[Truncated abstract] Reactive oxygen species (ROS) have been implicated to play a major role in many pathological conditions including heart attack and stroke. Their ability to modulate the extracellular signal-regulated protein kinase (ERK) and c-Jun Nterminal kinase (JNK) signalling pathways, thereby influencing cellular response has been well-documented. Recent studies implicate a central role for mitochondria in ERK and JNK activation by ROS although the mechanisms remained unresolved. Using Jurkat T-lymphocyte as a cell model, this study demonstrated increased mitochondrial ROS production as a result of decreased mitochondrial complex activities mediated by hydrogen peroxide treatment. This is the first study to show that mitochondria are not essential for activating ERKs, however damaged mitochondria producing ROS can be expected to cause sustained ERK activation . . . This study revealed that JNK and its upstream kinases MKK4, MKK7 and ASK1 are associated with the mitochondria. Furthermore, findings from this study imply that JNK resides in the mitochondrial matrix. This study is the first to demonstrate that mitochondrial JNK can be activated in a cell-free environment by signals originating from the mitochondria. Experimental work using isolated mitochondria demonstrated that mitochondrial JNK can be activated by ROS generated from the mitochondria themselves. Flavin-containing proteins appear to be the main sources of mitochondrial-ROS which signal through redoxsensitive proteins to activate mitochondrial JNK.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 192 pages. Includes vita. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 166-187). Also available online.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vi, 130 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Πολλές παθήσεις του καρδιαγγειακού συστήματος σχετίζονται με την λειτουργία του ενζύμου της διαλυτής γουανυλικής κυκλάσης (soluble guanylate cyclase, sGC). Η sGC εμπλέκεται στο μονοπάτι ΝΟ-sGC-cGMP το οποίο ενεργοποιείται από το βιολογικά διαθέσιμο μονοξείδιο του αζώτου (nitric oxide, ΝΟ). Πολλές παθολογικές καταστάσεις αντιμετωπίστηκαν για πάνω από 140 χρόνια με τη χρήση φαρμάκων που παρέχουν NO (ΝΟ-φάρμακα), χωρίς ωστόσο να είναι γνωστός ο μηχανισμός δράσης τους. Αν και τα φάρμακα αυτά συνεισέφεραν στη βελτίωση των παθολογικών καταστάσεων, ωστόσο παρουσίαζαν σημαντικά μειονεκτήματα. Για την αντιμετώπιση αυτών, το ενδιαφέρον στράφηκε στον σχεδιασμό και την σύνθεση ενώσεων των οποίων η δράση θα ήταν ανεξάρτητη από το ΝΟ. Μεταξύ αυτών, τα παράγωγα BAY 58-2667 και η HMR 1766 αποδείχθηκαν ενεργοποιητές της sGC. Στα πλαίσια της παρούσας μελέτης, σχεδιάσθηκαν και συντέθηκαν έξι νέα βενζοφουρανικά ανάλογα του HMR-1766, σε μία προσπάθεια ανακάλυψης νέων ενώσεων, ενεργοποιητών της sGC με ενισχυμένη δραστικότητα και εκλεκτικότητα δράσης. Η προσέγγιση που ακολουθήθηκε για την σύνθεση των τελικών προϊόντων περιελάμβανε την ανοικοδόμηση του βενζοφουρανικού δακτυλίου από υποκατεστημένα παράγωγα σαλικυλικού οξέος και την μετέπειτα σύζευξη αυτού με κατάλληλους δομικούς λίθους για τον σχηματισμό μίας σουλφοναμιδικής και μίας αμιδικής πλευρικής αλυσίδας. Στα πλαίσια της μελέτης, διερευνήθηκαν και βελτιστοποιήθηκαν όλα τα συνθετικά στάδια για την παραλαβή των ενδιάμεσων και των τελικών προϊόντων. Η μελλοντική αποτίμηση της βιολογικής δράσης των νέων ενώσεων αναμένεται να διευκρινίσει αν οι ενώσεις αυτές είναι ικανές να δράσουν ως ενεργοποιητές της sGC, αλλά και αν