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Identification of new tissue-specific interaction partners of chromatin remodelling ATPase Smarca5
Arishaka, Yuliia ; Kokavec, Juraj (advisor) ; Děd, Lukáš (referee)
The regulation of chromatin structure is fundamental to a wide range of cellular processes, including transcriptional regulation, cell division, differentiation and DNA damage repair, and ATP-dependent chromatin remodeling complexes have been established as essential components of this regulatory network. Smarca5, as an ATPase/Helicase enzyme, has been shown to regulate chromatin structure by interacting with bromodomain and DDT-WHIM domain-containing partners to control the binding of chromatin-associated proteins and transcription factors to their specific DNA target sequences. In this work we identify a previously undescribed protein with a conserved N-terminal bromodomain and ISWI protein binding DDT-WHIM domain through co-immunoprecipitation and mass spectrometry in mammalian cell lines and establish it as a novel interaction partner of chromatin remodeling ATPase Smarca5. Furthermore, we have pinpointed the region required for Smarca5 interaction that corresponds to DDT-WHIM domain. We have furthermore attempted to identify additional interaction partners which may hint on the potential function of this novel chromatin complex and validated its expression in embryonic and postnatal tissues. This discovery represents a unique opportunity for further investigation into its potential function in...
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Effect of estrogens on in vitro models of testicular tissue and spermatogenesis
Jursová, Pavlína ; Děd, Lukáš (advisor) ; Tlapáková, Tereza (referee)
Although estrogens are primarily known for their functions in female reproductive system, their effect on male reproductive functions has also been well established. Physiological estrogen concentration is essential for a proper spermatogenesis. Estrogens regulate many functions in testicular tissue, including proliferation and apoptosis of all testicular cell types, dynamic restructuring of cell-cell junctions in the testis, and post-translation modifications of histones. Hence, the aim of this thesis was to study effect of estrogens on in vitro models of testicular tissue and spermatogenesis and thus to address their functions in testicular tissue more deeply. This project includes testicular organoid cultivation for further usage as in vitro model of spermatogenesis. To addresss the effect of various avaliable estrogen forms, experiments on MCF-7 cell line were done. Finally experiments with in vitro model of testicullar tissue - TM4 Sertoli cell line were done. In order to fulfill the aims and verify the hypotheses, some advanced methods such as CLARITY volume confocal imaging and holographic microscopy were used. It was found that estrogens can affect Sertoli cell morphology and the expression of some genes involved in cell-cell junction dynamics. Furthermore the process of spermatogenesis was...
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The role of Prdm9 in the house mouse hybrid sterility model
Mukaj, Amisa ; Parvanov, Emil (advisor) ; Děd, Lukáš (referee) ; Reifová, Radka (referee)
(English) Hybrid sterility is a phenomenon representing reproductive isolation between closely related species and thus assuring the process of speciation. Hybrid sterility is a complex polygenic trait and the most significant advance in its study is achieved in Drosophila melanogaster. Nearly half century ago Forejt and Ivanyi mapped the first hybrid sterility genetic locus in vertebrates (Hybrid sterility 1, Hst1) in crosses of wild mice with laboratory inbred strains where the male offspring is sterile. Further mapping of the locus led to discovery of a gene called PR domain zinc finger protein 9 (Prdm9), a histone methyltransferase, whose deficiency causes meiotic arrest and sterility in mice. Furthermore, it was found that Prdm9 defines the hotspot placement in meiotic recombination by posting trimethylation marks on lysine residues 4 and 36 of histone 3 hence explaining the observed defects in its absence during meiosis. Nonetheless, its exact role in molecular mechanism of hybrid sterility is yet unclear. Prdm9 expression is present during the stage of formation of meiotic DNA double-strand breaks and is a hint about the time of hybrid sterility initiation. Phenotypically in the case of male mouse hybrid sterility is observed incomplete chromosome synapsis, meiotic arrest at mid-to-late...
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Detekce objektů na včelím plástu a spadových podložkách mobilním telefonem
DĚD, Lukáš
This thesis deals with the improvement of the adopted solutions, dealing with the detection of the bee queen and the calculation of the fall of the Varroa destructor mite. These advanced solutions are implemented in a mobile application for Android devices. Furthermore, this work deals with the investigation of methods for the detection of short-lived and long-lived bees. The theoretical part contains a description of the methods used in the field of computer vision and convolutional neural networks. The practical part describes the environment and technologies, which were used to implement the methods described in the theoretical part, as well as the process of porting the results to the mobile platform.
