De nombreuses affections chez l’enfant et l’adulte sont associées à une exposition in utero à des perturbateurs endocriniens (PE). Pour réduire cette exposition, des programmes d’éducation pour la santé environnementale périnatale se développent. Ils font évoluer des dimensions psychosociales que sont la perception du risque (PR) et la croyance en l’action de réduction de l’exposition (CAR), mais peu d’entre eux sont évaluésL’étude de recherche interventionnelle PREVED a pour objectif d’évaluer l’efficacité d’un programme d’éducation pour la santé environnementale pour des femmes enceintes, visant à diminuer leur exposition aux PE.Les objectifs de ces travaux métrologiques étaient de développer des outils (i) analytiques et (ii) épidémiologiques en vue d’évaluer l’efficacité du programme. Ainsi, (i) des méthodes analytiques ultrasensibles par LC-MS/MS permettant de doser les fractions non conjuguées de PE dans les urines et le colostrum ont été développées et validés sur des prélèvements recueillis dans le cadre de la cohorte périnatale EDDS; (ii) un questionnaire psychosocial explorant la PR, la CAR et les connaissances des femmes enceintes au moyen de scores a été conçu.Ces travaux s’inscrivent dans une approche interdisciplinaire de santé environnementale. Ils proposent des méthodes analytiques fiables pour mesurer l’exposition aux PE étudiés d’une part, et un questionnaire évaluant connaissances, attitudes et pratiques des femmes enceintes sur la question des PE d’autre part. Ces outils permettront de mesurer l’impact du programme d’éducation pour la santé environnementale périnatale. / Childhood and adulthood diseases are associated to in utero exposure to endocrine disrupting chemicals (EDCs). To reduce this exposure, environmental health education programs dedicated to perinatal period are implemented. These programs change psychosocial dimensions such as risk perception (RP) and belief in the action of exposure reduction (BAR), but few are assessed.The interventional research PREVED study aims to assess efficacy of a perinatal environmental health education program to reduce pregnant women exposure to EDC.The objectives of this work were to develop analytical (i) and (ii) epidemiological tools to evaluate the efficacy of the program. Thus, (i) ultrasensitive LC-MS/MS analytical methods were developed to determine unconjugated fractions of EDCs in urine and colostrum and validated using samples collected from the EDDS perinatal cohort; (ii) a psychosocial questionnaire exploring RP, BAR and knowledge of pregnant women by means of scores was developed.This work, part of an interdisciplinary approach to environmental health, proposes reliable analytical methods to assess exposure to the studied EDCs on the one hand, and a questionnaire assessing knowledge, attitudes and practices of pregnant women about EDCs on the other hand. The impact of the perinatal environmental health education program will be assessed using these tools.

O alumínio (Al) é o metal mais abundante na natureza e tornou-se importante poluente aquático trazendo prejuízos a reprodução de teleósteos, atuando como um desregulador endócrino. No entanto, em experimentos in vivo não é possível demonstrar que os efeitos do Al no eixo endócrino reprodutivo são devido a sua atuação direta sobre os órgãos que o compõem. Por isso, este trabalho teve como objetivo avaliar o efeito direto do Al sobre células foliculares ovarianas, gonadotrópicas e somatolactínicas hipofisárias de fêmeas maduras de Oreochromis niloticus. Para isso dois experimentos in vitro de exposição ao Al foram realizados: um utilizando-se ovários maduros e outro hipófises de fêmeas sexualmente maduras. Para os experimentos ovarianos, frações de ovários maduros foram incubadas por 4 horas formando os seguintes grupos: 1) Grupo controle (Ctr): tecido ovariano exposto somente à solução de Krebs-Ringer-glucose-HEPES; 2) Grupo gonadotropina coriônica humana (hCG): tecido exposto à 6 µg/ml de hCG (Ovidrel); 3) Grupo (hCG+Al): tecido exposto à 6 µg/ml de hCG (Ovidrel) + cloreto de alumínio (AlCl3) - 10mM; 4) Grupo (Al): exposto somente ao AlCl3 - 10mM. A concentração dos hormônios 17β-estradiol (E2) e 17α-hidroxiprogesterona (17αOHP) no meio de incubação foi determinada por ELISA. Para os experimentos hipofisários, hipófises de fêmeas sexualmente maduras foram incubadas por 24 horas formando os seguintes grupos: 1) Ctr: hipófise exposta somente ao meio L15 (controle interno); 2) GnRH: hipófise exposta somente ao GnRH (controle de liberação