projetos de pesquisas em desenvolvimento
TRANSCRIPT
Pedro F. C. Vasconcelos, MD, PhDPedro F. C. Vasconcelos, MD, [email protected]
INSTITUTO EVNDRO CHAGASINSTITUTO EVNDRO CHAGASMINISTÉRIO DA SAÚDEMINISTÉRIO DA SAÚDE
Department of Arbovirology and Hemorrhagic FeversDepartment of Arbovirology and Hemorrhagic FeversNational Reference Laboratory for ArbovirusesNational Reference Laboratory for Arboviruses
PAHO/WHO Collaborating Center for Reference and Research in ArbovirusesPAHO/WHO Collaborating Center for Reference and Research in Arboviruses
Projetos de pesquisas em desenvolvimento
Dados pessoaisDados pessoais
GRADUAÇÃO EM MEDICINA: Faculdade de Medicina, UFPA, 1982, Belém, PA.
ESPECIALIZAÇÃO EM MEDICINA TROPICAL: Instituto de Medicina Tropical de São Paulo, USP, 1987.
DOUTORADO: Faculdade de Medicina, UFBA, 1999.
PÓS-DOUTORADO: Department of Pathology, University of Texas Medical Branch, 2002-2003, Galveston, EUA.
MÉDICO PESQUISADOR DO IEC, 1983 até o presente.
DIRETOR DO IEC, 2014 até o presente.
PESQUISADOR DO CNPq – nível 1A
MEMBRO COMITÊ ASSESSOR DO CNPq – CA-MP
MEMBRO DE COMITÊS: CNPq, OPAS, OMS, MS, etc…
GRADUAÇÃO EM MEDICINA: Faculdade de Medicina, UFPA, 1982, Belém, PA.
ESPECIALIZAÇÃO EM MEDICINA TROPICAL: Instituto de Medicina Tropical de São Paulo, USP, 1987.
DOUTORADO: Faculdade de Medicina, UFBA, 1999.
PÓS-DOUTORADO: Department of Pathology, University of Texas Medical Branch, 2002-2003, Galveston, EUA.
MÉDICO PESQUISADOR DO IEC, 1983 até o presente.
DIRETOR DO IEC, 2014 até o presente.
PESQUISADOR DO CNPq – nível 1A
MEMBRO COMITÊ ASSESSOR DO CNPq – CA-MP
MEMBRO DE COMITÊS: CNPq, OPAS, OMS, MS, etc…
Estudo eco-epidemiológico e laboratorial sobre a emergência/reemergência de arbovírus em uma comunidade rural da Amazônia Ocidental Brasileira
Submetido à FAPEAM
Vigentes – FAPEAM/Fiocruz CPqLMD
Arboviroses emergentes no Amazonas: Dinâmica de transmissão e caracterização de agentes virais de importância em saúde pública
PROJETOS COM FINANCIAMENTO EXTERNO (coordenação)
Vigentes – CNPq/CAPES/FAPESPAVigentes – CNPq/CAPES/FAPESPA
INCT para Febres Hemorrágicas Virais (INCT-FHV)
Projeto Temático sobre Febre Amarela
Pesquisa de Febre Amarela e outras Arboviroses na Região de Caxiuanã
Pesquisa de arbovírus na região metropolitana de Belém e região nordeste do Pará
Vários projetos de dissertação e teses em andamento
Projeto em desenvolvimentoProjeto em desenvolvimento
Pedro F. C. Vasconcelos, MD, PhD Márcio R. T. Nunes, BMD, PhDPedro F. C. Vasconcelos, MD, PhD Márcio R. T. Nunes, BMD, PhDCoordinator Vice-CoordinatorCoordinator Vice-Coordinator
[email protected] [email protected]
INSTITUTO EVNDRO CHAGASINSTITUTO EVNDRO CHAGASBRAZILIAN MINISTRY OF HEALTHBRAZILIAN MINISTRY OF HEALTH
Department of Arbovirology and Hemorrhagic FeversDepartment of Arbovirology and Hemorrhagic FeversNational Reference Laboratory for ArbovirusesNational Reference Laboratory for Arboviruses
PAHO/WHO Collaborating Center for Reference and Research in ArbovirusesPAHO/WHO Collaborating Center for Reference and Research in Arboviruses
INCT para FEBRES HEMORRÁGICAS VIRAIS
INCT for VIRAL HEMORRHAGIC FEVERS
Why to study the Viral Hemorrhagic Fevers?
1.They are important to public health
2.High case-fatality rate worldwide
3.Physiopathology and pathogenesis are poorly understood
4.Are considered neglected tropical diseases
5.Several of them have great importance in Brazil. In the INCT-FHV we are studying Yellow Fever, Dengue, Brazilian Hantaviruses and Hepatitis B and D viruses.
INCT-FHV structureINCT-FHV structure
Coordinator
Executor institution IEC Collaborators
National
International
UFPA-ICB
UFPA-NMT
IBU-SP
USP-SP
UEPA-PA
U TEXAS MEDICAL BRANCH
U COLUMBIA
I PASTEUR
UMASS MED (NIH)
Vice-Coordinator
Steering committee
Local Coordinators
U GOTTIGEN
U SOUTHAMPTON
CPqRR
UFPA-ICB
IEC
USP-SP
UFPA-NMT
U PITTSBURGH
T C I Neuroscience
IAL
CENP
F LUSÍADAS
INCT-FHV
Genomics
Proteomics
INCT-Nanobiotecnologia
Generation of bioproductsApplicability in the heath sector (Diagnostic tolls)
Internal INCT-FHV branches
INCT-Inovação emTecnologia em Saúde
INC
T- D
oenç
as
Neg
ligen
ciad
as
INCT-DIAG.SAÚDE PÚBLICA
INCT-DENGUE
Capacity building through National / International Collaboration programs
1.1. Columbia University (CII)Columbia University (CII)
2.2. University of Texas Medical BranchUniversity of Texas Medical Branch
3.3. Institut Pasteur (Paris/Cayenne)Institut Pasteur (Paris/Cayenne)
4.4. University of SouthamptomUniversity of Southamptom
5.5. University of Massachusetts Medical School (NIH)University of Massachusetts Medical School (NIH)
6.6. UFPA (ICB/NMT)UFPA (ICB/NMT)
7.7. USP (Pathology/School of Medicine)USP (Pathology/School of Medicine)
8.8. University of GottigenUniversity of Gottigen
9.9. CPqRR (Belo Horizonte)CPqRR (Belo Horizonte)
10.10.University of PittsburghUniversity of Pittsburgh
11.11. Triniti College of NeuroscienceTriniti College of Neuroscience
The Network: 13 InstitutionsThe Network: 13 Institutions IEC campus Ananindeua, PA; May 2009IEC campus Ananindeua, PA; May 2009
National collaborators (08)National collaborators (08) International collaborators (08)International collaborators (08)
Europe
North America
South America
CU
CPqRR
NIHNIH
IALIAL
TCINTCIN
GUGU
INCT-FHV Facilities INCT-FHV Facilities
The IEC-Ananindeua campus (upper view)The IEC-Ananindeua campus (upper view)
Department of Arbovirology and Hemorrhagic Fevers
Financial support
Serology, Molecular Biology, P3 and P3 Serology, Molecular Biology, P3 and P3 Animal Care FacilitiesAnimal Care Facilities
Genomics and proteomics Branch (equipments)Genomics and proteomics Branch (equipments)
Solid 4 ™ System
GS Jr.System
GSFLX 454 System
Micro dissection Arcturus System
Mass spectrometry systemMaldi TOF/TOF & Qu-Trap
Sub-projectsSub-projectsSubproject 1. Development of tests for the rapid diagnosis of the Dengue virus using recombinant proteins of the Subproject 1. Development of tests for the rapid diagnosis of the Dengue virus using recombinant proteins of the Envelope (E) and monoclonal antibodies; Envelope (E) and monoclonal antibodies;
Subproject 2. Development of an ELISA test for detection of NS1 antigen for Yellow Fever virus; Subproject 2. Development of an ELISA test for detection of NS1 antigen for Yellow Fever virus;
Subproject 3. Development of an experimental model of dengue for study of infections of the liver and central Subproject 3. Development of an experimental model of dengue for study of infections of the liver and central nervous system (encephalitis and encephalomyelitis) caused by dengue virus: neuropathological and nervous system (encephalitis and encephalomyelitis) caused by dengue virus: neuropathological and comportamental aspects; comportamental aspects;
Subproject 4. Development of genetic studies for association of polymorphism of genes KIR and their ligant Subproject 4. Development of genetic studies for association of polymorphism of genes KIR and their ligant groups HLA-C with Dengue. groups HLA-C with Dengue.
