Sendo o GnRH a molécula capaz de iniciar a cascata hormonal reprodutiva, realizamos a clonagem, analisamos os efeitos da indução à reprodução e as funções dos GnRHs de A. altiparanae. Como resultados, obtivemos as sequências dos cDNAs do GnRH2 e GnRH3. Quando induzidos à reprodução, a desova foi às 20 h pós-estímulo (hpe) em fêmeas e às 16 hpe em machos, o aumento da expressão do mRNA de GnRH3 ocorreu às 8 hpe em fêmeas, e o aumento da expressão do mRNA de GnRH2 foi às 0 hpe em machos. Com relação ao efeito dos GnRHs, todos estimularam a expressão do mRNA de βLH, mas não de βFSH, e só o GnRH2 foi capaz de elevar o MIS e causar a desova. Como conclusão, temos que as sequências dos cDNAs dos GnRHs se mostraram conservadas; a indução à reprodução por redução do nível da água foi eficaz; em cativeiro, a espécie teve desenvolvimento assincrônico; o GnRH2 provavelmente está ligado ao comportamento reprodutivo, e o GnRH3 é a possível forma hipofisiotrópica. Por fim, só o GnRH2 desencadeou toda a cascata hormonal. / The study on reproduction in fish has been acquired great importance in last years, mainly for the benefit of threatened species. During the reproductive process, the hypothalamic neurons synthesize and release GnRH that stimulates the pituitary cells to release FSH and LH, which, in turn, promote the gonadal maturation. In fact, the morphological changes in gonads are the result of the endocrine action of the reproductive axis, in which the GnRH is the key molecule to starting the reproductive axis control. Thus, the knowledge about the GnRH, as well as about the gonadal morphological changes in the spawning might contribute to effectiveness of reproduction. Therefore, in this work, with Astyanax altiparanae as a model, we made the molecular characterization of the GnRHs, and we analyzed the gonadal morphological changes during the reproductive stimulus. In addition, we evaluated the role of injected GnRHs in vivo. As results, we obtain the cDNA complete sequence of preproGnRH2 (612bp) and preproGnRH3 (407bp) of A. altiparanae. Regarding the induction of reproduction by water level drawdown, the released of gametes occurred at 20 hours after stimulus in female and at 16 hours after stimulus in males, the mRNA expression of GnRH3 increased at 8 hours after stimulus in female and the mRNA expression of GnRH2 increased at 0 hours in males. Regarding the effects of injected GnRH, all of them stimulated the βLH but not βFSH mRNA expression, and only the GnRH2 was able to rise the MIS and stimulate the released of gametes. We conclude that the cDNAs sequences of preproGnRH2 and preproGnRH3 were conserved, although there is a change in the amino acid at the position 8 of the GnRH3 decapeptide of A. altiparanae. Furthermore, the induced reproduction by water level drawdown was effective, and in captivity, the A. altiparanae has an asynchronous development with splitted spawning during the breeding season. The analysis of the animals submitted to the reproductive stimulus allowed us to suggest that in A. altiparanae, the GnRH2 probably has a role in sexual behavior and the GnRH3 possibly is the hypophysiotropic form. Finally, analyzing the GnRH effects, we observed that only the GnRH2 was able to start the entire reproductive hormonal cascade, leading the animal to spawning.

