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Behavioural
Brain
Research
279
(2015)
123–128
Contents
lists
available
at
ScienceDirect
Behavioural
Brain
Research
jou
rn
al
hom
epage:
www.elsevier.com/locate/bbr
Research
report
Acute
stress
affects
the
global
DNA
methylation
profile
in
rat
brain:
Modulation
by
physical
exercise
Gelson
M.
Rodrigues
Jr.a,
Leandro
V.
Toffolia,
Marcelo
H.
Manfredoa,
José
Francis-Oliveirab,
Andrey
S.
Silvab,
Hiviny
A.
Raquelb,
Marli
C.
Martins-Pingeb,
Estefânia
G.
Moreirab,
Karen
B.
Fernandesa,
Gislaine
G.
Pelosib,
Marcus
V.
Gomesa,∗
aUniversidade
Norte
do
Paraná
(UNOPAR),
Londrina,
Brazil
bUniversidade
Estadual
de
Londrina
(UEL),
Londrina,
Brazil
h
i
g
h
l
i
g
h
t
s
•In
this
study,
acute
restraint
stress
induced
a
decrease
in
the
global
DNA
methylation
in
rat
brain.
•The
adaptive
changes
on
global
DNA
methylation
evoked
by
acute
restraint
stress
are
independent
of
the
expression
of
the
Dnmt1
gene.
•Physical
exercise
modulates
the
impacts
of
stress
on
global
DNA
methylation
profile.
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
29
July
2014
Received
in
revised
form
7
November
2014
Accepted
12
November
2014
Available
online
21
November
2014
Keywords:
Acute
stress
Epigenetics
DNA
methylation
Physical
exercise
Dnmt1
Bdnf
a
b
s
t
r
a
c
t
The
vulnerability
of
epigenetic
marks
of
brain
cells
to
environmental
stimuli
and
its
implication
for
health
have
been
recently
debated.
Thus,
we
used
the
rat
model
of
acute
restraint
stress
(ARS)
to
evaluate
the
impact
of
stress
on
the
global
DNA
methylation
and
on
the
expression
of
the
Dnmt1
and
Bdnf
genes
of
hippocampus,
cortex,
hypothalamus
and
periaqueductal
gray
(PAG).
Furthermore,
we
verified
the
potential
of
physical
exercise
to
modulate
epigenetic
responses
evoked
by
ARS.
Sedentary
male
Wistar
rats
were
submitted
to
ARS
at
the
75th
postnatal
day
(PND),
whereas
animals
from
a
physically
active
group
were
previously
submitted
to
swimming
sessions
(35–74th
PND)
and
to
ARS
at
the
75th
PND.
Global
DNA
methylation
profile
was
quantified
using
an
ELISA-based
method
and
the
quantitative
expression
of
the
Dnmt1
and
Bdnf
genes
was
evaluated
by
real-time
PCR.
ARS
induced
a
decrease
in
global
DNA
methylation
in
hippocampus,
cortex
and
PAG
of
sedentary
animals
and
an
increased
expression
of
Bdnf
in
PAG.
No
change
in
DNA
methylation
was
associated
with
ARS
in
the
exercised
animals,
although
it
was
associated
with
abnormal
expression
of
Dnmt1
and
Bdnf
in
cortex,
hypothalamus
and
PAG.
Our
data
reveal
that
ARS
evokes
adaptive
changes
in
global
DNA
methylation
of
rat
brain
that
are
independent
of
the
expression
of
the
Dnmt1
gene
but
might
be
linked
to
abnormal
expression
of
the
Bdnf
gene
in
the
PAG.
Furthermore,
our
evidence
indicates
that
physical
exercise
has
the
potential
to
modulate
changes
in
DNA
methylation
and
gene
expression
consequent
to
ARS.
©
2014
Elsevier
B.V.
All
rights
reserved.
1.
Introduction
Stress
has
become
an
important
imbalance
factor
in
per-
sonal
relationships
and
in
the
health
of
mankind
[1].
The
main
adaptive
mechanisms
of
stress
involve
activation
of
the
sympathoa-
drenomedulary
system
and
the
hypothalamic–pituitary–adrenal
(HPA)
axis
[2].
Moreover,
an
important
role
in
the
adaptive
response
∗Corresponding
author.
Tel.:
+55
43
3371
9859;
fax:
+55
43
3371
7721.
