Human mitochondrial ATP synthase is an enzyme containing 18 unlike subunits located in the inner mitochondrial membrane (IMM), where the catalytic F1 domain extends into the mitochondrial matrix and the FO domain, which contains the c8-ring rotor, the a-subunit and the supernumerary subunits, is anchored in the IMM. All the subunits, apart from the a- and A6L-subunits, are encoded in the nucleus and require transport into the mitochondria before being assembled. The a- and A6L-subunits are encoded on the mitochondrial genome. The respiratory complexes generate the proton motive force (PMF), which ATP synthase uses to generate ATP from ADP and Pi. Rotation of the α- and β-subunits with the central stalk γ-, δ- and ε-subunits is prevented by coupling the F1 domain to the FO domain via the peripheral stalk (the OSCP-, F6-, d- and b-subunits). ATP hydrolysis is prevented by the natural inhibitor of the enzyme, IF1, binding to the F1 domain. In addition to the aand, b-subunits, the FO domain contains the c8-ring and six supernumerary subunits not involved in the catalytic activity of ATP synthase. The roles of five of these subunits in the assembly of ATP synthase, the e-, f-, g-, DAPIT- and 6.8 kDa proteolipid-subunits, were investigated by suppressing or disrupting their expression individually. The e-subunit is the first of the supernumerary subunits to assemble, then the g-subunit followed by the f-, 6.8 kDa proteolipid- and DAPIT-subunits. All five supernumerary subunits investigated were required to facilitate the dimerisation and oligomerisation of ATP synthase. The e-, f- and g-subunits were found to be important for maintaining mitochondrial respiratory capacity.

A hemoglobina extracelular gigante do anelídeo Glossoscolex paulistus (HbGp) é homológa à hemoglobina da Lumbricus terrestris (HbLt). Baseado nos estudos de MALDI-TOF-MS foi determinada a massa molecular (MM) das subunidades da HbGp. Entretanto, ainda não era possível propor o valor exato da MM para a HbGp íntegra, pois a estequiometria deste oligômero ainda não era totalmente clara. Este trabalho objetiva avaliar a massa molecular do oligômero em dois estados de oxidação: oxi- e cianometa-HbGp, bem como avaliar a estabilidade desta proteína, ou seja, a dissociação e desnaturação em função do pH em meio ácido. O estudo por ultracentrifugação analítica permitiu uma avaliação independente da massa molecular da HbGp. Valores de MM de 3600 ± 100 e 3700 ± 100 kDa foram obtidos para a oxi- e cianometa-HbGp, respectivamente. Estes valores está de acordo com a massa esperada, assumindo a estequiometria proposta por Vinogradov para a HbLt. Os dados de ultracentrifugação para as amostras do monômero d puro mostraram um coeficiente de sedimentação de 1,95 ± 0,04 S para ambos os valores de pH 7,0 e 10,0. Além disso, as distribuições c (s) do monômero d puro indicaram que uma pequena contribuição (5%) de dímero de monômeros, d2, com valores de s020,w de 3,2 S estava presente em solução. Para a oxi-HbGp íntegra no pH 10,0 nenhuma contribuição em 58 - 59 S foi observada, sugerindo completa dissociação oligomérica. As distribuições c (s) mostraram dois picos adicionais em relação ao monômero puro: um pico em 4,2 - 4,4 S, que está associado ao trímero, abc; e um segundo pico em 5,8 - 6,0 S, que poderia ser associado ao tetrâmero, abcd. A adição de β-mercaptoetanol leva ao desaparecimento do pico em 4,2 S, consistente com a redução das pontes dissulfeto do trímero abc e produção dos monômeros a, b e c. Cerca de 19 % da forma cianometa-HbGp íntegra coexiste em equilíbrio com as subunidades dissociadas. Finalmente, estudos em meio ácido mostravam que na faixa de pH 5,0 - 7,0 as três formas de oxidação de HbGp apresentaram alta estabilidade oligomérica. Abaixo de pH 5,0 os dados de fluorescência mostrava que a estabilidade diminui na sequência cianometa > oxi > meta. Assim a estabilidade das formas oxi-, meta- e cianometa-HbGp foi avaliada, ficando evidenciada a maior estabilidade da forma cianometa-HbGp. / The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is homologous to Lumbricus terrestris (HbLt). Based on MALDI-TOF-MS the molecular masses (MM) of HbGp subunits were determined. However, the exact value of the MM for the HbGp oligomer is not known. This study has as a main goal to evaluate the molecular weight of the oligomer in two oxidation states: oxy- and cyanomet-HbGp. Also the stability of the protein dissociation and denaturation as a function of pH was monitored. The present analytical ultracentrifugation study allowed us to assess the molecular mass of the whole oligomer giving valores of MM of 3600 ± 100 and 3700 ± 100 kDa for the oxy- and cyanomet-HbGp, respectively. These values are in agreement with the expected mass based on Vinogradov model for HbLt. Data were obtained of so20, w for the pure monomer d as 1.95 ± 0.04 S for both pH values 7.0 and 10.0. C(s) distributions for pure monomer indicated that a small contribution of dimer of monomers (5%), d2, was also present with so20, w of 3.2 S in solution. For the whole oxy- HbGp at pH 10.0 no contribution at 58 - 59 S was observed, suggesting complete oligomeric dissociation. C(s) distribution showed two additional peaks as compared to pure monomer: a peak at 4.2 - 4.5 S, probably due to the trimer, abc; a second peak at 5.8 - 6.0 S, that could be associated to the tetramer, abcd. Addition of beta-mercaptoethanol leads to the disappearance of the peak at 4.2 S, consistent with the reduction of the trimer abc disulfide bridges and production of monomers a, b and c. It may be noted that about 19% of cyanomet-HbGp, undissociated, coexist in equilibrium with the isolated subunits. Finally, studies in acidic pH values show that in the pH range 5.0-7.0 the oligomeric stability for the three oxidation forms of HbGp is quite high. Below pH 5.0, fluorescence emission data suggest that the stability is reduced in the following order: cyanomet > oxy > met-HbGp. Thus the stability of the oxy-, meta- and cyanomet-HbGp forms was evaluated evidenced making it clear the higher stability of the cyanomet-HbGp.