Subproject 5. Development of immunoenzymatic, RT-PCR and real time RT-PCR tests for characterization of Subproject 5. Development of immunoenzymatic, RT-PCR and real time RT-PCR tests for characterization of infections by Brazilian hantaviruses; infections by Brazilian hantaviruses;
Subproject 6. Development and standardization of biomolecular techniques for detection, quantification and Subproject 6. Development and standardization of biomolecular techniques for detection, quantification and genotyping of the viruses of the hepatitis B and D in cases of striking hepatitis; genotyping of the viruses of the hepatitis B and D in cases of striking hepatitis;
Subproject 7. Viral hemorrhagic fevers: characterization of the immune innate tissue response Subproject 7. Viral hemorrhagic fevers: characterization of the immune innate tissue response in situ in situ and the role and the role of the vascular phenomena in human;of the vascular phenomena in human;
Subproject 8. Experimental Dengue virus infections in a marmoset model.Subproject 8. Experimental Dengue virus infections in a marmoset model.
Subproject 9. Development of diagnostic approaches for VHF using nanoparticles.Subproject 9. Development of diagnostic approaches for VHF using nanoparticles.
Subproject 1: : Development of rapid tests for the diagnosis of the Dengue virus using recombinant proteins of the Envelope (E) and
monoclonal antibodies by IgM-ELISA and Biochip Technology
Objective: Objective:
The aim of the project is the development of The aim of the project is the development of a rapid method for Dengue diagnostic using a rapid method for Dengue diagnostic using the Biochip technology and IgM-ELISA the Biochip technology and IgM-ELISA
Methodology and ResultsMethodology and Results
Nucleotide sequencing Nucleotide sequencing 75 DV strains 75 DV strains
34 DV-1 34 DV-1 33 DV-2 33 DV-2 31 DV-3 31 DV-3 17 DV-417 DV-4
Full-lenght sequences obtained by combination Full-lenght sequences obtained by combination of two sequencing platforms: Pirosequencing (GS of two sequencing platforms: Pirosequencing (GS FLX 454) and Sanger sequencing (gaps covered FLX 454) and Sanger sequencing (gaps covered by ABI 3130)by ABI 3130)
Nunes et al, 2012 - EIDNunes et al, 2012 - EID
Development of IgM ELISA Development of IgM ELISA Assay (Rapid MAC-ELISA)Assay (Rapid MAC-ELISA)
Transferred to SVS (CGLAB)
Sub-project 2: : Development of an ELISA test for detection of NS1 antigen for Yellow Fever virus
Objectives:Objectives: To develop a ELISA plate format assay for the YF To develop a ELISA plate format assay for the YF
diagnostic using the NS1 recombinant antigen.diagnostic using the NS1 recombinant antigen. Serologic tests to characterize YFV immune responseSerologic tests to characterize YFV immune response
Methodology and resultsMethodology and results Full-length sequencing of 12 Brazilian wild-type YFV Full-length sequencing of 12 Brazilian wild-type YFV
strains (first complete sequences obtained) using the strains (first complete sequences obtained) using the GS FLX 454 systemGS FLX 454 system
Multiple sequencing alignment and phylogeny for Multiple sequencing alignment and phylogeny for selection of candidate strains for NS1 recombinant selection of candidate strains for NS1 recombinant antigen and monoclonal antibody productionantigen and monoclonal antibody production
Selected YFV strains used Selected YFV strains used
15% nt sequence divergence and 8% aa sequence divergence
Development of molecular diagnostic tools SYBR green Development of molecular diagnostic tools SYBR green and TaqMan qRT-PCR and RT-Semi-Nested PCR and TaqMan qRT-PCR and RT-Semi-Nested PCR
80 oC± 1oC
Transferred to SVS (CGLAB)
