Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) ανήκουν στην υπερ-οικογένεια των πενταμερών ιοντικών καναλιών (LGICs), που περιλαμβάνει τους υποδοχείς σεροτονίνης (5-HT3), γλυκίνης (GlyR) και γ-αμινοβουτυρικού οξέος (GABAA, GABAB). Κάθε υπομονάδα του nAChR αποτελείται από μία αμινο-τελική εξωκυτταρική περιοχή (ΕΚΠ), στην οποία βρίσκεται η χαρακτηριστική Cys θηλιά της υπερ-οικογένειας, από τέσσερεις διαμεμβρανικές α-έλικες και από μία κυτταροπλασματική περιοχή. Οι nAChRs διακρίνονται σε μυϊκούς και νευρικούς. Οι μυϊκοί nAChRs βρίσκονται στα ηλεκτρικά όργανα ιχθύων Torpedo sp. και στις νευρομυϊκές συνάψεις σπονδυλωτών, όπου μεταβιβάζουν τις νευρικές ώσεις στους μείς. Σχηματίζουν ετεροπενταμερή με στοιχειομετρία υπομονάδων (α1)2β1γδ ή (α1)2β1εδ στα ενήλικα άτομα θηλαστικών, με δύο θέσεις πρόσδεσης χολινεργικών προσδετών μεταξύ των α1-ΕΚΠ και της γ(ε) και δ-ΕΚΠ. Στην α1-ΕΚΠ εδράζει επίσης η κύρια ανοσογόνος περιοχή (MIR), έναντι της οποίας κατευθύνονται αντι-nAChR αντισώματα στην περίπτωση της βαριάς μυασθένειας. Οι νευρικοί nAChRs βρίσκονται στο κεντρικό και περιφερικό νευρικό σύστημα, όπου διαβιβάζουν τις νευρικές ώσεις. Σχηματίζουν είτε ετεροπενταμερή, μεταξύ 2-3 α-υπομονάδων (υπότυποι α2-6) και 2-3 β-υπομονάδων (υπότυποι β2-4), ή ομοπενταμερή. Η α7 είναι η μόνη γνωστή υπομονάδα του νευρικού nAChR που σχηματίζει ομοπενταμερή μόρια στον άνθρωπο, με πέντε θέσεις πρόσδεσης χολινεργικών υποκαταστατών. Μέχρι στιγμής, έχουν πραγματοποιηθεί τέσσερα σημαντικά επιτεύγματα, όσον αφορά την κατανόηση της δομής των nAChRs: Α. Η λύση της δομής, με κρυσταλλογραφία ακτίνων-Χ, της ομόλογης προς τα ΕΚΠ τμήματα των α υπομονάδων του nAChR (25% αμινοξική ταύτιση με την α7), ομοπενταμερούς πρωτεΐνης δέσμευσης της ACh γαστεροπόδων (AChBP), καθώς και των δομών της με διάφορους χολινεργικούς προσδέτες. Προσεγγίστηκε έτσι για πρώτη φορά η διαμόρφωση των nAChR-θέσεων πρόσδεσης χολινεργικών υποκαταστατών. Β. Η λύση της δομής, με ηλεκτρονική μικροσκοπία, του Torpedo nAChR, η οποία αποκάλυψε την δευτεροταγή και τριτοταγή δομή του nAChR. Εντούτοις, λόγω της περιορισμένης ευκρίνειας (4 Å), δεν αποκάλυψε τη συγκρότηση των θέσεων πρόσδεσης της ACh σε ατομικό επίπεδο. Γ. Η λύση της δομής σε υψηλή ευκρίνεια (1,94 Å) του συμπλόκου της α1-ΕΚΠ επίμυος με τον nAChR- ανταγωνιστή α-μπουγκαροτοξίνη (α-bgtx). Η λυση αυτής της δομής αποκάλυψε σε ατομικό επίπεδο την MIR περιοχή, τις θηλιές Α, B και C της κυρίως πλευράς των θέσεων πρόσδεσης χολινεργικών προσδετών και τη χαρακτηριστική Cys θηλιά. Δεδομένου όμως, ότι η δομή της α1-ΕΚΠ αναπαριστά ένα μονομερές, η δομή μίας πλήρως διαμορφωμένης θέσης πρόσδεσης εξακολουθεί να απουσιάζει. Δ. Η λύση της δομής, με κρυσταλλογραφία ακτίνων-Χ, μίας προκαρυωτικής LGIC πρωτεΐνης (ELIC), η οποία θεωρείται μάλιστα πως αποτελεί τον πρόγονο όλων των ευκαρυωτικών LGICs, συμπεριλαμαβανομένου του nAChR, με την οποία αποκαλύφθηκε ο βασικός σκελετός της δομής των LGICs. Είναι φανερό, ότι παρόλα αυτά τα επιτεύγματα, απουσιάζει ακόμη η λύση της δομής ενός πενταμερούς nAChR. Η νευρική α7 υπομονάδα του nAChR είναι μία καλή υποψήφια για την επίτευξη αυτού του στόχου, αφού σχηματίζει πενταμερή in vivo. Επιπλέον, αυτή εμπλέκεται σε ένα μεγάλο αριθμό νευρικών παθήσεων (Alzheimer, Parkinson, επιληψία, σχιζοφρένεια, κλπ), και η λύση της δομής της θα οδηγήσει και στο σχεδιασμό θεραπευτικών προσεγγίσεων έναντι αυτών των ασθενειών. Μάλιστα, εφ’όσον στην α7- ΕΚΠ εδράζουν οι θέσεις πρόσδεσης χολινεργικών υποκαταστατών, είναι πιο ρεαλιστική η προσπάθεια κρυστάλλωσης αυτής της περιοχής αντί ολόκληρου του α7 nAChR, αφού οι εκτενείς υδρόφοβες διαμεμβρανικές περιοχές του τελευταίου περιορίζουν τη διαλυτότητά του. Στο παρελθόν είχαμε εκφράσει στο Εργαστήριό μας την ανθρώπινη α7-EKΠ αγρίου τύπου και ένα διπλό μετάλλαγμα αυτής, σε κύτταρα P. pastoris, με στόχο μελέτη της δομής τους. Τα μόρια αγρίου τύπου, βρέθηκαν όμως να είναι αρκετά υδρόφοβα, αφού εκφράσθηκαν ως συσσωματώματα πολύ υψηλού μοριακού βάρους (ΜΒ) και ως ολιγομερή μεγαλύτερα από πενταμερή. Τα μόρια του διπλού μεταλλάγματος, στα οποία