A descoberta de fármacos a partir de produtos isolados de organismos marinhos tem apresentado um grande crescimento nos últimos anos, principalmente devido aos avanços tecnológicos analíticos, síntese química e biotecnologia. Dentre estes, as esponjas representam uma das principais fontes de metabólitos protótipos para diversas atividades, destacando-se os efeitos antitumorais. Neste contexto, este trabalho teve como objetivo a investigação química e biológica de três esponjas coletadas na costa sul-brasileira: Haliclona tubifera, Polymastia janeirensis e Scopalina ruetzleri. Considerando a correlação entre câncer, distúrbios da coagulação e desbalanço de espécies reativas de oxigênio (EROs), foram realizados ensaios visando a aquisição destas atividades e a identificação de substâncias bioativas. Para a esponja H. tubifera foram observados interessantes efeitos antitumorais em células de glioma e neuroblastoma humano (IC50 < 20 μg/ml), além das atividades antioxidante e anticoagulante para a fração acetato de etila. O composto majoritário desta fração foi isolado como um derivado N-Boc e sua configuração foi estabelecida utilizando um novo protocolo de dicroísmo circular e semissíntese de derivados. Assim, este esfingosídeo de cadeia longa isolado (2R,3R,6R,7Z)-2-amino-7-octadecene-1,3,6-triol, foi denominado halisphingosine A. Um novo composto minoritário, halisphingosine B foi obtido usando técnicas de isolamento em escala nanomolar. Sua configuração absoluta foi estabelecida por comparação com o composto A. Da mesma forma, para a esponja S. ruetzleri, a fração acetato de etila demonstrou os resultados mais promissores. Um potencial efeito anticâncer e de inibição dos radicais peroxila foi observado. Além disso, um efeito modulador da peroxidação lipídica foi evidenciado em ensaio ex vivo de dienos conjugados. Através da análise por RMN de 1H, verificou-se que a fração era majoritariamente constituída por ácidos graxos, os quais foram derivatizados para caracterização por Cromatografia Gasosa (GC/FID). Foram identificados 32 ácidos graxos principalmente poli-insaturados (53%). Ácidos graxos minoritários não usuais para o ambiente marinho também foram caracterizados. A esponja P. janeirensis apresentou os efeitos citotóxicos mais promissores em células de glioma e neuroblastoma humano, com um IC50 < 1,0 μg/ml para o extrato aquoso (pH 6,8), sendo este efeito pH-dependente, uma vez que o extrato (pH 5,8) não alterou a viabilidade celular. Para P. janeirensis, foi também investigado seu potencial de defesa química em modelo de Zebrafish. Foi observado que o extrato aquoso desencadeia um efeito de fuga, alterando significativamente o comportamento espaço-temporal de peixes Danio rerio. Analisando em conjunto, os dados do presente trabalho representam uma nova contribuição para o estudo químico e biológico de espécies de esponjas marinhas da costa sul-brasileira e apontam as potencialidades destas esponjas na busca de moléculas protótipos para fármacos, especialmente relacionados à terapia do câncer. / Drug discovery from marine natural products has increased in the past few years, mainly due to technological advances in spectroscopy, chemical synthesis and biotechnology. Among all marine animals, sponges represent one of the major sources of prototype metabolites for several biological activities, highlighting the antitumor effects. In this context, this study carried out chemical and biological investigation of three sponges collected on the South Brazilian coastline: Haliclona tubifera, Polymastia janeirensis and Scopalina ruetzleri. Considering the correlation between cancer, clotting disorders and imbalance of reactive oxygen species (ROS), experiments were conducted for acquisition of these activities and identification of bioactive compounds. H. tubifera showed an interesting cytotoxic effect in human neuroblastoma and glioma cell lines (IC50 <20 μg/ml), antioxidant and anticoagulant effect for ethyl acetate (EtOAc) fraction. The major compound of EtOAc fraction was isolated as an N-Boc derivative and its configuration was established using a new circular dichroism protocol with the production of semi-synthetic derivatives. This long chain sphingoid base (2R,3R,6R,Z)-2-aminooctadec-7-ene-1,3,6-triol was named as halisphingosine A. A new minor compound, halisphingosine B was obtained using nanomol scale techniques and their absolute configuration was established by comparison with compound A. Likewise, for the sponge S. ruetzleri, the EtOAc fraction showed the most promising results. A potential anticancer effect, inhibition of peroxyl radicals and modulation effect of lipid peroxidation was observed. Fingerprint 1H NMR analysis showed that this fraction is mainly constituted of fatty acids. Through Fatty Acid Methyl Ester (FAMEs) analysis by GC/FID, it was possible to identify 32 fatty acids, of which around 50% were Polyunsaturated Fatty Acids (PUFAs). In addition, some minor unusual fatty acids for the marine biosphere were identified. It was observed for P. janeirensis the most promising cytotoxic effects on human glioma and neuroblastoma cells, with an IC50 <1.0 μg/ml to aqueous extract (pH 6.8), being this effect pH-dependent, since the extract (pH 5.8) did not affect the cell viability. Moreover, P. janeirensis was investigated along their potential chemical defense in Zebrafish model. Aqueous extract trigged an escape effect, significantly altering the spatio-temporal swimming activity of animals. Taken together, the data presented from this study represent a new contribution to chemical and biological research of marine sponge species from South Brazilian coastline, and point the potentialities of sponges to search chemical prototypes for drugs, especially related to cancer therapy.

Hydroxytyrosol, 3,4-dihydroxyphenolethanol, a naturally occurring polyphenol most common in olive tree (Olea europaea), is one of the most effective member of the polyphenols family, because of its remarkable antioxidant activity, its ability to inhibit oxidation of low density lipids (LDL), and its protection against DNA oxidative damage. Hydroxytyrosol, which is widely used in cosmetics and food supplements industries, can be purchased as an olive oil extract that contains low concentration of hydroxytyrosol besides other polyphenols. The price and low natural abundance of hydroxytyrosol make alternative synthetic sources very attractive. In this research, a novel method for the synthesis of pure hydroxytyrosol from a commercially inexpensive precursor catechol was developed; this can satisfy the increasing market demand and provide a more economical alternative source for this valuable polyphenol.

This work focuses on the design and synthesis of Au(I) and Ag(I) complexes from ligand systems that aim to combine both N-heterocyclic carbene (NHC) and phosphine ligand types. A number of synthetic approaches towards both the ligands and the prepared metal complexes have been developed, with a concerted effort on achieving the desired Au(I) or Ag(I) complexes with minimal reaction steps and synthetic style. The thesis body is divided into two main sections. The first section addresses the preparation of suitable ligand precursors of potential Au(I) and Ag(I) complexes in the form of halo- and phosphino-functionalised imidazolium salts. Several series of haloalkylimidazolium salts were prepared that encompass a range of halogens (Cl, Br, I), alkyl substituents (Me, i-Pr, t-Bu, n-Bu), differing alkyl linker length (n = 0-3), and a variety of organic spacers employed to bridge multi-imidazolium moieties. Novel bidentate and multidentate phosphinoalkylimidazolium salts were synthesised from the various haloalkylimidazolium salts, via the substitution of a halide with nucleophilic diphenylphosphide. A new approach towards rare methylene bridged phosphinomethylimidazolium salts was achieved from the reactions of halomethylimidazolium salts with diphenylphosphine. The second section investigates the preparation of Au(I) and Ag(I) complexes from the halo- and phosphino-functionalised imidazolium salts. A series of dicationic 10, 12, and 14-membered metallacyclic Ag(I) complexes were prepared from the bidentate phosphinoalkylimidazolium salts. The dinuclear Ag(I) metallacycles combine two phosphino-functionalised NHC ligands that are bridged by two coordinated Ag(I) ions in an exclusively head-to-head arrangement. A dinuclear Ag(I) metallacycle was investigated for transmetallation potential to a Au(I) complex and found to selectively transmetallate at the Ag(I) coordinated to the NHC ligands to form a bimetallic metallacycle. Unexpected phosphine oxidation of a 10-membered dinuclear Ag(I) metallacycle resulted in complex disproportionation to an isolable and rare silver(I) trimer. Metal-NHC complexes from haloalkylimidazolium salts have not been reported previously, a novel approach to the synthesis of a series of Au(I) complexes from haloalkylimidazolium salts and a respective gold source was developed and is reported herein. Different synthetic approaches towards Au(I) complexes with the phosphinoalkylimidazolium salts explored a variety of ways to generate the NHC from an imidazolium in the presence of the phosphine. A one-pot, high yielding synthesis of a dinuclear Au(I) complex from PPh3 was also devised, with controlled assembly of the complex resulting in a similar head-to-head ligand arrangement to the dinuclear Ag(I) metallacycles. As an aside, a family of mononuclear [Au(R2NHC)2]+ complexes (R = Me, i-Pr, t- Bu, n-Bu, Cy) prepared previously in our research group, was expanded because of the promising antimitochondrial activity shown by [Au(i-Pr2NHC)2]+. Two new [Au(R2NHC)2]+ complexes with simple alkyl chain functionality were prepared with fine-tuned lipophilicity in close proximity to that of [Au(i-Pr2NHC)2]+.

