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GENE EXPRESSION AND IMMUNOLOGICAL RESPONSE IN MICE EXPOSED TO ZnO NANOPARTICLES
Rössner ml., Pavel ; Vrbová, Kristýna ; Strapáčová, S. ; Rössnerová, Andrea ; Ambrož, Antonín ; Brzicová, Táňa ; Líbalová, Helena ; Javorková, Eliška ; Zajícová, Alena ; Holáň, Vladimír ; Kulich, P. ; Večeřa, Zbyněk ; Mikuška, Pavel ; Coufalík, Pavel ; Křůmal, Kamil ; Čapka, Lukáš ; Dočekal, Bohumil ; Šerý, Omar ; Machala, M. ; Topinka, Jan
We analyzed gene expression changes in the lungs and the immunological response in splenocytes of mice exposed by inhalation of ZnO nanoparticles - NP. Adult female ICR mice were treated for three days and three months, respectively. Analysis of differential expression in genes involved in oxidative stress was conducted using quantitative RT-PCR. The potential immunotoxic and immunomodulatory effects of ZnO NP were analyzed by phenotyping and cytokine production by splenocytes after three months exposure. Three days exposure resulted in down-regulation of GCLC, GSR, HMOX-1, NQO-1, NF-kB2, PTGS2 and TXNRD1 mRNA expression, three months exposure increased the expression of these genes. Three months exposure caused a significant decrease in the percentage of granulocytes in the spleen cells, and affected the production of IL-10 and IL-6 by lipopolysaccharide-stimulated leukocytes. In summary, our study revealed changes in the expression of genes involved in the oxidative stress response following acute ZnO NP exposure. Subchronic ZnO NP exposure induced immunomodulatory effects in the spleen.
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Regulation of gene expression at posttranscriptional levels.
Kollárová, Johana ; Kostrouch, Zdeněk (advisor) ; Macůrková, Marie (referee) ; Jindra, Marek (referee)
Regulation of gene expression in response to cellular and organismal needs is essential for sustaining organisms' survival and successful competition in the evolution of life forms. This regulation is executed at multiple levels starting with regulation of gene transcription, followed by regulation at multiple posttranscriptional levels. In this thesis, I focused on posttranscriptional mechanisms that contribute to gene expression regulation in the model organism Caenorhabditis elegans which enables powerful genetic and genomic techniques and allows the visualization of experimental genetic manipulations in toto, on the level of the complete organism during its life span. For this, we analysed the function of the orthologue of mammalian transcriptional corepressor NCOR, GEI-8. We used a functionally defective mutant gei-8(ok1671). I analysed the whole genome expression of homozygous gei- 8(ok1671) mutant and its link with observed mutant phenotype that includes defective gonad development and sterility and performed experiments leading to the proposition that disbalances in 21-U RNAs of piRNA class present in the most derepressed gene, the predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16, are associated with the gonadal phenotype. In the second part of the thesis, I focused on...
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Molecular mechanisms of mutagenesis and resistence in CML cell lineages
Karasová, Dominika ; Čuřík, Nikola (advisor) ; Savvulidi Vargová, Karina (referee)
Chronic myeloid leukemia is a clonal haematopoietic disease, with characteristic BCR-ABL1 fusion gene. Despite the significant improvement in patient treated with tyrosine kinase inhibitors (TKI), 20-30 % of patients develop resistance. One of the main causes of treatment failure are mutations in the BCR-ABL1 kinase domain (KD). The aim of this work was to elucidate the molecular mechanisms of resistance and mutagenesis development in CML using an in vitro CML model KCL-22. The main part of this work was focused on the identification of genes involved in DNA damage response and repair, that could play a role in the process of mutagenesis of BCR-ABL1. We used the RT2 Profiler PCR Arrays method for the group of selected genes regulating DNA damage response and repair. We identified the genes XRCC6 and PARP1 whose gene expression was significantly and specifically decreased during KD BCR-ABL1 mutagenesis. Products of these genes are involved in repairing DNA double-strand breaks through non-homologous end joining (NHEJ). During study of the KD BCR-ABL1 mutagenesis we also found that clones, which developed mutations, did not show the increased BCR-ABL1 expression in the beginning of the culture compared to the clones in which mutations have not evolved. Key words: myeloid leukemia, mutation,...
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The inhibitors of tyrosine kinases and their effect on the expression of biotransformation enzymes
Dvořák, Josef ; Dračínská, Helena (advisor) ; Moserová, Michaela (referee)
Tumor diseases are one of the most common causes of death in the human population. One of the many possible causes of tumor growth is abnormal function of tyrosine kinases, which are involved in signal transfer and regulation of the most important cell processes. These processes include the control of cell growth, division and cell differentiation and apoptosis. For the therapy of tumor diseases caused by the abnormal function of tyrosine kinases, their specific inhibitors are developed. For the targeted treatment of thyroid tumors, the tyrosine kinases vandetanib and lenvatinib are newly used. In this bachelor thesis, the effect of vandetanib and lenvatinib on the gene expression of the cytochrome P450 family of 2 (CYP2A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1) biotransformation enzymes has been investigated as they are involved in the biotransformation reactions of a large portion of the available drugs on the market. To examine the relative gene expression of CYP2, quantitative PCR of samples of cDNA, synthesized from isolated RNA from rat liver and kidney exposed to the above-mentioned tyrosine kinase inhibitors, was used. The results suggest, that vandetanib and lenvatinib do not have a significant effect on the gene expression of cytochrome P450 family 2 in rat liver and kidney tissue. KEY WORDS:...
