Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function.

A potential approach to in vivo gene therapy is to target retrovirus to specific receptors through a ligand-receptor interaction. Previous studies have placed a ligand at or close to the N-terminus of the ecotropic Moloney murine leukemia virus envelope and require co-expression of a wild type envelope on the pseudotyped virus for successful transduction of human cells. In this study, over forty chimeric envelopes were generated, which have single or multiple insertions of a 13 or 21 amino acid RGD containing sequence, flanked by cysteine residues, that target the cellular integrin receptors (Chapter III). Virus displaying only the chimeric envelopes was generated from packaging cell lines that express the gag and pol genes. Many of the mutant envelopes demonstrated the formation of syncytia when they were transfected into the XC indicator cell line, which is frequently used to determine envelope binding and fusion capabilities. Pseudotyped virus for several of the chimeric envelopes, transduced both NIH 3T3 mouse fibroblasts and human A375 melanoma cells. Ligands placed in the N-terminal region, within the VRA variable domain, and close to the N-terminus of the proline-rich region (PRR), demonstrated transduction into human melanoma cells. Ligands placed within the PRR and the C-terminus of the envelope did not demonstrate transduction into melanoma cells, although host cell transduction was demonstrated. Pseudotyped virus expressing an RGE containing target sequence, replacing the RGD sequence, had significantly lower transduction efficiency of melanoma cells. These data indicate that the MLV envelope tropism can be altered by insertion of short ligands at various locations throughout the envelope. These initial results were promising and helped to define regions within the envelope that could accommodate the insertion of small targeting ligands, that could redirect the tropism of pseudo typed virus to human cells. In the second part of this study, the focus shifted to targeting receptors that were expressed on specific cells, such as carcinoma cells. We inserted short ligands, flanked with cysteines, into the envelope to generate numerous targeting constructs that bind to receptors over-expressed on a variety of carcinoma cells. These pseudotyped retroviral vectors were generated by packaging cell lines that express only the viral Gag and Pol genes, with no wild-type envelope present. Select chimeric envelopes that express the 21 amino acid bombesin (BN)/gastrin releasing protein (GRP) binding sequence successfully transduced human melanoma cells, breast cancer cells, and cells that express the cloned GRP receptor gene. Nine additional chimeric envelopes were generated, that express a modified 56 amino acid heregulin sequence (HRG), that targets c-rbB-3 (Her-3) and c-erbB-4 (Her-4) receptors on breast carcinoma cells. Pseudotyped virus expressing only the BN/GRP mutant envelopes, transduced NIH 3T3 host cells, and two human carcinoma cell lines; A375 melanoma and MDA-MB-231 breast cells. The HRG chimeric envelopes demonstrated transduction of NIH 3T3 cells and human MDA-MB-453 breast carcinoma cells. Finally, a pseudotyped virus that expressed the chimeric BN/GRP envelopes and packaged the thymidine kinase gene, transduced melenoma and breast carcinoma cells and demonstrated ganciclovir cytotoxicity. Collectively, these data indicate that ligands of various sizes can be used to target pseudotyped virus to a variety of human cancer cells and transfer genes of interest. These findings may expand the feasibility and potential scope of gene therapy.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-02T18:00:17Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoClinicaDoadores.pdf: 39451144 bytes, checksum: ad6c8f89524624185ea1ef12eec7c179 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-03T14:06:54Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoClinicaDoadores.pdf: 39451144 bytes, checksum: ad6c8f89524624185ea1ef12eec7c179 (MD5) / Made available in DSpace on 2013-04-03T14:06:54Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoClinicaDoadores.pdf: 39451144 bytes, checksum: ad6c8f89524624185ea1ef12eec7c179 (MD5) Previous issue date: 2002 / Os vírus linfotrópicos de células T humanas do tipo I e II (HTLV-I/II) são retrovírus que podem ser transmitidos por transfusão de sangue. Estes vírus estão