This thesis entitled “Mechanism of Anticancer and Antimalarial Action of a Modulator of Heat Shock Proteins” describes the successful elucidation of the mechanism of anticancer and antimalarial action of 15-Deoxyspergualin (DSG). DSG, a relatively well known immunosuppressant and antitumor molecule has been demonstrated to kill the malaria parasite in vitro and in vivo (Midorikawa et al., 1997; Midorikawa et al., 1998). A highly polar molecule, DSG binds the carboxy terminal “EEVD” motif of heat shock proteins, Hsp70 and Hsp90, enhances the ATPase activity of Hsp70 (Nadler et al., 1992; Nadler et al., 1998), and modulates several seemingly unrelated cellular processes. DSG has also been demonstrated to inhibit protein synthesis and polyamine synthesis in cells (Kawada et al., 2002; Hibasami et al., 1991), and previously speculated to inhibit malaria parasite growth by inhibiting polyamine synthesis. The grim situation with regard to malaria infection and mortality, principally an offshoot of the emergence of chloroquine resistant strains of the causative agent of malaria - Plasmodium falciparum, calls for intense efforts towards developing efficacious antimalarial agents with few side effects. DSG, having been used already in graft rejection cases in man and demonstrated to potently inhibit malaria in mice (Midorikawa et al., 1997), offers promise in this regard. It was, therefore, of interest to solve the mystery of its mechanism of antimalarial action. Chapter 1 surveys literature related to DSG mechanism of action and presents the thesis objective. Chapter 1 also gives an overview of heat shock proteins and their role in cancer, and the biology of the malaria parasite (Plasmodium falciparum), the working of the principal metabolic pathways existing in it, and a description of processes related to the intriguing, relict plastid present in apicomplexans. The metabolic processes previously speculated to be targeted by DSG, and those later found to be involved in DSG mechanism of action – polyamine synthesis and transport, protein synthesis and apicoplast processes are dealt with in more detail. Though DSG has been speculated to kill the malaria parasite by inhibiting polyamine synthesis, that DSG could clear malaria infection in Plasmodium berghei infected mice did not corroborate with the observation that inhibitors of polyamine biosynthesis are incapable of inhibiting the malaria parasite in vivo probably because the parasites make do with polyamines salvaged from the host (Assaraf et al., 1984; Bitonti et al., 1987). On the other hand, DSG is known to bind heat shock proteins, and inhibit protein synthesis, and heat shock proteins are speculated to be involved in the activation of HRI (heme regulated inhibitor), a type of eIF2á kinase that phosphorylates the eukaryotic initiation factor, eIF2á in conditions of heme deficiency or other cellular stress. eIF2á phosphorylation leads to stalling of protein synthesis. It seemed likely that if HRI is activated upon sequestration of heat shock proteins by DSG, it would culminate in protein synthesis inhibition and ultimately, cell death. With the intention to investigate this line of thought, the PlasmodB database was mined for proteins essential to the existence of heme dependent protein synthesis in Plasmodium falciparum. Two Hsp70 proteins from Plasmodium falciparum, one with the carboxy terminal “EEVD” motif implicated in DSG binding, and one without, and an Hsp70 interacting protein were cloned and expressed in their recombinant form in Escherichia coli. The preliminary characterization of these heat shock proteins described in Chapter 2 revealed that they were functionally active. DSG did not inhibit either the chaperone activity of the Hsp70s or the interaction of Hsp70 with Hip, but stimulated their ATPase activity as anticipated. Chapter 3 gives a complete picture of the mechanism of protein synthesis inhibition by DSG in the standard protein synthesis system – reticulocyte lysate. The experiments carried out revealed that DSG inhibits protein synthesis precisely through the mechanism envisaged, i.e. through phosphorylation of HRI following sequestration of Hsp70. Experiments involving exogenous addition of heat shock protein to in vitro translation reactions confirmed this hypothesis. Moreover, DSG inhibited protein synthesis in cancer cells in vivo, too, and HRI knockdown cells were not affected by DSG. Interestingly, the Hsp70 levels in various cancer cell lines inversely correlated with the inhibitory activity of DSG, and modulation of Hsp70 levels through standard methods altered DSG inhibition of protein synthesis in these cells. It was thus confirmed that DSG did indeed inhibit mammalian cells through the pathway envisaged. Its previously reported antitumor property is probably through this outlined mechanism of interference with protein regulation. In the malaria parasite, too, DSG inhibited protein synthesis through eIF2 alpha phosphorylation following Hsp70 sequestration as outlined in Chapter 4. However, while the concentration of DSG required for inhibition of malaria parasite growth was in the nanomolar range, high micromolar concentrations of DSG were required to effect protein synthesis inhibition in the malaria parasite, indicating that yet another target for DSG existed in the malaria parasite. With protein synthesis no longer a candidate target of DSG, I looked into the previously implicated polyamine synthesis pathway. In the event of DSG inhibiting polyamine transport in addition to polyamine biosynthesis, it would be expected to clear malaria infection in vivo contrary to other inhibitors of polyamine biosynthesis. In Chapter 5, evidence for the polyamine synthesis pathway in the malaria parasite is provided. Experiments involving incorporation of radiolabeled precursors in the malaria parasite and in mammalian cells, however, revealed that only high micromolar concentrations of DSG inhibit polyamine synthesis. Polyamine transport was also studied in considerable detail in malaria parasite infected red blood cells. Though infected red blood cells demonstrated different kinetic parameters, implying that new polyamine transporters were employed by the parasite on the red blood cell upon infection, DSG did not potently inhibit polyamine transport, either. The mystery of the target of DSG in the malaria parasite was, however, close to solution, when the growth inhibition of the malaria parasite by DSG was studied carefully. DSG invoked “delayed death” – a phenomenon wherein death is invoked only one cycle after incubation with the inhibitor. “Delayed death” is typical of inhibitors that target apicoplast processes (Fichera and Roos, 1997). DSG did not inhibit either fatty acid synthesis or prokaryotic protein synthesis – processes that occur in the apicoplast, but effected a decrease in the amount of nucleus encoded proteins that are targeted to the apicoplast, suggesting that it inhibited the trafficking of nucleus encoded proteins to the apicoplast. Confocal microscopy of parasites transfected with GFP fusion protein confirmed these findings, and is described in Chapter 6. The thesis ends with a summary of the findings in Chapter 7. Apicoplast processes have always been considered to harbor immense potential in the development of antimalarial agents, thanks to the absence of an equivalent organelle and hence pathways, in the human host. Trafficking of nucleus encoded proteins to the apicoplast has remained unexplored however. The work done in this thesis not only serves to demystify DSG with regard to its mechanism of action, but also paves the way for further studies in this area of intracellular trafficking, which could help in the development of more efficacious antimalarial agents. It also adds a new dimension to previous work conducted with regard to the anticancer action of DSG. Appendix 1 revolves around inhibitors which target various apicoplast processes. Apicoplast processes have been conventionally linked to the intriguing but unfortunate (with respect to clinical application) “delayed death”. Results presented in this section demonstrate that not all apicoplast processes invoke “delayed death”. Inhibition of apicoplast processes such as fatty acid biosynthesis and heme synthesis evoke rapid death. Inhibitors designed to target these processes could, therefore, be highly efficacious.

