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Detection of resistance to echinocandins antifungal agents in Candida sp. using molecular biological methods
Vitáčková, Petra ; Chrenková, Vanda (advisor) ; Nyč, Otakar (referee)
Invasive diseases due to Candida sp., especially by C. albicans, represent very severe complication in immunocompromised patients. More over the presence of antifungal resistance leads to delay of targeted antifungal therapy and increases morbidity and mortality in this group of patients. Therefore the aim was to introduce a rapid method of caspofungin resistance detection by the mass spectrometry MALDITOF. The tests were performed by the use of reference strain C. albicans CCM8261 and caspofungin resistant strain C. albicans M30. Different settings of mass spectrometer were used for the measuring. The obtained spectra were evaluated by correlation and cluster analysis (dendrogram). By cluster analysis it was possible to differenciate the susceptible and the resistant strain. During the analysis we have found, that mass spectrometer settings are unique for each machine and we cannot use the published data. We did not succeed to determine the similarity by correlation analysis. The quality of obtained spectra was quite poor, probably because of non-suitable culture medium used in the test The cluster analysis confirmed the possibility of resistance detection by mass spectrometry; nevertheless more testing profiting from current experience is needed for introduction of this test in routine. Powered by TCPDF...
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Raman Microspectroscopy and Mapping of Single Cells
Gregorová, Šárka ; Mojzeš, Peter (advisor) ; Plášek, Jaromír (referee)
Raman microspectroscopy enables one to acquire spectra of Raman scattering with a spatial resolution in the order of a few μm3 and thus to study the natural composition of biological objects such as tissues, single cells and cellular organelles in a non-invasive way. In this work, we used Raman microspectroscopy to investigate vacuoles of the opportunistic human yeast pathogen Candida albicans. Large sets of Raman spectra of vacuoles were collected based on different cultivation protocols. The sets of the spectra were evaluated using the multivariate statistical method of singular value decomposition. Based on the spectral analysis, we characterized the chemical composition of the vacuoles. We found out that the vacuoles of cells cultured differently or in different media vary particularly in the concentration of polyphosphate, represented in the spectra by the peak near 1155 cm-1 . Interestingly, the wavenumber position of the polyphosphate peak may also be shifted by several cm-1 . We studied these shifts in vitro with sodium hexametaphosphate as a model of vacuolar polyphosphate. Based on these experiments, we suggest that the peak position is significantly influenced by the concentration of divalent cations.
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The study of mutual interaction between pathogenic yeasts of the genus Candida and bacterium Pseudomonas aeruginosa during cocultivation
Mynářová, Lenka ; Hodek, Petr (advisor) ; Papoušková, Klára (referee)
The genus Candida includes several opportunistically pathogenic species which are common causative agents of the yeast infections in humans. Although current medical research is focused mostly on cancer, AIDS or Alzheimer disease, the problem of systemic candidiases cannot be neglected. These infections represent a real threat to the immunocompromissed patients, they are connected with a high mortality rate and expensive medication with poor prognosis. Pseudomonas aeruginosa could be an inspiration in a way of how to eliminate the pathogenic yeasts. The bacterium can inhibit growth of the most common yeast species of the genus Candida, C. albicans. This effect is based on production of toxic substances by the bacterium and on interaction of the bacterium with the C. albicans cell wall, which leads to the lysis of the yeast cells and which is not fully understood. Nevertheless, coexistence of these microorganisms is also possible and their relationship is affected by various factors. Knowledge of these inter- microbial interactions was obtained from studies of diseases and pathologies, during which C. albicans + P. aeruginosa coinfections occur. In this thesis I studied mechanisms of interaction between pathogenic yeast C. albicans and bacterium P. aeruginosa by a) C. albicans gene expression...
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Klinicky významné druhy kvasinek a jejich interakce s hostitelem
Novosadová, Zuzana ; Palková, Zdena (advisor) ; Beranová, Jana (referee)
Pathogenic yeasts are nowadays a serious threat for mammalian host. They can cause dangerous diseases, which in many cases even result in death. Pathogens increase the chances of systemic infections by many virulence mechanisms. Experiments addressing the pathogenhost interactions are crucial for defeating these types of infections within the human body. Host-pathogen interactions are very complex and include all components of multicellular host organism. Recently, scientists have focused on the interaction of the mammalian immune system and pathogenic yeasts in more detail. This work summarises interactions of pathogen with selected host cells, especially with macrophages. Yeast pathogens, especially Candida albicans, are capable of influencing the gene expression in interacting cells. These pathogens are capable of modulating the expression while engulfed inside macrophages and other cells of the immune system. Pathogenic yeasts can also change the overall characteristics of their surrounding environment. C. albicans can sense pH and influence it. Therefore, it can increase its virulence by the changes of pH leading to autoinduction of morphological transitions. This work briefly reviews how selected yeast pathogens influence their surroundings while interacting within the host organism. Deeper...
