Dissertations / Theses: 'Puring'

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Kolososki, Jorge. "Estudo de sistema de canais para fundição de ligas de aluminio por gravidade." [s.n.], 2001. http://repositorio.unicamp.br/jspui/handle/REPOSIP/265179.

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Orientadores: Rezende Gomes dos Santos, Ricardo Fuoco Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica Made available in DSpace on 2018-08-08T07:46:15Z (GMT). No. of bitstreams: 1 Kolososki_Jorge_M.pdf: 7343986 bytes, checksum: 91fb9f7d500f8f650a42e7e445885c96 (MD5) Previous issue date: 2001 Resumo: Durante o vazamento dos moldes, o fluxo de metal através dos canais está sujeito a grande turbulência, gerando filmes de óxidos devido a exposição ao ar. Estes óxidos podem permanecer nas peças fundidas como descontinuidades, prejudicando as propriedades, em particular a resistência mecânica e a dutilidade. São apresentados os principais conceitos e as principais recomendações existentes na literatura para o projeto dos sistemas de canais de ligas com elevada tendência à oxidação, como as ligas de alumínio. Estes princípios e recomendações foram utilizados para projetar seis diferentes sistemas de canais para o preenchimento de placas na posição vertical, utilizando liga de alumínio UNS A03560. A avaliação do grau de turbulência alcançado em cada um dos sistemas de canais projetados foi feita através da maior ou menor geração de inclusões de óxidos, medida através de ensaios de flexão de corpos-de-prova retirados diretamente das placas. São apresentados os resultados destes ensaios de flexão e evidências de inclusões de óxidos nas fraturas dos corpos-de-prova ensaiados Abstract: During mould pouring, metal flow in runner system is submitted to large turbulence and air contact, producing oxide films. This oxide is incorporated to cast products as inclusions and discontinuities, reducing noticeably mechanical strength and ductility. The principal concepts and literature recommendations for the best runner system project applied to alloys with high oxidation tendency, like aluminum alloys, were presented in this work. This information was used to project six different runner systems for plate casting in vertical position, considering aluminum alloy UNS A03560 as the cast metal. The evaluation of turbulence degree reached in each one of the systems was made by oxide inclusion classification, comparing results of three-point bend tests in specimens obtained from the six plate cast procedures. Mechanical test results and oxide inclusion evidence in fracture surface of those broken specimens are presented. Mestrado Materiais e Processos de Fabricação Mestre em Engenharia Mecânica

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Silva, Diego Oliveira Nolasco da [UNESP]. "Estrutura cristalográfica da Purina nucleosídeo foslorilase do Mycobacterium tuberculosis." Universidade Estadual Paulista (UNESP), 2005. http://hdl.handle.net/11449/87508.

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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.

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Oliveira, Lisandre de. "Métodos em nutrição de ruminantes: uso de índices fecais para estimar consumo e estimativa da síntese proteica microbiana ruminal." Universidade Federal de Santa Maria, 2009. http://repositorio.ufsm.br/handle/1/10733.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico This study was carried out to evaluate methods in ruminant nutrition: use of fecal index to estimate intake and use of purine or purine derivatives to estimate rumen microbial protein synthesis. Data from three digestibility trials conducted with castrated male lambs fed ryegrass (Lolium multiflorum, Lam) or Cynodon (Cynodon dactylon var dactylon) at different levels of intake were compiled. Intake data were related to faecal excretion of different chemical compounds through regression analysis. Significant regressions with high R2 were used to calculate estimated intake values, which were compared to observed values by paired t test. Organic matter (OM) intake (g/day) had significant (P<0.01) relation to faecal excretion (g/day) of N and/or acid detergent fiber (ADF) in ryegrass trial (R2 varied from 0.69 to 0.85) while N, ADF and neutral detergent fiber (NDF) had significant (P<0.01) relation to OM intake in Cynodon trials (R2 varied from 0.51 to 0.56). It is concluded that faecal excretion of N and/or ADF had high potential to estimate intake by grazing animals. Rumen microbial protein synthesis estimated either by duodenal purines or urine purines derivatives was different (P<0.01). Moreover, these estimates were highly affected by purines:microbial N proportion used in calculations. O objetivo deste trabalho foi avaliar métodos em nutrição de ruminantes: o uso de índices fecais para estimar consumo e uso de purinas no duodeno ou de derivados de purinas na urina para estimar a síntese de nitrogênio microbiano ruminal. Foram compilados dados de três ensaios de digestibilidade in vivo utilizando ovinos machos castrados não fistulados ou fistulados no duodeno alimentados com azevém (Lolium multiflorum, Lam) ou capim-paulista (Cynodon dactylon var dactylon) fornecidos verde a diferentes níveis de consumo. Os dados de consumo foram relacionados à excreção fecal de diferentes componentes químicos. As equações de regressão com coeficientes de correlação mais significativos foram utilizadas para calcular consumos estimados, os quais foram comparados pelo teste t para dados pareados com os consumos observados. O consumo de MO foi significativamente (P<0,01) relacionado com a excreção fecal de N e/ou fibra em detergente ácido (FDA) no ensaio com azevém (R2 variou de 0,69 a 0,85) e com a excreção fecal de N, FDA ou fibra em detergente neutro (FDN) nos ensaios com Cynodon (R2 variou de 0,51 a 0,56). Conclui-se que a excreção fecal de FDA e/ou N tem um alto potencial para estimar consumo por animais em pastejo. A síntese de nitrogênio microbiano ruminal estimado pela metodologia das purinas duodenais ou dos derivados de purinas excretados na urina não foi similar (P<0,01). Adicionalmente, estas estimativas foram altamente influenciadas pela relação purinas:N microbiano utilizada nos cálculos.

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Worthington, Rebecca A. (Ann). "Structure-function studies of P2X receptors." Thesis, The University of Sydney, 2001. https://hdl.handle.net/2123/27719.

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P2X receptors are fast, ATP-gated cation ion channels. To date seven subtypes of P2X receptors have been cloned and identified, PZXH. The membrane topology of the P2X subunit consists of intracellular amino- and carboxy-termini, two transmembrane spanning domains and a large extracellular loop. Despite similar membrane topology, within this family of receptors the P2X subtypes possess different functional characteristics. They exhibit different sensitivities to agonists and antagonists, are modulated differently by extracellular ions, and have different pore forming abilities. The regions that are responsible for differences in function between P2X subtypes have not been elucidated. This thesis aims to further knowledge regarding the relationship between the structure of the P2X family and differences in the function of the various receptor subtypes. Examination of the primary structure of the P2X receptor family led to the identification of epitope regions suitable for antibody production. This suite of antibodies was tested for specificity and the distribution of P2X receptors was examined in a range of rat tissues and two cell lines. The pathophysiological involvement of the P2X7 receptor was examined in B-CLL patients. Two polymorphisms as well as a loss of function mutation were identified in both normal and leukaemic populations. The site of agonist binding is believed to be within the extracellular loop. Examination of the primary structure of the human cytolytic receptor P2X7 led to the identification of two noncontiguous regions that could potentially be involved in binding ATP. Three amino acid residues that lie within the extracellular loop were targeted and their involvement in ATP binding was determined. Two lysine residues at positions 193 and 31 1 and a proline residue at 210 were each exchanged with alanine. An abolition of function of human receptors with mutations at positions 193 or 311 was observed, consistent with a disruption of the ATP binding domain, although alterations in transduction or gating cannot be dismissed. The P2X receptor appears to be comprised of a trimeric subunit arrangement, and Hill coefficients of between 1 and 3 reported for ATP binding suggest that there is more than one ATP binding site per functional receptor. Modelling of the putative binding cleft of the hP2X7 subunit was performed and the residues important for ATP binding were highlighted. The fimctional trimeric receptor appears to possess three intersubunit ATP binding sites. In an attempt to isolate regions of the extracellular domain that contribute to or control various channel properties, chimaeras between subtypes P2X], P2X4 and P2X7 were constructed and their properties examined. Each of the six chimaeras has been shown to be correctly inserted into the cell membrane and functional. These constructs will continue to be investigated and form the basis for extensive future work.

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Aoki, Juliana Ide. "Ação do análogo de purina tóxico tubercidina em Leishmania ssp." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-30092010-130259/.

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A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de alguns desses compostos. Utilizando a estratégia que permite a indução de super-expressão após transfecção gênica, isolamos dois loci relacionados com resistência ao análogo tóxico de purina, tubercidina (TUB). Em um desses locus identificamos um ortólogo do gene TOR (TOxic nucleoside Resistance) em L. (L.) major (TOR-Lm), capaz de conferir altos níveis de resistência a TUB. A identificação e localização cromossomal do segundo locus foi obtida, mas os testes funcionais em presença de TUB não foram tão significativos quanto os obtidos após a transfecção do TOR-Lm. Na segunda parte desta dissertação avaliamos a eficácia da associação de TUB com um inibidor específico do transporte de nucleosídeos em mamíferos, nitrobenziltioinosina (NBMPR), visando reverter a toxicidade de TUB apenas no hospedeiro. Demonstramos que TUB tem uma potente ação anti-parasitária em culturas de Leishmania spp., e que o inibidor NBMPR é capaz de proteger células mamíferas de camundongos infectados da ação tóxica de TUB. Gene identification associated with drug resistance has contributed to a better understanding of the mechanism of action of anti parasitic compounds. Using transfection and over-expression selection strategy we isolated two loci related with the resistance of tubercidin (TUB), a toxic analog purine. In the first locus we identified an ortholog of the TOR gene (TOxic nucleoside Resistance) in L. (L.) major (TOR-Lm), capable to render wild cells resistance to TUB after transfection and over-expression. Chromosomal location and identification of the second locus was done, but functional tests in the presence of TUB were not as significant as those obtained after TOR-Lm transfection. In the second part of this work, we evaluate the effectiveness of the association of TUB with an inhibitor specific to the mammals nucleoside transport, as nitrobenzylthioinosine (NBMPR), aimed at reversing the TUB toxicity only on the host. We first demonstrate that TUB has a potent anti-parasitic action in cultures of Leishmania spp. Then, we discuss the capacity of the NBMPR inhibitor to protect infected macrophages from the toxic effects of TUB.

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Rodrigues, Elisandra Márcia. "Estudos moleculares das fosforribosil pirofosfato sintetases." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-18062008-085454/.

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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.

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Barbosa, Sara Isabel Cadinha. "Compostos que interferem no metabolismo dos purina- e pirimidina-nucleótidos: utilização como agentes terapêuticos." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5160.

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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas O conteúdo deste trabalho será desenvolvido em dois temas principais, um referente à utilização de compostos que interferem no metabolismo dos purina- e pirimidinanucleótidos como agentes antineoplásicos e outro referente à sua utilização como agentes antivirais. A síntese dos nucleótidos envolve a construção de ácidos nucleicos e a inserção dos derivados de nucleótidos noutras vias bioquímicas, sendo responsável por inúmeras funções do metabolismo celular. Existem patologias que envolvem enzimas essenciais do metabolismo dos nucleótidos, o que levou à síntese de novos fármacos. As doenças oncológicas continuam a matar milhares de pessoas e um tratamento eficaz e com sucesso tem sido um desafio. O mesmo se passa com algumas infeções virais, nomeadamente infeções provocadas pelo HIV. Para contornar os obstáculos enfrentados na terapia destas doenças têm sido usados análogos de nucleótidos e/ou nucleósidos como agentes terapêuticos. Estes têm o propósito de inibir a síntese de novo dos nucleótidos em determinadas etapas, estando envolvidos na replicação e síntese do RNA e DNA nas células em divisão. Atuam por inibição específica de enzimas no metabolismo dos nucleótidos/nucleósidos ou ainda por incorporação no DNA ou no RNA. This study will be developed into two main subjects; one related to the use of compounds which interfere with the metabolism of purine- and pyrimidine- nucleotides as antineoplastic agents; another related to their use as antiviral agents. The nucleotides’ synthesis involves the construction of nucleic acids and the introduction of the nucleotides’ derivatives into other biochemical pathways and it is responsible for numerous functions of cellular metabolism. There are pathologies involving key enzymes from the nucleotides’ metabolism, which led to the synthesis of new drugs. Cancer is a disease that continues killing thousands of people, an effective and successful treatment has been a challenge. The same happens with some viral infections, mainly infections caused by HIV. To overcome the obstacles faced in the therapy of these diseases it has been used nucleotide and/or nucleoside analogues as therapeutic agents. These agents have the purpose of inhibiting the de novo nucleotide synthesis in certain steps, by being involved in RNA and DNA replication and synthesis in dividing cells. They act by specific enzymes inhibition in nucleotide/nucleoside metabolism and by incorporation into DNA or RNA.

