La grande variété de propriétés biologiques associées au noyau purine en fait une structure privilégiée pour la conception et la synthèse de nouvelles molécules à visée thérapeutique. Cette spécificité est étroitement liée à la grande diversité de substituants pouvant être introduits sur les différentes positions du noyau purine et en particulier sur C2, C6, C8 et N9. Par conséquent, le développement de méthodes de fonctionnalisation rapides de cette famille de composés est d'un grand intérêt synthétique. Nous nous sommes focalisés sur la formation de liaisons C-C et C-N sur les positions 6 et 8 du noyau purine pour pouvoir présenter de nouveaux outils de synthèse permettant d'introduire une plus grande diversité fonctionnelle. D'une part, nous avons étudié la fonctionnalisation directe de liaisons C-H de purines, sujet encore peu exploré. En effet, de nos jours, le traditionnel couplage croisé (Negishi, Suzuki-Miyaura), utilisé pour la création de liaisons C-C, se voit de plus en plus concurrencé par ces réactions puisqu'elles ne nécessitent pas la préparation d'un partenaire organométallique. Ce sont des réactions dites à économie d'atomes. En nous basant sur l'expérience du laboratoire dans le domaine de la fonctionnalisation directe de liaisons C-H, nous avons envisagé l'alcénylation et l'alcynylation directes en position 8 de la purine, les motifs alcényle et alcynyle étant présents dans certaines purines d'intérêt biologique. D'autre part, nous nous sommes intéressés à deux méthodes de couplage croisé pallado-catalysé permettant la formation de liaisons C-N et C-C : le couplage de Buchwald - Hartwig entre une 8-iodopurine et des amides ou des amines aromatiques, et le couplage de Liebeskind - Srogl entre une 6-thioétherpurine et divers acides boroniques.

Thesis (PhD (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The current understanding of the regulation of carbohydrate accumulation is still under investigation despite the tremendous work done in this subject. Purine and pyrimidine nucleotides have been implicated in many biochemical processes in plants. Amongst others, they are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products such as sucrose, polysaccharides, phospholipids, as well as secondary products. With the aim of placing adenine and uridine nucleotides in the context of sucrose and starch metabolism and carbon partitioning in higher plant, we have investigated the transcripts, enzymes and metabolites in carbohydrate metabolism and both de novo and salvage of purine and pyrimidine nucleotides in both sugarcane and tobacco tissues. For that purpose, adenylate kinase (ADK) and UMP synthase were chosen for silencing and over expression as they are rate limiting steps of de novo adenine and uridine nucleotides biosynthesis, respectively. Sugarcane with repressed ADK activity showed significant increase in both the starch and adenylate pools. Increase in starch content was highly correlated with reduced ADK activity. As a result of decreased ADK activity, the salvage pathway was up regulated via the increased activity of both adenosine kinase (AK) and adenine phosphoribosyl transferase (APRTase) which positively correlated with increase in adenine nucleotide contents. In addition hexose phosphates and ADP glucose, the committed substrate for starch biosynthesis positively correlated with changes in starch content. A high ratio of ATP/ADP was observed in all transgenic lines compared with the untransformed wild type and suggested to favour starch synthesis. Over expression of cytosolic ADK in tobacco demonstrated an expression of the enzyme where 2/3 of the total activity was in the direction of ADP production. As a result of over expression of ADK, starch content increased in all transgenic plants and positively correlated with changes in the activity of ADK. Despite changes in adenine nucleotide content, the salvage pathway was not activated and no significant changes in both AK and APRTase acivities were found between the transgenic and the untransformed plants. Sucrose synthase (SuSy) activity in breakdown direction positively correlated with changes in starch content suggesting a contribution in the starch accumulation in tobacco plants. In addition the ratio of ATP/ADP was low in all transgenic lines compared with the untransformed wild type. This was in line with the higher content in ADP compare to ATP in all transgenic lines and was supported by the over expression of ADK, and predominantly in the direction of ADP production. Repressed UMP synthase in transgenic sugarcane resulted in increases in sucrose, starch and uridinylate. UDP-glucose, hexose phosphates and uridinylate content positively correlated with changes in sucrose content. Transgenic lines had increased sucrose phosphate synthase (SPS) activity and low activity in SuSy, which suggests alteration of carbon flux toward sucrose. As a result of decreased UMP synthase activity, an up regulation of the salvage pathway was observed and predominantly via increased activity of uridine kinase (UK) which positively correlated with changes in the uridinylate pool. In addition to repressed UMP synthase activity, starch content and adenine nucleotides increased in transgenic lines. Tobacco plants transformed with a cytosolic UMP synthase demonstrated an over expression of the enzyme in all transgenic lines. As a result of over expression of UMP synthase, key metabolites were up regulated, amongst them sucrose. Increase in sucrose content positively correlated with both hexoses and hexose phosphates but not the uridinylate pool. SPS activity positively correlated with increase in sucrose content, and accounted for most of the sucrose synthesized in transgenic lines. Despite the increase in the adenylate pool, no significant changes were observed in starch content. The depletion level of UDP-glucose in all transgenic lines was a mere reflection of the higher activity of UDP glucose pyrophosphorylase (UGPase) in the formation of glucose-1-phosphate. In addition, no salvage pathway was up regulated in transgenic lines. / AFRIKAANSE OPSOMMING: Die huidige beskikbare inligting in verband met die reguleering van koolhidraat akkumulasie word steeds ondersoek ten spyte van die groot hoeveelheid navorsing wat reeds in hierdie verband gedoen is. Purien en pirimidien nukleotide speel ‘n rol in baie biochemiese prosesse in plante. Onder andere is hulle boublokke vir nukleïensuur sintese, ‘n energie bron, voorlopers vir die sintese van primêre produkte