The recreation of the complicated tissue microenvironment is essential to reduce the gap between in vitro and in vivo research. Polysaccharide-based hydrogels form excellent scaffolds to allow for three-dimensional cell culture owing to the favorable properties such as capability to absorb large amount of water when immersed in biological fluids, ability to form “smart hydrogels” by being shear-thinning and thixotropic, and eliciting minimum immunological response from the host. In this study, the biodegradable shear-thinning polysaccharide, gellan-gum based hydrogel was investigated for the conditions and concentrations in which it can be applied for the adhesion, propagation and assembly of different mammalian cell types in an unmodified state, at physiological conditions of temperature. Cell studies, to show successful propagation and assembly into three-dimensional structures, were performed in the range of hydrogels which were deemed to be optimum for cell culture and the cell types were chosen to represent each embryonic germ layer, i.e., human neural stem cells for ectoderm, human brain microvasculature cells for mesoderm, and murine β-cells for endoderm, along with a pluripotent cell line of human induced pluripotent stem cells, derived from human foreskin fibroblasts. Three-dimensional cell organoid models, to allow for gellan gum based bioprinting, were also developed using human induced pluripotent stem cells and human neural stem cells.

This diploma thesis deals with the analysis of structural and dynamic organization of thyrotropin releasing hormone receptor (TRH-R) and δ-opioid receptor (DOR) within plasma membrane (PM) in relation to the specific sub-compartments of PM denominated as domains or membrane rafts. Modern fluorescence microscopy techniques FLIM, FRAP and RICS were used for this purpose. The experiments were performed on the live cells derived from HEK293 cell line. To reach the main goal of this work, the integrity of PM structure was altered by depletion of cholesterol which was performed by incubation of cells with β cyclodextrin. Results clearly support our previously suggested idea that the vast majority of TRH-R is localized in non-raft regions of plasma membrane. This work also compared different modes of performance of FRAP and results obtained by FRAP and RICS because these methods are to some extent analogous. This is one of the first works that used the RICS approach to characterize the G protein-coupled receptors. In the second part of this work, the setup of transient transfection of the HEK293 cells with DOR-ECFP and DOR EYFP constructs was established. Simultaneously, the functionality of these constructs, i.e. the ability of DOR to activate the cognate G protein was determined. Powered by TCPDF (www.tcpdf.org)

La synapse est un assemblage macromoléculaire dynamique : les protéines synaptiques sont constamment et rapidement échangées par un mouvement diffusif entre l'assemblage synaptique et la région extrasynaptique, alors que l'assemblage reste stable. Par ailleurs, dans le compartiment présynaptique, les cycles d'endo- et d'exocytose des vésicules synaptiques imposent une réorgani- sation dynamique de la membrane plasmique. La syntaxin1A est une protéine membranaire impliquée dans l'exocytose. Quelles sont les caractéristiques de la diffusion latérale de la syntaxin et ses implications pour la transmission sy- naptique ? Nous avons étudié la diffusion latérale de la syntaxin à l'échelle de la popu- lation (méthode de retour de fluorescence après photoblanchiment, FRAP) et à l'échelle de la molécule individuelle (suivi de particule unique, SPT). Nous avons montré que la syntaxin diffuse rapidement dans la membrane plasmique et présente des périodes d'immobilisation transitoire, qui reflètent des inter- actions moléculaires impliquées dans la formation du complexe exocytotique. Enfin, nous avons construit un modèle computationnel qui réconcilie les des- criptions de la diffusion latérale de la syntaxin à l'échelle de la population et de la molécule individuelle. Ce modèle a permis de caractériser les cinétiques des interactions entre la syntaxin et ses partenaires, qui conduisent à sa stabilisa- tion à la synapse.

Solute diffusion is critical to maintenance of cellular function and matrix integrity in articular cartilage. Nutrient deficiency due to transport limitations is thought to be one of the causes of the pathological degeneration of the cartilage tissue. Thus, a study of diffusion within cartilage will lead to a better understanding of the causes of cartilage degeneration. To accurately estimate diffusion coefficients in cartilage and other hydrated medium, we developed a finite-element based method, the Direct Diffusion Simulation Parameter Estimation method (DDSPE), to be used for quantitative determination of solute diffusivities from Fluorescence Recovery After Photobleaching data. Analyses of simulated and experimental FRAP data demonstrated that this method was more accurate than existing analytical methods, including having a low sensitivity to variations in the spot radius. Subsequently, the roles of extracellular matrix (ECM) composition and tissue orientation in solute diffusion within immature bovine cartilage were explored. Diffusivities were measured through the cartilage depth and in two different orientations (radial and transverse). Diffusivities were then correlated with ECM components. Matrix water content was found to be the best predictor of solute diffusion rates in immature cartilage. Although no specific experiments were done to measure the effect of structure, our results suggested that matrix structure did indeed modulate transport. Diffusional anisotropy, defined as the ratio of the diffusivities in both orientations, was observed to be significant in all the immature cartilage zones. As a consequence, the differences in solute diffusion between immature and mature bovine cartilage were investigated. Diffusion rates and diffusional anisotropy decreased in the mature cartilage superficial zone. The decrease in diffusivities observed in mature cartilage suggests that there may be a reduction in nutrient and growth factor supply to the cells. Nevertheless, healthy adult cartilage can still maintain its normal function even with a reduction in solute diffusion rates as nutrient diffusion distances are shorter in mature cartilage. However, any disruption in the mechanical or biological environment could cause an imbalance in tissue homeostasis, which when combined with decreased diffusivities, could trigger matrix degeneration. Thus, decreased diffusivity may be a necessary but not a sufficient prerequisite of matrix degeneration.