μπορούν να αποτελέσουν χρήσιμα χημικά εργαλεία για την διευκρίνιση δομικών πληροφοριών του ενζύμου. Το δεύτερο τμήμα της παρούσας εργασίας, αφορά στον σχεδιασμό και την σύνθεση νέων αναλόγων του φυσικού προιόντος Hymenialdesine (HMD). Η HMD είναι ένα φυσικό προϊόν το οποίο έχει αποδειχθεί αναστολέας πολλών πρωτεϊνικών κινασών, όπως των κυκλινο-εξαρτώμενων κινασών (CDKs), η υπερλειτουργία των CDKs ενέχεται στην εμφάνιση παθολογικών καταστάσεων (καρκίνος, νευροεκφυλιστικές παθήσεις, διαβήτης). Στόχος της μελέτης ήταν ο σχεδιασμός και η διερεύνηση της σύνθεσης νέων σπειρανικών ινδολοαζεπινικών αναλόγων της HMD, με ενισχυμένη ανασταλτική και εκλεκτική δράση έναντι των CDKs. Για το σκοπό αυτό, μελετήθηκε η μετατροπή της 5-κετονομάδας της αζεπινο[3,4-b]ινδολο-1,5-διόνης σε ένα αμινο-υποκατεστημένο στερεογονικό κέντρο μέσω νουκλεόφιλης προσβολής της πρόδρομης χειρόμορφης t-βουτυλοσουλφινυλ-ιμίνης. Διερευνήθηκαν ποικίλες πειραματικές συνθήκες για τη βελτιστοποίηση σχηματισμού τόσο της ενδιάμεσης σουλφινυλ-ιμίνης, όσο και της υποκατάστασης αυτής. Τα συνθετικά αυτά στάδια θεωρούνται κρίσιμα και η βελτιστοποίηση τους απαραίτητη για την ομαλή εξέλιξη του συνθετικού σχήματος. Τα αποτελέσματα που καταγράφηκαν στα πλαίσια της μελέτης αναμένεται να συμβάλλουν ουσιαστικά στην επιτυχή ολοκλήρωση της σύνθεσης των νέων σπειρανικών αναλόγων της HMD. / Many cardiovascular diseases are connected with the activity of soluble guanylate cyclase (sGC). sGC is part of the NO-sGC-cGMP pathway, which is activated by the biologically available nitric oxide (NO). Many drugs that release NO (NO-drugs) have been used for more than 140 years. Although these drugs have contributed to the treatment of these diseases, they have presented some disadvantages. Thus, new compounds have been discovered whose activity is independent of NO. Compounds BAY 58-2667 and HMR-1766 belong to this new class of compounds and are characterized as sGC activators. In the first part of this study, six new benzofuran derivatives of HMR-1766 were designed and synthesized, aiming at the discovery of new compounds, activators of sGC with enhanced activity and selectivity against sGC. The synthetic approach involves the initial formation of benzofuran ring from substituted derivatives of salicylic acid and its coupling with selected building blocks. The optimazation of all synthetic steps for the synthesis of the intermediate and final products was also part of this study. The biological evaluation of the new compounds is expected to reveal their biological activity as sGC activators and/or their role as chemical tools for the structural elucidation of the enzyme. The second part of this study, concerns the design and synthesis of new derivatives of Hymenialdesine (HMD). HMD is a natural product with inhibitory activity against many protein kinases, such as cyclin-dependent kinases (CDKs). Hypeactivation of CDKs is implicated in pathological disorders such as cancer, neurodegenerative diseases and diabetes. The aim of the study was the synthesis of new spiro-indolazepino derivatives of HMD with potential enhanced inhibitory activity and selectivity against CDKs. The transformation of the 5-ketogroup of the azepino[3,4-b]indol-1,5-dione to a new amino-substituted stereogenic center by nucleophilic attack of the intermediate chiral tert-sulfinylimine was the key-step of the synthetic approach. The results of this study are expected to contribute substantially to the synthesis of new spiro HMD derivatives.

