A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

Cancer is the second leading cause of death after cardiovascular diseases in the world. Many of the chemical carcinogens need metabolic activation that catalyzed by cytochrome P450 and Phase II enzymes in order to exert their genotoxic and carcinogenic effects. Hence one possible mechanism is that phenolic compounds may alter anticarcinogenic effects is through an interaction with these enzymes either by the inhibition or activation of certain forms, leading to a reduced production of the ultimate carcinogen. Therefore anti-carcinogen activity of tannic acid, a hydrolyzable plant polyphenol, has a crucial importance to prevent conversion of pro-carcinogens to their carcinogenic form. Tannic acid is produced from secondary metabolism of plants and is found in edible vegetables, fruits and nuts, especially tea, cocoa, coffee and wine. In the present work, modulation of rabbit liver and kidney microsomal P450 dependent aniline 4-hydroxylase, N-nitrosodimethylamine N-demethylase and p-nitrophenol hydroxylase activities and cytosolic phase II enzymes / glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase:1 (NQO1) were studied in the presence of tannic acid at concentrations ranging from 0.5 &micro / M to 150 &micro / M in the reaction medium. The results obtained in this study were shown that tannic acid significantly inhibited the activities of p-nitrophenol hydroxylase, aniline 4-hydroxylase, NDMA N-demethylase, glutathione S-transferase, NAD(P)H:quinine oxidoreductase 1. Tannic acid was found to be the most potent inhibitor of cytosolic glutathione S-transferase with IC50 of 0.33 &micro / M and the least potent inhibitor of microsomal aniline 4-hydroxylase.with IC50 of 60.26 &micro / M. Effect of tannic acid on enzyme activities was further studied for both mode and type of inhibition. For this purpose various concentrations of the substrate were examined at various tannic acid concentrations. Lineweaver-Burk and Dixon plots were then generated from the resulting data sets. The Km value and inhibition constants (KI) were determined from double reciprocal and Dixon plot of the enzyme activity versus substrate and inhibitor concentration, respectively. Tannic acid was shown to be a noncompetitive inhibitor for liver cytosolic GST, NQO1 and microsomal aniline 4- hydroxylase enzymes with KI of 0.3 &micro / M, 41 &micro / M and 54.7 &micro / M, respectively. On the other hand, in kidney tissues, tannic acid was an uncompetitive inhibitor of cytosolic GST, while it was noncompetitive inhibitor for cytosolic NQO1 with a KI of 12.6 &micro / M. These results indicate that tannic acid may modulate cytochrome P450 dependent and Phase II enzymes and influence the metabolic activation of xenobiotics mediated by these enzymes.

Stroke, a major cause of death and disability, is described as interruption or severe reduction of blood flow in cerebral arteries. Oxidative stress plays an important role in the pathogenesis of atherosclerosis and carotid atherosclerosis is a risk factor for stroke. Combination of multiple environmental and genetic risk factors is thought to increase susceptibility to the development of this disease. Therefore, investigation of the polymorphisms of drug metabolizing enzymes is of crucial importance to determine the molecular etiology of the disease. The main objective of this study was to investigate the possible association between polymorphisms of enzymes causing oxidative stress (CYP2E1, FMO3 and NOS3) and enzymes protecting against oxidative stress (GST and NQO1), and the pathogenesis of atherosclerosis and ischemic stroke risk. The study population consisted of 245 unrelated ischemic stroke patients and 145 healthy control subjects. There was no statistically difference between the patient and control groups in terms of age and gender. Hypertension, diabetes, smoking and obesity were found to be at least 2 times more common in stroke patients than controls. While total cholesterol, triglyceride and LDL-cholesterol level were higher in stroke patients, HDL-cholesterol level was lower in stroke patients when compared to controls. In the case-control analyses for the risk of ischemic stroke, CYP2E1*5B mutant allele, *5B was found to be associated with the development of disease (Odds Ratio / OR=7.876, 95%CI=1.025-60.525, P=0.019). In addition, significant difference was observed between stroke patients and controls with respect to CYP2E1*5B genotype distribution (OR=0.869, 95%CI=1.044-62.339, P=0.017). On the other hand, in the NQO1*2 polymorphism, together with NQO1 heterozygote (*1*2), NQO1 homozygote mutant (*2*2) genotype was found protective against ischemic stroke (OR=0.627, 95%CI=0.414-0.950, P=0.027). The risk of hypertensive individuals having stroke was highest in the FMO3 472GA group (OR=6.110, P=0.000). In diabetics, GSTP1 313AG genotype was found to be the highest risk factor for stroke (OR=3.808 P=0.001). On the other hand, NQO1 *1*2 heterozygote genotype was associated with 5 times increased risk for stroke in smokers (OR=5.000, P=0.000). In addition GSTM1 present genotype constituted 8 times increased stroke risk in obese individuals (OR=8.068, P=0.001). Logistic regression analysis revealed that hypertension, diabetes mellitus, obesity and smoking were significant risk factors for stroke. On the other hand, HDL-cholesterol and having NQO1 *1*2 heterozygote genotype were found to be protective factors against stroke.

