Thesis (Ph. D. in Mathematics)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

A esquistossomose humana, doença causada pelo S. mansoni e com 6 milhões de infectados somente no Brasil, possui uma única estratégia terapêutica eficiente atualmente disponível. Esta se baseia na utilização de praziquantel e relatos de cepas resistentes à essa droga tem despertado o interesse da comunidade científica sobre o desenvolvimento de novas estratégias terapêuticas. Uma melhor caracterização dos processos metabólicos do parasita podem auxiliar nestas buscas. Diante desse contexto, nosso grupo tem trabalhado na caracterização estrutural e funcional das enzimas que compõem a via de salvação de purinas e pirimidinas deste parasita, com dez enzimas já caracterizadas. Uma das enzimas remanescentes é a uridina fosforilase (UP) (EC 2.4.2.3), cujo a qual o genoma do parasita apresenta duas isoformas, a smUPa e smUPb (92% de identidade entre elas). Com o objetivo de caracterizar estruturalmente estas enzimas, ambas foram obtidas via expressão heteróloga, purificadas e submetidas a ensaios de cristalização e co-cristalização (para obtenção das estruturas interagindo com diferentes ligantes). Após coleta de dados de difração de raio-x, processamento e refinamento adequado foram obtidas seis estruturas da smUPa (smUPaapo, smUPa+Timidina, smUPa+timina, smUPa+uracil, smUPa-5fluorouracil) e duas da smUPb (smUPbapo e smUPb+citrato). A análise das estruturas revela que as duas isoformas apresentam essencialmente a mesma estrutura, no entanto, apesar das poucas divergências em nível de sequência de aminoácidos, existem diferenças significativas entre os sítios ativos. A smUPa apresenta o sítio com as mesmas características de UPs conhecidas, em contrapartida a smUPb apresenta duas mudanças significativas que elimina a capacidade de interagir com a base nitrogenada (Q201L) e a cavidade que acomoda a base nitrogenada (G126D), o que torna as smUPs um caso único de isoformas de UP em um mesmo organismo conhecidas. É plausível que a smUPb não seja capaz de catalisar a fosforólise reversível da uridina, sendo ou um pseudogene ou alguma outra enzima com atividade catalítica diferente da UP. Para a completa caracterização destas enzimas, testes de atividade enzimática serão realizados e deverão auxiliar a determinar a real função da smUPb. / Human schistosomiasis, a disease caused by Schistosoma sp., is estimated to affect six million individuals in Brazil alone and there is currently only one therapeutic strategy available. This is based on the use of praziquantel and reports of the appearance of strains resistant to the drug has motivated the scientific community towards the search for new possible therapies. Biochemical characterization of the parasites metabolism is essential for the rational development of new therapeutic alternatives. Based on this,reasoning our group has been working on the structural and functional characterization of the enzymes involved in the pyrimidine and purine salvage pathways of S. mansoni. One of the remaining enzymes to be characterized is uridine phosphorylase (UP) (EC 2.4.2.3), for which there are two isoforms present in the parasite genome, smUPa and smUPb, which share 92% sequence identity. In order to structurally characterize these enzymes, both smUPs were produced by heterologous expression, purified and submitted to crystallization e co-crystallization assays, in the latter case in order to obtain the structure of different enzyme-ligand complexes. After data collection, processing and refinement, five structures of smUPa (smUPaapo, smUPa+Timidina, smUPa+timina, smUPa+uracil and smUPa+5fluorouracil) and two structures of smUPb (smUPbapo and smUPb+citrato) were obtained. Analysis of the structures revealed that the isoforms have the same fold, but despite the high sequence identity, significant differences are observed at the active site probably profoundly affecting enzyme activity. Whilst SmUPa presents an active site similar to that of other known UPs, smUPb is predicted to lack the ability to interact with the nucleoside base due to the presence of a leucine in place of a glutamine at position 201 and an aspartatic acid in place of glycine at position 126. These differences turn the smUPs into a unique case of UP isoforms. It is plausible that smUPb is unable to catalyze the reversible phosphorolysis of uridine and could be either a pseudogene or a different enzyme altogether of unknown catalytic activity. A complete functional characterization in vitro will be necessary in order to determine its real function.

