Sulfiredoxin (Srx) is an antioxidant enzyme that can be induced by oxidative stress. It promotes oncogenic phenotypes of cell proliferation, colony formation, migration, and metastasis in lung, skin and colon cancers. Srx reduces the overoxidation of 2-cysteine peroxiredoxins in cells, in addition to its role of removing glutathione modification from several proteins. In this study, I explored additional physiological functions of Srx in lung cancer through studying its interacting proteins. Protein disulfide isomerase (PDI) family members, thioredoxin domain containing protein 5 (TXNDC5) and protein disulfide isomerase family A member 6 (PDIA6), were detected to interact with Srx. Therefore, I proposed that TXNDC5 and PDIA6 are important for the oncogenic phenotypes of Srx in lung cancer. In chapter one, I presented background information about the role of Srx as an antioxidant enzyme in cancer. I also explained the functional significance of PDIs as oxidoreductase and chaperones in cells. In chapter two, I verified the Srx-TXNDC5/PDIA6 interaction in HEK293T and A549 cells by co-immunoprecipitation and other assays. In TXNDC5 and PDIA6, the N-terminal thioredoxin-like domain (D1) is determined to be the main platform for interaction with Srx. The Srx-TXNDC5 interaction was enhanced by H2O2 treatment in A549 cells. Srx was determined to localize in the endoplasmic reticulum (ER) of A549 cells along with TXNDC5 and PDIA6. This localization was confirmed by both subcellular fractionation and immunofluorescence imaging experiments. In chapter three I focused on studying the physiological function of Srx interacting proteins in the ER. A549 subcellular fractionation results showed that TXNDC5 facilitates Srx retention in the ER. Moreover, TXNDC5 and Srx were found to participate in chaperone activities in lung cancer. Both proteins contributed in the refolding of heat-shock induced protein aggregates. In addition, TXNDC5 and PDIA6 were found to enhance the protein refolding in response to H2O2 treatment. Conversely, Srx appeared to have an inhibitory effect on protein folding under same treatment conditions. Downregulation of Srx, TXNDC5, or PDIA6 significantly reduced cell viability in response to tunicamycin treatment. TXNDC5 knockdown decreased the time required for the splicing of X-box binding protein-1 (XBP-1). In either knockdown Srx or TXNDC5 cells, there was an observable decrease in the expression of GRP78 and the splicing of spliced XBP-1. These results suggest a possible role of Srx in unfolded protein response signaling. TXNDC5 and PDIA6, similar to Srx, contribute to the proliferation, anchorage independent colony formation and migration of lung cancer cells. In this dissertation I concluded that Srx TXNDC5, and PDIA6 proteins participate in oxidative protein folding in lung cancer. Srx and TXNDC5 can modulate unfolded protein response (UPR) sensor activation and growth inhibition. Furthermore, TXNDC5 and PDIA6 can promote tumorigenesis of lung cancer cells. Therefore, the molecular interaction of Srx with TXNDC5/PDIA6 has the potential to be used as novel therapeutic targets for lung cancer treatment.

INTRODUÇÃO: A ativação da NADPH oxidase de neutrófilos requer o acoplamento das subunidades citosólicas p47phox, p67phox, p40phox e Rac2 ao componente de membrana citocromo b558. Em trabalhos anteriores, nós mostramos que as isoformas vasculares da oxidase são reguladas pela proteína dissulfeto isomerase (PDI), uma chaperona redox. Neste trabalho, nós utilizamos um sistema cellfree semirecombinante como ferramenta para investigar o papel da PDI na ativação da NADPH oxidase do neutrófilo. RESULTADOS: Inibidores da PDI, scrambled RNAse (100g/mL) ou bacitracina (1mM), praticamente suprimiram a geração de superóxido. Para avaliação dos efeitos do estado redox da PDI sobre a atividade da oxidase, amostras de PDI foram previamente oxidadas (H2O2; 0,5 mM) ou reduzidas (DTT; 0,5 mM). A PDI oxidada (100 nM) aumentou a produção de superóxido em aproximadamente 30%, enquanto a mesma concentração de PDI reduzida promoveu efeito inverso, inibindo a atividade do complexo. A adição de um peptídeo contendo a seqüência peptídica do sítio ativo da PDI inibiu a produção de superóxido em 70%. Dados de imunolocalização e colocalização demonstraram que a interação da