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Preparation and characterization of surface-modified nanoliposomes using click chemistry techniques
Frydrychová, Aneta ; Bartoš,, Milan (referee) ; Turánek, Jaroslav (advisor)
Over the past decades the liposomes have been intensively studied for their unique properties which predispose them for use as drug delivery systems or for constructions of vaccines. This diploma thesis provides an overview of their most important properties, preparation options and their surface modification. The aim of this thesis is thus a preparation and characterization of the nanoliposomes and their surface modification. The liposomes were prepared by lipid film hydration method and mannan polysacharide was used for surface modification. Due to the use of a lipid with an N-oxy group, the modification was carried out via an oxime ligation via click chemistry. Nanoliposomes were characterized by series of physicochemical methods such as TEM, DLS, FT-IR or nano-flow cytometry. Part of the thesis is a study of the interactions of liposomes accomplished on selected cell lines to verify whether they stimulate immune response pathways. Its results confirmed activation of the NLRP3 inflammasome leading to the production of pro-inflammatory cytokines (IL-1). Thus, these polymer coated nanoliposomes are potentially useful as vaccine adjuvants.
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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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Photoaffinitive conjugation of biomolecules
Masárová, Petra ; Slanina, Tomáš (advisor) ; Vrábel, Milan (referee)
Photoclick chemistry represents a class of photo-activated click reactions generally characterised by precise spatial and temporal control, high selectivity, and quantitative yields. The following thesis will focus on several classes of light triggered click reactions and attempt to outline their chemistry in reversible nucleophilic addition, irreversible cycloaddition and Diels-Alder reactions initiated upon irradiation of photoactive precursors. The absorption of such precursors can be shifted towards visible light by derivatization of their chromophoric moieties, which allows for a non-harmful excitation in vivo and therefore offers promising prospects in biological applicability. The importance of click photochemistry resides in a remarkable selectivity that enables reactions to take place in highly complex, non-controlled biological environments. The photo-inducible precursors can be conjugated with various moieties of biological significance - a method most notably used in photoaffinity labelling. Considerable efforts have been made to explore the reactivity of photoactivatable compounds towards biological nucleophiles in various spheres of applied research which will all be discussed and summarised.
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Synthesis of a combinatorial compound library based on the double click reaction
Krutáková, Mária ; Zitko, Jan (advisor) ; Kučerová, Marta (referee)
(English) Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Author: Mária Krutáková Supervisor: Assoc. Prof. PharmDr. Jan Zitko, Ph.D. Title: Synthesis of a combinatorial compound library based on the double click reaction Click chemistry is a powerful tool in drug discovery. It is very efficient in creating compound libraries through the combinatorial methodology. The copper(I)-catalyzed 1,2,3-triazole- forming reaction between azides and terminal alkynes has become the gold standard of click chemistry due to high reaction efficiency, mild reaction conditions, chemo- and regioselectivities. The molecules with triazole moiety display a broad spectrum of favorable properties and have been used in the development of antibacterial, antiviral, anti-inflammatory, anticancer, and anti-tubercular agents. This work focused on preparing a compound library using double-click reactions. Firstly, we synthesized several compounds with two alkyne groups ("alkyne cores") and a diverse group of structurally simple azides. In the next step, one equivalent of alkyne core was reacted with two equivalents of all prepared azides. The reaction between each alkyne core and ten azides yielded 100 compounds. The prepared libraries of compounds were...
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Studying protein interactions with expanded genetic code
Tekel, Andrej ; Obšil, Tomáš (advisor) ; Fuertes Vives, Gustavo (referee)
Nature, using proteins composed of approximately 20 amino acids repertoire, is able to perform a large number of admirable things, many of which we are still not quite able to mimic. However, it has to be said, that the set of chemical scaffolds available to the nature is somewhat limited. Therefore, it seems plausible to assume, that we could make things better simply by introducing novel scaffolds and exploring chemical space so far unavailable. It is exactly this idea upon which expanded genetic code is based on. To set ourselves free from limitations imposed by standard genetic code. This thesis will aim to provide an overview of various attempts to this problem and will try to do so in a distinct context of studying protein interactions. I will thus try to highlight methods by which we are currently able to expand genetic code, specific chemistries and physical properties of non-canonical amino acids and biophysical methods which can profit from genetic code expansion. Keywords: expanded genetic code, non-canonical amino acid, amber codon, fluores- cence, magnetic resonance, crosslinking, click chemistry
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Preparation and characterization of surface-modified nanoliposomes using click chemistry techniques
Frydrychová, Aneta ; Bartoš,, Milan (referee) ; Turánek, Jaroslav (advisor)
Over the past decades the liposomes have been intensively studied for their unique properties which predispose them for use as drug delivery systems or for constructions of vaccines. This diploma thesis provides an overview of their most important properties, preparation options and their surface modification. The aim of this thesis is thus a preparation and characterization of the nanoliposomes and their surface modification. The liposomes were prepared by lipid film hydration method and mannan polysacharide was used for surface modification. Due to the use of a lipid with an N-oxy group, the modification was carried out via an oxime ligation via click chemistry. Nanoliposomes were characterized by series of physicochemical methods such as TEM, DLS, FT-IR or nano-flow cytometry. Part of the thesis is a study of the interactions of liposomes accomplished on selected cell lines to verify whether they stimulate immune response pathways. Its results confirmed activation of the NLRP3 inflammasome leading to the production of pro-inflammatory cytokines (IL-1). Thus, these polymer coated nanoliposomes are potentially useful as vaccine adjuvants.
