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The role of cohesin genes in the meiosis of male house mouse
Šebestová, Lenka ; Trachtulec, Zdeněk (advisor) ; Král, Jiří (referee)
Cohesin genes play an important role in cell division. They ensure proper chromosome segregation during mitosis and meiosis. This study is focused on the role of cohesin genes during meiosis in male house mouse (Mus musculus). At first, this study introduces key processes of mammalian meiosis. Next, the structure of cohesin complex is described; it consists of a heterodimer SMC proteins - SMC3 and SMC1α or SMC1β, which are enclosed to the ring by cleavable subunit RAD21, RAD21L or REC8. Fourth subunit - a STAG protein (STAG1, STAG2 or STAG3) associates with the cleavable subunit. Meiotic function of specific cohesin proteins (SMC1β, RAD21L, REC8 and STAG3) as deduced from the phenotypes of the deficiencies of their genes in male mouse is depicted. All these four genes are necessary for many processes during meiosis, - e.q. sister chromatid cohesion maintenance, synapsis and recombination. STAG3, SMC1β, and REC8 are necessary for centromeric cohesion. STAG3 and RAD21L are important for the assembly of the remaining cohesin subunits. The most important phenotype of deficiency of all four genes is the complete meiotic arrest in male prophase I. Therefore, cohesin research is important for the investigation of the causes of sterility in mammals. key words: cohesin, meiosis, spermatogenesis, mouse,...
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CRISPR/Cas9-based genome editing in mice: state of the art and future perspectives
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Čáp, Michal (referee)
Mutant mice are crucial tools for understanding gene functions in vivo. Recently, generation of mouse mutants was revolutionized by rapid developement of programmable nucleases, predominantly by the CRISPR/Cas9 system. Genome editing based on introduction of CRISPR/Cas9 components into early stage mouse embyros allows fast and inexpensive generation of gene-deficient animal models, especially when compared to the traditional techniques based on modification of embryonic stem cells (ESCs). The ability of CRISPR/Cas9 to induce double-strand break (DSB) at a given location of genomic DNA enables effective gene-ablation by random modification of the coding sequences or by complete ablation of the gene. However, precise modification of the gene sequences, such as incorporation of a DNA fragment into specific loci, are still difficult to make. In this work, I present a review of CRISPR/Cas9 system, its use in production of mutant mice and possible modifications of the system to increase the efficiency of precise gene-targeting. Keywords: CRISPR/Cas9, mouse, transgenesis, homologous recombination
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Species-specific structural differences of mammalian sperm and function of their key proteins during fertilization.
Dobrodinská, Anna ; Frolíková, Michaela (advisor) ; Kuntová, Barbora (referee)
The fertilization is a process during which a male and a female gamete merge so that a new organism may come into being. The sperm-egg fusion is preceded by several essential processes, such as the capacitation, acrosome reaction, the sperm binding to the zona pellucida and oolemma, and membrane fusion of the gametes. Numerous proteins, which are located in both sperm and eggs, are major actors in controlling the listed, essential processes. During the process of fertilization these proteins fulfil one or more functions. In mammalian sperm, significant species-specific differences may be found both in their morphology and at the protein level. A complex understanding of species-specific distinctions in sperm structure and functions of key sperm proteins would contribute to a better insight into the process of fertilization, thereby enabling us to better diagnose and subsequently treat the causes of infertility in humans. This bachelor's thesis summarizes the current knowledge of sperm structure and its key proteins that has been acquired through the studies of the following model mammal species: bull, boar, mouse, and human. Further, this thesis brings an interspecific comparison between the studied species. Keywords: sperm, fertilization, acrosome reaction, capacitation, sperm proteins, bull,...
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Methods and consequences of some neurosurgical interventions in animal model and in human clinic. Pinealectomy and intracerebroventricular application.
