Sur-exprimée par les cellules endothéliales des néo-vaisseaux tumoraux, l'intégrine alphaV béta3 (aVb3) constitue une cible judicieuse pour atteindre les foyers tumoraux et empêcher la vascularisation des tumeurs. À ces fins, nos travaux ont été consacrés à la conception, la synthèse et la caractérisation biologique de nouveaux vecteurs synthétiques ciblant l'intégrine aVb3. Le squelette du vecteur est un cyclodécapeptide RAFT, présentant deux faces d'adressage indépendantes, permettant la séparation dans l'espace du domaine des ligands, assurant le ciblage du vecteur, de celui supportant les molécules à vectoriser. La fonction de ciblage est assurée par la présentation de quatre motifs cyclopentapeptidiques c[-RGDfK-], ligands de l'intégrine aVb3, greffés sur la face supérieure du RAFT. L'architecture multivalente a été synthétisée de manière convergente par formation de liens éthers d'oxime, stables in vitro et in vivo, grâce à des réactions chimiosélectives hautement efficaces. La conjugaison du vecteur RAFT(c[-RGDfK-])4 à diverses molécules de détection a permis d'étudier ses propriétés biologiques in vitro et in vivo. Son interaction avec les cellules HEK293(b3) induit le clustering des intégrines aVb3 et conduit à un phénomène d'endocytose récepteur-dépendante. Chez un modèle animal murin, le vecteur (Cy5)RAFT(c[-RGDfK-])4, administré par voie systémique, détecte des tumeurs localisées ou métastatiques. Pour accroître son efficacité anti-tumorale, nous avons conjugué notre vecteur à différentes drogues cytotoxiques. Le peptide (KLAKLAK)2, la doxorubicine et la chaîne A de la ricine ont été couplés par des liens disulfures favorisant leur libération intracellulaire.

L'objectif de cette thèse est de développer l'imagerie de transfert de saturation des agents de contraste lipoCEST pour la détection de l'angiogenèse dans un modèle souris de tumeur cérébrale U87. Un lipoCEST offrant un seuil de sensibilité in vitro de 100 pM est optimisé afin de répondre aux contraintes de l'imagerie CEST in vivo. Grâce à la mise en place d'un dispositif expérimental dédié à l'imagerie CEST, nous évaluons les performances des lipoCEST pour détecter de façon spécifique l'angiogenèse tumorale. Nous montrons pour la première fois qu'il est possible de détecter un lipoCEST in vivo dans un cerveau de souris suite à une injection intraveineuse. De plus, l'utilisation d'un lipoCEST fonctionnalisé avec un peptide RGD permet de cibler spécifiquement l'intégrine ανβ3 surexprimée lors de l'angiogenèse tumorale. L'association spécifique du RGD-lipoCEST est confirmée grâce à des données d'immunohistochimie et de microscopie de fluorescence. Enfin, dans le but de tendre vers un protocole d'imagerie moléculaire par IRM-CEST, nous mettons en place un outil de quantification des lipoCEST. Cet outil repose sur la modélisation des processus d'échange de protons in vivo. Grâce à la prise en compte des inhomogénéités de champs B0 et B1 qui peuvent se révélées être délétères pour le contraste CEST, nous démontrons que la précision de notre outil de quantification est de 300 pM in vitro. La quantification des données CEST acquises chez la souris U87 permet d'estimer à 1,8 nM la concentration maximale en RGD-lipoCEST liés à leur cible moléculaire.

