Bei der Entdeckung der Glutathionperoxidase-2 (GPx2) wurde zunächst davon ausgegangen, dass die Funktion dieses Enzyms im Kryptengrund des Colons einzig in der Reduktion von H2O2 besteht. Im Laufe der weiteren Erforschung zeigte sich, dass GPx2 auch in verschiedenen Tumorgeweben vermehrt exprimiert wird. Dabei wird diskutiert, ob die Wirkung von GPx2 im Tumor eher als pro- oder als antikarzinogen einzustufen ist. Mehrere Experimente in vitro und in vivo zeigten antiinflammatorische Eigenschaften der GPx2. Aufgrund dieser Befunde wird derzeit über weitere Funktionen der GPx2 spekuliert. In dieser Arbeit wurde die physiologische Funktion von GPx2 näher erforscht, dazu wurden Wildtyp- und GPx2-Knockout-Mäuse in Hinblick auf Veränderungen der Enzymexpression und der Colonmorphologie untersucht. Es wurden drei verschiedene Selendiäten verfüttert: selenarmes, selenadäquates und selensupplementiertes Futter. Unter physiologischen Bedingungen ist am Kryptengrund des Colons, innerhalb der proliferierenden Zone, die Mitoserate am höchsten. Der Großteil der apoptotischen Zellen ist hingegen an der Kryptenspitze vorzufinden. Durch den Knockout von GPx2 kam es zu einer signifikanten Erhöhung der Apoptoserate am Kryptengrund. Dabei war der größte Effekt auf selenarmem Futter zu verzeichnen. Hierbei wurde sogar eine Veränderung der Colonmorphologie dokumentiert, da die Verschiebung der Proliferationszone in Richtung Kryptenspitze eine Verlängerung der Krypten nach sich zog. Im Wildtyp wurden keine Apoptosen im Kryptengrund detektiert. GPx1 wird unter physiologischen Bedingungen im Gegensatz zur GPx2 in der Kryptenspitze exprimiert und ist im Selenmangel nicht mehr detektierbar. Der Knockout von GPx2 erhöhte die GPx1-Expression im Kryptengrund auf allen drei Selendiäten. Diese Überexpression von GPx1 am Kryptengrund soll vermutlich den Verlust von GPx2 an dieser Stelle kompensieren. Da jedoch dort die massive Apoptoserate detektiert wurde, kann die GPx1 nicht die komplette Funktion von GPx2 kompensieren. Diese Ergebnisse deuten darauf hin, dass die Funktion von GPx2 nicht nur in der Reduktion von H2O2 liegt. Vielmehr kann eine Rolle bei der Aufrechterhaltung der Homöostase von Zellen postuliert werden. Ein weiterer Bestandteil dieser Arbeit war die Klärung der Frage, welchen Einfluss GPx2 auf die entzündungsassoziierte Colonkarzinogenese ausübt. In dem hierfür verwendeten AOM/DSS-Model wird der karzinogene Prozess durch Entzündung vorangetrieben. Es erfolgte sowohl im Wildtyp als auch im GPx2-Knockout zum einen die Bewertung des Entzündungsstatus des Colons und zum anderen wurde die Anzahl von ACF und Tumoren verglichen. Das Colon im GPx2-Knockout war wesentlich stärker entzündet als im Wildtyp. Diese Ergebnisse bestätigen die für die GPx2 postulierte antiinflammatorische Funktion. Normalerweise führt eine Erhöhung der Mitoseanzahl zur Regeneration des entzündeten Gewebes. Jedoch beeinflusst der Verlust von GPx2 vermutlich den Ablauf der Entzündung, indem beispielsweise die Regeneration des Gewebes durch die enorm hohe Apoptoserate am Kryptengrund verlangsamt wird. Des Weiteren hatten sich im GPx2-Knockout tendenziell mehr Tumore entwickelt. Somit korrelierte die Entzündung des Colons mit der Entwicklung von Tumoren. Der Verlust von GPx2 begünstigte vermutlich sowohl die Tumorinitiation als auch die Tumorprogression. Allerdings stimulierte die Expression von GPx2 ebenfalls das Tumorwachstum. Es kann geschlussfolgert werden, dass eine adäquate GPx2-Expression vor Entzündung schützt und somit das Risiko für Colonkrebs senkt. Ob GPx2 aber insgesamt pro- oder antikarzinogen wirkt, hängt vermutlich vom Stadium des Colonkarzinogenese ab. / Since the detection of glutathione peroxidase-2 (GPx2) it was assumed that reducing hydroperoxides is the only function of this enzyme in the crypt ground of the colon. But further studies showed that GPx2 is also highly expressed in tumor tissue. However, it is not known whether it acts a pro- or anticarcinogenic manner at this site. In vitro and in vivo experiments elucidate antiinflammatory features of GPx2, based on these findings additional functions of GPx2 are discussed. In this dissertation the physiological function of GPx2 was investigated. For this purpose in wild type and