7 research outputs found
Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide
The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM
Oral administration of linoleic acid induces new vessel formation and improves skin wound healing in diabetic rats
Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide
The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM
An Asp49 Phospholipase A2 From Snake Venom Induces Cyclooxygenase-2 Expression And Prostaglandin E2 Production Via Activation Of Nf- κ B, P38mapk, And Pkc In Macrophages
Phospholipases A2 (PLA2) are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PG)E2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2). Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE 2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins. © 2014 Vanessa Moreira et al.2014Astiz, M., Saha, D., Lustbader, D., Lin, R., Rackow, E., Monocyte response to bacterial toxins, expression of cell surface receptors, and release of anti-inflammatory cytokines during sepsis (1996) Journal of Laboratory and Clinical Medicine, 128 (6), pp. 594-600Schaloske, R.H., Dennis, E.A., The phospholipase A2 superfamily and its group numbering system (2006) Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1761 (11), pp. 1246-1259. , DOI 10.1016/j.bbalip.2006.07.011, PII S1388198106002204Kaiser, I.I., Gutierrez, J.M., Plummer, D., Aird, S.D., Odell, G.V., The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper (1990) Archives of Biochemistry and Biophysics, 278 (2), pp. 319-325. , DOI 10.1016/0003-9861(90)90266-2Zuliani, J.P., Fernandes, C.M., Zamuner, S.R., Gutierrez, J.M., Teixeira, C.F.P., Inflammatory events induced by Lys-49 and Asp-49 phospholipases A 2 isolated from Bothrops asper snake venom: Role of catalytic activity (2005) Toxicon, 45 (3), pp. 335-346. , DOI 10.1016/j.toxicon.2004.11.004Zuliani, J.P., Gutierrez, J.M., Casais E Silva, L.L., Sampaio, S.C., Lomonte, B., Pereira Teixeira, C.D.F., Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A2 (2005) Toxicon, 46 (5), pp. 523-532. , DOI 10.1016/j.toxicon.2005.06.017, PII S0041010105002266Chaves, F., Leon, G., Alvarado, V.H., Gutierrez, J.M., Pharmacological modulation of edema induced by Lys-49 and Asp-49 myotoxic phospholipases A2 isolated from the venom of the snake Bothrops asper (terciopelo) (1998) Toxicon, 36 (12), pp. 1861-1869. , DOI 10.1016/S0041-0101(98)00107-X, PII S004101019800107XChacur, M., Longo, I., Picolo, G., Gutierrez, J.M., Lomonte, B., Guerra, J.L., Teixeira, C.F.P., Cury, Y., Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: Pharmacological mediation and molecular determinants (2003) Toxicon, 41 (6), pp. 667-678. , DOI 10.1016/S0041-0101(03)00007-2Moreira, V., Gutiérrez, J.M., Soares, A.M., Zamunér, S.R., Purgatto, E., Teixeira, C.D.F.P., Secretory phospholipases A2 isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E2 and D2 and expression of cyclooxygenases (2008) Toxicon, 52 (3), pp. 428-439. , 2-s2.0-50549090862 10.1016/j.toxicon.2008.06.012O'Neill, G.P., Ford-Hutchinson, A.W., Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues (1993) FEBS Letters, 330 (2), pp. 156-160Herschman, H.R., Prostaglandin synthase 2 (1996) Biochimica et Biophysica Acta - Lipids and Lipid Metabolism, 1299 (1), pp. 125-140. , DOI 10.1016/0005-2760(95)00194-8Sheng, H., Williams, C.S., Shao, J., Liang, P., DuBois, R.N., Beauchamp, R.D., Induction of cyclooxygenase-2 by activated Ha-ras oncogene in rat-1 