Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of upper and lower motor neurons, resulting in muscle weakness and death from respiratory failure within 3-5 years after onset. The incidence is 1.5-2.7/100,000 inhabitants. 5-10% of all cases are hereditary. The aetiology of sporadic ALS is still unknown. The only neuroprotective drug approved for the treatment of ALS is riluzole, a glutamate-antagonist, which has shown to improve survival. We evaluated if riluzole sales statistics can be used as a method for estimating the prevalence of ALS/motor neuron disease in Sweden. We found that this method, which is less time consuming than conventional methods, could be used as a crude marker for the prevalence. In a longitudinal study of overall Quality of Life (QoL) in ALS we found that QoL changes only slightly over time despite disease progression. ALS does not necessarily result in a low QoL. Growth factors are important for the survival of neurons. In ALS we found increased or normal levels of GDNF mRNA and BDNF mRNA in muscle biopsies, VEGF in serum and spinal cord and FGF-2 in serum and cerebrospinal fluid. There is thus no deficit of these growth factors although there may be a relative lack because of high demands of the motor neurons. Polyamines are small aliphatic molecules that are important for the function of cells. The level of the polyamines spermidine and spermine were increased in red blood cells in both patients with ALS and patients with Parkinson’s disease, suggesting that polyamines may have a role for the neurodegenerative process. Polyamines in spinal cord were of the same level in the patients with ALS and in controls, indicating a maintained regulation of polyamines at the end-stage of the disease.

No description available.

Η κλινδαμυκίνη αποτελεί μέλος της οικογενείας των MLS αντιβιοτικών, με ευρύτατες εφαρμογές στην Ιατρική. Προσδεδενόμενη στο κέντρο της πεπτιδυλοτρανσφεράσης, δρα ως αναστολέας της πρωτεϊνικής σύνθεσης. Η ακριβής θέση δράσης της δεν έχει πλήρως διασαφηνισθεί. Διάφορες μελέτες έχουν δείξει πως δρα στην Α θέση, ενώ άλλες στην Ρ θέση της μεγάλης ριβοσωματικής υπομονάδας. Στην παρούσα εργασία γίνεται λεπτομερής κινητική ανάλυση της αναστολής του σχηματισμού του πεπτιδικού δεσμού από την κλινδαμυκίνη σε ιοντικό περιβάλλον που πλησιάζει το φυσιολογικό του κυττάρου (4,5 mM Mg2+, 150 mM NH4+). Συγκεκριμένα, η δράση της κλινδαμυκίνης μελετήθηκε σε ένα σύστημα ελεύθερο-κυττάρων του εντεροβακτηρίου Escherichia coli, όπου ένας πεπτιδικός δεσμός σχηματίζεται μεταξύ πουρομυκίνης και AcPhe-tRNA, προσδεδενόμενου στην Ρ-θέση ριβοσωμάτων προγραμματισμένων με poly(U). Η πουρομυκίνη δρα ως ανάλογο του 3΄ άκρου ενός αμινοακυλο-tRNA ενώ το τριμερές AcPhe-tRNA∙ poly(U)∙ ριβόσωμα, σύμπλοκο C, ως ανάλογο του εναρκτήριου μεταφραστικού συμπλόκου. Η κινητική ανάλυση αποκάλυψε ότι η κλινδαμυκίνη συμπεριφέρεται ως αναστολέας βραδείας δεσμεύσεως. Μετά από μια παροδική αλληλεπίδραση με την Α-θέση, εγκαθίσταται κοντά στην Ρ-θέση του ριβοσώματος με αποτέλεσμα να επηρεάζει την ταχύτητα σχηματισμού του πεπτιδικού δεσμού. Στην συνέχεια μελετήθηκε η επίδραση των πολυαμινών στην αλληλεπίδραση της κλινδαμυκίνης με το σύμπλοκο C. Τα αποτελέσματα έδειξαν πως η σπερμίνη παρεμποδίζει την επίδραση της κλινδαμυκίνης στην Ρ-θέση, όμως επηρεάζει ευνοϊκά και σε μεγαλύτερο βαθμό, την αρχική δέσμευση του φαρμάκου στην Α-θέση ελαττώνοντας το εντροπικό κόστος. Η επίδραση αυτή δεν μεταβλήθηκε όταν αντί της σπερμίνης χρησιμοποιήθηκαν ριβοσώματα επισημασμένα μ’ένα φωτοδραστικό ανάλογο της σπερμίνης, την Ν1-αζιδοβενζαμιδινο-σπερμίνη, ή όταν στο διάλυμα επώασης προστέθηκε μείγμα σπερμίνης και σπερμιδίνης. Πειράματα σταυρο-σύνδεσης έδειξαν ότι η σπερμίνη προσδένεται πλησίον της θέσης δέσμευσης της κλινδαμυκίνης. Η παρατήρηση αυτή οδήγησε στην υπόθεση, ότι οι πολυαμίνες δεσμευόμενες πλησίον της θέσης πρόσδεσης της κλινδαμυκίνης επηρεάζουν την αλληλεπίδραση του αντιβιοτικού με το ριβόσωμα, επάγοντας αλλαγές διαμόρφωσης στο ριβοσωμικό σύμπλοκο. Η υπόθεση αυτή βρίσκεται σε συμφωνία με πειράματα χημικής προστασίας, που έδειξαν, ότι οι πολυαμίνες επηρεάζουν σημαντικά την τριτοταγή δομή του ριβοσώματος. Από άποψη φαρμακευτικών εφαρμογών η παρούσα μελέτη εισηγείται ότι, κάθε φορά που ένα αντιβιοτικό, με μοριακό στόχο το ριβόσωμα, είναι προς σχεδιασμό, η επίδραση του ιοντικού περιβάλλοντος πρέπει να λαμβάνεται σοβαρά υπόψη. / Clindamycin is a representative antibiotic of the MLS family, widely used in clinical practice. It inhibits protein synthesis by binding to the peptidyltransferase center of the ribosome. Clindamycin’s exact site of action is not known. Several studies have shown that it is an inhibitor of the A-site. However, there are also stydies that suggest that clindamycin acts at the P-site of the large ribosomal subunit. In this study, we re-examined the mechanism by which the antibiotic inhibits the formation of peptide bond in an ionic environment that resembles in vivo conditions (4.5 mM Mg2+, 150 mM NH4+). Clindamycin was investigated in a cell-free system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly (U) –programmed ribosomes. Puromycin can be considered as an analogue of the 3’-end of aminoacyl-tRNA, while the ternary complex AcPhe-tRNA∙poly(U)∙ ribosome (complex C) as an analogue of the initiation translation complex. Kinetics revealed that clindamycin behaves as a slow – binding inhibitor. After a transient interaction with the A-site of ribosomes, it slowly accommodates near the P-site so that peptide bond is still formed but with a lower velocity. Next, we investigated the influence of polyamines to the interaction of clindamycin with complex C. It was found that spermine hinders the accommodation of clindamycin to the P-site, but exerts a beneficial, more pronounced, effect on the potency of the drug by lowering the entropic cost of clindamycin binding to the A-site. Polyamine effect was not substantially altered when ribosomes labeled with a photoreactive analogue of spermine, N1-azidobenzamidino-spermine, were used or when a mixture of spermine and spermidine was added in the incubation mixture, instead of spermine alone. Cross-linking experiments have demonstrated that spermine binds to the vicinity of the antibiotic binding pocket. This observation temped us to suppose that polyamines bound adjacently to the binding site of clindamycin modulate the interaction of this drug with the ribosome by inducing conformational changes in the elongating ribosomal complex. Such a hypothesis is in agreement with chemical protection data revealing that polyamines influence significantly the tertiary structure of ribosomes. From the stand point of pharmaceutical applications, the present work postulates that when a drug is designed to target to the ribosome, the influence of the ionic environment should be taken into account.

Coconut (Cocos nucifera L.) is native to the regions between 20oN and 20oS of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore the coconut palm is grown in about 90 tropical countries on more than 10 millions ha of land (Hamon et al., 1999). Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999). Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time however one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998). These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for aim that explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore the latter was chosen to be used as a starting protocol for this study. New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those however have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use in the next steps of the current study. Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process. Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future. The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants. As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

Coconut (Cocos nucifera L.) is native to the regions between 20oN and 20oS of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore the coconut palm is grown in about 90 tropical countries on more than 10 millions ha of land (Hamon et al., 1999). Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999). Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time however one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998). These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for aim that explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore the latter was chosen to be used as a starting protocol for this study. New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those however have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use in the next steps of the current study. Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process. Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future. The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants. As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [73]-84).

Thesis (Ph. D.)--University of California, Riverside, 2010. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed May 17, 2010). Includes bibliographical references. Also issued in print.

Τα ρετινοειδή αποτελούν μια μεγάλη οικογένεια οργανικών μορίων που μοιάζουν δομικά με τη βιταμίνη Α. Το all-trans ρετινοϊκό οξύ (atRA) συμμετέχει σε μεγάλο εύρος βιολογικών διεργασιών μέσω της πρόσδεσής του και της ενεργοποίησης των υποδοχέων του, τους υποδοχείς ρετινοϊκού οξέος (RAR) και τους υποδοχείς ρειτινοειδών Χ (RXR). Κάθε κατηγορία υποδοχέων περιλαμβάνει τρία μέλη, α, β και γ. Τα ρετινοειδή χρησιμοποιούνται για τη θεραπεία πολλών ασθενειών, από την κοινή ακμή ως την οξεία προμυελωτική λευχαιμία. Λόγω των σοβαρών ανεπιθύμητων ενεργειών τους, γίνεται προσπάθεια να συντεθούν ανάλογα με λιγότερες ανεπιθύμητες δράσεις ή/και καλύτερη αποτελεσματικότητα. Προς αυτήν την κατεύθυνση έχουν συντεθεί πολλά ανάλογα, ανάμεσα τους και αυτά που χρησιμοποιήθηκαν στην παρούσα εργασία: RA2SPM (ένα σύζευγμα δύο μορίων atRA με σπερμίνη), ACI2SPM (ένα σύζευγμα δύο μορίων ασιτρετίνης με σπερμίνη), TRX2SPM (ένα σύζευγμα δύο μορίων τριοξαλενίου με σπερμίνη), ACI-SPM-TRX (σύζευγμα ασιτρετίνης και τριοξαλενίου με σπερμίνη), RA-SPM-ACI (σύζευγμα atRA και τριοξαλενίου με σπερμίνη). Τα ανάλογα αυτά συντέθηκαν από την ερευνητική ομάδα του καθηγητή Δ. Παπαϊωάννου, στο Τμήμα Χημείας του Πανεπιστημίου Πατρών. Στην παρούσα εργασία μελετήθηκε η δράση των αναλόγων αυτών στην ανάπτυξη καρκινικών κυττάρων προστάτη PC3 in vitro και έγινε μια αρχική διερεύνηση του μηχανισμού δράσης και της πιθανής εμπλοκής των υποδοχέων ρετινοειδών. Ως πρότυπη ένωση με την οποία συγκρίθηκαν όλες οι άλλες ενώσεις χρησιμοποιήθηκε το ανάλογο RA2SPM, Το ανάλογο ACI2SPM έχει την καλύτερη δράση σε σύγκριση με τα άλλα ανάλογα, αφού προκάλεσε δοσο-εξαρτώμενη μείωση του αριθμού των κυττάρων PC3 σε ποσοστό παρόμοιο με τη δράση του RA2SPM και είναι πιο δραστικό στη συγκέντρωση 10-6Μ σε σχέση με τα άλλα