O objetivo desse estudo foi avaliar o efeito do tratamento com a aldosterona (Aldo), por 21 dias, sobre a função e a morfologia renal de ratos, procurando correlacionar as alterações com a expressão do EGFR e da ERK1/2 fosforilados. A Aldo não alterou os parâmetros fisiológicos, a PA e a função renal dos ratos, mas verificou-se uma tendência para o aumento das concentrações plasmáticas de K+. No córtex e na medula renal, a Aldo aumentou a expressão do EGFR e da ERK1/2 fosforilados, sendo que este efeito foi abolido pelo tratamento concomitante com a espironolactona ou o RU 486 (antagonistas dos receptores da Aldo). O tratamento hormonal também induziu um aumento na marcação imuno-histoquímica para essas proteínas no córtex e medula; contudo, este efeito foi reduzido, principalmente, com o tratamento conjunto com a espironolactona. Observou-se um aumento na área glomerular e na porcentagem da área intersticial, após o tratamento com a Aldo. As alterações na morfometria renal foram parcialmente reduzidas pelo tratamento com a espironolactona ou o RU 486. Esses resultados indicam que a Aldo pode induzir alterações morfológicas no rim, aparentemente, de maneira dependente dos receptores MR e GR e da via do EGFR-ERK1/2, mas sem o comprometimento da função renal. / The purpose of this study was to evaluate the effect of 21 days of treatment with aldosterone (Aldo) in the renal function and morphology of rats, correlating changes with the expression of EGFR and ERK1/2 phosphorylated. The Aldo did not alter physiological parameters, blood pressure and renal function of rats, but there was a tendency to increased K+ plasma concentrations. In renal cortex and medulla, Aldo increased the expression of EGFR and ERK1/2 phosphorylated and this effect was abolished by treatment Aldo plus spironolactone or RU 486 (Aldo receptor antagonists). Hormonal treatment also induced an increase in immunohistochemical staining for these proteins in the cortex and medulla; however, this effect was limited mainly to treatment Aldo plus spironolactone. There was an increase in glomerular area and the percentage of interstitial area after treatment with Aldo. Changes in renal morphology were partially reduced by treatment Aldo plus spironolactone or RU 486. These results indicate that Aldo may induce morphological changes in the kidney of dependent manner of MR and GR receptors and EGFR-ERK1/2 signaling, but without the impairment of renal function.

O objetivo desse estudo foi avaliar o efeito do tratamento com a aldosterona (Aldo), por 21 dias, sobre a função e a morfologia renal de ratos, procurando correlacionar as alterações com a expressão do EGFR e da ERK1/2 fosforilados. A Aldo não alterou os parâmetros fisiológicos, a PA e a função renal dos ratos, mas verificou-se uma tendência para o aumento das concentrações plasmáticas de K+. No córtex e na medula renal, a Aldo aumentou a expressão do EGFR e da ERK1/2 fosforilados, sendo que este efeito foi abolido pelo tratamento concomitante com a espironolactona ou o RU 486 (antagonistas dos receptores da Aldo). O tratamento hormonal também induziu um aumento na marcação imuno-histoquímica para essas proteínas no córtex e medula; contudo, este efeito foi reduzido, principalmente, com o tratamento conjunto com a espironolactona. Observou-se um aumento na área glomerular e na porcentagem da área intersticial, após o tratamento com a Aldo. As alterações na morfometria renal foram parcialmente reduzidas pelo tratamento com a espironolactona ou o RU 486. Esses resultados indicam que a Aldo pode induzir alterações morfológicas no rim, aparentemente, de maneira dependente dos receptores MR e GR e da via do EGFR-ERK1/2, mas sem o comprometimento da função renal. / The purpose of this study was to evaluate the effect of 21 days of treatment with aldosterone (Aldo) in the renal function and morphology of rats, correlating changes with the expression of EGFR and ERK1/2 phosphorylated. The Aldo did not alter physiological parameters, blood pressure and renal function of rats, but there was a tendency to increased K+ plasma concentrations. In renal cortex and medulla, Aldo increased the expression of EGFR and ERK1/2 phosphorylated and this effect was abolished by treatment Aldo plus spironolactone or RU 486 (Aldo receptor antagonists). Hormonal treatment also induced an increase in immunohistochemical staining for these proteins in the cortex and medulla; however, this effect was limited mainly to treatment Aldo plus spironolactone. There was an increase in glomerular area and the percentage of interstitial area after treatment with Aldo. Changes in renal morphology were partially reduced by treatment Aldo plus spironolactone or RU 486. These results indicate that Aldo may induce morphological changes in the kidney of dependent manner of MR and GR receptors and EGFR-ERK1/2 signaling, but without the impairment of renal function.

