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Biosynthesis of terpenoid secondary metabolites in termites
Jungwirth, Daniel ; Heidingsfeld, Olga (advisor) ; Buček, Aleš (referee)
Terpenoids are natural compounds, which can be found in the form of tens of thousands diverse molecules in all forms of life. Terpenoids are mostly represented in plants, microorganisms,and animals. It should not be a surprise that terpenoids are often found in insects, the most species-rich class of Metazoa. In insects terpenoids are mostly used for communication as pheromones and for chemical defence. In comparison to widespread knowledge of terpene (and terpenoid) biosynthesis in plants we lack thorough knowledge in insects. Studies conducted in past few years showedthat the ability to synthesize terpenoids has occurred independently and repeatedly in various lineages of insects. It was also found that terpene synthase genes evolved via duplication and neofunctionalization of specific genes, which were responsible for isoprenyldiphospate synthesis. Despite these discoveries the terpenoid synthesis in insects is mostly unknown and remains to be elucidated. This thesis aims to functionally characterize three candidate terpene synthases from the termite Nasutitermes takasagoensis. Expression of selected proteins was done in two bacterial systems. For the purification of recombinant proteins immobilized metal ion affinity chromatography was used. Detection of products from assays with purified...
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Vacuolar aspartic protease of Candida albicans
Hirko, Dominik ; Heidingsfeld, Olga (advisor) ; Dostál, Jiří (referee)
Aspartic proteases (APs) are crucial for diverse cellular processes. This thesis delves into the complexities of protein expression and characterization of vacuolar aspartic endoprotease Apr1p from Candida albicans, comparing it to its Saccharomyces cerevisiae ortholog, Pep4p. Recombinant expression of Apr1p in Escherichia coli yielded the inactive proenzyme, proApr1p. Extensive refolding efforts failed to produce mature, active Apr1p, suggesting a reliance on intricate cellular machinery or specific post-translational modifications for activation. Attempts to leverage vacuolar enzymes or cell lysates for proApr1p activation were unsuccessful, potentially due to the fragility of isolated vacuoles and the complex mixture of enzymes in cell lysates. Positive results emerged when Apr1p was expressed in S. cerevisiae, where fractionated cell lysates exhibited specific proteolytic activity at acidic pH after inhibiting serine and metalloproteases proteases. The eukaryotic system can probably produce active Apr1p. However, after preliminary small-scale experiments, upscaling of Apr1p expression in S. cerevisiae will be necessary in order to obtain sufficient amount of protein for further characterization. A reciprocal gene swap experiment, exchanging PEP4 in S. cerevisiae with APR1 and vice versa,...
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Study of the binding interaction of tyrosine kinase inhibitors with serum albumin
Rodová, Marie ; Indra, Radek (advisor) ; Heidingsfeld, Olga (referee)
Sunitinib and vandetanib are anti-cancer medications prescribed for medullary thyroid cancer (in the case of vandetanib) and for renal cell carcinoma, gastrointestinal stromal tumor, and pancreatic cancer (in the case of sunitinib). They belong to the group of tyrosine kinase inhibitors and act by exhibiting anti-angiogenic effects and by inhibiting tumor cell proliferation and survival through VEGFR. Additionally, vandetanib also inhibits tumor cell survival via EGFR and RET. In the presented thesis, we investigated the binding interaction between serum albumin and the TKIs vandetanib and sunitinib using BSA, HSA, and blood plasma. We examined the differences in interaction between the TKIs and various serum albumins, including pure BSA, pure HSA, and blood plasma, as well as the nature and location of the binding interaction. Additionally, we studied the influence of other ligands on this interaction and the photosensitivity of sunitinib itself. Utilizing spectroscopic techniques, including UV-VIS absorption and fluorescence quenching, we have determined the Stern-Volmer and binding constants, as well as the thermodynamic parameters, for the binding interactions of sunitinib and vandetanib with BSA and HSA. Our results indicate that complex formation occurs between BSA and sunitinib, BSA and...
