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Development of a non-contact blood rheometer using acoustic levitation and laser scattering techniquesAnsari Hosseinzadeh, Vahideh 04 June 2019 (has links)
Coagulopathy, a condition in which blood coagulation is impaired, can be inherited or result from a variety of conditions including severe trauma, illness or surgery. Perioperative monitoring of a patient’s coagulation status is important to identify coagulopathic patients. Thromboelastography or TEG remains the gold standard for whole blood coagulation monitoring. However, TEG suffers from certain well-documented drawbacks such as contact containment and manipulation of the blood sample, large and uncontrolled strain, and the inability to distinguish the contribution of elasticity and viscosity during blood coagulation. We developed a non-contact blood rheometer which uses a single drop of blood to measure its viscoelastic properties. Small sample size (typically 5-15 μL), low shear strain (linear viscoelasticity), and non-contact manipulation and containment of samples make this technique unique for real-time monitoring of blood coagulation.
In the first part of this work, we addressed the development of the technique, benchmarking the results against known material properties standards. We observed large amplitude oscillations of the levitated drop results in multiple resonance modes and excessive dissipation. We suggested upper bound limits for drop oscillation amplitudes required to satisfy the Lamb theoretical expressions for drop frequency and damping. In the second part, we applied our technique to study sickle-cell disease. Our technique showed that the shape oscillation of blood drops was able to assess an abnormally increased viscosity in sickle cell patients when compared with normal controls over a range of hematocrit. Furthermore, the technique was sensitive enough to detect viscosity changes induced by hydroxyurea treatment. The third part of this work focused on blood coagulation monitoring. The technique showed sensitivity to coagulation parameters, such as platelet count, calcium ion concentration, and hematocrit. A comparison of the results with TEG showed coagulation started sooner in the levitation technique, but with a lower rate and lower maximum stiffness. Thus, the technique developed can be used as a monitoring tool to assess blood mechanical properties sensitively enough to be of use in clinical diagnostic settings. / 2020-06-04T00:00:00Z
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Efeitos da fotobiomodulação na adesão e proliferação das células-tronco da papila apical humana em scaffold de quitosana com incorporação de coágulo sanguíneo. Estudo in vitro / Effects of photobiomodulation on adhesion and proliferation of stem cells from human apical papilla in chitosan scaffold with blood clot incorporation. In vitro studyAbe, Gabriela Laranjeira 06 September 2016 (has links)
Revascularização é uma técnica utilizada em dentes jovens, que apresentem rizogênese incompleta e danos irreversíveis ao tecido pulpar, necessitando de tratamento endodôntico, para formar novo tecido em lugar da polpa perdida. Clinicamente os resultados mostram a continuidade da rizogênese e a devolução da vitalidade dental. Porém, pouco se sabe sobre o novo tecido formado e não está estabelecido se este é capaz de desempenhar todas as funções da polpa dentária. Para melhorar as características do tecido formado pela técnica da revascularização, podemos utilizar ferramentas de engenharia tecidual, como célulastronco, fatores de crescimento e arcabouços de sustentação celular (scaffolds). As célulastronco (CTs) já estão presentes quando o sangue invade o canal radicular, e utilizar essa reserva de CTs que o hospedeiro possui, procedimento conhecido como homing, é uma vantagem em comparação com outras técnicas que injetam CTs obtidas por cultivo em laboratório. Entretanto os aspectos físicos do coágulo sanguíneo formado no interior do canal radicular podem ser melhorados com a adição de hidrogel de quitosana, que interage quimicamente com o sangue e forma um scaffold híbrido mais estável. Então, o objetivo deste estudo foi testar a hipótese de que o scaffold híbrido, composto por hidrogel de quitosana e sangue, ofereceria maior estabilidade física estrutural, bem como condições