Le sang est un fluide complexe mis en écoulement par la pompe très peu puissante qu'est le cœur (environ 1 W), dans un réseau branché de plusieurs milliers de kilomètres de vaisseaux. Pour que cela soit réalisable, il se peut que les propriétés mécaniques du sang contribuent à l'entretien de l'écoulement. Malgré le nombre important d'études sur la rhéologie du sang, sa viscoélasticité n'a jamais été caractérisée en cisaillement simple. Le rôle physiologique du caillot est, lui, d'éviter un épanchement excessif de sang en présence d'une brèche vasculaire. Une de ses fonctions principales est donc de résister aux contraintes générées par l'écoulement sanguin, c'est-à-dire d'avoir une résistance mécanique appropriée. Que ce soit pour la caractérisation mécanique du sang ou du caillot, le principal verrou est l'absence de méthode de mesure adaptée à un matériau peu consistant, et dont les propriétés mécaniques sont en évolution rapide. Il est donc nécessaire de produire une méthode de mesure adéquate, couplée à un système de mesure assez sensible. Dans ce travail, nous présentons la méthode de rhéométrie que nous avons développée dans ce but, baptisée Optimal Fourier Rheometry (OFR). Cette technique a été validée avec succès sur différents matériaux modèles de plus en plus complexes : une huile newtonienne, une gomme viscoélastique (PDMS), une suspension de micelles vermiformes (CpCl Nasal) et enfin un alginate dentaire tout au long de sa gélification. Nous montrons ainsi que l'OFR est une technique de mesure fonctionnelle, fiable et optimale temporellement. Elle permet le suivi de grandeurs mécaniques dont le temps caractéristique de mutation est très inférieur à la minute. En raison de la sédimentation des globules rouges, le sang est un fluide évoluant dans le temps. Par conséquent l'OFR est bien adaptée pour la mesure de ses propriétés viscoélastiques. Pour nous affranchir de la variabilité très importante du sang de témoins, nous avons balayé de façon systématique la concentration en les composants sanguins les plus abondants sur des suspensions de globules rouges lavés. De façon a priori surprenante, nous montrons qu'en présence de fibrinogène, le sang présente une élasticité importante, du même ordre de grandeur, voire plus grande que sa viscosité. Cette élasticité augmente avec la concentration en fibrinogène et l'hématocrite et provient du réseau percolé de globules rouges agrégés de dimension fractale 2.08 qui existe dans la suspension lorsqu'elle est peu cisaillée. L'OFR a également été appliquée au suivi de la coagulation activée par voie intrinsèque et extrinsèque. Cela a permis de montrer que le procédé d'activation n'avait d'effet que sur la cinétique de la réaction, mais que cela ne changeait pas les étapes mécaniques observées. L'OFR permet grâce à sa résolution fréquentielle élevée et son temps de mesure minimal, d'affirmer que le processus de coagulation du sang n'est pas une transition sol-gel. / Blood is a complex fluid set into flow by the heart, which is a very low power pump (approximately 1 W), in a connected network consisting of several thousand kilometers of vessels. To do so, it seems reasonable that the mechanical properties of blood contribute to the maintenance of the flow. In spite of the important number of studies on blood rheology, the viscoelasticity of blood has never been characterized in simple shear. The physiological role of the blood clot is to avoid an excessive effusion of blood in the presence of a vascular breach. Thus, it has to resist to the stress induced by the blood flow. So, one of its essential functions is this mechanical resistance. Whether it is for the mechanical characterization of the blood or the clot, the main obstacle is the absence of viscoelasticity measurement techniques adapted to a low viscosity material evolving rapidly in time. So, it is necessary to provide an adapted measurement method, coupled with a sensitive enough measurement. In this work, we present the new rheometry method we developed, named Optimal Fourier Rheometry (OFR) as it is optimal both in duration and signal to noise ratio This method was successfully validated on materials of increasing complexity: a Newtonian oil, a viscoelastic gum (PDMS), a suspension of wormlike micelles (CpCl Nasal) and a dental alginate during its gelation. Because of the sedimentation of red blood cells, the mechanical properties of blood are evolving in time. Consequently the use of the OFR is well suited for the measurement of its viscoelastic properties. A systematic scanning of the concentrations in the most abundant blood components added to washed blood allowed to highlight the most important parameters. Our results show that blood has a surprisingly large elasticity, which is of the same order of magnitude as the viscosity of the material. This elasticity increases with fibrinogen concentration and hematocrit. When these two parameters are in the physiological range, a percolated network of aggregated red blood cells exists in the suspension of fractal dimension 2.08. The, OFR was applied to the monitoring of blood clot formation. The activation by intrinsic and extrinsic pathway was used on whole blood. It showed that the process of activation affects only the kinetics of the reaction, but does not change the observed mechanical s. Due to its high frequency resolution and minimal measurement time, OFR shows that coagulation is not a gelation process.