soos byvoorbeeld sukrose, polisakkariede, fosfolipiede, asook sekondêre produkte. Met die vooruitsig om adenine- en uridiennukleotide in verband te plaas met sukrose en stysel metabolisme en koolstof afskorting in plante, ondersoek ons hier die transkripte, ensieme en metaboliete in koolhidraat metabolisme in beide de novo en berging van purien en pirimidien nukleotide in suikerriet asook tabak weefsel. Vir hierdie doel is adenilaatkinase (ADK) en UMP-sintase gekies vir uitskakeling en ooruitdrukking, juis omdat hulle tempo vermindering stappe van de novo adenine- en uridiennukleotide biosintese is. Suikerriet met onderdrukte ADK aktiwiteit wys betekenisvolle vermeerdering in beide die stysel en adenilaat poele. Verhoging in styselinhoud was hoogs gekorreleerd met verminderde ADK aktiwiteit. As gevolg van ‘n vermindering in ADK aktiwiteit, is die bergingspad opwaards gereguleer via die vermeerdering van beide adenosienkinase (AK) en adenien-fosforibosieltransferase (APRTase) aktiwiteit wat positief korreleer met die vermeerdering in adeniennukleotied-inhoud. Addisioneel word hexosefosfate en ADP-glukose, die toegewysde substraat vir stysel biosintese, positief gekorreleer met veranderinge in styselinhoud. ‘n Hoë verhouding van ATP/ADP was geobserveer in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe en blyk stysel sintese te begunstig. Ooruitdrukking van sitologiese ADK in tabak demonstreer die uitdrukking van die ensiem waar 2/3 van die totale aktiwiteit in die rigting van ADP produksie was. As ‘n resultaat van ooruitdrukking van ADK, word stysel inhoud vermeerder in alle transgeniese plante en positief gekorreleer met die verandering in die aktiwiteit van ADK. Ten spyte van veranderinge in adeniennukleotide inhoud was die bergingspad nie geaktiveer nie en geen betekenisvolle veranderinge in beide AK en APRTase aktiwiteit was gevind tussen die transgeniese en nie-transgeniese plante nie. Sukrose sintese (SuSy) aktiwiteit tydens afbreking korreleer positief met die veranderinge in stysel inhoud en dui moontlik op ‘n bydrae in die stysel akkumulasie in tabak plante. Verder was die verhouding van ATP/ADP laag in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe. Hierdie bevinding word ondersteun deur die hoër inhoud in ADP in vergelyking met ATP in alle transgeniese lyne en word verder ondersteun deur die ooruitdrukking van ADK, hoofsaaklik in die rigting van ADP produksie. Onderdrukte UMP-sintase in transgeniese suikerriet lei tot verhogings in sukrose, stysel en uridienilaat. UDP-glukose, hexose-fosfate en uridienilaat inhoud korreleer positief met die verandering in sukrose inhoud. Transgeniese lyne het verhoogde sukrose-fosfaatsintase (SPS) aktiwiteit en lae SuSy aktiwiteit wat dui op ‘n verandering in koolstof vloei in die rigting van sukrose. As gevolg van die afname in UMP-sintese aktiwiteit, word ‘n verhoogde reguleering van die bergingspad gesien, en dít hoofsaaklik via verhoogde aktiwiteit in uridienkinase (UK) wat positief korreleer met veranderinge in die uridienilaat poel. Addisioneel tot die onderdrukking van UMP-sintase was stysel inhoud en adenine- nucleotides in transgeniese lyne verhoog. Tabak plante wat getransformeer is met sitologiese UMP-sintase demonstreer verhoogde uitdrukking van die ensiem in al die transgeniese lyne. As ‘n resultaat van ooruitdrukking van UMP-sintase is sleutel metaboliete, onderandere sucrose, oorgereguleer. ‘n Verhoging in sukrose inhoud korreleer positief met beide hexose en hexose-fosfate maar nie met die uridienilaat poel nie. SPS aktiwiteit korreleer positief met die verhoging in sukrose inhoud en verklaar die meeste van die sukrose vervaardig in transgeniese lyne. Ten spyte van die verhoging in die adenilaat poel word geen noemenswaardige veranderinge gesien in die stysel inhoud nie. Die uitputtingsvlak van die UDP-glukose in alle transgeniese lyne was slegs ‘n aanduiding van die hoër aktiwiteit van UDP-glukose pirofosforilase (UGPase) in die formasie van glukose-1-fosfaat. Verder was geen bergingspad opgereguleer in die transgeniese lyne nie. / The South African Sugarcane Research Institute and the Gabonese Government who provided the financial support for this work

Folic acid is an essential vitamin utilized in the one-carbon metabolism pathway for the synthesis of purine and thymidine nucleotides, which are necessary for cell growth and proliferation. As a result, the enzymes that participate in the metabolism of folic acid have been good targets for cancer chemotherapy. Folylpoly-γ-glutamate synthetase (FPGS) is an enzyme in the folate metabolism pathway that catalyzes the addition of glutamic acid to the naturally occurring folates, thereby allowing the retention of folate cofactors in cells. Similarly, in the case of cancer chemotherapy, antifolates, such as Lometrexol and Tomudex are retained in cells through the activity of FPGS. Consequently, any single nucleotide polymorphisms (SNPs) that exist in the fpgs gene may decrease or increase the cytotoxicity of antifolates and, ultimately, the clinical response rate to antifolate therapy. The goal of this project is to define the position and frequency of single nucleotide polymorphisms (SNPs) in the mRNA made from the fpgs gene from peripheral blood of one hundred normal individuals. Six Polymerase Chain Reaction (PCR) primers were designed to amplify the gene as three overlapping pieces and four primers were designed for sequencing of the three PCR products. In this study, we found polymorphic sites at nucleotides 64, 123, 253, 423, 1334 and 1781. The majority of the samples (49/88) expressed rnRNA with point mutations on at least one allele at base 64, while 8 samples had a SNP at base 123. At nucleotide 123, 6 samples expressed the heterozygote GIA genotype, and one sample expressed the homozygote A/A allele at this site. At nucleotide 423, two samples expressed a G allele and also the common C allele. While the SNPs at nucleotide 64, 123, and 423 caused a silent or conservative mutation in the gene, in sample 82, a mutation C253T produced an amino acid change from an arginine to tryptophan, which may cause a functional change in the fpgs protein, due to the significant change in size and charge of the wild type amino acid. Similarly, sample 26 expessed a homozygote T/T allele at nucleotide 1334 instead of the common C/C allele expressed in the remaining samples. This point mutation caused a valine to alanine amino acid change. We also detected a SNP that is expressed after the stop codon in sample 40.

