Techn. Hochsch., Diss., 2003--Aachen.

Techn. Hochsch., Diss., 2004--Aachen.

Universiẗat, Diss., 2003--Marburg.

Techn. University, Diss., 2004--Berlin.

Univ., Diss., 2006--Frankfurt (Main) / Zsfassung in engl. und dt. Sprache

Το υδρόθειο (H2S) είναι ένα άχρωμο, εύφλεκτο αέριο με τη χαρακτηριστική οσμή του κλούβιου αυγού. Τα τελευταία χρόνια έχει αναγνωριστεί ο ρόλος του ως ένα σηματοδοτικό μόριο για το καρδιαγγειακό και το νευρικό σύστημα και είναι το τρίτο μέλος της οικογένειας των αέριων διαβιβαστών. Παράγεται ενδογενώς μέσω του μεταβολισμού της L-κυστεΐνης από δυο ένζυμα: τη β-συνθετάση της κυσταθειονίνης (CBS) και τη γ-λυάση της κυσταθειονίνης (CSE). Ένας τρίτος τρόπος σύνθεσης του H2S από L-κυστεΐνη που ανακαλύφθηκε πρόσφατα είναι μέσω της συνδυασμένης δράσης της σουλφοτρανσφεράσης του 3-μερκαπτοπυροσταφυλικού και της αμινοτρανσφεράσης της κυστεΐνης (3MST/CAT). Στην παρούσα εργασία μελετήσαμε την εκλεκτικότητα των διαθέσιμων φαρμακολογικών αναστολέων της σύνθεσης του H2S που επιδρούν στα ένζυμα παραγωγής του, CBS και CSE. Για την υλοποίηση της μελέτης έγινε η έκφραση των ενζύμων CSE και CBS ως χιμαιρικές πρωτεΐνες με GST σε βακτήρια E.coli και ακολούθησε η απομόνωση αυτών με χρωματογραφία συγγένειας με τη χρήση στηλών που έχουν ακινητοποιημένη σε σεφαρόζη τη γλουταθειόνη. Έπειτα ελέγχθηκε η δραστικότητα των ανασυνδυασμένων ενζύμων με την μέθοδο του μπλε του μεθυλενίου και στη συνέχεια εξετάσθηκαν οι αναστολείς έναντι και των δυο ενζύμων CSE και CBS. Τα αποτελέσματα μας επιβεβαιώνουν την εκλεκτικότητα της προπαργυλογλυκίνης (PAG) και της β-κυανοαλανίνης (BCA) για το CSE. Από τη μελέτη μας βρέθηκε ότι η β-κυανοαλανίνη (BCA) είναι ισχυρότερος αναστολέας του CSE από την προπαργυλογλυκίνη (PAG) με IC50 14μΜ και 750μΜ, αντίστοιχα. Το αμινο-οξικό οξύ (ΑΟΑΑ) βρέθηκε ότι είναι πιο ισχυρός αναστολέας για το CSE (IC50 1,1μΜ) παρά για το CBS (IC50 8,5μΜ), παρόλο που θεωρητικά είναι εκλεκτικός αναστολέας του CBS. Μάλιστα είναι ισχυρότερος των PAG και BCA, για το CSE. Η τριφθοροαλανίνη και η υδροξυλαμίνη ανέστειλαν και τα δυο ένζυμα, με την τριφθοροαλανίνη να είναι πιο ισχυρός αναστολέας του CBS, ενώ η υδροξυλαμίνη του CSE. Στην παρούσα εργασία εξετάστηκαν επίσης κάποιες νέες ουσίες ως πιθανοί αναστολείς του CSE, οι οποίες δε φαίνεται να είχαν την ικανότητα να προκαλέσουν σημαντική αναστολή του ενζύμου αυτού. Συμπερασματικά, παρόλο που οι αναστολείς PAG και BCA εμφανίζουν εκλεκτικότητα ως προς το CSE έναντι του CBS, δεν υπάρχει, προς το παρόν, κανένας εκλεκτικός αναστολέας για το CBS. / Hydrogen sulphide (H2S) is a colorless, flammable gas with the characteristic smell of rotten eggs. H2S is now recognized as an important signalling molecule in the cardiovascular and nervous systems and is the third member of the gasotransmitter family. H2S is synthesized via two pyridoxal-5′-phosphate-dependent enzymes responsible for the metabolism of L-cysteine: cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE), as well as by a recently identified third pathway that catalyzes the production of H2S from L-cysteine via the combined action of 3-mercaptopyruvate sulphurtransferase and cysteine aminotransferase (3MST/CAT). In the present study we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS. To address this question human CSE or CBS enzymes were expressed and purified from E. coli as fusion proteins with Glutathione-S-Transferase (GST). After purification the activity of the recombinant enzymes was tested using the methylene blue method. We found that β-cyanoalanine (BCA) was a more potent CSE inhibitor than propargylglycine (PAG) (IC50 14μΜ and 750μΜ, respectively), and that aminooxyacetic acid (AOAA) was even more potent than BCA and PAG towards CSE (IC50 1.1μΜ), although it is claimed to be a CBS-selective inhibitor; AOAA inhibited CBS with an IC50 8.5μΜ. Trifluoroalanine and hydroxylamine inhibited both enzymes with the former being a more potent inhibitor of CBS, while the later of CSE. We also examined some new synthesized substances as CSE inhibitors. In conclusion, although PAG and BCA exhibit selectivity in inhibiting CSE vs CBS, no selective CBS inhibitor is currently available.

