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Necrosis is considered to be an unregulated and chaotic cell death. However, recent advances in cell death strategies support necroptosis as a form of regulated programmed necrotic cell death. In response to TNF-a or Fas ligands, necroptosis can be induced by cell death receptors in multiple cell lines in the presence of a caspase inhibitor z-VAD; necroptotic cell death has been found to play an important role in normal development, immunity, inflammation, cancer, and human diseases. In this chapter, the molecular mechanisms governing necroptosis, recent findings about the upstream and downstream schema of necroptosis, and potential therapeutic targets in neurological disorders are discussed. After being activated by TNF-a (or Fas ligands) and death receptors, receptor-interacting proteins 1 and 3 (RIP1 and RIP3) form a complex, which play a central role in the induction of necroptosis. RIP3 phosphorylates and activates mitochondrial proteins mixed lineage kinase domain-like protein (MLKL) and PGAM5, resulting in the execution of necroptosis by dynamin-related protein 1, the GTPase that controls mitochondrial fission. Some small molecules such as necrostain-1 and necrosulfonamide target different steps of necroptosis and impede the progress of necroptosis. FADD, caspase-8, CLIP, and CYLD positively or negatively regulate RIP1-/RIP3-dependent necroptosis by different mechanisms. Recent studies demonstrate the involvement of necroptosis in many neurological disorders including stroke, trauma, neonatal hypoxic-ischemic encephalopathy, and Huntington's disease. As a potential therapeutic target, the understanding of necroptotic mechanisms will provide new insights to develop more potent neuroprotectants and specific therapeutic strategies for clinical treatments of neurological disorders.

Antibodies directed against AMPAR and GABA_BR subunits have been implicated in forms of limbic encephalitis (LE), a disease characterised by memory loss and seizures. Patients with LE show clinical improvement with immunomodulatory treatment, suggesting that the associated antibodies are pathogenic. To explore further the AMPAR and GABA_BR antibodies, an in house cell based assay (CBA) was established for screening and potential pathogenicity was explored using a series of in vitro experiments. Human embryonic kidney (HEK) cells transfected with AMPAR and GABA_BR subunits and primary neuronal cultures were used to detect antibodies in patient sera and CSF. In total, 15/1361 (1.1%) AMPAR antibody positive samples and 24/1438 (1.7%) GABABR antibody positive samples were identified. The predominant antibody subclass for AMPAR and GABA_BR antibodies was shown to be IgG1. Interestingly, on transfected cells, only AMPAR antibodies showed complement deposition, and therefore had the potential to activate the classical pathway of the complement cascade. Application of IgG purified from AMPAR antibody positive patients, but not GABA_BR antibody positive patients caused a down regulation of the receptor from the cell surface of transfected HEK cells and primary hippocampal cultures. Electrophysiological analysis showed changes in Up state duration and spike rate in the entorhinal cortex following application of purified GABA_BR antibody IgG on brain slices. These findings suggest that GABABR antibodies are having a direct short term effect on GABA_BRs, and by extension cortical networks. Finally, we attempted to study whether or not viral infection could be a trigger for antibody production to known and novel antigen targets in a cohort of Japanese encephalitis viral (JEV) samples. A JEV cohort of 44 children was screened for antibodies to neuronal surface proteins by CBA. Twenty-seven percent of patients had antibodies to known neuronal surface antigens, with NMDAR and CASPR2 as the most common antigen targets. Interestingly, screening the cohort on primary neuronal cultures revealed that 65.9% of children with JEV have neuronal surface antibodies. The identification of the novel antigen target was attempted using immunoprecipitation and mass spectrometry techniques. In total, 4 neuronal surface proteins were identified that could be potential targets in JEV patients. In summary, antibodies directed against previously described antigenic targets were explored further for pathogenic potential, and a cohort of patients with a viral infection with potential novel antigen targets was investigated.

Reliable biomarkers of axonal damage are urgently needed in neurological diseases. Neurofilaments (Nf) are specific structural elements of neurons composed of at least three subunits: Nf light chain (NfL), Nf medium and Nf heavy chain (NfH). This PhD aimed to characterise NfL levels and their correlation with clinical features in patients with neurological diseases with a different rate of progression and following and under different treatment regimes. An important aim was also to develop a bioassay for NfL measurements in blood. Cerebrospinal fluid (CSF) NfL levels discriminated patients with a clinically isolated syndrome (CIS) (p=0.001) or multiple sclerosis (MS) (p=0.035) from healthy controls more efficiently, and was more sensitive to change after natalizumab therapy (p<0.0001) than CSF NfH (p=0.002). Further, CSF NfL levels decreased in fingolimodtreated MS patients (p=0.001), but not in those receiving placebo (p=0.433). Based on these findings, a sensitive method for the detection of NfL in serum was developed and validated. Patients with neurological diseases had higher serum NfL values than controls. In acute spinal cord injury (SCI), serum NfL levels correlated with injury severity and long-term motor outcome, and Minocycline treatment was associated with decreased NfL levels in complete SCI patients compared to placebo. Finally, I found that serum NfL levels were higher in CIS patients than in healthy controls but did not predict conversion to clinically definite MS (CDMS). Independent predictors of CDMS were instead oligoclonal bands, number of T2 lesions and age at CIS. Lower 25-OHvitamin D levels were associated with CDMS in univariate analysis, but this was attenuated in the multivariate model. In conclusion, NfL proved to be an analytically stable protein which is an important prerequisite for biomarkers. The role of NfL quantification as a surrogate measure of neuroaxonal damage is corroborated by my findings and further supports the usefulness of NfL as a putative biomarker of axonal damage in various neurological diseases.

We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer's disease (AD), Parkinson's disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained similar to 106 EVs/mu L by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.

O uso de técnicas de mineração de dados tem produzido resultados importantes em diversas áreas, tais como bioinformática, atividades de transações bancárias, auditorias de computadores relacionados à segurança, tráfego de redes, análise de textos, imagens e avaliação da qualidade em processos de fabricação. Em medicina, métodos de mineração de dados têm se revelado muito eficazes na realização de diagnósticos automáticos, ajudando na tomada de decisões por equipes médicas. Além do uso de mineração de dados, dados médicos podem ser representados por redes complexas, de modo a incluir conexões entre seus elementos. Por exemplo, no caso do cérebro, regiões corticais podem representar vértices em um grafo e as conexões podem ser definidas através das atividades corticais. Com isso, pode-se comparar a estrutura do cérebro de sujeitos sadios com a de pacientes que apresentam doenças mentais de modo a definir métodos para diagnóstico e obter conhecimento sobre como a estrutura do cérebro está relacionada com alterações comportamentais e neurológicas. Nesse trabalho, estamos interessados em usar métodos de mineração de dados e redes complexas para classificar pacientes portadores de quatro diferentes tipos de doenças mentais, isto é, esquizofrenia, autismo, déficit de atenção/desordem de hiperatividade e paralisia progressiva nuclear. / A data mining and knowledge discovery is in a field of research, with applications in different areas such as bioinformatics, customer transaction activity, security related computer audits, network traffic, text analysis and quality evaluation in manufacturing. In medicine, data mining methods have proven very effective in performing automatic diagnostics, helping in making decisions by medical teams. In addition to the use of data mining, medical data can be represented by complex networks in order to include connections between its elements. For example, in the case of the brain, cortical regions can represent vertices in a graph and the connections can be defined through cortical activities. Thus, we can compare the brain structure of healthy patients with those of patients with mental disorder in order to define methods for diagnosis and to obtain knowledge about how the structure of the brain is related to behavioral and neurological changes. Here, we are interested in using data mining methods and complex networks to classify patients with four different types of mental desorders, that is, schizophrenia, autism, attention deficit / hyperactivity disorder, and progressive supranuclear paralysis.

O uso de técnicas de mineração de dados tem produzido resultados importantes em diversas áreas, tais como bioinformática, atividades de transações bancárias, auditorias de computadores relacionados à segurança, tráfego de redes, análise de textos, imagens e avaliação da qualidade em processos de fabricação. Em medicina, métodos de mineração de dados têm se revelado muito eficazes na realização de diagnósticos automáticos, ajudando na tomada de decisões por equipes médicas. Além do uso de mineração de dados, dados médicos podem ser representados por redes complexas, de modo a incluir conexões entre seus elementos. Por exemplo, no caso do cérebro, regiões corticais podem representar vértices em um grafo e as conexões podem ser definidas através das atividades corticais. Com isso, pode-se comparar a estrutura do cérebro de sujeitos sadios com a de pacientes que apresentam doenças mentais de modo a definir métodos para diagnóstico e obter conhecimento sobre como a estrutura do cérebro está relacionada com alterações comportamentais e neurológicas. Nesse trabalho, estamos interessados em usar métodos de mineração de dados e redes complexas para classificar pacientes portadores de quatro diferentes tipos de doenças mentais, isto é, esquizofrenia, autismo, déficit de atenção/desordem de hiperatividade e paralisia progressiva nuclear. / A data mining and knowledge discovery is in a field of research, with applications in different areas such as bioinformatics, customer transaction activity, security related computer audits, network traffic, text analysis and quality evaluation in manufacturing. In medicine, data mining methods have proven very effective in performing automatic diagnostics, helping in making decisions by medical teams. In addition to the use of data mining, medical data can be represented by complex networks in order to include connections between its elements. For example, in the case of the brain, cortical regions can represent vertices in a graph and the connections can be defined through cortical activities. Thus, we can compare the brain structure of healthy patients with those of patients with mental disorder in order to define methods for diagnosis and to obtain knowledge about how the structure of the brain is related to behavioral and neurological changes. Here, we are interested in using data mining methods and complex networks to classify patients with four different types of mental desorders, that is, schizophrenia, autism, attention deficit / hyperactivity disorder, and progressive supranuclear paralysis.

Submitted by admin tede (tede@pucgoias.edu.br) on 2018-05-03T12:58:27Z No. of bitstreams: 1 Husys Cardoso Souto.pdf: 1103920 bytes, checksum: 1a37590505c8ec6568fe48b8acbb758f (MD5) / Made available in DSpace on 2018-05-03T12:58:27Z (GMT). No. of bitstreams: 1 Husys Cardoso Souto.pdf: 1103920 bytes, checksum: 1a37590505c8ec6568fe48b8acbb758f (MD5) Previous issue date: 2018-03-15 / Introduction: Physiotherapeutic rehabilitation and the Takkyu Volley therapeutic workshop are two very important modalities for the treatment of neurological patients. Objective: To evaluate the impact of conventional physiotherapy treatment and the Takkyu Volley therapeutic workshop on the functional independence and quality of life of patients in neurological rehabilitation. Method: This is a longitudinal, prospective, analytical study with 30 patients, performed at the Rehabilitation Clinic, from 2016/June to 2017/November, in Goiânia/GO. Of these patients, 10 were submitted only to conventional physiotherapy and 20, besides conventional physiotherapy, were also directed to the Takkyu Volley therapeutic workshop. Three questionnaires were applied: sociodemographic, SF-36v2 quality of life and Barthel Index. Descriptive and comparative statistics were performed. Results: The evaluated group had a mean age of 52.4 years; it consisted mainly of men, lived with the spouse, with family income up to a minimum wage. The evaluation of the quality of life showed greater impact on the dimensions "Functional Capacity", "General Health Status" and "Pain". The Takkyu Volley Therapy Workshop excelled at some items compared to conventional physiotherapy. In the first phase they achieved better scores in the dimensions: mental component, social aspect. And in the second phase: physical component, mental component, functional capacity, physical aspect, general state of health and emotional aspect. All the data evaluated by the Barthel index questionnaire did not show statistically significant differences in the comparison between the two modalities evaluated. Conclusion: Both treatments had positive results in improving functional disability and patients' quality of life, with special emphasis on the Takkyu Volley workshop, which achieved more expressive results. / Introdução: A reabilitação fisioterapêutica e a oficina terapêutica Takkyu Volley são duas modalidades importantíssima para o tratamento de pacientes neurológicos. Objetivo: Avaliar o impacto do tratamento de fisioterapia convencional e da oficina terapêutica Takkyu Volley na independência funcional e na qualidade de vida dos pacientes em reabilitação neurológica. Método: Trata-se de um estudo analítico, longitudinal, prospectivo, com 30 pacientes, realizado na Clínica Reabilitar, no período de junho/2016 a novembro/2017, em Goiânia/GO. Deste pacientes, 10 foram submetidos apenas à fisioterapia convencional e 20, além da fisioterapia convencional, foram também direcionados à oficina terapêutica Takkyu Volley. Foram aplicados três questionários: sociodemográfico, de qualidade de vida SF-36v2 e Índice de Barthel. Foram realizadas estatísticas: descritiva e comparativa. Resultados: O grupo avaliado tinha a média de idades de 52,4 anos, era constituído principalmente de homens, morava com o cônjuge, com renda familiar de até um salário mínimo. A avaliação da qualidade de vida apontou maior impacto sobre as dimensões "Capacidade Funcional", "Estado Geral de Saúde" e "Dor". A Oficina Terapêutica Takkyu Volley sobressaiu em alguns itens em relação à fisioterapia convencional. Na primeira fase alcançaram melhores escores nas dimensões: componente mental, aspecto social. E na segunda fase: componente físico, componente mental, capacidade funcional, aspecto físico, estado geral de saúde e aspecto emocional. Todos os dados avaliados pelo questionário do índice de Barthel não mostraram diferenças estatisticamente significantes na comparação entre as duas modalidades avaliadas. Conclusão: Ambos os tratamentos tiveram resultados positivos na melhorar a incapacidade funcional e da qualidade de vida dos pacientes, com destaque especial para a oficina Takkyu Volley, que atingiram resultados mais expressivos.

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.