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Marsac, Roxane. "La déficience en Adénylosuccinate Lyase - de la déficience métabolique aux défauts musculaires en utilisant le Caenorhabditis elegans comme modèle animal." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0321.

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La voie de biosynthèse des purines est un réseau métabolique conservé de procaryotes à l'Homme, assurant l'homéostasie de l'ATP et du GTP. Les purines peuvent être synthétisées de novo, réutilisées ou produites par interconversion de métabolites existants à l'aide de la voie dite de recyclage. De plus, les intermédiaires peuvent agir en tant que métabolites signals régulant l'expression génique. Cette voie est bien caractérisée chez des micro-organismes tels la levure ou la bactérie, mais peu de choses sont connues chez les métazoaires. Différentes maladies sont associées à des déficiences enzymatiques de la voie de biosynthèse des purines conduisant à des anomalies neuro-musculaires, des comportements du spectre autistique et à un retard psychomoteur chez l'Homme. Nous nous sommes particulièrement intéressés à la déficience en Adénylosuccinate Lyase (ADSL), une enzyme impliquée dans la voie de novo et dans la voie de recyclage des purines, responsable des symptômes neuronaux et musculaires chez les patients.Pour mieux comprendre les mécanismes sous-jacents à cette déficience, nous avons établi C. elegans comme organisme modèle métazoaire pour étudier la voie de la biosynthèse des purines, et en particulier le déficit en ADSL. Dans notre étude, par alignement de séquence, profil HPLC et complémentation fonctionnelle chez la levure, nous avons montré que la voie de novo et la voie de recyclage sont fonctionnellement conservées chez C. elegans. Grâce à notre étude, nous sommes en mesure d’attribuer des phénotypes développementaux et tissus spécifiques à des étapes séparables du métabolisme des purines dans un organisme modèle métazoaire. Notre analyse montre que l'activité ADSL dans la voie de recyclage joue un rôle crucial pour le maintien de la lignée germinale, pour l'intégrité musculaire et pendant le développement post-embryonnaire The purine biosynthesis pathway is a metabolic network conserved from prokaryotes to humans, ensuring ATP and GTP homeostasis. Purines can either be synthesized de novo, reused, or produced by interconversion of extant metabolites using the so-called recycling pathway. Moreover, intermediates can act as signal metabolites regulating gene expression. This pathway is well characterized in microorganisms such as heat or bacteria, but little is know about its regulation in metazoans. Different diseases are associated with deficiencies in purine synthesis enzymes leading to neuromuscular defects, autistic spectrum behaviors and psychomotor delay in humans. We focused our analysis on the deficiency of Adenylosuccinate Lyase (ADSL), which is an enzyme involved in the purine de novo and the recycling pathways causing neuronal and muscular symptoms in patients. To better understand mechanisms underlying this deficiency, we have established C. elegans as a metazoan model organism to study the purine biosynthesis pathway, specially the ADSL deficiency. In our study, by sequence alignment, HPLC profiling and functional complementation in yeast, we have shown that both the de novo and the recycling pathway are functionally conserved in C. elegans. Thanks to our study, we are able to ascribe developmental and tissue specific phenotypes to separable steps of the purine metabolism network in a metazoan model organism. Our analysis shows that ADSL activity in the recycling pathway plays a crucial role for germline maintenance, for muscle integrity and during the post-embryonic development

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Aoki, Juliana Ide. "Identificação de um gene que confere resistência a tubercidina em Leishmania (Leishmania) major." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/99/99131/tde-21022014-151125/.

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A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de compostos antileishmania. Pouco se sabe sobre o mecanismo de ação do análogo de purina tubercidina (TUB) em Leishmania. Utilizando a estratégia de superexpressão após transfecção gênica, isolamos um locus de Leishmania (Leishmania) major, de 31 kb, capaz de conferir níveis de resistência quatro vezes maior que o parasita selvagem. Várias deleções desse locus foram geradas e a construção de 3 kb (pSNBR/3kbClaI-EcoRI) também conferiu níveis de resistência quando comparado ao parasita elvagem. Através de análises no genoma de L. (L.) major, localizamos esse locus no cromossomo 31 e, no fragmento de 3 kb, um gene que codifica para uma proteína com função desconhecida até o momento (LmjF.31.2010). Esta proteína foi relacionada com resistência a TUB em todas as linhagens transfectadas analisadas (cosTUB2 e pSNBR/3kbClaI-EcoRI), assim denominamos LmjF.31.2010, de proteína relacionada com resistência a TUB (PRRT). A quantificação relativa de transcritos de mRNA na construção pSNBR/3kbClaI-EcoRI apresentou níveis altos de transcritos da PRRT. Foram gerados ainda mutantes de L. (L.) major e L. (L.) amazonensis resistentes a TUB e estes se apresentaram bem adaptados a concentrações altas de TUB, apresentando razão de resistência maior que 200 vezes, quando comparado com os respectivos parasitas selvagens. A PRRT também foi relacionada na resistência a TUB nos mutantes gerados, pois houve amplificação gênica de prrt. Os resultados obtidos neste trabalho fornecem dados para inferir a importância da PRRT no mecanismo relacionado com resistência a TUB. The identification of genes associated with resistance to antiparasitic compounds has contributed to a better understanding of the mechanism of action of compounds against Leishmania. Little is known about the mechanism of action of purine analog tubercidin (TUB) in Leishmania. Using a strategy of gene overexpression after transfection, we isolated a locus of Leishmania (Leishmania) major, 31 kb, capable of conferring fold resistance four times greater than the wild type parasite. A set of deletions of this locus were generated and a 3 kb construction (pSNBR/3kbClaI-EcoRI) conferred fold resistance twice than the wild type. Analysis of L. (L.) major genome, located this locus on chromosome 31 and on 3 kb fragment we identified a gene encoding a protein with unknown function (LmjF.31.2010). This protein has been related to TUB resistance in all strains analyzed (cosTUB2 and pSNBR/3kbClaI-EcoRI), so we named mjF.31.2010 of protein related with resistance to TUB (PRRT). Relative quantification of mRNA transcripts in the construction pSNBR/3kbClaI-EcoRI showed high levels of PRRT transcripts.Mutants of L. (L.) major and L. (L.) amazonensis resistant to TUB were also generated and these were well adapted to high TUB concentrations, presenting fol resistance greater than 200 times when compared with their respective wild type. The PRRT was also related to TUB resistance mutants generated by PRRT gene amplification. Despite the high fold resistance presented by TUB resistant mutants, the ratio of expression of these mutant PRRT transfected and wild was similar to the wild type.

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Stefanello, Cristiano Miguel. "Avaliação de marcadores internos para estimativa de fluxo de digesta e proteína microbiana no duodeno de ruminantes." Universidade Federal de Santa Maria, 2014. http://repositorio.ufsm.br/handle/1/10849.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The aim of this study was to evaluate the use of internal markers to estimate duodenal flow of digesta and duodenal microbial protein synthesis in cattle and sheep. Data and samples of in vivo digestibility trials, eight trials with sheep (n=204) and two with cattle (n=31) were used. All animals were cannulated in the duodenum, procedure previously conducted at the Laboratory of Food Science and Ruminants Nutrition at the Federal University of Santa Maria and Laboratory of Animal Nutrition and Food Science at the University of the State of Santa Catarina. Internal markers, acid detergent fiber (ADF) and acid detergent lignin (ADL) were compared to estimate the flow of duodenal digesta. Purines in duodenal digesta were compared with purine derivatives (PD) excreted in urine as markers to estimate the flow of rumen microbial protein in the duodenum. In the latter case, different equations available in literature were tested to estimate the flow of microbial protein from urinary PD. It was concluded that ADF may be used as an internal marker to estimate duodenal flow of digesta in experimental animals cannulated in the duodenum where total fecal excretion is measured, and that the use of DP as a marker of duodenal flow of microbial protein is acceptable for cattle but not for sheep. In sheep, regardless of the equation used, the DP underestimated duodenal availability of rumen microbial protein. O objetivo do presente estudo foi avaliar o uso de marcadores internos para estimativa de fluxo de digesta duodenal e de síntese de proteína microbiana duodenal em bovinos e ovinos. Foram utilizados dados e amostras de oito ensaios de digestibilidade in vivo com ovinos (n=204) e dois com bovinos (n=31), canulados no duodeno, previamente conduzidos no Laboratório de Bromatologia e Nutrição de Ruminantes da Universidade Federal de Santa Maria e no Laboratório de Nutrição Animal e Bromatologia da Universidade do Estado de Santa Catarina. Para estimativa do fluxo de digesta duodenal, foram comparados os seguintes marcadores internos: fibra em detergente ácido (FDA) e lignina em detergente ácido (LDA). Para estimar o fluxo de proteína microbiana ruminal no duodeno, foi comparado o uso de purinas na digesta duodenal com os derivados de purinas (DP) excretados na urina como marcadores. Neste último caso, foram testadas diferentes equações disponíveis na literatura para estimar o fluxo de proteína microbiana a partir dos DP urinários. Concluiu-se que a FDA pode ser utilizada como marcador interno para estimar o fluxo duodenal de digesta em ensaios com animais canulados no duodeno, onde é medida a excreção total de fezes, e que o uso dos DP como marcadores do fluxo duodenal de proteína microbiana é aceitável em bovinos, mas não em ovinos. Em ovinos, independentemente da equação utilizada, os DP subestimam a disponibilidade duodenal de proteína microbiana ruminal.

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Denis, Valérie. "Régulation des gènes de la biosynthèse des purines chez la levure Saccharomyces Cerevisiae." Bordeaux 2, 1999. http://www.theses.fr/1999BOR28660.

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Souza, Juliana Roberta Torini de. "Caracterização estrutural e funcional de duas Nucleosídeo Fosforilases de Schistosoma mansoni." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-26102016-153439/.

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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.

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Petrilli, Camila Lopes. "Regulação da atividade da glândula pineal por estimulação purinérgica." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/41/41135/tde-01052013-140517/.