La protéine kinase activée par AMP (AMPK) est un senseur ubiquitaire du statut énergétique de la cellule eucaryote. Elle est exprimée sous la forme d'un complexe hétérotrimèrique comprenant les sous unités catalytique (α) et régulatrices (β et γ). Ce large complexe protéique (130kDa), fonctionne comme un hub central de la signalisation cellulaire, régulateur du métabolisme énergétique et au-delà. La (dé)régulation de l'AMPK est impliquée dans de nombreuses pathologies et l'AMPK apparait comme une cible de choix pour développer de nouveaux médicaments contre le diabète de type 2. Une fois activée, l'AMPK va restaurer l'homéostasie énergétique en diminuant le métabolisme demandeur d'énergie (anabolisme) et en stimulant le métabolisme produisant le l'énergie (catabolisme). In vivo, l'AMPK est activée par des mécanismes multiples et complexes permettant la fine régulation de son activité lors de différentes situations de stress métaboliques. Premièrement, l'activité de l'AMPK est modulée de manière systémique par phosphorylation et déphosphorylation de la sous unité α (par des kinases et phosphatases en amont respectivement). De plus, l'attachement d'AMP et d'ADP à la sous unité γ augmente la phosphorylation de l'AMPK. Deuxièmement, l'AMPK est activée de manière allostérique par l'AMP qui se lie à sous unité γ lors de chutes du ratio ATP/AMP. Tous ces mécanismes requièrent une communication entre les sous unités α et γ, mais un modèle consensus complet de l'activation de l'AMPK est toujours manquant. Se basant sur différentes études structurales, d'autres et nous-mêmes avons proposé un changement de conformation induit par AMP au sein de l'hétérotrimère AMPK. Afin de mieux élucider ce mécanisme, nous avons tiré profit de ces changements conformationels pour imaginer et créer un hétérotrimère d'AMPK permettant de suivre directement et en temps réel l'état de conformation de l'AMPK par FRET. Une limite importante lors du développement de complexes multiprotéiques est l'augmentation exponentielle de la quantité de travail liée à la modification et la combinaison de nombreux gènes hétérologues lors du remaniement de ces complexes protéiques et de leurs productions. Nous avons utilisé la technologie ACEMBL, qui exploite des techniques de recombinaisons homologues, pour faciliter la révision rapide et itérative de la production et de l'analyse fonctionnelle, après ingénierie, de complexes multi protéiques. Le senseur fluorescent génétiquement codé ainsi crée, et nommé AMPfret, a la propriété de rapporter les changements de conformation induits par les nucléotides ayant lieu au sein de l'AMPK. De plus, les changements de signal FRET corrèlent avec l'activation allostérique de l'AMPK. Le senseur répond à de faible concentrations en AMP (micromolaire) et a démontré la capacité exclusive qu'a l'ATP, et non l'ATP-Mg, à concurrencer l'AMP. De plus, son utilisation a permis une meilleure compréhension du rôle des sites CBS lors de l'activation allostérique. AMPfret peut aussi être considérer comme un outil de choix pour le criblage de molécules ciblant l'AMPK, et pour le monitoring de l'état énergétique intracellulaire. / AMP-activated protein kinase (AMPK) is a ubiquitous sensor of cellular energy and nutrient status in eukaryotic cells. It is expressed as heterotrimeric complexes comprising catalytic (α) and regulatory (β and γ) subunits. This large protein complex (130kDa), conserved from yeast to plants and mammals, functions as a central signaling hub and master regulator of energy metabolism and beyond. (Dys)regulation of AMPK signaling has been implicated in various pathologies. In particular, AMPK emerged as a suitable target to develop novel drugs for type II diabetes. Once activated AMPK will attempt to restore the energy homeostasis by down-regulating energy demanding pathways (anabolism) and up-regulating the energy producing ones (catabolism). AMPK is activated in vivo by multiple, complex mechanisms allowing fine tuning of AMPK activity in different situations of metabolic stress. First, AMPK activity is systemically modulated via activating phosphorylation at the α-subunit (by upstream kinases) and inactivating dephosphorylation (by upstream phosphatases). In addition, AMP and ADP binding to the γ-subunit increase AMPK phosphorylation. Second, AMPK is allosterically activated by AMP binding to the γ-subunit when the ATP/AMP ratio is falling. All these mechanisms require close communication between the γ- and α subunits, but a complete consensus model for AMPK activation is still lacking. We and others have proposed an AMP-induced conformational