Nitric oxide (NO), a reactive diatomic gas and a potent signaling molecule, is required for proper cardiovascular functioning. Soluble guanylate cyclase (sGC), a heterodimeric heme protein, is the key intracellular NO receptor protein which, upon NO binding, undergoes conformational changes leading to catalysis and the cGMP signaling cascade. Several small molecules that allosterically stimulate sGC have been developed for treatment of pulmonary hypertension, but little is known about their binding site or how they stimulate activity. This dissertation describes experiments designed to uncover the molecular basis for signal transduction in sGC by NO and small molecule stimulators. The crystal structure of the α-subunit PAS domain from Manduca sexta (Ms) sGC was solved at 1.8 Å resolution revealing the expected PAS fold but with an additional β strand and a shorter Fα helix. CO binding measurements on different Ms sGC N-terminal constructs and the β₁ (1-380) construct revealed that the α-subunit keeps the β₁ H-NOX domain in an inhibited conformation and this inhibition is relieved by removal of the α-subunit or by addition of stimulatory compounds such as compound YC-1. Linked-equilibria measurements on the N-terminal constructs show that YC-1 binding affinity is increased in the presence of CO. Surface plasmon resonance (SPR) studies on the in-vitro biotinylated constructs showed that YC-1 binds near or directly to the β₁ H-NOX domain. Computational and mutational analysis of the β₁ H-NOX domain revealed a pocket important in allostery and drug action. Finally, we show that the coiled coil domain plays an important role in allosteric regulation of the β₁ H-NOX domain and possibly in signal transduction. Our data are consistent with a model of allosteric activation in which the α-subunit and the coiled coil domains function to keep heme in a low affinity conformation while YC-1 binding to the β₁ H-NOX domain switches heme to a high affinity conformation, and sGC to its high activity form.

Πολλές παθήσεις του καρδιαγγειακού συστήματος σχετίζονται με την λειτουργία του ενζύμου της διαλυτής γουανυλικής κυκλάσης (soluble guanylate cyclase, sGC). Η sGC εμπλέκεται στο μονοπάτι ΝΟ-sGC-cGMP το οποίο ενεργοποιείται από το βιολογικά διαθέσιμο μονοξείδιο του αζώτου (nitric oxide, ΝΟ). Πολλές παθολογικές καταστάσεις αντιμετωπίστηκαν για πάνω από 140 χρόνια με τη χρήση φαρμάκων που παρέχουν NO (ΝΟ-φάρμακα), χωρίς ωστόσο να είναι γνωστός ο μηχανισμός δράσης τους. Αν και τα φάρμακα αυτά συνεισέφεραν στη βελτίωση των παθολογικών καταστάσεων, ωστόσο παρουσίαζαν σημαντικά μειονεκτήματα. Για την αντιμετώπιση αυτών, το ενδιαφέρον στράφηκε στον σχεδιασμό και την σύνθεση ενώσεων των οποίων η δράση θα ήταν ανεξάρτητη από το ΝΟ. Μεταξύ αυτών, τα παράγωγα BAY 58-2667 και η HMR 1766 αποδείχθηκαν ενεργοποιητές της sGC. Στα πλαίσια της παρούσας μελέτης, σχεδιάσθηκαν και συντέθηκαν έξι νέα βενζοφουρανικά ανάλογα του HMR-1766, σε μία προσπάθεια ανακάλυψης νέων ενώσεων, ενεργοποιητών της sGC με ενισχυμένη δραστικότητα και εκλεκτικότητα δράσης. Η προσέγγιση που ακολουθήθηκε για την σύνθεση των τελικών προϊόντων περιελάμβανε την ανοικοδόμηση του βενζοφουρανικού δακτυλίου από υποκατεστημένα παράγωγα σαλικυλικού οξέος και την μετέπειτα σύζευξη αυτού με κατάλληλους δομικούς λίθους για τον σχηματισμό μίας σουλφοναμιδικής και μίας αμιδικής πλευρικής αλυσίδας. Στα πλαίσια της μελέτης, διερευνήθηκαν και βελτιστοποιήθηκαν όλα τα συνθετικά στάδια για την παραλαβή των ενδιάμεσων και των τελικών προϊόντων. Η μελλοντική αποτίμηση της βιολογικής δράσης των νέων ενώσεων αναμένεται