Les anticorps anti-phospholipides (aPL), tels que les anticoagulants lupiques (LAC), sont associés au développement récurrent de thromboses chez les patients atteints du lupus érythémateux disséminé (LED). Il a été observé que des titres élevés d’auto-anticorps antilamine B1 (anti-LB1), chez des patients porteurs de LAC, diminuent le risque de ces manifestations thrombotiques. Toutefois, la relation existant entre la lamine B1 (LB1), les anti-LB1 et la thromboprotection n’est toujours pas expliquée. Dans cette étude, nous avons donc cherché à comprendre comment la LB1 et les anti-LB1 induisent cette thromboprotection. Nous avons testé les effets d'anti-LB1 purifiés et de LB1 recombinante sur l'activation des cellules endothéliales et des plaquettes. Nous avons été en mesure de déterminer que la LB1, contrairement aux anti-LB1, possède une activité anti-plaquettaire. En effet, la LB1 réduit l’activation et l’agrégation plaquettaires in vitro et in vivo. Cette activité est due à une liaison directe de la LB1 aux plaquettes, suivie par une internalisation rapide dans des vésicules de clathrine. Par co-immunoprécipitation, nous avons découvert que la LB1 interagit avec le récepteur de l’insuline situé sur la membrane plaquettaire. La liaison de la LB1 à ce récepteur entraîne vraisemblablement son internalisation et l'inhibition d'une des cascades de signalisation normalement induite par le récepteur de l’insuline, menant éventuellement à l’inhibition des fonctions plaquettaires. L’ajout d’anti-LB1 purifiés dans nos expériences a permis d'augmenter de façon significative la persistance de la LB1 dans les plaquettes, une observation confirmée par la détection de LB1 uniquement dans les lysats de plaquettes prélevées chez des patients anti-LB1 positifs. iv Nos résultats suggèrent que la LB1 prend part aux mécanismes régulateurs des processus d’hémostase chez des sujets sains et que la présence d’anti-LB1, chez les patients lupiques, prolonge la persistance de cet auto-antigène dans les plaquettes, les empêchant ainsi de s’activer. Ce mécanisme expliquerait la diminution du risque de thrombose chez les patients LAC positifs porteurs d’anti-LB1 circulants. / Anti-phospholipid antibodies such as lupus anticoagulant antibodies (LAC) are associated with recurrent thrombotic events in systemic lupus erythematosus (SLE) patients. However, the risk of thrombosis in LAC positive patients is markedly reduced in the presence of high titers of autoantibodies to lamin B1 (anti-LB1). To date, the implication of lamin B1 (LB1) and anti-LB1 in thromboprotection remains unclear. Our objective was to examine the mechanism whereby LB1 and anti-LB1 induced thromboprotection. Functional platelet and endothelial cell activation assays were used to determine the effects of recombinant LB1 and affinity purified anti-LB1 on these two cell types. LB1, contrarily to anti-LB1, was found to possess an intrinsic anti-platelet activity. This protein reduced the activation and aggregation of platelets in vitro and in vivo. This activity was likely due to the direct binding of LB1 to platelets, followed by its rapid internalization within clathrin coated-pits. Coimmunoprecipitation revealed that LB1 interacted with the insulin receptor located within the platelet membrane. The binding of LB1 to this receptor induced its internalization and inhibited at least one of the phosphorylation cascade stimulated by the receptor, which in turn inhibited platelet functions. The addition of affinity-purified anti-LB1 in our model markedly increased the persistence of LB1 within platelets, a finding supported by the detection of LB1 only in platelets from anti-LB1 positive SLE patients. Our results suggest that LB1 regulates haemostasis in normal subjects. The presence of anti-LB1 in SLE patients prolongs the persistence of LB1 within platelets, thus possibly vi preventing further platelet activation. This mechanism likely explains the reduced risk of thrombotic events in LAC positive SLE patients with circulating anti-LB1 autoantibodies.

