This project focuses on two important topics which may pose hazards to human health. Firstly, drinking water disinfection by-products (DBPs), which are generated by the chemical disinfection of water have been investigated. What has not been shown is the effect of DBPs in human germ cells as well as somatic cells and whether oxidative stress is involved in the mechanism of genotoxic action. Three different DBPs (halo acetic acids: HAAs), together with the antioxidants – catalase and butylated hydroxyanisole (BHA), were investigated in peripheral blood cells and sperm from healthy individuals using the Comet assay and lymphocytes only using the micronucleus assay. Secondly, nanoparticles of the non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and ibuprofen, have been investigated in patients with respiratory diseases, in the micronucleus assay and the Comet repair assay. NSAIDs inhibit cyclooxygenase enzyme activity, which plays part in tumour progression. In the Comet assay, BHA and catalase were able to reduce DNA damage in both cell types compared to HAAs alone. Similarly, in the micronucleus assay, micronuclei were reduced with the antioxidants, suggesting oxygen radical involvement in both assays. With the NSAIDs, reductions were seen for DNA damage in the micronucleus assay with aspirin and ibuprofen nanoparticles compared to their bulk forms. Using the Comet repair assay, aspirin and ibuprofen nanoparticles aided repair of DNA to a greater extent than their bulk counterparts, which in turn showed better repair compared to samples repaired without NSAIDs. These observations show the importance of DBPs and NSAIDs in genotoxic public health issues. / United Kingdom India Education and Research Initiative (UKIERI).

Yes / Chronic obstructive pulmonary disease (COPD) in humans, describes a group of lung conditions characterised by airflow limitation that is poorly reversible. The airflow limitation usually progresses slowly and is related to an abnormal inflammatory response of the lung to toxic particles. COPD is characterised by oxidative stress and an increased risk of lung carcinoma. The 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) is one of a number of mutagenic/carcinogenic heterocyclic amines found mainly in well-cooked meats which are thus part of the regular diet. Antioxidants are very important in order to protect the cells against oxidative damage. The aim of the present study was to assess the effects of IQ on the level of DNA damage and susceptibility to a potent mutagen in peripheral blood cells of COPD patients. DNA damage and the frequency of micronuclei (MNi) were evaluated using the Comet and micronucleus assays, respectively. Differential expressions of both mRNA and protein of the endogenous antioxidant enzyme catalase were evaluated with quantitative polymerase chain reaction (qPCR) and Western blot analysis, respectively. Furthermore, the effect of bulk and nano forms of quercetin and their combination with IQ were examined. Results of the present study clearly demonstrated that MNi frequency in the peripheral blood lymphocytes exhibited a positive correlation with the DNA damage as evident from the different Comet assay parameters. Increase of the endogenous antioxidant catalase also showed there was a stimulation of this enzyme system by IQ. Whereas, the endogenous antioxidant quercetin significantly reduced oxidative stress in COPD patients and healthy individuals. / Libyan Government

Diabetes mellitus is a chronic autoimmune disease in which the immune system attacks the insulin-secreting ß-cells in the pancreas. It leads to an absolute deficiency of insulin. Chronic hyperglycemia induces increased production of reactive oxygen species, which leads to a decrease of natural antioxidant level in blood, and it contributes to genesis of diabetes complications (e.g. vascular or pulmonic). Moreover, the oxidative stress results in onset of pancreas inflammations and the damage of its ß-cells. Aims: Our aim was to assess whether or not certain genotypes or their combinations occur with higher frequency among groups of patients of type 1 diabetes (T1D) and type 2 diabetes and in a control group of healthy individuals. Methods: The study included groups of 40 T1D patients, 40 T2D patients and 45 healthy individuals. The polymorphisms of genes involved in the oxidative stress response were analyzedby using RFLP, PCR with TaqMan probes and allele specific PCR. The target genes involved superoxide dismutase SOD1 and SOD3 genes; glutathione-S-transferase GSTM1, GSTT1, GSTP1 genes; glutathioneperoxidase gene GPX1 and catalase gene CAT. The levels of plasma malondialdehyde were measured by using liquid chromatography. Results: Statistically significant differences were found in the...

