Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-07-21T18:05:28Z No. of bitstreams: 1 Alan Moreira de Araújo, Detecção de espécies... 2013.pdf: 1675349 bytes, checksum: 82f7886b065bdb7c68db0c1fb5d46f67 (MD5) / Made available in DSpace on 2014-07-21T18:05:28Z (GMT). No. of bitstreams: 1 Alan Moreira de Araújo, Detecção de espécies... 2013.pdf: 1675349 bytes, checksum: 82f7886b065bdb7c68db0c1fb5d46f67 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Leptospirose é uma zoonose que pode levar a graves complicações, como a síndrome de Weil e a síndrome pulmonar hemorrágica, porém os mecanismos patogênicos que levam ao desenvolvimento das formas graves da doença ainda são desconhecidos. Após a penetração no indivíduo, as leptospiras invadem a corrente sanguínea e se disseminam para os órgãos. Dessa forma, a leptospirose apresenta características semelhantes as da sepse, doença que tem o estresse oxidativo como um dos principais responsáveis pelo seu agravamento. Entretanto, pouco se sabe sobre o envolvimento do estresse oxidativo na leptospirose. O presente estudo teve como objetivo avaliar se a produção de espécies reativas de oxigênio (ROS) e os níveis do antioxidante glutationa (GSH) estão relacionados com as manifestações clínicas mais graves de pacientes hospitalizados com leptospirose. A produção de ROS e os níveis de GSH foram avaliados nas amostras de sangue de doze pacientes e nove indivíduos saudáveis através dos ensaios de quimioluminescência e de absorbância, respectivamente. Nós observamos que os níveis de ROS estavam aumentados (p=0.0012) e os de GSH diminuídos (p=0.0002) nos pacientes quando comparados com os indivíduos saudáveis. Dentre os pacientes, a diminuição de GSH estava correlacionada com a trombocitopenia (r=0.63) e com elevados níveis de creatinina (r= -0.64), enquanto que a produção de ROS estava fortemente correlacionada com os níveis elevados de potássio sérico (r=0.8). A compreensão da importância biológica de ROS e do GSH na leptospirose faz-se necessária, pois uma investigação mais detalhada pode levar ao desenvolvimento de terapias adjuvantes focadas no estresse oxidativo. / Leptospirosis is a zoonotic disease that causes severe manifestations such as Weil’s disease and pulmonary hemorrhage syndrome, however the underlying mechanisms that lead to the development of severe forms are not clear. Leptospires penetrate through skin, reach the bloodstream and disseminate to the organs. Thus, leptospirosis and sepsis have similar characteristics. Although there is vast literature demonstrating that oxidative stress play an important role in the severity of sepsis, none is known about it in leptospirosis. The aim of this study was to evaluate whether reactive oxygen species (ROS) production and antioxidant reduced glutathione (GSH) levels are related to complications in patients hospitalized with leptospirosis. ROS production and GSH levels were measured in blood samples of twelve patients and nine healthy controls using chemiluminescence and absorbance assays. We found that ROS production was higher (p=0.0012) and GSH levels were lower (p=0.0002) in leptospirosis patients compared with healthy individuals. Among patients, GSH depletion was correlated with thrombocytopenia (r=0.63) and elevated serum creatinine (r= -0.64), while a strong positive correlation was observed between ROS production and elevated serum potassium (r=0.8). Additional investigation of the biological significance of ROS production and GSH levels is warranted as they may guide the development of novel adjuvant therapies for leptospirosis targeting oxidative stress.