L'usage des pesticides au sein de l'Union européenne est de plus en plus réglementé, et les rodonticides actuellement disponibles sur le marché sont responsables de nombreuses intoxications secondaires chez les prédateurs des rongeurs. Il est donc crucial aujourd'hui de trouver une alternative plus écologique aux molécules commerciales. Les travaux de recherches décrits dans cette thèse s'inscrivent dans ce contexte et présentent la mise au point de nouvelles voies d'accès à des structures coumariniques et leurs études biologiques. La molécule anticoagulante ciblée se devait d'être active sur rats sensibles et résistants, et d'avoir une rémanence faible dans l'organisme. Les synthèses chimiques ont été menées conjointement avec les tests biologiques, conduisant l'ensemble des études de façon convergente vers la production d'un lead. Trois nouveaux outils moléculaires ont été mis au point et ont permis la synthèse et l'évaluation d'un grand nombre de candidats. Les deux premières en catalyse homogène, sous micro-onde, ont permis de réduire le temps réactionnel nécessaire à la synthèse de 4-hydroxycoumarine substituée sur le carbone 3. La troisième méthodologie conduit par une approche séquentielle aux mêmes types de composés à l'échelle du gramme. Cette dernière ouvre également la porte à de nombreuses possibilités réactionnelles permettant d'envisager plus de diversité. Toutes les molécules ont été évaluées in vitro, sur différentes souches d'enzymes VKORC1 et ont offert une meilleure compréhension des interactions enzyme/inhibiteur. Après cette première évaluation, des tests in vivo ont été conduits sur une sélection de composés, et ont apporté des informations cruciales sur les relations structure/activité in vivo et structure/rémanence. Le meilleur composé synthétisé à ce jour semble répondre parfaitement aux différentes contraintes liées au cahier des charges établi initialement qui se basait sur une approche singlefeeding. Une stratégie multifeeding est aujourd'hui envisagée afin de mieux correspondre à la réalité du terrain. Sur la base de celle-ci, le nombre de composés décrit dans ce manuscrit potentiellement utilisable en tant que rodonticide se retrouve largement augmenté / To reduce the ecological impact of pesticides in UE many new legislations were put in place, in other hand, most of secondary intoxications of rodent's predators are due to rodenticides available on the market. That why it’s crucial to find alternative rodenticide more eco-friendly. This work describes optimization of new coumarinics compounds synthesis and their biological studies. The new anticoagulant should be active on wild and mutant rat, and must have a low hepatic persistence in the rat body. Organic syntheses were driven with biological studies and have converged to discover the lead. Three different new molecular tools were optimized and have allowed the synthesis and the evaluation of a large number of candidates. The first two through homogeneous catalysis by using micro-waves have reduced the time needed for the alkylation of 4-hydroxycoumarin on the carbon 3. The third methodology allows the synthesis of same kind of compounds in large scale. This methodology opens news potentials reactions to add structural diversity. All the molecules were evaluated in vitro on different types of VKORC1 and have participated to a better understanding of the enzyme/inhibitor interactions. After this first evaluation, in vivo tests were performed on a selection of candidates, and have brought a crucial structural relationship between structure and in vivo persistence/activity. The best compound produced by now seems to answer at all specifications established linked to the single-feeding strategy. Multiple-feeding strategy is today planned to better correspond to the field reality. On the base of this one the number of candidates usable as rodenticides is increased

A ce jour, les anticoagulants commerciaux souffrent de deux inconvénients majeurs : leur rémanence avec, pour certains d’entre eux, une demi-vie hépatique proche des 300 jours causant des intoxications secondaires sur les prédateurs des rongeurs, ainsi que le développement de nombreuses mutations génétiques causé par l’utilisation intensive de ces composés, rendant inopérant l’utilisation de certains AVKs commerciaux. Face à ce constat, l’Union Européenne envisage d’interdire l’utilisation de tels composés. La mission prioritaire est donc de trouver un anticoagulant capable de gérer les populations de rongeurs sans affecter leurs prédateurs. Les recherches mises en avant par Adrien Montagut (Thèse 2011-2014) ont permis d’aboutir à une structure type d’anticoagulants, dérivés de la 4-hydroxycoumarine. Actuellement, AMR361 a été testé in vitro sur l’ensemble des mutations de VKORC1 et in vivo sur rats sauvages, et constitue le premier AVK développé qui répond à l’ensemble des caractéristiques du cahier des charges initial. La première partie de mon projet de thèse consistait à compléter l’étude biologique sur le noyau 4-hydroxycoumarine en amenant de la diversité fonctionnelle sur la position para du noyau aromatique. D’un point de vue biologique, l’allongement du bras espaceur sur la chaîne latérale par l’utilisation de fonctions telles que les amides ou amides inversés ou l’introduction d’un groupement diméthyle sur le pont méthylène ont été étudiés afin d’analyser les paramètres d’efficacité et de rémanence. Cependant, la plupart des composés synthétisés appartenant à la famille des 4-hydroxycoumarines font déjà l’objet d’un brevet déposé par l’entreprise Liphatech en 1999. L’étude de nouveaux noyaux, dont certains sont analogues à la 4-hydroxycoumarine, de même que la fonctionnalisation du noyau 4-hydroxycoumarine sur la partie aromatique, a permis l’accès à des structures plus diverses. Ces perspectives originales pour l’innovation ont été introduites pour contourner les brevets déjà existants. / To date, commercial anticoagulants suffer from two major inconveniences: their persistence causing secondary poisoning of rodent predators and the development of many genetic mutations caused by the intensive use of these compounds. As a result, the European Union plans to prohibit the use of such compounds. Consequently, the priority task is to find an anticoagulant that can control the rodent populations without affecting their predators. The research of Dr. Adrien Montagut (PhD, 2011-2014) have led to the structure type of an anticoagulant derived from 4-hydroxycoumarin. Currently, AMR361 was tested in vitro on all VKORC1 mutations and in vivo on wild rats. It is the first AVK developed that responds to all the characteristics of the initial specification. The first part of my PhD was to complete the biological study on 4-hydroxycoumarin core by bringing functional diversity on the para position of the aromatic ring. From a biological point of view, the lengthening of the spacer arm on the side chain by use of various functions or the introduction of a dimethyl group on the methylene bridge were studied in order to analyze the effectiveness and persistence parameters. However, most of the synthesized compounds belonging to the family of 4-hydroxycoumarins are already described in a patent filed by Liphatech company in 1999. The study of new cores which are similar to the 4-hydroxycoumarin or the functionalization of the aromatic part of the 4-hydroxycoumarin has provided access to more diverse structures. These original possibilities for innovation have been introduced to circumvent existing patents.

