<p>Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives & Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury.</p><p>PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.</p>

Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives &amp; Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury. PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.

PNP is a crucial enzyme in purine metabolism, and its inherited defects result in severe T-lineage immune deficiency in humans. I hypothesized that PNP deficiency disrupts the development of late CD4-CD8- double negative (DN) thymocytes and induces mitochondrial-mediated apoptosis of CD4+CD8+ double positive (DP) thymocytes. By using PNP-deficient (PNP-/-) mice as well as an OP9-DL1 co-culture system simulating PNP-deficient conditions, I demonstrated that PNP deficiency interferes with the maturation of DN thymocytes at the transition from DN3 to DN4 stage. Although PNP deficiency does not affect the generation or proliferation of DP thymocytes, PNP-/- DP thymocytes were observed to undergo apoptosis at a higher rate. My results suggest that apoptosis is induced through a mitochondrial mediated pathway. Additionally, re-introduction of PNP into PNP-/- thymocytes protected the cells from the toxic effects of deoxyguanosine by preventing the formation of deoxyguanosine triphosphate, indicating that the toxic metabolite in PNP deficiency is deoxyguanosine.

PNP is a crucial enzyme in purine metabolism, and its inherited defects result in severe T-lineage immune deficiency in humans. I hypothesized that PNP deficiency disrupts the development of late CD4-CD8- double negative (DN) thymocytes and induces mitochondrial-mediated apoptosis of CD4+CD8+ double positive (DP) thymocytes. By using PNP-deficient (PNP-/-) mice as well as an OP9-DL1 co-culture system simulating PNP-deficient conditions, I demonstrated that PNP deficiency interferes with the maturation of DN thymocytes at the transition from DN3 to DN4 stage. Although PNP deficiency does not affect the generation or proliferation of DP thymocytes, PNP-/- DP thymocytes were observed to undergo apoptosis at a higher rate. My results suggest that apoptosis is induced through a mitochondrial mediated pathway. Additionally, re-introduction of PNP into PNP-/- thymocytes protected the cells from the toxic effects of deoxyguanosine by preventing the formation of deoxyguanosine triphosphate, indicating that the toxic metabolite in PNP deficiency is deoxyguanosine.

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.

Orientador: Walter Filgueira de Azevedo Júnior / Banca: Luis Fernando Delboni / Banca: Fernanda Canduri / Resumo: A Purina Nucleosídeo Fosforilase (PNP) catalisa a fosforólise de nucleosídeos de purina para suas respectivas bases e açucares (ribose ou desoxirribose) 1-fosfato. A PNP desempenha uma função central no metabolismo das purinas, normalmente operando na via de recuperação do DNA das células. Mais ainda, a PNP cliva ligações glicosídicas com inversão da configuração para produzir a-ribose 1-fosfato. Acredita-se que no organismo do Mycobacterium tuberculosis a PNP desempenha tarefas similares, o que levanta o interesse em desenvolver ciência que dê suporte para o desenvolvimento de drogas baseadas na estrutura desta proteína. A proteína é um homotrímero simétrico com um arranjo triangular das subunidades, similar às PNPs triméricas de mamíferos. Cada monômero consiste de um enovelamento a/ß formado por onze fitas ß circundadas por oito hélices a. O estudo desta PNP visa proporcionar comparações com outras estruturas, na intenção de identificar as bases estruturais de possíveis diferenças ou similaridades funcionais entre esta e outras PNPs, num esforço para desenvolver pesquisa que dê suporte para o desenho de novas drogas mais seletivas e poderosas contra a tuberculose. / Abstract: The Purine nucleoside phosphorylase (PNP) catalyses the phosphorolysis of purine nucleosides to corresponding bases and ribose 1-phosphate. PNP plays a central role in purine metabolism, normally operating in the purine salvage pathway of cells. Moreover, PNP cleaves glycosidic bond with inversion of configuration to produce á-ribose 1-phosphate. It is believed that in the MtPNP is responsible for the same labor in the Mycobacterium tuberculosis organism, which arouses the interest in developing science for giving support to the development of structure based drugs. The protein is a symmetrical homotrimer with triangular arrangement of the subunits, similar to the trimeric mammalian PNPs. Each monomer consist of a á/â folding formed by eleven â sheet surrounded by eight á helices. The study of this PNP aims the possibility of caring out comparisons with other structures, in order to identify the structural basis of possible differences or functional similarities between this and other PNPs, in an effort to develop research which gives support to the design of more selective and powerful new drugs against tuberculosis. / Mestre

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.