E-mail
addresses:
mvmgomes@gmail.com,
marcus@unopar.br
(M.V.
Gomes).
to
stress
has
been
attributed
to
the
hippocampus
[3],
periaqueduc-
tal
gray
matter
(PAG)
[4,5],
and
cortex
[6,7].
From
a
molecular
point
of
view,
recent
data
have
indicated
the
participation
of
several
genes
in
the
adaptive
response
to
stress,
including
Bdnf
(brain-derived
neurotrophic
factor),
which
is
involved
in
neuronal
plasticity
[8],
and
Dnmt1
(DNA
methyl-
transferase
1),
which
plays
a
general
role
in
maintaining
DNA
methylation
[9].
The
participation
of
epigenetic
changes
in
molecular
adaptive
responses
to
behavioral
stress
have
been
strongly
supported
by
recent
evidence
[10,11],
however,
despite
the
rapidly
expanding
knowledge
in
this
field
little
is
known
about
the
contribution
of
http://dx.doi.org/10.1016/j.bbr.2014.11.023
0166-4328/©
2014
Elsevier
B.V.
All
rights
reserved.
124
G.M.
Rodrigues
Jr.
et
al.
/
Behavioural
Brain
Research
279
(2015)
123–128
DNA
methylation
changes
to
the
modulation
of
neurophysiological
responses
of
brain
cells
evoked
by
stress.
Thus,
the
present
study
aimed
to
identify
the
effect
of
acute
restraint
stress
(ARS)
on
the
global
DNA
methylation
profile
and
on
the
expression
of
the
Dnmt1
and
Bdnf
genes
in
rat’s
brain
cells.
In
addition,
considering
a
previous
report
of
the
effects
of
physical
exercise
on
brain-specific
epigenetic
patterns
[3,12],
we
evaluated
the
potential
of
physical
activity
(swimming)
in
modu-
lating
the
impact
of
ARS
on
the
DNA
methylation
profile
and
on
Dnmt1
and
Bdnf
gene
expression.
2.
Materials
and
methods
2.1.
Animals
and
experimental
groups
Experimental
procedures
were
carried
out
following
protocols
approved
by
the
ethical
review
committee
of
the
University
of
Lon-
drina,
Londrina,
Parana,
Brazil
(CEUA
no.
14441.2013.18)
and
all
efforts
were
made
to
minimize
suffering.
Non-related
male
Wistar
rats
were
kept
under
standard
condi-
tions
(temperature
25
±
1◦C,
photoperiod
12
h
light/12
h
dark)
and
with
water
and
food
ad
libitum
in
the
Central
Animal
House
of
the
University
of
Londrina.
The
animals
were
arranged
into
three
groups,
containing
4–6
animals
each:
(1)
stress
group
(ST):
animals
that
were
submitted
to
ARS
at
the
75th
postnatal
day
(PND);
(2)
physical
activity
group
(EX-ST):
animals
that
were
submitted
to
physical
exercise
(swim-
ming
for
60
min/day)
from
the
35th
PND
(periadolescent)
to
the
74th
PND
(young
adult)
followed
by
ARS
at
the
75th
PND;
and
(3)
control
group
(CTL):
animals
that
were
not
submitted
to
interven-
tions.
The
animals
from
group
two
(EX-ST)
were
assisted
during
the
whole
swimming
sessions
in
order
to
avoid
passive
floatation
and
minimize
bias
related
to
differential
intensity
of
physical
exer-
cise.
All
the
experimental
protocols
were
conducted
in
the
morning
period
between
07:00
and
12:00
h
in
the
light
phase.
2.2.
Acute
restraint
stress
(ARS)
Animals
were
transported
in
the
morning
period
(07:00–9:00am)
to
the
experimental
room
in
their
home
cages
and
allowed
to
adapt
to
this
environment
for
at
least
60
min
[13].
After
this
period,
the
animals
were
submitted
to
the
protocol
of
ARS
[14],
being
placed
in
a
metal
cylinder
6.5
cm
in
diameter
and
15
cm
in
length
with
holes
to
allow
ventilation
where
they
remained
closed
during
the
whole
period
(60
min).
2.3.
Physical
exercise
The
animals
of
the
EX-ST
group
were
submitted
to
physi-
cal
training
(swimming)
without
stimuli
or
tasks,
according
to
Martins-Pinge
et
al.
