In addition to their conventional role as hormones, studies have shown that steroids can act as pheromones in mammals. Emphasis has been placed on evaluating the physiological and behavioural effects of male, urinary 17β- estradiol (E2) exposure in pheromone phenomena including the prevention of embryo implantation and induced precocious puberty in females. Steroids have also been observed to transfer between female mice, leading to changes in the duration of their estrous cycle. Progesterone (P4), a crucial female sex steroid, promotes pro-social sexual reproductive behaviour and the growth of the endometrium in preparation for ovum implantation. Few studies have investigated the effects of P4 in a pheromonal context. Big brown bats (Eptesicus fuscus) are ideal models for pheromone research because they are evolutionarily distinct from rodents, live in highly social and sexually-competitive harems, and are regularly exposed to conspecific secretions in the close confines of their roost. Experimental analysis revealed absorption of tritium-labeled progesterone (3H-P4) (10 μCi) 1 h after cutaneous and intranasal application to adult females. Additionally, radioactivity was observed in mature female bats caged for 48 h with an adult female conspecific that had been intraperitoneally-injected with 3H- P4 (50 μCi). Using the same paradigm, 3H-E2 transfer was not observed between females. Enzyme-linked immunosorbent assays revealed measurable levels of unconjugated P4 and E2 present in the urine of female bats, suggesting urine as one likely vector for P4 transfer. Given corroborative findings in mice, progesterone transfer during cohabitation is likely a mammalian-wide phenomenon that could have evolved to prime conspecifics—and more specifically kin—for sexual reproduction. / Thesis / Master of Science (MSc) / Steroid molecules are conventionally assumed to act solely within the individual that produced them; however, recent experiments have demonstrated that the sex steroid 17β-Estradiol (E2) can be excreted in the urine of adult male mice and taken up into the neural, reproductive, and peripheral tissues of cohabitating females. This exogenous E2 can result in changes to female physiology and behaviour. Our lab has observed E2 to transfer between male and female captive big brown bats during the mating season. The current project aimed to determine whether E2 transfers between captive cohabitating female bats. We also examined whether progesterone (P4), an important female sex steroid involved in the preparation and maintenance of pregnancy, transfers between female bats. We determined that P4 reliably transfers between female bats, but E2 does not. Bioactive E2 and P4 were measured in the urine of non- pregnant female bats and propose urine as one likely vector of P4 transfer between cohabitating individuals.

La photodégradation du bêta-estradiol (E2), un perturbateur chimique endocrinien, est réalisée en présence d'un complexe de fer, d'argiles et d'un oxyde de fer. L'impact d'un agent complexant du fer l'acide éthylénediamine-N,N'-disuccinique (EDDS) est aussi étudié. Après la détermination des propriétés physico-chimiques du complexe Fe(III)-EDDS, les rendements quantiques de formation des °OH et de dégradation de E2 ont été évalués en fonction de différents (pH, [O2], [Fe(III)-EDDS], [Fe(III)]. Pour la première fois, les rendements quantiques de production d'°OH ont été mesurés via la photolyse du complexe Fe(III)-EDDS en utlilisant l'acide téréphtalique comme sonde. Dans une seconde partie, les processus d'adsorption et de dégradation photocatalytique de E2 dans des suspensions de Montmorillonite KSF, de Montmorillonite naturel et de Goethite sont étudiés. L'adsorption de E2 sur les minéraux est rapide et faible. La vitesse de photodégradation de E2 est influencée par la concentration en minéraux et le pH. Le processus de dégradation photocatalytique de E2 dans ces systèmes a par la suite été étudié en présence d'EDDS. Dans les trois suspensions et en présence d'EDDS, la photodégradation de E2 augmente significativement dans la zone de pH neutre et basique (de 5,0 à 9,0). Au contraire, sans EDDS le pH optimal est limité aux pHs acides ( entre 3,0 et 4,0). Les cinétiques de dégradation de E2 suivent une loi de vitesse du modèle Langmuir-Hinshelwood pour les trois systèmes. Sur la base de nos résultats, il est possible de conclure que les systèmes EDDS-Fe(III)/minéraux sont photocatalytiquement efficaces pour l'élimination de polluants dans l'eau

