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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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Preparation and characterization of modern wound covers
Balášová, Patricie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
This diploma thesis is focused on the study of bioactive wound dressings. During the thesis, hydrogel, lyophilized and nanofiber wound dressings were prepared. Hydrogel and lyophilized wound dressings were prepared on basis of two polysaccharides – alginate and chitosan. Nanofiber wound dressings were prepared by spinning polyhydroxybutyrate. All prepared wound dressings were enriched with bioactive substances, which represented analgesics (ibuprofen), antibiotics (ampicillin) and enzymes (collagenase). Into hydrogel and lyophilized wound dressings were all the mentioned active substances incorporated, whereas nanofiber wound dressings were only with ibuprofen and ampicillin prepared. The theoretical part deals with the anatomy and function of human skin. There was explained the process of wound healing and also there were introduced available modern wound dressings. The next chapter of the theoretical part deals with materials for preparing wound dressings (alginate, chitosan, polyhydroxybutyrate) and with active substances, which were used during the experimental part of this thesis. In the theoretical part, the methods of preparation of nanofiber wound dressings and also the methods of cytotoxicity testing used in this work were presented. The first part of the experimental part of this thesis was focused on preparing already mentioned wound dressings. Then, their morphological changes over time and also the gradual release of incorporated active substances into the model environment were monitored. The gradual release of ampicillin was monitored not only spectrophotometrically, but also by ultra-high-performance chromatography. In wound dressings, in which collagenase was incorporated, was also the final proteolytic activity of this enzyme monitored. The effect of the active substances was observed on three selected microorganisms: Escherichia coli, Staphylococcus epidermidis and Candida glabrata. The cytotoxic effect of the active substances on the human keratinocyte cell line was monitored by MTT test and LDH test. A test for monitoring the rate of wound healing – a scratch test – was also performed.
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Properties of aminoclay complexes and biologically active substances
Dušek, Jakub ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
This paper builds on previous research of aminoclay complexes in undergraduate studies. Theoretical part deals with study of current problems of aminoclay complexes with bioactive substances and the choice of substances for complexing with aminoclay. The experimental part consists of preparation of aminoclay complexes with selected bioactive agents at various concentrations. Verification of binding of bioactive agents to the aminoclay matrix was performed by Elemental Analysis (EA) and Fourier-transform infrared spectroscopy (FT-IR). For finding of the bound amount of bioactive substance were used the Ultra Performance Liquid Chromatography (UPLC®) and analysis of the cytotoxic properties of the formed complexes by used by the MTT assay. The main motivation of this study is to create new complexes with improved characteristics that would replace existing forms of substances used in pharmaceutical and biomedical applications.
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Vliv proteinové korony na toxicitu částic
Jemelíková, Vendula
When foreign particles enter the organism, they come into contact with the physiological environment and subsequent adsorption of naturally occurring biomolecules on the surface of the particles. Protein corona is formed during the adsorption of proteins, which fundamentally changes the physico-chemical properties of the particles. The aim of this work was to focus on the change in properties and toxicity of particles caused by the formation of a protein corona around selected particles. The formation of protein corona around different particles based on hydroxyapatites (CaNPs), iron (FeNPs), platinum (PtNPs) and titanium (TiNPs), was characterized and proved using electrophoretic methods, and subsequently the change in size, zeta potential and cytotoxicity was monitored depending on the formation of a protein corona. Based on the results, it is possible to claim that in all cases particles with a protein corona were less toxic to breast tumor cells, than particles without a corona and CaNPs were the most cytotoxic. In general, it is possible to say that the formation of protein corona around the tested nanoparticles affects their properties and reduces cytotoxicity and oxidative stress.
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Comparison of in vitro methods for the study of cytotoxicity
Eliášová, Pavla ; Maixnerová, Jana (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Pavla Eliášová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Comparison of in vitro methods for the study of cytotoxicity People are exposed to a growing number of toxic substances from the environment. Endocrine disrupting chemicals (EDCs) are a broad category of molecules that are thought to cause adverse effects on the endocrine system by interfering with the synthesis, transport, degradation or action of endogenous ligands. One of the aims of this thesis was to determine the in vitro toxicity of 17 selected endocrine disruptors on the human hepatocellular carcinoma HepG2 cell line. Cell viability was determined using the CellTiter96® AQueous One Solution Cell Proliferation Assay colorimetric method, the principle of which is the reduction of MTS to the colored product formazan by mitochondria in viable cells. The cytotoxic potential of the compounds was expressed by using the toxicological parameter IC50, which was measured in three time intervals (6, 12 and 24 hours). For 14 substances: Atrazine, DHEP, Bisphenol A, Carbofuran, 3-hydroxycarbofuran, Cypermethrin, DDE, DES, MEHP, PCB 118, PCB 153, PFOA, PFOS, Propiconazole, IC50 > 100 µM (respectively > 250 µM) was...
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