project mutamba for hiv\aids

1. THE EFFICIENCY OF THE TANINN
EXTRACTED FROM THE Guazuma
ulmifolia IN THE REVERSE
TRANSCRIPTASE INHIBITION OF THE
HIV VIRUS PAULO ANTONIO RODRIGUES
GOUVEIA
PAULO ANTONIO RODRIGUES GOUVEIA
Contents
1. INITIAL
CONSIDERATIONS..................................................................
...................... 2
1.1 GUIDING
QUESTIONS ......................................................................
..................... 4
1.2
OBJECTIVES .....................................................................
....................................... 4
General: .......................................................................
................................................. 4
Specific: ......................................................................
................................................. 4
1.3
JUSTIFICATIVE ..................................................................
..................................... 4
2 THEORETICAL
FUNDAMENTS .....................................................................
.............. 6
3.
METHODOLOGY ....................................................................
.................................... 16
REFERENCES .....................................................................
............................................. 17

2. 1. INITIAL CONSIDERATIONS
Virus is the smallest particle that exists; it only can be seen by the
microscope. The virus,
so that it can live and multiply itself, needs to be inside of the cells of an
organism where it can
assume the command, making them work to it(Adesokan et al, 2004).
The AIDS/SIDA “Acquired Immunodeficiency Syndrome“ is caused by HIV, a virus
that,
firstly, attacks the immune system; then, it can install itself in several parts
of the body. When the
virus affects the immune system, its action becomes inefficient and the body
becomes more
vulnerable to any disease that may occur (Barre-Sinoussi et al., 1983; Brown,
2000).
The HIV is a retrovirus, which means that its genetic material is in RNA shape
instead of
DNA shape, and to replicate itself it must, firstly, create a DNA copy from its
genetic material
(Buckheit Jr et al., 2001).
The HIV virion has a complex structure and it is grossly spherical, with a
diameter of
approximately 1/10.000 mm. The virus external coverage is a double layer of
lipidic molecules
sprinkled with proteins (the envelope proteins, gp120 and gp41).Inside of it, a
layer of protein
matrix surrounds the conical capsid, or nucleus, that contains the RNA of the
HIV(Brown, 2000).
A cell infection occurs when the HIV virion connects itself to a cellular
receptor,
generally the CD4, through its protein gp120; then, the virus merges itself to
the cellular
membrane and the content of the capsid is liberated in the cellular cytoplasm.
The HIV enzyme,
the reverse transcriptase, catalyses the production of a DNA copy of the RNA
from the HIV and
the component ribonuclease-H from the reverse transcriptase, finally, removes
the RNA helix,
now redundant. The DNA copy of single helix is, then, converted by the reverse
transcriptase
into a DNA copy of double helix, which is transported to the cellular nucleus
where a second

3. HIV enzyme, the integrase, catalyses the incorporation of the viral DNA to the
host genetic
material (Adesokan, 2004).
The antiretroviral therapy is aimed to the prevention of the viral replication,
with
different drugs addressed to various stages of the replication cycle. The
antiretroviral drugs
currently available to treat the HIV infection are a series of inhibitors of the
reverse transcriptase,
that act previously to the incorporation of the viral genetic material to the
host chromosome and
the inhibitors of the protease, which act subsequently to this step and prevent
the virion
formation with functional proteins, that is, of infectious virus. The cocktail
is an association of
the two kinds of medicines (Chi et al., 2004).
The reverse transcriptase inhibitors prevent the virus transforming its genetic
code from
ARN into ADN, a required operation to multiply itself inside the cells (Costi et
al, 2004).
Several natural compounds are being studied about the retroviral inhibition of
the HIV
reverse transcriptase; among them are the tannins, which are phenolic compounds,
characterized
by their capacity of combining themselves with proteins and other polymers, such
as
polysaccharides (Buolamwini e Assefa, 2002).
Studies have shown that the bark and the bast of the Guazuma ulmifolia (mutamba)
are
rich in components with pharmacologic properties. It presents triterpenes,
alkaloids and
reasonable amount of tannins (Costi et al., 2004).
The tannin properties are linked to its capacity of forming complexes with the
proteins
that participate of the tissues in relation to the microbiological attacks.
Besides, they confer taste
properties united under the term astringency, have anti-free radical power, and
have the capacity
of consuming dissolved oxygen, that is, they have interesting antioxidant
properties to the
pharmacologic function, and also show powerful activity against the HIV
replication (Cocuzza,

