Οι σύγχρονες στρατηγικές στην ανακάλυψη νέων βιοδραστικών ενώσεων στοχεύουν, κατά κύριο λόγο, σε καλά χαρακτηρισμένους μοριακούς στόχους με σκοπό την επαγωγή ή την αναστολή συγκεκριμένων βιολογικών δράσεων και τον περιορισμό ανεπιθύμητων παρενεργειών. Έναν ανάλογο μοριακό στόχο αποτελεί η διαλυτή γουανυλική κυκλάση (sGC), η οποία βρίσκεται στο επίκεντρο του ερευνητικού ενδιαφέροντος την τελευταία δεκαετία. Παρόλο που ανακαλύφθηκε περίπου πριν από τέσσερις δεκαετίες ως μέλος της οικογένειας των κυκλασών, η έρευνα που ακολούθησε την ανακάλυψή της δεν χαρακτηρίστηκε από την ίδια πρόοδο που χαρακτήρισε έναν άλλο εκπρόσωπο της ίδιας οικογένειας, την αδενυλική κυκλάση. Μερικοί από τους λόγους της περιορισμένης μελέτης της sGC συνοψίζονται στην χαμηλή ενδοκυτταρική της συγκέντρωση, που συνεπάγεται δυσκολίες στην απομόνωση, στον καθαρισμό και το χειρισμό της, και στην έλλειψη ενός αποτελεσματικού συστήματος έκφρασής της. Επιπρόσθετα, μόνο μετά την ανακάλυψη του μονοξειδίου του αζώτου (ΝΟ), την αναγνώρισή του ως σηματοδοτικό μόριο καθώς και την ταυτοποίησή του ως τον EDRF στα τέλη της δεκαετίας του ’80, πραγματοποιήθηκε ουσιαστική πρόοδος στον τομέα των βιολογικών δράσεων της sGC. Ο καθαρισμός του ετεροδιμερούς ενζύμου και η ανακάλυψη ενός μορίου αίμης ανά διμερές έθεσε τη βάση για την αποσαφήνιση του μηχανισμού ενεργοποίησης της sGC από το ΝΟ και την ακόλουθη κατάλυση της μετατροπής του GTP σε κυκλικό GMP, αν και ο ακριβής μηχανισμός παραμένει μέχρι σήμερα άγνωστος. Η ταυτοποίηση, επομένως, του σηματοδοτικού μονοπατιού NO/sGC/cGMP αποτέλεσε τη βάση για την ανακάλυψη ότι φάρμακα όπως τα οργανικά νιτρώδη, που χρησιμοποιούνται στη θεραπεία της στηθάγχης για περισσότερο από έναν αιώνα, δρουν στην ουσία ως δότες μονοξειδίου του αζώτου μέσω βιομετατροπής τους και ασκούν τη δράση τους μέσω ενεργοποίησης της sGC και παραγωγής, ακολούθως, του δεύτερου αγγελιοφόρου cGMP. Tα παραπάνω δεδομένα οδήγησαν στον προσδιορισμό του ΝΟ ως σημαντικού σηματοδοτικού μορίου στη φυσιολογία καταρχήν του καρδιαγγειακού συστήματος. Η πρόοδος που ακολούθησε τη μελέτη του sGC-cGMP μονοπατιού αποκάλυψε την ευρεία παρουσία του ενζύμου σχεδόν σε όλα τα κύτταρα των θηλαστικών και την εμπλοκή του σε ουσιαστικές φυσιολογικές λειτουργίες, όπως την καρδιακή ομοιόσταση, τη χάλαση αγγειακών και μη λείων μυικών ινών, την αναστολή της συσσώρευσης/συγκόλλησης αιμοπεταλίων, την περιφερειακή και κεντρική νευροδιαβίβαση, την ανοσολογική απόκριση, τη μεταγωγή του οπτικού ερεθίσματος, ενώ παρουσιάζει και ρυθμιστική δράση στο γαστρεντερικό και ουρογεννητικό σύστημα. Ταυτόχρονα, απορρύθμιση του σηματοδοτικού μονοπατιού έχει διαπιστωθεί να εμπλέκεται στην φυσιολογία και την εξέλιξη συγκεκριμένων παθολογικών καταστάσεων όπως αρτηριακή και πνευμονική υπέρταση, καρδιακή ανεπάρκεια, αρτηριοσκλήρυνση και θρόμβωση, διαβήτης, νεφρική ίνωση και ανεπάρκεια, φλεγμονώδεις και νευροεκφυλιστικές παθήσεις, γαστρεντερικές διαταραχές, σήψη και καρκίνος, όπου εντοπίζεται είτε να υπερλειτουργεί είτε να υπολειτουργεί. Εξαιτίας της σπουδαιότητας και της σοβαρότητας των καρδιαγγειακών παθήσεων, όπου το σηματοδοτικό μονοπάτι υπολειτουργεί, το μεγαλύτερο κομμάτι της έρευνας που ακολούθησε σχετικά με την ανακάλυψη νέων βιοδραστικών ενώσεων εστίασε κυρίως στην ανάπτυξη αγωνιστών της sGC, παρέχοντας όχι μόνο ΝΟ-δότες με βελτιωμένες ιδιότητες αλλά, επιπρόσθετα, δύο νέες κατηγορίες ΝΟ-ανεξάρτητων ενεργοποιητών που περιλαμβάνουν διακριτές δομικά ενώσεις. Η πρόοδος, ωστόσο, στην αναστολή του ενζύμου κρίνεται εμφανώς υποδεέστερη, παρόλη την προφανή σπουδαιότητα ανάλογων ενώσεων στη διερεύνηση των cGMP-εξαρτώμενων βιολογικών δράσεων. Μεταξύ διάφορων ενώσεων, όπως οργανικών φωσφορικών ενώσεων, νουκλεοτιδικών αναλόγων, πορφυρινικών παραγώγων και άλλων, ο περισσότερο ισχυρός και εκλεκτικός αναστολέας που έχει αναφερθεί