να διευκρινίσει αν οι ενώσεις αυτές είναι ικανές να δράσουν ως ενεργοποιητές της sGC, αλλά και αν μπορούν να αποτελέσουν χρήσιμα χημικά εργαλεία για την διευκρίνιση δομικών πληροφοριών του ενζύμου. Το δεύτερο τμήμα της παρούσας εργασίας, αφορά στον σχεδιασμό και την σύνθεση νέων αναλόγων του φυσικού προιόντος Hymenialdesine (HMD). Η HMD είναι ένα φυσικό προϊόν το οποίο έχει αποδειχθεί αναστολέας πολλών πρωτεϊνικών κινασών, όπως των κυκλινο-εξαρτώμενων κινασών (CDKs), η υπερλειτουργία των CDKs ενέχεται στην εμφάνιση παθολογικών καταστάσεων (καρκίνος, νευροεκφυλιστικές παθήσεις, διαβήτης). Στόχος της μελέτης ήταν ο σχεδιασμός και η διερεύνηση της σύνθεσης νέων σπειρανικών ινδολοαζεπινικών αναλόγων της HMD, με ενισχυμένη ανασταλτική και εκλεκτική δράση έναντι των CDKs. Για το σκοπό αυτό, μελετήθηκε η μετατροπή της 5-κετονομάδας της αζεπινο[3,4-b]ινδολο-1,5-διόνης σε ένα αμινο-υποκατεστημένο στερεογονικό κέντρο μέσω νουκλεόφιλης προσβολής της πρόδρομης χειρόμορφης t-βουτυλοσουλφινυλ-ιμίνης. Διερευνήθηκαν ποικίλες πειραματικές συνθήκες για τη βελτιστοποίηση σχηματισμού τόσο της ενδιάμεσης σουλφινυλ-ιμίνης, όσο και της υποκατάστασης αυτής. Τα συνθετικά αυτά στάδια θεωρούνται κρίσιμα και η βελτιστοποίηση τους απαραίτητη για την ομαλή εξέλιξη του συνθετικού σχήματος. Τα αποτελέσματα που καταγράφηκαν στα πλαίσια της μελέτης αναμένεται να συμβάλλουν ουσιαστικά στην επιτυχή ολοκλήρωση της σύνθεσης των νέων σπειρανικών αναλόγων της HMD. / Many cardiovascular diseases are connected with the activity of soluble guanylate cyclase (sGC). sGC is part of the NO-sGC-cGMP pathway, which is activated by the biologically available nitric oxide (NO). Many drugs that release NO (NO-drugs) have been used for more than 140 years. Although these drugs have contributed to the treatment of these diseases, they have presented some disadvantages. Thus, new compounds have been discovered whose activity is independent of NO. Compounds BAY 58-2667 and HMR-1766 belong to this new class of compounds and are characterized as sGC activators. In the first part of this study, six new benzofuran derivatives of HMR-1766 were designed and synthesized, aiming at the discovery of new compounds, activators of sGC with enhanced activity and selectivity against sGC. The synthetic approach involves the initial formation of benzofuran ring from substituted derivatives of salicylic acid and its coupling with selected building blocks. The optimazation of all synthetic steps for the synthesis of the intermediate and final products was also part of this study. The biological evaluation of the new compounds is expected to reveal their biological activity as sGC activators and/or their role as chemical tools for the structural elucidation of the enzyme. The second part of this study, concerns the design and synthesis of new derivatives of Hymenialdesine (HMD). HMD is a natural product with inhibitory activity against many protein kinases, such as cyclin-dependent kinases (CDKs). Hypeactivation of CDKs is implicated in pathological disorders such as cancer, neurodegenerative diseases and diabetes. The aim of the study was the synthesis of new spiro-indolazepino derivatives of HMD with potential enhanced inhibitory activity and selectivity against CDKs. The transformation of the 5-ketogroup of the azepino[3,4-b]indol-1,5-dione to a new amino-substituted stereogenic center by nucleophilic attack of the intermediate chiral tert-sulfinylimine was the key-step of the synthetic approach. The results of this study are expected to contribute substantially to the synthesis of new spiro HMD derivatives.

Soluble guanylyl/guanylate cyclase (sGC), the primary receptor for nitric oxide (NO), is a heme containing heterodimeric enzyme involved in numerous physiological events in animals. The small molecule YC-1 stimulates sGC, but the mechanism behind and the location of binding are unknown. I have developed a prokaryotic expression system for insect ( <italic>Manduca sexta</italic>) sGC. The recombinant holoenzyme, like its mammalian counterpart, is responsive to NO, CO and YC-1, displaying a 175-fold increase in activity on binding. Truncated constructs containing the N-terminal two-thirds of both subunits (msGC-NT) were designed to facilitate expression. With the highly pure material, we investigated NO and CO binding, reaction kinetics and regulation. Binding of NO to msGC-NT heme forms a six-coordinate intermediate followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex. The conversion rate is insensitive to nucleotides, YC-1 and changes in NO concentration up to ~30 micromolar. In contrast, NO release from msGC-NT is biphasic in the absence of YC-1, while binding of YC-1 eliminates the fast phase but has little effect on the slower phase. CO binding to msGC-NT is also regulated by YC-1. The CO release rate is reduced by YC-1 while the on rate remains unchanged, which leads to an ~50-fold increase in binding affinity. YC-1 binding leads to a substantial geminate recombination of CO to msGC-NT upon photolysis. Our data are consistent with a model for allosteric activation in which (1) YC-1 binds away from the catalytic site and (2) sGC undergoes a conformational switch between two states of an open and a closed heme pocket. The final catalysis results from the integration of the influence of numerous allosteric effectors on the equilibrium between these two states.<italic>S </italic>-nitrosoglutathione (GSNO) exists <italic>in vivo </italic> and plays important roles in NO signaling. We have developed a model cell line, in which inducible NO synthase and human sGC genes were included. GSNO stimulation of sGC has been investigated using recombinant insect and human enzymes. GSNO can activate sGC as efficiently as gaseous NO, but apparently with a distinct mechanism. GSNO or endogenous NO could <italic>S </italic>-nitrosylate sGC, which might regulate the enzyme function.

No presente trabalho avaliamos o potencial do BAY 41-2272 e sua via, como uma ferramenta para modulação da função dos linfócitos. Para isso, realizamos tratamentos farmacológicos com BAY 41-2272, avaliando a produção de citocinas e observamos que este fármaco, como ativador direto, não induz produção de IFN, IL-4 e IL-10 nos linfócitos. No entanto, o prétratamento por 24 horas com BAY 41-2272, com posterior ativação com PMA, mostrou que esta droga tem efeito inibitório na produção das citocinas. Em vista disto, avaliamos se este fármaco seria capaz de ativar estas células através da expressão de CD69. Vimos que por si só esta droga não foi capaz de aumentar a expressão de CD69, no entanto o pré-tratamento com BAY 41-2272 inibiu a ativação dos linfócitos T CD4. Assim, avaliamos se o fármaco seria capaz de inibir a expressão de fatores de transcrição FOXP3, RORT, Tbet e GATA3. Vimos que o BAY 41-2272 não induziu expressão desses fatores de transcrição e o pré-tratamento com este fármaco não alterou a expressão de FOXP3, RORT e GATA3, mas inibiu a expressão de Tbet quando comparado ao estimulado com PMA e Ionomicina sem o prétratamento. Observamos também que o pré-tratamento com BAY 41-2272 inibiu a linfoproliferação. Estes resultados sugerem que o BAY 41-2272 e sua via, têm um perfil inibitório sobre os linfócitos T CD4, e potencialmente podem ser utilizados como imunomodulador em pacientes com comprometimento