For clinical translation of a pancreatic substitute, long-term storage is essential, and cryopreservation is a promising means to achieve this goal. The two main cryopreservation methods are conventional freezing and vitrification, or ice-free cryopreservation. However, as both methods have their potential drawbacks for cryopreservation of a pancreatic substitute, they must be systematically evaluated in order to determine the appropriate method of cryopreservation. Furthermore, previous studies have indicated benefits to encapsulation in 3-D adhesive environments for pancreatic substitutes and that adhesion affects cell response to cryopreservation. Thus, the overall goal of this thesis was to investigate cryopreservation effects on model pancreatic substitutes consisting of cells encapsulated in non-adhesive and adhesive 3-D alginate hydrogels. Murine insulinoma betaTC-tet cells encapsulated in unmodified alginate hydrogels were chosen as the model pancreatic substitute in a non-adhesive 3-D environment. Murine myoblast C2C12 cells, stably transfected to secrete insulin, encapsulated in partially oxidized, RGD-modified alginate hydrogels were chosen as the model pancreatic substitute in a 3-D adhesive environment. With respect to cryopreservation effects on intermediary metabolism of betaTC-tet cells encapsulated in unmodified alginate, results indicate that relative carbon flow through the tricarboxylic acid cycle pathways examined is unaffected by cryopreservation. Additionally, insulin secretory function is maintained in Frozen constructs. However, vitrification by a cryopreservation cocktail referred to as DPS causes impairment in insulin secretion from encapsulated betaTC-tet cells, possibly due to a defect in late-stage insulin secretion. Results from Stable C2C12 cells encapsulated in RGD vs. RGE-alginate indicate that up to one day post-warming, cell-matrix interactions do not affect cellular response to cryopreservation after vitrification or freezing. Although there are differences in metabolic activity and insulin secretion immediately post-warming for DPS-vitrified RGD-encapsulated Stable C2C12 cells relative to Fresh controls, metabolic activity and insulin secretion are maintained at all time points assayed for Frozen constructs. Overall, due to results comparable to Fresh controls and simplicity of procedure, conventional freezing is appropriate for cryopreservation of betaTC-tet cells encapsulated in unmodified alginate or Stable C2C12 cells encapsulated in partially oxidized, RGD-modified alginate.

L'objectif du travail présenté dans cette thèse était de développer des nanoparticulesfonctionnelles ayant la capacité d'induire l'adhésion et la migration de kératinocyteshumains normaux. L'utilisation de systèmes particulaires pour favoriser l'adhésion etla migration cellulaire dans les processus de cicatrisation constitue une nouvelleapproche de l'ingéniérie tissulaire. Dans cette optique, un dérivé hydrosoluble du chitosan rendu fonctionnel par l'ajoutde peptides RGD a été développé. Les nanoparticules furent développées parcoacervation complexe entre le dérivé cationique du chitosan et le sulfate dechondroïtine anionique. La capacité du système particulaire à induire unchangement cellulaire phénotypique a été évaluée in vitro.Lors de l'évaluation de ce nouveau polymère, le succès de la synthèse a été montrépar l'absence de cytotoxicité et par la préservation de son activité biologique médiéepar les séquences RGD. Aussi bien les polymères que les nanoparticules ont induitl'adhésion et la mobilité de fibroblastes dermiques humains, confirmant le conceptde nanoparticules bio-actives. Cependant, concernant l'étude des interactions entreles nanoparticules et les kératinocytes, aucune conclusion n'a pu être tirée etd'autres travaux sont nécessaires. Pour résumer, un système particulaire bio-actif a été développé. Le choix despeptides RGD pour induire la migration des kératinocytes doit être réévalué, etl'utilisation de concentrations plus importantes, de mélange de peptides d'adhésionou l'utilisation de peptides d'adhésion différents doit être envisagée pour laréalisation d'études ultérieures.

