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Study of plasma interaction with yeast Candida glabrata
Trebulová, Kristína ; Lehocký, Marián (referee) ; Krčma, František (advisor)
The main subject of this diploma thesis is the study of low temperature discharges´ effects on the yeast Candida glabrata (C. glabrata). Cold atmospheric-pressure plasma (CAPP) technology has achieved a great deal of attention in recent years, due to its positive attributes, including high efficiency against a wide range of microorganisms, ease of operation, economic simplicity and also environmental friendliness. The research of CAPP focuses mainly on the applications involving the treatment of cancer and superficial infections as well as sterilization technologies in medicine, food and agriculture. Various effects and also some mechanisms of inactivation of bacteria and tumor cells have been explained over the last decade but the effects and mechanisms of fungi inactivation have not yet been sufficiently investigated. The aim of this work is to verify the effects of chosen low-temperature discharges on the model yeast example C. glabrata, in order to establish basic plasma set-up for further studies and experiments in vivo and to expand a global knowledge about the antifungal effects of CAPP. In this study, 5 different discharges, namely: microwave (unipolar and surface wave) in the form of plasma torch, transient spark, corona discharge in the form of corona pen and dielectric barriere discharge (DBD) in the form of DBD pen were tested. All discharges were tested by direct application onto a 24-hour inoculum of C. glabrata inoculated on agar plates. The results were evaluated based on the size and stability of the inhibition zones formed in the post treatment cultivation. From the individual measurements optimal conditions for testing the inhibitory effects of low temperature discharges on the selected yeast were determined. The microwave discharges (unipolar and surface wave) were chosen to be the most effective in the inhibition of C. glabrata. The measurements were therefore pursued with these discharges under selected conditions corresponding to chosen application of sterilization of surface infections in living organisms. The unipolar microwave discharge was chosen to be the most effective discharge for the potential treatment of superficial candidiasis. The indirect effect of plasma was also investigated using plasma activated liquid (PAL) on inoculated yeast culture. PAL was prepared using 3 different discharges: Dielectric Barrier Discharge (DBD), Unipolar Microwave Discharge and Electric Discharge in Liquid. The results of this work prove significant inhibitory effects of CAPP on the yeast C. glabrata, within the limits of in vitro cultivation and the so-called flat biology. The overall inhibitory effects are directly proportional to the irradiation time, the magnitude of applied power and also the area covered by the discharge. In terms of the initial cell concentration an indirect dependency can be observed, but it can also be stated that this factor is less significant compared to the other examined factors. Observations of the effects of plasma activated liquid (PAL) indicate that a single application of PAL under selected conditions is not alone enough for successful inhibition of C. glabrata.
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Physical hydrogels based on biopolymers and surfactants
Velcer, Tomáš ; Lehocký, Marián (referee) ; Kráčalík, Milan (referee) ; Pekař, Miloslav (advisor)
This doctoral thesis studies the properties and behaviour of phase-separated hydrogels. These can be prepared by interaction of polyelectrolytes with oppositely charged surfactants. Negatively charged polysaccharide hyaluronan and cationic surfactant carbethopendecinium bromide (Septonex), whose properties, behaviour and utilization are described in the first two chapters of theoretical part, were selected for this role. Hyaluronan is naturally-occuring in living organisms and is known for his specific targeting to the tumour cells. Septonex is used as antiseptic and disinfectant. Experimental part of this work is focused on examination of the structure and behaviour of these types of hydrogels especially from rheological point of view. The crucial part of this study was to establish mechanical properties and their dependence on environment. This led to design further studies. Structural analysis was held using the methods of ATR-FTIR, ionic chromatography and ICP-OES helping to measure the inner content of entry materials in the gels and supernatants respectively. The last chapter deals with antimicrobial activity. The results of this study indicate a potential usage of these substances in the field of medical applications.