do sistema imunológico e síndromes linfoproliferativas. / In this work, we evaluated the potential of BAY 41-2272 and its pathway as a tool for modulating lymphocyte function. For this, we performed pharmacological treatments with BAY 41-2272, evaluating the production of cytokines and observed that this drug, as a direct activator, does not induce production of IFN, IL-4 e IL-10. However, pre-treatment for 24 hours with BAY 41-2272 and subsequente activation with PMA showed that this drug has an inhibitory effect on cytokine production. Thus, we evaluated if this drug would be able to activate these cells through the CD69 expression. We saw that alone, BAY 41-2272 was not able to increase CD69 expression, however, pre-treatment inhibited activation of CD4 T lymphocytes. Then, we evaluated if this chemical compound would be able to inhibite the expression of transcription factors FOXP3, RORT, Tbet and GATA3. We have seen that BAY 41-2272 did not induce expression of these transcription factors and pretreatment with this drug did not alter expression of FOXP3, RORT and GATA3, but inhibited Tbet expression. We also observed that pre-treatment with BAY 41-2272 inhibited lymphoproliferation. These results suggest that BAY 41-2272 and its pathway have an inhibitory profile on CD4 T lymphocytes and can potentially be used as an immunomodulator in patients with impaired imune system and lymphoproliferative syndromes.

Thrombospondin-1 (TSP-1) binding to its membrane receptor CD47 results in an inhibtion of the nitric oxide (NO) receptor soluble guanylate cyclase (sGC) and a decrease in intracellular cGMP levels. This causes physiologic effects such as vasoconstriction and a rise in blood pressure. The mechanism by which TSP-1 binds to CD47 at the membrane to decrease sGC activity is largely unknown. CD47 can physically associate with a number of binding partners, including α(v)β₃ and vascular endothelial growth factor receptor 2 (VEGFR2). Binding of a C-terminal fragment of TSP-1 called E3CaG1 to CD47 leads to a rise in intracellular calcium ([Ca²⁺](i)), which decreases sGC activity via a phosphorylation event. Binding of E3CaG1 is also known to disrupt the interaction between CD47 and VEGFR2, leading to a decrease in endothelial nitric-oxide synthase (eNOS) activity and cGMP levels through an Akt signaling pathway. However, it is not known whether other membrane proteins associated with CD47 are required for E3CaG1 binding and a subsequent [Ca²⁺](i) increase. Plasmon-waveguide resonance (PWR) spectroscopy was employed to elucidate the mechanism of TSP-1 inhibition of sGC activity through membrane complexes involving CD47. Using PWR, I found E3CaG1 can bind specifically to CD47 within native Jurkat membranes with picomolar and nanomolar dissociation constants (K(d)), suggesting multiple CD47 complexes are present. Among these complexes, CD47/VEGFR2 was found to bind E3CaG1 with a picomolar K(d)and CD47/α(v)β₃ was found to bind E3CaG1 with a nanomolar K(d). In addition, the presence of an anti-VEGFR2 antibody inhibited the E3CaG1-induced calcium response, which suggested CD47 in complex with VEGFR2 was responsible for TSP-1 reduction of sGC activity. I show that when both CD47 and VEGFR2 are returned to a HEK 293T cell line that does not contain these receptors, an increase in [Ca²⁺](i) upon E3CaG1 binding is restored. Interestingly, E3CaG1 was also found to bind to VEGFR2 in complex with the integrin α(v)β₃ on CD47-null cell lines and their derivations, causing a decrease in [Ca²⁺](i) levels. Therefore, the third type 2 repeat and C-terminal domains of TSP-1 can cause both increases and decreases in calcium based upon the availability of protein complexes to which it binds.