Bioinorganic chemistry strives to understand the roles of metals in biological systems, whether in the form of naturally occurring or addition of non-essential metals to natural systems. Metal ions play vital roles in many cellular functions such as gene expression/regulation and DNA transcription and repair. The study of metal-protein-DNA/RNA interactions has been relatively unexplored. It is important to understand the role of metalloprotein interactions with DNA/RNA as this enhanced knowledge may lead to better understanding of diseases and therefore more effective treatments. A major milestone in the development of this field was the discovery of the cytotoxic properties of cisplatin in 1965 and its FDA approval in 1978. Since then, two other chemotherapeutic drugs containing platinum, carboplatin and oxaliplatin, have been used in the clinic. These three compounds are all bifunctional with the ligands surrounding platinum In the cis conformation and rearrangement of the ligands to the trans orientation results in a loss of cytotoxic properties due to rapid deactivation through binding to S-containing proteins. This enhanced reactivity yields new opportunities to study the reactions between proteins and DNA. One of the first crosslinking experiments used transplatin to crosslink NCp7 to viral RNA in order to understand how/where the protein bound to RNA. We have studied the interaction between cis and trans dinuclear platinum complexes and the C-terminal zinc finger (ZF). The trans complex reacts at a faster rate than the cis isomer and causes N- terminal specific cleavage of the ZF. The dinuclear structure plays a critical role in the peptide cleavage as studies with transplatin (the mononuclear derivative) does not result in cleavage. Monofunctional trans platinum-nucleobase complexes (MPNs) serve as a model for the binding of transplatin to DNA. This provides an interesting opportunity to study their reactions with S-containing proteins, such as HIV1 NCp7. MPNs have been shown to bind to the C-terminal ZF of HIV1 NCp7, resulting in zinc ejection. This occurs through a two-step process where the nucleobase π-stacks with Trp37 on the ZF, followed by covalent binding at the labile Cl site to Cys. MPNs have also shown antiviral activity in vitro. The labile Cl on MPNs reduces specificity of these compounds, as it leaves an available coordination site on the platinum center for binding to other S-proteins or DNA. Therefore, we have moved to an inert PtN4 coordination sphere, [Pt(dien)L]2+ (dien= diethylenetri- amine). Due to the strong bond between platinum and nitrogen, covalent reactions are highly unlikely to occur at rapid rates. The strength of the pi-stacking interaction between nucleobases (free and platinated) and the aromatic amino acid, tryptophan (Trp), showed an enhanced binding constant for platinated nucleobases. This was confirmed by density functional theory (DFT) calculations as the difference in energy between the HOMO of Trp and the LUMO of the nucleobase was smaller for the platinum complex. The studies were extended to the Trp-containing C-terminal ZF of HIV1 NCp7 and an increase in association constant was seen compared to free Trp. Reaction of PtN4 nucleobases compounds with a short amino acid sequence con- taining either Ala (no pi-stacking capabilities) or Trp (pi-stacking interactions) revealed an enhanced rate of reactivity for the Trp-containing peptide. This result supports the theory of a two-step reaction mechanism where the platinum-nucleobase complex recognizes the pep- tide through a pi-stacking interaction with Trp followed by covalent binding to the platinum center. The [Pt(dien)L]2+ motif allows for systematic modification of the structural elements surrounding platinum in a search for the most effective compound. Methylation of the dien ligand should, in theory, increase lipophilicity of the compounds, however, due to 2+ charge of the compounds, this simple association does not hold true. Analysis of the cellular accumulation profiles showed little change in the uptake with the addition of methyl groups to the dien ligand, in agreement with the non-linear change in lipophilicity. Modification of L using different nucleobases allows for the tuning of the strength of the π-stacking interaction between Trp and the platinum complex. The addition of inosine (which lacks a H-bonding donor/acceptor at the C2 position) resulted in a lower association constant with both N-AcTrp and the C-terminal zinc finger of HIV1 NCp7. Interestingly, the addition of xanthosine resulted in an ehanced pi-stacking interaction with the C-terminal zinc finger of HIV1 NCp7; likely as a results of the addition of a H-bonding donor (double-bonded O) at the C2 position. The ability of PtN4 nucleobase complexes to inhibit formation of the NCp7 complexation with viral RNA was studied by mass spectrometry and gel electrophoresis. Dissociation of the NCp7-RNA complex was seen upon addition of PtN4 compounds. These compounds were also able to retard formation of the NCp7-RNA complex when pre-incubated with the protein. These results have important implications as inhibition of complex formation between NCp7 and viral RNA has negative implications for viral replication. Despite the success of platinum-nucleobase compounds, it is important to evaluate all potential pi-stacking ligands. A series of pyridine- and thiazole-based compounds were evaluated for the strength of the pi-stacking interaction with N-AcTrp and the C-terminal ZF of HIV1 NCp7. There was notable increase in association constant for the platinum- DMAP (4-dimethylaminopyridine) complex compared to other ligands studied. This result highlights the importance of exploring multiple avenues for the design of specifically targeted inhibitors and further confirms the viability of the medicinal chemistry dual approach of target recognition (non-covalent) followed by target fixation (covalent).

