There are a large number of natural and synthetic environmental contaminants (ECs) known or suspected to mimic or interfere with male reproductive endocrine functions. Our current knowledge is largely addressed to ECs that induce oestrogen-induced feminization. However, there are several ECs that cause alternations in androgen production similar to oestrogenic ECs, but they do not induce vitellogenin-induced feminization. The mechanisms of action of these chemicals are still unclear due to the fact that androgen receptor (AR) functions in androgen-mediated male reproductive physiology are largely unknown. In the present study, we examined the effects of vinclozolin (VZ) (100, 400 and 800 ?g/L) and di-(2-ethylhexyl)-phthalate (DEHP) (1, 10 and 100 ?g/L) on male reproductive performance in goldfish following 30 days of exposure. Since both VZ and DEHP act as agonists and/or antagonists of hormonal receptors, estrogen receptor subtypes (er) and androgen receptor (ar) mRNA levels were studied. For studying their oestrogenic activity, one group of goldfish were exposed to 17?-estradiol (E2). In DEHP treated goldfish, sperm production, motility and velocity were decreased at 1, 100 and 10 ?g/L, respectively. Our previous study has shown that sperm production, motility and velocity were decreased in goldfish exposed to 800 ?g/L VZ. These suggest that DEHP and VZ are capable of interfering with spermatogenesis and sperm maturation. In E2 treated goldfish, none of the males produced sperm, indicating E2-suppressed spermatogenesis. Similar to E2 treated goldfish, 11-KT levels were decreased in goldfish exposed to ?10 and ?1 ?g/L DEHP at day 15 and 30, respectively. In VZ treated goldfish, 11-KT levels were decreased in goldfish exposed to 800 ?g/L VZ following 7 d, but increased in goldfish exposed to 100 ?g/L VZ following 30 d of exposure. E2 levels remained unchanged and increased in DEHP and E2 treated goldfish, respectively. In VZ treated goldfish, LH levels were increased at 100 ?g/L. In contrast, LH levels were decreased in DEHP and E2 treated goldfish following 15 d of exposure. There were also positive relationships between LH and 11-KT levels suggesting that inhibition or stimulation of androgen production were mediated by DEHP- or VZ-induced alternations in pituitary function. In VZ treated goldfish, gnrh3 mRNA levels were decreased following 7 d of exposure and increased at low dose following 30 d of exposure. kiss1 or kiss2 mRNA levels were also increased in VZ treated goldfish, while gpr54 mRNA remained unchanged. In DEHP treated goldfish, gnrh3, kiss1 and its receptor (gpr54) mRNA levels did not change during the period of exposure. In E2 treated goldfish, gnrh3 mRNA levels were decreased at day 7, but kiss1 and gpr54 mRNA levels were increased at day 30 of exposure. These results suggest that, in contrast to DEHP, VZ effects on pituitary and testicular functions are mediated by disruption of hypothalamus function and upstream neuroendocrine regulators. The brain ar and testicular lhr mRNA levels were changed in VZ and E2 treated goldfish depending on dose and period of treatment, however they remained unchanged in DEHP treated goldfish. These differences suggest that DEHP may act through an independent hormonal receptor pathway, while VZ acts through a receptor pathway. vtg, er and cyp19a1b remained unchanged in DEHP and VZ treated goldfish but increased in E2 treated goldfish. These indicate that neither VZ nor DEHP acts as oestrogenic compound to impair male fertility. In conclusion, DEHP and VZ reduced sperm quality in goldfish due to stimulation and inhibition of 11-KT production which were mediated by alternations in pituitary function to produce LH or by disruption of the transfer of Cholesterol to steroidogenesis. Upstream neuroendocrine regulators (gnrh3 and kiss-1/gpr54) were disrupted in VZ treated goldfish. Taken together, VZ and DEHP differentially act on brain and testis to impair fertility endpoints.

