Introdução: Embora a imagem com segunda harmônica esteja largamente disponível na maioria dos aparelhos de ultra-sonografia, sua acurácia para avaliar a morfologia e a função do apêndice atrial esquerdo (AAE) permanece precariamente caracterizada. Objetivos: Explorar o desempenho diagnóstico da ecocardiografia transtorácica com segunda harmônica (ETTsh) na avaliação do AAE após eventos neurológicos agudos. Métodos: Realizamos um estudo transversal em pacientes com eventos neurológicos isquêmicos agudos, encaminhados para realização de ETTsh e Ecocardiografia Transesofágica (ETE). As análises da área longitudinal máxima e do pico da velocidade de esvaziamento de fluxo do AAE foram realizadas por observadores cegos. Resultados: Foram avaliados 51 pacientes (49% femininas, 62 ± 12 anos) com eventos neurológicos isquêmicos agudos. Contraste ecocardiográfico espontâneo foi observado em 11 (22%) pacientes no AE, em 7(14%) no AAE e em 3 (6%) na aorta torácica descendente. Trombo no AAE foi identificado em apenas 2 (4%) pacientes. O mapeamento e a análise do AAE foi factível na maioria dos casos (98%), tanto para o estudo com Doppler quanto para avaliação da área do AAE. Observamos uma associação positiva e significativa entre o ETTsh e o ETE, tanto para a avaliação das velocidades máximas de esvaziamento do AAE (r=0,63; p<0,001) quanto para a área longitudinal máxima do AAE (r=0,73; p<0,001). Ademais, todos os pacientes com trombos no AAE ou contraste espontâneo (n=7) tiveram velocidade de esvaziamento inferior a 50 cm/s no mapeamento transtorácico (valor preditivo negativo de 100%). Na análise multivariada ajustada para diversos potenciais preditores transtorácicos de risco, a velocidade máxima de esvaziamento do AAE permaneceu independentemente associada com trombos no AAE ou contraste espontâneo. Conclusão: ETTsh pode fornecer informações relevantes a respeito da morfologia e dinâmica do AAE. Em particular, pacientes com velocidades altas de esvaziamento do AAE podem não necessitar de avaliação adicional com ETE. / Background. Although second harmonic imaging is widely available in most ultrasound systems, its accuracy to evaluate the left atrial appendage (LAA) morphology and function remains poorly characterized. Objectives. To explore the performance of second harmonic transthoracic echocardiography (shTTE) in the assessment of LAA after acute neurologic events. Methods. We conducted a cross-sectional survey of patients with acute ischemic neurologic events that underwent both shTTE and transesophageal echocardiography (TEE). Blinded off-line analysis of LAA maximal area and peak emptying velocities were performed. Results. Fifty-one consecutive patients (49% females, 62 ± 12 years) with acute ischemic neurologic events were evaluated. Spontaneous echocardiographic contrast was observed in 11(22%) patients in the LA, in 7(14%) in the LAA and in 3 (6%) in the thoracic aorta. LAA thrombus was identified in only 2(4%) patients. Scanning and analysis of the LAA was feasible in most patients (98%) both for Doppler and LAA area assessment. We observed a positive and significant association between shTTE and TEE assessment of both LAA peak emptying velocities (r=0.63, p<0.001) and LAA maximum area (r=0.73, p<0.001). In addition, all patients (n=7) with LAA thrombus or spontaneous contrast had peak emptying velocities inferior to 50 cm/s on transthoracic scanning (negative predictive value of 100%). In multivariate analysis adjusted for several potential transthoracic predictors of risk, LAA peak-emptying velocity remained independently associated with LAA thrombus or spontaneous contrast. Conclusions. shTTE can provide valuable information of LAA morphology and dynamic. In particular, patients with high peak emptying velocities might not need additional TEE imaging.