fibroblasts and the role of mitogen-activated protein kinase pathway (1998) Journal of Biological Chemistry, 273 (34), pp. 22120-22127. , DOI 10.1074/jbc.273.34.22120Barrios-Rodiles, M., Tiraloche, G., Chadee, K., Lipopolysaccharide modulates cyclooxygenase-2 transcriptionally and posttranscriptionally in human macrophages independently from endogenous IL- 1β and TNF-α (1999) Journal of Immunology, 163 (2), pp. 963-969Caivano, M., Cohen, P., Role of mitogen-activated protein kinase cascades in mediating lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1β in RAW264 macrophages (2000) Journal of Immunology, 164 (6), pp. 3018-3025Minghetti, L., Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases (2004) Journal of Neuropathology and Experimental Neurology, 63 (9), pp. 901-910Chen, C.-C., Chiu, K.-T., Chan, S.-T., Chern, J.-W., Conjugated polyhydroxybenzene derivatives block tumor necrosis factor-α-mediated nuclear factor-κB activation and cyclooxygenase-2 gene transcription by targeting IκB kinase activity (2001) Molecular Pharmacology, 60 (6), pp. 1439-1448De Gregorio, R., Iñiguez, M.A., Fresno, M., Alemany, S., Cot kinase induces cyclooxygenase-2 expression in T cells through activation of the nuclear factor of activated T cells (2001) The Journal of Biological Chemistry, 276 (29), pp. 27003-27009. , 2-s2.0-0035920233 10.1074/jbc.M100885200Yamamoto, K., Arakawa, T., Ueda, N., Yamamoto, S., Transcriptional roles of nuclear factor B and nuclear factor-interleukin-6 in the tumor necrosis factor α -dependent induction of cyclooxygenase-2 in MC3T3-E1 cells (1995) The Journal of Biological Chemistry, 270 (52), pp. 31315-31320. , 2-s2.0-0029595394Dayong, W., Marko, M., Claycombe, K., Paulson, K.E., Meydani, S.N., Ceramide-induced and age-associated increase in macrophage COX-2 expression is mediated through up-regulation of NF-κB activity (2003) Journal of Biological Chemistry, 278 (13), pp. 10983-10992. , DOI 10.1074/jbc.M207470200Chang, M.-Y., Ho, F.-M., Wang, J.-S., Kang, H.-C., Chang, Y., Ye, Z.-X., Lin, W.-W., AICAR induces cyclooxygenase-2 expression through AMP-activated protein kinase-transforming growth factor- β -activated kinase 1-p38 mitogen-activated protein kinase signaling pathway (2010) Biochemical Pharmacology, 80 (8), pp. 1210-1220. , 2-s2.0-77956229580 10.1016/j.bcp.2010.06.049Lo, H.-M., Chen, C.-L., Tsai, Y.-J., Wu, P.-H., Wu, W.-B., Thrombin induces cyclooxygenase-2 expression and prostaglandin E 2 release via PAR1 activation and ERK1/2- and p38 MAPK-dependent pathway in murine macrophages (2009) Journal of Cellular Biochemistry, 108 (5), pp. 1143-1152. , 2-s2.0-72449173512 10.1002/jcb.22341Misra, U.K., Pizzo, S.V., Induction of cyclooxygenase-2 synthesis by ligation of the macrophage α2-macroglobulin signalling receptor (2001) Cellular Signalling, 13 (11), pp. 801-808. , DOI 10.1016/S0898-6568(01)00202-9, PII S0898656801002029Freudenburg, W., Moran, J.M., Lents, N.H., Baldassare, J.J., Buller, R.M.L., Corbett, J.A., Phosphatidylinositol 3-kinase regulates macrophage responses to double-stranded RNA and encephalomyocarditis virus (2010) Journal of Innate Immunity, 2 (1), pp. 77-86. , 2-s2.0-77953430564 10.1159/000243785Lin, W.-W., Chen, B.