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Studying of Gene Expression Involved in Hyaluronic Acid Synthesis in Streptococcus Equi Subsp. Zooepidemicus Using DNA Microarrays and Real-Time PCR
Hrudíková, Radka ; Šeda,, Ondřej (referee) ; Bobek,, Jan (referee) ; Velebný, Vladimír (advisor)
Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246.
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Gene regulatory network inference based on mutual information in non-model organisms
Pirkl, Petr ; Sedlář, Karel (referee) ; Musilová, Jana (advisor)
The thesis is focused on summary of laboratory methods for determining gene expression, data preprocessing procedures and possible tools used to infere gene regulatory networks. Furthermore, the thesis handles with the pre-processing of data. It means create count table and normalize it. It was use data from the non-model organism Clostridium beijerinckii NRRL B-598. The main parts of the thesis are designed an algorithm for the creation of a gene regulatory network using mutual information and its implementation in the R language. This include testing the algorithm on data from the non-model organism and the gold standard.
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Long term sublethal stress in colonies of Saccharomyces cerevisiae deleted in WHI3
Krampotová, Ester ; Schierová, Michaela (advisor) ; Dušková, Michala (referee)
This diploma thesis is devoted to the topic of the development of natural non-pathogenic yeast colonies in non-laboratory diploid strain of Saccharomyces cerevisae with deletion WHI3, during long-term mild stress. The absence of Whi3 causes significantly higher senzitivity to medium composition relative to the parental strain, thus whi3Δ /whi3Δ colonies are a good model for study of the virulence induction due to environmental stress in pathogenic yeasts. Deletion of WHI3 in the BR-F strain results in a significant suppression of the ability to form structured colonies caused mainly by the reduced level of Flo11. The absence of Whi3 also has a negative effect on the expression of other genes involved in the stress response. The aim of the work is to determine whether changes in the expression of genes encoding stress proteins induced by sublethal doses of the inhibitor are dependent on Whi3, Yap6 and Mpt5 proteins. To induce stress in yeast, we used NaCl or CdSO4. The YAP6 and MPT5 genes encode regulatory proteins involved in the stress response, which expression is under control of Whi3. In whi3Δ /whi3Δ , the level of Hsp26, Tsa1, Pab1, and Gre2 was lower than in the parental strain. The Yap6 protein affects the response to the presence of Cd2+ in the medium, although according to SGD, only its...
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Účinky vybraných peptidů na jednobuněčnou řasu Chlamydomonas reinhardtii
Avdeeva, Olga
In the presented work, the modified novicidin peptides NVCAA and NVCHH were applied to the unicellular alga Chlamydomonas reinhardtii. The aim of the study was to determine the minimum inhibitory concentrations of the tested peptides on Escherichia coli bacteria with subsequent application on the unicellular algae Chlamydomonas reinhardtii. The assessment of the effect of the tested peptides on the algae was carried out using spectrophotometric analyses: chlorophyll A and chlorophyll B content, total flavonoid content, total antioxidant capacity and using the DPPH ASSAY method. Chlamydomonas reinhardtii molecular changes were determined by monitoring the amount of isolated RNA and the expression of APX1, APX2, CAT1, CAT2 genes using the Real-time PCR method. Statistically processed data showed that the application of NVCAA and NVCHH peptides induced considerable stress in the cilia.
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Potenciální vliv tkáňově specifických transkripčních faktorů na expresi metalothioneinů
Michálková, Nikola
Metallothioneins represent a group of intracellular proteins capable of binding metal ions, and their function includes the regulation of cellular levels of these metal ions. Transcription factors are central regulators of gene expression and play an important role in regulating the expression of metallothionein genes, which represents the basic mechanism for the cellular response to changes in metal ion levels. Although transcription factors that bind to the promoter regions of metallothioneins are known, their role in inducing the expression of metallothionein genes is far from clear. The subject of this thesis was the induction of gene expression of metallothioneins by zinc ions in HUH7 (hepatocellular carcinoma) and T47D (breast cancer) cell lines, and the subsequent analysis of gene expression of selected transcription factors. The selection of transcription factors was based on TF ChIP-seq analysis of ENCODE database. For the experimental part, the transcription factors FOXA1, FOXA2, HNF4A, and HNF4G were chosen, whose high levels are typical for liver tissue. Furthermore, gene expression of the ubiquitinated transcription factors JUND, NR3C1, RXRA, STAT3, SP1, and MTF1 was monitored. Statistically significant changes in gene expression (RT-qPCR) after short-term exposure (4-6 h) to 100 μM ZnSO4 were observed for FOXA1, JUND, and MTF1. In the case of FOXA1, a decrease in gene expression was observed in both tumor lines compared to the control, whereas an increase in expression was observed in JUND and MTF1 after ZnSO4 treatment. In the case of MTF1, a significant increase was observed only in the HUH7 tumor line. The results show that, in addition to the validated zinc-sensitive transcription factor MTF1, the transcription factors FOXA1 and JUND could also be involved in the regulation of metallothionein gene expression. FOXA1 could represent a potential repressor and inducer of JUND metallothionein expression in response to increased exposure to zinc ions.
