Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Filosofia e Ciencias Humanas / Made available in DSpace on 2018-07-13T20:13:23Z (GMT). No. of bitstreams: 1 Brito_MariaNoemiCastilho_M.pdf: 5356989 bytes, checksum: 194d317ab6d356a76f0d73bdbc0f5dd7 (MD5) Previous issue date: 1985 / Resumo: Não informado / Abstract: Not informed / Mestrado / Mestre em Antropologia

Orientador: Marianne Hoeltgebaum. / Dissertação (mestrado) - Universidade Regional de Blumenau, Centro de Ciências Sociais Aplicadas, Programa de Pós-Graduação em Administração.

Nitric oxide (•NO) is a molecule with diverse biological effects involved in both signaling and defense mechanisms in mammalian systems. The production of •NO is catalyzed from L-Arginine by a family of enzymes known as nitric oxide synthases (NOS). All mammalian isoforms contain an active site oxygenase domain and an electron-donating reductase domain joined by a calmodulin binding region. Recently, prokaryotic homologues to the oxygenase domain of mammalian NOS enzymes have been identified. Although several bacterial NOS (bNOS) enzymes have been characterized, their function is still unknown. Possible roles for this enzyme could include: (i) intercellular signaling to coordinate cellular/infectious activity, (ii) regulation via •NO-mediated posttranslational modifications, or (iii) nitration of compounds in different biosynthetic pathways. Efforts, contained in this thesis, to determine a probable role for this enzyme are two-fold: (i) via a search for possible interacting protein partners, and (ii) via a proteomic analysis of the effects of a knockout of the bNOS gene. Bacterial-NOS knockouts in Bacillus subtilis and Bacillus cereus have been created. 2D-Differential in gel electrophoresis (DIGE) has been used to analyze the proteomic effects of the expression of this gene in B. subtilis. Thus far 16 proteins which exhibited significant changes in expression, with a p value ≤ 0.05 and fold change ≥│2│, have been isolated and identified by peptide mass fingerprinting (PMF) in order to shed light on the relevance of this bacterial NOS homologue. The proteins which have been identified thus far are involved in cellular metabolism, amino acid metabolism and nitrogen metabolism. The identification of further proteins is required for a broader view of the impact of the expression of this gene on the proteome.

Nitric oxide (•NO) is a molecule with diverse biological effects involved in both signaling and defense mechanisms in mammalian systems. The production of •NO is catalyzed from L-Arginine by a family of enzymes known as nitric oxide synthases (NOS). All mammalian isoforms contain an active site oxygenase domain and an electron-donating reductase domain joined by a calmodulin binding region. Recently, prokaryotic homologues to the oxygenase domain of mammalian NOS enzymes have been identified. Although several bacterial NOS (bNOS) enzymes have been characterized, their function is still unknown. Possible roles for this enzyme could include: (i) intercellular signaling to coordinate cellular/infectious activity, (ii) regulation via •NO-mediated posttranslational modifications, or (iii) nitration of compounds in different biosynthetic pathways. Efforts, contained in this thesis, to determine a probable role for this enzyme are two-fold: (i) via a search for possible interacting protein partners, and (ii) via a proteomic analysis of the effects of a knockout of the bNOS gene. Bacterial-NOS knockouts in Bacillus subtilis and Bacillus cereus have been created. 2D-Differential in gel electrophoresis (DIGE) has been used to analyze the proteomic effects of the expression of this gene in B. subtilis. Thus far 16 proteins which exhibited significant changes in expression, with a p value ≤ 0.05 and fold change ≥│2│, have been isolated and identified by peptide mass fingerprinting (PMF) in order to shed light on the relevance of this bacterial NOS homologue. The proteins which have been identified thus far are involved in cellular metabolism, amino acid metabolism and nitrogen metabolism. The identification of further proteins is required for a broader view of the impact of the expression of this gene on the proteome.

