This diploma thesis is concerned about the PCR-RFLP method for identification of yeasts genus Saccharomyces from leaves, grapes and must of white wine. The thesis describes an obtaining of pure cultures of yeasts, isolation DNA and amplification of specific segments of DNA using PCR method. Splitting of this segments by restriction endonukleases and a detection of final fragments by zone electrophoresis are mentioned too. Length of fragments and numbers of restriction fragments, which are characteristic of every species, enable a determination of microorganisms and their inclusion into the system. The basic informations on yeasts and molecular methods for their identification are named in the theoretical part of the thesis. The main part of the thesis deals with the processing of grapes of grape-wine and the manufacturing of white wine.

Carotenoids are important industrial pigments present practically in all living organisms. The aim of presented work is the study of regulation of carotenoid production in yeasts of the genus Cystofilobasidium in presence of exogenous stress factors. Growth curve of C. capitatum exhibited typical two-stage course with prolonged stationary phase similar to other carotenogenic yeasts. Maximal production of biomass and beta-carotene occurred in 103rd hour. Applied stress factors (2-5% NACl, 2-5 mM H2O2, 0,01-1 mM Se(IV), 0,1-5 mM Cr(III)) exhibited no significant influence on biomass production, which reached on average 8-9 g/l. Positive effect was observed in presence of 5mM Cr where 10 g/L of biomass was produced. Beta-carotene formation was positively influenced by many applied stress factors, the highest yield (695 g/g) was reached in presence of 0,1 mM Se(IV). No simultaneous regulation of ergosterol and carotenes was observed in Cystofilobasidium cells. Production properties of yeast strain C. capitatum CCY 10-1-1 wee compared with those of other carotenogenic yeasts of the genes Rhodotorula and Sporobolomyces. C. capitatum produced similar biomass yield as Rhodotorula sp. in presence of salt. Production of beta-carotene by C. capitatum was slightly higher than in Rhodotorula glutinis, but lower than in Sporobolomyces strains which exhibited substantially lower biomass production. Karyotype of C. capitatum is relatively different when compared with karyotype of other carotenogenic yeasts. Based on summary of our results in seems that yeasts C. capitatum exhibit similar physiological as well as production properties as some Rhodotorula strains. Thus, yeasts of the genus Cystofilobasidium could be potentially used to industrial production of carotenoid pigments as well as yeast biomass rich in carotenoids and some biogenic elements.

Carotenoids are naturally occurring pigments of plants also produced by microbes. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. Currently, the isolation of carotenoids from plants is markedly regulated by legislation, so the study of their production is greatly emphasised, where the microbiological, instead of the synthetic, production of carotenoids is being prioritized. This work was made as a comparative study of carotenogenic yeasts of the genes Rhodotorula, Sporobolomyces, and Cystofilobasidium. Their ability to use various waste substrates as a carbon and nitrogen source and source of other nutrition factors was tested. In this work, conditions of random mutagenesis were optimized. Particular yeast strains were also subjected to the effect of mutagen ethyl methanesulfonate (EMS) in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. Random mutagenesis and mutant strain selection was performed using waste subtrates as glycerol, pasta and some pasta hydrolyzed by fungal extracellular enzymes. Subsequently, a control of specific DNA sequences in pigments overproducing mutants was analyzed by PCR/DGGE (denaturating gradient gel electrophoresis). Increased production of -carotene was achieved in a mutant of Sporobolomyces roseus strain growing on glycerol, pasta, and hydrolyzed pasta. Overproduction of carotenoids by mutant strain of Rhodotorula glutinis was observed in glucose medium only. Mutants of Cystofilobasidium capitatum exhibited a decrease of biomass production; on the other hand, the production of carotenoids increased especially in pasta medium hydrolyzed by enzyme preparative from Fusarium solani. In this work it was confirmed that using random mutagenesis strains capable to utilize waste substrates can be selected. In mutant strains increased carotenoids biosynthesis was observed, which enables effective use of cheap substrates and reduction of the negative effects of wastes on the environment.

The aim of this diploma thesis was the selection of appropriate indigenous yeasts for wine production. Tested yeasts were isolated from the grapes from the winery Maňák Žádovice. The yeasts isolated within theses in 2009 – 2012 have been also tested, and commercial wine yeast have been tested for comparison. In the theoretical part the focus is on the technology of wine, in particular fermentation. The work is also focused on yeasts metabolism and taxonomy. There was described the principle of PCR-RFLP, and methods used to characterize the properties of isolated yeasts. In the experimental part was made isolation of yeasts, theirs identification using PCR-RFLP and to select the most suitable yeast in wine making proces were performed physiological tests.

The aim of this work was to isolate the Saccharomyces cerevisiae yeast from the surface of wine grapes of the Malverina and Sauvignon varieties. Isolated yeasts were identified by the PCR-RFLP method. 5,8S ITS rDNA was amplified using PCR and restricition endonucleases HaeIII, HhaI, HinfI and TaqI were used for the restriction analysis which followed. The results were processed by UPGMA cluster analysis using the BioNumerics programme. The dominant genus on the surface of the Malverina variety grapes was Brettanomyces/Dekkera, while on the surface of the Sauvignon grapes we found mainly yeasts of the Pichia genus. The Saccharomyces cerevisiae species was not isolated from any of the two grape varieties.