L’estradiol (E2) est une hormone femelle qui joue un rôle essentiel, à la fois dans la régulation et dans la détermination de certaines conditions physiologiques in vivo, telle que la différenciation et la prolifération cellulaire. Lorsque l’E2 est donné en supplément, par exemple dans le cas de thérapie hormonale, deux effets sont observés, un effet génomique et un effet non-génomique, de par son interaction avec les récepteurs à œstrogène du noyau ou de la membrane cellulaire, respectivement. L’effet non-génomique est plus difficile à étudier biologiquement parce que l’effet se produit sur une échelle de temps extrêmement courte et à cause de la nature hydrophobe de l’E2 qui réduit sa biodisponibilité et donc son accessibilité aux cellules cibles. C’est pourquoi il est nécessaire de développer des systèmes d’administration de l’E2 qui permettent de n’étudier que l’effet non-génomique de l’œstrogène. Une des stratégies employée consiste à greffer l’E2 à des macromolécules hydrophiles, comme de l’albumine de sérum bovin (BSA) ou des dendrimères de type poly(amido)amine, permettant de maintenir l’interaction de l’E2 avec les récepteurs d’œstrogène de la membrane cellulaire et d’éviter la pénétration de l’E2 dans le noyau des cellules. Toutefois, ces systèmes macromolécules-E2 sont critiquables car ils sont peu stables et l’E2 peut se retrouver sous forme libre, ce qui affecte sa localisation cellulaire. L’objectif de cette thèse est donc de développer de nouvelles plateformes fonctionnalisées avec de l’E2 en utilisant les approches de synthèses ascendantes et descendantes. Le but de ces plateformes est de permettre d’étudier le mécanisme de l’effet non-génomique de l’E2, ainsi que d’explorer des applications potentielles dans le domaine biomédical. L’approche ascendante est basée sur un ligand d’E2 activé, l’acide 17,α-éthinylestradiol-benzoïque, attaché de façon covalente à un polymère de chitosan avec des substitutions de phosphorylcholine (CH-PC-E2). L’estradiol est sous forme de pro-drogue attachée au polymère qui s’auto-assembler pour former un film. L’effet biologique de la composition chimique du film de chitosan-phosphorylcholine a été étudié sur des cellules endothéliales. Les films de compositions chimiques différentes ont préalablement été caractérisés de façon physicochimique. La topographie de la surface, la charge de surface, ainsi que la rhéologie des différents films contenant 15, 25, ou 40% molaires de phosphorylcholine, ont été étudiés par microscopie à force atomique (AFM), potentiel zêta, résonance plasmonique de surface et par microbalance à cristal de quartz avec dissipation (QCM-D). Les résultats de QCM-D ont montré que plus la part molaire en phosphorylcholine est grande moins il y a de fibrinogène qui s’adsorbe sur le film de CH-PC. Des cellules humaines de veine ombilicale (HUVECs) cultivées sur des films de CH-PC25 et de CH-PC40 forment des amas cellulaire appelés sphéroïdes au bout de 4 jours, alors que ce n’est pas le cas lorsque ces cellules sont cultivées sur des films de CH-PC15. L’attachement de l’estradiol au polymère a été caractérisé par plusieurs techniques, telles que la résonance magnétique nucléaire de proton (1H NMR), la spectroscopie infrarouge avec transformée de Fourier à réfraction totale atténuée (FTIR-ATR) et la spectroscopie UV-visible. La nature hydrogel des films (sa capacité à retenir l’eau) ainsi que l’interaction des films avec des récepteurs à E2, ont été étudiés par la QCM-D. Des études d’imagerie cellulaires utilisant du diacétate de diaminofluoresceine-FM ont révélé que les films hydrogels de CH-PC-E2 stimulent la production d’oxyde nitrique par les cellules endothéliales, qui joue un rôle protecteur pour le système cardiovasculaire. L’ensemble de ces études met en valeur les rôles différents et les applications potentielles qu’ont les films de type CH-PC-E2 et CH-PC dans le cadre de la médecine cardiovasculaire régénérative. L’approche descendante est basée sur l’attachement de façon covalente d’E2 sur des ilots d’or de 2 μm disposés en rangées et espacés par 12 μm sur un substrat en verre. Les ilots ont été préparés par photolithographie. La surface du verre a quant à elle été modifiée à l’aide d’un tripeptide cyclique, le cRGD, favorisant l’adhésion cellulaire. L’attachement d’E2 sur les surfaces d’or a été suivi et confirmé par les techniques de SPR et de QCM-D. Des études d’ELISA ont montré une augmentation significative du niveau de phosphorylation de la kinase ERK (marqueur important de l’effet non-génomique) après 1 heure d’exposition des cellules endothéliales aux motifs alternant l’E2 et le cRGD. Par contre lorsque des cellules