The three isoforms of nitric oxide synthase (NOS) are classified based on their mode of regulation by calmodulin (CaM). The "calcium-independent" inducible isoform (iNOS) contains tightly-bound CaM and is active at all levels of intracellular calcium. In contrast, the two calcium-dependent isoforms (neuronal, nNOS and endothelial eNOS) are activated by CaM-binding following stimulus-evoked increases in intracellular calcium. However, both the structural basis for these differences in regulation and the molecular basis for CaM-induced cNOS activation remained unclear. Given that the regulation of calmodulin regulated enzyme systems often involves the displacement of an intrinsic autoinhibitory domain, we attempted to identify regions of eNOS which may fulfill this autoinhibory function. Herein, I describe the identification of two autoinhibitory control elements (ACEs) in eNOS, one within the FMN binding domain (ACE-I) and one located at the C-terminus (ACE-2). Together with CaM, these form a tripartite system for the regulation of eNOS. ACE-lIACE-2 exerting negative effects to attenuate catalytic activity, which are overcome by the conformational changes induced by CaM-binding. The mechanism of ACE-lIACE-2-mediated inhibition and the alleviation of this inhibition were investigated.

Made available in DSpace on 2014-06-12T17:23:15Z (GMT). No. of bitstreams: 2 arquivo7258_1.pdf: 725882 bytes, checksum: c243982e76ff59fb957a8f2a62180228 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002 / A Tortura nos presídios paraibanos, estudada no presente trabalho, representa um grande desafio a todo pesquisador, em especial no âmbito do Direito, quanto à perquirição da sua sobrevida apesar de extirpada das legislações há mais de dois séculos. O tema da tortura é uma discussão permanente, bem como precisa ser uma preocupação cotidiana dos cidadãos, pois a tomada de consciência acerca deste tema representa um compromisso social inarredável. Assim, longe de ser uma conduta ignóbil de nossos antepassados, a tortura continua viva no relacionamento poder político/cidadão, merecendo postura enérgica das instituições estatais, no sentido de ao menos atenuá-la, já que o desejo do homem de oprimir seu semelhante é um mal que sempre acompanhará a raça humana. Este trabalho, realizado sob o enfoque jurídicopenal da prática da tortura nos presídios da Paraíba, revela que a tortura, a partir da antiguidade, percorreu linha temporal de evolução humana, transmutando-se de instrumento legal de sustentação de poder e destinado à instrução criminal a ilícito penal. No entanto, continua a ser praticada na clandestinidade ou semiclandestinidade, tanto no Estado da Paraíba como em todo o Brasil, sob a estrutura dos governos tanto despóticos como naqueles estruturados à luz da democracia. Neste contexto, objetiva-se, no presente trabalho, demonstrar a existência da prática da tortura nos presídios paraibanos e apresentar soluções para o problema. O relato de casos no interior dos cárceres demonstra a total inaplicabilidade da Lei de Tortura, como forma de não reconhecimento dos direitos humanos dos aprisionados, demonstrando a falência do sistema prisional. O cumprimento da lei supra citada implica que o Estado combine aspectos fundamentais na sua relação com o problema, devendo ir mais além da abstenção da prática da tortura, cumprindo a obrigação que tem de dar garantias e criar procedimentos de combate contra a tortura e maus tratos às pessoas privadas de sua liberdade

