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Use of cell cultures to testing of natural substances of plant origin
Ručková, Michaela ; Kostovová, Iveta (referee) ; Márová, Ivana (advisor)
The presented bachelor thesis is focused on preparing aqueous and oil extracts of plant origin, their characterization and testing of their cytotoxicity and genotoxicity. The theoretical part contains basic information about plants and kinds of spices or tea that were used. Description of used methods of liposome preparation and encapsulation of active substances, measurement of antioxidant activity, particle characterization, cultivation of cell cultures and the principle of cytotoxicity and genotoxicity tests used in this thesis is included. Phenolics and flavonoids content was determined using the spectrophotometry. Antioxidant activity was evaluated spectrophotometrically as well. By ultrasonic homogenization method, liposomes were prepared and active agents were encapsulated into these vesicles. Encapsulation efficiency and stability of active substances were measured by spectrophotometry. By DLS method, size of particles was determined. Particle stability was derived from the zeta potential value. Important part of the bachelor thesis was cultivation of human keratinocytes. The cytotoxicity of extracts and liposomes was assessed by MTT assay on human cell cultures. The genotoxicity of extracts and liposomes was defined using SOS Chromotest on model organism, genetically modified E. coli. In conclusion, the potential use of prepared liposomes in the cosmetic or pharmaceutical industry has been proposed.
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Biocomposite material for 3D print in the field of regenerative medicine
Chaloupková, Kateřina ; Obruča, Stanislav (referee) ; Přikryl, Radek (advisor)
The presented thesis deals with preparation of material for use in regenerative medicine based on poly(3-hydroxybutyrate) and its characterization. In addition to poly (3-hydroxybutyrate), there were used other materials lactic acid (PLA), tricalcium phosphate (TCP) and two types of plasticizers Citroflex®B-6 (CB6) and Syncroflex3114 (S3114). These materials were selected based on their biocompatibility and, in the case of TCP, also bioactivity. TCP allows new bone to grow on the surface of the scaffold. PLA was used to improve the mechanical properties of the material. Both plasticizers have been used to improve the processability of the material. Theoretical part of this work contains a literature review describing basic information about used materials. Aim of the experimental part is to prepare the material, characterization of properties and determination of printability on a 3D printer. The material is examined for thermal properties by thermogravimetric analysis and differential scanning calorimetry. This work also deals with the matter of 3D printing, especially FDM technology. It has been found that materials containing the syncroflex plasticizer are better processed and therefore printed on a 3D printer. The printability tests performed are temperature towers and filling studies. Printed samples were subjected to mechanical tests of tensile and bending tests. Experiments of cytotoxicity and biocompatibility of the material were also performed. Within the work, TCP particles were characterized using a particle size analyzer. The average TCP particle size is 10,76 µm. Using SEM-EDX, the distribution of TCP in sample filaments was subsequently observed, where it was found that by mixing TCP particles with the remaining components of materials, TCP particles agglomerate into formations up to 20 µm in size. Roughness of materials was determined by confocal microscopy. Cytotoxicity was also tested in the extracts of samples on mouse fibroblasts. Cytotoxicity was determined by metabolic activity assay and light microscopy. The metabolic activity test proved the biocompatibility of the observed materials; therefore, it was possible to perform cell proliferation and biocompatibility tests directly on the samples. Assays were performed using human mesenchymal stem cells. DNA quantification was used to determine cell proliferation. Shape of cells was subsequently observed by confocal microscopy. Tests confirmed growth of cells and their appropriate shape. Stem cell differentiation into bone was performed by measuring alkaline phosphatase activity.
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Analysis of active substances and biological effects of some non-tradizional cereals
Pecháček, Michal ; Vysoká, Marie (referee) ; Márová, Ivana (advisor)
This thesis deals with the characterization of active substances and biological effects of non-traditional cereals. Basic substances of these non-traditional cereals such as carbohydrates or proteins are characterized in this work. Active compounds such as antioxidants, polyphenols or -glucans are also characterized. These substances were mainly analysed by spectrophotometric methods. The theoretical part describes cereals in general. It focuses on grain morphology, cereal products and mostly on chemical composition. The summary of substances appearing in the cereal grain such as carbohydrates, proteins, vitamins, minerals etc. was processed in this part. In the practical part, the samples of the non-traditional cereals such as amaranth, sorghum, millet, kamut, buckwheat, quinoa, Job’s tears and teff were analysed. Amaranth, millet and buckwheat were also analysed in the form of flakes. Teff was analysed only in the form of flakes. Sorghum and quinoa were analysed also in their coloured variations. Firstly, the water extracts were prepared, then they were used for determination of antioxidant activity, the content of polyphenols compounds and flavonoids. The hydrolysed samples were used for determination of the carbohydrates. For other basic analysis, samples were used in the form of powder. Best results were measured for quinoa, amaranth and buckwheat. Besides basic analysis, the content of -glucans was measured. However, the content of -glucans in these non-traditional cereals was very low. Selected cereals were tested for cytotoxicity on human cells. Cytotoxicity was evaluated by using the MTT cytotoxicity test on human keratinocytes HaCaT and human caucasian colon adenocarcinoma CaCO-2. The sensory analysis was carried out in the last part of the thesis. There were tested 6 samples. From these samples were made muffins that were analysed and sensorically evaluated.
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Influence of liposomal platinum cytostatics on cancer cells – voltammetric study
Laníková, Petra ; Prášek, Jan (referee) ; Hynek, David (advisor)
Aim of this thesis is voltammetric study influence of liposomal platinum cytostatics on cancer cells. One of the goals is summarize available informations about influence of cisplatine on cancer cells, its encapsulation into liposome and affection of this cytostatic cisplatin encapsulated in liposome on cancer cell lines. In literary recherche is detail description of these issues. Than is there specification of voltammetric methods, which serve to electrochemical detection of cisplatin. Based on literary recherche was chosen the best method for detection and subsequently the method was optimalized and than was applied to measuring itself.
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Study of interactions of biomaterials with human cell cultures.
Maslonková, Ivana ; Kostovová, Iveta (referee) ; Márová, Ivana (advisor)
The bachelor thesis was focused on introducing of techniques of cultivation of human cells in vitro and optimization of their uses in variety of cytotoxicity tests. Further, in this work were studied also interactions of cells with biomaterials and prepared organic-inorganic nanoparticles. In the theoretical part the basic conditions of cell cultivation, the characteristics of keratinocytes as cell cultures, basic characteristics of particles and also short search about the most used tests of cytotoxicity on various cells systems were described. The experimental part was focused on the optimizing the preparation of silver particles, where maltose was chosen as the most appropriate reduction agent. The average size of these prepared particles was 35 nm. The possibility of preparation of combined organic-inorganic particles including liposome particles, PHB particles, alginate particles and chitosan particles was also tested in the thesis. Later, the silver nanoparticles were encapsulated into these organic-inorganic particles as an inorganic component. The liposome particles prepared by ultrasound were evaluated as the most stable of all the other prepared particles. Human keratinocytes were chosen as the cell cultures that were needed for the observation of the particle interactions with human cells. In this work basic methods of cell cultivation and passage were introduced. Conditions of experimental work in cell culture laboratory were setting and possible application of cell cultures to cytotoxicity testing by MTT was tested.
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Analysis of beta-glucans in some fungi
Phan, Huong Tra ; Veselá, Mária (referee) ; Márová, Ivana (advisor)
The main goal of this thesis was to analyse the amount of beta glucans in selected mushroom species. The overall characterization of the composition and content of bioactive compounds in mushrooms was studied too. The theoretical part was focused on description of Fungi, especially the Basidiomycota phyla. The overview of fungal morphology and chemical composition was elaborated. Further, theoretical basis of analytical methods used was introduced. In the experimental part, extracts from selected species of mushrooms (shiitake, oyster mushroom, white champignon, brown champignon and jew’s ear) were prepared. Overall group parameters such as polyphenols, flavonoids, antioxidant activity were determined by spectrophotometry. The amount of beta-glucans in the fungi was measured after enzyme hydrolysis using commercial kit. The content and composition of fatty acids was measured by gas spectrometry. Using emission spectrometry (ICP/OES) the presence of heavy metals was analysed in fungal extracts. Cytotoxicity was tested by MTT test on human cell lines. In the last part the antimicrobial activity against Gram-positive bacterium Micrococcus luteus, Gram-negative Serratia marcescens and yeast Candida labrata was tested. In the thesis, a complex characterization of composition and biological effect of some mushrooms was given.
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Encapsulation of active substances into nanofibers and possibilities of their application
Procházková, Lucie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
The master thesis was based on the optimization of the production of nanofiber covers and to gaine the product for subsequent functional use. The production of nanofiber covers was made by electrospinning and forcespinning from selected materials. Polyhydroxybutyrate, gelatin, chitosan and alginate were used as starting materials. After successful optimization, these materials were enriched with active ingredients ampicillin and ibuprofen for the functionalized use of covers for more effective wound healing. The theoretical part was focused on the issue of skin, healing processes, types of wounds and nanofibers, the characterization of selected starting materials for the formation of nanofibers was also mentioned. The practical part was based on the lengthy optimization of the preparation of fiber covers and later enriched with active ingredients. Furthermore, combined covers made of different materials with contents of both active ingredients were designed. This was followed by the characterization of all prepared covers from the point of view of stability in the short and long term. The gradual release of active ingredients was determined spectrophotometrically and by hifh performance liquid chromatography. It was also important to determine the antimicrobial activity of selected active substances. At the end of all testing, combined coatings containing both active ingredients were used for safety testing with human keratinocyte cells (HaCaT). Safety testing was based on determining the viability of human cells using the MTT test, to verify the LDH test. A scratch test was also performed, a wound healing test after the application of devised combined covers.