favoráveis à adesão e proliferação de células-tronco da papila apical humana (SCAPs; do inglês, Stem Cell from Apical Papila). Para isso, investigamos in vitro se a incorporação do sangue ao hidrogel de quitosana gera um scaffold mais estável, se este é favorável à adesão e proliferação de células-tronco da papila apical e se a fotobiomodulação potencializa essas características celulares. Para isso, SCAPs foram isoladas e caracterizadas por citometria de fluxo, tempo de dobra populacional, e contagem de unidades formadoras de colônias fibroblásticas (CFU-F; do inglês, Colony Forming Units - Fibroblastic). Ensaios de incorporação sanguínea, dissolução e embebição foram realizados para determinar o comportamento dos scaffolds híbridos. A adesão celular foi observada pela coloração PHK26® (do inglês, Red Fluorescent Cell Linker) e por microscopias eletrônicas de varredura (MEV); e a proliferação foi investigada pelo ensaio de alamarBlue®. Adicionalmente, a sobrevivência das SCAPs após a degradação do scaffold híbrido foi avaliada pela coloração Live/Dead®. A população celular estudada apresentou características de células tronco. O scaffold híbrido, constituído de densa rede alveolar com poros interconectados, embebeu e dissolveu rapidamente. De acordo com o PKH26® e alamarBlue® SCAPs aderiram e proliferaram no scaffold híbrido. SCAPs fotobiomoduladas exibiram maior taxa de proliferação e o ensaio Live/Dead® mostrou células vivas após 12 dias de cultivo. Concluiu-se que o scaffold híbrido apresenta biocompatibilidade e condições favoráveis de sobrevivência para SCAPs, que são potencializadas pela fotobiomodulação. / Revascularization is a technique used to form a new tissue, replacing the lost pulp, in young permanent teeth presenting incomplete rhizogenesis and irreversible damage, where endodontic treatment is needed. Clinically, the results show the continuity of the root formation and the return of dental vitality. However, little is known about the newly formed tissue and it has not been established if it is able to perform all functions of the dental pulp. To improve the characteristics of the newly formed tissue by the technique of revascularization, tissue engineering tools can be used, represented by stem cells, growth factors and scaffolds for cell supportting. Stem cells (SCs) are already present when the blood invades the root canal, and to use this SCs reserve that the host possesses, procedure known as homing, is an advantage compared with other techniques that inject SCs obtained by cultivation in the laboratory. However the physical aspects of blood clot in the root canal can be improved with the addition of chitosan hydrogel that chemically interacts with the blood and forms a more stable hybrid scaffold. So the aim of this study was to test the hypothesis that the hybrid scaffold, composed of hydrogel chitosan and blood clot, provides greater structural physical stability as well as favorable conditions for adhesion and proliferation of Stem Cells from Apical Papilla (SCAPs). For this, we investigated in vitro if the incorporation of blood to chitosan hydrogel, generates a more stable scaffold and if it supports the stem cell adhesion and proliferation, in addition, if photobiomodulation potentiates these cell characteristics. For this, SCAPs were isolated and characterized by flow cytometry, population doubling time, and counting colony forming units - fibroblastic (CFUF). Blood incorporation assays, dissolution and swelling were conducted to determine the behavior of hybrid scaffolds. Cell adhesion was observed by PHK26® (Red Fluorescent Cell Linker) and scanning electron microscopy (SEM); and proliferation was investigated by alamarBlue® assay. In addition, the survival of SCAPs after degradation of the scaffold was assessed by Live/Dead® staining. The cell population showed stem cell characteristics. The hybrid scaffold, constituted of dense cellular network with interconnected pores, soaked and dissolved quickly. According to PKH26® and alamarBlue® assays, the SCAPs adhered and proliferated in the hybrid scaffold. Photobiomodulation leads to SCAPs higher proliferation rate and the Live/Dead® test showed live cells after 12 days of cultivation. It was concluded that the hybrid scaffold is biocompatible and favors survival of SCAPs, which was enhanced by photobiomodulation.