La mucoviscidose est la maladie génétique la plus fréquente dans la population caucasienne. Le genre Scedosporium se situe au deuxième rang parmi les champignons filamenteux isolés des expectorations dans ce contexte. Au niveau pulmonaire, les colonisations/infections entraînent le recrutement de phagocytes qui induisent un stress oxydatif normalement délétère pour les pathogènes. Pour se défendre, ceux-ci ont développé des systèmes antioxydants, notamment diverses enzymes. Ce travail de thèse visait à étudier la réponse au stress oxydatif chez Scedosporium. Tout d’abord, la capacité à germer en présence d’oxydants a été évaluée. Par la suite, trente-trois gènes potentiellement impliqués dans la défense contre le stress oxydatif ont été identifiés. Leur expression en présence d’oxydants et en co-cultures avec des phagocytes suggère un rôle majeur, notamment pour une catalase, une peroxyrédoxine et deux thiorédoxine réductases. Par ailleurs, un mutant défectif pour un gène codant une superoxyde dismutase (SOD) pariétale et spécifique des spores a été produit. L’auranofin, un inhibiteur des thiorédoxine réductases, présente une activité vis-à-vis des Scedosporium et un effet additif avec des triazolés. Un test ELISA a été développé pour le sérodiagnostic des scédosporioses, utilisant une catalase et une Cu/Zn-SOD recombinantes. Ce test sensible et spécifique permet de distinguer les infections à Scedosporium de celles à Aspergillus fumigatus et des colonisations à Scedosporium. Au final, ces résultats indiquent un rôle majeur des enzymes antioxydantes chez Scedosporium, qui pourraient être de véritables facteurs de virulence et donc de nouvelles cibles thérapeutiques. / Cystic fibrosis (CF) is the most common genetic disease in Caucasian populations. The Scedosporium genus ranks the second among the filamentous fungi colonizing the airways of CF patients. In the respiratory tract, colonizations/infections lead to the recruitment of phagocytes which produce an oxidative stress, usually deleterious for pathogens. To defend themselves, pathogens have developed protective antioxidant systems, especially various enzymes. This thesis aimed to study the oxidative stress response in Scedosporium species. First, capacity of several Scedosporium isolates to germinate upon oxidative stress conditions was evaluated. Then, thirty-three genes potentially involved in protection against the oxidative stress were identified. Their overexpression in response to oxidants and in co-cultures with phagocytes suggested a crucial role, especially for one catalase, one peroxiredoxin and the two thioredoxin reductases. A mutant defective for the gene encoding a superoxide dismutase (SOD) anchored to the cell wall and specific for the conidia was produced. Auranofin, a thioredoxin reductase inhibitor, exhibits little anti-Scedosporium activity and an additive effect with triazole drugs. An ELISA was developed for serodiagnosis of scedosporiosis, using recombinant proteins derived from one catalase and a Cu/Zn-SOD. This sensitive and specific assay allows to differentiate Scedosporium infections from Aspergillus fumigatus infections and Scedosporium colonizations. Finally, these results indicate a crucial role of antioxidant enzymes in Scedosporium species, which could therefore be considered as virulence factors and as possible new therapeutic targets.

Ce travail de thèse s'articule selon deux axes principaux et vise à modéliser, au moyen de la Théorie de la Fonctionnelle de la Densité (DFT), des grandeurs spectroscopiques (Mössbauer, RPE et magnétisme) mesurées pour des systèmes bioinorganiques à base de fer. La première partie de ce travail est relative au calcul théorique de deux quantités caractéristiques issues de la spectroscopie Mössbauer : le déplacement isomérique Δ et de l'éclatement quadripolaire ΔEQ. Dans un premier temps, des corrélations calculs-expérience ont été mises en place pour l'étude de simples complexes de fer. Dans un second temps, nous nous sommes intéressée à l'étude d'un système biologique, la catalase, qui est un composé héminique à base de fer, afin d'en caractériser les différentes formes. La seconde partie de ce travail a été dédiée à la rationalisation des variations conjointes de deux grandeurs spectroscopiques RPE : la valeur moyenne de tenseur , gav (= (∑gi)/3), et la constante d'échange effective, Jeff. Nous avons considéré un ensemble d'agrégats de type [2Fe-2S] issus de systèmes biologiques (ferrédoxines, protéines Rieske, xanthine oxidases, etc) et nous avons modélisé ces derniers paramètres via un modèle phénoménologique mettant en compétition deux termes antagonistes : le terme B de double-échange traduisant la délocalisation électronique entre les sites de fer et le terme de piégeage ΔE.contribuant à la localisation préférentielle de l'électron réducteur sur l'un des deux sites de fer. Nous avons pu exprimer analytiquement gav et Jeff en fonction du ratio ΔE/B et expliquer la source des variations observées.