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The effects of epigenetic factor PRDM9 on the fertility of rodent females
Gašić, Srdan ; Trachtulec, Zdeněk (advisor) ; Děd, Lukáš (referee) ; Fulková, Helena (referee)
The Prdm9 gene encodes a histone-3-lysine-4,36-trimethyltransferase that specifies meiotic recombination sites and guides programmed double-strand breaks (DSBs) in mice, rats, and humans. Some vertebrates lost Prdm9 but not fertility throughout evolution, while the removal of Prdm9 caused sterility in some mouse strains, such as C57BL/6 (B6). The reasons for such species-specific fertility differences are unknown. To resolve these different requirements for PRDM9 in fertility, we produced Prdm9 mutants in another mammalian species, Rattus norvegicus, strain SHR/OlaIpcv. The removal of Prdm9 function did not completely abolish fertility in rats (as in B6 mice). Here I demonstrate that the loss of rat PRDM9 delayed female meiosis and caused synapsis and DSB repair defects that lead to a significant oocyte loss. However, unlike Prdm9-deficient B6 mouse oocytes, about 10-15% of pachytene-like mutant rat oocytes synapsed their chromosomes and repaired DSBs to the levels similar to controls. Because of this, female rats lacking PRDM9 maintained some oocytes until adulthood and yielded offspring, while B6 mice lost oocytes around the time of birth. Nevertheless, the adult rat mutant oocytes were exhausted earlier than the control adult rat oocytes. Therefore, PRDM9-lacking female rats suffered from...
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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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The Influence of Selected Environmental Pollutants on Gametogenesis and Early Embryonic Development
Fenclová, Tereza ; Nevoral, Jan (advisor) ; Děd, Lukáš (referee) ; Sovadinová, Iva (referee)
The influence of selected environmental pollutants on gametogenesis and early embryonic development Ing. Tereza Fenclová Idiopatic infertility is a serious problem, which can be caused by exposure to endocrine disruptors, e.g. bisphenols. Bisphenol A, the most widely used bisphenol, is now being replaced in manufacturing processes due to its toxicity by its analogues, bisphenol S and F. While most of the scientific experiments examine the effect of toxic doses after direct exposure, this study provides results for exposure to real low doses after direct and indirect exposure. We assumed a negative effect of direct and indirect exposure to low doses of alternative bisphenols BPS and BPF on male and female reproduction at the level of gametes and embryos. The aim of this work is to evaluate the effect of direct exposure to mouse oocytes and sperm, to evaluate the effect of indirect exposure through breast milk to mouse oocytes, sperm, testicular tissue and the extent of damage to early embryonic development. Experimental ICR laboratory mice were exposed to low doses of bisphenols directly via drinking water or an oral gavage, or indirectly through the breast milk. Direct exposure to low doses of bisphenols affects the reproductive abilities of females mainly by malformations of the dividing spindle of...
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Male infertility and DNA germ cell breaks affected by the epigenetic factor PRDM9
Kusari, Fitore ; Trachtulec, Zdeněk (advisor) ; Děd, Lukáš (referee) ; Liška, František (referee)
DNA-binding histone-3-lysine-4,36-trimethyltransferase PRDM9 specifies meiotic recombination hotspots in mice, rats and humans. Interallelic variation at the Prdm9 locus plays a role in hybrid male sterility. Sterile mouse F1 hybrid male offspring from the PWD × C57BL/6J (B6) cross exhibit meiosis breakdown reminiscent of that observed in Prdm9-deficient B6 sterile mice. However, reciprocal (B6 × PWD)F1 hybrids and some rodent models lacking PRDM9, i.e., PWD and SHR rat males execute meiotic recombination, produce sperm, raising the possibility that PRDM9's role may extend beyond meiosis. Here I demonstrate that PRDM9 is important for post- meiotic male gamete development and release. Unlike their parents, (B6 × PWD)F1 generated spermatozoa of lower quantity and motility but higher percentage of deformations, thus resembling oligoasthenoteratozoospermic (OAT) (semi)sterile men. Histopathological and (ultra)structural analysis revealed compromised spermiogenesis characterized by acrosome detachment and aberrant nucleus elongation in (B6 × PWD)F1 hybrids. Consequently, F1 spermatozoa had malformed acrosomes and nuclear DNA breaks with elevated base oxidation. While deletion of one Prdm9 copy improved sperm phenotypes in (B6 × PWD)F1, copy number gains of the surrounding genes had the opposite effect....
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Effect of endocrine disruptor bisphenol S on human health and reproduction
Procházková, Bára ; Petr, Jaroslav (advisor) ; Děd, Lukáš (referee)
Bisphenol S (BPS) is an environmental pollutant that has replaced bisphenol A (BPA) in plastic and paper products since 2011 as a safer "BPA-free" alternative. However, due to its rapid replacement, its potential adverse effects have not been investigated in detail, and due to its structural similarity to BPA, many studies suggest that it acts, like BPA, as an endocrine disruptor. Its use is wide and BPS is globally widespread, found in the environment and has been detected in both animal and human tissues. Nevertheless, an overall effect of BPS on an organism remains unclear. This work is focusing on gathering sources for its effect on reproductive ability and its association with metabolic disorders.
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