The interaction of materials with biological systems is of critical importance to a vast number of applications from medical implants, tissue engineering scaffolds, blood-contacting devices, cell-culture products, as well as many other products in industries as diverse as agriculture. This thesis describes a method for the modification of biomaterial surfaces and the generation of tissue engineering scaffolds that utilises the self assembly of poly (styrene)-block-poly (ethylene oxide) (PS-PEO) block copolymers. Block copolymers consist of alternating segments of two or more chemically distinct polymers. The salient feature of these materials is their ability to self organise into a wide range of micro-phase separated structures generating patterned surfaces that have domain sizes in the order of 10-100nm. Further, it is also possible to specifically functionalise only one segment of the block copolymer, providing a means to precisely locate specific biological signals within the 10-100nm domains of a nano-patterned surface, formed via the programmed micro-phase separation of the block copolymer system. The density and spatial location of signalling molecules can be controlled by altering several variables, such as block length, block asymmetry, as well as processing parameters, providing the potential to authentically emulate the cellular micro to nano-environment and thus greatly improving on existing biomaterial and tissue engineering technologies. This thesis achieved several aims as outlined below; Developed methods to control the self-assembly of PS-PEO block copolymers and generate nano-patterned surfaces and scaffolds with utility for biomaterials applications. PS-PEO diblock copolymers were blended with polystyrene (PS) homopolymer and spin cast, resulting in the rapid self-assembly of vertically oriented PEO cylinders in a matrix of PS. Due to the kinetically constrained phase-separation of the system, increasing addition of homopolymer is shown to reduce the diameter of the PEO domains. This outcome provides a simple method that requires the adjustment of a single variable to tune the size of vertically oriented PEO domains between 10-100nm. Polymeric scaffolds for tissue engineering were manufactured via a method that combines macro-scale temperature induced phase separation with micro-phase separation of block copolymers. The phase behaviour of these polymer-solvent systems is described, and potential mechanisms leading to this spectacular structure formation are presented. The result is highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available technologies. Characterised the properties of these unique nano-structured materials as well as their interaction with proteinaceous fluids and cells. Nano-patterned PS-PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control PS surfaces. The adhesion of NIH 3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-islands, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface. In order to understand the mechanism by which these surfaces resisted protein and cellular adsorption we utilised neutron reflection to study their solvation and swelling properties. The results indicate that the PEO domains are highly solvated in water; however, the PEO chains do not extend into the solvent but remain in their isolated domains. The data supports growing evidence that the key mechanism by which PEO prevents protein adsorption is the blocking of protein adsorption sites. Control the nano-scale presentation of cellular adhesion and other biological molecules via the self-assembly of functionalised PS-PEO block copolymers Precise control over the nano-scale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, including differentiation and apoptosis. We present the self-assembly of maleimide functionalised PS-PEO copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By controlling the phase separation of the functional PS-PEO block copolymer we alter the nano-scale (on PEO islands of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from ~ 450 to ~ 900 islands per um2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between islands of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces were utilised to immobilise poly-histidine tagged proteins and ECM fragments. The technologies developed in this thesis aim to improve on several weaknesses of existing biomaterials, in particular, directing cellular behaviour on surfaces, and within tissue engineering scaffolds, but also, on the prevention of fouling of biomaterials via non-specific protein adsorption. The application of block copolymer self-assembly for biomaterial and tissue engineering systems described in this thesis has great potential as a platform technology for the investigation of fundamental cell-surface and protein-surface interactions as well as for use in existing and emerging biomedical applications.

