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Study of genome of Metschnikowia yeasts by molecular methods
Schneiderwindová, Nicole ; Skoumalová, Petra (referee) ; Němcová, Andrea (advisor)
Yeasts of the genus Metschnikowia belonging to the family Metschnikowiacea are yeasts characterized by vegetative propagation through multilateral budding. These are yeasts widely distributed in nature. More than 35 species occurring have been defined in the wild. They most often occur on flowers, fruits, but also on insects or human skin. They have a wide range of uses due to their antifungal effects in agriculture and the cosmetics industry. This bachelor thesis deals with the study of usage of molecular methods to characterize selected species of yeasts of the genus Metschnikowia. It focuses on a detailed description of the yeast cell structure, karyotype and methods of reproduction in the theoretical part of the work. In the practical part on optimization and description of molecular methods including pulse gel electrophoresis methods used to separate the yeast genome and their subsequent observation of changes in individual parts of genome. First, the yeast was cultured under special conditions that are characteristic of Metschnikowia yeasts, then yeast DNA was isolated using methods suitable for DNA isolation, which was further examined by the PFGE molecular method. The DNA isolation procedure was first optimized for individual yeast strains, as it was necessary to verify the required ratio of low melting agarose to isolated DNA. That was because of it was important for the resulting gel blocks to be suitable for measurement by PFGE analysis. By optimizing the method was possible to create ideal blocks of isolated yeast DNA, which were subsequently subjected to PFGE analysis. Several measurements of PFGE analysis were performed at different time intervals in order to separate small and large yeast chromosomes. The CHEF standard of the yeast Hansenula wingei and the standard of the yeast Schizosaccharomyces pombe were used for the measurements. According to the measurement results, it can be determined that the yeast DNA isolation procedure and subsequent analysis by pulsed gel electrophoresis were successful, as the number of chromosomes of all used yeast species of the genus Metschnikowia was determined.
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Microbial profile of model cheese
Lavičková, Ivana ; Veselá, Mária (referee) ; Vítová, Eva (advisor)
This Diploma thesis deals with the identification of microorganisms in samples of experimentally produced cheese. Model samples of cheese were produced in association with a private manufacturer. Raw organic milk was used to make the cheese. The theoretical part provides an overview of the issues such as the characteristics of the cheese, its properties and compounds. A special chapter is dedicated to molecular diagnostic methods, which serve for identification of microorganisms. In the experimental part of the thesis were identified some microorganisms in the samples. A polymerase chain reaction was used. The DNA was isolated from coarse lysates using phenol extraction; it was amplified using specific primers and demonstrated by gel electrophoresis. DNA of lactic acid bacteria (Lactobacillus spp., Lactococcus spp.) and yeast was ascertained in the cheese. The samples do not contain DNA of pathogenic genera Bacillus and Salmonella.
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Isolation, Identification and Characterisation of Microbial Communities of Wine and Selected Foods
Šuranská, Hana ; Španová, Alena (referee) ; Jarošová, Alžběta (referee) ; Omelková, Jiřina (advisor)
Proposed dissertation thesis deals with wine and artisanal cheeses microbiology. The first part is focused on identification of yeasts isolated from grapes and musts during production of white and red wines. The grape varieties were grown under the integrated and organic farming on Moravian vineyard. Yeasts were identified by ITS-PCR-RFLP method (amplifying internal transcribed spacer ITS: ITS1, ITS2 and 5.8S rDNA) and unknown species were subjected to partial sequencing of ITS rDNA region. In total, 524 isolates were divided into 14 different species belonging to six genus were identified from. The first stages of fermentation process were characterised by predominance of non-saccharomyces species especially H. uvarum. Due to increased ethanol concentration strains of S. cerevisiae prevailed in the later phases of the process. Further, partial aim of this study was to isolate and to apply selected autochthonous S. cerevisiae strains as starter culture during controlled industrial wine fermentation process. Genus Saccharomyces was distinguished from other non-saccharomyces species by ITS-PCR-RFLP. Further, in order to distinguish Saccharomyces genus at the species and the strain level, several molecular methods were applied including PCR-fingerprinting (rep- and RAPD-PCR), species-specific primers (multiplex and touchdown PCR), LSU-DGGE and interdelta PCR. Species-specific primers enabled us to distinguish some species of the Saccharomyces sensu stricto complex. Furthermore, interdelta PCR seems to be useful tool for S. cerevisiae strains identification. Among 120 isolated autochthonous strains belonging to Saccharomyces genus, 45 different strains were identified. Based on its sufficient technological properties (osmo- and ethanol tolerance, low H2S production etc.), S. cerevisiae 1-09 strain isolated from grape berries coming from moravian vineyard was chosen. Strain S. cerevisiae 1-09 was tested in small amount of must and after that also during industrial fermentation of red and white wine production. Based on the results of chemical and sensorial analysis, the strain seems to be suitable for application as the starter culture for winemaking process. The final part of this thesis is focused on quantification and identification of the yeasts isolated from artisanal cheeses and their by-products coming from Western Balkan Countries. Isolated species were identified by ITS-PCR-RFLP, partial sequencing and by physiological tests. Among the 20 yeast species found, D. hansenii, C. zeylanoides and Y. lipolytica were found to be predominant. Moreover, we developed culture-independent, semi-quantitative technique based on construction of ITS-clone library from metagenomic DNA to investigate complex fungal communities associated with artisanal cheeses and their by-products. Novel technique is based on direct extraction of total DNA from the sample. This was compared with culture-dependent ITS-PCR-RFLP and culture-independent LSU-DGGE methods. The results highlighted the discrepancies among these methods. Finally, the divergences among applied methods were confirmed by correlation analysis and by indices of general biodiversity and dominance of species. ITS-clone library approach combines the advantages of cultivation-based analysis and LSU-DGGE with semi-quantification of fungal species without the requirement of their cultivation. This study might open new perspectives in direct and complex analysis of yeasts and moulds in food matrices.