associados com paraparesia espástica tropical (PET), leucemia/linfoma de células T do adulto (L/LTA) e outras doenças sistêmicas imunomediadas. O presente estudo teve como objetivo investigar sinais clínicos de patologias associadas a esses vírus para utilizar na triagem clinica de candidatos à doação de sangue. Usou procedimentos padronizados para avaliação clinica de 30 doadores de sangue soropositivos para HTLV-I/II confirmados pela técnica de reação em cadeia da polimerase (PCR) matriculados no ambulatório do Núcleo de Medicina Tropical da UFPA. Paralelamente, através de interrogatório clínico complementar, estudou grupo controle com 40 candidatos à doação de sangue escolhidos aleatoriamente, que tiveram resultados sorológicos negativos. Dos 30 pacientes examinados, verificou-se que 23 eram portadores de HTLV-I e 07 HTLV-II. Na avaliação clínica, 15 pacientes (50%) não referiram queixas, sendo que 12 pacientes com queixas exclusivamente neurológicas. Observou 05 pacientes com formigamentos; 05 com diminuição da força muscular; 04 com constipação intestinal; 02 com parestesia; 02 com nódulos subcutâneos; 01 com incontinência urinária; 01 com visão borrada; 01 com diminuição do libido. No grupo controle, 05 candidatos (12,5%) referiram queixas. Os resultados indicam que diminuição da força muscular e formigamento devem ser questionados na triagem clínica prévia à doação de sangue para reduzir risco de infecção transfusional. / The human T-cell lymphotropic virus type I and II (HTLV-IIII) are retrovirus that can be transmitted through blood transfusion. These virus are associated with tropical spastic paraparesis (TSP), adult T-cell leukemia/lymphoma (ALT/L) and other immunomediated systemic diseases. In this study, clinic symptoms related to these virus have ivestigated to use in clinical screening of the candidates for blood donation. It used standard procedures to clinical evaluation from 30 blood donors in Tropical Medicine ambulatory of the Universidade Federal do Para, seropositives for HTLV-UII, confirmated by the polymerase chain reation method. Fourty blood donors candidates, that was chosen ramdomily, It studied as a control group through complementary clinical inquiry. Thirty patients studied, 23 of these were HTLV-I positive and 07 HTLV-II positive. The symptons referred in the evaluation, some patients referred more thar one, 15 patients (50%) did not referred any symptoms, and the other (50%) presented many kinds of symptoms, however, 12 presented only neurological symptoms. Five patients presented tingles, 05 muscular force decrease, 04 constipation, 02 paresthesia, 02 subcutaneous nodes, 01 urinary incontinence, 01 smudgy vision, and 01 libido decrease. In the control group, 05 candidates referred neurological symptoms. The results of this study indicate that symptoms such as, muscular force decrease and tingles have to asked to blood donors candidates in order to reduce the risk of transfusional infection.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-03T18:42:05Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoPresencaVirus.pdf: 47528252 bytes, checksum: 2dca4a913fcafe90e46766875717bdd6 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-08T15:29:40Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoPresencaVirus.pdf: 47528252 bytes, checksum: 2dca4a913fcafe90e46766875717bdd6 (MD5) / Made available in DSpace on 2013-04-08T15:29:40Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoPresencaVirus.pdf: 47528252 bytes, checksum: 2dca4a913fcafe90e46766875717bdd6 (MD5) Previous issue date: 2003 / Os vírus linfotrópicos de células T humanas do tipo I e II (HLTV-I/II) apresentam genoma de ácido ribonucléico (RNA) e infectam geralmente células CD4+, com relação endêmica em determinadas áreas como Japão e Caribe com predomínio maior ou menor em outras regiões; na Amazônia brasileira as pesquisas estão correlacionadas principalmente à população indígena. Estes vírus estão associados a doenças malígnas, desordens neurológicas e imunodeficiências, ocasionando viremia por longo período, sem manifestações clínicas. O HTLV é considerado agente etiógico da Leucemia/ linfoma de célula T do adulto (L/LTA) e Parapasemia espática tropical/Mielopatia associada ao HTLV-I (PET/HAM) dentre outras. Este estudo tem como objetivo investigar a presença de HTLV e determinar o tipo mais freqüente (HTLV-I ou HTLV-II) em crianças com Leucemia Linfóide Aguda, matriculadas no