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Gasotransmise v epigenetických regulacích gametogeneze a embryogeneze
BRICHCÍN, Jiří
As the problems with the reproduction of livestock and humans starting to increase, the need for knowledge of mechanisms involved in regulating the correct process of gametogenesis and embryogenesis also rises. For the experimental part of this work, two components from two different, mostly separately explored fields, i.e. gasotransmitters and epigenetic mechanisms, which are necessary for the correct process of gametes production and early embryonic evolution were chosen. Hydrogen sulfide was chosen from the series of gasotransmitters, and histone deacetylase Sirtuin 1 (SIRT1) was chosen as its possible substrate. Confirmation of the presence of these components was carried out on oocytes and embryos of laboratory mice (Mus musculus).
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NAD+-dependent histone deacetylase SIRT1 in the process of oogenesis, fertilization and early embryonic development
Valentová, Iveta ; Nevoral, Jan (advisor) ; Drutovič, David (referee)
SIRT1 is a histone deacetylase from the sirtuin family that affects epigenetic and non- epigenetic targets. We can assume that the known SIRT1 substrates are involved in the regulation of gametogenesis and early embryonic development. Our hypotheses say SIRT1 is present in oocytes and early embryos and it plays a physiological role in oocyte maturation, fertilization and early embryonic development. A mouse model of a conditional knock-out line producing Sirt1-deficient oocytes was developed to verify our hypotheses. Oocytes and embryos were analyzed for SIRT1, its selected substrates and other markers by immunocytochemistry. We found out that the presence of SIRT1 contributes to oocyte quality through modulation of the chromatin histone code and stabilization of the spindle. Furthermore, the purely maternal origin of SIRT1 presents in both zygote pronuclei. Last but not least we discovered a significant effect of SIRT1 on early embryonic development, probably mainly due to its role in the activation of the embryonic genome. The results confirm our hypothesis that SIRT1 is present in oocytes and embryos mainly around chromatin. The results show that SIRT1 is a maternal factor determining oocyte quality and it is necessary for the embryonic genome activation.
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Relation between n-3 polyunsaturated fatty acids and cellular sensors of energetic state
Zouhar, Petr ; Flachs, Pavel (advisor) ; Pecina, Petr (referee)
The important factor in regulation of metabolic processes is regulatory proteins, which are able to react by feed-back to energetic state of the cell. Big attention is focused on the AMP activated kinase (AMPK) and NAD+ activated deacetylase SIRT1. These enzymes interact together and their stimulation increases mitochondrial biogenesis and fatty acid oxidation. Due to this it functions beneficially against the onset of obesity, insulin resistance and ageing. Fasting, exercise and some antidiabetogenic drugs act by these regulators. n-3 polyunsaturated fatty acids (PUFA) are also known because of their stimulative effects on mitochondrial biogenesis and -oxidation. Previous work of our group have showed that intake of higher dose of n-3 polyunsaturated fatty acids (PUFA) in diet lead to increase in activity of AMPK in white adipose tissue. New results presented in this thesis show that SIRT1 is essential for increase in expression of stimulators of -oxidation (PPAR etc) in response to n-3 PUFA in diet. n-3 PUFA futher improve the metabolic profile synergistically with calorie restriction probably through SIRT1.
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The impact of SIRT1 inhibition on zebrafish morphology and behavior
Faustová, Zuzana ; Červený, Lukáš (advisor) ; Mladěnka, Přemysl (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Faustová Supervisor: Prof. Doutor Jorge Miguel de Ascenção Oliveira PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: The impact of SIRT1 inhibition on zebrafish morphology and behavior After discovery of connection between yeast Silent Information Regulator 2 (Sir2) and its ability to alter lifespan, Sir2 and its seven mammalian orthologs became very attractive therapeutic target. These so called sirtuins are members of a histone deacetylase family. They possess unique catalytic activity having nicotinamide adenine dinucleotide as a cofactor and their function can be influenced by environmental factors. The aim of this diploma thesis was to extend knowledge of Sirtuin 1 (SIRT1), which is from all mammalian sirtuins considered to have the closest relation to yeast Sir2. At first we tested the impact of SIRT1 inhibition on early developmental stages of zebrafish (Danio rerio) embryos and larvae, finding out that SIRT1 is important for normal development and SIRT1 inhibition or malfunction result in cardiovascular defects, delayed development, and death. Additionally, we tried to learn more about SIRT1 and its connection with Parkinson's disease by combining nontoxic doses...