No description available.

No description available.

INTRODUÇÃO. O termo Lesão Pulmonar Aguda (LPA) é usado para descrever a resposta pulmonar à lesão que ocorre diretamente ou indiretamente nos pulmões. A quebra da barreira alvéolo-capilar determina o influxo de líquido rico em proteínas para dentro dos espaços alveolares, sendo necessária a reabsorção desse líquido no processo de resolução da LPA. A infusão intravenosa de Ácido Oleico (AO) em ratos provoca agudamente edema alveolar difuso e focos hemorrágicos intra-alveolares, sendo um bom modelo de indução. Estudos relatam que o Azul de Metileno (AM) atenua tais lesões, com efeito protetor, no tecido pulmonar, e reduz o edema presente na LPA em animais com sepse através da inibição da guanilato ciclase solúvel (GCs), uma enzima ativadora da via NO-GMPc. OBJETIVO. Estudar a repercussão da inibição da GCs pelo AM na permeabilidade capilar pulmonar ministrando-o antes e após a indução da lesão pulmonar por AO. MÉTODO. Ratos Wistar foram divididos em 5 grupos: Sham com infusão de salina em bolus; AM com infusão de AM por 2h; AO com infusão de AO em bolus, AM/AO com infusão de AM por 2h, sendo que, após 5 min do início, recebeu AO simultaneamente em bolus e AO/AM com infusão de AO em bolus e, após 2h, infusão de AM por mais 2h. Após 4h foi realizada a coleta de materiais (sangue, lavado bronco-alveolar e tecido pulmonar) para análise do NO plasmático e tecidual, gasometria arterial, cálculo do peso úmido/peso seco (PU/PS) e histologia do tecido pulmonar de todos os grupos. A estatística utilizada foi a análise de variância (one-way ANOVA) com p<0.05. RESULTADO. Não foi encontrado hipoxemia grave após 4h de lesão. O grupo AO apresenta um aumento no número de proteínas no lavado bronco-alveolar e na relação PU/PS comparado aos grupos controle: Sham e AM, confirmando a presença de lesão e alteração de permeabilidade pulmonar. Os grupos tratados com AM apresentaram melhora na permeabilidade pulmonar, porém, apenas o grupo pré-tratamento (AM/AO) apresentou diferença estatística na redução do extravasamento de proteínas no lavado. Não foram encontradas diferenças significativas no NO plasmático e tecidual. Na microscopia, a congestão capilar foi intensa, acompanhada de múltiplos focos de edema alveolar, exsudato intra-alveolar proteico, áreas de hemorragia e infiltrado inflamatório neutrofílico, tanto no interstício quanto nos septos alveolares.Os grupos tratados com AM apresentaram diminuição das áreas de edema, exsudação e hemorragia, porém, com maior evidência no grupo AM/AO. CONCLUSÃO. O AM diminui a permeabilidade pulmonar quando administrado de maneira precoce amenizando os danos causados pela LPA induzida por AO. / BACKGROUND. The term Acute Lung Injury (ALI) is used to describe the response to lung injury that occurs directly or indirectly in the lungs. The rupture of the alveolar - capillary barrier determines the influx of protein-rich fluid into the alveolar spaces, the reabsorption of this fluid in the process of resolving the ALI is required. Intravenous infusion of oleic acid (OA) in rats acutely causes diffuse alveolar edema and intra-alveolar hemorrhagic foci, being a good role model induction. Studies have reported that Methylene Blue (MB) attenuates such injuries, with a protective effect in lung tissue and reduce edema present in the ALI present in with sepsis by inhibition of soluble guanylate cyclase (sGC), an activator of the enzyme NO- cGMP pathway. OBJECTIVE. To study the effect of inhibition of sGC