abstract: Thrombus (blood clot) formation is at the roots of hemostasis and pathological thrombosis. Although many studies have successfully elucidated the cellular and molecular mechanisms underlying thrombus formation, there is still a void in understanding the processes limiting thrombus growth beyond that needed for stabilization. As a hemostatic thrombus grows, its surface consisting primarily of platelets changes to that composed of fibrin, which mechanically stabilizes the thrombus. Formation of fibrin ceases after some time; however, it is unclear why this fibrin is non-thrombogenic. This is puzzling since fibrin is known to support strong integrin-mediated adhesion of both platelets and leukocytes in vitro. Therefore, it would be expected that the fibrin surface of hemostatic thrombi in the circulation also support accumulation of these cells and thus continuous thrombus growth or degradation. Nevertheless, many in vivo studies did not detect any accumulation of blood cells including platelets at the fibrin surfaces of thrombi. This finding suggests the existence of natural processes that modulate the adhesive properties of fibrin to ensure proper regulation of thrombus growth, stability and degradation. In this dissertation, I document and discuss the findings supporting the existence of anti-adhesive mechanisms and their physiological relevance in surface-mediated control of thrombus growth and stability. The studies discussed in my dissertation have the potential to establish a novel aspect of hemostasis. Furthermore, it may provide new insights into the intricate and dynamic interplay between the mechanisms underlying hemostatic balance, which is essential to understanding the dysfunction of this process during pathological conditions. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2016

Introdução: Embora a imagem com segunda harmônica esteja largamente disponível na maioria dos aparelhos de ultra-sonografia, sua acurácia para avaliar a morfologia e a função do apêndice atrial esquerdo (AAE) permanece precariamente caracterizada. Objetivos: Explorar o desempenho diagnóstico da ecocardiografia transtorácica com segunda harmônica (ETTsh) na avaliação do AAE após eventos neurológicos agudos. Métodos: Realizamos um estudo transversal em pacientes com eventos neurológicos isquêmicos agudos, encaminhados para realização de ETTsh e Ecocardiografia Transesofágica (ETE). As análises da área longitudinal máxima e do pico da velocidade de esvaziamento de fluxo do AAE foram realizadas por observadores cegos. Resultados: Foram avaliados 51 pacientes (49% femininas, 62 ± 12 anos) com eventos neurológicos isquêmicos agudos. Contraste ecocardiográfico espontâneo foi observado em 11 (22%) pacientes no AE, em 7(14%) no AAE e em 3 (6%) na aorta torácica descendente. Trombo no AAE foi identificado em apenas 2 (4%) pacientes. O mapeamento e a análise do AAE foi factível na maioria dos casos (98%), tanto para o estudo com Doppler quanto para avaliação da área do AAE. Observamos uma associação positiva e significativa entre o ETTsh e o ETE, tanto para a avaliação das velocidades máximas de esvaziamento do AAE (r=0,63; p<0,001) quanto para a área longitudinal máxima do AAE (r=0,73; p<0,001). Ademais, todos os pacientes com trombos no AAE ou contraste espontâneo (n=7) tiveram velocidade de esvaziamento inferior a 50 cm/s no mapeamento transtorácico (valor preditivo negativo de 100%). Na análise multivariada ajustada para diversos potenciais preditores transtorácicos de risco, a velocidade máxima de esvaziamento do AAE permaneceu independentemente associada com trombos no AAE ou contraste espontâneo. Conclusão: ETTsh pode fornecer informações relevantes a respeito da morfologia e dinâmica do AAE. Em particular, pacientes com velocidades altas de esvaziamento do AAE podem não necessitar de avaliação adicional com ETE. / Background. Although second harmonic imaging is widely available in most ultrasound systems, its accuracy to evaluate the left atrial appendage (LAA) morphology and function remains poorly characterized. Objectives. To explore the performance of second harmonic transthoracic echocardiography (shTTE) in the assessment of LAA after acute neurologic events. Methods. We conducted a cross-sectional survey of patients with acute ischemic neurologic events that underwent both shTTE and transesophageal echocardiography (TEE). Blinded off-line analysis of LAA maximal area and peak emptying velocities were performed. Results. Fifty-one consecutive patients (49% females, 62 ± 12 years) with acute ischemic neurologic events were evaluated. Spontaneous echocardiographic contrast was observed in 11(22%) patients in the LA, in 7(14%) in the LAA and in 3 (6%) in the thoracic aorta. LAA thrombus was identified in only 2(4%) patients. Scanning and analysis of the LAA was feasible in most patients (98%) both for Doppler and LAA area assessment. We observed a positive and significant association between shTTE and TEE assessment of both LAA peak emptying velocities (r=0.63, p<0.001) and LAA maximum area (r=0.73, p<0.001). In addition, all patients (n=7) with LAA thrombus or spontaneous contrast had peak emptying velocities inferior to 50 cm/s on transthoracic scanning (negative predictive value of 100%). In multivariate analysis adjusted for several potential transthoracic predictors of risk, LAA peak-emptying velocity remained independently associated with LAA thrombus or spontaneous contrast. Conclusions. shTTE can provide valuable information of LAA morphology and dynamic. In particular, patients with high peak emptying velocities might not need additional TEE imaging.