[15].
The
swimming
sessions
were
performed
in
a
glass
tank
filled
with
lukewarm
water
(31
±
1◦C)
with
a
4000
cm2surface
area
and
60
cm
depth.
The
training
consisted
of
4
weeks
(20
sessions)
of
swimming
being
held
5
days
a
week
and
60
min/day.
During
the
first
week,
the
training
was
graded,
begin-
ning
with
15
min
on
the
first
day,
30
min
on
the
second
day,
and
45
min
on
the
third
day,
for
adaptation
to
the
training
process.
From
the
fourth
day
on,
each
session
consisted
of
60
min
of
swimming
until
the
74th
PND.
After
each
swimming
session,
the
animals
were
dried
with
a
towel
and
returned
to
their
cages.
2.4.
Sample
collection
Sample
collections
were
performed
in
the
morning
(between
08:00
and
12:00
h)
in
order
to
keep
the
circadian
rhythm
for
all
the
groups.
The
animals
were
killed
by
decapitation
and
blood
sample
was
obtained
from
trunk.
Brain
was
rapidly
removed
from
the
skull
and
samples
from
hypothalamus,
frontal
region
of
cortex
(approx-
imately
2
mm
including
the
cingulate
cortex
area
and
excluding
the
corpus
callosum),
PAG
and
hippocampus
were
immediately
obtained.
Brain
areas
were
sectioned
according
to
the
anatomical
atlas
of
Paxinos
and
Watson
[16]
and
the
tissues
were
stored
at
−80 ◦C.
2.5.
Global
DNA
methylation
profile
Genomic
DNA
was
obtained
by
a
standard
salting
out
pro-
tocol
[17].
Evaluation
of
purity
and
concentration
of
the
DNA
was
performed
by
analysis
of
absorbance
in
a
spectrophotometer
(NanoDrop
ND-2000—Thermo
Scientific)
at
260
nm
and
280
nm.
The
global
DNA
methylation
profile
was
evaluated
by
dosage
(percentage)
of
methyl
groups
(CH3)
using
the
Imprint
Methyl-
ated
DNA
Quantification
Kit
(Sigma-Aldrich®)
according
to
the
manufacturers’
recommendations
and
as
previously
described
[18].
Briefly,
the
methylation
status
of
each
sample
was
cal-
culated
by
the
amount
of
methylated
cytosines
in
the
sample
(5mC)
relative
to
global
cytidine
(5mC
+
dC)
in
a
positive
con-
trol
(100%
methylated)
that
had
been
previously
methylated
and
a
no
template
control
sample
(0%
methylated)
using
absorbance
readings
at
450
nm
and
following
the
formula:
(A450sample
−
A450NTC)/(A450met
−
A450NTC)
×
100.
All
sam-
ples
were
analyzed
in
triplicate.
2.6.
Quantitative
gene
expression
RNA
samples
were
obtained
using
Trizol
(Invitrogen®)
and
reverse
transcription
was
performed
using
the
High
Capacity
Kit
(Applied
Biosystems®,
Foster
City,
CA,
USA),
according
to
the
man-
ufacturers’
recommendations.
Expression
levels
of
Dnmt1
and
Bdnf
genes
were
evaluated
by
Real-Time
PCR
using
a
StepOne
Plus
machine
(Applied
Biosystems®,
Foster
City,
CA,
USA).
Amplifica-
tions
were
obtained
using
on
demand
Taqman
probes
(Applied
Biosystems®,
Foster
City,
CA,
USA)
for
both
Dnmt1
(Assay
I.D.
Rn00709664
M1)
and
Bdnf
(Assay
I.D
Rn
01027162
G1)
genes.
For
the
normalization
of
differences
in
the
amount
of
cDNA
we
used
the
Gapdh
(Assay
I.D
Rn01775763
G1)
gene
as
an
endogenous
control.
The
experiments
were
performed
in
triplicate.
2.7.
Corticosterone
levels
Blood
samples
were
centrifuged
at
2300
rpm
(830
rcf)
at
4◦C
for
20
min
and
the
plasma
frozen
for
subsequent
analysis
by
radioim-
munoassay
as
previously
described
[19].
2.8.
Statistical
analysis
Analyses
of
data
normality
were
performed
using
the
Shapiro–Wilk
test.