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Atherosclerosis is a chronic inflammatory condition associated with an overproduction of oxidant species. Since low density lipoprotein (LDL) oxidation plays a key role in the pathogenesis of atherosclerosis, antioxidants that can inhibit this oxidative process might be useful in preventing atherosclerosis-related pathological conditions, such as cardiovascular diseases and stroke. Here, we investigated the potential toxicological effects and the anti-atherogenic properties of diphenyl diselenide (PhSe)2, a simple organoselenium compound that presents important antioxidant properties. Toxicological studies, revealed that (PhSe)2, orally administered, produced minor toxicological effects in rabbits exposure for a long term. The toxic potential of (PhSe)2 was evidenced by a decrease in the ascorbic acid contents in blood, liver and brain of rabbits. Nevertheless, the prolonged exposition with (PhSe)2 was not hepato or renotoxic and not modified the others oxidative stress parameters. The consumption of (PhSe)2 by hipercholesterolemic rabbits significantly decreased the serum cholesterol levels and also diminished the serum and hepatic levels of TBARS, as well as the production of free radical species in blood and brain. In vitro studies demonstrated that (PhSe)2 showed a dosedependent protective effect against Cu2+ and 2,2-azobis-2-amidinopropane (AAPH)-induced oxidation of LDL or parinaric acid (PnA) incorporated into isolated human LDL. Interestingly, (PhSe)2 also protected protein moieties from human isolated LDL against Cu2+-induced oxidation. These effects were related to its thiol-peroxidase activity. In experiments with primary cultures of bovine aortic endothelial cells (BAEC), (PhSe)2 protected endothelial cells from the damage promoted by peroxynitrite exposure, in a more effective way than ebselen. The intracellular levels of glutathione (GSH) were almost completely consumed by peroxynitrite and although the compounds did not restore the normal levels, (PhSe)2 per se increases significantly GSH in a concentration-dependent manner. This effect may be related with the significant increase in cellular glutathione peroxidase (GPx) activity promoted by this compound. Taken together, the results indicated that (PhSe)2 is relatively safe for rabbits even after a prolonged time of ingestion and suggest a new role for diphenyl diselenide as a potential anti-atherogenic agent. / A aterosclerose é uma condição inflamatória crônica associada à produção de espécies oxidantes. A oxidação da lipoproteína de baixa densidade (LDL) parece ter um papel fundamental na patogênese da aterosclerose, desta forma os antioxidantes, substâncias capazes de inibir o processo oxidativo, podem ser úteis na prevenção das condições patológicas associadas à aterosclerose, como doenças cardiovasculares e acidentes vasculares cerebrais. Nesta tese, investigou-se os potenciais efeitos toxicológicos e as propriedades anti-aterogênicas do disseleneto de difenila (PhSe)2, um simples composto orgânico de selênio que apresenta importantes propriedades antioxidantes. Os estudos toxicológicos revelaram que o (PhSe)2 oralmente administrado produziu poucos efeitos tóxicos nos coelhos expostos ao composto por um longo período de tempo. O principal efeito tóxico