Made available in DSpace on 2014-06-11T19:27:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-29Bitstream added on 2014-06-13T20:17:05Z : No. of bitstreams: 1 oliveira_gjpl_me_arafo.pdf: 645436 bytes, checksum: 36e8a12c99620e4820f3710a3bb8b0d3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Foram utilizados 60 dentes humanos nessa pesquisa. No primeiro experimento, 15 dentes extraídos por doença periodontal foram selecionados para avaliar a influência da irradiação com laser de Er,Cr: YSGG sobre a morfologia e adesão de células sanguíneas sobre as superfícies radiculares. As 60 amostras provenientes desses dentes foram divididos em 3 grupos de acordo com o tipo de tratamento aplicado: Grupo 1- RAR; Grupo 2- Irradiação com laser de Er,Cr: YSGG; Grupo 3- RAR e irradiação com o laser de Er,Cr: YSGG. 10 amostras de cada grupo foram avaliados quanto a adesão de elementos sanguíneos, e as outras 10 amostras foram avaliados quanto a morfologia da superfície radicular por MEV. Os testes de Kruskall-Wallis e Mann-Whitney foram utilizados para avaliar os resultados. Em relação à adesão de elementos sanguíneos, este estudo não demonstrou diferenças estatísticas entre os grupos (p=0.359), a análise morfológica demonstrou que as superfícies radiculares irradiadas com o laser de Er-Cr:YSGG foram mais rugosas que as do grupo controle (G2-G1: p=0.0003 e G3-G1: p=0.0003). No segundo experimento, 20 dentes foram utilizados para avaliar a influência do ângulo de irradiação do laser de Er,Cr:YSGG sobre a rugosidade e o desgaste das superfícies radiculares. Cada face proximal foi dividida em 3 áreas, sendo que a área superior foi tratada com raspagem e alisamento radicular, a área média não foi submetida a nenhum tipo de tratamento e a área inferior foi irradiada com o laser de Er,Cr:YSGG. Os dentes foram divididos em 4 grupos ,com 5 dentes cada, a depender da angulação da aplicação da irradiação do laser de Er,Cr:YSGG na área ( 30º, 45º, 60º, 90º). A rugosidade das áreas foram avaliadas através de um rugosímetro e posteriormente os dentes foram submetidos a processamento histológico... / 60 human teeth were used in that research. In the first experiment, 15 extracted teeth for periodontal disease were selected to evaluated the effect of Er,Cr:YSGG irradiation on root surfaces for adhesion of blood components and morphology. 60 root surface specimens were obtained by selecting four from each tooth. Samples were divided into three groups of 20 each, according to treatments. Group 1 (G1) was treated by scaling and root planing (SRP), Group 2 (G2) was irradiated by Er,Cr:YSGG laser and Group 3 (G3) was treated by SRP and Er,Cr:YSGG laser irradiation. Blood was placed on each of 10 specimens from each of the three groups, to evaluate adhesion of blood components to the root surfaces. A morphological analysis was made of the root surfaces of the other 10 specimens from each group by scanning electron microscope (SEM). Statistical processing was done with the Kruskal-Wallis and Mann-Whitney tests. No statistical differences for adhesion of blood components to root surfaces were found between the groups (p = 0.359). However, morphological analysis disclosed that all root surfaces irradiated by Er,Cr:YSGG laser (100%) were rougher than surfaces that were not irradiated (G1-G2: p = 0.0003 and G1-G3: p = 0.0003). In the second experiment, 20 teeth were used to evaluated the effect of the working tip angulations on root roughness and substance removal using Er,Cr:YSGG radiation. The distal and mesial surfaces of each tooth was divided in 3 areas. The upper area was treated with scaling and root planing. The medium area was not submitted to any treatment and the lower area was irradiated with Er,Cr:YSGG laser. The teeh were divided in 4 groups, with 5 teeth each depending the working tip angulations using Er,Cr:YSGG at the lower area (30º, 45º, 60º, 90º).The roughness surfaces were evaluated by a profilometer, and... (Complete abstract, click electronic access below)