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Metabolic and biophysical characterization of bacterial cells capable of PHA accumulation
Slaninová, Eva ; Lehocký, Marián (referee) ; Doškař, Jiří (referee) ; Chodak, Ivan (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the characterization of bacterial cells capable of polyhydroxyalkanoates (PHA) accumulation. The dissertation thesis is written in the form of a discussed published publications which are attached to the thesis as appendixes. The work develops a study of the current topic of the protective functions of PHA and clarifies protective mechanisms against selected stressors. Firstly, we focused on the protective effects of PHA granules against UV radiation and osmotic stress, specifically hypotonic conditions. In the case of UV exposition, the cells protected themselves by scattering UV radiation on the intracellular granules protecting especially nucleoid. When exposed to osmotic stress, the amorphous state of PHA granules is very important since it is capable of stabilization of cell membranes under hypertonic stress, afterwards, bacterial cells can maintain their integrity during the subsequent hypotonic challenge. In general, the amorphous state of PHA granules is key to ensure the proper biological functions of PHA whether as storage or protective polymer. Therefore, in the next part of this work, we focused on the core of the stabilization mechanism that protects native PHA granules from crystallization and thus the intracellular polymer maintains in a thermodynamically unfavorable amorphous phase state. Based on experimental work, we applied selected stresses because we proposed a new model of stabilization of the amorphous state of PHA granules in vivo. It consists of two mechanisms, where small volumes of PHA granules reduce the rates of crystallization and at the same time the water present in the granules plays the role of a low molecular plasticizer. Due to the metabolic apparatus of bacterial cells, PHA are simultaneously synthesized and degraded which leads to an increment of intracellular concentration of monomers that also figure in the protective effect of PHA. In this context, we aimed at the description of the mechanism of cryoprotective effects of 3-hydroxybutyrate, the monomer of the most common of PHA, poly(3-hydroxybutyrate). Hence, we constructed an equilibrium and non-equilibrium phase diagram of the 3HB-water system to prove that 3HB is a very effective cryoprotectant. This fundamental understanding of the protective properties of PHA monomers could be also used in the food industry or cryopreservation of biological samples.
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Comparison of various amination methods of polycaprolactone concerning their effectivity in tissue engineering
Kováč, Ján ; Lehocký, Marián (referee) ; Zajíčková, Lenka (advisor)
This diploma thesis dealt with the comparison of different methods of amination of polycaprolcatone in terms of their effectiveness for tissue engineering. A polycaprolactone membrane was prepared by an electrospinning method, which was subsequently modified by three different amination methods. Selected types of amination were plasma polymerization with cyclopropylamine monomer, hybrid modification using plasma and N-allylmethylamine monomer, and chemical amination using aminolysis with diaminohexane. Surface amines were subsequently characterized by electron scanning microscopy (SEM), X-ray photoelectron spectroscopy (XPS), attenuated total reflection infrared spectroscopy (ATR-FTIR) and contact angle measurement. A cell culture designated A375 (Human malignant melanoma cell lines A375® CRL-1619®) was cultured on the thus modified membranes, which was analyzed by optical microscopy, and a proliferation assay was performed by determining the relative amount of ATP. Based on the experimental results, we can confirm the success for all types of amination. In terms of efficiency for tissue engineering, the amination method by plasma polymerization with the monomer cyclopropylamine has the most satisfactory results.
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Morphogenesis and Viscoelastic Properties of Dimethacrylate Networks
Bystřický, Zdeněk ; Lehocký, Marián (referee) ; Bradna, Pavel (referee) ; Jančář, Josef (advisor)