μέχρι σήμερα είναι το ODQ, το οποίο θεωρείται ότι δρα μέσω οξείδωσης του σιδήρου της αίμης με αποτέλεσμα την απώλεια της ικανότητας ενεργοποίησης του ενζύμου από το ΝΟ. Το μειονέκτημα του ODQ, ωστόσο, να δρα in vivo και σε άλλες αιμοπρωτεΐνες περιορίζει την εφαρμογή του και καθιστά επιτακτική την ανάγκη ανακάλυψης νέων εκλεκτικών αναστολέων. Σε αυτά τα πλαίσια, στόχο της παρούσας μελέτης αποτελεί ο σχεδιασμός, η σύνθεση και η βιολογική αποτίμηση νέων πιθανών αναστολέων της sGC. Με βάση τη χημική δομή του ODQ, ως την ένωση-οδηγό, σχεδιάστηκαν νέα τρικυκλικά ανάλογα του βασικού σκελετού του ινδολίου και του διϋδροϊνδολίου. Η επιλογή του ινδολικού δακτυλίου έναντι του κινοξαλινικού σκελετού του ODQ πραγματοποιήθηκε με στόχο να διερευνηθεί κατά πόσο ο νέος σκελετός θα έχει επίδραση στη διατήρηση ή/και την ενίσχυση της ανασταλτικής δράσης έναντι της sGC. Επιπρόσθετα, η επιλογή του διϋδροϊνδολικού σκελετού αποσκοπούσε στη διερεύνηση της επίδρασης που μπορεί να έχουν μη επίπεδες ενώσεις έναντι του επίπεδου ODQ. Ταυτόχρονα, σχεδιάστηκε μία σειρά ετεροκυκλικών δακτυλίων προσδεδεμένων στους βασικούς σκελετούς, διαφορετικών από το δακτύλιο οξαδιαζολόνης του ODQ, ώστε να εξετασθούν πιθανές διαφορές της τελικής δράσης σε συνάρτηση με δομικές διαφορές των τρίτων δακτυλίων. Ακολούθως, πραγματοποιήθηκε διερεύνηση της συνθετικής μεθοδολογίας για την παραλαβή τόσο των τελικών προϊόντων όσο και σημαντικών ενδιάμεσων ενώσεων ως συνθετικών ενδιαμέσων για τη μελλοντική σύνθεση τροποποιημένων παραγώγων. Η σύνθεση των τελικών διϋδροϊνδολικών αναλόγων πραγματοποιήθηκε με κυκλοποίηση μέσω επίδρασης υδροξυλαμίνης ή διάφορων υδραζινών σε α-υδροξυμεθυλεν-κετο- ενδιάμεσα παράγωγα, τα οποία παραλήφθηκαν από τα αντίστοιχα 4-κετο-τετραϋδροϊνδολικά παράγωγα με την επίδραση μυρμηκικού αιθυλεστέρα. Τα αντίστοιχα ινδολικά τρικυκλικά προϊόντα συντέθηκαν από τα διϋδροϊνδολικά υπό συνθήκες αφυδρογόνωσης με επίδραση DDQ. Εξαιτίας των πειραματικών συνθηκών που εφαρμόζονται για την παραλαβή των συνθετικών ενδιαμέσων, κρίθηκε απαραίτητη για την εξέλιξη του συνθετικού σχήματος η προστασία, προηγουμένως, του πυρρολικού αζώτου με την κατάλληλη ομάδα. Με στόχο τη διερεύνηση της επίδρασης που μπορεί να έχει στην τελική βιολογική δράση η παρουσία ελεύθερου –ΝΗ στον πυρρολικό δακτύλιο, κρίθηκε σκόπιμη η αποπροστασία των τελικών προϊόντων. Παρόλο που εξετάσθηκε ποικιλία προστατευτικών ομάδων και συνθηκών αποπροστασίας, τα διϋδροϊνδολικά τρικυκλικά προϊόντα αποδείχτηκαν ασταθή. Κατέστη εφικτό να απομακρυνθεί μόνο μία προστατευτική ομάδα, η SEM ομάδα, και μόνο από τα πλήρως αρωματοποιημένα ινδολικά προϊόντα, τα οποία απδείχτηκαν σταθερότερα. Σύμφωνα με την παραπάνω μεθοδολογία συντέθηκαν 24 τελικά προϊόντα, από τα οποία 14 είναι διϋδροϊνδολικά παράγωγα και 10 ινδολικά, ενώ από τα τελευταία τα τρία φέρουν ελεύθερο πυρρολικό –ΝΗ. Εκτός από τα τελικά προϊόντα κατέστη εφικτό να συντεθούν και σημαντικά συνθετικά ενδιάμεσα. Τα α-μεθοξυ και α-αιθοξυκαρβονυλο- 4- ή 7-οξοτετραϋδροϊνδολικά παράγωγα παραλήφθηκαν με εφαρμογή διαφορετικής πειραματικής μεθοδολογίας και συντέθηκαν, επιπρόσθετα, τα α-υδροξυμεθυλεν-4-οξο-τετραϋδροϊνδολικά παράγωγα. Τα προαναφερθέντα συνθετικά ενδιάμεσα δύνανται να οδηγήσουν σύμφωνα με διαφορετικά συνθετικά σχήματα σε νέα τρικυκλικά ανάλογα. Επιπλέον, διερευνήθηκαν οι πειραματικές συνθήκες κάθε σταδίου της μεθοδολογίας με σκοπό τη βελτιστοποίηση των τελικών αποδόσεων. Μερικά από τα τελικά τρικυκλικά ανάλογα, τα οποία δεν έφεραν υποκατάσταση στις θέσεις 2- και 3- του πυρρολικού δακτυλίου, προωθήθηκαν σε in vitro βιολογικές μελέτες. Εξετάστηκαν, καταρχήν, όσον αφορά την ικανότητά τους να αναστέλουν την επαγόμενη από νιτροπρωσσικό νάτριο δραστηριότητα της sGC σε δύο διαφορετικές συγκεντρώσεις, 1 μΜ και 100 μΜ. Όλες οι υπό μελέτη ενώσεις αποδείχτηκαν αναστολείς του