The interaction of materials with biological systems is of critical importance to a vast number of applications from medical implants, tissue engineering scaffolds, blood-contacting devices, cell-culture products, as well as many other products in industries as diverse as agriculture. This thesis describes a method for the modification of biomaterial surfaces and the generation of tissue engineering scaffolds that utilises the self assembly of poly (styrene)-block-poly (ethylene oxide) (PS-PEO) block copolymers. Block copolymers consist of alternating segments of two or more chemically distinct polymers. The salient feature of these materials is their ability to self organise into a wide range of micro-phase separated structures generating patterned surfaces that have domain sizes in the order of 10-100nm. Further, it is also possible to specifically functionalise only one segment of the block copolymer, providing a means to precisely locate specific biological signals within the 10-100nm domains of a nano-patterned surface, formed via the programmed micro-phase separation of the block copolymer system. The density and spatial location of signalling molecules can be controlled by altering several variables, such as block length, block asymmetry, as well as processing parameters, providing the potential to authentically emulate the cellular micro to nano-environment and thus greatly improving on existing biomaterial and tissue engineering technologies. This thesis achieved several aims as outlined below; Developed methods to control the self-assembly of PS-PEO block copolymers and generate nano-patterned surfaces and scaffolds with utility for biomaterials applications. PS-PEO diblock copolymers were blended with polystyrene (PS) homopolymer and spin cast, resulting in the rapid self-assembly of vertically oriented PEO cylinders in a matrix of PS. Due to the kinetically constrained phase-separation of the system, increasing addition of homopolymer is shown to reduce the diameter of the PEO domains. This outcome provides a simple method that requires the adjustment of a single variable to tune the size of vertically oriented PEO domains between 10-100nm. Polymeric scaffolds for tissue engineering were manufactured via a method that combines macro-scale temperature induced phase separation with micro-phase separation of block copolymers. The phase behaviour of these polymer-solvent systems is described, and potential mechanisms leading to this spectacular structure formation are presented. The result is highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available technologies. Characterised the properties of these unique nano-structured materials as well as their interaction with proteinaceous fluids and cells. Nano-patterned PS-PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control PS surfaces. The adhesion of NIH 3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-islands, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface. In order to understand the mechanism by which these surfaces resisted protein and cellular adsorption we utilised neutron reflection to study their solvation and swelling properties. The results indicate that the PEO domains are highly solvated in water; however, the PEO chains do not extend into the solvent but remain in their isolated domains. The data supports growing evidence that the key mechanism by which PEO prevents protein adsorption is the blocking of protein adsorption sites. Control the nano-scale presentation of cellular adhesion and other biological molecules via the self-assembly of functionalised PS-PEO block copolymers Precise control over the nano-scale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, including differentiation and apoptosis. We present the self-assembly of maleimide functionalised PS-PEO copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By controlling the phase