ανάλογα. Αυτή η δράση φαίνεται να σχετίζεται με αύξηση των επιπέδων του mRNA του ογκοκατασταλτικού γονιδίου RARβ, που όμως είναι μικρότερη από αυτήν που προκαλεί το ανάλογο RA2SPM. Ο εκλεκτικός ανταγωνιστής του RARα, Ro-41-5253, βρέθηκε ότι επίσης προκαλεί αύξηση των επιπέδων του mRNA του RARβ, ενώ δεν επηρεάζει τη δράση του ACI2SPM. Επιπλέον, το ανάλογο ACI2SPM μείωσε τα επίπεδα της πρωτεΐνης πλειοτροπίνης (PTN), η οποία είναι γνωστό πως παίζει σημαντικό ρόλο στην ανάπτυξη των καρκινικών κυττάρων προστάτη PC3. Η μείωση είναι μικρότερη από αυτήν που προκαλεί το ανάλογο RA2SPM και δεν επηρεάζεται από τον ανταγωνιστή Ro 41-5253, ο οποίος έχει από μόνος του ανασταλτική δράση. Συμπερασματικά, τα αποτελέσματα της παρούσας εργασίας υποδεικνύουν ότι το ανάλογο ACI2SPM είναι αποτελεσματικό στη μείωση του αριθμού των καρκινικών κυττάρων προστάτη PC3 και στη μείωση των επιπέδων της (PTN), αλλά η δράση του είναι μικρότερη από αυτήν του RA2SPM. Επίσης, καταδεικνύουν ότι ο εκλεκτικός αναστολέας του RARα Ro-41-5253, όπως έχει αναφερθεί ξανά στη διεθνή βιβλιογραφία, παρουσιάζει προβλήματα εκλεκτικότητας και πρέπει να χρησιμοποιείται με προσοχή. / Retinoids constitute a large family of organic compounds structurally related to the naturally occurring vitamin A. All-trans-retinoic acid (atRA) is known to modulate a wide range of cellular biological processes through binding to and activation of its specific receptors, retinoic acid receptors (RAR) and retinoid X receptors α, β and γ. Retinoids are being used for the treatment of various diseases, ranging from acne vulgaris to acute promyelocytic leukemia. However, due to serious adverse effects, there has been a great effort to synthesize analogues with better efficacy or/and minimized adverse effects. Towards this direction, many analogues have been synthesized, among which those that have been used in the present work: RA2SPM (a conjugate of all-trans retinoic acid with spermine), ACI2SPM (a conjugate of 2 molecules of acitretin with spermine), TRX2SPM (a conjugate of 2 molecules of trioxalen with spermine), ACI-SPM-TRX (a conjugate of trioxalen and acitretin with spermine), RA-SPM-ACI (a conjugate of all-trans retinoic acid and acitretin with spermine). These analogues have been synthesized by the research group of Prof. D. Papaioannou at the Department of Chemistry of the University of Patras. The present work represents an initial investigation of the in vitro effects of these analogues on prostate cancer cell growth (PC3), as well as of their mechanism of action and the possible involvement of retinoid receptors. The analogue RA2SPM was used as a reference control. The analogue ACI2SPM decreased the number of prostate cancer PC3 cells in a concentration-dependent manner, being as effective as RA2SPM and more effective compared with the rest of the tested analogues. This effect seems to correlate with an increase of the mRNA levels of the RARβ tumour repressor gene; however, the RARβ tumour repressor mRNA levels decrease is smaller than that observed with RA2SPM. The RARa selective antagonist Ro 41-5253 also increases the RARβ mRNA levels, while it does not affect the ACI2SP-induced increase. Furthermore, the ACI2SPM analogue was found to decrease the protein levels of the growth factor pleiotrophin (PTN), which is known to play an important role in the growth of the prostate cancer cell line PC3. Similarly to the effect on RARβ, the decrease of the PTN levels achieved by ACI2SPM is smaller than the one achieved by RA2SPM and is unaffected by the antagonist Ro 41-5253. Ro 41-5253 decreased PTN levels by itself, supporting the increasing notion that it should be used with caution as a RARα antagonist. In conclusion, the results of this work suggest that the ACI2SPM analogue is efficient in decreasing the prostate cancer PC3 cell numbers and PTN protein levels but seems to be less effective than RA2SPM action. More work is required in order to define the mechanism(s) of action of the tested analogues, as well as to better specify their effectiveness and toxicity.

