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Characterization of 32,33-didehydroroflamycoin - secondary metabolite from Streptomyces durmitorensis
Koukalová, Alena ; Černý, Jan (advisor) ; Konopásek, Ivo (referee)
Streptomycetes are soil filamentous Gram-positive bacteria that produce wide variety of pigments and biologically active substances including macrolides. Some of them are used as very efficient antibiotics and strong antifungal agents in medicine, others have became useful tools for staining biomembranes and detecting cholesterol via their internal fluorescence. Actinomycete Streptomyces durmitorensis (wild type strain MS405T ) is a bacteria isolated from Durmitor National Park in Montenegro soil samples. It produces secondary metabolite that has been identified as 32,33-didehydroroflamycoin (DDHR) closely related to the macrolides roflamycoin and generaly used filipin. DDHR exhibits cytototoxic activity against mammalian cells and yeast Saccharomyces cerevisiae strain EGY48. In addition it has interesting fluorescence properties allowing visualization of some membrane components. DDHR interacts with biomembranes, causes their disintegration leading to changes of the actin and tubulin cytoskeleton organization and in higher concentrations it causes cells necrosis. DDHR-sterol interaction in cell membranes decreases fluorescence intensity of DDHR. The compound is able to fluorescently stain aberrant lysosomes and could be therefore potentially used in diagnostics of some lysosomal storage disease.
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Lipid Membranes at the Nanoscale: Single-Molecule Fluorescence Approach
Koukalová, Alena ; Černý, Jan (advisor) ; Malínský, Jan (referee) ; Benda, Aleš (referee)
The complexity of cell membranes is far from being only a simple assembly of lipids and proteins separating cells from the surrounding environment. Each of the thousands of different membrane components performs its specific role in cellular functions, since a multitude of biological processes is mediated by membranes. The understanding of the molecular basis of these processes is one of the important aims of current biological research. Our research employing single- molecule fluorescence methods (e.g. FCS, FCCS, FLIM-FRET) has made a contribution to the knowledge of membrane lateral organization or mechanism of membrane fusion. Furthermore, we revealed the mechanism of membrane activity of a small natural compound. As native cell membranes are very complex structures, we performed the experiments on simplified model lipid membranes that allow studying lipid-lipid or lipid-protein interactions at the molecular level in a controlled way. The first part of this thesis deals with the mode of action of a membrane active secondary metabolite didehydroroflamycoin (DDHR). We demonstrated that DDHR is a pore-forming agent and that this activity is influenced by the presence of cholesterol. Direct visualization of intrinsic fluorescence of DDHR revealed its preferential partitioning into membrane areas...
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Functional membrane microdomains in the bacterial cytoplasmic membrane
Švecová, Magdaléna ; Mikušová, Gabriela (advisor) ; Koukalová, Alena (referee)
Functional membrane microdomains are structural heterogeneities in bacterial cytoplasmic membrane with up to few tens of nanometers in size. Same as in the case of eukaryotic lipid rafts the lipid and protein composition and fluidity of bacterial membrane microdomains differ from the rest of the membrane. Membrane microdomains contain the structural analogues of eukaryotic flotilin as well as hopanoids and carotenoids as functional analogues of cholesterol in eukaryotic lipid rafts. In functional membrane microdomains there are located proteins associated with membrane trafficking, signaling, secretion, biofilm formation, and sporulation. Functional membrane microdomains are specific sites for the entry of certain antibiotics into cells. What is more, disassembly of functional membrane microdomains might be regarded as a possible novel mechanism of bacterial infections suppression that is caused by antibiotic resistant bacterial pathogens. In the absence of membrane domains proteins which require for their functions the membrane domain localization lose their activity. This may result in inhibition of bacterial cell growth. Key words: Functional membrane microdomains, bacterial cytoplasmic membrane, cardiolipin, hopanoids, flotilins, antibiotic resistance
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Lipid Membranes at the Nanoscale: Single-Molecule Fluorescence Approach
Koukalová, Alena ; Černý, Jan (advisor) ; Malínský, Jan (referee) ; Benda, Aleš (referee)
The complexity of cell membranes is far from being only a simple assembly of lipids and proteins separating cells from the surrounding environment. Each of the thousands of different membrane components performs its specific role in cellular functions, since a multitude of biological processes is mediated by membranes. The understanding of the molecular basis of these processes is one of the important aims of current biological research. Our research employing single- molecule fluorescence methods (e.g. FCS, FCCS, FLIM-FRET) has made a contribution to the knowledge of membrane lateral organization or mechanism of membrane fusion. Furthermore, we revealed the mechanism of membrane activity of a small natural compound. As native cell membranes are very complex structures, we performed the experiments on simplified model lipid membranes that allow studying lipid-lipid or lipid-protein interactions at the molecular level in a controlled way. The first part of this thesis deals with the mode of action of a membrane active secondary metabolite didehydroroflamycoin (DDHR). We demonstrated that DDHR is a pore-forming agent and that this activity is influenced by the presence of cholesterol. Direct visualization of intrinsic fluorescence of DDHR revealed its preferential partitioning into membrane areas...
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Characterization of 32,33-didehydroroflamycoin - secondary metabolite from Streptomyces durmitorensis
Koukalová, Alena ; Černý, Jan (advisor) ; Konopásek, Ivo (referee)
Streptomycetes are soil filamentous Gram-positive bacteria that produce wide variety of pigments and biologically active substances including macrolides. Some of them are used as very efficient antibiotics and strong antifungal agents in medicine, others have became useful tools for staining biomembranes and detecting cholesterol via their internal fluorescence. Actinomycete Streptomyces durmitorensis (wild type strain MS405T ) is a bacteria isolated from Durmitor National Park in Montenegro soil samples. It produces secondary metabolite that has been identified as 32,33-didehydroroflamycoin (DDHR) closely related to the macrolides roflamycoin and generaly used filipin. DDHR exhibits cytototoxic activity against mammalian cells and yeast Saccharomyces cerevisiae strain EGY48. In addition it has interesting fluorescence properties allowing visualization of some membrane components. DDHR interacts with biomembranes, causes their disintegration leading to changes of the actin and tubulin cytoskeleton organization and in higher concentrations it causes cells necrosis. DDHR-sterol interaction in cell membranes decreases fluorescence intensity of DDHR. The compound is able to fluorescently stain aberrant lysosomes and could be therefore potentially used in diagnostics of some lysosomal storage disease.
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The tools for study of cholesterol in biological membranes
Koukalová, Alena ; Nováková, Olga (referee) ; Černý, Jan (advisor)
Cholesterol is one of the major membrane component in most eukaryotic cells, where it performs multiple functions. Cholesterol regulates the fluidity of cell membranes, influences the activity of several membrane proteins and is the precursor of many steroid hormones and bile acids. Cholesterol is not a fluorophor or chromophor on its own, therefore it is necessary to visualize it to study its localization and distribution in cells. Reporter molecules that can be used for this purpose can be classified in two groups: specific cholesterol binding probes and cholesterol analogues. Among specific binding cholesterol probes are cholesterol oxidase enzyme that catalyses the reaction of cholesterol and oxygen into the products cholest-4-en-3-one and hydrogen peroxide and is used to study membrane organization and cholesterol flip-flop kinetics in plasma membrane, the polyene filipin forms fluorescent complex with cholesterol and is usually used to gain information about cholesterol distribution and localization in cells. Thiol-activated cytolysins and a derivate of perfringolysin O - BCθ-toxin - are suitable for recognizing cholesterol-rich domains and localization cholesterol in membrane. Cyclodextrins (especially methyl-β-cyclodextrin) selectively extract cholesterol from membranes and they can be used as...
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Czech Business Environment from the Point of View of Swiss Investors
Kracík, Josef ; Müllerová, Františka (advisor) ; Koukalová, Alena (referee)
The aim of my thesis was to analyze the business environment in the Czech Republic and to evaluate the possibilities for Swiss entrepreneurs on this market. My second aim was to provide the necessary information to the companies from Switzerland who currently consider possible enter on the Czech market. The business environment in the Czech Republic was analyzed on the basis quantitative analysis of particular factors, which determine the business environment in the Czech Republic. Furthermore I made the qualitative analysis of the Swiss point of view by conducting three individual interviews with the Swiss ambassador in the Czech Republic, with the CEO of the Chamber of Commerce Switzerland -- Czech Republic and the marketing director of a Swiss company operating in the Czech Republic.
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