TOXIMED, 2001-2006MSc, 2003

UO, 1995-2005Graduated, 2000

UFPR, 2005-2006

IQ-USP, 2006 –PhD, 2009

Post Doctoral, 2010

Unifesp, 2010 –Professor, 2010-

LABITA, 2011

TIME OF FORMATION AND EVOLUTION

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Can the manganese to be related with these topics?

Genes Decide Why Some People Love or it have Music ability and dance preference http://dx.doi.org/10.3389/fpsyg.2014.00658

http://www.biomedcentral.com/1471-2148/8/210

The molecular basis of color vision in fish and cognitive ability test in this model that can extrapolated for human

The genetic basis of cognition.http://dx.doi.org/10.1093/brain/122.11.2015

10.1007/s12311-013-0511-xThe Cerebellum's Role in Movement and Cognition

Dementia is classified as a neurocognitive disorder, with various degrees of severity, that cause a long term and often gradual decrease of cognitive ability and some time motor ability as well.

Avdesh et. al, 2012; Ahmad and Richardson, 2013http://www.nature.com/nature/journal/v496/n7446/full/nature12111.html

Prof. Dr Raúl Bonne HernándezBioinorgânica e Toxicologia Ambiental. Departamento de Ciências Exatas e da Terra

Instituto de Ciências Químicas, Farmacêuticas e Ambientais Universidade Federal de São Paulo

Toxicogenomics analysis of zebrafish (danio rerio) embryos reveals pathways involved in

manganese-induced dementia

• Neurological risks associated with occupational exposures is well established… Couper (1837) was the first that observed and reported the manganism (Parkinsonian symptoms)

• The environmental guideline have been little studied and its are based on neurotoxic effects observed in occupationally exposed workers and unclear animal data (low exposures ≠ elevated exposures), which suggest more studies for risk assessment of chronic low-level manganese exposure to humans (Gwiazda et al., 2007).

• Organisms during development are the more vulnerable for Mn and the neurotoxicity mechanisms are unclear. Hence, are necessaries more studies to identify effective biomarkers and to improve the neurotherapies available.

Mn neurotoxicity: 178 years about an unclear history

Manganese (Mn) from earth to human

Human dietary requirement for Mn

Upper Intake Level (UL)Adequate Intake (AI)

Mn is a naturally occurring metallic element (#25 in the period table) throughout the earth’s crust, found naturally in water, air , soil and several foods, being an essential trace element of metalloproteins for human health, in different metabolic processes (e.g., gluconeogenesis, energy metabolism, and antioxidant defenses (Crossgrove and Zheng, 2004).

Mn in airborne(MnA) > drinking water (MnW) for Mn NeurotoxicityAuthors/Localities Guideline MnA/MnW Neurologic symptoms

Baldwin et al., 1999; Hudnell, 1999mean (n = 297) women (n = 156)Quebec (Canada)

a0.05µg/m3

b0.15µg/m3

MnTP0.009-0.035µg/m3

MnPM100.007- 0.019 µg/m3

motor deficits and mood disorders, similar to those seen in occupationally exposed workers

Menezes-Filho et al., 200983 children aged 6 - 12 years Bahia (Brazil)

MnPM2.5 0.15 µg/m3

poorer cognitive performance,

He et al., 199492 children 11–13 years of age from Shanxi (China)

300 μg/La

400 μg/Lb

1000 μg/Lc

240–350 µg/L impaired manual dexterity and speed, short-term memory, and visual identification…

Wasserman et al., 2006142 children of 10 years of age from Bangladesh

800 µg/L lower intelligence quotient (IQ)

Bouchard et al., 2011362 children of 6–13 years of age from Quebec (Canada)

34 µg/L (1–2,700 µg/L)

MnW 10-fold ↑ 2.4 IQ

Total particulates (TP); PM10 and PM2.5 (particles measuring 10µm, 2.5 µm or less)aUSEPA, 2004; b Air quality guidelines for Europe, 2000/ bWHO, 2006; cPortaria-MS No 518/2004.Brazil

Despite airborne exposure for Mn (>1 mg/m3) is the most important to Mn neurotoxicity, the increasing number of studies reporting a relationship between Mn exposure for “safe levels in airborne and drinking water” and children’s health suggests further investigations about environmental neurotoxicity risk assessment for this metal.

The environmental “safe levels” for Mn should be reviewed!, specially in aquatic systems.

