by Mo-Lung Chen. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 70-75). / Abstract also in Chinese. / ABBREVIATIONS --- p.iv / LIST OF TABLES --- p.v / LIST OF FIGURES --- p.vii / ACKNOWLEDGEMENT --- p.ix / ABSTRACT --- p.xi / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- BACKGROUND --- p.4 / Chapter 2.1 --- Production of pleural fluid --- p.4 / Chapter 2.2 --- Pathophysiology of pleural effusion --- p.5 / Chapter 2.3 --- Separating exudate from transudate --- p.8 / Chapter 2.4 --- Receiver operating characteristic curve --- p.9 / Chapter CHAPTER 3. --- ADENOSINE DEAMINASE --- p.12 / Chapter 3.1 --- Background --- p.12 / Chapter 3.2 --- Differentiation of tuberculous and non-tuberculous pleural effusion --- p.12 / Chapter CHAPTER 4. --- MATERIALS AND METHODS --- p.17 / Chapter 4.1 --- Patients --- p.17 / Chapter 4.2 --- Collection and handling of specimens --- p.17 / Chapter 4.3 --- Diagnostic criteria --- p.18 / Chapter 4.4 --- Methods --- p.19 / Chapter 4.4.1 --- Routine chemistries --- p.19 / Chapter 4.4.2 --- Protein zone electrophoresis --- p.19 / Chapter 4.4.3 --- Adenosine deaminase --- p.19 / Chapter 4.4.3.1 --- Instrumentation --- p.22 / Chapter 4.4.3.2 --- Optimization of reaction time --- p.24 / Chapter 4.4.4 --- Analytical performance --- p.24 / Chapter 4.4.4.1 --- Imprecision --- p.24 / Chapter 4.4.4.2 --- Recovery --- p.26 / Chapter 4.4.4.3 --- Lowest detection limit --- p.26 / Chapter 4.4.4.4 --- Linearity --- p.26 / Chapter 4.4.4.5 --- Interference by ammonia --- p.26 / Chapter 4.4.4.6 --- Interference by turbidity --- p.28 / Chapter 4.4.4.7 --- Interference by haemoglobin --- p.28 / Chapter 4.4.4.8 --- Interference by bilirubin --- p.29 / Chapter 4.4.4.9 --- Storage stability of ADA at -80°C --- p.29 / Chapter 4.4.5 --- Statistical analysis --- p.30 / Chapter CHAPTER 5. --- RESULTS OF OPTIMIZATION AND EVALUATION EXPERIMENTS --- p.31 / Chapter 5.1 --- Optimization of reaction time --- p.31 / Chapter 5.2 --- Analytical performance --- p.31 / Chapter 5.2.1 --- Imprecision --- p.31 / Chapter 5.2.1.1 --- Within-run --- p.31 / Chapter 5.2.1.2 --- Between-run --- p.31 / Chapter 5.2.2 --- Recovery --- p.31 / Chapter 5.2.3 --- Lowest detection limit --- p.34 / Chapter 5.2.4 --- Linearity --- p.34 / Chapter 5.2.5 --- Interference by / Chapter 5.2.5.1 --- ammonia --- p.34 / Chapter 5.2.5.2 --- turbidity --- p.34 / Chapter 5.2.5.3 --- haemoglobin --- p.37 / Chapter 5.2.5.4 --- bilirubin --- p.37 / Chapter 5.2.6 --- Storage stability of ADA at -80°C --- p.37 / Chapter CHAPTER 6. --- TRANSUDATIVE AND EXUDATIVE PLEURAL EFFUSION --- p.39 / Chapter 6.1 --- Results of routine chemistries --- p.39 / Chapter 6.2 --- Decision thresholds by ROC curve --- p.39 / Chapter 6.3 --- Discussion --- p.39 / Chapter 6.4 --- Results of protein zone electrophoresis --- p.49 / Chapter 6.5 --- Discussion --- p.51 / Chapter 6.6 --- Comparison of protein zone electrophoresis and Light's criteria --- p.55 / Chapter 6.7 --- Discussion --- p.55 / Chapter CHAPTER 7. --- TUBERCULOUS AND NON-TUBERCULOUS EXUDATIVE PLEURAL EFFUSION --- p.59 / Chapter 7.1 --- Results of adenosine deaminase assay --- p.59 / Chapter 7.2 --- Combinations of analysis --- p.59 / Chapter 7.3 --- Decision thresholds by ROC curve --- p.64 / Chapter 7.4 --- Discussion --- p.64 / Chapter CHAPTER8. --- GENERAL DISCUSSION --- p.69 / REFERENCES --- p.70