-C., Induction of cyclo-oxygenase-2 expression by methyl arachidonyl fluorophosphonate in murine J774 macrophages: Roles of protein kinase C, ERKs and p38 MAPK (1999) British Journal of Pharmacology, 126 (6), pp. 1419-1425Lomonte, B., Gutierrez, J.M., A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo) (1989) Toxicon, 27 (7), pp. 725-733. , DOI 10.1016/0041-0101(89)90039-1Takayama, K., Mitchell, D.H., Din, Z.Z., Mukerjee, P., Li, C., Coleman, D.L., Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages (1994) Journal of Biological Chemistry, 269 (3), pp. 2241-2244Leiguez, E., Zuliani, J.P., Cianciarullo, A.M., Fernandes, C.M., Gutiérrez, J.M., Teixeira, C., A group IIA-secreted phospholipase A2 from snake venom induces lipid body formation in macrophages: The roles of intracellular phospholipases A2 and distinct signaling pathways (2011) Journal of Leukocyte Biology, 90 (1), pp. 155-166. , 2-s2.0-79959814270 10.1189/jlb.0510263Rong, Y., Baudry, M., Seizure activity results in a rapid induction of nuclear factor-κB in adult but not juvenile rat limbic structures (1996) Journal of Neurochemistry, 67 (2), pp. 662-668Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding (1976) Analytical Biochemistry, 72 (1-2), pp. 248-254. , 2-s2.0-0017184389Vinolo, M.A.R., Rodrigues, H.G., Hatanaka, E., Sato, F.T., Sampaio, S.C., Curi, R., Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils (2011) Journal of Nutritional Biochemistry, 22 (9), pp. 849-855. , 2-s2.0-80051471073 10.1016/j.jnutbio.2010.07.009Lin, Y.-C., Brown, K., Siebenlist, U., Activation of NF- B requires proteolysis of the inhibitor i B- α: Signal-induced phosphorylation of i B- α alone does not release active NF- B (1995) Proceedings of the National Academy of Sciences of the United States of America, 92 (2), pp. 552-556. , 2-s2.0-0028981050 10.1073/pnas.92.2.552Baek, S.H., Lim, J.H., Park, D.W., Kim, S.Y., Lee, J.H., Kim, J.H., Group IIA secretory phospholipase A2 stimulates inducible nitric oxide synthase expression via ERK and NF-kB in macrophages (2001) European Journal of Immunology, 31 (9), pp. 2709-2717Hernandez, M., Burillo, S.L., Crespo, M.S., Nieto, M.L., Secretory phospholipase A2 activates the cascade of mitogen-activated protein kinases and cytosolic phospholipase A2 in the human astrocytoma cell line 1321N1 (1998) Journal of Biological Chemistry, 273 (1), pp. 606-612. , DOI 10.1074/jbc.273.1.606Moreira, V., Souto, P.C.C., Vinolo, M.A.R., Lomonte, B., Gutiérrez, J.M., Curi, R., Teixeira, C., A catalytically-inactive snake venom Lys49 phospholipase A2 homolog induces expression of cyclooxygenase-2 and production of prostaglandins through selected signaling pathways in macrophages (2013) European Journal of Pharmacology, 708 (1-3), pp. 68-79Viatour, P., Merville, M.-P., Bours, V., Chariot, A., Phosphorylation of NF-κB and IκB proteins: Implications in cancer and inflammation (2005) Trends in Biochemical Sciences, 30 (1), pp. 43-52. , DOI 10.1016/j.tibs.2004.11.009, PII S0968000404002993Chen, K.-C., Kao, P.-H., Lin, S.-R., Chang, L.-S., P38 MAPK activation and mitochondrial depolarization mediate the cytotoxicity of Taiwan cobra phospholipase A2 on human neuroblastoma SK-N-SH cells (2008) Toxicology Letters, 180 (1), pp. 53-58. , 2-s2.0-47049088244 10.1016/j.toxlet.2008.05.014Craig, R., Larkin, A., Mingo, A.M., Thuerauf, D.J., Andrews, C., McDonough, P.M., Glembotski, C.C., P38 MAPK and NF-κB collaborate to induce Interleukin-6 Gene expression and release: Evidence for a cytoprotective Autocrine signaling pathway in a cardiac myocyte model system (2000) Journal of Biological Chemistry, 275 (31), pp. 23814-23824. , DOI 10.1074/jbc.M909695199Fan, Y., Wang, J., Wei, L., He, B., Wang, C., Wang, B., Iron deficiency activates pro-inflammatory signaling in macrophages and foam cells via the p38 MAPK-NF- B pathway (2011) International Journal of Cardiology, 152 (1), pp. 49-55. , 2-s2.0-78751538345 10.1016/j.ijcard.2010.07.005Liu, S., Feng, G., Wang, G.