switch within the full-length heterotrimeric AMPK complex based on different, complementary structural studies. To further elucidate this mechanism, we have profited from these structural rearrangements to imagine and engineer an AMPK complex that allows a direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). A definite bottleneck in engineering multiprotein complexes is the exponential increase in work-load if several heterologous genes need to be altered, engineered and combined for revised protein complex production experiments. We used the ACEMBL technology which harnesses site-specific and homologous recombination techniques in tandem to facilitate rapid, iterative revision of multi-protein complex expressions after engineering and functional analysis of multiprotein complex. The resulting genetically encoded fluorescent biosensor, named AMPfret, can report conformational changes within the AMPK heterotrimer induced by nucleotide binding and the monitored FRET correlates with AMPK allosteric activation. The sensor responds to low micromolar concentrations of AMP, shows the exclusive ability of ATP, but not Mg-ATP, to compete with AMP, and allows insight into the role of CBS domains for allosteric AMPK activation. It may also be a tool of choice for AMPK targeted drug screening, and reporting the intracellular energy state.

The c-Jun NH2-terminal kinase (JNK) group of kinases include ten members that are created by alternative splicing of transcripts derived from Jnk1, Jnk2 and Jnk3 genes. The JNK1 and JNK2 protein kinases are ubiquitously expressed while JNK3 is expressed in a limited number of tissues. The JNK signaling pathway is implicated in multiple physiological processes including cell transformation. There is growing evidence that JNK signaling is involved in oncogenesis. Nevertheless, the role that JNK plays in malignant transformation is still unclear. The aim of this thesis is to examine the role of JNK in malignant transformation. For this purpose, I used the Bcr/Abl oncogene as a transforming agent. Bcr/Abl is a leukemogenic oncogene that is created by reciprocal translocation between chromosome 9 and 22. The translocation breakpoint is variable and several different Bcr/Abl isoforms have been identified such as Bcr/AblP185 and Bcr/AblP210, whose expression is associated with different types of leukemia. Bcr/Abl activates the JNK signaling pathway in hematopoietic cells and increases AP-1 transcription activity. Furthermore, dominant negative approaches demonstrate that inhibition of c-Jun or JNK prevents Bcr/ Abl-induced cell transformation in vitro. These data implicate the JNK signaling pathway in Bcr/Abl transformation although the role that JNK might have in this process is unclear. Thus, I examined the importance of JNK signaling in Bcr/Abl-induced lymphoid or myeloid transformation. For this purpose I compared Bcr/AblP185- and Bcr/AblP210- induced transformation of wild-type and JNK1-deficient cells using three approaches: in vitro, in vivo and ex vivo. The results obtained with the in vitro approach suggest that both Bcr/AblP185 and Bcr/AblP210 require JNK activity to induce lymphoid transformation. While JNK1-deficiency inhibits Bcr/AblP210 oncogenic potential in lymphoid cells both in vitro and in vivo, pharmacological inhibition of JNK activity (JNK1 and/or JNK2) blocked Bcr/AblP185 induced malignant proliferation in vitro. The differential requirement for JNK observed in the two Bcr/Abl isoforms can be ascribed to the presence in Bcr/AblP210 of the Dbl domain which can activate the JNK pathway in vitro. In the case of Bcr/AblP210, JNK1 is critical for the survival of the ex vivo derived transformed lymphoblasts upon growth factor removal. This result correlates with the fact that mice reconstituted with Bcr/AblP210 transformed Jnk1-l- bone marrow showed normal malignant lymphoid expansion in the bone marrow yet they had reduced numbers of lymphoblast in the bloodstream and lacked peripheral organ infiltration. Thus JNK1 is essential for the survival of the transformed lymphoblast outside the bone marrow microenvironment in Bcr/AblP210induced lymphoid leukemia. Interestingly, while JNK1 is essential for lymphoid transformation, it is dispensable for the proliferation of transformed myeloblasts. Taken together these results indicate that the JNK signaling pathway plays an essential role in the survival of Bcr/AblP210 lymphoblasts and that JNK-deficiency decreases the leukomogenic potential of Bcr/AblP210 in vivo. Thus, cell survival mediated by JNK may contribute to the pathogenesis of proliferative diseases.