ολόκληρη η Cys θηλιά του α7 nAChR αντικαταστάθηκε από την πιο υδρόφιλη Cys θηλιά της AChBP σε συνδυασμό με την μετάλλαξη Cys116Ser, εμφάνισαν μεν αυξημένη διαλυτότητα και ικανότητα πρόσδεσης α-bgtx, διατήρησαν όμως δε ένα σημαντικό βαθμό δημιουργίας συσσωματωμάτων υψηλού ΜΒ, οδηγώντας σε αποτυχείς προσπάθειες κρυστάλλωσής τους. Στην παρούσα μελέτη, στηριχθήκαμε στο τρισδιάστατο μοντέλο της ανθρώπινης α7- ΕΚΠ, το οποίο κατασκευάσαμε χρησιμοποιώντας ως εκμαγεία τις, μόνες γνωστές έως τότε, δομές της AChBP και της α-EKΠ του Torpedo nAChR, προκειμένου να σχεδιάσουμε νέες μεταλλάξεις για την ενίσχυση της διαλυτότητας και της πενταμερούς συγκρότησης των α7- ΕΚΠ μορίων. Έτσι, μεταλλάξαμε τα εκτεινόμενα, στο εξωτερικό περιβάλλον του μοντέλου, υδρόφοβα αμινοξικά κατάλοιπα προς λιγότερο υδρόφοβα, με στόχο την αύξηση της διαλυτότητας των εκφραζόμενων α7-ΕΚΠ μορίων, καθώς και μερικά κατάλοιπα της διεπιφάνειας μεταξύ γειτονικών α7-ΕΚΠ πρωτομερών προς μεγαλύτερα ή φορτισμένα, με στόχο την εισαγωγή επιπλέον υδρογονικών ή ηλεκτροστατικών δεσμών με αντικρυστά κατάλοιπα της γειτονικής α7-ΕΚΠ υπομονάδας. Η μελέτη των χρωματογραφημάτων μοριακής διήθησης και δυναμικής σκέδασης του φωτός όλων των προκύπτοντων α7-ΕΚΠ μεταλλαγμάτων υπέδειξε ότι αυτά που έφεραν μεταλλάξεις σε κατάλοιπα της διεπιφάνειας εκφράσθηκαν όπως τα μόρια αγρίου-τύπου, ενώ αυτά που έφεραν την αντικατάσταση των εκτειθέμενων υδρόφοβων αμινοξικών καταλοίπων από λιγότερο υδρόφοβα, εμφάνισαν σημαντικά αυξημένη διαλυτότητα σε σχέση με τα μόρια αγρίου τύπου. Μάλιστα, ένα τουλάχιστον τέτοιο μετάλλαγμα (mut-10), εμφάνισε σημαντικά αυξημένη διαλυτότητα και ως προς το προϋπάρχον διπλό μετάλλαγμα, αφού εκφράσθηκε αποκλειστικά υπό τη μορφή ολιγομερών με κοντινό στο θεωρητικά αναμενόμενο ΜΒ για πενταμερή μόρια. Επιπλέον, εμφάνισε μία σημαντικά αυξημένη συγγένεια πρόσδεσης για τον σημασμένο ανταγωνιστή 125I-α-bgtx (Kd = 24 nM), σε σχέση με τα μόρια αγρίου τύπου (Kd = 70 nM) και διπλού μεταλλάγματος (Kd = 52 nM), η οποία πρόσδεση βρέθηκε να αναστέλλεται από μη σημασμένα μόρια α-bgtx, d-τουμποκουραρίνης ή νικοτίνης (Ki = 21,5 nM, Ki = 127 μM, Ki = 17,5 mM, αντίστοιχα). Οι τιμές αυτές των σταθερών για το mut-10 είναι χαμηλότερες από αυτές των μορίων αγρίου τύπου και άλλων μεταλλαγμάτων, ενώ είναι αρκετά κοντινές προς αυτές του φυσικού μορίου α7 nAChR, υποδηλώνοντας ταυτόχρονα την αύξηση στην ικανότητα πρόσδεσης χολινεργικών υποκαταστατών, αλλά και την κοντινή διαμόρφωση του mut-10 στο χώρο με αυτό των φυσικών μορίων. Επιπρόσθετα, μελέτες κυκλικού διχρωϊσμού, αποκάλυψαν την ύπαρξη καλά ορισμένης τριτοταγούς δομής στα mut-10 μόρια, καθώς και τοπικές αλλαγές στη διαμόρφωσή τους κατά την πρόσδεση διάφορων χολινεργικών προσδετών. Επίσης, αποκάλυψαν τη σύσταση της δευτεροταγούς δομής των ανθρώπινων α7-ΕΚΠ μορίων, η οποία βρέθηκε να είναι ~45% β-πτυχωτή επιφάνεια και 5% α-έλικα, σε συμφωνία με αυτή των ομόλογων ΕΚΠ τμημάτων του Torpedo nAChR, της AChBP και της α1-EKΠ του nAChR επίμυος. Τέλος, με ηλεκτρονική μικροσκοπία αποκαλύφθηκε ένας υψηλός βαθμός ομοιογένειας των εκφραζόμενων mut-10 μορίων, και η συγκρότησή τους πιθανότατα σε πενταμερή με εμφανή το χαρακτηριστικό κεντρικό πόρο, ο οποίος αποτελεί την απαρχή του ιοντικού καναλιού σε ολόκληρο τον α7 nAChR. Συμπερασματικά, το μετάλλαγμα mut-10 της παρούσης μελέτης είναι κατάλληλο για δομικές μελέτες υψηλής ανάλυσης, απαραίτητες για την έναρξη ενός ορθολογικού σχεδιασμού φαρμάκων έναντι των ασθενειών που συνδέονται με τον α7 nAChR. Επιπλέον, δεδομένου ότι τα απογλυκοζυλιωμένα mut-10 μόρια διατήρησαν την υδροφιλικότητα, την ικανότητα πρόσδεσης χολινεργικών υποκαταστατών και τη δευτεροταγή τους δομή, αυτά είναι επίσης κατάλληλα για κρυσταλλώσεις, αφού μάλιστα σε αυτή την περίπτωση αυξάνεται πιθανότατα και ο βαθμός ομοιογένειάς τους. Ένας παράλληλος σκοπός της παρούσης διατριβής ήταν η ανίχνευση με μελέτες κυκλικού διχρωϊσμού, της δευτεροταγούς και τριτοταγούς δομής των α1-, β1-, γ- και ε- ΕΚΠ μορίων του ανθρώπινου μυϊκού nAChR, τα οποία είχαν επίσης εκφρασθεί σε κύτταρα P. pastoris. Οι