A miostatina é um agente regulador negativo do crescimento muscular. Na terapia de suplementação com testosterona observou-se diminuição na expressão da miostatina. Este trabalho tem como objetivo determinar a influência do esteróides anabolizantes na expressão do gene da miostatina em camundongos normais, C57BL e no modelo distrófico Largemyd. Utilizamos a técnica de PCR em tempo real, para determinarmos a expressão relativa dos genes. Os animais tratados apenas com esteróide apresentaram aumento significativo em sua massa corpórea, com melhora de desempenho nas avaliações funcionais no Largemyd. Não foram observadas diferenças significativas na expressão do genes da miostatina no músculo normal e distrófico. Concluímos que o uso do esteróide anabolizante foi benéfico para o aumento na força do modelo Largemyd, mas o aumento de massa corpórea nestes animais, como no camundongo normal, não deveu-se a inibição da expressão da miostatina. / Myostatin is a negative regulator agent of muscle growth. In the testosterone supplementation therapy we observed decreased myostatin expression. The aim of this project is to determine the influence of anabolic steroids in the expression of myostatin gene in normal C57BL mice and in the dystrophic model Largemyd. We used the Real Time PCR assay to determine the relative expression of genes. Animals treated only with steroids presented significant increase in body mass and Largemyd showed improvement in the functional evaluations. There werent significant differences in the myostatin gene expression in the normal and dystrophic muscle. We concluded that the use of anabolic steroid was benefic to the increase of the strength in the Largemyd model, but the increase of body mass in these animals, as in the normal mice, is not related to the inhibition of myostatin expression.

O gênero Croton possui aproximadamente 1300 espécies amplamente distribuídas em zonas tropicais e subtropicais do novo e velho mundo e é o segundo maior gênero de Euphorbiaceae. No presente trabalho, foram analisadas a composição e a morfologia das ceras foliares cuticulares de 13 espécies de Croton. Para a maioria das espécies, foram amostrados três indivíduos. A morfologia das ceras foi analisada por microscopia eletrônica de varredura. Diversos tipos de depósitos cerosos foram observados (amorfos, plaquetas, grânulos), principalmente na face adaxial e caracterizando grupos de espécies. A observação dos padrões de depósitos cerosos de várias espécies foi prejudicada na face abaxial, devido à alta densidade de tricomas. As ceras foram extraídas por três imersões consecutivas das folhas em diclorometano e a separação das classes dos constituintes foi feita por cromatografia em camada delgada preparativa. A análise da distribuição dos constituintes foi realizada por cromatografia a gás acoplada a espectrometria de massas. n-Alcanos e álcoois primários foram detectados em todos os indivíduos analisados. Triterpenos também são muito comuns, não tendo sido detectados em apenas uma espécie. Os esteroides são constituintes raros nas espécies analisadas. Os resultados da distribuição de n-alcanos, álcoois primários e triterpenos foram utilizados para o estabelecimento de afinidades químicas por meio de análises de agrupamento pelo método UPGMA, empregando-se distâncias euclidianas (n-alcanos e álcoois) e coeficiente de DICE (triterpenos), baseando-se na distribuição de cada classe de constituinte isoladamente e combinando-se as distribuições de n-alcanos e álcoois primários. Não se notou congruência entre as topologias dos dendrogramas de afinidades químicas e a filogenia molecular do gênero. Observou-se coerência entre algumas afinidades químicas e características morfológicas, como tipos de tricomas foliares e presença de glândulas peciolares. A distribuição de n-alcanos, álcoois primários e triterpenos mostraram-se úteis como caracteres da maioria das espécies analisadas. Entre os constituintes analisados, distribuição de álcoois primários revelou-se o melhor marcador para a caracterização de espécies. A variação intraespecífica, no entanto, impede que esses caracteres sejam úteis como marcadores taxonômicos de algumas espécies de Croton / Croton comprises nearly 1300 species distributed in tropical and subtropical areas of either the New or Old Worlds, making up the largest genus of Euphorbiaceae. In the present work, the chemical composition and the morphology of the foliar cuticular waxes of 13 species of Croton were analized. Three individuals were sampled for analyses of most species. The morphology of the waxes was analyzed by scanning electron microscopy. Several types of wax deposits were observed (amorph, platlets, granules), chiefly on the adaxial