by MB in pulmonary capillary permeability ministering to the before and after induction of lung injury by OA. METHOD. Wistar rats were divided into 5 groups: Sham infused with saline bolus, MB infused with MB for 2hrs, OA infused with OA bolus, MB/OA infused with MB for 2hrs, and after 5 min from the beginning, simultaneously received OA bolus and OA/MB infused with OA bolus and after 2hrs, MB infusion for 2hrs. After 4hrs the collection of materials was performed (blood, bronchoalveolar lavage and lung tissue) for analysis of plasma and tissue NO, arterial blood gases, calculation of the wet weight/dry weight (WW/DW) and histology of lung tissue from all groups. The statistic used was analysis of variance (one-way ANOVA) with p<0.05. RESULTS. Not found severe hypoxemia after 4hrs of injury. The OA group shows an increase in the number of proteins in bronchoalveolar lavage and in WW/DW ratio compared to the control groups: Sham and MB, confirming the presence of injury and alterations of lung permeability. The groups treated with MB showed improvement in lung permeability, however, only the pretreatment group (MB/OA) showed statistical significance in reducing the leakage of protein in the lavage. No significant differences were found in plasma and tissue NO. In microscopy, capillary congestion was intense, accompanied by multiple foci of alveolar edema, intra-alveolar proteinaceous exudates, areas of hemorrhage and neutrophilic inflammatory infiltrate in both the interstitium and in the alveolar septa. The groups treated with MB showed reduction in areas of edema, exudation and hemorrhage, however, most obviously in MB/OA group. CONCLUSION. The MB decreases lung permeability when administered as early as possible, mitigating the damage caused by OA-induced ALI.

INTRODUÇÃO. O termo Lesão Pulmonar Aguda (LPA) é usado para descrever a resposta pulmonar à lesão que ocorre diretamente ou indiretamente nos pulmões. A quebra da barreira alvéolo-capilar determina o influxo de líquido rico em proteínas para dentro dos espaços alveolares, sendo necessária a reabsorção desse líquido no processo de resolução da LPA. A infusão intravenosa de Ácido Oleico (AO) em ratos provoca agudamente edema alveolar difuso e focos hemorrágicos intra-alveolares, sendo um bom modelo de indução. Estudos relatam que o Azul de Metileno (AM) atenua tais lesões, com efeito protetor, no tecido pulmonar, e reduz o edema presente na LPA em animais com sepse através da inibição da guanilato ciclase solúvel (GCs), uma enzima ativadora da via NO-GMPc. OBJETIVO. Estudar a repercussão da inibição da GCs pelo AM na permeabilidade capilar pulmonar ministrando-o antes e após a indução da lesão pulmonar por AO. MÉTODO. Ratos Wistar foram divididos em 5 grupos: Sham com infusão de salina em bolus; AM com infusão de AM por 2h; AO com infusão de AO em bolus, AM/AO com infusão de AM por 2h, sendo que, após 5 min do início, recebeu AO simultaneamente em bolus e AO/AM com infusão de AO em bolus e, após 2h, infusão de AM por mais 2h. Após 4h foi realizada a coleta de materiais (sangue, lavado bronco-alveolar e tecido pulmonar) para análise do NO plasmático e tecidual, gasometria arterial, cálculo do peso úmido/peso seco (PU/PS) e histologia do tecido pulmonar de todos os grupos. A estatística utilizada foi a análise de variância (one-way ANOVA) com p<0.05. RESULTADO. Não foi encontrado hipoxemia grave após 4h de lesão. O grupo AO apresenta um aumento no número de proteínas no lavado bronco-alveolar e na relação PU/PS