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Diversity and Ecological Role of Cyanobacterial Lipopeptides
GALICA, Tomáš
The presented study focuses on the biosynthesis and distribution of lipopeptides in the context of evolutionary history and life strategies of cyanobacteria. Cyanobacteria are ancient organisms often naturally exposed to situations in which other bacteria, such as Bacillus or Pseudomonas, were shown to employ lipopeptides to facilitate colony expansion or iron acquisition. The study explores these analogies and maps the distribution and diversity of lipopeptides across cyanobacteria with various lifestyles. Furthermore, the study provides evidence that widespread cyanobacterial lipopeptides containing a particular structural motif with two -hydroxy aspartate residues are a part of iron stress response and likely facilitate iron acquisition. The gathered information aims to integrate and deepen our knowledge of the ecophysiological roles of cyanobacterial lipopeptides.
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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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Optimization of lipopeptide analysis by LC-MS
Kadeřábková, Marta ; Hubálek, Martin (advisor) ; Novák, Petr (referee)
Modification of proteins by lipid structure is relatively common post-translational modification that affects the properties of proteins directly and has a forthright effect on the binding of modified proteins to cell membranes. Some lipoproteins play key role in various pathological processes. Before the proteomic analysis of these proteins, the sample of interest is digested using a protease. The resulting hydrolysate contains both unmodified peptides and peptides bearing a lipid modification. During the subsequent chromatographic separation, the lipopeptides differ significantly from the unmodified peptides. For this reason, the analysis of lipopeptides, lipoproteins respectively, is problematic in terms of separation and detection. The subject of the study of this diploma thesis was the optimization of the method of lipoprotein analysis using liquid chromatography and mass spectrometry. The procedures were tested on lipoprotein Cya A (bifunctional adenylate-cyclase toxin from Bordetella pertussis) and MMTV (matrix protein of mouse mammary tumour virus). First, various sample preparation procedures involving proteolytic cleavage were tested. When enzymatic digestion using trypsin on filter (eFASP) was used, the lipid modification was detected with high degree of reliability. In the next step,...
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Separation and identification of lipopeptides by chromatography and mass spectrometry
Pravdová, Adéla ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
5 Abstract Protein lipidation occupies an important place in the post-translational modification group. It plays an important role in the processes of cell differentiation or synaptic transmission. Changes in the presence of lipidated proteins may in some cases indicate the appearance of human diseases such as Huntington's disease, schizophrenia, or cancer. For this reason, their analysis has become desirable, but also problematic due to its specific characteristics. This work is focused on the development of conditions for measurement of lipopeptides produced by cleavage of proteins containing lipomodification. Two separation procedures were tested, in which the aim of work was to enrich the lipopeptides and remove as many other molecules as possible from the samples. The first test method - phase interface separation, using water and chloroform, was able to concentrate the lipopeptides on the phase interface and separate some of the added unmodified peptides from the mixture. The second method of separation was a solid phase using a C18 stationary phase. As a more suitable solid phase extraction variant, elution with solutions containing acetonitrile with trifluoroacetic acid was chosen as compared to solutions containing isopropyl alcohol. With the modified elution procedure, it was possible to separate...
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Detekce a monitoring potenciálně toxických sinicových lipopeptidů
BÁRTOVÁ, Marie
The aim of this study was to design and optimize new PCR primers for detection of potential cyanobacterial producers of cytotoxic lipopeptides puwainaphycins and minutissamides in environmental samples. Samples from two distinct localities were tested, as suggested based on preliminary data. The first set of samples consisted of cyanobacterial soil biofilms from sheep pastures affected by Alveld illness in Norway. The other one contained samples of planktic cyanobacaterial blooms from Protected Landscape Area Třeboň and its vicinity. Three different approaches were used for evaluation of the presence of cyanobacterial lipopeptide producers: microscopy, PCR with the designed primeres, and liquid chromatography-mass spectrometry analysis. Results of this study confirmed the specificity of the newly designed PCR primers. The presence of producers of puwainaphycins/minutissamides was proven at both tested localities.
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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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Mode of action of antimicrobial lipopeptides produced by Bacillus subtilis
Pinkas, Dominik ; Seydlová, Gabriela (advisor) ; Žíla, Vojtěch (referee)
Increasing bacterial resistance to classical antibiotics and emergence of multi-resistant strains impose a constant threat. Antimicrobial compounds of bacterial origin are an important source of new antibacterial therapeutic agents needed to answer this issue. Three families of lipopeptides produced by Bacillus subtilis - surfactins, fengycins and iturins represent an interesting class of such compounds. They exert a wide range of biological activities and possess a good potential for modifications and improvement of their structure and function. Lipopeptides produced by B. subtilis are surface active compounds capable of reducing surface/interface tension. The variety of their biological activities stems from their ability to insert into lipid membranes leading to disruption and permeabilization of the membrane. Specific mode of action differs between the three families but the common feature is that it is concentration dependent. First, lipopeptides induce ion leakage, pore formation and then the increasing concentration eventually causes complete solubilisation of the membrane in a detergent-like manner. In addition, surfactin can inhibit some enzymes by chelating divalent cations required for their activity. These properties make the B. subtilis lipopeptides promising compounds for commercial applications.
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