-L., Liu, G.-J., P38MAPK inhibition attenuates LPS-induced acute lung injury involvement of NF- B pathway (2008) European Journal of Pharmacology, 584 (1), pp. 159-165. , 2-s2.0-41149147968 10.1016/j.ejphar.2008.02.009Lin, M.-L., Lu, Y.-C., Chung, J.-G., Wang, S.-G., Lin, H.-T., Kang, S.-E., Tang, C.-H., Chen, S.-S., Down-regulation of MMP-2 through the p38 MAPK-NF- B-dependent pathway by aloe-emodin leads to inhibition of nasopharyngeal carcinoma cell invasion (2010) Molecular Carcinogenesis, 49 (9), pp. 783-797. , 2-s2.0-77956196134 10.1002/mc.20652Chang, Y.-C., Li, P.-C., Chen, B.-C., Chang, M.-S., Wang, J.-L., Chiu, W.-T., Lin, C.-H., Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor- B pathways (2006) Cellular Signalling, 18 (8), pp. 1235-1243. , 2-s2.0-33646358491 10.1109/TCSI.2006.875167Han, W.K., Sapirstein, A., Hung, C.C., Alessandrini, A., Bonventre, J.V., Cross-talk between cytosolic phospholipase A2α (cPLA2α) and secretory phospholipase A2 (sPLA 2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: SPLA2 regulates cPLA2α activity that is responsible for arachidonic acid release (2003) Journal of Biological Chemistry, 278 (26), pp. 24153-24163. , DOI 10.1074/jbc.M300424200Gambero, A., Thomazzi, S.M., Cintra, A.C.O., Landucci, E.C.T., De Nucci, G., Antunes, E., Signalling pathways regulating human neutrophil migration induced by secretory phospholipases A2 (2004) Toxicon, 44 (5), pp. 473-481. , DOI 10.1016/j.toxicon.2004.06.004, PII S0041010104002582Giroux, M., Descoteaux, A., Cyclooxygenase-2 expression in macrophages: Modulation by protein kinase C-α1 (2000) Journal of Immunology, 165 (7), pp. 3985-3991Sun, J., Ramnath, R.D., Tamizhselvi, R., Bhatia, M., Role of protein kinase C and phosphoinositide 3-kinase-Akt in substance P-induced proinflammatory pathways in mouse macrophages (2009) The FASEB Journal, 23 (4), pp. 997-1010. , 2-s2.0-65349103540 10.1096/fj.08-121756Bansal, K., Holla, S., Verma-Kumar, S., Sharma, P., Balaji, K.N., ESAT-6 induced COX-2 expression involves coordinated interplay between PI3K and MAPK signaling (2012) Molecular Immunology, 49 (4), pp. 655-663. , 2-s2.0-84555217911 10.1016/j.molimm.2011.11.011De Oliveira, A.C.P., Candelario-Jalil, E., Langbein, J., Wendeburg, L., Bhatia, H.S., Schlachetzki, J.C.M., Biber, K., Fiebich, B.L., Pharmacological inhibition of Akt and downstream pathways modulates the expression of COX-2 and mPGES-1 in activated microglia (2012) Journal of Neuroinflammation, 92. , 2-s2.0-84855239318 10.1186/1742-2094-9-2Bachelor, M.A., Cooper, S.J., Sikorski, E.T., Bowden, G.T., Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model (2005) Molecular Cancer Research, 3 (2), pp. 90-99. , DOI 10.1158/1541-7786.MCR-04-0065Zhang, Q.-H., Zu, X.-Y., Cao, R.-X., Liu, J.-H., Mo, Z.-C., Zeng, Y., Li, Y.-B., Yi, G.-H., An involvement of SR-B1 mediated PI3K-Akt-eNOS signaling in HDL-induced cyclooxygenase 2 expression and prostacyclin production in endothelial cells (2012) Biochemical and Biophysical Research Communications, 420 (1), pp. 17-23. , 2-s2.0-84859106262 10.1016/j.bbrc.2012.02.10
Protein-energy malnutrition alters histological and ultrastructural characteristics of the bone marrow and decreases haematopoiesis in adult mice
Protein-energy malnutrition (PEM) decreases
resistance to infection by impairing a number of
physiological processes, including haematopoiesis. The
aim of this study was to evaluate the microanatomical
aspects of bone marrow (BM) in mice that were
subjected to PEM, in particular, with respect to the
components of the local extracellular matrix and the
proliferative activity of haematopoietic cells. For this,
histological, histochemical, immunohistochemical and
ultrastructural techniques were used. Two-month old
male Swiss mice were fed with a low-protein diet
containing 4% protein and control mice fed a 20%
protein diet. When the experimental group had attained a
25% loss of their original body weight, we collected the
different biological samples. Malnourished mice had
presented severe BM atrophy as well as a reduction in
proliferating cell nuclear antigen and gelatinous
degeneration. The malnourished mice had more
fibronectin accretion in paratrabecular and endosteal
regions and more laminin deposition in perisinusal sites
than controls. Endosteal cell activation and hyperplasia
were found, suggesting their participation in the process.
Additionally, we have observed a decrease in the
capacity of malnourished haematopoietic stroma to
support the growth of haematopoietic stem cells
(CD34+) in vitro. These findings point to a structural impairment of the haematopoietic microenvironments in
mice with PEM, possibly hampering the interactions
between cells and cellular signalling
Reduced graphene oxide: nanotoxicological profile in rats
We have previously demonstrated that reduced graphene oxide (rGO) administered intravenously in rats was detected inside the hippocampus after downregulation of the tight and adherens junction proteins of the blood-brain barrier. While down-regulators of junctional proteins could be useful tools for drug delivery through the paracellular pathway, concerns over toxicity must be investigated before clinical application. Herein, our purpose was to trace whether the rGO inside the hippocampus triggered toxic alterations in this brain region and in target organs (blood, liver and kidney) of rats at various time points (15 min, 1, 3 h and 7 days). Results: The assessed rGO-treated rats (7 mg/kg) were clinically indistinguishable from controls at all the time points. Hematological, histopathological (neurons and astrocytes markers), biochemical (nephrotoxicity and hepatotoxicity assessment) and genotoxicological based tests showed that systemic rGO single injection seemed to produce minimal toxicological effects at the time points assessed. Relative to control, the only change was a decrease in the blood urea nitrogen level 3 h post-treatment and increases in superoxide dismutase activity 1 h and 7 days post-treatment. While no alteration in leukocyte parameters was detected between control and rGO-treated animals, time-dependent leukocytosis (rGO-1 h versus rGO-3 h) and leukopenia (rGO-3 h versus rGO-7 days) was observed intra-treated groups. Nevertheless, no inflammatory response was induced in serum and hippocampus at any time. Conclusions: The toxic effects seemed to be peripheral and transitory in the short-term analysis after systemic administration of rGO. The effects were self-limited and non-significant even at 7 days post-rGO administration.141CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305099/2011-6 ; 486142/2012-42012/24782-