In the 1980's, Nicholas Farrell developed a range of structurally distinct multinuclear Pt complexes that form long-range interstrand crosslinks (IXLs) in DNA. The dinuclear complex [{trans-PtCl2(NH3)}2-µ-(H2N(CH2)6NH2)]2+ (1,1/t,t) was the first of this series to show promising results, however, it was the trinuclear complex [{trans-PtCl2(NH3)}2-µ-trans-Pt(NH3)2(H2N(CH2)6NH2)2]4+ (1,0,1/t,t,t or BBR3464) that was chosen for clinical trials based on significantly increased cytotoxicity compared to 1,1/t,t and cisplatin. Molecular biology experiments have shown that 1,1/t,t exclusively forms IXLs in DNA in the 5'¿ 5' direction, whilst 1,0,1/t,t,t can form IXLs in both the 5'¿5' and 3'¿3' directions. Previously, 2D [1H,15N] HSQC NMR has been used to study the formation of 5'–5' 1,4–GG IXLs. The formation of 3'–3' 1,4–GG IXLs have been studied as part of this thesis. More recently, Pt complexes such as [{trans–PtCl2(NH3)}2{H2N(CH2)6(NH2(CH2)2NH2)(CH2)6NH2}]4+ (1,1/t,t–6,2,6) and [{trans–PtCl2(NH3)}2{H2N(CH2)6(NH2)(CH2)6NH2}]3+ (1,1/t,t–6,6), where the charged central Pt moiety of 1,0,1/t,t,t is replaced by a polyamine linker, have been developed in the Farrell group and show increased potency compared to 1,0,1/t,t,t. The complex 1,1/t,t 6,2,6 is a lead candidate currently undergoing Phase I clinical trials. Prior to the work presented in this thesis, little was known about the aquation chemistry or kinetics of DNA binding of these novel complexes. Reported in Chapter 3 is the study of the formation of 3'–3' 1,4–GG IXLs by both 1,0,1/t,t,t and 1,1/t,t in the duplex 5' {d(TATACATGTATA)2} (33–14XL) (pH 5.4, 298K). A combination of 1D 1H and 2D [1H, 15N] HSQC NMR experiments was used to directly compare the results with the stepwise formation of the 5'–5' 1,4–GG IXL with the previously studied duplex, 5' {d(ATATGTACATAT)2} (55–14XL), under the same conditions. Preassociation as well as aquation were similar, however, differences were observed at the monofunctional binding step with evidence for numerous monofunctional adducts. Both reactions did not yield a single 3'–3' 1,4–GG IXL, rather several adducts that could not be characterised. Molecular dynamics simulations of the 3'–3' 1,4–GG IXLs showed highly distorted lesions that may have implication in cellular repair processes.

Στη παρούσα εργασία, μελετήθηκαν διάφοροι τύποι αρσονολιποσωμάτων, ως προς την επίδραση της λιπιδικής τους σύστασης στην σταθερότητα τους, την αλληλεπίδραση τους με φυσιολογικά και καρκινικά κύτταρα και την βιοκατανομή τους. Παρασκευάστηκαν sonicated αρσονολιποσώματα με 1,2-rac-διάκυλοοξυπροπυλ-αρσονικό οξύ με παλμιτόυλ- παράπλευρες αλυσίδες (αρσονολιπίδιο, Ars), χοληστερόλη (Chol) και φωσφατιδυλοχολίνη (PC) [PC-based αρσονολιποσώματα] ή 1,2-διστεαρόυλ-sn-φωσφατιδυλοχολίνη (DSPC) [DSPC-based αρσονολιποσώματα], με αναλογίες PC/Ars/Chol και DSPC/Ars/Chol ίσες με 12:8:10 mol:mol:mol. Επίσης παρασκευάστηκαν παρόμοια αρσονολιποσώματα με αναλογίες PC/Ars/Chol και DSPC/Ars/Chol ίσες με 17:3:10 mol:mol:mol. Παρασκευάστηκαν επίσης αρσονολιποσώματα που, εκτός από αρσονολιπίδιο, χοληστερόλη, φωσφατιδυλοχολίνη ή 1,2-διστεαρόυλ-sn-φωσφατιδυλοχολίνη περιείχαν στην σύσταση τους και PEG2000-λιπίδιο (πολυαιθυλενογλυκόλη, Μ.Β. 2000, συζευγμένη με 1,2-διστεαρόυλ-φωσφατιδυλοαιθανολαμίνη, DSPE-PEG2000) [PEGylated PC- και DSPC-based αρσονολιποσώματα]. Εκτός από τα παραπάνω αρσονολιποσώματα, παρασκευάστηκαν και sonicated λιποσώματα με χοληστερόλη και PC ή DSPC σε αναλογίες PC:Chol και DSPC:Chol 2:1 mol:mol («συμβατικά» λιποσώματα, χωρίς αρσονολιπίδιο). Μελετήθηκε αρχικά η τοξικότητα όλων των παραπάνω λιποσωμάτων έναντι διάφορων τύπων καρκινικών κυττάρων: ανθρώπινων λευχαιμικών κυττάρων (ΝΒ4), κυττάρων από καρκίνο του προστάτη (PC3), ανθρώπινων κυττάρων από καρκίνο του μαστού (MDA-MB-468) και Τ-λεμφοκυττάρων από ασθενείς με λευχαιμία (ΜΤ-4), καθώς και έναντι φυσιολογικών ενδοθηλιακών κυττάρων από φλέβες ανθρώπινου ομφάλιου λώρου (HUVEC). Η τοξικότητα των αρσονολιποσωμάτων υπολογίσθηκε με μετρήσεις της βιωσιμότητας των κυττάρων μετά από αλληλεπίδραση τους με τα λιποσώματα με την μέθοδο MTT. Από τις καμπύλες βιωσιμότητας υπολογίσθηκαν και οι IC50 τιμές, οι συγκεντρώσεις δηλαδή στις οποίες κάθε λιπόσωμα προκαλεί 50% μείωση της κυτταρικής βιωσιμότητας. Σύμφωνα με τα αποτελέσματα όλοι οι τύπο αρσονολιποσωμάτων είναι τοξικοί έναντι των καρκινικών κυττάρων (PC3, NB4 και MT-4), με εξαίρεση τα MDA-MB-468 κύτταρα, ενώ αντιθέτως δεν επηρεάζουν την βιωσιμότητα των φυσιολογικών HUVEC κυττάρων. Ωστόσο, για τον ίδιο τύπο κυττάρων, υπάρχουν σημαντικές διαφορές μεταξύ των διαφορετικών τύπων αρσονολιποσωμάτων, οι οποίες δεν είναι ανάλογες με τις διαφορές της σταθερότητας των αρσονολιποσωμάτων, γεγονός που υποδηλώνει ότι η ακεραιότητα της μεμβράνης των αρσονολιποσωμάτων δεν αποτελεί ανασταλτικό παράγοντα για την εμφάνιση τοξικότητας. Είναι φανερό επομένως πως η σύσταση των αρσονολιποσωμάτων πρέπει να προσαρμόζεται κατάλληλα, ανάλογα με την in vivo κινητική τους και την επιθυμητή βιοκατανομή τους. Μελετήθηκε επίσης ο μηχανισμός αλληλεπίδρασης των λιποσωμάτων με NB4 και MDA-MB-468 κύτταρα, καθώς αυτές οι δυο κυτταρικές σειρές ήταν οι σειρές που επέδειξαν την περισσότερη και την λιγότερη ευαισθησία αντίστοιχα έναντι των αρσονολιποσωμάτων στις μελέτες βιωσιμότητας, με χρήση μιας φθορισμομετρικής μεθόδου. Η μέθοδος επιτρέπει την παρακολούθηση της ενδοκυττάρωσης, της δέσμευσης και διαφυγής των λιποσωμάτων από τα κύτταρα. Το HPTS, μια υδατοδιαλυτή, έντονα φθορίζουσα ουσία, της οποίας ο φθορισμός εξαρτάται από το pH, εγκλωβίστηκε στο εσωτερικό των λιποσωμάτων. Η τιμή του φθορισμού του HPTS στα 413 nm εξαρτάται από το pH, ενώ είναι ανεξάρτητη από το pH στα 454 nm, με μήκος κύματος διέγερσης 512 nm. Η ύπαρξη ισοασβεστικού σημείου στα 413 nm επιτρέπει την «μετάφραση» του φθορισμού σε ποσότητα της ουσίας. Τα NB4 και MDA-MB-468 κύτταρα επωάστηκαν με λιποσώματα που είχαν εγκλωβίσει φθορίζουσα στο εσωτερικό τους (HPTS-λιποσώματα) στους 37 oC για διάφορα χρονικά διαστήματα και υπολογίστηκε ο λόγος των τιμών φθορισμού στα 413 nm και τα 454 nm (Ι454/Ι413), που λειτουργεί ως ένδειξη της ενδοκυττάριας εντόπισης των λιποσωμάτων. Με σκοπό να διευκρινιστεί ο εντοπισμός των λιποσωμάτων στα κύτταρα μετά την επώαση τους, τα κύτταρα επωάστηκαν με NH4Cl, που αλκαλοποιεί το όξινο περιβάλλον των υποκυτταρικών διαμερισμάτων, με αποτέλεσμα αύξηση της τιμής φθορισμού στα 454 nm, εφόσον η φθορίζουσα βρίσκεται στα ενδοσώματα (π.