P2X-Rezeptoren treten aufgrund ihrer Präsenz in verschiedensten Organsystemen des menschlichen Körpers zunehmend in den Mittelpunkt zahlreicher Forschungsansätze. Besonderes Interesse gilt dabei u.a. den P2X2/3-Rezeptoren, da in ihnen ein neuer Angriffspunkt für die Entwicklung von Schmerztherapeutika gesehen wird. Trotz enormer Fortschritte in diesem Bereich, bleiben die Vorgänge und strukturellen Gegebenheiten, die zur Öffnung der Ionenkanäle führen, weiterhin spekulativ. In der vorliegenden Arbeit wurden mithilfe einer Mutagenese einzelne Aminosäuren des hP2X2-Rezeptors, welche sich in geringer Entfernung zueinander zwischen zwei Untereinheiten befanden, durch Cysteine substituiert. Die Auswahl der Aminosäuren erfolgte dabei anhand eines Homologiemodells des hP2X2-Rezeptors und des Aminosäureabgleichs zwischen den hP2X2- und hP2X3-Rezeptoren. Auf diese Weise sollte deren Interaktion über eine mögliche Ausbildung von Disulfidbrücken zwischen zwei Untereinheiten untersucht werden. Die Rezeptorfunktion wurde anschließend mittels der whole-cell patch-clamp-Technik charakterisiert. Der Rezeptor reagierte bei allen untersuchten Varianten mit einem Funktionsverlust, ein spontan öffnender Kanal konnte somit nicht generiert werden. Durch die Kombination der verschiedenen hP2X2-Rezeptor-Cysteinmutanten mit einer hP2X3-Rezeptor-Cysteindoppelmutante, konnte gezeigt werden, dass sich die verschiedenen Untereinheiten im heterotrimeren hP2X2/3-Rezeptor nicht soweit annähern, dass eine Disulfidbrücken-Bildung zwischen den Untereinheiten möglich wird. Es konnte allerdings verdeutlicht werden, dass für die Aktivierung des heterotrimeren hP2X2/3-Rezeptors zwei funktionelle Bindungsstellen zur Kanalaktivierung ausreichen.

Ο σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη της έκφρασης των υπομονάδων του GABAΑ υποδοχέα σε τομές από τον διαφραγματικό και τον κροταφικό ιππόκαμπο αμέσως μετά την θανάτωση του ζώου και κατά την διάρκεια της ανάπτυξης των "in vitro" οξέων κυμάτων που έχουν παρατηρηθεί μόνο σε τομές του κροταφικού ιππόκαμπου. Επιπλέον, μελετήθηκε η ποσοτική και τοπογραφική κατανομή των Α1 υποδοχέων αδενοσίνης με την χρήση του ραδιενεργού ιχνηθέτη [3H]-CHA (αγωνιστής των Α1 υποδοχέων) στον κροταφικό και τον διαφραγματικό ιππόκαμπο αμέσως μετά την θανάτωση του επίμυος. Η μελέτη της κατανομής των Α1 υποδοχέων αδενοσίνης έδειξε ότι η δέσμευση της [3H]-CHA ήταν μικρότερη στον κροταφικό σε σύγκριση με τον διαφραγματικό ιππόκαμπο με την μεγαλύτερη διαφορά να εντοπίζεται στην CA1 περιοχή. Το παραπάνω αποτέλεσμα έρχεται σε συμφωνία με την υπόθεση, ότι οι συνάψεις του κροταφικού ιππόκαμπου εμφανίζουν μεγαλύτερη πιθανότητα απελευθέρωσης γλουταμινικού οξέος σε σύγκριση με αυτές του διαφραγματικού ιππόκαμπου, καθώς οι Α1 υποδοχέων αδενοσίνης εντοπίζονται στην CA1 περιοχή κυρίως προσυναπτικά όπου ελέγχουν την απελευθέρωση γλουταμινικού οξέος. Στη συνέχεια της παρούσας μελέτης δείξαμε ότι η έκφραση του mRNA και των πρωτεϊνών για τις κυριότερες υπομονάδες του GABAA υποδοχέα είναι διαφορετική μεταξύ του διαφραγματικού και του κροταφικού ιπποκάμπου. Ειδικά, στην CA1 περιοχή του ιπποκάμπου η έκφραση των α1, β2 και γ2 υπομονάδων ήταν μικρότερη, ενώ αντίθετα η έκφραση των α2 και β1 υπομονάδων ήταν μεγαλύτερη στον κροταφικό ιπποκάμπο σε σύγκριση με τον διαφραγματικό ιππόκαμπο. Σύμφωνα με προηγούμενες μελέτες που αφορούν την συνέκφραση των υπομονάδων στο σύμπλοκο του GABAA υποδοχέα τα αποτελέσματα μας υποδηλώνουν ότι ο α1β2 υποτύπος του GABAA υποδοχέα επικρατεί στον διαφραγματικό ιππόκαμπο, ενώ ο α2β1 υπότυπος κυριαρχεί στον κροταφικό ιππόκαμπο. Η διαφορετική κατανομή των υποτύπων στους δυο πόλους του ιπποκάμπου που είναι εντονότερη στην CA1 περιοχή, μπορεί