side, characterizing groups of species. The observation of wax deposits on the abaxial side was hampered in several species due to the high density of trichomes. The cuticular wax was extracted by three successive immersions of the leaves in dichloromethane, and the separation of the constituent classes was achieved by preparative thin layer chromatography. The analyses of the distribution of the constituents were performed by gas chromatography coupled to mass spectrometry. n-Alkanes and primary alcohols were detected in all analyzed individuals. Triterpenes were also very common, having not been detected in only one species. Steroids are rare constituents in the analyzed species. The results of the distribution of n-alkanes, primary alcohols and triterpenes were used for the establishment of chemical affinities using cluster analysis by the UPGMA method and Euclidean distances (n-alkanes and alcohols) and DICE coefficient, based on the distribution of each constituent class alone and combining the distribution of n-alkanes and primary alcohols. No congruence was noted between the topologies of the dendrograms of chemical affinity and the molecular phylogeny of the genus. Coherence was observed between chemical affinities and morphologic characteristics, such as types of foliar trichomes and petiolar glands. The distribution of n-alkanes, primary alcohols and triterpenes were shown to be useful as characters for most analyzed species. Among the constituents analyzed, the distribution of primary alcohols was the best marker for species characterization. Intraspecific variation, however, precludes the use of these characters as taxonomic markers of some Croton species

Cette thèse s’inscrit dans le cadre de la thématique du Laboratoire de Pharmacognosie de l’UFR Pharmacie, au sein de l’Université de Bourgogne. Elle vise essentiellement la recherche de molécules d’origine végétale issues de la biodiversité tropicale dont principalement des saponines. Ces composés suscitent un grand intérêt de par leur large éventail d’applications pharmacologiques. Dans ce contexte l’étude de trois espèces végétales appartenant à deux familles, à savoir Chlorophytum blepharophyllum Schweinf. ex Baker, Ornithogalum dubium Houtt (Asparagaceae) et Weigela × « kosteriana variegata » (Caprifoliaceae), a conduit à l’isolement de 16 glycosides naturels par les techniques chromatographiques (Chromatographie liquide sous vide, Chromatographie d’exclusion moléculaire, Chromatographie liquide moyenne pression). Les structures de ces derniers ont été élucidées principalement par les techniques spectroscopiques de RMN1D et -2D, et de spectrométrie de masse. Il s’agit de 4 glycosides phénoliques de structure connue, 8 saponines stéroïdiques parmi lesquelles 6 sont de structure nouvelle, ainsi que 4 saponines triterpéniques dont une nouvelle. 5 saponines stéroïdiques ont été testées en vue d’évaluer leur activité cytotoxique sur deux lignées cellulaires cancéreuses (A549 et HL 60). Les résultats ont montré une faible sensibilité de ces deux lignées cellulaires à ces saponines. Les effets toxiques et tératogènes des 3 saponines triterpéniques ont été également déterminés à l’aide d'un test in vivo de poisson-zèbre (zebrafish). Les résultats ont montré un effet létal à de faibles concentrations des 3 saponines. Des relations structure/activité ont été ainsi proposées. / This thesis was carried out in the Laboratory of Pharmacognosy, in health department of the University of Burgundy. The principal theme of this Laboratory is the research of natural compounds from tropical biodiversity, mainly saponins. These molecules are known for their various pharmacological activities. The study of 3 species belonging to 2 different families: Chlorophytum blepharophyllum Schweinf. ex Baker, Ornithogalum dubium Houtt (Asparagaceae) and Weigela × « kosteriana variegata » (Caprifoliaceae), led to the isolation and characterization of 16 natural glycosides by column chromatography, medium pressure liquid chromatography, and vacuum liquid chromatography. The spectral analysis was achieved using mainly 2D NMR and mass spectrometry. Among them, 4 were phenolic glucosides, 8 were steroidic saponins with 6 new structures and 4 were triterpenic saponins with one new structure. The cytotoxic activities of 5 isolated steroidic saponins were evaluated on 2 strains cancer cells (A549 and HL 60). The results showed a low sensitivity of these two cell lines to these saponins. The toxic and teratogenic effects of 3 triterpenic saponins were also determined in by using an in vivo zebrafish assay. The results showed a lethal effect at low concentrations of these 3 saponins. Structure / activity relationships have been proposed.