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Viscoélasticité du sang et du caillot / Blood and blood clot viscoelasticityGhiringhelli, Etienne 21 May 2014 (has links)
Le sang est un fluide complexe mis en écoulement par la pompe très peu puissante qu'est le cœur (environ 1 W), dans un réseau branché de plusieurs milliers de kilomètres de vaisseaux. Pour que cela soit réalisable, il se peut que les propriétés mécaniques du sang contribuent à l'entretien de l'écoulement. Malgré le nombre important d'études sur la rhéologie du sang, sa viscoélasticité n'a jamais été caractérisée en cisaillement simple. Le rôle physiologique du caillot est, lui, d'éviter un épanchement excessif de sang en présence d'une brèche vasculaire. Une de ses fonctions principales est donc de résister aux contraintes générées par l'écoulement sanguin, c'est-à-dire d'avoir une résistance mécanique appropriée. Que ce soit pour la caractérisation mécanique du sang ou du caillot, le principal verrou est l'absence de méthode de mesure adaptée à un matériau peu consistant, et dont les propriétés mécaniques sont en évolution rapide. Il est donc nécessaire de produire une méthode de mesure adéquate, couplée à un système de mesure assez sensible. Dans ce travail, nous présentons la méthode de rhéométrie que nous avons développée dans ce but, baptisée Optimal Fourier Rheometry (OFR). Cette technique a été validée avec succès sur différents matériaux modèles de plus en plus complexes : une huile newtonienne, une gomme viscoélastique (PDMS), une suspension de micelles vermiformes (CpCl Nasal) et enfin un alginate dentaire tout au long de sa gélification. Nous montrons ainsi que l'OFR est une technique de mesure fonctionnelle, fiable et optimale temporellement. Elle permet le suivi de grandeurs mécaniques dont le temps caractéristique de mutation est très inférieur à la minute. En raison de la sédimentation des globules rouges, le sang est un fluide évoluant dans le temps. Par conséquent l'OFR est bien adaptée pour la mesure de ses propriétés viscoélastiques. Pour nous affranchir de la variabilité très importante du sang de témoins, nous avons balayé de façon systématique la concentration en les composants sanguins les plus abondants sur des suspensions de globules rouges lavés. De façon a priori surprenante, nous montrons qu'en présence de fibrinogène, le sang présente une élasticité importante, du même ordre de grandeur, voire plus grande que sa viscosité. Cette élasticité augmente avec la concentration en fibrinogène et l'hématocrite et provient du réseau percolé de globules rouges agrégés de dimension fractale 2.08 qui existe dans la suspension lorsqu'elle est peu cisaillée. L'OFR a également été appliquée au suivi de la coagulation activée par voie intrinsèque et extrinsèque. Cela a permis de montrer que le procédé d'activation n'avait d'effet que sur la cinétique de la réaction, mais que cela ne changeait pas les étapes mécaniques observées. L'OFR permet grâce à sa résolution fréquentielle élevée et son temps de mesure minimal, d'affirmer que le processus de coagulation du sang n'est pas une transition sol-gel. / Blood is a complex fluid set into flow by the heart, which is a very low power pump (approximately 1 W), in a connected network consisting of several thousand kilometers of vessels. To do so, it seems reasonable that the mechanical properties of blood contribute to the maintenance of the flow. In spite of the important number of studies on blood rheology, the viscoelasticity of blood has never been characterized in simple shear. The physiological role of the blood clot is to avoid an excessive effusion of blood in the presence of a vascular breach. Thus, it has to resist to the stress induced by the blood flow. So, one of its essential functions is this mechanical resistance. Whether it is for the mechanical characterization of the blood or the clot, the main obstacle is the absence of viscoelasticity measurement techniques adapted to a low viscosity material evolving rapidly in time. So, it is necessary to provide an adapted measurement method, coupled with a sensitive enough measurement. In this work, we present the new rheometry method we developed, named Optimal Fourier Rheometry (OFR) as it is optimal both in duration and signal to noise ratio This method was successfully validated on materials of increasing complexity: a Newtonian oil, a viscoelastic gum (PDMS), a suspension of wormlike micelles (CpCl Nasal) and a dental alginate during its gelation. Because of the sedimentation of red blood cells, the mechanical properties of blood are evolving in time. Consequently the use of the OFR is well suited for the measurement of its viscoelastic properties. A systematic scanning of the concentrations in the most abundant blood components added to washed blood allowed to highlight the most important parameters. Our results show that blood has a surprisingly large elasticity, which is of the same order of magnitude as the viscosity of the material. This elasticity increases with fibrinogen concentration and hematocrit. When these two parameters are in the physiological range, a percolated network of aggregated red blood cells exists in the suspension of fractal dimension 2.08. The, OFR was applied to the monitoring of blood clot formation. The activation by intrinsic and extrinsic pathway was used on whole blood. It showed that the process of activation affects only the kinetics of the reaction, but does not change the observed mechanical s. Due to its high frequency resolution and minimal measurement time, OFR shows that coagulation is not a gelation process.