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Methods of molecular diagnostics of parasitic helminths
Dejmková, Tereza ; Leontovyč, Roman (advisor) ; Škorpíková, Lucie (referee)
Helminthiases represent a significant health risk, therefore the use of effective and rapid diagnostic methods is essential, including molecular methods, which are summarized in presented thesis. The biology of important human helminth pathogens is summarized in the introductory part. The DNA isolation, visualisation and molecular methods are described in the following parts. The section about molecular methods is divided into two parts. The first part is focused on PCR and its modifications, the second part describes methods of isothermal DNA amplification, which represent more practical and effective alternative to PCR. The advantages, disadvantages of all methods and examples of helminthiases to which they have been applied are also discussed. The final part is about the impact of the COVID-19 pandemic on the development of molecular diagnostic methods, during which were previously established protocols modified or new molecular methods were applied to increase the efficiency. The presented thesis brings an overview of molecular methods that were used to this day for diagnosis of helminthiases. At the same time the thesis discusses the impact of the COVID-19 pandemic on the development of molecular diagnostics and shows the possible future development of molecular diagnostic methods in the field of...
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Střevní paraziti ptáků na Svalbardu
ŽÁNOVÁ, Veronika
The study aimed to obtain essential knowledge about the distribution of intestinal parasites of birds occurring in Svalbard. Noninvasive coprological techniques were used to detect coccidian, cryptosporidian and microspordian parasites. The research included field collection of faecal samples and laboratory examination by microscopic and molecular methods.
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Variabilita druhu \kur{Calamagrostis varia}
NAKLÁDALOVÁ, Jana
Calamagrostis varia is a critically endangered species in Czechia. It has an interesting distribution range comprising the main area in calcareous parts of Alps and Carpathians and several isolated distribution areas on the northern edge of its range. The ecology of these sites is very heterogeneous. We therefore decided to explore this species' variability. In this study DNA ploidy level by FCM, morphometric characters and 2 genetic markers of 50 populations across the distribution range of C. varia were studied.
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Application of molecular methods in population genetic studies
Šurinová, Mária
A vast range of factors shape the genetic structure of plant populations. In this thesis, I focus on two of them. The first factor, polyploidization, is a process of chromosome set multiplication through whole-genome duplication within a single species (autopolyploids) or hybridization of two different species (allopolyploids). It rapidly brings changes into genomes, allowing species to occupy distinct niches, adapt to new habitats, colonize them, or adapt to changing environment in their native range. But it comes at a certain cost - difficulties in mitosis and meiosis, changes in cellular architecture. Furthermore, after cytotype establishment, new individuals have to deal with cytotype exclusion effect, competition with parental individuals and higher nutrition requirements. In this thesis, I present the effect of polyploidization on populations of three species- Arabidopsis arenosa, Aster amellus and Festuca rubra. The second factor changing the population genetic structure presented in this thesis is the fragmentation of populations. Population fragmentation can be caused by natural or anthropogenic activities and often leads to overall reduction in population size and reduced connectivity among fragments. Restricted gene flow may threaten long-term population survival due to inbreeding...
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Conspecific brood parasitism: a case study on the common pochard and the barn swallow
Petrželková, Adéla ; Hořák, David (advisor) ; Musil, Petr (referee) ; Guillemain, Matthieu (referee)
Presented thesis is mainly concerned with the conspecific brood parasitism (CBP) in birds. CBP is an alternative reproductive strategy when a parasitic female lay egg or eggs to other (host) female's nest of the same species. Then the parasitic female leaves the host's nest and does not provide any energetic investment in a subsequent parental care. Distinguish parasitic eggs or young is problematic because there are no obvious morphological differences. Thus, the use of molecular method is crucial for proper determination of CBP. CBP can be used as 'a best-of-bad-job' when female does not have her own nest or lost her nest for example through predation. Other option is that female can increase her fecundity ('fecundity enhancement' hypothesis) when she lay parasitic egg/eggs and care about her own clutch afterward. Two different model species were studied. The first one was a precocial diving duck - the common pochard (Aythya ferina; Anseriformes; Anatidae). Protein fingerprinting was used for detection of CBP and for distinguishing between eggs of individual females. It was found that the rate of CBP was relatively high in this species (91%, 72%; Chapter 1, 4). Further results indicated that the host clutch size decreased with the number of parasitic eggs in the clutch. The study of individual...
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