Acute lymphoblastic leukemia (ALL) is the most common type of cancer affecting children in the world and in our country. The exact molecular etiology of the disease still remains to be elucidated. This study hypothesized that four genes, namely CYP2E1*5B, *6, and *7B, NQO1*2 SNPs, GSTM1 null and GSTT1 null, alone or in combination, could contribute to the risk of development of childhood ALL. Also interactions of these polymorphisms with non-genetic risk factors were investigated. The genotyping of these polymorphisms were done on 209 healthy subjects, and 185 patients with childhood ALL, in Turkish population. Venous blood samples were collected and genomic DNA was isolated from these samples. Genotyping was done by PCR-RFLP techniques. In the case-control analyses for the risk of development of childhood ALL, only GSTT1 null was found to be associated with the development of disease (OR= 1.8, p=0.01). CYP2E1*5B and *6 combination showed an increased risk of 2.7 fold (p= 0.04). Also co-presence of CYP2E1*6-GSTT1 and CYP2E1*7B-GSTT1 polymorphisms increased the risk significantly above 4.0 fold. The risk increased more to 7.6 fold, when CYP2E1*5B,*6 and GSTT1 null were considered together, with borderline significance (p=0.04). When interaction of exposure to cigarette smoke and genetic polymorphisms were investigated, NQO1*2 and GSTM1 null were turned out to be significant risk factors for the development of disease when the parental or child&rsquo / s postnatal exposure to cigarette smoke was considered. This study presented several new findings to the literature in terms of genetic epidemiology of childhood ALL. The present work would also contribute to public health in determining the susceptibility of the Turkish population to childhood ALL.

No / NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone "trigger" by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-beta,beta,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (V(max) and K(m) values of 11.86 +/- 3.09 micromol/min/mg and 2.70 +/- 1.14 micromol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time- and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase. In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-29T21:11:40Z No. of bitstreams: 1 Silvana Sousa Paz Avaliação de marcadores....pdf: 756990 bytes, checksum: 5aac886be232eac44d86b25a30837ac4 (MD5) / Made available in DSpace on 2012-08-29T21:11:40Z (GMT). No. of bitstreams: 1 Silvana Sousa Paz Avaliação de marcadores....pdf: 756990 bytes, checksum: 5aac886be232eac44d86b25a30837ac4 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / As leucemias são malignidades hematopoiéticas, caracterizadas por subgrupos biologicamente distintos, sendo os tipos mais frequentes de cânceres em crianças e adolescentes. Polimorfismos em genes de enzimas que metabolizam xenobióticos podem estar relacionados com a inserções/deleções, polimorfismos de nucleotídeo simples (SNP’s) e variações no número de cópias e têm sido relacionados com a patogênese de algumas neoplasias hematológicas, como a leucemia linfóide aguda (LLA). O objetivo deste estudo foi o de determinar as frequências de polimorfismos em genes associados ao estresse oxidativo e metabolismo de xenobióticos (GSTT1, GSTM1, CYP2E1, NQO1 e MPO), em pacientes pediátricos com LLA, associando-as a aspectos clínicos e marcadores de evolução da doença. A casuística foi composta por 37 pacientes pediátricos seguidos na clínica ONCO e tratados pelo protocolo GBTLI-LLA 93. O perfil hematológico dos pacientes foi realizado ao diagnóstico e durante o tratamento e os polimorfismos gênicos foram investigados por reação da polimerase em cadeia - polimorfismo de tamanho de fragmento de restrição (PCR-RFLP) e por reação da polimerase em cadeia multiplex (PCR