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.

Polychlorinated biphenyls (PCBs) decrease thyroid function in rats and mice by inducing activity of a liver enzyme, uridine diphosphate-glucuronosyltransferase (UDP-GT), thereby increasing thyroxine (T4) clearance. This loss of T4 can lead to hypothyroidism. In this study, an assay was validated for measuring UDP-GT activity toward T4 in Japanese quail (Coturnix japonica). Then UDP-GT induction by Aroclor 1254 was evaluated in quail, and quail and mice were compared in their responses to Aroclor 1254. In Experiment 1, Japanese quail and Balb/c mice were dosed orally with vehicle or Aroclor 1254 (250 or 500 mg/kg) and sacrificed five days later. In Experiment 2, Japanese quail were dosed orally with vehicle or Aroclor 1254 (500 mg/kg) and sacrificed either five or 21 days later. Total liver UDP-GT capacity increased with Aroclor 1254 exposure in all treatment groups of both species. Enzyme induction led to a trend to decreased plasma T4 concentrations at both doses and exposure times in quail and significantly decreased plasma T4 concentrations at both doses in mice. PCBs altered thyroid function in quail, but they did not become hypothyroid. This was in contrast to mice, which did become hypothyroid. It is unclear how PCBs affect the hypothalamic-pituitary-thyroid (HPT) axis in quail, and activation of the HPT axis appears to be inhibited in mice. Overall, quail showed a lesser response than mice to equivalent doses of Aroclor 1254, so it appears that birds may be less vulnerable to PCBs than mammals. / Master of Science

Les édifices multi-porphyriniques retiennent de plus en plus l'attention des chercheurs et leur compréhension n'a cessé de croître ces dernières années en raison de leur vaste domaine d'application, que ce soit pour l'élaboration de complexes collecteurs d'énergie lumineuse, pour la construction de fils moléculaires photoniques ou électroniques ainsi que pour la formation de complexes supramoléculaires originaux.<br />Dans l'optique de synthétiser des pinces bis-porphyriniques capables de former des complexes supramoléculaires stables avec des bases bidentates de tailles variées, nous avons synthétisé plusieurs architectures moléculaires de type bis- et tris-porphyrines dans lesquelles les chromophores sont liés entre eux par des espaceurs nucléosidiques flexibles dérivés de l'uridine. Nous avons donc fait varier les positions d'ancrage des deux porphyrines sur l'uridine en accrochant soit les deux porphyrines en des positions différentes du sucre, soit en greffant un chromophore sur le ribose et l'autre sur l'uracile. <br />La caractérisation par spectroscopie UV-visible de l'ensemble des architectures moléculaires nous a permis d'observer l'existence d'interactions électroniques entre les différents chromophores au sein de ces composés. Pour deux des trois dimères ainsi que pour les trimères, l'espaceur nucléosidique bloque la conformation de ces molécules sous la forme de pinces comportant une cavité pré-organisée. Ainsi, la complexation de bases bidentates de tailles variées a été suivie par spectroscopie UV-visible, et des constantes d'association supérieures de 1,5 à 2 ordres de grandeur par rapport à la complexation des mêmes bases par une mono-porphyrine ont été trouvées. La pré-organisation des cavités bis-porphyriniques augmente donc la stabilité des complexes supramoléculaires de type hôte/récepteurs formés. En outre, l'ajustement de la cavité à la taille de l'hôte complexé a pu être démontré. Un piégeage efficace de la luminescence de la porphyrine de Zn(II) a également été observé au sein des deux dimères porteurs à la fois d'une porphyrine de Zn(II) et d'une porphyrine base-libre, et attribué à un transfert d'énergie ayant lieu vers la porphyrine base-libre.<br />Enfin, ces espaceurs nucléosidiques offrent la possibilité d'établir des liaisons hydrogène avec un synthon complémentaire de l'uracile fonctionnalisé par une porphyrine ou deux, et ainsi d'augmenter le nombre de chromophores collecteurs de photons.

Mutations found in genes encoding human Elongator complex subunits have been linked to neurodevelopmental disorders such as familial dysautonomia (FD), rolandic epilepsy and amyotrophic lateral sclerosis. In addition, loss-of-function mutations in genes encoding Elongator complex subunits cause defects in neurodevelopment and reduced neuronal function in both mice and nematodes. The Elongator complex is a conserved protein complex comprising six subunits (Elp1p-Elp6p) found in eukaryotes. The primary function of this complex in yeast is formation of the 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side chains found on wobble uridines (U34) in tRNAs. The aim of this thesis is to investigate the physiological consequences of Elongator complex inactivation in humans and in the yeast Saccharomyces cerevisiae. Inactivation of the Elongator complex causes widespread defects in a multitude of different cellular processes in S. cerevisiae. Thus, we investigated metabolic alterations resulting from Elongator complex inactivation. We show that deletion of the S. cerevisiae ELP3 gene leads to widespread metabolic alterations. Moreover, all global metabolic alterations observed in the elp3Δ strain are not restored in the presence of elevated levels of hypomodified tRNAs that normally have the modified nucleoside mcm5s2U. Collectively, we show that modified wobble nucleosides in tRNAs are required for metabolic homeostasis. Elongator mutants display sensitivity to DNA damage agents, but the underlying mechanism explaining this sensitivity remains elusive. We demonstrate that deletion of the S. cerevisiae ELP3 gene results in post-transcriptional reduction of Ixr1p levels. Further, we show that the reduced Ixr1p levels prevent adequate Rnr1p levels upon treatment with DNA damage agents. These findings suggest that reduced Ixr1p levels could in part explain why Elongator mutants are sensitive to DNA damage agents. Depletion of Elongator complex subunits results in loss of wobble uridine modifications in plants, nematodes, mice and yeast. Therefore, we investigated whether patients with the neurodegenerative disease familial dysautonomia (FD), who have lower levels of the ELP1 protein, display reduced amounts of modified wobble uridine nucleosides. We show that tRNA isolated from brain tissue and fibroblast cell lines derived from FD patients have 64–71% of the mcm5s2U nucleoside levels observed in total tRNA from non-FD brain tissue and non-FD fibroblasts. Overall, these results suggest that the cause for the neurodegenerative nature of FD could be translation impairment caused by reduced levels of modified wobble uridine nucleosides in tRNAs. Thus, our results give new insight on the importance of modified wobble uridine nucleosides for neurodevelopment.

Transfer RNAs (tRNAs) act as adaptor molecules in decoding messenger RNA into protein. Frequently found in tRNAs are different modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U). Although modified nucleosides are present at many positions in tRNAs, two positions in the anticodon region, position 34 (wobble position) and position 37, show the largest variety of modified nucleosides. In Saccharomyces cerevisiae, the xm5U type of modified uridines found at position 34 are 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2´-O-methyluridine, (ncm5Um), 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U). Based on the complex structure of these nucleosides, it is likely that their formation requires several synthesis steps. The Elongator complex consisting of proteins Elp1p - Elp6p, and the proteins Kti11p - Kti14p, Sit4p, Sap185p, and Sap190p were shown to be involved in 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side-chain synthesis at position 34 in eleven tRNA species. The proteins Urm1p, Uba4p, Ncs2p, Ncs6p, and Yor251cp were also identified to be required for the 2-thio (s2) group formation of the modified nucleoside mcm5s2U at wobble position. Modified nucleosides in the anticodon region of tRNA influence the efficiency and fidelity of translation. The identification of mutants lacking ncm5-, mcm5-, or s2-group at the wobble position allowed the investigation of the in vivo role of these nucleosides in the tRNA decoding process. It was revealed that the presence of ncm5-, mcm5- or s2-group promotes reading of G-ending codons. The concurrent presence of the mcm5- and the s2-groups in the wobble nucleoside mcm5s2U improves reading of A- and G-ending codons, whereas absence of both groups is lethal to the yeast cell. The Elongator complex was previously proposed to regulate polarized exocytosis and to participate in elongation of RNA polymerase II transcription. The pleiotropic phenotypes observed in Elongator mutants were therefore suggested to be caused by defects in exocytosis and transcription of many genes. Here it is shown that elevated levels of hypomodified tRNALys [mcm5s2UUU] and tRNAGln[mcm5s2UUG] can efficiently suppress these pleiotropic phenotypes, suggesting that the defects in transcription and exocytosis are indirectly caused by inefficient translation of mRNAs encoding proteins important in these processes.