serviço de referência para Câncer em Belém, observando a via de transmissão pelo aleitamento materno, os sintomas neurológcas relacionados com a infecção a revisão bibliográfica pertinente. A pesquisa dos vírus foi realizada pela técnica de PCR (Reação em Cadeia da Polimerase), que permite a distinção entre HTLV-I e HTLV-II. Foram observados os parâmetros de idade, sexo, lesões cutâneas, marcha e transfusão sanguínea através de porcentagens. O HTLV-I foi positivo em uma criança do sexo feminino, sem relação com transmissão por aleitamento materno, e não houve o envolvimento do HTLV como agente etiológico de neoplasia de células linfóide na faixa etária pediátrica. / The lymphotropic virus of human T-cell I and II (HTLV-I/II) present genoma of ribonucleic acid (RNA) and generally infect CD4+ cell, with endemic frequency in some areas like Japan and Caribe with greater or minor predominancy in other places; in Brasilian Amazonia, the research is mainly connected to the native population. These virus are relationed with malign deseases, neurological disturbances and il11unodeficiencies, which cause viremia for long time without clinical effects. The HTLV is regarded as an ethiological agent of adult T-cell leukemia/lymphoma (L/LTA) and tropical spastic paraparesis / HTLV-I associated myelopathy (PET/HAM) among. This analisys has the mais purpose of researching the presence of HTLV and situate the most frequent (HTLV-I/II) in children with Acute Lymphoblastic Leukemia treated in the authorized Cancer Assistence in Belem, Para State, Brazil, observing the transmition way by lactation, the neurological symptoms with infections and its bibliografic revise. The rear search of virus was accomplish through the PCR (Polimerase Chain Reaction) tecnique that shows differences among HTLV-I and HTLV-II. Parameters among age, sex, skin lesions, progression and blood transfusion by porcentage had been remarked in a little girl, without relationship to transmition by breastfeeding, and there was no conection with HTLV as na ethiological agent of lymphoidal cells of neoplasia in childhood.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-10T16:44:47Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EpidemiologiaMolecularVirus.pdf: 1170321 bytes, checksum: da4e03bfc36fd6fcd133e64edcce5f7d (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-13T17:24:06Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EpidemiologiaMolecularVirus.pdf: 1170321 bytes, checksum: da4e03bfc36fd6fcd133e64edcce5f7d (MD5) / Made available in DSpace on 2014-02-13T17:24:06Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EpidemiologiaMolecularVirus.pdf: 1170321 bytes, checksum: da4e03bfc36fd6fcd133e64edcce5f7d (MD5) Previous issue date: 2006 / A distribuição geográfica da infecção pelo Vírus Linfotrópico de Células T HTLV 1/2 humanas 1 e 2 é ampla, porém existem áreas de maior endemicidade e também particularidades de acordo com o tipo de HTLV. O HTLV-1 apresenta maior soroprevalência no sudoeste do Japão, no Caribe, na América Central, nas diferentes regiões da América do Sul e nas porções centrais e ocidentais da África e Melanésia. Enquanto o HTLV-2 parece acometer grupos populacionais distintos, como as populações nativas de indígenas das Américas do Norte, Central e Sul, pigmeus da África Central, mongóis na Ásia e também usuários de drogas injetáveis. O trabalho realizado teve como objetivo descrever a epidemiologia molecular do HTLV em três populações distintas do estado do Amapá, que foram: pacientes HIV/AIDS infectado, população afro-descendente e finalmente indivíduos atendidos no Laboratório Central de Saúde Pública do Amapá (LACEN-AP), encaminhados para diagnóstico de HTLV. As amostras foram avaliadas para a presença do vírus por métodos sorológicos (ELISA e Western blot) e moleculares (amplificação gênica e caracterização de segmentos das regiões pX e env pela análise de polimorfismo de fragmentos de restrição por ação de endonuclease. Os resultados obtidos nas diferentes populações foram na população de indivíduos infectados pelo HIV/AIDS, todas as amostras foram negativas, na população afro-descendente, apenas uma amostra apresentou positividade na sorologia pelo método de ELISA, porém foi negativa no Western blot e quando submetida ao método molecular, não houve amplificação. No entanto, entre os indivíduos encaminhados para diagnóstico de HTLV, 06 (seis) amostras foram positivas, e dessas, 05 (cinco) foram confirmadas por Western blot e pelo método molecular. O resultado molecular demonstrou a presença de HTLV-1. / The geographical distribution of the infection by human T-cell lymphotropic virus 1/2 – HTLV 1 / 2 is extensive, nevertheless, there are areas that are more endemic and have more particularities depending on the HTLV type. The HTLV 1 shows a bigger occurrence in south west of Japan, Caribbean, Central America, in different regions of South America and in parts of Central, as well as in western Africa, while HTLV-2, seems to assault distinct groups of people, such as Indians native people from North, Central and South America, Pigmies from Central Africa, Mongols in Asia as well as in infecting drug users. The present work had as objective describing HTLV molecular epidemiology, in three different populations the State of Amapá, they are: HIV/IDS positive patients, Afro-descenig population and individuals assisted of Public Health Center Laboratory of Amapá – LACEN-AP, directed for diagnosis of HTLV. The samples were tested for the presence of virus using serological (ELISA and Western blot) and molecular assays (gene amplification and restriction fragment length polymorphism from pX and env for the analysis of polimorfismo o restriction fragments for endonuclease action. The obtained results in different populations are In the population of HIV infected people, all the samples were negative; in the Afro-desceding population, only one sample was positive confirmed by serological test (ELISA), but negative according to western blot test and submitted to the molecular analysis, there wast not amplification. However, among samples of individuals directed for diagnosis of HTLV, 06 (six) were positive, 5(five) out of 6/them were confirmed by western blot test. The molecular result demonstrated the presence of HTLV-1.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-07T15:35:43Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoFatoresRisco.pdf: 443800 bytes, checksum: d37908afb422ab24a201164564bfd50e (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-14T16:58:38Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoFatoresRisco.pdf: 443800 bytes, checksum: d37908afb422ab24a201164564bfd50e (MD5) / Made available in DSpace on 2014-02-14T16:58:38Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoFatoresRisco.pdf: 443800 bytes, checksum: d37908afb422ab24a201164564bfd50e (MD5) Previous issue date: 2006 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Com o objetivo de definir o perfil epidemiológico da infecção pelos Vírus linfotrópico de células T humanas (HTLV-1 e HTLV-2), na população de doadores de sangue inaptos, da Fundação HEMOPA, na cidade de Belém do Pará, analisaram-se 113 fichas, em relação a fatores de risco associados à transmissão destes retrovírus, entre portadores e não portadores dos HTLV. Observou-se infecção em 76% (n=50) dos doadores inaptos pelo HTLV-1 e em 24% (n=16) pelo HTLV-2; 62% (n=70) dos portadores eram do sexo masculino e 38% (n=43) do sexo feminino, havendo uma maior tendência da infecção por indivíduos deste sexo (p=0,007). Os fatores de risco que exibiram resultados significativos foram: ter recebido transfusão sangüínea (p=0,0003), mais especificamente para HTLV-2 (p=0,02); ter sido amamentado por ama de leite (p=0,006), mais especificamente para HTLV-1 (p=0,04); ter sido submetido à cirurgia (p=0,01), discriminadamente para HTLV-1 (p=0,03) e HTLV-2 (p=0,04); compartilhar lâmina/barbeador (p=0,02), mais especificamente para HTLV-1 (p=0,02); não usar preservativo nas relações sexuais (p=0,0003), discriminadamente para HTLV-1 (p=0,001) e HTLV-2 (p=0,002). Apesar das diversas etapas existentes no processo de triagem de doadores de sangue, cujo objetivo é eliminar potenciais candidatos portadores de doenças transmissíveis pelo sangue, em especial as de curso crônico e assintomático, existem vieses que impossibilitam um processo isento de falhas. / In order to define the epidemiological profile of the Human T-cell Lymphotropic Virus (HTLV-1 and HTLV-2) among inapt blood donors population, at the HEMOPA Foundation, in Belém, state of Pará, we analyzed 113 epidemiological forms, related with risk factors associated with these retrovirus transmission, among carriers and noncarriers of HTLV. We observed that 76% (n=50) of the inapt blood donors were infected by HTLV-1 and 24% (n=16) by HTLV-2; 62% (n=70) of the carriers were