In the course of this work a novel strategy has been developed for linking the adjuvant Escherichia coli heat-labile enterotoxin subunit B (LTB) to Simian Immunodeficiency Virus (SIV) proteins via an antibody bridge and the systemic and mucosal immunogenicity of such SIV-MAb-LTB complexes have been investigated. A short peptide tag, termed Pk, was joined to the 3'-end of the gene coding for LTB and expression studies revealed that the gene product, LTB-Pk, could be efficiently synthesised and secreted from non-pathogenic Vibrio sp.60. Analysis of the functional properties of LTB-Pk demonstrated that LTB-Pk , like native LTB, was a heat-labile oligomer, that could bind to the glycolipid GM1-ganglioside and was immunogenic in vivo. In attempts to purify LTB-Pk for immunisation studies, both hydrophobic and ion-exchange chromatography schedules were analysed, the latter procedure being more efficient. Strategies were developed for joining LTB-Pk to one arm of an anti-Pk MAb, (MAb SV5-P- k) and Pk-linked SIV proteins to the other arm, and such SFV-MAb-LTB complexes bound to GM1 -ganglioside in vitro. Systemic immunisation studies suggested that SIV-MAb-LTB complexes, using recombinant p17 as the target antigen, promoted both humoral and cell- mediated immunity to the recombinant p17. In addition, it was later shown that conjugation of LTB-Pk to recombinant SIV proteins via an antibody bridge, resulted in a more efficent presentation of the recombinant SIV protein to the immune system, than co-administration of LTB-Pk with the recombinant SIV protein. However, intranasal administration of p17-MAb-LTB complexes did not induce immunity to recombinant p17. Subsequently it was shown that the recombinant p17 was highly susceptible to mucosal degradation, suggesting the poor mucosal immunogenicity of p17-MAb-LTB complexes may be related to the instability of recombinant p17 in the mucosal environment. Further investigations into the stability of other recombinant SIV proteins in the mucosa, revealed that recombinant p27 was more resilient to mucosal degradation. p27-MAb-LTB complexes were constructed and initial intranasal immunisation studies revealed that both systemic and cell-mediated immunity to recombinant p27, could be induced following intranasal administration. Furthermore, mucosal immunity to recombinant p27 was evident in the lungs of vaccinated mice, with anti-recombinant p27 IgG-secreting cells predominating.

abstract: Animals must learn to ignore stimuli that are irrelevant to survival, which is a process referred to as ‘latent inhibition’. This process has been shown to be genetically heritable (Latshaw JS, Mazade R, Sinakevitch I, Mustard JA, Gadau J, Smith BH (submitted)). The locus containing the AmTYR1 gene has been shown through quantitative trait loci mapping to be linked to strong latent inhibition in honey bees. The Smith lab has been able to show a correlation between learning and the AmTYR1 receptor gene through pharmacological inhibition of the receptor. In order to further confirm this finding, experiments were designed to test how honey bees learn with this receptor knocked out. Here this G-protein coupled receptor for the biogenic amine tyramine is implemented as an important factor underlying latent inhibition in honey bees. It is shown that double-stranded RNA (dsRNA) and Dicer-substrate small interfering RNA (dsiRNA) that are targeted to disrupt the tyramine receptors specifically affects latent inhibition but not excitatory associative conditioning. The results therefore identify a distinct reinforcement pathway for latent inhibition in insects. / Dissertation/Thesis / Masters Thesis Biology 2017

The aim of this study was to provide an account of the power relations that are implicit in the experience of clients who initially withhold but eventually disclose a sensitive issue in the psychotherapeutic dialogue. Mainstream psychotherapeutic literature has maintained that clients who withhold sensitive material implicitly express a psychological powerlessness. The literature review also turned attention to an alternative view, not arising from within the psychotherapeutic literature. Specific reference was made to the work of Foucault who suggests that although clients may appear to be empowered through self-disclosure, they are in fact constrained, since disclosing themselves constitutes. an appropriation of selfunderstanding which forecloses openness to other forms of self-understanding. The tension between these conflicting accounts about the relation of self-disclosure to empowerment was discussed as an issue requiring further exploration through clinical research. A phenomenologically orientated research method was used to describe the experiences of five clients who withheld and subsequently disclosed sensitive issues in psychotherapy. These descriptions yielded a thematically differentiated process of psychological change. The structure of client inhibition and self-disclosure was seen to correspond to the concepts of powerlessness and empowerment outlined in the psychotherapeutic literature. The apparent empowerment of clients during self-disclosure casts doubt on Foucault's perspective. However, on further reflection and through a review of the research method, it became apparent that the lack of support for Foucault's perspective was a consequence of the particular research method used rather than an indication of the non-existence of constraint. Ricoeur's hermeneutic phenomenology was used to develop the above methodological critique. Using this alternative approach the researcher critically evaluated the findings of the phenomenological study. This facilitated a reinterpretation of the clinical material. It emerged that the experience of empowerment represents a particular form of selfunderstanding, and it was shown, in relation to the clinical material, how this can indeed as Foucault suggests (because of its very specificity) constrain the client from understanding him/herself in alternative ways. It was revealed that the experience of empowerment is a necessary but limited component of successful client disclosure. This does not, however, go far enough. It was suggested that ideally, critical reflection on the constraints of self-understanding, rather than self-disclosure per se, should be regarded as the destination of the urge to self-disclosure.

Made available in DSpace on 2014-10-09T12:35:31Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:02Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP