Senescence and prostate : steroid hormone and growth factors interactions in the glandular microenvironment

Orientador: Valeria Helena Alves Cagnon QuiteteTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A senescência é fator determinante para a ocorrência de alterações morfofuncionais da próstata. O objetivo desse estudo foi caracterizar e correlacionar as interações entre os receptores dos fatores de crescimento fibroblásticos (FGFR2, FGFR7, FGFR8), fator de crescimento epidermal (EGFR), ?-actina e vimentina e os receptores androgênicos (AR), estrogênicos ? e ? (ER?, ER?) e de prolactina (PR) nos compartimentos epiteliais e estromal frente à condição de senilidade e variações hormonais. Além disso, caracterizar e correlacionar o AR, ER?, ER? e PR com os FGFs nos compartimentos epitelial e estromal de amostras humanas com adenocarcinoma de alto grau e baixo grau. 50 ratos machos senis (10 meses de idade) e 10 ratos machos jovens (4 meses de idade) foram divididos em grupos: Jovem (JOV) e Senil (SE): óleo de amendoim por 30 dias; Castrado (CAS): castração cirúrgica e química; Tamoxifeno-Letrozol (TAM): tamoxifeno e de letrozol por 30 dias; Castrado+estrógeno (REEST): tratamento similar ao CAS, e posteriormente recebeu injeções de 17?-estradiol por 30 dias; Tamoxifeno-Letrozol+Andrógeno (RETEST): após tratamento similar ao grupo TAM, os animais receberam injeções de Cipionato de Testosterona por 30 dias. Os animais foram sacrificados e amostras do lobo ventral foram coletadas e submetidas às análises de Microscopia de Luz, imunohistoquímicas, western blotting e dosagem hormonal. 30 amostras prostáticas humanas foram divididas em grupos: Adenocarcinoma de alto grau e Adenocarcinoma de baixo grau. As amostras foram submetidas às análises de Microscopia de luz e imunohistoquímicas. Após a administração estrogênica, presença de microácinos, células inflamatórias e hipertrofia do estroma prostático foram observados. A hiperandrogenização levou à recuperação epitelial. No SE houve aumento de vimentina, ER? e PR em relação ao JOV. No CAS observou-se localização diferencial da prolactina e ?-actina em relação ao SE. No RETEST, observou-se recuperação do padrão de distribuição de reatividade da ?-actina e da prolactina em relação ao SE. No REEST foi observado aumento de ER? e ER? e localização diferencial destes, somando-se a diminuição da ?-actina e vimentina em relação ao SE. No TAM foi observada diminuição de ER? e ?-actina, e aumento de prolactina no compartimento estromal, em relação ao SE. Em humanos, os FGFR2 e FGFR8 apresentaram-se aumentados no estágio inicial do câncer prostático, sugerindo essas moléculas como bons alvos terapêuticos. Pode-se concluir que o envolvimento do ER? na ativação do estroma reativo tornou o microambiente favorável à progressão do câncer, devido à potencialização do desequilíbrio estromal, e o ER? contribuíram para a inibição das lesões précancerosas em homens na senescência. Já, o desequilíbrio causado pela ablação e/ou reposição hormonal não somente alterou o feedback entre os hormônios esteróides como modificou a localização da reatividade das moléculas nos compartimentos prostáticos, provavelmente interferindo nas sinalizações autócrinas e parácrinas dos estrógenos, EGF e prolactina, apontando esses como deflagradores da formação do estroma reativo. A ablação hormonal nos animais senis levou ao aumento da reatividade dos FGFs, sugerindo interações entre os hormônios e suas vias de sinalização e o microambiente prostático senil. As vias dos FGFs podem ser ativadas também de maneira andrógeno-independente, uma vez que os FGFs apresentaram níveis de detecção aumentados mesmo diante da intensa depleção androgênica imposta pela castraçãoAbstract: Senescence is a determining factor for morphological and functional prostatic alterations. The objective of this study was to characterize and correlate the interactions among fibroblast growth factor receptors (FGFR2, FGFR7, FGFR8), epidermal growth factor (EGFR), ?-actin and vimentin and the androgen receptor (AR), estrogen ? and ? (ER?, ER?) and prolactin (PR) in epithelial and stromal prostatic compartments in elderly rats on hormonal variation. Also, the objective was to characterize and correlate the AR, ER?, ER? and PR with the FGFs in the human prostatic samples, presenting high grade and low grade adenocarcinoma. Fifty male rats (10 months old) and 10 young male rats (4 months old) were divided into groups: Young (JOV) and Senile Groups (SE)- peanut oil injections for 30 days, Castrated Group (CAS)- surgical and chemical castration; Tamoxifen-Letrozole Group (TAM)- tamoxifen and letrozole injections in period of 48 hours for 30 days; Castrated + estrogen Group (REEST)- surgical and chemical castration and subsequently the animals received 17?-estradiol injections for 30 days; Tamoxifen- Letrozole + Androgen Group (RETEST): after treatment similar to the TAM group, the animals received testosterone cypionate injections for 30 days. After the treatment, the animals were sacrificed and the ventral lobe samples were collected and analyzed for the Light Microscopy, immunohistochemistry and Western blotting. Thirty human prostatic samples were collected from elderly men and divided into High-grade and Low-grade Adenocarcinoma Groups. The samples were submitted to light microscopy and immunohistochemical analyses. After estrogen administration, epithelial atrophy, microacini, inflammatory cells and stromal hypertrophy were observed. The hyperandrogenization led to the recovery of epithelium. The vimentin, ER? and PR increase was verified in the SE group in relation to JOV one. Differential localization of PR and ?-actin was seen in the CAS group in relation to SE one. Recovery of the distribution pattern of ?-actin and prolactin reactivities was observed in the RETEST group in relation to SE. In the REEST group, it was observed the ER? and ER? increase and differential localization of these receptors, and the ?-actin and vimentin decrease in relation to SE. In the group TAM, it was observed the ER? and ?-actin decrease and the prolactin increase in the stromal compartment in relation to SE group. Regarding to human samples, increased FGFR2 and FGFR8 were observed in the early stages of prostate cancer, suggesting these molecules as good therapeutic targets. Thus, it can be concluded that the involvement of ER? in activation of reactive stromal led to the favorable microenvironment to cancer progression considering the strong stromal imbalance, and the ER? contributed to the inhibition of precancerous lesions in elderly men. The imbalance caused by ablation and/or hormone therapy not only changed the feedback between steroid hormones but also changed the reactivity localization of molecules in prostatic compartments, probably interfering in the autocrine and paracrine signaling of estrogen, prolactin and EGF, and pointing these molecules as possible triggers of the formation of reactive stroma. The present results demonstrated that hormone ablation in senile rats led to increased reactivities of the FGFs, suggesting interactions among hormones and their signaling pathways and senile prostatic microenvironment. Furthermore, it can be concluded that the ways of FGFs can be activated also androgen-independent manner, considering that the FGFs showed increased levels in the severe androgen depletion characterized by castrationDoutoradoAnatomiaDoutora em Biologia Celular e Estrutura