Nitric oxide synthase (NOS) catalyses the production of nitric oxide (NO). A cytochrome P450-like oxygenase, it uses two monooxygenation steps to convert L-arginine (L-arg) first to N -hydroxy-L-arginine (NOHA), a stable intermediate, and then to L-citrulline and NO. Mammalian NOSs are homodimeric enzymes. Each monomer is composed of an oxygenase domain (containing the L-arg binding site, a heme ligated by a cysteine thiolate, and a tetrahydrobiopterin (H4B)) and a reductase domain (binding NADPH, FAD, and FMN). NOS substrates are O2, L-arg, and NADPH. NADPH is the source of electrons required for oxygen activation. H4B is a vital cofactor that aids dimerisation and acts as a reducing/oxidising agent. Controversy still exists as to the final oxygenating species in the NOS mechanism, but the general reaction scheme is known. The ferric heme is reduced to the ferrous state by an electron from the reductase domain. Then oxygen binds to form the oxy-ferrous species. Then H4B donates an electron to form a peroxy-ferric species. It is likely this then forms a compound 1 (Fe(IV)+.=O) species that is the final oxygenating species. This thesis probes the mechanism of NOS to further define the mechanistic intermediates involved. The role of H4B in NO synthesis has been probed in both normal turnover conditions and special case reactions. To elucidate this mechanism further a mutant with a residue capable of stabilising the activated oxygen species was created, G586S, where glycine 586 of nNOS was replaced with a serine. This serine was within hydrogen bonding distance of the oxy-heme. A stabilised intermediate was observed by stopped flow reaction in the presence of H4B, but not aH4B (an inactive pterin analogue). Here single turnover reactions, each following either the reaction of L-arg to NOHA or NOHA to citrulline, were performed on the mutant using an external source of electrons. The reaction products were observed by HPLC. The mutant appears capable of the conversion of NOHA to citrulline, but not L-arg to NOHA. The WT enzyme appears capable of both. The intermediate is observed with either L-arg or NOHA bound, suggesting both reactions proceed via the same active oxygenating species. The inability of the mutant to catalyse the conversion of L-arg to NOHA may be due to protonation of the substrate hindering reaction such that the active oxygenating species decays before reaction can occur. This mutation, in allowing separation of the two monooxygenation steps, deserves further study. H4B binds at the dimer interface of NOS. Here the -systems of the pterins are only 13Å apart. This is within allowed distances for efficient electron transfer. Electron transfer between hemes, via the pterins, would allow a route for the breakdown of a dead end, ferrous-NO, species. Stopped flow monitoring of the decay of the ferrous-heme NO complex with nNOSoxy dimers with varying proportions of the hemes in the ferrous heme-NO complex showed no electron transfer between hemes of the dimer. The rate of decay of the ferrous heme-NO complex in oxygenated buffer is 0.12 s-1 for all conditions tested here. H4B-deficiency leads to several diseases. H4B makes a poor drug due to instability and cost, the search for druggable analogues of it is ongoing. H4B analogues blocked at the 6,7-positions in the dihydropterine-form have been screened here for catalytic activity. Several have shown comparable ability to catalyse NO production in vitro. Structure function analysis of these analogues has revealed the extent extension is tolerated at the C6 and C7 positions of the pterin.

Nitric oxide (NO) is produced in different mammalian tissues by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All NOS isoforms contain two domains, an oxygenase domain and a reductase domain. NO is an important transmitter of information between cells in many physiological processes; however, overproduction of this molecule may lead to health problems. Therefore, selective inhibition of NOS isoforms has useful therapeutic potential for treatment of certain diseases that can appear because of the pathological overproduction of nitric oxide. Producing useful isoform selective-inhibitors that bind to the active site in the oxygenase domain has proven to be difficult when based solely on the structure of these enzymes. Biophysical studies in combination with structural properties should provide better insights into isoform-specific inhibitor development. The first step of this study was to produce and purify truncated versions of NOS isozymes consisting of the oxygenase domain as they contain the active site of the enzyme. As a result of differences between humans and other mammals in the amino acids found in the second and third shells/layers surrounding the active site, all the experiments were performed with genes coding for human proteins. The major result of this project was the development of an Escherichia coli (E. coli) expression system to produce large amounts of pure protein. This system will allow for the testing of inhibitors that bind to the active site of NOS enzymes.

