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Factor XI (FXI) is the zymogen of the coagulation protease factor XIa (FXIa) that contributes to thrombin generation through the intrinsic pathway. FXI is activated by the contact pathway protease, factor XIIa (FXIIa), in a high molecular weight kininogen-dependent manner. It is also known to be activated by thrombin in a positive feedback reaction, however, the mechanism of this activation is not yet completely understood. Therefore, our objectives were to identify the role of polyanions in the thrombin mediated FXI activation, and the role of the thrombin exosites in the activation. To study this activation, we assessed the activation of FXI by thrombin in the presence and absence of the polyanions, dextran sulfate and polyphosphate (polyP). We utilized surface plasmon resonance to determine whether FXI and thrombin bind to the polyanions, and how the exosites effect thrombin’s ability to bind using thrombin exosite 1 and 2 variants. To investigate the role of the exosites in FXI activation, we analyzed the activation of FXI by the thrombin variants. In addition, we explored how inhibiting the thrombin exosites using DNA aptamers affects thrombin’s ability to bind to polyanions and activate FXI. We found that polyanions are required as a cofactor for the activation of FXI by thrombin, and stimulate the activation in a concentration dependent fashion, suggesting a template mechanism. Our findings also show that exosite 1 and 2 are required for thrombin to bind to polyanions, however, exosite 2 plays the predominant role in FXI activation. Our aptamer data showed that either exosite can be targeted to inhibit FXI activation. These findings enrich our understanding of the mechanism of FXI activation by thrombin and provides further insight on how to attenuate the activation for potential antithrombotic therapies. / Thesis / Master of Science (MSc)

Water utilities can experience problems from bubble formation during conventional treatment, including impaired particle settling, filter air binding, and measurement as false turbidity in filter effluent. Coagulation processes can cause supersaturation and bubble formation by converting bicarbonate alkalinity to carbon dioxide by acidification. A model was developed to predict the extent of bubble formation during coagulation which proved accurate, using an apparatus designed to physically measure the actual volume of bubble formation. Alum acted similar to hydrochloric acid for initializing bubble formation, and higher initial alkalinity, lower final solution pH, and increased mixing rate tended to increase bubble formation. Lastly, the protocol outlined in Standard Methods for predicting the degree of supersaturation was examined, and when compared to this work, the Standard Methods approach produces an error up to 16% for conditions found in water treatment. Air entrainment and ozonation are the key causes of dissolved gas supersaturation and eventual bubble formation in water treatment plants. Total dissolved gas probes (TDGP) are now available to directly measure supersaturation and have many advantages compared to conventional techniques. Bubble formation during coagulation-flocculation hindered particle sedimentation, producing settled turbidities double that of solutions without dissolved gases. In a filtration study, run time to one half of initial flow was decreased by 54% when the source water was increased from 0.1 to 0.2 atm supersaturation. Indeed, even at 0.05 atm supersaturation, run length was only 21 hours in solutions without added particulate matter. A case study confirmed that bubble formation can interfere with coagulation and filtration processes at conventional treatment plants. / Master of Science

Removal of organic matter is increasingly important to drinking water utilities and consumers. Organic matter is a significant precursor in the formation of disinfection by-products (DBPs). The maximum contaminant levels for (DBPs) are decreasing and more DBPs are believed carcinogenic. Traditional coagulation focuses on the removal of particulate matter and in the last decade soluble species have also been targeted with high coagulant doses. However, colloidal matter is smaller than particulate matter and therefore not easily removed by conventional drinking water treatment. This research focused on the conversion of soluble organic matter to colloids using relatively low doses of ferric sulfate coagulant and the subsequent removal of the colloids by filtration during drinking water treatment. The goal is to achieve enhanced removal of soluble organic matter with