Řezáčová, Lenka ; Tichý, Michal (advisor) ; Szárszoi, Ondrej (referee) ; Přibáň, Vladimír (referee)
Clinical surgical techniques of pinealectomy and intracerebroventricular (i.c.v) cannulation are still used in the clinic in indicated cases. In research, there is an effort to replace these classical surgical techniques by other means. However, these various new models often do not reflect the complexity of the functions taking place in the living organism as a whole. The thesis shows that these surgical techniques should be also a part of biomedical research in the future, as they still bring new important information. In the experiment, the pinealectomy can serve as a model of resection or depletion (removal of natural hormone secretion), while i.c.v. application of the active substance to the ventricles on the contrary as a model of addition or substitution. The practical part of the thesis is divided into 4 areas: A) pinealectomy - an animal models, B) pinealectomy - in the experiment, C) pinealectomy - in the clinical practice and D) intracerebroventricular application - in the experiment. The work describes in detail the surgical techniques and discuss possible consequences of pinealectomy in 6 animal species (and their comparison) and in humans. It also includes an experiment with pinealectomy in a rat and a clinical study in patients with pineal cyst and subsequent pinealectomy. The work...
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Early tooth development of Tabby mice
Smrčková, Lucie ; Peterková, Renata (advisor) ; Černý, Robert (referee)
The developing mouse dentition is a very useful tool to study molecular regulation of odontogenesis and also organogenesis. The embryonic mouse dentition comprises developing functional tooth primordia as well as rudimentary tooth primordia. These rudiments arrest their growth during development and either degenerate or become a part of a functional tooth. Mice with gene defects also allow elucidation of a function of genes, their products and signalling pathways. The protein ectodysplasin is essential for development of ectodermal derivatives - skin, hair, glands and teeth. The Tabby mice have a mutation in the Eda gene, which encodes the protein ectodysplasin, and they display a number of dentition anomalies. Early development of the lower jaw dentition in Tabby embryos has been already morphologically described. As a prerequisite for understanding regulatory mechanisms of odontogenesis in Tabby mice, it is also necessary to map the spatiotemporal dynamics of signalling centres that express Shh in both the rudimentary and functional tooth primordia. The aim of this thesis was to compare the signalling centres based on the Shh expression and its spatiotemporal dynamics in the lower jaw of Tabby and WT mouse embryos. Then the Shh data were correlated with known morphological data to clarify the...
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Sexual transmission of Toxoplasma gondii from males to females: experimental verification using laboratory animal model
Navrátil, Jiří ; Kodym, Petr (advisor) ; Votýpka, Jan (referee)
Toxoplasma gondii is cosmopolitly living parasite which prevalence in human extends to tens of percent. In its life cycle it uses any homoiothermic vertebrate as an intermediate host. The definitive host are felines from Felidae family. The acute phase of infection is medically important in immunocompromised pacients and by its risk of congenital toxoplasmosis in pregnant women who never suffered from this illness before. Infection could have serious and rarely even lethal consequences in both cases. This thesis focuses on experimental verification of theory of sexual transmission of toxoplasmosis from male to female on laboratory mice. Possible transmission was tested in acute phase and latent phase of infection. The result was negative in both cases. Moreover, we observed the parasite's affinity to tissue of organs in male mice by PCR technique. Particularly, our interest was in comparing genital organs with others. It was discovered that lungs and spleen are the most infected organs in acute phase of infection. Toxoplasma was also present in genital organs (especially in epididymis) but not more frequently than in others. We observed statistically significant difference between sexual and non-sexual organs in acute and latent toxoplasmosis - non-sexual organs were more infected in both phases....
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Role of Nkx2.5 in development and electrophysiology of the mouse heart
Hámor, Peter ; Sedmera, David (advisor) ; Neckář, Jan (referee)
Role of Nkx2.5 in development and electrophysiology of the mouse heart Prague 2016 Peter Hámor, B.S. ABSTRACT The objective of this thesis is to investigate the role of Nkx2.5 gene dosage on electrophysiology of the mouse heart in prenatal stage of its development. The main goal of this work is to search for differences in conduction of electric impulses through the embryonic mouse hearts of different genotype. Special method of capturing the conduction of electric impulse through myocardium, called optical mapping, was used to visualize the electrical activity. Thanks to this method I was able to construct images and videos capturing the spread of the impulse with identification of the beginning of the activation and its direction in the heart. These outputs, or optical maps, help to define anomalies and defects in mutants compared with a normal functioning heart. The thesis focuses on the expression of the transcription factor Nkx2.5 and regulatory components related with the correct formation and physiology of the heart until 9.5 days post coitum. Embryos at this developmental stage were optically mapped and analysed according to their genotype. While the wild type and heterozygote mouse embryos exhibited high degree of similarity, the homozygous mutants were dramatically different. Considering this work...