Πρόσφατες μελέτες παρέχουν ενδείξεις για τη συμμετοχή του οξειδωτικού στρες στην ανάπτυξη καρδιαγγειακών νοσημάτων. Το οξειδωτικό στρες έχει συσχετιστεί ισχυρά με τον κυτταρικό θάνατο και διαδικασίες καρδιακής αναδόμησης, που αποτελούν χαρακτηριστικά της καρδιακής ανεπάρκειας. Μύες χωρίς δεσμίνη, σημαντική πρωτεΐνη των μυϊκών ενδιαμέσων ινιδίων, αναπτύσσουν διατατική μυοκαρδιοπάθεια και καρδιακή ανεπάρκεια, η οποία χαρακτηρίζεται από μιτοχονδριακές ανωμαλίες και κυτταρικό θάνατο μαζί με εκτεταμένες εναποθέσεις ασβεστίου και ίνωση, προσφέροντας έτσι ένα πολύ καλό μοντέλο καρδιακής ανεπάρκειας.Διάφορες κυτταρικές και βιοχημικές αλλοιώσεις στην καρδιά των μυών αυτών υποδηλώνουν έντονα ότι το οξειδωτικό στρες είναι ένας σημαντικός μηχανισμός που συμβάλλει στην παθογένεση αυτού του φαινότυπου. Οι ανωμαλίες στη μιτοχονδριακή δομή και λειτουργία, οι οποίες χαρακτηρίζουν το φαινότυπο του μυός χωρίς δεσμίνη, προσφέρουν τις πιο σημαντικές ενδείξεις για την ύπαρξη οξειδωτικού στρες, καθώς η αναπνευστική αλυσίδα είναι η πιο σημαντική πηγή δραστικών Ενώσεων Οξυγόνου (ΔΕΟ ή Reactive Oxygen Species - ROS) στα μυοκαρδιοκύτταρα. Προκειμένου να διασαφηνιστεί η ύπαρξη οξειδωτικού στρες στο μυοκάρδιο απουσία δεσμίνης και συνεπώς η συμμετοχή του στην εξέλιξη του μυοεκφυλισμού, επιχειρήθηκαν τόσο in vitro, όσο και in vivo προσεγγίσεις. Η ύπαρξη οξειδωτικής καταπόνησης διερευνήθηκε σε πρωτογενείς καλλιέργειες ενήλικων μυοκαρδιοκυττάρων. Επιχειρήθηκε η ενίσχυση του αντιοξειδωτικού αμυντικού συστήματος, έτσι ώστε να αποτιμηθεί, τόσο η συμβολή του οξειδωτικού στρες στο μυοεκφυλισμό, όσο και η πιθανή θεραπευτική δράση των αντιοξειδωτικών στρατηγικών. Προκειμένου να αποτιμηθούν τα επίπεδα ενδοκυτταρικής οξειδωτικής καταπόνησης αναπτύχθηκε νέα μέθοδος απομόνωσης ενήλικων μυοκαρδιοκυττάρων από μυ. Επειδή τα μιτοχόνδρια αποτελούν το κύριο στόχο των παρατηρούμενων αλλοιώσεων, επιχειρήθηκε πρώτα ο προσδιορισμός των γενικών αλλαγών που παρατηρούνται στο μιτοχονδριακό πρωτέωμα. Πράγματι, οι παρατηρούμενες αλλαγές στα επίπεδα πρωτεϊνικής έκφρασης ενίσχυσαν την αρχική υπόθεση. Στη συνέχεια εκτιμήθηκαν τα ενδοκυτταρικά επίπεδα ROS σε καλλιέργειες ενήλικων 8 μυοκαρδιοκυττάρων χρησιμοποιώντας φθορίζοντες ιχνηθέτες. Ανάλυση των αποτελεσμάτων έδειξε ότι υπάρχουν αυξημένα επίπεδα ROS στα μυοκαρδιοκύτταρα απουσία δεσμίνης. Επιπλέον, διερευνήθηκε το μιτοχονδριακό μεμβρανικό δυναμικό, το οποίο είναι ενδεικτικό της σωστής μιτοχονδριακής λειτουργίας, χρησιμοποιώντας ειδικό φθορίζοντα ιχνηθέτη. Διαπιστώθηκε ότι υπάρχουν σημαντικές αλλοιώσεις σε αρκετά μυοκαρδιοκύτταρα απουσία δεσμίνης. Με σκοπό (1) να επιβεβαιωθεί η ύπαρξη οξειδωτικού στρες in vivo, (2) να αποτιμηθεί η συμβολή του στο φαινότυπο του μυός χωρίς δεσμίνη και (3) να εκτιμηθεί η θεραπευτική δυνατότητα της προστασίας έναντί του, το αντιοξειδωτικό αμυντικό σύστημα ενισχύθηκε in vivo, χρησιμοποιώντας το μυ χωρίς δεσμίνη ως μοντέλο καρδιακής ανεπάρκειας. Για το σκοπό αυτό δημιουργήθηκαν διαγονιδιακοί μύες που υπερεκφράζουν στο μυοκάρδιο τα αντιοξειδωτικά ένζυμα καταλάση και υπεροξειδική δυσμουτάση (MnSOD). Η καταλάση αποτοξινώνει τα κύτταρα από το H2O2 μετατρέποντας το σε νερό και οξυγόνο. Η μυοκαρδιακή υπερέκφραση καταλάσης μελετήθηκε σε υπόβαθρο απουσίας δεσμίνης. Το επίπεδο υπερέκφρασης αποτιμήθηκε σε επίπεδο τόσο πρωτεϊνικό, όσο και ενζυμικής ενεργότητας. Ο καρδιοπροστατευτικός ρόλος της καταλάσης αποτιμήθηκε ως συνιστώσα των επιπέδων ινωδών αλλοιώσεων, της υπερδομής και της καρδιακής συστολικής λειτουργίας. Η υπερέκφραση καταλάσης στο μυοκάρδιο μυών χωρίς δεσμίνη οδηγεί σε σημαντική μείωση των ενδοκυτταρικών επιπέδων ROS και της έκτασης ινωδών αλλοιώσεων, μειώνει το μυοεκφυλισμό και βελτιώνει την καρδιακή συστολική λειτουργία. Τα αποτελέσματα αυτά επιβεβαιώνουν τη συμβολή του οξειδωτικού στρες και ειδικά του H2O2 στην ανάπτυξη μυοκαρδιοπάθειας και καρδιακής ανεπάρκειας στο μυ χωρίς δεσμίνη και υπογραμμίζουν τη θεραπευτική δυνατότητα της υπερέκφρασης καταλάσης. Η MnSOD εντοπίζεται στη μιτοχονδριακή μήτρα και μετατρέπει το υπεροξειδικό ανιόν σε υπεροξείδιο του υδρογόνου. Η καρδιακή υπερέκφραση MnSOD μελετήθηκε σε υπόβαθρο απουσίας δεσμίνης. Υπερέκφραση της MnSOD μόνο σε ενδιάμεσα επίπεδα οδηγεί σε μείωση των επιπέδων του υπεροξειδικού ανιόντος και των ινωδών αλλοιώσεων στο μυοκάρδιο απουσία