O vitiligo é uma doença dermatológica de causa desconhecida. O aparecimento se dá através de manchas branco-nacaradas na pele, ocorridas pela morte ou redução na funcionalidade das células epidérmicas, os melanócitos, que produzem a melanina, pigmento cutâneo. As células Natural Killer (NK) fazem parte do sistema imune inato e através de seus receptores KIR (Killer immunoglobulin-like-receptors) reconhecem moléculas de HLA (Human leukocyte antigen) classe I presentes nas células. Quando não há o reconhecimento do HLA classe I, como em células tumorais ou infectadas por vírus, a célula NK induz a morte da célula alvo. Uma das teorias para essa doença é a imunológica, a qual admite que o vitiligo seja doença autoimune pela formação de anticorpos antimelanócitos, podendo ser associado a outras doenças autoimunes. O objetivo deste estudo foi investigar polimorfismos dos genes KIR e HLA e sua associação com pacientes com vitiligo comparando com um grupo controle. Foram genotipados 112 pacientes com diagnóstico de vitiligo e 250 indivíduos saudáveis para 16 genes KIR e seus ligantes HLA por PCR-SSO e PCR-SSP respectivamente. Nossos resultados mostraram um fator de risco para a doença na interação do gene ativador KIR2DS1 com o seu ligante C2 (P=0,015; OR: 2,06). Também houve uma associação significativa do gene ativador KIR2DS1 com o ligante heterozigoto C1/C2 (P=0,025; OR: 2,26). A interação KIR2DS1/C2 está presente em 52,8% dos pacientes com vitiligo e em 35,2% do grupo controle, já a interação KIR2DS1/C1/C2 está presente em 54,7% dos pacientes com vitiligo e 34,9% do grupo controle. Nossos resultados sugerem um possível fator de risco do gene ativador KIR2DS1 com o seu ligante C2, sendo essa combinação uma possível susceptibilidade à doença. / Vitiligo is a skin disease of unknown cause. The main symptom of vitiligo is white patches on the skin. Which are caused by destruction of pigment-forming cells (melanocytes). Natural killer (NK) cells are part of the innate immune system and they recognize class I HLA molecules (human leukocyte antigen) through their KIR receptors (killer-cell immunoglobulin-like-receptors). When class I HLA molecules are not recognized, e.g.: tumour cells or virus-infected cells, NK cells induce the death of target cells. One of the possible aetiologies for this disease is the immune cause. According to this theory, vitiligo is an autoimmune disease caused by the production of anti-melanocyte antibodies and it may be associated with other autoimmune diseases. The objective of the present study was to investigate KIR and HLA gene polymorphisms and their association with vitiligo comparing with a control group. We genotyped 112 patients diagnosed with vitiligo and 250 healthy individuals for 16 KIR genes and their HLA ligands using PCR-SSO and PCR-SSP respectively. Our findings showed a risk factor for vitiligo in the interaction between the activating KIR2DS1 gene and its C2 ligand (P=0.015; OR: 2.06). There was also a significant association of the activating KIR2DS1 gene with the heterozygous C1/C2 ligand (P=0.025; OR: 2.26). The KIR2DS1/C2 interaction was found in 52.8% of vitiligo patients and in 35.2% of the control group; whereas the KIR2DS1/C1/C2 interaction was found in 54.7% of vitiligo patients and 34.9% of the control group. These findings suggest a possible risk factor related to the interaction between the activating KIR2DS1 gene and its C2 ligand, since this combination may be a disease susceptibility factor.

