L’objectif principal de cette thèse a été le développement, la caractérisation et l’évaluation in vitro et in vivo de différents nanovecteurs plus spécifiquement les nanoparticules spécifiques pour le traitement de la mélanome et β-lapachone-lipossomale incorporée dans des hydrogels de biopolymère pour la cicatrisation de blessures topiques. La première étape de cette thèse a consisté en la révision de la littérature liées aux résentes avancées sur les nanoparticules pour le ciblage d’agents thérapeutiques pour des cellules circulantes et des mésenchymateuses de mélanomes. De plus, cette révision a approfondi la connaissance sur les principaux biomarcateurs qui étaient déjà identifiés dans ces cellules et quelles caractéristiques des nanovecteurs peuvent influencer leur performance in vivo. Dans la phase expérimentale, les nanoparticules ont été développées en utilisant la méthode de nanoprécipitation de polymères dérivés du poli(γ-benzyl-L-glutamate). Ensuite, des immuno-nanoparticules combinées avec l’anticorps MART-1 spécifique pour les cellules de mélanome, ont été obtenues par le lien streptavidine-biotine. La combinaison de l’anticorps sur la superficie des nanoparticules a été évaluée par western blot. Les nanoparticules ont été caractérisées et évaluées in vitro dans des cellules de mélanome B 16-GFP et des cellules endothéliales de la veine ombilicale humaine (HUVECs), l’activation de complément a été recherchée par la technique d’immunoélectrophorèse bidimensionnelle. Les nanoparticules ont présenté des tailles comprises entre 20 et 100 nm et une charge négative (-3 à -30 mV). La combinaison de l’anticorps sur la superficie des nanoparticules a été observée par la technique western blot et confirmée par les altérations de la taille et de la charge de superficie des particules. Les nanoparticules développées n’ont pas été capables d’activer le systhème complémentaire étant considérées de longue circulation sanguine. Pour l’analyse in vitro, les nanoparticules n’ont pas présenté de cytotoxixité lorsqu’elles ont été testées dans des cellules de mélanome ou dans des cellules normales endothéliales. Lors des tests de capture cellulaire, les immuno-nanoparicules, qui contenaient l’anticorps spécifique pour la reconnaissance de l’antigène surexprimé dans des cellules de mélanome, ont présenté une augmentation de 40 à 50% lors de la capture pour ces cellules, cela indiquant une plus grande spécificité de ce nanovecteurs. La deuxième partie de cette thèse a été le développement, la caractérisation et l’évaluation de l’activité cicatrisante in vivo de β-lapacho encapsulé dans des liposomes multilamellaires incorporés dans de l’hydrogel de biopolymère produit par la bactérie Zoogloea sp. (β-lap-Lipo/ZBP/HEC). β-lap-Lipo/ZBP/HEC a présenté un pH et un comportement rhéologique approprié pour l’application topique, ainsi qu’un profil de libération plus lent de la β-lapachone à partir de l’hydrogel. Les analyses hystopotologiques de l’activité cicatrisante in vivo ont montré que le véhicule hydrogel de biopolymère a été capable de stimuler les réparations du tissu, augmenter la cellularité locale, les fibroblastes, les cellules inflamatoires, les vaisseaux sanguins et les fibrilles de collagène pendant la phase proliférative de la cicatrisation. De plus, β-lap-Lipo/ZBP/HEC a favorisé une augmentation de l’angiogenèse locale et une dimminution de l’inflamation de la blessure. Ces résultats ont montré le potentiel de l’application topique de β-lap-Lipo/ZBP/HEC pour la thérapie des lésions. Pour conclure, cette thèse a contribué au développement de nanovecteurs promisseurs ayant différentes applications biologiques et formes d’administration, comme le traitement systémétique de la mélanome et l’action topique lors de la cicatrisation des blessures / This thesis had as general objective the development, characterization and evaluation in vitro or in vivo of different nanocarriers, specifically site-specific nanoparticles for the treatment of melanoma and liposomal-hydrogel containing β-lapachone for topical wound healing. The first part of this thesis consisted in a literature review about the recent advances in nanoparticles for the targeting of therapeutic agents to circulating and mesenchymal melanoma cells. In addition, this review deepened the knowledge about the main biomarkers