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Control of metabolism of carotenogenic yeasts on molecular level
Pokrývková, Zuzana ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.
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Study of red yeast metabolism on molecular level
Roubalová, Monika ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This master thesis is focused on the molecular characterization of the eight red yeasts species. For molecular characterisation, the most variable rDNA regions ITS1, 5,8S ITS2 and the region encoding the large ribosomal subunit (26S) were amplified. This long region of the yeasts DNA was sequenced and compared by NCBI database for identification. The red yeasts identification was confirmed by data from DGGE method. Another aim of this thesis was to select the best yeasts producer of carotenoids and triacylglycerols. Rhodosporidium toruloides was found as the best producer and, thus, this strain was subjected to random mutagenesis by UV irradiation. The results of the production of metabolites by R. toruloides were compared with mutant strains, which were also adapted to the glycerol and waste whey substrates. The mutant strain G33 was found as the best producer of total carotenoids with a yield of 7.14 mg.g-1 of biomass. The highest production of ergosterol was demonstrated by the mutant strain Y34, the ergosterol yield was 47.72 mg.g-1 of biomass. The wild type of R. toruloides was able to produce the highest amount of both carotene (2.42 mg.g-1 of biomass) and TAG (76.32 mg.g-1 of biomass) on glucose medium.
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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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Avian schistosomes and cercarial dermatitis in Czech Republic: distribution, diverzity and factors influencing their occurrence
Pokrupová, Zuzana ; Bulantová, Jana (advisor) ; Soldánová, Miroslava (referee)
Cercarial dermatitis (CD) is manifested as a strong itchy rash that usually occurs after bathing in the natural water bodies where it makes recreation uncomfortable. As a consequence, the natural swimming areas and be closed because of inconvenient water quality, what subsequently leads to the financial losses. The infection can also affect people working in the natural water bodies as lifeguards (at the natural swimming areas), people monitoring water organisms or water quality etc.). Nowadays, CD in Czech Republic occurs more frequently than in the past. For this reason, the topic of avian schistosomes and CD is very attractive not only for scientists, but newly also for employees of hygienic stations and health institutes. Based on the Act No. 258/2000 Coll. on protection of public health and related executive Decree No. 238/2011 Coll. approved this year their duty will be regular monitoring of official natural swimming areas for causative agents of CD. For the comprehensive overview about the occurrence of the avian schistosomes and CD, up to now, at the localities in the Czech Republic the specialized overview map with the marked catches of avian schistosomes and CD was created in the program ArcGIS Online. This map was made with use the records obtained from the scientific articles, final...
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Study of red yeast metabolism on molecular level
Roubalová, Monika ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This master thesis is focused on the molecular characterization of the eight red yeasts species. For molecular characterisation, the most variable rDNA regions ITS1, 5,8S ITS2 and the region encoding the large ribosomal subunit (26S) were amplified. This long region of the yeasts DNA was sequenced and compared by NCBI database for identification. The red yeasts identification was confirmed by data from DGGE method. Another aim of this thesis was to select the best yeasts producer of carotenoids and triacylglycerols. Rhodosporidium toruloides was found as the best producer and, thus, this strain was subjected to random mutagenesis by UV irradiation. The results of the production of metabolites by R. toruloides were compared with mutant strains, which were also adapted to the glycerol and waste whey substrates. The mutant strain G33 was found as the best producer of total carotenoids with a yield of 7.14 mg.g-1 of biomass. The highest production of ergosterol was demonstrated by the mutant strain Y34, the ergosterol yield was 47.72 mg.g-1 of biomass. The wild type of R. toruloides was able to produce the highest amount of both carotene (2.42 mg.g-1 of biomass) and TAG (76.32 mg.g-1 of biomass) on glucose medium.
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Control of metabolism of carotenogenic yeasts on molecular level
Pokrývková, Zuzana ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.