Les anticorps anti-phospholipides (aPL), tels que les anticoagulants lupiques (LAC), sont associés au développement récurrent de thromboses chez les patients atteints du lupus érythémateux disséminé (LED). Il a été observé que des titres élevés d’auto-anticorps antilamine B1 (anti-LB1), chez des patients porteurs de LAC, diminuent le risque de ces manifestations thrombotiques. Toutefois, la relation existant entre la lamine B1 (LB1), les anti-LB1 et la thromboprotection n’est toujours pas expliquée. Dans cette étude, nous avons donc cherché à comprendre comment la LB1 et les anti-LB1 induisent cette thromboprotection. Nous avons testé les effets d'anti-LB1 purifiés et de LB1 recombinante sur l'activation des cellules endothéliales et des plaquettes. Nous avons été en mesure de déterminer que la LB1, contrairement aux anti-LB1, possède une activité anti-plaquettaire. En effet, la LB1 réduit l’activation et l’agrégation plaquettaires in vitro et in vivo. Cette activité est due à une liaison directe de la LB1 aux plaquettes, suivie par une internalisation rapide dans des vésicules de clathrine. Par co-immunoprécipitation, nous avons découvert que la LB1 interagit avec le récepteur de l’insuline situé sur la membrane plaquettaire. La liaison de la LB1 à ce récepteur entraîne vraisemblablement son internalisation et l'inhibition d'une des cascades de signalisation normalement induite par le récepteur de l’insuline, menant éventuellement à l’inhibition des fonctions plaquettaires. L’ajout d’anti-LB1 purifiés dans nos expériences a permis d'augmenter de façon significative la persistance de la LB1 dans les plaquettes, une observation confirmée par la détection de LB1 uniquement dans les lysats de plaquettes prélevées chez des patients anti-LB1 positifs. iv Nos résultats suggèrent que la LB1 prend part aux mécanismes régulateurs des processus d’hémostase chez des sujets sains et que la présence d’anti-LB1, chez les patients lupiques, prolonge la persistance de cet auto-antigène dans les plaquettes, les empêchant ainsi de s’activer. Ce mécanisme expliquerait la diminution du risque de thrombose chez les patients LAC positifs porteurs d’anti-LB1 circulants. / Anti-phospholipid antibodies such as lupus anticoagulant antibodies (LAC) are associated with recurrent thrombotic events in systemic lupus erythematosus (SLE) patients. However, the risk of thrombosis in LAC positive patients is markedly reduced in the presence of high titers of autoantibodies to lamin B1 (anti-LB1). To date, the implication of lamin B1 (LB1) and anti-LB1 in thromboprotection remains unclear. Our objective was to examine the mechanism whereby LB1 and anti-LB1 induced thromboprotection. Functional platelet and endothelial cell activation assays were used to determine the effects of recombinant LB1 and affinity purified anti-LB1 on these two cell types. LB1, contrarily to anti-LB1, was found to possess an intrinsic anti-platelet activity. This protein reduced the activation and aggregation of platelets in vitro and in vivo. This activity was likely due to the direct binding of LB1 to platelets, followed by its rapid internalization within clathrin coated-pits. Coimmunoprecipitation revealed that LB1 interacted with the insulin receptor located within the platelet membrane. The binding of LB1 to this receptor induced its internalization and inhibited at least one of the phosphorylation cascade stimulated by the receptor, which in turn inhibited platelet functions. The addition of affinity-purified anti-LB1 in our model markedly increased the persistence of LB1 within platelets, a finding supported by the detection of LB1 only in platelets from anti-LB1 positive SLE patients. Our results suggest that LB1 regulates haemostasis in normal subjects. The presence of anti-LB1 in SLE patients prolongs the persistence of LB1 within platelets, thus possibly vi preventing further platelet activation. This mechanism likely explains the reduced risk of thrombotic events in LAC positive SLE patients with circulating anti-LB1 autoantibodies.

Les rodenticides anticoagulants (RA) constituent le principal moyen de lutte contre les rongeurs. L’exposition aux RA du renard roux et l’impact des RA sur les populations de ce prédateur sont étudiés. En France, on distingue un contexte « biocide » (BCD) où les RA sont principalement utilisés près des bâtiments et un contexte « phytopharmaceutique » (PP) où la bromadiolone (un RA) est également utilisée en plein champs contre le campagnol terrestre. La contamination des rongeurs aux RA est mesurée : 5 RA sont détectés en contexte BCD alors que la bromadiolone est la molécule majoritaire en contexte PP ; les espèces de rongeurs non ciblées par les RA étant exposées dans les2 contextes. L’exposition est maximale chez les espèces ciblées ou celles au mode de vie similaire.L’exposition du renard est évaluée par la mesure des résidus de RA dans des fèces collectées in situ.La bromadiolone est retrouvée dans 97 % des fèces positives et les RA sont plus retrouvés dans les fèces en cas de traitements PP. En contexte PP, le ratio de fèces positives augmente non linéairement avec la surface traitée dans un rayon d’1 km autour des fèces. L’impact des traitements PP sur les populations de renards est évalué (période 2003-2009, département du Doubs). Les indices d’abondance de renard mesurés sur une commune le printemps d’une année n diminuent avec l’augmentation des quantités d’appâts utilisées les années n-1 et n-2. Pendant la période suivie,la mise en place d’une lutte intégrée contre le campagnol terrestre s’est traduite par une diminution des quantités d’ AR utilisées et donc par une diminution des impacts sur les populations de renards. / Rodents are mainly controlled with anticoagulant rodenticides (AR). AR exposure and impact were studied for red fox populations. In France, we distinguish 2 contexts of AR use: “biocide” (BCD) where AR are used next to buildings and “phytopharmaceutical” (PP) where bromadiolone (an AR) is also applied in the field against the water vole. Rodent contamination to AR was characterized: 5 differentAR were detected in rodents in the BCD context while bromadiolone was predominantly found in thePP context; non target rodents being exposed in both contexts. AR contamination was the highest fortarget species or species with similar lifestyle. Red fox exposure was assessed through themeasurement of AR residues in faeces sampled in the field. ARs were detected more frequently where PP treatments occurred. Every positive faeces contained only bromadiolone except one (BCD context)with chlorophacinone. In PP context, the ratio of positive faeces varied non-linearly with the area of PPtreatments within a 1km-radius around faeces. The impact of bromadiolone PP treatments on red fox populations was assessed (period 2003–2009, Doubs department). Kilometric Abundance Index offoxes measured a year n decreased with higher treatment intensities the years n-1 and n-2. Moreoverwe have shown that a shift to preventive treatments with reduced AR use is less harmful to fox populations.