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Phosphorylation of eukaryotic initiation factor 2α by alternative phosphate sources catalyzed by heme-regulated kinase: kinetic analysis
Ovad, Tomáš ; Martínková, Markéta (advisor) ; Heidingsfeld, Olga (referee)
master thesis Ing. Bc. Tomáš Ovad Phosphorylation of eukaryotic initiation factor 2α by alternative phosphate sour- ces catalyzed by heme-regulated kinase: kinetic analysis This master thesis focuses on the phosphorylation of the eukaryotic initiation factor 2α (eIF2α) by the heme-regulated inhibitor (HRI), a key reaction in the regulation of eukary- otic protein synthesis. Kinetic parameters of this reaction in the presence of ATP as the phosphate donor were determined previously. However, there are no reports on the potential of alternative NTPs (GTP, UTP, CTP) to serve as the sources of phosphate for this reaction. In this thesis, the wild-type HRI enzyme was produced by heterologous expression in E. coli and its kinase activity was assayed in the presence of ATP, GTP, UTP, and CTP. Kinetic parameters and heme half-maximal inhibitory concentrations for the HRI kinase reaction with the use of each NTP as a phosphate donor were determined. To validate these kinetic experiments, contaminations by ATP in the solutions of GTP, UTP, and CTP were excluded with the aid of ion-pair reversed-phase high-performance liquid chromatography. It has been shown that ATP is the most efficient phosphate donor for the HRI kinase reaction, although the remaining NTPs (GTP, UTP, CTP) may be utilized as the sources...
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Antagonistic regulation by global transcription factors Tup1p, and Cyc8p of Flo11 and Flo11 -dependent phenotypes in wild yeast
Nguyen Van, Phu ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee) ; Malcová, Ivana (referee)
Biofilms are a common mode of yeast growth in which cells adhere to each other and adhere to abiotic surfaces to form complex multicellular structures. Living together in biofilms provides cells with several beneficial features compared to planktonic cells. Undoubtedly, protection and resistance are advantages of life inside colony biofilms. Biofilms are found in many environments and play many important roles in commercial industries. However, biofilms can also be extremely dangerous in clinical settings. There is thus great interest in studying biofilms and how to eliminate them. In this study, we used wild yeast Saccharomyces cerevisiae colony biofilm as an ideal system to investigate potential functions of the yeast Cyc8-Tup1 transcriptional corepressor complex in the regulation of yeast adhesion, and biofilm formation on agar and at solid-liquid interfaces. Unexpectedly, we have found that Cyc8p and Tup1p antagonistically control the formation of structured biofilm colonies on agar and FLO11 expression. Cyc8p itself acts as a key repressor of FLO11, whereas Tup1p promotes the formation of biofilm colonies and induces FLO11 expression by inhibiting the repressive function of Cyc8p and preventing Flo11p degradation possibly by inhibiting an extracellular protease. In addition, other features...
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Production and characterisation of human C1 inhibitor and Plasmodium falciparum PfMSP3.1 recombinant proteins for structural studies
Čápová, Kateřina ; Konvalinka, Jan (advisor) ; Heidingsfeld, Olga (referee)
PfMSP3.1 is one of the surface proteins of the intracellular parasite Plasmodium falciparum, which causes malaria. As one of the evasion strategies of the immunity system of the human host this protein interacts with one regulator of the complement system - C1 inhibitor. Determining the exact binding site and its structural assessment would help to better understand the interaction between the parasite and the host, which is necessary for the disease progression and thus for the development of a potential therapy. In the theoretical part of the thesis, the life cycle of Plasmodium falciparum, the role of the parasite stage called merozoite, the role of its surface proteins, including merozoite surface protein 3, in the attack of red blood cells by the parasite, are described in more detail. It also briefly describes the complement system, its activation pathways and the regulation of these pathways. The experimental part includes the cloning of plasmids to produce C1 inhibitor and various forms of merozoite surface protein PfMSP3.1, transfection of S2 insect cells with these plasmids, subsequent protein expression in S2 cells and their purification. In the second half of the experimental part, we tried to create complexes of C1 inhibitor with individual PfMSP3.1 forms and an attempt to crystallize...
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Chicken antibodies against the pathogenic yeast Candida albicans
Konečná, Lucia ; Heidingsfeld, Olga (advisor) ; Kubíčková, Božena (referee)
5 Abstract Candida albicans belongs to the most widespread pathogenic yeasts. As an opportunistic pathogen, it mainly colonises the mucous membranes of the host and causes infection, especially in individuals with a compromised immune system. The most common infection caused by C. albicans is vaginal mycosis, which at least 3 out of 4 women will experience during their lifetime. Antifungal drugs are used to treat this condition, but the yeasts are becoming less susceptible. For this reason, there is an effort to develop new treatment options for (not only) infections caused by C. albicans. Although vaginal mycosis is not a severe condition in most cases, it is a widespread infection that occurs repeatedly in many women, sometimes several times a year. Since vaginal mycosis reduces the quality of life in many women, we decided to focus on this disease in this paper. This project used hen antibodies, which have a number of advantages over mammalian antibodies. In oviparous animals, antibodies are not only found in the blood serum, as seen in mammals but are also secreted into their eggs. This makes the isolation of hen antibodies non-invasive and ethical. Moreover, the methods of isolating antibodies from eggs are also simpler than isolation from blood serum. Ultimately, hen antibodies are also more...