να επηρεάζει τις ιδιότητες του διαύλου (αγωγιμότητα, πλάτος και διάρκεια των IPSCs), δείχνοντας ότι οι υπότυποι του GABAA υποδοχέα που εντοπίζονται στον κροταφικό ιππόκαμπο έχουν μικρότερη ανασταλτική αποτελεσματικότητα, η οποία συμφωνεί με την μικρότερη GABAA-προερχόμενη αναστολή που έχει δειχθεί στην CA1 περιοχή του κροταφικού ιππόκαμπου. Επιπλέον θα μπορούσε να εξηγήσει την μεγαλύτερη επιρρέπεια του κροταφικού ιππόκαμπου στην επιληψία. Η χαμηλότερη έκφραση του mRNA για τις α4, β3 και δ υπομονάδες του GABAA υποδοχέα στην περιοχή της οδοντωτής έλικας του κροταφικού ιπποκάμπο υποδεικνύει ότι η έκφραση του α4β3δ υποτύπου είναι μικρότερη στον κροταφικό σε σύγκριση με τον διαφραγματικό ιππόκαμπο. Καθώς έχει δειχθεί ότι ο α4β3δ υπότυπος παίζει σημαντικό ρόλο στην τονική αναστολή στα κοκκιώδη κύτταρα της οδοντωτής έλικας, τα παραπάνω αποτέλεσμα μας πιθανώς σημαίνει ότι η τονική αναστολή είναι διαφορετική στους δυο πόλους του ιππόκαμπου. Η αύξηση της έκφρασης του mRNA της α5 υπομονάδας στην CA1 περιοχή του κροταφικού ιπποκάμπου μπορεί να επηρεάζει την ικανότητα για συναπτική βραχυ- και μακρο-χρόνια πλαστικότητα η οποία έχει βρεθεί να είναι διαφορετική μεταξύ του κροταφικού και του διαφραγματικού ιπποκάμπου καθώς έχει δειχθεί ότι οι α5-υπότυποι παίζουν ρόλο σε διαδικασίες μνήμης και μάθησης. Επίσης, οι α5-υπότυποι του GABAA υποδοχέα στην CA1 περιοχή του ιππόκαμπου συμμετέχουν στην τονική αναστολή. Τα υψηλότερα επίπεδα στην έκφραση του mRNA για την α5 υπομονάδα στον κροταφικό ιππόκαμπο σε σύγκριση με τον διαφραγματικό ιππόκαμπο πιθανώς υποδεικνύουν ότι η τονική αναστολή είναι διαφορετική στην CA1 περιοχή των δυο πόλων του ιππόκαμπου. Στο δεύτερο μέρος της παρούσας διατριβής μελετήσαμε την πιθανή συσχέτιση του GABAεργικού συστήματος με την οργάνωση των "in vitro" οξέων κυμάτων η οποία έχει παρατηρηθεί, σε κανονικές "in vitro" συνθήκες, μόνο σε τομές του κροταφικού ιππόκαμπου. Για το λόγο αυτό πραγματοποιήσαμε αναλυτική μελέτη της έκφρασης του mRNA των υπομονάδων (α1, α2, α5, β1, β2, β3, γ2) του GABAΑ υποδοχέα σε διάφορα χρονικά διαστήματα κατά την κανονική "in vitro" διατήρηση των τομών (15min, 1, 3, 5 και 8h). Αρχικά μελετήσαμε την έκφραση των πρώιμων γονιδίων (c-fos, zif-268), που είναι δείκτες της νευρωνικής ενεργότητας, μετά από 5 ώρες κανονικής "in vitro" διατήρησης των τομών με σκοπό τη πιθανή συχέτιση της έκφρασης τους με την οργάνωση των "in vitro" οξέων κυμάτων. Τα αποτελέσματα μας έδειξαν και στους δυο πόλους του ιπποκάμπου παρόμοια αύξηση της έκφρασης του mRNA τόσο για το c-fos όσο και για zif-268 γεγονός που υποδηλώνει ότι γονιδιακή ενεργότητα είναι παρόμοια και όσο αφορά τα συγκεκριμένα πρώιμα γονίδια ανεξάρτητη της ανάπτυξης των "in vitro" οξέων κυμάτων. Στην CA1 περιοχή του κροταφικού ιππόκαμπου παρατηρήθηκε σημαντική αύξηση της έκφρασης του mRNA των α1, β2 και γ2 υπομονάδων του GABAΑ υποδοχέα η οποία ξεκινάει την 1η ώρα, δηλαδή πριν την οργανωμένη εμφάνιση της αυθόρμητης δραστηριότητας, γίνεται μέγιστη στις 4 ώρες παραμονής των τομών σε Τεχνητό Εγκεφαλονωτιαίο Υγρό (ΤΕΝΥ) και συμβαδίζει χρονικά με την οργάνωση των "in vitro" οξέων κυμάτων. Δεν παρατηρήθηκαν σημαντικές αλλαγές σε τομές που προέρχονται από τον διαφραγματικό ιππόκαμπο. Έχει δειχθεί ότι οι α1-υπότυποι παίζουν σημαντικό ρόλο στην ανασταλτική ικανότητα του υποδοχέα. Επίσης η παρουσία της β2 υπομονάδας στον δίαυλο χαρακτηρίζει μεγαλύτερα σε πλάτος και διάρκεια ανασταλτικά ρεύματα συγκρινόμενη με τις β1 υπομονάδες. Φαίνεται λοιπόν ότι ο υπότυπος α1β2γ2, του οποίου η έκφραση αυξάνει πριν την έναρξη της οργανωμένης ρυθμικής δραστηριότητας, λόγω της συγκρότησης του από τις συγκεκριμένες υπομονάδες, έχει μεγάλη ανασταλτική αποτελεσματικότητα η οποία μπορεί να συμμετέχει