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Biological effects of complex extracts from yeast and algae biomass
Bočán, David ; Byrtusová, Dana (referee) ; Skoumalová, Petra (advisor)
This bachelor thesis was focused on optimalization of extractions of active compounds from microalgae and carotenogenic yeasts. Other part of this work was focused on characterization of these extracts and their tests of cytotoxicity on human keratinocytes. In theoretical part a literary research which summarizes the basic properties and cultivation conditions of used microorganisms was conducted. Then there were listed an examples of antioxidant compounds found in these microorganisms as well as their properties. Finally the term cytotoxicity and methods of its measurement were clarified. The practical part of this thesis consists of series of extractions from mentioned microorganisms. These extracts were characterized by the content determination of phenolic and flavonoid compounds and also chlorophyll and carotenoid compounds. Determination of antioxidant capacity and SPF were done too. Lastly the cytotoxicity of chosen extracts was measured using the MTT test.
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Catecholamine modified polymer-composite bone cement: preparation, characterization and in-vitro evaluation
Raszková, Alena ; Vojtová, Lucy (referee) ; Michlovská, Lenka (advisor)
Tato bakalářská práce je zaměřena na přípravu, charakterizaci a in-vitro hodnocení cytotoxicity katecholaminem modifikovaných fosfátových cementů. Teoretická část práce se zabývá charakteristikou cementů na bázi fosforečnanu vápenatého a jejich materiálových vlastností. Obsahuje také přehled aditiv používaných pro modifikaci cementů za účelem zlepšit jak jejich chemofyzikální, tak biologické vlastnosti. Rovněž je zde diskutováno, dnes stále častější, využití katecholaminu v tkáňovém inženýrství. První část experimentální práce se zaměřuje zejména na samotnou optimalizaci přípravy a charakterizaci katecholaminem modifikovaný fosfátový cement. Kinetika fázového složení byla hodnocena pomocí rentgenové difrakční analýzy a chemické složení pomocí infračervené spektroskopie. Druhá část práce je poté zaměřena na optimalizaci a hodnocení in-vitro cytotoxicity modifikovaného cementu. V závěru bylo zjištěno, že modifikace katecholaminem zrychluje míru konverze cementu během vytvrzování. U takto připravených vzorků byl stupeň konverze vypočten na 93 % po 3 dnech vytvrzování, zatímco u nemodifikovaných vzorků dosahoval ve stejný čas pouze 79 %. Pro in-vitro testování byla navržena nová metoda přípravy vzorků. Vzhledem k výsledkům testování je nutná další optimalizace této nové metody a podmínek testů. Díky svým atraktivním vlastnostem se katecholaminem modifikovaný fosfátový cement zdá být zajímavým materiálem pro regeneraci kostí.
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Preparation of bioactive wound dressings and testing their interaction with human cells
Bendová, Agáta ; Márová, Ivana (referee) ; Skoumalová, Petra (advisor)
The thesis was focused on the preparation and optimization of the preparation of wound dressing from materials with bioactive ingredients. In this work were prepared nanofiber dressings based on polyhydroxybutyrate and non-fibrous dressings from alginate and chitosan. Nanofibers were prepared by electrospinning and forcespinning methods. The bioactive components, which were used to functionalize the prepared dressings, were plant extracts, clotrimazole, ampicillin, lysozyme, and proteolytic enzymes. The theoretical part is focused on the description of the use of nanofibrous and non-fibrous materials in medicine, characterization of materials for the production of wound dressings and bioactive components. Furthermore, this section describes the methods used to prepare and characterize wound dressings. In the practical part were prepared aqueous and oil extracts from selected plants. Extracts were characterized for polyphenols content and antioxidant activity. PHB-based nanofibers were prepared using electrospinning and forcepinning methods. Nanofibers were enriched with selected plant oil extracts and clotrimazol. Modified nanofibres were detemined for antioxidant activity, short-term and long-term stability. Non-fibrous wound dressings were prepared from alginate and chitosan. These dressings were functionalized by the addition of selected aqueous extracts, ampicillin, lysozyme, papain, bromelain, and collagenase. Non-fibrous wound dressings were determined for antioxidant activity, short-term stability and proteolytic activity. The prepared wound dressings were tested for their antimicrobial effects on cultures of Micrococcus luteus, Serratia marcescens, Staphylococcus epidermidis and Escherichia coli. In conclusion, successfully prepared bioactive wound dressings with antioxidant and antimicrobial agents were tested for safety on human cells. The determination was performed using the MTT cytotoxicity test on human keratinocytes.
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