P1B-type ATPases transport a number of monovalent and divalent heavy metals (Cu+, Cu2+, Ag+, Zn2+, Cd2+, Pb2+ and Co+2) across biological membranes. These ATPases are found in archea, bacteria and eukaryotes and are one of the key elements required for maintaining metal homeostasis. Plants have an unusually high number of P1B-type ATPases with distinct metal selectivity compared to other eukaryotes that usually have one or two Cu+-ATPases. Higher plants are the only eukaryotes where Zn2+-ATPases have been identified. Towards understanding the physiological roles of plant Zn2+-ATPases, we characterized Arabidopsis thaliana HMA2. We expressed HMA2 in yeast and measured the metal dependent ATPase activity in membranes. We showed that HMA2 is a Zn2+-ATPase that is also activated by Cd2+. Zn2+ transport determinations showed that this enzyme drives the efflux of metal from the cytoplasm. Analysis of HMA2 mRNA levels showed that the enzyme is present in all plant organs. We analyzed the effect of removal of HMA2 full-length transcript in whole plants by gene knock out. Although hma2 mutants did not show a different visible phenotype from the wild type plants, we observed increased levels of Zn2+ or Cd2+. The observed phenotype of hma2 mutants and plasma membrane location of HMA2, mainly in vasculature (Hussain et al., 2004), indicates that this ATPase might have a central role in Zn2+ uploading into the phloem. P1B-type ATPases have cytoplasmic regulatory metal binding domains (MBDs) in addition to transmembrane metal binding sites (TMBDs). Plant Zn2+-ATPases have distinct sequences in both their N- and C-termini that might contribute to novel metal binding sites. These ATPases contain long C-terminal sequences rich in CC dipeptides and His repeats. Removal of the C-terminus (C-MBD) of HMA2 leads to a 50% reduction in the enzyme turnover suggesting a regulatory role for this domain. Atomic Absorption Spectroscopy (AAS) analysis showed that Zn2+ binds to C-MBD with a stoichiometry of three (3 Zn/C-MBD). Chemical modification studies and Zn K-edge X-ray Absorption Spectroscopy (XAS) of Zn-C-MBD showed that Zn2+ is likely coordinated by His in two sites and the third site slightly differs from the others involving a Cys together with three His. All plant Zn2+-ATPases lack the typical CXXC signature sequences observed in Cu+-ATPases and some bacterial Zn2+-ATPases N-terminus metal binding domains (N-MBDs). Instead, these have conserved CCXXE sequences. Truncation of HMA2 N-MBD results in a 50% decrease in enzyme Vmax suggesting that N-MBD is also a regulatory domain. The results indicate that the N-MBD binds Zn2+ with a stoichiometry of one (1 Zn/N-MBD). Metal binding analysis of individual N-MBD mutants Cys17Ala, Cys18Ala and Glu21Ala/Cys prevented Zn+2 binding to HMA2 N-MBD suggesting the involvement of all these residues in metal coordination. ATPase activity measurements with HMA2 carrying the mutations Cys17Ala, Cys18Ala and Glu21Ala/Cys showed a reduction in the enzyme activity similar to that observed the truncated protein indicating that the enzyme activity reduction observed in the N-terminus truncated forms of the enzyme is related to the removal of the metal binding capability. Summaryzing, these studies show the central role of HMA2 in plant Zn2+ homeostasis. They also describe the mechanism and direction of Zn2+ transport. Finally, they establish the presence of novel metal binding domains in the cytoplasmic portion of the enzyme. Metal binding to these domains is required for full enzymatic activity.