Un apport élevé de vitamine K a été associé à une meilleure stabilité du traitement à la warfarine. Toutefois, l’effet du gène VKORC1, codant pour une enzyme impliquée dans le métabolisme de la vitamine K et inhibée par la warfarine, sur cette association a été très peu étudié. De plus, il a été suggéré que les patients anticoagulés sont fréquemment conseillés de restreindre leur consommation d’aliments riches en vitamine K dans le contexte clinique. Néanmoins, l’effet de cette recommandation sur l’apport de vitamine K n’est pas établi. Afin d’examiner ces questions, 317 Québécois anticoagulés à la warfarine provenant de 17 sites hospitaliers ont été sondés sur les recommandations alimentaires reçues en début de traitement. L’apport alimentaire habituel de vitamine K a été évalué rétrospectivement sur une période de 12 mois. La stabilité du traitement a été mesurée par le pourcentage de temps passé dans l’intervalle thérapeutique (n=246) du 3e au 12e mois suivant l’initiation du traitement. La majorité des patients (68%) ont rapporté avoir été conseillés de restreindre leur consommation d’aliments riches en vitamine K. L’adhérence à cette recommandation était associée à de plus faibles apports alimentaires de vitamine K. De plus, l’apport alimentaire de vitamine K était positivement associé à la stabilité du traitement, et cette relation n’était pas modulée par le génotype de VKORC1. Ces données ont permis d’illustrer des lacunes dans l’éducation nutritionnelle prodiguée aux patients anticoagulés à la warfarine, et ont contribué à la recherche portant sur l’interaction entre l’apport de vitamine K et la warfarine. / Recent studies suggest that higher vitamin K intake is associated with better warfarin therapy stability. However, whether the VKORC1 gene, encoding an enzyme involved in vitamin K metabolism and inhibited by warfarin, modulates this association is not well studied. Moreover, it has been suggested that warfarin-treated patients are often instructed to limit their consumption of vitamin K-rich foods in the clinical setting. Yet, the impact of this advice on usual dietary vitamin K intakes is unknown. To gain insight in these issues, 317 warfarin-treated patients from 17 hospital sites in the province of Quebec were questioned on the dietary recommendations they had received at warfarin initiation. Usual dietary vitamin K intake was assessed retrospectively over a 12-month period. Stability of warfarin therapy was measured by the percentage of time in the therapeutic range (n=246) from the 3rd to 12th month following warfarin initiation. Most patients (68%) reported being advised to limit their consumption of vitamin K-rich foods, particularly green vegetables. Adherence to this recommendation was associated with lower vitamin K intakes. Moreover, usual dietary vitamin K intake was positively associated with warfarin therapy stability. This association was not modulated by VKORC1 genotype. These data highlighted the need for better nutritional education in warfarin users, and contributed to the research on the interaction between dietary vitamin K intake and warfarin.