Tato dizertační práce se zabývá studiem morfogeneze dimethakrylátových sítí. V práci byly využity zjednodušené systémy založené na monomerech, které bývají typicky využívány jako složky matric pryskyřičných kompozitních materiálů využívaných v oblasti záchovné stomatologie. Kinetika a mechanismy formování polymerních sítí byly studovány především s ohledem na strukturu jednotlivých monomerů, jejich vzájemný molární poměr a koncentraci iniciačního systému využitého pro radikálovou polymeraci. Vypočtené profily konverze funkčních skupin a reakčních rychlostí byly využity jako základ pro pochopení a interpretaci mechanismů morfogeneze sítí a porovnání se známými modely. Dále byla studována kinetika termické degradace, která je s morfologií vytvrzených sítí přímo spjata. V rámci takto charakterizovaných systémů byla stanovena teplotní závislost dynamického modulu a byl popsán vztah mezi supra-molekulární strukturou dimethakrylátových sítí a jejich viskoelastickou odezvou v daném teplotním rozmezí. Kinetika polymerace byla studována pomocí diferenční kompenzační foto-kalorimetrie (DPC) a infračervené spektroskopie (FTIR). Proces termické degradace byl analyzován pomocí termo-gravimetrické analýzy (TGA). Viskoelastické parametry byly charakterizovány pomocí dynamicko-mechanické analýzy (DMA). Reaktivita jednotlivých systémů je přímo odvozena od molekulární struktury monomerů, která ovlivňuje mobilitu reagujících složek v průběhu polymerace. Kinetika polymerace je řízena především difúzí, přičemž její rychlost je dána tuhostí monomerní páteře, koncentrací funkčních skupin a vlivem fyzikálních interakcí. Omezená mobilita rostoucích řetězců, postranních funkčních skupin i samotných monomerů vede k monomolekulární terminaci makro-radikálů a omezení stupně konverze funkčních skupin. Vzhledem k tomu, že k zásadnímu omezení mobility dochází již v počáteční fázi polymerace, tj. v bodu gelace, je případná termodynamická nestabilita vedoucí k fázové separaci polymerujícího systému potlačena a proces kopolymerace je ve své podstatě náhodný. To bylo prokázáno i prostřednictvím identifikace jedné teploty skelného přechodu u charakterizovaných kopolymerů. Heterogenní charakter morfogeneze je spjat s rozdílnou reaktivitou postranních funkčních skupin. V počátečních fázích polymerace dochází k propagaci reakcí postranní funkční skupiny s radikálem na stejném rostoucím řetězci, což vede ke vzniku tzv. primárního cyklu. Pravděpodobnost cyklizace souvisí především s flexibilitou monomerní páteře. Heterogenita polymerace je charakterizována vznikem vnitřně zesítěných struktur, tzv. mikrogelů, a jejich následným spojováním. Tuhost monomeru naopak přispívá k vyšší efektivitě zesítění a více homogenní morfologii vytvrzené sítě. Heterogenita dimethakrylátových sítí se odráží v mechanismu termické degradace, přičemž přítomnost strukturně odlišných domén vede k rozkladu ve dvou krocích. Průběh soufázového modulu a teplota skelného přechodu korelují s tuhostí polymerních sítí, efektivitou zesítění a přítomností fyzikálních interakcí, které vyztužují strukturu sítě nad rámec kovalentního zesítění. Heterogenní morfologie sítí se projevuje rozšiřováním spektra relaxačních časů. Experimentální data jsou v kvalitativní shodě s existujícími numerickými modely popisujícími kinetiku radikálové polymerace multifunkčních monomerů.
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Hybrid Blends of Recycled Plastics
Černý, Miroslav ; Lehocký, Marián (referee) ; Balgová,, Vendula (referee) ; Jančář, Josef (advisor)
Work describes the relationship between structure and mechanical properties in case of hybrid mixtures. They are composed from polyurethane matrices (eventually polyurethane-urea or polyurethane-inorganic filler mixtures), different rubber fractions as filler and eventually reinforcement made from PET monofilaments. Mechanical behaviour was studied by tensile testing. Studied mechanical properties include tensile modulus, ultimate strength and strain and also specific energy need neccessary for ultimate strength achievement. Prepared materials are naturally porous. Therefore their mechanical behaviour has to be described generally for porous composites. The porosity occurence means, that it is difficult to describe them by models valid for mechanical behaviour (mainly elastic modulus) of nonporous composites. Models are also based on idealized structures on microscopic level. In case of porous composites, it is very difficult to create any idealized structure. The structure is changed with modifications of composition (change of components or their rates). These conditions have lead to utilizing of different approach how to describe the relationship between structure and mentioned mechanical properties. The chosen approach comes from knewledge about structural parametres coming from porosity. Structural parameters describe the structure on macroscopic level. They include interspace volume (volume lying between filler particles), interspace filling (how the matrix fills the interspace volume) and matrix volume fraction. Proposed parameters are used in relations, where they are fitted by exponents to interlay values of chosen mechanical properties for composites containing discrete matrix and filler. Found exponents are then described by properties of matrices to obtain relations describing properties of filled porous materials. Very similar approach was chosen for description in the case of reinforced composites. Proposed relation are potentially valid for materials containing binding matrix, infinity count of fillers and one kind of reinforcement.