O objetivo deste trabalho foi avaliar o efeito dos diferentes níveis de nucleotídeos sobre o desempenho de frangos de corte alimentados com probióticos. Para o trabalho, foi utilizado um delineamento inteiramente casualizado com cinco repetições dentro de cada tratamento. Foram utilizados 1050 frangos machos da linhagem Ross 308 totalizando trinta e cinco aves por boxe. As aves foram criadas até 42 dias de idade que receberam as rações experimentais a base de milho e farelo de soja contendo seis diferentes níveis de nucleotídeos (0; 100; 200; 300; 400 e 500 gramas por tonelada de ração). Os diferentes níveis de nucleotídeos foram utilizados na fase inicial (1 a 21 dias de idade) e crescimento (22 a 35 dias de idade). Durante a fase final (36 a 42 dias de idade) foi fornecido rações sem nucleotídeo para todos os tratamentos. Os resultados experimentais demonstraram que houve melhora linear no desempenho dos frangos de corte no período de 1 a 21 dias de idade, indicando que, quanto maior o nível de nucleotídeos na dieta de frangos de corte, maior foi o peso corporal das aves. A conversão alimentar também é melhorou linearmente no período de 1 a 21 dias de idade à medida que aumentou o nível de nucleotídeos na ração. O peso no período de 35 dias de idade, também teve um comportamento linear, semelhante ao período de 1 a 21 dias, indicando que, quanto maior o nível de nucleotídeos na dieta de frangos de corte, maior o desempenho das aves. Quanto aos níveis plasmáticos de ácido úrico, pôde-se observar efeito quadrático no período de 1 a 21 dias de idade, indicando o valor de 231,59 gramas de nucleotídeos por tonelada de ração, como o melhor, em níveis mínimos de ácido úrico, por outro lado, no período de 35 dias de idade, estima-se o nível de 208,99 g de nucleotídeos por tonelada de ração; como o melhor em níveis mínimos de ácido úrico no sangue. No período final (35 a 42 dias de idade) e período total (1 a 42 dias de idade) não foi possível o observar efeito dos contrastes testados em nenhum dos parâmetros avaliados. / The objective of this experiment was to evaluate the influence of different nucleotides levels in the rations of broilers containing probiotics on response of the birds and its influence on the performance. Birds were allocated in a randomized experimental design with five replications of each treatment. It was used 1,050 chicks of a day of age, males, distributed in 30 experimental boxes with 35 birds each. The chickens were reared from 1 to 42 days of age and the diets contained corn and soybean meal with one of six different nucleotídes levels (0; 100; 200; 300; 400 and 500 grams for ton of ration). The different nucleotides levels were used in the initial phase (1 to 21 days of age) and growth (22 to 35 days of age). During the final phase (36 to 42 days of age) it was supplied rations without nucleotides for all of the treatments. The experimental results demonstrated that there was improvement on broilers performance in the period from 1 to 21 days of age, demonstrating proportionality between nucleotides level in the diet of broilers and body weight of the birds. Feed conversion at 21 days of age was directly proportional to nucleotides level in the diet. Body weight at 35 days of age, also had a linear behavior, similar to the period from 1 to 21 days, indicating that, as higher the nucleotides level in the diet of broilers, higher the acting of the birds. Acid plasmatic uric levels, demonstrated quadratic effect at 21 days of age, indicating 231,59 grams of nucleotides for ton of ration, and at 35 days of age, it was considered the level of 208,99 g of nucleotides for ton of ration. In the final period (35 to 42 days of age) and total period (1 to 42 days of age) it was not demonstrated effect of the contrasts tested in none of the appraised parameters.