Estudo exploratório, de delineamento longitudinal, com 81 pacientes em uso de anticoagulante oral e que foram avaliados durante internação e dois meses após a alta. Os objetivos foram acompanhar a evolução quanto á terapia de anticoagulação oral e adesão ao tratamento; comparar o estado geral de saúde e a presença de sintomas de ansiedade e depressão. Foram utilizados instrumentos específicos para avaliar adesão ao tratamento medicamentoso (Medida de Adesão aos Tratamentos), estado geral de saúde (Escala Visual Analógica) e presença de sintomas de ansiedade (HADS-Ansiedade) e depressão (HADS- Depressão). As análises estatísticas realizadas foram: Teste t de Student pareado para comparar as médias do estado geral de saúde e da HADS; Modelo Linear de Efeitos Mistos para analisar a associação entre estado geral de saúde, ansiedade, depressão e tempo de avaliação no estudo. O nível de significância adotado foi 0,05. Entre os participantes, 54,3% eram mulheres, com idade média de 59,5 anos e nível médio de instrução de 5,1 anos. A principal indicação clínica para uso do medicamento foi formação de trombos (34,6%), sendo a varfarina o anticoagulante oral mais utilizado (97,5%). Dois meses após a alta, todos os pacientes foram classificados como aderentes ao tratamento e 42% mantiveram o INR na faixa terapêutica. As diferenças entre as médias do estado geral de saúde, HADS-Ansiedade e HADS-Depressão durante a internação e dois meses após a alta não foram estatisticamente significantes (p=0,78; p=0,27 e p=0,40, respectivamente). Em relação á presença de ansiedade, quando associamos as duas variáveis de forma categórica, com e sem sintomas, e o tempo de avaliação, 38 (46,9%) pacientes foram classificados como \"sem sintomas\" de ansiedade e 22 (27,1%) \"com sintomas\", sendo esta associação estatisticamente significante (p<0,001). Para depressão, a maioria (55; 67,9%) foi classificada como \"sem sintomas\" e 11 (13,6%) \"com sintomas\", sendo tal associação estatisticamente significante (p<0,001). Ao analisarmos as médias do estado geral de saúde segundo tempo e grupo de sintomas, resultados estatisticamente significantes foram obtidos apenas quando comparamos os valores entre os grupos sem sintomas de depressão (M=80,5; D.P.=23,46) e com sintomas (M=62,5; D.P.=26,07) (p=0,021), dois meses após a alta e quando comparamos as médias do grupo com sintomas de depressão, na internação (M= 75,37; D.P.=25,02) e dois meses após a alta (M=62,5; D.P.=26,07) (p=0,046). Identificar o perfil clínico de pacientes em uso de anticoagulante oral, desde a internação até dois meses de seguimento ambulatorial; conhecer sua percepção sobre estado de saúde, presença de sintomas de ansiedade e de depressão e a adesão ao tratamento são ações importantes a serem consideradas no atendimento desses pacientes. Tais resultados poderão ser utilizados para nortear mudanças na organização do ambulatório de anticoagulação oral e no planejamento da assistência de enfermagem a tais pacientes. / This exploratory and longitudinal research involved 81 patients under oral anticoagulation treatment, who were evaluated during hospitalization and two months after discharge. The objectives were to follow the patients\' clinical evolution, considering the oral anticoagulation therapy and treatment adherence; and to compare the general health condition and the presence of anxiety and depression symptoms. Specific instruments were used to assess adherence to medication treatment (Treatment Adherence Measure), general health condition (visual analogue scale) and the presence of anxiety (HADS-Anxiety) and depression symptoms (HADS-depression). For the sake of statistical analysis, the following were applied: Student\'s paired t-test to compare the mean scores for the general health condition and HADS scores; the Linear Fixed Effects Model to analyze the association between general health condition, anxiety, depression and research moment. Significance was set at 0.05. Among the participants, 54.3% were women, with a mean age of 59.5 years and a mean education time of 5.1 years. The main clinical indication for medication use was the formation of thrombi (34.6%), with warfarin as the most used oral anticoagulant drug (97.5%). AT two months after discharge, all patients were classified as adherent to the treatment and 42% maintained their INR within the therapeutic range. The differences between the mean general health, HADS-Anxiety and HADS-Depression scores during hospitalization and two months after discharge were not statistically significant (p=0.78; p=0.27 and p=0.40, respectively). As regards the presence of anxiety, when the two variables are associated categorically, with and without symptoms, and the research moment, 38 (46.9%) patients were classified as \"without symptoms\" of anxiety and 22 (27.1%) \"with symptoms\", with a statistically significant association (p<0.001). As for depression, the majority (55; 67.9%) was classified as \"without symptoms\" and 11 (13.6%) \"with symptoms\", a statistically significant association (p<0.001). The analysis of the mean general health condition scores according to the research moment and group of symptoms only revealed statistically significant results when comparing the groups without (M=80.5; S.D.=23.46) and with symptoms (M=62.5; S.D.=26.07) (p=0.021) two months after the discharge and when comparing the mean scores for the group with depression symptoms during hospitalization (M= 75.37; S.D.=25.02) and two months after the discharge (M=62.5; S.D.=26.07) (p=0.046). Identifying the clinical profile of patients under oral anticoagulant therapy since hospitalization and after two months of outpatient monitoring and getting to know their perceived health condition, presence of anxiety and depression symptoms and treatment adherence are important actions for consideration in care delivery to these patients. These results can be used to guide changes in the organization of the oral anticoagulation outpatient clinic and in nursing care planning for these patients.