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A biossíntese de MEL pela pineal envolve a conversão da serotonina a NAS pela enzima AA-NAT, seguida demetilação da NAS pela enzima HIOMT gerando a MEL. A ativação β-adrenérgica é essencial e a co-estimulação de receptores P2Y1 potencia a produção de NAS induzida por noradrenalina. Neste trabalho avaliamos o efeito da estimulação de receptores P2Y1 sobre a produção de MEL induzida pela estimulação β-adrenérgica. A co-estimulação purinérgica com ADP, levou a potenciação da produção de NAS induzida por ISO e a inibição do conteúdo de MEL de maneira dependente de concentração. Em extratos nucleares de pineais estimuladas com ADP, a translocação nuclear de AP-1 foi observada, não havendo alteração significativa na translocação nuclear dos dímeros NF-κB p50/p50 e p50/RelA. PDTC, inibidor de NF-κB, não alterou o conteúdo de NAS e MEL em pineais em cultura estimuladas com ISO e ADP. A expressão gênica e a atividade enzimática de AA-NAT e HIOMT não foram alteradas pela co-estimulação com ISO e ADP. O bloqueio seletivo dos receptores P2Y1 por A3\'P5\'P inibiu de maneira dependente de concentração o efeito potenciador do ADP sobre a produção de NAS induzida por ISO, mas não reverteu a inibição observada nos níveis de melatonina. Estes resultados apontam para um mecanismo diferencial de modulação da produção de NAS e MEL abrindo novas perspectivas ao estudo dos mecanismos regulatórios da produção de MEL e seus metabólitos The biosynthesis of MEL by the pineal gland involves the conversion of serotonin to NAS by the enzyme AA-NAT, followed by methylation of NAS by the enzyme HIOMT generating MEL. The activation of β-adrenergic receptors is essential and the co-stimulation of P2Y1 receptors potentiates noradrenaline-induced NAS production. In this study we evaluated the effect of P2Y1 receptors stimulation on the production of MEL synthesis induced by β-adrenergic stimulation. Purinergic co-stimulation with ADP potentiated ISO-induced NAS production and inhibited melatonin content in a concentration dependent manner. In nuclear extracts from stimulated pineal glands with ADP, the nuclear translocation of AP-1 was observed, with no significant change in the nuclear translocation of the NF-κB dimers p50/p50 and p50/RelA. PDTC, an inhibitor of NF-κB, did not alter the content of NAS and MEL in cultured pineal glands co-stimulated with ISO and ADP. ISO and ADP co-stimulation did not alter the transcript and enzyme activity of AA-NAT and HIOMT. The selective blockade of P2Y1 receptors by A3\'P5\'P inhibited, in a concentration-dependent manner, the potentiating effect of ADP on ISO-induced NAS production but did not change the inhibition observed on MEL levels. These results suggest a differential mechanism on the modulation of NAS and MEL production opening new perspectives to the study of the regulatory mechanisms involved in the biosynthesis of MEL and its metabolites

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Mantovani, Monique. "Adenylosuccinato lyase (ADSL) de leishmania major friedlin: sua relevância na via de recuperação de purino-nucleotídeos." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-09042008-133909/.

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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.

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Spiegel, Erin Kathleen. "Psychomotor deficits in mice transgenic for a mutant adenylosuccinate lyase associated with autism in humans /." Connect to full text via ProQuest. IP filtered, 2006.

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Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006. Typescript. Includes bibliographical references (leaves 127-143). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

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16

Black, Duncan Arthur. "Aspects of purine and pyrimidine metabolism." Doctoral thesis, University of Cape Town, 1989. http://hdl.handle.net/11427/26590.

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In Chapter 1 a review of the literature concerning aspects of erythrocyte membrane transport and metabolism, and purine and pyrimidine metabolism is presented. The effects of pH, pO₂ and inorganic phosphate (Pi) on the uptake and metabolism of hypoxanthine by erythrocytes has been studied in Chapter 2. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low pO₂. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favouring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate (PRPP). Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3- bisphosphoglycerate (2,3-DPG). These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely PRPP synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue. In Chapter 3 the kinetics and mechanism of transport of orotate across the human erythrocyte membrane and the effect of pH and inorganic phosphate on its metabolism (in the erythrocyte) have been studied. It has been shown that orotate enters erythrocytes with non-saturable kinetics and with a capacity of 190 μmoles/1 packed cells/min at a concentration of 4-6 mmolar. The presence of competition for transport by a number of anions and the lack of competition by uridine is indicative of transport by a general anion transporter, with the ability for concentrative uptake in the absence of other external anions being compatible with transport via a ping-pong mechanism. Inhibition of transport by the specific band 3 inhibitors DIDS and CHCA confirm that transport is via the band 3 anion transporter. This explains the lack of significant uptake of orotate by most differentiated tissues which lack the intact band 3 protein. However, the demonstration of band 3 in rat hepatocytes (Cheng and Levy, 1980) provides a mechanism for the orotate transport which has been observed in liver (Handschumacher and Coleridge, 1979). Changes in pH and inorganic phosphate (Pi) concentrations have been shown to have marked effects on the relative quantities of metabolic products produced by the erythrocyte from orotate. There was an increase in orotate metabolised with increasing Pi, an effect augmented by lowering the pH, and most easily explained by the allosteric activation of PRPP synthetase by Pi. The increase in UTP levels with decreasing pH may be the consequence of both increased PRPP availability for the formation of uridine nucleotide from orotate, and decreased conversion of UMP to uridine by pyrimidine 5'-nucleotidase, which is known to be inhibited by phosphate. The accumulation of UDP sugars is optimal at a phosphate concentration of 10 mmolar, which is unexplained but would be compatible with an inhibitory effect of Pi on CTP synthetase. A PRPP wasting cycle at alkaline pH values is proposed to explain the apparent paradox where no PRPP was observed to accumulate in erythrocytes (Chapter 2) at pH values of 7.6 and above in the presence of 10 mmolar phosphate and no added hypoxanthine, yet the metabolism of orotate, which is a PRPP utilising reaction, at alkaline pH values was readily demonstrable here. This (apparent paradox) can be resolved if one assumes that even in the absence of added hypoxanthine and demonstrable intracellular IMP there are sufficient quantities of hypoxanthine and/or IMP to maintain a PRPP wasting cycle at alkaline pH values. The cycle is interrupted at acidic pH values as phosphate levels rise and inhibit 5'-nucleotidase, an effect augmented by the decreasing levels of 2,3-DPG which accompany decreasing pH. This wasting cycle has recently been confirmed by P. Berman (unpublished). The kinetics of orotate uptake by erythrocytes and its eventual release as uridine provides a role for the erythrocyte in the transport and distribution of pyrimidines to peripheral tissues. A model is proposed and involves the de novo production of orotate in the liver. In the next step erythrocytes take up the orotate secreted by the liver into the circulation, convert it into an intermediate buffer store of uridine nucleotides, whose distribution is a function of pH and phosphate concentration, and eventually release it as uridine, which is a readily available form of pyrimidine for utilisation by peripheral nucleated cells. The enhancement of uptake of labelled orotate into nucleic acids of cultured cells is demonstrated here. The degradative half of the cycle proposes that uracil and palanine are the predominant degradative forms of pyrimidines produced by peripheral cells, and their ultimate metabolic fate is complete catabolism in the liver to CO₂ and water. In the final chapter the possible role of the human erythrocyte in the prevention of reperfusion injury has been investigated. The development of a model of renal ischaemia in the rat is described. The ability of human erythrocytes, "primed" by preincubating in acid medium of high Pi concentration and low pO₂, to take up hypoxanthine in a concentrative manner when perfused through ischaemic rat kidney is demonstrated. Attempts to demonstrate improved survival and renal function in rats with "primed" human erythrocytes prior to reperfusion were, however, unsuccessful. It is further demonstrated that "unprimed" human erythrocytes, resident in ischaemic rat kidney for 3 hours, take up hypoxanthine and convert it to IMP. that erythrocytes could play a physiological prevention of reperfusion injury.

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Silva, Júnior Jarbas Miguel da. "Excreção urinária de derivados de purinas e de compostos nitrogenados de zebuínos em pastejo." Universidade Federal de Viçosa, 2014. http://locus.ufv.br/handle/123456789/5825.

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Made available in DSpace on 2015-03-26T13:55:26Z (GMT). No. of bitstreams: 1 texto completo.pdf: 876963 bytes, checksum: 2254ce6044753867875b6bfab3b2249e (MD5) Previous issue date: 2014-07-21 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior This study aimed evaluating the excretion of purine derivatives and nitrogen compounds in zebu cattle grazing, on different days and times within days. The experiment was conducted in the cattle department of the Federal University of Viçosa / MG, using five Nelore heifers with an average body weight of 300 ± 15 kg and 20 months of age, in 5x5 Latin square design. The experimental treatments were defined to represent those commonly used in the dry season, as follows: control (mineral salt), concentrated with 20.31% crude protein (CP) on dry matter (DM) being offered (OF) level of 0.5 to 1% of body weight fasted (BWF) OF5 and OF10, respectively; and two concentrated self- regulating (SR) consumption, containing 69.38% CP on a DM basis (20% urea and 20% salt) offered ad libitun (SR70) and other concentrate containing 39.73% CP based on MS being offered ad libitum (SR40). The experimental periods was 18 days, with one day to perform 14 hours of fasting for weighing and adjustment of the quantities supplied, 12 days for adaptation to the experimental diets and five for total collection of urine and stool sample at the times of 0h00a.m. to 4h00a.m, 4h00a.m. to 8:00a.m., 8:00a.m. to 12:00p.m., 12:00p.m. to 4:00p.m., 4:00p.m. to 8:00p.m. and 20:00p.m. to 0:00a.m. For total collection of urine was used probe Folley number 26, coupled to polyethylene hose leading to a urine collection bag for urine closed system, which was emptied every two hours in the range of 8:00a.m. to 8:00p.m., and every four hours in the range from 8:00p.m. to 8:00a.m. and subsequently homogenized and cooled. The collected urine sampling was performed every four hours, measuring the volume, and withdrawing one sample were diluted in H 2 SO 4 at 0,036N and another don't diluted. To estimate fecal output, used the titanium dioxide, provided the total daily amount of 15g, between 9 th and 18 th day of each period. To estimate the intake of pasture, used the indigestible neutral detergent fiber (iNDF) as internal indicator. Was performed by collecting pasture technique for determining the square potentially digestible dry matter (PDDM) on the third day of each experimental period, and on days 14 th and 18 th was held grazing simulation to estimate the consumption of constituents of diets. In urine samples the concentrations of creatinine, total nitrogen, urea, uric acid and allantoin. For statistical analysis we used the statistical program SAS Proc Mixed. Dry matter intake 10was higher (P<0.05) for the treatment OF10 compared to SR70, SR40 and control treatments but was not different (P>0.05) treatment OF5. The CP intake increased by supplementation (P<0.05), which caused no effect on DM intake from pasture. Excretions of creatinine did not change treatment, day and sampling period (P>0.05) and had a mean of 23.03 ± 0.30 mg / kgPC. Urinary relations of allantoin (Al) and uric acid (UA) with creatinine were not affected (P>0.05) by treatments, collection days and times of collection. The total nitrogen relations:creatinine and urea nitrogen:creatinine in urine showed interaction (P<0.05) between treatment and sampling period. The relationship between urea nitrogen:total nitrogen was influenced (P<0.05) only at time of collection. The nitrogen balance (NB) in g/day did not differ between treatments OF10, SR70 and SR40, however these had higher retention of N (P<0.05) than treatments OF5 and control, which were not different. The NB, in g/ging, showed differences (P<0.05) between treatments with concentrated, which did not differ (P> 0.05), and control treatment, with the lowest NB. The production of microbial N was not affected (P>0.05) by treatments. The microbial efficiency gPBmic/kgMOD and gPBmic/kgNDT was affected (P<0.05) by supplementation, being higher (P<0.05) for OF5, OF10 and SR70 treatments, which did not differ. The control and OF5, treatments had the lowest values were similar. The lack of effect of day and the collection period on allantoin and uric acid compared with creatinine has wide practical application, enabling use spot urine sample to calculate the excretion of purine derivatives at any time of day or night, and consequently the microbial production. Depending on the variations observed for total nitrogen and urea nitrogen relations with creatinine over 24 hours is not recommended the use of a single spot urine sample for determination of these nitrogen compounds. O presente trabalho foi desenvolvido com os objetivos de avaliar a excreção dos derivados de purinas e de compostos nitrogenados em zebuínos em pastejo, em diferentes dias e períodos dentro de dias. O experimento foi conduzido no setor de gado de corte da Universidade Federal de Viçosa/MG, utilizando-se cinco novilhas Nelore com peso corporal médio de 300 ± 15kg e 20 meses de idade, distribuídas em quadrado latino 5x5. Os tratamentos experimentais foram definidos para representar aqueles normalmente utilizados na época seca do ano, sendo eles: controle (sal mineral), concentrado com 20,31% de proteína bruta (PB) com base na matéria seca (MS) sendo oferecido (OF) em nível de 0,5 e 1% do peso corporal em jejum (PCJ), OF5 e OF10, respectivamente; e dois concentrados autorreguladores (AR) de consumo, um contendo 69,38% PB com base na MS (20% de ureia e 20% de sal) ofertado ad libitun (AR70) e outro concentrado contendo 39,73% PB com base na MS sendo ofertado ad libitum (AR40). Os períodos experimentais possuíram 18 dias, sendo o dia um para realização de jejum de 14 horas para pesagem e ajuste das quantidades fornecidas, 12 para adaptação dos animais às dietas experimentais e cinco para a coleta total de urina e amostral de fezes, nos horários das 0h00 às 4h00, 4h00 às 8h00, 8h00 às 12h00, 12h00 às 16h00, 16h00 às 20h00 e 20h00 às 24h00. Para a coleta total de urina utilizou-se sonda de Folley no26, acoplada a mangueira de polietileno que conduziu a urina até uma bolsa coletora de urina por sistema fechado, que foi esvaziada a cada duas horas no intervalo das 8h00 às 20h00, e a cada quatro horas no intervalo das 20h00 às 8h00, sendo posteriormente homogeneizadas e resfriadas. A amostragem da urina coletada foi realizada a cada 4 horas, medindo-se o volume e retirando-se duas amostras, uma diluída com solução H2SO4 0,036N e não diluida. Para determinação da excreção fecal, utilizou-se o dioxido de titânio, fornecido na quantidade total diária de 15g, entre os dias 9 e 18 de cada período. Para estimativa do consumo de pasto, utilizou-se a fibra indigestível em detergente neutro (FDNi), como indicador interno. Realizou-se coleta de pasto pela técnica do quadrado para determinação da matéria seca potencialmente digestível (MSpd) no terceiro dia de cada período experimental, e nos dias 14o e 18o realizou-se simulação de pastejo para estimar os consumos dos constituintes das dietas. Nas amostras de urina foram determinadas as concentrações de creatinina, nitrogênio total, ureia, acido úrico e alantoína. Para análise estatística utilizou-se o programa estatístico Proc Mixed do SAS. O consumo de MS foi superior (P<0,05) para o tratamento OF10 em relação aos tratamentos AR70, AR40 e controle, mas não diferiu (P>0,05) do tratamento OF5. O consumo de PB aumentou com a suplementação (P<0,05), que não causou efeito sobre o consumo de MS do pasto. As excreções de creatinina não sofreram efeito de tratamento, dia e período de coleta (P>0,05) e apresentaram média de 23,03 ± 0,30 mg/kgPC. As relações urinárias da alantoína (Al) e do ácido úrico (AU) com a creatinina não foram influenciadas (P>0,05) pelos tratamentos, dias de coleta e horários de coleta. As relações nitrogênio total:creatinina e nitrogênio ureico:creatinina na urina apresentaram interação (P<0,05) entre tratamento e período de coleta. A relação entre nitrogênio ureico:nitrogênio total foi influenciada (P<0,05) apenas pelo horário de coleta. O balanço de compostos nitrogenados (BN), em g/dia, não diferiu entre os tratamentos OF10, AR70 e AR40, contudo esses apresentaram maiores retenções de N (P<0,05) que os tratamentos OF5 e controle, que não foram diferentes. O BN, em g/ging, apresentou diferença (P<0,05) entre os tratamentos com concentrado, que não diferiram entre si (P>0,05), e tratamento controle, que apresentou o menor BN. A produção de compostos nitrogenados microbianos não foi alterada (P>0,05) pelos tratamentos. A eficiência microbiana, em gPBmic/kgMOD e gPBmic/kgNDT foi afetada (P<0,05) pela suplementação, sendo maior (P<0,05) para os tratamentos OF5, OF10 e AR70, que não diferiram entre si. Os tratamentos controle e OF5, apresentaram os menores valores e foram semelhantes entre si. A ausência de efeito de dia e do período de coleta sobre a relação alantoína e ácido úrico com a creatinina tem grande aplicação prática, possibilitando utilizar a amostra spot de urina para calcular a excreção de derivados de purinas em qualquer horário do dia ou da noite, e consequentemente a produção microbiana. Em função das variações observadas para as relações nitrogênio ureico e nitrogênio total com a creatinina ao longo do período de 24 horas não se recomenda o uso de uma única amostra spot de urina para determinação destes compostos nitrogenados.