As doenças parasitárias são uma das maiores causas de morte em países em desenvolvimento, e recebem pouca ou nenhuma atenção das indústrias farmacêuticas para o desenvolvimento de terapias. Causada pelo parasita Schistosoma mansoni a esquistossomose mansônica afeta aproximadamente 259 milhões de pessoas no mundo sendo aproximadamente 6 milhões somente no Brasil. O S. mansoni não possui a via \"de novo\" para a biossíntese de bases púricas e depende integralmente da via de salvação para o suprimento dessas bases, portanto, essa via é um alvo em potencial. Agentes capazes de bloquear a atividade das enzimas participantes desta via atuam de forma inespecífica e são quase sempre tóxicos ao homem e por isso o estudo minucioso das pequenas diferenças encontradas entre as enzimas do hospedeiro e do parasita são de extrema importância. Uma diferença marcante entre a via de salvação de purinas do parasita e do hospedeiro humano é a presença de atividade para adenosina fosforilase, que no parasita é exercida por duas entidades distintas: pela enzima Metiltioadenosina fosforilase de S. mansoni (SmMTAP) e por uma enzima até então desconhecida. A enzima SmMTAP naturalmente converte 5\'-deoxi-5\'-metiltioadenosina (MTA) em adenina livre, mas ao contrário do que é visto no hospedeiro, no parasita essa enzima atua preferencialmente na conversão de adenosina. Substituições encontradas no sítio ativo dessa enzima, podem explicar tamanha preferência pelo substrato alternativo, revelando mecanismos distintos da enzima humana. A enzima Purina nucleosídeo fosforilase de S. mansoni (SmPNP) converte inosina e guanosina à hipoxantina e guanina, respectivamente, mas não possui atividade catalítica para adenosina. No entanto, no genoma de S. mansoni é descrita uma isoforma para a SmPNP (SmPNP2), cuja atividade catalítica é desconhecida e, portanto, essa enzima pode também atuar na conversão de adenosina juntamente com a SmMTAP. Assim, os objetivos deste trabalho foram realizar estudos bioquímicos da ação da enzima SmMTAP e realizar a caracterização estrutural e funcional da enzima SmPNP2. Para isso, foram realizadas mutações no sítio ativo da SmMTAP (S12T, N87T, Q289L, S12T/N87T e S12T/N87T/Q289L), as mutantes da SmMTAP juntamente com a enzima SmPNP2 foram clonadas, expressas de forma heteróloga e purificadas. Foram realizados ensaios de cristalização e cinéticos por espectrofotometria utilizando um sistema acoplado. A atividade da SmPNP2 foi ainda avaliada por calorimetria e HPLC. Foram determinadas as constantes catalíticas da forma nativa e para os cinco mutantes da SmMTAP para cinco diferentes substratos. Foi determinada atividade catalítica da SmPNP2 por 3 diferentes substratos: adenosina, inosina e citidina, as constantes catalíticas foram determinadas para os três substratos. Foram obtidos cristais para os mutantes da SmMTAP e da SmPNP2, que foram submetidos à difração de raios X nas linhas I04-1 e I02 do laboratório de radiação síncrotron Diamond Light Source (DLS). Foram resolvidas 9 estruturas dos mutantes da SmMTAP e 4 da proteína SmPNP2. Os dados cinéticos, juntamente com os dados estruturais, permitiram compreender mecanismos catalíticos e de interação das proteínas estudadas, complementando o conhecimento do metabolismo do parasita Schistosoma mansoni e revelando alvos em potencial para o desenvolvimento de fármacos específicos. / The parasitic illness are the leading cause of deaths in developing countries, and receives little or no attention from drug companies to develop therapies. The schistosomiasis is caused by Schistosoma mansoni parasite and affects approximately 259 million people worldwide with 6 million only in Brazil. The Schistosoma mansoni parasite does not possess the \"de novo\" pathway for purine bases biosynthesis and depends entirely on salvage pathway for its purine requirement, therefore this pathway is a potential target. Compounds able to block the enzymes from this pathway, are not specific and are often toxic to humans, thus the thorough study about the particularity found between enzymes from host and parasite are extremely important. A notable difference between human and parasite metabolism, is the activity existence to Adenosine phosphorylase that in parasite is carried out by two distinct entities: by Methylthioadenosine phosphorylase (SmMTAP) and by a hitherto unknown enzyme. The SmMTAP enzyme, naturally converts 5\'-deoxy-5\'-methylthioadenosine (MTA) to free adenine and in opposition to host, in the parasite this enzyme acts manly in adenosine conversion. Substitutions found in the active site from SmMTAP, can explain the huge preference by alternative substrate and to expose a distinct mechanisms from human enzyme. The Purine nucleoside Phosphorylase from S. mansoni (SmPNP) converts inosine and guanosine to hypoxanthine and guanine, respectively, but it not possess catalytic activity to adenosine conversion. However in the S. mansoni genome there is a isoform to SmPNP, whose activity is unknown, thus is possible that SmPNP2 enzyme also can to convert adenosine. This study aimed to perform biochemical studies to investigate the SmMTAP enzyme action and perform the structural and functional characterization of SmPNP2. For this propose was made site-directed mutagenesis (S12T, N87T, Q289L, S12T/N87T e S12T/N87T/Q289L). The SmMTAP mutants and SmPNP2 enzyme were cloned, expressed by heterolog process and purified. Were perform kinetic assays by spectrophotometric method in a coupled system. The SmPNP2 activity was also available by calorimetry and HPLC methods. Were determined the catalytic constants to wild and mutants SmMTAP to five different substrate. Was determinated to SmPNP2 catalictical activity and kinetics parameters to three substrate: adenosine, inosine e cytidine. Were obtained crystals from SmMTAP mutants and SmPNP2 enzyme, those crystals were submitted to X-rays diffractions in the I04-1 and I02 beamlines from Diamond Light Source (DLS). Nine structures were obtained from SmMTAP mutants and four from SmPNP2 enzyme. The kinetics and structural data allowed understanding the catalytic and interaction mechanisms about the protein studied, supplementing the knowledge around Schistosoma mansoni metabolism and reporting potential targets for the specific drugs development.