Submitted by ANGELICA AUGUSTA GRIGOLI DOMINATO null (angelica@unoeste.br) on 2017-02-16T18:51:51Z No. of bitstreams: 1 Angélica A. Grigoli Dominato.pdf: 4485568 bytes, checksum: 4abc8fe39b8c64a5ed8be37f2be077de (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-02-20T20:26:27Z (GMT) No. of bitstreams: 1 dominato_aag_dr_sjrp.pdf: 4485568 bytes, checksum: 4abc8fe39b8c64a5ed8be37f2be077de (MD5) / Made available in DSpace on 2017-02-20T20:26:27Z (GMT). No. of bitstreams: 1 dominato_aag_dr_sjrp.pdf: 4485568 bytes, checksum: 4abc8fe39b8c64a5ed8be37f2be077de (MD5) Previous issue date: 2017-01-20 / A atividade metabólica fúngica, especialmente nos endofíticos, favorece a secreção de moléculas como antibióticos, pigmentos, enzimas e polissacarídeos, que podem ser aplicadas nas indústrias alimentícia, cosmética, farmacêutica, entre outras. A diversidade química dos exopolissacarídeos (EPS) bem como a possibilidade de sua utilização como substrato para diferentes modificações químicas (carboxilação, sulfatação e metilação) aumentam seu espectro de aplicação. Realizar o cultivo submerso dos fungos endofíticos Neofusicoccum parvum, Fusarium sp e Colletotrichum gloeosporioides para a produção de EPS foi o primeiro objetivo deste trabalho. Uma vez obtidos, os EPS foram purificados e quimicamente caracterizados. Sulfatação e ensaio da atividade anticoagulante foram realizados. Os parâmetros concentração de inóculo e tempo de cultivo foram estabelecidos para maior produção dos EPS, por fermentação submersa. Etapas de purificação, por centrifugação, foram efetuadas após análises por cromatografia de exclusão estérica a alta pressão, com detecção por índice de refração (HPSEC/RID). Os EPS purificados [precipitado do C. gloeosporioides (C. gloeosporioidesprec) e os três sobrenadantes] mostraram-se praticamente isentos de proteínas. A hidrólise ácida e subsequente análise dos hidrolisados por cromatografia de troca aniônica a alta pressão com detecção por amperometria pulsada (HPAEC/PAD) indicaram que apenas o C. gloeosporioidesprec era uma glucana. A análise por cromatografia gasosa acoplada à espectrometria de massa (GC/MS) mostrou um único derivado, 1,3,5 tri-O-acetil, 2,4,6-tri-O-metil glucitol com fragmentos de massa (m/z 118, 161, 203, 234.1) condizente com uma glucana do tipo (1→3). Os espectros de FT-IR apresentaram sinais na região de impressão digital, 926 cm-1 e 820 cm-1, típicos de polissacarídeos em configuração . Esses resultados foram confirmados por ressonância magnética nuclear bidimensional (HSQC), com um único acoplamento C1/H1, em 99,3/5,18 ppm e um sinal deslocado para campo baixo, 82,8/3,74 ppm, característico de C-3 substituído, indicando que o EPS é uma α-(1→3)-glucana. A sulfatação desta molécula resultou em α-(1→3)-D-glucanasulf com DS de 0,75 que foi utilizada nos ensaios de atividade anticoagulante. O aumento do tempo para a coagulação, nos testes do APTT (Tempo de Tromboplastina Parcial Ativada) e TT (Tempo de Trombina), foi concentração-dependente, indicando que [α-(1→3)-D-glucanasulf] pode atuar como um inibidor da via intrínseca da coagulação sanguínea e da conversão do fibrinogênio em fibrina, caracterizando-a como um potencial anticoagulante. / Fungal metabolic activity, especially in the endophytic, favors secretion of molecules such as antibiotics, pigments, enzymes and polysaccharides, which can be applicable by food, cosmetic and pharmaceutical industries, and others. The chemical diversity of the exopolysaccharides (EPS), as well as the possibility of their use as substrate for different chemical modifications (carboxylation, sulfation and methylation) increases their application spectrum. Submerged cultivation of the endophytic fungi Neofusicoccum parvum, Fusarium sp and Colletotrichum gloeosporioides for the production of EPS was the first aim of this study. Once the EPS were obtained, they were purified and chemically characterized. Chemical modification by sulfation and anticoagulant activity assays were performed. Cultivation to obtain EPS were performed in submerged fermentation. The inoculum concentration and incubation time parameters were set in order to obtain a higher amount of EPS. Purification by centrifugation was performed after analysis by high pressure steric exclusion chromatography with refractive index detection (HPSEC / RID). Purified EPS [precipitate of C. gloeosporioides (C. gloeosporioidesprec) and the three supernatants] proved to be virtually free of protein polysaccharides. Acid hydrolysis and subsequent analysis of the hydrolysates with high performance anionic exchange chromatography with amperometric detection (HPAEC/PAD) indicated that only the C. gloeosporioidesprec was a glucan. Analysis from gas chromatography and mass spectrometry showed a single derivative, 1,3,5-tri-Oacetyl, 2,4,6-tri-O-methyl glucitol with mass fragments (m/z 118, 161, 203, 234.1) consistent with a (1→3)-glucan. FT-IR spectra showed absorption bands typical from carbohydrate and signals in the digital region at 926 cm-1 and 820 cm-1, typical of polysaccharides in the α- configuration. These results were confirmed by two-dimensional nuclear magnetic resonance (HSQC), with a single C1/H1, in 99.2/4.98 ppm, typical of one α bonding, and low-field 82.6/3.55 ppm signal displacement, characteristic of substituted C-3, indicating that EPS is an α-(1→3)-glucan. Sulfation of this molecule resulted in α- (1→3)-glucansulf with DS of 0.75 which was used in the anticoagulant activity assays. The increase in coagulation reaction time in the APTT (Activated Partial Thromboplastin Time) and TT (Thrombin Time) tests was concentration-dependent, indicating that [α-(1→3)-D-glucansulf] might act as an inhibitor of the intrinsic via of blood clotting and conversion of fibrinogen into fibrin, characterizing it as a potential anticoagulant.