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Production of Selected Yeast Metabolites Applicable to Food Supplements
Němcová, Andrea ; Čertík, Milan (referee) ; Vávrová, Milada (referee) ; Márová, Ivana (advisor)
Carotenoids are naturally occurring pigments of plants also produced in many bacteria, and fungi. They represent one of the widest group of natural antioxidants with significant biological effects and numerous of industrial applications. There is an increased interest in carotenoids as natural antioxidants for their ability to reduce chronic diseases, various pathological stages and aging. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. One possibility is study of potential of red yeasts that are able to convert various substrates into carotenoid pigments. In presented thesis carotenogenic yeast belonging to the genus Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates and also random mutagenesis in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. As alternative nutrient sources derived from waste substrates from agricultural and food production (rapeseed substrate, rice, wheat, apple fiber, pasta and lignocellusic materials) were tested. To selected production media extracellular hydrolytic enzymes or commercial enzymes degrading polysaccharide were added. All tested red yeast strains were able to utilize these substrates as the only carbon source and simultaneous produce carotenoid enriched biomass. In this work, characterization of carotenogenic yeast using molecular techniques was studied. For this usage, interspecific variables of strongly conserved sequences of genomic DNA, especially rDNA D1/D2 large ribosomal subunit and ITS1 and 5,8-ITS2 rDNA regions were amplified. These sequences were subjected analysed by DGGE method to compare differences of carotenogenic yeasts. Isolation procedure of the intact DNA were optimized for caryotypic yeast characterization by pulsed field gel electrophoresis (PFGE). The karyotype of tested yeasts contain visible differences between yeast species and genera.
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Molecular characterization of selected strains of amoebae of the genus Naegleria, the potential human parasites.
ZÍTKOVÁ, Klára
The genus Naegleria is a eukaryotic organism, one of those we can call "amoeba" a general name of creatures changing their shape during their locomotion. Amoebae comprise a huge number of various different genera some of which contain species able to infect humans similar to Naegleria fowleri. Two important exaples are the genera Acanthamoeba and Entamoeba, causative agents of acanthamoeba keratitis or diarrhoea, respectively, and other serious symptoms. Although superficially similar, they are not close relatives of Naegleria: they belong to Amoebozoa, whereas Naegleria is a Heterolobosea. The most important species of this speciose genus is the amoeba Naegleria fowleri. Is is the only Naegleria which is a human pathogen. After infection, it causes primary amoebic meningoecephalitis. The invasive stage of Naegleria is an amoebic trophozoite. Under certain circumstances, it can transform in another form, the flagellate bearing undullipodia. When the conditions worsen, it changes in the resistant cyst stage. Major part of my work is related to Naegleria fowleri and primary amebic meningoencephalitis, which causes human (PAM). They clarify here the source of infection and risk factors for assault. The length of incubation period and the first appearance of symptoms. The next chapter concerns the mechanism of intrusion into the body, how can this infection properly diagnose and detect and also a treatment option, resulting in early detection of symptoms. Possibilities prevention and epidemiology, including presentation of biggest epidemic, that happened in the Czech Republic. In the practical part of this thesis summarize my own study and methods used, as well as the results and their interpretation. The aim of the experimental part was the analysis of Naegleria DNA maintained in the collection of the laboratory of fish pathogens. To reach my goal and test the hypotheses of this thesis, I identified the Naegleria isolates. The final phylogenetic analysis was enriched with samples sequenced earlier, but so far not analyzed in any previous work. The first step in the workflow was the DNA isolation. The next one was to PCR amplify and sequence the ITS sequences to confirm they belong to Naegeria species, and to analyze the sequences phylogeneticaly and identify the species. The obtained sequences were edited in BioEdit software, program ClustalX was used to align them. The phylogenetic trees were computed in PAUP. Sequencing was provided by Seqme company. During the interpretation of the phylogenetic trees, I found which species were present in studied samples. They were as follows: two samples were N. americana, twelve others N. canariensis, three were N. dobsoni and there were also N. pagei and N. tihangensis one sample each. Several samples (GERK, MSED4, ALM1A, 62K4 and GG1BV) were different from any known species and it is therefore possible to consider them new, undescribed species. On the basis of this fact we can confirm the hypothesis nr. two: the species diversity is understudied and the recognition of new species probable. The data seem to support hypothesis 3: in a given locality, a single dominant species becomes dominant. In some localities, however, we could not fully exclude the opposing hypothesis 4.
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