The chicken hepatoma cell line, LMH, was evaluated with respect to its usefulness for studies of the regulation of heme metabolism. Levels of δ-aminolevulinate synthase mRNA arid accumulation of porphyrins were used to evaluate the heme biosynthetic pathway. Regulation of heme oxygenase-1 by known inducers was used as a measure of heme degradation. The induction of heme oxygenase-1 by sodium arsenite was characterized. AP-1 transcription factor elements and MAP kinase signal transduction pathways that modulate expression of endogenous heme oxygenase-1 and transfected heme oxygenase-1 reporter gene constructs in response to arsenite were delineated. In initial studies, the drug glutethimide was used alone or in combination with ferric nitrilotriacetate to induce δ-aminolevulinate synthase mRNA. Levels of porphyrins, intermediates in the heme biosynthetic pathway, and levels of δ-aminolevulinate synthase mRNA were increased by these treatments in a manner similar to those previously observed in the widely used model system, primary chick embryo liver cells. The iron chelator, deferoxamine, gave a characteristic shift in the glutethimide induced porphyrin accumulation in primary hepatocytes, but was found to have no, effect on LMH cells. Heme mediated repression of δ-aminolevulinate synthase mRNA levels was similar among primary hepatocytes and LMH cells. Heme oxygenase-1 was regulated by heme, metals, heat shock, and oxidative stress-inducing chemicals in LMH cells. Heat shock induction of heme oxygenase-1 mRNA levels was observed for the first time in primary chick embryo liver cells. These data supported the further use of LMH cells to elucidate mechanisms responsible for modulating heme oxygenase-1 gene expression in response to inducers. The remainder of the studies focused on the role of heme oxygenase-1 as a stress response protein. The oxidative stress inducer, sodium arsenite was used to probe the cellular mechanisms that control the expression of heme oxygenase-1. A series of promoter-reporter constructs were used to search the heme oxygenase-1 promoter for arsenite responsive elements. Several activator protein-1 (AP-1) transcription factor binding elements were identified by computer sequence analysis. Three of these sites, located at -1578, -3656, and -4597 base pairs upstream of the transcription start site, were mutated. The arsenite responsiveness of the reporter constructs containing mutated AP-1 elements was less than that of the same constructs containing wild type AP-1 elements. At least part of the arsenite-mediated induction of heme oxygenase-1 required the activity of AP-1 transcriptional elements. The MAP kinase signal transduction pathways and heme oxygenase-1 are activated by similar stimuli, including cellular stress. MAP kinases have been shown to exert control over gene expression through effects on the AP-1 family of transcription factors. The MAP kinases ERK, JNK, and p38 were activated by arsenite in LMH cells. Constitutively activated components of the ERK and p38 pathways increased expression of heme oxygenase-1 promoter-luciferase reporter constructs. Arsenite-mediated induction of heme oxygenase-1 was blocked by dominant negative ERK or p38 pathway components, and by specific inhibitors of MEK (upstream ERK kinase) or p38. In contrast, reporter gene expression was unchanged in the presence of constitutively activated JNK pathway components. Dominant negative JNK pathway components had no effect on arsenite induced heme oxygenase-1 gene activity. In summary, LMH cells were characterized as a new model system for the study of heme metabolism. This cell line was then used to delineate promoter elements and signaling pathways involved in the arsenite responsiveness of heme oxygenase-1 gene expression. Three AP-1 transcription factor binding sites in the heme oxygenase-1 promoter region were required for responsiveness to arsenite. The MAP kinases ERK and p38 were shown to play an integral role in arsenite-mediated induction of heme oxygenase-1. These studies elucidate one facet of heme oxygenase-1 regulation, and provide tools that will be useful in delineating additional regulatory mechanisms.