4. 2001).
1.1 GUIDING QUESTIONS
. Does the tannin extracted from the Guazuma ulmifolia show the best inhibitors
pharmacokinetic profiles of the HIV reverse transcriptase with fewer side
effects?
. Which molecular weight of the tannin has the retroviral inhibitor action of
the
HIV reverse transcriptase with fewer toxicity?
1.2 OBJECTIVES
General:
. To identify the efficiency of the Guazuma ulmifolia plant substracted as a
viral
duplication inhibitor of the HIV reverse transcriptase with a lower toxicity
risk.
Specific:
. To identify the tannin molecular weight that has retroviral inhibitor action
of the
HIV reverse transcriptase with lower toxicity risk.
. To promote the reduction of the viral load preservating the function of the
immunologic system.
. To postpone the illness evolution changing the HIV natural history.
1.3 JUSTIFICATIVE
Although the therapy appearance, which reduces the viral particles to
undetectable levels,
the virus persists in reservoirs in the organism, as T sleepy lymphocytes. The
emergence and the
appearance velocity of resistant strains to different medicine combinations
available in the
market also is a limitant factor. But one of the factors that makes the anti-HIV
therapy more

5. difficult is the high incidence of side effects caused by the medicines
currently available.
Searches realized by Ligani Jr. and partners showed to be this one the main
cause of faults in the
antiretroviral therapy adherence (20,5%), characterized mainly by vomits,
diarrhea, nauseas and
abdominal pain, beyond headaches and, even so, changes in skin color.
Despite several laboratories and search groups are working in the HIV inhibitors
development, no compound has arisen that can be effectively used in the
therapeutic.
This fomenta great need of discovery and/or development of efficient drugs that
act on
the reverse transcriptase (RT), on the protease (PR) and on other points of the
viral replication
cycle. Then the great challenge is to find possible targets that effectively
interrupt the virus cycle,
without harming the normal cell (Dayam, 2003).
The use of herbal medicines is the result of the secular accumulation of
empirical
knowledge about the herbal action by different ethnic groups. However, there are
questions about
the standardization of production techniques and herbal medicines marketing (De
Clercq, 2000).
Some studies have treated about the anti-nutritional aspect of cultivars with
high levels of
tannins and the resistance to pests and seasonality has been considered as an
interesting factor to
some species (De Clercq, 2002).
It“s found, in the vegetables, relatively important amounts of phenolic
compounds. Their
role is essentially to protect the issues against the attacks of insects,
or bacteria. It“s
considered a passive defense system relatively efficient. The plants also
produce great
amounts of phenols based in an alteration in the live cells surface: it“s
active defense. The
best example is given by the bite from insects in the leaves that are the
of the galls
formation.
fungi,
can
the
A mutamba tree had been observed for ten years and it was realized that in
certain period
source

6. of the year it was notorious the presence of dry galls nearly reaching 70% of
length and as the
days went by, the plant recovered itself showing resistance to the pest that had
affected it.
In 1946, in Faina “ GO, the researcher Francisco Cabral de Melo, a pharmacist
graduated
from UFRJ, and partners realized some tests, undocumented, in patients with
yellow fever using
the mutamba tannin that has a low molecular weight, having favorable outcome,
there was a cure
in 100% of the cases, with no toxicity demonstration.
Tannin molecules are being tested with the intention of discovering an efficient
drug
against HIV. Kilkuskie and partners had observed that galotannins showed
inhibitory activity
only in toxic concentrations, elagitannins and condensed tannins poorly
inhibited the viral
replication and the complex tannins showed powerful activity against the HIV
replication.
It justifies the great importance of a survey with tannins of low molecular
weight and
smaller toxicity.
So, the interest in the survey begins because there is no cellular equivalent,
what is an
advantage in relation to smaller chances of occurring side effects, and it has a
social and
scientific relevance because there is no endogenous mediator which chemical
structure can be
used as a prototype.
2 THEORETICAL FUNDAMENTS
The viruses are involved in a big variety of chronic and degenerative diseases,
being
responsible for more than 60% of the human diseases (Korolkovas, 1988). The
fight against viral
infections is hard, because the viral replication is an intracellular process,
being closely related to
the metabolism of the infected cells (Barre-Sinoussi, 1983). One of the most
studied virus
nowadays is named “Human Immunodeficiency Virus“ (HIV), from retrovirus family
(ANR

7. compound); it is able to be parasitic upon man’s immune system, leading to an
infectious disease
known as ’Acquired Immuno Deficiency Syndrome’ (AIDS) (Johnson, 1989).
HIV is different from the other viruses because it attacks and damages the
immune
system, that is its main target in the human organism. One of the components of
the immune
system are the T lymphocytes, that attack directly the invader microorganism.
Among the T
lymphocytes there is a class called T4 (CD4+ T or T-helpers), which has a very
important role in
the initiating of an immune response and in the coordination of the same
response, being the HIV
main target (Gupta, 1979). This virus, when infecting the CD4+ T, lead to the
lack of
coordination of the immune system and to its gradual in effectiveness,
establishing an
immunodeficiency (De Clercq, 2000).
In Brazil, from 1980 to December 2002, 257.771 cases of the disease were
reported, with
113.840 known deaths. Throughout serological tests conducted by the Brazilian
Department of
Health, at least 536.000Brazilians are expected to be infected with HIV (Souza,
2003).
It’s worth highlighting three big stages in the epidemic’s evolution:
. 1 ’ an initial stage, characterized by the infection among men that have
sexual
relations with other men, and by a patient’s high level of education;
. 2 ’ a second stage, characterized by an increase of cases due to the
transmission
caused by injecting drug use, as the consequent reduction of the age group and
greater dissemination among heterosexual individuals;
. 3- third and current stage, when there is a growing trend to dissemination
among
the heterosexuals, mainly the women (Goldgur at al., 1999).
This last observation deserves highlighting, because it has been reported in
several
countries a process of the ’feminization’ of the HIV/SIDA epidemic. Regarding to
the

8. distribution of the cases according to age group, it was observed in the last
decade an ageing of
it, with a persistent relevant growing of the age group over the age of 35, in
both sexes. The age
group from 35 to 39 and 40 to 49, with an increase of the incidence among women
(from about
6, 5 men to 1 woman, in the 1980s, to 1, 7 men to 1 woman in 2001) (De Clercq.
2001).
The morphological structures of the HIV viruses (1 and 2) include structural and
functional proteins and an RNA genome protected by the viral envelope. The
envelope is made
of a lipid bilayer and contains a complex protein, known as env (Ferreira,
2002).
In the interior face, the HIV has a viral protein called p17 (matrix) and,
enclosed by this
protein, there is the capsule composed by the p24 protein. In the most internal
part, it’s found the
most important elements: two simple filaments of RNA, the p7 protein
(nucleocapside) and three
essential enzymes, p51 (reverse transcriptase), p11 (protease) and p31
(integrase) (Goldgur, et
al., 1999).
The transcriptase is an enzyme that performs a contrary transcription process
related to
the cellular standard. This enzyme polymerizes DNA molecules from RNA molecules,
exactly
the opposite of what happens in the cells, where RNA is produced from DNA
(Gupta, 1979;
2003).
That’s exactly for having this enzyme, which it acts ’contrary’, that the HIV
and other
similar viruses are called retroviruses. After being in the host cell, the
reverse transcriptase uses
the nucleotides found in the cytoplasm to compose a DNA strand together with the
virus RNA
strand. The RNA se-H enzyme is responsible for unbundling the RNA strand by
hydrolysis and
let the RNA simple strand be free in the cytoplasm. The reverse transcriptase
comes back to
complete this DNA strand, thus becoming the nucleotides double helix to be
integrated in the
host-cell DNA helped by the integrase enzyme (Fox, 2002).