δεσμίνης. Επιπλέον, παρατηρήθηκε βελτίωση της μυοκαρδιακής υπερδομής, καθώς και μέτρια βελτίωση της καρδιακής συστολικής λειτουργίας. Η υποβολή μυών χωρίς δεσμίνη που υπερεκφράζουν MnSOD σε υποχρεωτική άσκηση είχε ως αποτέλεσμα το θάνατο. Αυτή η κατάληξη δεν παρατηρήθηκε όταν στο μυοκάρδιο χωρίς δεσμίνη 9 υπερεκφράζονταν τόσο η καταλάση, όσο και η MnSOD. Αυτό το αποτέλεσμα υποδηλώνει ότι το H2O2 είναι σημαντικός διαμεσολαβητής της παρατηρούμενης θνησιμότητας. Είναι ενδιαφέρον ότι η MnSOD έχει «μεικτή» συμβολή στην αποτοξίνωση από ΕΜΟ, καθώς διασπά μία δραστική ένωση δημιουργώντας ταυτόχρονα μία άλλη. Είναι συνεπώς πολύ σημαντικό η υπερέκφραση αυτού του αντιοξειδωτικού ενζύμου να πραγματοποιείται με επίγνωση των επιβλαβών συνέπειών του. Συνολικά, τα αποτελέσματα που παρουσιάζονται εδώ επιβεβαιώνουν τη συμβολή της οξειδωτικής καταπόνησης στην ανάπτυξη κληρονομικής μυοκαρδιοπάθειας και καρδιακής ανεπάρκειας, καθώς και τη θεραπευτική ικανότητα των διαφορετικών αντιοξειδωτικών στρατηγικών και του συνδυασμού τους. / Recent studies support the contribution of oxidative stress in the development of cardiovascular diseases. Oxidative stress has been strongly linked to cell death and cardiac remodeling processes, all hallmarks of heart failure. Mice null for desmin, which is the major muscle specific intermediate filament protein, develop dilated cardiomyopathy and heart failure characterized by mitochondrial defects and cardiomyocyte death accompanied by extensive calcification and fibrosis, thus providing a very good model for heart failure. Several cellular and biochemical alterations in the hearts of these mice strongly suggested that oxidative stress is one of the mechanisms contributing to the pathogenesis of the phenotype. The defects in mitochondrial structure and function, hallmarks of the desmin null mouse phenotype, provide the most important indications for the existence of oxidative stress, as the respiratory chain is the most important source of reactive oxygen species (ROS) in cardiomyocytes. In order to delineate the existence of oxidative stress in the desmin null myocardium and therefore its participation in the development of the myocardial degeneration we sought both in vitro and in vivo approaches. The existence of oxidative stress was addressed in primary adult cardiomyocytes. The reinforcement of the antioxidant defense system was pursued, in order to assess the contribution of oxidative stress in the myocardial degeneration, as well as the therapeutic potential of antioxidant strategies. To assess intracellular oxidative stress a new method for the isolation of adult mouse cardiomyocytes was developed. Since mitochondria were the target of pathology, we wanted to first determine global changes in the mitochondrial proteome. The observed changes in protein levels reinforced the original hypothesis. Intracellular reactive oxygen species were measured using fluorescent probes in adult cardiomyocyte cultures. Analysis of the above data showed that there are increased levels of ROS in desmin null cardiomyocytes. Furthermore, the mitochondrial membrane potential, which is indicative of proper mitochondrial function, was investigated using a fluorescent probe. It was found altered in a subset of the desmin null cardiomyocytes. In order to (1) verify the existence of oxidative stress in vivo, (2) assess its contribution to the phenotype of desmin null mice and (3) evaluate the therapeutic 5 potential of protecting against it, the antioxidant defense system was fortified in vivo using the desmin null mouse as a heart failure model. Towards this goal transgenic mice overexpressing the antioxidant genes catalase and manganese superoxide dismutase (MnSOD) were created. Catalase detoxifies the cells from hydrogen peroxide by converting it to water and oxygen. Cardiac specific overexpression of catalase was brought to a desmin null background. The level of overexpression was assessed by measuring protein levels and enzyme activity. The cardioprotective effect of catalase was assessed in terms of fibrotic lesion extent, ultrastructure and cardiac systolic function. Overexpression of catalase in the heart of desmin null mice leads to marked decrease in intracellular ROS levels and significant decrease in fibrotic