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...

This dissertation describes efforts to improve the oxidative capacity of n-alkane- induced Beauveria bassiana; a fungus and a versatile whole cell biocatalyst used in the biotransformation of steroids. n-Hexadecane was used as the carbon source during the growth of B. bassiana, presumably to induce the expression of oxidative enzymes, thus enhancing the oxidation of unactivated carbons. Dehydroepiandrosterone (DHEA) is an essential endogenous male-hormone and serves as a metabolic intermediate in the production of more potent androgens. Using DHEA as a substrate also provides the opportunity to study the hydroxylation of an unfunctionalized carbon, an attractive reaction that produces valuable intermediates for chemical synthesis. Results showed that exposing and inducing cells in n-hexadecane improves the synthesis of 11α-hydroxy derivatives. Reactions were carried out with cells grown on n-hexadecane, resulting in 65 ± 6.3 % conversion of DHEA to androstenediol (40.3% mM) and 3β,11∝,17β- trihydroxyandrost-5-ene (22.8% mM), as determined by HPLC, NMR and LCMS analyses. However, experiments with non-induced cells resulted in a poor substrate conversion (17%). To extend use of B. bassiana to pharmaceutical applications, it was necessary to optimize reaction conditions such as biocatalyst preparation, substrate concentration, agitation reaction temperature and pH. Higher substrate conversion, selectivity and yield of desired product were achieved with the reactor arrangement of “Resting Cells”. The apparent rate of reaction fits a Michaelis-Menten kinetic model with a maximum reaction rate of 4.45 mM/day, revealing that the transformation of intermediate androstenediol to desired 3β,11∝,17β-trihydroxyandrost-5-ene is the limiting step in the reaction. Interestingly, when a diluted amount of substrate was used, a higher yield of 11∞-hydroxy steroid was achieved. Also, reactions at 26°C with pH ranges between 6.0 and 7.0, resulted in the highest conversion (70%) and the higher product yield (45.8%). The maximum conversion of DHEA (71%) was achieved in experiments with high biomass loading, and the increment of desired product yield (11∝-hydroxy) was directly proportional to the amount of biomass used. Moreover, a high VMax/KM value was achieved with high biomass yields. Interestingly, the changes in biomass yield did not have a considerable effect on reaction selectivity. The main drawbacks of biocatalysis for production of steroids were addressed and approaches to minimize the drawbacks have been presented. The production of desired product (11∝-DHEA) was significantly improved using cells previously adapted to n-hexadecane.

La consommation frauduleuse de nandrolone est détectée dans les urines par la présence de ses métabolites isomères norandrostérone et norétiocholanolone. Afin de contrôler si, chez la femme, une activité sportive peut être responsable de l'augmentation de leurs productions endogènes, ainsi que de celles de la déhydroépiandrostérone et de l'androstérone, une méthode d'analyse urinaire faisant intervenir trois extractions sur phase solide suivies d'une analyse par GC-MS a été mise au point. Une correction des concentrations brutes en analytes a été appliquée par rapport au taux de créatinine, marqueur de la dilution des urines. Le traitement statistique des résultats obtenu à partir de 400 urines n'a montré aucun effet de l'exercice sur les concentrations en métabolites de la nandrolone, mais un effet modéré de l'exercice aérobie sur les concentrations en androstérone. Le statut hormonal des individus (phase du cycle menstruel et prise de contraceptif) a également été évalué. <br />Une amélioration de l'extraction a été ensuite proposée par la synthèse d'une phase stationnaire polymérique à impression moléculaire (MIP). Le MIP a été synthétisé dans des conditions favorisant la mise en place d'interactions non covalentes avec l'androstérone, ces mêmes interactions étant ensuite mises à contribution dans la rétention de la norandrostérone et de ses isomères lors de la percolation d'un échantillon sur la phase d'extraction. Conduisant à des résultats prometteurs, la technique semble intéressante en vue de l'analyse de traces de métabolites de la nandrolone grâce à une amélioration de la sélectivité de la méthode globale d'analyse