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18

Pinson, Benoît. "Purification et caractérisations chimique et fonctionnelle de la purine-cytosine perméase de la membrane plasmique de la levure Saccharomyces cerevisae." Bordeaux 2, 1996. http://www.theses.fr/1996BOR28443.

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L'objectif de ce travail est l'étude biochimique et bioénergétique du mécanisme moléculaire de fonctionnement de la Purine-Cytosine Perméase (PCP), protéine intrinsèque de la membrane plasmique de la levure Saccharomyces cerevisiae qui catalyse l'entrée dans la cellule de trois bases purines (adenine, hypoxanthine et guanine) et de la cytosine. La PCP solubilisée par un détergent non ionique a pu être purifiée par chromatographie d'affinité, soit par une colonne de chélate de nickel en travaillant avec une forme modifiée de la protéine (six résidus histidyles ajoutés à son extrémité C-terminale), soit sur une colonne d'immunoaffinité construite avec un anticorps anti-C terminal, mais en travaillant cette fois-ci avec la forme sauvage du transporteur. Ces deux approches ont permis d'obtenir des échantillons de PCP pure à 90 %, et l'analyse de la séquence de la protéine purifiée nous a montré que cette protéine avait son extrémité N-terminal bloquée. L'immunoprécipitation de la PCP après des marquages in vivo des proréines cellulaires avec de l'ortophosphate radioactif a permis de mettre en évidence que la PCP est phosphorylée. Cette phosphorylation se produit sur des résidus séryles au cours du trafic des protéines entre l'appareil de Golgi et la membrane plasmique ou dans cette membrane elle même. La fusion (par congélation décongélation et extrusion) de préparations enrichies en membranes plasmiques de souches surexprimant la PCP et de protéoliposomes contenant la cytochrome c oxydase a permis d'obtenir des vésicules closes dans lesquelles nous avons pu générer une différence de potentiel électrochimique transmembranaire en protons. Ce système artificiel a permis de prouver que la PCP est un transporteur actif secondaire qui catalyse un symport proton/base et qui utilise principalement la composante chimique de la force proton-motrice The aim of this work is the biochemical and bioenergetical analyses of the uptake mechanism of the Purine-Cytosine Permease (PCP), a protein located in the plasma membrane of the yeast Saccharomyces cerevisiae which catalyses adenine, guanine, hypoxantine and cytosine transport in cells. After solubilisation with a non ionic detergent, PCP was purified by affinity chromatography, either on nickel chelate affinity column using a recombinant PCP carrying a carboxy-terminal affinity tag for metallic ions (six successive histidyl residues), or on immunoaffinity column obtained with anti-C-terminal peptide) IgG using a wild-type PCP. By means of these two chromatographies, we have obtained a PCP enriched fraction containing 90 % of this protein. Determination of the PCP saquence shows a blocked N-terminal extremity. By means of in vivo radioactive ortophosphate labelling of cells proteins and PCP immunoprecipitation, we demonstrate that this protein is phosphorylated. This phosphorylation occurs on seryls residues in the secretory pathway either between the Golgi apparatus and the plasma membrane or in the plasma membrane itself. Plasma membrane enriched fractions containing PCP were fused with proteoliposomes containing cytochrome oxidase to obtain closed vesicles in which proton motive force could be generated and maintained. By means of this artificial system, we demonstrate that PCP is a secondary active carrier that catalyses a proton/base symport using mainly the chemical component of the proton motive force

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19

Martins, Nádia Helena 1982. "Estudos estruturais e biofísicos da enzima purina nucleosídeo fosforilase hexamérica de Bacillus subtilis." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317143.

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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

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20

Simic, Aleksandra. "Le Corbusier’s Purist Period and the Concept of Truth in Architecture." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1155728763.

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21

Deharvengt, Sophie. "Etude du système gène suicide/prodrogue : Purine nucléoside phosphorylase/6-méthyle purine désoxyribose et de son ciblage thérapeutique dans les carcinomes pancréatiques." Université Louis Pasteur (Strasbourg) (1971-2008), 2003. http://www.theses.fr/2003STR13209.

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Le cancer du pancréas reste une maladie peu ou pas curable, nécessitant le développement de nouveaux moyens thérapeutiques, tel que la thérapie génique. Une approche prometteuse est l'utilisation de systèmes gène suicide/prodrogue, consistant à insérer dans les cellules tumorales un gène codant pour une enzyme exogène capable de convertir une prodrogue, relativement non toxique, en un métabolite cytotoxique. Son avantage majeur est son effet de voisinage (bystander), qui permet une éradication de la tumeur sans que la totalité des cellules exprime le gène suicide, surmontant ainsi le faible taux de transfection rencontré dans les expériences in vivo. L'objectif de ce travail est d'étudier un nouveau système gène suicide/prodrogue : la Purine Nucléoside Phosphorylase d'Escherichia Coli (ePNP)/6-méthyle-purine désoxyribose (MePdR) dans le traitement des adénocarcinomes pancréatiques. EPNP converti le MePdR non toxique en un métabolite toxique, le 6-méthyle-purine (MeP). Le MeP est capable de tuer les cellules quiescentes, ce qui le distingue de la plupart des agents anti-tumoraux et permet son utilisation dans le traitement de tumeurs solides. Après clonage du gène et établissement de clones stables, la toxicité et l'effet bystander de ce système ont été vérifiés in vitro sur des modèles digestifs et in vivo sur des xénogreffes humaines chez des souris immunodéficientes. Le transfert d'ePNP par électropration a été évalué sur le même modèle de tumeurs localisées en sous cutané. Afin de mieux cibler l'expression d'ePNP dans les cellules tumorales, des promoteurs de marqueurs tumoraux ont été utilisés. Ils permettent l'expression sélective d'ePNP dans les cellules néoplasiques exprimant ces marqueurs. Enfin, pour prendre en compte le contexte clinique avec présence de métastases dans lequel l'électroporation s'avère difficilement applicable, une technique de transfert systémique par l'utilisation de parvovirus murins présentant des propriétés d'oncotropisme, a été utilisée. Tous les résultats obtenus mettent l'accent sur les potentialités du système ePNP/MePdR dans le traitement des adénocarcinomes pancréatiques humains Pancreatic cancer is a disease poorly or not curable that requires the development of new therapeutic tools, such as the gene therapy. A promising approach is the use of suicide gene/prodrug systems, which consists in transducing in the tumoral cells a gene encoding for an exogenous enzyme able to convert a prodrug, relatively safe, in a cytotoxic metabolite. The major advantage of this approach is its bystander effect (on the vicinity), which allows tumour eradication even when all cells are not expressing the suicide gene. It offers the opportunity to overcome the poor experimental rate of transfection encountered in vivo. The objective of the current work is to study a new suicide gene /prodrug system, Escherichia Coli Purine Nucleoside Phosphorylase (ePNP)/6-methylpurine deoxyribose (MePdR) for pancreatic adenocarcinoma treatment. EPNP converted non-toxic MePdR into a toxic metabolite 6-methyl-purine (MeP). The ability of MeP to kill the quiescent cells, on the contrary of the majority of the antitumour agents, allows its use for the treatment of solid tumours. After gene cloning and establishment of stable clones, toxicity and bystander effect of this system were checked in vitro on digestive models and in vivo on human subcutaneous tumour xenografts in immunocompromised mice. Transfer of ePNP gene by electroporation was evaluated on the same tumour model. We used promoters of tumour markers to improve the targeting of ePNP expression within tumour cells. They allow the selective expression of ePNP into the neoplastic cells expressing such markers. Lastly, taking in account a clinical situation with presence of metastases in which electroporation cannot be easily applied, we used murine parvovirus with oncotropic properties to perform a systemic transfer. Thus, all these results highlight the potentialities of the MePdR/ePNP system to treat human pancreatic adenocarcinoma

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22

Kulikov, Vladislav [Verfasser], Gerd [Akademischer Betreuer] Meyer, Leroy [Akademischer Betreuer] Cronin, Bernhard [Akademischer Betreuer] Lippert, and Hans-Günther [Akademischer Betreuer] Schmalz. "Hybrid Materials Consisting of Silver(I) Purine Complexes, Protonated Purines and Polyoxometalates / Vladislav Kulikov. Gutachter: Gerd Meyer ; Leroy Cronin ; Bernhard Lippert ; Hans-Günther Schmalz." Köln : Universitäts- und Stadtbibliothek Köln, 2013. http://d-nb.info/1044679964/34.