Revascularização é uma técnica utilizada em dentes jovens, que apresentem rizogênese incompleta e danos irreversíveis ao tecido pulpar, necessitando de tratamento endodôntico, para formar novo tecido em lugar da polpa perdida. Clinicamente os resultados mostram a continuidade da rizogênese e a devolução da vitalidade dental. Porém, pouco se sabe sobre o novo tecido formado e não está estabelecido se este é capaz de desempenhar todas as funções da polpa dentária. Para melhorar as características do tecido formado pela técnica da revascularização, podemos utilizar ferramentas de engenharia tecidual, como célulastronco, fatores de crescimento e arcabouços de sustentação celular (scaffolds). As célulastronco (CTs) já estão presentes quando o sangue invade o canal radicular, e utilizar essa reserva de CTs que o hospedeiro possui, procedimento conhecido como homing, é uma vantagem em comparação com outras técnicas que injetam CTs obtidas por cultivo em laboratório. Entretanto os aspectos físicos do coágulo sanguíneo formado no interior do canal radicular podem ser melhorados com a adição de hidrogel de quitosana, que interage quimicamente com o sangue e forma um scaffold híbrido mais estável. Então, o objetivo deste estudo foi testar a hipótese de que o scaffold híbrido, composto por hidrogel de quitosana e sangue, ofereceria maior estabilidade física estrutural, bem como condições favoráveis à adesão e proliferação de células-tronco da papila apical humana (SCAPs; do inglês, Stem Cell from Apical Papila). Para isso, investigamos in vitro se a incorporação do sangue ao hidrogel de quitosana gera um scaffold mais estável, se este é favorável à adesão e proliferação de células-tronco da papila apical e se a fotobiomodulação potencializa essas características celulares. Para isso, SCAPs foram isoladas e caracterizadas por citometria de fluxo, tempo de dobra populacional, e contagem de unidades formadoras de colônias fibroblásticas (CFU-F; do inglês, Colony Forming Units - Fibroblastic). Ensaios de incorporação sanguínea, dissolução e embebição foram realizados para determinar o comportamento dos scaffolds híbridos. A adesão celular foi observada pela coloração PHK26® (do inglês, Red Fluorescent Cell Linker) e por microscopias eletrônicas de varredura (MEV); e a proliferação foi investigada pelo ensaio de alamarBlue®. Adicionalmente, a sobrevivência das SCAPs após a degradação do scaffold híbrido foi avaliada pela coloração Live/Dead®. A população celular estudada apresentou características de células tronco. O scaffold híbrido, constituído de densa rede alveolar com poros interconectados, embebeu e dissolveu rapidamente. De acordo com o PKH26® e alamarBlue® SCAPs aderiram e proliferaram no scaffold híbrido. SCAPs fotobiomoduladas exibiram maior taxa de proliferação e o ensaio Live/Dead® mostrou células vivas após 12 dias de cultivo. Concluiu-se que o scaffold híbrido apresenta biocompatibilidade e condições favoráveis de sobrevivência para SCAPs, que são potencializadas pela fotobiomodulação. / Revascularization is a technique used to form a new tissue, replacing the lost pulp, in young permanent teeth presenting incomplete rhizogenesis and irreversible damage, where endodontic treatment is needed. Clinically, the results show the continuity of the root formation and the return of dental vitality. However, little is known about the newly formed tissue and it has not been established if it is able to perform all functions of the dental pulp. To improve the characteristics of the newly formed tissue by the technique of revascularization, tissue engineering tools can be used, represented by stem cells, growth factors and scaffolds