minimal chemical costs and residual formation. This study investigated the effects of pH, iron coagulant dose, turbidity, organic matter concentration, and temperature on colloid formation. Characterization of the colloidal organic matter was attempted using zeta potential and sizing analyses. Cationic low molecular weight, nonionic high molecular weight, and cationic medium molecular weight polymers were evaluated on their removal of colloidal organic matter. Colloidal organic matter formation was affected by changes in coagulation pH, coagulant dose, and organic matter concentration, whereas turbidity and temperature did not significantly impact colloid formation. Decreased coagulation pH caused increased organic carbon removal. As coagulant dose was increased, colloid formation initially increased to maximum and subsequently rapidly decreased. Colloid formation was increased as the organic matter concentration increased. Due to low sample signal, the colloids could not be characterized using zeta potential and sizing analyses. In addition, polymers were ineffective for aggregating colloidal organic matter when used as flocculant aids. / Master of Science

This research evaluated natural organic matter (NOM) dissolved-solid phase separation resulting from alum coagulation under the following sourcewater conditions: pH, initial NOM concentration, initial turbidity, and temperature. The solid phase was partitioned into two operationally defined size fractions; colloidal matter was defined as organic carbon (OC) retained by a 30 kilodalton ultrafiltration membrane, and particulate matter was defined as OC retained by a 1μm glass-fiber filter. Coagulation pH had a considerable impact on residual OC colloid formation, signified by more colloids formed as a function of alum dose at pH 6.8 as compared to pH 5.8. Initial NOM concentration strongly influenced the alum dose range over which OC colloid formation occurred and was found to be a proportional relationship. The presence of bentonite clay (used as the initial turbidity source) somewhat affected OC colloid formation by exerting some amount of coagulant demand, signified by decreasing OC colloid formation with increasing initial turbidity. Coagulation temperature had a considerable impact on particulate matter formation, as there was an increase in the dose at which particle formation first occurred at 4 ºC when compared to 25 ºC. Phase separation of OC from dissolved to colloidal matter was very similar at both 4 ºC and 25 ºC. The ability for low doses of polymers to replace a large portion of alum in order to further aggregate colloids during flocculation was unsuccessfully investigated. OC phase separation resulting from alum and iron sulfate coagulation was compared on a molar coagulant metal basis. The amount of residual OC associated with colloidal matter was similar, while the critical coagulant dose at which particulate matter formed was shifted to a much higher dose for iron. / Master of Science

A stability theory for lyophobic colloids was put forth in the 1940’s by Derjaguin, Landau, Verwey, and Overbeek. This theory, known as DLVO theory, has gone through the test of time and survived as a pillar of colloid science. In the present work, this theory has been used for describing the behavior of fine coal and silica particles in aqueous media. It has been found, however, that the classical DLVO theory is applicable only to weakly hydrophobic solids but not to very hydrophobic ones. The coagulation experiments conducted with very hydrophobic particles suggest that there exists a strong attractive force that has not been considered in the theory. This non-DLVO force has been estimated in the present work based on the data obtained from coagulation experiments. Contributions from the non-DLVO force, which is referred to as hydrophobic interaction energy (V_H), have been related to the nondispersion component of work of adhesion of water on solids (W^nd_a). An expression for V_H which is now a function of W^nd_a, has been added as a third term in the DLVO equation in order to better describe the stability of colloidal suspensions regardless of the hydrophobicity of the particles involved. A population balance model for a system of isotropic turbulent flow has been developed. Both aggregate growth and breakage have been considered in the model and their rate constants have been derived from a phenomenological approach. Numerical procedures have been proposed for solving the coagulation kinetic equations. Computer simulations show that the model is fairly flexible and the results are in reasonable agreement with experiment. / Ph. D.