de gonadotropinas); 3) GnRH+Al: hipófise exposta ao GnRH + AlCl3 10mM e 4) Al: hipófise exposta somente AlCl3 10mM. Após o período experimental, foram realizadas análises de qPCR (PCR quantitativo), análises de imunohistoquímica, e de microscopia eletrônica. Os resultados do experimento ovariano mostraram que os fragmentos ovarianos do grupo exposto à hCG apresentaram um aumento significativo da liberação dos hormônios E2 e 17αOHP em relação aos demais grupos, confirmando o efeito desta gonadotropina sintética na liberação destes esteroides gonadais. No entanto, a administração conjunta da hCG com Al (hCG+Al) não gerou este aumento da produção dos esteroides em relação ao grupo controle. Estes dados evidenciam que o Al inibiu a resposta celular das células esteroidogênicas ovarianas às gonadotropinas. Os resultados dos experimentos hipofisários mostraram que o Al (GnRH+Al e Al) afetou a expressão gênica dos genes estudados (βFSH, (βLH, SL) inclusive dos normalizadores (EF1α e (βAc), o que tem sido comum em experimentos de ecotoxicologia. Os dados de microscopia eletrônica mostraram desestruturação celular nas hipófises que foram expostas ao Al e as análises de imunohistoquímica apontaram que o Al (GnRH+Al e Al) não interferiu sobre a quantidade de grânulos de βLH e SL, enquanto o grupo Al indicou uma diminuição na quantidade de grânulos de βFSH, sugerindo que o Al afeta a dinâmica de síntese/liberação desta gonadotropina. Estes dados evidenciam a toxicidade do Al diretamente sobre ambos os órgãos estudados, tanto em nível de resposta celular quanto em nível estrutural confirmando o potencial do Al como um DE e amplia as perspectivas de estudo sobre o mecanismo de ação do Al como um DE / Aluminum (Al) is the most abundant metal in nature and has become an important water pollutant impairing reproduction of teleosts, acting as an endocrine disrupter. However, in vivo experiments cannot demonstrate that the effects of Al on the reproductive endocrine axis are due to direct action on the organs that compose it. Therefore, this study aimed to assess the direct effect of Al on ovarian follicular cells, gonadotropic and somatolactin pituitary cells of mature females of O. niloticus. For this, two in vitro exposure experiments of Al were performed: one using mature ovaries and other using pituitaries of sexually mature females. For ovarian experiments, fractions of mature ovaries were incubated for 4 hours to obtain the following groups: 1) control group (Ctr): ovarian tissue only exposed to Krebs- Ringer-HEPES-glucose; 2) human chorionic gonadotropin (hCG) group: tissue exposed to 6 mg/ml hCG (Ovidrel); 3) hCG + Al group: tissue exposed to 6 mg/ml hCG (Ovidrel) + aluminum chloride (AlCl3) - 10mM; 4) Al group: only exposed to 10 mM AlCl3. The concentration of the hormones 17β-estradiol (E2) and 17α-hydroxyprogesterone (17αOHP) in the incubation medium was determined by ELISA. For pituitary experiments, pituitaries of sexually mature females were incubated for 24 hours to form the following groups: 1) Ctr: pituitary exposed only to L15 (internal control); 2) GnRH: only exposed to the pituitary GnRH (gonadotropin releasing hormone); 3) GnRH + Al: exposed to the pituitary GnRH AlCl3 + 10 mM and 4) Al: 10mM AlCl3 only exposed pituitary. After the assay period, analysis of qPCR (quantitative PCR), analysis of immunohistochemistry and electron microscopy were performed. The results of the experiment showed that the ovarian exposed to hCG group showed a significant increase in the release of E2 and 17αOHP compared to the other groups, confirming the effect of synthetic gonadotropin in the release of these gonadal steroids. However, the administration combined of hCG with Al (Al + hCG) did not generate this increased production of steroids compared with the control group. These data show that Al inhibited the cellular response of the ovarian steroidogenic cells to gonadotropins. The results of the experiments with pituitaries showed that Al (GnRH + Al and Al) affected the gene expression of the genes studied (βFSH, (βLH, SL) including the house keeping genes (EF1α and βAc), what has been common in ecotoxicology experiments. Data from electron microscopy showed cell disruption in the pituitary glands that were exposed to Al and immunohistochemical analyzes showed that Al (GnRH + Al and Al) did not affect the amount of granules of βLH and SL, while the Al group indicated a decrease the amount of βFSH granules, suggesting that Al affects the dynamics of the synthesis/release of this gonadotropin. These data show the toxicity of Al directly on both organs studied, at both the cellular response as for structural level confirming the potential of Al as a DE and increases the perspectives of study on the mechanism of action of Al as a DE

