<p>Cilj ove doktorske disertacije je bio da se ispita mogućnost dobijanja prirodnih<br />koagulanata iz zrna pasulja koji bi se koristili u obradi različitih voda i otpadnih voda. U okviru izvedenih eksperimenata su utvrđeni najbolji uslovi za ekstrakciju prirodnih koagulanata iz pasulja, određen je hemijski sastav sirovih ekstrakata dobijenih na različite načine, proverena je trajnost i određen način čuvanja sirovih ekstrakata i ispitan uticaj različitih parametara na koagulacionu aktivnost sirovih ekstrakata.<br />S obzirom na to da su prirodni koagulanti organskog porekla, oni povećavaju sadržaj organskih materija u tretiranoj vodi, pa su u okviru rada ispitane i upoređene različite metode preči&scaron;ćavanja sirovog ekstrakta pasulja, a zatim određen uticaj sirovog ekstrakta i preči&scaron;ćenih koagulanata na sadržaj organskih materija u obrađenoj vodi.<br />Kako bi se ispitala efikasnost prirodnih koagulanata iz zrna pasulja u realnim vodama,<br />oni su bili primenjeni u otpadnim vodama od proizvodnje bioetanola. Pored toga, ispitana je mogućnost njihove primene u kombinaciji sa konvencionalnim koagulantima, i na samom kraju je ispitan sastav pasulja koji preostane nakon ekstrakcije prirodnih koagulanata kako bi se utvrdilo da li je pogodan za kori&scaron;ćenje kao<br />dodatak stočnoj hrani.<br />Dobijeni rezultati su pokazali da se iz zrna pasulja može dobiti ekstrakt visoke koagulacione aktivnosti, koji se dalje može efikasno prečistiti tako da ne povećava sadržaj organskih materija u obrađenoj vodi, već ga naprotiv smanjuje. Takođe, prirodni koagulanti iz pasulja se mogu uspe&scaron;no primeniti za tretman otpadnih voda od proizvodnje bioetanola, mogu biti pomoćni koagulanti aluminijum-sulfatu, a pasulj nakon ekstrakcije prirodnih koagulanata sa destilovanom vodom se može iskoristiti kao<br />dodatak stočnoj hrani.</p> / <p>The aim of this PhD thesis was to investigate the possibility to obtain natural coagulants from common bean which could be used for different waters and wastewaters treatment. The optimal conditions for extraction of natural coagulants from common bean, the chemical composition of crude extracts obtained in different ways and the influence of various parameters on coagulation activity of crude extracts were determined within the performed experiments. The durability of crude extracts was checked and the way of their storage defined.<br />Considering the fact that natural coagulants are of organic origin, they increase the content of organic matter in treated water. Hence, the different methods of purification of common bean crude extract were investigated and compared, and thereafter the influence of crude extract and purified coagulants on organic matter content in treated water was determined.<br />Natural coagulants obtained from common bean were applied in bioethanol stillages in order to explore their efficiency in real waters. Besides, the possibility of their usage combined with conventional coagulants was investigated. At the end, the composition of common bean that remains after extraction of natural coagulants was determined in order to to prove if it was appropriate as addition to feed.<br />Obtained results showed that extract of high coagulation activity can be obtained from common bean. It can be efficiently purified so as not to increase the organic matter content of the water, but on the contrary, to decrease it. Natural coagulants from common bean can be successfully applied for treatment of wastewaters remained after bioethanol production, they can be used as coagulant aid with alum, and the common bean remained after extraction of natural coagulants with distilled water can be utilized as addition to feed.</p>

Heparin, a glycosaminoglycan (GAG), is a complex biopolymer of varying chain length and consisting of uronic acid and glucosamine residues, which are sulfated at various positions. The interaction of heparin with antithrombin is the basis for anticoagulation therapy. Heparin accelerates the antithrombin mediated inhibition of factor Xa and thrombin by a conformational activation mechansism and bridging mechanism, respectively. The sequence specific pentasaccharide DEFGH in full length heparin is the most important fragment for high affinity and activation of antithrombin, without which the heparin is incapable of binding to antithrombin. Although heparin is a commonly used anticoagulant, it