Neste trabalho relata-se pela primeira vez a determinação de adenosina por um biossensor baseado em tirosinase. O biossensor foi desenvolvido mediante a modificação de um eletrodo de carbono impresso (SPE) com nanopartículas de ouro (AuNPs), tirosinase (Tyr) e Nafion, denominado biossensor Nafion/Tyr/AuNPs/SPE. As AuNPs sintetizadas possuem diâmetro médio de 15,0 ± 1,1 nm e sua função é melhorar a via de condução de elétrons entre a enzima e o eletrodo. Utilizou-se o aprisionamento com filme Nafion® para evitar a lixiviação enzimática da superfície do eletrodo. A tirosinase imobilizada apresentou boa atividade frente ao substrato catecol. Verificou-se que a adenosina atua como um inibidor do tipo não-competitivo. O biossensor é estável durante pelo menos 45 dias. Além disso, foi realizada a eletro-oxidação da adenosina para sua determinação. O biossensor apresenta sensibilidade superior em comparação com SPE, AuNPs/SPE e Nafion/AuNPs/SPE. As curvas de calibração revelaram duas faixas lineares para as concentrações de adenosina, de 1,0 × 10-5 mol L-1 até 5,0 × 10-5 mol L-1 e entre 6,0 × 10-5 mol L-1 e 1,2 × 10-4 mol L -1. O limite de detecção (3 × (desvio padrão + média dos brancos)/coeficiente angular da curva) foi de 7,0 × 10-7 mol L-1. / In this work we report for the first time the determination of adenosine by a biosensor based on tyrosinase. The biosensor was developed by modifying a screen-printed carbon electrode (SPE) with gold nanoparticles (AuNPs), tyrosinase (Tyr) and Nafion, denoted as Nafion/Tyr/AuNPs/SPE biosensor. The synthesized AuNPs have a mean diameter of 15.0 ± 1.1 nm and their function is to improve the electron conduction pathway between the enzyme and the electrode. The entrapment with Nafion® film was selected to prevent the enzyme lixiviation from the electrode surface. Immobilized tyrosinase showed good activity with the catechol substrate. It was found that adenosine acts as a non-competitive type inhibitor. The biosensor is stable for at least 45 days. In addition, the electro-oxidation of adenosine was performed for its determination. The biosensor has superior sensitivity compared to SPE, AuNPs/SPE and Nafion/AuNPs/SPE. Calibration curves revealed two linear ranges for adenosine concentrations of 1,010-5 mol L-1 up to 5,010-5 mol L-1 and from 6,010-5 mol L-1 to 1,210-4 mol L-1. The detection limit (3 × (standard deviation + mean of blanks)/slope of the curve) was 7,010-7 mol L-1.

