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Interaction of phospholipids with polyelectrolytes in aqueous medium
Maivaldová, Iva ; RNDr.Miroslav Gál, Ph.D. (referee) ; Klučáková, Martina (advisor)
This diploma thesis is focused on determination of aggregation behavior of selected phospholipids (lecithin; 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) in water and on the effect of native hyaluronan addition of various molecular weights and concentrations on this behavior. The behavior has been investigated with fluorescence spectroscopy using pyrene and perylene as fluorescence probes being able to penetrate into hydrophobic cavities of formed aggregates. Critical aggregation concentration and the concentration at which lecithin begins to aggregate have been determined. Regarding 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, it was possible to determine only the beginning of aggregation value. The values of this parameter for lecithin and for 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine correspond in the order. It has been investigated, that the addition of native hyaluronan has only in some systems slight effect on the aggregate behavior of selected phospholipids.
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Hemoglobin-mediated oxidation of marine liposomes
Škrabalová, Lada ; Mozuraityte, Revilija (referee) ; Rustad, Turid (advisor)
Cílem této práce bylo studium mechanismu oxidace lipidů katalyzované hovězím methemoglobinem a zhodnocení účinků různých experimentálních podmínek a antioxidantů (EDTA, askorbová kyselina, kávová kyselina, a-tokoferol, d-tokoferol, astaxanthin a L-askorbyl-6-palmitát) na methemoglobinem zprostředkovanou oxidaci lipidů v modelovém systému liposomů připravených z fosfolipidů. K monitorování oxidace lipidů při pH 5,5 a teplotě 30 °C bylo použito spotřeby kyslíku. Pro zhodnocení antioxidační aktivity v modelovém systému liposomů se ukázaly být důležitými faktory typ prooxidantu a koncentrace prooxidantu a antioxidantu. Dalšími důležitými faktory jsou struktura molekuly antioxidantu, jeho hydrofilita/lipofilita a umístění v systému. Všechny testované antioxidanty ve všech koncentracích (kromě koncentrace 0.1 % astaxanthinu and 0.1 % askorbyl palmitátu) inhibovaly oxidaci vyvolanou methemoglobinem. Účinnost antioxidantu stoupala s jeho zvyšující se koncentrací. Koncentrace 0.1 % astaxanthinu neměla žádný vliv na oxidaci liposomů. Koncentrace 0.1 % askorbyl palmitátu měla prooxidační efekt, který lze vysvětlit prooxidačním působením radikálu askorbylu, který může urychlit štěpení hydroperoxidů. Volné železo uvolněné z methemoglobinu se podílelo jen velmi málo na oxidaci liposomů, zatímco část prooxidační aktivity methemoglobinu byla přisouzena tvorbě singletového kyslíku (methemoglobin jako fotosenzitizátor). Antioxidační aktivita astaxanthinu, askorbyl palmitátu a tokoferolu byla z části přisouzena schopnosti zhášet singletový kyslík. Ovšem hlavním prooxidačním mechanismem methemoglobinu se ukázal být rozklad lipidových hydroperoxidů, tvorba volných radikálů a hypervalentních forem hemoglobinu. EDTA utlumila oxidaci liposomů díky chelataci přechodných kovů obsažených v liposomech a chelataci volného železa přítomného v methemoglobinovém roztoku. Velmi důležitým antioxidačním mechanismem (který vykazují askorbyl palmitát, askorbová a kávová kyselina) se ukázala být redukce hypervalentních forem hemoglobinu. Askorbová kyselina, kávová kyselina, tokoferoly a astaxanthin inhibovaly methemoglobinem zprostředkovanou oxidaci lipidů odstraňováním volných radikálů. Při použití peroxidu vodíku nebyl pozorován žádný vliv na oxidaci liposomů vyvolanou methemoglobinem. Působení vysoké teploty (tepelná denaturace) mírně utlumilo oxidaci. Významná inhibice oxidace byla pozorována u liposomů obsahujících TPP (triphenylphosphin), což značí, že je methemoglobinem vyvolaná oxidace liposomů závislá na přítomnosti již vzniklých lipidových peroxidů. Výsledky této práce přispívají k hlubšímu pochopení prooxidačních a antioxidačních mechanismů a faktorů, které ovlivňují oxidaci liposomálních roztoků, buněčných membrán a emulzí typu olej ve vodě stabilizovaných fosfolipidy.