Made available in DSpace on 2014-06-11T19:31:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-13Bitstream added on 2014-06-13T19:20:14Z : No. of bitstreams: 1 fregonezi_pag_dr_arafcf.pdf: 955927 bytes, checksum: 05e673cbc28acbfcfcaeefd8ebec47bc (MD5) / Universidade Estadual Paulista (UNESP) / As células tumorais infectadas pelo HPV podem apresentar estratégias para escapar da resposta imunológica resultando na persistência da infecção viral e na transformação celular maligna. O objetivo do presente estudo foi investigar o papel das células T e, indiretamente, das células Natural Killer (NK), em lesões orais causadas pelo HPV. A avaliação imunohistoquímica dos receptores de células T em infiltrado linfocitário foi realizada nas 79 biópsias de mucosa oral divididas de acordo com sua diferenciação histológica. Tipos de HPV de alto risco foram mais freqüentes em todas as lesões orais e infecções por diferentes tipos de HPV foram observados na mesma lesão. Diminuição da expressão de CD4/CD25 e CD25/CD28 ( P=0,037) foi observada nas lesões malignas e em todas as lesões com HPV. Menor expressão de CD4 e maior expressão de CD28 foram observadas em lesões orais malignas quando comparadas com as lesões benignas (P=0,005). Em 51 pacientes, elevada expressão de HLA-G foi observada em lesões orais benignas (p<0,01) e progressivamente diminuiu nas lesões pré-malignas e malignas.Correlação inversa (r = - 0,3944, p<0,05) foi observada em lesões orais com HPV, com alta expressão de HLA-G e baixa expressão de HLA-E. Em conclusão, a diminuição da expressão de receptores de células T e de moléculas HLA-E, bem como a maior expressão de moléculas HLA-G pode ser uma estratégia do HPV para escapar da imunovigilância do hospedeiro, resultando na infecção viral persistente e na carcinogênese oral. / HPV-infected tumor cells could present strategies to evade from the immune responses, resulting in the persistence of viral infection and to malignant cell transformation. The aim of the present study was to investigate the role of T cells, and, indirectly, natural killer cells, in HPV-infected oral lesions. To investigate T cell receptors in lymphocyte infiltrate, immunohistochemistry evaluation was performed in 79 oral biopsies, stratified according histological differentiation. High-risk HPV types were more frequent among all oral lesions and multiple HPV type infection was observed in the same oral lesions. Downregulation of CD4/CD25 and CD25/28 (P=0.037) was observed in malignant oral lesions and in all oral lesions groups HPV-infected. Lower expression of CD4 and higher expression of CD28 were observed in malignant oral lesions, when compared with benign lesions (P=0.005). In 51 patients, HLA-G overexpression was observed in benign oral lesions (p < 0.01) and progressively decreased in premalignant to malignant oral lesions. An inverse correlation (r = - 0.3944, p< 0.05) was observed in HPV-infected oral lesions, with high HLA-G expression and low HLA-E expression. We can conclude that downregulation of T cell receptors and HLA-E tissue expression as well as upregulation of HLA-G molecule might be an HPV strategy to escape from host immune surveillance, which could result in HPV persistent infection and oral carcinogenesis.

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-18Bitstream added on 2014-06-13T19:49:53Z : No. of bitstreams: 1 cabestre_cw_me_botfm.pdf: 3064043 bytes, checksum: bb4557e1fc26c57d13076134662046a8 (MD5) / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / Doença falciforme (DF) é a denominação usada para caracterizar uma doença causada pela presença de hemoglobina S (HbS) nas hemácias, ocorrido pela troca do aminoácido Valina pelo aminoácido Ácido Glutâmico na estrutura da cadeia β da Hb, originando uma Hb anormal, denominada HbS. Os indivíduos heterozigotos (que possuem uma cópia da HbS e outra HbA normal), geralmente, são assintomáticos. As formas sintomáticas da doença ocorrem em indivíduos homozigotos para a HbS (chamada de Anemia Falciforme) ou em casos de combinação da HbS com anormalidades hereditárias: hemoglobinopatia SC, hemoblobinopatia SD, S/betatalassemia (Sβ). A transfusão é um componente vital no tratamento de algumas complicações agudas e crônicas da doença falciforme. No entanto, o uso crônico de transfusões de hemácias pode levar à aloimunização eritrocitária, o que pode dificultar o encontro de hemácias para novas transfusões e aumentar o risco de reações hemolíticas. Situados no braço curto do cromossomo 6, o Complexo Principal de Histocompatibilidade (MHC) compreende uma região de genes altamente polimórficos. As proteínas resultantes destes loci formam as moléculas do complexo HLA que são responsáveis pela apresentação de antígenos aos receptores de linfócitos T e B. Isto posto, o objetivo do estudo foi relacionar o perfil genotípico do sistema HLA de classe II (HLA-DRB1 e HLA-DQB1) com a aloimunização eritrocitária em pacientes com Doença Falciforme. Foram coletadas amostras de 47 pacientes com Doença Falciforme (18 adultos e 29 crianças), com faixa etária entre 1 a 58 anos, que apresentavam, ou não, anticorpos eritrocitários, e que são acompanhados no Hemocentro da Faculdade de Medicina de Marília (FAMEMA). Os exames imunohematológicos foram realizados no Laboratório de Imunohematologia