Wong Lai Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 162-173). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Historical Background --- p.2 / Chapter 1.2. --- Heterogeneity of mast cells --- p.3 / Chapter 1.3. --- Mast cell mediators --- p.5 / Chapter 1.3.1. --- Performed and granule associated mediators --- p.5 / Chapter 1.3.2. --- Newly synthesized mediators --- p.8 / Chapter 1.3.3. --- Cytokines --- p.10 / Chapter 1.4. --- Mast cell activation --- p.10 / Chapter 1.4.1. --- Aggregation of IgE Receptors (FcεRI) --- p.10 / Chapter 1.4.2. --- Activation of Phospholipase C --- p.11 / Chapter 1.4.3. --- Activation of Adenylate cyclase --- p.13 / Chapter 1.5. --- Adenosine --- p.14 / Chapter 1.5.1. --- Adenosine receptors --- p.14 / Chapter 1.5.2. --- Selective agonists and antagonists --- p.17 / Chapter 1.5.3. --- Physiological and pathological roles of adenosine --- p.20 / Chapter 1.6. --- Role of adenosine receptors in mast cell activation --- p.21 / Chapter 1.7. --- Aims of the study --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.31 / Chapter 2.1.1. --- Mast cells secretagogues --- p.31 / Chapter 2.1.2. --- Anti-allergic compounds --- p.31 / Chapter 2.1.3. --- Adenosine receptor agonists and antagonists --- p.31 / Chapter 2.1.4. --- Materials for buffers --- p.32 / Chapter 2.1.5. --- Materials for rat sensitization --- p.32 / Chapter 2.1.6. --- Materials for histamine assay --- p.33 / Chapter 2.1.7. --- Miscellaneous --- p.33 / Chapter 2.2. --- Buffers and stock solutions --- p.34 / Chapter 2.2.1 --- Buffer ingredients --- p.34 / Chapter 2.2.2 --- Stock solutions --- p.34 / Chapter 2.3. --- Source of mast cells --- p.35 / Chapter 2.3.1. --- Animals --- p.35 / Chapter 2.3.2. --- Sensitization of animals --- p.35 / Chapter 2.3.3. --- Isolation of rat peritoneal mast cells --- p.35 / Chapter 2.3.4. --- Mast cells purification --- p.36 / Chapter 2.3.5. --- Cell counting --- p.36 / Chapter 2.4. --- General protocol for histamine release --- p.37 / Chapter 2.4.1. --- Histamine assay --- p.37 / Chapter 2.4.2. --- Antagonist studies --- p.38 / Chapter 2.4.3. --- Determination of histamine contents --- p.38 / Chapter 2.4.4. --- Calculation of histamine levels --- p.39 / Chapter 2.5. --- Statistical analysis --- p.40 / Chapter Chapter 3 --- "Effects of adenosine, adenosine deaminase and adenosine receptor agonists on mast cell activation" --- p.42 / Chapter 3.1. --- Introduction --- p.43 / Chapter 3.2. --- Materials and methods --- p.44 / Chapter 3.3. --- Results --- p.45 / Chapter 3.3.1. --- Effects of adenosine on anti-IgE induced histamine release in HEPES buffer --- p.45 / Chapter 3.3.2. --- Effects of NECA on anti-IgE induced histamine release in HEPES buffer --- p.46 / Chapter 3.3.3. --- Effects of CCPA on anti-IgE induced histamine release in HEPES buffer --- p.47 / Chapter 3.3.4. --- Effects of CPA on anti-IgE induced histamine release in HEPES buffer --- p.47 / Chapter 3.3.5. --- Effects of CGS21680 on anti-IgE induced histamine release in HEPES buffer --- p.48 / Chapter 3.3.6. --- Effects of Cl-MECA on anti-IgE induced histamine release in HEPES buffer --- p.49 / Chapter 3.3.7. --- Effects of adenosine deaminase on anti-IgE induced histamine release from rat peritoneal mast cells --- p.50 / Chapter 3.3.8. --- Effects of NECA on anti-IgE induced histamine release with and without adenosine deaminase --- p.50 / Chapter 3.3.9. --- Effects of Cl-MECA on anti-IgE induced histamine release with and without adenosine deaminase --- p.53 / Chapter 3.3.10. --- Effects of CV1808 on anti-IgE induced histamine release in HEPES buffer --- p.55 / Chapter 3.4. --- Discussion --- p.76 / Chapter 3.5. --- Conclusion --- p.83 / Chapter Chapter 4 --- Effects of adenosine receptor antagonists on mast cell activation --- p.88 / Chapter 4.1. --- Introduction --- p.89 / Chapter 4.2. --- Materials and methods --- p.90 / Chapter 4.3. --- Results --- p.91 / Chapter 4.3.1. --- Effects of A1 receptor antagonist DPCPX on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.91 / Chapter 4.3.2. --- Effects of A2A receptor antagonist ZM241385 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.91 / Chapter 4.3.3. --- Effects of A2B receptor antagonist MRS 1706 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.92 / Chapter 4.3.4. --- Effects of A3 