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Effect of abacavir on the expression of nucleoside transporters, adenosine receptors, and enzymes involved in adenosine synthesis and biodegradation in trophoblasts
Gala, Viktor ; Červený, Lukáš (advisor) ; Čečková, Martina (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Viktor Gala Supervisor: doc. PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Effect of abacavir on the expression of nucleoside transporters, adenosine receptors, and enzymes involved in adenosine synthesis and biodegradation in trophoblasts The nucleoside reverse transcriptase inhibitor (NRTI) abacavir (ABC) is now the mainstay of combination antiretroviral therapy (cART) for HIV in pregnant women. The introduction of cART, along with several other measures, has reduced mother-to-fetus transmission of HIV to less than 1% in recent years. The placenta is a key organ for the health of both the fetus and the mother. Imbalances in placental development can result in adaptive changes and fetal programming errors. cART recommended in pregnancy is known for its good safety profile, but some epidemiological studies suggest a higher risk of reduced fetal weight, preterm birth, etc. The placenta is a rapidly growing organ dependent on the supply of building materials that resembles tumor growth in certain aspects. Nucleosides are promoters of tumor proliferation and are involved in the development of immunotolerance. The placenta is complexly equipped for nucleoside synthesis, uptake,...
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Study of the expression and toxicity of catechol derivates in MCF-7 cell line
Kleplová, Dominika ; Carazo Fernández, Alejandro (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Dominika Kleplová Supervisor: PharmDr. Alejandro Carazo, PhD. Title of the Diploma thesis: Study of the expression and toxicity of catechol derivatives in MCF-7 cell line Estrogens perform many important functions in the human body. They act by binding to estrogen receptors (ER) and thus regulate reproduction, the menstrual cycle, bone density, cholesterol metabolism or brain function. They also play an important role in the development and onset of breast cancer, where the amount of ER expressed is used as a very important biomarker in patients suffering from this disease. Despite ongoing research, breast cancer is considered to have the highest mortality rate. Catechols are organic compounds. In the human body, they can occur as metabolites in the degradation of benzene and estrogens or other endogenous compounds such as neurotransmitters and their precursors. Catechols are known to be involved in redox processes in the body, to exert antioxidant and toxic effects, to interfere with protein function and to cause DNA strand breaks. Their positive effect on breast cancer therapy is the subject of research, but it has not yet been fully explored. The subject of this thesis is to study the...
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Effects of peripheral inflammation on gene expression modulation in passerines and parrots
Kuttiyarthu Veetil, Nithya ; Vinkler, Michal (advisor) ; Hyršl, Pavel (referee) ; Harazim, Markéta (referee)
(English) Birds have well-defined roles in maintaining the ecological balance as predators, seed dispersers, nutrient cyclers, and pollinators making them an integral part of many ecosystems. Birds are often the flag-ship species and are important for wildlife preservation. Some of the avian populations are very well connected across the globe through their annual migration, increasing risks of epidemics of infections. Birds also face different levels of existence encounters in challenging living conditions like deserts and cold mountains. To cope with these diverse environments not only need physiological adaptations, but also a very well-equipped immune system, optimised to challenges common to the environment they inhabit. How well a host immune system responds to pathogens determines the overall fitness of the organism and its survival. Insight into the avian immune system functions is of great significance as birds are reservoirs of innumerable pathogens. They have been the primary source of several major epidemics' onset leading to worldwide human and animal fatalities (e.g., COVID-19, Avian influenza, or West Nile virus outbreaks). Similar to all living beings, avian hosts and pathogens are always in a continuous adaptational arms race. This coevolution of hosts and their pathogens forms the...
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Functional analysis of the ERK signaling pathway in epithelial cells
Galvánková, Kristína ; Vomastek, Tomáš (advisor) ; Rösel, Daniel (referee)
The MAPK/ERK pathway, which is evolutionarily conserved in eukaryotes is one of the most intensively studied signaling pathways and consists of a three-tier cascade of Raf- MEK-ERK protein kinases. A variety of extracellular signals are transduced from receptors to hundreds of substrates by a series of sequential phosphorylations leading from Raf to MEK to ERK. The ERK pathway regulates a plethora of cell- and extracellular signal- specific responses such as gene expression, proliferation, differentiation, migration, and apoptosis. The proper execution of these physiological processes requires a precise temporal and spatial regulation of the pathway and disruption of the regulatory mechanisms leads to pathological consequence such as tumor transformation. Specificity and regulation of signal transduction are provided in part by the presence of isoforms at each level of the ERK signaling pathway. The functional differences between the effector protein kinases ERK1 and ERK2 have been controversial for a long time, but it is still unclear how important they are in achieving an appropriate cellular response. In this work, we focused on the functional characterization of ERK1 and ERK2 isoforms in MDCK epithelial cells. Specifically, we examined the effects of ERK2 inactivation on cell morphology and...
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Gene expression in chicken embryo: micromanipulation and visualization methods
Bendová, Michaela
The aim of this work was to obtain better insight into the principles of cell structures and organs in the chicken embryo development. To reach this goal special methods of micromanipulations and visualization in vitro, ex vivo, ex ovo and in ovo were implemented and adjusted. These methods were used to study gene expression in neural crest development and eye development. In the course of long term research in our laboratory we observed that oncoprotein v-Myb influences the development of the neural crest and has the capacity to change natural cell fate. We performed a series of experiments to investigate v-Myb protein influence on neural crest cells differentiation, especially melanocyte lineage development, and its influence on gene expression in the neural crest. Therefore we focused on Gremlin 2 (PRDC), the gene upregulated by v-Myb in the neural crest. The established procedure of electroporation in ovo was adjusted to transfect cells of the developing eye and used to study gene expression during lens induction. The results obtained from chicken embryo experiments endorsed the study performed on mouse embryos. Futhermore, the electroporation technique was slightly modified for manipulations of the neural retina in the developing eye in ovo. Thereafter, the retinas were processed ex vivo and...
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