suffers from serious side effects including bleeding complications, heparin-induced thrombocytopenia, and intra- and inter-patient dose response variability. Desai and co-workers have shown that it is possible to replace the GAG skeleton by small, non-saccharide sulfated molecules as antithrombin activators. However, the designed molecules were found to be weak activators of antithrombin due to their binding to the extended heparin-binding site (EHBS), instead of the pentasaccharide-binding site (PBS), of antithrombin. To design better non-saccharide antithrombin activators, a virtual screening-based approach was employed. Combinatorial virtual screening of 24576 molecules based on tetrahydroisoquinoline core scaffold resulted in 92 hits that were predicted to bind preferentially in the PBS of activated antithrombin with good affinity. The work resulted in a predicted pharmacophore consisting of a 5,6-disulfated bicyclic tetrahydroisoquinoline and a 2′,5′-disulfated unicyclic phenyl ring connected by a 4- to 5-carbon linker. The work has led to several hypotheses, which are being tested in the laboratory through synthesis and biochemical evaluation. To understand the mechanism of heparin binding to thrombin in greater detail, structural biology and molecular modeling approaches were used. More specifically, the nature of the heparin binding to thrombin was studied with a special focus on understanding the specificity of recognition. Comparative analysis was performed with heparin–antithrombin interaction to assess similarities and differences between the two heparin binding systems. In antithrombin, three important amino acids are involved in heparin pentasaccharide binding, while in thrombin, at least seven basic amino acids are predicted to be involved. For biological systems, one would expect greater specificity with more interacting points. However, the heparin–thrombin system interestingly displays a lack of specificity. The molecular basis for this lack of specificity is not clear. A study of antithrombin and thrombin crystal structures with regard to surface exposure, flexibility, and geometry of basic amino acids present in the respective heparin binding site provides the basis for the specificity of recognition (or lack thereof) in the two systems. Interestingly, analysis of thrombin exosite-II showed that Arg101, Arg165 and Arg233 are spatially conserved and form a local asymmetric center. Using in-silico docking techniques, selected tetrasaccharide sequences were found to specifically recognize this triad of amino acids indicating the possibility of specific recognition of thrombin. This hypothesis led to the design of a putative lead sequence that is 50% smaller in size and contains 62.5% fewer charges in comparison to the literature reported known exosite II sequence. The design of novel putative ‘specific’ exosite II sequence challenges the idea that the thrombin–heparin interaction is completely non-specific and gives rise to novel opportunities of designing specific thrombin exosite-II ligands.

Chez le chien, les thromboses représentent une complication majeure de nombreuses conditions qui sont revues dans ce manuscrit. L’arsenal thérapeutique actuel présente certaines limites: des effets anticoagulants variables d’un patient à l’autre, des hémorragies et une administration par voie sous-cutanée pour l’héparine. Le rivaroxaban est un nouvel anticoagulant oral approuvé pour la prévention et le traitement des thromboses chez l’humain. C’est un inhibiteur direct du facteur Xa. La présente étude a pour objectif d’évaluer les effets hémostatiques du rivaroxaban chez des chiens en santé, en utilisant les tests de coagulation suivants: temps de prothrombine (PT), temps partiel de thromboplastine (aPTT), activité anti-facteur X, génération de thrombine (GT) et thromboélastographie (TEG®). Tout d’abord, l’effet anticoagulant du rivaroxaban a été évalué in vitro : le plasma citraté pauvre en plaquettes provenant de 20 Beagle en santé a été aliquoté et enrichi avec des solutions de rivaroxaban à des concentrations de 0 à 1000 mg/L d’anticoagulant. Une prolongation concentration-dépendante de tous les tests de coagulation a été notée. Les concentrations de 0.024 et 0.053 mg/L diminuent respectivement de 50% la vitesse de propagation de la GT et la densité optique de l’activité anti-facteur X. Ces derniers tests sont les plus sensibles