χ. λυσοσώματα). Το φαινόμενο αυτό μπορεί να αναστραφεί με απομάκρυνση του NH4Cl. Υπολογίσθηκαν οι λόγοι Ι454/Ι413 πριν και μετά την επώαση τους με NH4Cl, καθώς και μετά από την απομάκρυνση του. Προσδιορίστηκε επίσης το % ποσοστό πρόσληψης αρσονολιποσωμάτων από τα κύτταρα. Η τιμή του φθορισμού στα 413 nm αποτελεί μέτρο της συγκέντρωσης αρσονολιποσωμάτων στα κύτταρα, ανεξάρτητα από το σημείο εντόπισης τους (ανεξάρτητα από το pH). Με σύγκριση των τιμών φθορισμού με πρότυπες καμπύλες αναφοράς προσδιορίστηκε η συγκέντρωση φθορίζουσας σε NB4 και MDA-MB-468 κύτταρα, μετά από επώαση τους με HPTS-λιποσώματα στους 37 oC για διάφορα χρονικά διαστήματα. Οι ίδιες μετρήσεις πραγματοποιήθηκαν και μετά από επώαση NB4 και MDA-MB-468 κυττάρων με τα ίδια HPTS-αρσονολιποσώματα στους 4 oC, με σκοπό να διευκρινιστεί αν ο μηχανισμός αλληλεπίδρασης τους εξαρτάται από την θερμοκρασία, αν είναι δηλαδή ενεργειακά εξαρτώμενη διαδικασία. Για τις μορφολογικές μελέτες που πραγματοποιήθηκαν, προστέθηκε στα λιποσώματα επισημασμένο με φθορίζουσα λιπίδιο στην μεμβράνη τους (ροδαμίνη συζευγμένη με φωσφατιδυλοαιθανολαμίνη, Rho-PE) και η κατανομή των λιποσωμάτων παρατηρήθηκε μικροσκοπικά μετά από επώαση τους στους 37 oC για διάφορα χρονικά διαστήματα με NB4 και MDA-MB-468 κύτταρα. Η επίδραση αναστολέων της ενδοκυττάρωσης μελετήθηκε με επώαση των NB4 και MDA-MB-468 κυττάρων με χλωροπρομαζίνη (CPZ, αναστολέας της ενδοκυττάρωσης μέσω κλαθρίνης) και με μεθυλο-β-κυκλοδεξτρίνη (ΜeβCD, αναστολέας της ενδοκυττάρωσης μέσω caveolae) πριν την επώαση τους με HPTS-λιποσώματα. Προσδιορίστηκαν στην συνέχεια οι λόγοι Ι454/Ι413 και τα % ποσοστά πρόσληψης των αρσονολιποσωμάτων από τα κύτταρα, με σκοπό να διευκρινιστεί ο μηχανισμός της ενδοκυττάρωσης. Όλα τα αρσονολιποσώματα επέδειξαν σχεδόν την ίδια συμπεριφορά σε σχέση με τον τρόπο που αλληλεπιδρούν με τα MDA-MB-468 κύτταρα. Ο κύριος μηχανισμός ενδοκυττάρωσης τους φαίνεται να περιλαμβάνει συμμετοχή υποδοχέων, όπως κλαθρίνη ή/και caveolae. Σε σύγκριση με τα αποτελέσματα των μελετών βιωσιμότητας προκύπτει ότι η έλλειψη τοξικότητας αυτών των αρσονολιποσωμάτων μπορεί να οφείλεται στην αποικοδόμηση τους στο εσωτερικό των λυσοσωμάτων ως φυσικό επακόλουθο της ενδοκυττάρωσης μέσω κλαθρίνης, γεγονός που συμφωνεί και με τον προσδιορισμό αρσενικού που έγινε στα MDA-MB-468 κύτταρα κάτω από τις ίδιες συνθήκες επώασης και ανιχνεύτηκαν ποσοστά αρσενικού μόνο στην περίπτωση των πιο σταθερών DSPC-based λιποσωμάτων (PEGylated και μη). Στην περίπτωση των NB4 κυττάρων, προκύπτει πως η τοξικότητα των αρσονολιποσωμάτων μπορεί να συνδέεται επίσης με το ποσοστό συμμετοχής υποδοχέων, όπως κλαθρίνη ή/και caveolae. Λιποσώματα όπως τα PEGylated PC-based αρσονολιποσώματα (που είναι τα πιο τοξικά), δεν αλληλεπιδρούν καθόλου με τα NB4 κύτταρα μέσω υποδοχέων, ενώ αντίθετα τα PEGylated DSPC-based αρσονολιποσώματα, που αποδείχθηκαν τα λιγότερο τοξικά έναντι των NB4 κυττάρων, φαίνεται να εισέρχονται στα κύτταρα αποκλειστικά μέσω κλαθρίνης ή/και caveolae. Κάτω από τις ίδιες συνθήκες επώασης, δεν ανιχνεύτηκαν παρ’ όλα αυτά ποσοστά αρσενικού στα κύτταρα, γεγονός που θέτει πολλά ερωτηματικά για την ενδοκυττάρια τύχη των αρσονολιποσωμάτων και συγκεκριμένα των αρσονολιπιδίων, δεδομένου ότι γίνεται ενδοκυττάρωση ολόκληρων των λιποσωμάτων αφού «control» (χωρίς αρσενικό) λιποσώματα δεν αποδείχθηκαν τοξικά για τα κύτταρα. Τέλος, με σκοπό να μελετηθεί η βιοκατανομή των αρσονολιποσωμάτων, πραγματοποιήθηκαν in vivo πειράματα. Σε ένα πρώτο σετ πειραμάτων, ενέθηκαν ενδοπεριτοναϊκά (i.p.) DSPC-based και PEGylated DSPC-based αρσονολιποσώματα σε balb-c ποντίκια και η κατανομή του αρσενικού στα διάφορα όργανα 1, 2, 5, 12 και 24 ώρες μετά την χορήγηση μετρήθηκε με Φασματομετρία Ατομικής Απορρόφησης με χρήση φούρνου γραφίτη (GFAAS). Ένα υψηλό ποσοστό της δόσης που χορηγήθηκε αποβλήθηκε ταχέως, καθώς στην πρώτη ώρα μετά την χορήγηση σχεδόν 30-40% της δόσης ανιχνεύθηκε στις ιστούς των ζώων. Από το χρονικό σημείο αυτό και μετά, η απομάκρυνση του αρσενικού ήταν βραδεία με χρόνους ημιζωής 27.6 h για τα PEGylated DSPC-based αρσονολιποσώματα και 83 h για τα DSPC-based. Και για τους δύο τύπους αρσονολιποσωμάτων, η κατανομή αρσενικού ήταν υψηλότερη στα έντερα, μετά στο ήπαρ και μειωνόταν κατά την ακόλουθη σειρά: δέρμα + τρίχωμα, στομάχι, σπλήνα, νεφρά, πνεύμονες και καρδιά. Από τα αποτελέσματα αυτά, αν συγκριθούν με τα αποτελέσματα από παρόμοιες μελέτες που έχουν γίνει με PC-based και PEGylated PC-based αρσονολιποσώματα, φαίνεται πως τα DSPC-based και PEGylated DSPC-based αρσονολιποσώματα χαρακτηρίζονται από καλύτερη βιοδιαθεσιμότητα. Από το γεγονός αυτό γίνεται φανερό πως η λιπιδική σύσταση (και κατ’ επέκταση η σταθερότητα) των λιποσωμάτων επηρεάζει το φαρμακοκινητικό τους προφίλ και συνεπώς, η σωστή επιλογή της λιπιδικής σύστασης των λιποσωμάτων είναι πολύ σημαντική ανάλογα με την εφαρμογή για την οποία προορίζονται τα λιποσώματα. Παρόμοιες in vivo μελέτες πραγματοποιήθηκαν τόσο με DSPC-based και PEGylated DSPC-based, όσο και με PC-based και PEGylated PC-based αρσονολιποσώματα. Τα λιποσώματα χορηγήθηκαν ενδοφλέβια (i.v.) σε balb-c ποντίκια και η κατανομή του αρσενικού στα διάφορα όργανα προσδιορίστηκε με Φασματομετρία Ατομικής Απορρόφησης με χρήση φούρνου γραφίτη (GFAAS) 1, 3 και 24 ώρες μετά την χορήγηση. Στην περίπτωση των DSPC-based, τα επίπεδα αρσενικού στους ιστούς των ζώων ήταν γενικά υψηλότερα συγκριτικά με τα επίπεδα μετά από χορήγηση PC-based αρσονολιποσωμάτων. Η επικάλυψη των λιποσωμάτων με PEG επηρέασε περισσότερο την συμπεριφορά των DSPC-based. Τα PEGylated PC-based αρσονολιποσώματα επέδειξαν παρόμοια συμπεριφορά με τα PEGylated «συμβατικά» (χωρίς αρσενικό) λιποσώματα. Μετά από σύγκριση με τα αποτελέσματα των πειραμάτων με i.p. χορήγηση, προκύπτει πως ο ρόλος της οδού χορήγησης είναι σημαντικός στην κινητική των αρσονολιποσωμάτων, όπως και στην περίπτωση των «συμβατικών» λιποσωμάτων. Ενδιαφέρον παρουσιάζει το γεγονός ότι, μετά από την i.v. χορήγηση, βρέθηκαν ανιχνεύσιμες ποσότητες αρσενικού στον εγκέφαλο, που αποτελεί ένδειξη πως τα αρσονολιποσώματα μπορούν να περάσουν τον αιματεγκεφαλικό φραγμό σε κάποιο ποσοστό, όταν βρίσκονται σε υψηλές συγκεντρώσεις στο αίμα. / In this study we investigated the behavior of arsonolipid containing liposomes, in respect with their lipid composition and its effect on their stability, their interactions with normal and cancer cells and their biodistribution. Sonicated arsonoliposomes were prepared using arsonolipid with palmitic acid acyl chain (Ars), mixed with cholesterol (Chol) and phosphatidylcholine (PC-based arsonoliposomes) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC-based arsonoliposomes), with PC/Ars/Chol and DSPC/Ars/Chol 12:8:10 molar ratio. Arsonoliposomes