PDI com a subunidade p47phox parece ser intensificada pelo estímulo com PMA e envolvem modificações do estado redox de ambas as proteínas. CONCLUSÕES: Nossos dados confirmam a associação física e funcional entre a PDI e o complexo NADPH oxidase. Além disso, sugerem que a PDI exerça um importante papel como fator de regulação redox da ativação da oxidase. / Activation of the leukocyte NADPH oxidase requires the assembly of the cytosolic subunits p47phox, p67phox and p40phox and Rac2 with the membranebound cytochrome b558. We have previously shown that the vascular oxidase is regulated by the redox chaperone protein disulfide isomerase (PDI). Taking advantage of the semirecombinant cellfree system, we sought to investigate the role of PDI in the activation of neutrophil NADPH oxidase. The PDI thiol inhibitors scrambled RNase (100g/mL) or bacitracin (1mM), almost suppressed superoxide generation. In order to investigate if the redox status of PDI thiols could modulate superoxide generation, PDI was oxidized or reduced by treatment with H2O2 (0.5mM) or DTT (1mM), respectively. Oxidized PDI increased by 30% superoxide production, while reduced PDI diminished superoxide generation also in 30%. The addition of a peptide (1M) containing PDI´s exact active site sequence inhibited superoxide production by 70 %. Immuno and colocalization data demonstrated the interaction of PDI with the subunit p47phox to be intensified by PMA stimulation and to involve redox status exchange of both proteins. Our data confirm the physical and functional association between PDI and the oxidase complex. Moreover, we show a relevant role for PDI as a redoxdependent supportive factor for NADPH oxidase activation.

A oxidação do ácido úrico (7,9-diidro-1H-purina-2,6,8(3H)-triona) pela mieloperoxidase (MPO) gera o radical de urato que, em presença do radical ânion superóxido combinam para formar o hidroperóxido de urato (HOOU). Considerando os altos níveis de MPO, ácido úrico e superóxido na placa de ateroma espera-se que o ácido úrico seja oxidado a HOOU neste microambiente inflamatório. O HOOU é um oxidante mais forte que o peróxido de hidrogênio e pode oxidar grupos tiólicos de proteínas como a proteína dissulfeto isomerase (PDI). Como consequência à oxidação da PDI, ocorre uma modulação positiva sobre a NADPH oxidase (Nox) com aumento da produção de superóxido por neutrófilos. Sendo assim, a formação do HOOU no leito vascular poderia elucidar os mecanismos moleculares pelos quais o urato colabora para a progressão da aterosclerose em pacientes com hiperuricemia. Para investigar a interação do HOOU com a PDI, padronizou-se a síntese química do HOOU através de um sistema de fotooxidação do tipo I, utilizando urato, riboflavina (fotossensibilizador) e luz UVA. Inicialmente padronizou-se o tipo de irradiação e tempo de reação com o melhor rendimento para a síntese do HOOU. O HOOU formado e seu produto de redução o álcool 5-hidroxiisourato foram separados, identificados e caracterizados através de cromatografia liquida acoplada à espectrometria de massa (LC/MS). Após a purificação, determinou-se o coeficiente de absortividade molar em 308 nm (ε308 = 6537 ± 377 M-1.cm-1) e o tempo de meia-vida, aproximadamente 41 minutos à 22°C do HOOU. O HOOU foi capaz de reagir seletivamente com o aminoácido metionina e com o tripeptídeo glutationa. Além disso, o HOOU não forma adutos estáveis com a glutationa, sendo que toda glutationa consumida foi transformada em glutationa dissulfeto. Quando incubado com a PDI (10 µM), cerca de 70 e 100% do total de HOOU (3 µM) foi consumido após 30 e 120 segundos, respectivamente, enquanto que a PDI (23 µM) teve seus grupos tiólicos oxidados após a incubação com 140 µM HOOU por 30 min a 22°C. O HOOU oxidou os resíduos de cisteína dos dois sítios catalíticos da PDI com uma constante de velocidade da reação de 1,5 ± 0,04 x 103 M-1.s-1, demonstrando uma interação favorável com essa proteína no meio biológico e um possível papel modulatório do HOOU sobre a via PDI-Nox. Interessantemente, o ácido úrico aumentou a produção de superóxido e o consumo de oxigênio de células HL-60 diferenciadas em neutrófilos (dHL-60) e ativadas com acetato de miristato de forbol (PMA). Essa regulação poderia ser mediada através da formação do HOOU durante o \"burst\" oxidativo dos neutrófilos que oxidaria a enzima PDI induzindo um consequente aumento da atividade da Nox. / The oxidation of the uric acid (7,9-dihidro-1H-purine-2,6,8(3H)-trione) by myeloperoxidase (MPO) generates the urate radical. In inflammatory conditions the superoxide reacts with urate radical to form the urate hydroperoxide (HOOU). Taking into account the high amount of MPO, urate and superoxide in the atheroma plaque, it is likely that HOOU is being formed in this inflammatory environment. The HOOU is a strong oxidizing agent and can react with thiol groups from proteins, like the protein disulfide isomerase (PDI). As a consequence of its oxidation, PDI positively modulates NADPH oxidase (Nox) and increases superoxide production by neutrophils. Therefore, the formation of HOOU in the vascular sheet would contribute to tissue damage and would explain the positive correlation between hyperuricemia and the risk for cardiovascular disease. To investigate the interaction of HOOU with PDI, we performed the chemical synthesis of the compound by the Type I photooxidation, using UVA irradiation and riboflavin as a photosensitizer. Initially, we standardized the irradiation light and the time of the reaction that produced the highest income. The HOOU and its reduced product 5-hydroxiisourate were separate, identified and characterized by liquid chromatography coupled to mass spectrometry (LC/MS). We also determine the molar extinction coefficient of HOOU at 308 nm (ε308 = 6537 ± 377 M-1.cm-1). The half-life of the compound was 41 min at 22°C. The HOOU selectively oxidized methionine and glutathione. The reaction of HOOU with glutathione did not form any stable adducts. Thus, all consumed glutathione generated glutathione disulfide. When incubated with PDI (10 µM), 70 and 100% of the total amount of HOOU (3 µM) was consumed within 30 and 120 seconds, respectively. Besides, the PDI (23 µM) had its thiol groups oxidized after incubation with 140 µM HOOU for 30 min at 22°C. The HOOU oxidized the cysteine residues from the catalytical sites of PDI with a second order rate constant of 1.5 ± 0.04 x 103 M-1.s-1. This result suggests a favorable interaction of HOOU with this protein in the biological system, as well as a possible modulatory role of HOOU on the PDI-Nox pathway. Interestingly, urate increased superoxide production and oxygen consumption by neutrophil-like cells (differentiated HL-60 cells). This effect could be mediated by the formation of HOOU during the neutrophil oxidative burst, followed by the oxidation of PDI, a positive regulation of Nox and an increase in superoxide production.

Os acidentes ofídicos são considerados um grande problema de saúde pública e no Estado de São Paulo a serpente Bothrops jararaca é o agente considerado mais relevante epidemiologicamente. O antiveneno é o único medicamento oficialmente aprovado para o tratamento de picadas de serpentes e, apesar de ser eficaz em diversos aspectos, mostra-se ineficaz em relação a complicações secundárias do envenenamento, como o estresse oxidativo/nitrosativo, caracterizado por um desequilíbrio no estado redox, que pode ser extremamente deletério aos pacientes. Portanto, vê-se necessária a busca por terapias complementares que possam combater o estresse oxidativo/nitrosativo. Ademais, distúrbios da hemostasia observados em envenenamentos pela B. jararaca, tais como a plaquetopenia, a hipofibrinogenemia e o aumento da atividade do fator tissular (TF) plasmático podem ter também como causa indireta o estresse oxidativo/nitrosativo, induzido por componentes do veneno de B. jararaca (BjV). Recentemente, demonstrou-se que a regulação da atividade biológica do TF, a proteína responsável pela iniciação da cascata de coagulação in vivo, pode ser controlada pela isomerase de dissulfeto proteico (PDI) e que a atividade da PDI é inibida in vivo pela quercetina-3-rutinosídeo (rutina), um antioxidante natural encontrado em plantas e alimentos. Considerando essas premissas, o presente trabalho objetivou investigar em camundongos injetados com o veneno de B. jararaca (BjV): (a) a ocorrência de distúrbios do estado redox; (b) as alterações hematológicas e hemostáticas (em plasma e tecidos) associadas ao desequilíbrio do estado redox; (c) a atividade da rutina como agente modulador sobre essas alterações. Para isso, camundongos