for cell supportting. Stem cells (SCs) are already present when the blood invades the root canal, and to use this SCs reserve that the host possesses, procedure known as homing, is an advantage compared with other techniques that inject SCs obtained by cultivation in the laboratory. However the physical aspects of blood clot in the root canal can be improved with the addition of chitosan hydrogel that chemically interacts with the blood and forms a more stable hybrid scaffold. So the aim of this study was to test the hypothesis that the hybrid scaffold, composed of hydrogel chitosan and blood clot, provides greater structural physical stability as well as favorable conditions for adhesion and proliferation of Stem Cells from Apical Papilla (SCAPs). For this, we investigated in vitro if the incorporation of blood to chitosan hydrogel, generates a more stable scaffold and if it supports the stem cell adhesion and proliferation, in addition, if photobiomodulation potentiates these cell characteristics. For this, SCAPs were isolated and characterized by flow cytometry, population doubling time, and counting colony forming units - fibroblastic (CFUF). Blood incorporation assays, dissolution and swelling were conducted to determine the behavior of hybrid scaffolds. Cell adhesion was observed by PHK26® (Red Fluorescent Cell Linker) and scanning electron microscopy (SEM); and proliferation was investigated by alamarBlue® assay. In addition, the survival of SCAPs after degradation of the scaffold was assessed by Live/Dead® staining. The cell population showed stem cell characteristics. The hybrid scaffold, constituted of dense cellular network with interconnected pores, soaked and dissolved quickly. According to PKH26® and alamarBlue® assays, the SCAPs adhered and proliferated in the hybrid scaffold. Photobiomodulation leads to SCAPs higher proliferation rate and the Live/Dead® test showed live cells after 12 days of cultivation. It was concluded that the hybrid scaffold is biocompatible and favors survival of SCAPs, which was enhanced by photobiomodulation.

OBJECTIVES: Clot in transit represents a life-threatening manifestation of venous thromboembolism of which we have limited understanding. This study was designed to describe the risk factors, clinical characteristics and outcomes associated with the development of a clot in transit as well as death within clot in transit patients. METHODS: We enrolled 1,093 consecutive patients into our single-center Pulmonary Embolism Response Team Registry. We compared 76 patients who had a clot in transit to 589 pulmonary embolism patients who did not have a clot in transit. RESULTS: Clot in transit was present in 11.4% of patients who received an echocardiogram to look for it. Multivariate analysis showed congestive heart failure (OR 2.954, 95% CI 1.349 – 6.467, P = 0.0068), a pre-existing inferior vena cava filter (OR 2.777, 95% CI 1.204 – 6.407, P = 0.0167), and hemodynamic collapse (OR 3.495, 95% CI 1.129 – 10.823, P = 0.0300) to be independent predictors of clot in transit. All-cause mortality by 30 days was higher in clot in transit patients (24.3% vs 9.7%, P < 0.001). All-cause mortality by 7 days within clot in transit patients was associated with hemodynamic collapse (45.5% vs 12.3%, P = 0.018) and mental status change (63.6% vs 21.5%, P = 0.008). CONCLUSIONS: The presence of congestive heart failure, a pre-existing inferior vena cava filter, and hemodynamic collapse are independent predictors of clot in transit and should alert physicians to patients who may require an echocardiogram. The mortality for clot in transit is high even when compared to a more severe pulmonary embolism population. Clot in transit represents a high-risk finding that may require more aggressive interventions. / 2020-06-14T00:00:00Z

No description available.