στην ανάπτυξη των "in vitro" οξέων κυμάτων. Η δέσμευση της [3H]–muscimol αυξάνει σε τομές που προέρχονται μόνο από τον κροταφικό ιππόκαμπο και έχουν παραμείνει σε κανονικές "in vitro" συνθήκες για 8 ώρες σε σύγκριση με αντίστοιχες τομές που προέρχονται αμέσως μετά την θανάτωση του ζώου. Καμία αλλαγή δεν παρατηρήθηκε στην δέσμευση της [3H]–muscimol σε τομές που προέρχονται από τον διαφραγματικό ιππόκαμπο. Η αύξηση της δέσμευσης της [3H]–muscimol μόνο στις τομές που προέρχονται από τον κροταφικό ιππόκαμπο είναι σε συμφωνία με την αύξηση της έκφρασης του α1β2γ2-υποτύπου καθώς έχει δειχθεί ότι η θέση δέσμευσης της muscimol στο δίαυλο του GABAA υποδοχέα είναι μεταξύ των α1 και β2 υπομονάδων. Συμπερασματικά, η εκλεκτική αύξηση της έκφρασης του α1β2γ2-υπότυπου μόνο στην CA1 περιοχή του κροταφικού δηλώνει μεγαλύτερη ανασταλτική αποτελεσματικότητα των GABAA υποδοχέων, δεδομένου ότι ο α1β2γ2-υπότυπος προκαλεί μεγαλύτερα ανασταλτικά μετασυναπτικά ρεύματα. Το παραπάνω μπορεί να σχετίζεται με την "in vitro" εμφάνιση των οξέων κυμάτων καθώς η αυθόρμητη δραστηριότητα προέρχεται από GABAA-επαγόμενες υπερπολώσεις των πυραμιδικών κυττάρων, ενώ και η αύξηση στην έκφραση του α1β2γ2-υποτύπου συμπίπτει χρονικά με την εμφάνιση των "in vitro" οξέων κυμάτων. / The hippocampus in the rat appears grossly as an elongated structure with its long axis bending in a C-shaped manner from the septal nuclei rostrodorsally to the incipient temporal lobe caudoventrally. The long axis of the hippocampal formation is referred as the dorsoventral axis. Although hippocampus has been traditionally thought as a homogeneous structure, several studies have been demonstrated differences at several organization levels (from the behavioural to the cellular) between its dorsal (DH) and ventral (VH) pole. In the present study, we examined whether the recently reported differences in the GABA-mediated somatic inhibition between the DH and VH could be related to variations in the GABAA receptors. We therefore studied the quantitative distribution, the kinetic parameters and the subunit composition of the GABAA receptors in the two parts of hippocampus. We also studied the A1 adenosine receptors in order to examine the involvement of the adenosinergic system in the glutamate release between the two hippocampal poles. The study of [3H]-CHA binding on A1 adenosine receptors by using "in vitro" quantitative autoradiography, revealed a weaker A1 receptor binding in VH compared to DH in all regions we examined. Taken into consideration that the A1 adenosine receptors are localized in the CA1 glutamatergic terminals, these results may to some extend explain our hypothesis that synapses in the VH have greater probability of glutamate release compared to those in the DH counterpart. Recent data have demonstrated a weaker somatic GABAergic inhibition in CA1 region of VH compared to DH. We therefore examined possible differences in the GABAA receptor subunit composition and receptor binding parameters between DH and VH by using "in situ" hybridization, western blotting and the specific binding of the GABAA receptor agonist [3H]-muscimol using quantitative autoradiography and saturation experiments. The experiments demonstrated that the VH compared to DH displayed: 1) lower levels of mRNA expression for α1, β2, γ2 but higher levels for α2 and β1 subunits in CA1, CA2 and CA3, with the differences being more pronounced in CA1 region. Western blot analysis confirmed the mRNA expression data, showing lower levels for α1, β2 and higher levels for α2 subunits’ protein. Only in the CA1 region the mRNA levels of α5 were higher, while those of α4 subunit were slightly lower; in dentate gyrus, the