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23

Märschenz, Katrin. "Neue Purine mit gerinnungsphysiologischer Aktivität." [S.l.] : [s.n.], 2003. http://www.diss.fu-berlin.de/2003/255/index.html.

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24

Quashie, Neils Benjamin. "Purine transport in plasmodium falciparum." Thesis, Thesis restricted. Connect to e-thesis to view abstract, 2008. http://theses.gla.ac.uk/165/.

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Thesis (Ph.D.) -- University of Glasgow, 2008. Ph.D. thesis submitted to the Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

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25

Carrique, Loic. "Études des relations structure-fonctionactivité d’enzymes de Plasmodium falciparum pour la conception et la synthèse de nouvelles molécules antipaludiques." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1109.

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Plasmodium falciparum est responsable de la forme la plus grave de paludisme avec plus de 600 000 décès par an. L'absence de vaccin efficace, combinée à l'émergence de résistances aux traitements récurrents, exige le développement de nouvelles molécules. Afin de limiter ces résistances, il est nécessaire de cibler de nouvelles voies métaboliques indispensables à la survie du parasite. Ce travail de thèse repose sur l'étude de deux voies métaboliques essentielles au parasite que sont la voie de recyclage des bases puriques et la voie de biosynthèse des ancres glycosylphosphatidylinositol (GPI).En ce qui concerne la voie de recyclage des bases puriques, la détermination des structures cristallines de l' « IMP specific 5‘-nucleotidase » (PfISN1) associée aux études biochimiques et biophysiques (SAXS, EM, MALS…), a permis de préciser le mécanisme d'action fournissant ainsi une base solide pour la mise au point d'inhibiteurs. Une banque de plus 3000 composés a été criblée par Fluorimétrie à Balayage Différentiel et les effets des molécules sélectionnées seront évalués sur l'enzyme et sur la croissance du parasite en culture.Quatre cibles thérapeutiques potentielles appartenant à la voie de biosynthèse des ancres GPI ont été sélectionnées. L'utilisation de plusieurs systèmes d'expression disponibles au laboratoire (bactérie, levure, acellulaire en germe de blé) ou via des plateformes européennes pour l‘expression en cellules de mammifères HEK293T (Oxford), de cellules BHK21 transfectées avec le virus de la vaccine modifié, T7-MVA, (Strasbourg) ou la plateforme ESPRIT (Grenoble) ont permis de passer outre les difficultés rencontrées pour exprimer les protéines d'intérêt. L'une des quatre cibles, la mannose-1-phosphate guanylyltransférase (PfMPG), a pu être exprimée de manière suffisante quantitativement et qualitativement pour une caractérisation biochimique et structurale. Une analyse par SAXS et cristallographie aux rayons X a été réalisée Plasmodium falciparum is responsible for the most severe form of malaria with more than 600,000 deaths per year. The lack of an effective vaccine, combined with the emergence of drug resistant parasites, necessitates the development of new drugs. In order to limit these resistances, it is necessary to target new metabolic pathways essential for parasite survival. This thesis work is based on the study of two metabolic pathways essential to the parasite, the purine salvage pathways and the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway.Concerning the purine salvage pathway, the determination of the crystal structures of the IMP -specific 5'-nucleotidase (PfISN1) associated with biochemical and biophysical studies (SAXS, EM, MALS, etc.) have allowed to propose a reaction mechanism, thereby providing a solid basis for the conception and development of inhibitors. A library of more than 3000 compounds was screened by Differential Scanning Fluorimetry and the selected molecules will be evaluated for their inhibitory effect on the enzyme and on the growth of parasites in culture.Four potential therapeutic targets belonging to the GPI anchor biosynthesis pathway were selected. The use of several in-house available expression systems (bacteria, yeast, and acellular wheat germ) as well as European platforms for the expression in HEK293T mammalian cells (Oxford), in BHK21 cells transfected with the modified vaccinia virus, T7-MVA, (Strasbourg) or the ESPRIT platform (Grenoble) has allowed us to overcome the difficulties encountered on obtaining the selected protein targets. One of the four targets has been expressed in sufficient amount and quality for biochemical- and structural characterization, namely the mannose-1-phosphate guanylyltransferase (PfMPG). SAXS and X-ray crystallography analyses have been carried out

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26

Monsalve, Monica Soto. "Determinação estrutural por difração de raios X de pirrolidinas poliidroxiladas com potencial atividade inibidora de purina nucleosídeo fosforilase." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/75/75135/tde-02102017-145525/.

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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···π. 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···π. 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.

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27

Wong, Christopher. "Purine-based Inhibitors of Nek2 Kinase." Thesis, University of Newcastle Upon Tyne, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612618.

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Protein kinases are involved in many important regulatory processes throughout the cell cycle. In particular, several kinase families (i.e. cyclin-dependent kinases, aurora, polo, Nek and checkpoint kinases) have been identified to play important roles in centrosome regulation and cell cycle progression. Consistent errors in cell duplication can often result in defective cells, possessing aneuploidy and/ or chromosome instability (CIN); a common trait of cancer cells. Overexpression and increased kinase activity is often observed in a diverse number of human cancers. Numerous drug discovery programs targeting CDKs, aurora, Polo and Chk kinases are currently ongoing, with evidence of anti-tumour activity observed by using selective, small molecule inhibitors.

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28

Leigh, Maria. "Non purine inhibitors of xanthine oxidase." Thesis, University of York, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.550273.

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The increase in the occurrence of hyperuricemia and gout in recent years, coupled with side- effects associated with use of the main anti-gout therapeutic, allopurinol, have triggered the search for non-purine alternatives. The first such inhibitor to be made available is Adenurlc", 2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylic acid. The aim of this work was to synthesise a small library of non-purine compounds and evaluate their inhibitory activity against the enzyme, xanthine oxidase. Successful inhibitors are marked as those that gave a comparable or lower ICso value than allopurinol (3 ± 0 J.1M), under conditions employed in this work. A range of five-membered heterocyclic compounds, including 1,2,4-triazole-3-thiones, 2- amino-1,3,4-thiadiazoles and 1,3-thiazoles were synthesised. On testing, the heterocyciic compounds did not reveal significant inhibition of XO. SAR analysis marked the presence of alkyl chains and para-cyano substituents as contributors to inhibition. The most successful heterocyciics investigated were highlighted as [17b), 4-(4-cyanophenyl)-1,3-thiazole-2- carboxylic acid, (ICso= 150 ± 14 J.1M) and [15), [4-(4-bromophenyl)-1,3-thiazole-2- sulfanyl]acetonitrile, (ICso= 29 ± 4 J.1M). A concise library of Schiff base compounds was then investigated, incorporating thiosemicarbazones, dithiocarbazates and hydrazones, with varied number and positioning of aromatic substituents. SAR analysis revealed important structural features affecting inhibition, such as a para-hydroxyl benzaldehyde substituent and a para-hydroxyl/cyano substituent on hydrazide/thiosemicarbazide rings. The position rather than the number of hydroxyl substituents proved significant for inhibition. The most potent compounds were thiosemicarbazones 8-Tz3 (0.6 ± 0.2 IlM), 10-Tz2 (0.5 ± 0 ~LM), 10-Tz3 (0.6 ± 0.1 IlM), 11- Tz2 (O.B ± 0.1 J.1M), 11-Tz3 (0.6 ± 0 J.1M) and 11•DTz1 (0.7 ± 0.1 J.1M), giving four times lower ICso than allopurinol. Potent uncomplexed Schiff bases as XO inhibitors have not been previously reported. Steady-state kinetic studies on 8-Tz3, 11-Tz3, 8-DTz1 and 11•DTz1 revealed mixed-type inhibition. As 11•DTz1 gave the lowest Kj value (0.4 ± 0 IlM), coupled with its low ICso value, highlight it as a promising lead compound. Upon reduction of the imine bond, 11•DTz1 reduced retained improved activity over allopurinol, though less potent than its parent Schiff base. Complexation to Mo{VI) and Cu{lI) centres occurred, with the ligands acting as tridentate ONS/ONO donors. The distorted octahedral coordination geometry of Mo(Vl) complexes was confirmed via crystal structure determination. The Cu(lI) complexes, [Cun(l10-TZ2)n) and [Cun(L S-HS)n), revealed a 3-fold increase in potency over their free ligands. The presence of Cu(lI) therefore appears to have a cooperative effect and warrants further investigation as means of improving potency and hydrolytic stability.

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Matt, Alissa Anne Haedt. "Ecological momentary assessment of purging disorder." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/3348.

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Purging Disorder (PD) is characterized by purging after normal or small amounts of food among individuals who are not underweight. Several studies indicate that PD is associated with distress and impairment, underscoring the need for intervention. However, little is known about factors that trigger and maintain purging in PD. This study examined antecedents and consequences of purging using Ecological Momentary Assessment (EMA), a design that involved repeated assessments of current psychological states in participants' natural environments. Women with PD (N = 24) were recruited from the community to make multiple daily ratings of affect, shape/weight concerns, violation of dietary rules, and stomach discomfort using random-, interval-, and event-contingent recordings over a two-week period. Multilevel model analyses were used to examine between-day differences (purge versus non-purge day) and within-day changes in psychological variables relative to purging behavior. Results supported study hypotheses that negative affect and shape/weight concerns would be higher and positive affect would be lower on days when participants purged compared to days they did not purge. In addition, antecedent analyses supported within-day increases in negative affect, shape/weight concerns, and stomach discomfort prior to purging; however, only changes in positive affect and shape/weight concerns on purge days differed from naturally-occurring changes observed on non-purge days. For consequence analyses, negative affect, shape/weight concerns, and stomach discomfort decreased following purging on purge days, and trajectories of change were significantly different from non-purge days. Finally, exploratory analyses suggested that lower levels of impulsivity enhanced associations between antecedent affect and purging. These data are crucial to understand why women with PD purge after consuming normal or small amounts of food and may point to specific targets for the development of effective interventions.

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Souza, Marcos Michel de [UNESP]. "Estudos estruturais da Purina Nucleosídeo Fosforilase do Mycobacterium tuberculosis complexada com ligantes." Universidade Estadual Paulista (UNESP), 2007. http://hdl.handle.net/11449/87511.