Haemostasis is a complex physiological process involving cellular and plasma protein components that interact to keep the blood fluid under normal conditions and prevent blood loss after vessel injury by promoting clot formation. Primary haemostasis encompasses the activation and aggregation of platelets and is supported by secondary haemostasis, in which the coagulation factors of the plasma interact in a complex series of reactions. Secondary haemostasis is initiated by the exposure of tissue factor (TF) to the blood after vessel injury. TF forms a complex with activated factor VII (FVIIa), which in turn activates factor X (FXa) and ultimately results in fibrin formation. The TF-FVIIa complex and FXa are tightly regulated by tissue factor pathway inhibitor (TFPI), a trivalent Kunitz-type protease inhibitor. The endothelium, consisting of endothelial cells (ECs), constitutes the inner lining of all blood vessels. As such, it is in constant contact with the blood and plays a major role in haemostasis by synthesising and storing both pro- and anti- coagulant substances, including TF and TFPI. Release of TFPI from ECs is increased after exposure to both unfractionated and low molecular weight heparins, though the mechanisms are not clearly defined. TFPI circulates in plasma, predominantly bound to lipoproteins, though the effect of the three major lipoproteins [low density (LDL), very low density (VLDL) and high density (HDL)] on the release of TFPI from ECs is not well established. Furthermore, previous studies have not systematically investigated the effect of these lipoproteins on both TF and TFPI. The initial aim of this project was to establish assays for the measurement of TF activity and TFPI antigen to supplement the TFPI activity assay that is well established in our laboratory. / These assays were then used to determine the effects of heparin and the major lipoproteins on the expression of TF and the release of TFPI on/from ECs. Human umbilical vein endothelial cells (HUVECs) were used as the EC model because their collection and isolation is well established and they have biochemical and physiological properties representative of in vivo conditions. A TF activity assay, based on a previously published method, was successfully modified and validated for the measurement of cell surface TF (standard curve R2 = 0.997). Despite exhaustive attempts, adaptation of this assay for plasma TF was unsuccessful, raising doubts regarding the plasma fractionation procedure of the originally published assay [Fukuda, C., Iijima, K. and Nakamura, K. (1989). "Measuring tissue factor (factor III) activity in plasma." Clinical Chemistry 35(9): 1897‐1900]. A novel insect cell expression system was used to produce well defined recombinant TFPI standards for use in TFPI activity and antigen assays. For the first time, truncated TFPI variants, containing the first Kunitz domain only, the first and second Kunitz domains only, and the first through third Kunitz domains minus the carboxyl terminus, were successfully produced in insect cells, though the full length molecule was not. Possible reasons for this include codon bias, protein instability and/or the signal peptide used. An ELISA to measure TFPI antigen was designed using a monoclonal anti‐TFPI antibody directed against the N‐terminus for protein capture and a polyclonal anti‐ TFPI antibody for detection. The assay was successfully optimised (standard curve R2 = 0.978, intra‐assay CV = 4.8%), however it produced inaccurate results (normal range = 498.7 ± 156.3 ng/mL), probably due to the antibody combination used. / TF and TFPI activity assays were used to determine the effect of both unfractionated and low molecular weight heparins (UFH and LMWH, respectively) on the release of TFPI and the expression of TF from/on ECs. A significant increase in the secretion of functional TFPI from ECs due to heparin (0 U/ml vs 1 and 10 U/mL) was demonstrated only in the presence of serum (UFH: 9.0 mU/mL vs 18.3 and 18.4 mU/mL, p < 0.0001; LMWH: 8.8 mU/mL vs 13.3 and 21.4 mU/mL, p < 0.05), suggesting, for the first time, that a component of serum is required for the heparin‐dependent release of TFPI. The effect of LDL, VLDL and HDL on the release of TFPI and the expression of TF from/on ECs was also investigated. All three lipoprotein