separation of the functional PS-PEO block copolymer we alter the nano-scale (on PEO islands of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from ~ 450 to ~ 900 islands per um2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between islands of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces were utilised to immobilise poly-histidine tagged proteins and ECM fragments. The technologies developed in this thesis aim to improve on several weaknesses of existing biomaterials, in particular, directing cellular behaviour on surfaces, and within tissue engineering scaffolds, but also, on the prevention of fouling of biomaterials via non-specific protein adsorption. The application of block copolymer self-assembly for biomaterial and tissue engineering systems described in this thesis has great potential as a platform technology for the investigation of fundamental cell-surface and protein-surface interactions as well as for use in existing and emerging biomedical applications.

The interaction of materials with biological systems is of critical importance to a vast number of applications from medical implants, tissue engineering scaffolds, blood-contacting devices, cell-culture products, as well as many other products in industries as diverse as agriculture. This thesis describes a method for the modification of biomaterial surfaces and the generation of tissue engineering scaffolds that utilises the self assembly of poly (styrene)-block-poly (ethylene oxide) (PS-PEO) block copolymers. Block copolymers consist of alternating segments of two or more chemically distinct polymers. The salient feature of these materials is their ability to self organise into a wide range of micro-phase separated structures generating patterned surfaces that have domain sizes in the order of 10-100nm. Further, it is also possible to specifically functionalise only one segment of the block copolymer, providing a means to precisely locate specific biological signals within the 10-100nm domains of a nano-patterned surface, formed via the programmed micro-phase separation of the block copolymer system. The density and spatial location of signalling molecules can be controlled by altering several variables, such as block length, block asymmetry, as well as processing parameters, providing the potential to authentically emulate the cellular micro to nano-environment and thus greatly improving on existing biomaterial and tissue engineering technologies. This thesis achieved several aims as outlined below; Developed methods to control the self-assembly of PS-PEO block copolymers and generate nano-patterned surfaces and scaffolds with utility for biomaterials applications. PS-PEO diblock copolymers were blended with polystyrene (PS) homopolymer and spin cast, resulting in the rapid self-assembly of vertically oriented PEO cylinders in a matrix of PS. Due to the kinetically constrained phase-separation of the system, increasing addition of homopolymer is shown to reduce the diameter of the PEO domains. This outcome provides a simple method that requires the adjustment of a single variable to tune the size of vertically oriented PEO domains between 10-100nm. Polymeric scaffolds for tissue engineering were manufactured via a method that combines macro-scale temperature induced phase separation with micro-phase separation of block copolymers. The phase behaviour of these polymer-solvent systems is described, and potential mechanisms leading to this spectacular structure formation are presented. The result is highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available technologies. Characterised the properties of these unique nano-structured materials as well as their interaction with proteinaceous fluids and cells. Nano-patterned PS-PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control PS surfaces. The adhesion of NIH 3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-islands, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface. In order to understand the mechanism by which these surfaces resisted protein and cellular adsorption we utilised neutron reflection to study their solvation and swelling properties. The results indicate that the PEO domains are highly solvated in water; however, the PEO chains do not extend into the solvent but remain in their isolated domains. The data supports growing evidence that the key mechanism by which PEO prevents