receptor antagonist VUF5574 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.93 / Chapter 4.3.5. --- Further characterization of adenosine mediated potentiation of anti-IgE histamine release using VUF5574 and ZM241385 --- p.93 / Chapter 4.3.6. --- Effects of theophylline on anti-IgE induced percentage potentiation in HEPES buffer --- p.95 / Chapter 4.4. --- Discussion --- p.130 / Chapter 4.5. --- Conclusion --- p.135 / Chapter Chapter 5 --- Further characterization of the effects of adenosine on mast cells --- p.138 / Chapter 5.1. --- Introduction --- p.139 / Chapter 5.2. --- Materials and methods --- p.141 / Chapter 5.3. --- Results --- p.142 / Chapter 5.3.1. --- Effects of anti-IgE induced histamine release in calcium free and HEPES buffers --- p.142 / Chapter 5.3.2. --- Effects of adenosine on anti-IgE induced histamine release in calcium free buffer --- p.143 / Chapter 5.3.3. --- Effects of adenosine deaminase on compound48/80 induced histamine release from rat peritoneal mast cells --- p.143 / Chapter 5.3.4. --- Effects of adenosine on compound 48/80 induced histamine release in HEPES buffer --- p.144 / Chapter 5.3.5. --- Effects of adenosine deaminase on A23187 induced histamine release from rat peritoneal mast cells --- p.144 / Chapter 5.3.6. --- Effects of adenosine on calcium ionophore A23187 induced histamine release in HEPES buffer --- p.145 / Chapter 5.3.7. --- Effects of adenosine receptor antagonists on inosine mediated enhancement of anti-IgE induced histamine release --- p.145 / Chapter 5.4. --- Discussion --- p.157 / Chapter 5.5. --- Conclusion --- p.160 / References --- p.162

Estudos têm demonstrado que o microambiente tumoral é rico em ATP e adenosina, sugerindo o envolvimento da sinalização purinérgica no desenvolvimento e/ou manutenção do câncer. Ainda, o receptor purinérgico P2X7, conhecido pelo seu papel na indução de apoptose, encontra-se reduzido em alguns tecidos tumorais em comparação aos tecidos saudáveis, indicando que a sua redução possa ser um mecanismo de resistência celular à apoptose. Dessa forma, compreender o papel da sinalização purinérgica no contexto do câncer se torna indispensável e permite que novas abordagens terapêuticas sejam implementadas. Nesse trabalho, avaliamos a função dos nucleotídeos e nucleosídeos da adenina, bem como do receptor P2X7 na indução da morte celular em células de câncer cervical. Também verificamos o efeito do heat shock na potencialização da atividade do receptor P2X7 frente à curta exposição ao ATP em células de câncer de cólon. De acordo com os nossos resultados, o efeito citotóxico do ATP extracelular nas linhagens de câncer cervical é mediado principalmente pela ação do seu metabólito adenosina, que ao entrar no interior das células, promove o aumento dos níveis intracelulares de AMP, ativação de AMPK, aumento da p53 e indução de autofagia. O papel do receptor P2X7 nesse contexto parece ser apenas coadjuvante, visto que o seu bloqueio ou silenciamento impediu em apenas 20% a morte celular. Além disso, utilizando células de câncer de cólon, nós demonstramos que o heat shock aumenta a funcionalidade do receptor P2X7, independente da interação com heat shock proteins ou canais do tipo conexina/panexina, potencializando o efeito citotóxico do ATP. Esse efeito parece estar relacionado à mudanças na composição e arquitetura da membrana celular, visto que o uso do agente fluidizador de membrana benzil álcool foi capaz de mimetizar o efeito do heat shock na potencialização do receptor P2X7 a 37ºC. Este estudo fornece evidências adicionais sobre o papel da sinalização purinérgica no contexto da biologia celular tumoral e abre novas perspectivas para o uso dos nucleotídeos de adenina associados a hipertermia como agentes adjuvantes na terapia do câncer. / The tumor microenvironment is rich in ATP and adenosine, suggesting an involvement for purinergic signaling in cancer development and surveillance. The P2X7 receptor, among the P2 purinergic receptors, is broadly recognized as the “death receptor”, because it promotes cell apoptosis when exposed to high levels of extracellular ATP. Researches have been shown that P2X7 protein levels are decreased at the tumor site in comparison to adjacent healthy tissue, suggesting a mechanism of tumor escape to cell death. Thus, understanding