comparado aos grupos controle: Sham e AM, confirmando a presença de lesão e alteração de permeabilidade pulmonar. Os grupos tratados com AM apresentaram melhora na permeabilidade pulmonar, porém, apenas o grupo pré-tratamento (AM/AO) apresentou diferença estatística na redução do extravasamento de proteínas no lavado. Não foram encontradas diferenças significativas no NO plasmático e tecidual. Na microscopia, a congestão capilar foi intensa, acompanhada de múltiplos focos de edema alveolar, exsudato intra-alveolar proteico, áreas de hemorragia e infiltrado inflamatório neutrofílico, tanto no interstício quanto nos septos alveolares.Os grupos tratados com AM apresentaram diminuição das áreas de edema, exsudação e hemorragia, porém, com maior evidência no grupo AM/AO. CONCLUSÃO. O AM diminui a permeabilidade pulmonar quando administrado de maneira precoce amenizando os danos causados pela LPA induzida por AO. / BACKGROUND. The term Acute Lung Injury (ALI) is used to describe the response to lung injury that occurs directly or indirectly in the lungs. The rupture of the alveolar - capillary barrier determines the influx of protein-rich fluid into the alveolar spaces, the reabsorption of this fluid in the process of resolving the ALI is required. Intravenous infusion of oleic acid (OA) in rats acutely causes diffuse alveolar edema and intra-alveolar hemorrhagic foci, being a good role model induction. Studies have reported that Methylene Blue (MB) attenuates such injuries, with a protective effect in lung tissue and reduce edema present in the ALI present in with sepsis by inhibition of soluble guanylate cyclase (sGC), an activator of the enzyme NO- cGMP pathway. OBJECTIVE. To study the effect of inhibition of sGC by MB in pulmonary capillary permeability ministering to the before and after induction of lung injury by OA. METHOD. Wistar rats were divided into 5 groups: Sham infused with saline bolus, MB infused with MB for 2hrs, OA infused with OA bolus, MB/OA infused with MB for 2hrs, and after 5 min from the beginning, simultaneously received OA bolus and OA/MB infused with OA bolus and after 2hrs, MB infusion for 2hrs. After 4hrs the collection of materials was performed (blood, bronchoalveolar lavage and lung tissue) for analysis of plasma and tissue NO, arterial blood gases, calculation of the wet weight/dry weight (WW/DW) and histology of lung tissue from all groups. The statistic used was analysis of variance (one-way ANOVA) with p<0.05. RESULTS. Not found severe hypoxemia after 4hrs of injury. The OA group shows an increase in the number of proteins in bronchoalveolar lavage and in WW/DW ratio compared to the control groups: Sham and MB, confirming the presence of injury and alterations of lung permeability. The groups treated with MB showed improvement in lung permeability, however, only the pretreatment group (MB/OA) showed statistical significance in reducing the leakage of protein in the lavage. No significant differences were found in plasma and tissue NO. In microscopy, capillary congestion was intense, accompanied by multiple foci of alveolar edema, intra-alveolar proteinaceous exudates, areas of hemorrhage and neutrophilic inflammatory infiltrate in both the interstitium and in the alveolar septa. The groups treated with MB showed reduction in areas of edema, exudation and hemorrhage, however, most obviously in MB/OA group. CONCLUSION. The MB decreases lung permeability when administered as early as possible, mitigating the damage caused by OA-induced ALI.