This research will investigate techniques to create a sensor that is responsive to methane at 23°C. The approach will use the integration of a very thin film, which changes its resistive properties when methane gas is applied, deposited atop the surface of a piezoelectric substrate. An aluminum thin film interdigital transducer will launch a surface acoustic wave (SAW) that travels under the sensor’s gas-sensitive resistive thin film. The SAW/resistive film interaction changes the SAW amplitude, phase and delay. For this work, three films, tin dioxide (SnO2), zinc oxide (ZnO) and palladium (Pd) [1, 2] will be studied. Gas detection will be shown when combining ZnO and Pd, and, observable change in SAW propagation loss is measured when methane gas is present at the film.

Whether a patient presents with deep vein thrombosis (DVT) or pulmonary embolism (PE) varies based on clinical factors. Patients with factor V Leiden (FVL) typically present with DVT while cancer patients present with PE. The biological mechanisms that determine DVT stability in the progression of DVT to PE are not known. Thus, little is known about the mechanism of thrombus stability, the factors involved, or the effect of anticoagulants on embolization and PE burden. In order to answer these questions, we first need to (i) develop a mouse model to evaluate DVT stability and its relationship with PE burden when treated with anticoagulants, (ii) determine if anticoagulants, by inhibiting thrombin, require FXIII to decrease thrombus stability, (iii) determine the effects of attenuating fibrinolysis, using epsilon aminocaproic acid (ε-ACA or EACA), supplemental FXIII and α2-AP, on clot stability and (iv) utilize our model to explain the FVL paradox. For our thrombus stability model, the femoral vein of C57BL/6, FXIII deficient (FXIII-/-), FVL heterozygous, or FVL homozygous female mice was subjected to ferric chloride (FeCl3) injury to initiate a non-occlusive thrombus. Treatment with saline, dalteparin, dabigatran, EACA or FXIII was administered 12 minutes after thrombus formation. Intravital videomicroscopy recorded the thrombus sizes and embolic events leaving the thrombus for 2 hours. Lungs were harvested, sectioned and stained for the presence of PE. Total and large embolic events were highest after dabigatran treatment compared to saline or dalteparin in wild-type (WT) mice. Variations in amounts of embolic events were not attributed to variations in thrombus size since thrombus size was similar between the groups. The number of emboli per lung slice was higher in dabigatran-treated mice. Large embolic events correlated positively with the number of emboli per lung slice independent of treatment. Dabigatran treatment in FXIII-/- mice did not alter embolization patterns suggesting that FXIII is required for dabigatran to decrease thrombus stability. EACA increases thrombus size significantly and therefore would not be a feasible alternative to IVC filters, as it will increase DVT size. FXIII marginally increased thrombus size. Treatment with FXIII decreases total and large embolic events in saline-, dalteparin- or dabigatran-treated mice, similar to EACA-treated mice. The number of emboli per lung slice was reduced after treatment with FXIII and EACA compared to non-treated mice. PE burden was not significantly different between FXIII anticoagulated mice or EACA-treated mice. The large embolic events correlate positively with PE burden. FVL heterozygous and homozygous mice had significantly reduced embolization and thrombus size grew significantly over time, this contrasted with WT mice, where thrombus size remained similar to the initial injury. PE burden was significantly reduced in the FVL mice compared to WT. Collectively, these data shows that we have successfully developed a mouse model of acute venous thrombus stability that can quantify emboli and PE burden. Consistent with clinical data, dabigatran, a DTI, was shown to acutely decrease thrombus stability and increase PE burden compared to LMWH or saline; an effect that was FXIII-dependent. Also, attenuating fibrinolysis with EACA, but not FXIII, increases thrombus size; but both increase DVT stability and decrease PE burden. Supplementing α2-AP did not alter thrombus stability. This suggests that administration of FXIII may be a better treatment option for DVT patients who are bleeding than EACA, since EACA may increase DVT size. Lastly, our model can explain the FVL paradox. Those with FVL have stable thrombus formation leading to an increased incidence of symptomatic DVT and a decreased risk of PE. / Thesis / Doctor of Philosophy (PhD)

No description available.