The thesis focuses on selected, yet unsolved question of the role of less common steroids and SHBG as a junction of three endocrine systems. Answering these questions may help to understand the complex mechanism of action of these hormones on the human organism. This thesis is based on five author and co-written studies mostly published in foreign scientific journals. In the case of studies of metabolites 16α-OH-DHEA and 7-oxo-DHEA (Zamrazilová et al., 2007; Kazihnitková et al., 2007) were focused on the development of appropriate methodological approach because recently used methods showed a low sensitivity and specificity. We developed and statistically evaluated new RIA methods which are rapid, sensitive and inexpensive. Both of them can be used in other research even in routine practice. New methods were also used to determine the metabolites in a statistically significant sample of healthy human population. In another study (Zamrazilová et al., 2010) we examined the relationships of selected steroid metabolites and SHBG in patients with CAH. We assumed that SHBG may act as a non-steroidal laboratory parameter reflecting the effectiveness of substitution therapy in these patients. Our assumption was not confirmed. We observed lower levels of SHBG which at least in women reflect effects of...

Normotensive hydrocephalus (NPH) is a neurodegenerative disease that occurs mainly in patients of high age. The disorder causes accumulation of cerebrospinal fluid (CSF), which leads to enlargement of ventricles and pressure exerted on cerebral structures. Clinical manifestations (gait disorders, development of dementia, incontinence) can be easily confused with symptoms of other neurodegenerative diseases; unlike other such disorders, however, NPH can be treated by surgery, if diagnosed in time. Patients are indicated for the procedure by a lumbar drainage test. There are currently no reliable laboratory biomarkers known that could be the basis of NPH diagnostics. In the past, steroids proved to be linked to neuronal activity in neurodegenerative diseases with the help of specific diagnostic markers. An instrumental method has been developed for the purposes of this thesis and it was used to gauge the level of certain steroids in CSF in a sample group of NPH patients and a control sample group of healthy individuals. A significant difference has been found in levels of aldosterone and cortisone. Aldosterone was higher in NPH sufferers, while cortisone levels were higher in the control group. It is crucial to differentiate patients with NPH from patients with similar clinical manifestations during...

Maternal smoking causes serious health danger for a mother but especially for a baby. Cigarette smoking produces complex steroidogenesis changes during the whole life of a woman. To study the influence of smoking on fetoplacental unit focusing on steroid hormons it was important first to concentrate on changes of the chosen steroids around the delivery. The first part of the thesis is dedicated to observe some chosen steroid hormons in peripartal period (37th week of the pregnancy, first stage of labor of mothers and mixed umbilical blood of their neonates) and to look for relations to the age of mother, the increase of the weight during the pregnancy, the type of the delivery and the sex of the baby. It was interesting to compare steroids in the relation to the type of the delivery: vaginal delivery versus planned caesarean section. Non-smoking women who delivered a boy spontaneously had significantly higher level of 17-OH-pregnenolone, progesterone, cortisol, corticosterone and significantly lower level of estradiol in comparison with non-smoking women who delivered a boy by a planned Caesarean section. In the maternal blood in the 37th week of the pregnancy there were found differences between steroids in accordance to the sex of the fetus but they were not found in the neonates' case. The age...

Farnesoid X receptor is mostly expressed in liver cells and its activation may be used for the treatment of cirrhosis causing diseases, especially biliary cirrhosis and nonalcoholic steatosis. These two latter diseases are most common in developed countries and, as of date, no effective treatments are available. Therefore, the aim of this project is the design and synthesis of novel bile acid analogues with subsequent biological evaluation towards farnesoid X receptor. Thus, a series of new compounds were designed using computational modeling studies and chemical synthesis was done to develop structure-activity relationships. Chemical structure analysis and purity was confirmed by conventional analytical methods. Finally, synthetic compounds were profiled against farnesoid X receptor in collaboration with the Pharmaceutical faculty of Charles University in Hradec Králové. Keywords: farnesoid X receptor, FXR, bile acids

The theoretical part of the Master´s thesis describes steroids, their features, and the methods for their determination in waters. In experimental part three steroid hormones, namely 1,4-androstadiene-3,17-dione (ADD), 1,4-androstadiene-3-one-17(beta)-ol (boldenone) and 17(beta)-estradiol were selected. The compounds were concentrated from water by LLE and SPE extraction and determined by gas chromatography coupled to mass spectrometry. Several experiments were conducted to assess the recovery of specific extraction for studied compound.