Structural and molecular characterization of elderly men's prostate with and without diagnosis of prostatic lesions

Orientador: Valéria Helena Alves Cagnon QuiteteDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A senescência é fator determinante para a ocorrência de alterações na biologia molecular da próstata, considerando-se tanto o desequilíbrio hormonal quanto o das interações epitélioestroma. Assim, os objetivos desse estudo foram caracterizar e comparar a biologia celular e molecular da zona periférica prostática de homens senis, com e sem diagnóstico de lesões prostáticas, através da morfologia e imunolocalização dos receptores androgênicos (AR), estrogênicos ? e ? (ER?, ER?), metaloproteinases de matriz 2 e 9 (MMP2, MMP9), distroglicanas ? e ? (DG?, DG?), laminina e fator de crescimento homólogo à insulina (IGFR-1). Para tal, foram utilizadas 60 amostras das zonas periféricas prostáticas provenientes de necrópsias e/ou prostatectomias de homens (60-90 anos), as quais foram divididas em 4 grupos (15 amostras cada): Grupo Senil (sem lesões prostáticas); Grupo Neoplasia Intraepitelial Prostática (NIP); Grupo Hiperplasia Benigna Prostática (HBP); Grupo Câncer Prostático (CP). O diagnóstico das lesões prostáticas baseou-se em critérios morfológicos e por imunomarcação para citoqueratina de alto peso molecular. Os resultados foram avaliados por microscopias de luz e eletrônica de transmissão, métodos morfométricos, imunohistoquímicas e Western Blotting. Os resultados mostraram danos morfológicos no estroma e organelas celulares envolvidas no processo secretor da próstata de homens senis. As diferentes lesões prostáticas na senescência demonstraram desequilíbrio nas taxas de proliferação/apoptose, indicando predomínio do processo proliferativo. Além disso, a imunolocalização para AR se manteve similar em todos os grupos estudados, com predominância da imunolocalização no compartimento epitelial. Já o ER? foi preferencialmente localizado no compartimento epitelial, sendo ausente no compartimento estromal do grupo CP. A imunolocalização do ER? foi preferencialmente no compartimento epitelial dos grupos estudados, sendo intensa nos grupos CP e NIP em relação aos demais. Também, intensa imunoreatividade para IGFR-1, MMP2 e MMP9 foi verificada nos grupos CP e NIP em relação aos demais grupos. As imunolocalizações para DG?, DG? e laminina foram mais fracas nos grupos CP e NIP em relação aos grupos HBP e Senil. Assim, pode-se concluir que o desequilíbrio da interação epitélio-estroma prostático causado pelas mudanças estruturais é fator decisivo para o desenvolvimento e progressão da carcinogênese na senescência. Ainda, a diminuição dos níveis protéicos do complexo das DGs e o aumento dos níveis das MMPs estão associados com a gravidade da lesão prostática e tem importante papel na manutenção do fenótipo maligno. A ocorrência de IGFR-1 sinalizou o grau de comprometimento glandular e a perda da imunolocalização da laminina provavelmente propiciou a progressão das lesões. Também, a localização diferencial dos receptores esteróides nas diferentes lesões prostáticas entre os compartimentos epitelial e estromal de homens senis certamente indicou diferentes sinalizações parácrinas para a dinâmica prostática, indicando ação diferencial desses receptores a partir dos compartimentos prostáticos e apontaram a importância das vias estrogênicas nos mecanismos de ativação dessas alterações. Finalmente, a interação do IGFR-1 com as MMP2 e MMP9 apontaram arelevância dessas moléculas nas investigações e tratamentos das doenças prostáticas.Abstract: Senescence is a determining factor for the occurrence of morphological changes in the prostate, considering hormonal imbalance and paracrine signaling disturbance between the epithelium and the stroma. Thus, the objective of this work was to characterize and correlate the ? and ?-dystroglycans (?-DG, ?-DG), androgen receptor (AR), ? and ?-estrogen receptors (?ER, ?ER), matriz metalloproteinases 2 and 9 (MMP-2, MMP-9) and insulin-like growth factor receptor (IGFR-1) reactivities in both the stroma and epithelium compartments in the prostatic peripheral zone of elderly men showing Benign Prostatic Hyperplasia (BPH), Prostatic Intraepithelial Neoplasia (PIN) and Prostatic Cancer (PC). Sixty prostate samples from 60-90 year old patients were divided into 4 groups: The Senile Group (without prostatic lesions); The Prostatic Intraepithelial Neoplasia (PIN) Group; The Prostatic Cancer (PC) Group and The Prostatic Benign Hyperplasia (BPH) Group. The lesion diagnosis was based on architectural disturbance according to morphological criteria and by immunostaining for cytokeratin. The results were evaluated by light microscopy and transmission electron, morphometric methods, immunohistochemical and Western Blotting. The different prostatic lesions in senescence showed an imbalance in the rates of proliferation/apoptosis, indicating predominance of proliferative process. The results showed more intense IGFR-1, MMP-2 and MMP-9 immunoreactivities in the PC and PIN groups than in the other groups. The ?-DG and ?-DG immunolocalization were weaker in the PC and PIN groups than in the BPH and Senile groups. Intensified AR reactivity was verified in the epithelial compartment in all studied groups. ?ER immunoreactivity was more intense in the epithelial compartment in the PC and PIN groups than in the other groups. ?ER immunoreactivity was weaker in the epithelial compartment of the PIN and PC groups than in the BPH and Senile groups. Also, this receptor was absent in the stromal compartment in the PC group. The different prostatic lesions showed abnormal epithelial-stromal interaction, due to decreased ?-DG and ?-DG levels and increased IGFR-1, MMP-2 and MMP-9 levels, which are essential elements for the maintenance of glandular paracrine signaling, contributing to disease progression in the senescence. The differential steroid hormone receptor reactivity in the different lesions in both prostatic compartments certainly indicated different paracrine signals to the dynamics of the prostate and pointed out the importance of estrogenic pathways in the activation mechanisms of these changes. Also, these findings indicated that there was a direct correlation between IGFR-1, MMPs and steroid hormone receptors, pointing towards IGFR-1 as a molecule target in prostate therapy, as well as being a possible factor for MMPs positive signaling.MestradoAnatomiaMestre em Biologia Celular e Estrutura