Rapid increases in plasma androgens are generally associated with short-term aggressive challenges in many breeding vertebrates. However, some animals such as song sparrows (Melospiza melodia) are aggressive year-round, even during the non-breeding season, when gonads are regressed and systemic testosterone (T) levels are non-detectable. In contrast, levels of the prohormone dehydroepiandrosterone (DHEA) are elevated year-round in the plasma and brain. The local conversion of brain DHEA to potent androgens may be critical in regulating non-breeding aggression. 3β-hydroxysteroid dehydrogenase/Δ4-Δ5 isomerase (3β-HSD) catalyzes DHEA conversion to androstenedione (AE) and the cofactor NAD⁺ assists in this transformation. In this thesis, I asked whether brain 3β-HSD activity is regulated by social encounters in seasonally breeding male songbirds. In Experiment 1, I looked at the long-term seasonal regulation of brain 3β-HSD activity. 3β-HSD activity was highest in the non-breeding season compared to the breeding season and molt. In Experiment 2, I hypothesized that brain 3β-HSD activity is rapidly regulated by short-term social encounters during the non-breeding season. A 30 min social challenge increased aggressive behavior. Without exogenous NAD⁺, there was ~355% increase in 3β-HSD activity in the caudal telencephalon and ~615% increase in the medial central telencephalon compared to controls (p<0.05). With exogenous NAD⁺, there was no effect of social challenge on 3β-HSD activity. These data suggest that endogenous cofactors play a critical role in the neuroendocrine response to social challenges. The increase in brain DHEA conversion to AE during social challenges may be a mechanism to rapidly increase local androgens in the non-breeding season, when there are many costs of systemic T.

Classically, testosterone (T) was considered the principal regulator of aggression. However, recent studies in birds have found aggression and T uncoupled during the non-breeding season. Circulating testosterone comes with costs such as immunosuppression and energy expenditure. Instead, the pro-hormone, dehydroepiandrosterone (DHEA), is circulated and activated within the brain via conversion into oestradiol (E2), avoiding the costs associated with T. At present the site of DHEA synthesis is unknown. My thesis investigated the existence of an analogous pathway in non-breeding red squirrels (Tamiasciurus hudsonicus) with two studies: (a) a field study investigating the effects of E2 on aggression and (b) a laboratory study that attempted to determine the site of DHEA synthesis. I conclude that E2 regulates non-breeding aggression in red squirrels and that the adrenals are not the site of DHEA synthesis. My results suggest the existence of a mammalian analogue to the regulatory pathway found in birds.

Classically, testosterone (T) was considered the principal regulator of aggression. However, recent studies in birds have found aggression and T uncoupled during the non-breeding season. Circulating testosterone comes with costs such as immunosuppression and energy expenditure. Instead, the pro-hormone, dehydroepiandrosterone (DHEA), is circulated and activated within the brain via conversion into oestradiol (E2), avoiding the costs associated with T. At present the site of DHEA synthesis is unknown. My thesis investigated the existence of an analogous pathway in non-breeding red squirrels (Tamiasciurus hudsonicus) with two studies: (a) a field study investigating the effects of E2 on aggression and (b) a laboratory study that attempted to determine the site of DHEA synthesis. I conclude that E2 regulates non-breeding aggression in red squirrels and that the adrenals are not the site of DHEA synthesis. My results suggest the existence of a mammalian analogue to the regulatory pathway found in birds.

Rapid increases in plasma androgens are generally associated with short-term aggressive challenges in many breeding vertebrates. However, some animals such as song sparrows (Melospiza melodia) are aggressive year-round, even during the non-breeding season, when gonads are regressed and systemic testosterone (T) levels are non-detectable. In contrast, levels of the prohormone dehydroepiandrosterone (DHEA) are elevated year-round in the plasma and brain. The local conversion of brain DHEA to potent androgens may be critical in regulating non-breeding aggression. 3β-hydroxysteroid dehydrogenase/Δ4-Δ5 isomerase (3β-HSD) catalyzes DHEA conversion to androstenedione (AE) and the cofactor NAD⁺ assists in this transformation. In this thesis, I asked whether brain 3β-HSD activity is regulated by social encounters in seasonally breeding male songbirds. In Experiment 1, I looked at the long-term seasonal regulation of brain 3β-HSD activity. 3β-HSD activity was highest in the non-breeding season compared to the breeding season and molt. In Experiment 2, I hypothesized that brain 3β-HSD activity is rapidly regulated by short-term social encounters during the non-breeding season. A 30 min social challenge increased aggressive behavior. Without exogenous NAD⁺, there was ~355% increase in 3β-HSD activity in the caudal telencephalon and ~615% increase in the medial central telencephalon compared to controls (p<0.05). With exogenous NAD⁺, there was no effect of social challenge on 3β-HSD activity. These data suggest that endogenous cofactors play a critical role in the neuroendocrine response to social challenges. The increase in brain DHEA conversion to AE during social challenges may be a mechanism to rapidly increase local androgens in the non-breeding season, when there are many costs of systemic T.

In our study, we analyzed four stress markers (cortisol, cortisone, DHEA and DHEAS) in blood serum in young sows using minimally invasive heart catheterisation as the stress factor. The marker levels were assessed in four defined periods of the experiment, beginning with the baseline level on the day before intervention (1), the second period was after the introduction of anaesthesia (2), the third was after conducting tissue stimulation or ablation (3), and the final period was after the end of the catheterisation (4). Cortisol and cortisone were detected using HPLC method, DHEA(S) by commercial kits. For statistical analyses non-parametric tests were used (due to non standard Gaussian data distribution). In our study we arranged these experiments: 1. Diurnal variability in these markers concentration during heart catheterisation was tested. 2. Are there differences between stress markers concentration the day before experiment (sampling 1) and in the day of the catheterisation (samplings 2, 3, 4)? 3. Are there differences between these markers concentration during operation (the first sampling was excluded)? 4. The cortisol/cortisone ratio was calculated. We found only minimal statistical differences in studied markers between the morning and afternoon group (p > 0.05) in experiment 1. For tested...

Rapid increases in plasma androgens are generally associated with short-term aggressive challenges in many breeding vertebrates. However, some animals such as song sparrows (Melospiza melodia) are aggressive year-round, even during the non-breeding season, when gonads are regressed and systemic testosterone (T) levels are non-detectable. In contrast, levels of the prohormone dehydroepiandrosterone (DHEA) are elevated year-round in the plasma and brain. The local conversion of brain DHEA to potent androgens may be critical in regulating non-breeding aggression. 3β-hydroxysteroid dehydrogenase/Δ4-Δ5 isomerase (3β-HSD) catalyzes DHEA conversion to androstenedione (AE) and the cofactor NAD⁺ assists in this transformation. In this thesis, I asked whether brain 3β-HSD activity is regulated by social encounters in seasonally breeding male songbirds. In Experiment 1, I looked at the long-term seasonal regulation of brain 3β-HSD activity. 3β-HSD activity was highest in the non-breeding season compared to the breeding season and molt. In Experiment 2, I hypothesized that brain 3β-HSD activity is rapidly regulated by short-term social encounters during the non-breeding season. A 30 min social challenge increased aggressive behavior. Without exogenous NAD⁺, there was ~355% increase in 3β-HSD activity in the caudal telencephalon and ~615% increase in the medial central telencephalon compared to controls (p<0.05). With exogenous NAD⁺, there was no effect of social challenge on 3β-HSD activity. These data suggest that endogenous cofactors play a critical role in the neuroendocrine response to social challenges. The increase in brain DHEA conversion to AE during social challenges may be a mechanism to rapidly increase local androgens in the non-breeding season, when there are many costs of systemic T. / Science, Faculty of / Zoology, Department of / Graduate

In our study, we analyzed four stress markers (cortisol, cortisone, DHEA and DHEAS) in blood serum in young sows using minimally invasive heart catheterisation as the stress factor. The marker levels were assessed in four defined periods of the experiment, beginning with the baseline level on the day before intervention (1), the second period was after the introduction of anaesthesia (2), the third was after conducting tissue stimulation or ablation (3), and the final period was after the end of the catheterisation (4). Cortisol and cortisone were detected using HPLC method, DHEA(S) by commercial kits. For statistical analyses non-parametric tests were used (due to non standard Gaussian data distribution). In our study we arranged these experiments: 1. Diurnal variability in these markers concentration during heart catheterisation was tested. 2. Are there differences between stress markers concentration the day before experiment (sampling 1) and in the day of the catheterisation (samplings 2, 3, 4)? 3. Are there differences between these markers concentration during operation (the first sampling was excluded)? 4. The cortisol/cortisone ratio was calculated. We found only minimal statistical differences in studied markers between the morning and afternoon group (p > 0.05) in experiment 1. For tested...