ενζύμου και παρουσίασαν ισχυρότερη δράση, με μία εξαίρεση, στην υψηλότερη συγκέντρωση. Οι ενώσεις 74 και 81 αποδείχτηκαν οι ισχυρότεροι αναστολείς με δοσοεξαρτώμενη δράση. Η ένωση 81 εξετάστηκε στη συνέχεια όσον αφορά στην εκλεκτικότητά της για την sGC σε μία μελέτη που περιελάμβανε επαγόμενη από τον ANF παραγωγή cGMP. Από την τελευταία μελέτη αποδείχτηκε ότι η παραπάνω ένωση δεν αναστέλει τις pGCs, υποδεικνύοντας ότι πιθανότατα η νέα σειρά ενώσεων δεν επιδρά στα άλλα μέλη της οικογένειας των κυκλασών, τις pGCs και τις ACs, και παρέχοντας μια ένδειξη για πιθανό μηχανισμό δράσης παρόμοιο με αυτό του ODQ. Η τελευταία υπόθεση ενισχύεται και από το γεγονός ότι οι υπό μελέτη ενώσεις δεν μεταβάλλουν τη βασική δραστηριότητα της sGC. Τα αποτελέσματα των πρωταρχικών βιολογικών μελετών επαληθεύουν τον κύριο στόχο της παρούσας μελέτης που ήταν η σύνθεση νέων αναστολέων της sGC. Η επιλογή του ινδολικού δακτυλίου, ως τον βασικό σκελετό των νέων ενώσεων, επιβεβαιώνει την υπόθεση του αρχικού σχεδιασμού, παρέχοντας αναστολείς με νέα δομικά χαρακτηριστικά. Πρώιμες μελέτες δομής-δράσης επισημαίνουν τη βελτιωμένη δράση που παρουσιάζουν τα επίπεδα ινδολικά προϊόντα έναντι των μη επίπεδων διϋδροϊνδολικών, αν και δεν είναι ακόμη δυνατό να εξαχθούν ασφαλή συμπεράσματα σχετικά με την επίδραση της δομής του τρίτου ετεροκυκλικού δακτυλίου. Περαιτέρω βιολογική αποτίμηση των υπόλοιπων προϊόντων της μελέτης καθώς και σύνθεση νέων τροποποιημένων αναλόγων θα εξυπηρετήσει την εξαγωγή λεπτομερέστερων συμπερασμάτων από μελέτες χημικής δομής-βιολογικής δραστικότητας και, πιθανότατα, την ανακάλυψη νέας ένωσης-οδηγού στο πεδίο της αναστολής της sGC. Τα συμπεράσματα που προέκυψαν από την παρούσα μελέτη όσον αφορά στη χημική σύνθεση ενδιαμέσων και τελικών προϊόντων αλλά και στη βιολογική αποτίμηση επιλεγμένων τελικών ενώσεων μπορούν να αποδειχτούν χρήσιμο εργαλείο για το μελλοντικό σχεδιασμό, εφαρμογή και σύνθεση νέων ενώσεων με τα απαραίτητα χαρακτηριστικά για αποτελεσματική αναστολή της sGC. Ανάλογες ενώσεις είναι δυνατό να αποδειχτούν όχι μόνο χρήσιμα πειραματικά εργαλεία αλλά και βιοδραστικά μόρια με πιθανή κλινική εφαρμογή σε συγκεκριμένες παθολογικές καταστάσεις, όπου η sGC υπερλειτουργεί, όπως στη σήψη. / Modern strategies in the field of drug discovery and development of new therapeutic agents are aiming mostly at well-characterized molecular targets in order to stimulate or inhibit specific biological actions without undesirable side-effects. Soluble guanylate cyclase (sGC) is such a molecular target that has been, among others, intensively reviewed during the last decade. Although it has been discovered as a member of the cyclase family four decades ago, the research regarding the enzyme hasn’t followed the progress made with its sibling, the adenylate cyclase. Some of the reasons for this delayed progress are summarized in the difficulties concerning the isolation, purification and manipulation of the protein as a result of its low intracellular concentration and the lack of an efficient expression system. In addition, only after the discovery of nitric oxide (NO) as a signaling molecule and its recognition as the endothelium-derived relaxing factor (EDRF) at the late ’80s, was any significant progress made in the field of sGC biological activities. The purification of the heterodimeric enzyme and the discovery of the presence of one molecule of heme per dimer shed light in the mechanism of activation of sGC by NO and the consequent catalysis of the conversion of GTP into cyclic GMP, although no exact mechanism has been proved until today. The identification of the NO/sGC/cGMP signaling pathway served the discovery that drugs such organic nitrates, which have been used for the treatment of angina pectoris for over a century, practically act as NO-donors