Swiss (30-35 g) foram divididos em 4 grupos experimentais: controle salina (salina, controle negativo), controle rutina (rutina+salina), BjV+salina (BjV+salina, controle positivo) e BjV+rutina (BjV+rutina, tratamento). Após 3, 6 e 24 h da administração dos tratamentos (via s.c.), foram coletados sangue e fragmentos de tecidos para posteriores análises. O envenenamento induziu um aumento de espécies reativas, diminuição da capacidade antioxidante total, alterações hematológicas (plaquetopenia, neutrofilia e diminuição de eritrócitos), distúrbios hemostáticos (hipofibrinogenemia, prolongamento do tempo de protrombina e aumento da atividade de TF no plasma), além de diminuir a expressão proteica de PDI no coração. A rutina não inibiu in vitro a atividade biológica de proteínas presentes no BjV (metaloproteinases, serinaproteases, fosfolipases A2 e L-aminoácido oxidases) e nem mesmo a agregação plaquetária induzida pelo veneno. No entanto, a administração do veneno incubado com rutina foi capaz de reduzir os níveis de espécies reativas, impedir a queda de eritrócitos, normalizar os níveis de fibrinogênio e o tempo de protrombina e alterar a atividade de TF e expressão proteica de TF e PDI em tecidos. Desse modo, concluímos que a rutina foi capaz de favoravelmente modular importantes alterações hemostáticas e de estado redox no envenenamento por B. jararaca, o que indica seu grande potencial como agente terapêutico complementar em envenenamentos / Snakebites are a major public health issue and in São Paulo State, and Bothrops jararaca snakes are considered the most important agents epidemiologically. Antivenom is the only officially approved treatment for snakebites and although effective in many aspects, it is ineffective in combating secondary complications induced by envenomation, e.g. oxidative/nitrosative stress (ONS), which can be extremely deleterious to patients. Therefore, it is necessary to search for new complementary therapies that could attenuate ONS. Furthermore, the hemostatic disturbances in B. jararaca envenomation - characterized by the presence of thrombocytopenia, hypofibrinogenemia and increased tissue factor (TF) activity in plasma - could be indirectly evoked by ONS induced by B. jararaca venom (BjV) components. Recently, the biological activity of TF - the protein responsible for initiating the extrinsic pathway of the coagulation cascade - was demonstrated to be regulated by protein disulfide isomerase (PDI), which in turn, can be inhibited in vivo by quercetin-3-rutinose (rutin), a natural antioxidant found in plants and diet. Based on that, the present study aimed to investigate in mice injected with BjV: (a) the alterations in redox status in plasma and tissues; (b) the hematological/hemostatic disturbances (in plasma and tissues) associated with the imbalance in redox status; (c) the activity of rutin as a modulatory agent on those alterations. Swiss mice (30-35 g) were divided in four experimental groups: saline control (saline, negative control), rutin control (rutin+saline), BjV+saline (BjV+saline, positive control) and BjV+rutin (BjV+rutin, treatment) and after 3, 6 and 24 h of the injection of treatments (s.c. route), blood and tissues were collected to further analyses. Envenomation induced an increase in reactive species, a decrease in total antioxidant capacity, hematological alterations (thrombocytopenia, neutrophilia and a decrease in red blood cell counts), hemostatic disturbances (hypofibrinogenemia, prolonged prothrombin time and an increase in the TF activity in plasma), and a decrease in protein expression of PDI in the heart. In vitro, rutin failed to inhibit the biological activity of the main BjV enzymes (metalloproteinases, serine proteases, phospholipases A2 and L-amino acid oxidases) and BjV-induced platelet aggregation. However, when the venom was incubated with rutin, there were reduced reactive species levels, less intense red blood cell drops, recovery of fibrinogen levels and prothrombin time, and alteration of TF activity and protein expression of TF and PDI in tissues. Therefore, we conclude that rutin favorably modulated important hemostatic and redox status alterations in B. jararaca envenomation, indicating its great potential as a complementary therapeutic agent for snakebites

No description available.

