T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha<q/11>-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha<q/11>-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha<q/11> signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha<q/11>-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs.

Doctor of Philosophy(PhD) / This thesis addresses the neurochemistry and function of specific nuclei in the autonomic nervous system that are crucial mediators of cardiorespiratory regulation. The primary aim is to build on previous knowledge about muscarinic cholinergic mechanisms within cardiorespiratory nuclei located in the ventrolateral medulla oblongata. The general focus is characterisation of gene expression patterns of specific muscarinic receptor subtypes in central nuclei involved in blood pressure control and respiratory control in normal rats. The findings were subsequently extended by characterisation of muscarinic receptor gene expression patterns in 1) a rat model of abnormal blood pressure control (hypertension) (Chapter 3) 2) a rat model of cholinergic sensitivity (Chapter 5) 3) the rat ventral respiratory group (Chapter 6) The results of a series of related investigations that ensued from the initial aims more finely characterise the neurocircuitry of the ventrolateral medulla, from a specifically cholinoceptive approach. All five muscarinic receptor subtypes are globally expressed in the ventrolateral medulla but only the M2R mRNA was significantly elevated in the VLM of hypertensive animals compared to their normotensive controls and in the VLM of animals displaying cholinergic hypersensitivity compared to their resistant controls. Surprisingly, M2R mRNA is absent in catecholaminergic cell groups but abundant in certain respiratory nuclei. Two smaller projects involving gene expression of other neurotransmitter / neuromodulators expressed in cardiorespiratory nuclei were also completed during my candidature. Firstly, the neurochemical characterisation of enkephalinergic neurons in the RVLM, and their relationship with bulbospinal, catecholaminergic neurons in hypertensive compared to normotensive animals was carried out (Chapter 4). A substantial proportion of sympathoexcitatory neurons located in the RVLM were enkephalinergic in nature. However, there was no significant difference in preproenkephalin expression in the RVLM in hypertensive compared to normotensive animals. Secondly, the identification and distribution of components of the renin-angiotensin aldosterone system (RAAS) within the brainstem, and differences in gene expression levels between hypertensive and normotensive animals was also investigated. The RAAS data was not included in this thesis, since the topic digresses substantially from other chapters and since it is published (Kumar et al., 2006). The mRNA expression aldosterone synthase, mineralocorticoid receptor (MR1), 12-lipoxygenase (12-LO), serum- and glucocorticoid- inducible kinase and K-ras) were found to be present at all rostrocaudal levels of the ventrolateral medulla. Expression of MR1 mRNA was lower in the RVLM of SHR compared with WKY rats and 12-LO mRNA levels were lower in the CVLM in SHR compared with WKY rats. Otherwise, there was no difference in gene expression level, or the method of detection was not sensitive enough to detect differences in low copy transcripts between hypertensive and normotensive animals.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.

Ο νικοτινικός υποδοχέας της ακετυλοχολίνης ανήκει στην υπερ-οικογένεια των ιοντικών διάυλων ενεργοποιούμενων μέσω προσδέτη (ligand-gated ion channels). Οι υπομονάδες των υποδοχέων που ανήκουν στην οικογένεια αυτή, φέρουν στο αμινοτελικό τους άκρο τη χαρακτηριστική κυστεϊνική θηλιά (Cys-loop) μεταξύ δύο συντηρημένων καταλοίπων κυστεΐνης, οι οποίες συνδέονται με δισουλφιδικό δεσμό. Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης είναι μεγάλες διαμεμβρανικές πρωτεΐνες που αποτελούνται από πέντε ομόλογες υπομονάδες και χωρίζονται σε δυο κατηγορίες, τους νευρικούς και τους μυϊκούς. Οι νευρικοί υποδοχείς εκφράζονται κυρίως στο κεντρικό και περιφερικό νευρικό σύστημα, καθώς και σε μη-νευρικούς ιστούς, όπως τα επιθηλιακά κύτταρα και τα κύτταρα του ανοσοποιητικού συστήματος. Επιπλέον, χωρίζονται σε δύο υποκατηγορίες, τους ετεροπενταμερείς, όπως είναι η (α4)x(β2)y, και τους ομοπενταμερείς, όπως η (α7)5. Οι μυϊκοί υποδοχείς απαντώνται στις μετασυναπτικές μεμβράνες των νευρομυϊκών συνάψεων των σπονδυλωτών και στα ηλεκτρικά όργανα ορισμένων ψαριών και εμφανίζουν στοιχειομετρία (α1)2β1γδ (στα έμβρυα) ή (α1)2β1εδ (στους ενήλικες). Ο μυϊκός υποδοχέας της ακετυλοχολίνης σε σχέση με τα υπόλοιπα μέλη της υπερ-οικογένειας, είναι ο καλύτερα μελετημένος ως προς τα χαρακτηριστικά και τη λειτουργία του. Ένας από τους κύριους παράγοντες που συνέβαλε στον εκτεταμένο χαρακτηρισμό του μυϊκού υποδοχέα είναι η δυνατότητα απομόνωσης, σε μεγάλες ποσότητες και σε λειτουργική μορφή, υποδοχέων της ακετυλοχολίνης παρόμοιων με αυτούς της νευρομυϊκής σύναψης από τα ηλεκτρικά όργανα των ψαριών Torpedo και Electrophorus. Οι πρώτες πληροφορίες για την τρισδιάστατη δομή του υποδοχέα δόθηκαν από ένα μοντέλο ηλεκτρονικής μικροσκοπίας, από υποδοχέα που απομονώθηκε από το ηλεκτρικό όργανο του ψαριού Torpedo marmorata. Στη συνέχεια, η λύση της δομής ενός ομολόγου του εξωκυτταρικού τμήματος του υποδοχέα, της acetylcholine binding protein (AChBP) από το σαλιγκάρι Lymnaea stagnalis έφερε και τις πρώτες πληροφορίες υψηλής ανάλυσης. Η AChBP είναι μια υδατοδιαλυτή πρωτεΐνη, η οποία σχηματίζει σταθερά ομοπενταμερή, με ομολογία 20%-24% με το εξωκυτταρικό τμήμα των υπομονάδων του υποδοχέα. Πρόσφατα, η λύση της δομής της εξωκυτταρικής περιοχής της α1 υπομονάδας του υποδοχέα ποντικού, στην