purinergic signaling in a cancer context becomes urgent and opens a new field for therapeutic strategies. Here, we evaluated adenine nucleotides and nucleosides cytotoxicity, as well as P2X7 role in cell death induction using cervical cancer cell lines. Indeed, we investigated heat shock effect on P2X7 functionality through exposing colon cancer cell shortly to ATP at 40ºC. According to our data, adenosine uptake formed from ATP metabolism is the main responsible for the extracellular ATP cytotoxicity in cervical cancer cells. While inside of the cell, adenosine is converted to AMP, leading to AMPK activation, p53 increase and autophagy induction. ATP induced cell death per se through P2X7 in this context seems to be less important, since P2X7 blockage or knocking down reduced only 20% of cell death. In colon cancer cells, we found that heat shock stress was able to increase P2X7 pore formation independently of heat shock protein interaction or native pore-forming transporters association (e.g pannexin-or connexin-type channels), thus leading to an increase ATP cytotoxicity. The mechanism enrolled in this process seems to be related to changes in the lipid composition and architecture of membrane, as the membrane fluidizer benzyl alcohol could reproduce heat stress effect in potentiating P2X7 activation at 37ºC. In conclusion, our work provides further evidence for a purinergic signaling role in the cancer biology context and opens new perspectives for the utility of purine-based drugs associated to hypertermia as adjunctive agents in cancer therapy.

Les mécanismes de défense de l’organisme contre des pathogènes ou des particules toxiques sont regroupés sous le terme d’Immunité. L’Immunité a développé plusieurs familles de récepteurs dont les NLR (Nod Like Receptors) spécialisés dans la reconnaissance de motifs de pathogènes (PAMP), de signaux de danger endogènes (DAMP) et de désordres métaboliques et capables d’engager une réponse inflammatoire. L’inflammation est dite stérile si elle n’est pas déclenchée par des agents infectieux mais par des molécules endogènes en excès comme l’acide urique et l’ATP ou par des polluants environnementaux tels que la silice et les nanoparticules. Nous nous sommes d’une part intéressés aux mécanismes d’activation de l’inflammasome NLRP3 qui permet la maturation d’une cytokine pro-inflammatoire, l’Interleukine (IL)-1β. Nous montrons que les cristaux d’acide urique, de silice et d’Alum ainsi que les nanoparticules de titane et de silice induisent une libération active d’ATP dépendante d’hémicanaux par les cellules. Nous mettons en évidence un nouveau mécanisme d’activation de l’inflammasome NLRP3 qui fait intervenir l’ATP et ses différents métabolites, en particulier l’adénosine, via des familles de récepteurs purinergiques. Nous nous sommes d’autre part focalisés sur l’inflammation pulmonaire induite par l’administration de nanoparticules dans un modèle murin. Nous avions montré dans une étude précédente que cette inflammation est dépendante des IL-1α et IL-1β et de leur récepteur IL-1R1. Nos résultats montrent que l’adénosine est impliquée in vivo dans la réponse inflammatoire aux nanoparticules et que l’inflammation des voies respiratoires est modulée par l’IL-33 et son récepteur ST2. / The mechanisms of defense of the organism against pathogens or oxious particles are termed Immunity. The Immunity developed several receptors’ families among which the NLR (Nod-Like Receptors), specialized in the recognition of pathogen patterns (PAMPs), of endogenous danger signals (DAMPs) and of metabolic disorders, and capable of triggering an inflammatory response. The inflammation is named “sterile” if it is not activated by infectious agents but by endogenous molecules in excess, as uric acid and ATP, or by environmental pollutants, such as silica and nanoparticles. We first became interested in the mechanisms of activation of the NLRP3 inflammasome which allows the maturation of a pro-inflammatory cytokine, the Interleukine (IL) -1β. We show that uric acid, silica or Alum crystals but also titanium and silica nanoparticles induce an active release of ATP by cells, dependent of hemichannels. We highlight an innovative mechanism of activation of the NLRP3 inflammasome which involves ATP and its metabolites, in particular adenosine, through purinergic receptor families. We then focused on the nanoparticle-induced lung inflammation in a murine model. We had shown in a previous study that this inflammation is dependent on IL-1α, IL-1β and their common receptor IL-1R1. Our in vivo results show that adenosine is involved in nanoparticle-induced inflammatory response and that airway inflammation is modulated by IL-33 and its receptor ST2.