Currently there is a spate of interest in the presence of pharmacologically active substances in the environment. These substances are excreted in active or metabolized form and with wastewater pass the wastewater treatment plant. Current treatment technologies do not always eliminate all pharmaceuticals effectively and therefore they enter the environment. One of these active groups is the group of steroid hormones. Steroid hormones belong to the group of endocrine disruptors and they are considered to be dangerous for the ecosystems. Due to the hydrophobic character of steroid hormones they undergo partial or total sorption from wastewater to sludge. Sewage sludge is the by product of wastewater treatment and contains heavy metals, organic contaminants and pathogenic bacteria. A part of produced sewage sludge is used as a fertilizer in the Czech Republic. This issue follows the regulation 347/2016 – conditions of agricultural use of sewage sludge. Waste policy of EU will alter the conditions of sludge disposal, so it is necessary to obtain data about the concentration levels, fate and behaviour of those pollutants. This thesis was focused on five natural female hormones and four synthetic, which are used as a part of contraceptive pills and substitutional hormonal therapy. Determination of hormones was performed in four steps including ultrasonic assisted extraction, clean up by solid phase extraction, derivatization and final analysis by gas chromatography with mass spectrometry on triple quadrupole in MS/MS mode.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease that causes a disorder of steroidogenesis in the adrenal cortex. This disease is a part of a panel of diseases searched in preclinical nationwide neonatal screening. The methodology is based on measuring the concentration of 17-hydroxyprogesterone (17-OHP) in a dried blood spot using fluorescence immunoassay (FIA). However, this determination is not entirely specific and generates a high rate of false positive results (up to 4.3 %). In this diploma thesis the LC-MS / MS method was developed. This method measures selected steroid hormones involved in cortisol metabolism with respect to the diagnosis of CAH disease. The method was validated and applied to clinical samples, it identified CAH patients from negative controls and significantly reduced the false positivity of neonatal screening results. Compared to the FIA results, the LC-MS / MS method reduced false positivity up to 50 % by evaluating the concentration of 17-OHP. Moreover, by extending the diagnostic algorithm with other measured markers, the reduction was enhanced up to 98%. The developed method is also applicable for the measurement of serum and plasma samples, respectively, and has become a part of the confirmation tests for suspected CAH screening findings. Key...

MT nucleation from γ-tubulin complexes, located at centrosome, is an essential step in the formation of MT cytoskeleton. In mammalian cells, -tubulin is encoded by two genes. We functionally characterized two γ-tubulin proteins and have found that both are functionally equivalent. γ-Tubulin 2 is able to substitute for γ-tubulin 1 in MT nucleation. However, we revealed that unlike TUBG1, TUBG2 expression is downregulated in mouse preimplantation development. Mast cells represent effectors of the allergy reaction. Their activation by antigen induces number of cellular processes such as degranulation, proliferation and cytoskeleton rearrangements. The regulatory mechanisms of MT reorganization during mast cell activation are unknown. We identified new signaling proteins, GIT1 and PIX that interact with - tubulin. Depletion of GIT1 or PIX leads to changes in MT nucleation. GIT1 is phosphorylated on tyrosine and associates with γ-tubulin in a Ca2+ -dependent manner. Our data suggested a novel signaling pathway for MT rearrangement in mast cells where tyrosine kinase-activated GIT1 and βPIX work in concert with Ca2+ signaling to regulate MT nucleation. We tested the capability of GIT1 and PIX to influence -tubulin function in more cell types. We found out that GIT1/βPIX signaling proteins together...

The thesis focuses on the use of passive sampling during an analysis of hormones in drinkable water. In the theoretical part there are described selected passive samplers and the sample of the type of POCIS is described at fuller length. The next chapter engages with steroid hormones and an input of estrogenic hormone into the environment. The thesis contains chapters dealing with possibilities of the determination of hormones in water. It is specialized on the analysis of drinkable and surface water. In the last chapter of the theoretical part there are introduced water works where samples for an experimental assessment were collected from. The experimental part of the thesis presents the specification of the extraction of analytes and an adjustment of used device. In the thesis there are specific calibrations for individual assessment of hormones. The results of the assessment for each collection point are ordered to a well-arranged table. Increased levels of targeted estrogens were not approved in most of sampling points.