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Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-23Bitstream added on 2014-06-13T20:10:04Z : No. of bitstreams: 1 souza_mm_me_sjrp.pdf: 836354 bytes, checksum: 11c4cceceb0b5cfc0fbfbee0acea3de5 (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) De acordo com a OMS, a tuberculose mata 5000 pessoas por dia, e se não controlada, são estimados 35 milhões de novos casos nos próximos vinte anos. A alta susceptibilidade dos infectados por HIV para a tuberculose, bem como a proliferação da tuberculose multidroga-resistente (MDR), tem criado um grande interesse mundial de expansão nos programas atuais de pesquisas sobre a tuberculose. A Purina Nucleosídeo Fosforilase do Mycobacterium tuberculosis (MtPNP) é um potencial alvo para novas drogas anti-tuberculose visto que é responsável pela síntese de novo de ribonucleotídeos de purina. A Inibição específica da PNP poderia potencialmente levar o M. tuberculosis ao estado latente. O objetivo desde trabalho é resolver a estrutura da MtPNP complexada com adenina, e analisar as interações com os ligantes. A MtPNP foi cristalizada utilizando condições experimentais descritas posteriormente, e o ligante (adenina) foi adicionado por soaking. Os dados de difração de raios X foram coletados no detector CCD utilizando fonte de radiação síncotron (Laboratório Nacional de Luz Síncrotron, LNLS, Campinas, Brazil). O programa MOSFLM foi utilizado para o processamento, e os dados foram escalonados através do programa SCALA, apresentando o grupo espacial ortorrômico P21212 (a=118,96Å, b=134,65 Å, c=44,42 Å). O cristal foi determinado utilizando o método de substituição... In according to the WHO, the tuberculosis kills 5000 people every day, if does not controlled, are esteemed 35 millions of new cases in next 20 years. The high susceptibility of human immunodeficiency virus infected persons to the disease and the proliferation of multidrug-resistant (MDR) strains have created a worldwide interest in expanding current programs in tuberculosis research. The Purine Nucleoside Phosphorylase from Mycobacterum tuberculosis (MtPNP) is a potential target for new drug anti-tuberculosis, whereas is responsible for de novo synthesis of purine ribonucleotides. The specific inhibition of M. tuberculosis PNP could potentially lead to the latent state of M. tuberculosis. The objective of this work is to solve the structure from MtPNP complexed with adenine, and to analyze their interactions with the ligand. MtPNP was crystallized using the experimental conditions described elsewhere, and the ligand (adenine) was added by soaking. The X-ray diffraction data were collected on a CCD detector using synchrotron radiation source (Laboratório Nacional de Luz Síncrotron, LNLS, Campinas, Brazil). It was used the MOSFLM program to processing, and the data were scaled through the program SCALA, presenting orthorhombic spatial group P21212 (a=118,96Å, b=134,65 Å, c=44,42 Å). The crystal was determined by molecular replacement methods using the program AMoRe. The final model has Rfree and Rfactor of 23.87% and 17.51% respectively, and maximum resolution of 1.86Å. It was observed a large...(Complete abstract click electronic access below)

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Retamoso, Leandro Thies. "PAPEL DO SISTEMA PURINÉRGICO E DOS RECEPTORES DE POTENCIAL TRANSITÓRIO VANILOIDE 1 (TRPV 1) NA DOR MUSCULAR TARDIA APÓS EXERCÍCIO EXCÊNTRICO EM RATOS." Universidade Federal de Santa Maria, 2014. http://repositorio.ufsm.br/handle/1/6715.

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Chronic exercise has been recommended as a strategy for preventing several diseases associated with lifestyle such as heart disease, hypertension, osteoporosis and type II diabetes. Although regular physical exercise has benefits for health, all sports practitioners, and even sedentary people, have already feel delayed onset muscle soreness (DOMS) once, characterized by discomfort in skeletal muscle. As the most DOMS generator, acute eccentric exercise induce fatigue, strength reduction and performance impairment. Despite some researches demonstrating reactive oxygen species (ROS) in this context, there are few information about purine degradation as well as transient receptor potential vanilloid 1 (TRPV1) on DOMS development. In this context, animals performed a downhill running test (eccentric exercise) on treadmill until exhaustion for histologic evaluation, mechanical allodynea, strength force test and biochemical analysis. The results showed an increase in mechanical allodynea and ADP, AMP, uric acid and TRPV1 immunoreactivity levels. In conclusion, the results support the contribution of ROS and the participation of purine and TRPV1 on delayed onset muscle soreness. O exercício físico crônico tem sido recomendado como estratégia para a prevenção de diversas doenças associadas ao estilo de vida, como doenças cardíacas, hipertensão, osteoporose e diabetes tipo 2. Embora o exercício físico regular traga benefícios para a saúde, todos os praticantes de atividade física e esporte e, até mesmo indivíduos sedentários, já experimentaram alguma vez na vida um episódio de dor muscular tardia (DMT), caracterizada pela sensação de desconforto na musculatura esquelética. Como grande gerador de DMT, destaca-se o exercício excêntrico agudo que induz fadiga, redução de força e perda de desempenho. Apesar de diversos estudos demonstrando a participação das espécies reativas de oxigênio neste quadro, pouco se sabe sobre a participação da degradação das purinas bem como a participação dos receptores de potencial transitório vaniloide 1 (TRPV1) no desenvolvimento da dor muscular tardia. Para tanto, os ratos wistar machos realizaram teste de downhill em esteira (exercício excêntrico) até a exaustão. Após foram analisados os danos histológicos nos músculos gastrocnêmio e sóleo, Outro set de animais após a exaustão foram avaliados nos testes de alodinea mecanica na pata traseira direita, teste funcional de força nas pastas dianteiras e análises bioquímicas no músculo gastrocnêmio. Os resultados demonstram aumento na alodinea, na carbonilação protéica, nos níves de ADP, AMP, ácido úrico, além de elevar os níveis de immureatividade do receptor TRPV1 e atividade da xantina oxidase. Esses dados apontam uma possível contribuição das espécies reativas de oxigênio, da degradação de purinas e dos receptores TRPV1 na dor muscular tardia.

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Rico, Eduardo Pacheco. "A utilização do zebrafish como modelo para avaliar a influência da exposição crônica ao etanol nos sistemas glutamatérgico, purinérgico e níveis de BDNF." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2011. http://hdl.handle.net/10183/31701.

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O zebrafish (Danio rerio) é uma espécie utilizada como modelo experimental em diversas áreas, tais como neurociências toxicologia. Seu genoma já está praticamente sequenciado e estudos demonstraram que muitos genes deste peixe são similares aos de mamíferos. Além disso, o zebrafish é um excelente modelo para estudar a função de diferentes sistemas de neurotransmissão. O consumo do etanol exerce diversas mudanças na coordenação motora, percepção sensorial e cognição promovendo um amplo espectro de alterações bioquímicas e fisiológicas nas células nervosas. Aqui, nós investigamos o efeito promovido pela exposição crônica de etanol nos sistemas purinérgico e glutamatérgico, e níveis de BDNF no SNC de zebrafish. Os transportadores de alta afinidade de aminoácidos (EAAT) regulam os níveis extracelulares de glutamato. Nós identificamos e descrevemos o padrão de expressão dos genes relacionados aos transportadores e as propriedades de captação de glutamato nas três importantes estruturas cerebrais de zebrafish (telencéfalo, tecto óptico e cerebelo). As pesquisas nos bancos de dados do seu genoma através de análise filogenética confirmaram a presença de diversos EAATs (EAAT2, EAAT3, três EAAT1 parálogos e duas sequências parálogas para EAAT5). Também, a captação de glutamato dependente de sódio foi significativamente maior no tecto óptico, indicando diferenças funcionais entre as estruturas cerebrais. Os EAATs pertencem à família dos carreadores de soluto 1 (SLC1), que constitui os transportadores de alta afinidade de aminoácidos e transportadores de aminoácidos neutros. Recentemente, foi demonstrada uma análise filogenética e clonagem dos genes SLC1/EAAT identificando distintos membros desta família de transportadores. No sentido de unificar a designação dos genes SLC1/EAAT em zebrafish, foi proposta uma nomenclatura comum para estes grupos. O etanol promoveu uma diminuição significativa na captação de glutamato após 7 e 14 dias de exposição (30% e 54%, respectivamente), enquanto que após 28 dias, não foram observadas alterações. Na,K-ATPase, a enzima responsável pelo controle do gradiente iônico, não teve sua atividade alterada após todos os períodos de exposição testados. Além disso, os peixes expostos ao etanol durante 7 e 14 dias tiveram uma redução nos níveis de mRNA para SLC1A1/EAAT3. Entretanto, a expressão gênica do SLC1A8a,b/EAAT6a,b aumentou após todos os períodos testados, enquanto que SLC1A3a,b/EAAT1b aumentou somente após 28 dias. A prolongada exposição ao etanol não foi capaz de alterar as atividades da glutamina sintetase e glutaminase. Da mesma forma, etanol não alterou a hidrólise de ATP e GTP. Entretanto, foi verificada uma diminuição na hidrólise de ADP (46% e 34%) e GDP (48% e 36%) após 7 e 14 dias respectivamente. Após 7 e 14 dias de exposição ao etanol, também foi observada uma significativa alteração na hidrólise de AMP (48% e 36%), enquanto que a hidrólise de GTP foi inibida somente após 7 dias (46%). Os níveis de transcritos das nucleosídeo trifosfato difosfohidrolase (NTPDases) foram alteradas após 7, 14 e 28 dias. Em contraste, a expressão da 5′-nucleotidase não foi alterada. A atividade da adenosina deaminase (ADA) na fração solúvel não foi modificada, mas uma redução da atividade na fração de membrana após 28 dias de exposição ao etanol (44%) foi observada. A análise da expressão gênica demonstrou que ADA1 permaneceu inalterada, enquanto que os transcritos de ADAL, ADA2-2, ADA2-1, e sua isoforma truncada de ―splicing‖ alternativo (ADA2-1/T) foram alteradas após a ação prolongada do etanol. Após 14 e 28 dias, etanol aumentou a expressão gênica do BDNF, mas não alterou os níveis de transcritos para trkB. A determinação da proteína BDNF através dos métodos de ELISA indicou um aumento de (51%), sendo confirmando imunohistioquímicamente. Estes resultados sugerem que a homeostase da função neurotrófica pode ser alterada pelo prolongado consumo de etanol. Esta tembém inclui uma revisão sobre o papel de diferentes neurotransmissores excitatórios e inibitórios em zebrafish, tais como, dopaminérgico, serotoninérgico, colinérgico, glutamatérgico, purinérgico, histaminérgico, nitrérgico, glicinérgico, gabaérgico, enfatizando aspectos farmacológicos e toxicológicos. Em suma, esta tese demonstra o efeito da exposição crônica ao etanol afeta o sistema glutamtérgico e purinérgico, expressão de BDNF em cérebro de zebrafish. O aumento do conhecimento global sobre os sistemas de neurotransmisão em zebrafish e o esclarecimento de efeitos farmacológicos e toxicológicos poderia contribuir para novas estratégias de pesquisa em ciências básicas e biomédicas. The zebrafish (Danio rerio) is a species used as experimental models in various fields such as neurosciences, toxicology. Its genome has already been sequenced and studies have shown that many genes are similar to those of mammals. Furthermore, zebrafish provides an excellent model to study the function of different neurotransmitter systems. The ethanol consumption exerts several changes in motor coordination, sensory perception and cognition, promoting a wide-spectrum of biochemical and physiological alterations on nervous cells. Here we investigated the effects promoted by chronic ethanol exposure on glutamatergic and purinergic systems, and BDNF levels in zebrafish CNS. High-affinity excitatory amino acid transporters (EAATs) regulate extracellular glutamate levels. We identified and described the expression profile of EAATs-related genes and the functional properties of glutamate uptake in three major brain structures from zebrafish (telencephalon, optic tectum and cerebellum). Searches on zebrafish genome databases and a phylogenetic analysis confirmed the presence of several EAAT-related genes (EAAT2, EAAT3, three EAAT1 paralogs and two EAAT5 sequences). Moreover, the glutamate uptake was significantly higher in optic tectum, which indicates functional differences within zebrafish brain structures. EAATs belong to the solute carrier family 1 (SLC1), that constitute high-affinity glutamate and neutral amino acid transporters. Recently, the phylogenetic analysis and cloning reporting of SLC1/EAAT genes from zebrafish identified distinct members of this transporter family. In order to unify the nomenclature of SLC1/EAAT genes in zebrafish, it was proposed a common nomenclature for these groups. The actions of ethanol on glutamate uptake showed a significant decrease in glutamate transport (30% and 54%) after 7 and 14 days of exposure, whereas after 28 days, no significant changes were detected. Na,K-ATPase, the enzyme responsible to generate ion gradients, did not alter after all exposure periods. Moreover, fish exposed to ethanol during 7 and 14 days exhibit a decrease of mRNA levels for SLC1A1/EAAT3. However, the gene expression of SLC1A8a,b/EAAT6a,b increased after all exposure periods, whereas SLC1A3a,b/EAAT1b increased only after 28 days. The prolonged ethanol exposure did not significantly change the glutamine synthetase and glutaminase activities. In the same way, ethanol did not alter the ATP and GTP hydrolysis. However, a decrease in ADP (46% and 34%) and GDP (48% and 36%) hydrolysis was verified after 7 and 14 days, respectively. After 7 and 14 days of ethanol exposure, a significant decrease in AMP hydrolysis (48% and 36%) was also observed, whereas GMP hydrolysis was inhibited only after 7 days (46%). Furthermore, nucleoside triphosphate diphosphohydrolase (NTPDase) transcript levels were altered after 7, 14, and 28 days. In contrast, 5′-nucleotidase expression was not altered. Adenosine deaminase (ADA) activity from soluble fraction was not modified, but a decrease of ADA activity in membrane fraction after 28 days (44%) of ethanol exposure was observed. Gene expression analysis demonstrated that ADA1 remained unaltered, whereas ADAL, ADA2-2, ADA2-1 transcripts, and its truncated alternative splice isoform (ADA2-1/T) were altered after prolonged ethanol exposure. After 14 and 28 days, ethanol increased the BDNF gene expression, but did not change the levels of trkB transcripts. The measurement of BDNF protein through ELISA kit anti-BDNF showed increased amounts after 28 days of exposure (51%), which was also confirmed by immunohistochemstry. These results suggest that the homeostasis of neurotrophic functions may be altered by prolonged ethanol consumption. Moreover, we present a review about the role of different excitatory and inhibitory neurotransmitters systems in zebrafish, such as dopaminergic, serotoninergic, cholinergic, glutamatergic, purinergic, histaminergic, nitrergic, glycinergic, and GABAergic systems, emphasizing pharmacological and toxicological aspects. In conclusion, this thesis demonstrates that chronic ethanol exposure affects the glutamatergic and purinergic systems, and BDNF expression in zebrafish brain. The significant increase in the global knowledge about the neurotransmitters systems in zebrafish and the elucidation of pharmacological and toxicological effects could lead to new strategies and appropriate priorities in research in order to support complementary insights on basic sciences and biomedical research.