GPx2-knockout mice, changes of enzyme expression and colon morphology were analyzed. The mice were fed three diets containing different selenium concentrations: selenium deficient, selenium adequate and selenium supplemented. Under physiological conditions the mitosis rate is highest in the proliferating zone in the crypt ground of the colon. The majority of apoptotic cells are located at the tip of the crypt. The knockout of GPx2 significantly increased the rate of apoptosis in the crypt ground. The greatest effect was documented on the selenium deficient diet. Here, changes of the colonic morphology were detectable, because the shift of the proliferating zone towards the tip of the crypt lead to an extension of the crypts. In the wild type mice no apoptotic cells were detected on the crypt ground. Under physiological conditions GPx1, in contrast to GPx2, is mainly expressed on the top of the crypt, and this enzyme is no longer detectable under selenium deficiency. The knockout of GPx2 increased the expression of GPx1 in the crypt ground of the colon on all three selenium diets. It is likely that this over expression of GPx1 compensates for the loss of GPx2. However the massive apoptotic rate in the crypt ground shows that GPx1 can not compensate the complete function of GPx2. These results elucidate that GPx2 not only functions as a hydroperoxide reducer, but that it is also important for the maintenance of the stem cell character and the homeostasis of cells. The question if GPx2 influences the inflammation triggered by the coloncarcinogenic process was next assessed in this dissertation. Therefore the AOM/DSS model was used to trigger the carcinogenic process through inflammation. The amount of aberrant crypt foci (ACF) and tumors in the colon were analyzed in both wild type and GPx2-knockout mice. However initially the inflammation status was compared between the two genotypes. The inflammation of the colon was stronger in the GPx2-knockout mice than in wild type. These results support the postulated antiinflammatory features of GPx2. The loss of GPx2 may influence the inflammation process by decelerating the regeneration of the tissue caused by the increased apoptotic rate in the proliferating zone. Additionally, the GPx2-knockout mice developed more tumors in the colon. Therefore the inflammation of the colon correlated with the development of tumors. The loss of GPx2 may have enhanced both tumor initiation and progression. But the expression of GPx2 also stimulated the growth of tumors. These results indicate that an adequate GPx2-expression can protect from colonic inflammation, and therefore decrease the risk of developing colon cancer. Whether GPx2 acts in a pro- or anticarcinogenic manner appears to depend on the state of the carcinogenic process.

The thesis focuses on the development of new homogeneous gels for the separation of drug enantiomers, peptides, DNA and virus by electrophoresis and electrochromatography in capillaries and microchips. This type of separation media offers high resolution and small zone broadening. Compared to particulate beds the resolution in this type of separation media is high because the eddy diffusion is zero and the resistance to mass transfer is small, since the diffusional distance between two polymer chains in the gel is considerably shorter than that between two beads in a packed bed. The gels have been characterized in terms of plate heights, plate numbers, resolution, etc. Gels of agarose, polyvinyl alcohol, albumin and polyacrylamide have been employed for electrochromatography or electrophoresis. N,N’-methylene-bisacrylamide, the most widely used crosslinker in polyacrylamide gels, was exchanged for allyl-β-cyclodextrin to get a multi-purpose gel, i.e., a separation medium the separation properties of which is determined not only by the polyacrylamide chains, but also by β-cyclodextrin with its complexation power. A cost-effective, hybrid microdevice has been designed for fast electrophoretic and electrochromatographic analyses as well as for microchromatography. It consists of a fused silica capillary mounted on a supporting plate which integrates most of the compartments necessary for automation and sensitive detection at short UV wavelengths.

S.absconditiflora is one of the endemic Salvia species grown in Turkey, which is consumed as a herbal tea. Because of the presence of high amounts of vesicles on their leaves, S.absconditiflora is very rich in active compounds. S.absconditiflora water extract was investigated for its antioxidant capacity by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. Total phenolic and total flavonoid contents were quantified by spectrophotometric methods. LC-MS/MS analyses revealed the presence and quantities of caffeic acid, luteolin rutin and coumaric acid. Cytotoxic effects of water extract of S.absconditiflora on breast cancer cell lines (MCF-7 and MDA-MB-231) were examined via XTT colorimetric assay and Trypan Dye Exclusion cell viability assay. IC50 values for each cell line at 24 and 48 hours were determined. The results indicated that water extract of leaves of S.absconditiflora could inhibit cell proliferation in MCF-7 and MDA-231 cells in dose dependent but not in time dependent manner. Effects of S.absconditiflora water extract on the expression of glutathione-S-transferases (GSTs) in MCF-7 and MDA-MB-231 cells were investigated with qRT-PCR technique. IC50 values calculated in XTT experiment for 24h incubation was used as cytotoxic extract concentration. It was found that treatment of MCF-7 cells with 1,558 mg/ml of extract enhanced an increase in expression as 2 and 2,8 fold in GSTO1 and GSTZ1 genes, respectively. Treatment of MDA-MB-231 cells with 1,131 mg/ml of extract resulted in 1,57 fold increase for GSTO1 and 1,56 fold increase for GSTZ1.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 113-137).

Στόχος της παρούσας διατριβής ήταν η μελέτη της σκληρωτιακής διαφοροποίησης όπως εκφράζεται στις τέσσερις βασικές μορφές της στους μυκηλιακούς μύκητες S. rolfsii, R. solani, S. sclerotiorum και S. minor σε σχέση με τη θειολική οξειδοαναγωγική κατάσταση (ΘΟΚ), αλλά και με το οξειδωτικό στρες, στo πλαίσιο της θεωρίας της οξειδωτικώς επαγόμενης σκληρωτικής διαφοροποίησης (που προτάθηκε από το εργαστήριό μας το 1997). Ως μάρτυρες για τους υπό μελέτη μύκητες χρησιμοποιήθηκαν αντίστοιχα μη σκληρωτιογόνα στελέχη. Στη διαμόρφωση της ΘΟΚ συμμετέχουν πολλά μη πρωτεϊνικά, πρωτεϊνικά και μεικτά θειολικά και δισουλφιδικά συστατικά, ανάμεσα στα οποία η γλουταθειόνη θεωρείται ο κύριος συνδετικός κρίκος. Επειδή στη διεθνή βιβλιογραφία δεν υπήρχε κατάλληλη μεθοδολογία για τον υπολογισμό όλων των παραγόντων της ΘΟΚ για την εκπλήρωση του στόχου της μελέτης, αναπτύχθηκε μια νέα μέθοδος εφαρμόσιμη εκτός από τους υπό μελέτη μύκητες σε όλους τους οργανισμούς. Παράλληλα, αναπτύχθηκε και μια νέα μέθοδος για την εξειδικευμένη ποσοτικοποίηση του μικρο-κατακερματισμένου DNA, καθώς στη διεθνή βιβλιογραφία οι περισσότερες υπάρχουσες μέθοδοι ήταν ποιοτικές και όχι εξειδικευμένες ως προς το μέγεθος των θραυσμάτων DNA. Επιπρόσθετα, η εκτίμηση των οξειδωτικών βλαβών στο DNA έγινε και με τη μέτρηση ενός γενικού δείκτη αυτών των βλαβών (εγκοπές και σπασίματα) με υπάρχουσα μέθοδο ύστερα από σημαντική τροποποίησή της. Επιπλέον, χρησιμοποιήθηκε η ποσοτικοποίηση της ειδικής ενεργότητας των βασικών ενζύμων που μετέχουν στη ρύθμιση της ΘΟΚ και της σχετιζόμενης με τη ΘΟΚ ενδογενούς βιταμίνης C, καθώς και η ποσοτικοποίηση της