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-08-18Bitstream added on 2014-06-13T20:29:19Z : No. of bitstreams: 1 silva_don_me_sjrp.pdf: 1054782 bytes, checksum: 832047dd271670cc0bc6debdbd5dc8d8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A Purina Nucleosídeo Fosforilase (PNP) catalisa a fosforólise de nucleosídeos de purina para suas respectivas bases e açucares (ribose ou desoxirribose) 1-fosfato. A PNP desempenha uma função central no metabolismo das purinas, normalmente operando na via de recuperação do DNA das células. Mais ainda, a PNP cliva ligações glicosídicas com inversão da configuração para produzir a-ribose 1-fosfato. Acredita-se que no organismo do Mycobacterium tuberculosis a PNP desempenha tarefas similares, o que levanta o interesse em desenvolver ciência que dê suporte para o desenvolvimento de drogas baseadas na estrutura desta proteína. A proteína é um homotrímero simétrico com um arranjo triangular das subunidades, similar às PNPs triméricas de mamíferos. Cada monômero consiste de um enovelamento a/ß formado por onze fitas ß circundadas por oito hélices a. O estudo desta PNP visa proporcionar comparações com outras estruturas, na intenção de identificar as bases estruturais de possíveis diferenças ou similaridades funcionais entre esta e outras PNPs, num esforço para desenvolver pesquisa que dê suporte para o desenho de novas drogas mais seletivas e poderosas contra a tuberculose. / The Purine nucleoside phosphorylase (PNP) catalyses the phosphorolysis of purine nucleosides to corresponding bases and ribose 1-phosphate. PNP plays a central role in purine metabolism, normally operating in the purine salvage pathway of cells. Moreover, PNP cleaves glycosidic bond with inversion of configuration to produce á-ribose 1-phosphate. It is believed that in the MtPNP is responsible for the same labor in the Mycobacterium tuberculosis organism, which arouses the interest in developing science for giving support to the development of structure based drugs. The protein is a symmetrical homotrimer with triangular arrangement of the subunits, similar to the trimeric mammalian PNPs. Each monomer consist of a á/â folding formed by eleven â sheet surrounded by eight á helices. The study of this PNP aims the possibility of caring out comparisons with other structures, in order to identify the structural basis of possible differences or functional similarities between this and other PNPs, in an effort to develop research which gives support to the design of more selective and powerful new drugs against tuberculosis.

Orientador: Mário Tyago Murakami / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T16:32:04Z (GMT). No. of bitstreams: 1 Martins_NadiaHelena_D.pdf: 33217097 bytes, checksum: 6cbd9ad5dcfddd06e2357b3c680cd3f6 (MD5) Previous issue date: 2011 / Resumo: A enzima purina nucleosídeo fosforilase hexamérica de Bacillus subtilis (BsPNP233) c uma nucleosídeo fosforilase do tipo 1 , responsável pela catalise reversível da reação de guebra de urn nucleosídeo em base nitrogenada e ribose-1-fosfato na via de salvação de purinas. Essa enzima possui interesses biomédicos e biotecnológicos devido ao uso na terapia gênica em canceres sólidos e na biossíntese de análogos de nucleosídeos...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The hexamcric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) is a nucleoside phosphorylase typc-1 involved in purine salvage pathway by the nucleoside phosphorolysis resulting in purine base and ribose-1-phosphatc. The interest about this enzyme involves gene therapy application in solid cancers treatment and nucleoside analogs biosynthesis...Note: The complete abstract is available with the full electronic document / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Foram determinadas por meio de difração de raios x as estruturas de cinco compostos azaçúcares. Foram estudadas as interações envolvidas na formação das redes cristalinas em cada um dos compostos analisados. Foi encontrado que nos compostos azaçúcares estudados, as interações principais são as ligações de hidrogênio do tipo C-H···O e C-H···&pi;. Este comportamento foi verificado usando ferramentas como as superfícies de Hirshfeld e os gráficos de impressão digital. Realizou-se o estudo de docking molecular dos compostos azaçúcares com respeito à enzima purina nucleosídeo fosforilase (PNP). Foi determinado que estes compostos têm a capacidade de entrar no sitio ativo da PNP. O estudo das interações dos cinco azaçúcares com a PNP mostrou que estes compostos apresentam as mesmas interações presentes em inibidores da PNP já reportados. / Structures of five azasugars were determined by X-ray diffraction. Crystal network interactions were analyzed for each compound. The main interaction found for these azasugar compounds is hydrogen bond as C-H···O e C-H···&pi;. This behavior was verified by tools as Hirshfeld surface and 2D finger print plots. Molecular docking was performed for azasugar compounds in Purine Nucleoside phosphorylase (PNP). This study confirmed that these compounds are available to enter to the PNP active site. Interactions exploration showed the same interactions for the azasugars studied and for already known PNP inhibitors.