do Hemocentro da FAMEMA, através das seguintes técnicas: Tipagem... / Sickle Cell Disease (SCD) is the denomination used to characterize a disease caused by the presence of hemoglobin S (HbS) in erythrocyte occurred by the exchange of the valine amino acid to acid glutamic amino acid in the structure of chain B of the Hb, originating a diferent Hb, denominade HbS. Heterozygous people (having a HbS copy and other normal HsA) usually are asymptomatic. The symptomatic forms of the disease occur in homozygous individuals for HbS (called Sickle Cell Anemia) or in cases of conjuction of HbS with hereditary abnormalities: SC hemoglobinopathy, SD hemoglobinopathy, Sβ-thalassemia. The transfusion is a vital component in the treatment of some acute and chronic complications of sickle cell disease. However, the chronic use of red blood cell (RBC) transfusions may lead to RBC alloimmunization, which can impede the meeting of the RBCs for new blood transfusions and increase the risk of hemolytic reactions. Situated in the short arm of chromosome 6, the genes of Major Histocompatibility Complex (MHC) comprises a region of genes highly polymorphic. The proteins resulting from these loci form the HLA complex molecules of which are responsible for presentation of antigens to T and B lymphocyte receptors. The objective of this study was to relate the profile system genotypic of HLA class II (HLA-DRB1 and HLA-DQB1) with RBC alloimmunization in patients with SCD. Samples were collected from 47 patients with sickle cell disease (18 adults and 29 children), with ages between 1 to 58 years, which presented, or not, erythrocyte antibodies, and which are accompanied by the hemocenter of “Faculdade de Medicina de Marília” (FAMEMA). Immunohematological testes were performed in the immunohematology laboratory of FAMEMA’s hemocenter, through the following techniques: ABO/RhD blood typing, irregular antibodies search, identification of the irregular antibodies, RBC phenotyping... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:23:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-30Bitstream added on 2014-06-13T18:51:17Z : No. of bitstreams: 1 monfre_erm_me_arafcf.pdf: 1147104 bytes, checksum: 534bfc5215b124d9a99b52e9e8577b57 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / O câncer cervical é um problema de saúde pública mundial, especialmente nos países em desenvolvimento representando 12% de todos os tipos de neoplasias malignas que acometem as mulheres, configurando a segunda neoplasia ginecológica mais freqüente no mundo. Estudos epidemiológicos e moleculares demonstram a forte associação entre a etiologia do câncer cervical associado à infecção pelo HPV, especialmente para os tipos de alto risco oncogênico (HP-V16 e HPV-18). A cancerologia experimental, utilizando cultura de células, é de grande valia para se estudar os diversos aspectos relacionados aos processos neoplásicos cervicais. Alguns fármacos usados na quimioterapia são oriundas de espécies vegetais ou derivadas de um protótipo natural. Os produtos naturais têm contribuído intensamente para o desenvolvimento da terapêutica moderna. A quimioprevenção envolve o uso de substâncias naturais ou sintéticas para reduzir o risco de desenvolvimento de câncer. Por esta razão, o presente estudo foi inserido no Projeto de Bioprospecção BIOTA-FAPESP para a avaliação de dois compostos isolados da planta Pterogyne nitens, uma planta com conhecida atividade citotóxica, antioxidante e antifúngica que está distribuída do sudeste ao sul do Brasil, principalmente na Mata Atlântica e no Cerrado. No presente estudo, o potencial inflamatório, antitumoral e quimiopreventor de pteroginina e pteroginidina foram estudados em cultura de células normais e tumorais de câncer cervical (HeLa, SiHa e C33A), queratinócito normal de pele (HaCaT), corioncarcinoma (JEG-3), hepatocarcinoma murino (Hepa1c1c7) e hepatocarcinoma murino mutado (TAOc1BPrc1). Portanto foram realizados ensaios de citotoxicidade por MTT, de determinação de citocinas: IL1-β, IL-4, IL-6, TNF-α e IFN-γ por ELISA, da atividade antitumoral... / Cervical cancer is a public health problem worldwide, mainly in developing countries, representing 12% of all types of malignancies that affect women, and the second common gynecologic malignancy in the world. Molecular and epidemiological research showed a strong association between infection, with HPV, especially high-risk HPV, and the etiology and progression of cervical cancer. Cell culture based on experimental oncology is valuable for study of various features of neoplasic processes. Several drugs used in chemotherapy were isolated from plants species or derived from a natural prototype. Natural products have contributed to the development of modern therapeutics. Chemoprevention involves the use of natural or synthetic substances to reduce the risk of developing cancer. Thus, this study, Bioprospecting Project BIOTA-FAPESP was aimed at assessing two compounds from Pterogyne nitens, a plant with known antiinflammatory and antioxidant activities, is distributed from southeast to southern Brazil, mainly in the Forest Atlantic and Cerrado biomes. The anti-inflammatory, antitumor and quimiopreventor potentials of the compounds pterogynine and pterogynidine was studied in normal and tumor cell lines: cervical cancer (HeLa, SiHa and C33A), normal skin keratinocytes (HaCaT), chorioncarcionoma (JEG-3), murine hepatocellular carcinoma (Hepa 1c1c7) and mutated murine hepatocellular carcinoma (TAOc1BPrc1). These compounds were assesmet for of cytotoxicity (MTT), determination of inflammatory cytokines IL1-β, IL-4, IL-6, TNF-α and IFN-γ (ELISA), antitumor activity with sHLA-G molecule (ELISA) and chemoprevention with quinone reductase (QR). The results showed that both tested compounds exhibited high concentration-dependent cytotoxicity to the three cervical carcinoma cell... (Complete abstract click electronic access below)