do (PhSe)2 foi a diminuição dos níveis de ácido ascórbico no sangue, fígado e cérebro dos coelhos. Todavia, a exposição prolongada ao (PhSe)2 não alterou os marcadores clássicos de lesão hepática e renal e os demais marcadores do estresse oxidativo. O consumo de (PhSe)2 por coelhos hipercolesterolêmicos diminuiu significativamente os níveis sorológicos de colesterol total e ainda reduziu os níveis sanguíneos e hepáticos de TBARS, bem como a produção de espécies oxidativas no sangue e no cérebro destes animais. Estudos in vitro demonstraram que o (PhSe)2 protegeu LDLs humanas isoladas contra a oxidação induzida por Cu2+ e AAPH e a oxidação do ácido parinárico (PnA) incorporado nas LDLs de maneira concentração dependente. O (PhSe)2 também foi capaz de prevenir a oxidação da porção protéica das LDLs. Estes efeitos protetores do (PhSe)2 foram associados a sua atividade tiol peroxidase. Em experimentos utilizando cultura primária de células endoteliais de aorta bovina (BAEC), evidenciou-se o efeito protetor do (PhSe)2 contra a morte celular induzida pelo peroxinitrito, de forma mais efetiva que o ebselen. Os níveis intracelulares de glutationa (GSH) foram completamente consumidos pelo peroxinitrito. O (PhSe)2 per se aumentou significativamente os níveis de GSH de maneira concentração dependente, todavia, a adição dos compostos não preveniu o consumo de GSH pelo peroxinitrito. Este efeito pode ser relacionado com o significativo aumento na atividade celular da glutationa peroxidase (GPx) promovido pelo (PhSe)2. Em conclusão estes resultados indicam que a suplementação oral com (PhSe)2 demonstrou baixa toxicidade nos coelhos mesmo após uma ingestão prolongada do composto e sugere-se um novo papel para o (PhSe)2 como um potencial agente anti-aterogênico.

"Το τσάι του βουνού" (στο οποίο ανήκουν πολλά είδη του γένους Sideritis) καταναλώνεται παραδοσιακά στην Ελλάδα ως ηρεμιστικό αλλά και ενάντια του κρυολογήματος και αλλεργιών. Η παρούσα μελέτη διερευνά την πιθανή νευροπροστατευτική δράση του ανωτέρω φυτού και εστιάζεται: α) στην επίδραση του αφεψήματος του φυτού Sideritis clandestina subsp. clandestina σε συμπεριφερικές παραμέτρους ενηλίκων μυών (άγχος/φόβος, μνήμη/μάθηση), β) στην επίδραση του αφεψήματος του ανωτέρω φυτού σε βιοχημικές παραμέτρους και πιο συγκεκριμένα στη συγκέντρωση της ανηγμένης γλουταθειόνης, στην υπεροξείδωση λιπιδίων και στην ενεργότητα δυο ισομορφών του ενζύμου της ακετυλοχολινεστεράσης (AChE) και γ) στην in vitro πιθανή αντιχολινεστερασική και αντιαμυλοειδική δράση του υδατικού εκχυλίσματος του φυτού. Το φυτικό αφέψημα σε συγκέντρωση 4% w/v χορηγoόταν καθημερινά σε αρσενικούς Balb-c μύες για περίοδο 40 ημερών. Για την εκτίμηση του άγχους/φόβου χρησιμοποιήθηκε α) η δοκιμασία του Υπερυψωμένου Λαβυρίνθου (χρόνος παραμονής των μυών στους ανοιχτούς βραχίονες ως προς το συνολικό χρόνο παραμονής στους ανοιχτούς και κλειστούς βραχίονες της συσκευής) και β) η δοκιμασία του Θιγμοτακτισμού (τάση παραμονής των μυών πλησίον των τοιχωμάτων του ειδικού κλωβού). Η μνήμη-μάθηση μελετήθηκε με τη δοκιμασία της Παθητικής Αποφυγής η οποία βασίζεται στην παρατήρηση ότι τα πειραματόζωα θα θυμούνται ότι μια συγκεκριμένη αντίδρασή τους θα έχει αρνητικές συνέπειες (εφαρμογή επώδυνου ηλεκτρικού ερεθίσματος στα άκρα). Οι οξειδωτικές/αντιοξειδωτικές ιδιότητες του φυτικού αφεψήματος προσδιορίστηκαν με εκτίμηση α) της συγκέντρωσης του αντιοξειδωτικού δείκτη της ανηγμένης γλουταθειόνης, η οποία στηρίζεται στο σχηματισμό ενός φθορίζοντος