identified in these cells and which characteristics of nanocarriers may influence on their in vivo performance. In the experimental phase, nanoparticles were developed through the nanoprecipitation method of polymers derived from poly (γ-benzyl-L-glutamate). Next, immunonanoparticles conjugated with MART-1 antibody specific for melanoma cells were obtained through the streptavidin-biotin binding. The conjugation of this antibody on the nanoparticles surface was evaluated by western blot. The nanoparticles were characterized and evaluated in vitro in B16-GFP melanoma cells and human umbilical vein endothelial cells (HUVECs) and the complement activation was investigated by bidimensional immunoelectrophoresis. The nanoparticles presented sizes between 20 and 100 nm and negative surface charge (-3 to -30 mV). The conjugation of antibody on the nanoparticle surfaces was detected by the western blot technique and confirmed by the changes in particle size and surface charge. The developed nanoparticles were not able to activate the complement system being considered long blood circulation. Regarding the in vitro analysis, the particles did not show cytotoxicity when tested in melanoma cells or normal endothelial cells. In the cell capture assays, the immunonanoparticles, containing a specific antibody for the recognition of the overexpressed antigen in melanoma cells, showed an increase of 40 to 50% in the uptake for these cells, indicating a specificity of this nanocarrier. The second part of this thesis consisted of the development, characterization and evaluation of the in vivo wound healing activity of β-lapachone encapsulated in multilamellar liposomes and incorporated in a biopolymer hydrogel produced by Zoogloea sp (β-lap-Lipo/ZBP/HEC). β-lap-Lipo/ZBP/HEC presented pH and rheological behavior suitable for topical application, as well as a slower release profile of β-lapachone through the hydrogel. Histopathological analyzes of the healing activity in vivo, showed that the biopolymer hydrogel vehicle was able to stimulate tissue repair, with the increase of local cellularity, fibroblasts, inflammatory cells, blood vessels and collagen fibers, during the proliferative phase of wound healing. In addition, β-lap-Lipo/ZBP/HEC promoted an increase in local angiogenesis and a decrease of inflammation at the wound site. These results demonstrate a promising topical application of β-lap-Lipo/ZBP/HEC for wound therapy. In conclusion, this thesis contributed for the development of promising nanocarriers with different biological applications and administration routes, such as systemic treatment of melanoma and topical action in wound healing.

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23848号 / 薬博第855号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 掛谷 秀昭, 教授 高須 清誠, 教授 大野 浩章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24965号 / 医博第5019号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 波多野 悦朗, 教授 中本 裕士 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Plants have developed a number of ways how to minimise negative influence of the environment. As a consequence of stress action, plants carbohydrate metabolism is quite often influenced, esp. on the level of expression and activities of different enzymes and also several metabolites concentration. One of key enzymes of carbohydrate metabolism is invertase. The aim of this work was to find out, whether the activity of its isoforms (cytoplasmic, vacuolar and extracellular) in tobacco plants is influenced by Potato virus Y (PVY). It was shown, that activity of cytoplasmic invertase was not affected, but the activity of vacuolar and extracellular isoform was enhanced during potyviral infection. Hence, it is likely, that vacuolar and extracellular invertases are related to plant antiviral defence. The effect of PVY on other enzymes of carbohydrate metabolism and several metabolites content was studied. Activity of α-amylase and phosphorylase, starch-degrading enzymes, was strongly enhanced during potyviral infection. That is probably how plant cells get glucose, which is a key source of energy and metabolites for biosynthesis of different compounds. It may also serve as a signal molecule. Activity of other hydrolytic enzymes, β-glucosidase and β-hexosaminidase, was also slightly increased. There was no...