μελέτες αυτές αποκάλυψαν ότι το κυρίαρχο δευτεροταγές δομικό χαρακτηριστικό όλων αυτών των μορίων ήταν η β-πτυχωτή επιφάνεια (~40%), ενώ παρατηρήθηκε και μία μικρή συμμετοχή α-έλικας (~5%), σε συμφωνία με την δευτεροταγή δομή των ομόλογων ΕΚΠ τμημάτων του Torpedo nAChR και της AChBP και με τη μεταγενέστερη των αποτελεσμάτων αυτών, λύση της δομής της α1-ΕΚΠ του nAChR επίμυος. Επίσης, επιβεβαίωσαν την ύπαρξη καλά ορισμένης τριτοταγούς δομής στα μόρια αυτά, κάτι που είχε στο παρελθόν υπαινιγχθεί από βιοχημικές και ανοσοχημικές μελέτες. Τα αποτελέσματα αυτά υποδηλώνουν την κοντινή προς τα φυσικά μόρια διαμόρφωση των ΕΚΠ μορίων του ανθρώπινου μυϊκού nAChR που εκφράζονται στο Εργαστήριό μας, και ενισχύουν τη χρήση τους για περαιτέρω δομικές μελέτες υψηλής ανάλυσης, αλλά και ως ειδικούς ανοσοπροσροφητές των αντι-nAChR αντισωμάτων ορών μυασθενικών σε μία, υπό ανάπτυξη από το Εργαστήριό μας, αντιγονο-ειδική θεραπευτική προσέγγιση της βαριάς μυασθένειας. / Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of pentameric ligand-gated ion channels (LGICs), also including serotonin (5-HT3), glycine and γ- aminobutyric acid receptors (GABAA and GABAC). Each nAChR subunit consists of an Nterminal extracellular domain (ECD), harbouring the signature Cys-loop, four transmembrane α-helices and a small cytoplasmic loop. nAChRs are classified into muscle and neuronal types. Muscle-type nAChRs are found in fish electric organs and at the vertebrate neuromuscular junctions, where they mediate neuromuscular transmission. They form heteropentamers with a stoichiometry (α1)2β1γδ or (α1)2β1εδ in adult mammalian nAChR and bear two ligand-binding sites formed between α1- and γ(ε)- or δ-ECDs. The α1-ECD hosts the main immunogenic region (MIR), against which a large number of anti-nAChR antibodies are directed in the autoimmune disease, myasthenia gravis. Neuronal nAChRs are widely distributed in the central and peripheral nervous system and play key roles in neuron-neuron interactions. They exist either as heteropentamers of 2-3 α subunits (subtypes α2–6) plus 2-3 β subunits (β2–4) or as homopentamers. α7 is the only human neuronal subunit known to form a homopentamer with five ligand-binding sites between its ECDs. Regarding the atomic structure of nAChR, four major breakthroughs have been achieved so far: A. The X-ray crystal structure of the homologous to nAChR α-ECDs (25% sequence identity with α7), molluscan homopentameric acetylcholine-binding protein (AChBP) and its complexes with various cholinergic ligands, which approached the structure of the nAChR ligand-binding site in atomic detail for the first time. Β. Τhe 4Å electron microscopy (EM) structure of the Torpedo nAChR, which revealed the architecture of the muscle-type nAChR-ECDs and the fivefold symmetry of the receptor. Nevertheless, given the relatively low-resolution, no atomic details for the ligand-binding site could be observed. C. The high-resolution X-ray crystal structure of the complex of mouse muscle α1-ECD with the nAChR antagonist α-bungarotoxin (α-bgtx), which revealed atomic details for the MIR epitope, the loops A, B and C, forming the principal side of the nAChR ligandbinding pocket and the Cys-loop. However, since the structure of mouse α1-ECD is a monomer, the nAChR ligand-binding site is still missing. D. The high-resolution X-ray crystal structure of a prokaryotic LGIC (ELIC protein) considered to be the ancestor of eukaryotic LGICS, including the nAChR, which revealed the core structure of the LGICs. Apparently, it still remains essential to obtain the structure of a pentameric nAChR-ECD in high-resolution, so as to look deep into the details of the complete nAChR ligand-binding pockets. Neuronal α7 nAChR is a good candidate for achieving this goal, as it forms homopentamers. Furthermore, since α7 nAChR is implicated in neurological diseases and disorders (Alzheimer’s, Parkinson’s, epilepsy, schizophrenia, etc), elucidation of its structure will also lead to the rational drug-design towards these