fractions increased the secretion of functional TFPI after one hour incubation (LDL: 12.5 μg/mL, p < 0.01; 25 μg/mL, p < 0.05; VLDL: 50 μg/mL, p < 0.01; HDL: 50 μg/mL, p < 0.05). This is the first data to demonstrate a HDL‐dependent increase in released TFPI. After 24 hours, both LDL and VLDL decreased levels of secreted functional TFPI (LDL: 25 μg/mL, p < 0.01; 50 μg/mL, p < 0.01; VLDL: 12.5 μg/mL, p < 0.01), probably due to the oxidation and subsequent association of both lipoprotein species with TFPI. Surprisingly, both LDL and VLDL decreased cell surface TF, though this effect was not dose dependent. These results suggest that the major lipoproteins have a short term anticoagulant effect which is reversed in the longer term due to lipid oxidation. In summary, this thesis describes the successful adaptation of a chromogenic assay for the measurement of cell surface TF activity and the production of truncated TFPI variants. / Both will be used for the measurement of TF and TFPI, their association with thrombus formation and propagation, and investigations into potential therapeutic applications of TFPI. The results presented in this thesis extend the current knowledge on the expression and release of TF and TFPI on/from ECs by heparin, highlighting the importance of serum in the heparin dependent release of TFPI in vitro. Furthermore, it describes for the first time the effects of the major lipoprotein fractions on TFPI release and TF expression. The data support novel mechanisms by which LDL and VLDL are procoagulant, and HDL anticoagulant. This study provides a foundation for future research of the TF pathway in cellular models, which is critical in increasing the understanding of the pathogenesis and treatment of thrombotic disease. vitro. Furthermore, it describes for the first time the effects of the major lipoprotein fractions on TFPI release and TF expression. The data support novel mechanisms by which LDL and VLDL are procoagulant, and HDL anticoagulant. This study provides a foundation for future research of the TF pathway in cellular models, which is critical in increasing the understanding of the pathogenesis and treatment of thrombotic disease.

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.

Orientador: Rosemary Adriana Chiérici Marcantonio / Banca: Márcio Zafalon Casati / Banca: Estela Sasso Cerri / Resumo: Foram utilizados 60 dentes humanos nessa pesquisa. No primeiro experimento, 15 dentes extraídos por doença periodontal foram selecionados para avaliar a influência da irradiação com laser de Er,Cr: YSGG sobre a morfologia e adesão de células sanguíneas sobre as superfícies radiculares. As 60 amostras provenientes desses dentes foram divididos em 3 grupos de acordo com o tipo de tratamento aplicado: Grupo 1- RAR; Grupo 2- Irradiação com laser de Er,Cr: YSGG; Grupo 3- RAR e irradiação com o laser de Er,Cr: YSGG. 10 amostras de cada grupo foram avaliados quanto a adesão de elementos sanguíneos, e as outras 10 amostras foram avaliados quanto a morfologia da superfície radicular por MEV. Os testes de Kruskall-Wallis e Mann-Whitney foram utilizados para avaliar os resultados. Em relação à adesão de elementos sanguíneos, este estudo não demonstrou diferenças estatísticas entre os grupos (p=0.359), a análise morfológica demonstrou que as superfícies radiculares irradiadas com o laser de Er-Cr:YSGG foram mais rugosas que as do grupo controle (G2-G1: p=0.0003 e G3-G1: p=0.0003). No segundo experimento, 20 dentes foram utilizados para avaliar a influência do ângulo de irradiação do laser de Er,Cr:YSGG sobre a rugosidade e o desgaste das superfícies radiculares. Cada face proximal foi dividida em 3 áreas, sendo que a área superior foi tratada com raspagem e alisamento radicular, a área média não foi submetida a nenhum tipo de tratamento e a área inferior foi irradiada com o laser de Er,Cr:YSGG. Os dentes foram divididos em 4 grupos ,com 5 dentes cada, a depender da angulação da aplicação da irradiação do laser de Er,Cr:YSGG na área ( 30º, 45º, 60º, 90º). A rugosidade das áreas foram avaliadas através de um rugosímetro e posteriormente os dentes foram submetidos a processamento histológico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 60 human teeth were used in that research. In the first experiment, 15 extracted teeth for periodontal disease were selected to evaluated the effect of Er,Cr:YSGG irradiation on root surfaces for adhesion of blood components and morphology. 