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Study of retention and separation of peptides in HPLC
Boudová, Hana ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
This diploma thesis deals with the study of retention and separation of structurally different peptides and digest products of cytochrome c using reverse phase and mixed modes of liquid chromatography. As the model peptides, five unmodified dipeptides, nine dipeptides containing a protective benzoyl group on the N-terminal amino acid and eight therapeutic peptides containing five to ten amino acids were selected. The chromatographic behaviour of the model peptides was tested on three different columns: XBridge C18, XSelect CSH C18 and Atlantis Premier BEH C18 AX under different conditions. The influence of the pH of the aqueous part of the mobile phase, i.e. 2,1; 3,0; 4,7 and 6,8, and the amount of acetonitrile in the mobile phase on retention and separation of model peptides was tested. The effect of the concentration of ammonium acetate buffer, pH = 6,8 in the mobile phase on the retention and peak symmetry in the concentration range of 10-50 mmol L-1 was assessed. The chromatographic conditions for the baseline separation of 20 different model peptides were optimized. The conditions were as follows: XSelect CSH C18 column, mobile phase composed of acetonitrile/10mM ammonium acetate, pH = 6,8 (v/v) under gradient elution. The suitable chromatographic conditions for the analysis and identification...
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Transport of charged and neutral particles across the model biomembranes
Parisová, Martina ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
This work was focused on the preparation of model stabilized phospholipid membranes formed on porous polycarbonate carrier. 1,2-dipalmitoyl-sn-glycero-3-phosphocholin was used for their formation in hydrophilic pores of polycarbonate carrier. For characterization of the formation of phospholipid layers, their changes and a study of transport processes, electrochemical impedance spectroscopy and voltammetry were used. Transport of cadmium and copper ions was studied in the presence and in the absence of ionophore calcimycin which was incorporated into the formed of phospholipid membrane. Because these ions are often bound in complexes with various substances, such as low molecular weight organic acids (LMWOAs), this work was also focused on the transport of copper and cadmium ions across the model phospholipid membranes in the presence of malic acid, citric acid and oxalic acid at different pH. Besides the use of ionophore, some pilot experiments were performed to realize the transfer of copper ions using two peptides, nisin and transportan 10. Formation of phospholipid membranes and the transport processes were characterized by two proposed electric equivalent circuits which correspond to the covered and to the uncovered polycarbobate carrier. Keywords: Phospholipids, Membranes, Ionophore, Peptid....
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Multidimensional characterization of polyelectrolytes and interpolyelectrolyte complexes in aqueous solutions
Murmiliuk, Anastasiia ; Štěpánek, Miroslav (advisor) ; Šachl, Radek (referee) ; Hoffmann, Ingo (referee)
Multidimensional characterization of polyelectrolytes and interpolyelectrolyte complexes in aqueous solutions Abstract: This PhD thesis is dedicated to the study of polyelectrolytes and their self-assembly in aqueous solutions. The morphology and ionization state of individual polymer chains were investigated as well as their co-assembly with oppositely charged species. First, the charge regulation of short and long weak polyelectrolytes in aqueous solution was investigated to deeper understand their pH-responsive properties. The changes of ionization degree and charge of oligopeptides composed of 5 amino acids with acid side-chains and 5 with basic side chains were followed upon varying the pH in order to reveal the effect of interactions between the like-charged and oppositely charged groups. It was shown that intra-molecular electrostatic interactions and conformational flexibility caused the suppression of the total charge and enhancement of ionization of the peptides. To get an insight into the distribution of local H+ concentration in the proximity of a polyelectrolyte chain, a modified polyelectrolyte structure was used with a fluorescent pH- indicator, covalently attached at the end of the chain. Ionization of the pH-responsive fluorophore revealed its effective pK which was compared with the local...
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Nanostructured Modifcation of Material Surfaces for Controlled Cell Cultivation
Kotelnikov, Ilya ; Proks, Vladimír (advisor) ; Chytil, Petr (referee) ; Šebestová Janoušková, Olga (referee)
Commercial media and surfaces for cell cultivation do not promote conditions for cell cultivation and proliferation with specific interactions cell-material surface. The aim of this thesis is developing surfaces for cell cultivation which mimic conditions in living tissues. The approach introduced in this thesis is based on applying biomimetic peptide ligands on inert supporting materials with non-fouling properties. Considering that a choice of a ligand sequence and distance between peptides can dramatically influence the outcome, a few model peptides with varying parameters were synthesized and investigated. The cell adhesive peptides were synthesized by solid phase peptide synthesis and scrambled peptides were synthesized and tested as well. Spatial deposition of peptides is another highly important point of study. The 'click'- reaction was used to successfully immobilize the peptides on produced surfaces. The distance between the peptide molecules on the surfaces was controlled by their concentration in a reaction mixture. The reference samples were immobilized with radiolabeled peptides for quantitative estimation of the peptides present. Then, the materials with different types of peptides and range of concentrations were examined via cultivation of cell cultures. The experiments were focused on...
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Multidimensional characterization of polyelectrolytes and interpolyelectrolyte complexes in aqueous solutions
Murmiliuk, Anastasiia ; Štěpánek, Miroslav (advisor) ; Šachl, Radek (referee) ; Hoffmann, Ingo (referee)
Multidimensional characterization of polyelectrolytes and interpolyelectrolyte complexes in aqueous solutions Abstract: This PhD thesis is dedicated to the study of polyelectrolytes and their self-assembly in aqueous solutions. The morphology and ionization state of individual polymer chains were investigated as well as their co-assembly with oppositely charged species. First, the charge regulation of short and long weak polyelectrolytes in aqueous solution was investigated to deeper understand their pH-responsive properties. The changes of ionization degree and charge of oligopeptides composed of 5 amino acids with acid side-chains and 5 with basic side chains were followed upon varying the pH in order to reveal the effect of interactions between the like-charged and oppositely charged groups. It was shown that intra-molecular electrostatic interactions and conformational flexibility caused the suppression of the total charge and enhancement of ionization of the peptides. To get an insight into the distribution of local H+ concentration in the proximity of a polyelectrolyte chain, a modified polyelectrolyte structure was used with a fluorescent pH- indicator, covalently attached at the end of the chain. Ionization of the pH-responsive fluorophore revealed its effective pK which was compared with the local...