diseases. Furthermore, since the α7-ECD is of the main pharmacological interest as this bears the ligand-binding sites, it seems more realistic to perform crystallization trials on this domain, rather than on the intact α7 nAChR, due to the large hydrophobic transmembrane domains of the latter. Our laboratory has previously expressed the wild-type and a double mutant of human α7-ECD in yeast P. pastoris, with the aim to proceed to their detailed structural analysis. However, the wild-type was expressed in the form of microaggregates and oligomers larger than the expected pentamers, whereas the double mutant, carrying the mutation Cys116Ser and the replacement of its Cys-loop by the more hydrophilic AChBP Cys-loop, appeared to be more soluble than the wild-type and capable of binding α-bgtx with an increased affinity, relatively close to that of the native α7 nAChR. However, this mutant was still aggregationprone to some extent, thus leading to unsuccessful crystallization trials. Therefore, in the present study, we based on the model of human α7-ECD constructed using as templates the X-ray crystal structure of L-AChBP and the electron microscopy structure of the Torpedo nAChR α1-ECD, and introduced several mutations in α7-ECD so as to enhance both its solubility and assembly to pentamers. The hydrophobic amino acid residues found exposed to the environment of α7-ECD model were mutated to less hydrophobic ones, with the aim to reduce the considerably high hydrophobicity of α7-ECD molecules, while various residues facing the interface between two adjacent α7-ECD protomers were mutated to larger or charged residues, with the aim to introduce additional hydrogen or electrostatic bonds with facing residues of the adjacent protomer. Gel filtration and dynamic light scattering analysis for the novel α7-ECD mutants under study, suggested that the mutants carrying mutations in the interface-located amino acid residues were expressed similarly to the wild-type, whereas the mutants carrying the substitution of external hydrophobic residues by less hydrophobic ones, all appeared significantly more water-soluble than the wild-type molecules. Moreover, at least one mutant (mut-10) presented enhanced solubility compared to both the wild type and the previously studied soluble double mutant, as it was expressed exclusively to oligomers close to a pentameric form. Furthermore, it displayed a significantly improved binding affinity for the nAChR antagonist 125I-α-bgtx (Kd = 24 nM), compared to the wild type (Kd = 70 nM) and to the double mutant (Kd = 52 nM), the binding being inhibited by unlabelled α-bungarotoxin, d-tubocurarine or nicotine (Ki = 21.5 nM, Ki = 127 μM, Ki = 17.5 mM, respectively). These values for mut-10 are comparable to those for the native α7 nAChR, denoting its native-like conformation. Circular dichroism (CD) studies on mut-10 suggested a well-defined tertiary structure and special local conformational changes upon binding of several cholinergic ligands. They also revealed a secondary structure composition (~5% α- helix, ~45% β-sheet) similar to that of the homologous Torpedo nAChR-ΕCDs, AChBP and mouse muscle α1-nAChR-ECD. Finally, electron microscopy studies revealed a high degree of homogeneity and well-assembled particles of the expressed mut-10, probably pentameric, with the characteristic formation of the central hole, which in the case of the intact α7 nAChR continues to the central pore. In conclusion, the mutagenesis strategy followed in the current study, towards a crystallisable α7-ECD form, led to the construction and expression of at least one novel mutant (mut-10), which is a promising starting material for atomic-resolution studies, essential for rational drug design towards diseases related to the α7-nAChR. Furthermore, since its deglycosylated form maintained its solubility, ligand-binding properties and secondary structure, it is even more appropriate for crystallization, as it appears to be more homogeneous than the glycosylated molecules. Another