60 root surface specimens were obtained by selecting four from each tooth. Samples were divided into three groups of 20 each, according to treatments. Group 1 (G1) was treated by scaling and root planing (SRP), Group 2 (G2) was irradiated by Er,Cr:YSGG laser and Group 3 (G3) was treated by SRP and Er,Cr:YSGG laser irradiation. Blood was placed on each of 10 specimens from each of the three groups, to evaluate adhesion of blood components to the root surfaces. A morphological analysis was made of the root surfaces of the other 10 specimens from each group by scanning electron microscope (SEM). Statistical processing was done with the Kruskal-Wallis and Mann-Whitney tests. No statistical differences for adhesion of blood components to root surfaces were found between the groups (p = 0.359). However, morphological analysis disclosed that all root surfaces irradiated by Er,Cr:YSGG laser (100%) were rougher than surfaces that were not irradiated (G1-G2: p = 0.0003 and G1-G3: p = 0.0003). In the second experiment, 20 teeth were used to evaluated the effect of the working tip angulations on root roughness and substance removal using Er,Cr:YSGG radiation. The distal and mesial surfaces of each tooth was divided in 3 areas. The upper area was treated with scaling and root planing. The medium area was not submitted to any treatment and the lower area was irradiated with Er,Cr:YSGG laser. The teeh were divided in 4 groups, with 5 teeth each depending the working tip angulations using Er,Cr:YSGG at the lower area (30º, 45º, 60º, 90º).The roughness surfaces were evaluated by a profilometer, and... (Complete abstract, click electronic access below) / Mestre

The impact storage has on function of platelet concentrates is not completely known, although some factors have been discovered and measures have been taken to counteract them, such as adding platelet additive solution. There are several methods for analysing platelet function. In this study, the aim was to analyse change of platelet function in platelet concentrates over time and to see what effect fibrogen has on the coagulation. A technique using free oscillation rheometry (FOR), ReoRox®, was used to analyse function in platelet concentrates, both over time and after addition of fibrinogen. The platelets were analyzed at a concentration of 800 x109 Ptl/L and activated with thrombin receptor antigen peptide (TRAP). For fibrinogen efect analysis, four different concentrations were used, 10 g/L, 2,25 g/L, 1,0 g/L and 0,1 g/L. The results showed no statistically significant change in the function over time. However an increase in elasticity and decrease in the decline of elasticity could be seen. While analysing the platelets with fibrinogen it showed that up to 2,25 g/L the aggregation increased, while it decreased significantly at 10 g/L. In conclusion, the platelet concentrates retained a good clotting function from day one to day seven of storage, while the clot became stronger and fibrinolysis decreased. Fibrinogen proved important for coagulation, however a too high concentration inhibits coagulation.

Ventricle Assist Devices (VADs), which are typically either axial or centrifugal flow pumps implanted on the aortic arch, have been used to support patients who are awaiting cardiac transplantation. Success of the apparatus in the short term has led to long term use. Despite anticoagulation measures, blood clots (thrombi) have been known to form in the device itself or inside of the heart. The Ventricle Assist Devices supply blood flow via a conduit (cannula) implanted on the ascending aorta. Currently, the implantation angle of the VAD cannula is not taken into consideration. Since the VADs supply a significant amount of blood flow to the aorta, the implantation angle can greatly affect the trajectory of the formed thrombi as well as the cardiac flow field inside of the aortic arch. This study aims to vary the implantation angle of a pediatric Left Ventricle Assist Device (LVAD) through a series of computational fluid dynamics (CFD) software simulations focusing on the aortic arch and its branching arteries of a 20 kg pediatric patient in order to reduce the occurrence of stroke.