οξειδωτικής καταστροφής των μεμβρανικών λιπιδίων. Επιπλέον, χρησιμοποιήθηκαν συγκεκριμένοι εξωγενείς τροποποιητές της ΘΟΚ. Τα αποτελέσματα της μελέτης επιβεβαίωσαν την αναγκαιότητα της ανάπτυξης των νέων μεθόδων για την ποσοτικοποίηση των παραμέτρων της ΘΟΚ και των οξειδωτικών βλαβών στο DNA ως προς την εξαγωγή πιο ασφαλών συμπερασμάτων για τη συσχέτιση της ΘΟΚ με το οξειδωτικό στρες. Συγκεκριμένα, δείχθηκε ότι οι τέσσερις μορφές σκληρωτιακής διαφοροποίησης εξαρτώνται άμεσα από το οξειδωτικό στρες, και ότι η σχετιζόμενη με αυτό ΘΟΚ μεταβάλλεται με διαφορετικό τρόπο σε κάθε μορφή σκληρωτιακής διαφοροποίησης. Το συμπέρασμα αυτό βασίστηκε στη διαπίστωση (α) της ύπαρξης διαφορετικών για κάθε μορφή διαφοροποίησης ενδογενών θειολικών παραμέτρων της ΘΟΚ και αντιοξειδωτικών ενζύμων και (β) διαφορετικών προφίλ-διαβαθμίσεων των συγκεντρώσεων των παραμέτρων της ΘΟΚ και των δεικτών οξειδωτικού στρες. Επιπρόσθετα, ο άμεσος αντιοξειδωτικός ρόλος των ενδογενών μη πρωτεϊνικών θειολικών (με –SH ομάδα) παραμέτρων της ΘΟΚ στη σκληρωτιακή διαφοροποίηση επιβεβαιώθηκε και από τη μείωση της τελευταίας κατά την τεχνητή αύξηση αυτών των παραμέτρων ύστερα από τη χορήγηση τροποποιητών της ΘΟΚ, γεγονός που θα μπορούσε να αποδοθεί στην εξ αυτών αύξηση της ενδογενούς γλουταθειόνης και της κυστεΐνης. Το συμπέρασμα αυτό ενισχύεται και από την ανάλογη αντιοξειδωτική επίδραση των χορηγούμενων θειολών-τροποποιητών της ΘΟΚ Ν-ακετυλοκυστεΐνη (που ανιχνεύτηκε και ενδοκυτταρίως) και γλουταθειόνη και της μη πρωτεϊνικής θειολικής ουσίας-μάρτυρα διθειοθρεϊτόλη. Αξίζει να σημειωθεί ότι παρόλο που τα επίπεδα της ενδογενούς γλουταθειόνης και του δισουλφιδίου της καθώς και η μεταξύ τους αναλογία συσχετίζονται συνήθως με το οξειδωτικό στρες στη διεθνή βιβλιογραφία, στην παρούσα μελέτη διαπιστώθηκε ότι δεν συμβαδίζουν με τους αποδεδειγμένους δείκτες του οξειδωτικού στρες υπεροξείδωση των λιπιδίων και οξειδωτικές βλάβες του DNA. Έτσι, ο ρόλος των ενδογενών θειολικών παραμέτρων της ΘΟΚ στη σκληρωτιακή διαφοροποίηση αποδεικνύεται περίπλοκος, ένεκα του ότι δεν δρουν μόνο ως άμεσοι αντιοξειδωτές αλλά και ως υποστρώματα των βασικών ενζύμων που εμπλέκονται στη ρύθμιση της ΘΟΚ. Τέλος, και στους τέσσερις τύπους σκληρωτίων ανιχνεύθηκαν υψηλές συγκεντρώσεις θειολικών παραμέτρων της ΘΟΚ και βιταμίνης C, καθώς και ενζύμων που μετέχουν στη ρύθμιση της ΘΟΚ, υποδηλώνοντας ότι ο πιθανός στόχος της συσσώρευσης όλων των παραπάνω παραμέτρων της ΘΟΚ στα σκληρώτια είναι η αξιοποίησή τους για αντιοξειδωτική προστασία των υφών του μυελού του πυρήνα τους κατά το χρονικό διάστημα της αναπτυξιακής τους στασιμότητας, μέχρι να βρεθούν σε κατάλληλες συνθήκες για βλάστηση. / The aim of this dissertation was the study of sclerotial differentiation, represented by four basic sclerotial types expressed by the filamentous fungi S. rolfsii, R. solani, S. sclerotiorum and S. minor in relation to thiol redox state (TRS) and oxidative stress under the theory of the oxidative induction of sclerotiogenesis (proposed by our lab in 1997). Non-sclerotium producing fungi were used as controls of the corresponding wild type strains. TRS is known to be comprised of many non-protein, protein and mixed thiol and disulfide components, with glutathione being the central component. Since there