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Epignetic Modifications of the Sperm and the Application in Clinical Practice of Human Assisted Reproduction Therapy
Štiavnická, Miriama ; Nevoral, Jan (advisor) ; Kloudová, Soňa (referee) ; Vašíček, Jaromír (referee)
Basement of healthy embryo development comes from quality of oocytes and spermatozoa. Today, when percentage of couples suffering infertility together with assisted reproductive therapy (ART) is increasing, understanding to gamete biology and heritable epigenetic code is crucial. The study is focused on promising epigenome based markers that could serve as indicators of gamete quality for either their screening or selection for ART. Accordingly selected markers were used for the investigation of environmental pollutant bisphenol S (BPS) effect on gametes quality. To obtain these aims, we have used human semen samples, boar semen samples and ICR mice gametes. Samples were analyzed by flow cytometry, immunocytochemistry and western blot analysis. All experimental work was in accordance with Ethics committee University Hospital in Pilsen and approved experimental designs for appropriate experimental animal project. In the study, we detected the dimethylation of histone H3 on lysine K4 (H3K4me2) as potential epigenetic marker of sperm quality and chromatin immaturity. Secondly, we observed the role of the gasotransmitter hydrogen sulphide (H2S) as anti-capacitating agents, slowing down capacitation possibly through post-translational modification of proteins. Thirdly, SIRT1 histone deacetylase was...
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The role of SIRT1 during in vitro maturation of oocytes
Landsmann, Lukáš ; Nevoral, Jan (advisor) ; Šolc, Petr (referee)
SIRT1 histone deacetylase acts towards many epigenetic and non-epigenetic targets. The involvement of SIRT1 in oocyte maturation is assumed and the importance of ooplasmic SIRT1 pool for further destiny of matured oocyte is strongly suggested. We hypothesized that SIRT1 play role of the signal molecule in mature oocyte through selected epigenetic and non- epigenetic regulation. We observed SIRT1 re-localization in mature oocyte and the association with spindle microtubules. In matured oocyte, SIRT1 shows a spindle-like pattern and spindle- specific SIRT1 action is supported decreasing α-tubulin acetylation. Based on the observation of histone code in immature and matured oocytes, we suggest that SIRT1 is mostly predestined for epigenetic mode of action in germinal vesicle (GV) of immature oocyte. Accordingly, SIRT1- driven trimethylation of histone H3 on lysine K9 in matured oocyte is considered to be an inheritance of GV epigenetic transformation. Taken together, our observations point out the dual spatiotemporal SIRT1 action in oocyte capable to be switched from epigenetic to the non- epigenetic mode of action readily depending on meiosis progress. Keywords: oocyte, SIRT1, histone, developmental competence, tubuline, epigenetics
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The role of new profibrotic molecules in the pathogenesis of systemic sclerosis.
Šumová, Barbora ; Šenolt, Ladislav (advisor) ; Funda, David (referee) ; Soukup, Tomáš (referee)
Systemic sclerosis (SSc) is immune-mediated fibrotic disease of unknown aetiology. Among the dominant pathogenic manifestations of SSc belong vascular changes, production of autoantibodies, activation of innate and adaptive immune responses and fibrotic processes. Transforming growth factor beta (TGF-β) has been identified as a central profibrotic factor stimulating fibroblasts to produce collagen. There are, however, a number of other mediators involved in the pathogenesis of SSc. Mutual activation and amplification of these molecules and their cascades may be a central mechanism of the SSc pathogenesis. Hedgehog (Hh) canonical signalling pathway plays an important role in the development and progression of fibrotic diseases. Expression of Hh target genes can be regulated through a canonical or non-canonical signalling cascade. The non-canonical activation of GLI transcription factors by TGF-β has not yet been investigated in SSc. The substantial part of this thesis is focused on the study of the mutual interaction of TGF-β and Hh signalling pathway. In vitro analysis confirmed TGF- β/SMAD3 dependent activation of GLI2 in dermal fibroblasts. Fibroblasts specific knockout of GLI2 prevented the development of experimental fibrosis in vivo. Combined targeting of canonical and non-canonical Hh...
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Circadian system in mitochondria
Šemíková, Johana ; Bendová, Zdeňka (advisor) ; Moravcová, Radka (referee)
The rhythms of circadian clocks throughout our bodies are not governed only by the light/dark cycle, but in many peripheral tissues circadian clocks are reset based on availability of nutrients and timing of food intake. The circadian system responds to changes in the levels of two metabolites (AMP and NAD+ ) that are central to biochemical reactions involved in energy production, storage, and utilization through the metabolic sensors AMPK and SIRT1. The aim of this review is to summarize mechanisms for energetic metabolism known to date that are connected with the regulation of circadian rhythms and explain their function in maintaining their stability. Last but not least, to show possible dysregulation of these mechanisms and their impact on the circadian system.
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