Human pathogenic yeasts of the genus Candida and 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 are responsible for about 10% of hospital-acquired infections. In addition, drug-resistant yeasts are rapidly emerging with the use of anti-fungal drugs. Common drugs such as Fluconazole and Amphotericin B target the ergosterol pathway in yeast. Learning about other metabolic differences in yeasts may also give a new understanding to their role as pathogens. Metabolomics is a field of study about the large spectrum of metabolites necessary for the growth and survival of an organism. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the metabolome of different yeasts in three different studies. The first study was a targeted analysis of the ergosterol pathway in 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 and a double drug-resistant mutant was found to have several changes in its sterol composition while Fluconazole-resistant strains were similar to the wild-type. In the second study, a comprehensive analysis of the polar and lipid metabolite profiles of six pathogenic yeasts revealed that lipid profiles were more conserved than polar profiles, thereby better reflecting their taxonomical relationship according to 265 rRNA sequences. However, there were several potential species-specific metabolites and short regions in the metabolite profiles with enough peak differentiation that could be used to rapidly distinguish between these yeasts by visual inspection. In the third study, the metabolic phenotypes of three strains of 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 were analysed to determine the extent of contribution of the metabolite phenotypes from two parents to their hybrid offspring. While the lipid metabolite phenotypes of all strains resembled each other, the polar metabolite phenotype of the hybrid offspring strongly resembled one parent but not the other. / Thesis / Master of Science (MS)

Nos últimos anos, têm ocorrido um aumento considerável das infecções fúngicas em ambiente hospitalar. As estimativas da incidência das infecções causadas por fungos não correspondem à realidade, devido às dificuldades diagnósticas destes microrganismos. Embora as infeções fúngicas, principalmente leveduras do gênero Candida, estejam ganhando importância nos pacientes hospitalizados, a grande maioria das instituições ainda não está preparada para o diagnóstico destes episódios. O presente trabalho teve como objetivo realizar um estudo epidemiológico, determinando a ocorrência leveduras isoladas de diversos materiais, de diferentes setores em pacientes internados de um Hospital Universitário da região do Vale do Paraíba-SP. Neste estudo foram isoladas 55 cepas de leveduras das 558 amostras analisadas, no período de fevereiro a agosto de 2002. O gênero Candida ocupou a 2ª posição (9,85%) em relação aos 10 microrganismos mais isolados em amostras positivas com suspeita clínica de infecção hospitalar. Das amostras positivas para leveduras, a espécie mais freqüente foi C.albicans com 40,0 %, seguida de C.tropicalis com 25,5%, C.parapsilosis com 23,6%, C.glabrata com 9,1% e C. norvagensis com 1,8%. A clínica médica foi o setor que apresentou maior número de amostras positivas para leveduras (63,6%), seguido por UTI neonatal ( 20,0%) e UTI adulto ( 16,4%).Dentre os espécimes clínicos analisados, as secreções ( 47,3%), de um modo geral, foram os que apresentaram maior número de amostras positivas para leveduras, em 2º lugar ponta de sonda ( 29,1%) e em 3º lugar sangue( 12,7%), com predomínio de C. albicans. Dos 38 pacientes que apresentaram cultura positiva para leveduras do gênero Candida, 26 casos de colonização foram identificados (68,4%) e 22 casos foram classificados como casos de infecção hospitalar ( 57,8%), onde 50% destes casos de pacientes foram a óbito . Os resultados obtidos demonstram a importância da identificação correta dos episódios de infecção, na caracterização do perfil epidemiológico,auxiliando assim, no diagnóstico e terapêutica destes agentes emergentes. / In recent years, there have been a considerable increase of fungal infections in hospitals. Estimates of the incidence of fungal infections do not correspond to reality, due to diagnostic difficulties of these microorganisms. Although fungal infections, especially Candida species, are gaining importance in hospitalized patients, the vast majority of institutions are not yet prepared for the diagnosis of these episodes. The present work aims to perform an epidemiological study, determining the prevalence of these strains isolated from different materials, different sectors in inpatients of a university hospital in the region of Vale do Paraíba-SP. In this study we isolated 55 yeast strains from 558 samples in the period from February to August 2002. The genus Candida occupied the 2nd position (9.85%) for the 10 microorganisms isolated from positive samples with clinical suspicion of nosocomial infection. Samples were positive for yeasts, the most frequent species was C. albicans with 40.0%, followed by C. tropicalis 25.5% C. parapsilosis with 23.6% and 9.1% with C. glabrata and C. norvagensis 1.8%. The medical clinic was the sector that had the greatest number of samples positive for yeast (63.6%), followed by neonatal ICU (20.0%) and adult ICU (16.4%). Among the clinical specimens examined, the secretions (47.3%), in general, presented the largest number of samples positive for yeast, in 2nd place probe tip (29.1%) and blood in 3rd place (12.7%), predominantly C. albicans. Of the 38 patients who presented positive culture for Candida species, 26 cases of colonization were identified (68.4%) and 22 cases were classified as cases of nosocomial infection (57.8%), where 50% of these cases were patients died. The results demonstrate the importance of correct identification of episodes of infection, to characterize the epidemiological profile, thereby aiding in the diagnosis and treatment of these emerging agents.