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Small signaling molecules in yeast
Putalová, Tereza ; Váchová, Libuše (advisor) ; Horníková, Lenka (referee)
Yeasts excrete metabolitesinto the environment some of which may have a signal function. The small signalling molecules include ammonia, alcohols, esters, acids and CO2 beside other molecules. These substances may be formed as waste products of metabolism, such as some alcohols in the catabolism of amino acids. After exclusion they influence other / surrounding cells by binding to receptors or they affect their target in the cell or may form a concentration gradient or a pH gradient. New findings show that using ammonia yeasts can communicate and may diversify within colonies. Farnesol, tyrosol and other molecules use the yeasts to quorum sensing. Yeasts also secrete aromatic esters and fatty acids. High concentrations of CO2 trigger switch from yeasts to hypha. This paper summarizes existing information on the occurrence and impact of selected molecules (ammonia, alcohols, esters, acids and CO2) signaling in the yeast Saccharomyces cerevisiae and Candida albicans.
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Role of MDR transporters in yeast biofilm resistance
Urbanová, Daniela ; Palková, Zdena (advisor) ; Seydlová, Gabriela (referee)
This work is focused on multidrug resistance transporters (MDR) and their role in the drug resistance of yeast biofilms. Biofilms are structured microbial communities that are markedly different from planktonic cells. Biofilm cells produce extracellular matrix and display other typical characteristics related to their enormous resistance to antimicrobial agents. MDR pumps contribute to higher resistance of biofilms only during early phases of biofilm development; later, MDR pumps are substituted by many other mechanisms. Cdr1p, Cdr2p and Mdr1p are the most important MDR transporters of Candida albicans. Cdr1p and Cdr2p cause resistance to azoles - fluconazole, ketoconazole and itraconazole, which have been widely used as drugs against yeast infections. Mdr1p contributes also to the resistance to fluconazole. Drug resistance causes considerable problems in the treatment of fungal infections. For this reason, it is so important to understand drug-resistance mechanisms of yeast communities. Keywords: resistance, MDR transporters, Candida albicans, biofilms
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CUG Codon in Pathogenic Yeasts of the Genus Candida
Marečková, Lucie ; Půta, František (referee) ; Heidingsfeld, Olga (advisor)
2. Abstract In many Candida species the standard leucine CUG codon is translated as a serine, although not in 100% cases. This dual specifity of the CUG codon has evolved through a mechanism that required codon ambiguity mediated by a unique tRNACAG, which is in vitro aminoacylated more often by serine than by leucine. This codon ambiguity has been tolerated for more than 170 million years. The explanation at least for now is that the CUG codon reassignment could have generated genetic diversity that facilitated occurrence of new phenotypes resistant to stress. Beside this, an important step was to reduce negative impact of the codon ambiguity by crucial mutations in the structure of the ser-tRNACAG. Candida species became a valuable experimental model for elucidation of the genetic code changes. While consequences of the CUG codon reassignment have been extensively studied in Candida albicans, this topic has not yet been addressed in Candida parapsilosis. Solving the structure of C. parapsilosis secreted proteinase Sapp1p provided a tool to carry out a "case study" of possible effects of the CUG codon ambiguity. The SAPP1 gene contains one CUG codon, and the respective serine is located on the loop in the close proximity of the active site of the proteinase.
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Expression and characterization of Phr2 protein of Candida albicans
Vlčková, Zuzana ; Ing.Zuzana Firáková (referee) ; Kováčová, Kristína (advisor)
Transglycosylases have an import function in yeast the cell wall. These enzymes cleave a molecule of donor, which is followed by transferring a newly created reducing end to a non-reducing end of a molecule of an acceptor. This process enables a growth, expansion and shaping of the yeast cell walls during their life. The aim of this Bachelor Thesis is to characterise the role of Phr2 recombinant protein of Candida albicans in a transglycosylase activity. Using fluorescent paper method, optimal pH and optimal temperature of transglycosylase activity of Phr2 protein were found. The same method was used to establish the acceptor and donor specificity of Phr2p was performed as another method for substrate specificity measurement. High donor and acceptor specificity of Phr2 protein to -1,3-glucan was confirmed. HPLC products of transglycosylase reaction catalysed by Phr2p with laminarin as a donor and L5-SR as an acceptor containing a fluorescent marker were detected by TLC and MALDI-TOF.
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