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  1. 1. Pedro F. C. Vasconcelos, MD, PhDPedro F. C. Vasconcelos, MD, PhD pedrovasconcelos@iec.pa.gov.brpedrovasconcelos@iec.pa.gov.br INSTITUTO EVNDRO CHAGASINSTITUTO EVNDRO CHAGAS MINISTÉRIO DA SAÚDEMINISTÉRIO DA SAÚDE Department of Arbovirology and Hemorrhagic FeversDepartment of Arbovirology and Hemorrhagic Fevers National 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
  2. 2. 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…
  3. 3. 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
  4. 4. Projeto em desenvolvimentoProjeto em desenvolvimento
  5. 5. Pedro F. C. Vasconcelos, MD, PhD Márcio R. T. Nunes, BMD, PhDPedro F. C. Vasconcelos, MD, PhD Márcio R. T. Nunes, BMD, PhD Coordinator Vice-CoordinatorCoordinator Vice-Coordinator pedrovasconcelos@iec.pa.gov.brpedrovasconcelos@iec.pa.gov.br marcionunes@iec.pa.gov.brmarcionunes@iec.pa.gov.br INSTITUTO EVNDRO CHAGASINSTITUTO EVNDRO CHAGAS BRAZILIAN MINISTRY OF HEALTHBRAZILIAN MINISTRY OF HEALTH Department of Arbovirology and Hemorrhagic FeversDepartment of Arbovirology and Hemorrhagic Fevers National 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
  6. 6. 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.
  7. 7. 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
  8. 8. INCT-FHV Genomics Proteomics INCT-Nanobiotecnologia Generation of bioproducts Applicability in the heath sector (Diagnostic tolls) Internal INCT-FHV branches INCT-Inovação em Tecnologia em Saúde INCT-Doenças Negligenciadas INCT-DIAG. SAÚDE PÚBLICA INCT-DENGUE
  9. 9. 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
  10. 10. 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 NIH IAL TCIN GU
  11. 11. INCT-FHV FacilitiesINCT-FHV Facilities The IEC-Ananindeua campus (upper view)The IEC-Ananindeua campus (upper view) Department of Arbovirology and Hemorrhagic Fevers Financial support
  12. 12. Serology, Molecular Biology, P3 and P3Serology, Molecular Biology, P3 and P3 Animal Care FacilitiesAnimal Care Facilities
  13. 13. 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 system Maldi TOF/TOF & Qu-Trap
  14. 14. Sub-projectsSub-projects Subproject 1. Development of tests for the rapid diagnosis of the Dengue virus usingSubproject 1. Development of tests for the rapid diagnosis of the Dengue virus using recombinant proteins of the Envelope (E) and monoclonal antibodies;recombinant proteins of the Envelope (E) and monoclonal antibodies; Subproject 2. Development of an ELISA test for detection of NS1 antigen for YellowSubproject 2. Development of an ELISA test for detection of NS1 antigen for Yellow Fever virus;Fever virus; Subproject 3. Development of an experimental model of dengue for study of infections ofSubproject 3. Development of an experimental model of dengue for study of infections of the liver and central nervous system (encephalitis and encephalomyelitis) caused bythe liver and central nervous system (encephalitis and encephalomyelitis) caused by dengue virus: neuropathological and comportamental aspects;dengue virus: neuropathological and comportamental aspects; Subproject 4. Development of genetic studies for association of polymorphism of genesSubproject 4. Development of genetic studies for association of polymorphism of genes KIR and their ligant groups HLA-C with Dengue.KIR and their ligant groups HLA-C with Dengue. Subproject 5. Development of immunoenzymatic, RT-PCR and real time RT-PCR testsSubproject 5. Development of immunoenzymatic, RT-PCR and real time RT-PCR tests for characterization of infections by Brazilian hantaviruses;for characterization of infections by Brazilian hantaviruses; Subproject 6. Development and standardization of biomolecular techniques forSubproject 6. Development and standardization of biomolecular techniques for detection, quantification and genotyping of the viruses of the hepatitis B and D in casesdetection, quantification and genotyping of the viruses of the hepatitis B and D in cases of striking hepatitis;of striking hepatitis; Subproject 7. Viral hemorrhagic fevers: characterization of the immune innate tissueSubproject 7. Viral hemorrhagic fevers: characterization of the immune innate tissue responseresponse in situin situ and the role of the vascular phenomena in human;and the role 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.