Nitric oxide (NO) is produced in different mammalian tissues by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All NOS isoforms contain two domains, an oxygenase domain and a reductase domain. NO is an important transmitter of information between cells in many physiological processes; however, overproduction of this molecule may lead to health problems. Therefore, selective inhibition of NOS isoforms has useful therapeutic potential for treatment of certain diseases that can appear because of the pathological overproduction of nitric oxide. Producing useful isoform selective-inhibitors that bind to the active site in the oxygenase domain has proven to be difficult when based solely on the structure of these enzymes. Biophysical studies in combination with structural properties should provide better insights into isoform-specific inhibitor development. The first step of this study was to produce and purify truncated versions of NOS isozymes consisting of the oxygenase domain as they contain the active site of the enzyme. As a result of differences between humans and other mammals in the amino acids found in the second and third shells/layers surrounding the active site, all the experiments were performed with genes coding for human proteins. The major result of this project was the development of an Escherichia coli (E. coli) expression system to produce large amounts of pure protein. This system will allow for the testing of inhibitors that bind to the active site of NOS enzymes.

FAÇANHA NETO, Pedro Brasil. Assimetria de informação e impactos na estrutura de custo ao varejo. 2005. 102p. Dissertação (mestrado profissional). Programa de Pós Graduação em Economia, CAEN, Universidade Federal do Ceará, CAEN, Fortaleza, 2005. / Submitted by Mônica Correia Aquino (monicacorreiaaquino@gmail.com) on 2013-11-22T20:13:46Z No. of bitstreams: 1 2005_dissert_pbfacanhaneto.pdf: 692049 bytes, checksum: a897d14186faacd213ffa4af3cc17310 (MD5) / Approved for entry into archive by Mônica Correia Aquino(monicacorreiaaquino@gmail.com) on 2013-11-22T20:13:55Z (GMT) No. of bitstreams: 1 2005_dissert_pbfacanhaneto.pdf: 692049 bytes, checksum: a897d14186faacd213ffa4af3cc17310 (MD5) / Made available in DSpace on 2013-11-22T20:13:55Z (GMT). No. of bitstreams: 1 2005_dissert_pbfacanhaneto.pdf: 692049 bytes, checksum: a897d14186faacd213ffa4af3cc17310 (MD5) Previous issue date: 2005 / The present dissertation has as its main objective to investigate an optimized financial structure, through its forms of payments, that softens the effects of asymmetric information on the costs of transactions. To reduce transaction costs for retailer companies is justified for the fact of today’s era of competitiveness, where companies, more and more, look for alternatives linked to high productivity, and for new management models. In a stable economy, where production costs do not oscillate to the point of harming the companies, the “war” is to focus on price administration. This, with prices always in fall to more and more conquer customers, the consequence is unavoidably the loss of profitability of the firms operation. The counter-point of that context is the systematic and continuous reduction of operational and financial costs. This dissertation focus on the relationship between ways of payment and the finantial cost structure. It is recognized the importance of asymmetry information as a major factor that increass the sales cost. / A presente dissertação tem como objetivo principal investigar uma estrutura financeira otimizada, através de suas formas de pagamentos, que amenize os efeitos decorrentes da assimetria de informações nos custos de transações. Reduzir custo de transação para empresas varejista se justifica pelo fato de se estar vivendo a era da competitividade, onde empresas, cada vez mais, buscam por alternativas de alta produtividade e por novos modelos de gestão. Em uma economia estável, onde os custos de produção não oscilam a ponto de comprometer as empresas, a “guerra” se foca para a administração de preços. Assim, com políticas de redução de preços para conquistar cada vez mais clientes, a conseqüência é inevitavelmente a perda da rentabilidade da operação como um todo. O contra-ponto desse contexto é a redução sistemática e continuada dos custos operacionais e financeiros. É com este enfoque que serão abordadas as relações entre as diversas formas de pagamentos dos clientes aos lojistas e seus impactos na estrutura de custos, assim como sobre um dos prováveis causadores destes distúrbios, reconhecido aqui como a assimetria de informação entre os agentes envolvidos nas transações varejistas.