male and 38% (n=43) were female, with a tendency of infection in this gender (p=0,007). The risk factors which exhibited significant results were: have received blood transfusion (p=0,0003), more specifically to HTLV-2 (p=0,02); have been breastfeeding from non-mother (p=0,006), more specifically to HTLV-1 (p=0,04); have been submitted to surgery (p=0,01), discriminately to HTLV-1 (p=0,03) and HTLV-2 (p=0,04); share blades/shavers (p=0,02), more specifically to HTLV-1 (p=0,02); do not use condoms during sexual intercourse (p=0,0003), discriminately to HTLV-1 (p=0,001) and HTLV-2 (p=0,002). Despite of the diverse stages existing in the process of selection of blood donors, which the main objective is to eliminate potentials candidates carrying transmissible blood diseases, in special of chronic and asymptomatic course, exist bias that disable an exempt process of fails.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-13T11:58:16Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_SoroepidemiologiaCaracterizacaoMolecular.pdf: 950016 bytes, checksum: 0dab8c38100d1541aefa71f47bdae8a0 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-01T13:25:54Z (GMT) No. of bitstreams: 2 Dissertacao_SoroepidemiologiaCaracterizacaoMolecular.pdf: 950016 bytes, checksum: 0dab8c38100d1541aefa71f47bdae8a0 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-01T13:25:54Z (GMT). No. of bitstreams: 2 Dissertacao_SoroepidemiologiaCaracterizacaoMolecular.pdf: 950016 bytes, checksum: 0dab8c38100d1541aefa71f47bdae8a0 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2007 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A infecção pelo HTLV-1/2 já foi investigada em diversas populações, mostrando prevalência variável conforme a área geográfica, grupos étnicos e subpopulações analisadas. Em estudos brasileiros foi observada uma maior prevalência nos Estados da Bahia e do Pará. O presente estudo teve como objetivo avaliar a prevalência do HTLV em mulheres grávidas na cidade de Belém, Pará, por meio de técnicas sorológicas e moleculares. Foram coletadas 1.027 amostras de sangue e alíquotas de plasmas foram testadas para a presença de anticorpos anti-HTLV-1/2, utilizando o método imunoenzimático do tipo ELISA. A confirmação da infecção e diferenciação dos tipos e subtipos virais foi realizada por meio da Reação em Cadeia mediada pela Polimerase em duas etapas (Nested PCR) através da amplificação gênica das regiões pX, env e 5’LTR seguido da análise de RFLP e seqüenciamento. Foram detectados seis (0,58%) casos de sororreatividade para o HTLV que posteriormente foram confirmados pela amplificação de um fragmento de 159 pb da região pX. A análise de RFLP com a enzima TaqI mostrou que quatro (66,7%) amostras eram positivas para HTLV-1 e duas (33,3%) para HTLV-2. Uma das amostras HTLV-2 teve o fragmento de 630 pb do gene env amplificado. A análise com a endonuclease XhoI mostrou a presença de um perfil de RFLP característico dos subtipos HTLV-2a/2c. Duas amostras HTLV-1 e uma HTLV-2 tiveram a região 5’LTR amplificada e seqüenciada. Por meio da análise filogenética foi possível classificar as amostras HTLV-1 como Cosmopolita Transcontinental e a HTLV-2 como pertencente ao subtipo HTLV-2c. Esse estudo mostra a relevância de se introduzir o teste para HTLV na triagem pré-natal, a fim de reduzir transmissão vertical e horizontal em nosso estado. / HTLV-1/2 infection was already investigated in several populations, showing variable prevalence according to the geographical area, ethnic groups and analyzed subpopulations. In Brazilian studies a larger prevalence was observed in the States of Bahia and Pará. The present study aimed to evaluate the prevalence of HTLV in pregnant women in the Belém city, Pará, through sorologics and molecular assays. One thousand and twnty-seven (1.027) blood samples were collected and plasma aliquots were tested for the presence of antibodies anti-HTLV-1/2, using enzyme immunoassay ELISA (Ortho Diagnostic Systems Inc., USA). The confirmation of the infection and types and subtypes differentiation was determined by a Nested Polimerase Chain Reaction (Nested PCR) targeting the pX, env and 5'LTR regions followed by RFLP analysis and sequencing of the products amplified. It were detected six (0,59%) cases of serorreactivity for anti-HTLV that were confirmed by the amplification of a 159 pb fragment from the pX region. The RFLP analysis with the enzyme TaqI showed that four (66,7%) samples were positive for HTLV-1 and two (33,3%) for HTLV-2. One out of the HTLV-2 samples had a fragment of 630 pb of the env gene amplified. The analysis with the endonuclease XhoI showed the presence of a profile characteristic of the HTLV-2a/2c subtypes. Two samples HTLV-1 and one HTLV-2 had the 5'LTR region amplified and sequenced. The phylogenetic analysis classified the samples HTLV-1 as Cosmopolitan subtype, subgroup Transcontinental and HTLV-2 as belonging to the HTLV-2c subtype. That study shows the relevance of introducing the test for HTLV in the prenatal selection, in order to reduce vertical and horizontal transmission in our State.