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-07-03T12:25:38Z No. of bitstreams: 1 arquivototal.pdf: 3177649 bytes, checksum: b09cbb264ec1b770f1aa0bb04cc0fb8f (MD5) / Made available in DSpace on 2017-07-03T12:25:39Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3177649 bytes, checksum: b09cbb264ec1b770f1aa0bb04cc0fb8f (MD5) Previous issue date: 2017-04-04 / Systems using Unmanned Aerial Vehicles (UAV) are typical examples of cyber-physical systems. Designing such systems is not a trivial task because it brings the challenge of dealing with the uncertainty that is inherent to this type of system. Therefore, it is important the usage of appropriate tools for design that can ensure implementation of these systems with a certain level of confiability. Thus, the purpose of this work is to integrate two simulators via HLA in order to simulate and evaluate different flights strategies. For this, it is presented a simulation environment that can execute flight plans in order to evaluate different strategies in uncertain scenarios. The simulator was developed in Ptolemy and integrated with SITL/ArduPilot via HLA. The results show that with the use of the approach presented in this paper it is possible to obtain results closer to reality, thus more efficient flight strategies can be developed and evaluate. / Sistemas que utilizam Veículos Aéreos Não-Tripulados (VANT) são exemplos típicos de sistemas ciber-físicos. Projetar tais sistemas não é uma tarefa trivial porque traz consigo o desafio de lidar com a incerteza, que é algo inerente a este tipo de sistema. Por isso, é importante que o projeto seja feito com ferramentas apropriadas que possam viabilizar a execução desses sistemas com um certo nível de confiança para seus usuários. Deste modo, a proposta deste trabalho é unir dois simuladores, através do HLA, com o objetivo de simular e avaliar estratégias de voos mais próximas do ambiente de voo real. Para isso, foi construído um simulador onde é possível realizar diversos planos de voo com a finalidade de analisar diferentes estratégias em um ambiente provido de incertezas. O simulador foi desenvolvido na ferramenta Ptolemy e integrado, através do HLA, com o simulador SITL/ArduPilot. Os resultados mostram que com a utilização da abordagem defendida neste trabalho é possível obter resultados mais próximos da realidade, assim estratégias mais eficientes de voo podem ser desenvolvidas e avaliadas.

Resumo: Doença falciforme (DF) é a denominação usada para caracterizar uma doença causada pela presença de hemoglobina S (HbS) nas hemácias, ocorrido pela troca do aminoácido Valina pelo aminoácido Ácido Glutâmico na estrutura da cadeia β da Hb, originando uma Hb anormal, denominada HbS. Os indivíduos heterozigotos (que possuem uma cópia da HbS e outra HbA normal), geralmente, são assintomáticos. As formas sintomáticas da doença ocorrem em indivíduos homozigotos para a HbS (chamada de Anemia Falciforme) ou em casos de combinação da HbS com anormalidades hereditárias: hemoglobinopatia SC, hemoblobinopatia SD, S/betatalassemia (Sβ). A transfusão é um componente vital no tratamento de algumas complicações agudas e crônicas da doença falciforme. No entanto, o uso crônico de transfusões de hemácias pode levar à aloimunização eritrocitária, o que pode dificultar o encontro de hemácias para novas transfusões e aumentar o risco de reações hemolíticas. Situados no braço curto do cromossomo 6, o Complexo Principal de Histocompatibilidade (MHC) compreende uma região de genes altamente polimórficos. As proteínas resultantes destes loci formam as moléculas do complexo HLA que são responsáveis pela apresentação de antígenos aos receptores de linfócitos T e B. Isto posto, o objetivo do estudo foi relacionar o perfil genotípico do sistema HLA de classe II (HLA-DRB1 e HLA-DQB1) com a aloimunização eritrocitária em pacientes com Doença Falciforme. Foram coletadas amostras de 47 pacientes com Doença Falciforme (18 adultos e 29 crianças), com faixa etária entre 1 a 58 anos, que apresentavam, ou não, anticorpos eritrocitários, e que são acompanhados no Hemocentro da Faculdade de Medicina de Marília (FAMEMA). Os exames imunohematológicos foram realizados no Laboratório de Imunohematologia do Hemocentro da FAMEMA, através das seguintes técnicas: Tipagem... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sickle Cell Disease (SCD) is the denomination used to characterize a disease caused by the presence of hemoglobin S (HbS) in erythrocyte occurred by the exchange of the valine amino acid to acid glutamic amino acid in the structure of chain B of the Hb, originating a diferent Hb, denominade HbS. Heterozygous people (having a HbS copy and other normal HsA) usually are asymptomatic. The symptomatic forms of the disease occur in homozygous individuals for HbS (called Sickle Cell Anemia) or in cases of conjuction of HbS with hereditary abnormalities: SC hemoglobinopathy, SD hemoglobinopathy, Sβ-thalassemia. The transfusion is a vital component in the treatment of some acute and chronic complications of sickle cell disease. However, the chronic use of red blood cell (RBC) transfusions may lead to RBC alloimmunization, which can impede the meeting of the RBCs for new blood transfusions and increase the risk of hemolytic reactions. Situated in the short arm of chromosome 6, the genes of Major Histocompatibility Complex (MHC) comprises a region of genes highly polymorphic. The proteins resulting from these loci form the HLA complex molecules of which are responsible for presentation of antigens to T and B lymphocyte receptors. The objective of this study was to relate the profile system genotypic of HLA class II (HLA-DRB1 and HLA-DQB1) with RBC alloimmunization in patients with SCD. Samples were collected from 47 patients with sickle cell disease (18 adults and 29 children), with ages between 1 to 58 years, which presented, or not, erythrocyte antibodies, and which are accompanied by the hemocenter of "Faculdade de Medicina de Marília" (FAMEMA). Immunohematological testes were performed in the immunohematology laboratory of FAMEMA's hemocenter, through the following techniques: ABO/RhD blood typing, irregular antibodies search, identification of the irregular antibodies, RBC phenotyping... (Complete abstract click electronic access below) / Orientador: Valéria Nogueira Dias Paes Secco / Coorientador: Antônio Fabron Júnior / Banca: Wilson Baleotti Júnior / Banca: Rosana Rossi Ferreira / Mestre

O vitiligo é uma doença dermatológica de causa desconhecida. O aparecimento se dá através de manchas branco-nacaradas na pele, ocorridas pela morte ou redução na funcionalidade das células epidérmicas, os melanócitos, que produzem a melanina, pigmento cutâneo. As células Natural Killer (NK) fazem parte do sistema imune inato e através de seus receptores KIR (Killer immunoglobulin-like-receptors) reconhecem moléculas de HLA (Human leukocyte antigen) classe I presentes nas células. Quando não há o reconhecimento do HLA classe I, como em células tumorais ou infectadas por vírus, a célula NK induz a morte da célula alvo. Uma das teorias para essa doença é a imunológica, a qual admite que o vitiligo seja doença autoimune pela formação de anticorpos antimelanócitos, podendo ser associado a outras doenças autoimunes. O objetivo deste estudo foi investigar polimorfismos dos genes KIR e HLA e sua associação com pacientes com vitiligo comparando com um grupo controle. Foram genotipados 112 pacientes com diagnóstico de vitiligo e 250 indivíduos saudáveis para 16 genes KIR e seus ligantes HLA por PCR-SSO e PCR-SSP respectivamente. Nossos resultados mostraram um fator de risco para a doença na interação do gene ativador KIR2DS1 com o seu ligante C2 (P=0,015; OR: 2,06). Também houve uma associação significativa do gene ativador KIR2DS1 com o ligante heterozigoto C1/C2 (P=0,025; OR: 2,26). A interação KIR2DS1/C2 está presente em 52,8% dos pacientes com vitiligo e em 35,2% do grupo controle, já a interação KIR2DS1/C1/C2 está presente em 54,7% dos pacientes com vitiligo e 34,9% do grupo controle. Nossos resultados sugerem um possível fator de risco do gene ativador KIR2DS1 com o seu ligante C2, sendo essa combinação uma possível susceptibilidade à doença. / Vitiligo is a skin disease of unknown cause. The main symptom of vitiligo is white patches on the skin. Which are caused by destruction of pigment-forming cells (melanocytes). Natural killer (NK) cells are part of the innate immune system and they recognize class I HLA molecules (human leukocyte antigen) through their KIR receptors (killer-cell immunoglobulin-like-receptors). When class I HLA molecules are not recognized, e.g.: tumour cells or virus-infected cells, NK cells induce the death of target cells. One of the possible aetiologies for this disease is the immune cause. According to this theory, vitiligo is an autoimmune disease caused by the production of anti-melanocyte antibodies and it may be associated with other autoimmune diseases. The objective of the present study was to investigate KIR and HLA gene polymorphisms and their association with vitiligo comparing with a control group. We genotyped 112 patients diagnosed with vitiligo and 250 healthy individuals for 16 KIR genes and their HLA ligands using PCR-SSO and PCR-SSP respectively. Our findings showed a risk factor for vitiligo in the interaction between the activating KIR2DS1 gene and its C2 ligand (P=0.015; OR: 2.06). There was also a significant association of the activating KIR2DS1 gene with the heterozygous C1/C2 ligand (P=0.025; OR: 2.26). The KIR2DS1/C2 interaction was found in 52.8% of vitiligo patients and in 35.2% of the control group; whereas the KIR2DS1/C1/C2 interaction was found in 54.7% of vitiligo patients and 34.9% of the control group. These findings suggest a possible risk factor related to the interaction between the activating KIR2DS1 gene and its C2 ligand, since this combination may be a disease susceptibility factor.