οποία είχε προσδεθεί ο ανταγωνιστής της ακετυλοχολίνης α-μπουγκαροτοξίνη, προσέφερε επιπλέον πληροφορίες για τα χαρακτηριστικά του υποδοχέα, όπως είναι η κύρια ανοσογόνος περιοχή (MIR) και η συντηρημένη Cys-loop περιοχή. Παρόλα αυτά δεν έχουν δημοσιευτεί υψηλής ανάλυσης πληροφορίες για τη δομή του ανθρώπινου υποδοχέα. Επιπλέον από το φυσιολογικό ρόλο του μυϊκού υποδοχέα, ο οποίος είναι άμεσα υπεύθυνος για τη διαβίβαση της ώσης στη νευρομυϊκή σύναψη, παράλληλα ο υποδοχέας αυτός αποτελεί και στόχο για πολλά κληρονομικά και επίκτητα νοσήματα, στα οποία ανήκει και το αυτοάνοσο νόσημα μυασθένεια. Στη νόσο αυτή, αυτοαντισώματα έναντι του μυϊκού υποδοχέα προκαλούν μείωση των διαθέσιμων λειτουργικών υποδοχέων στη νευρομυϊκή σύναψη με συνέπεια να παρεμποδίζεται η δράση της ακετυλοχολίνης. Αυτοαντισώματα έναντι του υποδοχέα της ακετυλοχολίνης εντοπίζονται στο 80%-90% των μυασθενών και η παρουσία τους θεωρείται ο κύριος παθογόνος παράγοντας για τη μυασθένεια. Ο σημαντικός ρόλος λοιπόν του υποδοχέα σε φυσιολογικές και παθολογικές καταστάσεις, τον καθιστά αντικείμενο εντατικής έρευνας. Η λεπτομερής ανάλυση της δομής και της λειτουργίας του θα μπορούσε να συμβάλλει στην κατανόηση της δομής και της λειτουργίας των υπόλοιπων υποδοχέων-μελών της υπερ-οικογένειας καθώς επίσης και στην εξιχνίαση του αυτοάνοσου μηχανισμού της μυασθένειας. Ωστόσο, το μεγάλο μέγεθος του μορίου του υποδοχέα, ο υδρόφοβος χαρακτήρας του και η αδυναμία απομόνωσής του από φυσικές πηγές, δυσχεραίνει την πραγματοποίηση δομικών μελετών. Για το λόγο αυτό, είναι αναγκαίο να προκύψουν πρωτεϊνικά μόρια με δομή η οποία να πλησιάζει αρκετά τη φυσική διαμόρφωση του νικοτινικού υποδοχέα της ακετυλοχολίνης και σε ποσότητες ικανές ώστε να είναι εφικτές οι δομικές μελέτες τους. Προηγούμενα, είχαν εκφραστεί στο εργαστήριό μας οι εξωκυτταρικές περιοχές (ΕΚΠ) των υπομονάδων α1, β1, γ και ε (α1ΕΚΠ, β1ΕΚΠ, γΕΚΠ και εΕΚΠ) του ανθρώπινου μυϊκού υποδοχέα, χρησιμοποιώντας ως σύστημα έκφρασης το ζυμομύκητα Pichia pastoris. Οι ανασυνδυασμένες αυτές πρωτεΐνες, απομονώθηκαν σε διαλυτή και γλυκοζυλιωμένη μορφή, αναγνωρίζονταν από μονοκλωνικά αντισώματα έναντι της κάθε υπομονάδας, αλλά και από αντισώματα προερχόμενα από ορούς μυασθενών. Ωστόσο, τα επίπεδα έκφρασης και η διαλυτότητα δεν ήταν ικανοποιητικά για δομικές μελέτες. Παρόλα αυτά, οι ανασυνδυασμένες αυτές πρωτεΐνες, χρησιμοποιήθηκαν επιτυχώς για μια καινούρια αντιγονοειδική θεραπευτική προσέγγιση για τη μυασθένεια, η οποία έγκειται στην ειδική αφαίρεση των αυτοαντισωμάτων από ορούς μυασθενών, χρησιμοποιώντας τις συγκεκριμένες πρωτεΐνες ως ανοσοπροσροφητές. Η χρήση της α1ΕΚΠ του ανθρώπινου μυϊκού υποδοχέα ως ανοσοπροσροφητή, είχε ως αποτέλεσμα την αφαίρεση μεγάλου ποσοστού αυτοαντισωμάτων από ορούς μυασθενών. Παρόμοια ήταν και τα αποτελέσματα με τη χρήση των εξωκυτταρικών τμημάτων των υπομονάδων β1, γ και ε ως ανοσοπροσροφητές. Ωστόσο, με τον τρόπο αυτό δεν επιτυγχάνεται πλήρης απομάκρυνση των παθογόνων αυτοαντισωμάτων. Η παρούσα εργασία είχε στόχο τη βελτίωση της δομής, της διαλυτότητας και των επιπέδων έκφρασης των υπομονάδων του μυϊκού υποδοχέα, έτσι ώστε να είναι κατάλληλες για δομικές μελέτες και κυρίως για να συμβάλλουν στις προσπάθειες βελτίωσης της αντιγονοειδικής θεραπείας για τη μυασθένεια. Ειδικότερα, το πρώτο μέρος αφορά μελέτες σχετικές με την έκφραση της γΕΚΠ, η οποία σχημάτιζε ολιγομερή και είχε πολύ χαμηλά επίπεδα έκφρασης. Σχεδιάστηκαν λοιπόν 4 μεταλλάγματα, με την προοπτική να προκύψουν πιο διαλυτά και υδρόφιλα μόρια. Οι μεταλλάξεις που σχεδιάστηκαν αφορούσαν στην αντικατάσταση συγκεκριμένων κυστεϊνών, που θα μπορούσαν να σχηματίζουν ακατάλληλους ένδο- ή δια-μοριακούς δεσμούς (μέσω δισουλφιδικών δεσμών), καθώς επίσης και στην αντικατάσταση της υδρόφοβης Cys-loop περιοχής με την αντίστοιχη, πιο υδρόφιλη της AChBP. Στα δύο μεταλλάγματα που έφεραν τη Cys-loop περιοχή της AChBP, παρατηρήθηκε δραματική βελτίωση στα επίπεδα έκφρασης και στη διαλυτότητα των μορίων, ενώ ακόμη προσεγγίστηκε το αναμενόμενο μοριακό μέγεθος, όπως φανέρωσαν τα αποτελέσματα από τη χρωματογραφία μοριακής διήθησης καθώς και οι μετρήσεις δυναμικής σκέδασης φωτός. Όταν τα δύο βελτιωμένα μεταλλάγματα χρησιμοποιήθηκαν ως ανοσοπροσροφητές για την αντιγονοειδική θεραπεία που προαναφέρθηκε, παρουσίασαν βελτιωμένη ικανότητα πρόσδεσης αυτοαντισωμάτων από ορούς μυασθενών, συγκριτικά με την αγρίου τύπου γΕΚΠ. Τα αποτελέσματα αυτά έδειξαν ότι η υδρόφοβη Cys-loop περιοχή της αγρίου τύπου γΕΚΠ συμβάλλει σημαντικά στη δημιουργία συσσωματωμάτων κατά την έκφραση της ΕΚΠ και για το λόγο αυτό ακολούθησε η αντικατάσταση αυτής της περιοχής και στις υπόλοιπες ΕΚΠ των υπομονάδων του μυϊκού υποδοχέα. Προκειμένου να βελτιωθεί περαιτέρω η αντιγονοειδική θεραπευτική προσέγγιση για τη μυασθένεια, πραγματοποιήθηκαν προσπάθειες να συνεκφραστούν όλες οι ΕΚΠ των υπομονάδων του μυϊκού υποδοχέα, με σκοπό να δημιουργηθούν σύμπλοκα που διαθέτουν και τις διεπιφάνειες μεταξύ των ΕΚΠ του υποδοχέα, οι οποίες πιστεύεται ότι είναι και αυτές ανοσογόνες. Στις προσπάθειες αυτές χρησιμοποιήθηκαν οι μεταλλαγμένες ΕΚΠ των υπομονάδων που φέρουν τη Cys-loop περιοχή της AChBP. Οι αρχικές συνεκφράσεις των ΕΚΠ όλων των υπομονάδων του ανθρώπινου μυϊκού υποδοχέα ήταν ανεπιτυχείς όσον αφορά στη δημιουργία συμπλόκων. Προκειμένου λοιπόν, να πραγματοποιηθεί ο συνδυασμός όλων των ΕΚΠ των υπομονάδων και η δημιουργία ενός λειτουργικού μορίου, το επόμενο βήμα ήταν η σύνδεση των υπομονάδων με ένα πεπτιδικό συνδέτη επαναλαμβανόμενων αμινοξέων. Ο συνδέτης που χρησιμοποιήθηκε αποτελείται από οκτώ επαναλαμβανόμενες τριάδες των αμινοξέων αλανίνη – γλυκίνη – σερίνη (AGS)8. Αρχικά κατασκευάστηκαν όλα τα ζεύγη (συγκαταμερή) που χρειάζονται, για να επακολουθήσουν πολλαπλά στάδια υποκλωνοποιήσεων, προκειμένου να σχηματιστεί το πενταμερές. Όλα τα συγκαταμερή