Le Tanfloc est un coagulant organique naturel à base d’extrait de tanin. Ce produit est issu du métabolisme de certaines plantes, dont l’Acacia mearnsii, qui est utilisé pour sa production. Selon certaines études, le Tanfloc présenterait des performances d'abattement de la matière organique dissoute comparables à celles de l’alun dans le traitement des eaux de surface, avec l’avantage de ne pas être influencé par le pH. Il pourrait être également utilisé comme l’alun, dans le traitement des eaux usées, pour l'abattement de la DCO (Demande chimique en Oxygène), de la MES (Matière En Suspension) et de la demande biologique en oxygène. Contrairement aux autres coagulants organiques naturels, tels que le Moringa, le Tanfloc est présenté comme le coagulant organique qui pourrait être un alternatif aux coagulants inorganiques tels que l’alun, dont l’utilisation présente quelques inconvénients. Les objectifs de cette étude, sont d’améliorer les connaissances sur ce coagulant : ses mécanismes de coagulation, ses performances, et d’étudier les possibilités de son utilisation dans le traitement des eaux usées et dans la potabilisation en comparaison avec l’alun. En eau potable, les résultats de séries de jar tests réalisées avec des sources d’eau différentes (Fleuve Saint Laurent et la Rivière Chaudière), montrent que le résiduel de COD (Carbone organique dissous) du Tanfloc en plus de dégrader la qualité de l’eau traitée, implique une demande en chlore plus élevée. Cependant, il semble que cet apport de COD ne soit pas précurseur des sous-produits de désinfection. En outre, tout comme l’alun, ses performances sont influencées par la variation du pH, de la dose et des caractéristiques de l’eau brute. En eaux usées, bien qu’il ne permette pas l’abattement de l’azote total et du phosphore total, le Tanfloc pourrait être utilisé dans le traitement des eaux usées (traitement primaire), car il permet selon nos résultats un meilleur abattement de la DCO et de la MES que l’alun. En outre, le caractère organique des boues produites suite à l’utilisation du Tanfloc pourrait améliorer leurs valorisations. / Tanfloc is a tannin extracts-based natural and organic coagulant. Tannin is derived from the metabolism of some plants like Acacia mearnsii which is used in its production. Some studies suggest that Tanfloc offers a reduction performance of dissolved organic matter similar to that of alum for the treatment of surface waters, with the advantage that the pH has no impact on it. Like alum, it can be used in waste water treatment to reduce Chemical Oxygen Demand (COD), Suspended Solids (SS), and Biological Oxygen Demand. Unlike other natural organic coagulants such as Moringa, Tanflloc is seen as the organic coagulant which might be a suitable alternative to inorganic coagulants like alum whose use presents some disadvantages. The objectives of this research were to improve on the knowledge about this coagulant: how it works, its performance; and to investigate its potential use in waste and potable water treatment as compared to alum. As far as potable water is concerned, the results of jar tests on different water sources (St. Lawrence River and Chaudière River) show that Tanfloc’s DOC (Dissolved Organic Carbon) residual does not only degrade treated water quality, but also involves a higher chlorine demand. However, it appears that this COD residue is not a harbinger of disinfection by-products. Moreover, as it is the case with alum, pH variation, dosing, and raw water characteristics have an impact on Tanfloc’s performance. As far as waste water is concerned, although Tanfloc does not reduce total nitrogen and total phosphorus, it can be used in waste water treatment (primary treatment) because our results suggest that it enables a better reduction of COD and SS than alum. Furthermore, the resulting sludge from Tanfloc’s use is organic and this can add to its valuation.

Thrombin is a key enzyme of the coagulation cascade exhibiting important roles in both pro-coagulation and anti-coagulation processes. Most clinically used anticoagulant drugs, including polymeric heparin, warfarin, hirudin, argatroban and the recently approved dabigatran, aim to reduce thrombin activity. There are several binding domains on thrombin including the active site, anion-binding exosites I and II, and the sodium binding site. We hypothesized that thrombin may be better regulated through an allosteric process mediated by small molecules binding to either exosite I or II. An appropriately designed allosteric regulator that reduces the procoagulant signal in a finely tuned manner may maintain a delicate balance between procoagulant and anticoagulant signals in blood resulting reduced bleeding complications. In this work, we synthesized and studied a library of potent, small, aromatic molecules as allosteric inhibitors of thrombin. Of the 28 potential inhibitors, 11 molecules inhibited thrombin with reasonable potency. Structure activity relationship studies showed that sulfation at the 5-position of the benzofuran scaffold was essential for targeting thrombin. Michaelis-Menten kinetic studies indicated a non-competitive, allosteric mechanism of inhibition. Site-directed mutagenesis, competitive binding and molecular modeling studies led to the identification of the most plausible binding pose for a potent sulfated dimer. To further improve the potency, a small library of sulfated benzofuran trimers was synthesized and studied for thrombin inhibition. Further, to find new scaffold to inhibit thrombin allosterically, docking-based virtual screening approach was used. All these molecules were found to be moderately potent thrombin inhibitors and can serve as lead to develop allosteric inhibitor. Overall, this work presents the first small, synthetic, sulfated aromatic molecules as potent allosteric modulators of thrombin. Finally, this work also highlights the opportunity of exploring allosteric modulators of other coagulation enzymes, e.g., factors Xa, IXa and XIa, based on the sulfated benzofuran scaffold.