9. In Brazil, the Law 9113/96 ensured everyone the access, without its cost, to the
cocktail
of drugs. Introduced in 1996, this is a combination of drugs able to inhibit two
stages of the viral
replication, with a possibility of reduction in 100 times of the virus
production compared to the
mono-therapies used so far (Souza et al., 2003). Depending on the conditions and
the age of the
patient, it can be composed by two or more different medicines of the following
classes: (i)
nucleoside reverse transcriptase inhibitors (NRTIs); (ii) non-nucleoside reverse
transcriptase
inhibitors (NNRTIs); (iii) protease inhibitors (PI) and, more recently, (iv)
fusion inhibitors (FI),
and the latter was recently released to sale in Brazil (Goldgur, 1999).
The transcription process consists in the RNA synthesis, realized by an
enzymatic
complex which key enzyme is the RNA polymerase, able to produce the viral
proteins in the
polyproteins precursor’s form, long units composed of viral enzymes and
assembled structural
proteins (Li, 1999).The fluoroquinolones are a class of synthetic compounds with
strong
antimicrobial activity. Nowadays, this class of compounds also has been
described as able to
interfere in the viral transcription process, thus preventing the replication.
The K-37
fluoroquinolone has shown good results in the inhibition of that enzyme. The
temacrazine and
the flavopiridol are other compounds able to inhibit this transcription enzyme,
even in
chronically-infected cells (Cocuzza, 2001).
The protease inhibitors have the function of blocking one of the HIV components,
the
protease. This way, the new copies of the virus don’t infect the new cells. The
reverse
transcriptase inhibitors and the protease inhibitors act inside of the CD4 cell
(Hazuda et al.,
2004).
The nucleocapside protein (NCp7) is an essential protein in different stages of
the viral
replication, being important in the stage that involves the reverse
transcriptase enzyme,

10. participating of the RNA ringing (De Clercq, 2002).The AZDC (azodicarbonamide),
is a
compound in clinical testing phase II and it’s able to become inactive that
protein, by
complexation with its zinc atoms, preventing the viral replication of the
integrase inhibitors. The
integrase enzyme is fundamental in the viral replication process, being
responsible for the viral
DNA integration to the host chromosome, thus allowing for the continuation of
the viral
replication cycle. The ’L ’ quicórico" acid is able to inhibit the integrase
enzyme activity of
different kinds of powerful HIV (De Clercq, 2001).
The fusion inhibitors (FI) represent a new approach in the combat strategy to
the capacity
of the HIV replication in the organism. So that the HIV can complete its
reproductive cycle, it
needs to merge itself with T lymphocytes, where it deposits its genetic
information, giving rise to
a new virus. While the protease inhibitors (PIs) and the reverse transcriptase
inhibitors (NRTIs
and NNRTIs) aim at interrupting the viral replication cycle in a stage that the
HIV had already
infected lymphocytic target cell, the FIs were designed to prevent the virus
penetration in the
lymphocytes and the beginning of an infection (Ikemoto, et al., 2001).
The virus needs to merge itself with a T lymphocyte to multiply, and it’s
exactly this
action that the fusion inhibitors prevent. With this kind of medicine, the HIV
can’t complete its
reproduction cycle, because it can’t infect the T lymphocytes and can’t create
new virus copies.
The fusion inhibitors act outside the host cell (CD4 lymphocyte) at an earlier
stage in the virus
reproduction cycle than the protease and the reverse transcriptase inhibitors
(Heralth et al.,
2004).
To achieve more powerful new drugs, with better pharmacokinetic profiles, fewer
sideeffects and wide range of activity to different resistant HIV viruses, new
strategies have been
created. These strategies are based on the conception of new compounds able to
inhibit different

11. points of the viral replication (Jayasuriya et al., 2003).
In a memorandum from a WHO meeting, recommendations were made about the
research with natural substances for the treatment of AIDS. Several natural
compounds that
belong to different structures have been appointed as reverse transcriptase
inhibitors, such as
coumarins, flavonoids, tannins, lignins, alkaloids, terpenes, nafto, and
anthraquinones and
polysaccharides (Kannan et al., 2001).
Compounds of natural
of
diseases. These also
of more
powerful derivatives
property that
allows to be used as
source can be used as therapeutic agents to a great amount
can become excellent prototype-compounds to the development
or with improvement of some biological or physical-chemical
drugs (Huang et al., 2001).
The bark and the bast of the Guazuma ulmifolia (mutamba) is rich in components
with
pharmacological properties. In addition, these isolated principles have been
treated together with
the treatment of several diseases. So, the betasitosterol act against the
hypoproteinemias; the
triterpenes are used as anti-inflammatories (pneumonia and bronchitis); the
caffeine acts as
diuretics and stimulant of the CNS (Central Nervous System) and cardiac muscles;
the alkaloids
are treated as anti-microbials, painkillers, antispasmodics and CNS stimulants,
the tannins are
excellent in the combat of dysentery processes (Windholz, 1983; Almeida et al.,
1998; Rizzo et
al., 1990; Rizzo et al. 1999; Tridente, 2002).
Guazuma ulmifolia, popularly known as mutamba, has been used by the population
as
natural medicine in almost every place that it occurs. Generally, the used parts
are the bark and
the leaves; however, there are some reports that the fruit can also be useful.
The bark tea is used
in Brazil as a sudorific, also used in cases of fever, cough, bronchitis,
asthma, pneumonia, and
liver problems. Several authors show some activities with mutamba extracts,
among them they