et précis pour détecter l’effet anticoagulant du rivaroxaban. Ensuite, 24 Beagle en santé ont été répartis aléatoirement en 3 groupes (n=8). Chaque groupe a reçu par voie orale un placebo, ou 20 mg de rivaroxaban une ou deux fois à 8h d’intervalle. Quinze échantillons sanguins ont été prélevés pour chaque chien sur 30 heures. Pour tous les tests de coagulation excepté la TEG®, une différence significative a été notée dans les résultats entre les groupes traités et le groupe placebo (p<0.0001). La durée de l’effet anticoagulant du rivaroxaban était de 7.9-18.7h dans le groupe traité une fois; et de 17.5-26.8h dans le groupe traité deux fois. Le pic d’action de l’effet anticoagulant était d’environ 2h. Seul le paramètre R de la TEG® était significativement affecté dans les groupes traités. En conclusion, le rivaroxaban exerce un effet anticoagulant chez le chien à la dose de 2 mg/kg. Une administration biquotidienne semble appropriée pour un effet de 24h. / In dogs, thrombosis is a major complication detected in many conditions. The limits of the current available anticoagulants in veterinary medicine are their variable effects from one patient to another, bleeding complications and subcutaneous injections for heparin administration. Rivaroxaban is a novel oral anticoagulant approved for the prevention and treatment of thrombosis in humans. It is a direct factor Xa inhibitor. The objectives of the study were to determine the haemostatic effects of rivaroxaban in healthy dogs by evaluating the following coagulation assays: prothrombin time (PT), activated partial thromboplastin time (aPTT), anti-factor X activity, thrombin generation (TG) and thromboelastography (TEG®). An in vitro study was conducted: citrated platelet poor plasma from 20 healthy Beagles was aliquoted and mixed with rivaroxaban to obtain solutions ranging from 0 to 1000 mg/L of the anticoagulant. Rivaroxaban exerted a concentration-dependent anticoagulant effect. Rivaroxaban solutions at 0.024 and 0.053 mg/L cause 50% inhibition of the propagation of TG and of the optical density of anti-factor X activity respectively. These assays were the most sensitive to detect the anticoagulant effect of rivaroxaban. Secondly, 24 healthy Beagles were randomly divided in 3 groups (n=8) and received one placebo pill orally, or 20 mg rivaroxaban once or twice at 8h interval. Fifteen citrated blood samples were collected from each dog over 30h. For each coagulation assay except for TEG®, there was a significant difference in assay results between placebo and rivaroxaban groups (p<0.0001). The duration of the rivaroxaban anticoagulant effect was 7.9-18.7h in the group receiving rivaroxaban once, and 17.5-26.8h in the group receiving rivaroxaban twice. The peak of action of rivaroxaban appeared 2h after the dose. Only R parameter of TEG® was significantly affected by rivaroxaban administration. To conclude, rivaroxaban is an efficient anticoagulant in healthy dogs at 2 mg/kg. A twice daily administration seems appropriate to exert a 24h anticoagulation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The aim of this diploma thesis is to explore the coagulation phase in water treatment process from two perspectives, the removal of cyanotoxin microcystin and the responses of ecotoxicological indicator species Daphnia magna to different concentration of this toxin, contained in a sample of cyanobacterial water bloom, which was extracted from a dam and was dominated by cyanobacteria Microcystis aeruginosa. The sample was administered in three environmentally relevant concentrations to 6 clones of Daphnia magna, 3 of which had previous experience with M. aeruginosa. Coagulation process was performed under optimal conditions: pH = 6.36; KNK4,5 = 0.26 mmol.l-1 ; Fe = 0.162 mg.l-1 ; DOC = 2.83 mg.l-1 using 10 ml of 0.125M NaHCO3 in two litres of ultrapure water. Individual forms of microcystin were detected in this ratio: 31.6 % MC-LR, 53.6 % MC-RR and 14.8 % MC-YR. The study has shown that under these conditions coagulation does not remove microcystin, e.g. the efficiency of the process is zero. In ecotoxicological study, with growing concentration of cyanobacterial mixture the negative impact on Daphnia magna increased. We have found interclonal variability in responses of D. magna, however, the previous experience with M. aeruginosa had no effect. With growing concentration of cyanobacterial water...