through bioconversion and exert their actions through activation of sGC and generation of the second messenger cGMP. The above results led to the establishment of NO as a significant signaling molecule in the physiology of cardiovascular system. The following progress concerning the study of the sGC-cGMP signaling cascade revealed a broad presence in almost all the mammalian cells and an implication in many substantial physiological processes, such as cardiac homeostasis, vascular and non-vascular smooth muscle relaxation, inhibition of platelet accumulation and coagulation, peripheral and central neurotransmission, immune response, transduction of light signals and regulating function in the gastrointestinal and urogenital systems. Apart from the physiological function, the NO/sGC/cGMP pathway is implicated in the pathophysiology of certain conditions as well, such as arterial and pulmonary hypertension, heart failure, arteriosclerosis and thrombosis, diabetes, kidney fibrosis and failure, inflammatory and neurodegenerative diseases, gastrointestinal disorders, sepsis and cancer, where it could be down- or up-regulated. Due to the significance and the severity of cardiovascular disorders where the expression or the activity of the pathway substituents is downregulated, most of the following drug discovery research focused on developing new agents that activate sGC, affording not only improved NO-donors but, additionally, two new categories of NO-independent activators, involving structurally diverse compounds. Regarding the inhibition of the enzyme, progress could not be considered as high, in spite of the tremendous utility of inhibitors in elucidating the cGMP-dependent biological processes. Among other compounds such as methylene blue, LY83583, organic phosphates, nucleotide derivatives, porphyrin analogues, the most potent and selective sGC inhibitor is ODQ, which is suggested to act through oxidation of the heme iron and consequent abolishment of the enzyme NO-activation ability. ODQ’s selectivity disadvantage, though, to inhibit in vivo not only the soluble guanylate cyclase but other hemoproteins as well, results in an urgent need for new selective sGC-inhibitors. According to the above facts, the aim of the present study involves the design, synthesis and biological evaluation of new possible sGC-inhibitors. Using the chemical structure of ODQ as a lead-compound, new tricyclic analogues were designed, bearing the basic indole or dihydroindole skeleton. The choice of the indole ring against the quinoxaline framework of ODQ aimed at the evaluation of how the new heterocyclic skeleton could contribute in the maintenance or the enhancement of the biological activity of the lead-compound. Moreover, the impact of non-flat molecules against ODQ was intended to be examined by the use of dihydroindole ring. Additionally, a series of different heterocyclic rings, diverse from the oxadiazolone ring of ODQ, fused to the basic skeletons were designated in order to study any possible influence based on the structure in inhibitory activity. Consequently, the synthetic methodologies for the synthesis of the final products were investigated, as well as the preparation of key molecules which could be used in the future as synthetic intermediates for the synthesis of additional modified derivatives. The synthesis of final dihydroindole tricyclic analogues was accomplished under cyclization conditions reacting hydroxylamine or various hydrazines with the α-hydroxymethylen-keto-intermediates, which were prepared from 4-keto-tetrahydroindole derivatives and ethyl formate. The corresponding indole final products were prepared from the dihydroindole ones under dehydrogenation conditions using DDQ. Due to the synthetic conditions applied for the synthesis of the key intermediate compounds, the substitution of the pyrrole nitrogen atom with a proper protecting group was considered crucial for the progress of the synthetic plan. Aiming to study the impact of the unsubstituted indole ring on the biological activity, the cleavage of the protecting group from the final products was necessary. Although, a number of protecting groups and of cleavage conditions were examined, the dihydroindole tricyclic products proved unstable. We only attempted to cleave one protecting group, the SEM group, and only in fully aromatized products, which proved more stable. 24 final products were synthesized following the aforementioned procedures, fourteen dihydroindole derivatives, ten indole derivatives among of which three bear unsubstituted pyrrole nitrogen atom. Apart from the final products, some significant synthetic intermediates were managed to be prepared. The α-methoxy- or α-ethyoxycarbonyl- 4- or 7-oxo-tetrahydroindole derivatives were synthesized according to different experimental procedures, as well as the a-hydroxymethylen-4-oxo-tetrahydroindole derivatives. The above synthetic intermediates may lead following different synthetic pathways to new tricyclic indole final compounds. In addition, the experimental conditions in each single step of the synthetic plan were investigated and determined on the purpose of improved yields. Some of the final tricyclic analogues, bearing no substituent at positions 2- and 3- of the pyrrole ring, were initially tested in in vitro biological assays. Firstly, they were tested concerning their inhibitory ability against SNP-induced sGC-activity using two different concentrations (1 μΜ and 100 μΜ). All the tested compounds proved to be sGC-inhibitors, being more potent at the higher concentration, except of one compound. Derivatives 74 and 81 were the most potent inhibitors and their inhibitory activity was dose-responded. Product 81 was then examined regarding its selectivity towards sGC by an in vitro assay involving ANF-induced cGMP production. The last assay proved that the above compound does not inhibit pGCs, suggesting that possibly the new series of compounds does not have any impact on either pGC or AC, determining a possible ODQ mechanism of action. Another conclusion from the biological assays that reinforces the last hypothesis is that the new compounds do not inhibit the basal sGC activity. The preliminary results of the biological evaluation confirm the main purpose of the present study which is the synthesis of new sGC-inhibitors. The choice of the indole ring as the basic skeleton of the new compounds verifies the initial design suggesting inhibitors with new structural characteristics. Preliminary SAR studies highlight the improved potency of flat fully aromatized indole derivatives in comparison with the non-flat dihydroindole ones, while there could not yet be safe conclusions derived regarding the structure of the third heterocyclic ring. Further biological evaluation of the rest of the products as well as the synthesis of new additional modified compounds will serve more detailed structure-activity relationship studies and possibly the development of a new lead-compound in the field of sGC-inhibition. Conclusions related with the chemical synthesis of the intermediate and final products, as well as conclusions regarding the biological evaluation of selected tricyclic derivatives might be proved useful for the future design, application and synthesis of new molecules with all the appropriate structural characteristics for effective sGC-inhibition. Such compounds not only could be proved useful experimental tools but possible pharmacological modulators as well in certain conditions, like sepsis, where sGC is overexpressed.

