Transcapillary exchange is the movement of fluid and molecules through the porous capillary wall, and is important in maintaining homeostasis of bodily tissues. The classical view of this process is that of Starling's hypothesis, in which the forces driving filtration or absorption are the hydrostatic and osmotic pressure differences across the capillary wall. However, experimental evidence has emerged suggesting the importance of the capillary wall ultrastructure, and thus rather than the global differences between capillary and tissue, it is the local difference across a structure lining the capillary wall known as the endothelial glycocalyx that determines filtration. Hu and Weinbaum presented a detailed cellular level microstructural model of this phenomenon which was able to explain some experimental discrepancies. In this Thesis, rather than describing the microstructural details, the capillary wall is treated as a poroelastic material. The assumptions of poroelasticity theory are such that the detailed pore structure is smeared out and replaced by an idealized homogeneous system in which the fluid and solid phases coexist at each point. The advantage of this approach is that the mathematical problem is greatly simplified such that analytical solutions of the governing equations may be obtained. This approach also allows calculation of the stress and strain distribution in the tissue. We depart from classical poroelasticity, however, due to the fact that since there are concentration gradients within the capillary wall, the filtration is driven by both hydrostatic and osmotic pressure gradients. The model predictions for the filtration flux as a function of capillary pressure compares favourably with both experimental observations and the predictions of the microstructural models. An important factor implicated in transcapillary exchange is the endothelial glycocalyx, which was shown experimentally to protect against edema formation. Using our theory in combination with the experimental measurements of glycocalyx thickness and pericapillary space dimension (PSD), we make a quantitative comparison for the excess flow as a result of a deteriorated glycocalyx, which shows reasonably good agreement with the data. Since many of the parameters in the model are difficult to measure, a sensitivity analysis was performed on the most important of these. Finally, since there was variation in the measurements of glycocalyx thickness and PSD, we used probability distributions to represent the data, and performed further calculations to obtain ranges of likely values for the various parameters. This work could find applications in cardiovascular disease, where the glycocalyx is degraded or absent, and in cancer research, where the abnormal vasculature is an impediment to the efficient delivery of anti-cancer drugs.

Ovarian cancer (OC) accounts for 4% of all cancer cases and 4.2% of all cancer deaths worldwide. OC is the most lethal gynecological cancer because it lacks early disease symptoms and does not have a specific diagnostic marker. As a result, more than 70% of OC patients are diagnosed in later stages when the disease has already metastasized and the 5-year survival rate has decreased to less than 20% compared with approximately 90% survival for women diagnosed with early stage disease. Therefore, I initiated my studies with a computational analysis of the 27 most commonly reported literature-derived ovarian cancer (LDOC) protein biomarkers. I found that LDOC protein biomarkers share many biochemical features including a preponderance for a stable protein structure, the ability to be secreted, and functionality related to extracellular matrix (ECM) modification, immune response and/or energy production. Subsequently, I analyzed the human proteome to identify proteins that also share these biochemical features. Of the 70,616 proteins in the human proteome, 683 proteins were found to have similar biochemical features to the 27 LDOC proteins. I also identified a subset of 21 potential additional protein regulators of ovarian cancer (APROC) that interact with LDOCs. Three of the APROCs identified were amylase proteins AMY1A, AMY2A, and AMY2B which cleaves alpha 1, 4-glycosidic bonds in polysaccharides. Amylase is reportedly overexpressed in and secreted by ovarian tumors but its functional contribution to OC remains unknown[1]. In this thesis, I posit that amylase contributes to OC invasion. I initiated my studies by computational characterizing the different amylase isozymes to predict which amylase isozyme(s) is most likely overexpressed in and contributory to OC invasion. I found that AMY1 and AMY2B have unique regions of disorder and unique phosphorylation sites indicating that AMY1 and AMY2B would be more likely to interact with other proteins, and to be easily secreted. Using OC patient serum samples, I was able to validate AMY1 and AMY2B overexpression by western immunoblotting. I then developed an in vitro model system to study the molecular contribution of amylase to OC invasion using normal ovarian surface epithelial (IOSE) and OC cell lines. I showed that OC cells generally overexpress and secrete metabolically active amylase isozymes AMY1 and AMY2B. Abrogating amylase activity using siRNA silencing technology decreased the capacity of OC cells to invade collagen coated Boyden chambers and increased sulfated glycosaminoglycans (sGAG) production. Since a survey of OC cell lines indicated that cancer cells have a bulkier glycocalyx compared to IOSE cells and immunogold labeling studies indicated the presence of amylase within the immediate OC microenvironment, my data suggest that, by cleaving alpha 1, 4-glycosidic bonds in glycoconjugates present within ECM, amylase may remodel the ECM to promote an invasive cancer phenotype. Amylase is therefore a target for therapeutic intervention in OC patients with hyperamylasemia. I established Spirulina, a dietary supplement, as a novel transcriptional inhibitor of amylase. Spirulina inhibited amylase expression in OC cell lines at both the message and protein levels. Spirulina reduced OC cell invasion and migration in vitro, putatively by decreasing amylase expression.