Subproject 3:Development of an experimental model of :Development of an experimental model of dengue for study of infections of the liver and central nervous dengue for study of infections of the liver and central nervous system (encephalitis and encephalomyelitis) caused by system (encephalitis and encephalomyelitis) caused by dengue virus: neuropathological and comportamental aspects; dengue virus: neuropathological and comportamental aspects;
ObjectivesObjectives to study the impact of the infectious disease to study the impact of the infectious disease
on the CNS produced by the virus of the on the CNS produced by the virus of the dengue fever so as to develop abilities to face dengue fever so as to develop abilities to face the multidimensional challenge imposed by the multidimensional challenge imposed by the epidemic on the way the epidemic on the way
Two other papers submittedTwo other papers submitted
Figure 4. Hematoxlin-eosin staining to illustrate significant pathological changes in the lungs and liver. A: Hypertrophy and discrete hyperplasia of bronchic epithelium. B: Vascular congestion in sinusoids and regeneration signs (bi-nuclear hepatocytes). C: Hepatocytic necrosis focus with limphocyte and macrophage infiltrations, discrete tumefaction and steatosis. D: Mononuclear infiltration in the subendothelial space peri-efferent vein. Magnification B, D: 500x; A, C:125x.
Diniz et al. 2012, Mem Inst O CruzDiniz et al. 2012, Mem Inst O Cruz
Subproject 4: : Development of genetic studies for association of polymorphism of genes KIR and their ligant groups HLA-C with Dengue.
Evaluation of possible association of polymorphism of KIR genes and their ligant groups HLA-C with dengue 220 individuals were analyzed
131 dengue fever patients
89 oligosymptomatic patients.
All patients were clinically and laboratorially (HI and MAC-ELISA) screened
Results for KIR genes Results for KIR genes polymorphism evaluationpolymorphism evaluation
Frequency of the KIR genes in the studied population.
*Represents results statistically significant
(χ2 = 10.89 e p= 0.0012) for gene frequencies presented by
carriers of the KIR2DS5 gene among oligosymptomatic and symptomatic
individuals (Dengue fever patients).
Polyacrilamide gel stained with silver:Presence of band demonstrate that the
individual is a carrier of the KIR gene, while absence of bands (arrows) indicated that the individual does not possess a given KIR gene on its genotype.
Another paper submittedAnother paper submitted
Subproject 5: : Development of immunoenzymatic, RT-PCR and real time PCR tests for characterization of infections
by Brazilian hantaviruses
55 samples 29 blood and sera human samples 26 wild rodents tissue samples
were tested by RT-PCR method
20 out 29 and 16 out 26 samples were positive
for hantavirus genome detection
Positive samples were sequenced: GS FLX 454
Complete genome sequences of all Brazilian Amazon hantaviruses
Phylogeny of the studied hantavirusesPhylogeny of the studied hantaviruses
HPS associated strains
Rodent associated strains
New proposal for characterization and classification of New World Hantaviruses
New proposal for characterization and classification of New World Hantaviruses
Subproject 6: Development and standardization of biomolecular : Development and standardization of biomolecular techniques for detection, quantification and genotyping of the techniques for detection, quantification and genotyping of the viruses of the hepatitis B and D in cases of striking hepatitis; viruses of the hepatitis B and D in cases of striking hepatitis;
The aim of this study was characterize the HBV and HDV genotypes from outbreaks of fulminant hepatitis in Boca do Acre and Sena Madureira counties, Western Amazon basin of Brazil.
Five papers accepted/submittedFive papers accepted/submitted
Proposed to be transferred to SVS (CGLAB)
HDV genotype 3 were found in all samples while HBV genotypes were F (n=7), A (n=3) and D (n=3).
