Aaueous polymer two phase systems containing dextran T500, PEG 8000, and buffer are widely used to separate and analyse cells and other biological material based on the way they partition between the two phases and their interface. The behaviour of human erythrocytes in such two phase systems was studied in order to characterize some of the physico-chemical interactions important in determining cell partition. Two aspects were studied: the role of electrostatic and affinity ligand effects in determining the relative affinity of the cell for the two phases, and the relationship of this relative affinity to the cell partition. The potential difference produced by the unequal affinity of the buffer cations and anions for each phase was related to the salt partition by a thermodynamic model, which agreed with experimental results obtained in single and mixed salt systems. A thermodynamic theory for the effects of an affinity ligand on the cell surface free energy difference between the phases was derived, and found to agree quantitatively with experimental results using the affinity ligand PEG-palmitate. The change in cell surface free energy difference as a function of potential and ligand concentration was determined by contact angle measurements. This change was very small, based either on previous estimates of the surface charge density, or on the amount of PEG-palmitate bound to the cell surface as determined by adsorption experiments. This was attributed to partial exclusion of the phases from the cell glycocalyx. Cell partition into the upper PEG rich phase increased as this phase was made more positive with respect to the lower phase, or as the amount of an affinity ligand, PEG-palmitate, in the system was increased. Contact angle measurements were used to determine the energy of erythrocyte attachment to the interface between the two phases. The dependence of the cell partition on this parameter showed that thermal energies are far too small to partition cells in these systems. The cell partition was unaffected by the density difference between the phases. This and other results led to the hypothesis that droplet coalescence is the primary process by which large particles (>1 µm dia.) such as cells are distributed between the interface and one of the phases. / Science, Faculty of / Chemistry, Department of / Graduate

The aim of this dissertation is to develop theoretical models for the motion of rigid and flexible particles through very tight spaces. The geometries of conduits which will be investigated are cylindrical tubes, parallel plane walls and rectangular channels. This work is motivated by an interest in the flow and deformation of single red blood cells in very narrow capillaries, in spleen and in bone marrow. Mammalian red cells are highly flexible, but their deformations satisfy two significant constraints. They must deform at constant volume, because the contents of the cell are incompressible, and also at nearly constant surface area, because the red cell membrane strongly resists dilation. Consequently, there exists a minimal tube diameter below which passage of intact cells is not possible. A cell in a tube with this diameter has its critical shape: a cylinder with hemispherical ends. The motion of red cells is analysed using lubrication theory. When the tube diameter is slightly larger than the minimal value, the cell shape is close to its shape in the critical case. However, the rear end of the cell becomes flattened and then concave with a relatively small further increase in the diameter. The changes in cell shape and the resulting rheological parameters are analysed using matched asymptotic expansions for the high-velocity limit and using numerical solutions. A rapid decrease in the apparent viscosity of red cell suspensions with increasing tube diameter is predicted over the range of diameters considered. The red cell velocity is found to exceed the mean bulk velocity by an amount which increases with increasing tube diameter. The same type of analysis is applied to the flow and deformation of red blood cells between two parallel plates with near-minimal spacings. First, the critical shape of the particle and the minimum gap width are determined using calculus of variations. In this case, it is a disk with a rounded edge. The flow in the plasma layers between the cell and the plates is described using lubrication theory. Approximate solutions can be obtained using a locally two-dimensional analysis at each point of the rim of the cell. Cell shapes, pressure distributions, membrane stresses and particle velocities are deduced as functions of geometrical parameters. One significant finding is that the gap width between the cell and the wall decreases with distance from the axis of symmetry parallel to the flow direction. The red cell velocity may be smaller or larger than the mean fluid velocity far from the cell, depending on the spacing of the plates, with equality when the width of the red cell is about ninety percent of the spacing between plates. The same procedure is also applied to rectangular channels.

