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MALDI - TOF MS identification of filamentous fungi.
ŠNAJDROVÁ, Martina
The method of mass spectrometry with laser desorption / ionization time of flight in the presence of the matrix analyzer ( MALDI-TOF MS ) is using a technique called soft ionization. This is a fast and ever-evolving method, which has found application for the analysis of proteins, glycoproteins, nucleic acids, lipids and high molecular weight and low molecular weight substances. This method has brought inovation to the routine microbiology laboratories. MALDI-TOF mass spectrometry can be used for identification aerobic and anaerobic bacteria, yeast, mycobacteria, but also to determine the genus and species of filamentous fungi. Except identification of microorganisms, is possible to detect the mechanisms of antibiotic resistance in bacteria (e.g., - lactamases ability for the inactivation of - lactam antibiotics ). MALDI TOF MS is an easy to use method more accurate, faster and more cost efficient than commonly used biochemical tests and comparing macroscopic and microscopic appearance. Mass spectrometry MALDI TOF generate mass spectra of proteins (bacteria) and glycoproteins (fungi), which are species -specific for identification of strain. Identification of the strain by comparing the obtained mass spectra with reference spectra databases . In the period from January 2013 to February 2014 were in the practical part examined 114 isolates of filamentous fungi, 80 strains as agent of a superficial mycoses and 34 strains as agents of systemic and organ fungal infections. All 114 samples were determined by conventional methods and also were identified by MALDI TOF MS. The selected group of isolates was compared to molecular genetic methods (PCR) followed by the sequence of the amplicon. MALDI TOF mass spectrometry is a method for easily identifying bacteria. During testing of filamentous fungi is a main importance on the stage of preparation of the sample before testing. It was tried several workflows whose success is summarized in this thesis.
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Soil micromycetes - the potential source of N.sub.2./sub.O in soils
Jirout, Jiří ; Šimek, Miloslav ; Elhottová, Dana
The aim of this paper is to summarize knowledge on potential production of N.sub.2./sub.O by fungi. The processes involved in the global nitrogen cycle are suggested to have an important role in the global environmental changes. Nitrification and denitrification processes cotribute to the production of N.sub.2./sub.O, which belongs to the group of highly active greenhouse gases. Denitrification - the successive reduction of nitrate to N.sub.2./sub. - was supposed to occur only in prokaryotes, but recently the evidence for dissimilation of nitrates and formation of nitrogen oxides and N.sub.2./sub. was reported also in fungi. N.sub.2./sub.O of fungal origin is formed from nitrate or nitrite by reduction under anaerobic or microaerobic conditions. Since fungi showed the capability to use denitrification and oxygen respiration simultaneously under various pO.sub.2./sub. conditions, the N.sub.2./sub.O production by fungi is expected in a wide range of soils with different aeration status.
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Diversity of anaerobic ciliates
Nováková, Ludmila ; Čepička, Ivan (advisor) ; Kostka, Martin (referee)
Ciliates (Ciliata) are single-celled eukaryotic organisms belonging to the large group Alveolata. Ciliates are classified to eleven classes. Anaerobic/microaerophilic representatives belong to classes Armophorea, Litostomatea, Plagiopylea, Oligohymenophorea, and Prostomatea. The mitochondrion of the anaerobic ciliates has been tranformed to hydrogenosome. The anaerobic ciliates are characteristic for the presence of methanogenic symbionts in the cytoplasm near hydrogenosomes. Anaerobic flagellates are free-living or they live as commensals, mutualists or parasites in the digestive tract of animals including humans. The true diversity of anaerobic ciliates is still not fully understood. The reason is that only sequences of described ciliate species are usually included into phylogenetic analyses. However, many environmental sequences representing considerable part of known molecular diversity of ciliates have been published as well. The aim of this work was to obtain and analyze sequence data of anaerobic free-living ciliates. We have determined SSU rDNA sequences of 32 different freshwater and marine strains of ciliates. Phylogenetic analyses showed that the strains belonged mostly to the classes Armophorea, Plagiopylea and Oligohymenophorea. The strain LIVADIAN belonged, together with genera...