Nous avons caractérisé cinq protéines bovines qui sont potentiellement impliquées dans la reproduction.Un travail de clonage a été initié qui permettra à terme de purifier les GPA2 et GPB5 recombinantes puis naturelles pour étudier leurs structures. GPA2 et GPB5 sont considérés comme les ancêtres moléculaires des sous-unités α et β des hormones glycoprotéiques. Nous avons montré la relative fragilité thermique de la structure quaternaire de la FSH bovine par rapport aux FSH ovine et humaine et nous avons étudié les propriétés enzymatiques de la PDI (Protein Disulfide Isomerase) en préalable à l’étude de l’activité PDI de GPA2/GPB5. Nous avons aussi purifié la phosphatidyl-ethanolamine-binding protein (PEBP) et l’ubiquitine testiculaires par chromatographie hydrophobe à très haute concentration de sulfate d’ammonium. A partir de la PEBP purifiée, on a produit des anticorps spécifiques chez le lapin qui nous ont permis d’être les premiers à développer un dosage ELISA fiable pour cette protéine. / We characterized five bovine proteins that are potentially involved in reproduction. We started with the cloning of gpa2 and gpb5 cDNAs in order to eventually purify recombinant and natural GPA2 and GPB5 to study their possible quaternary structure. GPA2 and GPB5 are the evolutionary ancestors of Glycoprotein hormones α and β subunits respectively. Meanwhile, we have shown the relative quaternary structure fragility of bovine FSH compared to human and sheep FSH. We also studied the effect of endocrine disruptors on PDI (Protein Disulfide Isomerase) before addressing GPA2/GPB5 PDI activity of GPA2/GPB5 once purified.We succeeded to purify the phosphatidyl-ethanolamine-binding protein (PEBP) and ubiquitin from bovine testis by hydrophobic interaction chromatography at very high ammonium sulfate concentration and we produced specific antibodies (anti-PEBP) in rabbits that allowed us to be the first to develop a reliable Elisa assay for this protein.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects are described in this dissertation, and they are: (i) discovery of piperidine derivatives as T-type calcium channel inhibitors for the treatment of epilepsy and neuropathic pain and as protein disulfide isomerase inhibitors for the treatment of influenza viral infection; (ii) discovery of peptide-based calcitonin gene-related peptide receptor antagonists for the treatment of inflammatory pain; and (iii) synthesis of substituted 6-(dimethylamino)-2-phenylisoindolin-1-ones for the inhibition of luciferase. T-type calcium channels are important regulators of nervous system, and upregulated T-type calcium channel activities have been found to link to various types of neurological disorders, such as epilepsy and neuropathic pain. To discover novel T-type calcium channel blockers, a series of 1,4-disubstituted piperidine derivatives were designed and synthesized. Among them, compound 1-4 was found to be a good T-type calcium channel inhibitor with an IC₅₀ of 1 nM for Ca[subscript v]3.2 inhibition. It also showed 86% suppression of seizure induced death in mice and good in vivo analgesic effects on both thermal and mechanical pain thresholds in Spared Nerve Injury rat models. Therefore 1-4 can potentially be used as a T-type calcium channel blocker in the treatment of epilepsy and neuropathic pain. Influenza is a respiratory viral infection. Since viruses rely on host cell proteins for their entry, survival and replication, development of drugs targeting host cell proteins has identified as an effective strategy in controlling viral infections. We synthesized a series of 1,4-disubstituted piperidine derivatives for the inhibition of protein disulfide isomerase enzyme and influenza. Among them, 1-29 was found to possess strong anti-influenza activity (EC₅₀ = 2.5 µM). This suggests the potential use of piperidine scaffold in designing anti-influenza drugs in future. Calcitonin gene-related peptide (CGRP) receptor antagonism has been identified as a successful approach for the treatment of inflammatory pain. Therefore, a novel class of peptide antagonists of CGRP receptor was synthesized and screened for their binding affinities to the CGRP receptor and their analgesic effects on inflammatory-induced pain in rats. Among them, peptide 2-3 showed a higher binding affinity towards the CGRP receptor than previously reported peptide antagonists and exhibited analgesic effects up to 2 h in both Aδ and c-fiber pain tests. Therefore 2-3 indicates its potential use as a CGRP receptor antagonist in the treatment of inflammatory pain. Firefly luciferase is commonly used as a reporter in cells expressing a luciferase gene or its enzymatic activity under the control of a promoter of interest to assess its transcriptional activity. It has been found that some molecules such as molecules with carboxylic acid moiety can directly inhibit luciferase activity in cells. However, it is suggested that carboxylic acid moiety of the compounds may also be associated with side reactions in cells. Therefore, to study whether carboxylic acid moiety causes side effects, we designed two probe molecules, 3-1 and 3-2. Synthesis of probe molecule 3-2 is discussed. Synthesis of probe molecule 3-1 and further investigation of its luciferase inhibition will therefore be useful to understand the toxicity of carboxylic acid containing drugs in future.