Multiplex). As análises estatísticas apresentaram significância para os valores de leucócitos totais nos D1 e D7 (p= 0,0016) e nos D1 e D14 (p= 0,0059); linfócitos nos D1 e D7 (p= 0,0088) e D1 e D14 (p= 0,0101); segmentados neutrófilos nos D1 e D7 (p= 0,0033) e D1 e D14 (p= 0,0252); blastos periféricos D1 e D7 (p< 0,0001) e D1 e D14 (p< 0,0001) e; para a contagem de blastos na medula óssea (MO) nos D1 e D15 (p<0,0001), D1 e D28 (p< 0,0001) e D15 e D28 (p= 0,0005). As frequências alélicas e genotípicas para os genes estudados estavam em equilíbrio de Hardy-Weinberg. A mutação do gene MPO foi associada a infiltração da MO (p= 0,0473) e presença de blastos no líquor (p= 0,0473). O polimorfismo do gene GSTT1 foi associado à contagem de leucócitos (p= 0,014) e plaquetas (p= 0,0034) no D1 e a contagem de leucócitos (p=0,037) e segmentados neutrófilos (p= 0,0008) no D7. A presença do polimorfismo no gene NQO1 foi associado à infiltração da MO (p= 0,0410) e a presença de blastos no líquor (p= 0,0410). Entretanto, o polimorfismo NQO1 apresentou associação com a presença de palidez (p=0,0096). Os dados encontrados corroboram em parte com dados encontrados na literatura, sendo necessária a realização de um estudo com numero maior de pacientes para confirmação dos achados relacionados aos genes investigados e a LLA. / Leukemia is characterized by biologically distinct subgroups and is the most frequent hematological malignity in childhood. Polymorphisms in genes of enzymes that metabolize xenobiotics may be related to insertions/ deletions, single nucleotide polymorphisms (SNP's) and gene copies variation and have been related to the pathogenesis of some hematologic malignancies, including acute lymphoblastic leukemia (ALL). The aim of this study was to investigate genes polymorphisms associated with the oxidative stress and xenobiotic metabolism (GSTT1, GSTM1, CYP2E1, NQO1 and MPO) in a group of childhood ALL patients, associating them with clinical evolution and prognostic markers. The casuistic was compound by 37 pediatric patients followed and treated at the clinic ONCO with the protocol GBTLI-LLA 93. The hematological profile of patients was performed at diagnosis and during treatment and gene polymorphisms were investigated by Polimerase Chain Reaction - Restriction Fragment Length Polymorfism (PCR-RFLP) and Polimerase Chain Reaction Multiplex (Multiplex PCR). Statistical analyses were significant for values of total leukocytes in D1 and D7 (p= 0.0016) and in D1 e D14 (p= 0.0059); lymphocytes in D1 and D7 (p= 0.0088), D1 and D14 (p= 0.0101); neutrophils in D1 and D7 (p= 0.0033), D1 and D14 (p= 0.0252). It was also find statistical significance at the number of peripheral blasts in D1 and D7 (p< 0.0001), D1 and D14 (p< 0.0001); the blast count in bone marrow (BM) in D1 and D15 (p<0.0001), D1 and D28 (p< 0.0001) and D15 and D28 (p= 0.0005). The allelic and genotypic frequencies of all gene polymorphism investigated were in Hardy-Weinberg equilibrium. The MPO gene mutation was associated with infiltration of the BM in D28 (p= 0.0473) and the presence of blasts in the CSF (p= 0.0473). The GSTT1 gene polymorphism was associated with leukocyte (p= 0.014) and platelet counts (p= 0.0034) in D1 and with leukocytes (p=0,037) and neutrophils counts (p= 0.0008) in D7. The NQO1 gene polymorphism presence was associated with BM infiltration at D28 (p= 0.0410) and the presence of blasts in the CSF (p= 0.0410). However, the NQO1 polymorphism was associated with the presence of pallor (p=0.0096). Result described here corroborated in part with previous described data, being necessary to carry out additional study with a larger number of patients to confirm the finding related to genes polymorphism investigated and the clinical evolution of ALL patients.