Το φυσικό προϊόν αρτεμισινίνη και τα παράγωγά της αποτελούν σήμερα φάρμακα επιλογής για την αντιμετώπιση της ελονοσίας ενώ πολλά απ’αυτά παρουσιάζουν και ιδιαίτερα σημαντική αντικαρκινική δράση. Στο πλαίσιο της παρούσας διπλωματικής εργασίας έγινε χημική τροποίηση του μορίου της αρτεμισίνης, προκειμένου να συντεθούν διμερή συζεύγματά της με πολυαμίνες (πουτρεσκίνη, σπερμιδίνη, σπερμίνη). Αρχικά, η αρτεμισινίνη τροποποιήθηκε κατάλληλα έτσι ώστε να φέρει συνδέτη με δεσμό C-O (10-oξo) ή C-C (10-καρβο) στη θέση-10 και N-C με αλλαγή του ετεροατόμου στην θέση-11, προκειμένου στη συνέχεια να προσδεθεί στις πολυαμίνες μέσω δεσμού ουρεθάνης. Για το λόγο αυτό, συντέθηκαν ενεργοποιημένα ανάλογα της αρτεμισινίνης, τα οποία μετά από αντίδραση με κατάλληλα προστατευμένες πολυαμίνες, οδηγούν στο σχηματισμό διμερών πολυαμινικών συζευγμάτων της αρτεμισινίνης. Επιπλέον, για προκαταρκτικές βιολογικές μελέτες, συντέθηκε ένα ασύμμετρο πολυαμινικό σύζευγμα της αρτεμισινίνης με το αντικαρκινικό φάρμακο χλωραμβουκίλη, χρησιμοποιώντας το 10-oξo ενεργοποιημένο ανάλογο και την πουτρεσκίνη. / The natural product Artemisinin and its derivatives are currently the drugs of choice for the treatment of malaria, which some of them showing important anticancer activity. In the context of the present dissertation, three chemical modifications of the molecule of artemisinin were accomplished towards the synthesis of several Artemisinin dimer conjugates with polyamines (putrescine, spermidine, spermine). Initially, artemisinin was modified at position 10 and 11, in order to synthesize analogues bearing suitable linkers, through C-O (10-oxo), C-C (10-carbo) and C-N (11-aza, by replacement of O with N in the ring A of Artemisinin) bonds, able to form carbamate bonds with amino groups of polyamines. For this purpose, the corresponding activated intermediates were synthesized, which upon reaction with suitably protected polyamines afforded the Artemisinin symmetric conjugates. In addition, for the sake of preliminary biological evaluation a new asymmetric conjugate consisted of an 10-oxo Artemisinin and a chlorambucil moiety, using putrescine as a polyamine-type linker, was synthesized.