National Repository of Grey Literature 31 records found  beginprevious22 - 31  jump to record: Search took 0.01 seconds. 
Heterologous expression and purification of human cytochrome b5
Kostelanská, Marie ; Černá, Věra (advisor) ; Bořek Dohalská, Lucie (referee)
The metabolism of xenobiotics and endogenous substances is mediated by a mixed function oxidase system which includes cytochrome b5 participating in catalytic activities of CYP. The mechanism of action of the cytochrome b5 has not been fully elucidated yet. But it is assumed that cytochrome b5 is involved either in direct electron transfer within the mixed function oxidase system or in induction of conformational changes in CYPs. So it is important to gain the pure form of apo-cytochrome b5, devoid of heme, which is not capable of electron transfer and further study the effect of this form on CYP-catalyzed reactions. The obtained results can contribute to understanding the mechanism of cytochrome b5 effects. The transformation of bacterial cells of Escherichia coli BL-21 (DE3) Gold was performed by expression vector pET22b which contained genes for microsomal and erythrocyte cytochrome b5. In order to produce a high level of apoprotein form, the heterologous expression of cytochrome b5 was induced by addition of higher amount of IPTG. Expression was performed at 37řC. This bachelor thesis is primarily engaged in purification of both microsomal and erythrocyte form of cytotochrom b5, especially in its apo-form. However, the productions of holo-cytochrome b5 form always occur in a greater or lesser...
Preparation of recombinant cytochrome P450 1A1
Dvořák, Martin ; Svášková, Dagmar (advisor) ; Ingr, Marek (referee)
5 Abstract Cytochrome P450 1A1 (CYP1A1) is one of the major isoforms of the cytochrome P450 superfamily. It is primarily an extrahepatic enzyme which is responsible for oxidation of many polycyclic aromatic hydrocarbons and other xenobiotics. Besides of the role in detoxification metabolism CYP1A1 is the one most important isoform involved in activation of procarcinogens. The main aim of this project was preparation of two modifications of the rat CYP1A1 gene with codon optimization for expression in E. coli by gene synthesis. One was wild type (wt1A1) and the other was with modified N-terminal anchor (mod1A1) - for both modifications with or without His Tag at the C-end of CYP1A1. Furthermore, an aim was to compare their level of expression in different strains of E. coli and try to purify and assess enzymatic activity of the gene's products. From pre-prepared oligonucleotides 2 "syntons" (parts of gene) were synthetized and separately inserted into pUC19. After verified sequence of the "syntons" they were cleaved from pUC19 and inserted together into pET-22b. These vectors were prepared for transformation of 3 strains of E. coli (BL-21 (DE3) GOLD, RIL a RIPL). For production of proteins many conditions were tested: temperature (18, 22, 24, 27 a 37 řC), time of production (untill 48 hours), concentration...
Hydrogels based on cationic polyelectrolytes
Jarábková, Sabína ; Lehocký,, Marián (referee) ; Pekař, Miloslav (advisor)
This diploma thesis deals with the study of physical hydrogels based on positively-charged polyelectrolyte. The study is to investigate the interaction of selected positively-charged polyelectrolytes with oppositely charged surfactants in water and in physiologicla saline solution (0.15 M NaCl). The influence of the process for preparing hydrogels. Hydrogels were prepared by dry or wet. Were tested solubilization abilities hydrogels prepared using the hydrophobic dye oil red O were also measured rheological properties of the hydrogels prepared using frequency oscillatory tests and flow tests, depending on the concentration of the polymer or surfactant. For the rheological experiments suggest that chitosan is capable of forming rigid hydrogels with better mechanical properties than the dextran in the presence of both surfactants. Selected samples were also carried out experiments using fluorescent sold prodan, nile red and -naphthol as fluorescent probes. And in selected experiments was determined by solids content in percentage depending on the concentration used polyelectrolytes or surfactants.