Θεωρητικό υπόβαθρο: Το οξειδωτικό στρες (ΟΣ) ορίζεται ως το παθολογικό αποτέλεσμα που προκύπτει από τη διαταραχή της ισορροπίας των κυτταρικών συγκεντρώσεων των οξειδωτικών, δραστικών ενώσεων και των αντιοξειδωτικών μορίων. Εκτός από τη βλάβη που υπόκεινται οι πρωτεΐνες και τα λιπίδια, το ΟΣ μπορεί επίσης να προκαλέσει μεταλλάξεις και επιγενετικές μεταβολές καταστρέφοντας τόσο το DNA όσο και τις πρωτεΐνες που τροποποιούν τη χρωματίνη. Παρ' όλα αυτά, στα θυρεοειδικά θυλακικά κύτταρα παράγονται σε καθημερινή βάση υψηλές ποσότητες υπεροξειδίου του υδρογόνου (H2O2), οξειδωτικής ουσίας απαραίτητης για την πραγματοποίηση της θυρεοειδικής ορμονογένεσης. Δεδομένου ότι ένα ελάχιστο ποσό οξειδωτικού φορτίου αποτελεί προϋπόθεση αφ’ενός για τη φυσιολογική λειτουργία των θυλακικών κυττάρων και αφ’ετέρου για την ανάπτυξη του θυρεοειδούς αδένα, πρόσφατα αποδείχτηκε ότι ο θυρεοειδής αδένας παρουσιάζει αυξημένη αμυντική ανταπόκριση έναντι του ΟΣ. Ωστόσο, οι ακριβείς μηχανισμοί με τους οποίους τα θυλακικά κύτταρα αντιλαμβάνονται και απαντούν στο ΟΣ παραμένουν ασαφείς. Ο NFE2-related factor 2 (Nrf2), ο οποίος κωδικοποιείται από το γονίδιο NFE2L2, είναι ένας μεταγραφικός παράγοντας ο οποίος απαντά σε σήματα κυτταρικού στρες και ανταποκρίνεται επιδρώντας στη μεταγραφή γονιδίων σε διάφορους τύπους ιστών. Σε βασικές συνθήκες, ο Nrf2 οδηγείται σε πρωτεασωματική αποικοδόμηση μέσω του κυτταροπλασματικού του αναστολέα, Keap1, ενώ σε συνθήκες ΟΣ, η αποικοδόμηση του Nrf2 δεν είναι δυνατή και ο Nrf2 εισέρχεται στον πυρήνα ώστε να ενεργοποιήσει τη μεταγραφή αντιοξειδωτικών γονιδίων όπως του γονιδίου Nqo1. Καθώς η οξειδοαναγωγική ομοιοστασία κατέχει κεντρικό ρόλο στην φυσιολογία του θυρεοειδούς αδένα και ο Nrf2 πρόσφατα χαρακτηρίσθηκε ως μεσολαβητής στην αντίσταση θυρεοειδικών καρκινικών κυτταρικών σειρών σε πρωτεασωμικούς αναστολείς, το αντιοξειδωτικό μονοπάτι Nrf2 μπορεί να θεωρηθεί ως εξαιρετικός υποψήφιος της διαμεσολάβησης της απόκρισης του θυρεοειδούς αδένα στο ΟΣ. Παρ 'όλα αυτά, ο ρόλος του μονοπατιού Nrf2 στον ανθρώπινο θυρεοειδικό καρκίνο παραμένει άγνωστος. Στόχος: Στόχοι της παρούσας μελέτης ήταν η εκτίμηση της δραστηριότητας του μονοπατιού Νrf2 στο διαφοροποιημένο καρκίνωμα του θυρεοειδούς αδένα και η διερεύνηση σωματικών μεταλλάξεων των γονιδίων NFE2L2 και Keap1. Yλικά και Μέθοδοι Ασθενείς: Στη μελέτη συμμετείχαν 90 περιστατικά εκ των οποίων τα 42 αφορούσαν θηλώδη καρκινώματα (papillary thyroid carcinomas, PTCs), τα 6 θυλακιώδη καρκινώματα (follicular thyroid carcinomas, FTCs) και τα υπόλοιπα 42 καλοήθεις όγκους (24 αδενώματα και 18 οζώδης υπερπλασία). Κυτταρικές σειρές: Στα πλαίσια της παρούσας μελέτης χρησιμοποιήθηκαν κυτταρικές σειρές PTC (K1, TPC-1, XTC-1), κυτταρική σειρά φτωχά διαφοροποιημένου PTC (T243), κυτταρικές σειρές αδιαφοροποίητου καρκινώματος (C643, 8505C, Hth74) και τέλος κυτταρικές σειρές αναπλαστικού καρκινώματος (T235 , T241, T238). Μέθοδοι: Αναδρομική ανοσοϊστοχημική ανάλυση δειγμάτων PTC και FTC, παρακείμενου φυσιολογικού ιστού και καλοηθών βλαβών. Ανάλυση αλληλουχίας DNA των κυτταρικών σειρών και PTC δειγμάτων. Κύριες μετρήσεις και