão logística atingiu 90,5% para acurácia de classificação para o conjunto de trabalho e 58% para o conjunto de teste após a análise do domínio de aplicabilidade do modelo. O modelo para árvore de classificação alcançou 95% para acurácia de classificação para o conjunto de trabalho e 83% para o conjunto de teste. Para a série dos antifúngicos foi utilizado um modelo de regressão linear múltipla onde a energia eletrônica e a área da superfície polar se mostraram importantes para a atividade leishmanicida da série. Os valores previstos exibem 98% de correlação com os valores experimentais. Os valores encontrados para o conjunto de teste também estão de acordo com a literatura. Finalmente, novos compostos foram propostos para síntese e avaliação da atividade leishmanicida. / Abstract: The object of study of this thesis is the most severe and lethal form of leishmaniasis: visceral leishmaniasis, LV, whose main etiological agent is the species Leishmania donovani. The goal of this work is the development of Quantitative Structure-Activity Relationship models for two series of compounds presenting antileishmanial activity against amastigotes of Leishmania donovani. The first series includes twenty-one analogues of pyrazolo-pyrimidine nucleosides and the second series comprises eight antifungals. To ensure the robustness of the models, in addition to the compounds that make up their series, molecules with known activity against LV were selected to compose a testset and evaluate the predictive power of their models. For the series of nucleosides logistic regression models and tree classification were developed. In both approaches, the Mor26v and GAP(HOMO, HOMO-1) descriptors were relevant to explain the leishmanicidal activity of these compounds. The logistic regression model reached 90.5% for classification accuracy to the workingset and 58% for testset after analysis of the domain of applicability of the model. The classification tree model reached 95% for classification accuracy of the workingset and 86% for the testset. A multiple linear regression model was applied to the series of antifungal agents. The electron energy and polar surface area were important for the leishmanicidal activity of this series. The predicted values showed 98% of correlation with the experimental values. The values found for the testset are also in agreement with the literature. Finally, new compounds were proposed for synthesis and evaluation of leishmanicidal activity. / Doutorado / Físico-Química / Doutor em Ciências

The diverse biological properties exhibited by uridine analogues modified at carbon-5 of the uracil base have attracted special interest to the development of efficient methodologies for their synthesis. This study aimed to evaluate the possible application of vinyl tris(trimethylsilyl)germanes in the synthesis of conjugated 5-modified uridine analogues via Pd-catalyzed cross-coupling reactions. The stereoselective synthesis of 5-[(2-tris(trimethylsilyl)germyl)ethenyl]uridine derivatives was achieved by the radical-mediated hydrogermylation of the protected 5-alkynyluridine precursors with tris(trimethylsilyl)germane [(TMS)3GeH]. The hydrogermylation with Ph3GeH afforded in addition to the expected 5-vinylgermane, novel 5-(2-triphenylgermyl)acetyl derivatives. Also, the treatment with Me3GeH provided access to 5-vinylgermane uridine analogues with potential biological applications. Since the Pd-catalyzed cross-coupling of organogermanes has received much less attention than the couplings involving organostannanes and organosilanes, we were prompted to develop novel organogermane precursors suitable for transfer of aryl and/or alkenyl groups. The allyl(phenyl)germanes were found to transfer allyl groups to aryl iodides in the presence of sodium hydroxide or tetrabutylammonium fluoride (TBAF) via a Heck arylation mechanism. On the other hand, the treatment of allyl(phenyl)germanes with tetracyanoethylene (TCNE) effectively cleaved the Ge-C(allyl) bonds and promoted the transfer of the phenyl groups upon fluoride activation in toluene. It was discovered that the trichlorophenyl,- dichlorodiphenyl,- and chlorotriphenylgermanes undergo Pd-catalyzed cross-couplings with aryl bromides and iodides in the presence of TBAF in toluene with addition of the measured amount of water. One chloride ligand on the Ge center allows efficient activation by fluoride to promote transfer of one, two or three phenyl groups from the organogermane precursors. The methodology shows that organogermanes can render a coupling efficiency comparable to the more established stannane and silane counterparts. Our coupling methodology (TBAF/moist toluene) was also found to promote the transfer of multiple phenyl groups from analogous chloro(phenyl)silanes and stannanes.

Les O-glycoconjugués et les dérivés glycosidiques sont des composés naturels impliqués dans de nombreux processus biologiques. Cependant, leurs propriétés sont grevées par la médiocre stabilité in vivo de la liaison osidique. Il est donc intéressant de développer des mimes non hydrolysables. Nous nous sommes intéressés au remplacement de l’oxygène intracyclique par un groupement gem-difluorométhylène.La synthèse d’analogues difluorocarbocycliques de 5-désoxypentofuranoses et de 1-amino-5-désoxypentofuranoses a été décrite. La synthèse comporte une addition dePhSeCF2TMS sur des aldéhydes dérivés de sucres ou sur les tert-butanesulfinylimines correspondantes, suivie d’une cyclisation radicalaire. La diastéréosélectivité de ces deux étapes-clés a été étudiée, puis cette stratégie de synthèse a été appliquée à la synthèse deCF2-carbasucres, notamment l’analogue CF2-carbocyclique du D-arabinose. / O-Glycoconjugates and carbohydrate-based molecules are natural compoundsimplied in many biological processes. However, their properties are burdened by the low invivo stability of the osidic bond. It is thus interesting to develop non hydrolyzable mimetics.We were interested in the replacement of the intracyclic oxygen by a gem-difluoromethylenegroup.The synthesis of difluorinated carbocyclic analogues of 5-deoxypentofuranoses and1-amino-5-deoxypentofuranoses is described. The sequence involves an addition ofPhSeCF2TMS to carbohydrate-derived aldehydes or their corresponding tertbutanesulfinyliminesfollowed by a radical cyclization. The stereochemical outcome of these two key steps was studied, and then this strategy was applied to CF2-carbasugars, inparticular of the CF2-carbocyclic analogue of D-arabinose.

Untersuchung zur Eignung renal ausgeschiedener modifizierter Nukleoside in der Frühdiagnostik und in der Verlaufskontrolle von nicht-kleinzelligen Bronchialkarzinomen

Face à l’explosion démographique mondiale actuelle, les rendements agricoles se doivent d’être les plus élevés possible. Afin de relever ce défi, l’utilisation de pesticides efficaces et éco-compatibles est devenue indispensable. Dans le cadre de la recherche de nouveaux composés répondants à ces objectifs, les produits naturels continuent de retenir l’attention des chercheurs. Parmi eux, nous nous sommes intéressés à la gougérotine, un peptidonucléoside découvert au sein d’une bactérie dans les années 60 et possédant un large spectre d’activités biologiques. Dans le but d’optimiser l’activité antifongique et de diminuer la toxicité de la molécule, plusieurs analogues ont été synthétisés en jouant notamment sur la nature de la base nucléique. Une des réactions clé au cœur de la synthèse de nucléoside est la N-glycosylation permettant la liaison glycosidique entre la base nucléique et un donneur de glycosyle. Au cours de la voie de synthèse des analogues peptidonucléosidiques, une étude méthodologique de la N-glycosylation a été réalisée mettant en jeu différents donneurs de glycosyle et bases nucléiques. Des tests préventifs ont ensuite été réalisés sur les composés obtenus afin d’évaluer leur potentiel antifongique. Grâce aux analogues de la gougérotine et aux intermédiaires synthétisés, une étude de relation structure-activité a été menée. / In the current context of the world demographic explosion, agricultural yields need to be as high as possible. The use of efficient and environment friendly pesticides has therefore become essential. In the framework of the discovery of new compounds, natural products remain an important source of inspiration. Among them, we focused on gougerotin, a peptidylnucleoside isolated from a bacteria in the 60’s which possess a broad spectrum of biologic activities. In order to improve antifungal potential and decrease the phytotoxicity of gougerotin, several analogues have been synthesized replacing the natural nucleobase. N-glycosylation is one of the key-reaction in peptidylnucleoside synthesis. During the synthesis of the analogues, a study of the N-glycosylation was carried out with three different donors and several bases. Protectives tests were realized with many pathogens to evaluate antifungal activity of our compounds and a structure-activity relationship was established.

Nucleic acid aptamers are "short single-stranded DNA- or RNA-based oligonucleotides that can selectively bind to small molecular ligands or protein targets with high affinity and specificity, when folded into their unique three-dimensional structure" (1). Aptamers have shown promising ability for detection in the subnanomolar range of nucleic acid targets with specificity down to single nucleotide variations (2). The selectivity, low limit of detection, and cost-effectiveness make these nucleic acid aptamers optimal bio-sensors. Previous work by our group has been done to optimize the signal of the dapoxyl-binding aptamer (DAP), which is a light-up aptamer. Here, we propose to organize this aptamer into a split-aptamer system and determine the limit of detection and selectivity for a target gene in Mycobacterium tuberculosis.

No description available.