<p>Uvod: Ftalati su vrsta endokrinih disruptora koji se &scaron;iroko koriste kao plastifikatori i rastvarači ali i kao aditivi brojim proizvodima koji se svakodnevno koriste. Prema dosada&scaron;njim studijama na eksperimentalnim životinjama, ali i u humanoj populaciji, diestri ftalne kiseline utiču na reproduktivni sistem, učestvuju u nastanku insulinske rezistencije i gojaznosti. Sindrom policističnih ovarijuma (PCOS) je najče&scaron;ći endokrinolo&scaron;ki poremećaj reproduktivnog sistema žena u generativnom periodu. Insulinska rezistencija i centralna gojaznost, kao faktori kardiometaboličkog sindroma imaju značajnu ulogu u etiologiji PCOS. Ciljevi: Utvrditi prisustvo metabolita ftalne kiseline kod žena u reproduktivnom periodu u na&scaron;oj sredini, kao i da li postoji povezanost ovih endokrinih disruptora sa antropometrijskim parametrima, parametrima glikoregulacije, lipidima i lipoproteinima seruma, gonadotropinima, polnim hormonima, leptinom i indeksima kardiometaboličkog rizika kod žena sa sindromom policističnih jajnika. Metode: Istraživanje je obuhvatalo 61 ženu sa sindromom policističnih jajnika koje su podeljene u dve podgrupe: 31 ispitanica sa PCOS i prisutnim ftalatnim metabolitima u urinu i 30 ispitanica sa PCOS bez ftalatnih metabolita u urinu. Kontrolnu grupu je činilo 30 zdravih žena. Kod svih ispitanica vr&scaron;ena su antropometrijska merenja (TV, TM, OS) i određivane vrednosti parametara glikoregulacije (glikemija i insulinemija, izačunat je HOMA index), lipidi i lipoproteini seruma (ukupan holesterol, trigliceridi, LDL i HDL holesterol), gonadotropini (LH i FSH), polni hormoni (estradiol i testosteron) i leptin. U proceni kardiometaboličkog rizika kori&scaron;ćena su dva indeksa LAP i VAI. Iz jutarnjeg uzorka urina određivano je prisustvo i koncentracija 10 ftalatnih metabolita: Mono-metil ftalat- MMP, Mono-etil ftalat - MEP, Mono-n-butil ftalat - MBP, Mono-n-propil ftalat- MPP, Mono-izo-amil ftalat - MiAP, Mono-n-amil ftalat - MnAP, Mono-cikloheksil ftalat - MCHP, Monobenzil ftalat- MBzP, Mono-(2-etilheksil) ftalat- MHEP, Mono-n-octil ftalat-MOP. Za statističku obradu podataka kori&scaron;ćeni su odgovarajući parametarski i neparametarski testovi. Rezultati: Kod 51 % ispitanica sa PCOS potvrđeno je prisustvo ftalatnih metabolita u urinu. Najzastupljeniji ftalatni metabolit je bio MEHP, zatim MEP, ali je potvrđeno i prisustvo MMP, MPP i MOP. Kod ispitanica sa PCOS su potvrđene statistički značajne korelacije između ukupnih ftalatnih metabolita i BMI, obima struka, glikemije, insulinemije, HOMA indeksa, ukupnog holesterola, triglicerida, LDL holesterola, leptina i testosterona. Analizom pojedinačnih ftalatnih metabolita utvrđene su pozitivne korelacije između MMP i obima struka, parametara glukoznog metabolizma, ukupnog holesterola, LDL holesterola, triglicerida, LAP i VAI indeksa. Zaključak: Kod žena sa PCOS u reproduktivnom periodu u na&scaron;oj sredini je potvrđeno prisustvo ftalanih metabolita. Najvi&scaron;e koncentracije su verifikovane za MEHP i MEP, ukazujući na visoku ekspoziciju di-etilheksil ftalatu (DEHP) i di-etil ftalatu (DEP). Ukupni ftalati značajno povećavaju vrednosti parametara koje učestvuju u razvoju metaboličkog sindroma kod PCOS, ali i povećavaju kardiovaskularni rizik ovih bolesnica. Direktna, značajna veza je potvrđena između MMP i testosterona, parametara i indeksa kardiometaboličkog sindroma.</p> / <p>Introduction: Phthalates are a type of endocrine disruptor widely used as plasticizers and solvents but also as additives to many products that are used daily. According to previous studies in experimental animals, but also in the human population, phthalic diesters affect the reproductive system, participate in the onset of insulin resistance and obesity. Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder of the reproductive system of women in the generative period. Insulin resistance and central obesity, as factors of cardiometabolic syndrome, have a significant role in the etiology of PCOS. Objectives: To determine the presence of phthalic acid metabolites in women in the reproductive period in our environment, and whether there is a connection between these endocrine disruptors with anthropometric parameters, glycoregulation parameters, lipids and serum lipoproteins, gonadotrophins, sex hormones, leptin and indexes of cardiometabolic risk in women with polycystic ovarian syndrome. Methods: The study included 61 women with polycystic ovarian syndrome divided into two subgroups: 31 subjects with PCOS and present phthalate metabolites in urine and 30 subjects with PCOS without phthalate metabolites in urine. The control group consisted of 30 healthy women. In all subjects, anthropometric measurements were carried out (TV, TM, WC) and the values of glycoregulation parameters (glycemia and insulinemia, HOMA index), lipids and serum lipoproteins (total cholesterol, triglycerides, LDL I HDL cholesterol), gonadotropins FSH), sex hormones (estradiol and testosterone) and leptin. In the assessment of cardiometabolic risk LAP and VAI indexes were determined. From the morning urine sample, the presence and concentration of 10 phthalate metabolites were determined: Mono-methyl phthalate-MMP, Mono-ethyl phthalate-MEP, Mono-n-butyl phthalate-MBF, Mono-n- propyl phthalate- MPP, Mono-iso-amyl phthalate &ndash; MiAP, Mono-n-amyl phthalate &ndash; MnAP, Mono-cyclohexyl phthalate-MCHP, Monobenzyl phthalate-MBzP, Mono- (2-ethylhexyl) phthalate-MHEP, Mono-n-octyl phthalate-MOP. For statistical data processing, appropriate parametric and non-parametric tests were used. Results: 51% of subjects with PCOS confirmed the presence of phthalate metabolites in urine. The most common phthalate metabolite was MEHP, then MEP, but the presence of MMP, MPP and MOP also was confirmed. In subjects with PCOS, a statistically significant correlations between total phthalate metabolites and BMI, waist circumference, glycemia, insulinemia, HOMA index, total cholesterol, triglyceride, LDL cholesterol, leptin and testosterone were confirmed. By analyzing individual phthalate metabolites, a positive correlations between MMP and waist circumference, glycoregulation parameters, total cholesterol, LDL cholesterol, triglyceride, testosterone and LAP and VAI index were determined. Conclusion: In women with PCOS in the reproductive period, the presence of phthalic metabolites in our environment was confirmed. The highest concentrations were verified for MEHP and MEP, indicating a high exposure of DEHP and DEP. Total phthalates significantly increase the values of parameters involved in the development of metabolic syndrome in PCOS, but also increase the cardiovascular risk of these patients. A direct, significant association was confirmed between MMP and testosterone, parameters and index of cardiometabolic syndrome.</p>

Analytical Characterization of complex mixtures, such as crude oil, environmental samples, and biological mixtures, is challenging because of the large diversity of molecular components. Mass spectrometry based techniques are among the most powerful tools for the separation of molecules based on their molecular composition, and the coupling of ion mobility spectrometry has enabled the separation and structural elucidation using the tridimensional structure of the molecule. The present work expands the ability of analytical chemists by furthering the development of IMS-MS instrumentation by coupling Trapped Ion Mobility Spectrometry to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (TIMS-FT-ICR MS). The TIMS-FT-ICR MS platform combines the high-resolution separation of TIMS, which has mobility resolving powers up to 400, and ultra-high mass resolution of FT-ICR MS, with mass resolving power over 1,000,000. This instrumentation allows the assignment of exact chemical composition for compounds in a complex mixture, as well as measurement of the collision cross-section of the molecule. Herein, the principles of the TIMS separation and its coupling to FT-ICR MS are described, as well as how the platform can be applied to targeted analysis of molecules, and untargeted characterization of complex mixtures. Molecular standards were analyzed by TIMS-MS in order to develop a computational workflow that can be utilized to elucidate molecular structure, using the measured collision cross-section of the ion. This workflow enabled identification of structural, cis/trans isomers, and chelated molecules and provides the basis for unsupervised structural elucidation of a complex mixture, and in particular for the elucidation of hydrocarbons from fossil fuels. In summary, this work presents the coupling of TIMS-FT-ICR MS and provides examples of applications as a proof of concept of the potential of this platform for solving complex analytical challenges.