υπολογισμοί: Αξιολογήθηκε η ένταση της ανοσοαντίδρασης των δειγμάτων των ιστών σε αντισώματα για τα Nrf2, Nqo1, Keap1 και 4-HNE. Μελετήθηκε η αλληλουχία του εξονίου 2 του γονιδίου NFE2L2 καθώς και του γονιδίου Keap1. Αποτελέσματα: O μεταγραφικός παράγοντας Nrf2 καθώς και ο στόχος του, η πρωτεΐνη Nqo1 ήταν μη ανιχνεύσιμα σε φυσιολογικό ιστό θυρεοειδούς αδένα. Τα επίπεδά τους ήταν σημαντικά υψηλότερα στα PTC δείγματα από ό,τι στα δείγματα καλοηθών βλαβών. Η έκφραση του Keap1 εμφάνισε διακύμανση στα δείγματα PTC με τα επίπεδά του να μην εμφανίζουν συσχέτιση με τα αντίστοιχα του Nrf2, ενάντια στη θεωρία πως τα μειωμένα επίπεδα του Κeap1 συνιστούν μηχανισμό ενεργοποίησης του Nrf2. Ο δείκτης ΟΣ, 4-HNE βρέθηκε αυξημένος στη πλειοψηφία των δειγμάτων PTC σε σχέση με το φυσιολογικό ιστό αναδεικνύοντας την ύπαρξη αυξημένου ΟΣ στο PTC. Επιπλέον, όσον αφορά τα δείγματα FTC, ο μεταγραφικός παράγοντας Nrf2 και η πρωτεΐνη Nqo1 ήταν ανιχνεύσιμα σε όλα τα δείγματα, ενώ τα επίπεδα του 4-ΗΝΕ ήταν αυξημένα. Όσον αφορά την ανάλυση αλληλουχίας DNA στις καρκινικές σειρές και σε 11 δείγματα PTC με υψηλή έκφραση Nrf2, καμία μετάλλαξη δεν ανευρέθηκε στο εξόνιο 2 του γονιδίου NFE2L2 και στο γονίδιο Keap1. Συμπεράσματα: Τα αποτελέσματα της μελέτης μας σε συνδυασμό με περαιτέρω μελέτες από το εργαστήριο Ενδοκρινολογίας και Ανατομικής του Πανεπιστημίου Πατρών καθώς και από το BC κέντρο έρευνας καρκίνου (Vancouver, Canada) αποδεικνύουν ότι το μονοπάτι Nrf2 ενεργοποιείται σε PTC και κατέχει ρυθμιστικό ρόλο στην αντιοξειδωτική απόκριση και τη βιωσιμότητα των θυρεοειδικών καρκινικών κυττάρων. Συνεπώς, αναδεικνύεται το Nrf2 μονοπάτι ως νέο “σήμα κατατεθέν” του PTC. Παρά το γεγονός ότι δεν ήταν δυνατή η πραγματοποίηση στατιστικών συσχετίσεων στη μελέτη του FTC λόγω του περιορισμένου αριθμού δειγμάτων, το μονοπάτι Nrf2 φαίνεται να ενεργοποιείται επίσης στο FTC. Η σταθερή ενεργοποίηση του Nrf2 στο PTC και ενδεχομένως στο FTC δίνει το έναυσμα για περαιτέρω διερεύνηση του μονοπατιού αυτού σε όλα τα είδη θυρεοειδικού καρκίνου καθώς και της πιθανής διαγνωστικής, προγνωστικής, και/ή θεραπευτικής χρησιμότητας του μονοπατιού στο διαφοροποιημένο καρκίνο του θυρεοειδούς αδένα. / Scientific background: Oxidative stress (ΟS) is experienced by cells when pro-oxidant and electrophilic reactive species overwhelm the cell’s antioxidant and detoxification proteins. In addition to causing protein and lipid damage, oxidative stress can cause mutations and epigenetic perturbation by damaging DNA and proteins that modify chromatin. Nevertheless, in thyrocytes a daily basis high amounts of the oxidant hydrogen hyperoxide (H2O2) was generated due to the fact that H2O2 is a reactive oxygen species required for thyroid hormonogenesis. A minimal oxidative load is a prerequisite for normal thyroid cell function and development and it was recently shown that the thyroid has increased capacity for defending itself against OS. However, precise mechanisms by which thyrocytes sense and respond to OS remain obscure. NFE2-related factor 2 (Nrf2), encoded by NFE2L2 gene, is a transcription factor that integrates