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Pyruvate:ferredoxin oxidoreductase homologues from Trichomonas vaginalis hydrogenosomes
Zedníková, Věra ; Hrdý, Ivan (advisor) ; Doležal, Pavel (referee)
Oxidative decarboxylation of pyruvate is a fundamental reaction of living organisms in general, leading to energy conservation. In some anaerobic or microaerophilic eukaryotic or prokaryotic organisms pyruvate decarboxylation is carried out by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFO). PFO contains Fe-S clusters and thiamin pyrophosphate cofactor (TPP). In the reaction catalyzed by PFO, from pyruvate and Co-A arise acetyl-CoA, CO2, and two electrons are released. Those electrons are accepted by low molecular carrier proteins. Most frequently these proteins are ferredoxins or flavodoxins such as in nitrogen fixating bacteria. PFO can perform a reversible reaction. Trichomonads are mostly parasitic or endosymbiotic organisms with mitochondria-like organelles, hydrogenosomes. These organelles possess PFO which is one of the key enzymes in the metabolism of Trichomonas vaginalis hydrogenosomes. PFO of T. vaginalis, a sexually transmitted pathogen of man, plays also a role in a term of medical importance. PFO is, by a universally accepted concept, one of the key proteins acting in the activation of antimicrobial drugs against trichomoniasis 5-nitroimidazoles, including metronidazole. In the genome of T. vaginalis seven PFO genes were identified. They were named PFO A, B1, B2, C, D, E and...
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Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist
Rada, Petr
Charles University in Prague, Faculty of Science Department of Parasitology Ph.D. study program: Parasitology Abstract of the Ph.D. Thesis Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist Petr Rada Supervisor: Prof. RNDr. Jan Tachezy,Ph.D. Advisor: Doc. RNDr. Ivan Hrdý, Ph.D. Prague, 2011 ABSTRACT Trichomonas vaginalis and Giardia intestinalis are parasitic protists of the Excavata group. Both contain anaerobic forms of mitochondria called hydrogenosomes (Trichomonas) and mitosomes (Giardia). Hydrogenosomes produce hydrogen and ATP by substarte level phosphorylation and mitosomes represent the highly-reduced form of mitochondria that do not participate in cellular energy metabolism and ATP generation. Both types of organelles lost the majority of mitochondrial pathways and their genomes during the mitochondrion to hydrogenosome transition. Consequently, hydrogenosomes and mitosomes facilitate translocation of nuclearly encoded proteins into the matrix of the organelle as well as exchange of metabolites and ions across their membranes. Little is known about the membrane machineries required for the biogenesis of the organelle and metabolite exchange and the limited knowledge of mitosomal proteomes has been mostly gained from genomic analysis and localization studies of a few...
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Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist
Rada, Petr ; Tachezy, Jan (advisor) ; Embley, Martin (referee) ; Eliáš, Marek (referee)
Charles University in Prague, Faculty of Science Department of Parasitology Ph.D. study program: Parasitology Abstract of the Ph.D. Thesis Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist Petr Rada Supervisor: Prof. RNDr. Jan Tachezy,Ph.D. Advisor: Doc. RNDr. Ivan Hrdý, Ph.D. Praha, 2011 1 ABSTRACT Trichomonas vaginalis and Giardia intestinalis are parasitic protists of the Excavata group. Both contain anaerobic forms of mitochondria called hydrogenosomes (Trichomonas) and mitosomes (Giardia). Hydrogenosomes produce hydrogen and ATP by substarte level phosphorylation and mitosomes represent the highly-reduced form of mitochondria that do not participate in cellular energy metabolism and ATP generation. Both types of organelles lost the majority of mitochondrial pathways and their genomes during the mitochondrion to hydrogenosome transition. Consequently, hydrogenosomes and mitosomes facilitate translocation of nuclearly encoded proteins into the matrix of the organelle as well as exchange of metabolites and ions across their membranes. Little is known about the membrane machineries required for the biogenesis of the organelle and metabolite exchange and the limited knowledge of mitosomal proteomes has been mostly gained from genomic analysis and localization studies of a few...
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Cryptic diversity of free-living trichomonads and their phylogenetic position within Parabasalia
Céza, Vít ; Čepička, Ivan (advisor) ; Hampl, Vladimír (referee)
Trichomonads (Parabasalia) are anaerobic microeukaryotes classified in the supergroup Excavata. Inclusion of parabasalids within Excavata is exclusively based on the molecular- phylogenetic evidence. Over 400 species of parabasalids have been described so far, and the vast majority of them are endobiotic. In contrast, only few species of free-living parabasalids forming four independent lineages have been described (Pseudotrichomonas keilini, Ditrichomonas honigbergii, Monotrichomonas carabina, Honigbergiella sp., Tetratrichomonas undula, and Lacusteria cypriaca). Lacusteria cypriaca is a new species and genus described in our recent paper. In this paper we published the first two sequences of SSU rDNA from Pseudotrichomonas keilini as well. All of these lineages are likely secondarily free-living, and they developed from endobiotic ancestors. In addition to the already published Lacusteria cypriaca and Pseudotrichomonas keilini strains, we have recently obtained seven another isolates of free-living trichomonads (LAGOS2D, E2NT, CK, LAGOS2M, GR8, GOU23 LIVADIAN, and VAV1A1); from all of these isolates we sequenced SSU rDNA and performed phylogenetic analyses. These isolates split into four independent evolutionary lineages, which indicate that free-living parabasalids are more diversed and...
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