L'objectif de ce travail est de contribuer à une meilleure prédiction de la rupture des Anévrismes de l'Aorte Abdominale. Pour ce faire, des simulations par éléments finis ont été mises en oeuvre sur des anévrismes modèles dans des conditions proches de la réalité physiopathologique, i.e. en tenant compte de l'anisotropie de la paroi anévrismale, du caractère poreux du thrombus et des Interactions Fluide-Structure. Dans la première partie, une étude statique en l'absence du thrombus a permis de mettre en évidence l'influence de la géométrie de l'anévrisme et du comportement mécanique (isotrope ou anisotrope) de la paroi sur la distribution des contraintes, i.e. la rupture potentielle de l'anévrisme, ainsi que sur l'évolution du module de Peterson. Dans la seconde partie, une modélisation poro-hyperélastique du thrombus est proposée, en s'appuyant sur des données expérimentales de la littérature. La prise en compte de ce comportement et des Interactions Fluide-Structure montre que la pression intra-thrombus demeure du même ordre de grandeur que la pression intra-luminale, conformément à des mesures in vivo réalisées par ailleurs. Enfin, nous montrons que ceci n'est pas en contradiction avec une réduction du risque de rupture potentielle de l'anévrisme. / The aim of this work is to contribute to a better prediction of the Abdominal Aortic Aneurysm rupture (AAA). For that purpose, finite elements simulations have been performed on idealized AAA models under physiopathological like conditions, by taking into account the aneurysmal wall anisotropy, the intra-luminal thrombus porosity and the Fluid-Structure Interactions. In the first part, the influence of the aneurysm geometry and its wall properties (isotropic or anisotropic hyperelasticity) on the wall stress distribution and the Peterson's modulus has been studied in a static analysis and without taking into account the thrombus. In the second part, based on the experimental results from the litterature, a porohyperelastic model has been proposed for the thrombus. By considering such behavior for the thrombus and the Fluid-Structure Interactions, we observe that the intra-thrombus pressure is the same order as the intra-luminal pressure, which is consistent with in vivo measurements. Our results show that despite this unchanged pressure, the maximum wall stress decreases leading to a decrease of the aneurysm potential rupture.

L'élévation de la concentration du calcium (Ca2+) cytosolique est responsable de l’activation plaquettaire. Cette élévation est due à l'entrée du Ca2+ à partir du milieu extérieur (influx) ou sa translocation (mobilisation) dans le cytosol depuis ses réserves internes. Les SERCAs (Sarco/Endoplasmic Reticulum Ca2+ ATPases) pompent le Ca2+ depuis le cytosol vers les réserves internes, maintenant le Ca2+ cytosolique bas (100 nM) et les plaquettes au repos. D'autre part elles assurent une concentration calcique élevée (≥1 mM) dans les réserves calciques permettant sa mobilisation, et enfin modulent l'intensité et la forme du signal calcique lors de l'activation. Mais les rôles respectifs des SERCAs plaquettaires, SERCA2b et SERCA3, sont encore mal définis. D’où l’intérêt de mon projet qui a été de déterminer si SERCA3 avait un rôle fonctionnel précis et spécifique. Nous avons observé sur des souris SERCA3-/- un défaut de l'hémostase et s’accompagne d’une résistance à la thrombose dû à un déficit de sécrétion d'ADP, entrainant un défaut d'agrégation et d'adhérence. SERCA3 semble contrôler une voie de sécrétion initiale d'ADP capable d’agir en synergie avec une faible activation plaquettaire, aboutissant à un renforcement de la sécrétion et de l'agrégation. De plus, l’utilisation des inhibiteurs pharmacologiques spécifiques de SERCA2b (thapsigargine) ou SERCA3 (tBHQ), a montré que la sécrétion initiale d'ADP n'était pas dépendante de la mobilisation des réserves SERCA2b mais dépendait spécifiquement des réserves SERCA3. Nous avons retrouvé la même voie de sécrétion d'ADP dépendante de SERCA3 dans les plaquettes humaines. Nous avons en particulier montré par le suivi d'une cohorte de