aim of the present study was to probe the structure of the α1-, β1-, γ- and ε- ECDs of the human muscle nAChR, previously expressed in yeast P. pastoris, by performing CD studies. These studies demonstrated that the dominant structural feature of all these ECDs is β-sheet structure (~40%) with a small contribution of α-helical content (~5%). This was the first time direct experimental evidence appeared for the secondary structure composition of human nAChR-ECDs, which seems to be in very good agreement with that of the similar Torpedo muscle-type nAChR-ECDs and in considerable, though lower, agreement with that of the less homologous AChBPs. The subsequent structure solution of the α1-ECD of the mouse muscle nAChR in high resolution, further confirmed the native-like conformation of the human muscle nAChR-ECDs expressed in our laboratory, since their secondary structure composition was also in considerable agreement with that of mouse α1-ECD (47% β-sheet; 7% α-helix). The CD studies also suggested well-defined tertiary structures and considerable folding for all human muscle nAChR-ECDs under study, as this was previously implied by biochemical and immunochemical studies. In conclusion, these results strongly suggest that the ECDs of the human muscle nAChR under study fold to a near-native conformation, confirming their suitability for more detailed structural studies and for their use as specific immunoadsorbents in an under development antigen-specific therapeutic strategy to remove pathogenic anti-nAChR antibodies from myasthenic patients’ sera.

No description available.

The objective of this study was to contribute evidence regarding the use of the SF6D and HUI3 in persons with neurological conditions. The data of 776 individuals from the LINC Study was analyzed. The mean utility score of the HUI3 was 0.47 (95% CI 0.45, 0.49) and SF6D was 0.62 (95% CI 0.62, 0.63). Even though the SF6D and HUI3 were sensitive to a variety of HRQoL domains relevant to persons with neurological conditions, they showed only marginal agreement (ICC of 0.41) with a mean utility difference of 0.15 (95% CI 0.13, 0.17). Discordance varied systematically with HRQoL status and was consistent regardless of the participant or impairment characteristics present. Despite sharing a common purpose, the substantial and clinically important differences found between the SF6D and HUI3 cast doubt on whether the utility estimates produced by these instruments are directly comparable or universally valid.

The aim of this thesis is to develop a novel technique for the estimation of firing rate dynamics from single-unit recordings of neural pulse trains. This method applies an offline digital filtering technique to extract information transmitted by a neuron in teens of a rate code. While there is increasing evidence that the traditional rate coding cannot account for all the information transmitted by a cell, and that information may also be contained in the precise timing of spikes, the firing rate signal remains the benchmark by which the vast majority of electrophysiological studies relating neural activity to functional behaviour have been interpreted. Nevertheless, there does not seem to be an agreement on a single definition of a rate code let alone a consensus on an optimal estimation method. This study raises significant concerns about the validity of some of the most common methods in systems neuroscience, and proposes a simple yet more robust alternative. This latter is based on the convolution of the spike train with an optimally designed Kaiser window. Using computer-simulated as well as experimental data obtained from single-unit recordings of vestibular canal afferents, the proposed technique is shown to consistently outperform the current methods and even to permit robust estimations under time-varying conditions. These results suggest that estimates acquired with the conventional methods are biased and hence models of neural dynamics based on these latter may not be reliable.