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Development od methods for analysis of interactions of the antiviral drug ritonavir with lanosterol-14- α-demethylase of pathogenic yeasts
Burdová, Tereza ; Heidingsfeld, Olga (advisor) ; Dostál, Jiří (referee)
Ritonavir is an HIV protease inhibitor, and has been used as a part of drug cocktails for highly active antiretroviral therapy (HAART). Oropharyngeal candidiasis is one of the most common opportunistic infections among AIDS patients. The research showed that those who were treated with cocktails containing HIV protease inhibitors including ritonavir suffered from oropharyngeal candidiasis to a much lesser extent than patients who received drugs without protease inhibitors. The reason may be the fact that HIV protease inhibitors also inhibit the proteases of pathogenic yeasts, despite high Ki. Another explanation may be that some of these inhibitors block lanosterol-14-α- demethylase, a key enzyme in the biosynthesis of ergosterol, which is an important component of yeast membranes. Aim of the thesis is the isolation of the microsomal fraction of the pathogenic yeasts Candida albicans and Candida parapsilosis and optimization of methods for analysis of the inhibition of yeast lanosterol-14-α- demethylase by ritonavir. Key words: ritonavir, Candida albicans, Candida parapsilosis, lanosterol-14-α- demethylase, microsomal fraction
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Trk1 Potassium Importers - key transport systems for yeast cell fitness and stress tolerance
Masaryk, Jakub ; Sychrová, Hana (advisor) ; Heidingsfeld, Olga (referee) ; Malínský, Jan (referee)
One of the key prerequisites for yeast cell growth is the uptake of essential compounds, such as potassium. Potassium is a vital monovalent cation and its sufficient intracellular concentration is crucial for various processes, for instance: regulation of membrane potential and cell turgor, enzymatic activity, and protein synthesis. A sufficient internal concentration of potassium is also one of the pivotal signals for cell division. However, as also excess of potassium might lead to unfavourable physiological consequences in yeast, such as deacidification of vacuoles and depolarization of plasma membrane, it is imperative for the yeast cells that the whole process of potassium acquisition is a tightly regulated affair, in order to maintain proper potassium homeostasis. In yeast Saccharomyces cerevisiae, uniporter Trk1 is considered a key player in potassium uptake. The presented thesis aimed to provide novel knowledge regarding Trk1, more specifically to study its ability to modify its capacity for potassium uptake, putative regulation by phosphorylation, and involvement in the survival of glucose-induced cell death (GICD). Additionally, potassium-uptake systems in selected non-conventional species were characterized as well. The most distinctive feature of Trk1 is its alleged ability to switch...
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Development and differentiation of different types of yeast colonies: Regulation of metabolic diversification and development of cells with novel properties
Maršíková, Jana ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee) ; Demnerová, Kateřina (referee)
Yeasts are unicellular organisms, but on a solid substrate they are capable of forming complex organized structures that behave like primitive multicellular organisms. Examples of these structures include colonies and biofilms, whose cells interact with each other, coordinate their growth and development, differentiate spatially and form specialized cell subpopulations in which specific processes and regulatory pathways occur. The basis of cellular differentiation and specialization is the formation of gradients of nutrients, metabolites and signaling molecules. Thus, multicellular yeast communities differ significantly from planktonic populations in their characteristics. The aim of this work is to increase knowledge related to the development and differentiation of both smooth and structured colonies of the yeast Saccharomyces cerevisiae. The literature introduction of the thesis provides an overview of the current knowledge on the development of yeast colonies and biofilms, especially of S. cerevisiae species, and also includes selected regulations important for the formation of multicellular populations. The thesis provides insights into the antagonistic function of the transcriptional regulators Cyc8p and Tup1p in the development of structured biofilm colonies. Genome-wide transcriptomic...
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