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A adenosina desaminase (ADA â E.C.3.5.4.4.) à uma enzima fundamental no catabolismo das purinas. Ela catalisa a desaminaÃÃo da adenosina ou 2âdeoxi-adenosina produzindo amÃnia e inosina ou 2â-deoxi-inosina, respectivamente. Sua atividade à expressa por 2 isoenzimas presentes em 3 isoformas. A ADA1 (36kDa) ou ADA1 ligada ao CD26 (280kDa) sÃo amplamente distribuÃdas nos tecidos. Sua aÃÃo à particularmente importante porque altos nÃveis de 2âdeoxi-adenosina sÃo tÃxicos para as cÃlulas do sistema imunolÃgico. A ADA2 (100kDa) à normalmente encontrada no soro e sintetizada somente pelo sistema monocÃtico-macrofÃgico. A importÃncia biolÃgica da ADA2 ainda nÃo està totalmente estabelecida, principalmente devido as suas caracterÃsticas cinÃticas. O presente trabalho teve como objetivo discriminar as isoenzimas da adenosina desaminase humana atravÃs de eletroforese em gel de agarose e pelo modelo proposto por Vale e Almeida (1998), bem como realizar um estudo descritivo retrospectivo sobre o perfil dos exames de ADA no Estado do CearÃ. As amostras de lÃquido ascÃtico, pleural e pericÃrdico foram submetidas à eletroforese em agarose a 1% a 80 V por 7 horas. O gel foi fatiado e cada fatia foi incubada em adenosina (22 ou 0,55mM) por 20 horas para a detecÃÃo da amÃnia liberada pela reaÃÃo enzimÃtica. Os resultados encontrados a partir da eletroforese foram comparados com os resultados achados pelo modelo de Vale e Almeida (1998). O lÃquido pleural à o fluido que à mais frequentemente solicitado para a determinaÃÃo da ADA, seguido pelos lÃquidos ascÃtico, cefalorraquidiano, pericÃrdico e soro. Observamos que os valores de atividade enzimÃtica sÃo influenciados pelo tipo de lÃquido corporal onde a enzima se encontra, podendo estar relacionada Ãs barreiras corporais, tais como a barreira hematoencefÃlica. A partir dos resultados obtidos, podemos concluir que o modelo matemÃtico proposto pode ser usado em laboratÃrios clÃnicos para discriminar as isoenzimas da ADA. / Adenosine deaminase (ADA â E.C.3.5.4.4.) is a fundamental enzyme in the catabolism of the purines. It catalyzes the deamination of adenosine or 2âdeoxy-adenosine producing ammonium and inosine or 2â-deoxyinosine, respectively. Its activity is expressed by two isoenzymes presented in three isoforms. ADA1 (36 kDa) and ADA1 bound to CD26 (280kDa) are widely distributed in the body tissues. Their action is particularly important because high levels of 2âdeoxy-adenosine are toxic for the immune system cells. ADA2 (100kDa) is normally found in serum and is synthesized only in monocyte-macrophage system. The biological importance of ADA2 is not yet fully clear, especially for its kinetics characteristics. The objective of the present work was to discriminate the isoenzymes of human adenosine deaminase using agarose electrophoresis and by mathematical model proposed by Vale and Almeida (1998). In addition, we performed a study of the profile of ADA tests in State of Ceara (Brazil). Samples of of ascites, pleural and pericardial effusion were submitted to electrophoresis in 1% agarose at 80V for 7 hours. The gel was sliced and each slice was incubated in adenosine (22 or 0,55mM) for 20 hours to detect the ammonium released by enzymatic reaction. The results found from electrophoresis were compatible with the model proposed by Vale and Almeida (1998). The pleural fluid is the most frequently requested for the determination of ADA, followed by ascitic fluid, cerebrospinal fluid, pericardial fluid and serum. We observed that the value of enzymatic activity is influenced by corporal fluid type where the enzyme is localized. These data can be associated with the corporal barrier, like brain barrier. We concluded that the proposed mathematical model could be used in clinical laboratories to discriminate ADA isoenzymes to improve the diagnostic method.