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-11T11:53:27Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EpidemiologiaMolecularVirusLinfotropico.pdf: 2896905 bytes, checksum: fa8db903bb0b36528bb6eae32168175f (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-03-31T14:12:21Z (GMT) No. of bitstreams: 2 Dissertacao_EpidemiologiaMolecularVirusLinfotropico.pdf: 2896905 bytes, checksum: fa8db903bb0b36528bb6eae32168175f (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-03-31T14:12:21Z (GMT). No. of bitstreams: 2 Dissertacao_EpidemiologiaMolecularVirusLinfotropico.pdf: 2896905 bytes, checksum: fa8db903bb0b36528bb6eae32168175f (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2010 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os HTLV-1/2 pertencem à família Retroviridae, a qual inclui o HIV. O HHV-8 pertence à família Herpesviridae. Os HTLV-1/2, o HHV-8 e o HIV apresentam as mesmas formas de transmissão, resultando em fatores comuns de risco e isso pode justificar a coinfecção HIV/HTLV e HIV/HHV-8. O presente estudo teve como objetivo descrever a epidemiologia molecular das infecções causadas pelos HTLV-1/2 e o HHV-8 em indivíduos portadores do HIV-1 com ou sem SIDA/AIDS, da cidade de Belém, Pará. Das 520 amostras incluídas no estudo, 515 foram testadas para a presença de anticorpos anti- HTLV-1/2 e 499 para a presença de anticorpos anti-HHV-8, pelo método de ELISA. As amostras reativas para o HTLV e para o HHV-8 foram submetidas à métodos moleculares. A soroprevalência da co-infecção HIV/HTLV foi de 2,3%, enquanto que da co-infecção HIV/HHV-8 foi de 35,9%. Nove amostras do HTLV foram seqüenciadas e 1 classificada como HTLV-1 pertencente ao subtipo Cosmopolita, subgrupo Transcontinental e 3 como HTLV-2 do subtipo HTLV-2c, enquanto que a do HHV-8 agrupou-se ao subtipo B. Foi verificada a heterossexualização, menor escolaridade e pauperização entre os portadores do HIV-1 e não houve associação com fatores de risco. Não houve associação da co-infecção HIV/HTLV com fatores de risco e nem com a contagem de células CD4+ e CD8+ e Carga Viral do HIV-1. Houve associação da co-infecção HIV/HHV-8 com a Carga Viral do HIV- 1. Ocorreu maior taxa da carga viral plasmática do HIV-1 no intervalo 1000|—100000 cópias/mL no grupo dos co-infectados HIV/HHV-8. / The HTLV-1/2 belongs to the family Retroviridae, which includes HIV. The HHV-8 belongs to the family Herpesviridae. The HTLV-1/2, HHV-8 and HIV have the same modes of transmission, resulting in common risk factors that can justify the co-infection HIV/HTLV and HIV/HHV-8. The present study had as objective to describe the molecular epidemiology of infections caused by HTLV-1/2 and HHV-8 in individuals with HIV-1 with or without AIDS in Belém, Pará. Of 520 samples included in the study, 515 were tested for the presence of anti-HTLV-1/2 and 499 for the presence of anti-HHV-8 by ELISA. Samples reactive for HTLV, and HHV-8 were subjected to molecular methods. The seroprevalence of co-infection HIV/HTLV was 2.3%, while co-infection HIV/HHV-8 was 35.9%. Nine samples of the HTLV were sequenced and one classified as HTLV-1 belonging to the Cosmopolitan subtype, Transcontinental subgroup, and 3 as HTLV-2 of the subtype HTLV-2c, while the HHV-8 are clustered to subtype B. Was found to heterosexual, less educated and impoverishment among the carrier of the HIV-1 and had not association with risk factors. There was no association of the co-infection HIV/HTLV with the risk factors neither with the count of the CD4+ and CD8+ T cells and viral load of HIV-1 in relation the mono-infected. There is association of co-infection HIV/HHV-8 with viral load of HIV-1. There was a higher rate of plasma viral load of HIV-1 in the interval 1000|—100000 copies / mL in the group of coinfected HIV/HHV-8.