Une brosse de polymères est une matrice dense de macromolécules greffées à une surface donnée. Au-delà des brosses synthétiques réalisées en laboratoire, on trouve des exemples très variés dans la nature: un exemple emblématique est le glycocalyx endothélial, décorant la surface interne des vaisseaux sanguins des mammifères. L'interaction de cette structure avec le plasma et les cellules sous écoulement n'est encore que très partiellement explorée. La présente thèse propose, grâce à des simulations de "Dissipative Particle Dynamics", un modèle coarse-grained pour une analyse auto-cohérente d'une brosse polymérique dense sous écoulement parabolique. Cette étude mésoscopique met en évidence l'importance des effets collectifs entre molécules, entraînée par l'hydrodynamique, et propose des nouvelles interprétations à la phénoménologie du système brosse-écoulement. Des résultats préliminaires sont également produits pour l'interaction sous écoulement entre un objet mésoscopique déformable (prototype d'un globule rouge) et les polymères greffés. / Polymer brushes are dense matrices of grafted macromolecules. In addition to brushes finely designed in laboratory, various examples are offered by Nature, as the endothelial glycocalyx, decorating the lumen of mammalian blood vessels. The interaction of such network with the flowing plasma and cells is still partially unknown.The present thesis, by mean of Dissipative Particle Dynamics simulations, proposes a coarse-grained model for the self-consistent analysis of a dense polymer brush under parabolic flow. Our mesoscale investigation highlights the relevance of collective effects, driven by hydrodynamics, and proposes novel interpretations regarding the rich phenomenology of the brush-flow system.Preliminary results are also provided for the interplay between a mesoscopic deformable flowing object (prototype of a red blood cell) and the grafted polymers.

Vascular diseases, such as stroke and heart attacks, account for more than 50% of abnormal death worldwide. The cause of these diseases is linked to malfunctions of vascular endothelial cells, in particular the endothelial glycocalyx. This study investigates the location and stability of the endothelial glycocalyx under different flow conditions in vitro. AFM (Atomic Force Microscopy) micro indentation is carried out on endothelial cell membrane to determine its Young's modulus. The Young's modulus of the glycocalyx layer is then deduced from measurements on cell membranes with, and those without, the glycocalyx layer. Heparan sulphate (HS) is an important component of the glycocalyx and can be removed by the enzyme heparinase-III (Hep-III). Our results show the glycocalyx on cultured Human Umbilical Vein Endothelial Cells (HUVECs) has a Young's modulus of ~0.64Kpa. We further observe how the Young's modulus of the endothelial cell membrane decreases with time, as the glycocalyx layer redevelops, following its removal by Hep-III. Steady and oscillatory shear stimulations are used in flow chamber experiments. Under 24 hours' steady shear stimulation (12.6 dyn/cm2), cells are seen to elongate and reorient parallel to the flow direction. The glycocalyx is seen to shift to the peripheral region of the cell surface. With actin depolymerisation treatment, significant shedding of the glycocalyx from the luminal surface of the cell is observed. This occurs together with the loss of focal adhesions on the basal membrane. When endothelial cells are subjected to 24 hours' oscillating shear stress, the size of the cell increases as the oscillatory reversal time (time between changes in oscillatory flow direction) increases. Measurements are taken with oscillatory flow reversal programmed at 5s, 10s and 15s. The angle (between the long axis of the cell and the flow direction) and the aspect ratio (long axis vs short axis) change from 41.57° and 1.72 : 1 (static) to 40.18° and 3.26 : 1 (5s), 36.71° and 4.17 : 1 (10s), 26.5° and 4.39 : 1 (15s). Both the height and the area of the cell increase. The Young's modulus of the endothelial cell membrane is measured under oscillatory flows with different reversal time and compared to that under static flow conditions. An increase in the Young's modulus is observable under oscillatory flows, with the most significant change occurring at the edge (i.e. periphery) of the cell membrane area. As the oscillatory reversal time increases from 5s to 15s, the Young's modulus of the cell membrane increases. In the apical areas of the cell membrane, the increase is less significant. These results indicate that the thickness of the glycocalyx decreases as cells are exposed to oscillatory flows, and the loss is most significant in the peripheral region of the cell membrane. As the oscillatory reversal time increases from 5s to 15s, so the loss in the glycocalyx increases.