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Barriere layers for culture herritage objects preservation
Procházka, Michal ; Lehocký, Marián (referee) ; Zahoranová, Anna (referee) ; Krčma, František (advisor)
Every year, many archeological findings are discovered. It is necessary to document and conserve these items dug up from the ground. However, archeologists and conservators cannot handle such a big amount of newly found items. This work offers an alternative approach to standard conservation techniques, increasing the processing capacity and lowering the cost on items’ conservation. Studied alternative, mainly for protection of metallic artefacts, includes thin films based on parylene and organosilicons. Thin films were prepared on two experimental apparatuses. Parylene films were deposited by chemical vapour deposition (CVD). Final product was a thin film of parylene C. Organosilicon thin films were deposited via plasma enhanced chemical vapour deposition (PECVD). PECVD apparatus operates with capacitively coupled radiofrequently initiated plasma. Using hexamethyldisiloxane, thin films very similar to silicon dioxide were produced, thus called SiOx. Thin films were characterized by several methods and compared to standard treatment used by conservators – tannate layer, acrylic furnish Paraloid B72 and microcrystalline wax Revax 30. Parylene films showed excellent conformity and resistance to corrosion on iron substrate. First signs of corrosion were observed on layer of 5 µm thickness after 24 hours in salt fog. On samples coated by SiOx films, corrosion was spreading wide even during 1st hour of the corrosion test. Most probable cause was that SiOx film has thermal expansion coefficient different from iron substrate and due to this fact cracking occurs during cooling down of the treated substrate. On samples coated by standard treatment, corrosion occurred after 1-3 hours of the test. Oxygen transmission rate (OTR) measurements (performed on polypropylene substrate) confirmed good barrier properties of parylene C. Best parylene thin films had OTR approximately 170 cm3•m-2•atm-1•day-1. SiOx films reached good results with OTR 300 cm3•m-2•atm-1•day-1, compared to clean polypropylene substrate with OTR 1700 cm3•m-2•atm-1•day-1. Standard conservation layers could not be applied on polypropylene substrate, thus their OTR was not measured. We did not succeed in finding a substrate which is compatible for all types of treatments. Next, the study focused on combinations of thin films forming sandwich structures. All types let the corrosion attack the substrate during the 1st hour of the test. The cause was insufficient film thickness as well as crack ing of SiOx films due to different thermal expansion properties from the iron substrate. Although SiOx thin films were not deposited on substrate directly, they have excellent adhesion to parylenu and thus they could tear parylenu films due to thermal expansion. However, OTR measurements showed improvement in barrier properties. Multilayer parylene C–SiOx–parylene C–SiOx had OTR 5 cm3•m-2•atm-1•day-1. Final result is that parylene C thin films have better barrier properties than standard coatings and are suitable for conservation of metallic archaeological artefacts. SiOx films and multilayers showed poor anticorrosion protection of metallic substrates but they have good barrier and chemical properties in combination with other types of substrates. Mainly deposited on polymers, there is great potential for their application in many fields. Further research would be focused on new substrates for SiOx thin films and on the improvement of UV stability of parylene films.
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Reactive Modification of Polypropylene
Matláková, Jana ; Žídek, Jan (referee) ; Lehocký, Marián (referee) ; Kučera, František (advisor)
The theoretical part describes the principles of the free-radical induced grafting and the influence of various parameters on the reactions course. The literature research summarizes the latest knowledge in the field of the reactive modification of polypropylene (PP), it is mainly focused on the PP modification using an unsaturated acid anhydrides. Theoretical part describes the various procedures and modification of technology in order to increase the grafting yield of maleation. The experimental part is determined by the influence of the stabilizers concentration, the peroxide structure, and the binary mixture of monomers on the grafting yield and the reaction course. In the first part, the critical stabilizers concentration was determined based on the experimentally obtained relationship between the grafting yield and the stabilizers concentration. A kinetic schema of grafting of MAH onto PP in the presence of stabilizers has been proposed and compared with the experimental results. The effect of the stabilizers on the extent of the undesirable b-scission was evaluated based on the melt flow rate (MFR) and the rheological curves of PP-g-MAH. In the second part, the effect of the peroxide structure and the concentration of reactants on the grafting yield of PP modification with MAH and itaconic anhydride (IAH) was observed. The initial grafting yield Rg has been experimentally determined and compared with a defined area of theoretical values of Rg. The extent of b-scission was significantly influenced by the structure and the concentration of peroxide, as shown by the results of MFR and the rheological curves of PP-g-MAH. The last part of the doctoral thesis is focused on the assessment of the combination of MAH and IAH as comonomers on the grafting yield. At first, the reference polymers were prepared using the solution polymerization of MAH, and IAH, and copolymerization of MAH with IAH. The reference polymers were analyzed by FTIR, DSC, WAXS to confirm the probable copolymerization of MAH and IAH. The dependence of the reaction enthalpy on the reaction time was observed using simulations of bulk polymerization of MAH, IAH and mixtures MAH and IAH "in situ" in isothermal calorimeter. PP was subsequently modified with a combination of MAH and IAH as comonomers in order to assess its effect on the grafting yield
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