Fosforribosil pirofosfato (PRPP) sintetases são enzimas de central importância em diversas vias metabólicas em todas as células. A enzima PRPP sintetase humana é constituída por um complexo composto de três subunidades catalíticas (PRSI, PRSII e PRSIII) e por proteínas homólogas de 39 e 41 kDa denominadas PRS Associated Proteins (PAPs) cuja função é desconhecida. A importância da PRPP sintetase em humanos, tem sido documentada pela identificação de uma desordem associada ao cromossomo X, resultando em uma atividade aumentada da PRPP sintetase.Em conseqüência se percebem concentrações elevadas na dosagem de ácido úrico, purino nucleotídeos levando ao desenvolvimento de patogenias como a gota e problemas neurológicos. Nesse sentido, realizaram-se estudos moleculares com o complexo enzimático que compõem as PRPP sintetases. Foram obtidos clones do gene hprsI em vetor de expressão pET29a(+) e a enzima foi expressa em bactérias Escherichia coli cepa BL21 (DE3). A proteína recombinante hPRSI foi purificada depois de fracionamento com sulfato de estreptomicina, sulfato de amônio e em coluna cromatográfica de troca iônica. O raio hidrodinâmico e o pI da enzima foram determinados usando respectivamente a técnica de espalhamento dinâmico de luz e o sistema Fast de eletroforese. A enzima foi caracterizada quanto ao seu enovelamento através da técnica de Dicroísmo Circular, apresentando um espectro característico de estrutura enovelada, composta predominantemente por a-hélices. Os genes hprsII e hpap4l-1 que codificam respectivamente para as proteínas hPRSI1 e HPAP41-1 foram clonados no vetor de clonagem pCR4-TOPO. A proteína recombinante hPAP39 foi clonada em vetor pMAL-c2X em fusão com a proteína Maltose Binding Protein (MBP) e expressa em E. coli. A proteína hPAP39 está em fase de purificação e foi submetida ao experimento de Imunoblotting. Investigações estruturais destas enzimas poderão trazer informações fundamentais para o conhecimento da via biossintética, assim como para o desenvolvimento de inibidores específicos visando o tratamento das desordens a elas associadas. / Phosphoribosyl pyrophosphate (PRPP) synthetases are enzymes of central importance in several metabolic pathways in all cells. The enzyme PRPP synthetase complex is composed of three catalytic subunitis (PRSI, PRSII and PRSIII) and homologous 39 and 41 kDa proteins termed PRPP synthetase-Associated Proteins (PAPs) which function is unknown. The importance of PRPP synthetase function in humans has been documented by the identification of an X chromosome-linked disorder associated with super activity of PRPP synthetase. As a consequence uric acid overproduction, purine nucleotide are observed resulting in the development of diseases such as gout and neurodevelopment impairment. In this line, molecular studies were done with the enzyme complex that constitutes the PRPP synthetases. Clones were obtained from the hprsI gene in the pET29a(+) expression vector and the enzyme was expressed in Escherichia coli BL21(DE3) bacterial. The recombinant human PRSI enzyme was purified, after streptomicine and ammonium sulfate fractionation and by anion exchange chromatography. The hPRSI hydrodynamic radius and pI were determined using, respectively, measures of Dynamic Light Scattering (DLS) and isoeletrophocusing electrophoresis. In addition, according to circular dichroism spectroscopy, hPRSI prevalent secondary structure is a-helix. The hprsí and hpap41-1 genes that codify, respectively, to hPRSII and hPAP41-1 proteins were cloned in pCR4-TOPO cloning vector. The recombinant protein hPAP39 was cloned in the pMAl-c2X expression vector in fusion with the Maltose Binding Protein (MBP) and expressed in E.coli. A purification protocol is been establish for the hPAP39 protein and is submitted by imunoblotting technique. Structural investigation of these enzymes will provide information about the biosynthetic pathway de novo of purine nucleotides, as well as to development of specific inhibitors aiming at the treatment of the associated disorders.

Muitas espécies de Leishmania são responsáveis por sérias doenças cutâneas e viscerais, que exibem alta incidência em regiões tropicais e subtropicais. Os medicamentos disponíveis são potencialmente carcinogênicos, requerem múltiplas administrações e a hospitalização do paciente. Um programa efetivo de desenvolvimento de novos fármacos requer a validação genética e bioquímica dos alvos. Neste contexto, uma das diferenças metabólicas mais marcantes entre os protozoários parasitas e o hospedeiro mamífero encontra-se na cadeia de síntese de purino-nucleotídeos. A enzima adenilosuccinato liase (ADSL), no hospedeiro mamífero, atua em duas etapas no metabolismo de purino-nucleotídeos: uma na via de síntese de novo e outra na via de recuperação. Esta característica particular da ADSL nos motivou a investigar como esta enzima poderia nos fornecer evidências sobre a evolução desta via metabólica em Kinetoplastida. Assim, os objetivos do presente trabalho foram validar a ADSL como potencial alvo terapêutico, através da técnica de RNA de interferência (RNAi), usando Trypanosoma brucei como modelo, bem como, caracterizar molecularmente o gene adsl-Lm e caracterizar bioquímica e estruturalmente a enzima recombinante de Leishmania major Friedlin (ADSL-Lm). Os resultados de RNAi demonstraram que a ADSL pode ser considerada um potencial alvo, uma vez que ela se apresentou essencial a viabilidade do parasita. Em relação à caracterização molecular do gene adsl-Lm suas regiões não traduzidas 5\'UTR e 3\'UTR foram definidas