A l'interface physico-chimie/biologie, ce travail s'intéresse à l'interaction de colloïdes lipidiques (virus, vecteurs modèles...) en contact avec des cellules vivantes en culture (HeLa). Par une approche cinétique statistique complétée d'une mesure de force locale, nous décrivons l'adhésion de particules fonctionnalisées présentant une affinité spécifique pour des récepteurs cellulaires. En raison de son intérêt (ciblage de tumeurs, voie rétrograde), le couple ligand/récepteur de la toxine de Shiga et de son récepteur Gb3 a été utilisé pour le ciblage des colloïdes (chapitre 1). Les mesures cinétiques réalisées sur ces colloïdes fonctionnalisés par la toxine de Shiga, confrontées à plusieurs modèles cinétiques originaux, ont alors permis de mettre en évidence les mécanismes impliqués dans l'accrochage des objets sur la surface cellulaire. En particulier, un modèle inspiré des mécanismes de polymérisation et s'appuyant sur des effets de coopérativité des récepteurs (recrutement par diffusion) a été retenu et permet de proposer une description microscopique des processus mis en oeuvre (2). De plus, des modifications physico-chimiques de la surface des colloïdes (polymères PEG, nature et densité du ligand) ont été envisagées (3 et 4). L'étude a permis notamment de quantifier l'effet du PEG sur l'adhésion des colloïdes et de proposer une interprétation de l'origine microscopique (répulsion par le glycocalyx). Par ailleurs, nous avons pu, en utilisant une technique de micromanipulation (Biomembrane Force Probe) mesurer localement la force de la liaison colloïde/cellule. Cette mesure a révélé une quantification des forces de rupture, suggérant l'existence de liens multiples (5). Enfin, l'étude de l'entrée et du devenir des particules à l'intérieur des cellules a été amorcée (6). Ce travail souligne ainsi de quelle façon des mesures physiques quantitatives peuvent permettre de proposer une description microscopique de mécanismes biologiques.

La radiothérapie constitue l’un des principaux traitements pour l’éradication des cancers. Cependant, elle présente un risque d’effets secondaires aux tissus sains environnant la tumeur. Dans ce processus, le système vasculaire et plus particulièrement l’endothélium jouent un rôle clé. Les cellules endothéliales activées favorisent le recrutement chronique des thrombocytes et des leucocytes, contribuant ainsi aux effets secondaires. D’autre part, dans les maladies inflammatoires, les glycanes exprimés à la surface des cellules endothéliales sont modifiés et influencent le recrutement des cellules immunitaires. Dans cette étude, nous avons évalué la modification des glycanes endothéliaux en réponse à une irradiation à forte dose, et étudié les effets fonctionnels de ces modifications sur le recrutement des leucocytes en utilisant un modèle de cellules endothéliales (HUVECs) in vitro. Nos résultats apportent les premières preuves d’une modification du glycome des cellules endothéliales en réponse à l’irradiation. Les N-glycanes hautement mannosylés, les O-glycanes et les motifs sialylées sont surexprimés. Parallèlement, le glycocalyx endothélial semble subir une dégradation. Nous avons évalué l’effet fonctionnel des modifications glycanique des cellules endothéliales irradiées sur l’adhésion d’une lignée de monocyte (THP-1). Nos résultats montrent que l’adhésion radio-induite est en partie due à la surexpression endothéliale des N-glycanes hautement mannosylés. Nous avons aussi évalué le glycome sur un modèle de souris irradiées et sur des pièces opératoires de patients traités par radiothérapies. Nos résultats de transcriptomiques sur la souris suggèrent l’existence de modifications glycaniques radio-induites in vivo. L’intégration de la composante glycanique permet de porter un regard nouveau sur le continuum d’évènement qui conduit aux lésions tissulaires radio-induites. A l’avenir, l’étude du glycome pourrait ouvrir de nouvelles pistes thérapeutiques pour une meilleure prise en charge des effets secondaires de la radiothérapie. / Radiotherapy is one of the main treatments against cancers. However, it presents a risk of adverse effects for the normal tissues surrounding the tumors. The vascular network and especially the endothelium are considered as main targets to limit normal tissue damages and prevent side effects of radiotherapy. Activated endothelial cells are involved in the chronic recruitment of thrombocytes and leukocytes, resulting in tissue complications. On the other hand, in inflammatory diseases, the glycans expressed on the surface of endothelial cells are modified and lead to immune cells recruitment. We sought to evaluate changes in endothelial glycome in a context of exposure to high dose of radiation, and studied the functional consequences on the recruitment of leukocytes. In vitro, the characterization of the glycome was performed on a primary endothelial cell model (HUVEC). Our results provide the first evidences of an endothelial modification of the glycome after exposure to ionizing radiation. We report an overexpression of high mannose N-glycans, O-glycans and syalilated motifs. At the same time, endothelial glycocalyx appeared to be damaged by exposure to radiation. Next, we evaluated these radiation-induced modifications of endothelial glycans on monocyte adhesion. We show that the radiation induced adhesion was mediated by overexpression of high mannose N-glycans. We also investigated changes in glycome in an irradiated mouse model of enteropathy and in resections of patients treated with radiotherapy. In mice, a transcriptomic study suggests changes in glycans following radiation exposure. Collectively, these findings on glycome changes provide a new perspective of the continuum of events leading to normal tissue complications. In the future, the study of the glycome should open new therapeutics opportunities for better management of tissue damages induced by radiation.