cancéreuses sont déposées sur les surfaces présentant des motifs d’E2, ces cellules ne croissent pas, ce qui suggère que l’E2 n’exerce pas d’effet génomique. Les résultats de l’approche descendante montrent le potentiel des surfaces présentant des motifs d’E2 pour l’étude des effets non-génomiques de l’E2 dans un modèle in vitro. / Estradiol (E2) is an essential female hormone in the regulation and determination of various physiological conditions in vivo, such as cell proliferation and differentiation. When supplementing exogenous E2 as a clinical strategy for hormone therapy, it generates genomic and non-genomic effect simultaneously via binding to the estrogen receptors in the cell nucleus or membrane site. Compared to the genomic effect, it is quite difficult to monitor the E2-induced non-genomic biological behavior because this effect occurs in extremely transient time scale and the bioavailability and accessibility of E2 to target cells is very low due to the hydrophobic nature of E2. As a result, it is indispensable to develop E2 delivery systems to specifically understand estrogenic non-genomic nature. One of strategies is to graft E2 to the hydrophilic macromolecules, e.g. bovine serum albumin (BSA) or poly(amido)amine dendrimer, to maintain E2 interacting with membrane estrogen receptors instead of penetrating into the cell nucleus. However, the instability of those E2-macromolecules systems, either containing free E2 leaching or discrepancies of cellular localizations, led to controversies. Herein, the objective of present thesis is to develop novel E2-functionlized platforms by the principle of bottom-up and top-down approaches for understanding the mechanism of estrogenic non-genomic effect, and further, to explore their potential applications in the biomedicine. As a bottom-up approach, an activated E2 ligand, 17α-ethinylestradiol-benzoic acid was covalently conjugated onto a phosphorylcholine substituted chitosan polymer (CH-PC-E2) as a prodrug strategy for the fabrication of self-assembled films. Through a series of combined physicochemical and cellular investigations, the relationship between various chemical compositions of chitosan-phosphorylcholine (CH-PC) films and cellular responses was also evaluated. Based on atomic force microscopy (AFM) examination, zeta-potential measurements, surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D) measurements, surface topography, charge, and rheology of CH-PC films with 15, 25, and 40 mol% PC contents were characterized. Moreover, QCM-D measurements indicated that the amount of fibrinogen adsorbed on CH-PC films decreased significantly with increasing PC content. Finally, it was also showed that human umbilical vein endothelial cells (HUVECs) form spheroids on CH-PC25 and CH-PC40 films, but not on CH-PC15 films cultured over 4 days. In addition, the CH-PC-E2 polymer conjugates were prepared and characterized by several techniques, such as 1H nuclear magnetic resonance (1H NMR), Fourier transformed infrared-attenuated total refraction (FTIR-ATR) and UV/Vis spectra measurements. The hydrogel nature of CH-PC-E2 film as well as its interactions to estrogen receptors was further extensively investigated by QCM-D study. In the cellular study, CH-PC-E2 hydrogel films can significantly stimulate the production of nitric oxide, a protective molecule in the cardiovascular system, in the endothelial cells by a diaminofluorescein-FM diacetate imaging study. The studies above demonstrated the different roles and potential applications of CH-PC-E2 and CH-PC surfaces in the cardiovascular regenerative medicine. As a top-down approach, micropatterned substrates were used for E2 functionalization, which were prepared by photolithography via aligning ~ 2 μm in diameter gold arrays onto a glass substrate. After that, a cell adhesive peptide, cyclic RGD was introduced to the glass surface in order to induce the attachment of cells. Meanwhile, estradiol was covalently immobilized on the gold surface and the process was monitored and validated by combining SPR and QCM-D studies. In the micropatterned substrate-coupled cell ELISA study, a phosphorylation level of extracellular signal-regulated kinase (ERK), which is an important non-genomic marker, was significantly elevated by this E2-functionalized micropatterned surface after 1 hour incubation. Furthermore, E2-functionalized micropatterned substrate didn’t proliferate cancer cells indicating the absence of genomic effect stimulation. Based on these results, our E2-functionalized micropatterned substrates can function as an in vitro model for the elucidation of estrogenic non-genomic behaviors.