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33

Sharma, Ravi Janak. "Uptake and metabolism of circulating inosine and adenosine in the rat." Thesis, St George's, University of London, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295171.

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Buckley, Ragan. "The purine world: experimental investigations into the prebiotic synthesis of purine nucleobases and intercalation of homopurine DNA duplexes." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/48971.

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Formamide is a solvent of great interest to prebiotic chemists because it is liquid over a wide range, it is less volatile than either water or HCN, and it possesses a versatile reactivity. When formamide is heated in the presence of minerals or inorganic catalysts, a variety of products including purine nucleobases are generated. Irradiation of formamide reaction solutions with ultraviolet light increases the yield and diversity of products, and eliminates the need for a mineral catalyst. We have also performed formamide reactions in the presence of pyrite, a mineral which is likely to have been available on the primordial Earth, under a variety of atmospheric conditions. Our results indicate the greatest yield and diversity of products result from the combination of a pyrite mineral catalyst, heat, UV irradiation, and a carbon dioxide atmosphere. Purine nucleobases are simple to synthesize in model reactions and they stack well in aqueous solution; it has been hypothesized that the first nucleic acids were composed of only purine bases, and that water-soluble, cationic, aromatic molecules with large stacking surfaces (“”molecular midwives””) may have aided the assembly of the earliest nucleic acid analogs. We have characterized the interactions of various intercalators with a standard DNA duplex as well as with an antiparallel homopurine DNA duplex and have determined that molecules which possess four or more rings and a curved shape interact selectively with all-purine DNA; such molecules can serve as models for putative prebiotic midwives.

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Clingen, Peter H. "Biochemical studies of purine photodamage in DNA." Thesis, Queen's University Belfast, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241386.

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36

Whittaker, Steven Robert. "The molecular pharmacology of purine CDK inhibitors." Thesis, Institute of Cancer Research (University Of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404908.

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Alexiou, Maria. "Purine metabolism in the mouse preimplantation embryo." Thesis, University of York, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317658.

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Sen, Gupta Soma. "Purine and pyrimidine transport in Candida spp." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296640.

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Shapiro, Andrew P. "Electroosmotic purging of contaminants from saturated soils." Thesis, Massachusetts Institute of Technology, 1990. http://hdl.handle.net/1721.1/13674.

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40

Payne, Tiffany Anne. "Analysis of dirhenium carboxylate : purine dinucleotide adducts." Scholarly Commons, 2006. https://scholarlycommons.pacific.edu/uop_etds/629.

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The discovery of cisplatin, cis-[pt(NH3)2Cl2], as an anticancer agent in 1969 by Rosenbert and his colleagues sparked interest in the area of metal complexes as chemotherapeutic agents. Anticancer dimetal complexes such as Re2(O2CCH2CH3)2Br4·2H2O are proposed to prevent replication of cancel cells by coordinating to the purine nucleobases in DNA. To investigate the interaction between dimetal compounds and DNA, dirhenium complexes coordinated to purine dinucleotides were isolated and analyzed. LC/MS, HPLC, 1H NMR, and UV-Visible spectroscopy were used to characterized complexes of Re2(O2CR)2X4·2H2O (R = CH3, CH2CH3; X= Cl, Br) with the purine dinucleotides dApG and dGpG. HPLC, UV-Vis, and 1H NMR are used to investigate the aquation of Re2(O2C2H3)2Cl4μ2H2O which may contribute to its biological activity. Upon reaction of Re22C2H3)2Cl4μ2H2O with dApG or dGpG, the intact dirhenium:dinucleotide complex is observed by LC/MS after two days. In both of these reactions, dirhenium:GMP complexes are also observes. 1H NMR studies show the appearance of new resonances in the aromatic region that cannot be attributed to starting material or hydrolyzed DNA fragments. These resonances are proposed to result from the formation of dirhenium:dinucleotide complexes. Additionally, MS spectra support the conclusion that a complex between the dinuclear rhenium complex and the purine dinucleotides of dApG and dGpG is formed after two days. A dirhenium:nucleotide product is also formed as a result of the dinucleotide hydrolysis.

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Briheim, Ludvig, and Dennis Ståhl. "Researching and Developing Universal Gas Purging Solutions." Thesis, KTH, Maskinkonstruktion (Avd.), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-276080.

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In the welding of more sensitive metals such as stainless steel, titanium and some specific nickel alloys, the presence and application of a protective gas is essential. Without the use of a nonreactive shielding gas, defects occur in or adjacent to the weld joint, which vary from light discoloration of the weld bead to the direct onset and propagation of cracks. The supply of shielding gas is usually via the welding tool. Unfortunately, when it comes to welding pipes, the problem arises when the root bead comes into contact with the oxygen present within the pipe. Since the shielding gas supplied from the welding tool has no possibility of penetrating into the pipe and protecting the melt from the oxygen, the result consequence will be defects in the weld bead. To prevent this from happening, the pipes are sealed before welding and the oxygen in the pipe is purged and replaced with protective gas. Due to the fact that pipe welding operations concern a vast spectrum of pipe dimensions, complications arise for the welding operator as the solutions available have a low degree of adaptability. Thus, the purpose of this project is to investigate the possibility of designing and developing a new method of supplying root gas protection that can be adapted to several pipe diameters. Field studies at welding companies resulted in the clarification of which diameter spans the root gas protection solution should comply with, the pipe diameter span to cater for was identified as 25-100 mm in diameter. Requests were also made regarding the robustness of the product, with expressed desire for the product to withstand the strenuous working environment often encountered during welding operations. Solution proposals were produced which later developed into six separate concepts. From these six concepts, one was selected as the foremost solution and further developed for construction. The concept was sketched out and reproduced using CAD and a model was made using 3D printers. The model showed a need for modifications as it was not of a sufficiently adaptable nature. A modified model was thus developed and manufactured using a 3D printer, this modified model showed better results of adaptation to different pipe diameters. Furthermore, a flow analysis of the gas as it enters via the root gas plug and into the pipe was conducted, to verify total distribution of the gas within the pipe. The material with which to construct the concept with will be silicone infused with additives to make it more heat resistant and more resistant to wear. However, the exact mixture of silicone and additives in question was not available for this project but is something that is intended for future work. The concept meets the specified requirements of being applicable to multiple pipe diameters, more specifically so meeting the wishes of those active within the industry, covering a pipe span of 25- 100 mm. Vid svetsning av mer känsliga metaller såsom rostfritt stål, titan och vissa specifika nickellegeringar är närvaron och tillämpningen av en skyddsgas väsentligt. Utan nyttjandet av en icke reaktiv skyddsgas uppkommer defekter i eller intill svetsfogen, vilka varierar från lätta missfärgningar av svetssträngen till den direkta uppkomsten och propageringen av sprickor. Tillförseln av skyddsgas sker vanligtvis via svetsverktyget. När det kommer till svetsning av rör uppstår dessvärre problemet att rotsträngen kommer i kontakt med syret som finns på insidan av röret. Eftersom att den skyddsgas som tillförs från svetsverktyget inte har någon möjlighet att tränga sig in i röret och skydda smältan från syret kommer det resultera i en svetssträng med defekter. För att förhindra detta från att ske tätas rören innan svetsning och syret i röret ersätts med skyddsgas. Eftersom att de rör som skall sammanfogas förekommer i flera dimensioner uppstår komplikationer för svetsoperatören ty de lösningar som finns tillgängliga har låg anpassningsgrad. Därmed är syftet med detta projekt att undersöka möjligheten att designa och utveckla ett nytt rotgasskydd som kan anpassas till flera diametrar. Fältstudier hos svetsföretag resulterade i ett tydligt spann för vilka diametrar som bör tillgodoses med sagda rotgasskydd, detta spann av rördiametrar var 25-100 mm. Det uppgavs även önskemål om säkerställning av att produkterna är robusta nog att klara den påfrestande arbetsmiljö som ofta förekommer vid svetsning. Lösningsförslag lades fram vilket senare utvecklades till sex separata koncept. Från dessa sex koncept utsågs en som den främsta lösningen och togs vidare till konstruktion. Konceptet skissades upp i CAD och en modell gjordes i 3D-skrivare. Modellen visade upp behov på modifieringar då den inte var av tillräckligt anpassningsbar karaktär. En modifierad modell togs därmed fram och tillverkades i en 3D-skrivare, denna modifierade modell visade bättre prov på anpassning till olika rördiametrar. Vidare gjordes en flödesanalys av gasen då den färdas in via rotgaspluggen, vilket visade att gasen fyller röret som önskat. Materialet som konceptet kommer tillverkas i är silikon med tillförda additiv för att göra det mer värmebeständigt och mer resistent mot slitage. Den exakta blandningen av silikon och additiv i fråga var dock inte tillgängliga till detta projekt utan är något som får gå till vidare arbete. Konceptet uppfyller kraven på anpassning till flera rördiametrar och möter mer specifikt även önskemålet från branschaktiva med dess spann på 25-100 mm.

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Snider, Gordon L. "Cleansing in Psalm 51 cultic or ethical? /." Theological Research Exchange Network (TREN), 1995. http://www.tren.com.

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43

Vabre, Roxane. "Fonctionnalisation directe de liaisons C-H et couplages croisés pour la formation de liaisons C-C et C-N : synthèse de purines 6,8,9-trisubstituées." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00923198.

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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.

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44

Hatamzadeh-Dehboneh, Abdulla. "The regulation of caffeine production in Camellia sinensis L. cv. Iranian and Darjeeling tea." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298069.

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45

Hutchings, Matthew Ian. "Transcriptional regulation at two divergent gene promoters in Escherichia coli K-12." Thesis, University of Southampton, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241963.

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46

Humphries, Mark Jonathon. "Synthesis and chemistry of 5-aminoimidazole ribonucleosides." Thesis, Keele University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363753.