  15. 15. 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 ofThe aim of the project is the development of a rapid method for Dengue diagnostic usinga rapid method for Dengue diagnostic using the Biochip technology and IgM-ELISAthe Biochip technology and IgM-ELISA
  16. 16. Methodology and ResultsMethodology and Results Nucleotide sequencingNucleotide sequencing  75 DV strains75 DV strains 34 DV-134 DV-1 33 DV-233 DV-2 31 DV-331 DV-3 17 DV-417 DV-4  Full-lenght sequences obtained by combinationFull-lenght sequences obtained by combination of two sequencing platforms: Pirosequencing (GSof two sequencing platforms: Pirosequencing (GS FLX 454) and Sanger sequencing (gaps coveredFLX 454) and Sanger sequencing (gaps covered by ABI 3130)by ABI 3130)
  17. 17. Nunes et al, 2012 - EIDNunes et al, 2012 - EID
  18. 18. Development of IgM ELISADevelopment of IgM ELISA Assay (Rapid MAC-ELISA)Assay (Rapid MAC-ELISA) Transferred to SVS (CGLAB)
  19. 19. 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 YFTo 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 YFVFull-length sequencing of 12 Brazilian wild-type YFV strains (first complete sequences obtained) using thestrains (first complete sequences obtained) using the GS FLX 454 systemGS FLX 454 system  Multiple sequencing alignment and phylogeny forMultiple sequencing alignment and phylogeny for selection of candidate strains for NS1 recombinantselection of candidate strains for NS1 recombinant antigen and monoclonal antibody productionantigen and monoclonal antibody production
  20. 20. Selected YFV strains usedSelected YFV strains used 15% nt sequence divergence and 8% aa sequence divergence
  21. 21. Development of molecular diagnostic tools SYBR greenDevelopment of molecular diagnostic tools SYBR green and TaqMan qRT-PCR and RT-Semi-Nested PCRand TaqMan qRT-PCR and RT-Semi-Nested PCR 80 o C± 1o C Transferred to SVS (CGLAB)
  22. 22. Subproject 3:Development of an experimental model of:Development of an experimental model of dengue for study of infections of the liver and central nervousdengue for study of infections of the liver and central nervous system (encephalitis and encephalomyelitis) caused bysystem (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 diseaseto study the impact of the infectious disease on the CNS produced by the virus of theon the CNS produced by the virus of the dengue fever so as to develop abilities to facedengue fever so as to develop abilities to face the multidimensional challenge imposed bythe multidimensional challenge imposed by the epidemic on the waythe epidemic on the way Two other papers submittedTwo other papers submitted
  23. 23. 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 Cruz
  24. 24. 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
  25. 25. Results for KIR genesResults 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
  26. 26. 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
  27. 27. 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
  28. 28. Subproject 6: Development and standardization of biomolecular: Development and standardization of biomolecular techniques for detection, quantification and genotyping of thetechniques 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)
  29. 29. 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).
  30. 30. 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. Objectives Systematized 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.
  31. 31. Methods and resultsMethods and results Samples 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 submittedTwo other papers submitted
  32. 32. Figure 4Quaresma et al., 2013 – Rev Med Virol – published online
  33. 33. Human Resources M Sc. PhD. Post-Doc. DTI IC Active Concluded
  34. 34. 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
  35. 35. 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
  36. 36. THANK YOU www.iec.pa.gov.br/inct-fhvwww.iec.pa.gov.br/inct-fhv

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