A oxidação do ácido úrico (7,9-diidro-1H-purina-2,6,8(3H)-triona) pela mieloperoxidase (MPO) gera o radical de urato que, em presença do radical ânion superóxido combinam para formar o hidroperóxido de urato (HOOU). Considerando os altos níveis de MPO, ácido úrico e superóxido na placa de ateroma espera-se que o ácido úrico seja oxidado a HOOU neste microambiente inflamatório. O HOOU é um oxidante mais forte que o peróxido de hidrogênio e pode oxidar grupos tiólicos de proteínas como a proteína dissulfeto isomerase (PDI). Como consequência à oxidação da PDI, ocorre uma modulação positiva sobre a NADPH oxidase (Nox) com aumento da produção de superóxido por neutrófilos. Sendo assim, a formação do HOOU no leito vascular poderia elucidar os mecanismos moleculares pelos quais o urato colabora para a progressão da aterosclerose em pacientes com hiperuricemia. Para investigar a interação do HOOU com a PDI, padronizou-se a síntese química do HOOU através de um sistema de fotooxidação do tipo I, utilizando urato, riboflavina (fotossensibilizador) e luz UVA. Inicialmente padronizou-se o tipo de irradiação e tempo de reação com o melhor rendimento para a síntese do HOOU. O HOOU formado e seu produto de redução o álcool 5-hidroxiisourato foram separados, identificados e caracterizados através de cromatografia liquida acoplada à espectrometria de massa (LC/MS). Após a purificação, determinou-se o coeficiente de absortividade molar em 308 nm (ε308 = 6537 ± 377 M-1.cm-1) e o tempo de meia-vida, aproximadamente 41 minutos à 22°C do HOOU. O HOOU foi capaz de reagir seletivamente com o aminoácido metionina e com o tripeptídeo glutationa. Além disso, o HOOU não forma adutos estáveis com a glutationa, sendo que toda glutationa consumida foi transformada em glutationa dissulfeto. Quando incubado com a PDI (10 µM), cerca de 70 e 100% do total de HOOU (3 µM) foi consumido após 30 e 120 segundos, respectivamente, enquanto que a PDI (23 µM) teve seus grupos tiólicos oxidados após a incubação com 140 µM HOOU por 30 min a 22°C. O HOOU oxidou os resíduos de cisteína dos dois sítios catalíticos da PDI com uma constante de velocidade da reação de 1,5 ± 0,04 x 103 M-1.s-1, demonstrando uma interação favorável com essa proteína no meio biológico e um possível papel modulatório do HOOU sobre a via PDI-Nox. Interessantemente, o ácido úrico aumentou a produção de superóxido e o consumo de oxigênio de células HL-60 diferenciadas em neutrófilos (dHL-60) e ativadas com acetato de miristato de forbol (PMA). Essa regulação poderia ser mediada através da formação do HOOU durante o \"burst\" oxidativo dos neutrófilos que oxidaria a enzima PDI induzindo um consequente aumento da atividade da Nox. / The oxidation of the uric acid (7,9-dihidro-1H-purine-2,6,8(3H)-trione) by myeloperoxidase (MPO) generates the urate radical. In inflammatory conditions the superoxide reacts with urate radical to form the urate hydroperoxide (HOOU). Taking into account the high amount of MPO, urate and superoxide in the atheroma plaque, it is likely that HOOU is being formed in this inflammatory environment. The HOOU is a strong oxidizing agent and can react with thiol groups from proteins, like the protein disulfide isomerase (PDI). As a consequence of its oxidation, PDI positively modulates NADPH oxidase (Nox) and increases superoxide production by neutrophils. Therefore, the formation of HOOU in the vascular sheet would contribute to tissue damage and would explain the positive correlation between hyperuricemia and the risk for cardiovascular disease. To investigate the interaction of HOOU with PDI, we performed the chemical synthesis of the compound by the Type I photooxidation, using UVA irradiation and riboflavin as a photosensitizer. Initially, we standardized the irradiation light and the time of the reaction that produced the highest income. The HOOU and its reduced product 5-hydroxiisourate were separate, identified and characterized by liquid chromatography coupled to mass spectrometry (LC/MS). We also determine the molar extinction coefficient of HOOU at 308 nm (ε308 = 6537 ± 377 M-1.cm-1). The half-life of the compound was 41 min at 22°C. The HOOU selectively oxidized methionine and glutathione. The reaction of HOOU with glutathione did not form any stable adducts. Thus, all consumed glutathione generated glutathione disulfide. When incubated with PDI (10 µM), 70 and 100% of the total amount of HOOU (3 µM) was consumed within 30 and 120 seconds, respectively. Besides, the PDI (23 µM) had its thiol groups oxidized after incubation with 140 µM HOOU for 30 min at 22°C. The HOOU oxidized the cysteine residues from the catalytical sites of PDI with a second order rate constant of 1.5 ± 0.04 x 103 M-1.s-1. This result suggests a favorable interaction of HOOU with this protein in the biological system, as well as a possible modulatory role of HOOU on the PDI-Nox pathway. Interestingly, urate increased superoxide production and oxygen consumption by neutrophil-like cells (differentiated HL-60 cells). This effect could be mediated by the formation of HOOU during the neutrophil oxidative burst, followed by the oxidation of PDI, a positive regulation of Nox and an increase in superoxide production.

Os acidentes ofídicos são considerados um grande problema de saúde pública e no Estado de São Paulo a serpente Bothrops jararaca é o agente considerado mais relevante epidemiologicamente. O antiveneno é o único medicamento oficialmente aprovado para o tratamento de picadas de serpentes e, apesar de ser eficaz em diversos aspectos, mostra-se ineficaz em relação a complicações secundárias do envenenamento, como o estresse oxidativo/nitrosativo, caracterizado por um desequilíbrio no estado redox, que pode ser extremamente deletério aos pacientes. Portanto, vê-se necessária a busca por terapias complementares que possam combater o estresse oxidativo/nitrosativo. Ademais, distúrbios da hemostasia observados em envenenamentos pela B. jararaca, tais como a plaquetopenia, a hipofibrinogenemia e o aumento da atividade do fator tissular (TF) plasmático podem ter também como causa indireta o estresse oxidativo/nitrosativo, induzido por componentes do veneno de B. jararaca (BjV). Recentemente, demonstrou-se que a regulação da atividade biológica do TF, a proteína responsável pela iniciação da cascata de coagulação in vivo, pode ser controlada pela isomerase de dissulfeto proteico (PDI) e que a atividade da PDI é inibida in vivo pela quercetina-3-rutinosídeo (rutina), um antioxidante natural encontrado em plantas e alimentos. Considerando essas premissas, o presente trabalho objetivou investigar em camundongos injetados com o veneno de B. jararaca (BjV): (a) a ocorrência de distúrbios do estado redox; (b) as alterações hematológicas e hemostáticas (em plasma e tecidos) associadas ao desequilíbrio do estado redox; (c) a atividade da rutina como agente modulador sobre essas alterações. Para isso, camundongos Swiss (30-35 g) foram divididos em 4 grupos experimentais: controle salina (salina, controle negativo), controle rutina (rutina+salina), BjV+salina (BjV+salina, controle positivo) e BjV+rutina (BjV+rutina, tratamento). Após 3, 6 e 24 h da administração dos tratamentos (via s.c.), foram coletados sangue e fragmentos de tecidos para posteriores análises. O envenenamento induziu um aumento de espécies reativas, diminuição da capacidade antioxidante total, alterações hematológicas (plaquetopenia, neutrofilia e diminuição de eritrócitos), distúrbios hemostáticos (hipofibrinogenemia, prolongamento do tempo de protrombina e aumento da atividade de TF no plasma), além de diminuir a expressão proteica de PDI no coração. A rutina não inibiu in vitro a atividade biológica de proteínas presentes no BjV (metaloproteinases, serinaproteases, fosfolipases A2 e L-aminoácido oxidases) e nem mesmo a agregação plaquetária induzida pelo veneno. No entanto, a administração do veneno incubado com rutina foi capaz de reduzir os níveis de espécies reativas, impedir a queda de eritrócitos, normalizar os níveis de fibrinogênio e o tempo de protrombina e alterar a atividade de TF e expressão proteica de TF e PDI em tecidos. Desse modo, concluímos que a rutina foi capaz de favoravelmente modular importantes alterações hemostáticas e de estado redox no envenenamento por B. jararaca, o que indica seu grande potencial como agente terapêutico complementar em envenenamentos / Snakebites are a major public health issue and in São Paulo State, and Bothrops jararaca snakes are considered the most important agents epidemiologically. Antivenom is the only officially approved treatment for snakebites and although effective in many aspects, it is ineffective in combating secondary complications induced by envenomation, e.g. oxidative/nitrosative stress (ONS), which can be extremely deleterious to patients. Therefore, it is necessary to search for new complementary therapies that could attenuate ONS. Furthermore, the hemostatic disturbances in B. jararaca envenomation - characterized by the presence of thrombocytopenia, hypofibrinogenemia and increased tissue factor (TF) activity in plasma - could be indirectly evoked by ONS induced by B. jararaca venom (BjV) components. Recently, the biological activity of TF - the protein responsible for initiating the extrinsic pathway of the coagulation cascade - was demonstrated to be regulated by protein disulfide isomerase (PDI), which in turn, can be inhibited in vivo by