patientes atteintes d'obésité morbide, un déficit d'agrégation, une faible mobilisation calcique et un taux faible de SERCA3 plaquettaire, revenus à la normale après retour à un poids normal après chirurgie bariatrique. Surtout nous avons retrouvé, dans les plaquettes de ces patientes obèses, un défaut de sécrétion d'ADP associé au défaut de SERCA3. Il s'agit du premier défaut de SERCA3 plaquettaire lié à une pathologie humaine. Nous avons ensuite montré que la sécrétion initiale d'ADP était rapide (5 sec) et entièrement dépendante de SERCA3. A l'aide d'une sonde calcique fluorescente membranaire (FURA2-NearMem-AM), nous avons démontré l’existence d’une mobilisation calcique juxta-membranaire spécifique de SERCA3, indépendante de l'ADP, correspondant donc à une sécrétion primaire. Cette mobilisation SERCA3 s'est avérée indépendante d'IP3, mais dépendante du NAADP, qui mobilise spécifiquement les réserves SERCA3 et non SERCA2b. En conclusion, nous avons mis en évidence une nouvelle voie d'activation plaquettaire, indépendante de l'IP3 mais dépendante du NAADP qui libère le Ca2+ des stocks internes dépendants de SERCA3 et spécifiquement engagés dans la libération précoce d'ADP lors de l'activation plaquettaire. Ces données identifient de nouvelles cibles avec un intérêt thérapeutiques anti-thrombotiques potentiel. / The elevation of cytosolic calcium (Ca2 +) is responsible for platelet activation. This elevation is due to the entry of Ca2 + from the extracellular medium (influx) where its translocation (mobilization) into the cytosol from its Ca2+ stores. SERCAs (Sarco / EndoplasmicReticulumCa2 + ATPases) pump Ca2 + from the cytosol to the Ca2+ stores, maintaining low cytosolic Ca2 + (100 nM) and platelets at resting state. On the other hand, they ensure a high calcium concentration (≥ 1 mM) in the Ca2+ stores allowing its mobilization, and finally modulate the intensity and the shape of the Ca2 signal during the activation. However, the respective roles of SERCA platelets, SERCA2b and SERCA3, are still poorly defined. Hence the interest of my project which was to determine if SERCA3 had a precise and specific functional role. We observed in SERCA3 - / - mice a defect in hemostasis that is accompanied by resistance to thrombosis due to ADP secretion deficiency, resulting in a lack of aggregation and adhesion. SERCA3 seems to control an initial pathway of ADP secretion able to acting in synergy with low platelet activation, resulting in increased secretion and aggregation. In addition, the use of specific pharmacological inhibitors of SERCA2b (thapsigargine) or SERCA3 (tBHQ) showed that the initial secretion of ADP was not dependent on the mobilization of SERCA2b stores, but was specifically dependent on SERCA3 stores. We found the same SERCA3-dependent ADP secretion pathway in human platelets. In particular, we observed a defect of platelet aggregation, low Ca2+ mobilization and low platelet SERCA3 levels in a cohort of patients with morbid obesity compared to control subjects. Platelet functions and SERCA3 levels are restored after weight loss by a surgery bariatric. Above all, we found in the platelets of these obese patients, a defect of secretion of ADP associated with the defect of SERCA3. This is the first defect of platelet SERCA3 related to human pathology. We then showed that the initial secretion of ADP was rapid (5 sec) and entirely dependent on SERCA3. Using a membrane fluorescent Ca2+ probe (FURA2-NearMem-AM), we have demonstrated the existence of a juxta-membrane-specific calcium mobilization specific to SERCA3, independently of ADP, corresponding to primary secretion. This SERCA3 mobilization proved to be independent of IP3, but dependent on the NAADP, which specifically mobilizes the SERCA3 and not SERCA2b reserves. In conclusion, we have demonstrated a new platelet activation pathway, independent of IP3 but dependent on NAADP, which releases Ca2 + from SERCA3 dependent stores and specifically involved in the early release of ADP during platelet activation. These data identify new targets with a potential interest of anti-thrombotic therapy.