Submitted by Gislaine Aparecida Querino (gislainequerino@hotmail.com) on 2018-09-28T01:32:53Z No. of bitstreams: 1 Querino,GA.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-09-28T12:55:41Z (GMT) No. of bitstreams: 1 querino_ga_dr_bot.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5) / Made available in DSpace on 2018-09-28T12:55:41Z (GMT). No. of bitstreams: 1 querino_ga_dr_bot.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5) Previous issue date: 2018-07-25 / A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae que pode se manifestar sob diferentes formas clínicas a depender da resposta imune celular do hospedeiro. Vários marcadores genéticos têm sido definidos e, devido à participação dos alelos HLA na resposta imune, estes têm sido amplamente estudados na hanseníase. O HLA-DP constitui uma das moléculas clássicas de classe II, com poucos estudos em hanseníase. O objetivo deste trabalho foi conduzir um estudo de associação genética de base populacional em hanseníase para os genes HLA-DPA1 e HLA-DPB1 com duas amostras caso-controle: a primeira de Rondonópolis-MT, uma região hiperendêmica para hanseníase, e a segunda do Estado de São Paulo, uma região com índices epidemiológicos controlados. Foram realizados estudos com 9 tag SNPs na região dos genes HLA-DPA1 e HLA-DPB1 na população de Rondonópolis–MT (411 casos e 357 controles). O SNP rs9277341 que apresentou dados de associação com significância estatística após a correção de Bonferroni foi então testado na população de São Paulo (570 casos e 380 controles). Para avaliar o efeito funcional deste marcador foi determinada estudoa produção das citocinas IL-10 e TNF após estímulo com antígeno sonicado de M. leprae em 21 controles saudáveis. Na população de Rondonópolis-MT foi realizada a tipificação dos alelos HLA-DPA1* e HLA-DPB1* através da técnica de PCR-SSO empregando-se o kit Labtype-SSO (One Lambda, CA, USA). Os resultados de genotipagem mostraram associação com hanseníase per se para o marcador rs9377341 do gene HLA-DPA1 e dois marcadores do gene HLA-DPB1 (rs1431402 e rs9277469). Associações de proteção para a forma multibacilar da hanseníase foram encontradas para os marcadores rs9277341 e rs2301220 do gene HLA-DPA1 e para o marcador rs31350221 do gene HLA-DPB1. O SNP rs9277341, com dados significativos após a correção de Bonferroni e testado na população do Estado de São Paulo, não apresentou associação com hanseníase. A avaliação funcional mostrou que os carreadores do alelo G do rs9277341 apresentaram níveis maiores de IL-10 e TNF. Na tipificação dos alelos HLA, o alelo HLA-DPB1*03 foi associado à hanseníase per se e o genótipo HLA-DPB1*02/03 foi associado à hanseníase per se e à proteção para a forma MB. Esses dados sugerem a participação dos genes HLA-DPA1 e HLA-DPB1 nos desfechos da hanseníase. / Leprosy, a chronic infectious disease caused by Mycobacterium leprae, displays different clinical manifestations depending on the immune response of the host. Several genetic markers have been defined and due to the involvement of the HLA alleles in the immune response they were extensively studied in leprosy. However, HLA-DP is a classical class II molecule with few studies on leprosy. The objective of this study was to conduct a population-based genetic association study in leprosy for the HLA-DPA1 and HLA-DPB1 genes with two case-control samples: the first from Rondonópolis - Mato Grosso (MT), a hyperendemic region in leprosy and the second from the state of São Paulo, a region with controlled epidemiological indices. Studies were carried out with 9 Tag Single Nucleotide Polymorphism (SNP) in HLA-DPA and HLA-DPB genes in the population from Rondonópolis – MT (411 cases and 357 controls). The SNP rs9277341 who presented association data with statist significance after the Bonferroni correction was tested in the São Paulo population (570 cases and 380 controls). To evaluate the functional effect of this marker was carried a study of the production of IL-10 and TNF cytokines after stimulation with the sonicated antigen of M. leprae in 21 healthy controls. In the Rondonópolis populations was performed the HLA-DPA1* and HLA-DPB1* allele typing by Sequence-Specific Oligonucleotide – Polymerase Chain Reaction (SSO- PCR) using the Labtype-SSO kit (One Lambda, CA, USA). The genotyping results showed association with leprosy per se for the SNP rs9277341 in the HLA-DPA1 gene and two markers in the HLA-DPB1 gene (rs1431402 and rs9277469).The protections associations for the multibacilar form the leprosy was found for the markers rs9277341 and rs2301220 in the HLA-DPA1 gene and the marker rs 31350221 in the HLA-DPB1 gene. The rs9277341 with significatif datas after the Bonferroni correction and tested in the São Paulo state showed no association with leprosy. The functional study showed significantly increased IL-10 and TNF levels in G carriers. HLA typing analysis the HLA-DPB1*03 allele was associated with leprosy per se and the HLA-DPB1*02/03 genotype was associated with leprosy per se and the protection with MB form. These data suggest an association of HLA-DPA1 and HLA-DPB1 genes with leprosy.