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Function of nuclear phosphoinositides and their binding partners in gene expression
Uličná, Lívia ; Hozák, Pavel (advisor) ; Šolc, Petr (referee) ; Macůrková, Marie (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...
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The importance and role of reverse transcriptases in gene expression analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Španielová, Hana (referee)
The continuously advancing field of gene expression analysis enables the evaluation of even the slightest changes that occur in the cell transcriptome. In order to ensure accuracy of the observed biological variances, it is fundamentally important to be aware of the possible biases introduced during sample processing. In gene expression research, the methods of reverse transcription−quantitative PCR (RT−qPCR) and RNA- Sequencing (RNA-Seq) are often the primary choice, mostly because of their high precision and reproducibility. Since these both methods require DNA template, they are coupled with the same initial step - reverse transcription (RT), a reaction producing DNA complementary to its RNA template. It is well known that RT introduces bias. As a result, it is therefore of importance to thoroughly evaluate the effects of these biases. One such annotated source of artifacts is the reverse transcriptase (RTase) itself. However, it has been shown that the enzyme does not account for most of the variance alone. Surprisingly, choice of primers or RNA template may influence the reaction outcome even more than the bias introduced from the enzyme. This is especially the case with recent advances in protein engineering. Production of highly efficient RTases may pronounce the variation originating from...
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Eukaryotic translation initiation factor 3 and its role in plant translation regulation
Raabe, Karel ; Michailidis, Christos (advisor) ; Retzer, Katarzyna (referee)
After transcription, mRNA translation is another highly regulated process in gene expression. In plants, translation regulation plays an important role during progamic phase, fertilization and seed development, where synthesized transcripts are stored and selectively translated later in development. Translation regulation is also broadly used in stress responses as a fast and flexible tool to change gene expression; therefore, it plays an essential role in the survival strategy of sessile organisms like plants. Both regulation of the global translational rate as well as selective regulation of specific transcripts modulate the final gene expression response. Most of the regulatory mechanisms are concentrated in the stage of initiation, which is facilitated by several translation initiation factors. Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these factors, consisting of 12 conserved subunits. Its key function in the initiation is to scaffold the formation of the translation initiation complex and in the scanning mechanism accuracy. In past decades, additional eIF3 functions were discovered acting upon the whole translation cycle, including its importance in global and specific translation regulation. The aim of this work is to review eIF3 functions and to...
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Study of dysregulation of DLX1 protein in myeloid leukemia cells in in vitro and in vivo models
Jelínková, Alena ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
The heterogeneous nature of acute myeloid leukemia (AML) worsens the results of patients treated with standard therapy. Understanding the processes of leukemogenesis can contribute to identification of more appropriate treatment. Family of DLX genes (Distal-less homeobox), belonging to the homeobox genes, are associated with haematological malignancies and solid tumors. In the analysis of expression data, the low level of the DLX1 gene was associated with a worse prognosis of patients with AML. In this work we studied phenotypic changes of cell lines with different expression of the DLX1 gene. We silenced the DLX1 gene in AML cell line (sh cells) and compared it to the parental line with higher expression of DLX1 (NSC cells). By cell cycle analysis and apoptosis assays in vitro and in vivo, we have observed the arrest of sh cells in the G0 phase and a lower number of apoptotic cells. Differences were found when measuring the absolute number of cells in time. In in vitro conditions there were less sh cells, in in vivo environment there was significantly higher number of sh cells engrafted in comparison to NSC cells. Further results have shown that sh cells have lower levels of pro-apoptotic proteins and exhibit a higher level of TGF-β targeting PAI-1 gene that activates replicative senescence. We...
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Physiological basis of extended longevity in termite kings and queens - is activation of telomerase mechanism linked with extended longevity of termites?
Pangrácová, Marie ; Hanus, Robert (advisor) ; Kodrík, Dalibor (referee)
- Extended longevity and high fecundity are two phenomena typical for reproductive castes (queens, eventually kings) of eusocial insects. In my thesis, we explore the hypothesis that the longevity of reproductives in the termite Prorhinotermes simplex is linked with the activation of the telomerase enzyme complex. Telomerase is well known for its life-extension functions, due especially to its capacity to prolong the telomeric ends of chromosomes. Therefore, we studied here the gene expression of: (1) the gene TERT coding for the catalytic subunit of the telomerase and (2) the genes of the main endocrine regulatory pathways, known to be responsible for the control of reproduction and longevity in insects. Expression dynamics of these genes were measured in sterile and reproductive castes of P. simplex during their development and sexual maturation. Based on our results obtained from the TERT expression analyses and their comparison with telomerase enzyme activities, we assume that the telomerase action in the long-lived reproductive individuals is regulated at a post-transcription level. Furthermore, we observed in reproductive castes a simultaneous upregulation of some transcription variants of vitellogenin and the genes for insulin signalling pathways. We can, therefore, conclude that in...
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