<p>Förekomsten av Extended Spectrum β-Lactamases (ESBL) och Vancomycinresistenta enterokocker (VRE) har ökat hos gramnegativa bakterier under de senaste åren. Måsfåglar kan användas som biologiska indikatorer för att se prevalensen av antibiotikaresistens i naturen, vilken sedan kan jämföras med den hos humana populationer.</p><p>Syftet var att med odling av fekalprover på selekterande och differentierande agarplattor analysera det allmänna resistensläget hos <em>Escherichia coli</em> samt analysera förekomst av Extended Spectrum β-Lactamases hos enterobakterier och Vancomycinresistenta enterokocker i 300 prover från måsar på Irland. För att artbestämma bakterierna utfördes även biokemiska analyser.</p><p>Den allmänna resistensen analyserades med diskdiffusionstest på Muller-Hintonagarplattor. <em>E. coli</em> användes som indikatorart. För analys av ESBL-isolat och VRE odlades fekalproverna i selekterande anrikningsbuljong och på selekterande och differentierande agarplattor. Isolaten fenotypades med hjälp av biokemiska tester. För verifiering av ESBL-isolat utfördes synergitest med klavulansyra.</p><p>Låg allmänresistens erhölls för <em>E. coli.</em> Två isolat var resistenta mot båda β-laktamantibiotika ampicillin och cefadroxil. Tjugo stycken ESBL-producerande isolat erhölls. <em>E. coli</em> var den vanligaste förekommande ESBL-producerande bakteriearten i analyserade prover. Fem enterokockisolat med resistens mot Vancomycin och Aztreonam erhölls. Slutsatsen är att analysen av dessa 300 prover visar att det allmänna antibiotikaresistensläget är lågt samt att ESBL och eventuellt VRE förekommer hos måsfåglar på Irland. </p>

Förekomsten av Extended Spectrum β-Lactamases (ESBL) och Vancomycinresistenta enterokocker (VRE) har ökat hos gramnegativa bakterier under de senaste åren. Måsfåglar kan användas som biologiska indikatorer för att se prevalensen av antibiotikaresistens i naturen, vilken sedan kan jämföras med den hos humana populationer. Syftet var att med odling av fekalprover på selekterande och differentierande agarplattor analysera det allmänna resistensläget hos Escherichia coli samt analysera förekomst av Extended Spectrum β-Lactamases hos enterobakterier och Vancomycinresistenta enterokocker i 300 prover från måsar på Irland. För att artbestämma bakterierna utfördes även biokemiska analyser. Den allmänna resistensen analyserades med diskdiffusionstest på Muller-Hintonagarplattor. E. coli användes som indikatorart. För analys av ESBL-isolat och VRE odlades fekalproverna i selekterande anrikningsbuljong och på selekterande och differentierande agarplattor. Isolaten fenotypades med hjälp av biokemiska tester. För verifiering av ESBL-isolat utfördes synergitest med klavulansyra. Låg allmänresistens erhölls för E. coli. Två isolat var resistenta mot båda β-laktamantibiotika ampicillin och cefadroxil. Tjugo stycken ESBL-producerande isolat erhölls. E. coli var den vanligaste förekommande ESBL-producerande bakteriearten i analyserade prover. Fem enterokockisolat med resistens mot Vancomycin och Aztreonam erhölls. Slutsatsen är att analysen av dessa 300 prover visar att det allmänna antibiotikaresistensläget är lågt samt att ESBL och eventuellt VRE förekommer hos måsfåglar på Irland.