Nitric oxide (NO) is the principal mediator of erectile function. NO is released from the nerves and endothelium of small arteries in the penis and diffuses into surrounding smooth muscle to vasodilate through activation of soluble guanylate cyclase (sGC). Erectile dysfunction (ED) occurs in 50% of men between the ages of 40 and 70. It is likely that the pathology of ED results from impairment of NO formation or bioavailability in penile tissue. Iatrogenic nerve damage occurring during prostatectomy can attenuate neurotransmission and release of vasodilators from cavernosal nerves. Oxidative stress from chronic conditions such as diabetes and cardiovascular disease generates reactive oxygen species that can oxidize NO and decrease the molecule's bioavailability. The "gold standard" treatment for ED involves use of oral PDE-5 inhibitors that rely on an intact NO-signaling mechanism for efficacy. Although these therapies are easy to use, they are not effective in many patients suffering from ED associated with pathological conditions of decreased NO bioavailability. Rho-kinase inhibitors, sGC stimulators and sGC activators offer three new interventions that may demonstrate efficacy in treating ED associated with low NO bioavailability. Our results suggest that erectile responses to Rho-kinase inhibitors are not modulated by muscarinic receptor blockade, soluble guanylate cyclase inhibition or cavernosal nerve injury in the rat and that Rho-kinase inhibitors are additive and do not potentiate the endogenous NO-mediated erectile response. Our results with BAY 41-8543 show that this sGC stimulator has significant erectile activity and can potentiate erectile responses to low levels of exogenous and endogenously released NO. These results suggest that BAY 41-8543 would be useful in the treatment of ED occurring following nerve damage from prostatectomy. The sGC activator BAY 60-2770 has very potent erectile activity that is enhanced significantly in conditions of oxidative stress when erectile responses to endogenous NO or sGC stimulators are severely diminished. In oxidizing conditions erectile activity of sGC activators may be enhanced further with concomitant PDE-5 inhibitor therapy, providing evidence that sGC activators may be used alone and in combination with existing treatments to improve erectile function in patients who are non-responsive to standard therapeutic options for ED. / acase@tulane.edu

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Os neutrófilos estão entre as principais células da imunidade inata e são as primeiras células a migrarem para o sítio de infecção. O BAY 41-2272, estimulador de guanilato ciclase solúvel, é capaz de ativar fagócitos mononucleares e em neutrófilos diminuir migração in vivo e in vitro. O presente trabalho teve como objetivo avaliar o potencial do BAY 41-2272, e sua via, como uma ferramenta farmacológica para modulação da função dos neutrófilos. Para isso foi realizado o tratamento in vitro dos PMNs com o BAY 41-2272. A viabilidade celular, quimiotaxia e funções efetoras como burst oxidativo e produção de citocinas foram avaliadas, observando-se que o BAY 41-2272, como ativador direto, não altera estas funções. No entanto, o pré-tratamento com BAY 41-2272 nas doses de 3 M e 30M por uma hora, com subsequente ativação com PMA, mostrou perfil inibitório na quimiotaxia, produção da citocina IL-8 e no burst oxidativo . Foram avaliados também a expressão de moléculas como CD15, CD31, CD35, CD49d, CD63, CD66b, CD162 e a produção de GMPc. As moléculas de superfícies não mostraram alterações após tratamento direto com BAY 41-2272. A produção de GMPc foi induzida na dose de 30 M de BAY 41-2272 e pelo SNAP (doador de NO). Esses dados sugerem um potencial inibitório do BAY 41-2272 sobre a ação dos neutrófilos, e uma possível alternativa a ser explorada em seus aspectos translacionais na busca por novas terapias destinadas ao controle de doenças ligadas a inflamações crônicas e doenças auto-imunes. / Neutrophils are the major cells of innate immunity and are the first cells to migrate to the site of infection. BAY 41-2272, a soluble guanylate cyclase stimulator, is capable of activating mononuclear phagocytes and in neutrophils decreasing migration in vivo and in vitro. The present work aimed to evaluate the potential of BAY 41-2272, and its pathway, as a pharmacological tool for modulating neutrophil function. For this, the in vitro treatment of PMNs with BAY 41-2272 was performed. Cell viability, chemotaxis and effector functions such as oxidative burst and cytokine production were evaluated, observing that BAY 41-2272, as a direct activator, does not alter these functions. However, pretreatment with BAY 41-2272 at the doses of 3 M and 30 M for one hour, with subsequent activation with PMA, showed an inhibitory profile in chemotaxis, IL-8 cytokine production and in the \"oxidative burst\". We also evaluated the expression of molecules such as CD15, CD31, CD35, CD49d, CD63, CD66b, CD162 and cGMP production. Surface molecules did not show changes after direct treatment with BAY 41-2272. The cGMP production was induced at the dose of 30 M BAY 41-2272 and for SNAP (NO donor). These data suggest an inhibitory potential of BAY 41-2272 on the action of neutrophils, and a possible alternative to be explored in its translational aspects in the search for new therapies aimed at the control of diseases linked to chronic inflammation and autoimmune diseases.