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Efeitos da fotobiomodulação na adesão e proliferação das células-tronco da papila apical humana em scaffold de quitosana com incorporação de coágulo sanguíneo. Estudo in vitro / Effects of photobiomodulation on adhesion and proliferation of stem cells from human apical papilla in chitosan scaffold with blood clot incorporation. In vitro studyGabriela Laranjeira Abe 06 September 2016 (has links)
Revascularização é uma técnica utilizada em dentes jovens, que apresentem rizogênese incompleta e danos irreversíveis ao tecido pulpar, necessitando de tratamento endodôntico, para formar novo tecido em lugar da polpa perdida. Clinicamente os resultados mostram a continuidade da rizogênese e a devolução da vitalidade dental. Porém, pouco se sabe sobre o novo tecido formado e não está estabelecido se este é capaz de desempenhar todas as funções da polpa dentária. Para melhorar as características do tecido formado pela técnica da revascularização, podemos utilizar ferramentas de engenharia tecidual, como célulastronco, fatores de crescimento e arcabouços de sustentação celular (scaffolds). As célulastronco (CTs) já estão presentes quando o sangue invade o canal radicular, e utilizar essa reserva de CTs que o hospedeiro possui, procedimento conhecido como homing, é uma vantagem em comparação com outras técnicas que injetam CTs obtidas por cultivo em laboratório. Entretanto os aspectos físicos do coágulo sanguíneo formado no interior do canal radicular podem ser melhorados com a adição de hidrogel de quitosana, que interage quimicamente com o sangue e forma um scaffold híbrido mais estável. Então, o objetivo deste estudo foi testar a hipótese de que o scaffold híbrido, composto por hidrogel de quitosana e sangue, ofereceria maior estabilidade física estrutural, bem como condições favoráveis à adesão e proliferação de células-tronco da papila apical humana (SCAPs; do inglês, Stem Cell from Apical Papila). Para isso, investigamos in vitro se a incorporação do sangue ao hidrogel de quitosana gera um scaffold mais estável, se este é favorável à adesão e proliferação de células-tronco da papila apical e se a fotobiomodulação potencializa essas características celulares. Para isso, SCAPs foram isoladas e caracterizadas por citometria de fluxo, tempo de dobra populacional, e contagem de unidades formadoras de colônias fibroblásticas (CFU-F; do inglês, Colony Forming Units - Fibroblastic). Ensaios de incorporação sanguínea, dissolução e embebição foram realizados para determinar o comportamento dos scaffolds híbridos. A adesão celular foi observada pela coloração PHK26® (do inglês, Red Fluorescent Cell Linker) e por microscopias eletrônicas de varredura (MEV); e a proliferação foi investigada pelo ensaio de alamarBlue®. Adicionalmente, a sobrevivência das SCAPs após a degradação do scaffold híbrido foi avaliada pela coloração Live/Dead®. A população celular estudada apresentou características de células tronco. O scaffold híbrido, constituído de densa rede alveolar com poros interconectados, embebeu e dissolveu rapidamente. De acordo com o PKH26® e alamarBlue® SCAPs aderiram e proliferaram no scaffold híbrido. SCAPs fotobiomoduladas exibiram maior taxa de proliferação e o ensaio Live/Dead® mostrou células vivas após 12 dias de cultivo. Concluiu-se que o scaffold híbrido apresenta biocompatibilidade e condições favoráveis de sobrevivência para SCAPs, que são potencializadas pela fotobiomodulação. / Revascularization is a technique used to form a new tissue, replacing the lost pulp, in young permanent teeth presenting incomplete rhizogenesis and irreversible damage, where endodontic treatment is needed. Clinically, the results show the continuity of the root formation and the return of dental vitality. However, little is known about the newly formed tissue and it has not been established if it is able to perform all functions of the dental