Philosophiae Doctor - PhD / In this work, DNA aptamer biosensor systems were developed for the detection of l7p-estradiol - an estrogeneous endocrine disrupting chemical (EDC). Endocrine disrupting chemicals are group of compounds that impact negatively on the endocrine system of humans and wildlife. High concentrations of l7p-estradiol in water or food chain disrupts the physiology of the endocrine system of various animal species, leading to feminisation in fish and stimulates the proliferation of cancer cells in humans. Aptasensor systems for the determination of l7pestradiol were prepared with three immobilization platforms: (i) poly(3,4- ethylenedioxythiophene) {PEDOT} doped with gold nanoparticles (AuNPs) to form PEDOTIAuNPs polymeric nanocomposite, (ii) generation 1 poly(propylene thiophenoimine)-copoly( 3 ,4-ethy lenedioxythiophene) dendritic star copolymer (G 1PPT -co-PEDOT), and (iii) generation 2 poly (propylene thiophenoimine)-co-poly(3,4-ethylenedioxythiophene) dendritic star copolymer (G2PPT-co-PEDOT). The morphological properties of the sensor platforms were interrogated by scanning emission microscopy (SEM) and atomic force microscopy (AFM), while their spectroscopic characteristics were studied by Fourier transform infra red spectroscopy (FTIR) and fluorescence spectroscopy. The electrochemical behaviour of the platforms and the aptasensors were studied by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square wave voltammetry (SWV). The DNA aptamer developed for detecting 17~-estradiol and which was used in the fabrication of all aptamer biosensors in this study is a 76-mer biotinylated aptamer (5'-BiotinGCTTCCAGCTTATTGAATTACACGCAGAGG TAGCGGCTCTGCGCATTCAATGCTGCGCGCTGAAGCGCGGAAGC-3'). AulPEDOTIAuNPslAptamer (platform 1) was obtained by covalently attaching streptavidin to the polymeric nanocomposite platform using carbodiimide chemistry and the aptamer immobilized via streptavidin-biotin interaction. The electrochemical signal generated from the aptamer-target molecule interaction was monitored electrochemically using cyclic voltammetry and square wave voltammetry in the presence of [Fe(CN)6J 3-/4- as a redox probe. The signal current observed was inversely proportional to the concentration of 17Bestradiol. The aptasensor demonstrated specificity toward 17~-estradiol. The detectable concentration range of the 17B estradiol was 0.01 nM-O .09 nM with a detection limit of 3.2 pM. The 76-mer biotinylated aptamer for 17~-estradiol was incorporated into a generation 1 poly(propylenethiophenoimine )-co-poly(3 ,4-ethylenedioxythiophene) dendritic star copolymer modified Au electrode via biotin-avidin interaction (platform 2). The Bode plot shows that the charge transfer dynamics of the nanoelectrode can be frequency modulated while the AulG 1PPTco- PEDOT nanoelectrode exhibited greater semi-conductor behavior (higher phase angle value) than AulG 1PPT due to the incorporation of charged functionalized dendrimer at low frequencies (100 mHz). The biosensor response to 17~-estradiol was based on the decrease in the SWV current as the EDC binds to the ssDNA aptamer on the biosensor. The dynamic linear range of the sensor was 0.01-0.07 nM with a detection limit of7.27 pM. Synthesis of electro synthetic generation G2PPT-co-PEDOT (platform 3) was performed by copolymerization of PEDOT with G2PPT dendrimer modified electrode immersed in a solution of 0.1 M LiCI04 containing 0.1 M EDOT monomer and 0.1 M sodium dodecyl sulphate (SDS) for ten (10) cycles. The electrochemical behaviour of the dendritic star copolymer was investigated with CV and EIS in LiCI04 and phosphate buffer solutions. The results show that the electrochemical deposition of G2PPT-co-PEDOT on gold electrode decreased the electrochemical charge transfer resistance when compared to AuiPEDOTILiCI04 and AuiLiCI04 interfaces. Bode impedimetric analysis indicates that G2PPT-co-PEDOT is a semiconductor. The fabrication of two novel aptasensors (based on platforms 2 and 3) simultaneously on a screen printed micro array electrode of 96-well multichannel electrochemical robotic sensor testing system for the detection of endocrine disrupting l7~-estradiol, was also carried out. The aptasensors responses to l7~-estradiol, based on the decrease in the SWV current, were evaluated.

Hormonaly active compounds in wastewaters represent nowdays a serious problem. Proceses currently used in watewater treatment plants (WWTP) are unefficient in removing these compounds from contaminated wastewaters. The compounds are supposed to sorb onto solid sludge elements and sediments. In this work seven endocrine disruptors were detected in the sludge samples from WWTPs. A new sensitive method for detection of seven selected endocrine disruptors (4-nonylphenol, bisphenol A, estriol, 17β-estradiol, estrone, 17α- ethynylestradiol, irgasan) was developed. The method is based on accelerated solvent extraction (ASE), gel permeation chromatography (GPC) and solid phased extraction. For final extract analysis, gas chromatography coupled with mass spectrometry (GC/MS) was used. The efficiency of this method was tested using artificially contaminated sludge and the method was used to analyse real samples from several WWTPs in Czech Republic. The effect of sludge age on detection of individual analytes was assessed as well. The concentrations of endocrine disruptors measured in the samples reached up to 1 µg/g. The results are comparable or higher then those reported in other works and they show the necessity of further research on endocrine disruptors in the environment.