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Modification and analysis of low-molecular chitosans
Moravcová, Dagmar ; Bosáková, Zuzana (advisor) ; Strašák, Tomáš (referee)
4 Abstract Chitosan is a natural biopolymer derived from chitin, found in the exoskeletons of crustaceans and fungi. It has gained considerable attention due to its biodegradability, biocompatibility, antimicrobial properties, and potential antitumor activities. Chitosan has cationic amino groups that enable interactions with various anionic compounds. Therefore, it is an ideal candidate for conjugation with monovalent bioactive compounds to form efficient multivalent conjugates. It was found that galectins, especially Gal-1 and Gal-3, are overexpressed in tumor cells. Several monovalent inhibitors derived from the parent inhibitor thiodigalactoside, also those modified with coumaryl groups, have been synthesized, which show a high selectivity and affinity towards Gal-3. The main goal of this work was to synthesize a chitosan-based carrier and conjugate it with ligands exhibiting selectivity and inhibitory effect towards Gal-3. The first step involved the modification of chitosan using a unique effective method based on solid-phase hydrolysis, which significantly reduces the consumption of chemical materials and, in addition, excessive losses during purification. At the same time, the chitosan carrier was functionalized with pentynoic acid. Furthermore, monovalent galectin ligands,...
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In vivo biotinylation as a new method of galectin labeling
Balogová, Soňa ; Bojarová, Pavla (advisor) ; Kavan, Daniel (referee)
Galectins are animal lectins with affinity to terminal β-galactosides. These lectins are characterized by an evolutionarily conserved carbohydrate recognition domain (CRD). Galectins are involved in many processes in vivo, such as cell adhesion; signaling; cell proliferation and carcinogenesis. They can be used as markers in certain types of cancer and also for the targeted binding of therapeutics in the diagnosis and therapy of cancer. Galectins have been defined as a protein family composed of 15 members (11 of which are known in humans). They are classified into three subgroups, according to their structure: prototypical; chimeric; tandem-repeat type. Galectin-8 belongs to tandem-repeat galectins, which consist of two different subunits connected with a peptide linker; they contain two different CRD domains, and are therefore bivalent. Galectin-glycan interactions are based on the affinity of galectin CRDs to glycans containing a terminal β-galactoside, and this affinity is formed by the set of conserved amino acids within each CRD. These interactions can be investigated by many methods, such as X-ray crystallography; competitive ELISA; biolayer interferometry. Due to the extensive involvement of galectins in biological processes, there are many methods of labeling galectins for their detection....
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Advanced methods of assaying lectin-carbohydrate interaction
Červený, Jakub ; Bosáková, Zuzana (advisor) ; Pavlíček, Jiří (referee)
A reliable and reproducible quantification and detailed characterization of the interactions between a biomedically relevant protein and its synthetic carbohydrate ligand can provide new information for the design of better diagnostic and therapeutic tools. A high sensitivity and specificity are essential requirements for the feasibility of an analytical method. These parameters can be difficult to achieve for highly complex systems such as lectin-saccharide complexes. On the other hand, carbohydrate-based diagnostics and therapeutics offer the great advantage of detecting biologically active lectin receptors, which cannot be achieved with antibodies. In this work we demonstrate the use of the BLI technique to monitor lectin- carbohydrate interactions for a multivalent system. Using functionalization of a novel construct of galectin-1 with an in vivo biotinylated AVI tag, suitable immobilization on a biosensor was achieved without the loss of its lectin activity. Due to the high sensitivity of this technique, kinetic parameters of interaction with prepared multivalent neoglycoproteins based on LacNAc epitopes were obtained in the micro- to nanolar KD range. The complementarity of this method was demonstrated by parallel ITC and competitive ELISA measurements. The neoglycoprotein with a lower LacNAc...
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Glycocalixarenes and their affinity to human galectins
Bálková, Denisa ; Bojarová, Pavla (advisor) ; Kavan, Daniel (referee)
Calix[n]arenes are macrocyclic compounds synthesized by the oligomerisation of phenol and formaldehyde. They can have different numbers of phenol rings and can adopt various conformations. As they can be easily modified, they are widely used in many ways, especially in industry or medicine. Modifications can comprise many functional groups such as hydroxyl, amine or carboxyl. Moreover, they can be functionalized with carbohydrates. In such a case, glycocalyx[n]arenes are formed. Galectins, S-type lectins, are capable of specific recognition and subsequent binding of a number of glycoconjugates. However, it is possible only when the molecule presents β-D-galactosides. Galectins have different binding affinities to particular ligands. It has been proven that if the β-D-galactosides are conjugated to a multivalent carrier, galectins have higher affinity to the resulting glycoconjugate. With these ligands it is possible to affect many functions of galectins such as cell adhesion, regulation of apoptosis, proliferation, cell signalling pathways and immune response. Through these pathways, galectins can also cause serious diseases such as autoimmune diseases or carcinogenesis. Thus, the goal of many studies is to find such galectin ligands that could prevent these disorders or help with their treatment....
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New trends in cell and molecular biology of the head and neck cancer
Fík, Zdeněk ; Chovanec, Martin (advisor) ; Komínek, Pavel (referee) ; Ludvíková, Marie (referee)
Head and neck squamous cell carcinomas are still challenging despite progress in the oncological treatment. Study of the molecular biology allows to deeply characterize tumor properties and to predict the prognosis for affected patients. Nowadays there are many drugs clinically tested in the group of targeted therapy medicine Experimental work comprised both in vitro and in situ assays, being performed thanks to the collaboration between a number of departments of the 1st Faculty of Medicine of the Charles University in Prague, Academy of Sciences of the Czech Republic, Institute of Hematology and Blood Transfusion and Faculty of Veterinary Medicine of the Ludwig-Maxmillian University Munich. Galectin-1 is important inductor of the myofibroblasts/cancer associated fibroblasts. These fibroblasts are regarded as negative prognostic markers thanks to their capability of invasive cancer cells induction. On the other hand, Galectin-9 is not present in the carcinoma and in the case of dysplasia, its expression indicate aberrant features together with aberrant expression of keratin 14 and 19. Except from galectins using as prognostic markers, we focused on the galectins as a therapeutics instruments as well. Presented work with mutant variants of galectin-2 proved their effect on both pharmacodynamics and...
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Healing wound as a model for the study of cell interactions
Gál, Peter ; Smetana, Karel (advisor) ; Motlík, Jan (referee) ; Brábek, Jan (referee)
Healing wound as a model for the study of cell interactions Abstract Galectins play an important role in the processes of cell proliferation, differentiation, migration and extracellular matrix formation. Furthermore, galectins are able to transfer cellular signals and to participate in cell interaction. It has been proven that galectins play an important role in the microenvironment formation of a tumor and/or healing wound. This study demonstrated significant role of galectins, in particular Galectin-1, in wound healing and cell interactions (endothelial cells, fibroblasts and keratinocytes) forming a part of the granulation tissue and tumor stroma. We have demonstrated that the extracellular matrix rich on Galectin-1 creates a suitable environment for the cultivation of keratinocytes. Galectin-1 also induces differentiation of fibroblasts into myofibroblasts. The knowledge of above mentioned processes is important to better understand the complexity of cancer biology and its parallel to wound healing. Key words: tissue repair, regeneration, galectin, tumor
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Glycobiology of the head and neck cancer
Valach, Jaroslav ; Smetana, Karel (advisor) ; Dřízhal, Ivo (referee) ; Říhová, Blanka (referee)
iii Abstract Glycobiology represents a very progressive subject of cell biology. Protein-saccharide interactions play not only supporting and cell organization role, but they also represent medium for information storage and its decoding. Galectins, group of animal lectins (saccharide-binding proteins), which have selective affinity to ß-galactosides, are multifactorial molecules. They participate in cell-cell and cell-matrix interaction, transmembrane signaling, apoptosis, pre- mRNA splicing and are also present in various types of carcinomas. High expression of galectin-1 has been detected in cancer stroma originated from squamous cell epithelium. In the previous study we established that the fibroblasts - myofibroblasts transition, apart from the known TGF- beta, is also induced by galectin-1. We compared relationship between galectin-1 expression, presence of myofibroblasts and gene expression in tissue samples from patients with head and neck squamous cell carcinoma. Cancer stroma with myofibroblasts was rich in galectin-1 expression in comparison with stroma without myofibroblasts. Moreover, we used microarray analysis (ILLUMINA) to compare the whole genome transcriptome from samples with and without presence of galectin-1. High expression of galectin-1 in tissue samples corresponded with expression...
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Glycobiology of the head and neck cancer
Szabo, Pavol ; Smetana, Karel (advisor) ; Říhová, Blanka (referee) ; Brábek, Jan (referee)
Povrch buněk je bohatě pokryt oligosacharidy, které jsou v plazmalemě ukotvené pomocí proteinů a lipidů. Oligosacharidy zprostředkují vzájemnou vazbu mezi buňkami nebo vazbu buněk k složkám extracelulární matrix. Galektiny jsou živočišné lektinů které mají afinitu k oligosacharidům obsahujícím β-galaktózu. Jsou to multifaktoriální proteiny, které se účastňují řady reakcí v organizmu, jako jsou mezibuněčné interakce, interakce buněk s mezibuněčnou hmotou, proliferace i apoptóza a sestřih pre-mRNA. Proteiny po translaci procházejí různými strukturálnimy úpravami, které mají vliv na jejich funkci. Galektin-3 je možný prognostický ukazatel u nádorů vycházejících z vrstevnatých dlaždicových epitelů je fosforylován na N-konci. Prokázali jsme, že tato posttranslační modifikace nemá vliv na jeho vazebnou reaktivitu. Jiný endogenní lektin, galektin-1 je charakteristickou molekulou nádorového stromatu a granulační tkáně hojícího se poranění. Zjistili jsme, že galektin-1 indukuje na TGF-β nezávislý in vitro přechod normálních fibroblastů na myofibroblasty včetně produkce sítě extracelulární matrix bohaté fibronektinem a galektinem-1. Tento poznatek je využitelný v terapii hojení ran a v tkáňovém inženýrství. Dnes je jasné, že nádorové stroma ovlivňuje i biologické vlastnosti nádoru (lokální agresivita,...
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