A thesis submitted to the Faculty of Medicine, University of the Witwatersrand, for the degree of Doctor of Philosophy in Medicine. / Despite having been initially described more than fifty years ago, sclerotherapy of oesophageal varices has only relatively recently became regarded as one of the primary modalities both to control bleeding oesophageal varices and to prevent recurrent bleeding. Sclerotherapy, however, ts associated with numerous complications and its exact mechanism of action, particularly that pertaining to its effect on haemostasis, has to date been poorly documented. One of the problems of comparing the various trials has been the diversity of both the technique and the type and concentration of the sclerosants used. (Abbreviation abstract) / Andrew Chakane 2018

A proposta deste trabalho foi utilizar quatro diferentes coagulantes - sulfato de alumínio, cloreto férrico, hidroxicloreto de aluminio e sulfato ferrico; construir os diagramas de coagulação; e assimilar as regiões de maior ou menor eficiência da remoção de turbidez ou da cor. Para permitir uma avaliação de custos na utilização dos coagulantes, observo-se nos diagramas as regiões onde os valores de turbidez remanescente são aproximadamente iguais para diferentes coagulantes. Com essa consideração, foi stabelecida a relação entre consumo e custo dos produtos quimícos utilizados. Os coagulantes que apresentaram particularidades para as águas estudadas foram: o cloreto ferrico que mostrou melhor desempenho para valores de ph baixos; o sulfato ferrico que mostrou-se mais economico; e o hidroxicloreto de aluminio que atua numa grande faixa de ph. Na conclusão dos estudos, verifica-se que cada água a ser tratada deve ser analisada através de diagramas de coagulação, visando a otimização dos parâmetros de ph versus dosagem e buscando o melhor coagulante pelo menor custo. / The purpose of this study was to use four different coagulants - aluminum sulphate, iron chloride, polyaluminum hidroxichloride and ferric sulphate; to build the coagulation diagrams; and to mark the regions of higher or lower efficiency in the removal of turbidity or color. To allow a cost appraisal in the use of the coagulants, the region where the values of remaining turbidity, which are approximately the same for different coagulants, was examined in the diagrams. This being taken into consideration, the relation between consumption and cost of the chemical products was established. The coagulants that presented certain peculiarities for the studied waters were: iron chloride, that showed better performance for low pH values; the ferric sulphate, that showed itself as more economical; and the polyaluminum hidroxichloride, that functions in a large interval of pH. In the conclusion of the study, it was observed that each type of water to be treated must be analyzed through coagulation diagrams, aiming at the optimization of pH parameters versus dosages and looking for the best coagulant with lower cost.

O ato cirúrgico sempre deve ser precedido de uma avaliação das condições de saúde local e sistêmica do paciente, sendo os exames complementares, o subsídio utilizado para avaliar e confirmar as suspeitas clínicas e hipóteses diagnósticas, adequando o paciente à terapêutica proposta. A literatura é ampla no que diz respeito a testes de hemostasia por meio de diferentes métodos, contudo, não há estudos na literatura revisada que comparem o tempo de sangramento pelo método de Ivy com o tempo de sangramento aferido na mucosa bucal. A proposta deste estudo é de avaliar a técnica de aferição do tempo de sangramento da mucosa bucal e comparar com o tempo de sangramento pelo método de Ivy convencional, em pacientes com história de diátese hemorrágica relacionada a procedimentos cirúrgicos anteriores. Tais pacientes apresentaramse no ambulatório de Cirurgia da Faculdade de Odontologia da Universidade de São Paulo com necessidade de procedimentos relacionados à cirurgia oral menor. Foi realizado o tempo de sangramento pelo método de Ivy previamente ao teste do tempo de sangramento da mucosa bucal em 30 pacientes. O tempo de sangramento da mucosa bucal não apresentou diferença estatística quando comparado ao tempo de sangramento de Ivy (p=0,755). Os resultados avaliados pelo método de KOLMOGOROV-SMIRNOV seguiram uma distribuição normal em ambas amostras (p>0,15) sendo que o tempo médio de sangramento na pele e na mucosa bucal foi de 295 segundos e 291 segundos respectivamente, demonstrando semelhança entre os tempos aferidos. / Surgical procedures must be preceded by an accurate evaluation of the local and systemic health status and complementary exams can confirm or not clinical suspect and diagnosis hypothesis in order to adequate the patient to the proposed surgical treatment. The world literature is generous with respect of hemostasia tests employing several methodologies although there are not studies that compare the bleeding time test by Ivy?s method with bleeding time accessed at the oral mucosa. We propose at this study the evaluation of a bleeding time method in the oral mucosa comparing the results with the conventional Ivy?s test in patients with bleeding disorders history in past oral minor surgical procedures. The patients were those selected in the Oral Surgery Clinic of the Dental School of University of São Paulo that underwent to oral minor surgery. The Ivy?s bleeding time test were previously obtained before bleeding time of the mucosa in 30 patients and it was concluded that the bleeding time of the mucosa did not present statistically difference significant comparing the Ivy?s test (p=0,755). The results evaluated by KOLMOGOROV-SMIRNOV?s method followed a normal distribution in both samples (p>0,15) and that the mean bleeding time at the skin and at the oral mucosa was 295 seconds and 291 seconds respectively showing similarity between the groups.