mRNA levels of α4, β3 and δ subunits were significantly lower in VH compared to DH presumably suggesting a lower expression of the α4/β3/δ receptor subtype; 2) lower levels of [3H]-muscimol binding in the VH, with the lowest value observed in CA1, apparently resulting from weaker affinity for GABA and not from a decreased receptor density, since the KD values were higher in VH, while the Bmax values were similar between DH and VH. In conclusion, the differences in the subunit mRNA and protein expression and the lower affinity of GABAA receptor observed predominantly in CA1 region of VH, suggest that the α1 subunit-containing GABAA receptors dominate in the DH, while the α2 subunit-containing receptors prevail in VH. This could underlie the lower GABAA mediated somatic inhibition observed in VH and, to some extent, explain: a) the higher liability of VH for epileptic activity and b) the differential involvement of DH and VH in cognitive and emotional processes. Recent electrophysiological experiments have been shown that slices from the VH of adult rats generate rhythmical activity during their maintenance in the recording chamber. This activity is fully organized during the first 3 hours of in vitro maintenance and resembles the in vivo recorded hippocampal "sharp waves", therefore called "in vitro sharp waves". The field manifestation of this spontaneous activity results from GABAA receptor-mediated hyperpolarizations in pyramidal cells. The aim of the second part of the present thesis focused on the possible relationship between the characteristics of GABAA receptors and the development of "in vitro sharp waves". Using the "in situ hybridisation" technique, we examined the mRNA expression of the alpha1/2/5,beta1/2/3 and gamma2 subunits of GABAA receptor and the binding of GABAA receptor agonist [3H]-muscimol in a time course including periods before and during the development of the "in vitro sharp waves". Six sets of transversely cut DH and VH slices were prepared: slices frozen immediately after killing the animal (naive slices), and slices maintained in vitro and frozen at different time points (15min, 1, 3, 5 and 8h) during the electrophysiological experiment. The results showed: A) Upregulation of alpha1, beta2 and gamma2 subunits mRNA in VH but not in DH slices at 1h of their maintenance, which became significant at 3h as compared to the respective naive slices; B) Increase in [3H]-muscimol binding only in VH slices, obtained at 8h compared to the respective naive ones. The upregulation of the α1β2γ2 GABAA receptor subtype (starting at 1h) in VH but not in DH presumably suggests an increase in GABAergic activity, which could be related with the appearance of "in vitro sharp waves" observed only in VH; C) Τhe similar mRNA expression of the early genes c-fos and zif-268 in the two hippocampal poles showing a comparable general gene activity in DH and VH. In conclusion, the α1β2γ2 subtype dominates in DH while the α2β2-subtype prevails in VH and this could be related to the weaker somatic inhibition observed in the CA1 region of VH, and also to the distinct involvement of DH and VH in cognitive and emotional processes. Moreover, the higher expression of the GABAA receptor subtype α4β4δ in the DG of DH compared to VH may imply a higher tonic inhibition in the former hippocampal pole. The upregulation of the α1β2γ2- subtype only in VH slices during their in vitro maintenance may reflect an increase in the impact of GABAA receptor-mediated transmission, which is required for the organization of "in vitro" sharp waves.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 100 pages. Includes Vita. Includes bibliographical references.