was not available any appropriate method for the determination of all TRS components, a new method was developed for the purpose of this study and its applicability was extended to any organism. Additionally, another new method was developed for the quantification of small-sized fragmented DNA, since the existing methods were qualitative and not discriminating DNA size. Complementarily, oxidative damage of DNA was also estimated by a general DNA damage (nicks and fragments) marker by a published method that was significantly modified. TRS was also evaluated by the measurement of the specific activity of certain enzymes that regulate it, by the quantification of the TRS-related antioxidant vitamin C, as well as by the determination of oxidative damage on membrane lipids. Certain exogenous TRS modulators were also used. The results of this study verified the need for the development of the new methods for the determination of TRS parameters and oxidative damage of DNA, since their use allowed a more accurate estimation of the relation of TRS with oxidative stress. Specifically, it was found that the four studied types of sclerotial differentiation are directly related with oxidative stress, and that TRS components are variously formed and dependent on the type of sclerotial differentiation. This conclusion was based on (a) the existence of different (for each type of sclerotial differentiation) endogenous TRS thiol parameters and TRS-related antioxidant enzymes and (b) the different profiles-concentration gradients of TRS parameters and oxidative markers. Moreover, the direct antioxidant role of the endogenous TRS non-protein thiol parameters in sclerotial differentiation was verified by the decrease of the latter during the endogenous increase of those parameters after administration of the specified TRS-modulating substances. This could be attributed to the resulting by those substances increase of the endogenous glutathione and cysteine. This result was also supported by the antioxidant effect of the administered TRS-modulating thiol substances N-acetylcysteine (which was traced also intracellularly) and glutathione, and of the non-protein control-thiol dithiothreitol. It is worth noting that in spite of the fact that endogenous glutathione, its disulfide, and their resulting ratio are usually considered as oxidative stress markers, it was found that in this study these markers are not in accordance with the specific markers of oxidative stress lipid peroxidation and DNA damage in the evaluation of the role of oxidative stress and TRS in sclerotial differentiation. Thus, the role of the thiol parameters of TRS in sclerotial differentiation is very complicated in light of the fact that they do not act only as direct antioxidants but also as substrates of the studied TRS-related enzymes. Finally, in all sclerotial types have been found high levels of intracellular thiol parameters of TRS and vitamin C as well as enzymes that participate to TRS regulation, implying that their possible role is to provide antioxidant protection of the hyphae of the sclerotial medulla until the environmental conditions become appropriate for their germination.

No description available.

No description available.