Prolactin, Egfr, Vimentin And Alpha-actin Profiles In Elderly Rat Prostate Subjected To Steroid Hormonal Imbalance

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The aim of this study was to characterize and relate the prolactin (PR), epidermal growth factor receptor (EGFR), alpha-actin and vimentin immunoreactivity in the prostate of elderly rats subjected to steroid hormonal imbalance. Senile and young rats were divided into the young group (YNG), the senile group (SE), the castrated group (CAS), the estrogen-deficient group (ED), the castrated + estrogen group (CASE), and the estrogen-deficient + androgen group (EDTEST). PR and EGFR increased in the estrogen and androgen ablation groups. In addition, EGFR influenced the immunolocalization by changing it from the prostatic stroma to the epithelium in elderly rats. Hormone ablation in elderly rats, not only related to androgen but also estrogen, led to increased stromal EGFR immunolocalization. The alpha-actin pattern decreased in the groups with estrogenic imbalance. Moreover, vimentin increased in the senile and estrogen deficient group. To conclude, we can suggest that EGFR contributed towards the proliferative process in the prostate, by means however, of different mechanisms, considering the androgenic and estrogenic pathways. Also, our results indicated that prolactin could be activated not only in an androgen-independent pathway but also in an estrogen independent pathway. Finally, PR and vimentin immunolocalization increase, in the prostatic stroma in the group showing estrogenic ablation, could be one of the factors which contribute to the reactive stroma formation. (C) 2016 Elsevier Ltd. All rights reserved.483189196Sao Paulo Research Foundation (FAPESP) [2010/01739-1, 2009/50396-2]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Prostatic Angiogenic Responses In Late Life: Antiangiogenic Therapy Influences And Relation With The Glandular Microenvironment In The Transgenic Adenocarcinoma Of Mouse Prostate (tramp) Model.

Aging is considered one of the main predisposing factors for the development of prostate malignancies. Angiogenesis is fundamental for tumor growth and its inhibition represents a promising therapeutic approach in cancer treatment. Thus, we sought to determine angiogenic responses and the effects of antiangiogenic therapy in the mouse prostate during late life, comparing these findings with the prostatic microenvironment in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. Male mice (52 week-old FVB) were submitted to treatments with SU5416 (6 mg/kg; i.p.) and/or TNP-470 (15 mg/kg; s.c.). Finasteride was administered (20 mg/kg; s.c.), alone or in association to both inhibitors. The dorsolateral prostate was collected for VEGF, HIF-1α, FGF-2 and endostatin immunohistochemical and Western Blotting analyses and for microvessel density (MVD) count. Senescence led to increased MVD and VEGF, HIF-1α and FGF-2 protein levels in the prostatic microenvironment, similarly to what was observed in TRAMP mice prostate. The angiogenic process was impaired in all the treated groups, demonstrating significantly decreased MVD. Antiangiogenic and/or finasteride treatments resulted in decreased VEGF and HIF-1α levels, especially following TNP-470 administration, either alone or associated to SU5416. The combination of these agents resulted in increased endostatin levels, regardless of the presence of finasteride. Prostatic angiogenesis stimulation during senescence favored the development of neoplastic lesions, considering the pro-angiogenic microenvironment as a common aspect also observed during cancer progression in TRAMP mice. The combined antiangiogenic therapy was more efficient, leading to enhanced imbalance towards angiogenic inhibition in the organ. Finally, finasteride administration might secondarily upregulate the expression of pro-angiogenic factors, pointing to the harmful effects of this therapy. Prostate 75: 484-499, 2015. © 2014 Wiley Periodicals, Inc.75484-9