LC50-CGC (Neurotoxicity) 1.9 0.6 23LC50-CTX (Neurotoxicity) 8,8 4 12

Gene-DarT 186 116 6LC50-DarT (Mortality) 267 117 6

EC50-DarT (Neurotoxicity) 42 6 6RAC 76.7 30 12FII-A 0.08 0.06 12

H2O-CETESB 0.1-0.5H2O-WHO 0.4

LEL 460

Risk Parameter level (ppm) sd n

Hernández et al., JEM 2009, Hernández, 2009, Hernández et al., 2015

is essential for normal prenatal and neonatal development...

for bone mineralization, protein and energy metabolism, cellular

protection from freeradical species, etc

The brain remain small amount of Mn by long time because it elimination is the lowest of the whole body, specially during development… neurotransmitters alteration (Glutamate,

GABA, Dopamine…) motor and cognitive dysfunction

Hernández et al., 2011 Hernández et al, manuscript in eleboration

What is Toxicogenomics and which its role in the studies about Mn Neurotoxicity?

?

?

??

?

?

Results in vitro suggest that neurotoxicity:is the major problem for Mn exposure, Hernández, 2009Mn(II) is more toxic than Mn(III) Hernández et al., 2011

Mn is accumulated mainly in the basal ganglia and the cerebellum tooBurton et al., 2009;Fitsanakis et al., 2011; García et al., 2006; Sotogaku et al., 2000a;Yoon et al., 2009.

Differential expression of several metal transport systems within the developmental period Aschner et al., 2007

Mn disrupted proteins involved in glycolysis, excitotoxicity and cytoskeletal dynamics.Wegrzynowicz et al., 2012

Biochemical changes identified in manganese-exposed monkeys included endpoints relate to oxidative stress (e.g., oxidized glutathione) and neurotransmission (aminobutyrate, glutamine, phenylalanine).Dorman et al., 2008

Neurobehavioral deficits, characterized by locomotor and emotional perturbations, and nigral glial activation associated with significant brain Mn deposition are among the early signs of Mn neurotoxicity inexperimental animals caused by drinking water (DW)OverexposureKrishna et al., 2014

Objectives and justification for studies aboutMn Neurotoxicity in vivo using the zebrafish embryo model

(1) Fish represent an important species of economic value and are commonly used as model organism in environmental risk assessment and its embryos are considered as refinement, if not replacement to experiments with adult fish and higher vertebrates (Nagel, 2002; EFSA, 2005).

(2) The zebrafish is one of the most popular model species used in genetics, developmental biology, pharmacology research, and (eco)toxicology providing a rich source of available information and its embryonic stages appear to represent the most sensitive life stages for manganese toxicity and to other chemical too.

(3) Due to the principal similarities among vertebrates the zebrafish embryos also allow to unravel basic principles of toxicity important for human health (Eimon and Rubinstein, 2009; Brittijn, et al., 2009).

To compare the toxicity of several and representing common aquatic chemical species of manganese, and

To determine by toxicogenomics approaches the potential neurotoxicological mechanism of the manganese and its linking with dementia, using the wild-type zebrafish embryos model.

Toxicological results suggest that the (Mn(II) > Mn(III)) induced significant (p<0.05) lethality and reduced or

altered motility in zebrafish embryos

Hernández, 2009; Hernández et al., 2015

A-8 -4

0

50

100

-4 -3 -2 -1 0

2-50 hpf24-72 hpf

24-72 hpf, dc48-96 hpf72-120 hpf48-120 hpf2-122 hpf

MnCl2 (log mM)

% d

eath

of t

he c

ontr

ol

2-50 24-72 24-72dC 48-96 72-120 48-120 2-1220

10

20

30

40

50

60

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90

100

110

120

a

a

b

c

d

b

d

Embryo developmental stages-hpf

LC

50

(mM

)

B

Manganese-induced embryotoxicity depends on developmental stage and of the time exposure

Hernández et al, 2015

Concentration-response curves for lethality zebrafish embryos exposed to manganese chloride (MnCl2) and B) comparison of the LC50 of the MnCl2 in zebrafish embryos at different development stages and with different exposure durations (dC – chorion manually removed). Hatching occurs between 48 and 72 hpf (hours post fertilization). Treatments that do not share a common letter are significantly different from each other (p<0.05). Bars represent mean of the LC50 from three independent experiments ± standard deviation.