συμπλόκου μετά την αντίδραση της ο-φθαλαλδεϋδης με τη γλουταθειόνη και υστιδύλ-ενώσεις και β) τα επίπεδα λιπιδικής υπεροξείδωσης μέσω προσδιορισμού των επιπέδων του οξειδωτικού δείκτη της μηλονικής διαλδεΰδης που στηρίζεται στο σχηματισμό του φθορίζοντος συμπλόκου που δημιουργείται όταν η μηλονική αλδεΰδη αντιδρά με το θειοβαρβιτουρικό οξύ. Η ενεργότητα του ενζύμου της ακετυλοχολινεστεράσης (AChE) τόσο ex vivo όσο και in vitro προσδιορίστηκε με τη χρήση της χρωματομετρικής μεθόδου του Ellman, όπου ως υπόστρωμα του ενζύμου χρησιμοποιήθηκε η ιωδιούχος ακετυλοθειοχολίνη. Κατά τον προσδιορισμό της ενεργότητας του ενζύμου in vitro ως εσωτερικό πρότυπο χρησιμοποιήθηκε η γαλανταμίνη, ένας ισχυρός αναστολέας του ενζύμου. Η πιθανή αντιαμυλοειδκή δράση του φυτικού εκχυλίσματος μελετήθηκε με τη μέθοδο της θειοφλαβίνης-Τ. Τα αποτελέσματα της δοκιμασίας του Υπερυψωμένου Λαβύρινθου έδειξαν ότι ο χρόνος παραμονής στους ανοιχτούς βραχίονες ως προς το συνολικό χρόνο παραμονής στους ανοιχτούς και κλειστούς βραχίονες της συσκευής είναι στατιστικώς σημαντικά αυξημένος για την ομάδα των ζώων που κατανάλωσαν το φυτικό αφέψημα σε σύγκριση με τους μάρτυρες. Από τη δοκιμασία του Θιγμοτακτισμού για την πειραματική ομάδα φάνηκε ότι ο χρόνος θιγμοτακτισμού μειώνεται και ο αριθμός των εισόδων στην κεντρική περιοχή του ανοιχτού πεδίου αυξάνεται για κάθε 5 λεπτά παραμονής (εκ του συνολικού διαστήματος των 30 λεπτών) στη συσκευή εν συγκρίσει με την ομάδα των μαρτύρων. Τέλος, κατά τη δοκιμασία της Παθητικής Αποφυγής ο αρχικός και τελικός λανθάνων χρόνος (IL, STL αντίστοιχα) φάνηκε να μη διαφέρουν μεταξύ των δυο ομάδων πειραματοζώων. Η πόση του φυτικού αφεψήματος επηρέασε με ιστοειδικό τρόπο τις υπό μελέτη βιοχημικές παραμέτρους, καθώς παρατηρήθηκε αύξηση των επιπέδων της ανηγμένης γλουταθειόνης και μείωση της υπεροξείδωσης λιπιδίων στον ολικό εγκέφαλο (-παρ/δας) των ενηλίκων μυών, η οποία συνοδευόταν από αλλαγές στις επιμέρους εγκεφαλικές περιοχές της παρεγκεφαλίδας και του μεσεγκεφάλου ενώ ο εγκεφαλικός φλοιός ακολούθησε το πρότυπο του ήπατος και έδειξε να μην επηρεάζεται. Σε σχέση με την ενεργότητα των δυο ισομορφών του ενζύμου της AChE παρατηρήθηκε αναστολή στον ολικό εγκέφαλο (-παρ/δας) της πειραματικής ομάδας συγκρινόμενη με τους μάρτυρες, η οποία συνοδεύτηκε από μείωση στις επιμέρους περιοχές του εγκεφαλικού φλοιού, του ραβδωτού και του ιπποκάμπου. Το υδατικό εκχύλισμα του Sideritis clandestina subsp. clandestina παρουσίασε: 10% αντιχολινεστερασική δράση in vitro σε αντίθεση με τον αναστολέα της γαλανταμίνης ο οποίος ανέστειλλε το ένζυμο σε ποσοστό 85%. Τέλος, το φυτικό εκχυλίσματος σε συγκέντρωση 0,3mg/mL ανέστειλε τη συσσωμάτωση του Αβ πεπτιδίου σε ποσοστό περίπου 85%. Με βάση τα ανωτέρω το «τσάι του βουνού» επιδεικνύει τάση για αγχόλυση, ενισχύει την αντιοξειδωτική άμυνα, έχει αντιχολινεστερασικές ιδιότητες και επιφέρει ανασταλτικό αποτέλεσμα στη συσσωμάτωση του Αβ αμυλοειδούς. Οι δράσεις του αυτές οφείλονται πιθανόν στην πολυφαινολική του σύσταση και οι μηχανισμοί που εμπλέκονται χρήζουν περαιτέρω διερεύνησης. / “Mountain tea” (various species of Sideritis) has been traditionally consumed in Greece as a calmative and against common cold and allergies. The aim of the present study was to examine the neuroprotective role of the above plant and focus on: a) the effect of tea drinking in behavioral parameters of adult mice (fear/anxiety, learning and memory), b) the effect of tea drinking in antioxidative biochemical parameters of adult mice