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Synthesis of block copolymers by ring-opening polymerization and post polymerization modification with phenylboronic acid
Dudičová, Dorota ; Uchman, Mariusz Marcin (advisor) ; Strachota, Adam (referee)
This thesis deals with the synthesis of propargyl-functionalized amphiphilic diblock copolymers, poly(ε-caprolactone)-b-poly(ethylene oxide), PgCL-PEO via Ring Opening Polymerization (ROP) using monohydroxyl-terminated PEO block as the macroinitiator and its post polymerization modification via Copper-Catalyzed Azide-Alkyne Click reaction (CuAAC) to introduce phenylboronic acid functionality into block copolymers. A reproducible synthetic protocol was developed for preparation of well-defined functional diblock copolymers. The amphiphilic block copolymers were characterized in terms of molecular weight and dispersity by various techniques: Nuclear Magnetic Resonance (NMR) spectroscopy, Gel Permeation Chromatography (GPC) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI ToF) Mass Spectrometry. Keywords: Amphiphilic block copolymers, CuAAC click, phenylboronic acid, poly-ε- caprolactone, ring-opening polymerization
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Synthesis of block copolymers by ring-opening polymerization and post polymerization modification with phenylboronic acid
Dudičová, Dorota ; Uchman, Mariusz Marcin (advisor) ; Strachota, Adam (referee)
This thesis deals with the synthesis of propargyl-functionalized amphiphilic diblock copolymers, poly(ε-caprolactone)-b-poly(ethylene oxide), PgCL-PEO via Ring Opening Polymerization (ROP) using monohydroxyl-terminated PEO block as the macroinitiator and its post polymerization modification via Copper-Catalyzed Azide-Alkyne Click reaction (CuAAC) to introduce phenylboronic acid functionality into block copolymers. A reproducible synthetic protocol was developed for preparation of well-defined functional diblock copolymers. The amphiphilic block copolymers were characterized in terms of molecular weight and dispersity by various techniques: Nuclear Magnetic Resonance (NMR) spectroscopy, Gel Permeation Chromatography (GPC) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI ToF) Mass Spectrometry. Keywords: Amphiphilic block copolymers, CuAAC click, phenylboronic acid, poly-ε- caprolactone, ring-opening polymerization
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Synthesis and characterization of new insulin analogs with a triazole bridge at the C-terminus of the B chain
Kuntová, Vendula ; Jiráček, Jiří (advisor) ; Ryšlavá, Helena (referee)
Insulin is a peptide hormone responsible for maintaining glucose homeostasis in the circulation. Insulin interacts with two isoforms of the insulin receptor, IR-A and IR-B, which have different tissue distribution. IR-A is supposed to have rather mitogenic function and IR-B rather metabolic function. The goal of this study was to develop insulin analog, which will be more selective for IR-B than human insulin. We prepared three new insulin analogs with a 1,2,3-triazole bridge at the positions B26 and B29. The triazole bridge was formed by Cu(I)- catalysed cycloaddition between side chains of azidopentanoic acid (N3Pent) at B26 and propargylglycine (Prg) at B29. The analogs differed in configurations on C carbons of unusual amino acids at the positions B26 and B29. Specifically, we prepared insulin analog 1 with D- N3PentB26 and D-PrgB29, insulin analog 2 with D-N3PentB26 and L-PrgB29 and insulin analog 3 with L-N3PentB26 and D-PrgB29. New analogs were tested for their binding to both isoforms (IR-A and IR-B) of the insulin receptor. Analogs 1 and 2 were less potent in binding than human insulin and had no selectivity for receptor isoforms. Analog 3 was 4-times more potent in binding to IR-B and 2-times more potent in binding to IR-A than human insulin. However, the binding selectivity of the...
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Optimization of methods for analysis of early steps of mouse polymavirus life cycle
Soukup, Jakub ; Španielová, Hana (advisor) ; Němečková, Šárka (referee)
Mouse polyomavirus is a type species of Polyomaviridae family and serves as model for studying viral infection of human pathogenic polyomaviruses. Minor proteins of viral capsid have been found to be necessary for effective initiation of infection. In order to study their role in the early steps of infection we utilized the novel Cre-LoxP system for production of the viral mutant lacking both minor proteins. Virus produced this way was compared with virus produced by standard method and we found that both systems facilitate production of mutant virus with the comparable quality and quantity. The mutant virus contained reduced amount of viral DNA and formed virions with impaired stability. For further studies of intracellular virion trafficking we prepared virions with genomes modified by thymidine analogues 5- bromo-2'-deoxyuridine (BrdU) and 5-Ethynyl-2'-deoxyuridine (EdU) and optimized the methods for analogue detection. The viral genome become accessible for detection 4 hours post infection. For ultramicroscopic analysis of translocation of virus to the nucleus we used freeze substitution. All this methods will be utilized for detailed study of distinct steps in viral infection. Key words: Mouse polyomavirus, minor proteins,...
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