with PC/Ars/Chol and DSPC/Ars/Chol 17:3:10 molar ratio was also studied. PEG2000-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (PEGylated arsonoliposomes, PC-based and DSPC-based) were also prepared. Sonicated liposomes were also prepared using phosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine, both mixed with cholesterol, PC/Chol and DSPC/Chol 2:1 molar ratio. The cytotoxicity of these arsonoliposomes towards different cancer cells (human promyelocytic leukemia (NB4), prostatic cancer (PC3), human breast adenocarcinoma (MDA-MB-468), human T-lymphocyte (MT-4) and also towards HUVEC (human umbilical vein endothelial cells) was evaluated by calculating the arsonoliposome-induced growth inhibition of the cells by the MTT assay. IC50 values were interpolated from cell number/arsonolipid concentration curves. The results reveal that all types of arsonoliposomes evaluated significantly inhibit the growth of most of the cancer cells studied (PC3, NB4 and MT-4) with the exception of the MDA-MB-468 breast cancer cells, which were minimally affected by arsonoliposomes in some cases even less than HUVEC. Nevertheless, for the same cell type the differences between the different types of arsonoliposomes were significant but never proportional to their stability, indicating that high rigidity of the arsonoliposome membrane is not a problem for their anticancer activity. Thereby it is concluded that arsonoliposome composition should be adjusted accordingly depending on their in vivo kinetics and the desired biodistribution. The interaction of liposomes with NB4 and MDA-MB-468 cells (cells with minimum and maximum viability after interaction with the arsonoliposomes accordingly) was monitored by a fluorescence method, which allows for the simultaneous monitoring of endocytosis, binding and leakage. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of liposomes. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm), which have a complementary pH dependence. The intensity of fluorescence of HPTS at 413 nm (the isosbestic point of HPTS) is pH-independent, while the intensity of fluorescence at 454 nm is pH-dependent. NB4 and MDA-MB-468 cells were incubated at 37 oC with HPTS-encapsulating liposomes for different time periods and the intracellular fate of liposomes was monitored by measuring the fluorescence, at excitation wavelengths of 413 nm and 454 nm and an emission wavelength of 512 nm. The ratio of the fluorescence intensities (Ι454/Ι413) was calculated and evaluated, since it serves as an indicator of liposomes intracellular fate. In order to gain quantitative proof concerning the localization of cell-associated liposomal fluorescence in endosomes or lysosomes of NB4 and MDA-MB-468 cells, after incubation with HPTS-encapsulating liposomes, the cells were incubated with NH4Cl. This results in an increase of pH in the acidic cellular compartments, which would induce an increase in HPTS fluorescence at 454 nm, if the dye is localized in cell endosomes (i.e. lysosomes). This effect can be reversed by removal of NH4Cl. The ratio Ι454/Ι413 was calculated, before and after NH4Cl incubation, as well as after its removal. The % uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was also monitored. The intensity at 413 nm serves as a measure of total number of liposomes associated with cells, regardless of their location along the endocytotic pathway. The concentration of dye associated with cells was determined by measuring fluorescence at 413 nm (pH independent point) and comparing the results to a standard curve, after incubation of NB4 and MDA-MB-468 cells at 37 oC with HPTS-encapsulating liposomes for different time periods. Fluorescence microscopy studies were performed to visualize the uptake of HPTS-encapsulating arsonoliposomes in NB4 and MDA-MB-468 cells. The intra- and extracellular distribution of Rho-PE-arsonoliposomes (arsonoliposomes with 0,2% rhodamine conjugated with phosphatidyl-ethanolamine in their lipid composition), was observed after incubation with cells at 37 oC for different time periods, by fluorescence microscopy, using appropriate excitation and barrier filters. Furthermore, in another set of experiments, pretreatment of cells with endocytosis inhibitors was performed. Chloropromazine (clathrin mediated endocytosis inhibitor, CPZ) and methyl-β-cyclodextrin (caveolae mediated endocytosis inhibitor, ΜeβCD) were preincubated with cells, HPTS- encapsulating liposomes were added and the Ι454/Ι413 ratio and the % uptake of liposomal contents by cells were measured for different time periods., in order to clarify in which cases the endocytosis is clathrin- or caveolae-dependent. The interaction of liposomes with cells was monitored at 4 oC as well, in order to study the time-course of the binding of liposomes to cells and clarify whether the mechanism of the association of liposomes with cells is energy dependent. NB4 and MDA-MB-468 cells were incubated at 4 oC with HPTS-encapsulating liposomes for different time periods in two different concentrations and the estimation of the intracellular fate and amount of bound liposomes on the cells was once again based on of Ι454/Ι413 ratio and uptake measurements. The mechanism of the association of the liposomes seems to be similar for all arsonoliposomes in the case of MDA-MB-468 cells. The results reveal that arsonoliposomes are uptaken by a receptor-mediated mechanism, probably clathrin- or/and caveolae-mediated. In comparison with the results of the viability studies, the fact that arsonoliposome did not induce growth inhibition of the MDA-MB-468 cells may correlate to their lysosomal degradation resulting from the clathrin-mediated endocytosis. Moreover, the fact that no arsenic was detected by Graphite Furnace Atomic Absorbance Spectrometry in the cells after incubation under the same conditions, except for the more rigid DSPC arsonoliposomes (PEGylated or not), comes in agreement with this previous hypothesis. In the case of NB4 cells, the mechanism of the association and the extent of receptor-mediated endocytosis role may affect the induced growth inhibition. More cytotoxic PEGylated PC-based arsonoliposomes are not endocytosed through receptors at all, while PEGylated DSPC-based seem to be endocytosed through clathrin- or/and caveolae-mediated mechanism. The fact though that no arsenic was detected, after both cancer cell lines were incubated with all arsonoliposomes studied under identical conditions, needs further investigation, given that whole