Les lymphomes T cutanés constituent un ensemble hétérogène de lymphomes non-hodkinniens. Les lymphomes T cutanés sont définis par une prolifération clonale de lymphocytes T malins et de cellules NK (natural killers) de localisation cutanée. Dans l'Union Européenne, l'indication du bexarotène par voie orale est le traitement des manifestations cutanées des lymphomes cutanés T épidermotropes (LCT), au stade avancé (ou dès un stade précoce aux États Unis). L'objectif de ce travail a été d'étudier les effets indésirables du bexarotène chez les patients atteints de lymphome cutané. Nous nous sommes plus particulièrement intéressés aux interactions du bexarotène avec le système de la coagulation et avec le métabolisme lipidique.Dans la première partie de nos travaux, nous avons étudié l'origine de la coagulopathie induite par le bexarotène. Nous avons montré que le bexarotène inhibe les facteurs IX et X de la coagulation, ce qui provoque le prolongement du temps de coagulation ; ces effets pourraient être à l'origine de la coagulopathie observée chez les patients traités au bexarotène.Dans la deuxième partie, nous avons montré que le bexarotène interagit également avec le métabolisme lipidique par l'activation du CETP et par l'inhibition de la lipoprotéine lipase, ce qui provoque une chlolésterémie et une triglyéridémie.Enfin, nous avons présenté les résultats préliminaires de l'essai clinique organisé au niveau de la région ouest de la France sur les effets indésirables du bexarotène.Les résultats de ce travail mettent en évidence l'importance de la structure chimique du bexarotène (effets de charges) dans la neutralisation des facteurs de coagulation d'une part et dans le mécanisme d'interaction avec le métabolisme lipidique (CETP et lipoprotéine lipase) d'autre part. La compréhension des mécanismes moléculaires conduisant à une coagulopathie et à la dyslipidémie représente un enjeu majeur pour prévenir la toxicité du bexarotène et pour permettre une meilleure prise en charge du patient. / The cutaneous T cell lymphomas are a heterogeneous group of non-lymphoma hodkinniens. The cutaneous T cell lymphomas are defined by a clonal proliferation of malignant T cells and NK (natural killer) cells from skin location. In the European Union, the indication of oral bexarotene is the treatment of cutaneous manifestations of cutaneous T-cell lymphomas epidermotropic (LCT) to advanced (or at an early stage in the United States). The objective of this work was to study the adverse effects of bexarotene in patients with cutaneous lymphoma. We are particularly interested in the interactions of bexarotene with the coagulation system and lipid metabolism.In the first part of our work, we studied the origin of the coagulopathy induced by bexarotene. We have shown that bexarotene inhibited factors IX and X of the coagulation causing the extension of clotting time and coagulopathy that was observed in bexarotene-treated patients. In a second part, we showed that Bexarotene induces in vitro cholesteryl ester transfer protein activity, and suppress lipoprotein lipase activity in human plasma.Finally, we presented the preliminary results of the clinical trial conducted at the western region of France on the adverse effects of bexarotene.The results of this study highlight the importance of the chemical structure of bexarotene (charge effects) in the neutralization of coagulation factors mechanisms on the one hand and in its interaction with lipid metabolism (CETP and lipoprotein lipase) on the other hand. Understanding the molecular mechanisms leading to coagulopathy and dyslipidemia is a major issue to prevent the toxicity of bexarotene and to elicit better management of the bexarotene-treated patient.