<p>U prvom delu ove doktorske disertacije je izvr&scaron;ena<br />sinteza novih derivata androstana sa D-laktonskom<br />funkcijom ili D-seko derivata, sa modifikacijama u Ai/<br />ili B-prstenovima. U drugom delu je ispitana<br />antiproliferativna aktivnost odabranih<br />novosintetizovanih jedinjenja prema humanim<br />tumorskim ćelijskim linijama (MCF-7, MDA-MB-231,<br />PC-3, HeLa, HT-29, K562) kao i prema zdravim<br />ćelijama fetalnih fibroblasta pluća (MRC-5).</p> / <p>In first part of this work was achieved synthesis of&nbsp;some new androstane derivatives with D-lactone&nbsp;function and new D-seco derivatives with modification&nbsp;in A and/or B rings. In the second part the&nbsp;antiproliferative activity of selected newly synthesized&nbsp;compounds by human carcinoma cell lines (MCF-7,&nbsp;MDA-MB-231, PC-3, HeLa, HT-29, K562) and the&nbsp;healthy cells of fetal lung fibroblasts (MRC-5 ) was&nbsp;examined.</p>

<p>U prvom delu ove doktorske disertacije je izvr&scaron;ena sinteza&nbsp;novih 17&alpha;-pikolil i 17(E)-pikoliniliden androstanskih&nbsp;derivata, sa modifikacijama u A- i/ili B-prstenovima. U&nbsp;drugom delu rada je ispitana antiproliferativna aktivnost&nbsp;odabranih novosintetizovanih jedinjenja prema humanim&nbsp;tumorskim&nbsp; ćelijskim linijama (MCF-7, MDA-MB-231,&nbsp;PC-3, HeLa, HT-29 i A549) kao i prema zdravim ćelijama&nbsp;fetalnih fibroblasta pluća (MRC-5).</p> / <p>In the first part of this dissertation, the synthesis of new&nbsp;17&alpha;-picolyl and 17(E)-picolinylidene androstane&nbsp;derivatives with modifications in A and/or B rings, were&nbsp;presented. The second part of the work consisted on&nbsp;investigation of antiproliferative activity of selected newly&nbsp;synthesized compounds toward human carcinoma cell lines&nbsp;(MCF-7, MDA-MB-231, PC-3, HeLa, HT-29 and A549),&nbsp;as well as the healthy fetal lung fibroblasts (MRC-5).</p>

Adrenal steroid hormones, which regulate a plethora of physiological functions, are produced via tightly controlled pathways. Adrenal hormone excess associates with clinical conditions impacting metabolism and cardiovascular and immune function. Aldosterone and cortisol producing enzymes, CYP11B2 and CYP11B1, share 93% homology requiring highly selective drugs for pharmacological treatment. Investigations of these pathways, based on experimental data, can be facilitated by computational modeling for calculations of metabolic rate alterations. Such systems can be utilized in a variety of applications including investigating effects of endocrine-disrupting chemicals, drugs, gene manipulations. On a human level, regression modelling involving use of clinical data can contribute in defining effects of such diseases or providing reference data for characterizing normal values of physiological processes. The main subject of this thesis was the development of modeling techniques that would benefit basic and clinical research by supplying means for investigating adrenal related dysfunctions and disease. As a first approach, we used a model system, based on mass balance and mass reaction equations, to kinetically evaluate adrenal steroidogenesis in human adrenal cortex-derived NCI H295R cells. For this purpose a panel of 10 steroids was measured by liquid chromatographic-tandem mass spectrometry. Time-dependent changes in cell incubate concentrations of steroids were measured after incubation with angII, forskolin and abiraterone. Model parameters were estimated based on experimental data using weighted least square fitting. Time-dependent angII- and forskolin-induced changes were observed for incubate concentrations of precursor steroids with peaks that preceded maximal increases in aldosterone and cortisol. Inhibition of 17-alpha-hydroxylase/17,20-lyase with abiraterone resulted in increases in upstream precursor steroids and decreases in downstream products. Derived model parameters, including rate constants of enzymatic processes, appropriately quantified observed and expected changes in metabolic pathways at multiple conversion steps. Our data from our first approach demonstrated limitations of single time point measurements and the importance of assessing pathway dynamics in studies of adrenal cortical cell line steroidogenesis. Despite the benefits from a computational approach in comparison to the single time point studies, this kind of modeling demonstrated certain