brain and liver, i.e. the concentration of reduced glutathione (GSH), the levels of lipid peroxidation and the activity of the two acetylcholinesterase (AChE) isoforms, and c) in vitro putative anticholinesterase and antiamyloid actions of Sideritis clandestina subsp. clandestina infusion. The beverage was provided (4g/100 mL, daily) for 40 days to adult male Balb-c/jice. Fear/anxiety was assessed by the measurement of i) the percentage of time spent in the open arms of the elevated plus maze apparatus and ii) the thigmotactic response (the tendency to remain close to vertical surfaces) of adult mice in an open-field. Learning and memory was assessed using the step-through passive avoidance task which is based on the obseravation that the experimental mice remember that a specific reaction has negative effects (electric foot shock). The oxidant/antioxidant properties of tea drinking was determined via measurement of a) the concentration of GSH (antioxidant marker), which is based on the formation of a fluorescent complex after the reaction of o-pthalaldehyde with glutathione and hystidyl compounds and b) the levels of malondialdehyde (MDA) (oxidant marker) by monitoring thiobarbituric acid reactive substance formation. The ex vivo and in vitro AChE activity was measured using the colorimetric method of Ellman where acetylthiocholine iodide was used as a substrate. Galantamine, a strong inhibitor of AChE, was used as a standard during in vitro determination of the enzyme activity. The effect of the infusion on amyloid-beta aggregation was studied in vitro with a thioflavine T - based fluorescence assay. Tea drinking caused statistically significant a) increase of the time percentage that animals spent into the open arms of the elevated plus maze apparatus and b) decrease of thigmotaxis time and increase of the time that animals entered to the central area of open field. Initial and Step-Through Latency showed no significant difference between two animal groups. The beverage also affected the biochemical parameters examined in the present study, in a tissue specific manner. More specifically, adult mice whole brain (-Ce) displayed increased reduced glutathione content and decreased lipid peroxidation levels compared to the controls. Similar changes were also observed in cerebellum and midbrain while cerebral cortex and liver were not affected. Regarding the activity of the two AChE isoforms, tea intake caused significant inhibition of these enzymes’ activity in whole brain (-Ce), compared with the control group. In agreement, cerebral cortex, striatum and hippocampus dispayed similar inhibition in both AChE isoforms activity after tea consuming. Sideritis clandestina subsp. clandestina infusion exhibited 10% in vitro anticholinesterase activity while galantamine inhibited the enzyme in a percentage of 85%. Moreover, tea infusion in a concentration of 0,3 mg/mL exhibited 85% inhibition of Ab1-40 fibrillogenesis in vitro, indicating, thus, strongly a putative antiamyloid action of Sideritis clandestina subsp. clandestinα. Conclusively, our results suggest that mountain tea infusion exhibits anxiolytic-like action, antioxidant and anticholintesterase properties and a strong, in vitro, inhibitory effect on amyloid-beta’s aggregation. These activities are most probably due to the polyphenols contained in Sideritis clandestina subsp. clandestina infusion; the underlying mechanisms need, though, further, in deep investigation.