cellular stress signals and responds by directing transcriptional program in various tissues. In basal conditions, Nrf2 is targeted for proteasomal degradation by its cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), while in oxidative stress Nrf2 degradation is abolished and Nrf2 accumulates in the nucleus where it transactivates protective genes such as NAD(P)H dehydrogonase quinone 1 (Nqo1). As redox homeostasis plays a principal role in thyroid gland’s physiology, and Nrf2 has recently been characterised as mediator of thyroid cancer cell lines’ resistance to proteasome inhibitors, the Nrf2 antioxidant pathway seems to be an excellent candidate for mediating the antioxidant response of the thyroid gland. Nevertheless, the activity status of the Nrf2 pathway in human thyroid cancer remains unknown. Objective: The aims of this study were to assess the activity status of the Nrf2 pathway in differentiated thyroid carcinoma and investigate somatic mutations in NFE2L2 and Keap1 genes. Μethods and Materials Patients: The study included 90 individual samples; 42 papillarz thyroid carcinomas (PTCs), 6 follicular thyroid carcinomas (FTCs) and 42 benign lesions (24 adenomas and 18 nodular hyperplasias). Cell lines: Ten thyroid cell lines are used for this study: The PTC cell lines, K1, TPC-1 and XTC-1; the poorly differentiated PTC cell line, T243; the undifferentiated carcinoma cell lines, C643, 8505C and Hth74; and the anaplastic carcinoma cell lines, T235, T241 and T238. Methods: We conducted retrospective immunohistochemical analyses of PTC and FTC specimens, adjacent normal tissue, and benign lesions; DNA sequencing in cell lines and PTC samples. Main Outcome Measures: We assessed the abundance of Nrf2, Nqo1, Keap1, and 4HNE; and the sequence of NFE2L2 gene’s exon 2 and of KEAP1 gene. Results: Nrf2 and its target Nqo1 were undetectable in normal tissue; their levels were significantly higher in PTC than in benign lesions. The Nrf2 inhibitor, Keap1 was variably abundant in PTC, and its levels did not correlate with Nrf2, arguing against decreased levels as the mechanism for Nrf2 activation. The oxidized lipid 4HNE was more abundant in PTC than normal tissue indicating oxidative stress. In addition, as far as FTC samples are concerned, Nrf2 and Nqo1 were detectable in all samples as well as the levels of 4-HNE were significantly high. No mutations were detectable in exon 2 of NFE2L2 gene and in Keap1 gene. Conclusions: Our study’s results supported by further studies in laboratories of Endocrinology and Anatomy at University of Patras and BC Cancer Research Center (Vancouver, Canada) demonstrate that the Nrf2 pathway is commonly activated in PTC and that it regulates antioxidant responses and viability of cancer cells. Thus, Nrf2 is highlighted as a new hallmark of PTC. Although, statistic correlations were not possible in FTC samples’ study because of small sample size, the Nrf2 pathway seems to be also activated in FTC. The high activity of Nrf2 in PTC and possibly in FTC warrants further exploration of this pathway’s potential diagnostic, prognostic, and/or therapeutic utility in differentiated thyroid carcinoma.