Hernández et al, 2015

Chemical speciation is important for manganese-induced embryotoxicity

Concentration-response curves for lethality zebrafish embryos exposed to manganese chloride (MnCl 2) and B) comparison of the LC50 of the MnCl2 in zebrafish embryos at different development stages and with different exposure durations (dC – chorion manually removed). Hatching occurs between 48 and 72 hpf (hours post fertilization). Treatments that do not share a common letter are significantly different from each other (p<0.05). Bars represent mean of the LC50 from three independent experiments ± standard deviation.

Hernández et al, 2015

Metallomics results confirmed that both the chemical speciation and the chemical fractionation are important for manganese-induced embryotoxicity

Schematic representation for chemical fractionation, speciation and total metal analysis ICP, inductively coupled plasma; OES, optical emission spectrometer; SEC, size exclusion chromatography and MS, mass spectrometry.

O-ISO

2H Cit-6.0 m

M-1.5mM

2MnCl

Mn(II)Cit-1

.5mM

Mn(III)Cit-1

.5mM

0

100

200

300

400

Mn Total Mn Ext Mn Pellet

*

*

***

AMn species

mg

/kg

(d

ry w

eig

ht)

O-ISO

2HCit-6

.0 mM

-1.5mM

2MnCl

Mn(II)Cit-1

.5mM

Mn(III)Cit-1

.5mM

0

20

40

60

80

Fe Total Fe Ext Fe Pellet

BMn species

mg

/kg

(d

ry w

eig

ht)

O-ISO

2H Cit-6.0 m

M-1.5mM

2MnCl

Mn(II)Cit-1

.5mM

Mn(III)Cit-1

.5mM

0

10

20

30

40

Cu Total Cu Ext Cu Pellet

CMn species

mg

/kg

(d

ry w

eig

ht)

O-ISO

2H Cit-6.0 m

M-1.5mM

2MnCl

Mn(II)Cit-1

.5mM

Mn(III)Cit-1

.5mM

0

100

200

300

Zn Total Zn Ext Zn Pellet

* **

D Mn species

mg

/kg

(d

ry w

eig

ht)

Hernández et al, 2015

Contrary to Mn(III), the Mn(II) is more accumulated in the pellet fraction (granule and membrane component) than the supernatant fraction (stable and denature

proteins, organelles and other cytoplasmic component). Mn appear in tissue majority as an inorganic specie and trace complexed with citrate in supernatant

A) Manganese fractionation in samples from whole zebrafish larvae tissues after exposure to manganese species, from 48 to 120 hpf. Total (total metal), Ext (metal in liquid extract) and Pellet (non-dissolved metal). Data are shown as g Mn/kg of dry weight. Bars represent means from 3-4 independent experiments ± standard deviation. *** = significant different from each other with respect to the control, H2O-ISO (T-test, p≤0.001), * = significant different among metal fraction in each treatment (T-test, p≤0.05). B) Representative SEC-ICP/MS spectra of manganese speciation in samples from whole zebrafish embryos tissues, after exposure from 48 to 120 hpf to water-ISO (A) and MnCl2 (B). cps = counts per second.

Mn(II) but not Mn(III) induced calcium disruption, which is a plausible cause of Mn-induced embryotoxicity

Hernández et al, 2015

Total content of the macronutrients Na, K, Mg, and Ca (B) was analyzed in samples of whole zebrafish larvae tissues after exposure to the manganese species from 48 to 120 hpf. Data are shown as %, m/m of dry weight. Bars represent means from 3-4 independent experiments ± standard deviation. () and () denote significantly different from each other with respect to the control (H2O-ISO) and 6 mM citrate, respectively (t-test, p≤0.05).

0.0

0.5

1.0

1.5

2.0 P Total S Total

A(%

, m

/m,

dry

we

igh

t)

0.0

0.5

1.0

1.5 Na Total K Total Mg Total Ca Total

* ** * *

B Mn species

(%, m

/m, d

ry w

eig

ht)

Mn(II)

Hernández et al, 2015 (unpublished data)

Transcriptomic results confirmed too that the chemical speciation is important for manganese-induced embrytoxicity

GENECHIP ZEBRAFISH TRANSCRIPTOME ARRAY

(AFFYMETRIX)

qRt-PCR: TaqMan® Gene Expression Assays Protocol

(Applied Biosystems), using preformulated primers of genes

selected of the microarray results

Hernández et al, 2015 (unpublished data)