limitations regarding effort, reproducibility and costs. Therefor a second study was conducted in order to address such limitations. As a second approach we introduced an effective in vitro assay for evaluation of steroidogenic enzyme kinetics based on intracellular flux calculations. H295RA cells were cultured in chambers (µ-Slide, Ibidi) under constant medium flow. Four hourly samples were collected (control samples), followed by collections over an additional four hours after treatment with either fadrozole (10nM), metyrapone (10uM), ASI_191 (5nM), a novel CYP11B2 inhibitor or ASI_254 (100nM), a newly synthesized CYP17 inhibitor. Mass spectrometric measurements of multiple steroids combined with linear system computational modeling facilitated calculation of intracellular flux rates at different steroidogenic pathway steps and assessment of the selectivity of drugs for those specific steps. While treatment with fadrozole, metyrapone and ASI_191 all resulted in reductions in fluxes of aldosterone, corticosterone and cortisol production, treatment with ASI_254 led to increased flux through the mineralocorticoid pathway and increased production of aldosterone with reduced production of steroids downstream of CYP17. Comparisons of changes in intracellular fluxes revealed much higher selectivity of ASI_191 for CYP11B2 over CYP11B1 compared to fadrozole or metyrapone. Our study demonstrates the advantages of continuous culture systems over static systems for studying effects of steroidogenic inhibitors. By culturing cells under perfusion the methodology establishes a more realistic model for investigating drug effects, provides for simple and rapid calculations of intracellular fluxes and offers a robust method for drug screening or in vitro investigations of metabolic mechanisms. As a third approach we utilized LC-MS/MS derived plasma concentrations for each of 525 normotensive and hypertensive volunteers with (n=227) and without (n=298) hypertension in combination with regression modeling for the extraction of age and gender-adjusted reference intervals. Values of 8 steroids (pregnenolone, 11-deoxycorticosterone, corticosterone, 17-hydroxyprogesterone, cortisone, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androstenedione) versus age and gender were modelled via multivariate fractional polynomial analysis successfully providing with 0.5 and 99.5% reference intervals as a function of age and gender.:Contents Acknowledgements 4 Publication Note 5 Contents 6 Abbreviations 9 1. Introduction 10 1.1. The adrenal gland 10 1.1.1. Adrenal cortex steroids regulation 10 1.1.2. Physiological metabolic processes of steroidogenesis 13 1.2. Adrenal cortical dysfunction and therapeutic challenges 15 1.3. The adrenocortical cell line NCI –H295R 16 1.4. Chemical regulators of adrenal steroidogenesis 17 1.5. Computational mechanistic modelling of adrenal metabolism 18 1.5.1. Static cell culture models for characterizing pathway dynamics 18 1.5.2. Steady state as a tool for intracellular fluxes estimation 19 2. Hypothesis and aims 20 3. Materials and methods 22 3.1. Experimental overview 22 3.1.1. Computational mechanistic modeling of steroid metabolism 22 3.1.2 A steady state system for in vitro evaluation of steroidogenic pathway dynamics 24 3.1.3 Calculation of age and gender adjusted reference intervals for plasma adrenal steroids for healthy population 27 3.2 Liquid chromatography – tandem mass spectrometry 29 3.3. Static cell culture mechanistic model 29 3.3.1. Cell culture conditions 29 3.3.2. Computational model representation for steroid metabolism and cell proliferation using ODE systems 29 3.3.3. Metabolic pathways 30 3.3.4. Transport processes 32 3.3.5. Parameter estimation 34 3.4. Steady state model system for computation of pathway kinetics 35 3.4.1. Cell culture conditions for continuous flow culture system 35 3.4.2 Steroid secretion rates and intracellular fluxes calculation under steady state conditions 37 3.4.3 Statistical analysis 40 3.5. Regression and classification models for diagnostic clinical studies 40 3.5.1. Age and sex adjusted reference intervals for adrenal steroids in plasma – patient data collection 40 3.5.2. Mathematical description of multivariate fractional polynomial analysis 41 4. Results 43 4.1. Computational analysis of steroid profiling in NCI H295R cells 43 4.1.1. Transport and metabolic pathway modeling 43 4.1.2. Angiotensin