12. emphasize: anti-glucose, anti-bacterial and anti-fungus, cytotoxic and antisecretory activities
(Johnson, 1989; Makhija, 2002).
Guazuma shows four species distributed through South America and Mexico, and two
of
them are in Brazil: Guazuma ulmifolia Lam and Guazuma crinita Mart., both known
as mutamba
(Huff, 1999).
Photochemical analysis realized with the mutamba bark showed positivity to the
flavonoid and tannin groups, among others. Spectrophotometers dosing 691 nm of
tannins, show
that the period of the year can increase or decrease the tenor in the mutamba
(Li, 1999).
Comparing the tenor of tannin in the leaves Crataegus oxyacantha L.
(cratego),about 3%,
and the ratany barks (Krameria triandra), about 10%, with the mutamba barks,
that present about
5%, the amount of tannins can be considered reasonable (Kim et al., 2005).
The tannins are phenolic compounds characterized by their capacity of
combination with
the proteins and other polymers as the polysaccharides. This characteristic
explains their
astringency caused by the precipitations of the proteins and by the spittle
glycoproteins.
Tannins (from the French tannin) are polyphenols of plant origin, with molecular
weights
generally between 500 and 3000. They inhibit the attack to the plants from
vertebrates or
invertebrates herbivorous (reduction of palatability, digestion difficulties,
toxic compounds
productions from tannin hydrolysis) and also from pathogenic microorganisms. The
term is
widely used to designate any big polyphenol compound containing enough hydroxyl
groups and
others (as carboxyl) to form strong complexes with proteins and other
macromolecules.
Generally, they are divided in two kinds: hydrolysable and condensed tannins
(protoantocianidines).
Tannins from Quercus suber L. and Q. coccifera L. species show gastroprotector
effect,

13. changing from 66 to 91%.The tannin antimicrobial properties are well-known and
documented.
Tannin molecules are being tested with the intention of finding a new efficient
drug against HIV.
Kilkuskie and partners observed that the galotannins showed inhibitory activity
only in toxic
concentrations, elagitannins and condensed tannins poorly inhibited the viral
replication and the
complex tannins showed powerful activity against the HIV replication. They
concluded that the
anti-HIV activity showed by the tannins is due to the reverse transcriptase
inhibition, turning
difficult the viral replication.
The tannin properties are linked to its capacity of forming complexes with the
proteins
that participate in this case, of the tissues protection in relation to the
microbiological attacks.
Besides, they confer taste properties united under the term astringency, have
anti-free radical
power, and have the capacity of consuming dissolved oxygen, that is, they have
interesting
antioxidant properties to the pharmacologic function and also show agri-food
activity
(Korolkovas, 1988; Makhija, 2001).
The link between tannins and proteins occurs, probably, through hydrogen bridges
between the tannins phenolic groups and certain proteins sites, lending a
lasting stability to these
substances. To the formation of these links it’s necessary that the tannin
molecular weight is
among clear limits; if it is very high, the molecule can’t be inserted among the
interfibrilar spaces
of the proteins or of the macromolecules; if it is very low, the phenolic
molecule can insert, but it
doesn’t form a sufficient amount of links that assure the combination stability.
The tannins have
been the aim of several studies, but the majority has discussed ecological
relationships between
vegetables and herbivorous, because it has been suggested that the tannin tenors
can reduce the
rate of predation for they become unpalatable, taking away their natural
predators. Researches on
tannins biological activities highlighted important action against certain
microorganisms, as