liposomes internalization is taking place and that control liposomes (with no arsenic in their lipid composition) have been found not to be toxic against cancer cells. In order to study the biodistribution of arsonoliposomes, DSPC-based and PEGylated DSPC-based arsonoliposomes, were administered by intraperitoneal (i.p.) injection in balb-c mice and the distribution of arsenic in the organs after 1, 2, 5, 12 and 24h post-injection was measured by atomic absorption spectroscopy. Results demonstrate that a high portion of the dose administered is rapidly excreted, since 1h post-injection only about 30–40% of the dose was detected cumulatively in animal tissues. After this, the whole body elimination of arsenic was a slow process with a half-life of 27.6 h for PEGylated DSPC-based arsonoliposomes, and 83 h for the DSPC-based ones. For both arsonoliposomes, arsenic distribution was greater in intestines, followed by liver, carcass + skin stomach, spleen, kidney, lung and heart. Different arsenic kinetics in blood between the two liposome types was observed. Compared to the results obtained previously with PC-based arsonoliposomes, both the DSPC-based and PEGylated-arsonoliposomes have better bioavailability. This proves that arsonoliposome lipid composition (and consequently their integrity) influences their pharmacokinetic profile. Thus, the proper arsonoliposome composition should be used according to the intended application. The same set of in vivo studies were also performed with DSPC-based and PC-based arsonoliposomes intravenous (i.v.) injection. In all cases PEG-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) was also added in the lipid membrane of the arsonoliposomes (PEGylated-arsonoliposomes) at 8 mol %. The liposomes were injected intravenously in balb-c mice. For all arsonoliposome types, the biodistribution of arsenic in mice organs was measured by atomic absorption spectroscopy, at 1, 3 and 24 h post-injection. DSPC-based arsonoliposomes give higher arsenic levels in most tissues (compared to PC-based ones) and are also affected to a higher extent by surface PEGylation. PEGylated DSPC arsonoliposomes behave similarly to PEGylated conventional liposomes that bear anionic charge. By comparison with the previous biodistribution values obtained after intraperitoneal administration of most of the vesicle types studied herein, it is concluded that the administration route has a significant effect on arsonoliposome kinetics, in some ways similar to that observed for conventional liposomes. Interestingly, after i.v. injection of all the arsonoliposome types tested, arsenic was found in detectable amounts in the animal brains, indicating that perhaps arsonoliposomes (intact or not) can pass the brain barrier to a certain extent, when high vesicle concentrations are present in the bloodstream.

La 20(S)-camptothécine (CPT) est un alcaloïde pentacyclique doté d'une activité anticancéreuse remarquable, agissant comme inhibiteur de la topoisomérase I (topo I). Le problème majeur de ce composé (et de la plupart de ses dérivés) est la fragilité de son cycle E qui s'hydrolyse rapidement à pH physiologique pour conduire au carboxylate correspondant inactif. L'une des approches permettant de pallier ce problème d'hydrolyse consiste à modifier le cycle E. Ce travail de thèse porte sur la synthèse totale de deux nouveaux analogues de la CPT modifiés au niveau du cycle E. Chacune de ces synthèses fait appel à une cycloaddition [3+2] afin de préparer l'hydroxypyridone de départ (cycles C et D), à un réarrangement de Claisen permettant de mettre en place le cycle E et à une condensation de Friedländer pour installer le motif quinoléinique (cycles A et B). Le premier analogue synthétisé, la (±) 17 norcamptothécine (17-norCPT), possède une α-hydroxy γ-lactone à la place de l'α-hydroxy δ-lactone de la CPT. Ce composé a été obtenu en neuf étapes avec un rendement de 4,4% à partir de l'hydroxypyridone de départ. Le test d'inhibition de la topo I a été réalisé, mais cette molécule s'est révélée totalement inactive. Cependant, une étude de la cinétique d'hydrolyse de la 17-norCPT, réalisée par spectroscopie de fluorescence, a permis de montrer que cet analogue était très instable en milieu aqueux. Le second composé préparé est en fait un homologue de la 17 norCPT possédant un méthylène entre l'oxygène et le carbonyle de la lactone. Cette molécule, comportant un motif céto éther, est donc un isomère de la CPT. Elle a été obtenue par deux voies de synthèse différant l'une de l'autre par l'ordre des réactions mises en œuvre. Chacune de ces approches permet d'obtenir le composé souhaité en neuf étapes à partir de l'hydroxypyridone de départ, avec un rendement de 16% et 10% respectivement. Les tests biologiques sur le composé final sont actuellement en cours. / 20(S)-Camptothecin (CPT) is a pentacyclic alkaloid endowed with remarkable anticancer activity, acting as an inhibitor of topoisomerase I (topo I). The major problem with this compound (and many of its derivatives) is the fragility of its E-ring, which suffers rapid hydrolysis at physiological pH to yield the inactive corresponding carboxylate. One of the approaches to overcome this problem of hydrolysis consists in modifying the E-ring. This thesis focuses on the total synthesis of two new E-ring modified CPT analogs. Each of the syntheses involves a [3+2] cycloaddition to prepare the starting hydroxypyridone (rings C and D), a Claisen rearrangement to realize the E-ring, and a Friedländer condensation to install the quinoline system (rings A and B). The first analog synthesized, (±) 17 norcamptothecin (17-norCPT), has an α-hydroxy γ-lactone in place of the α-hydroxy δ-lactone of CPT. This compound has been obtained in nine steps with a yield of 4.4% from the starting hydroxypyridone. Unfortunately, though, this molecule has proved to be totally inactive in the topo I assay. A kinetic hydrolysis study of 17-norCPT, carried out by fluorescence spectroscopy, has demonstrated, however, that this analog is, in fact, unstable in aqueous media. The second compound prepared is a homolog of 17-norCPT with a methylene between the lactone oxygen and carbonyl, thus a CPT keto ether isomer. It has been obtained through two synthetic routes that differ from each other in the order of the steps. Each of the approaches yields the desired compound in nine steps from the starting hydroxypyridone, with yields of 16% and 10% for the two syntheses. Biological tests on the final compound are currently underway.

OLIVEIRA, Jefferson Soares de. Caracterização bioquímica e atividade citotóxica in vitro e antitumoral in vivo de proteínas do látex de Calotropis procera. 2011. 149 f. : Tese (Doutorado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2011. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-20T14:23:08Z No. of bitstreams: 1 2011_tese_jsoliveira.pdf: 3281739 bytes, checksum: 7e9d2e21ed1c7ab719fb84e18c80c57c (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:35:56Z (GMT) No. of bitstreams: 1 2011_tese_jsoliveira.pdf: 3281739 bytes, checksum: 7e9d2e21ed1c7ab719fb84e18c80c57c (MD5) / Made available in DSpace on 2016-08-02T20:35:56Z (GMT). No. of bitstreams: 1 2011_tese_jsoliveira.pdf: 3281739 bytes, checksum: 7e9d2e21ed1c7ab719fb84e18c80c57c (MD5) Previous issue date: 2011 / Latex of Calotropis procera was described as a source of pharmacologically active proteins such as anti-inflammatory and analgesic activities. This study evaluated the cytotoxic activity in vitro of proteins (LP) recovered from the latex of the medicinal plant C. procera against human cancer cells and the in vivo growth inhibition of Sarcoma 180. LP exhibited significant cytotoxicity for cell lines with IC50 values ranging from 0.11 to 1.36 µg/ml for tested cell lines (HL-60, SF295, HCT-8 and MDA-MB-435). There were no visible effects on the viability or morphology of healthy mononuclear cells exposed to PL (10 µg / ml) for 72 h, showing that PL was selective for malignant cells. Fractionation of PL by ion exchange chromatography (pH 5.0) gave rise to three new protein fractions (PI, PII and PIII) and almost all cytotoxicity present in PL was retained in fraction PI. The cytotoxic effects of PL and PI were diminished when pre-treated with pronase or 2-mercaptoethanol, reinforcing the protein nature of active molecules. PI was absent on cysteine protease activity, indicating that this enzyme abundantly found in PL is not involved in cytotoxicity. Mechanistic studies of LP cytotoxicity using HL-60 cells revealed that PL induces apoptosis probably due to changes in DNA topology since PL interfered in the activity of topoisomerase I. The cytotoxic activity present in PI seems to be performed by the synergic action of different proteins. This hypothesis is suggested since PI subjected to gel filtration chromatography produced distinct protein peaks that shared cytotoxic activity, although with lower extent than PI. Studies on growth inhibition of Sarcoma 180 showed that animals treated with PL by oral (10 or 20 mg/kg) or intraperitoneal (2 or 5 mg/kg) rout reduced tumor growth significantly (up 51.83%, po) and increased life span of transplanted animals for up to four days. The inhibitory activity of tumor growth was lost when the LP was subjected to proteolysis, acidic treatment or collected in iodoacetamide. On the other hand, LP maintained its in vivo activity after heat treatment, suggesting that thermo stable proteins are involved in the suppression of tumor growth. Biochemical parameters such as the enzymatic activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the content of urea in serum were not affected in animals treated with LP. Treatment of animals with LP induced increasing of leukocyte numbers and protected from leukopenia induced by 5-FU administration. In addition, no significant changes in the histopathology of liver of animals treated with oral LP were seen. In vivo antitumor activity was retained in the PII and this activity was observed even when animals received a single dose of PII. It seems that the in vivo action of latex proteins is related to an immunestimulant event and not to the cytotoxic action of protein on cells Sarcoma 180. PII-3, recovered after PII fractionation on ion exchange column at pH 6.0, retained the tumor growth inhibition activity found in PII. PII-3 was shown to possess cysteine proteinase and papain inhibitor activities; however is not completely clear weather this molecules are involved in the antitumor activity. This study confirms the pharmacological potential of latex proteins from C. procera to control the development of tumor cells. / O látex de Calotropis procera foi descrito como uma fonte de proteínas farmacologicamente ativas como atividade antiinflamatória e analgésica. O presente trabalho avaliou a atividade citotóxica in vitro das proteínas (PL) recuperadas do látex da planta medicinal C. procera contra células de câncer humano e a inibição do crescimento do Sarcoma 180 transplantado em camundongos. PL apresentou significante citotoxicidade para as linhagens celulares com valores de IC50 variando entre 0,11 a 1,36 µg/ml para as linhagens celulares testadas (HL-60, SF295, HCT-8 e MDA-MB-435). Não foram observados efeitos visíveis sobre a viabilidade ou a morfologia de células mononucleares saudáveis expostas a PL (10 µg / ml) por 72 h, mostrando que PL apresentou seletividade para células tumorais. O fracionamento de PL por cromatografia de troca iônica (pH 5,0) deu origem a três novas frações (PI, PII e PIII) e quase toda citotoxicidade presente em PL ficou retida na fração PI. Os efeitos citotóxicos de PL e PI foram diminuídos quando previamente tratados com pronase, ou 2-mercaptoetanol, sugerindo uma natureza protéica de moléculas ativas. PI não apresentou atividade de proteinase cisteínica, indicando que esta enzima, encontrada em abundância em PL, não está envolvida na citotoxicidade. Estudos do mecanismo da ação citotóxica de PL utilizando células HL-60 revelou que PL induz apoptose celular provavelmente devido a alterações na topologia de DNA, já que PL interferiu na atividade de topoisomerase I. A atividade citotóxica presente em PI parece ser desempenhada pela ação combinada de diferentes proteínas uma vez que PI submetida à cromatografia de filtração em gel gerou picos protéicos distintos que compartilharam atividade citotóxica, embora com menor potência que PI. Estudo de inibição do crescimento do Sarcoma 180 revelou que animais tratados com PL por via oral (10 or 20 mg/kg) ou intraperitoneal (2 or 5 mg/kg) reduziram de modo significativo o crescimento do tumor (em até 51,83%; v.o.) e prolongou o tempo de sobrevivência dos animais transplantados por até quatro dias. A atividade inibitória do crescimento do tumor foi perdida quando a fração PL foi submetida à proteólise, tratamento ácido ou com iodoacetamida. No entanto, PL conservou a sua atividade in vivo após o tratamento térmico, sugerindo que proteínas termoestáveis estão envolvidas na supressão do crescimento tumoral. Os parâmetros bioquímicos, como a atividade enzimática da aspartato aminotransferase (AST) e alanina aminotransferase (ALT) e o teor de uréia no soro, não foram afetados nos animais tratados com PL. PL induziu aumento no número de leucócitos de animais tratados e ainda eliminou completamente a leucopenia induzida pela administração do 5-FU. Em adição, não foram observadas mudanças na histopatologia do fígado de animais tratados com PL por via oral. Atividade antitumoral in vivo ficou retida no PII e esta atividade foi observada mesmo quando animais transplantados receberam uma única dose de PII sugerindo que a ação in vivo de proteínas do látex está relacionada a um evento imunoestimunate de proteínas e não à ação citotóxica sobre as células do Sarcoma 180. PII-3, obtido após fracionamento de PII em coluna de troca iônica em pH 6,0 reteve a atividade de inibição do crescimento tumoral de PII. Esta fração possui atividade de proteinase cisteínica e atividade de inibidor de papaína, porém não é completamente claro o envolvimento dessas moléculas na atividade in vivo. Este estudo confirma o potencial farmacológico das proteínas do látex de C. procera para controlar o desenvolvimento de células tumorais.