Human red blood cells (RBCs) must undergo severe deformation to pass through narrow capillaries and submicronic splenic slits for several hundred thousand times in their normal lifespan. Studies of RBC biomechanics have been mainly focused on cell deformability measured from a single application of stress using classical biomechanical techniques, such as optical tweezers and micropipette aspiration. Mechanical fatigue effect on RBCs under cyclic loadings of stress that contributes to the membrane failure in blood circulation is not fully understood. This research developed a new experimental method for mechanical fatigue testing of RBCs using amplitude-modulated electro-deformation technique. Biomechanical parameters of individually tracked RBCs show strong correlations with the number of the loading cycles. Effects of loading configurations on the cellular fatigue behavior of RBCs is further studied. The results uniquely establish the important role of mechanical fatigue in influencing physical properties of biological cells. They further provide insights into the accumulated membrane damage during blood circulation, paving the way for further investigations of the eventual failure of RBCs in various hemolytic pathologies. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

La microcirculation est rapidement altérée dans le sepsis et la persistance de ces altérations est associée à un mauvais pronostic. La microcirculation est composée de vaisseaux invisibles à l’œil (< 100 µm), de l’endothélium, du glycocalyx, des cellules musculaires lisses et des éléments sanguins dont les GR. <p>De nombreuses études animales et humaines ont rapporté des altérations rhéologiques des GR dans le sepsis. Ces modifications comprennent une diminution de la déformabilité, une augmentation de l’agrégation et de l’adhérence globulaire. <p>De plus, l’altération de la déformabilité peut induire des altérations du flux microcirculatoire dans des modèles expérimentaux animaux. Ces mêmes altérations rhéologiques sont rapportées dans le diabète. Dans cette pathologie, les GR présentent une diminution du contenu membranaire en AS, comme dans les processus de sénescence. <p>La déformabilité des GR dépend des caractéristiques cellulaires incluant surtout les propriétés de la membrane, la géométrie cellulaire et dans une moindre mesure la viscosité cellulaire. Malgré la connaissance des altérations de la rhéologie dans le sepsis, peu de travaux, au contraire du diabète, s’interessent aux modifications de la membrane.<p>Nous avons étudié, par analogie aux altérations globulaires rapportées dans le diabète, la membrane des GR de patients admis en soins intensifs pour un sepsis, et comparé à des GR de patients non septiques et de volontaires sains. Le contenu membranaire en AS était significativement diminué chez les patients septiques par rapport aux patients non-septiques et aux volontaires sains. De plus, les GR des patients septiques, analysés par une technique de cytométrie en flux indépendante de la température de l’échantillon, étaient rapidement plus sphériques (dans les 24 heures du sepsis) et incapables de modifier leurs formes en hypoosmolalité. Cette technique de cytométrie a par ailleurs aussi été utilisée pour l’analyse de GR de patients diabétiques et en insuffisance rénale terminale. <p> La diminution du contenu en AS est aussi rapidement observée sur la transferrine, suggérant une augmentation de la concentration et/ou de l’activité de la neuraminidase, enzyme clivant l’AS. Dans un modèle de choc septique induit chez l’ovin, nous avons confirmé la rapidité de ce phénomène. En effet, la concentration en AS libre augmente dès la 15ième heure après induction du sepsis.<p>In-vitro, nous avons pu reproduire les modifications de forme des GR observés chez les patients septiques par incubation de GR de volontaire avec de la neuraminidase, et ce en 10 heures, quelles que soient les concentrations utilisées. Ces modifications de forme et de membrane s’accompagnent d’une augmentation significative du contenu en lactate, suggérant une stimulation de la glycolyse érythrocytaire et en 2,3-DPG, facilitant la libération de l’O2 de l’Hb vers les tissus. <p>Toutes ces modifications touchant la membrane des GR des patients de soins intensifs, surtout septiques, peuvent être responsables des altérations de rhéologie que nous avons observé grâce au LORCA sur une large population admis aux soins intensifs.<p>Une meilleure compréhension des mécanismes conduisant aux altérations rhéologiques des GR dans le sepsis, et ses effets potentiellement déletères sur la microcirculation, sont nécessaires avant d’envisager les GR comme cible thérapeutique.<p><p><p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished