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Preparation of model membranes to study their interactions with biopolymers using fluorescence correlation spectroscopy
Adamcová, Zuzana ; Márová, Ivana (referee) ; Mravec, Filip (advisor)
This diploma thesis is focused on preparation and characterization of supported lipid bilayers as simplified models of cell membranes. The bilayers were prepared from source system of lecithin liposomes in phosphate buffer using the vesicle fusion method on a cover glass sufrace hydrophilized by plasma. Three fluorescent probes – Nile red, Oregon Green DHPE and DiO – were utilized to characterize diffusion within the bilayer using fluorescence correlation spectroscopy. For this purpose Z-scan FCS, which is a method developed specially for planar samples, was used. After the process of preparation and characterization of supported lipid bilayer was optimalized, interaction between this artificial membrane and solution of hyaluronic acid in phosphate buffer was studied. It was found out, that addition of this biopolymer causes slowing the diffusion of the fluorescent probe within the bilayer.
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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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Advanced membrane systems
Gjevik, Alžběta ; Márová, Ivana (referee) ; Mravec, Filip (advisor)
The diploma thesis deals with cellular membrane model preparation on microfluidic devices. It summarizes means of microfluidic device fabrication, phospholipid bilayer formation mechanisms, optimization techniques and characterization methods of those systems. It focuses on free-standing planar lipid bilayers which are easily accessible by a number of different characterization methods and at the same time exhibit good stability and variability. The aim of this work is to design and prepare a microfluidic chip on which a planar lipid bilayer can be prepared. It therefore presents microfluidic device prepared by soft lithography of PDMS adapted for model membrane formation by self-assembly of phospholipids at the interface of aqueous and organic phases created by the architecture of the microfluidic device. Formation of the model membrane was visualized by optical microscopy and fluorescence-lifetime imaging microscopy.
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Structural and transport properties of hybrid hydrogels with application utilization
Zhurauliova, Darya ; Pekař, Miloslav (referee) ; Smilek, Jiří (advisor)
The presented diploma thesis deals with the preparation and subsequent characterization of structural and transport properties of hybrid hydrogels based on polyvinyl alcohol and sodium alginate concerning their application potential. The thesis aims to study the current state of the solved issue on the topic of characterization of structural and transport (release) properties of hybrid hydrogels about their use in various branches of human activity and also based on literature research to propose and optimize a suitable preparation procedure and to carry out a series of experiments for their characterization. With regard to the literature research, suitable polymers capable of gelation, such as polyvinyl alcohol (PVA) and natural polysaccharide – sodium alginate, were selected for the preparation of hydrogels. The preparation of this hybrid network involved the physical cross-linking of PVA by freezing and thawing and ionic cross-linking of sodium alginate with calcium ions. By combining these polymers, viscoelastic, swelling and transport properties were determined by using physicochemical methods. Rheological measurements were performed to study the mechanical properties - specifically oscillation and amplitude tests. The structural properties of hydrogels (or xerogels) were determined, by scanning electron microscopy. The transport (release) properties of gel systems were determined by diffusion experiments with the UV-VIS spectrometry detection method, in which an organic dye (methylene blue) was used as a model probe. Another aim of this diploma thesis is to modify the internal structure of hybrid hydrogels with suitable reagents, ie., lecithin. The presence of lecithin in the hybrid hydrogel three-dimensional network serves as a model of the phospholipid bilayer, forming the cell membrane or occurring in tissues, and thus has a significant effect on rheological and transport properties. Physicochemical methods also determined the mechanical, swelling and transport properties of hybrid hydrogels with the addition of lecithin. The conclusion of this work is a summary of measured data to provide a comprehensive overview of this issue. The results suggest that the presence of sodium alginate in the PVA structure reduces the mechanical properties, although the presence of lecithin increases the strength of the hybrid hydrogels. However, by comparing all measurement results, it is possible to determine that sodium alginate, resp. lecithin affects the properties and structure of the hydrogel, which makes it possible to correct the properties of hybrid gel systems according to the required applications.
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Characterization of liposomes as precursors for the preparation of models of cellular membrane using scattering techniques
Gjevik, Alžběta ; Kalina, Michal (referee) ; Mravec, Filip (advisor)
The bachelor thesis presents a cellular membrane design based on lecithin, cholesterol and POPG basis. It summarizes formation mechanisms, optimization techniques and characterization methods of model cellular membranes. It focuses on preparation of liposomes with various lipid compositions as precursors for model membranes preparation and characterization. Small unilamellar liposomes were formed by thin layer evaporation, thin layer rehydration in phosphate buffer and sonication. Size and stability (-potential) of formed liposomes were measured using dynamic light scattering. Successful supported lipid bilayer formation on glass surface by vesicular fusion was tested using the most stable lipid composition. SLB was characterized by Z-scan fluorescence correlation spectroscopy.
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Plant-based cosmetic products
Horváthová, Iveta ; Pavelková, Renata (referee) ; Skoumalová, Petra (advisor)
The aim of this bachelor thesis was studying an antimicrobial activity of selected herbal extracts and their encapsulated forms as liposomes. Furthemore, the thesis focuses on determonation of biologically active compounds, natural pigments and antioxidant activity. 6 kinds of herbs were studied - Humulus lupulus, Matricaria chamomilla, Taraxum officinale, Bellis Perennis, Cymbopogon citratus and Rubus Fruticosus. In teoretical part, the general characteristics of herbs and their use are described, it also includes the description of active compounds, microorganisms and used methods. Practical part is focused on characterisation of herbs from the perspective of antioxidant activity, determination of totaly phenolic content and flavonoids, chlorophyll and carotenoids. For analysis of antioxidant activity, concentration of phenolic compounds and flavonoids and antimicrobial activity aqueous and 20% ethanol extracts were prepared. Based on the obtained results, selected extracts were encapsulated into various types of liposomes consisting egg yolk, soy or sunflower lecithin. Generally, ethanol extracts showed higher content of active compounds. For chlorophyll and total carotenoid content determination the highest content of natural pigments was found in Taraxum officinale. Furthemore, antimicrobial activity of extracts and liposomes against 6 strains were determined - Micrococcus luteus, Serratia marcescens, Escherichia coli, Propionibacterium acnes, Staphylococcus epidermidis and Candida glabrata. The highest inhibitory effect was found in Matricaria chamomilla and Cymbopogon citratus samples. Encapsulation effectivity, size, stability and long-term stability of liposomes after a week and two months were determined. From the perspective of zeta potential all the particles were stable and reached a good encapsulation effectivity. It turned out that after two monts of storage the particles decomposed and active compounds into surroundings were released. Selected herbs generally had high content of antioxidants and active compouds. The most suitable lecithin for encapsulation was shown to be sunflower lecithin.
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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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Structural and transport properties of hybrid hydrogels with application utilization
Zhurauliova, Darya ; Pekař, Miloslav (referee) ; Smilek, Jiří (advisor)
The presented diploma thesis deals with the preparation and subsequent characterization of structural and transport properties of hybrid hydrogels based on polyvinyl alcohol and sodium alginate concerning their application potential. The thesis aims to study the current state of the solved issue on the topic of characterization of structural and transport (release) properties of hybrid hydrogels about their use in various branches of human activity and also based on literature research to propose and optimize a suitable preparation procedure and to carry out a series of experiments for their characterization. With regard to the literature research, suitable polymers capable of gelation, such as polyvinyl alcohol (PVA) and natural polysaccharide – sodium alginate, were selected for the preparation of hydrogels. The preparation of this hybrid network involved the physical cross-linking of PVA by freezing and thawing and ionic cross-linking of sodium alginate with calcium ions. By combining these polymers, viscoelastic, swelling and transport properties were determined by using physicochemical methods. Rheological measurements were performed to study the mechanical properties - specifically oscillation and amplitude tests. The structural properties of hydrogels (or xerogels) were determined, by scanning electron microscopy. The transport (release) properties of gel systems were determined by diffusion experiments with the UV-VIS spectrometry detection method, in which an organic dye (methylene blue) was used as a model probe. Another aim of this diploma thesis is to modify the internal structure of hybrid hydrogels with suitable reagents, ie., lecithin. The presence of lecithin in the hybrid hydrogel three-dimensional network serves as a model of the phospholipid bilayer, forming the cell membrane or occurring in tissues, and thus has a significant effect on rheological and transport properties. Physicochemical methods also determined the mechanical, swelling and transport properties of hybrid hydrogels with the addition of lecithin. The conclusion of this work is a summary of measured data to provide a comprehensive overview of this issue. The results suggest that the presence of sodium alginate in the PVA structure reduces the mechanical properties, although the presence of lecithin increases the strength of the hybrid hydrogels. However, by comparing all measurement results, it is possible to determine that sodium alginate, resp. lecithin affects the properties and structure of the hydrogel, which makes it possible to correct the properties of hybrid gel systems according to the required applications.
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Plant-based cosmetic products
Horváthová, Iveta ; Pavelková, Renata (referee) ; Skoumalová, Petra (advisor)
The aim of this bachelor thesis was studying an antimicrobial activity of selected herbal extracts and their encapsulated forms as liposomes. Furthemore, the thesis focuses on determonation of biologically active compounds, natural pigments and antioxidant activity. 6 kinds of herbs were studied - Humulus lupulus, Matricaria chamomilla, Taraxum officinale, Bellis Perennis, Cymbopogon citratus and Rubus Fruticosus. In teoretical part, the general characteristics of herbs and their use are described, it also includes the description of active compounds, microorganisms and used methods. Practical part is focused on characterisation of herbs from the perspective of antioxidant activity, determination of totaly phenolic content and flavonoids, chlorophyll and carotenoids. For analysis of antioxidant activity, concentration of phenolic compounds and flavonoids and antimicrobial activity aqueous and 20% ethanol extracts were prepared. Based on the obtained results, selected extracts were encapsulated into various types of liposomes consisting egg yolk, soy or sunflower lecithin. Generally, ethanol extracts showed higher content of active compounds. For chlorophyll and total carotenoid content determination the highest content of natural pigments was found in Taraxum officinale. Furthemore, antimicrobial activity of extracts and liposomes against 6 strains were determined - Micrococcus luteus, Serratia marcescens, Escherichia coli, Propionibacterium acnes, Staphylococcus epidermidis and Candida glabrata. The highest inhibitory effect was found in Matricaria chamomilla and Cymbopogon citratus samples. Encapsulation effectivity, size, stability and long-term stability of liposomes after a week and two months were determined. From the perspective of zeta potential all the particles were stable and reached a good encapsulation effectivity. It turned out that after two monts of storage the particles decomposed and active compounds into surroundings were released. Selected herbs generally had high content of antioxidants and active compouds. The most suitable lecithin for encapsulation was shown to be sunflower lecithin.
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Advanced membrane systems
Gjevik, Alžběta ; Márová, Ivana (referee) ; Mravec, Filip (advisor)
The diploma thesis deals with cellular membrane model preparation on microfluidic devices. It summarizes means of microfluidic device fabrication, phospholipid bilayer formation mechanisms, optimization techniques and characterization methods of those systems. It focuses on free-standing planar lipid bilayers which are easily accessible by a number of different characterization methods and at the same time exhibit good stability and variability. The aim of this work is to design and prepare a microfluidic chip on which a planar lipid bilayer can be prepared. It therefore presents microfluidic device prepared by soft lithography of PDMS adapted for model membrane formation by self-assembly of phospholipids at the interface of aqueous and organic phases created by the architecture of the microfluidic device. Formation of the model membrane was visualized by optical microscopy and fluorescence-lifetime imaging microscopy.
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