παρουσίασαν το αναμενόμενο μοριακό βάρος όπως φάνηκε από ηλεκτροφόρηση σε πήκτωμα πολυακρυλαμιδίου σε αποδιατακτικές συνθήκες (SDS-PAGE), γεγονός που σημαίνει ότι ο συνδέτης δεν πρωτεολύεται και είναι ικανός να συγκρατεί τις υπομονάδες ενωμένες. Επιπλέον, τα επίπεδα έκφρασης που παρουσίασαν τα συγκαταμερή ήταν ικανοποιητικά και όλα τα συγκαταμερή που έφεραν την α1ΕΚΠ διατήρησαν την ικανότητα να προσδένουν σημασμένη α-μπουγκαροτοξίνη, η οποία προσδένεται ειδικά στην α1ΕΚΠ με υψηλή συγγένεια. Ωστόσο, τα αποτελέσματα από τη χρωματογραφία μοριακής διήθησης, φανέρωσαν ότι όλα τα συγκαταμερή εκλούονται δίνοντας ένα ευρύ φάσμα μοριακών βαρών, από μονομερή μέχρι ολιγομερή και συσσωματώματα. Τέλος, η α1ΕΚΠ-(AGS)8-γΕΚΠ παρουσίασε αυξημένη ικανότητα πρόσδεσης 125Ι-α-Βgt συγκριτικά με την γΕΚΠ-(AGS)8-α1ΕΚΠ, γεγονός που σημαίνει ότι η σειρά με την οποία συνδέονται οι υπομονάδες, παίζει σημαντικό ρόλο στη διαμόρφωση των μορίων. Στην προσπάθεια βελτίωσης της διαμόρφωσης και των χαρακτηριστικών των συγκαταμερών, έγινε χρήση ενός μεγαλύτερου συνδέτη, αποτελούμενου από έντεκα επαναλαμβανόμενες τριάδες των αμινοξέων αλανίνη – γλυκίνη – σερίνη (AGS)11, ωστόσο δεν παρατηρήθηκαν διαφορές στα τελικά προϊόντα έκφρασης. Όσον αφορά στη χρήση των συγκαταμερών στη θεραπευτική προσέγγιση για τη μυασθένεια, τα αποτελέσματα έδειξαν ότι όλα τα συγκαταμερή διατήρησαν πλήρως την ικανότητα να προσδένουν αυτοαντισώματα από τους ορούς των μυασθενών, αν και δε βελτίωναν περαιτέρω την ικανότητα ανοσοπροσρόφησης των επιμέρους ΕΚΠ. Συμπερασματικά λοιπόν, καταλήγουμε ότι η αντικατάσταση της υδρόφοβης Cys-loop περιοχής των ΕΚΠ των υπομονάδων με την αντίστοιχη, υδρόφιλη περιοχή του ομολόγου AChBP, οδηγεί στη δημιουργία μορίων με βελτιωμένα χαρακτηριστικά, όπως είναι τα επίπεδα έκφρασης και η διαλυτότητα των μορίων. Τα μόρια αυτά μπορούν στη συνέχεια να συνδεθούν με ένα πεπτιδικό συνδέτη και να παραχθούν συγκαταμερή με σωστή διαμόρφωση, τα οποία διατηρούν τα λειτουργικά χαρακτηριστικά τους, όπως είναι η πρόσδεση σημασμένης α-μπουγκαροτοξίνης και η πρόσδεση αυτοαντισωμάτων από ορούς μυασθενών. Τα δεδομένα αυτά ενισχύουν την πεποίθηση ότι είναι εφικτή η κατασκευή του ετεροπενταμερούς του ανθρώπινου μυϊκού υποδοχέα που θα αποτελείται μόνο από τις ΕΚΠ των υπομονάδων του. / The nicotinic acetylcholine receptor (AChR) belongs to the superfamily of pentameric ligand-gated ion channels (LGIC), also known as the Cys-loop receptor family. The subunits of the receptors belonging to this superfamily carry at their N-terminal domain characteristic, highly conserved Cys-loop region, a string of 13 amino acids linked by a disulfide bond. AChRs are large, transmembrane glycoproteins, which are composed of five homologous subunits, arranged around a central ion channel and they are divided into two subgroups, the neuronal and the muscle type. The neuronal receptors are expressed mainly in the central and the peripheral nervous system, as well as in non-neuronal tissues, such as epithelial and immune cells. Moreover, the neuronal receptors are further subdivided into two categories, the heteropentamers, such as (α4)x(β2)y, and the homopentamers, such as (α7)5. The muscle AChRs are located on the postsynaptic membrane of the neuromuscular junction and have the subunit composition α2βγδ (embryonic) or α2βεδ (adult). The muscle AChR is the best studied model from the superfamily of the LGICs, as far as its characteristics and function are concerned. One of the major reasons which contributed to its extensive characterization is the ability to isolate, in great amounts and in a functional form, AChRs similar to these of the neuromuscular junction from the electric organs of the fishes Torpedo and Electrophorus. An electron microscopy model of the Torpedo marmorata, purified from the electric organ of the electric ray, provided the first information about the three-dimensional structure of the AChR. The first high resolution insight was provided by the solved crystal structure of the AChBP from the snail Lymnaea stagnalis, a homologue of the extracellular domain (ECD) of the AChR. The AChBP is a soluble protein, which forms stable homopentamers and shares high homology with the ECDs of the AChR (20%-24%). Recently, the solved crystal structure of a mutated mouse α1ECD bound to α-bungarotoxin provided further information for a number of receptor-specific elements, including the main immunogenic region (MIR) and the Cys-loop. Nevertheless, high resolution information about the three-dimensional structure of the human AChR has not been reported so far. In addition to the physiological role of the muscle AChR, which is responsible for mediating the neuromuscular transmission, the muscle AChR is the target for a number of acquired and hereditary diseases, including the autoimmune disease myasthenia gravis (MG). In MG, autoantibodies against the AChR cause loss of the available and functional AChRs in the neuromuscular junction, and as a consequence, the action of acetylcholine is blocked. Autoantibodies against AChR are found in nearly 80%-90% of patients with generalized MG and are considered the main pathogenic factor in MG. The important pathophysiological roles of the AChRs made them subject to intensive research. The detailed analysis of the structure and function of the AChR could contribute to the understanding of the structure and function of the rest of the members of the superfamily, as well as to the comprehension of the pathogenic mechanism of MG. However, the fact that the AChR is a big, hydrophobic molecule, difficult to purify from natural sources, make the accomplishment of structural studies hard. It is necessary to generate recombinant molecules with conformation similar to that of the native AChR, in