Les travaux de thèse présentés dans ce manuscrit portent sur l’étude chimique de plantes du genre Psiadia. Trois axes de recherche ont été menés parallèlement à savoir (1) une étude chimiotaxonomique à partir de 11 espèces du genre Psiadia endémiques de La Réunion, (2) un criblage biologique réalisé sur 16 espèces du genre Psiadia dont 11 endémiques de La Réunion et 5 endémiques de Maurice et (3) une étude phytochimique ciblée sur l’espèce Psiadia arguta endémique de Maurice. Le premier axe comportant l'étude chimiotaxonomique menée par une approche métabolomique avait pour objectif d'identifier des marqueurs chimiotaxonomiques. Cette étude a été effectuée à partir des analyses CG-SM et CG-DIF des composés volatils et des analyses RMN 1H des composés non volatils de 11 espèces endémiques de La Réunion récoltées sur différents lieux géographiques et au cours des saisons estivale et hivernale. Une analyse intra-espèce a permis d'étudier la variabilité saisonnière et/ou géographique de la composition chimique de chaque espèce. Une analyse inter-espèces a conduit à deux classifications différentes des 11 espèces selon leur composition en métabolites volatils et non volatils. Le deuxième axe avait pour objectif d'identifier parmi 11 espèces du genre Psiadia endémiques de La Réunion et 5 espèces endémiques de Maurice, les espèces présentant une ou des activités biologiques prometteuses. Les cibles biologiques choisies ont été le parasite Plasmodium falciparum responsable du paludisme, la lignée cellulaire humaine cancéreuse HeLa responsable du cancer du col de l'utérus et l'enzyme HRP (Horseradish peroxydase) intervenant dans la réponse inflammatoire. À l'issue de ce criblage, 5 espèces se sont révélées prometteuses : les espèces réunionnaises P. amygdalina et P. anchusifolia et les espèces mauriciennes P. arguta et P. lithospermifolia pour l'activité antiplasmodiale, ainsi que l'espèce réunionnaise P. dentata pour les trois activités testées. Le troisième axe consacré à une étude phytochimique de P. arguta réalisée par un fractionnement bioguidé de l'extrait brut a conduit à l'isolement et à l'identification de 16 terpénoïdes : 2 triterpènes et 14 diterpènes de structure labdane dont 4 sont de structure nouvelle. Cinq diterpènes se sont révélés particulièrement actifs contre le parasite P. falciparum : l'acétate de labda-13(E)-en-8α-ol-15-yle, l'acétate de labdan-8α-ol-15-yle, le 13-épi-sclaréol, le labda-13(E)-ène-8α,15-diol et le (8R,13S)-labdane-8,15-diol. Par ailleurs, une étude métabolomique menée par RMN 1H sur des plantules de P. arguta cultivées in vitro et acclimatées a permis l'étude des facteurs influençant la production de ces composés bioactifs. / The present work describes the chemical composition of the plant genus Psiadia and focuses on three research topics: (1) a chemotaxonomic study of 11 species endemic to Reunion island, (2) a biological screening of 16 Psiadia species among which 11 are endemic to Reunion and 5 are endemic to Mauritius and (3) a phytochemical investigation of Psiadia arguta, endemic to Mauritius. The aim of the chemotaxonomic study was to identify chemical markers by a metabolomic approach using GC-MS and GC-FID for volatiles compounds and 1H NMR for non-volatiles compounds. The 11 studied species were harvested in different locations and seasons in order to analyze the seasonal or geographical variability of the chemical profile of each species. This study led to two classifications of the 11 species in terms of the composition of volatiles and non-volatiles compounds. The objective of the second research topic was to identify within 11 species endemic to Reunion island and 5 species endemic to Mauritius, the most active species for the biological activities tested. The targeted activities were antiplasmodial against Plasmodium falciparum, anticancer against the human cancer cell lines HeLa and anti-inflammatory through the inhibition of the enzyme HRP (Horseradish Peroxidase). Four species, P. amygdalina and P. anchusifolia, endemic to Reunion, and P. arguta and P. lithospermifolia, endemic to Mauritius, were particularly active against P. falciparum. Besides, P. dentata (endemic to Reunion) displayed interesting antiplasmodial, anticancer and anti-inflammatory activities. The third research topic was devoted to a phytochemical investigation of P. arguta by a bioguided fractionation and led to purification and identification of 16 terpenoids: 2 triterpenes and 4 diterpenes including 4 new compounds. The evaluation of the antiplasmodial activity of all isolated compounds allowed to highlight activities of five diterpenes: labda-13(E)-en-8α-ol-15-yle acetate, labdan-8α-ol-15-yle acetate, 13-epi-sclareol, labda-13(E)-ene-8α,15-diol and (8R,13S)-labdane-8,15-diol. Furthermore, in order to identify factors influencing the production of bioactive compounds, P. arguta has been multiplicated using in vitro culture techniques and micropropagated plants were acclimatized.

A descoberta de fármacos a partir de produtos isolados de organismos marinhos tem apresentado um grande crescimento nos últimos anos, principalmente devido aos avanços tecnológicos analíticos, síntese química e biotecnologia. Dentre estes, as esponjas representam uma das principais fontes de metabólitos protótipos para diversas atividades, destacando-se os efeitos antitumorais. Neste contexto, este trabalho teve como objetivo a investigação química e biológica de três esponjas coletadas na costa sul-brasileira: Haliclona tubifera, Polymastia janeirensis e Scopalina ruetzleri. Considerando a correlação entre câncer, distúrbios da coagulação e desbalanço de espécies reativas de oxigênio (EROs), foram realizados ensaios visando a aquisição destas atividades e a identificação de substâncias bioativas. Para a esponja H. tubifera foram observados interessantes efeitos antitumorais em células de glioma e neuroblastoma humano (IC50 < 20 μg/ml), além das atividades antioxidante e anticoagulante para a fração acetato de etila. O composto majoritário desta fração foi isolado como um derivado N-Boc e sua configuração foi estabelecida utilizando um novo protocolo de dicroísmo circular e semissíntese de derivados. Assim, este esfingosídeo de cadeia longa isolado (2R,3R,6R,7Z)-2-amino-7-octadecene-1,3,6-triol, foi denominado halisphingosine A. Um novo composto minoritário, halisphingosine B foi obtido usando técnicas de isolamento em escala nanomolar. Sua configuração absoluta foi estabelecida por comparação com o composto A. Da mesma forma, para a esponja S. ruetzleri, a fração acetato de etila demonstrou os resultados mais promissores. Um potencial efeito anticâncer e de inibição dos radicais peroxila foi observado. Além disso, um efeito modulador da peroxidação lipídica foi evidenciado em ensaio ex vivo de dienos conjugados. Através da análise por RMN de 1H, verificou-se que a fração era majoritariamente constituída por ácidos graxos, os quais foram derivatizados para caracterização por Cromatografia Gasosa (GC/FID). Foram identificados 32 ácidos graxos principalmente poli-insaturados (53%). Ácidos graxos minoritários não usuais para o ambiente marinho também foram caracterizados. A esponja P. janeirensis apresentou os efeitos citotóxicos mais promissores em células de glioma e neuroblastoma humano, com um IC50 < 1,0 μg/ml para o extrato aquoso (pH 6,8), sendo este efeito pH-dependente, uma vez que o extrato (pH 5,8) não alterou a viabilidade celular. Para P. janeirensis, foi também investigado seu potencial de defesa química em modelo de Zebrafish. Foi observado que o extrato aquoso desencadeia um efeito de fuga, alterando significativamente o comportamento espaço-temporal de peixes Danio rerio. Analisando em conjunto, os dados do presente trabalho representam uma nova contribuição para o estudo químico e biológico de espécies de esponjas marinhas da costa sul-brasileira e apontam as potencialidades destas esponjas na busca de moléculas protótipos para fármacos, especialmente relacionados à terapia do câncer. / Drug discovery from marine natural products has increased in the past few years, mainly due to technological advances in spectroscopy, chemical synthesis and biotechnology. Among all marine animals, sponges represent one of the major sources of prototype metabolites for several biological activities, highlighting the antitumor effects. In this context, this study carried out chemical and biological investigation of three sponges collected on the South Brazilian coastline: Haliclona tubifera, Polymastia janeirensis and Scopalina ruetzleri. Considering the correlation between cancer, clotting disorders and imbalance of reactive oxygen species (ROS), experiments were conducted for acquisition of these activities and identification of bioactive compounds. H. tubifera showed an interesting cytotoxic effect in human neuroblastoma and glioma cell lines (IC50 <20 μg/ml), antioxidant and anticoagulant effect for ethyl acetate (EtOAc) fraction. The major compound of EtOAc fraction was isolated as an N-Boc derivative and its configuration was established using a new circular dichroism protocol with the production of semi-synthetic derivatives. This long chain sphingoid base (2R,3R,6R,Z)-2-aminooctadec-7-ene-1,3,6-triol was named as halisphingosine A. A new minor compound, halisphingosine B was obtained using nanomol scale techniques and their absolute configuration was established by comparison with compound A. Likewise, for the sponge S. ruetzleri, the EtOAc fraction showed the most promising results. A potential anticancer effect, inhibition of peroxyl radicals and modulation effect of lipid peroxidation was observed. Fingerprint 1H NMR analysis showed that this fraction is mainly constituted of fatty acids. Through Fatty Acid Methyl Ester (FAMEs) analysis by GC/FID, it was possible to identify 32 fatty acids, of which around 50% were Polyunsaturated Fatty Acids (PUFAs). In addition, some minor unusual fatty acids for the marine biosphere were identified. It was observed for P. janeirensis the most promising cytotoxic effects on human glioma and neuroblastoma cells, with an IC50 <1.0 μg/ml to aqueous extract (pH 6.8), being this effect pH-dependent, since the extract (pH 5.8) did not affect the cell viability. Moreover, P. janeirensis was investigated along their potential chemical defense in Zebrafish model. Aqueous extract trigged an escape effect, significantly altering the spatio-temporal swimming activity of animals. Taken together, the data presented from this study represent a new contribution to chemical and biological research of marine sponge species from South Brazilian coastline, and point the potentialities of sponges to search chemical prototypes for drugs, especially related to cancer therapy.