Mn(II)Cit is more important than MnCl2 for manganese-induced differential gene expression

Hernández et al, 2015 (unpublished data)

Probe set ControlDr.4244.1.A1_at 5.82 9.24 -10.64 0.00001 0.154887 cpa1Dr.21605.1.S1_at 5.98 8.73 -6.74 0.000022 0.175652 wu:fc25e04Probe set Control Citrato Fold Change (linear) Anova, p<0.05 pFDR GeneDr.3664.1.S1_at 9.7 5.74 15.52 0.000007 0.109028 fundc2Dr.9399.2.S1_at 9.09 5.78 9.93 0.000018 0.141985 ppp2r4Dr.15412.2.S1_at 9.5 5.95 11.71 0.000038 0.198802 tssc1Dr.14203.1.S1_at 5.42 9.24 -14.12 0.000058 0.22639 ppp1r14aaProbe set Citrato Mn(II)Cit 1.5 mM Fold Change (linear) Anova, p<0.05 pFDR GeneDr.14866.1.A1_at 5.16 9.89 -26.55 0.00001 0.159918 gnsaDr.14481.1.S1_at 9.41 5.87 11.6 0.000072 0.281294 LOC100536058Dr.16350.1.A1_at 5.92 10.19 -19.23 0.00005 0.281294 LOC100535214Dr.22485.1.A1_at 4.84 9.16 -19.87 0.000067 0.281294 slc14a2Probe set Citrato Mn(II)Cit 3 mM Fold Change (linear) Anova, p<0.05 pFDR GeneDr.209.1.S1_at 10.02 6.29 13.3 0.000044 0.23421 pitpnblDr.8744.1.A1_at 10.54 6.34 18.43 0.000045 0.23421 actr2aDr.14481.2.S1_x_at 5.55 9.7 -17.84 0.000037 0.23421 LOC100536018Dr.2720.1.A1_at 9.8 4.73 33.7 0.000136 0.236234 sgceDr.18285.1.A1_at 5.8 10.34 -23.25 0.000134 0.236234 ell2Dr.14849.1.S1_at 10.32 4.79 46.18 0.000136 0.236234 myl6Dr.12375.1.S1_at 5.61 10.19 -23.99 0.000127 0.236234 si:ch211-285f17.1Dr.399.1.A1_at 10.2 6.97 9.4 0.000134 0.236234 dda1DrAffx.2.100.A1_at 4.84 9.16 -19.91 0.000122 0.236234 dpp4Dr.20032.1.S1_at 9.21 4.91 19.62 0.000247 0.237002 mc5rbDr.9964.1.S1_at 5.55 9.59 -16.52 0.000201 0.237002Dr.3966.1.A1_at 8.83 5.04 13.87 0.000215 0.237002 tagln3bDr.4325.1.A1_at 9.82 4.88 30.79 0.000246 0.237002 bcat2Dr.1429.1.S1_at 10.29 5.29 31.85 0.000184 0.237002 zgc:193541Dr.15634.1.S1_at 5.43 9.85 -21.36 0.000192 0.237002 tcea3Dr.16312.1.S1_at 5.5 9.16 -12.61 0.00019 0.237002 sb:cb25Dr.14752.1.A1_at 10.11 6.13 15.83 0.000258 0.237002 fam100aa . LOC100536798Dr.26185.1.A1_at 9.14 4.92 18.53 0.000286 0.248387Dr.23818.1.A1_at 10.44 11.27 -1.77 0.000331 0.258301 zfr2Dr.23024.1.A1_at 10.44 5.54 29.88 0.000325 0.258301 etv5aDr.14300.1.S1_at 10.18 5.06 34.67 0.000359 0.266758 guca1cDr.13330.1.A1_at 5.36 7.79 -5.39 0.000574 0.277552 zgc:112153Dr.5480.1.A1_at 9.97 5.9 16.8 0.00059 0.277552 snx17Dr.1139.1.A1_at 5.41 8.93 -11.46 0.000493 0.277552 zgc:154054Dr.10109.1.A1_at 5.66 10.73 -33.69 0.000604 0.277552 zgc:92202Dr.10393.1.A1_at 10.21 5.69 22.93 0.000532 0.277552 ca5Dr.15501.1.S1_at 9.93 6.53 10.57 0.000477 0.277552 zgc:85866Dr.18241.1.A1_at 5.48 9.82 -20.25 0.000428 0.277552 cenpjDr.2408.2.S1_at 5.55 6.76 -2.32 0.000531 0.277552 mmp2Dr.19086.1.S1_at 5.35 8.72 -10.33 0.000589 0.277552 LOC553490Dr.17250.1.A1_at 9.24 5.6 12.46 0.000535 0.277552 erbb2ipDr.2028.1.A1_at 10.01 5.37 24.91 0.000572 0.277552 cnot3aDr.11831.1.S1_at 5.69 10.56 -29.14 0.000457 0.277552Dr.17651.1.A1_at 5.23 10.26 -32.61 0.000399 0.277552Dr.5541.1.S1_at 10.35 5.6 26.95 0.00067 0.290737 eif2s1Dr.7485.1.S1_at 9.92 5.14 27.41 0.000659 0.290737 LOC100536256 . pknox1.1