La question se pose de l'existence de domaines membranaires permettant de regrouper les différentes protéines nécessaires à la transmission du signal par les récepteurs couplés aux protéines G (RCPG). Nous avons donc analysé l'organisation dynamique du récepteur mu opioïde humain (hMOR) en relation avec ses paramètres pharmacologiques et les contraintes de son environnement. Après avoir vérifié la fonctionnalité de T7-EGFP-hMOR dans les SH-SY5Y, sa dynamique latérale a été étudiée par retour de fluorescence après photoblanchiment à rayon variable (FRAPrv) et par suivi de particule unique (SPT). A 22°C ces analyses ont révélé une double compartimentation des récepteurs : dans des domaines perméables de près de 1 µm de rayon et dans des domaines de rayon inférieur à 200 nm. De plus, près d'un tiers des récepteurs présente une diffusion dirigée. Les effets de la variation de température, de la déstabilisation du cytosquelette d'actine et du blocage de l'interaction protéine G/récepteur ont été observés afin de mieux comprendre l'origine de ces domaines. Bien que tous les paramètres qui y participent ne soient pas encore déterminés, les résultats indiquent que les protéines G ainsi que le cytosquelette influencent l'organisation membranaire de hMOR. La fixation d'antagonistes ne modifie pas la diffusion du récepteur. Au contraire celle des agonistes entraînent une diminution de la taille des domaines et un ralentissement des récepteurs en lien avec la transmission du signal et l'internalisation. Nos résultats soulignent l'importance de plusieurs paramètres sur l'organisation dynamique de hMOR et confortent l'intérêt de l'utilisation conjointe du FRAP et du SPT.

Critical infrastructure (CI) is one of the most important branches in crisis man-agement. In recent years, CI demonstrated its importance on many occasions. My work is focused on the analysis of risks which can be a possible threat to CI. This is because the most important part of securing CI is the prevention of, and prepara-tion for, interruptions or damage. The risks in this part were specified using a FRAP analysis. I also identify the main reciprocal dependencies of all CI sectors to demonstrate which sector is the most important one. Finally, I specify the process of recovering or restoring these CI sectors. A new method was intentionally used for analytical-synthetic models. This method, in conjunction with the use of a FRAP analysis, is new to the branch of crisis management and was used for first time. The main aim of my work is to improve knowledge about CI at a regional level {--} city of České Budějovice. I submit new ways of identifying risks and resolv-ing problems during the recovery phase {--} specialized in CI.

Masters of Science / The aim of this study was therefore to evaluate the antioxidant and anti-diabesity potential of a hot water extract of C. maculata in non-cell based assays and correlate the activities with phenolic composition. Total antioxidant capacity (TAC) was assessed in terms of free radical scavenging and iron reducing ability. The DPPH, ABTS, ORAC and FRAP assays were employed. Anti-diabesity potential was assessed in terms of the inhibition of the digestive enzymes, α-glucosidase and pancreatic lipase

In this work, we investigated the behavior of the -opioid receptor (MOR) and the transient receptor potential vanilloid 1 (TRPV1) ion channel in the plasma membrane and their mutual communication. Both these receptors are implicated in pain perception and analgesia. We observed that the lateral mobility of MOR was strongly affected by different biased opioid agonists. DAMGO and endomorphin-2 display opposite bias towards MOR. According to our results, they also have the opposite effects on the mobility of MOR. Morphine induced only small changes in the mobility of MOR. Moreover, cholesterol depletion and blockage of G protein signaling by pertussis toxin (PTX) affected the ability of different MOR agonists to alter MOR mobility in a unique manner. The effects of DAMGO and endomorphin-2 were compromised under these conditions. On the other hand, we observed increased movement of MOR after the addition of morphine. PTX alone did not affect receptor movement, but it completely disrupted the effect of cholesterol depletion on morphine induced changes the mobility of MOR. Next we studied the mobility of TRPV1. The TRPV1 agonist capsaicin changed the lateral mobility of TRPV1. Surprisingly, after adding the MOR antagonist naloxone, the apparent diffusion coefficient of TRPV1 but to a lower extent than...

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