Estudo exploratório, de delineamento longitudinal, com 81 pacientes em uso de anticoagulante oral e que foram avaliados durante internação e dois meses após a alta. Os objetivos foram acompanhar a evolução quanto á terapia de anticoagulação oral e adesão ao tratamento; comparar o estado geral de saúde e a presença de sintomas de ansiedade e depressão. Foram utilizados instrumentos específicos para avaliar adesão ao tratamento medicamentoso (Medida de Adesão aos Tratamentos), estado geral de saúde (Escala Visual Analógica) e presença de sintomas de ansiedade (HADS-Ansiedade) e depressão (HADS- Depressão). As análises estatísticas realizadas foram: Teste t de Student pareado para comparar as médias do estado geral de saúde e da HADS; Modelo Linear de Efeitos Mistos para analisar a associação entre estado geral de saúde, ansiedade, depressão e tempo de avaliação no estudo. O nível de significância adotado foi 0,05. Entre os participantes, 54,3% eram mulheres, com idade média de 59,5 anos e nível médio de instrução de 5,1 anos. A principal indicação clínica para uso do medicamento foi formação de trombos (34,6%), sendo a varfarina o anticoagulante oral mais utilizado (97,5%). Dois meses após a alta, todos os pacientes foram classificados como aderentes ao tratamento e 42% mantiveram o INR na faixa terapêutica. As diferenças entre as médias do estado geral de saúde, HADS-Ansiedade e HADS-Depressão durante a internação e dois meses após a alta não foram estatisticamente significantes (p=0,78; p=0,27 e p=0,40, respectivamente). Em relação á presença de ansiedade, quando associamos as duas variáveis de forma categórica, com e sem sintomas, e o tempo de avaliação, 38 (46,9%) pacientes foram classificados como \"sem sintomas\" de ansiedade e 22 (27,1%) \"com sintomas\", sendo esta associação estatisticamente significante (p<0,001). Para depressão, a maioria (55; 67,9%) foi classificada como \"sem sintomas\" e 11 (13,6%) \"com sintomas\", sendo tal associação estatisticamente significante (p<0,001). Ao analisarmos as médias do estado geral de saúde segundo tempo e grupo de sintomas, resultados estatisticamente significantes foram obtidos apenas quando comparamos os valores entre os grupos sem sintomas de depressão (M=80,5; D.P.=23,46) e com sintomas (M=62,5; D.P.=26,07) (p=0,021), dois meses após a alta e quando comparamos as médias do grupo com sintomas de depressão, na internação (M= 75,37; D.P.=25,02) e dois meses após a alta (M=62,5; D.P.=26,07) (p=0,046). Identificar o perfil clínico de pacientes em uso de anticoagulante oral, desde a internação até dois meses de seguimento ambulatorial; conhecer sua percepção sobre estado de saúde, presença de sintomas de ansiedade e de depressão e a adesão ao tratamento são ações importantes a serem consideradas no atendimento desses pacientes. Tais resultados poderão ser utilizados para nortear mudanças na organização do ambulatório de anticoagulação oral e no planejamento da assistência de enfermagem a tais pacientes. / This exploratory and longitudinal research involved 81 patients under oral anticoagulation treatment, who were evaluated during hospitalization and two months after discharge. The objectives were to follow the patients\' clinical evolution, considering the oral anticoagulation therapy and treatment adherence; and to compare the general health condition and the presence of anxiety and depression symptoms. Specific instruments were used to assess adherence to medication treatment (Treatment Adherence Measure), general health condition (visual analogue scale) and the presence of anxiety (HADS-Anxiety) and depression symptoms (HADS-depression). For the sake of statistical analysis, the following were applied: Student\'s paired t-test to compare the mean scores for the general health condition and HADS scores; the Linear Fixed Effects Model to analyze the association between general health condition, anxiety, depression and research moment. Significance was set at 0.05. Among the participants, 54.3% were women, with a mean age of 59.5 years and a mean education time of 5.1 years. The main clinical indication for medication use was the formation of thrombi (34.6%), with warfarin as the most used oral anticoagulant drug (97.5%). AT two months after discharge, all patients were classified as adherent to the treatment and 42% maintained their INR within the therapeutic range. The differences between the mean general health, HADS-Anxiety and HADS-Depression scores during hospitalization and two months after discharge were not statistically significant (p=0.78; p=0.27 and p=0.40, respectively). As regards the presence of anxiety, when the two variables are associated categorically, with and without symptoms, and the research moment, 38 (46.9%) patients were classified as \"without symptoms\" of anxiety and 22 (27.1%) \"with symptoms\", with a statistically significant association (p<0.001). As for depression, the majority (55; 67.9%) was classified as \"without symptoms\" and 11 (13.6%) \"with symptoms\", a statistically significant association (p<0.001). The analysis of the mean general health condition scores according to the research moment and group of symptoms only revealed statistically significant results when comparing the groups without (M=80.5; S.D.=23.46) and with symptoms (M=62.5; S.D.=26.07) (p=0.021) two months after the discharge and when comparing the mean scores for the group with depression symptoms during hospitalization (M= 75.37; S.D.