efficient amounts, in order to perform structural studies. We have previously expressed the human muscle AChR α1, β1, γ and ε ECDs using the yeast Pichia pastoris. These recombinant proteins were purified in a soluble and glycosylated form, recognizable by both monoclonal antibodies and a considerable percentage of anti-subunit antibodies in positive sera from MG patients. However, both the expression yield and the solubility of these proteins were not satisfactory for structural studies. Nevertheless, they were successfully used for a novel, antigen-specific therapeutic approach against MG, regarding the specific removal of the autoantibodies from MG sera, using these proteins as immunoadsorbents. The use of the human α1ECD as an immunoadsorbent resulted in the removal of a great percentage of autoantibodies from sera derived from MG patients. The results were similar when the β1, γ and ε ECDs were used as immunoadsorbents. However, the complete removal of the pathogenic autoantibodies from the MG sera was not achieved. We aimed at the improvement of the structure, solubility and expression yield of the ECDs of the muscle AChR, in order to render them suitable for structural studies and mainly to use them for the amelioration of the antigen-specific therapeutic approach against MG. For this, we selected the γECD, which formed oligomers and displayed a low expression yield and constructed four mutants of the protein. The mutations were concerning the replacement of certain cysteines, which could form inappropriate intra- or inter-molecular bonding and thus aggregation, as well as the substitution of the hydrophobic Cys-loop region by its counterpart, more hydrophilic AChBP Cys-loop. The two mutants which carried the Cys-loop of the AChBP displayed a dramatic improvement at the expression yield and solubility, while they approached the expected molecular size, according to the results from gel filtration and dynamic light scattering. When these two improved mutants were used as immunoadsorbents, they exhibited an increased ability to bind autoantibodies from MG sera, compared with the wild type γECD. These results indicate that the hydrophobic Cys-loop region of the γECD importantly contributes to the formation of aggregates during the expression of the ECD, and consequently followed the replacement of this region at the rest ECDs of the muscle AChR. In order to further ameliorate the antigen-specific therapeutic approach against MG, we carried out efforts to co-express all of the ECDs of the muscle AChR, targeting to create complexes which would carry the interfaces between the ECDs, which are thought to be also immunogenic. At these efforts, the mutant forms of the ECDs which carried the Cys-loop of the AChBP were used. These initial co-expressions of the ECDs were not successful, with the respect to the creation of complexes. In order to combine the ECDs of all the subunits and construct a functional molecule, the next step was the binding of these ECDs with a linker of repeated amino acids. The linker that was used, was consisted of eight repeated triplets of the amino acids alanine-glycine-serine (AGS)8. At the beginning were constructed the pairs (concatamers) which were needed in order to follow subclonings, preparative to form the pentamer. All of the concatamers displayed the expected molecular weight, according the results of the SDS-PAGE. This fact suggests that the linker does not undergo proteolysis, and thus is capable of retaining the subunits conjoint. Furthermore, the expression yield of the concatamers was satisfactory and they retained their ability to bind radiolabelled α-bungarotoxin, which is specifically binds to the α1ECD with high affinity. However, the results from the gel filtration indicated that all the concatamers eluted in a broad spectrum of molecular weights, from monomers to oligomers. In an effort to improve the conformation and the characteristics of the concatamers, a bigger linker which was consisted of eleven repeated triplets of the amino acids alanine-glycine-serine (AGS)11 was used, but the final products where shown to be similar. With respect to the use of the concatamers at the therapeutic approach against MG, the results showed that all the concatamers retain the ability to bind autoantibodies from MG sera, although there was no further improvement in the immunoadsorbing ability of the single ECDs. Conclusively, the substitution of the hydrophobic Cys-loop of the ECDs by the corresponding, more hydrophilic of the AChBP, results in the construction of molecules with improved characteristics, such as the expression yield and the solubility. The molecules are able to be linked together and form concatamers with the appropriate conformation, retaining their functionality, such as the binding of radiolabelled α-bungarotoxin and autoantibodies from MG sera. These results demonstrate that it is feasible to construct the heteropentamer of the human muscle AChR, which will consist only of the ECDs.

Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) είναι πενταμερή διαμεμβρανικά γλυκοπρωτεϊνικά μόρια τα οποία ανήκουν στην υπερ-οικογένεια των συνδεόμενων με προσδέτη ιοντικών καναλιών και ανάλογως με τη θέση τους στα σπονδυλωτά διακρίνονται σε νευρικού τύπου και μυϊκού τύπου. Ο μυϊκός nAChR συναντάται στη νευρομυΪκή σύναψη και έχει στοιχειομετρία (α1)2β1γδ ή (α1)2β1εδ. Στους νευρικού τύπου nAChRs, μεταξύ άλλων ανήκει και ο α4β2 υποδοχέας ο οποίος συναντάται σε υψηλά επίπεδα στον εγκέφαλο του ανθρώπου και εμφανίζεται με τη στοιχειομετρία (α4)2(β2)3 ή (α4)3(β2)2. Αποτελέσματα μελετών έχουν δείξει ότι ο υποδοχέας αυτός εμπλέκεται σε νευροεκφυλιστικές νόσους – Alzheimer, Parkinson, σχιζοφρένεια – καθώς και στον εθισμό στο κάπνισμα. Για το λόγο αυτό ο α4β2 υποδοχέας αποτελεί σημαντικό στόχο για το σχεδιασμό φαρμάκων και συνεπώς οι πληροφορίες που αφορούν τη δομή του και κυρίως το εξωκυτταρικό τμήμα του (ECD) – όπου συναντώνται οι θέσεις πρόσδεσης των προσδετών – είναι σημαντικές. Στο εργαστήριό μας έχει κατασκευαστεί και εκφράζεται στο ζυμομύκητα Pichia pastoris ένα συγκαταμερές το οποίο αποτελείται από τα ECDs των υπομονάδων β2 και α4 συνδεδεμένα σε σειρά μέσω ενός πεπτιδίου 24 αμινοξικών καταλοίπων (β2-α4). Η υψηλή υδροφιλικότητα και οι καλές ιδιότητες πρόσδεσης συνδετών αποτελούν σπουδαία πλεονεκτήματα που καθιστούν το συγκαταμερές αυτό σημαντικό μόριο για προσπάθειες κρυσταλλογραφικής ανάλυσης. Στηριζόμενοι σε αποτελέσματα μελετών που έχουν δείξει ότι μόρια που δεν κρυσταλλώνονται εύκολα μόνα τους, μπορούν να κρυσταλλωθούν ευκολότερα αν συνδεθούν με άλλα πρωτεϊνικά μόρια, έγινε η παραγωγή μονοκλωνικών αντισωμάτων (mAbs) έναντι του β2α4 ώστε τμήματα των mAbs που θα προκύψουν από πέψη αυτών με παπαΐνη (Fab τμήματα) να συγκρυσταλλωθούν μελλοντικά με το β2-α4. Στο πρώτο μέρος της εργασίας πραγματοποιήθηκε η παραγωγή mAbs έναντι του β2-α4. Για το σκοπό αυτό χρησιμοποιήθηκε η τεχνική της κυτταρικής σύντηξης μυελωματικών κυττάρων και σπληνικών κυττάρων αρουραίου ανοσοποιημένου έναντι του β2-α4. Αποτέλεσμα της μεθόδου αυτής είναι η παραγωγή υβριδωμάτων καθένα από τα οποία εκκρίνει ένα συγκεκριμένο mAb. Στη συνέχεια αυτής της διαδικασίας έγινε η επιλογή έξι υβριδώματων από τα οποία εκκρίνονταν αντίστοιχα έξι mAbs (mAbNR1-mAbNR6) με διαφορετικές ικανότητες πρόσδεσης. Πέντε από τα έξι mAbs αποδείχθηκε ότι προσδένουν είτε στη β2 είτε στην α4 υπομονάδα ενώ ένα από αυτά (mAbNR6) φαίνεται να προσδένει στη διεπιφάνεια των δύο υπομονάδων. Τα αντισώματα mAbNR2 και mAbNR3 παρουσιάζουν υψηλή ικανότητα πρόσδεσης αυστηρά για στην β2 και α4 υπομονάδα αντίστοιχα, ενώ τα υπόλοιπα αντισώματα πραγματοποιούν διασταυρούμενες αλληλεπιδράσεις και με άλλες υπομονάδες. Πειράματα με ολόκληρο τον ανθρώπινο υποδοχέα α4β2 έδειξαν ότι το mAbNR2 προσδένει και σε αυτόν, γεγονός που οδηγεί στο συμπέρασμα ότι το αντίσωμα αυτό θα μπορούσε να αποτελέσει χρήσιμο εργαλείο και για τον εντοπισμό του α4β2 υποδοχέα σε ανθρώπινο νευρικό ιστό. Στο δεύτερο μέρος της εργασίας πραγματοποιήθηκε απομόνωση και στη συνέχεια πέψη του mAbNR2 καθώς και άλλων δύο μονοκλωνικών αντισωμάτων του εργαστηρίου mAb73 (έναντι της β1 υπομονάδας του μυϊκού nAChR) και mAb198 (έναντι της α1 υπομονάδας του μυϊκού nAChR). Τα αντισώματα αυτά απομονώθηκαν από καλλιέργειες υβριδωμάτων και στη συνέχεια πραγματοποιήθηκε πέψη αυτών για τη δημιουργία Fab τμημάτων. Τα τμήματα Fab χρησιμοποιήθηκαν στη συνέχεια για τη δημιουργία συμπλόκων με τις αντίστοιχες υπομονάδες με σκοπό τη συγκρυστάλλωση. Τελικός σκοπός αυτής της διαδικασίας είναι η μελέτη της δομής των nAChRs και των υπομονάδων τους καθώς και η διευρεύνηση του τρόπου αλληλεπίδρασης αυτών με τα αντισώματα. / Nicotinic acetylcholine receptors (nAChRs) are pentameric transmembrane glycoproteins which belong to the super-family of ligand-gated ion channels. Depending on the location of the nAChRs they are categorized into two groups: muscle type and neuronal type. The muscle type nAChR is present in the neuromuscular junction with the stoichiometry (α1)2β1γδ or (α1)2β1εδ. The α4β2 receptor subtype belongs to the neuronal group, it is abundant in the human brain and its stoichiometry is (α4)2(β2)3 or (α4)3(β2)2. The α4β2 receptor is thought to be implicated in addiction to nicotine and in several neurological diseases including Alzheimer’s and Parkinson’s. For this reason this subtype is an attractive target for drug design and information concerning its extracellular domain (ECD) structure – where the ligand binding site is located – is invaluable. In our laboratory, the yeast Pichia pastoris expression system has been used for the expression of linked ECDs of α4 and β2 nAChR subunits (concatamer β2-α4). We managed to produce a hydrophilic molecule with near-native pharmacological profile for structural studies. Since several published data indicate that crystals of a molecule can be easier obtained when it is co-crystallized with an interaction partner, we produced monoclonal antibodies (mAbs) against β2-α4. Following mAb digestion with papain enzyme the produced Fab fragments will be co-crystallized with β2-α4. In the first part, mAbs against β2-α4 were produced. Rats were immunized against this molecule and their spleen cells were fused with myeloma cells. The result of this process was the production of hybridomas which secreted specific mAbs. Six hybridomas were selected for production of mAbs. These six mAbs (mAbNR1-mAbNR2) had different binding properties. Five of them (mAbNR1-mAbNR5) were anti-β2 or anti-α4 and one (mAbNR6) seemed to bind at the interface of the two subunits. mAb-NR2 and mAb-NR3 were highly specific for β2 and α4 respectively, whereas the other four mAbs exhibited some cross-reactivity with other nAChR subunits. Also, mAbNR2 could be useful for the detection of α4β2 subtype in human neuronal tissue as it shows high specificity for the human wild type α4β2 receptors. The second part of this project involved mAb purification and digestion to Fab. mAbNR2 and two other antibodies that have been previously produced in our lab (mAb73 and mAb198) were used. mAb73 binds to the β1 subunit of the muscle nAChR and mAb198 binds to α1 subunit of neuronal nAChR. These mAbs were isolated from hybridoma cultures and then digested to Fab fragments. The Fabs were then used to obtain complexes with the corresponding subunits for co-crystallization trials. The final aim of this process is to investigate the structure of nAChRs and its subunits as well as their interaction with the corresponding mAbs.