areas, ameliorates the myocardial degeneration and improves cardiac function. These data support the contribution of oxidative stress and in particular of the ROS hydrogen peroxide in the development of cardiomyopathy and heart failure in the desmin null mouse and underscore the therapeutic potential of catalase overexpression. MnSOD in localized in the mitochondrial matrix and converts superoxide anion to hydrogen peroxide. Cardiac specific overexpression of MnSOD was studied in a desmin null background. Overexpression of MnSOD only at moderate levels leads to a significant reduction of fibrotic lesion in the desmin null myocardium. Furthermore, an improvement of the myocardial ultrastructure was observed, as well as a moderate improvement of cardiac systolic function. These data suggest that another ROS, superoxide anion, contributes to the development of cardiomyopathy and heart failure in the desmin null mouse and that MnSOD, when overexpressed at moderate levels, offers cardioprotective effect. When the mice overexpressing MnSOD in the desmin null myocardium were challenged to exercise an absolute reduction of survival was observed. This defect was completely reversed when desmin null mice overexpressed both MnSOD and catalase. This suggests that hydrogen peroxide is an important mediator of the observed lethality. It is of note that MnSOD retains a contradictory antioxidant role, both breaking down and creating a specific ROS. It is therefore of paramount importance that this antioxidant enzyme is employed with caution and awareness of its deleterious effects. Overall, the data presented here demonstrate the contribution of oxidative stress in the development of inherited cardiomyopathy and heart failure, as well as the therapeutic potential of different antioxidant strategies, and their combination.

Catalase-peroxidases (KatGs), responsible for the activation of the anti-tubercular prodrug isoniazid (INH), are unusual members of the class I plant peroxidase family that possess strong catalase activity as well as peroxidase activity. Due to their strong catalase activity and their ability to activate INH, KatGs have been the subject of intense study for many years, and thus the goal of this work is to further characterize this enzyme in the hope of gaining a better understanding into these unusual reactions. Recent successful crystallization of a few representative KatGs revealed a unique covalent Met-Tyr-Trp cross-link joined to the conserved tryptophan in the heme active site, along with a nearby arginine that is in ionic association with the cross-linked tyrosine. Using the KatG from Burkholderia pseudomallei (BpKatG) as a model, site-directed mutagenesis to these residues revealed that they were essential for catalase, but not peroxidase activity. Structural and kinetic analysis revealed that Arg426 acts as a molecular switch, moving between 2 conformations, favoring heme oxidation when not in association with Tyr238 and favoring heme reduction when in association with Tyr238 by imparting its influence on the heme through the cross-link. Analysis of the reaction with peroxyacetic acid using stopped-flow spectrophotometry revealed an initial, rapidly formed enzyme-substrate complex before the formation of the oxoferryl compound I. Kinetic characterization revealed that formation of both the enzyme-substrate complex and the oxoferryl species were dependent on peroxyacetic acid concentration implying that 2 molecules of peroxyacetic acid are required to form the oxoferryl compound I intermediate. Successful co-crystallization with INH and its co-substrate, NAD+ has revealed their binding sites for the first time in a KatG. The NAD+ binding site is 20 Å from the entrance to the heme cavity, involving interactions primarily with the ADP portion of the molecule. The best defined INH binding site is located in a funnel shaped channel on the opposite side of the protein from the entrance channel that requires the movement of a glutamate residue for binding. The structures suggest that once INH is cleaved to the isonicotinoyl radical it diffuses to the NAD+ binding site to form the final active antimicrobial compound, IN-NAD, in a non-enzymatic reaction enhanced by the enzyme’s ability to bind NAD+.