Malgré des progrès significatifs dans le diagnostic précoce, on constate ces dernières années une nette tendance à l'augmentation du nombre de patients atteints de carcinome à cellules rénales (CCR) présentant une complication spécifique sous la forme d'un thrombus tumoral (TT) s'étendant le long de la lumière de la veine cave inférieure (VCI) au-dessus du niveau du diaphragme, jusqu'à l'atrium droit. Le seul traitement efficace du CCR avec TT reste la chirurgie. La chirurgie conventionnelle comprend la néphrectomie radicale avec thrombectomie, nécessitant des techniques de pontage cardio-pulmonaire et l'arrêt de la circulation sanguine, ce qui implique toujours des complications graves. Pour les éviter, diverses options d'accès chirurgical à la partie supradiaphragmatique de la VCI et à l'atrium droit ont été proposées exclusivement par le côté abdominal, en utilisant diverses options de diaphragmotomie et de péricardotomie avec isolement de la VCI dans la cavité péricardique. L'un des points les plus importants dans le traitement chirurgical du CCR avec TT supradiaphragmatique, est le contrôle de l'extrémité distale du TT. Cette étape dépend largement de l'accès choisi. Dans ce travail, afin d'optimiser les techniques chirurgicales pour les patients atteints de TT supradiaphragmatique, nous avons comparé la faisabilité et la sécurité des différents accès chirurgicaux transdiaphragmatiques. Nous avons également proposé un accès transdiaphragmatique transpéricardique combiné comprenant des diaphragmotomies en forme de T et circulaires, combinées à des péricardotomies longitudinales et circulaires. Nous avons utilisé quatre corps préservés avec la méthode Thiel pour réaliser les simulations chirurgicales. La néphrectomie radicale avec thrombectomie a été réalisée par deux experts de manière indépendante. Quatre approches chirurgicales de la VCI supradiaphragmatique ont été réalisées consécutivement. Nous avons utilisé des simulateurs de TT de différents diamètres et densités pour étudier la palpation de l'apex du TT et son déplacement. La faisabilité et la sécurité de chaque accès et de chaque procédure chirurgicale ont été évaluées à l'aide du test du Chi carré, avec correction des comparaisons multiples (Bonferroni). Notre travail a confirmé la faisabilité et la sécurité des approches transdiaphragmatiques. Les accès transpéricardiques sont préférables dans les cas où les TT sont plus élevés. Ils constituent une alternative adéquate à la chirurgie conventionnelle en cas de TT flottant supradiaphragmatique, jusqu’à l’atrium droit. / Despite significant progress in early diagnosis, there has been a clear trend in recent years towards an increasing number of renal cell carcinoma (RCC) patients with a specific complication in the form of a tumor thrombus (TT) extending along the lumen of the inferior vena cava (IVC) above the level of the diaphragm into the right atrium. The only effective treatment for RCC with TT remains surgery. Conventional surgery includes radical nephrectomy with thrombectomy, requiring cardiopulmonary bypass techniques and cessation of blood flow, which always involves serious complications. To avoid them, various options for surgical access to the supradiaphragmatic part of the IVC and the right atrium have been proposed exclusively from the abdominal side, using various options of diaphragmotomy and pericardotomy with isolation of the IVC in the pericardial cavity. One of the most important issues in the surgical treatment of RCC with supradiaphragmatic TT is the control of the distal end of the TT. This step depends largely on the access chosen. In this work, to optimize surgical techniques for patients with supradiaphragmatic TT, we compared the feasibility and safety of different transdiaphragmatic surgical accesses. We also proposed a combined transdiaphragmatic transpericardial access including T-shaped and circular diaphragmotomies combined with longitudinal and circular pericardotomies. We used four bodies preserved with the Thiel method to perform the surgical simulations. Radical nephrectomy with thrombectomy was performed by two experts independently. Four supradiaphragmatic ICV surgical approaches were performed consecutively. We used TT simulators of different diameters and densities to study palpation of the TT apex and its displacement. The feasibility and safety of each access and surgical procedure were evaluated using the Chi-square test with correction for multiple comparisons (Bonferroni). Our work confirmed the feasibility and safety of transdiaphragmatic approaches. Transpericardial accesses are preferable in cases with higher TT. They are an adequate alternative to conventional surgery in cases of supradiaphragmatic floating TT, up to the right atrium.

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