pulp. To improve the characteristics of the newly formed tissue by the technique of revascularization, tissue engineering tools can be used, represented by stem cells, growth factors and scaffolds for cell supportting. Stem cells (SCs) are already present when the blood invades the root canal, and to use this SCs reserve that the host possesses, procedure known as homing, is an advantage compared with other techniques that inject SCs obtained by cultivation in the laboratory. However the physical aspects of blood clot in the root canal can be improved with the addition of chitosan hydrogel that chemically interacts with the blood and forms a more stable hybrid scaffold. So the aim of this study was to test the hypothesis that the hybrid scaffold, composed of hydrogel chitosan and blood clot, provides greater structural physical stability as well as favorable conditions for adhesion and proliferation of Stem Cells from Apical Papilla (SCAPs). For this, we investigated in vitro if the incorporation of blood to chitosan hydrogel, generates a more stable scaffold and if it supports the stem cell adhesion and proliferation, in addition, if photobiomodulation potentiates these cell characteristics. For this, SCAPs were isolated and characterized by flow cytometry, population doubling time, and counting colony forming units - fibroblastic (CFUF). Blood incorporation assays, dissolution and swelling were conducted to determine the behavior of hybrid scaffolds. Cell adhesion was observed by PHK26® (Red Fluorescent Cell Linker) and scanning electron microscopy (SEM); and proliferation was investigated by alamarBlue® assay. In addition, the survival of SCAPs after degradation of the scaffold was assessed by Live/Dead® staining. The cell population showed stem cell characteristics. The hybrid scaffold, constituted of dense cellular network with interconnected pores, soaked and dissolved quickly. According to PKH26® and alamarBlue® assays, the SCAPs adhered and proliferated in the hybrid scaffold. Photobiomodulation leads to SCAPs higher proliferation rate and the Live/Dead® test showed live cells after 12 days of cultivation. It was concluded that the hybrid scaffold is biocompatible and favors survival of SCAPs, which was enhanced by photobiomodulation.
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Evaluation of a Viscosity/Elasticity Assay (ReoRox®) for Assessment of Platelet Storage Lesion and Fibrinogen Dependent CoagulationGuðjónsdóttir, Erla January 2016 (has links)
The impact storage has on function of platelet concentrates is not completely known, although some factors have been discovered and measures have been taken to counteract them, such as adding platelet additive solution. There are several methods for analysing platelet function. In this study, the aim was to analyse change of platelet function in platelet concentrates over time and to see what effect fibrogen has on the coagulation. A technique using free oscillation rheometry (FOR), ReoRox®, was used to analyse function in platelet concentrates, both over time and after addition of fibrinogen. The platelets were analyzed at a concentration of 800 x109 Ptl/L and activated with thrombin receptor antigen peptide (TRAP). For fibrinogen efect analysis, four different concentrations were used, 10 g/L, 2,25 g/L, 1,0 g/L and 0,1 g/L. The results showed no statistically significant change in the function over time. However an increase in elasticity and decrease in the decline of elasticity could be seen. While analysing the platelets with fibrinogen it showed that up to 2,25 g/L the aggregation increased, while it decreased significantly at 10 g/L. In conclusion, the platelet concentrates retained a good clotting function from day one to day seven of storage, while the clot became stronger and fibrinolysis decreased. Fibrinogen proved important for coagulation, however a too high concentration inhibits coagulation.
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Computational fluid dynamics investigation of the orientation of a pediatric left ventricle assist device cannula to reduce stroke eventsGuimond, Stephen 01 December 2012 (has links)
Ventricle Assist Devices (VADs), which are typically either axial or centrifugal flow pumps implanted on the aortic arch, have been used to support patients who are awaiting cardiac transplantation. Success of the apparatus in the short term has led to long term use. Despite anticoagulation measures, blood clots (thrombi) have been known to form in the device itself or inside of the heart. The Ventricle Assist Devices supply blood flow via a conduit (cannula) implanted on the ascending aorta. Currently, the implantation angle of the VAD cannula is not taken into consideration. Since the VADs supply a significant amount of blood flow to the aorta, the implantation angle can greatly affect the trajectory of the formed thrombi as well as the cardiac flow field inside of the aortic arch. This study aims to vary the implantation angle of a pediatric Left Ventricle Assist Device (LVAD) through a series of computational fluid dynamics (CFD) software simulations focusing on the aortic arch and its branching arteries of a 20 kg pediatric patient in order to reduce the occurrence of stroke.
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DEVELOPING IN-VITRO SYNTHETIC BLOOD CLOT MODELS FOR TESTING THROMBOLYTIC DRUGSZiqian Zeng (12441402) 21 April 2022 (has links)
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<p>Thrombosis is the pathological formation of a blood clot in the body that blocks blood circulation, leading to high morbidity and mortality rates. Thrombolytic drugs that offer rapid clot dissolution are promising treatments yet current drugs are often associated with limited efficacy and high bleeding risks. While numerous animal thrombosis models have been developed for drug screening, the translation of therapeutic agents into and through clinical trials remains limited. This is largely due to animal models’ poor reproducibility and distinctive physiology to that of humans. <em>In-vitro</em> flow models that utilize both human blood components and physiologically relevant flow conditions can provide for a more representative testing environment to screen thrombolytic drugs. Developing better <em>in-vitro</em> models may not eliminate the need for preclinical animal testing but can help exclude inefficient agents earlier in the drug development pipeline to expedite the drug evaluation process. Existing <em>in-vitro</em> thrombolysis flow models are not ideal as they either adopt over-simplified clot substrates or utilize small-length-scale geometries that insufficiently mimic native hemodynamics. Thus, we propose to first develop a static fluorescently labeled clot lysis assay for an initial high throughput screening of thrombolytic drugs, and ultimately engineer a highly reproducible, physiological scale, flowing clot lysis model for more human relevant drug efficacy evaluation. Developing the static clot lysis assay not only helps to understand the mechanism of how diversified clotting conditions affect clot properties but also offer a chance to well-characterize fluorescence conjugations to fibrins. The ultimate flow model combines an <em>in-vivo</em>-like fluorescence incorporated synthetic clot (FISC) and a human-relevant flow system. Guided by results from static clotting experiments diversified FISCs are fluorescently optimized and fabricated dynamically using a Chandler loop setup at various conditions. The flow system is a tubing-based structure that comprises of a peristaltic pump, and a well-controlled flow chamber to provide for physiological shear and pulsatile levels. Therefore, the proposed synthetic clot model is a versatile platform that can mimic a variety of thrombosis conditions and offer representative drug testing and dosing results across numerous thrombolytic agents.</p>
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Small Angle Scattering Of Large Protein Units Under Osmotic StressPalacio, Luis A. 05 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Large protein molecules are abundant in biological cells but are very difficult to study in physiological conditions due to molecular disorder. For large proteins, most structural information is obtained in crystalline states which can be achieved in certain conditions at very low temperature. X-ray and neutron crystallography methods can then be used for determination of crystalline structures at atomic level. However, in solution at room or physiological temperatures such highly resolved descriptions cannot be obtained except in very few cases. Scattering methods that can be used to study this type of structures at room temperature include small-angle x-ray and neutron scattering. These methods are used here to study two distinct proteins that are both classified as glycoproteins, which are a large class of proteins with diverse biological functions. In this study, two specific plasma glycoproteins were used: Fibrinogen (340 kDa) and Alpha 1-Antitrypsin or A1AT (52 kDa). These proteins have been chosen based on the fact that they have a propensity to form very large molecular aggregates due to their tendency to polymerize. One goal of this project is to show that for such complex structures, a combination of scattering methods that include SAXS, SANS, and DLS can address important structural and interaction questions despite the fact that atomic resolution cannot be obtained as in crystallography. A1AT protein has been shown to have protective roles of lung cells against emphysema, while fibrinogen is a major factor in the blood clotting process. A systematic approach to study these proteins interactions with lipid membranes and other proteins, using contrast-matching small-angle neutron scattering (SANS), small angle x-ray scattering (SAXS) and dynamic light scattering (DLS), is presented here. A series of structural reference points for each protein in solution were determined by performing measurements under osmotic stress controlled by the addition of polyethylene glycol-1,500 MW (PEG 1500) in the samples. Osmotic pressure changes the free energy of the molecular mixture and has consequences on the structure and the interaction of molecular aggregates. In particular, the measured radius of gyration (Rg) for A1AT shows a sharp structural transition when the concentration of PEG 1500 is between 33 wt% and 36 wt%. Similarly, a significant structural change was observed for fibrinogen when the concentration of PEG 1500 was above 40 wt%. This analysis is applied to a study of A1AT interacting with lipid membranes and to a study of fibrinogen polymerization in the presence of the enzyme thrombin, which catalyzes the formation of blood clots. The experimental approach presented here and the applications to specific questions show that an appropriate combination of scattering methods can produce useful information on the behavior and the interactions of large protein systems in physiological conditions despite the lower resolution compared to crystallography.
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SMALL ANGLE SCATTERING OF LARGE PROTEIN UNITS UNDER OSMOTIC STRESSLuis Palacio (8775689) 30 April 2020 (has links)
<div>Large protein molecules are abundant in biological cells but are very difficult to study in physiological conditions due to molecular disorder. For large proteins, most structural information is obtained in crystalline states which can be achieved in certain conditions at very low temperature. X-ray and neutron crystallography methods can then be used for determination of crystalline structures at atomic level. However, in solution at room or physiological temperatures such highly resolved descriptions cannot be obtained except in very few cases. Scattering methods that can be used to study this type of structures at room temperature include small-angle x-ray and neutron scattering. These methods are used here to study two distinct proteins that are both classified as glycoproteins, which are a large class of proteins with diverse biological functions. In this study, two specific plasma glycoproteins were used: Fibrinogen (340 kDa) and Alpha 1-Antitrypsin or A1AT (52 kDa). These proteins have been chosen based on the fact that they have a propensity to form very large molecular aggregates due to their tendency to polymerize. One goal of this project is to show that for such complex structures, a combination of scattering methods that include SAXS, SANS, and DLS can address important structural and interaction questions despite the fact that atomic resolution cannot be obtained as in crystallography. A1AT protein has been shown to have protective roles of lung cells against emphysema, while fibrinogen is a major factor in the blood clotting process. A systematic approach to study these proteins interactions with lipid membranes and other proteins, using contrast-matching small-angle neutron scattering (SANS), small angle x-ray scattering (SAXS) and dynamic light scattering (DLS), is presented here. A series of structural reference points for each protein in solution were determined by performing measurements under osmotic stress controlled by the addition of polyethylene glycol-1,500 MW (PEG 1500) in the samples. Osmotic pressure changes the free energy of the molecular mixture and has consequences on the structure and the interaction of molecular aggregates. In particular, the measured radius of gyration (Rg) for A1AT shows a sharp structural transition when the concentration of PEG 1500 is between 33 wt\% and 36 wt\%. Similarly, a significant structural change was observed for fibrinogen when the concentration of PEG 1500 was above 40 wt\%. This analysis is applied to a study of A1AT interacting with lipid membranes and to a study of fibrinogen polymerization in the presence of the enzyme thrombin, which catalyzes the formation of blood clots. The experimental approach presented here and the applications to specific questions show that an appropriate combination of scattering methods can produce useful information on the behavior and the interactions of large protein systems in physiological conditions despite the lower resolution compared to crystallography.</div>
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