-- / The nicotinic acetylcholine receptors (nAChRs) are transmembrane proteins, composed of five subunits and belong to the superfamily of ligand gated ion channels The nAChRs are distinguished according to their topological and pharmacological characteristics in muscle and nervous type. Both the muscle and the nervous type are involved in the execution of many physiological functions (eg, nerve impulse transmission) but respectively in the pathogenesis of many diseases (eg Myasthenia Gravis,Parkinson's,Alzheimer's).This makes imperative the need to design drugs that target specific to each type of receptor. A prerequisite for achieving this objective is to study the structure of the extracellular regions of the receptor, as it is known that the specific areas are recognised by the cholinergic ligands and the abnormal antibodies. The α7 subunit of the human nicotinic acetylcholine receptor, can be used as a model for this study as It is expressed as a homopentamer. Wanting therefore to avoid the large and hydrophobic transmembrane regions of the receptor that would hinder the achievement of the objective, we focused on the extracellular domain (ECD) of the receptor .So, according to the above, a recombinant form of the extracellular region of the receptor was constructed and expressed previously in our laboratory (Zouridakis et al., 2009). The recombinant protein was (α7-mut10-myc-His), expressed in soluble form, in sufficient concentration and showed about three times greater affinity for I125-a-bgtx compared to the wild type (α7-ΔCDwt). Furthermore, studies of dynamic light scattering and electron microscopy confirmed the formation of homopentamer molecules. Moreover, the deglycosylated form of the protein displayed all these enhanced features, allowing the entry of crystallization experiments with both the glycosylated and the deglycosylated form. In order to further improve the specific mutant, new recombinant forms of the extracellular region of the α7 subunit of the nAChR were constructed. The recombinant forms were expressed with different expression tags in their N-or C-terminal in order to improve the folding of the molecule. The FLAG-α7-mut10-myc-His was produced in greater quantity and Ηts deglycosylated form differs significantly, indicating probably a more homogeneous protein population. Also, analysis of the molecule bygel filtration showed the predominant formation of a homopentamer molecule and the absence of high molecular weight aggregates. This protein, has enhanced features compared to the α7-mut10-myc-His and thus can proceed to crystallization trials. The second part of the study refers to the construction concateremers of the α7ECD. Σwo peptide linkers varying in their length were used. The mutant which carried the smaller linker (AGS)8, showed greater solubility compared to the more extended one (AGS)11.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Organização Mundial da Saúde estima que existam aproximadamente 250 milhões de mulheres tabagistas no mundo. Em países em desenvolvimento, a prevalência do tabagismo pode variar de 11 a 35% em mulheres grávidas, constituindo um problema de saúde pública. Nicotina, um agonista colinérgico considerado o mais importante componente ativo da fumaça do cigarro, é capaz de causar déficits em um cérebro em desenvolvimento, no entanto, muitos estudos investigam estes efeitos durante a gestação de roedores, que corresponde aos dois primeiros trimestres de gestação em humanos. O presente estudo, foi focado nos efeitos da nicotina sobre o sistema colinérgico cerebral durante o equivalente ao terceiro trimestre de gestação em humanos. Ratas lactantes foram expostas a nicotina (NIC, 6 mg/Kg/dia) ou a salina (SAL) via mini-bombas osmóticas (s.c.) a partir do 2 dia ate o 16 dia pós-natal (PN). Filhotes NIC e SAL foram sacrificados durante a exposição, em PN15, e após a retirada em três momentos diferentes, PN21, PN30 e em PN90. Quatro biomarcadores foram considerados. Para avaliação dos efeitos sobre os receptores nicotínicos de acetilcolina (nAChRs), nós utilizamos [3H]citisina, que é um ligante seletivo para &#945;4&#946;2. Nos também medimos o marcador [3H]hemicholinium-3 (CH-3) de alta afinidade para o transportador pré-sináptico de colina, e a atividade das enzimas colina acetiltransferase (ChAT) e acetilcolinesterase (AChE) no córtex cerebral (CX), mesencéfalo (MB) e hipocampo (HP) na prole. O grupo NIC apresentou supra-regulação de nAChRs em todas as regiões durante a exposição, este efeito foi revertido pouco tempo após a retirada. Interessantemente, uma significante infra-regulação de nAChRs foi observada após um longo tempo da retirada somente em CX. A exposição à nicotina reduziu a marcação para HC-3 durante a exposição em todas as regiões e foi revertida em PN21. Já em PN30, o grupo NIC apresentou uma diminuição do HC-3. Em contraste, em PN90, foi observado um aumento de HC-3. Não foram observados efeitos para atividade da ChAT e da AChE em CX. No que diz respeito ao MB, o grupo NIC apresentou um aumento de atividade para ambas as enzimas, ChAT e AChE, em PN30. Para a mesma idade, nos observamos um decréscimo desta atividade somente em HP. E, após um longo tempo de retirada, somente HP apresentou um aumento na atividade da ChAT. Estes dados sugerem que a exposição à nicotina em ratos durante o equivalente ao terceiro trimestre de gestação em humanos promove alterações no sistema colinérgico dos filhotes. Somado a isto, nossos resultados indicam que os efeitos prejudiciais são observáveis mesmo muito tempo após a exposição ter sido interrompida. / The Word Health Organization estimates that there are approximately 250 million women smokers in the world. In developing countries, the prevalence of smoking varies from 11% to 35% in pregnant women, constituting a public health concern. Nicotine, a cholinergic agonist that is considered one the main active components of cigarette smoke, causes deleterious effects on brain development, however, most studies investigate its effects during rodents gestation, which corresponds to the first two trimesters of human gestation. In the present study, we focused on nicotine effects on the brain cholinergic system during the third trimester equivalent of human gestation. Lactating rats were exposed either to nicotine (NIC, 6 mg/Kg/day) or to saline (SAL) via osmotic minipumps (s.c.) from the 2nd to the 16th postnatal day (PN). NIC and SAL offspring were sacrificed during exposure (PN15), or at withdrawal (PN21, PN30 and PN90). Four cholinergic biomarkers were considered. For evaluation of effects on nicotinic acetylcholine receptors (nAChRs), we utilized [3H]cytisine, a ligand that binds selectively to the a4b2 nAChR. We also assessed the binding of [3H]hemicholinium-3 (HC-3) to the high-affinity presynaptic choline transporter, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the cerebral cortex (CX), midbrain (MB) and hippocampus (HP) of the offspring. NIC offspring presented nAChR upregulation in all regions during exposure that was reversed at short term withdrawal, in PN21. Interestingly, a significant nAChR downregulation was observed at long term withdrawal only in CX. Nicotine reduced HC-3 binding during exposure in all regions that was reversed on PN21. On PN30, NIC offspring presents a decrease HC-3 binding. In contrast, on PN90 was observed an increase on HC-3 binding. There were no effects for ChAT and AChE in the CX. Regarding MB, NIC offspring presents an increase in both ChAT and AChE activity on PN30. At the same age, we observed a decreased in both ChAT and AChE activity on HP. At long term withdrawal, only HP showed an increase in ChAT activity. These data indicate that maternal nicotine exposure during the third trimester equivalent of human gestation promotes cholinergic system alterations in the offsprings. In addition, our data indicate that detrimental effects are observable even long after the exposure has been interrupted.