No description available.

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Orientador: Luis Carlos Paschoarelli / Banca: João Pedro Albino / Banca: José Carlos Plácido da Silva / Resumo: As interfaces de sites institucionais devem basear-se em princípios de usabilidade, cujo propósito é garantir a informação de forma segura, eficiente e efetiva às audiências e a seus respectivos usuários. Os princípios teóricos sobre recomendações para o desenvolvimento de sites apresentam-se amplamente disponíveis, mas a aplicação destes, verificados com ferramentas especializadas, continuam sendo fundamentais para o alcance da efetiva usabilidade. O presente estudo objetivou testar a ferramenta CMS (Content Management System) durante o desenvolvimento de um site institucional do Programa de Pós-graduação em design da Universidade Estadual Paulista, visando ao melhoramento de sua usabilidade. Os procedimentos metodológicos caracterizaram-se por cinco etapas, iniciando-se pela revisão teórica e seguindo por aplicações laboratoriais e de simulações práticas, as quais se caracterizaram pela evolução alternada de etapas de análise, desenvolvimento de protótipo, e reanálise, resultando em um protótipo final. Esses resultados foram considerados satisfatórios, pois foi verificado que a aplicação de ferramentas CMS no desenvolvimento de sites institucionais melhora seus níveis de usabilidade. Entretanto, a principal contribuição do presente estudo está na conquista de novos conhecimentos decorrentes dos experimentos realizados, bem como no incremento dos temas ligados ao design de interfaces, à usabilidade e à ergonomia informacional, os quais colaboram de forma efetiva para o desenvolvimento do design ergonômico / Abstract: The interfaces of institutional sites must be based on usability principles, whose purpose is to ensure the security, efficient and effective information to audiences and their users. The theoretical principles of recommendations for site development have become widely available, but he implementation of them checked with specialized tools remain be fundamental to the achievement of effective usability. This study aimed to test the CMS tool (Content Management System) during the development of an institutional site of the Graduate Program in Design at the Universidade Estadual Paulista intended to improve its usability. The methodological procedures were characterized by five steps, starting by the review theory and following by laboratory applications and practices simulations, which were characterized by alternating stages of development testing, prototype development, and reanalysis, resulting in a final prototype. These results were considered satisfactory, because it was verified that the use of CMS tools in the development of institucional sites improved their levels of usability. However, the main contribution of this study is to acquire new knowledge resulting from experiments, as well as the increase in issued related to interface design, usability and ergonomic information, which collaborate effectively for the development of ergonomic design / Mestre

Asphalt mixture compaction is an important procedure of asphalt mixture construction and can significantly affect the performance of asphalt pavement. Many laboratory compaction methods (or devices), have been developed to study the asphalt mixture compaction. Nevertheless, the whole process from the selection of aggregate to laboratory compaction is still time-consuming and requires significant human and material resources. In order to better understand asphalt mixture compaction, some researchers began to use finite element method (FEM) to study and analyze mixture compaction. However, FEM is a continuum approach and lacks the ability to take into account the slippage and interlocking of aggregates during compaction. Discrete Element Method (DEM) is a discontinuum analysis method, which can simulate the deformation process of joint systems or discrete particle assembly under quasi-static and dynamic condition. Therefore, it can overcome the shortcomings of FEM and is a