Probe setDr.7503.1.A1_x_at 9.49 5.08 21.26 0.000021 0.166739Dr.20274.1.A1_at 9.22 5.39 14.26 0.000013 0.166739 ubqln4 ubiquilin 4Dr.8516.1.S1_at 9.38 5.22 17.87 0.000059 0.228736 sepp1b selenoprotein P, plasma, 1bDr.6353.1.A1_at 11.25 8.15 8.57 0.000055 0.228736 LOC100536584 , si:ch211-140m22.7 uncharacterized LOC100536584 ; si:ch211-140m22.7Dr.7313.1.A1_at 8.93 5.14 13.85 0.000082 0.255638 zgc:73340 zgc:73340Dr.515.1.A1_at 9.54 11.15 -3.05 0.000116 0.258932 gtpbp1 GTP binding protein 1

Dr.15843.1.A1_at 9.34 5.18 17.89 0.000113 0.258932 atrxl Alpha thalassemia/mental retardation syndrome X-linked, likeDr.11536.1.A1_at 5.98 5.12 1.81 0.000148 0.289704Dr.1712.1.A1_at 10.21 5.57 24.97 0.000172 0.299285 nap1l4b nucleosome assembly protein 1-like 4b

Gene namefun14 domain containing 2protein phosphatase 2A activator. regulatory subunit 4tumor suppressing subtransferable candidate 1protein phosphatase 1. regulatory (inhibitor) subunit 14Aa

Mn(II)Cit is more important than MnCl2

for manganese-induced differential gene expression

Representative Data of differential gene expression for one-way anova + pFDR analysis (significant results to pFDR < 0.3)

Hernández et al, 2015 (unpublished data)

Mn(II)Cit is more important than MnCl2 for manganese-induced differential gene expression

Representative Data of differential gene expression for one-way anova + pFDR analysis (significant results to pFDR < 0.3)

Hernández et al, 2015 (unpublished data)

Mn induced equivalent differential gene expression in both the pellet fraction (granule and membrane component) and the supernatant fraction (stable and denature proteins, organelles and other cytoplasmic component).

Chemical fractionation not appear to be important for Mn-induced gene expression in zebrafish embryos

qRT-PCR results confirmed micro-array findings and the major toxicity of the Mn(II)Cit

Hernández et al, 2015 (unpublished data)

Relative quantitative Gene expression., qRT-PCR (bcta2, cpa1, eif2s1a, mmp2, sgce e ubqln4) in zebrafish embryos exposed from 48-120 hours post fertilization for MnCl2 or Mn(II)Cit em embriões de Danio rerio. The data was normalized to the control water-ISO. Bars represent (mean ± SD, n = 3). = p < 0,05 denote significant difference between MnCl2 e Mn(II)Cit.

Hernández et al, 2015 (unpublished data)

Manganese induced calcium homeostasis disruption in zebrafish, followed of endoplasmic reticulum stress and protein metabolism impairment is a

plausible TOXICOLOGICAL MECHANISMS TO THIS METAL

Hernández et al, 2015 (unpublished data)

Manganese-induced protein metabolism impairment is followed by catastrophe of several biological process and consequently

neurological diseases, including potential motor and cognitive disorders

Hernández et al, 2015 (unpublished data)

Manganese-induced protein metabolism impairment is potentially associated with the development of dementia

C

Genes disrupted by Mn-species, which are potentially linked to neurodisorders and dementia, according to Comparative Toxicogenomics Database. At the same time, it could be biomarkers candidates for early diagnosis of these pathologies

Works in course (not showed here) about proteomics studies with both alternative animal models the zebrafish and the culture of

primary cerebellar granule cells exposed for manganese is suggesting too that this metal is associated with dementia.