Unpaired
t
test
was
used
to
compare
the
dosage
of
serum
corticosterone
from
the
groups
ST
and
CTL
considering
its
normal
distribution.
However,
the
non-parametric
Kruskal–Wallis
test
(post-test:
Dunn)
was
used
to
compare
the
weights
of
animals,
the
global
DNA
methylation
profile
and
the
quantitative
expression
of
the
Bdnf
and
Dnmt1
genes
among
the
ST,
CTL
and
EX-ST
groups.
The
GraphPad
Prism
6.0
software
and
the
Statistical
Package
for
Social
Sciences
(SPSS)
version
20.0
were
used
for
statistical
analy-
ses.
A
95%
confidence
interval
and
significance
level
of
5%
(P
<
0.05)
were
considered
for
all
testes
applied.
G.M.
Rodrigues
Jr.
et
al.
/
Behavioural
Brain
Research
279
(2015)
123–128
125
Fig.
1.
Serum
corticosterone
levels
in
CTL
and
ST
groups.
CTL:
control
group;
ST:
stress
group. *P
=
0.0043.
Unpaired
t
test.
Error
bar
represents
the
standard
error
of
the
mean.
3.
Results
No
statistically
significant
difference
was
observed
when
com-
pared
the
weights
of
animals
from
CTL,
ST
and
EX-ST
groups
at
the
75DPN
(CTL:
294
±
2.4
g;
ST:
292
±
2.8
g;
EX-ST:
300
±
6
g;
P
=
0.43).
3.1.
Corticosterone
levels
Increased
levels
of
serum
corticosterone
were
observed
in
the
animals
from
the
ST
group
in
comparison
to
the
CTL
group
(P
=
0.0043)
confirming
the
efficacy
of
the
ARS
protocol
in
inducing
stress
related-physiological
changes
(Fig.
1).
3.2.
Effect
of
stress
on
global
DNA
methylation
Comparative
analyses
of
the
percentages
of
the
global
DNA
methylation
profile
revealed
a
statistically
significant
decrease
in
the
hippocampus
(P
=
0.013),
cortex
(P
=
0.014),
and
PAG
(P
=
0.048)
of
animals
that
were
submitted
to
ARS
(ST
group)
when
compared
to
the
CTL
group
(Fig.
2).
In
contrast,
no
significant
change
in
DNA
methylation
was
observed
in
animals
from
the
EX-ST
group
in
comparison
to
the
animals
from
the
ST
group
and
CTL
group,
revealing
the
potential
of
physical
exercise
in
attenuating
the
effects
of
stress
on
the
global
methylation
profile
(Fig.
2).
3.3.
Quantitative
expression
of
the
Dnmt1
gene
No
significant
alteration
in
the
expression
of
the
Dnmt1
gene
was
associated
with
stress
when
comparing
the
ST
and
CTL
groups.
However,
a
statistically
significant
decrease
(P
=
0.008)
in
the
expression
of
Dnmt1
was
found
in
the
cortex
of
animals
from
the
EX-ST
group
in
comparison
to
CTL
while
an
increased
expression
(P
=
0.002)
was
evidenced
in
the
PAG
when
comparing
EX-ST
to
ST
groups
(Fig.
3).
3.4.
Quantitative
expression
of
the
Bdnf
gene
A
statistically
significant
increase
in
the
expression
of
the
Bdnf
gene
was
observed
in
the
PAG
of
animals
from
the
ST
group
in
comparison
to
the
CTL
group
(P
=
0.020).
Statistically
significant
decrease
in
the
expression
of
Bdnf
was
associated
with
stress
in
the
EX-ST
group
in
the
cortex
(P
=
0.008)
in
comparison
to
CTL.
Specif-
ically,
in
the
hypothalamus
a
significant
increase
in
the
expression
Fig.
2.
Global
DNA
methylation
profile
in
the
hippocampus,
hypothalamus,
cortex
and
PAG
in
the
CTL,
ST
and
EX-ST
groups.
CTL:
control
group;
ST:
stress
group;
EX-ST:
exercise
+
stress
group. *P
<
0.05.
Kruskal–Wallis
test
(post-test:
Dunn).
Error
bar
represents
the
standard
error
of
the
mean.
126
G.M.
Rodrigues
Jr.
et
al.
