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The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.

Hyaluronan is a polymer that among other things is used in fillers. Products containing Hyaluronan is sometimes discoloured over time and the mechanism behind this discolouration is still unknown. However, it was suspected that discolouration occurs during the degradation due to high pH values or with a phosphate buffer. The discolouration of Hyaluronan that occurs with phosphate buffer was studied in more detail in this project. The samples of Hyaluronan with different concentrations of phosphate buffer were left at 90 °C in an oven to speed up the discolouration. These samples were then analyzed byusing UV/Vis spectrophotometry to measure the absorption and capillary viscometry to measure the molecular weight. The results showed that the discolouration increased with time and that the samples with the higher concentration of buffer got more discoloured faster. The molecular weight showed a decreasing trend with time. It also suggested that the phosphate buffer had an impact on the molecular weight. The samples with the highest concentration of phosphate buffer had a lower molecular weight compared to samples with no phosphate buffer. The main conclusion from this study is that the phosphate buffer had an effect on the discolouration of Hyaluronan.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The research presented in this thesis focuses on the study of the reaction between boronic acids and diols and its evaluation as a possible "click" reaction, possibly applicable in bioconjugation and drug delivery. A key feature of this reaction is its reversibility at acidic pH, which could allow the release of a diol-containing drug from a bioconjugate in the acidic environment of late endosome/lysosome, possibly after undergoing receptor mediated endocytosis. Over the last two decades various studies have focused on the study of the conjugation of boronic acids to diols using Alizarin Red S as a fluorescence reporter. In this research we have presented an alternative method based on the batochromic shifts of Alizarin Red S absorbance; this method is particularly advantageous in complex systems with an elevated scattering, such as colloidal dispersions or for binding to complexed active compounds. We have therefore demonstrated that this method allows the determination of equilibrium constants between diols (e.g. catecholamines) and boronic acids. We have also demonstrated that the method allows to follow the kinetics of enzymatic reactions involving catechols; in particular, we have focused on cytochrome P450-mediated reactions such as the conversion of estradiol to 2-hydroxyestradiol using CYP1A2, or the demethylation of 3-methoxytyramine to dopamine using CYP2D6. Once we have established a reliable method for following this reaction on low molecular weight compounds, we have applied it to polymeric bioconjugates. Specifically, we have selected hyaluronic acid (HA) as a biocompatible and biodegradable polymeric backbone and produced derivatives containing boronic acids, catechols and dimethylated catechols (as negative controls). The resulting polymers where characterised via UV-Vis, 1H NMR and SLS, also qualitatively evaluating their cytotoxicity and enzymatic degradability. The conjugates with boronic acids showed the lowest cytotoxicity, and the highest degradability. The complexation of HA-boronic derivatives was then studied; using the same library of diols previously used with low molecular weight compounds, evaluating the effect of the presence of the polysaccharidic macromolecular chain.

This thesis pursues monitoring of pH changes and description of properties in phase–separated hydrogels prepared by interaction of polyelectrolyte and oppositely charged surfactants. For the purposes of this work, all hydrogels were prepared by the interaction of hyaluronan with Septonex. All hydrogels were exposed to pH changes (4–9). Changes in pH were being monitored using spectroscopic indicators, methyl orange, methyl red, bromothymol blue and phenolphtalein. The pre– and post– pH changes in properties were compared using rheological methods. Additional information was gathered through thermogravimetric analysis. By these methods it was proved that the changes in the pH can modify the mechanical properties and partly the internal structure of the hydrogels. Rheological tests show, that from the application point of view, the most interesting hydrogels are observed at pH 9 which have the strongest bonds but have low permeability and hydrogels described at pH 7 which are much softer and are capable of absorbing large amounts of water.

Předmětem tohoto studia bylo prozkoumat reologické chování vodných roztoků vysokomolekulárního hyaluronanu. Byl studován vliv zvyšující se koncentrace biopolymeru v roztoku, která se pohybovala v rozmezí od 1 do 3 hmotnostních procent, a také vliv vzrůstající iontové síly rozpouštědla, způsobené přídavkem chloridu sodného, na viskoelasticitu a stabilitu těchto roztoků. Pro obsáhlejší popis viskoelasických vlastností roztoků byla použita, vedle běžných oscilačních měření, také metoda ceepových testů, ze které bylo možno určit důležité veličity, jako je procentuální poměr viskozní a elastické složky vzorku, rovnovážná poddajnost, viskozita při nulovém smykovém napětí a retardační čas. Ty byly následně porovnávány s výstupy z jiných typů měření, jako jsou právě oscilační a tokové křivky, nebo nesly dopňující informace důležité pro detailnější popis viskoelastických vlastností těchto roztoků. Ke studiu stability vzorků během namáhání pak byla použita metoda peak-hold, která ukázala na velmi dobou mechanickou i časovou odolnost roztoků hyaluronanu a naznačila hranice, za kterými už dochází k trvalému poškození struktury a degradaci řetězců hyaluronanu a je s němi proto potřeba při manipulaci s roztoky tohoto biopolymeru pro jejich další použití v aplikacích počítat.

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