protein adsorption is the blocking of protein adsorption sites. Control the nano-scale presentation of cellular adhesion and other biological molecules via the self-assembly of functionalised PS-PEO block copolymers Precise control over the nano-scale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, including differentiation and apoptosis. We present the self-assembly of maleimide functionalised PS-PEO copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By controlling the phase separation of the functional PS-PEO block copolymer we alter the nano-scale (on PEO islands of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from ~ 450 to ~ 900 islands per um2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between islands of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces were utilised to immobilise poly-histidine tagged proteins and ECM fragments. The technologies developed in this thesis aim to improve on several weaknesses of existing biomaterials, in particular, directing cellular behaviour on surfaces, and within tissue engineering scaffolds, but also, on the prevention of fouling of biomaterials via non-specific protein adsorption. The application of block copolymer self-assembly for biomaterial and tissue engineering systems described in this thesis has great potential as a platform technology for the investigation of fundamental cell-surface and protein-surface interactions as well as for use in existing and emerging biomedical applications.

Η συγκόλληση των αιμοπεταλίων προάγεται από το ινωδογόνο, μια εξωκυττάρια πρωτεΐνη, η οποία δεσμεύεται εκλεκτικά στον υποδοχέα GP IIb/IIIa. Το τριπεπτίδιο RGD (Arg-Gly-Asp) συνιστά τη μικρότερη αλληλουχία, η οποία είναι απαραίτητη για την αναγνώριση και πρόσδεση του ινωδογόνου στον υποδοχέα και απαντάται και σε άλλες συγκολλητικές πρωτεΐνες, οι οποίες είναι παρούσες στον εξωκυττάριο χώρο και στο αίμα, όπως η ινοσυνδετίνη, το κολλαγόνο, ο παράγοντας Von Willebrand, κτλ. Η αντιπηκτική θεραπεία έχει βασιστεί σε δύο διαφορετικές προσεγγίσεις του προβλήματος. Η μία προσέγγιση αφορά την εμπόδιση της πρωταρχικής διέγερσης των αιμοπεταλίων από διάφορους αγωνιστές, όπως θρομβίνη, επινεφρίνη, κολλαγόνο, κτλ. Η άλλη προσέγγιση περιλαμβάνει την διακοπή του μηχανισμού μεταγωγής σήματος, ο οποίος ακολουθεί την πρόσδεση του αγωνιστή στην επιφάνεια των αιμοπεταλίων. Η ασπιρίνη, παράγωγο του σαλικυλικού οξέος, αναστέλλει το πρώτο βήμα στη βιοσύνθεση της θρομβοξάνης Α2 από αραχιδονικό οξύ μέσω ακετυλίωσης του ενζύμου κυκλοοξυγενάση 1. Στην παρούσα διατριβή πραγματοποιήθηκε ο σχεδιασμός και η σύνθεση γραμμικών και κυκλικών αναλόγων του τριπεπτιδίου RGD με ενσωματωμένο σαλικυλικό οξύ ή παράγωγά του. Τα διάφορα ανάλογα συντέθηκαν με κλασικές μεθόδους πεπτιδικής σύνθεσης σε υγρή και στερεά φάση. Τη σύνθεση των αναλόγων ακολούθησε καθαρισμός τους (HPLC) και προσδιορισμός της δομής τους με (ESI-MS). Στη συνέχεια, προσδιορίστηκε in vitro με φωτομετρική μέθοδο στους 37C και συνεχή καταγραφή της διερχόμενης ακτινοβολίας με ειδικό όργανο (Dual Channel Aggregometer) η ανασταλτική τους δράση στη συγκολλητικότητα των αιμοπεταλίων του ανθρώπου. Προς περαιτέρω επιβεβαίωση των πειραμάτων συσσώρευσης και μελέτη της πρόσδεσης των αναλόγων στις ιντεγκρίνες χρησιμοποιήθηκε η κυτταρομετρία ροής με μονοκλωνικά αντισώματα έναντι των υποδοχέων Gp Ia, Gp IIb/IIIa, Gp IIIa και GMp 140. Αναλύοντας τα αποτελέσματα των βιολογικών μελετών, τόσο της αναστολής της συσσωμάτωσης των αιμοπεταλίων του ανθρώπου in vitro όσο και της κυτταρομετρίας ροής σε ενεργοποιημένα αιμοπετάλια για τα δραστικά πεπτίδια, οδηγούμαστε στα επόμενα συμπεράσματα: 1. Από τη σειρά των RGD γραμμικών αναλόγων που μελετήθηκαν, βρέθηκαν δραστικά μόνο στην περίπτωση που τα πεπτίδια έχουν στο C-τελικό τους άκρο αμίδιο. 2. Η σύζευξη του σαλικυλικού οξέος στο τριπεπτίδιο - αμίδιο RGD ενισχύει την αντισυγκολλητική του δράση έναντι των αιμοπεταλίων in vitro. Από αυτά τα ανάλογα 26 (IC50= 50μΜ), 27 (38μΜ) και 28 (53μΜ) (ενσωματωμένο σαλικυλικό οξύ στο τριπεπτίδιο) έχουν την ισχυρότερη δράση, ενώ μόνο το τριπεπτίδιο 23 έχει IC50= 540μΜ 3. Η προστασία του β-καρβοξυλίου του Asp με βενζυλομάδα αυξάνει τη δράση του πεπτιδίου σε σχέση με την ύπαρξη ελεύθερου β-καρβοξυλίου. Αυτό διαπιστώνεται από το γεγονός ότι όλα τα βιολογικώς δραστικά ανάλογα έχουν το β-καρβοξύλιο προστατευμένο με βενζυλομάδα και αυτό έρχεται σε συμφωνία με βιβλιογραφικά δεδομένα άλλων ερευνητών περί αναγκαιότητας ύπαρξης λιπόφιλης ομάδας στο C-τελικό άκρο του πεπτιδίου. 