A morte celular necrótica é conhecida pelo seu caráter inflamatório, conferido pela grande quantidade de sinais de dano liberados. Durante a tuberculose primária progressiva é observada intensa lesão necrótica nos pulmões e disseminação do bacilo para outros órgãos. Neste estudo, nós hipotetizamos que a amplificação da necrose pulmonar, via reconhecimento de sinais de dano tecidual pelo receptor purinérgico P2X7 (P2X7R), poderia favorecer a progressão da doença, bem como a potencialização da resposta inflamatória. Vimos que o reconhecimento de extracelular ATP (eATP) via o receptor P2X7 é de suma importância para o desenvolvimento da doença grave, por favorecer a indução de necrose dos macrófagos infectados, o que facilitou o escape do bacilo. A adenosina, resultante da hidrólise do eATP, impactou na ativação da resposta imune adquirida. Nosso estudo provê uma nova perspectiva para o desenvolvimento de protocolos terapêuticos baseados na inibição do P2X7R e dos receptores de adenosina para evitar a indução de formas graves da doença. / Necrosis cell death is known as an inflammatory process due to the large amount of damage signals released. During the progressive primary tuberculosis, extensive necrotic lesions in the lung and intensive bacterial dissemination are observed. In this study, we hypothesized that the amplification of pulmonary necrosis through damage signals recognition by a purinergic receptor called P2X7 (P2X7R) could favor the progression of disease, as well as the augmentation of the inflammatory response. We found that the recognition of extracellular ATP (eATP) through P2X7R is crucial to the outcome of fatal tuberculosis by favoring the induction of necrosis of infected macrophages, which facilitated the bacterial escape. The adenosine, resulted from eATP hydrolyzation, impacted to the acquired immune response activation. Our study provides a new perspective to the development of therapeutic protocols based on the inhibition of P2X7R and adenosine receptor to avoid the induction of aggressive forms of tuberculosis.

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

Left coronary blood flow (LCBF), left ventricular oxygen, extraction [(a-v)O₂ ], and myocardial oxygen consumption (MVO₂) were monitored in 10 dogs. HR was paced at 120 bpm and then increased to 180 bpm to elicit a hyperemic response (ΔLCBF). During the hyperemia, the vaso-dilatory response to exogenous adenosine (F_AD) was tested. Twenty min. after injection of aminophylline (100 mg/i.v.), HR was again increased. F_AD was again tested. The pacing-induced increase in MVO₂ (ΔMVO₂) was not affected by aminophylline (P>0.05). However, the slope ΔLCBF/ΔMVO₂ was decreased, and the slope (a-v)O₂ /ΔMVO₂ was increased. F_AD was also decreased and the magnitude of the reduction was correlated with the decrease in ΔLCBF/ΔMVO₂ (r=0.82). These results suggest that adenosine may play an role in coronary functional hyperemia induced by increases in heart rate.