Feeding problems in preterm neonates stem from complications of early delivery. Attainment of independent feeding is a prerequisite for Neonatal Intensive Care Unit (NICU) discharges. Some quantitative studies of infant feeding involve excessive amounts of time for data processing. Multivariate spectral analysis was used to minimize time for investigation of variability in these rhythms. Auto and Cross-spectral parameters of the rhythms were correlated with Gestational Age (GA), Postmenstrual Age (PMA), Birthweight (BW), Days of Life (DOL), and Time Since First Nipple feeding (TSFN). Auto-spectral analysis showed 25.55% increase in Bandwidth of suck (bw-su) for a 2-week increase in GA (DOL fixed) and 8.99% increase in bw-su for a 10-day increase in DOL (GA fixed). Crossspectral analysis showed a decrease of 0.158Hz of Bandwidth of Suck-Swallow (bw-SS) for a 2-week increase in GA for GA later than 28 weeks. For GA earlier than 28 weeks, peak coherence decreased by 0.774 for a 2-week increase in GA (PMA fixed) and decreased by 0.126 for a 2-week increase in PMA (GA fixed). The method describes the progression of feeding rhythms through correlations with clinical indexes, thus providing clinicians with an understanding of the development of infant feeding and helps predict long-term developmental outcomes.

Background: Examinations with the intent to assess neurological functions and level of conciousness are common in neurocritical care, but the effect on the patient is not entirely researched. Aim: The purpose of this study was to describe the patient’s situation at the neurological wake-up test concerning the environment, nursing interventions and the possibility given the patient to become awake, and if these conditions affect the outcome of the wake-up test. Methods: A quantitative observational study took place at a neurocritical care unit, where 20 patients were observed from interruption of sedation to the neurological assessment, which was performed by a neurosurgeon or a specialized critical care registered nurse. Nursing interventions, environmental factors and physiological parameters were documented. Result: Twelve intervention varieties to control patients’ physiological parameters were used. CPP > 80 mmHg was found in 90 % of observations. Adjustment of the patient’s body position was found in 45 % of observations. The patients were considered sufficiently awake for the neurological assessment in 74 % of observations. The level of consciousness at the point of the neurological assessment was different depending on if it took place in the mornings, performed by neurosurgeons or in the afternoon, performed by nurses. The patients were not agitated and 60 % of patients appeared pain-free. Conclusion: The observed patients were given good conditions concerning environmental factors and nursing interventions for a neurological wake-up test with a fair neurological assessment. NWT as a method didn´t generally cause the observed patients discomfort, such as pain or agitation. / Bakgrund: Undersökningar för att avgöra neurologisk funktion och medvetandegrad är vanliga inom neurointensivvården, men hur patienten påverkas av undersökningarna är relativt utforskat. Syfte: Studiens syfte var att beskriva förhållandena kring patienten vid neurologiskt wake-up test med avseende på miljö, åtgärder som utförs och möjligheten patienten ges att bli tillräckligt vaken, samt om dessa förhållanden påverkar utfallet i wake-up-testet. Metod: En kvantitativ observationsstudie genomfördes på en neurointensivvårdsavdelning, där 20 patienter observerades från sederingsstopp till bedömning av neurologisk status, vilken utfördes av neurokirurg eller intensivvårdssjuksköterska. Omvårdnadsåtgärder, miljöfaktorer och fysiologiska parametrar dokumenterades. Resultat: Tolv varianter av omvårdnadsåtgärder användes för att hålla patienternas fysiologiska parametrar inom gränsvärden. Högt CPP > 80 mmHg förekom i 90 % av observationerna och den omvårdnadsåtgärd som noterades vid flest observationer var justering av patientens kroppsposition (45 %). Vid 74 % av bedömningarna av neurologisk status ansågs patienten ha hunnit vakna tillräckligt mycket. Vakenhetsgraden vid bedömning av neurologisk status skilde sig åt beroende på om bedömningen utfördes på morgon av läkare eller på eftermiddag av intensivvårdssjuksköterska. Patienterna var inte agiterade och bedömdes vid 60 % av observationerna inte visa tecken på smärta innan bedömning av neurologisk status. Slutsats: Patienterna i studien gavs goda förutsättningar med avseende på miljö och omvårdnadshandlingar för ett neurologiskt wake-up test med rättvisande bedömning av neurologisk status. För de observerade patienterna var NWT en metod som generellt sett inte verkade orsaka obehag som smärta eller agitation.