Os invertebrados impedem o estabelecimento de uma infecção através de uma resposta imune eficiente. Esta resposta envolve reações celulares e humorais. Existem poucos trabalhos sobre a imunidade das aranhas. O principal objetivo deste estudo foi verificar o papel dos hemócitos e dos peptídeos antimicrobianos na resposta imune da aranha Acanthoscurria gomesiana. Inicialmente, a localização relativa da gomesina e da acanthoscurrina foi determinada, mostrando que 58% dos hemócitos armazenam os dois peptídeos antimicrobianos. Além disso, foi verificado que a gomesina é direcionada aos grânulos dos hemócitos da forma de pró-peptídeo. Observou-se ainda, que após um desafio, os hemócitos migram para o sítio de infecção, onde deve secretar componentes da cascata de coagulação e peptídeos antimicrobianos. Além disso, os resultados mostraram que a fagocitose não é o principal mecanismo ativado após uma infecção. Assim sendo, as principais respostas imunes da aranha são através da coagulação e secreção dos peptídeos antimicrobianos. / Invertebrates avoid the infection establishment through an efficient immune response. This response consists in cellular and humoral reactions. Few data are available concerning the spider\'s immunity. The main aim of this study was to determine the role of hemocytes and antimicrobial peptides on the immunity of the spider Acanthoscurria gomesiana. Initially, the localization of gomesin and acanthoscurrin was determined, showing that 58% of hemocytes store both antimicrobial peptides. Moreover, our results show that gomesin is addressed to the hemocyte granules as a pro-peptide. We also demonstrate, by in vivo and in vitro experiments, that hemocytes migrate to the site of microbial infection. Once at the site of infection, hemocytes might secrete components of coagulation cascade and antimicrobial peptides. Besides, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore the main reactions involved in the spider defense might be through the coagulation and antimicrobial peptides secretion.

Leptospiras são bactérias espiroquetas altamente móveis dotadas de estratégias que possibilitam grande eficiência nos processos de invasão e disseminação no hospedeiro. Nosso grupo demonstrou previamente que leptospiras patogênicas secretam proteases capazes de clivar e inativar moléculas-chave do sistema complemento humano, o que confere a essas bactérias a capacidade de driblar os mecanismos de defesa do sistema imune inato. Dada a rápida disseminação das leptospiras durante o processo de infecção, aventou-se a hipótese de que essas proteases secretadas pudessem alvejar uma gama maior de moléculas do hospedeiro. No presente estudo, a atividade proteolítica de proteínas secretadas por leptospiras sobre um painel de moléculas da matriz extracelular e do plasma foi avaliada. O sobrenadante de cultura da estirpe virulenta L. interrogans sorovar Kennewicki Fromm degradou fibrinogênio humano, fibronectina plasmática, colágeno Tipo I, e as proteoglicanas decorina, biglicam e lumicam. A atividade proteolítica foi inibida por 1,10-fenantrolina, sugerindo o envolvimento de metaloproteases. Laminina, matrigel, plasminogênio e trombina não foram clivados por proteases presentes nos sobrenadantes. Ainda, os dados indicam que a produção de proteases deve ser um determinante de virulência importante, uma vez que os sobrenadantes de estirpes saprófitas ou patogênicas atenuadas em cultura não apresentaram atividade proteolítica sobre componentes da matriz ou do plasma. A análise dos genomas de leptospiras disponíveis nos levou à identificação de quatro termolisinas, metaloproteases presentes apenas nas espécies patogênicas. Uma delas, codificada pele LIC13322, foi produzida na forma recombinante e apresentou atividade proteolítica sobre fibrinogênio, biglicam e decorina. Em paralelo, foram também realizadas análises comparativas dos exoproteomas das estirpes Fromm e Patoc I. Algumas metaloproteases que podem estar envolvidas na degradação de moléculas do hospedeiro foram identificadas. A capacidade de clivar moléculas do tecido conjuntivo e proteínas da cascata de coagulação pode certamente contribuir para a invasão e a destruição tecidual observadas durante a infecção por Leptospira. / Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the human complement system, allowing these bacteria to circumvent host´s innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm degraded human fibrinogen, plasma fibronectin, collagen Type 1, and the proteoglycans decorin, biglycan, and lumican. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Laminin, matrigel, plasminogen and thrombin were not cleaved by proteases present in the supernatants. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic acticity against ECM or plasma components. A search against Leptospira genomes allowed identification of four thermolysins, which are metalloproteases found exclusively in pathogenic species. One of them, encoded by LIC13322, was produced in the recombinant form and displayed proteolytic activity against fibrinogen, biglycan and decorin. Comparative exoproteomic analyses using Fromm and Patoc I strains were also performed and allowed identification of a few metalloproteases that could be involved in the degradation of host components. The ability to cleave connective tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.