por RT-PCR, indicando que o RNA mensageiro maduro possui 2060 nucleotídeos. Análises com enzimas de restrição e eletroforese em campo pulsado (PFGE), seguidas de \"Southern blot\" revelaram que adsl-Lm trata-se de um gene de cópia simples e está localizado no cromossomo 4 deste parasita. O gene adsl-Lm foi clonado em um vetor de expressão e um protocolo de purificação da enzima recombinante foi estabelecido. A forma tetramérica da ADSL-Lm foi confirmada por eletroforese em gel nativo e por espalhamento dinâmico de luz (DLS). ADSL-Lm apresentou um pI experimental de 6,07 e exibiu atividade máxima no pH 8,5. Os parâmetros cinéticos de Km, Vmax , Kcat e eficiência catalítica (Kcat/Vm) foram determinados para o substrato adenilosuccinato. Experimentos de complementação funcional evidenciaram que ADSL-Lm foi capaz de complementar de maneira eficiente o genoma deficiente em ADSL da linhagem de E. coli JK268 (mutação purB58). Entretanto, esta complementação deve ocorrer na via de recuperação, uma vez que ensaios enzimáticos com o SAICAR (substrato da via de novo) mostraram que a enzima não retém atividade sobre este composto. Estes resultados indicam que provavelmente os tripanosomatídeos não representam um caso de perda da via de síntese de novo. Adicionalmente, ADSL-Lm foi cristalizada no grupo espacial tetragonal I4122, com parâmetros de cela unitária a= b= 130,023 \'angstron\', c= 316,826 \'angstron\', = = =90°. A estrutura cristalográfica da ADSL-Lm foi resolvida por SIRAS (substituição isomórfica simples com dispersão anômala) usando um cristal nativo (2.2 \'angstron\') e um derivado de Gadolínio. O monômero é composto por três domínios em um enovelamento típico das enzimas da superfamília de -eliminação e é constituído quase exclusivamente por hélices- . Para o sitio ativo, três subunidades são requeridas e os resíduos envolvidos são His 153 (ácido geral), His 231 (base geral), Gln 308, Asn 364 and Glu 369. Entretanto, sem um ligante (substrato ou inibidor) no sítio ativo torna-se difícil estudar detalhadamente as interações entre a enzima e seus dois substratos. Desta forma, experimentos de co-cristalização e modelagem molecular podem auxiliar nesta questão. / Many species of Leishmania are responsible for serious visceral or skin diseases that exhibit high incidence in tropical and subtropical regions. The drugs currently employed in the treatment of parasitic diseases are potentially carcinogenic, often require prolonged treatment and patient hospitalization. An effective program of drug design requires the validation of the potential target. In this context, one of the most striking metabolic discrepancies between Trypanosomatidae and their human hosts is the purine nucleotide biosynthesis pathway. Adenylosuccinate lyase (ADSL) is a bifunctional enzyme that catalyses two non-sequential steps in this cycle (one in the de novo purine pathway and another in the salvage pathway). This particular ADSL feature motivated us to investigate if ADSL could give us information about purine biosynthesis evolution in Kinetoplastida. Hence, the present work is aimed to validate ADSL as a potential target using the RNAi (RNAi) technique, as well as to characterize the adsl gene and the recombinant enzyme from Leishmania major Friedlin (ADSL-Lm). The RNAi results proved that ADSL can be considered a potential target, because it is shown to be essential for parasite viability. Regarding the molecular characterization of the adsl-Lm gene, the mature mRNA transcript containing 2060 nucleotides was defined by 5\' and 3\'RT-PCR. Restriction analysis and Pulse Field Gel Electrophoresis (PFGE) followed by Southern Blot hybridizations showed that adsl-Lm is a single copy gene and is located in chromosome 4 of this parasite. The adsl-Lm gene was cloned into an expression vector and a purification protocol of the recombinant enzyme was established. The tetrameric form of the recombinant ADSL-Lm enzyme was confirmed by native gel electrophoresis and Dynamic Light Scattering. ADSL-Lm has an experimental pI of 6.07 and exhibited maximum enzymatic activity at pH 8.5. The kinetic parameters of Km, Vmax , Kcat and kinetic efficiency (Kcat/Vm) were obtained for adenylosuccinate substrate. Functional complementation experiments showed that the adsl-Lm gene can effectively complement the E. coli JK268 purB58 mutation. However, this complementation must be in the salvage pathway, because enzymatic assays were performed using SAICAR (de novo pathway substrate) and ADSL-Lm did not convert this compound into product. This result indicates that probably Trypanosomatidae is not an example of de novo purine nucleotide cycle lost. In addition, ADSL-Lm was crystallized in the tetragonal I4122 space group, with unit cell parameters a= b= 130,023 \'angstron\', c= 316,826 \'angstron\', = = =90° and diffracted beyond 2.2\'angstron\'. ADSL-Lm crystal structure has been determined by SIRAS (Single Isomorphous Replacement with Anomalous Dispersion), using both native and Gadolinium derivative crystals. The ADSL-Lm monomer is composed of three domains arranged in the elongated manner typical of enzymes in the p-elimination superfamily, and is constituted almost exclusively by helices. Three subunits are necessary to form the active site cleft and the residues His 153, His 231 (general bases), Gln 308, Asn 364 and Glu 369 are involved. Without a bound inhibitor or substrate, the specific contacts made by the enzyme to its two substrates cannot be analyzed in detail. Hence, co-crystallization and docking can help in this question.