/
Behavioural
Brain
Research
279
(2015)
123–128
Fig.
3.
Quantitative
expression
of
the
Dnmt1
gene
in
the
CTL,
ST
and
EX-ST
groups.
CTL:
control
group;
ST:
stress
group;
EX-ST:
exercise
+
stress
group. *P
<
0.05.
Kruskal–Wallis
test
(post-test:
Dunn).
Error
bar
represents
the
standard
error
of
the
mean.
RQ
=
relative
quantification
according
to
the
expression
of
the
Gapdh
housekeeping
gene.
of
Bdnf
was
observed
in
the
EX-ST
group
in
comparison
to
the
ST
group
(P
=
0.021)
(Fig.
4).
4.
Discussion
The
present
study
revealed
that
significant
adaptive
changes
in
the
global
DNA
methylation
profile
of
brain
cells
are
evoked
by
ARS
in
rats.
Furthermore,
our
evidence
demonstrated
that
these
epige-
netic
changes
might
be
potentially
modulated
by
physical
exercise.
The
idea
of
DNA
methylation
as
an
epigenetic
mechanism
for
controlling
gene
expression
in
neurons
and
its
implication
to
the
neurobiology
is
relatively
recent,
although
the
role
of
the
enzyme
Dnmt1
in
the
acquisition
of
learning
and
memory
in
mature
neurons
is
already
known,
by
maintaining
DNA
methylation
and
post-
mitotic
stability
of
marks,
as
well
as
the
physiological
implications
of
the
expression
of
Dnmt1
in
neurogenesis,
neuronal
morphology,
synaptic
plasticity,
memory,
and
learning
[20–22].
The
correlation
between
DNA
hypomethylation
in
the
hip-
pocampus
and
the
etiology
of
posttraumatic
stress
was
previously
suggested
[23],
however,
as
far
as
we
know,
there
is
no
report
to
date
concerning
the
association
between
stress
and
abnormal
DNA
methylation
profile
in
the
cortex
or
PAG.
Intriguingly,
our
data
indicate
that
the
adaptive
decrease
in
DNA
methylation
evoked
by
ARS
in
brain
cells
is
not
dependent
to
significant
change
of
the
expression
of
the
Dnmt1
gene
(at
the
transcriptional
level)
and
suggests
the
participation
of
an
active
demethylating
process.
Previously
reported
in
plants
[24],
the
existence
of
a
mam-
malian
active
DNA
demethylation
has
been
under
intensive
debate.
Emerging
evidences
indicate
the
hydroxylation
of
5-
methylcytosine
(5mC)
to
5-hydroxymethylcytosine
(5hmC)
by
the
methylcytosine
dioxygenase
TET1
(TET1)
protein
and
the
base-excision
repair
pathways
as
potential
molecular
mechanisms
involved
in
the
mammalian
active
DNA
demethylation
[25–27].
However,
additional
studies
are
certainly
needed
for
a
complete
understanding
of
this
process
and
also
to
verify
its
association
with
the
herein
reported
stress-induced
DNA
hypomethylation
in
rat
brain.
The
Bdnf
gene
plays
a
recognized
role
in
mediating
synaptic
plas-
ticity
and
behavioral
responses
to
aversive
social
experiences
[8].
Moreover,
Bdnf
is
important
in
the
control
of
biological
activities
in
several
brain
areas
[28]
so
that
disorders
in
Bdnf
expression
are
related
to
the
course
and
development
of
several
neurological
and
psychiatric
diseases
[29].
Additionally,
increased
expression
of
the
Bdnf
gene
in
the
hypothalamus
is
considered
an
important
indica-
tor
of
the
involvement
of
this
gene
in
synaptic
plasticity
in
response
to
stress
[30].
In
the
present
study,
we
observed
a
significant
increase
in
the
expression
of
Bdnf
exclusively
in
the
PAG
of
animals
submitted
to
behavioral
stress,
indicating
the
participation
of
Bdnf
in
the
modu-
lation
of
PAG
activity
after
stress.
Little
is
known
about
the
role
of
Bdnf
in
PAG
although
previous
evidences
indicated
an
implication
in
the
mechanisms
of
pain
and
panic
behavior
[31,32].
Thus,
our
data
contribute
to
the
knowledge
on
this
field
and
provide
information
for
a
speculative
relation
between
the
Bdnf
and
changes
in
the
neurobiology
of
fear,
anxiety,
cardiovascu-
lar
control
and
analgesia
[33,34].