Hernández et al, 2015 (unpublished data)

Phenomics confirmed transcriptomics findings about the manganese-induced neuromotor and neurocognitive impairment

Esquematic respresentation of neurobehavioral studies with zebrafish embryos of 120 hours post fertilization (hpf), exposed for chemical species of manganese, from 48 to 120 hpf, using the zebrabox tracking system (Zebrabox/ Viewpoint – França), determining locomotor activity (distance, time and speed) under environmental complexity (diferente color), allowing to indentfy cognitive impairment too. Analysis of locomotor activity under neutral color (A) and under diferente color (B) as well as (C) the speed of identification of preference and avoid of areas colour-enrieched.

Hernández et al, 2015 (unpublished data)

Mn speciation and the environment complexity are important for Mn-induced neuromotor impairment (time in movement)

Independing of the color environment, the embryos have the major preference by freezing activity, and the exposed for Mn-species have a trend to be stopped

more time in color rich areas, specially for Mn(III)Cit, during the first 5 minutes.

Total activity (time in movement) of the zebrafish embryos per area and block time of the experiment. Bars represent means + sd, n=12.

Mn speciation is important for manganese-induced neurocognitive impairment (preference or avoid color)

COLOR PREFERENCE

COLOR AVOIDE

NEUTRAL

YELLOW

BLACK

ORANGE

H2O-ISO Mn(II)Cit

Citrate, MnCl2 e

Mn(III)Cit

AREAPREFERENCE

CENTRAL

PERIPHERY COLOUR

ENRICHED

The pattern of preference color area of zebrafish embryos, according to frequency of random visits of each area. Bars represent means + sd, n=12.

Mn speciation is important for manganese-induced Neuromotor (speed) and neurocognitive (colour recognizing) impairment

SPEED

NEUTRAL

YELLOW

BLACK

ORANGE

H2O-ISO Mn(II)Cit

Citrate, MnCl2 e

Mn(III)Cit

SPEED

Despite zebrafish embryos have major preference by

freezing activity, when exposed for species of Mn, Its have a trend to develop more speed in the colour-

enriched enviroment

The zebrafish embryos speed per area for both low and high activity. Bars represent means + sd, n=12.

Spee

d (m

m/s

)Sp

eed

(mm

/s)

Conclusions1) Mn-induced embroy- and neurotoxicity depends on developmental stage,

which is mediated by chemical speciation and chemical fractionation. Indeed, Mn(II), specially the citrate of Mn(II) appear be more toxic than Mn(III).

2) Mn-induced calcium homeostasis disruption, followed of endoplasmic reticulum stress and protein metabolism impairment can be an important mechanism of neuro(toxicity) for this metal, which can be provoking aggregation/accumulation of proteins and finally dementia (neurocognitive and neuromotor impairment), which was verified by phenomics approach (it can be extended for neurobehavioral analysis in human).

3) Toxicogenomics analysis of zebrafish (danio rerio) embryos revealing pathways involved in manganese-induced dementia suggest that these pathologies can be appear in young populations exposed for manganese in chronic manner, and consequently the methods of diagnostic of dementia must be developed and/or to improved for early life stages too.

4) These results all together can to improve the environmental manganese risk assessment and management.

TEAM

Ministery of Health,

Spain

Prof. Pannia Espósito - teamIQ-USP, Brazil, 2006-to day

Prof. Scholz Stefan - teamUFZ, Germany, 2009-to day

Prof. Suñol Cristina - teamIIBB, Spain, 2007-to day

Financing CollaboratorsThank you very much by your attention

Prof. DiogoGBB, UNifesp, Brazil,

2012-to day

Prof. Georgia, UNifesp, Brazil,

2012-to day

Prof. Iracilda – TeamUnesp, Brazil 2014 -to day

Prof. Souza-Pinto - teamIQ-USP, Brazil, 2009-to day

Prof. Barbosa Junior- - teamUSP-RP, Brazil, 2010-to day

Prof. Dr. B. Michalke TeamGerman Research Center for

Environmental Health, 2012- to day

Prof. Michael AschnerVanderbilt University,

USA. 2011-to day