Orientador: Ana Carla Pereira Latini / Resumo: A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae que pode se manifestar sob diferentes formas clínicas a depender da resposta imune celular do hospedeiro. Vários marcadores genéticos têm sido definidos e, devido à participação dos alelos HLA na resposta imune, estes têm sido amplamente estudados na hanseníase. O HLA-DP constitui uma das moléculas clássicas de classe II, com poucos estudos em hanseníase. O objetivo deste trabalho foi conduzir um estudo de associação genética de base populacional em hanseníase para os genes HLA-DPA1 e HLA-DPB1 com duas amostras caso-controle: a primeira de Rondonópolis-MT, uma região hiperendêmica para hanseníase, e a segunda do Estado de São Paulo, uma região com índices epidemiológicos controlados. Foram realizados estudos com 9 tag SNPs na região dos genes HLA-DPA1 e HLA-DPB1 na população de Rondonópolis–MT (411 casos e 357 controles). O SNP rs9277341 que apresentou dados de associação com significância estatística após a correção de Bonferroni foi então testado na população de São Paulo (570 casos e 380 controles). Para avaliar o efeito funcional deste marcador foi determinada estudoa produção das citocinas IL-10 e TNF após estímulo com antígeno sonicado de M. leprae em 21 controles saudáveis. Na população de Rondonópolis-MT foi realizada a tipificação dos alelos HLA-DPA1* e HLA-DPB1* através da técnica de PCR-SSO empregando-se o kit Labtype-SSO (One Lambda, CA, USA). Os resultados de genotipagem mostraram asso... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, displays different clinical manifestations depending on the immune response of the host. Several genetic markers have been defined and due to the involvement of the HLA alleles in the immune response they were extensively studied in leprosy. However, HLA-DP is a classical class II molecule with few studies on leprosy. The objective of this study was to conduct a population-based genetic association study in leprosy for the HLA-DPA1 and HLA-DPB1 genes with two case-control samples: the first from Rondonópolis - Mato Grosso (MT), a hyperendemic region in leprosy and the second from the state of São Paulo, a region with controlled epidemiological indices. Studies were carried out with 9 Tag Single Nucleotide Polymorphism (SNP) in HLA-DPA and HLA-DPB genes in the population from Rondonópolis – MT (411 cases and 357 controls). The SNP rs9277341 who presented association data with statist significance after the Bonferroni correction was tested in the São Paulo population (570 cases and 380 controls). To evaluate the functional effect of this marker was carried a study of the production of IL-10 and TNF cytokines after stimulation with the sonicated antigen of M. leprae in 21 healthy controls. In the Rondonópolis populations was performed the HLA-DPA1* and HLA-DPB1* allele typing by Sequence-Specific Oligonucleotide – Polymerase Chain Reaction (SSO- PCR) using the Labtype-SSO kit (One Lambda, CA, USA). The genotyp... (Complete abstract click electronic access below) / Doutor

Japanese encephalitis virus (JEV) causes viral encephalitis in new born and young adults that is prevalent in different parts of India and other parts of South East Asia with an estimated 6000 deaths per year. JEV is a single stranded RNA virus that belongs to the Flavivirusgenus of the family Flaviviridae. It is a neurotropic virus which infects the central nervous system (CNS). The virus follows a zoonotic life-cycle involving mosquitoes and vertebrates, chiefly pigs and ardeid birds, as amplifying hosts. Humans are dead end hosts. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral leukocytosis in the brain and damage to Blood Brain Barrier (BBB). The exact role of the endothelial cells during CNS infection is still unclear. However, disruption of this endothelial barrier has been shown to be an important step in entry of the virus into the brain. Humoral and cell mediated immune responses during JEV infection have been intensively investigated. Previous studies from our lab have shown the activation of cytotoxic T-cells (CTLs) upon JEV infection. MHC molecules play pivotal role in eliciting both adaptive (T-cells) and innate (NK cells) immune response against viral invasion. Many viruses such as HIV, MCMV, HCMV, AdV and EBV have been found to decrease MHC expression upon infection. On the contrary, flaviviruses like West Nile Virus (WNV) have been