The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

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.

Activation of mammalian stress responsive pathways by plant secondary metabolites may contribute to the protection against certain chronic diseases afforded by fruit and vegetable consumption. This work focuses on the identification of plant compounds that activate the stress-responsive enzyme quinone reductase (QR) by stabilizing the transcription factor NF-E2 related factor-2 (Nrf2). Screening methanolic extracts of plants from Western Canada for QR induction in a mouse hepatoma cell line (Hepa-1c1c7) led to the identification of twenty-one extracts capable of doubling the activity of QR. Bioassay-guided fractionation of six extracts led to the identification of novel classes of compounds with QR-inducing activity including fatty-acid derived polyacetylenes, phthalides, and cannabinoids. Studies using low molecular weight thiols and the recombinantly expressed protein Keap1, the principal negative regulator of Nrf2, supported a mechanism of QR activation involving covalent modification of Keap1 cysteines for the polyacetylenes and phthalides. Analysis of transcriptional changes in response to treatment with a panel of QR-inducing compounds provided strong support for Nrf2 activation by the polyacetylene (3S,8S)-falcarindiol and the isothiocyanate (R)-sulforaphane and weaker support for the compounds (3R,8S)-falcarindiol, 6-isovaleryl-umbelliferone (6-IVU) and (Z)-ligustilide. Additionally, transcript level analyses supported a role for the aryl-hydrocarbon receptor in QR-activation by (3R,8S)-falcarindiol, (Z)-ligustilide, (R)-sulforaphane, 6-IVU and cannabidiol and suggested that treatment with polyacetylenes with a (3R)-configuration, (Z)-ligustilide and 6-IVU causes substantial changes in the expression of genes associated with lipid homeostasis and energy metabolism. As a whole, this work provides evidence that compounds that activate QR (and Nrf2) are widely distributed in the Canadian flora. However, of these QR activators, few are active at concentrations that are expected to be achieved through dietary consumption. Nevertheless, the most exceptional compounds isolated in this work, the compounds (3S,8S)-falcarindiol and epoxyfalcarindiol are highly potent and appear to be or are expected to be specific for activating Nrf2 and thus warrant attention with respect to dietary implications and as drug candidate leads.

POPULAR SCIENTIFIC SUMMARY Systemic lupus erythematosus (SLE) is not your average disorder. It behaves like a mischievous troublemaker, wreaking havoc throughout the body, causing inflammation that affects multiple organs. SLE presents a puzzle that keeps health care professionals worldwide intrigued, searching for answers amidst its complex of immunologic manifestations and clinical symptoms. While we’ve made progress in understanding SLE, its specific cause remains a mystery. What we do know is that SLE triggers a fascinating interplay between genetic, hormonal, and environmental factors in susceptible individuals. Macrophages, specialized white blood cells, can be likened to moody actors on a stage wearing different masks and wielding functional props. Among them are M1 macrophages, fiery troublemakers who provoke pro-inflammatory responses, and M2 macrophages, peacemakers striving for balance by generating anti-inflammatory responses. Then there is NRF2, the vigilante, normally held by its captor, KEAP1. However, when cells stress NRF2 manages to break free from KEAP1 and spring into action, embarking on a crucial journey into the cell nucleus where DNA is stored. Once inside, NRF2 binds specific regions of the DNA, promoting genes associated with protective activities, including antioxidative responses and detoxification processes, thereby shielding cells from further harm. Now, let us envision a therapeutic strategy that utilizes this; if we can deliberately unleashNRF2 on command, triggering a powerful cascade of antioxidative responses throughout the body,such a treatment would offer tremendous promise and serve as a paradigm for patients sufferingfrom chronic inflammation. But the question remains: Is it possible? In this study, we investigated the effects of certain chemicals on macrophages in a controlledlab environment. Our goal was to explore their potential for therapeutic purposes. Excitingly, wediscovered that these chemicals can indeed influence macrophages to produce a stronger antiinflammatory and antioxidant response. These findings could be promising for developing futuretreatments, especially in patients diagnosed with conditions such as SLE. / ABSTRACT Systemic lupus erythematosus (SLE) is a multifaceted, chronic autoimmune disorder that leads to inflammation and affects various organs. A wide range of immunologic manifestations and clinical symptoms characterizes SLE. While the specific cause remains unknown, it is thought to result from a combination of genetic susceptibility and the intricate interplay between environmental and hormonal factors. A significant subset of SLE patients also experience renal manifestation, lupus nephritis (LN), characterized by distinct inflammatory responses in which macrophages play a role. Macrophages exhibit different functional characteristics depending on their environment, and generally display two contrasting phenotypes; M1, which elicits proinflammatory responses, and M2, which generates anti-inflammatory responses Homeostasis is vital, yet environmental stress is inevitable. NRF2, a transcription factor known for its involvement in oxidative stress response, plays a pivotal role. Under basal conditions, NRF2 resides in the cytoplasm and is targeted for degradation by the protein KEAP1. However, during cellular stress, the NRF2-KEAP1 complex dissociates, allowing NRF2 to translocate into the nucleus where it binds specific regulatory regions of genes that promote cytoprotective activities. The NRF2 pathway has gained attention as a potential target for therapeutic strategies in inflammatory conditions, including SLE. This study aimed to assess the effects of certain chemical NRF2 activators and a KEAP1 inhibitor on an in vitro model of M1 and M2 macrophage polarization. The objective was to investigate whether these compounds could enhance antioxidative response. To evaluate this, key genes and proteins involved in antioxidative pathways were analyzed. Gene expression was assessed using quantitative real-time PCR (qPCR), and protein presence was determined through immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). The findings of this study indicate that stimulation of macrophage subgroups with the selected compounds promotes a shift towards anti-inflammatory and antioxidative response. / <p>Rektor tilldelade Leo Svahn stipendie Österby för <em>välartade obemedlade studier</em>.</p>