Moreover,
when
comparing
our
G.M.
Rodrigues
Jr.
et
al.
/
Behavioural
Brain
Research
279
(2015)
123–128
127
Fig.
4.
Quantitative
expression
of
the
Bdnf
gene
in
the
CTL,
ST
and
EX-ST
groups.
CTL:
control
group;
ST:
stress
group;
EX-ST:
exercise
+
stress
group. *P
<
0.05.
Kruskal–Wallis
test
(post-test:
Dunn).
Error
bar
represents
the
standard
error
of
the
mean.
RQ
=
relative
quantification
according
to
the
expression
of
the
Gapdh
housekeeping
gene.
data
concerning
the
expression
of
Bdnf
gene
and
the
global
DNA
methylation
we
can
hypothesize
a
relationship
between
the
increase
in
the
expression
of
Bdnf
and
a
significant
decrease
in
global
DNA
methylation
in
the
PAG.
Future
studies
are
needed
to
clarify
this
preposition
and
also
to
verify
its
relationship
with
the
methylation
pattern
of
the
promoter
region
of
the
Bdnf
gene.
In
order
to
evaluate
the
potential
of
physical
exercise
in
modulating
stress-induced
epigenetic
changes
in
brain
cells
we
compared
the
adaptive
responses
of
sedentary
and
exercised
ani-
mals
(previously
submitted
to
swimming
sessions)
to
ARS.
Our
data
revealed
that
ARS
evoked
a
less
prominent
decrease
in
the
global
DNA
methylation
profile
in
exercised
animals.
Similarly,
dis-
tinct
patterns
of
expression
of
the
Dnmt1
and
the
Bdnf
genes
were
observed
when
comparing
the
responses
of
exercised
and
seden-
tary
animals
to
ARS.
Although
one
might
interrogate
about
potential
bias
involving
the
swimming
protocol
and
the
stress-related
changes,
previous
studies
shown
that
animals
that
are
submitted
to
the
forced-swim
stress
model
for
a
prolonged
period
(15
days)
present
a
recov-
ery
of
physiological
parameters
such
as
heart
rate,
weight
of
the
heart,
kidney
and
adrenal
glands
suggesting
an
adaptation
of
the
HPA
axis
[35,36].
Considering
these
evidences,
we
thought
that
the
prolonged
swimming
training
(20
sessions)
minimized,
in
the
present
study,
possible
bias
involving
the
physiological
stress-
related
changes
and
the
swimming
protocol.
Taken
together,
our
findings
suggest
the
potential
of
physical
exercise
to
attenuate
the
adaptive
changes
in
global
DNA
methyla-
tion
and
to
modulate
changes
in
gene
expression
that
are
evoked
in
brain
cells
by
ARS.
Although
the
data
herein
presented
do
not
provide
information
about
the
impact
of
physical
exercise
for
the
epigenetic
patterns
of
non-stressed
animals
our
evidences
corroborate
to
the
previous
association
of
epigenetic
changes
and
neurophysiological
benefits
elicited
by
physical
exercise
[3,37,38].
In
summary,
our
data
reveal
that
behavioral
stress
induces
DNA
hypomethylation
in
the
hippocampus,
cortex
and
PAG,
and
affects
the
control
of
expression
of
Dnmt1
in
the
cortex
and
PAG,
and
of
Bdnf
in
the
PAG.
Additionally,
our
data
indicate
the
potential
of
physical
activity
in
attenuating
the
intensity
of
stress-induced
global
DNA
methylation
in
the
hippocampus,
cortex
and
PAG,
besides
modu-
lating
the
effects
on
expression
of
Dnmt1
and
Bdnf
genes.
Conflict
of
interest
statement
The
authors
declare
no
conflict
of
interest.
Acknowledgments
The
authors
wish
to
thank
Dr.
Paulo
S.
Monzani
and
Dr.
Paulo
R.
Adona
from
Agropecuária
Laffranchi
for
technical
support.
This
work
was
supported
by
the
National
Foundation
for
Development
of
Private
Education
(Funadesp—Process
Number:
0030/13)
and
the
National
Council
of
Technological
and
Scientific
Development
(CNPq—Process
number:
478566/2013-1).
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