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Phenotyping of proteolytic activities enabled by fluorogenic libraries
Pospíšil, Šimon ; Míšek, Jiří (advisor) ; Hocek, Michal (referee)
This work deals with the preparation of combinatorial libraries of peptides serving as platforms for proteolytic phenotyping. The primary objective was to prepare a solid phase fluorogenic peptide library and screen proteases by fluorescence. Further, the possibility of preparing solid phase DNA-encoded libraries was studied. Due to the non-reactivity of the specific proteases with the solid phase peptides, the solid phase was completely abandoned and DNA-encoded peptide library was prepared in the solution. Using this model of DNA-encoded dipeptide with terminal biotin, the new principle of testing proteolytic activities of proteases was verified. A combinatorial library of DNA-encoded hexapeptides was also prepared. Despite the low yield of the library, the possibility of DNA encoding, the amplifiability of the prepared molecules and the possibility of biotin-based separation were verified. The integrity of the hexapeptide sequence and the protease testing is the subject of further study.
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Design and characterization of advanced polymer-coated upconversion nanoparticles
Kostiv, Uliana ; Horák, Daniel (advisor) ; Kaman, Ondřej (referee) ; Brožek, Jiří (referee)
Lanthanide-based upconversion nanoparticles are able to absorb low-energy near-infrared (NIR) photons and emit visible or ultraviolet light. This unique optical property enables luminescent detection without autofluorescence from biological samples that is crucial for bioimaging and diagnostics. Moreover, NIR irradiation allows to deliver light deep into the tissue, which can be used for drug or gene delivery and NIR-induced photodynamic therapy (PDT) of tumors. Despite huge effort during the last 10 years devoted to the upconversion nanoparticle synthesis and their surface engineering, preparation of the particles suitable for biomedical applications still remains a big challenge. The current work is focused on synthesis of the upconversion nanoparticles with controllable morphology, size, distribution, crystallinity, and high upconversion efficiency, as well as on desirable surface modification by biocompatible inorganic or organic polymers. Neat and functionalized homogeneous silica shell was introduced on the NaYF4:Yb3+ /Er3+ and NaGdF4:Yb3+ /Er3+ nanoparticles to enhance their biocompatibility and colloidal stability in water. To render the nanoparticles with targeting moieties, their surface was decorated with cell-adhesive RGD or cell-penetrating TAT peptides. The RGD- and TAT- conjugated...
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Radiolabelled antibodies - the perspective for diagnosis and therapy
Mejtská, Jana ; Bárta, Pavel (advisor) ; Ramos Mandíková, Jana (referee)
1 CHARLES UNIVERSITY FAKULTY OF PHARMACY IN HRADEC KRÁLOVÉ DEPARTMENT OF BIOPHYSICS AND PHYSICAL CHEMISTRY DISSERTATION THESIS RADIOLABELED ANTIBODIES - THE PERSPECTIVE FOR DIAGNOSIS AND THERAPY Supervizor: Mgr. PAVEL BÁRTA, Ph.D. HRADEC KRÁLOVÉ, 2017 Bc. JANA MEJTSKÁ 2 ABSTRACT EN Different types of tissues have a characteristic cell morphology. Each cell has typical molecules on its surface, which may be either of physiological or pathological type. The presence of these surface structures can be interesting for possible modulation of specific cell populations from neighboring cells. Utilization of this property is then essential particularly in a case of tumor cells. Targeting on tumor specific cell structures involves the use of receptor specific peptides or monoclonal antibodies. The discovery of the preparation of monoclonal antibodies has opened a new chapter in the treatment and diagnosis not only tumor diseases. The advantage of monoclonal antibodies is their specificity and also high affinity to the type of the target cell structures. This study is focused on the summary of monoclonal antibodies which are currently being applied on the treatment or diagnosis of a particular cancer. Furthermore, this work also includes antibodies which are under development for intended medical applications with...
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Transport of charged and neutral particles across the model biomembranes
Parisová, Martina ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
This work was focused on the preparation of model stabilized phospholipid membranes formed on porous polycarbonate carrier. 1,2-dipalmitoyl-sn-glycero-3-phosphocholin was used for their formation in hydrophilic pores of polycarbonate carrier. For characterization of the formation of phospholipid layers, their changes and a study of transport processes, electrochemical impedance spectroscopy and voltammetry were used. Transport of cadmium and copper ions was studied in the presence and in the absence of ionophore calcimycin which was incorporated into the formed of phospholipid membrane. Because these ions are often bound in complexes with various substances, such as low molecular weight organic acids (LMWOAs), this work was also focused on the transport of copper and cadmium ions across the model phospholipid membranes in the presence of malic acid, citric acid and oxalic acid at different pH. Besides the use of ionophore, some pilot experiments were performed to realize the transfer of copper ions using two peptides, nisin and transportan 10. Formation of phospholipid membranes and the transport processes were characterized by two proposed electric equivalent circuits which correspond to the covered and to the uncovered polycarbobate carrier. Keywords: Phospholipids, Membranes, Ionophore, Peptid....
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