Un certain nombre d’études épidémiologiques ont montré au cours des cinquante dernières années une augmentation des infertilités, des malformations de l’appareil reproducteur masculin et des cancers testiculaires. Une des hypothèses est que, l’exposition durant la vie foetale ou néonatale à des composés présents dans l’environnement capables d’interférer avec le système hormonal (perturbateurs endocriniens), serait responsable de l’augmentation de l’incidence de ces pathologies. Les molécules qui sont suspectées d’avoir des effets néfastes à long terme sur le tractus génital mâle possèdent des activités de type estrogénique ou antiandrogénique. Parmi les mécanismes impliqués dans l’effet à long terme, un certain nombre d’auteurs mettent en avant l’intervention de mécanismes de type épigénétiques. Dans ce contexte, nous avons utilisé deux types de modèles expérimentaux reposant sur l’exposition développementale de rats à ces composés : un modèle d’exposition néonatale à un estrogène (estradiol benzoate) et un modèle d’exposition foetale à un antiandrogène (flutamide). Les deux modèles expérimentaux induisent chez le rongeur un phénotype d’hypospermatogenèse. Dans le cas de l’exposition néonatale à l’estradiol benzoate nous montrons que l’hypospermatogenèse observée chez les animaux à l’âge adulte est due à l’activation chronique de l’apoptose des cellules germinales testiculaires. Cette apoptose mettrait en jeu, par un mécanisme post-transcriptionnel, la diminution à long terme de l’expression de protéines clés de la machinerie épigénétique de méthylation de l’ADN, les ADN méthyltransférases (DNMT 3A, 3B et 1), et du facteur antiapoptotique, MCL-1. D’un point de vue fonctionnel, la chute d’expression des DNMTs se traduit notamment par l’augmentation d’expression des éléments transposables LINE-1 et du gène Ibtk normalement contrôlés par méthylation de l’ADN. En amont, la chute d’expression des DNMTs et de MCL-1 serait dépendante de l’augmentation d’expression d’autres effecteurs épigénétiques, les microRNAs de la famille miR-29. Dans le cas de l’exposition in utero au flutamide, notre travail indique que l’apoptose chronique des cellules germinales serait liée à la diminution à long terme de l’expression des inhibiteurs d’apoptose cIAP1 et cIAP2, et une augmentation d’expression de leur inhibiteur SMAC/DIABLO. En revanche, l’absence de mort des cellules somatiques testiculaires (Sertoli et Leydig) dans ce modèle serait due à l’augmentation d’expression spécifiquement dans ces cellules des inhibiteurs d’apoptose XIAP et SURVIVIN. Par ailleurs, le phénotype d’apoptose observé à l’âge adulte impliquerait également une altération précoce de l’expression des DNMTs. En conclusion, nous apportons une réponse mécanistique au phénotype de programmation foetale/néonatale d’apoptose des cellules germinales testiculaires adultes. En effet, l’augmentation des miR-29s provoquerait : (1) une chute d’expression des DNMTs altérant ainsi le profil de méthylation des gènes, et (2) une chute d’expression de facteurs protégeant les cellules germinales contre l’apoptose comme le facteur MCL-1. / During the five past decades, a number of epidemiological studies have indicated a higher incidence of infertility problems, male reproductive tract abnormalities and testicular cancers. Among the different hypotheses proposed, fetal or neonatal exposure to environmental compounds that can interfere with endocrine system, termed endocrine disruptors, may be at the origin of the rising incidence of these diseases. The molecules that are supposed to have long term adverse effects on the male genital tract have estrogenic or antiandrogenic properties. Some authors have suggested that the long term effects of endocrine disruptors could be mediated through epigenetic mechanisms. In this context, we have used two types of experimental models based on developmental exposure to these compounds: one model of neonatal exposure to an estrogen (estradiol benzoate) and another model of fetal exposure to an antiandrogen (flutamide). The two experimental models induce a phenotype of hypospermatogenesis in rodents. In the context of neonatal exposure to estradiol benzoate we show that the hypospermatogenesis observed in adult animals is the consequence of a chronic activation of the apoptotic process of testicular germ cells. This cell death process seem to involve the decrease of the key epigenetic effectors of DNA methylation machinery DNA methyltransferases (DNMT3A, 3B & 1), and of the antiapoptotic protein MCL-1 through post transcriptional mechanisms. In term of functional consequences the decrease of DNMTs proteins leads to increased expression of transposable element LINE-1 and Ibtk gene that are normally controlled by DNA methylation. Upstream DNMTs and MCL-1 decrease may be triggered by the increase of other epigenetic factors, the microRNAs belonging to miR-29 family. Concerning in utero exposure to flutamide, our work indicate that the chronic apoptotic process of germ cells may be linked to long term decrease of the inhibitors of apoptosis cIAP1 & 2, and an increase of proapoptotic factors SMAC/DIABLO. On the other hand, the lack of testicular somatic cell death in this model may be the result of higher expression of inhibitors of apoptosis XIAP and SURVIVIN in these cells. Besides, the apoptotic process observed at the adult age may also involve a precocious alteration of DNMTs expression. In summary, our work provides a mechanistic view to the fetal/neonatal programming of adult germ cell death. Indeed, the increased levels of miR-29s may induce: (1) a decrease in DNMTs expression levels that consequently could alter the methylation pattern of some genes, and (2) a decrease in factors that normally prevent germ cells death such as MCL-1.