14. carcinogenic agents and the ones that cause hepatic toxicity. These last
effects, with no doubt,
depend on the dosage and the kind of consumed tannin. The intake of green tea
and diets rich in
fruit that contain tannins, for example, has been associated with
anticarcinogenic activity.
Besides, they can act as anti-inflammatories and healings, and even so, as HIV
reverse
transcriptase inhibitors.
This way, if the toxicity is due to its astringency, high toxicity is closely
associated to the
bigger molecular weight. However, it doesn’t happen frequently, for example, the
catechin shows
bigger toxicity than the tannins, although it has low affinity for proteins.
The complexes formed between tannins and proteins can be reversible or
irreversible.
The reversible ones are established by hydrogen bridges and hydrophobic
interactions, while the
irreversible ones occur in oxidative conditions by covalent bonds (Almeida,
1999; Buckheit Jr. et
al., 2001).
The hydrogen bridges probably are formed between the tannin phenolic hydroxyls
and
the protein amine grouping. The hydrophobic interactions occur between the
tannin aromatic
rings and the aliphatic side-chains or aromatic of the protein amino acids
(Nair, 2004).It is
believed that the hydrophobic interactions act as initial traction forces in the
complexation
between tannins and proteins in aqueous media. This initial association is
reinforced in a second
stage with the formation of a polyfunctional net of hydrogen connections, in
which each tannin
molecule can make a lot of connections with the protein, thus, acting like a
polytoothed binder
(Peçanha, 2002).It was still observed a correlation between the polyphenol
polarity and the kind
of interaction with the bovine serum albumin (BSA) (Almeida, 1999).
The reversible complexes can be soluble or insoluble, depending on the
tannin/protein
proportion, on the ph and on the environment ionic strength. The addition of few
quantities of

15. proteins to a tannin solution produces a precipitate that is dissolved with the
addition of more
protein (Young, 2001).Then, the maximum of precipitation occurs when there is an
excellent
proportion between tannin and protein (Li, 1999), what is, however, dependent on
the quantity of
bounding sites as phenolichydroxyls and galoia groups, inside the tannins
(Almeida, 1999;
Okamoto, 2000).
The different tannins capacity of complexing with the proteins vary according to
their
chemical structure. It was observed that the molecular weight and the molecule
flexibility are
important factors in the complexation process (Oliveira, 2000).Several studies,
comparing the
relative affinities of the galoia-steriles groups with a lot of proteins showed
the decreasing
tendency in the penta- > tetra- > tri- > di > mono-galoil-glucose sequence, that
is, the more
quantity of galoia groups, the more affinity for the proteins (Okamoto, 2000).On
the other hand,
some factors in the protein structures such as conformation and polymer size
also influence in
the affinity of these molecules with the tannins. Several studies show the great
tannins affinity,
hydrolysable ad condensed ones, for proteins rich in proline, such as the
proteins present in the
mammal’s spittle (Pluymers et al., 2002).
For these objectives to be achieved, it’s fundamental that the treatment must be
applied
strictly and according to the doctor’s indication. If the patient doesn’t take
the medication
adequately, the quantity of medicine that there is in the blood isn’t enough to
inhibit the increase
of the virus and to reduce the viral load. It allows the virus to continue
destroying the CD4 cells
and also to acquire resistance to the medicines that the patient is taking in a
wrong way. On the
other hand, when it happens, there is a great possibility of occurring
resistance to other
medicines that the patient isn’t taking, which belong to the same classes of
those that he is taking
’ it’s called Cross-Resistance (Pommier, 2000).

16. 3. METHODOLOGY
This subject is a qualitative approach, performed from a collection of data
related to the
theme, based on information found in books, articles, specialized scientific
magazines,
monographs, resource and illustration books and on observation.
To MINAYO (2002, 46):
The qualitative survey answers very private questions (...),
works with the deepest relation and phenomena universe that
can’t be reduced to several variable operationalizations.
The scene of the research is a private laboratory. Previously it will be asked
authorization
to the accomplishment of this scientific research; as well, it will be submitted
to the ethic and
survey committee of the institution.
To acquire data in this study, tests of the low molecular weight tannin dosages
will be
done, in mice, extracted from the Guazuma ulmifolia; then, depending on the
results, it will
address the possibilities of using it in human beings, considering the guiding
questions of the
survey involving human beings. RESOLUTION N° 196/96 FROM THE BRAZILIAN
DEPARTMENT OF HEALTH that regulates the research involving human beings and it
will be
put under evaluation by the Research Ethics Committee of the institution
responsible for the
research.
The search data will be grouped in categories aimed at the results, and will
suffer
analyses based on thematic benchmark about the subject.

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