quercetin-3-rutinose (rutin), a natural antioxidant found in plants and diet. Based on that, the present study aimed to investigate in mice injected with BjV: (a) the alterations in redox status in plasma and tissues; (b) the hematological/hemostatic disturbances (in plasma and tissues) associated with the imbalance in redox status; (c) the activity of rutin as a modulatory agent on those alterations. Swiss mice (30-35 g) were divided in four experimental groups: saline control (saline, negative control), rutin control (rutin+saline), BjV+saline (BjV+saline, positive control) and BjV+rutin (BjV+rutin, treatment) and after 3, 6 and 24 h of the injection of treatments (s.c. route), blood and tissues were collected to further analyses. Envenomation induced an increase in reactive species, a decrease in total antioxidant capacity, hematological alterations (thrombocytopenia, neutrophilia and a decrease in red blood cell counts), hemostatic disturbances (hypofibrinogenemia, prolonged prothrombin time and an increase in the TF activity in plasma), and a decrease in protein expression of PDI in the heart. In vitro, rutin failed to inhibit the biological activity of the main BjV enzymes (metalloproteinases, serine proteases, phospholipases A2 and L-amino acid oxidases) and BjV-induced platelet aggregation. However, when the venom was incubated with rutin, there were reduced reactive species levels, less intense red blood cell drops, recovery of fibrinogen levels and prothrombin time, and alteration of TF activity and protein expression of TF and PDI in tissues. Therefore, we conclude that rutin favorably modulated important hemostatic and redox status alterations in B. jararaca envenomation, indicating its great potential as a complementary therapeutic agent for snakebites

The redox state in the cell plays a major role in determining vital functions and its major imbalance can lead to severe cell injury or death. Redox active proteins and cytokines involved in this process includes thioredoxin (Trx), protein disulfide isomerase (PDI), and tumor necrosis factor (TNF) superfamilies. Trx is a multipotent protein and key regulator of cellular redox balance operating in synergy with Trx reductase and NADPH (the Trx system). Trx has gene regulatory activity of several transcription factors. It also controls in a fascinating way redox-sensitive “on-off” decisions for apoptotic or hypertrophic pathways. Trx protects against H2O2 and TNFmediated cytotoxicity, a pathway in which TNF receptor-binding generates ROS. TNF is an autocrine growth factor and survival factor in vitro and in vivo for B-type of chronic lymphocytic leukemia (B-CLL) cells. The overall aim of this study was to investigate the importance of redox active proteins and cytokines in inflammation and cancer. We focused on: i) the role of Trx, TrxR, and selenium in carcinogenesis and in resistant cancer cells. ii) the importance of Trx in cancer cells and the redox regulation of TNF and its receptors TNFR1 and TNFR2. iii) the potential role of Trx as a key regulator in cellular redox balance, in the pathogenesis of cardiac dysfunction; its relationship to stress response parameters. iv) whether unmutated CLL (UCLL) responses to PKC and ROS pathways were different from mutated CLL (M-CLL) responses. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy resistant cells and suggest that redox regulation through the Trx system is an important target for cancer therapy. Trx was strikingly elevated in heart failure cases compared with controls signifying an adaptive stress response that is higher the more severe the disease. TNF autocrine release was redox modulated and the TNF receptors interacted at the cell surface membrane with the redox-active PDI, which excerted a stringent redox-control of the TNFR signaling. The proliferative response as well as increase of autocrine TNF and Trx were higher in U-CLL than in M-CLL. The overall conclusion of the four papers included in this thesis is that redox-active proteins and cytokines plays an important role in control and regulation of cancer and inflammation. Furthermore, redox regulation via thioredoxin by selenium, may offer novel treatment possibilities for resistant tumors disease.