Un certain nombre d’études épidémiologiques font état d’une augmentation de l’infertilité masculine durant ces cinquante dernières années, en particulier dans les pays industrialisés, mais aussi d’une augmentation des malformations de l’appareil reproducteur masculin telles que la cryptorchidie (absence de migration des testicules dans les bourses) ou l’hypospadias (malformation du pénis), et des cancers testiculaires. Des données expérimentales suggèrent que ces anomalies du tractus génital mâle sont liées. Ces symptômes forment le syndrome de dysgénésie testiculaire. Les causes d’apparition ce syndrome semblent être d’origine environnementale. En effet, les évolutions relativement rapides de ce syndrome suggèrent des facteurs dynamiques, en lien avec le mode de vie ou l’environnement. Une des hypothèses est que, l’exposition durant la vie fœtale ou néonatale à des composés présents dans l’environnement capables d’interférer avec le système hormonal (perturbateurs endocriniens environnementaux, PEEs), serait responsable de l’augmentation de l’incidence de ces pathologies. Au banc des principaux accusés, les molécules qui possèdent des activités de type estrogénique ou antiandrogénique. A ce jour, les mécanismes d’action à l’origine du syndrome de dysgénésie testiculaire sont encore mal connus. Certaines études suggèrent des mécanismes de type épigenétique dans les effets à long terme des PEEs. L’objectif de notre travail était d’identifier et caractériser les mécanismes d’action de type épigenétique impliqué dans l’infertilité mâle. Pour cela, nous avons utilisé un modèle expérimental (rats nouveau-nés) reposant sur une exposition développementale à un estrogène (estradiol benzoate). Ce modèle induit chez le rat adulte un phénotype d’hypospermatogenèse liée à une à apoptose chronique des cellules germinales testiculaires. Nous montrons que ce phénotype est lié à l’altération de deux voies, impliquant en amont des effecteurs épigénétiques. La première voie implique la famille des miR-29s. Ainsi, nous observons une augmentation de l’expression des miR-29a, b, c qui provoque une diminution de deux de ses cibles: la protéine antiapoptotique MCL-1 et les enzymes de méthylation de l’ADN DNMTs. La chute des DNMTs entraine une hypométhylation globale (estimée à travers le gène Line-1) et spécifique du facteur de choc thermique HSF1. Ceci provoque une réexpression de ces facteurs entrainant l’apoptose des cellules germinales adultes. La deuxième voie implique le miR-18a. L’augmentation de son expression provoque une chute de l’expression de sa cible HSF2 qui régule la protéine de choc thermique HSP70/HSPA2. Le faible taux d’HSPA2 est une autre explication de l’apoptose des cellules germinales dans notre modèle. Nous montrons aussi que ce phénotype est irréversible lorsque l’exposition à lieu chez le nouveau-né alors qu’il est réversible quand l’exposition à lieu à l’âge adulte. Ces données suggèrent que l’exposition néonatale à l’estradiol benzoate induit une programmation développementale de l’hypospermatogenèse.Enfin, les anomalies tissulaires d’expression des miRNAs se retrouvent au niveau sanguin, suggérant leur utilisation potentielle comme biomarqueurs. Nous avons validé cet aspect chez l’homme en montrant que l’expression des miR29s et du miR-18a était plus élevée chez les patients oligo- ou azoospermiques que les chez patients normospermiques.En conclusion, nos résultats indiquent que l’hypospermatogenèse due à une apoptose chronique des cellules germinales observée chez l’animal adulte après exposition néonatale à l’EB met en jeu une modification d’expression de plusieurs effecteurs épigénétiques clés: miR-29s, miR-18a et DNMTs. De plus, les miR-29s et miR-18a pourraient être de nouveaux biomarqueurs circulants non invasifs de la stérilité masculine dans le contexte d’une oligo ou azoospermie chez l'homme. / Epidemiological studies have reported an increase in male infertility over the past fifty years, especially in industrialized countries, but also an increase in malformations of the male reproductive tract such as cryptorchidism (no migration of the testes into the scrotum) and hypospadias (malformation of the penis), and testicular cancers. Experimental data suggest that these abnormalities of the male genital tract are related. These symptoms form the testicular dysgenesis syndrome. The causes of the occurrence of this syndrome appear to be environmental in origin. Indeed, the relatively rapid evolution of this syndrome suggests dynamic factors related to lifestyle or environment. One hypothesis is that exposure during fetal or neonatal life to compounds present in the environment can interfere with the hormonal system (environmental endocrine disruptors), would be responsible for the increased incidence of these pathologies. Bench of the main accused, molecules that have estrogenic or anti-androgenic activity types. To date, the mechanisms of action behind the testicular dysgenesis syndrome are poorly understood. Some studies suggest that epigenetic mechanisms are at playThe objective of our work was to identify and characterize the epigenetic mechanisms of action involved in male infertility induced by neonatal exposure to xenoestrogen. For this, we used an experimental model based on a developmental exposure to estrogen (estradiol benzoate). This model induced in adult rats a hypospermatogenesis phenotype due to chronic apoptosis of germ cells.We show that this phenotype is related to an alteration of two pathways, involving upstream effectors epigenetic. The first pathway involves the family of miR- 29s. Thus, we observe an up-regulation of miR -29a, b, c, which causes a decrease in two of his targets: the anti-apoptotic protein MCL- 1 and the enzymes of DNA methylation DNMTs. Falling DNMTs leads to a global hypomethylation (estimated through the Line -1 gene) and to specific hypomethylation of the heat shock factor, HSF1. This causes a re-expression of factors that induce apoptosis in adult germ cells. The second pathway involves up-regulation of miR -18a that causes a down-regulation of its target HSF2 which regulates the heat shock protein HSP70/HSPA2. The down-regulation of HSPA2 is another explanation of germ cell apoptosis in our model. We also show that this phenotype is irreversible when the estrogen exposure takes place in the newborn whereas it is reversible when exposure takes place in adulthood, suggesting that neonatal exposure to estradiol benzoate induced a developmental programming of hypospermatogenesis.Finally, abnormal tissue expressions of miRNAs are found in the blood, suggesting their potential use as biomarkers. We validated this aspect in humans showing that the expression of miR29s and miR-18a was higher in patients with decrease or no sperm counts compared to normal sperm count. In conclusion, our results indicate that hypospermatogenesis due to chronic germ cell apoptosis observed in adult animals after neonatal exposure to EB involves a change in expression of several key epigenetic effectors: miR-29, miR-18a and DNMTs. In addition, miR-29 and miR-18a could be new non invasive circulating biomarkers of men infertility.