Nucleic acids are important bio-macromolecules in living systems. They are involved in important functions like gene expression and regulation. Nucleoside triphosphates serve as precursors for biochemical synthesis of modified nucleic acids and nucleotide coenzymes. The modification of nucleic acids, particularly at nucleobases, can expand the function and chemical properties of nucleic acid. Herein, we report the chemical synthesis of a novel thiol-modified nucleoside S-(3-(acetylthio)propyl)-5-(mercaptomethyl)-uridine and the corresponding nucleotide via a “new synthetic methodology” developed in our laboratory. The synthesized triphosphate was used for RNA transcription. The activity and nuclease resistance of the transcribed RNA is studied. The results showed that the properties of the nucleotide with thiol functionality are as good as the native. The modified RNA can be used for RNA/protein complex structure studies and gold nanoparticles stabilizer. They can also serve as a probe in DNA/RNA microchip surface functionalization for detection of various diseases and pathogens.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [87]-96).

Abstract The aim of this PhD-study was to address some of the overlapping bottlenecks in protein engineering and metagenomics by developing or applying new tools which are useful for both disciplines. Two enzymes were studied as an example: Triosephosphate Isomerase (TIM) and Uridine Phosphorylase (UP). TIM is an important enzyme of the glycolysis pathway and has been investigated via means of protein engineering, while UP is a key enzyme in the pyrimidine-salvage pathway. In this thesis TIM was used to address protein engineering aspects, while UP was used in regards to some metagenomic and bioinformatic aspects. The aspects of a structural driven rational design approach and its implications for further engineering of monomeric TIM variants are discussed. Process development based on a new technology, EnBase®, addresses the relative instability of new variants, compared to its ancestors, for further studies. EnBase® is then applied for the production of 15N isotope labeling of a monomeric TIM variant, A-TIM. Systematical function- and engineering studies on dimeric TIM and monomeric TIM in regards to the hinges of the catalytic loop-6 were conducted to investigate enzyme activity and stability. Both the A178L and P168A were proposed to induce loop-6 closure, a wanted feature for A-TIM variants. The P168A mutants are hardly active, but gave great insight into the catalytic machinery, while the A178L mutants did induce partial loop-6 closure, however in addition, monomeric A178L was destabilized. Homology driven genome mining and subsequent isolation- high throughput (HTP) overexpression of a thermostable UP from the Archaea Aeopyrum pernix was carried out as an example for the production of recombinant proteins. In addition an alternative kinetic method to study the kinetics of UP by means of NMR directly from cell lysate is discussed. The combination of expression libraries and EnBase® in a HTP manner may relieve up the gene-to-product bottleneck. The structural aspects of A. pernix UP are explored by means of simple bioinformatic tools in the last section of this thesis. A thermostable, truncated version of UP was created and its use for protein engineering in the future is explored. The long N-terminal and C-terminal ends of A. pernix UP seem to be involved in stabilizing the dimeric and hexameric structures of UP. However, deletion of the N-terminal end of A. pernix UP yielded a thermostable protein. Overall, the finding in regards to process optimization and HTP expression and optimization and the underlying methods used in the TIM studies and the UP studies are interchangeable.