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47

Heinzen, Eduardo Luiz. "VariÃveis nutricionais e fisiolÃgicas de ovinos da raÃa Morada Nova de diferentes classes sexuais submetidos Ã restriÃÃo alimentar." Universidade Federal do CearÃ, 2016. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=16929.

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nÃo hÃ Este estudo foi realizado com o objetivo de determinar os efeitos da classe sexual e restriÃÃo alimentar sobre o consumo de nutrientes, digestibilidade, derivados de purina, balanÃo de nitrogÃnio, comportamento ingestivo e parÃmetros fisiolÃgicos de cordeiros da raÃa Morada Nova. Trinta e cinco animais (11 machos inteiros, 12 castrados e 12 fÃmeas), com peso mÃdio inicial de 14,5 Â 0,89kg, foram usados em delineamento inteiramente casualizado, em esquema fatorial 3x3, sendo trÃs classes sexuais (machos inteiros, castrados e fÃmeas) e trÃs nÃveis de restriÃÃo alimentar (ad libitum, 30 e 60%). A classe sexual e a restriÃÃo alimentar influenciaram (P<0,05) o consumo de matÃria seca (MS), matÃria orgÃnica (MO), proteÃna bruta (PB), extrato etÃreo (EE), fibra em detergente neutro corrigida para cinzas e proteÃna (FDNcp), carboidratos totais (CT), carboidratos nÃo fibrosos (CNF), nutrientes digestÃveis totais (NDT) e de energia metabolizÃvel (EM) e o balanÃo dos compostos nitrogenados. O coeficiente de digestibilidade da MS, PB e EE foram influenciados (P<0,05) pela restriÃÃo alimentar, no entanto, nÃo houve efeito (P>0,05) da classe sexual sobre os coeficientes de digestibilidade da PB, EE e CNF. Houve interaÃÃo entre a classe sexual e a restriÃÃo alimentar (P<0,05) na digestibilidade da MO, FDNcp e CT. Os tempos de alimentaÃÃo (TAL) e ruminaÃÃo (TRU) foram influenciados pela restriÃÃo alimentar (P<0,05), no entanto, nÃo foram influenciados pela classe sexual (P>0,05). O volume urinÃrio, excreÃÃes de creatinina, alantoÃna, xantina e hipoxantina, absorÃÃo de derivados de purina (absDP), derivados de purinas totais (DP) e sÃntese de proteÃna microbiana (PBmic) nÃo foram influenciados (P>0,05) pela classe sexual. Volume urinÃrio, excreÃÃes de creatinina, xantina e hipoxantina nÃo foram influenciados pela restriÃÃo alimentar (P>0,05), no entanto, este fator influenciou as excreÃÃes de alantoÃna, Ãcido Ãrico, absDP e DP. Os parÃmetros fisiolÃgicos temperatura superficial (TS) do lado esquerdo, regiÃo peitoral e temperatura retal (TR) nÃo foram influenciados pela classe sexual (P>0,05). A restriÃÃo alimentar e o perÃodo de coleta das variÃveis fisiolÃgicas influenciaram (P<0,05) a TS do lado esquerdo, peito e TR. Houve interaÃÃo (P<0,05) entre classe sexual e restriÃÃo alimentar para TS do lado direito. Consumo e digestibilidade de nutrientes sÃo influenciados por restriÃÃes alimentares mais severas. Classe sexual nÃo influencia a excreÃÃo de derivados de purina e sÃntese de proteÃna microbiana. A restriÃÃo alimentar diminui as perdas de calor pela superfÃcie corporal de cordeiros. This study was accomplished with the objective of determining the effects of sexual class and feed restriction on nutrient intake, digestibility, purine derivatives, nitrogen balance, feeding behavior and physiological parameters in hair sheep Morada Nova. Thirty-five animals (11 intact males, 12 castrated and 12 females) with an initial average weight of 14.5 Â 0,89kg were used in a completely randomized design in a 3x3 factorial design with three sexual classes (intact males, castrated and females) and three levels of feed restriction (ad libitum, 30 and 60%). Sexual class and feed restriction influenced (P<0.05) the intake of dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), neutral detergent fiber corrected for ash and protein (NDFap), total carbohydrates (TC), non-fibrous carbohydrates (NFC), total digestible nutrients (TDN) and metabolied energy (ME) and the balance of nitrogen compounds. The digestibility coefficient of DM, CP and EE were influenced (P<0.05) by feed restriction, however there was no effect (P>0.05) the sexual class on the digestibility coefficients of CP, EE and NFC. There was interaction between sexual class and feed restriction (P<0.05) in digestibility of OM, NDFap and CT. Eating times (ET) and rumination times (RUT) were affected by feed restriction (P<0.05), however they were not affected by sexual class (P>0.05). The urinary volume, excretions of creatinine, allantoin, xanthine and hypoxanthine, absorption of purine derivatives (absPD), total purine derivatives (PD) and microbial protein synthesis (micCP) were not influenced (P>0.05) by sexual class. Urine output, creatinine excretion, xanthine and hypoxanthine were not influenced by feed restriction (P>0.05), however, this factor influenced the excretion of allantoin, uric acid, absPD and PD. Physiological parameters, surficial temperature (ST) on the left of pectoral region and rectal temperature (RT), were not affected by sexual class (P>0.05). Feed restriction and the period of collect of physiological variables influenced (P<0.05) ST the left side, chest and RT. There was interaction (P<0.05) between sexual class and feed restriction ST to the right side. Intake and digestibility of nutrients are influenced by more severe dietary restrictions. Sexual class does not influence the excretion of purine derivatives and microbial protein synthesis. Feed restriction reduces heat losses by body surface of lambs.

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48

Heinzen, Eduardo Luiz. "Variáveis nutricionais e fisiológicas de ovinos da raça Morada Nova de diferentes classes sexuais submetidos à restrição alimentar." reponame:Repositório Institucional da UFC, 2016. http://www.repositorio.ufc.br/handle/riufc/18895.

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HEINZEN, Eduardo Luiz. Variáveis nutricionais e fisiológicas de ovinos da raça Morada Nova de diferentes classes sexuais submetidos à restrição alimentar. 2016. 50 f. : Dissertação (Mestrado) - Universidade Federal do Ceará, Centro de Ciências, Departamento de Zootecnia, Programa de Pós-Graduação em Zootecnia. Fortaleza-CE, 2016. Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-08-03T13:37:27Z No. of bitstreams: 1 2016_dis_elheinzen.pdf: 1285825 bytes, checksum: 32955795d549452fb81768336e0dfffc (MD5) Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-08-03T13:51:24Z (GMT) No. of bitstreams: 1 2016_dis_elheinzen.pdf: 1285825 bytes, checksum: 32955795d549452fb81768336e0dfffc (MD5) Made available in DSpace on 2016-08-03T13:51:24Z (GMT). No. of bitstreams: 1 2016_dis_elheinzen.pdf: 1285825 bytes, checksum: 32955795d549452fb81768336e0dfffc (MD5) Previous issue date: 2016 This study was accomplished with the objective of determining the effects of sexual class and feed restriction on nutrient intake, digestibility, purine derivatives, nitrogen balance, feeding behavior and physiological parameters in hair sheep Morada Nova. Thirty-five animals (11 intact males, 12 castrated and 12 females) with an initial average weight of 14.5 ± 0,89kg were used in a completely randomized design in a 3x3 factorial design with three sexual classes (intact males, castrated and females) and three levels of feed restriction (ad libitum, 30 and 60%). Sexual class and feed restriction influenced (P<0.05) the intake of dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), neutral detergent fiber corrected for ash and protein (NDFap), total carbohydrates (TC), non-fibrous carbohydrates (NFC), total digestible nutrients (TDN) and metabolied energy (ME) and the balance of nitrogen compounds. The digestibility coefficient of DM, CP and EE were influenced (P<0.05) by feed restriction, however there was no effect (P>0.05) the sexual class on the digestibility coefficients of CP, EE and NFC. There was interaction between sexual class and feed restriction (P<0.05) in digestibility of OM, NDFap and CT. Eating times (ET) and rumination times (RUT) were affected by feed restriction (P<0.05), however they were not affected by sexual class (P>0.05). The urinary volume, excretions of creatinine, allantoin, xanthine and hypoxanthine, absorption of purine derivatives (absPD), total purine derivatives (PD) and microbial protein synthesis (micCP) were not influenced (P>0.05) by sexual class. Urine output, creatinine excretion, xanthine and hypoxanthine were not influenced by feed restriction (P>0.05), however, this factor influenced the excretion of allantoin, uric acid, absPD and PD. Physiological parameters, surficial temperature (ST) on the left of pectoral region and rectal temperature (RT), were not affected by sexual class (P>0.05). Feed restriction and the period of collect of physiological variables influenced (P<0.05) ST the left side, chest and RT. There was interaction (P<0.05) between sexual class and feed restriction ST to the right side. Intake and digestibility of nutrients are influenced by more severe dietary restrictions. Sexual class does not influence the excretion of purine derivatives and microbial protein synthesis. Feed restriction reduces heat losses by body surface of lambs. Este estudo foi realizado com o objetivo de determinar os efeitos da classe sexual e restrição alimentar sobre o consumo de nutrientes, digestibilidade, derivados de purina, balanço de nitrogênio, comportamento ingestivo e parâmetros fisiológicos de cordeiros da raça Morada Nova. Trinta e cinco animais (11 machos inteiros, 12 castrados e 12 fêmeas), com peso médio inicial de 14,5 ± 0,89kg, foram usados em delineamento inteiramente casualizado, em esquema fatorial 3x3, sendo três classes sexuais (machos inteiros, castrados e fêmeas) e três níveis de restrição alimentar (ad libitum, 30 e 60%). A classe sexual e a restrição alimentar influenciaram (P<0,05) o consumo de matéria seca (MS), matéria orgânica (MO), proteína bruta (PB), extrato etéreo (EE), fibra em detergente neutro corrigida para cinzas e proteína (FDNcp), carboidratos totais (CT), carboidratos não fibrosos (CNF), nutrientes digestíveis totais (NDT) e de energia metabolizável (EM) e o balanço dos compostos nitrogenados. O coeficiente de digestibilidade da MS, PB e EE foram influenciados (P<0,05) pela restrição alimentar, no entanto, não houve efeito (P>0,05) da classe sexual sobre os coeficientes de digestibilidade da PB, EE e CNF. Houve interação entre a classe sexual e a restrição alimentar (P<0,05) na digestibilidade da MO, FDNcp e CT. Os tempos de alimentação (TAL) e ruminação (TRU) foram influenciados pela restrição alimentar (P<0,05), no entanto, não foram influenciados pela classe sexual (P>0,05). O volume urinário, excreções de creatinina, alantoína, xantina e hipoxantina, absorção de derivados de purina (absDP), derivados de purinas totais (DP) e síntese de proteína microbiana (PBmic) não foram influenciados (P>0,05) pela classe sexual. Volume urinário, excreções de creatinina, xantina e hipoxantina não foram influenciados pela restrição alimentar (P>0,05), no entanto, este fator influenciou as excreções de alantoína, ácido úrico, absDP e DP. Os parâmetros fisiológicos temperatura superficial (TS) do lado esquerdo, região peitoral e temperatura retal (TR) não foram influenciados pela classe sexual (P>0,05). A restrição alimentar e o período de coleta das variáveis fisiológicas influenciaram (P<0,05) a TS do lado esquerdo, peito e TR. Houve interação (P<0,05) entre classe sexual e restrição alimentar para TS do lado direito. Consumo e digestibilidade de nutrientes são influenciados por restrições alimentares mais severas. Classe sexual não influencia a excreção de derivados de purina e síntese de proteína microbiana. A restrição alimentar diminui as perdas de calor pela superfície corporal de cordeiros.

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49

O'Brien, Stephen Gerard. "Haemopoietic stem cell purging in chronic myeloid leukaemia." Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300928.

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50

Mondon, K. J. "Enzyme inactivation by pyrimidine and purine free radicals." Thesis, Brunel University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.332433.

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Dissertations / Theses on the topic 'Puring'

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