Fosforribosil pirofosfato (PRPP) sintetases são enzimas de central importância em diversas vias metabólicas em todas as células. A enzima PRPP sintetase humana é constituída por um complexo composto de três subunidades catalíticas (PRSI, PRSII e PRSIII) e por proteínas homólogas de 39 e 41 kDa denominadas PRS Associated Proteins (PAPs) cuja função é desconhecida. A importância da PRPP sintetase em humanos, tem sido documentada pela identificação de uma desordem associada ao cromossomo X, resultando em uma atividade aumentada da PRPP sintetase.Em conseqüência se percebem concentrações elevadas na dosagem de ácido úrico, purino nucleotídeos levando ao desenvolvimento de patogenias como a gota e problemas neurológicos. Nesse sentido, realizaram-se estudos moleculares com o complexo enzimático que compõem as PRPP sintetases. Foram obtidos clones do gene hprsI em vetor de expressão pET29a(+) e a enzima foi expressa em bactérias Escherichia coli cepa BL21 (DE3). A proteína recombinante hPRSI foi purificada depois de fracionamento com sulfato de estreptomicina, sulfato de amônio e em coluna cromatográfica de troca iônica. O raio hidrodinâmico e o pI da enzima foram determinados usando respectivamente a técnica de espalhamento dinâmico de luz e o sistema Fast de eletroforese. A enzima foi caracterizada quanto ao seu enovelamento através da técnica de Dicroísmo Circular, apresentando um espectro característico de estrutura enovelada, composta predominantemente por a-hélices. Os genes hprsII e hpap4l-1 que codificam respectivamente para as proteínas hPRSI1 e HPAP41-1 foram clonados no vetor de clonagem pCR4-TOPO. A proteína recombinante hPAP39 foi clonada em vetor pMAL-c2X em fusão com a proteína Maltose Binding Protein (MBP) e expressa em E. coli. A proteína hPAP39 está em fase de purificação e foi submetida ao experimento de Imunoblotting. Investigações estruturais destas enzimas poderão trazer informações fundamentais para o conhecimento da via biossintética, assim como para o desenvolvimento de inibidores específicos visando o tratamento das desordens a elas associadas. / Phosphoribosyl pyrophosphate (PRPP) synthetases are enzymes of central importance in several metabolic pathways in all cells. The enzyme PRPP synthetase complex is composed of three catalytic subunitis (PRSI, PRSII and PRSIII) and homologous 39 and 41 kDa proteins termed PRPP synthetase-Associated Proteins (PAPs) which function is unknown. The importance of PRPP synthetase function in humans has been documented by the identification of an X chromosome-linked disorder associated with super activity of PRPP synthetase. As a consequence uric acid overproduction, purine nucleotide are observed resulting in the development of diseases such as gout and neurodevelopment impairment. In this line, molecular studies were done with the enzyme complex that constitutes the PRPP synthetases. Clones were obtained from the hprsI gene in the pET29a(+) expression vector and the enzyme was expressed in Escherichia coli BL21(DE3) bacterial. The recombinant human PRSI enzyme was purified, after streptomicine and ammonium sulfate fractionation and by anion exchange chromatography. The hPRSI hydrodynamic radius and pI were determined using, respectively, measures of Dynamic Light Scattering (DLS) and isoeletrophocusing electrophoresis. In addition, according to circular dichroism spectroscopy, hPRSI prevalent secondary structure is a-helix. The hprsí and hpap41-1 genes that codify, respectively, to hPRSII and hPAP41-1 proteins were cloned in pCR4-TOPO cloning vector. The recombinant protein hPAP39 was cloned in the pMAl-c2X expression vector in fusion with the Maltose Binding Protein (MBP) and expressed in E.coli. A purification protocol is been establish for the hPAP39 protein and is submitted by imunoblotting technique. Structural investigation of these enzymes will provide information about the biosynthetic pathway de novo of purine nucleotides, as well as to development of specific inhibitors aiming at the treatment of the associated disorders.

In our studies we found that stabilized expression of HIF-1α in heart led to better recovery of function and less tissue death after 30 minutes of global ischemia, via mechanisms that preserve the mitochondrial polarization. Our group previously showed that HIF-1α conferred ischemic tolerance by allowing cardiomyocytes to use fumarate as an alternative terminal electron acceptor to sustain anaerobic mitochondrial polarization. The source of fumarate was identified as the purine nucleotide cycle (PNC). Here we discovered that HIF-1α upregulates AMP deaminase 2 (AMPD2), the entry point to the PNC. The combination of glycolysis and the PNC may protect the heart's nucleotide resources. We subsequently examined the effects that HIF-1α exerts on nucleotide metabolism in the ischemic heart. We found that HIF-1α expression reduces adenosine accumulation in the ischemic heart. As ATP is depleted during ischemia, AMP accumulates. Our results suggest that AMP metabolism is shunted towards AMPD2 rather than the adenosine producing 5'-nucleotidase pathway. Subsequently, we treated hearts with the PNC inhibitor hadacidin followed by 30 minutes of global ischemia. Inclusion of hadacidin reduced ATP and adenylate energy charge in the hearts. These findings allow us to propose that activity of the PNC prevents the F0F1 ATP synthase from consuming glycolytic ATP in order to maintain mitochondrial polarization during ischemia. Thus, the PNC provides ATP sparing effects and preserves the energy charge in the ischemic heart. The fact that ATP and adenylate energy charge is better preserved during the initial 20 minutes of ischemia in HIF-1α expressing hearts is supportive of our observation that HIF-1α upregulates the PNC. HIF-1α also upregulates adenosine deaminase, which degrades adenosine. The limitation of adenosine accumulation may help HIF-1α expressing hearts avoid toxicity due to chronic adenosine exposure. Finally, we found that HIF-1α induces the expression of the nucleotide salvage enzyme hypoxanthine phosphoribosyl transferase (HPRT). Upon reperfusion HPRT serves to reincorporate the nucleotide degradation product, hypoxanthine, into the adenylate pool and may prevent the production of reactive oxygen species. Collectively, HIF-1α robustly protects the heart from ischemic stress and it upregulates several pathways whose cardioprotective role may extend beyond the remodeling of nucleotide metabolism.