Ischemia caused by the disruption of blood flow results in kidney damage and dysfunction. This study investigated the effects of 30, 60 or 120 minutes of renal ischemia on the levels of glutathione (GSH), the major antioxidant inside cells. Kidneys from anesthetized female Lewis rats (9 months old) were clamped to induce ischemia and then homogenized and separated into cytosolic and mitochondria fractions by differential centrifugation. The levels of GSH and oxidized glutathione (GSSG) in the fractions were measured spectrophotometrically or by capillary electrophoresis. A significant reduction in GSH levels in the cytosol and mitochondria was seen only after the kidney underwent 60 minutes of ischemia. The significant decrease in GSH was accompanied by an increase in the GSSG/GSH ratio and an alteration in the glutathione redox ratio (i.e., GSH/total glutathione). This study demonstrates that an ischemic time of 60 minutes or longer is necessary to cause depletion of GSH levels in the rat kidney. / Department of Physiology and Health Science

Fetal Alcohol Spectrum Disorders (FASD) are the most common cause of cognitive impairment in the United States (Sokol et al., 2003). In young school children in North America and some Western European countries, recent reports have estimated the prevalence of FASD to be as high as 2-5% (May et al., 2009). Currently there are no widely accepted treatment options for FASD, mainly due to the fact that the underlying neurological deficits that occur with prenatal ethanol exposure (PNEE) are still largely unknown. This thesis examines the long-lasting changes that occur in the hippocampus following PNEE using biochemical and electrophysiological techniques. We find that PNEE produces a reduction of the endogenous antioxidant glutathione (GSH), resulting in an increase in oxidative stress that is accompanied by long-lasting reductions in long-term potentiation (LTP) of synaptic efficacy. Interestingly, males exhibited greater deficits in synaptic plasticity than females, despite similar reductions in GSH in both sexes. By depleting GSH in control animals we determined that LTP in the DG of female animals is more resistant to changes in GSH, which may explain the sexual dichotomy observed in these studies of PNEE. Based on these findings, ethanol-exposed animals received postnatal dietary supplementation with either a precursor of GSH, N-Acetylcysteine (NAC) or Omega-3 fatty acids. These supplements helped to counteract the effects of PNEE and improved hippocampal function. The findings in this thesis support the hypothesis that increasing antioxidant capacity can enhance hippocampal function, which in turn may improve learning and memory in FASD, providing a therapeutic avenue for children suffering with these disorders. / Graduate / 0570 Nutrition / 0317 Neuroscience / anna.r.patten@gmail.com

Fetal Alcohol Spectrum Disorders (FASD) are the most common cause of cognitive impairment in the United States (Sokol et al., 2003). In young school children in North America and some Western European countries, recent reports have estimated the prevalence of FASD to be as high as 2-5% (May et al., 2009). Currently there are no widely accepted treatment options for FASD, mainly due to the fact that the underlying neurological deficits that occur with prenatal ethanol exposure (PNEE) are still largely unknown. This thesis examines the long-lasting changes that occur in the hippocampus following PNEE using biochemical and electrophysiological techniques. We find that PNEE produces a reduction of the endogenous antioxidant glutathione (GSH), resulting in an increase in oxidative stress that is accompanied by long-lasting reductions in long-term potentiation (LTP) of synaptic efficacy. Interestingly, males exhibited greater deficits in synaptic plasticity than females, despite similar reductions in GSH in both sexes. By depleting GSH in control animals we determined that LTP in the DG of female animals is more resistant to changes in GSH, which may explain the sexual dichotomy observed in these studies of PNEE. Based on these findings, ethanol-exposed animals received postnatal dietary supplementation with either a precursor of GSH, N-Acetylcysteine (NAC) or Omega-3 fatty acids. These supplements helped to counteract the effects of PNEE and improved hippocampal function. The findings in this thesis support the hypothesis that increasing antioxidant capacity can enhance hippocampal function, which in turn may improve learning and memory in FASD, providing a therapeutic avenue for children suffering with these disorders. / Graduate / 0570 Nutrition / 0317 Neuroscience / anna.r.patten@gmail.com