Nos últimos anos, têm ocorrido um aumento considerável das infecções fúngicas em ambiente hospitalar. As estimativas da incidência das infecções causadas por fungos não correspondem à realidade, devido às dificuldades diagnósticas destes microrganismos. Embora as infeções fúngicas, principalmente leveduras do gênero Candida, estejam ganhando importância nos pacientes hospitalizados, a grande maioria das instituições ainda não está preparada para o diagnóstico destes episódios. O presente trabalho teve como objetivo realizar um estudo epidemiológico, determinando a ocorrência leveduras isoladas de diversos materiais, de diferentes setores em pacientes internados de um Hospital Universitário da região do Vale do Paraíba-SP. Neste estudo foram isoladas 55 cepas de leveduras das 558 amostras analisadas, no período de fevereiro a agosto de 2002. O gênero Candida ocupou a 2ª posição (9,85%) em relação aos 10 microrganismos mais isolados em amostras positivas com suspeita clínica de infecção hospitalar. Das amostras positivas para leveduras, a espécie mais freqüente foi C.albicans com 40,0 %, seguida de C.tropicalis com 25,5%, C.parapsilosis com 23,6%, C.glabrata com 9,1% e C. norvagensis com 1,8%. A clínica médica foi o setor que apresentou maior número de amostras positivas para leveduras (63,6%), seguido por UTI neonatal ( 20,0%) e UTI adulto ( 16,4%).Dentre os espécimes clínicos analisados, as secreções ( 47,3%), de um modo geral, foram os que apresentaram maior número de amostras positivas para leveduras, em 2º lugar ponta de sonda ( 29,1%) e em 3º lugar sangue( 12,7%), com predomínio de C. albicans. Dos 38 pacientes que apresentaram cultura positiva para leveduras do gênero Candida, 26 casos de colonização foram identificados (68,4%) e 22 casos foram classificados como casos de infecção hospitalar ( 57,8%), onde 50% destes casos de pacientes foram a óbito . Os resultados obtidos demonstram a importância da identificação correta dos episódios de infecção, na caracterização do perfil epidemiológico,auxiliando assim, no diagnóstico e terapêutica destes agentes emergentes. / In recent years, there have been a considerable increase of fungal infections in hospitals. Estimates of the incidence of fungal infections do not correspond to reality, due to diagnostic difficulties of these microorganisms. Although fungal infections, especially Candida species, are gaining importance in hospitalized patients, the vast majority of institutions are not yet prepared for the diagnosis of these episodes. The present work aims to perform an epidemiological study, determining the prevalence of these strains isolated from different materials, different sectors in inpatients of a university hospital in the region of Vale do Paraíba-SP. In this study we isolated 55 yeast strains from 558 samples in the period from February to August 2002. The genus Candida occupied the 2nd position (9.85%) for the 10 microorganisms isolated from positive samples with clinical suspicion of nosocomial infection. Samples were positive for yeasts, the most frequent species was C. albicans with 40.0%, followed by C. tropicalis 25.5% C. parapsilosis with 23.6% and 9.1% with C. glabrata and C. norvagensis 1.8%. The medical clinic was the sector that had the greatest number of samples positive for yeast (63.6%), followed by neonatal ICU (20.0%) and adult ICU (16.4%). Among the clinical specimens examined, the secretions (47.3%), in general, presented the largest number of samples positive for yeast, in 2nd place probe tip (29.1%) and blood in 3rd place (12.7%), predominantly C. albicans. Of the 38 patients who presented positive culture for Candida species, 26 cases of colonization were identified (68.4%) and 22 cases were classified as cases of nosocomial infection (57.8%), where 50% of these cases were patients died. The results demonstrate the importance of correct identification of episodes of infection, to characterize the epidemiological profile, thereby aiding in the diagnosis and treatment of these emerging agents.

The theoretical part discusses the yeasts and their taxonomic classification using traditional methods and using modern methods. Detail the work is concerned with descriptions of modern molecular-biology methods. The practical part was analyzed DNA by PCR-fingerprinting (rep-PCR) type of yeasts, which we received from the CCY and subsequent analysis of yeast samples obtained from grape musts. One of the grape must was obtained in 2009 (white grape variety) and the second in 2010 (red grape variety). Both grape musts come as integrated vineyards and organic. Grape musts samples were obtained from the winery Holánek from Ivaň. The cross-comparison of images PCR-fingerprint type yeasts and yeasts PCR-fingerprint samples using BioNumerics was to evaluate the results and conclude that the diversity of yeast flora in grape must.

Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.