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The effects of endocrine disruptors on the expression and the activity of cytochromes P450 2B in laboratory rat as a model organism
Měkotová, Barbora ; Dračínská, Helena (advisor) ; Levová, Kateřina (referee)
Endocrine disrupting chemicals are compounds that interfere with natural hormones and they are responsible for functional changes which may lead to damage of the endocrine system. Their presence in the environment is associated with a number of diseases whose extent is hard to predict. As endocrine disrupting chemicals, a wide range of exogenous and endogenous compounds is present in the environment. Important exogenous endocrine disrupting chemicals include benzo[a]pyrene (BaP) and 17α-ethinylestradiol (EE2); the female sex hormone 17β-estradiol (E2) can act like endogenous endocrine disruptor. In this thesis, the effect of these three compounds and their combinations on the expression and the activity of rat biotransformation enzymes cytochromes P450 2B is studied. The gene expression was determined by quantitative PCR, the expression of the protein itself was studied using Western blot method and consecutive immunodetection. The results show that CYP2B expression is almost unchanged after BaP premedication, whereas estrogenic compounds, E2, EE2, their combination and their combinations with BaP, significantly decrease the expression. The enzyme activity of CYP2B was also studied in rat liver microsomes using the marker substrate 7-pentoxyresorufin. EE2, E2 and their combination decrease the...
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Inducible RNAi against essential genes of nitrogen metabolism as a tool for control of GM plants
Kobercová, Eliška ; Fischer, Lukáš (advisor) ; Tylová, Edita (referee)
Uncontrolled spreading of genetically modified (GM) plants is one of the main concerns about their cultivation. Inducible RNA interference against an essential gene could be a tool for control of GM plants. After spraying with a chemical inducer, the essential gene will be silenced so the treated GM plant will die. For testing this strategy we chose two key enzymes of nitrogen metabolism, glutamate synthase (GOGAT) and glutamine synthetase (GS). GS processes ammonium ions into glutamine, then GOGAT transfers the amide group from glutamine to 2-oxoglutarate to form two glutamates. GS/GOGAT cycle is the main pathway for assimilation of ammonium ions, which could be toxic to plants in a higher concentration. Disruption of ammonium assimilation during photorespiration causes a strong inhibition of photosynthesis. The aim of this work was to describe the effects of silencing GOGAT and GS genes in Arabidopsis thaliana. To induce silencing, RNAi hairpin constructs under a control of constitutive or estradiol-inducible promoter were prepared. In selected independent transformants with the inducible hairpin against GOGAT, chlorosis and reduced growth were observed after the estradiol treatment in in vitro conditions. However, the spraying with estradiol was tricky, at the whole plant level, the induction of...
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Sex reversion in the lizard with genotypic sex determination (Squamata: Acrodonta: Pogona vitticeps)
Ehl, Jan ; Kratochvíl, Lukáš (advisor) ; Landová, Eva (referee)
Sex determination among reptiles is a very variable matter across it's taxa. We meet there temperature sex determination and genotypic sex determination with many independent transitions between them. It is a group suitable to study evolution of sex determination, sex chromosomes and sex determination genes. Rare cases of sex reversal caused by extreme incubation temperature or exogenous hormones have been reported in recent years. In case of Acrodont lizard, Pogona vitticeps, was reported sex reversal caused by high incubation temperatures. Our purpose was to repeat the experiment, mainly due to insufficient conclusiveness of used methods. We wanted to expand the experiment by hormonal reversal, studying persistence of sex reversal to maturity and fertility of reversed individuals. We managed successfully to demonstrate sex reversal in both treatments by histological examination. Individuals with discordant phenotypic and genotypic sex were breed till one year of life, which demonstrate persistence of reversal. Our outcomes are concordant with most recent work on this species and show full functional phenomenon of sex reversal with reptiles, which studying could contribute to our understanding of evolution of sex determination.
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Hormonal control of aggressive behaviour in lizards
Rauner, Petr ; Kratochvíl, Lukáš (advisor) ; Gregorovičová, Martina (referee)
Aggression is a highly functional form of social behaviour, which can be observed in nearly all species of vertebrates including lizards. There are many forms of aggressive behaviour and there is very significant difference in the physiological basis among them. The main and best understood hormone affecting aggression, is testosterone, but there exists evidence that some aggressive behaviour can be influenced by other hormones as well for example by progesterone, estradiol and corticosterone. The effect of these hormones on the organism is traditionally divided into the activation and organizational effects. Organizational effects are permanent and occur usually in the earlier stages of development. Activation effects are temporary and occur during the entire life. Elevated testosterone levels usually stimulate aggressive behaviour in both males and females. Progesterone and estradiol affect aggressive behaviour similarly but less effective. It is uncertain whether their effects are direct, or whether they are only a side-effect of testosterone, which is a precursor of estradiol and which has progesterone as own precursor. Corticosterone affects aggressive behaviour indirectly by influencing levels of testosterone. Hormonal influence on aggression in lizards is not the sole factor, but it depends on...
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Study of alternatively spliced variants of estrogen receptor alpha in breast cancer cell lines
Lhota, Filip ; Kleibl, Zdeněk (advisor) ; Souček, Pavel (referee)
Filip Lhota: Study of alternatively spliced variants of estrogen receptor alpha in breast cancer cell lines Abstract: Estrogen receptor α (ER-α) is a transcription factor responsible for mediation of the activities of its natural ligand 17-β-estradiol (E2), the hormone that together with progesterone belongs to the key regulators of mammary epithelial as well as breast cancer cells proliferation. Except to the major gene product consisting of all eight coding exons of ER-α, numerous qualitatively and quantitatively different spliced variants originated from primary transcript by activity of alternative splicing is expressed. Despite that some of these spliced variants have been functionally characterized, their precise role on final ER-α cellular activity remains to be elucidated. The functional characterization of individual alternative forms of ER-α and description of its participation on the overall ER-α activity is important for our understanding of their biogenesis and is also critical for the delineation of molecular bases for ER-α regulation during anti cancer chemotherapy. This work aimed to study the influence of alternatively spliced ER-α variants on the growth characteristics of clones constructed from stable mammary tissue cell lines in regulation to cultivation conditions and cellular...
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