Solubilization in sonographic systems
Überall, Martin ; Klučáková, Martina (referee) ; Mravec, Filip (advisor)
The aim of this thesis was to determine the solubilizing capacity of microbubbles based on SonoVue®, and phospholipids SonoVue® is made of, by using the UV-VIS spectrophotometry. The concentrations of solubilized substances within these systems was further determined. In particular, the properties of natrium dipalmitoylphosphatidylglycerole and distearoyl¬phosphatidylcholine were investigated. The microbubbles were produced using these phospholipids with the addition of polyethyleneglycol and palmitic acid. The solubilizing capacity was determined using hydrophobic solutes Sudan Red G, Oil Red O, 4-Di-2-Asp and Nile Red in order to obtain a model system of solubilized drugs or other hydrophobic substances. The behavior of solutes in phospholipids and our prepared microbubbles were examined in a moderately polar medium – physiological saline solution (0.15 M NaCl). The vizualization of prepared microbubbles was performed using optical and fluorescence microscopy. 4-Di-2-Asp, as a fluorescence probe, was not suitable for microbubble vizualization. The size of microbubbles that were produced during the experiment was almost the same as the size of microbubbles of commercially made SonoVue®. The concentration of solubilized hydrophobic solutes inside the liposomes of phospholipids ranged from tens to hundreds of micromoles per liter. With increasing concentration of phospholipids the concentration of solubilized solutes also increased. The results of this experiment can be used for further research focused on the solubilization of drugs in microbubbles, and contrast agents which are used in ultrasonography.
Solubilization properties of polyelctrolyte - surfactant complexes
Malá, Michaela ; Pekař, Miloslav (referee) ; Venerová, Tereza (advisor)
In this diploma thesis has been studied system of hydrogels based on interaction between polyelectrolyte and surfactants, especially their solubilizing properties. As a surfactant was used ionic cationic cetyltrimethylammonium bromide (CTAB). As a polyelectrolyte was chosen native anionic hyaluronan with various molecular weight - 0,3; 0,9; 1,6 MDa and with different concentration - 2%, 4%, 6% wt. Solubilization experiments were realized with hydrophobic dye sudan red G. The whole system was prepared in the simpliest model system of physiological solution 0,15 M NaCl and in water. At the prepared gels was investigated their stability, influence of the environment, molecular weight and concentration on solubilization. Gels were prepared in rate hyaluronan-surfactant 1:1. The purpose of this diploma thesis was to find and optimize the appropriate procedure for determining solubilization capacity by using UV-VIS spectrophotometry.
Solubilizattion properties of polysaccharides
Lenartová, Radka ; Foglarová, Marcela (referee) ; Pekař, Miloslav (advisor)
In this diploma thesis were studied solubilization properties of polysaccharides by using hydrophobic solutes (Sudan Orange G, Sudan Red G, (±)-alpha-Tocopherol, Pyrene, Perylene, Nile red), which were represented by alkyl derivates of hyaluronan. At first, a behaviour of individual hydrophobic solutes was investigated in variously polar solvents (Methanol, 1 Propanol, Chloroforme, Cyklohexane, n Heptane) and in the environment of varying ionic strength (water, 0.1 M and 0.4 M NaCl). Afterwards, solubilization properties of Sodium Dodecyl Sulfate model solubilizated the hydrophobic solutes into a core of micelles was examinate. We were interested in the solubilization capacity as the mol of solubilized molecules per mol micelles of surfactant corresponding with a state of micelles saturation. In the case of the solubilization of (±)-alpha-Tocopherol into the core of micelles, it was not possible to determine the solubilization capacity. So we changed the determination of universally solubilization power. The solubilization power is defined as mol of molecules solubilized per mol surfactant relative to the quantity solubilizate at the micelles saturation. Model system of Sodium Dodecyl Sulfate as a simple surfactant carrying a negative charge as the alkyl derivates of hyaluronan was selected bacause of its characteristics.The surfactant forms unimolar micelles and its critical micelle concentrations and aggregation numbers are tabelated for the investigated microenvironment. The main aim of the study was investigating of hydrophobic domains of alkyl derivates of hyaluronan as free places for incorporation hydropbobic solutes in the microenvironment of varying ionic strength. The critical aggregation concentrations were determined by the Pyrene 1:3 ratio method. For the research of micropolarity of alkyl derivates hyaluronan’s domains were selected two concentrations of derivates for the next research of solubilization experiments - the first concentration near the critical aggregation concentration and the second concentration above it. The effect of concentration of Pyrene on a core polarity of derivates was investigated. We discovered the influence of the concentration and the other we found a stationary area of the concentration. In the end we investigated the influence of preparation of solutions of derivates of hyaluronan on the core polarity by the concentration of pyrene which corresponds to the stationary area. The study of solubilization properties of alkyl derivates of hyaluronan is not a simple case as we assumed. When we measured spectra of the absorbance, higher concentration of derivates of hyaluronan belittle absorbance of solubilizates. At the experiment of solubilization with Sudan Red G we found out that Sudan Red G is not able to solubilizate into the hydrophobic core of micelles of hyaluronan’s derivates because of lipophilic or steric effects. We had to change Perylene as a new solubilizate. From the measured emission spectra we found saturation micelles. We can express the solubilization power of hyaluronan’s derivates for the concentration of Perylene. The main aim of the diploma thesis was to determine optimal way of the preparation of hyaluronan’s derivates solutions with required degree of solubilization.