Prolactin, EGFR, vimentin and α-actin profiles in elderly rat prostate subjected to steroid hormonal imbalance

The aim of this study was to characterize and relate the prolactin (PR), epidermal growth factor receptor (EGFR), alpha-actin and vimentin immunoreactivity in the prostate of elderly rats subjected to steroid hormonal imbalance. Senile and young rats were divided into the young group (YNG), the senile group (SE), the castrated group (CAS), the estrogen-deficient group (ED), the castrated + estrogen group (CASE), and the estrogen-deficient + androgen group (EDTEST). PR and EGFR increased in the estrogen and androgen ablation groups. In addition, EGFR influenced the immunolocalization by changing it from the prostatic stroma to the epithelium in elderly rats. Hormone ablation in elderly rats, not only related to androgen but also estrogen, led to increased stromal EGFR immunolocalization. The alpha-actin pattern decreased in the groups with estrogenic imbalance. Moreover, vimentin increased in the senile and estrogen deficient group. To conclude, we can suggest that EGFR contributed towards the proliferative process in the prostate, by means however, of different mechanisms, considering the androgenic and estrogenic pathways. Also, our results indicated that prolactin could be activated not only in an androgen-independent pathway but also in an estrogen independent pathway. Finally, PR and vimentin immunolocalization increase, in the prostatic stroma in the group showing estrogenic ablation, could be one of the factors which contribute to the reactive stroma formation483189196FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2010/01739-1; 2009/50396-

Hormonal Therapy In The Senescence: Prostatic Microenvironment Structure And Adhesion Molecules.

Hormonal replacement has been utilized to minimize the harmful effects of hormonal imbalance in elderly men. The development and progression of prostatic diseases and their relation to hormone therapy is still unclear. Thus, the aim herewith was to characterize the structure and dystroglycan molecule (DGs) reactivities in the ventral prostatic lobe from elderly rats submitted to steroid hormone replacement. Male rats (Sprague-Dawley) were divided into one Young group and six senile groups. The Young group (YNG) (4 months old) received peanut oil (5mL/kg, s.c.). The senile rats (10 months old) were submitted to the following treatments: Senile group (SEN) (5mL/kg peanut oil, s.c.); Testosterone group (TEST) (5mg/kg testosterone cipionate, s.c.); Estrogen group (EST) (25μg/kg 17β-estradiol, s.c.); Castrated group (CAS) (surgical castration); Castrated-Testosterone (CT) (surgical castration and treatment similar to TEST group); and Castrated-Estrogen (CE) (surgical castration and treatment similar to EST group). After 30 days treatment, blood samples were collected for hormonal analysis and ventral prostate samples were processed for light and transmission electron microscopies, morphometrical analysis, immunohistochemistry and Western Blotting. The results showed decreased serum testosterone levels in the senescence and increased testosterone and estrogen plasmatic levels after hormone administration in the TEST and EST groups, respectively, highlighting the therapy efficiency. Hypertrophied stroma and inflammatory cells were verified in the SEN group. After hormone replacement in the senescence or following castration, atrophic epithelium, epithelial cells with clear cytoplasmic halo around the nucleus, microacini and maintenance of hypertrophied stroma were seen. Decreased DG levels were verified in the senescence. After hormonal therapy, increased protein levels of these molecules were observed, especially in those groups which received estradiol. Thus, the occurrence of inflammatory cells, stromal hypertrophy and the presence of cells with clear halo around the nucleus after hormonal therapy probably indicated prostatic paracrine signaling imbalance, suggesting a stromal reactive microenvironment favorable to the development of glandular lesions. However, the increase of DG levels characterized positive effect of steroid hormone replacement on the prostate in the senescence. Thus, it could be concluded that despite having positive effects on important molecules involved in the maintenance of epithelial-stromal interaction and glandular cytoarchitecture, such as DGs, hormonal therapy enhanced structural changes associated with senescence, probably due to increased hormonal imbalance between androgens and estrogens in the prostatic tissue.42642-5