Introdução: Os receptores ionotrópicos do glutamato, como o N-metil-D-Aspartato (NMDA), estão envolvidos em desordens psiquiátricas. NMDARs são complexos heteroméricos que incorporam tres diferentes subunidades: NR1, NR2 e NR3. Objetivos: quantificar os níveis plasmáticos das subunidades NR1 e NR2 NMDAR em pacientes em primeiro episódio psicótico (PEP), em comparação com os irmãos e controles saudáveis. Métodos: Este é um estudo transversal de PEP na região de Ribeirão Preto, Brasil, sendo o grupo controle composto por indivíduos saudáveis, pareados por idade, sexo e mesma área de abrangência dos casos. Foram coletados 5 mL de amostra de sangue próxima a data de diagnóstico de PEP. A quantificação plasmática das subunidades NR1 e NR2 foi realizada por ELISA. Os dados foram analisados por ANOVA (significante se p<0,05) e curva ROC. Resultados: Foram incluídos 166 pacientes em PEP (idade: x = 30,34 ± 12,2 anos; 64% homens), destes 84 com diagnóstico de psicose não afetiva, 51 com transtorno bipolar e 31 com transtorno depressivo. Foram tambem incluídos 76 irmãos e 166 controles saudáveis. Os níveis plasmáticos das subunidades NR1 e NR2 foram significativamente menores em pacientes com transtornos psicóticos (NR1: x = 71,0 ± 100,3 pg/mL, NR2: x = 2,5 ± 2 ng/ml), transtorno bipolar (NR1: x = 185,7 ± 319,5 pg/ml; NR2: x = 2,1 ± 2,2 ng/ml), transtorno depressivo (NR1: x = 83,2 ±185,0 pg/ml; NR2: x = 2,1± 2,1 ng/ml) em comparação com os irmãos (NR1: x = 140,6 ± 193,8 pg/ml; NR2: = 6,2 ± 1,5 ng/ml) e voluntários saudáveis (NR1: x = 146,7 ± 361,1 pg/ml; NR2: x = 4,8 ± 2,2 ng/ml) [NR1 e NR2, p < 0,001]. Indivíduos com valores plasmáticos de NR2 inferiores a 3,648 ng/mL apresentam um risco 14,72 vezes maior de estar doente (PEP) de quem não possui o NR2 abaixo deste valor. Conclusões: Este é o primeiro estudo relatando a quantificação e a redução das concentrações plasmaticas das subunidades NR1 e NR2 em transtornos psiquiátricos graves quando comparados aos irmãos e controles, podendo a subunidade NR2 ser um candidato a biomarcador plasmático em pacientes com PEP. / Background: Ionotropic glutamate receptors, such as N-Methyl-D-Aspartate (NMDA), are involved in pathophysiology of several psychiatric disorders. NMDARs are described as heteromeric complexes incorporating distincts subunits within a repertoire of three types: NR1, NR2 and NR3. Aim: to quantify the NR1 and NR2 subunits NMDAR plasma levels in patients with first episode psychosis (FEP), compared with siblings and healthy controls. Methods: This is a cross-sectional study of FEP conducted in Ribeirão Preto, Brazil. The control group were composed by healthy subjects matched for age, sex and same coverage area of cases. 5 ml of blood sample were collected next to the date of FEP diagnosis. NR1 and NR2 subunits plasmatic quantification was performed by ELISA. Data were analyzed by ANOVA (significant at p < 0.05) and ROC curve. Results: FEP sample comprised 166 patients (age: x = 30.34 ± 12.2 years; 64% men), of these 84 with a diagnosis of psychotic disorder, 51 with bipolar disorder and 31 with depressive disorder. It was also included 76 siblings and 166 healthy controls. NR1 and NR2 subunits plasma levels were significantly lower in patients with psychotic disorders (NR1: x = 71.0 ± 100.3 pg / ml, NR2: x = 2.5 ± 2 ng/ml), bipolar disorder (NR1: x = 185.7 ± 319.5 pg/mL; NR2: x = 2.1 ± 2.2 ng/ml), depressive disorders (NR1: x = 83.2 ± 185.0 pg/mL; NR2: x = 2.1 ± 2.1 ng/ml) compared with siblings (NR1: x = 140.6 ± 193.8 pg/mL; NR2: x = 6.2 ± 1.5 ng/ml) and healthy volunteers (NR1: x = 146.7 ± 361.1 pg / mL; NR2: x = 4.8 ± 2.2 ng/ml) [NR1 and NR2, p < 0.001]. Interestingly, individuals with NR2 plasma values less than 3.648 ng/ml present 14.72 times a higher risk to be in FEP than other patients. Conclusions: This is the first study reporting the measurement and reduction of NR1 and NR2 subunits plasma concentrations in severe psychiatric disorders when compared to siblings and controls. And highlighting that NR2 subunit can be a candidate for plasma biomarker in patients with FEP.