Les adénovirus (Ad) sont parmi les vecteurs les plus utilisés en thérapie génique. Cependant, les données d’essais pré-Cliniques et cliniques ont montré qu’ils induisaient une forte toxicité hépatique consécutive à leur tropisme hépatique, une réponse inflammatoire et une forte thrombocytopénie. Différents travaux avaient montré que l’interaction de l’Ad avec le facteur de la coagulation X (FX).était responsable de la transduction in vivo des hépatocytes après administration systémique des vecteurs Ad. Cependant, des résultats précédents du laboratoire avaient montré également que le pseudotypage d’une autre protéine de capside, la fibre, permettait de réduire la transduction hépatique. Dans le but de mieux comprendre le rôle de la fibre dans le tropisme et la toxicité des Ad, nous avons comparé des Ad recombinants pseudotypés pour tout (tige et tête de la fibre : AdF3) ou partie (tige : AdS3K5) de la fibre Ad3 avec un Ad5 à capside non modifiée (Adwt). Après administration systémique chez la souris, l’AdF3 et l’AdS3K5 induisent une plus faible expression du transgène dans le foie et la rate comparativement à l’Ad5wt. Cette réduction ne résulte ni d’un défaut de capture de ces vecteurs dans le foie ni de leur incapacité à utiliser le FX. Cependant, nos résultats ont révélé que les Ad pseudotypés par la fibre Ad3 étaient capturés de façon plus importante par les cellules de Kupffer. Nous avons montré que cette capture était une propriété intrinsèque de la fibre Ad3 puisqu’elle était observée également après administration systémique d’un Ad de sérotype 3. De façon intéressante, les Ad pseudotypés par la fibre Ad3 restent capables de transférer des gènes dans les tumeurs aussi efficacement que l’Adwt.Dans la deuxième partie de nos travaux, nous avons cherché à mieux comprendre les mécanismes de la thrombocytopénie consécutive à l’administration d’Ad. Nous avons défini la cinétique de la thrombocytopénie ainsi que l’effet de la dose virale. Nous avons montré que certains facteurs de l’hôte comme les facteurs de la coagulation ou la rate n’étaient pas impliqués dans la thrombocytopénie. De façon intéressante, nous avons montré que la fibre Ad5 jouait un rôle dans l’induction de la baisse plaquettaire puisque l’administration des virus à fibre Ad3 n’induisait plus de forte baisse plaquettaire. Parallèlement, nous avons observé un profil inflammatoire associé à l’administration des Ad à fibre modifiée beaucoup plus réduit que celui de l’Adwt. Nos travaux en cours évaluent l’existence possible d’une corrélation entre la production de cytokines/chimiokines et la thrombocytopénie.L’ensemble de ces résultats montre que le pseudotypage des Ad5 par la fibre de l’Ad3 permet de réduire leur toxicité et de limiter la réponse inflammatoire tout en conservant un transfert de gènes efficace dans les tumeurs. L’introduction de ce type de modification de capside dans les Ad oncolytiques devrait permettre de conserver leur capacité à se répliquer dans les tumeurs tout en limitant les toxicités liées à leur dissémination par voie systémique. / To date adenoviruses (Ad) are the most used vectors in gene therapy. However, Ad use is hampered by a strong liver tropism that leads to hepatotoxicity, a strong inflammatory response and the induction of thrombocytopenia. Binding of Ad hexon to coagulation factor X (FX) is responsible for hepatocyte transduction in vivo. As a consequence, mutation of hexon protein abrogates Ad interaction with FX and reduces liver transduction. However, previous results of our lab have demonstrated that Ad5 pseudotyping with fiber Ad3 also resulted in significant reduction of liver transduction. To understand how fiber modification affects in vivo Ad tropism, we used two pseudotyped viruses with whole (AdF3) or only the shaft (AdS3K5) of Ad3 fiber.Following systemic delivery of fiber-Modified Ads, a reduced transduction was observed 2 days p.i. in liver and spleen. This reduction was not due to the impairment of fiber-Modified Ads liver entry or FX use in vivo. Remarkably, after Kupffer cells depletion, a restored transgene expression level was observed, suggesting that fiber-Modified Ads are strongly uptaken by Kupffer cells. We have demonstrated that this strong uptake is an Ad3 intrinsic property since Ad3 was also strongly uptaken by Kupffer cells. Interestingly, fiber-Modified Ads transduce tumours as efficiently as Ad5. In the second part of this work, we aimed to better understand the mechanism of Ad-Induced thrombocytopenia. We first defined the kinetic and dose-Dependence of Ad-Induced thrombocytopenia. Then, we have shown that factors of the host such as the coagulation factors and the spleen were not involved in the thrombocytopenia development. Interestingly, we demonstrated o role for Ad5 in this platelet count reduction since fiber-Modified Ad induced only a modest thrombocytopenia. In parallel, we have observed a reduced production of inflammatory cytokine and chemokine following fiber-Modified Ad administration. Experiments are ongoing to investigate a possible correlation between inflammatory responses and thrombocytopenia. Altogether, our findings demonstrate that Ad5 pseudotyping with Ad3 fiber allows à reduced toxicity and inflammatory response while tumour transduction efficacy is remained. Therfore, oncolytic Ad pseudotyped with Ad3 fiber might be potent tool in tumor virotherapy while limiting risk of toxicity.