The human brain (lat. cerebrum) is the most complex and heterogeneous organ in the human body. It is involved in a great number of body functions like movement, touch sensing, vision, hearing, smelling, hormone regulation and many more. In no other organ, the molecular communication mechanisms between different cells are so poorly understood. Due to the extensive diversity of processes that are controlled by the brain, diseases and injuries of the nervous system affect the human body significantly. Because of the immense complexity of the brain, the molecular mechanisms underlying the pathology of the diseases remain largely unknown. Hence, there is an urgent need for the development of new analytical strategies in order to investigate these conditions on a molecular level. Here, a central focus lies in the study of protein and peptide expression profiles, which can provide an insight in ongoing molecular mechanisms underlying the pathophysiology of the diseases. A powerful approach for studying proteins and peptide dynamics is mass spectrometry based proteomics, which is defined as the comprehensive study of all proteins expressed in a biological matrix at a certain point of time. The central objective of this thesis was to develop and employ different mass spectrometric techniques to study protein and peptide dynamics in the central nervous system in different neurological diseases. The individual studies comprise different aspects of proteome research. The first two studies included clinical proteomic applications for investigating protein dynamics in traumatic brain injury and amyotrophic lateral sclerosis. A further study was focused on method development for MS analysis of intact neural cells. The final three projects described in this thesis comprised MS based protein and peptide imaging in brain and spinal cord tissue samples. Here, the aim was to elucidate topological changes in protein expression in ALS as well as neuropeptide alterations in distinct brain structures in L-DOPA induced dyskinesia (LID) in Parkinson’s disease. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 713

In order for animals to experience pain and to suffer from it, they have to be capable of conscious perception. Recent evidence suggests that the fetus is maintained in a sleeplike unconscious state and that conscious perception therefore only occurs after birth. The timing of the onset of conscious perception depends on the maturation of underlying neurological processes and is anticipated to be species dependent. Painspecific electroencephalographic (EEG) responses of lightly anaesthetised young of three species born at different levels of neurological development were investigated. The results of the present thesis are in agreement with published data on general neurological, EEG and behavioural development. This information, in addition to the present results, has been used to estimate the approximate time of the onset of conscious perception in tammar wallaby joeys, rat pups and newborn lambs. In wallaby joeys (extremely immature at birth), the EEG remained isoelectric until about 100-120 days of in-pouch age and became continuous by about 150-160 days, with electroencephalographic and behavioural signs of conscious perception apparent by about 160-180 days. In rat pups (immature at birth), the absence of a differentiated EEG suggests that the ability for conscious perception in pups younger than 10-12 days is doubtful. The marginal EEG responses to noxious stimulation in 12-14 day-old pups and the pronounced EEG responses in pups 18-20 days suggest that rats may be capable of conscious perception from 12-14 days onwards. In lambs (mature at birth), full conscious perception is probably not apparent before 5 minutes after birth and may take up to several hours or days to become fully established. Its modulation by the residual neuroinhibitor allopregnanolone, if that occurs, would be highest over the first 12 hours after birth. Overall, the onset of conscious perception does not seem to follow an “on-off phenomenon”, but seems to develop gradually, even in species born neurologically mature. Although conscious perception, and hence pain experience, may be qualitatively different in younger animals, on the basis of the precautionary principle, when significantly invasive procedures are planned, pain relief should be provided from those postnatal ages when pain may first be perceived – i.e. from about 120 days in the tammar wallaby joey, about 10 days in the rat pup and from soon after birth in the lamb.

Introdução: Penfigoide bolhoso (PB) resulta da produção de autoanticorpos contra proteínas hemidesmossomais BP (Bullous Pemphigoid) 180 e/ou 230, acomete os idosos, e está associado com doenças neurológicas (DN), especialmente com a demência (DEM). BP180/230 foram identificadas no Sistema Nervoso Central (SNC), aventando-se possível mimetismo antigênico entre moléculas da pele e do SNC. O fator neurotrófico derivado do cérebro (BDNF) participa da neurogênese, sinaptogênese e sobrevivência neuronal, e a sua diminuição sérica tem sido relacionada com DN. Objetivo: Quantificar o peptídeo BDNF e os anticorpos anti-BP180/230 na relação PB com DEM. Material e Métodos: Em estudo comparativo, 50 pacientes com PB, 50 com demência e 50 controles foram avaliados. A detecção dos anticorpos anti-BP180/P230 e do peptídeo BDNF foi determinada por ensaios ELISA. Imunofluorescência indireta (IFI) foi conduzida nas amostras de soro dos pacientes do grupo DEM e dos controles que apresentaram positividade para anti-BP180/230. Resultados: No grupo