Foi levado a efeito estudo sobre formas múltiplas de aldolase de músculo de coelho. A enzima foi purificada a pH 7,5 por eluição com substrato a partir de coluna de fosfocelulose. A enzima foi ainda cristalizada por diálise dessas preparações contra solução saturada de sulfato de amônio. Formas múltiplas de aldolase foram obtidas por fracionamento a diferentes pI por eletrofocalização em gradiente de Ampholine na faixa de pH entre 7,0 a 10,0. Nessas condições foram separados cinco híbridos resultantes da associação ao acaso das sub-unidades &#945; e &#946;, os quais foram analisados em estado de dissociação a partir de proteínas carboximetiladas e separadas por eletroforese em gel de poliacrilamida na presença de uréia 8M. Foi também estudado o aparecimento de sub-unidade &#945; e &#946; em músculo de coelhos de idades que variavam de 1 a 240 dias, verificando-se que em coelhos de 1 dia existia apenas a proteína &#945;4, surgindo sub-unidades &#946; já em animais de 10 dias. / Not available.

The nuclear matrix (NM) is a fibrogranular network of ribonucleoproteins upon which transcriptional complexes and regulatory genomic sequences are organized. A hallmark of cancer is the disorganization of nuclear architecture; however, the extent to which the NM is involved in malignancy is not well studied. The RUNX1 and RUNX2 proteins form complexes within the NM to promote hematopoiesis and osteoblastogenesis, respectively at the transcriptional level. RUNX1 and RUNX2 are both expressed in breast cancer cells (BrCCs); however, their genome-wide BrCC functions are unknown. RUNX1 and RUNX2 activate many tumor suppressor pathways in blood and bone lineages, respectively, including attenuation of protein synthesis and cell growth via suppression of ribosomal RNA (rRNA) transcription, which appears contrary to Runx-expression in highly proliferative BrCCs. To define roles for RUNX1 and RUNX2 in BrCC phenotype, we examined the involvement of RUNX1 and RUNX2 in rRNA transcription and generated a genome-wide model for RUNX1 and RUNX2-binding and transcriptional regulation. To validate gene expression patterns identified in our screen, we developed a Real-Time qPCR primer design program, which allows rapid, high-throughput design of primer pairs (FoxPrimer). In BrCCs, RUNX1 and RUNX2 regulate genes that promote invasiveness and do not affect rRNA transcription, protein synthesis, or cell growth. We have characterized in vitro functions of Runx proteins in BrCCs; however, the relationships between Runx expression and diagnostic/prognostic markers of breast cancer (BrCa) in patients are not well studied. Immunohistochemical detection of RUNX1 and RUNX2 in BrCa tissue microarrays reveals RUNX1 expression is associated with early, smaller tumors that are ER+ (estrogen receptor), HER2+, p53-, and correlated with androgen receptor (AR) expression; RUNX2 expression is associated with late-stage, larger tumors that are HER2+. These results show that the functions and expression patterns of NM-associated RUNX1 and RUNX2 are context-sensitive, which suggests potential disease-specific roles. Two functionally disparate genomic sequence types bind to the NM: matrix associated regions (MARs) are functionally associated with transcriptional repression and scaffold associated regions (SARs) are functionally associated with actively expressed genes. It is unknown whether malignant nuclear disorganization affects the functions of MARs/SARs in BrCC. We have refined a method to isolate nuclear matrix associated DNA (NM-DNA) from a structurally preserved NM and applied this protocol to normal mammary epithelial cells and BrCCs. To define transcriptional functions for NM-DNA, we developed a computational algorithm (PeaksToGenes), which statistically tests the associations of experimentally-defined NM-DNA regions and ChIP-seq-defined positional enrichment of several histone marks with transcriptome-wide gene expression data. In normal mammary epithelial cells, NM-DNA is enriched in both MARs and SARs, and the positional enrichment patterns of MARs and SARs are strongly associated with gene expression patterns, suggesting functional roles. In contrast, the BrCCs are significantly enriched in the silencing mark H3K27me3, and the NM-DNA is enriched in MARs and depleted of SARs. The MARs/SARs in the BrCCs are only weakly associated with gene expression patterns, suggesting that loss of normal DNA-matrix associations accompanies the disease state. Our results show that structural preservation of the in situ NM allows isolation of both MARs and SARs, and further demonstrate that in a disorganized, cancerous nucleus, normal transcriptional functions of NM-DNA are disrupted. Our studies on nuclear organization in BrCC, show that the disorganized phenotype of the cancer cell nucleus is accompanied by deregulated transcriptional functions of two constituents of the NM. These results reinforce the role of the NM as an important structure-function component of gene expression regulation.

