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The role of alternative splicing in plants
Földi, Marek ; Klodová, Božena (advisor) ; Fischer, Lukáš (referee)
Alternative splicing is a mechanism of gene expression regulation that maintains, regulates, and creates genomic diversity and tissue specificity in plants. It involves the differential joining of exons in precursor mRNAs, leading to multiple mRNA isoforms from a single gene. The formation of these isoform variants and their subsequent translation leads to subfunctionalization of proteins, generating diversity in structure and function. Therefore, alternative splicing is often important in various biological processes in plants, such as development, stress response, immunity, and reproduction. Key types of alternative splicing events include intron retention, exon skipping, alternative 5'/3' splice sites, and mutually exclusive exons. Regulation of alternative splicing involves cis-regulatory elements and trans- acting protein factors such as serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). This thesis aims to summarise the mechanisms and consequences of alternative splicing in plant development, including maturation of male and female gametophytes, meiosis, stress, and cell differentiation. It also describes methodological approaches that allow for a genome-wide study of alternative splicing, including microarrays, RNA-seq, and PCR. A better understanding of...
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Hif1a role in cardiovascular function and heart disease
Matějková, Kateřina ; Pavlínková, Gabriela (advisor) ; Holzerová, Kristýna (referee)
Hypoxia inducible factor 1 alpha (HIF1A) is a transcriptional factor, which plays a central role in the maintenance of homeostasis under hypoxic conditions. It regulates a wide variety of genes encoding proteins that influence metabolism, extracellular matrix composition, oxidoreductase activity or angiogenesis in response to reduced oxygen levels. When HIF1A protein function is impaired, the organism is unable respond appropriately to hypoxia. Altered HIF1A regulation can result in severe tissue damage and eventually lead to death. The heart, as an organ with a huge oxygen consumption, is susceptible to various pathologies caused by hypoxic stress. The role of HIF1A in the heart is rather ambiguous and remains to be elucidated. It plays a role in cardioprotective mechanisms but also promotes the development of inflammation and apoptosis. This thesis aims to clarify the role of HIF1A in maintaining physiological functions of the heart during adaptation to hypoxic conditions using a mouse model with heterozygous Hif1a+/- deletion. Experiments involving molecular and cell biology methods performed on left ventricular tissue were preceded by bioinformatic analysis of data obtained by RNA sequencing of isolated cardiomyocytes. RNA sequencing data were analyzed using the R scripting language (packages...
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Research of epigenetic aspects of hematopoietic and spermatogenesis stem cells.
Hybešová, Michaela ; Pimková, Kristýna (advisor) ; Děd, Lukáš (referee)
Stem cell differentiation is controlled by coordinated regulation of gene transcription. One of the regulatory factors is the loosening of chromatin and the accessibility of DNA to transcription factors. Chromatin remodeling is mediated by remodeling complexes. The ISWI chromatin remodeling ATPase Smarca5 (S5) is an important factor of remodeling complexes. It is a highly conserved chromatin-remodeling factor forming a catalytic subunit that can be found in several oligosubunit complexes. In these complexes, it actively regulates nucleosome structure and remodeling during DNA replication, repair and transcription. S5 has been identified as a key protein in embryonic development. Its deficiency leads to defects in hematopoiesis and male genital development. In the presented study, we focused on the role of S5 in hematopoiesis and spermatogenesis. Using a mouse model with transgenic expression of S5, co-immunoprecipitation and mass spectrometry, we identified S5 complexes in hematopoietic and testicular cells. We also studied the phenotypic consequences of S5 deficiency in mouse testes and found that it leads to impaired sperm development and male sterility. Using transcriptomic and proteomic analysis, we identified several molecular programs that could lead to reproductive disorders. Our work...
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Hematopoietic stem and progenitor cell defects in transgenic model of Diamond-Blackfan anemia
Holečková, Markéta ; Kokavec, Juraj (advisor) ; Valášek, Leoš (referee)
Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by deficient development of erythroid progenitors and accompanied by a variable set of developmental defects. About 25 % of patients have mutations of the small ribosomal subunit protein RPS19, and the precise mechanism of single aminoacidic mutations of RPS19 protein in the pathology of Diamond-Blackfan anemia remains largely unknown. To understand the interaction between of genotype and phenotypic variability we have created a mouse model with homozygous mutation in a highly conserved arginine 67 (Rps19R67Δ/R67Δ ). Mouse model with this mutation display many of the same phenotypical trades as patients with DBA. We decided to focus on hematopoiesis and erythropoiesis in this mouse model and tried to characterize those processes. We discovered that Rps19R67Δ/R67Δ mice similarly to DBA patients suffer from anemia and that the erythropoiesis process is disrupted at the stage of proerythroblasts. We also observed changes in hematopoiesis in stages as early as multipotent progenitors. The role of p53 protein as a modifier of DBA phenotype is well known. We created mouse model with p53 depletion to assess the role of p53 protein in relation with mutation in Rps19. Rps19R67Δ/R67Δ Trp53-/- mice show no signs of...
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Study of genome and transcriptome variability employing data processing from massive parallel DNA sequencing.
Vojta, Petr ; Hajdúch, Marián (advisor) ; Budinská, Eva (referee) ; Mokrejš, Martin (referee)
Massive parallel sequencing (MPS) data analysis tasks are often computationally demanding and their execution time would take too long using standard computing machines. Thus there is a need for parallelization of this tasks and ability to execute them on a sufficiently powerful computing machines. In the first chapter we describe a newly created platform for resequencing analysis of MPS data - MOLDIMED and novel annotation tool, which is ready to deploy on HPC infrastructure. The second chapter describes MPS approaches in Diamond-Blackfan anaemia (DBA), which is predominantly underlined by mutations in genes encoding ribosomal proteins (RP); however, its etiology remains unexplained in approximately 25% of patients. We performed panel sequencing of all ribosomal genes in DBA patient without previously known molecular pathology. A novel heterozygous RPS7 mutation coding RPS7 p.V134F was found in one female patient and subsequently confirmed in two asymptomatic family members, in whom mild anemia were detected on further examination. Subsequently, we performed whole transcriptome analysis in all family members and patient with RPS7 mutation in comparison with healthy control group and with DBA patients with known mutation in RPS19. We observed dysregulation mainly in signal pathways of translation,...
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