Introdução: Estudos em animais e em humanos demonstram que o alto consumo de cafeína pode promover riscos e complicações obstétricas podendo resultar em eventos teratogênicos na prole, como alterações esqueléticas, retardo no crescimento intrauterino e baixo peso ao nascer e a partos prematuros. Embora a relação entre o consumo de cafeína durante a gravidez e seus efeitos tóxicos sobre o desenvolvimento embrionário venham sendo alvo de vários estudos, seus mecanismos ainda não estão totalmente esclarecidos. Objetivos: avaliar os efeitos da utilização crônica de cafeína no período gestacional e neonatal sobre o desenvolvimento da prole, reflexos motores, comportamentos, resposta nociceptiva, atividade e expressão em hipocampo da acetilcolinesterase e, atividade de ectonucleotidases em medula espinhal de ratos. Métodos: o ciclo estral das ratas com aproximadamente 90 dias (peso 200-300 g) foi avaliado por meio de esfregaço de lavado vaginal. Sendo a prenhez confirmada, as ratas foram divididas em 3 grupos experimentais: (1) controle; (2) cafeína; e (3) abstido de cafeína. As ratas controles recebiam somente água, as do grupo cafeína recebiam 0,3 g/L de solução de cafeína diluída em água e as abstidas recebiam a mesma solução até o 7º dia de vida pós-natal (P7) da prole, sendo após substituída por água. O dia do nascimento foi considerado P0, padronizou-se 8 machos por ninhada. O comportamento de endireitamento postural e resposta a geotáxia negativa foram avaliados do P1 até P14 e utilizados como parâmetros dos reflexos motores da prole. No final do tratamento (P14) foram avaliados: (1) o limiar nociceptivo, utilizando aparelho de tail-flick; (2) a atividade locomotora no campo aberto (3) a funcionalidade dos receptores de adenosina, por meio da administração de agonistas e antagonistas adenosinérgicos; (4) hidrólise de nucleotídeos em sinaptossomas de medula espinhal; e (5) a atividade e a expressão gênica da enzima acetilcolinesterase em hipocampo. Os dados foram expressos em Média+Erro Padrão da Média (EPM), e analisados utilizando ANOVA de uma via/Tukey e ANOVA de duas vias de medidas repetidas/Tukey de acordo com cada experimento, as diferenças foram consideradas significativas quando P<0,05. Resultados: os animais dos grupos cafeína e abstido apresentaram um atraso no desenvolvimento dos reflexos neurológicos ao longo dos 14 dias de vida quando comparados ao grupo controle. No campo aberto, os animais do grupo cafeína apresentaram diminuição no tempo total de locomoção e cruzamentos externos em relação ao controle. O grupo abstido aumentou cruzamentos internos e diminuiu rearing. No teste de tail-flick, não observou-se diferença entre os grupos. Apesar de não haver diferença entre os grupos na resposta nociceptiva basal no teste de tail-flick, quando avaliamos a funcionalidade dos receptores de adenosina do tipo A1, observou-se que o grupo cafeína teve sua resposta diminuída ao agonista adenosinérgico (CPA) e o grupo abstido apresentou este efeito parcialmente revertido, demonstrando não ser um efeito de longa duração. No entanto, não foram observadas diferenças entre os grupos nas atividades de NTPDases e de 5’nucleotidase. Adicionalmente, observamos uma diminuição significativa na atividade da acetilcolinesterase (AChE) em hipocampo dos animais cafeínados comparados aos controles sem diferenças em sua expressão gênica, sugerindo assim que a baixa atividade desta enzima esta relacionada a mudanças pós-traducionais. Conclusão: Nossos resultados demonstram que o uso de cafeína durante a gestação e lactação pode trazer prejuízos ao desenvolvimento da prole, salientando a importância da restrição de alimentos e preparações que contenham esta substancia durante estes períodos. / Introduction: Studies in animals and humans show that high consumption of caffeine can promote risks and obstetric complications may result in teratogenic events in the offspring, such as skeletal abnormalities, intrauterine growth retardation and low birth weight and premature births. Although the relationship between caffeine consumption during pregnancy and its toxic effects on embryonic development may be the target of several studies, its mechanisms are not fully understood. Objectives: the aim was to evaluate the effects of gestational chronic caffeine intake and neonatal offspring development, neurological reflexes, behavior, nociceptive response, acetylcholinesterase activity and expression in hippocampus and ectonucleotidases activity in spinal cord of rats. Methods: adult female Wistar rats were performed vaginal lavage to verify the estrous cycle. Mating was confirmed by sperm presence in vaginal smears. On the first day of pregnancy, rats were divided into three groups: (1) control, (2) caffeine and (3) washout. Control animals received only water; caffeine group received caffeine solution 0.3 g / L diluted with water and the washout group received the same caffeine solution until the 7th day (P7), which was replaced by water. The birth date was considered P0, animals were standardized at 8 male animals per group. The righting reflex (RR) and negative geotaxis (NG) behaviors were measured from P0 to P14 and were using as motor reflexes. At P14 were evaluated: (1) the nociceptive response by tail-flick latency (TFL); (2) locomotor activity by open field test (OF); (3) functionality of A1 adenosine receptors by agonist and an antagonist administration of DPCPX and CPA; (4) nucleotides and nucleoside hydrolysis by spinal cord synaptosomes and (5) acetylcholinesterase (AChE) activity and gene expression in hippocampus. Data were expressed as mean ± SEM and analyzed using one-way ANOVA / Tukey and repeated measures ANOVA/Tukey, values were considered significant if P<0.05. Results: caffeine and washout groups presented a delay in the development of neurological reflexes over the 14 days of life when compared to the control group. In the open field, caffeine group showed a decrease in the total time of locomotion and outer crossing in relation to control. The washout group presented increase in inner crossing and decrease in rearing behavior. In the tail-flick test, no difference was observed between groups. Although there is no difference between the groups in baseline nociceptive response in the tail-flick test, when evaluating the functionality of A1 adenosine receptors, caffeine group presented a decreased response to adenosinergic agonists (CPA) and the washout group presented partially reversed of this effect, demonstrating it is not a long duration effect. However, no differences were observed between groups in the activities of NTPDase and 5'nucleotidase. Additionally, we observed a significant decrease in the activity of acetylcholinesterase in hippocampus of caffeine group compared to controls without differences in their gene expression, suggesting that the low activity of this enzyme is related to post-translational changes. Conclusion: Our results demonstrate that caffeine intake during pregnancy and lactation can bring harm to the offspring developing. Therefore, it becomes increasingly important restriction of food and caffeine-containing preparations during the embryonic period.