The Runx family of transcription factors supports cell fate determination, cell cycle regulation, global protein synthesis control, and genetic as well as epigenetic regulation of target genes. Runx1, which is essential for hematopoiesis; Runx2, which is required for osteoblast differentiation; and Runx3, which is involved in neurologic and gut development; are expressed in the growth plate during chondrocyte maturation, and in the chondrocytes of permanent cartilage structures. While Runx2 is known to control genes that contribute to chondrocyte hypertrophy, the functions of Runx1 and Runx3 during chondrogenesis and in cartilage tissue have been less well studied. The goals of this project were to characterize expression of Runx proteins in articular cartilage and differentiating chondrocytes and to determine the contribution of Runx1 to osteoarthritis (OA). Here, the expression pattern of Runx1 and Runx2 was characterized in normal bovine articular cartilage. Runx2 is expressed at higher levels in deep zone chondrocytes, while Runx1 is primarily expressed in superficial zone chondrocytes, which is the single cell layer that lines the surface of articular cartilage. Based on this finding, the hypothesis was tested that Runx1 is involved in osteoarthritis, which is a disease characterized by degradation of articular cartilage and changes in chondrocytes. These studies showed that Runx1 is upregulated in articular cartilage explants in response to mechanical compression. Runx1 was also expressed in chondrocytes found at the periphery of OA lesions in the articular cartilage of mice that underwent an OA-inducing surgery. Runx1 was also upregulated in cartilage explants of human osteoarthritic knees, and IHC data showed that Runx1 is mainly expressed in chondrocyte “clones” characteristic of OA. To ascertain the potential function of the upregulation of Runx1 in these cartilage stress conditions and disease states, the hypothesis was tested that Runx1 is upregulated in very specific chondrocyte populations in response to the cartilage damage in osteoarthritis. These studies addressed the properties of these cells that related to functions in cell growth and differentiation. In both the surface layer of normal articular cartilage, and in OA cartilage, Runx1 expression by IF co-localized with markers of mesenchymal progenitor cells, as well as markers of proliferation Ki-67 and PCNA. This finding indicated that Runx1 is found in a population of cells that represent a proliferative population of mesenchymal progenitor cells in osteoarthritis. To further address Runx1 function and identify downstream targets of Runx proteins, a promoter analysis of genes that are known to be either downregulated or upregulated during chondrocyte maturation was done. These studies found that many of these genes have 1 or more Runx binding sites within 2kb of their transcription start site, indicating that they are potential downstream Runx target genes. Lastly, some preliminary experiments were done to characterize novel roles of Runx proteins in the chondrocyte. Runx proteins have been shown to epigenetically regulate their target genes by remaining bound to them throughout mitosis, “poising” them for transcription upon exit from mitosis. The hypothesis that Runx proteins also function by remaining bound to their target genes throughout mitosis in chondrocytes was tested. It was demonstrated by immunofluorescense imaging of Runx proteins on metaphase chromosomes of ATDC5 cells, that Runx2 remains bound to chromosomes during mitosis. Cell proliferation and hypertrophy are both linked to increases in protein synthesis. Runx factors, which regulate rates of global protein synthesis, are expressed in both proliferating and hypertrophic chondrocytes. Thus, it was hypothesized that Runx proteins regulate rates of global protein synthesis during chondrocyte maturation. These studies showed that the overexpression of Runx proteins in a chondrocyte cell line (ATDC5) did not affect protein synthesis rates or levels of protein synthesis machinery. Additionally, Runx proteins did not affect proliferation rates in this chondrocyte cell line.

Indiana University-Purdue University Indianapolis (IUPUI) / Increased electrical activity in peripheral sensory neurons contributes to pain. A unique type of sodium current, fast resurgent current, is proposed to increase nerve activity and has been associated with pain pathologies. While sodium channel isoform Nav1.6 has been identified as the main carrier of fast resurgent currents, our understanding of how resurgent currents are modulated in sensory neurons is fairly limited. Thus the goal of this dissertation was to identify resurgent current modulators. In particular, we focused on sodium channel beta subunits (Navβs) and fibroblast growth factor homologous factors (FHFs) in dorsal root ganglion (DRG) neurons. We hypothesized that Navβ4 and FHF2B act as positive regulators by mediating resurgent currents and modulating Nav1.6 inactivation, respectively. In contrast, we hypothesized FHF2A negatively regulates resurgent current by increasing the probability of channels in inactivated states. Thus, the aims of this dissertation were to 1) determine if Navβ4 regulates fast resurgent currents in DRG neurons, 2) examine the effects of Navβ4 knockdown on resurgent currents, firing frequency and pain associated behavior in an inflammatory pain model and 3) determine if FHF2A and FHF2B functionally regulate Nav1.6 currents, including resurgent currents in DRG neurons. To examine the aims, we used biochemical, electrophysiological and behavioral assays. Our results suggest that Navβ4 is a positive regulator of resurgent currents: in particular, the C-terminus likely mediates these currents. Localized knockdown of Navβ4 decreased inflammation-induced enhancement of resurgent currents and neuronal excitability, and prevented the development of persistent pain associated behavior in an inflammatory pain model. FHF2B increased resurgent currents and delayed inactivation. In contrast, FHF2A limited resurgent currents; an effect that is mainly contributed by FHF2A's N-terminus activity that increased accumulation of channels in inactivated states. Interestingly, in an inflammatory pain model FHF2B was upregulated and FHFA isoforms were downregulated. Together these results suggest that FHF2A/B modulation might contribute to enhanced resurgent currents and increased neuronal excitability observed in the inflammatory pain model. Overall, our work has identified three resurgent current modulators FHF2A, FHF2B and Navβ4. Manipulation of these proteins or their activity might result in novel strategies for the study and treatment of pain.