L'asma és una malaltia d'etiologia complexa, formada per factors genètics i ambientals, on la interrelació de ambdós factors mitjançant interaccions gen-ambient juga un paper clau. L'objectiu d'aquesta tesi ha sigut aprofundir en el coneixement del paper dels polimorfismes genètics, i la seva interacció amb factors ambientals, en la ocurrència d'asma, atòpia i hiperreactivitat bronquial. Aquest objectiu ha estat desenvolupat a través de la replicació de variants genètiques prèviament identificades, l'avaluació d'interaccions gen-ambient i la identificació de nous gens de susceptibilitat mitjançant un disseny basat en el genotipatge de variants genètiques all llarg del genoma en pools d'ADN. La tesi ha estat majoritàriament duta a terme dins l'estudi European Community Respiratory Health Survey (ECRHS) que està comprès per 5.000 individus seguits durant 9 anys, pels quals es disposa d'un qüestionari complet sobre símptomes respiratoris, avaluacions clíniques, informació sobre exposicions ambientals i mostres de ADN. Aquesta tesi a replicat l'associació del polimorfismes dels gens TNFA i NPSR1 amb asma. A més s'han establert les interaccions entre TNFA i obesitat, NQO1 i contaminació atmosfèrica, i NPSR1 i edat d'inici d'asma. L'anàlisi de pools d' ADN ha permès associar la regió on es situa el gen SGK493 amb atòpia. Aquesta tesi contribueix al coneixement de l'etiologia d'asma amb la identificació i replicació d'associacions genètiques i interaccions gen-ambient. / Asthma is a disease with a complex etiology, involving multiple genetic and environmental factors, and with an important role of the interplay of these factors through gene-environment interactions. In this thesis I aimed to advance our knowledge on the importance of genetic polymorphisms and their interaction with environmental data for the occurrence of asthma and related phenotypes (atopy and bronchial hyperreactivity). This objective was developed through the replication of genetic associations previously reported, the assessment of gene-environment interactions and the identification of new susceptibility genes using genome-wide analysis based on a pooling DNA strategy. The thesis was, mostly, performed within the European Community Respiratory Health Survey (ECRHS). This cohort has information and DNA samples from approximately 5,000 adult subjects followed-up for 9 years, with extensive questionnaires on respiratory symptoms, clinical evaluations and information on environmental exposures. This thesis replicates previous effects on asthma of polymorphisms in TNFA and NPSR1 genes. In addition, interactions have been established between TNFA and obesity, NQO1 and air-pollution, and NPSR1 and age at onset of asthma. The approach based on genome-wide analysis of DNA pools identified the SGK493 region being associated with atopy. This thesis contributes to the understanding of the etiology of asthma through the identification and replication of genetic associations and gene-environment interactions.