PB, a frequência de DN foi de 26%: DEM 16%, acidente vascular cerebral 6%, e epilepsia 4% - 5/8 (63%) pacientes apresentaram demência vascular e 3/8 (38%) demência por doença de Alzheimer. Positividade para anti-BP180/230 foi observada no grupo PB (74% e 40%, respectivamente), no grupo DEM (10% e 10%) e nos controles (14% e 0%). No grupo DEM, em 2/10 pacientes que apresentaram positividade para antiBP180/230, a IFI evidenciou depósito de IgG e C3 no lado epidérmico da clivagem, configurando quadro subclínico de PB. A mediana do BDNF resultou menor no grupo DEM (25,41 pg/ml) comparado aos controles (38,21 pg/mL), e o grupo PB apresentou os menores valores de BDNF (16,88 pg/mL). Não houve correlação dos títulos de anticorpos antiPB180/230 com a concentração do peptídeo BDNF no grupo PB. Os pacientes do grupo DEM foram alocados de acordo com a escala de demência - CDR1, CDR2 e CDR3; a mediana de BDNF do subgrupo CDR3 (23,37 pg/mL) foi inferior ao CDR1 (30,17 pg/ mL). Não houve diferença na concentração do BDNF segundo o tipo de demência. O grupo PB, quando estratificado em - com DEM, outras DN e sem DN, aqueles com associação com DEM apresentaram menores níveis de BDNF (9,1 pg/mL), comparados ao grupo sem DN e ao subgrupo CDR3 do grupo DEM. Conclusão: Marcadores para PB não são úteis para o diagnóstico de DEM. Valores sorológicos baixos de BDNF no grupo PB podem sugerir associação com DEM. BDNF pode ser utilizado como biomarcador de gravidade da DEM. / Introduction: Bullous pemphigoid (BP) is characterized by autoantibodies against the hemidesmossomal proteins BP180 and/or BP230, affects the elderly people and has been strongly associated with neurological disorders (ND), especially dementia. A possible antigenic mimicry hypothesis between the skin and the nervous system molecules is strong reasonable because BP peptides have also been identified in the central nervous system (CNS). Brain-derived neurotrophic factor (BDNF) plays a role in the synaptogenesis, neurogenesis, and neuronal survival, and some studies have been correlated the decreased serum BDNF levels with ND. The aim of this study was to quantify the BDNF peptide and the anti-BP180 and anti-BP230 antibodies in the BP and DEM relationship. Methods: AntiBP180/230 and BDNF quantification were analyzed in three groups: 50 patients with BP, 50 patients with dementia and 50 elderly individuals comprised a case-control study. Serum IgG anti-180/230, and the BDNF peptide were evaluated by using ELISA commercial kits; and immunofluorescence allied to salt split skin technique (SSS) was conducted in serum samples from patients of the dementia group and from controls who showed positive anti-BP180/230. Results: In BP group, 26% were associated with ND, and dementia was the most frequent (16%), followed by stroke (6%) and epilepsy (4%) - 5/8 (63%) patients showed vascular dementia and 3/8 (38%) patients presented dementia due to Alzheimer\'s disease. AntiBP180/230 positivity was observed in BP group (74%, 40%, respectively), in dementia group (10%, 10%) and in controls (14%, 0%). In 2/10 patients of the dementia group with positive anti-BP180/230, IIF showed IgG and C3 deposition in the epidermal side of cleavage, configuring a subclinical BP dermatosis. The medians of BDNF resulted lower in the patients of the dementia group (25.41 pg/mL) compared with controls (38.21 pg/mL), and the BP group presented the lowest BDNF values (16.88 pg/mL). There was no correlation between the anti-BP180/230 antibodies titres and the BDNF levels in BP group. There was no difference of BDNF levels accordingly with the clinical type of dementia in the dementia group. Patients of the dementia group were sub grouped accordingly with the clinical dementia severity - CDR1, CDR2 and CDR3 - being the median of BDNF in CDR3 (23.37 pg/mL) lower than in CDR1 (30.17 pg/mL) subgroup. BP group had lower levels of BDNF compared to CDR3 subgroup. BP patients when stratified with dementia, other ND and without ND, those with association with dementia presented the lowest levels of BDNF (9.1 pg/mL) compared to the PB patients without DN and to the CDR3 subgroup. Conclusion: BP biomarkers are not useful for the diagnosis of dementia. Low BDNF levels seen in BP patients may suggest an association with dementia. BDNF may be used as a biomarker of severity of dementia.

The common human GNB3 825C > T variant, which is present in 50% of the world’s chromosomes, has previously been shown to predispose individuals to hypertension, cardiac and neural disorders. This variant causes the production of a stable and gain of function protein Gβ_3S- This thesis describes the discovery of a novel D153del mutation that produces an unstable, loss of function, protein Gβ_3D in the recessively inherited, retinopathy globe enlarged (rge) chickens. This thesis also demonstrates that the normal Gβ₃ downstream phosphorylation signalling pathways are significantly altered in a tissue specific manner in rge chicken organs and in a human GNB3 825TT lymphoblast cell line. In rge tissues expressing Gβ_3D protein, the cAMP induced GRK2 phosphorylation activity is significantly altered. Moreover MAPK1 (ERK2) phosphorylation is significantly decreased compared to normal tissues. In contrast human 825TT cell lines expressing the Gβ_3S protein, showed enhanced cAMP induced GRK2 and MAPK (ERK1 and ERK2) phosphorylation activity. These results confirm previous findings of 825C > T Gβ₃ studies, that Gβ_3S is indeed a hyper-activating structural variant, in contrast to the D153del Gp3D is a classical recessively inherited non-functional mutation.