II and forskolin stimulation 43 4.1.3. Abiraterone treatment 46 4.2. Steady state model for in vitro evaluation of steroidogenic pathway dynamics 48 4.2.1. Continuous flow culture steroid profiling 48 4.2.2. Secretion rates, intracellular flux rates and relative changes in rate constants 51 4.2.3. Cell number and viability 53 4.3. Reference intervals for adrenal steroid plasma concentrations 55 5. Discussion 61 Appendix A – Static culture model 70 A1. ODE system equations of the static culture model 70 A2. Jsim MML example code of the ODE system implementation of the static culture model 74 A3. Supplementary data for static culture model derived data 79 Appendix B – Steady state model 84 B1. Mathematical derivation of analytical solution for intracellular flux calculation of the steady state model 84 B2. Equations describing steroid production in cells and flowing medium of the steady state model 86 B3. Quadratic programming formulation and solution of upstream steroidogenic pathways system of the steady state model 91 B4. Calculation of reaction rate constant relative changes 92 B5. Python implementation of secretion rates, flux rates and rate constant relative changes calculation 94 B6. Numerical example of intracellular flux calculation. 98 B7. Supplementary material for steady state model derived data 103 Appendix C – Age and gender-adjusted reference intervals 110 Zusammenfasung 112 Summary 113 Literature 116

Kainate receptors belong to the family of glutamate receptors, which include NMDA, AMPA and δ receptors. Glutamate receptors are widely found in the brain and therefore they are very dynamically investigated, especially from view of pharmacology, because there is great potential for finding new and more specific modulators which could be used in the treatment of neurodegenerative diseases. The aim of this work was to extend the knowledge about the influence of neurosteroids on homomeric kainate receptors (GluK1, GluK2, GluK3) in which is the study of modulation by neurosteroids still at the beginning. We have investigated interactions of homomeric kainate receptors with selected neurosteroids (pregnenolone sulfate, pregnanolone sulfate, dehydroepiandrosterone, dehydroepiandrosterone sulfate) by using patch clamp method in the configuration of whole-cell recording and also by using microfluorometry. We have found out that the biggest modulating effect on homomeric kainate receptors is caused by pregnenolone sulfate, which inhibits glutamate responses of these receptors. Keywords kainate receptor, glutamate, neurosteroids, steroids, patch-clamp technique

Around the time of birth, the lung switches from a secretory- to a liquid absorptive organ to enable the fetus to transit from an intra-uterine to an air-breathing environment. This study concerns hormonal control of the liquid reabsorptive mechanism in the fetal lung which allows this transition to take place. Thyroidectomy in the fetal sheep at 118 days gestation (term = 147 days) prevented the development of adrenaline- or cyclic AMP-sensitivity which, in euthyroid fetuses, resulted in the capacity to absorb lung liquid from 130 days onwards. Studies in which T₃ and T₄ were infused to thyroidectornized fetal sheep showed that T₃ was required for the normal evolution of the reabsorptive response. However, infusion of this hormone to immature fetuses (110 days) did not advance the gestation at which adrenaline-sensitive absorption is first seen. Co-infusion of T₃ and hydrocortisone showed that these 2 hormones have a powerful synergistic effect on the absorption mechanism. Within a few hours of infusion of these 2 hormones to immature fetuses, a reabsorptive response to adrenaline similar to that normally seen in mature fetuses was observed. This response was fully reversible on withdrawal of T₃ and hydrocortisone infusion, and the hormonal effect was blocked by the protein synthesis inhibitor, cyclohexirnide. These findings suggest that the normal rise in T₃ and cortisol seen in the fetus in late gestation is responsible for maturation of the liquid absorption mechanism which allows the fetus to make a transition to an independent air-breathing existence. These observations may be of significance in the clinical management of infants born prematurely, who may have had insufficient pre-natal exposure to T₃ and cortisol.

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...