more effective tool than FEM to simulate asphalt mixture compaction. In this study, an open source 3D DEM code implemented with the C++ programming language was modified and applied to simulate the compaction of hot-mix asphalt (HMA). A viscoelastic contact model was developed in the DEM code and was verified through comparison with well established analytical solutions. The input parameters of the newly developed contact model were obtained through nonlinear regression analysis of dynamic modulus test results. Two commonly used compaction methods (Superpave gyratory compaction and asphalt vibratory compaction) and one linear kneading compaction based on APA machine were simulated using the DEM code, and the DEM compaction models were verified through the comparison between the DEM predicted results and the laboratory measured test results. The air voids distribution within the asphalt specimens was also analyzed by post processing virtual DEM compaction digital specimens and the level of heterogeneity of the air void distribution within the specimens in the vertical and lateral directions was studied. The DEM simulation results in this study were in a relatively good agreement with the experimental data and previous research results, which demonstrates that the DEM is a feasible method to simulate asphalt mixture compaction under different loading conditions and, with further research, it could be a potentially helpful tool for asphalt mix design by reducing the number of physical compactions in the laboratory.

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Made available in DSpace on 2014-06-11T19:24:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-08Bitstream added on 2014-06-13T20:51:32Z : No. of bitstreams: 1 nomiso_ls_me_bauru.pdf: 11920475 bytes, checksum: 59a967a2b3720648640f47aaa829e7da (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As interfaces de sites institucionais devem basear-se em princípios de usabilidade, cujo propósito é garantir a informação de forma segura, eficiente e efetiva às audiências e a seus respectivos usuários. Os princípios teóricos sobre recomendações para o desenvolvimento de sites apresentam-se amplamente disponíveis, mas a aplicação destes, verificados com ferramentas especializadas, continuam sendo fundamentais para o alcance da efetiva usabilidade. O presente estudo objetivou testar a ferramenta CMS (Content Management System) durante o desenvolvimento de um site institucional do Programa de Pós-graduação em design da Universidade Estadual Paulista, visando ao melhoramento de sua usabilidade. Os procedimentos metodológicos caracterizaram-se por cinco etapas, iniciando-se pela revisão teórica e seguindo por aplicações laboratoriais e de simulações práticas, as quais se caracterizaram pela evolução alternada de etapas de análise, desenvolvimento de protótipo, e reanálise, resultando em um protótipo final. Esses resultados foram considerados satisfatórios, pois foi verificado que a aplicação de ferramentas CMS no desenvolvimento de sites institucionais melhora seus níveis de usabilidade. Entretanto, a principal contribuição do presente estudo está na conquista de novos conhecimentos decorrentes dos experimentos realizados, bem como no incremento dos temas ligados ao design de interfaces, à usabilidade e à ergonomia informacional, os quais colaboram de forma efetiva para o desenvolvimento do design ergonômico / The interfaces of institutional sites must be based on usability principles, whose purpose is to ensure the security, efficient and effective information to audiences and their users. The theoretical principles of recommendations for site development have become widely available, but he implementation of them checked with specialized tools remain be fundamental to the achievement of effective usability. This study aimed to test the CMS tool (Content Management System) during the development of an institutional site of the Graduate Program in Design at the Universidade Estadual Paulista intended to improve its usability. The methodological procedures were characterized by five steps, starting by the review theory and following by laboratory applications and practices simulations, which were characterized by alternating stages of development testing, prototype development, and reanalysis, resulting in a final prototype. These results were considered satisfactory, because it was verified that the use of CMS tools in the development of institucional sites improved their levels of usability. However, the main contribution of this study is to acquire new knowledge resulting from experiments, as well as the increase in issued related to interface design, usability and ergonomic information, which collaborate effectively for the development of ergonomic design