Hormonal therapy in the senescence: Prostatic microenvironment structure and adhesion molecules

Hormonal replacement has been utilized to minimize the harmful effects of hormonal imbalance in elderly men. The development and progression of prostatic diseases and their relation to hormone therapy is still unclear. Thus, the aim herewith was to characterize the structure and dystroglycan molecule (DGs) reactivities in the ventral prostatic lobe from elderly rats submitted to steroid hormone replacement. Male rats (Sprague-Dawley) were divided into one Young group and six senile groups. The Young group (YNG) (4 months old) received peanut oil (5 mL/kg, s.c.). The senile rats (10 months old) were submitted to the following treatments: Senile group (SEN) (5 mL/kg peanut oil, s.c.); Testosterone group (TEST) (5 mg/kg testosterone cipionate, s.c.); Estrogen group (EST) (25 mu g/kg 17 beta-estradiol, s.c.); Castrated group (CAS) (surgical castration); Castrated-Testosterone (CT) (surgical castration and treatment similar to TEST group); and Castrated-Estrogen (CE) (surgical castration and treatment similar to EST group). After 30 days treatment, blood samples were collected for hormonal analysis and ventral prostate samples were processed for light and transmission electron microscopies, morphometrical analysis, immunohistochemistry and Western Blotting. The results showed decreased serum testosterone levels in the senescence and increased testosterone and estrogen plasmatic levels after hormone administration in the TEST and EST groups, respectively, highlighting the therapy efficiency. Hypertrophied stroma and inflammatory cells were verified in the SEN group. After hormone replacement in the senescence or following castration, atrophic epithelium, epithelial cells with clear cytoplasmic halo around the nucleus, microacini and maintenance of hypertrophied stroma were seen. Decreased DG levels were verified in the senescence. After hormonal therapy, increased protein levels of these molecules were observed, especially in those groups which received estradiol. Thus, the occurrence of inflammatory cells, stromal hypertrophy and the presence of cells with clear halo around the nucleus after hormonal therapy probably indicated prostatic paracrine signaling imbalance, suggesting a stromal reactive microenvironment favorable to the development of glandular lesions. However, the increase of DG levels characterized positive effect of steroid hormone replacement on the prostate in the senescence. Thus, it could be concluded that despite having positive effects on important molecules involved in the maintenance of epithelial-stromal interaction and glandular cytoarchitecture, such as DGs, hormonal therapy enhanced structural changes associated with senescence, probably due to increased hormonal imbalance between androgens and estrogens in the prostatic tissue. (C) 2011 Elsevier Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Antiangiogenic and finasteride therapies: Responses of the prostate microenvironment in elderly mice

Aims The aim of this study was to evaluate the structural and molecular effects of antiangiogenic therapies and finasteride on the ventral prostate of senile mice. Main methods 90 male FVB mice were divided into: Young (18 weeks old) and senile (52 weeks old) groups; finasteride group: finasteride (20 mg/kg); SU5416 group: SU5416 (6 mg/kg); TNP-470 group: TNP-470 (15 mg/kg,) and SU5416 + TNP-470 group: similar to the SU5416 and TNP-470 groups. After 21 days, prostate ventral lobes were collected for morphological, immunohistochemical and Western blotting analyses. Key findings The results demonstrated atrophy, occasional proliferative lesions and inflammatory cells in the prostate during senescence, which were interrupted and/or blocked by treatment with antiangiogenic drugs and finasteride. Decreased AR and endostatin reactivities, and an increase for ER-α, ER-β and VEGF, were seen in the senile group. Decreased VEGF and ER-α reactivities and increased ER-β reactivity were verified in the finasteride, SU5416 groups and especially in SU5416 + TNP-470 group. The TNP-470 group showed reduced AR and ER-β protein levels. Significance The senescence favored the occurrence of structural and/or molecular alterations suggesting the onset of malignant lesions, due to the imbalance in the signaling between the epithelium and stroma. The SU5416 + TNP-470 treatment was more effective in maintaining the structural, hormonal and angiogenic factor balance in the prostate during senescence, highlighting the signaling of antiproliferation via ER-β1061-25870FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2011/03116-4; 2012/03010-