<p>Iron oxide nanoparticles were prepared using co-precipitation method in the presence and absence of beta-cyclodextrin (&beta / -CD). Such materials were characterized using transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), attenuated total reflection Fourier transform infrared (ATR-FTIR), X-ray diffraction (XRD), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA). The TEM shows that the surface morphology has no difference between nanoparticles prepared in the presence and absence of beta-cyclodextrin (&beta / -CD), amorphous particles with high surface area and dimensions of about 100 nm by 500 nm. The amorphous states of nanoparticles are confirmed further by XRD. The ATR-FTIR analysis confirms inclusion complex between &beta / -CD and nanoparticles.</p>

<p>Iron oxide nanoparticles were prepared using co-precipitation method in the presence and absence of beta-cyclodextrin (&beta / -CD). Such materials were characterized using transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), attenuated total reflection Fourier transform infrared (ATR-FTIR), X-ray diffraction (XRD), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA). The TEM shows that the surface morphology has no difference between nanoparticles prepared in the presence and absence of beta-cyclodextrin (&beta / -CD), amorphous particles with high surface area and dimensions of about 100 nm by 500 nm. The amorphous states of nanoparticles are confirmed further by XRD. The ATR-FTIR analysis confirms inclusion complex between &beta / -CD and nanoparticles.</p>

Magister Scientiae - MSc / Iron oxide nanoparticles were prepared using co-precipitation method in the presence and absence of beta-cyclodextrin β-CD). Such materials were characterized using transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), attenuated total reflection Fourier transform infrared (ATR-FTIR), X-ray diffraction (XRD), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA). The TEM shows that the surface morphology has no difference between nanoparticles prepared in the presence and absence of beta-cyclodextrin β-CD), amorphous particles with high surface area and dimensions of about 100 nm by 500 nm. The amorphous states of nanoparticles are confirmed further by XRD. The ATR-FTIR analysis confirms inclusion complex between β-CD and nanoparticles. The nanoparticles synthesized were used to develop an electrochemical sensor for phenolic endocrine disruptors by modifying the surface area of glassy carbon electrode (GCE). Electrochemical characterization of the iron oxide β-CD nano-composites, studied in 0.1 M potassium chloride (KCl) using chronoamperometry,showed that the surface concentration of the adsorbed composite material was 8.5 x 10-8 mol/cm2. Sensor analysis of bisphenol A (BPA) was carried out using cyclic voltammetry (CV) and square wave voltammetry (SWV) based on amperometric techniques which gave a linear range of 0.50 × 10-6 M to 50 × 10-6 M; limit of detection of 0.156 x 10-6 M and order of magnitude of linearity of 2.03. Hence, the sensor was further used to study 4-tert-octylphenol (TOP); the results showed that the sensitivity and the limit of detection were 11.31 nA L/mol and 0.249 x 10-6 M, respectively and order of magnitude of linearity of 2.00. / South Africa

Made available in DSpace on 2016-06-02T20:35:03Z (GMT). No. of bitstreams: 1 6749.pdf: 21604971 bytes, checksum: ea6b5030f47becaef3fc6875b8d6bc0e (MD5) Previous issue date: 2015-03-27 / Universidade Federal de Minas Gerais / This work aimed to investigate the occurrence of 12 emerging contaminants (pharmaceuticals, hormones and triclosan) in samples of influents and effluents, as well the removal in the sewage treatment plant (STP) of São Carlos. It was also investigated the occurrence and spatiotemporal distribution of emerging contaminants in surface waters and sediments from the Monjolinho River, the receptor of effluents, and one of its tributaries, the Água Quente Stream (AQS), which receives the discharge of non treated sewage. Influent and effluent samples were collected weekly. Surface water and sediment samples were obtained every two and four months, respectively, in points since the source until the mouth of the Monjolinho River. The target compounds in dissolved fraction of aquous samples were extracted by solid phase extraction (SPE), while for sediments it was performed ultrassonic extrations with solvents followed by SPE. The analytical determinations were made by ultra high pressure liquid chromatography coupled to mass spectrometer equipped with triplequadrupole analyser (LC-MS/MS). The hormone 17-&#945;-ethynilestradiol was not detected in any sample. However, all the pharmaceuticals evaluated and triclosan were detected in influent samples. The STP with upflow anaerobic sludge blancket (UASB) and air dissolved flotation in São Carlos presented low removal for carbamazepine, diclofenac, propranolol and ibuprofen. On the other hand, triclosan presented the highest removal (91.2%). In surface waters and sediments from Monjolinho river the concentration of contaminants presented an increasing trend since the source of the river to the downstream of the STP effluent discharge and after the confluence of the AQS (which flows into the river at the same place), where it was observed the highest concentrations in the Monjolinho River. The AQS, in its turn, presented higher concentrations of pharmaceuticals and triclosan than the Monjolinho River, due to the discharge of non treated sewage. Carbamazepine and diclofenac were persistent in both STP and aquatic body. The strong correlations between this compounds and electrical conductivity (considered a conservative parameter) sugests that CMZ and DCF can be tracers of both treated and non treated sewage release in the study area. Ee conclude that the main sources of phamaceuticals and triclosan to the Monjolinho River are the discharge of non treated sewage (from the AQS) and effluent from STP, since their removal is very low. Unlike the studies involving endocrine disruptors, the effects of other classes of emerging contaminants have been yet little explained in literature. Therefore, more surveys on the occurrence of these compounds combined with ecotoxicological studies must be conducted to make clear the magnitude of the impact that these substances can cause in the environment. Based on this information, adjustments in the effluent treatment may be investigated in the future, if there is interest in effectively removing this compounds in STP. / Neste trabalho foi investigada a ocorrência de 12 contaminantes emergentes (fármacos, hormônios e triclosan) em amostras de esgoto bruto e tratado, bem como foi avaliada a remoção dos mesmos pela estação de tratamento de esgoto (ETE) de São Carlos. Também investigou-se a ocorrência e distribuição espaço-temporal desses compostos em águas superficiais e sedimentos do rio do Monjolinho, o corpo aquático receptor, e em um de seus afluentes, o Córrego da Água Quente (CAQ), o qual recebe esgoto sanitário não tratado. Amostras de esgoto, águas e sedimentos foram coletadas semanalmente, bimestralmente e a cada quatro meses, respectivamente. No rio do Monjolinho, os pontos de amostragem abrangeram desde a nascente até a foz. Os compostos alvo na fração dissolvida das amostras aquosas foram extraídos por extração em fase sólida (SPE), enquanto que para sedimentos foi realizada extração ultrassônica com solventes, seguida de SPE. As determinações analíticas foram feitas por cromatografia de ultra eficiência acoplada à espectrometria de massas com analisador triplo quadrupolo (UPLC-MS/MS). O hormônio 17-&#945;-etinilestradiol não foi detectado em nenhuma das matrizes avaliadas. Porém, todos os fármacos avaliados e triclosan foram detectados nas amostras de esgoto bruto. O tratamento de esgoto empregando reator anaeróbio de fluxo ascendente (UASB) seguido de flotação por ar dissolvido (FAD) na ETE de São Carlos apresentou baixa remoção para carbamazepina, diclofenaco, propranolol e ibuprofeno, enquanto o triclosan apresentou a maior remoção (91,2%). Nas águas superficiais e sedimentos do rio do Monjolinho as concentrações dos contaminantes foram crescentes desde a nascente até após a confluência do CAQ e o lançamento do efluente da ETE (os quais ocorrem no mesmo local), onde ocorreram as maiores concentrações encontradas no rio do Monjolinho. O CAQ apresentou concentrações mais elevadas de fármacos e triclosan do que o rio do Monjolinho, devido ao aporte de esgoto não tratado. Carbamazepina e diclofenaco foram persistentes tanto em amostras de esgoto quanto no corpo aquático. Fortes correlações entre ambos os compostos e condutividade elétrica (este considerado um parâmetro conservativo) sugerem que os dois fármacos são bons traçadores do aporte de esgoto sanitário tratado e/ou não tratado na área de estudo. Conclui-se que as principais fontes de fármacos e triclosan no rio do Monjolinho são o lançamento de esgoto não tratado (por meio do CAQ) e esgoto tratado, uma vez que a remoção dos mesmos na ETE, é bastante baixa. Ao contrário dos estudos envolvendo desreguladores endócrinos, os efeitos adversos de outras classes de contaminantes emergentes ainda têm sido pouco elucidados na literatura. Portanto, mais pesquisas sobre a ocorrência desses compostos aliados a análises ecotoxicológicas devem ser realizados para que se dimensione a magnitude do impacto que essas substâncias podem causar ao ambiente. Com base em tais informações, adequações no tratamento de esgoto podem ser investigadas, no futuro, caso haja interesse em remover efetivamente esses compostos na ETE.