=25.02) and two months after the discharge (M=62.5; S.D.=26.07) (p=0.046). Identifying the clinical profile of patients under oral anticoagulant therapy since hospitalization and after two months of outpatient monitoring and getting to know their perceived health condition, presence of anxiety and depression symptoms and treatment adherence are important actions for consideration in care delivery to these patients. These results can be used to guide changes in the organization of the oral anticoagulation outpatient clinic and in nursing care planning for these patients.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Operculina macrocarpa (L.) Urb. (âjalapa brasileiraâ) (Convolvulaceae) à uma espÃcie comum do Nordeste brasileiro, sendo popularmente utilizada pelas suas propriedades laxativa, purgativa e âdepurativaâ. A tintura do tubÃrculo de jalapa, matÃria-prima majoritÃria, ao lado da tintura de Convolvulus scammonia (TCS) compÃem o fitoterÃpico Aguardente Alemà(AAL) ou Tintura de Jalapa Composta, indicada como antitrombÃtico na medicina popular. O objetivo do presente trabalho foi investigar o potencial antiagregante plaquetÃrio e anticoagulante de O. macrocarpa em plasma humano, incluindo estudo de bioprospecÃÃo e determinaÃÃo do possÃvel mecanismo de aÃÃo. A avaliaÃÃo da atividade antiagregante plaquetÃria das drogas testes (tintura e fraÃÃes de O. macrocarpa, TCS e AAL) em plasma humano rico em plaquetas (PRP) foi mensurada por mÃtodo turbidimÃtrico, sendo a agregaÃÃo induzida por vÃrios agonistas como difosfato de adenosina (ADP), epinefrina (EPI), Ãcido araquidÃnico (AA), colÃgeno (COL) ou trombina (TROM). Ao contrÃrio da TCS e da AAL, tanto a tintura de O. macrocarpa (TOM/precipitado(P) e sobrenadante(S)) quanto uma das fraÃÃes orgÃnicas obtidas da planta (TOM/1-F4) mostraram atividade antiagregante plaquetÃria, onde a TOM/1-F4 (100 Âg/mL) apresentou efeito comparÃvel ao Ãcido acetilsalicÃlico (AAS). CaracterizaÃÃo fÃsico-quÃmica (CLAE e espectrofotometria) da TOM/PS (teor de resinas 1,38g%) e da TOM/1-F4 permitiu a identificaÃÃo de Ãcidos fenÃlicos (clorogÃnico, gÃlico e cafÃico), bem como a determinaÃÃo do teor de fenÃis totais da TOM (0,14g%). O efeito antiagregante plaquetÃrio da TOM/PS e da TOM/1F-4 parece resultar de vÃrias aÃÃes intracelulares. Contudo, a TOM/PS na presenÃa do AAS, L-ARG, ODQ, pentoxifilina (PTX) ou ticlopidina (TIC) teve seu efeito antiagregante plaquetÃrio modificado principalmente pela TIC, sugerindo um papel importante dos receptores purinÃrgicos na bioatividade da TOM/PS. A TOM/PS apresentou atividade antioxidante determinada atravÃs do teste do DPPH. AlÃm disso, a TOM/PS e a TOM/1F-4 nÃo mostraram aÃÃo anticoagulante em plasma humano, mas a TOM/PS aumentou o tempo de sangramento em camundongos. Foi observada uma toxicidade relativa da TOM/PS em neutrÃfilo humano. Dessa forma, o presente estudo comprovou, de maneira inÃdita, o potencial antiagregante plaquetÃrio da tintura e da fraÃÃo orgÃnica de jalapa, que estÃo pelo menos em parte relacionados à presenÃa de fenÃis, particularmente Ãcidos fenÃlicos, e resinas na planta. / Operculina macrocarpa (L.) Urb ("Brazilian jalapa") (Convolvulaceae) is a common species of the Brazilian Northeast. It is popularly used because of its laxative and purgative properties. The tincture of Operculina macrocarpa tubers (major constituent) and Convolvulus scammonia composes the raw materials of Aguardente AlemÃÂ, herbal medicine, referred to as antithrombotic in folk medicine. The objective of this study was to investigate the antiplatelet and anticoagulant potential for Operculina macrocarpa in human plasma, including the bioprospection study and determination of possible mechanism of action. The evaluation of antiplatelet activity of drugs (tincture and fractions of O. macrocarpa, tincture of C. scammonia and AAL) in human platelet-rich plasma (PRP) was measured by the turbidimetric method where the aggregation was induced by the addition of agonists (adenosine diphosphate (ADP), epinephrine (EPI), arachidonic acid (AA) collagen (COL) or thrombin (TROM). Unlike tincture of C. scammonia and AAL, both O. macrocarpa tincture (TOM/preciptade (P) and supernatant (S)) and one of the organic fractions obtained from O. macrocarpa (TOM/1-F4) showed antiplatelet activity in human plasma where TOM/1-F4 (100 Âg/mL) presented comparable effect to AAS. Physicochemical characterization (HPLC and spectrophotometer) of TOM/PS (content of resins 1,38g%) and TOM/1-F4 allowed the identification of phenolic acids (chlorogenic, galic and caffeic) as well as the determination of total phenols of TOM (0,14g%). The antiplatelet effect of TOM/PS and TOM/1-F4 seems to result from many intracellular actions. However, TOM/PS in presence of AAS, L-ARG, ODQ, pentoxifilin (PTX) or ticlopidine (TIC) had its antiplatelet effect modified mainly because of TIC, suggesting an important role of purinergic receptors in TOM/PS. TOM/PS presented antioxidant activity determined by DPPH test. Furthermore, TOM/PS and TOM/1-F4 did not showed anticoagulant action in human plasma but TOM/PS increased the bleeding time in mice. It was observed a relative toxicity of TOM/PS in human neutrophil. Therefore, this study demonstrated, as never before, the antiplatelet potencial of tincture and the organic fraction of "jalapa" and that at least related to the presence of phenols, particulary phenolic acids, and resins in plant.