Colloid properties of hyaluronane sols
Krouská, Jitka ; Mašek, David (referee) ; Pekař, Miloslav (advisor)
Předmětem této práce bylo popsat agregační vlastnosti kyseliny hyaluronové (HA) a jejích hydrofobně modifikovaných derivátů. Za tímto účelem byly připraveny vodné roztoky kyseliny a dvou derivátů, lišící se stupněm substituce a druhem anorganické soli jako prostředím. Koncentrační řady čisté kyseliny hyaluronové a jejích derivátů byly studovány dvěma metodami, tensiometricky a spektrometricky. Povrchové vlastnosti roztoků byly měřeny s použitím kroužku Du Noüy. Zkoumáno bylo také solubilizační chování ve vodě a v chloridu sodném pomocí dvou sond: Coomassie brilantní modři a sudanu III. Amfifilní deriváty hyaluronanu mohou skutečně tvořit hydrofobní agregáty v roztoku a adsorbovat se na rozhraní vzduch–voda. Tato vlastnost je důležitá v souvislosti s transportními systémy léčiv, protože právě hydrofobní domény agregátů hyaluronanu mohou být použity jako nosiče hydrofobních léčiv. Byly zjištěny kritické agregační koncentrace roztoků derivátů hyaluronanu (tensiometricky a spektrometricky), které odpovídají právě těm koncentracím, při kterých vznikají hydrofobní domény.
Transport of Biomedical Active Compounds Through Porous Membranes
Vašíčková, Kateřina ; Smilková, Marcela (referee) ; Smilek, Jiří (advisor)
This bachelor thesis deals with a study of transport of biomedical active compounds through porous membranes. The main goal is to optimize a penetration method of micellar solutions with a solubilized dye and subsequently with solubilized drug. Cetyltrimethylammonium bromide is chosen as a surfactant and a nile red dye is used as a diffusion and fluorescence probe. Selected active compound is a nonsteroidal anti-inflammatory drug ketoprofen with analgesic and antipyretic effects. The diffusion processes are realized using Franz diffusion cells with polycarbonate membrane with a 2 µm size of poruses. The samples are withdrawn in a chosen time intervals and afterwards evaluated by a fluorescence correlation spectroscopy. Using this method, it is possible to screen the diffusion in time even in nanomolar concentrations of a fluorescence probe chosen. This thesis provides an overview of nile red behaviour in the micellar solutions of surfactant during diffusion processes. The experiment can be extended using an active compound, for instance using a ketoprofen as it is mentioned above.
Study of system based on phospholipids for sonographical application.
Überall, Martin ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
The aim of this thesis was to determine solubilizing capacity of surfactants and phospholipids using the UV-VIS spectrophotometry. The concentrations of solubilized substances within these systems was further determined. From the surfactant group, three representatives were selected, cetyltrimethylammonium bromide (CTAB), sodium dodecylbenzenesulfonate (SDBS) and Triton X-100. From the phospholipid group, lecithin and commercially available SonoVue® were chosen. The solubilizing capacity was determined using hydrophobic solutes Sudan Red G (SRG), Oil Red O (ORO) and 1-naphtol in order to obtain a model system of solubilized drugs or vitamins which are also hydrophobic. The behavior of solutes in surfactants was determined in a moderate polar medium of physiological saline (0.15 M NaCl), the behavior of solutes in lecithin was conversely determined in Mili-Q water medium. The determined concentration of solubilized hydrophobic solutes inside the micelles of surfactants is within the range of tens to hundreds of micromoles per liter. With increasing concentration of surfactant increases the concentration of solubilized solutes. The results obtained in this work can be used for further experiments and research focused on solubilization of drugs in contrast agents used in ultrasonography.
Model delivery systems for poorly soluble drugs
Sapárová, Alica ; Holínková, Petra (referee) ; Pilgrová, Tereza (advisor)
The solubilizing capacity of micelle–hyaluronan system was studied using the UV-VIS spectrophotometry method. Septonex was chosen as a surfactant and four molecular weights of hyaluronan were used. Solubilization experiments with a hydrophobic dye Sudan Red G and Sudan III were performed in order to obtain a model system of solubilized drugs, which are also hydrophobic. The system was prepared in the environment of mili-Q water. The influence of the molecular weight of hyaluronan on the solubilizing ability has been studied. It was found that concentration of solubilized dyes increases by increasing concentration of micelles in the system and the molecular weight of hyaluronan affects the solubility of the dyes.

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