Trotz enormer Fortschritte in der Therapie, bleibt der kardiogene Schock die führende Todesursache im akuten Myokardinfarkt. Die pathophysiologischen Veränderungen umfassen dabei unter anderem Störungen der Mikrozirkulation, endotheliale Dysfunktion mit vaskulärer Leckage, sowie vermehrte Thrombozyten- und Leukozytenadhäsion an die Gefäßwand. Die endotheliale Glykokalyx wurde als zentraler Regulator dieser Prozesse identifiziert. Das Glykosaminoglykan Heparansulfat repräsentiert dabei den Hauptbestandteil der Endothelzelloberfläche und Syndecan-1 das am weitesten verbreitete Proteoglykan. Diesbezüglich konnte in Studien eine Assoziation zwischen Schädigung der endothelialen Glykokalyx und den zirkulierenden Membranbestandteilen im Patientenblut beobachtet werden. Ziel der Arbeit war die Analyse der Glykokalyxmarker bei 184 Patienten mit Infarkt-assoziiertem kardiogenen Schock. In den Serumproben zum Zeitpunkt der Aufnahme und nach einem Tag wurde mittels ELISA die Konzentration von Heparansulfat und Syndecan-1 bestimmt. Dabei zeigte sich ein signifikanter Konzentrationsabfall von Syndecan-1 innerhalb des Analysezeitraums. Des Weiteren hatten die Überlebenden an beiden Tagen signifikant niedrigere Syndecan-1-Serumwerte. Durch eine schrittweise Multiregressionsanalyse wurde Syndecan-1 bei Patienten mit akutem Myokardinfarkt und assoziiertem kardiogenen Schock als unabhängiger Prädiktor der 30-Tage- Mortalität identifiziert.

Metastatic cancer is more dangerous and difficult to treat than pre-metastatic cancer. Ninety percent of cancer-related deaths are caused by metastatic cancer. When cells go through metastases, they go through changes that allow them to break away from the primary tumor and invade secondary tissues. These changes, in lipid membrane composition and cellular glycocalyx, make the cell more resistant to therapeutics. Actin cytoskeleton contractility plays a major role in these changes, as increased contractility has been linked to upregulation of phosphoinositides and production of glycoproteins. Light induced molecular adsorption of proteins (LIMAP) was used to control the actin arrangement and cell shape in order to mimic and study metastatic cells. Negatively charged proteins electrostatically adhere to the surface in order to create patterns for the cells to stick. Neutravidin was conjugated to poly(glutamic acid) to improve attachment to the surface. We observed differences in cell shape and phosphoinositide behavior based on LIMAP patterning. Additionally, expression of key glycoproteins related to cancer metastasis increased with increased actin contractility. The actin cytoskeleton was the main driver of changes to the cell membrane and glycocalyx.

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37°C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G+C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carinii carinii isolated from infected rat lungs are haploid organisms.

UNIVERZITA KARLOVA Lékařská fakulta v Plzni Dizertační práce Endothelial glycocalyx - diagnostic approach and intervention assessment MUDr.Jiří Pouska ABSTRACT Endothelial glycocalyx (EG) is fine structure on the surface of endothelium. After extensive research in past years, revisited Starling principle was finally formulated. It describes fluid physiology in capillaries precisely. EG has pivotal role in keeping endothelium semipermeable and thus avoiding extensive filtration of fluids to interstitium. Assessment of EG is clinically difficult. Many pathological conditions lead to damage of EG (sepsis etc.). Intravenous fluid therapy is mainstay of treatment of such conditions. Our aim was to determine the changes of EG integrity depending on the choice of intravenous fluid and its infusion time in physiological and pathological conditions. Key words: Endothelial glycocalyx, infusion therapy, anaesthesia, sepsis, microcirculation.