La malaltia d’Alzheimer es caracteritza per l’acumulació de plaques extracel.lulars amiloides, formades pels anomenats pèptids amiloides (Aβ40). L’homeòstasi del coure (Cu) es veu afectada en l’etiologia de la malaltia d’Alzheimer, encara que el seu rol és un fet controvertit. Per a l’estudi de la influència del Cu(II) en l’agregació del pèptid amiloide, la morfologia i l’estructura secundària dels agregats amiloides formats en presència de Cu(II) s’han utilitzat AFM, TEM, SEM, SAXS, FTIR i espectrometria de fluorescència. A més, els efectes tòxics d’aquests agregats s’han estudiat en cèl.lules neuronals. Els resultats obtinguts mostren que aquests agregats són amorfos i presenten una toxicitat més alta que les fibres. Per a l’estudi dels dendrímers de maltosa com a possibles moduladors de l’agregació i de la toxicitat del pèptide amiloide. S’ha confirmat que aquests dendrimers no són tòxics en cèl.lules neuronals i que són capaços de modular l’agregació i toxicitat del pèptid amiloide. Aquests resultats permeten considerar als dendrimers de maltosa com a possibles eines per a reduir la toxicitat dels pèptids amilodies. / Senile plaques of Alzheimer’s disease patients are composed primarily of the amyloid-β-peptide (Aβ). Recent studies implicate Cu(II) in the aetiology of AD. The role of Cu(II) in ADis currently highly disputed. Influence of Cu(II) on Aβ aggregation and amyloidogenic properties of glycodendrimers were investigated in this thesis. AFM, TEM, SEM, SAXS, FTIR and fluorescence spectroscopy were used to study a morphology and a secondary structure of Aβ-Cu(II) aggregates. The toxic effects of Aβ40-Cu(II) amorphous aggregates was confirmed for neuronal cell lines. It was shown that maltose glycodendrimers can be efficiently used to modulate Alzheimer’s amyloid peptide aggregation and inhibit cell toxicity by facilitating the clustering of amyloid fibrils. These results show that glycodendrimers are promising non-toxic agents in the search for anti-amyloidogenic compounds. It was also suggested that fibril clumping may be anti-amyloid toxicity strategy.

Η επιθηλιακή προς μεσεγχυματική μετατροπή είναι ένα φαινόμενο που πιθανότατα εμπλέκεται στην παθογένεια του καρκίνου του λάρυγγα. Η ΕΜΤ εξελισσόμενη μέσα από δαιδαλώδη μονοπάτια μεταγωγής σήματος καταλήγει να προσδώσει στο καρκινικό κύτταρο δομικά και λειτουργικά χαρακτηριστικά που το καθιστούν ικανό να μπορεί να διεισδύει στους ιστούς και να μεθίσταται. Κεντρικό μόριο στα μοριακά μονοπάτια που διαμεσολαβούν την ΕΜΤ στον καρκίνο του λάρυγγα είναι μια κινάση, η ILK, που δέχεται σήματα από τις ιντεγκρίνες και τους υποδοχείς αυξητικών παραγόντων. Στους επιθηλιακούς καρκίνους αναφέρεται η εμπλοκή της σε λειτουργίες όπως ρύθμιση του κυτταρικού κύκλου, αποφυγή της απόπτωσης, νεοαγγειογένεση, απώλεια των δομών συνοχής του κυττάρου, έκφραση μεταλλοπρωτεασών και αναδιαμόρφωση του κυτταροσκελετού. Στο λαρυγγικό καρκίνο όμως κρίσιμα φαινόμενα για τον μεταστατικό-επιθετικό χαρακτήρα των κυττάρων, όπως, η εξαφάνιση των E-cadherin, η μετακίνηση των β-catenin στον πυρήνα και η συσχέτιση μεταξύ τους, που διαπιστώθηκαν, δεν βρέθηκε να συνδέονται με την υπερέκφραση της ILK καθιστώντας προφανώς άλλους μηχανισμούς υπεύθυνους για την επιτέλεση αυτών των λειτουργιών. Ιδιαίτερα ενδιαφέρουσα όμως ήταν και η εντόπιση της ILK στον πυρήνα των κυττάρων του καρκίνου του λάρυγγα δίνοντας μια νέα προοπτική στην έρευνα για τον ρόλο της ILK στον καρκίνο. Στο μονοπάτι μεταγωγής σήματος της ILK στο λαρυγγικό καρκίνο βρέθηκε πως συμμετέχει και η ενεργοποιημένη Akt με την οποία επίσης σχετίζονται πολλές κρίσιμες λειτουργίες για το καρκινικό κύτταρο. Ωστόσο η p-Akt στο λαρυγγικό καρκίνο φαίνεται πως έχει κάποιο ρόλο αντίθετο με την λειτουργία του καρκινικού κυττάρου καθώς χαρακτηρίζει όγκους καλύτερης διαφοροποίησης. Ο λαρυγγικός καρκίνος τέλος διαπιστώθηκε πως χαρακτηρίζεται από την έκφραση υποδοχέων ανδρογόνων και οιστρογόνων καθιστώντας πολύ πιθανό το ρόλο αυτών των μορίων στην παθογένεια της νόσου. Ενδιαφέρουσα για την πιθανότητα εμπλοκής των υποδοχέων στερεοειδών ορμονών του φύλου στην ΕΜΤ ίσως να είναι η συσχέτιση των υποδοχέων ανδρογόνων και οιστρογόνων με την ILK και p-Akt αντίστοιχα. Οι υποδοχείς οιστρογόνων μάλιστα χαρακτηρίζοντας όγκους λάρυγγα αρχικών σταδίων ίσως θα μπορούσε να αποδειχτεί μόριο με προγνωστική αξία αλλά και θεραπευτικός στόχος. Τέλος η μελέτη της έκφρασης της ILK, της p-Akt και των υποδοχέων στεροειδών ορμονών του φύλου δεν ανέδειξε μια διαφορετική έκφραση μεταξύ υπεργλωττιδικών και γλωττιδικών καρκίνων του λάρυγγος ώστε να υποστηρίξει την ύπαρξη ενός μοριακού υποβάθρου στην παρατήρηση της ανόμοιας κλινικής συμπεριφοράς μεταξύ όγκων από τις δύο αυτές ανατομικά διακριτές περιοχές. / Epithelial to mesenchymal transition (EMT) is a process possibly implicated in the pathogenesis of laryngeal cancer. EMT is a type of epithelial cell plasticity which is characterized by long lasting phenotypic and molecular modifications of the epithelial cell as a result of a transforming procedure leading to a cell of mesenchymal type. This molecular procedure seems to be pivotal for the metastasis of epithelial cancers and its attribution to the epithelial cells is the gain of structural and functional traits which enable them to invade the tissues and metastaze. In the current study ILK expression, which is a central molecule in the signal transduction pathways of EMT, seems to be implicated in human laryngeal carcinoma. Furthermore, localization of ILK in the nucleus possibly suggests novel nuclear functions of ILK. In addition, enhanced ILK expression in laryngeal carcinoma correlates with activation of Akt. Moreover, while activated Act seems to characterize well differentiated tumors, loss of E-cadherin and activation of β-catenin were correlated with high grade carcinomas. Finally, in laryngeal cancer, mechanisms other than ILK overexpression seem to account for downregulation of E-cadherin and activation of β-catenin. Additionaly this study concluded that estrogen and androgen receptors are expressed in laryngeal carcinomas, indicating their possible role in the pathogenesis of this disease. It is interesting that the receptors of gender-related steroid hormones could have a possible relation to epithelial to mesenchymal transition phenomenon since a correlation between androgen and estrogen receptors with ILK and p-Akt respectively, was revealed. Moreover estrogen receptors characterize primary TMN-stage laryngeal carcinomas. This can be very important as it makes a prospect of using ER as a prognostic factor but even more as a novel hormonal therapy for laryngeal carcinomas. Finally the study of molecules like ILK, p-Akt, estrogen and androgen receptors did not reveal any differentiantal expression between glotic and supraglotic laryngeal carcinomas in order to support the existence of a molecular background, at least as far as those molecules are concerned, to the distinct clinical outcome of those different anatomically-derived laryngeal carcinomas. Metastasis is a rapid development in the ominous course of cancer. The effort to interpret the molecular basis of this phenomenon is not a subject of simple academic interest since the exploit of the molecular mechanisms so as to gain the control of metastasis could be the ‘‘bet’’ for a futuristic ‘‘molecular surgery’’.

Modifying substrate specificity of β-glucuronidase (GUS) would be helpful in various enzyme prodrug systems in delivering drug dose to the site of action in the cancer treatment. Due to the presence of endogenous enzyme in human tissues, GUS-based Antibody-Directed Enzyme Prodrug Therapy (ADEPT) requires a novel substrate to avoid undesirable systemic activation. GUS is a glycosyl hydrolase, highly specific towards the glucuronide derivatives. It catalyzes the glycosidic cleavage of β-D-glucuronides to β-D-glucuronic acid and aglycone moiety. In order to gain insight on the substrate specificity of GUS, C6 carboxyl group of glucuronic acid was modified to C6 carboxamide (amide derivative). We have examined amide derivatized substrates with a variety of different aglycone groups including p-nitrophenyl, phenyl and 4-methylumbelliferone to further probe the activity profile of GUS. In an effort to optimize GUS activity, docking studies have been performed which indicated that amino acid point mutations near C6 carboxyl group of glucuronic acid could improve binding of the derivatized substrates. As a result point mutations to Arg-562 and Lys-568 which make the active site less positively charged either by glutamine or glutamate lead to an enzyme with much lower native substrate activity but abolished activity for the amide-derivatized substrate. This research study showed that there is still a further need of finding appropriate mutations required to make glucuronamide a better substrate for the mutated version of GUS.

Yaku’amide A (YA) is a linear anticancer peptide that is rich in bulky dehydroamino acids (ΔAAs) and β-hydroxyamino acids (β-OHAAs). In our recent total synthesis of YA, we featured a one-pot anti dehydration–azide reduction–O→N acyl transfer process for the stereospecific construction of Z- and E- ∆Ile residues. Despite previous total syntheses and our efforts, the synthesis of YA remains lengthy. Via computational studies, we identified two analogue peptides that closely resemble the conformation of YA. The use of simpler and symmetrical bulky ΔAAs such as dehydrovaline (ΔVal) and dehydroethylnorvaline (ΔEnv) as surrogates of ∆Ile, along with azlactone chemistry for their incorporation, significantly decreased the overall number of synthetic steps. Biological studies revealed that our analogues exhibited very similar activity to that of the natural product YA, demonstrating their suitability as mimics and consistency with our computational model. Despite its utility in the construction of YA analogues, azlactone chemistry is sluggish and moderate to low yielding. For this reason, we have explored strategies to streamline the synthesis of peptides containing Z-dehydroaminobutyric acid (∆Abu), ∆Val, and Z-dehydrophenylalanine (∆Phe). The key process is to form the alkene moiety via elimination of a β-sulfonium or β-OHAA embedded within a peptide, avoiding the need to form the alkene moiety via azlactone-dipeptide dehydration and bypassing sluggish amidation/ring opening steps. β-sheet disruption of Tau-model hexapeptides is a key type of inhibition for modulating Alzheimer’s disease progression. Previous studies replaced key residues with proline, due to its rigidity and lack of amide proton, to inhibit β-sheet formation. Similar to proline, ∆AAs are also known for their rigidity and ability to favor other conformations (e.g. β-hairpin, 310-helix) along with increasing peptide half-life. We have incorporated ∆Abu, ∆Val and dehydrocyclohexylglycine (∆Chg) in a highly aggregative hexapeptide sequence, using previously studied methods, to assess their capabilities as putative β-sheet breakers and to stabilize against proteolysis. Studies are continuing.