Catalase-peroxidases (KatGs), responsible for the activation of the anti-tubercular prodrug isoniazid (INH), are unusual members of the class I plant peroxidase family that possess strong catalase activity as well as peroxidase activity. Due to their strong catalase activity and their ability to activate INH, KatGs have been the subject of intense study for many years, and thus the goal of this work is to further characterize this enzyme in the hope of gaining a better understanding into these unusual reactions. Recent successful crystallization of a few representative KatGs revealed a unique covalent Met-Tyr-Trp cross-link joined to the conserved tryptophan in the heme active site, along with a nearby arginine that is in ionic association with the cross-linked tyrosine. Using the KatG from Burkholderia pseudomallei (BpKatG) as a model, site-directed mutagenesis to these residues revealed that they were essential for catalase, but not peroxidase activity. Structural and kinetic analysis revealed that Arg426 acts as a molecular switch, moving between 2 conformations, favoring heme oxidation when not in association with Tyr238 and favoring heme reduction when in association with Tyr238 by imparting its influence on the heme through the cross-link. Analysis of the reaction with peroxyacetic acid using stopped-flow spectrophotometry revealed an initial, rapidly formed enzyme-substrate complex before the formation of the oxoferryl compound I. Kinetic characterization revealed that formation of both the enzyme-substrate complex and the oxoferryl species were dependent on peroxyacetic acid concentration implying that 2 molecules of peroxyacetic acid are required to form the oxoferryl compound I intermediate. Successful co-crystallization with INH and its co-substrate, NAD+ has revealed their binding sites for the first time in a KatG. The NAD+ binding site is 20 Å from the entrance to the heme cavity, involving interactions primarily with the ADP portion of the molecule. The best defined INH binding site is located in a funnel shaped channel on the opposite side of the protein from the entrance channel that requires the movement of a glutamate residue for binding. The structures suggest that once INH is cleaved to the isonicotinoyl radical it diffuses to the NAD+ binding site to form the final active antimicrobial compound, IN-NAD, in a non-enzymatic reaction enhanced by the enzyme’s ability to bind NAD+.

Made available in DSpace on 2014-06-11T19:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-18Bitstream added on 2014-06-13T20:46:13Z : No. of bitstreams: 1 maia_ms_dr_botfmvz.pdf: 678528 bytes, checksum: 3bcfd108f831025fba94f0aefc916387 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Objetivo deste estudo foi determinar o efeito da adição do surfactante lauril sulfato de sódio (Orvus es Paste OEP) e dos antioxidantes Trolox-C (6- hidroxi-2, 5,7,8-tetrametilcroman-2-acido carboxílico) e catalase ao meio diluidor, na motilidade espermática, integridade de membrana plasmática, acrossomal e mitocondrial, estádio de capacitação e geração de radicais livres no espermatozóide ovino pós-descongelação. Para isso, foram realizados dois experimentos. No Experimento 1, avaliou-se o efeito da adição do surfactante ao diluidor. O sêmen de 10 carneiros foi coletado por meio de vagina artificial e diluído para uma concentração final de 400x106 espermatozóides/mL no meio Tris-gema contendo OEP (0; 0,5 e 1%) ou no diluidor glicina-gema-leite (controle). A motilidade foi determinada pelo sistema computadorizado de análise de sêmen (CASA), a integridade de membrana pela combinação dos corantes fluorescente iodeto de propídio e diacetato de carboxifluoresceína (IP+DIC) e o estádio de capacitação pela técnica da CTC. Houve efeito significativo do tratamento em todos os parâmetros da motilidade, na integridade de membranas e na capacitação espermática. A adição de 0,5% ou 1% de OEP ao diluidor TRIS-gema aumentou significativamente (P<0,05) a motilidade, a integridade de membranas e o percentual de espermatozóides não capacitados, comparado ao controle. O diluidor TRIS 1 foi selecionado para utilização como meio base para o experimento 2. No Experimento 2, foi determinado o efeito da adição dos antioxidantes ao diluidor. O sêmen foi coletado e os seguintes tratamentos foram aplicados: T1- TRIS (controle), T2- TRO+ (diluidor T1 + Trolox, 50mM/108 espermatozóides), T3-CAT+ (diluidor T1 + catalase, 50mg/mL), T4- TRO/CAT+ (diluidor T1 + Trolox e Catalase, nas mesmas concentrações utilizadas no T2 e T3)...