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Inflammation and cancer in germ-free vs. conventionally reared animals
Čaja, Fabián ; Vannucci, Luca Ernesto (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Smrž, Daniel (referee)
Inflammation is considered as one of the main defence mechanisms of the immune system against threats that occur in the body. When present in its acute form, minimal or no detectable subsequent damage of original affected tissue exists. The more pathological form, chronic inflammation, is associated with permanent damage of the tissue and typically a hallmark of various diseases such as ulcerative colitis or colon carcinogenesis. These two pathologies are evolving in the unique colon microenvironment, where intensive interaction between the host cells and bacteria is present. The aim of our study was to investigate the immunological (ELISA, FACS, RT-PCR) and structural (histology, confocal microscopy) changes in the colon mucosa of Wistar-AVN rats induced by dextran sodium sulphate (DSS) to produce colon colitis and by azoxymethane (AOM) to produce colon carcinogenesis. Conventional (CV) and also germ-free (GF) reared animals were used to investigate the effects of the mucosal inflammation activated by the administered inducers as well as the role of colon microbiota - as promoters of a continuous immune activation - in the modulation of immunity and collagen scaffold remodelling. Our results showed that even in the early period after the induction, both inducers produced a smouldering...
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The effect of stress on regulation and regeneration of glucocorticoids in animal models differing in response of hypothalamo-pituitary-adrenal axis
Vodička, Martin ; Pácha, Jiří (advisor) ; Svoboda, Jan (referee) ; Bendová, Zdeňka (referee)
Stress reaction is usually activated by the brain, when homeostasis is or perceived to be threatened. The stress signals are transmitted from the brain by two main branches; the sympathoadrenomedullary and the hypothalamo-pituitary-adrenal (HPA) axes and employ neural, humoral and immune pathways to cope with the stressor. Because of its potency, the stress reaction has to be precisely regulated. The HPA axis is regulated by feedback loops where its end product, corticosterone in laboratory rat and mouse, inhibits its activity. The effect of corticosterone does not depend only on the concentration of corticosterone but also on local metabolism of glucocorticoids via oxo-reduction catalyzed by the enzyme 11β -hydroxysteroid dehydrogenase 1 (encoded by the Hsd11b1 gene), which intracellularly regenerates active corticosterone from inactive 11-dehydrocorticosterone, or by extra-adrenal de novo steroidogenesis of glucocorticoids. We focused on analysis of stress response in experimental animals differing in HPA axis responsivity (Fischer 344 rats (F344) vs. Lewis rats (LEW) and germ-free (GF) vs. specific pathogen free mice (SPF)) with special emphasis on regulation of stress response, glucocorticoid regeneration and influence of gut microbiota. We found that stress modulated local regeneration of...
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Mechanisms of microbiota regulation during the estrous cycle of the house mouse.
Dodoková, Alica ; Stopková, Romana (advisor) ; Janotová, Kateřina (referee)
There is a very few papers to provide an overview of the characteristics of the estrous cycle, the relationship of the estrous cycle to physiological manifestations such as the pH of the vaginal environment, as well as the dynamics of the vaginal microbiota in wild mice. The aim of this thesis is to contribute to the understanding of the dynamic relationship between external influences and the physiology of the female reproductive system, to develop a reliable methodology for measuring the pH of the vaginal microenvironment in mice as well as to quantify the overall abundance of some bacterial taxons by comparing sequencing and qPCR methods. The results suggest that the physical presence of the male in the cage has the most significant effect on the prolongation of the estrus phase, in contrast to non-significant olfactory stimulation of the urine. Fluctuation in the pH of the vaginal environment have also been shown to be cyclic, and the qPCR method shows that the composition of the vaginal microbiota, during the estrus phase, differs significantly from other phases of the estrous cycle, as we confirmed by 16S rRNA sequencing. Thus, these results provide a comprehensive view of the variability of the estrous cycle with an emphasis on the variability of the vaginal microbiota and the change in the...
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