Cette thèse est consacrée à la régulation de la guanylate cyclase soluble (sGC), le récepteur endogène du monoxyde d'azote (NO) chez les mammifères qui est impliqué dans la transduction du signal. L'enzyme sGC est activée par la fixation du NO sur son hème et catalyse alors la formation du cGMP à partir du GTP. Alors que la sGC est présente dans de nombreuses cellules de mammifères, le domaine hémique bactérien homologue (H-NOX) est impliqué dans la détection du NO et la régulation du métabolisme. Un objectif important est la découverte d'inhibiteurs de la sGC pour ralentir la progression tumorale.Le criblage de composés naturels d'une chimiothèque mesurant l'activité de la sGC purifiée a révélé six inhibiteurs actifs (Ki = 0.2 – 1 µM). Avec deux autres composés actifs en photothérapie (hypericin et hypocrellin) nous avons démontré que ces inhibiteurs sont des effecteurs allostériques qui ne se fixent ni sur l'hème, ni sur le site catalytique ou de fixation des activateurs, découvrant une nouvelle classe de composés pharmacologiques ciblant la voie de signalisation NO/cGMP.La transition structurale induite par l'activateur riociguat en synergie avec le CO a été étudiée par spectroscopie d'absorption résolue en temps pour démontrer des changements de coordination de l'hème. Deux états d'activation distincts de la sGC par le CO existent simultanément avec les coordiantions 6c-hème et 5c-hème en présence de l'activateur qui induit la rupture de la liaison Fe-His de l'hème, à l'instar de l'activateur naturel NO. De plus, nous montrons que l'isoliquiritigénine, commercialisée comme activateur de la sGC, et en réalité un inhibiteur de la sGC.La dynamique ds ligands CO, NO, and O2 a été mesurée sur 12 ordres de grandeur temporelle pour le type sauvage et un mutant du transporteur bactérien du NO (AXCP). La simple mutation Leu16Ala augmente l'afinité pour le CO 108 fois, celle du NO 106 fois et rend cette protéine réactive à O2. Dans le cas de CO et NO dont les affinités pour L16A-AXCP sont les plus grandes jamais mesurées, la recombinaison bimoléculaire n'est pas détectable. Des simulations de dynamique moléculaire ont démontré que le CO dissocié est contraint de rester à 4 Å du Fe2+ par Ala16, contrairement au type sauvage Leu16.La dynamique de O2 a été mesurée dans la protéine senseur Tt H-NOX par spectroscopie d'absorption transitoire et confirme l'hypothèse que Tt H-NOX n'est sans doute pas un senseur de NO stricto sensu mais un senseur redox. Les propriétés de Tt-H-NOX ne sont pas compatibles avec le rôle d'un simple transporteur de NO. / This thesis is devoted to the regulation of soluble guanylate cyclase (sGC), the endogenous nitric oxide (NO) receptor in mammals involved in signal transduction. The enzyme is activated by the binding of NO to its heme and catalyzes the formation of cGMP from GTP. While sGC is present in many mammalian cells, the homologous bacterial domain (H-NOX) is involved in NO detection and metabolism regulation. An important objective was to find sGC inhibitors to slow down tumor progression.The screening of natural compounds from a chemical library, tested on purified sGC activity, revealed six active inhibitors (Ki = 0.2 – 1 µM). Together with two agents for photodynamic therapy (hypericin and hypocrellin) we demonstrated that these inhibitors are allosteric modulators which bind neither to the heme nor to the catalytic and activator sites, revealing a new class of pharmacological compounds targetting the NO/cGMP signaling pathway.The structural transition induced in sGC by stimulator riociguat in synergy with CO was studied by transient absorption spectroscopy to demonstrate coordination changes of the heme. Two different activation states of sGC with CO 6c-heme and 5c-heme exist simultaneously in the presence of the stimulator which induces the breaking of the heme Fe-His bond, as does the sGC natural effector NO. In addition, the effect of isoliquiritigenin, which is sold as a sGC activator, was shown to be actually an inhibitor of sGC.The dynamics of the ligands CO, NO and O2 were measured over 12 orders of magnitude in time in wild type and mutant of a bacterial NO transporter (AXCP). The single mutation Leu16Ala increased 108-fold the CO affinity, ~106-fold the NO affinity and makes this protein reactive to O2. In the case of CO and NO, whose affinities for L16A-AXCP are the largest ever measured, the bimolecular rebinding was absolutely not detectable. Molecular dynamic simulations demonstrated that dissociated CO is constrained to stay within 4 Å from Fe2+ by Ala16, contrarily to wild-type Leu16.The dynamics of O2 in Tt-H-NOX proteins measured by transient absorption spectroscopy confirmed the hypothesis that Tt-H-NOX may not be a NO-sensor stricto sensu but a redox sensor. The properties of the Tt-H-NOX protein are not compatible with the role a mere NO-carrier.