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Fosfolipidy v potravinách
Zahradníková, Nikol
The main goal of this thesis is to highlight the benefits of phospholipids, which are currently underappreciated. The first part is devoted to a description of their basic characteristics, digestion and biological importance in living organism. Phospholipids differ in the benefits they provide to our organism. The next part focuses on the importance of their use in the food industry. In this sector, phospholipids are widely used as additives. Further, the work highlights the main sources and manufacturing animal and vegetable lecithin. Produced lecithin then undergoes a changing process of its chemical structure called modification. The modified lecithins produced this way have various applications in food. Subsequently, the thesis deals with methods for the determination of phospholipids in food. Specifically described is the most popular method for detection phospholipids in egg yolk and soy, HPLC-ELSD. The conclusion is devoted to the explanation of phospholipid fortification and functional foods. The increasing popularity of nanoliposomes in the food industry and the legislative requirements for lecithin are also mentioned.
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Development of analytical methods for determination of phosphorylated components of bacterial cell membranes
Mikulecká, Jana ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee)
Phospholipids are dominant components of bacterial cell membranes, where they create double layers. Bacteria differ in their phospholipid composition determination of which can help in identification of important groups of microorganisms. Phospholipid composition of bacteria is influenced by many environmental factors, therefore its variation can be observed within one bacterial stem also. Because of its simplicity, thin layer chromatography is usually applied to identification and determination of bacterial phospholipids. Disadvantage of this method are the high demands of time, carefulness and skills of the analytical personnel. The increasing interest in the phospholipid double-layer promotes the detailed investigation of their fatty acid composition because the more detailed analyses allows for more information yield about bacteria. Gas chromatography hyphenated with mass spectrometry seems to be the best choice for these purposes. Fatty acid identity and total fatty acid content in phospholipid molecules could be determined by this method. Additionally, number, position and isomerism of double bonds and presence of other functional groups on hydrocarbon chain could be determined. Whereas a suitable and...
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Effect of surfactin on the lipid moiety of Bacillus subtilis cytoplasmic membrane
Sklenářová, Petra ; Seydlová, Gabriela (advisor) ; Lichá, Irena (referee)
Surfactin, a secondary metabolite produced by Bacillus subtilis, is a surface active compound and antibiotic permeabilizing membrane bilayer. The aim of this study was to reveal the self-resistance strategy at the level of the lipid moiety of cytoplasmic membrane, which B. subtilis employs to combat surfactin in concentrations that are lethal for other bacterial species. Non-producing strain B. subtilis 168 was cultivated in the presence of two different sublethal concentrations of surfactin (350 a 650 µg/ml), which was isolated from the culture broth of B. subtilis ATCC 21332. Presence of surfactin in the medium resulted in a concentration dependent lag phase, which took 40 min (350 µg/ml) and 3 h (650 µg/ml), respectively. Afterwards, the culture grew with the altered doubling time of 44 min (350 µg/ml) and 126 min (650 µg/ml), respectively. Surfactin induced substantial changes in the phospholipid composition of the cytoplasmic membrane. The proportion of the major phospholipid component phosphatidylglycerol decreased and inversely, the level of phosphatidylethanolamine increased. Interestingly, the content of phosphatidic acid rose considerably in the presence of surfactin concentration causing stimulation of B. subtilis growth (350 µg/ml). Liposome leakage assay using phospholipids mimicking...
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Analysis of biologically active substances by modern separation methods
Bierhanzl, Václav ; Čabala, Radomír (advisor) ; Pacáková, Věra (referee) ; Sýkora, David (referee)
The thesis is dedicated to the phospholipids and their polar headgroups analysis by gas chromatography, capillary electrophoresis and mass spectrometry. Phospholipids are the most important polar lipids and they are classified into phospholipid classes according to their phosphorylated groups. Phospholipids can be found in cell membranes and the changes in their ratio are monitored to research the impact of external conditions on cells. Actually thin layer chromatography is still used for phospholipid class ratio analyses. It is not suitable for microbiological research due to its time demandingness. The presented compendium of papers engaged in phospholipid classification is targeted on Bacillus subtilis strain, which produces potential antibiotics with detergent effect - surfactin. Published methods can be used for research of optimal conditions for producing microbe cultivation. Because non-polar parts of the phospholipid molecule (fatty acids) can affect the analysis methods on spliced polar headgroups have to be designed. Capillary electrophoresis and gas chromatography methods were developed and the latter one was further optimized for simultaneous analysis with fatty acids. Additional part deals with an alternative approach which consists in direct injection on mass spectrometer of intact...
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Separation of phospholipids by high pressure liquid chromatography
Erdeová, Karolína ; Čabala, Radomír (advisor) ; Sobotníková, Jana (referee)
Fosfolipidy jsou součástí buněčných membrán všech živých organismů. Jedná se o nejvýznamnější skupinu polárních lipidů, vyznačující se amfifilními vlastnostmi. Tyto vlastnosti vyplývají z jejich molekulární struktury, která se skládá z hydrofobních mastných řetězců a polární hlavice. Podle polární hlavice se dělí fosfolipidy do příslušných tříd. Poměr fosfolipidových tříd, zastoupených v bakteriální membráně, značně odráží životní pochody mikroorganismu. Pomocí změn v zastoupení těchto tříd lze sledovat vliv vnějšího prostředí, čehož se využívá v mikrobiologickém výzkumu. Pro sledování fosfolipidového složení vyšlechtěného kmene Bacillus subtilis, který je intenzivně zkoumán pro produkci antibiotik, se stále využívá tenkovrstvá chromatografie. Její časová náročnost výzkum značně zpomaluje, a proto byly podmínky tohoto uspořádání přeneseny na HPLC. Pro tyto účely byla metoda optimalizována formou nelineárního gradientu. Vhodnost metody byla testována na vzorku bakteriálních membránových lipidů, který obsahoval pět fosfolipidových tříd, z čehož dvě majoritní. Jejich poměrné zastoupení lze stanovit, nicméně je nutno pečlivě volit objem nastřikovaného vzorku, aby se zabránilo překročení úzkého koncentračního rozsahu metody. Klíčová slova: fosfolipidy, HPLC, optimalizace, validace, HILIC, NP-HPLC
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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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The importance of lipid composition of membranes for the development of Alzheimer's disease
Novotná, Natálie ; Rudajev, Vladimír (advisor) ; Hanousková, Barbora (referee)
Lipids are an essential components of cell membranes and their homeostasis plays an important role in the development of Alzheimer's disease. The aggregation and neurotoxic effects of amyloid β, mainly Aβ42, on the neural cell membrane are crucial for pathological changes in the brain tissue which leads to its degeneration and loss of cognitive functions. The complex relationship between amyloid β and lipids is also supported by fact that membrane lipids do not only support the amyloid binding to the membrane, but also they regulate the splicing of the amyloid precursor protein, therefore the biosynthesis of β amyloid. The most important binding partners of Aβ42 include gangliosides, especially the ganglioside GM1, but also sphingomyelin and cholesterol. In contrast, glycerophospholipids primarily affect the process of the protein production.
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The Role of Lipids and Lipid Metabolizing Enzymes in Plant Autophagy
Krupař, Pavel ; Martinec, Jan (advisor) ; Hála, Michal (referee)
Plant autophagy is a crucial evolutionary conserved process for recycling cytoplasmic material under stress conditions or during development. The autophagic pathway is negatively regulated by TOR kinase, a versatile molecule modulating a wide range of cellular processes. In mammals, TOR kinase may be activated by phosphatidic acid, a vital signalling lipid. This thesis aims to prove the possible involvement of phospholipids in plant autophagy. I analysed the rate of primary root inhibition in knock-out mutants coding phospholipases in A. thaliana with induced autophagy, measured activity of lipid metabolising enzymes in wild type and atg10 mutant and observed autophagosome formation in selected mutants. Autophagosomes were labelled by fluorescent protein in vivo and by indirect immunolabelling in fixed samples. Using advanced stereological approach, I optimized a method for obtaining an unbiased estimate of autophagosome number in plant root cells.
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