4. Αντίθετα, η ενσωμάτωση σαλικυλο-παραγώγων (βρώμο-, χλώρο-, νίτρο-, άμινο-, κτλ) στα ανάλογα δίνει πολύ μικρή αντισυγκολλητική δράση στα αιμοπετάλια του ανθρώπου in vitro σε σχέση με το σαλικυλικό οξύ. 5. Από τα συντεθέντα κυκλικά ανάλογα μόνο το ανάλογο 61, που φέρει δισουλφιδικό δεσμό μεταξύ της κυστεΐνης και του θειοσαλικυλικού οξέος, επέδειξε ισχυρή αντισυγκολλητική δράση έναντι των αιμοπεταλίων του ανθρώπου in vitro με τιμή IC50= 8μΜ, που είναι και η καλύτερη τιμή IC50 για όλα τα ανάλογα που συντέθηκαν (γραμμικά και κυκλικά). 6. Και στην περίπτωση των κυκλικών πεπτιδίων, τα ανάλογα με το προστατευμένο β-καρβοξύλιο εμφανίζουν ισχυρότερη ανασταλτική δράση έναντι εκείνων που φέρουν το β-καρβοξύλιο ελεύθερο. 7. Από όλα τα γραμμικά ανάλογα που περιέχουν παράγωγα του σαλικυλικού οξέος το ανάλογο 39 που περιέχει το 5-χλωρο σαλικυλικό οξύ εμφανίζει ισχυρή ανασταλτική δράση έναντι του υποδοχέα Gp Ib. 8. Τέλος, θα πρέπει να αναφερθεί ότι είναι η πρώτη φορά που συνθετικά πεπτιδικά ανάλογα του RGD εμφανίζουν ισχυρή πρόσδεση στον υποδοχέα Gp Ib, o οποίος ευθύνεται για την προσκόλληση των αιμοπεταλίων στο κυτταρικό τοίχωμα. / Integrins constitute a large family of heterodimeric cell-surface, transmembrane receptors, which play a major role in cell/cell and cell/matrix adhesive interactions. The Arg-Gly-Asp (RGD) sequence is known to be the integrin recognition site of many extracellular matrix proteins such as fibronectin, osteopontin, collagen, fibrinogen, von Willebrand factor, laminin, etc. On the other hand, it is well known that low doses of aspirin (acetyl salicylic acid) decrease platelet aggregation by causing an inhibitory effect on thromboxane A2 production by platelets. Several antiplatelet strategies have already been developed and are under preclinical or clinical investigation. In the present thesis, the synthesis of linear and cyclic RGD analogs incorporating salicylic acid derivatives is reported. The syntheses of the new analogs were carried out by using classic methods of peptide synthesis in liquid or solid phase. The synthesized compounds were purified by RP-HPLC and lyophilised to give fluffy solid, identified by ESI-MS spectra. These compounds were tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents to citrated platelet rich plasma (PRP). The aggregation was determined using a dual channel electronic aggregometer by recording the increase of light transmission. Their specificity for the Gp receptors was checked by using flow cytometry with monoclonal antibodies against Gp Ib, Gp IIb/IIIa, Gp IIIa and GMP140 receptors. Based on the results of the biological studies we could report the next inferences: 1. From the studied synthetic RGD analogs only peptides – amides are active against human platelet aggregation in vitro. 2. The coupling of salicylic acid with the RGD peptides enforces the antiplatelet activity in vitro of the single tripeptide. From the above peptides, the analog 26 (tripeptide incorporating salicylic acid) shows strong antiplatelet activity (IC50=50 μΜ), whereas the analog 23 (only tripeptide) has IC50= 540μΜ. 3. The protection of the β-carboxy group of Asp as benzylester increases the activity of the peptides in comparison with those having the β-carboxy group unprotected. Thus, our results ensure the theory of necessity of the existence a lipophile center on the C-terminal side of the peptide. 4. The incorporation of salicylic acid derivatives in the RGD peptide does not increase further the antiplatelet activity than the incorporation of salicylic acid does. 5. Among the cyclic RGD peptides only the analog 61, having the disulfide bridge between the cysteine and the thiosalicylic acid, shows strong antiplatelet activity in vitro (IC50= 8μΜ). 6. Most of the analogs show high binding affinity for the Gp Ib receptor. The cyclic analog 61 shows special selectivity for this receptor at concetrations of 110 μΜ. 7. The analog 39, although it shows low antiplatelet activity, has high binding affinity for the Gp Ib receptor. Probably, this activity is due to the atom of Cl at the 5 position of aromatic ring of salicylic acid. 8. According to the literature data, it is the first time that synthetic RGD peptides show strong binding affinity for the Gp Ib receptor, which is responsible for the platelet adhesion to the subenthothelium.