A: Phylogenetic tree of HDV partial delta antigen genomic region. B: Phylogenetic tree of HBV S gene, partial region. Neighbour-Joining analyses were performed
with other sequences from different HDV or HBV genotypes described previously using MEGA. The Western Amazon isolates are show in red and the HBV and
HDV sequences obtained in GenBank are designated by their accession number or denominate according to the respective authors. Bootstrap values from 1000
replicates are shown for the genotype branches (shown as a percentage).
Subproject 7: : Viral hemorrhagic fevers: characterization of the immune innate tissue response in situ and the role of the vascular
phenomena in human and experimental infections in a mice model.
ObjectivesSystematized evaluation of the target-organs of the VHF through qualitative and semi quantitative analyses of the histopathological events. Evaluation of the contribution of the innate immunity in situ for pathogenesis of the VHF in the target-organs, through:
quantification by immunohistochemical assy of the NK, CD4, CD8, CD68, CD20, S-100, CD1a FOxp3 cells;
expression of the IFN for the NK cells through immunohistochemistry technique of double demarcation
quantification of the expression of dendrytic cells S-100+ and DC-SIGN tissue expression of TLR2 3 and 4 and C3 Cytocine expression: IL1, IL-2R, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-18, TNFα,
TGβ, IFNγ
Evaluation and characterization of the structural alterations and of the possible mechanisms of activation of the vascular endothelia in the VHF through:
ultrastructural analysis quantification of the expression of endoteliaI cells CD34 + tissue expression of VCAM expression of caspase 3, iNOS
Development of molecular methods for diagnosis of viral hemorrhagic fevers in humans from paraffin embedded specimens.
Methods and resultsMethods and resultsSamples
It was retrospectively and prospectively analyzed cases of biopsies, necropsies or viscerotomy previously diagnosed as VHF
Selection of cases: based on clinical and laboratorial data, serologic data and/or identification of specific antigenic material present in the tissues, demonstrated through immunohistochemical assay.
Two other papers submitted
Two other papers submitted
Figure 4Quaresma et al., 2013 – Rev Med Virol – published online
Human Resources
MSc.
PhD.
Post-D
oc.
DTI IC
Active
Concluded
2009-2011 M.SC. & Ph.D. CONCLUDED
of the INCT-FHV :contribution on
scientific development and
formation of people to science
Samir Mansour Moraes Casseb – Dissertação apresentada ao Curso de Pós Graduação em Genética e Biologia Molecular, UFPA – Título da dissertação: Desenvolvimento de um pipeline para montagem de genomas virais sequenciados por pirosequenciamento. Mestrado em Genética e Biologia Molecular, Belém, Orientador: Artur Luiz da Costa da Silva - 2011
Adriana Ribeiro Carneiro – Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Caracterização molecular de cepas do vírus Dengue 1 isoladas no Brasil entre os anos de 1994 a 2008. Mestrado em Biologia de Agentes Infecciosos e Parasitários, Belém, Orientador: Pedro Fernando da Costa Vasconcelos - 2009
Maíra Catherine Pereira Turiel - Dissertação de mestrado apresentado ao curso de pós-graduação em Neurociências e Biologia Celular, UFPA – Título da dissertação: Encefalite viral induzida pelo vírus da dengue (DENV3, genótipo III) em camundongos suíços albinos: a resposta inflamatória do SNC do hospedeiro neonato. Mestrado em Neurociências e Biologia Celular. Belém. Orientdador: Cristovam Wanderley Picanço Diniz – 2011
Maíra Catherine Pereira Turiel - Dissertação de mestrado apresentado ao curso de pós-graduação em Neurociências e Biologia Celular, UFPA – Título da dissertação: Encefalite viral induzida pelo vírus da dengue (DENV3, genótipo III) em camundongos suíços albinos: a resposta inflamatória do SNC do hospedeiro neonato. Mestrado em Neurociências e Biologia Celular. Belém. Orientdador: Cristovam Wanderley Picanço Diniz – 2011
Clayton Pereira Silva de Lima - Dissertação de mestrado apresentado ao curso de pós-graduação em Genética e Biologia Molecular, UFPA – Título da dissertação: Associação de KIR2DS5 com manifestações clínicas oligossintomáticas de Dengue 2009. Mestrado em Genética e Biologia Molecular, Belém, Orientador: Eduardo José Melo Santos- 2009
Lívia Carício Martins - Tese apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da tese: Correlação da Evolução Clínica de Isolamentos do Vírus da Febre Amarela com Diferente Expressão Fenotípica a partir da Caracterização Genética e Estudo Exeprimental em Hamsters Dourados. Doutorado em Biologia de Agentes Infecciosos e Parasitários. Belém, Orientador: Pedro Fernando da Costa Vasconcelos – 2009.
Mayra de Oliveira e Silva - Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Caracterização Genética do Virus Dengue 2 Circulante no Brasil. Mestrado em Biologia de Agentes Infecciosos e Parasitários, Belém, Orientador: Ana Cecília Ribeiro Cruz - 2010
Darlene Brito Simith - Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Caracterização genética de Hantavírus isolados na Amazônia Brasileira. 2010. Mestrado em Biologia de Agentes Infecciosos e Parasitários – Belém, Orientador: Marcio Roberto Teixeira Nunes - 2011
2012-2013 M.Sc. & Ph.D. CONCLUDED
of the INCT-FHV :contribution on
scientific development and
formation of people to science
Antonio Gregorio Dias Junior - Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Avaliação da expressão in vitro de Micro RNA e RNA de interferência em sorotipos de dengue isolados no Brasil. Mestrado em Biologia de Agentes Infecciosos e Parasitários), Belém, Orientador: Pedro Fernando da Costa Vasconcelos - 2013
Layanna Freitas de Oliveira. Dissertação apresentada ao Curso de Pós Graduação em Genética e Biologia Molecular, UFPA. – Título da dissertação: Polimorfismo -336 A/G no promotor de DC-SIGN (CD209) modula os sintomas na fase aguda inicial da Dengue clássica. Mestrado em Genética e Biologia Molecular, Belém, Orientador Eduardo José Melo Santos - 2012
Rodrigo Paes de Menezes. Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Análise retrospectiva da situação epidemiológica e das relações sócio-climáticas dos casos notificados de dengue no Estado de Rondônia no período de 2000 a 2009. Mestrado em Biologia de Agentes Infecciosos e Parasitários – Belém, Orientador: Ana Cecília Ribeiro Cruz - 2012
Ana Letícia Scaldelai Bernardi - Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Pesquisa de Arenavírus em amostras de roedores silvestres da Amazônia brasileira. Mestrado em Biologia de Agentes Infecciosos e Parasitários), Belém, Orientador: Pedro Fernando da Costa Vasconcelos - 2013
Ana Letícia Scaldelai Bernardi - Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Pesquisa de Arenavírus em amostras de roedores silvestres da Amazônia brasileira. Mestrado em Biologia de Agentes Infecciosos e Parasitários), Belém, Orientador: Pedro Fernando da Costa Vasconcelos - 2013
Jamilla Augusta de Sousa Pantoja - Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Epidemiologia Molecular do Vírus Dengue Sorotipo 3 no Brasil. Mestrado em Biologia de Agentes Infecciosos e Parasitários) – Belém, Orientador: Marcio Roberto Teixeira Nunes - 2012
Milene Silveira Ferreira – Dissertação apresentada ao Curso de Pós Graduação em Doenças Tropicais, UFPA - Título da dissertação: Estudo experimental sobre a resposta imunológica em infecções seqüenciais pelo vírus Dengue 3 e vírus Dengue 2 em primatas não humanos da espécie Callithrix penicillata. Mestrado em Doenças Tropicais, Orientador: Pedro Fernando da Costa Vasconcelos – 2012
Dafne Silva Furtado de Mendonça - Dissertação apresentada ao Curso de Pós Graduação em Biologia dos Agentes Infecciosos e Parasitários, UFPA – Título da dissertação: Associação de genes KIR com manifestações clínicas da dengue. Mestrado em Biologia de Agentes Infecciosos e Parasitários, Belém, Orientador Eduardo José Melo Santos - 2012
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