(Rsumo completo, clicar acesso eletrônico abaixo) / The aim of this study was to determine the effect of addition of surfactant sodium lauryl sulfate (Orvus es Paste OEP), and antioxidants, Trolox-C (6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxilic acid) and catalase to an extender on the motility, plasmatic and acrosomal and mitochondrial membrane integrity, capacitation status and free radicals generation of post thaw ram spermatozoa. Two experiments were performed. In Experiment 1, the effect of addition of surfactant was evaluated. The semen was collected from ten rams by artificial vagina and it was diluted to a final concentration of 400x 106 sperm/mL in egg yolk-Tris extender containing OEP (0, 0.5 and 1%) or glycinegg yolk-milk extender (control). The sperm motility was evaluated using a computer-assisted sperm analysis (CASA); the membrane integrity was verified using the fluorescent stains (propidium iodide and carboxyfluorescein diacetate) and the capacitation status determined by CTC technique. There was a significant treatment effect in all parameters involving sperm motility, membrane integrity and spermatic capacitation. The addition of OEP 0.5% or 1% to the egg yolk-Tris extender improved (P<0.05) the motility and membrane integrity and the percentage of uncapacited spermatozoa compared to the control. Then, the TRIS 1 extender was selected to be used at experiment 2. Experiment 2: the Exp 2 aimmed to determine the effect of antioxidants addition to the extender. Semen was collected and these treatments were applied: T1 - egg yolk-Tris extender (control), T2- TRO+ (T1 extender +Trolox, 50mM/108 spermatozoa), T3- CAT+ (T1 extender + catalase, 50mg/mL) and T4 TRO/CAT+ (T1 extender + trolox and catalase on the concentrations used in T2 and T3). Motility was determined by CASA system, plasmatic and acrosomal 4 membrane integrity and mitochondrial function were evaluated with PI, FITCPSA and JC-1 association...(Complete abstract, click electronic address below)

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-05-19T20:41:03Z No. of bitstreams: 1 TeseJCTR.pdf: 2688877 bytes, checksum: ce817179772a53321d1ef825e400760f (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-23T20:36:10Z (GMT) No. of bitstreams: 1 TeseJCTR.pdf: 2688877 bytes, checksum: ce817179772a53321d1ef825e400760f (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-23T20:36:17Z (GMT) No. of bitstreams: 1 TeseJCTR.pdf: 2688877 bytes, checksum: ce817179772a53321d1ef825e400760f (MD5) / Made available in DSpace on 2017-05-25T20:00:24Z (GMT). No. of bitstreams: 1 TeseJCTR.pdf: 2688877 bytes, checksum: ce817179772a53321d1ef825e400760f (MD5) Previous issue date: 2016-05-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) / Tissue repair in diabetic patients is hampered by several factors, including secretion of insulin deficiency that leads to hyperglycemia and production of free radicals. Mitochondrial dysfunction has been identified as one of the probable factors of DM. It is known that phototherapy has proliferative effects in tissues with some pathologies. This study aimed to evaluate the effects of phototherapy using laser and low power LED on tissue regeneration, oxidative stress and mitochondrial markers in diabetic animals. Methodology: 20 Swiss mice (30g, 6 weeks) for the study relating to oxidative stress and 20 Wistar rats (250 g, 8 weeks) were subjected to induction of DM (streptozotocin injection 70 mg / kg) and after 120 days follow up, suffered skin lesions and were treated with Low Level Laser Therapy and LED for 5 consecutive days and on day 6 the animals were killed and the skin removed. Another group of animals (20 male Wistar rats weighing 250 g, 8 weeks) was subjected to laser irradiation and in the LED region of the pancreas for 5 consecutive days and on day 6 animals were killed and the pancreas, liver, and gastrocnemius muscle were removed. Animals were divided into four groups: Sham (healthy control), NID (non-irradiated diabetic), DLED (diabetic irradiated by LED) and DLASER (diabetic irradiated by LED). Levels of catalase, thiobarbituric acid (TBARS), nitrite, collagen content, vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX2) Mitofusin 2 (MFN2), Mitochondrial fission 1 protein (FIS1) liver and muscle glycogen and functional insulin and glucose tests were measured. Results: It was observed in the skin of these animals, increase in collagen content and angiogenesis in irradiated diabetic animals, TBARS levels and lower nitrite in irradiated diabetic. Furthermore, COX-2 expression was higher among NID compared to SHAM and DLED groups. DLED showed the highest scores against MFN2 and FIS1 expression was higher in DLASER group. Laser and LED Therapy altered the expression of VEGF and COX2 and stimulated the proliferation of mature collagen fibers in diabetic animal wounds. About irradiation over pancreas results showed that the NID group, hepatic and muscle glycogen content were statistically lower than DLASER and DLED groups. Adding to these data, the density of the pancreatic duct and pancreatic islets were significantly different when compared to NID. These results suggest that both the low-level laser LED obtained as potential therapeutic effects on the skin and pancreas under conditions hyperglycemic and that both therapies acted modulating histological, angiogenic and mitochondrial parameters on pancreas and skin of diabetic animals. / O reparo tecidual em pacientes diabéticos é prejudicado por vários fatores, dentre eles a deficiência da secreção de insulina que conduz à hiperglicemia e consequente produção de radicais livres. A disfunção mitocondrial tem sido apontada como um dos prováveis fatores etiopatogênicos do DM mellitus. Sabe-se que a Fototerapia tem efeitos proliferativos em tecidos com algumas patologias. O presente estudo teve como objetivo avaliar os efeitos da Fototerapia utilizando-se Laser e LED de baixa Potência sobre a regeneração tecidual, estresse oxidativo e marcadores mitocondriais em animais diabéticos. Metodologia: 20 camundongos Swiss, pesando entre 30g , 6 semanas, para o estudo relacionado ao estresse oxidativo e 20 ratos Wistar pesando 250 g, 8 semanas, foram submetidos a indução do DM (injeção de streptozotocina 70mg/kg) e após 120 dias de seguimento, sofreram lesão cutânea e foram tratados com Terapia Laser de Baixa Intensidade e LED durante 5 dias consecutivos e no 6º dia os animais foram mortos e retirada a pele. Um outro grupo de animais (20 ratos Wistar pesando 250 g, 8 semanas) foi submetido à irradiação Laser e LED na região do pâncreas durante 5 dias consecutivos e no 6º dia os animais foram mortos e retirado o pâncreas, fígado e músculos gastrocnêmio. Foram divididos em quatro grupos: Sham (controle saudável), NID (diabético não irradiado), DLED(diabético irradiado pelo LED) e DLASER (diabético irradiado pelo LED). Mensurou-se níveis de catalase, àcido tiobarbitúrico (TBARS), nitrito, conteúdo de colágeno, fator de crescimento endotelial vascular (VEGF), Ciclo-oxigenase-2 (COX2) Mitofusin 2 (MFN2), Mitochondrial fission 1 protein (FIS1), glicogênio hepático e muscular bem como testes funcionais de glicose e insulina. Resultados: Observou-se na pele desses animais, aumento no conteúdo de colágeno e angiogênese nos animais diabéticos irradiados pela Terapia Laser de Baixa Intensidade 904nm bem como níveis de TBARS e nitrito menores nos diabéticos irradiados. Além disso, a expressão da COX-2 foi maior entre NID quando comparados aos grupos SHAM e DLED. DLED apresentou os maiores scores em relação a MFN2 e a expressão da FIS1 foi maior no grupo DLASER. As Terapias Laser e LED alteraram a expressão de VEGF e COX2 e estimularam a proliferação das fibras colágenas maduras em feridas de animais diabéticos. Os resultados relacionados à irradiação sobre o pâncreas demonstraram que no grupo NID, o conteúdo de glicogênio hepático e muscular foram estatisticamente menores que os grupos DLASER e DLED. Somando-se a esses dados, a densidade dos ductos pancreáticos e ilhotas pancreáticos diferiram significativamente quando comparados ao grupo diabético controle.Constatamos com esses resultados que tanto o Laser de Baixa Intensidade quanto o LED obtiveram potenciais efeitos terapêuticos sobre a pele e o pâncreas sob condições hiperglicêmicas e que ambas as terapias atuaram modulando parâmetros histológicos, angiogênicos e mitocondriais no pâncreas e pele dos animais diabéticos. / CAPES: 006648/2015-00. / FAPESP: 2010/07194-7.