Made available in DSpace on 2016-06-02T20:21:18Z (GMT). No. of bitstreams: 1 DissCLSP.pdf: 1835751 bytes, checksum: 94d92e1d4b14f4f8befcd6cb0b350de1 (MD5) Previous issue date: 2006-11-23 / Universidade Federal de Sao Carlos / Disintegrins are snake venom protein, of low molecular weight, rich in cysteines and RGDcontaining peptides that bind specifically to integrins αIIbβ3, α5β1, and αvβ3 expressed on platelets, endothelial and tumor cells. The biological effects of these peptides are related with biological process of cellular adhesion where receptors called integrins are presents. This dissertation describes the optimization of purification and additional characterization of a recombinant RGD-disintegrin of Bothrops alternatus, DisBa-01.. The DisBa-01 is a recombinant RGD-disintegrin, which interacts with αIIbβ3 integrin, inhibiting platelet aggregation and proliferation of endothelial and some tumor cells. In this work, a new protocol of purification was proposed for most efficient purification of the DisBa-01. The recombinant disintegrin, DisBa-01, was expressed in an optimizedbacterial system (Escherichia coli BL21(DE3) pET28a+DisBa-01) and purified by affinity chromatography. In this new protocol, the product of the purification in nickel column is submitted ion exchange chromatography. The use of the ion exchange chromatography as second step for purification increased the purity degree and the protein yield. The purified protein had its N-Terminal portion sequenced in 20 amino acids residues and its molecular mass determined by mass spectrometry. The mass spectrometry assay confirmed the mass of the DisBa-01 as 11.658 Da. The RGD-disintegrin pure had the tag of polihistidinas removed with thrombin. The cleavage of the tag of histidinas with thrombin was efficient. Polyclonal antibodies against DisBa-01 have also been produced in mice. The reactivity of these antibodies was observed through imunoblotting. These results provided one better form of obtain the pure protein as well as significant additional information for a better characterization of recombinant RGDdisintegrin, DisBa-01, which could be useful for the study of RGD-disintegrin, recombinant or native, and integrin. / Desintegrinas são proteínas de veneno de serpentes, de baixo peso molecular, ricas em cisteína e em geral um peptídeo contendo o motivo RGD que liga especificamente a integrinas αIIbβ3, α5β1, e αvβ3 de plaquetas, células endoteliais e células tumorais. Os efeitos biológicos desses peptídeos estão relacionados com processos biológicos de adesão celular onde receptores denominados integrinas estão presentes. Esta dissertação descreve a otimização da purificação de uma desintegrina-RGD recombinante de Bothrops alternatus, DisBa-01, e a caracterização adicional desta proteína. A DisBa-01 é uma desintegrina-RGD recombinante que interage com a integrina αIIbβ3 inibindo agregação plaquetária e proliferação de células endoteliais e algumas células tumorais. Neste trabalho, um novo protocolo de purificação foi proposto para a purificação mais eficiente da DisBa-01. A desintegrina, DisBa-01, foi expressa em um sistema bacteriano otimizado (Escherichia coli BL21(DE3)pET28a+DisBa-01) e purificada em coluna de afinidade. Neste novo protocolo, o produto da purificação na coluna de níquel é submetido à cromatografia de troca-iônica. O uso da cromatografia de troca-iônica aumentou o grau de pureza e o produto protéico final. A proteína purificada teve o N-terminal seqüenciado em 20 resíduos de aminoácidos. A massa molecular foi determinada por espectrometria de massa que confirmou como 11.658 Da a massa da proteína. A desintegrina-RGD pura teve a cauda de polihistidinas eficientemente removida com proteólise com trombina. Anticorpos policlonais contra DisBa-01 foram produzidos em camundongos. A reatividade desses anticorpos foi observada através de imunoblotting. Estes resultados estabelecem uma nova forma de obtenção da proteína pura, assim como informações adicionais significativas de uma melhor caracterização da desintegrina-RGD recombinante, DisBa-01, que pode ser útil para o estudo de desintegrinas- RGD, recombinantes ou nativas, e interações com integrinas.

La recherche actuelle sur le cancer se tourne vers des « stratégies ciblées » afin de développer de nouvelles méthodes diagnostiques plus sensibles et performantes, ainsi que de nouvelles thérapies plus efficaces mais aussi mieux tolérées. Dans ce contexte, nos travaux sont consacrés à la conception de vecteurs synthétiques ciblant un récepteur cellulaire surexprimé par les tumeurs, l'intégrine alphaVbeta3. Ce ciblage permet de concentrer les drogues ou les éléments de détection au niveau tumoral. L'outil utilisé pour la construction chimique de nos vecteurs est un châssis décapeptidique cyclique RAFT (Regioselectively Addressable Functionalized Template) présentant deux domaines indépendants permettant de séparer les deux fonctions du vecteur. Sur un domaine, la fonction de ciblage est assurée par la présentation multivalente de ligands -RGD- spécifiques du récepteur. L'autre domaine du vecteur supporte les molécules d'intérêt à vectoriser : agents thérapeutiques pour limiter la prolifération du foyer malin ou agents de détection pour l'imagerie médicale.