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Organização Mundial da Saúde estima que existam aproximadamente 250 milhões de mulheres tabagistas no mundo. Em países em desenvolvimento, a prevalência do tabagismo pode variar de 11 a 35% em mulheres grávidas, constituindo um problema de saúde pública. Nicotina, um agonista colinérgico considerado o mais importante componente ativo da fumaça do cigarro, é capaz de causar déficits em um cérebro em desenvolvimento, no entanto, muitos estudos investigam estes efeitos durante a gestação de roedores, que corresponde aos dois primeiros trimestres de gestação em humanos. O presente estudo, foi focado nos efeitos da nicotina sobre o sistema colinérgico cerebral durante o equivalente ao terceiro trimestre de gestação em humanos. Ratas lactantes foram expostas a nicotina (NIC, 6 mg/Kg/dia) ou a salina (SAL) via mini-bombas osmóticas (s.c.) a partir do 2 dia ate o 16 dia pós-natal (PN). Filhotes NIC e SAL foram sacrificados durante a exposição, em PN15, e após a retirada em três momentos diferentes, PN21, PN30 e em PN90. Quatro biomarcadores foram considerados. Para avaliação dos efeitos sobre os receptores nicotínicos de acetilcolina (nAChRs), nós utilizamos [3H]citisina, que é um ligante seletivo para &#945;4&#946;2. Nos também medimos o marcador [3H]hemicholinium-3 (CH-3) de alta afinidade para o transportador pré-sináptico de colina, e a atividade das enzimas colina acetiltransferase (ChAT) e acetilcolinesterase (AChE) no córtex cerebral (CX), mesencéfalo (MB) e hipocampo (HP) na prole. O grupo NIC apresentou supra-regulação de nAChRs em todas as regiões durante a exposição, este efeito foi revertido pouco tempo após a retirada. Interessantemente, uma significante infra-regulação de nAChRs foi observada após um longo tempo da retirada somente em CX. A exposição à nicotina reduziu a marcação para HC-3 durante a exposição em todas as regiões e foi revertida em PN21. Já em PN30, o grupo NIC apresentou uma diminuição do HC-3. Em contraste, em PN90, foi observado um aumento de HC-3. Não foram observados efeitos para atividade da ChAT e da AChE em CX. No que diz respeito ao MB, o grupo NIC apresentou um aumento de atividade para ambas as enzimas, ChAT e AChE, em PN30. Para a mesma idade, nos observamos um decréscimo desta atividade somente em HP. E, após um longo tempo de retirada, somente HP apresentou um aumento na atividade da ChAT. Estes dados sugerem que a exposição à nicotina em ratos durante o equivalente ao terceiro trimestre de gestação em humanos promove alterações no sistema colinérgico dos filhotes. Somado a isto, nossos resultados indicam que os efeitos prejudiciais são observáveis mesmo muito tempo após a exposição ter sido interrompida. / The Word Health Organization estimates that there are approximately 250 million women smokers in the world. In developing countries, the prevalence of smoking varies from 11% to 35% in pregnant women, constituting a public health concern. Nicotine, a cholinergic agonist that is considered one the main active components of cigarette smoke, causes deleterious effects on brain development, however, most studies investigate its effects during rodents gestation, which corresponds to the first two trimesters of human gestation. In the present study, we focused on nicotine effects on the brain cholinergic system during the third trimester equivalent of human gestation. Lactating rats were exposed either to nicotine (NIC, 6 mg/Kg/day) or to saline (SAL) via osmotic minipumps (s.c.) from the 2nd to the 16th postnatal day (PN). NIC and SAL offspring were sacrificed during exposure (PN15), or at withdrawal (PN21, PN30 and PN90). Four cholinergic biomarkers were considered. For evaluation of effects on nicotinic acetylcholine receptors (nAChRs), we utilized [3H]cytisine, a ligand that binds selectively to the a4b2 nAChR. We also assessed the binding of [3H]hemicholinium-3 (HC-3) to the high-affinity presynaptic choline transporter, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the cerebral cortex (CX), midbrain (MB) and hippocampus (HP) of the offspring. NIC offspring presented nAChR upregulation in all regions during exposure that was reversed at short term withdrawal, in PN21. Interestingly, a significant nAChR downregulation was observed at long term withdrawal only in CX. Nicotine reduced HC-3 binding during exposure in all regions that was reversed on PN21. On PN30, NIC offspring presents a decrease HC-3 binding. In contrast, on PN90 was observed an increase on HC-3 binding. There were no effects for ChAT and AChE in the CX. Regarding MB, NIC offspring presents an increase in both ChAT and AChE activity on PN30. At the same age, we observed a decreased in both ChAT and AChE activity on HP. At long term withdrawal, only HP showed an increase in ChAT activity. These data indicate that maternal nicotine exposure during the third trimester equivalent of human gestation promotes cholinergic system alterations in the offsprings. In addition, our data indicate that detrimental effects are observable even long after the exposure has been interrupted.

Orientador: José Vanderlei Menani / Banca: Eduardo Colombari / Banca: Lisandra Brandino de Oliveira / Resumo: Estudos recentes mostraram a existência de importantes mecanismos de controle da ingestão de sódio e água no núcleo parabraquial lateral (NPBL), uma estrutura pontina localizada dorsolateralmente ao pedúnculo cerebelar superior. Lesões eletrolíticas ou neurotóxicas do NPBL aumentam a ingestão de água induzida por administração central ou periférica de angiotensina II (ANG II). O bloqueio dos receptores serotoninérgicos, colecistocinérgicos ou glutamatérgicos com injeções bilaterais no NPBL de metisergida, proglumide ou DNQX, respectivamente, aumentam a ingestão de solução hipertônica de NaCl induzida por tratamento com o diurético furosemida combinado com doses baixas do inibidor da enzima conversora de angiotensina captopril injetados subcutaneamente. Metisergida injetada no NPBL ainda aumenta a ingestão de NaCl induzida por ANG II administrada no ventrículo lateral (VL) ou no órgão subfornical (OSF) e por privação hídrica por 24 h, depleção de sódio ou DOCA. Além disso, a ativação de receptores adrenérgicos a2 com injeções de moxonidina ou noradrenalina no NPBL aumenta a ingestão de NaCl induzida por furosemida + captopril. Sendo assim, esses estudos mostram que o bloqueio dos mecanismos inibitórios do NPBL com injeções de antagonistas de receptores serotoninérgicos, colecistocinérgicos e glutamatérgicos ou agonistas de receptores adrenérgicos aumenta a ingestão de NaCl e água induzida por tratamentos prévios. No entanto, mais recentemente mostrou-se que a ativação gabaérgica do NPBL induz ingestão de NaCl e água em ratos saciados e normovolêmicos que não receberam nenhum tratamento prévio. Essa resposta é reduzida pelo bloqueio dos mecanismos colinérgicos centrais. No presente estudo, investigamos o efeito da desativação de mecanismos prosencefálicos facilitatórios na ingestão de NaCl e água induzida pela ativação gabaérgica...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Recent studies have shown important mechanisms for the control of sodium and water intake in the lateral parabrachial nucleus (LPBN), a pontine structure localized dorsolaterally to the superior cerebellar peduncle. Electrolytic or neurotoxic lesions of the LPBN increase water intake induced by central or peripheral administration of angiotensin II (ANG II). The blockade of serotonergic, cholecystokinergic or glutamatergic receptors with bilateral injections of methysergide, proglumide or DNQX, respectively, into the LPBN increases hypertonic NaCl intake induced by the treatment with subcutaneous injections of diuretic furosemide combined with low dose of angiotensin converting enzyme inhibitor captopril. LPBN injections of methysergide also increases NaCl intake induced by ANG II injected in the lateral ventricle (LV) or into the subfornical organ (SFO) and 24 h water deprivation, sodium depletion or DOCA. Moreover, activation of α2-adrenoceptors with injections of moxonidine or noradrenaline into the LPBN also increases NaCl intake induced by furosemide + captopril. Therefore theses studies have shown that blockade of LPBN inhibitory mechanisms with injections of serotonergic, cholecystokinergic or glutamatergic antagonists or adrenergic agonists increase NaCl and water intake induced by treatments that induce water and/or NaCl intake. However, recently it has been shown that GABAergic activation of the LPBN induces NaCl and water intake in satiated and normovolemic rats that received no previous treatment, a response reduced by the blockade of central cholinergic mechanisms. In the present study, we investigated the effects of deactivation of forebrain facilitatory mechanisms on NaCl and water intake induced by gabaergic activation of the LPBN. Rats with stainless steel cannulas implanted into the LV or SFO and bilaterally into the LPBN were used to test the effects of the blockade... (Complete abstract click electronic access below) / Mestre