found to increase MHC-I and MHC-II expression. More recently, data from our lab has shown that JEV infection can lead to upregulation of mouse non-classical MHC class Ib molecules like Qb1, Qa1 and T-10 along with classical MHC molecules. Non-classical MHC molecules are important components of the innate and adaptive immune systems. Non-classical MHC molecules differ from their classical MHC class I counterparts by their limited polymorphism, restricted tissue distribution and lower levels of cell surface expression. Human classical MHC class I molecules are HLA-A, -B and –C while non-classical MHC Class Ib molecules are HLA-E, -G and –F. HLA-E, the human homologue of the mouse non-classical MHC molecule, Qa-1b has been shown to be the ligand for the inhibitory NK, NKG2A/CD94 and may bridge innate and adaptive immune responses. In this thesis, we have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecule, HLA-E in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial like cell line ECV304 (ECV), human glioblastoma cell line U87MG and human foreskin fibroblast cells (HFF). We observed an upregulation of classical HLA molecules and HLA-E mRNA in endothelial and fibroblast cells upon JEV infection. This mRNA increase also resulted in upregulation of cell surface classical HLA molecules and HLA-E in HFF cells but not in both the human endothelial cell lines, ECV and HBMECs. Release of soluble classical HLA molecules upon cytokine treatment has been a long known phenomenon. Recently HLA-E has also been shown to be released as a 37 kDa protein from endothelial cells upon cytokine treatments. Our study suggests that JEV mediated upregulation of classical HLA and HLA-E upregulation leads to release of both Classical HLA molecules and HLA-E as soluble forms in the human endothelial cell lines, ECV and HBMEC. This shedding of sHLA-E from human endothelial cells was found to be mediated by matrix metalloproteinase (MMP) proteolytic activity. MMP-9, a protease implicated in release of sHLA molecules was also found to be upregulated upon JEV infection only in endothelial cell lines but not in HFF cells. Our study provides evidence that the JEV mediated solubilisation of HLA-E could be mediated by MMP-9. Further, we have tried to understand the role of the MAPK pathway and NF-κB pathway in the process of HLA-E solubilisation by using specific inhibitors of these pathways during JEV infection of ECV cells. Our data suggests that release of sHLA-E is dependent on p38 and JNK pathways while ERK 1/2 and NF-κB pathway only had a minor role to play in this process. Treatment of endothelial cells with TNF-α, IL-1β and IFN-γ is known to result in release of sHLA-E. In addition to TNF-α and IFNtreatment, we observed that activating agents like poly (I:C), LPS and PMA also resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Treatment of endothelial cells with IFN-β, a type-I interferon also led to release of sHLA-E. IFN-γ, a type II interferon and TNF-α are known to show additive increase in solubilisation of HLA-E. We studied the interaction between type I interferon, IFN-β and TNF-α with regard to shedding of sHLA- E. Both IFNand TNF, when present together caused an additive increase in the shedding of sHLA-E. These two cytokines were also found to potentiate the HLA-E and MMP-9 mRNA expression. Hence, our data suggest that these two cytokines could be working conjunctly to release HLA-E, when these two cytokines are present together as in the case of virus infection of endothelial cells. HLA-E is known to be a ligand for NKG2A/CD94 inhibitory receptors present on NK and a subset of T cells. Previous reports have suggested that NKG2A/CD94 mediated signaling events could inhibit ERK 1/2 phosphorylation leading to inhibition of NK cell activation. IL-2 mediated ERK 1/2 phosphorylation is known to play a very important role in maintenance and activation of NK cells. We studied the effects of sHLA-E that was released, either by JEV infection or IFN-γ treatment on IL-2 mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited IL-2 and PMA induced phosphorylation of ERK 1/2 in NKL and Nishi cells. Virus infected or IFN-γ treated ECV cell culture supernatants containing sHLA-E was also found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. CD25 is a component of the high affinity IL-2 receptor and hence could play an important role in proliferation and activation of NK cells. sHLA-E was also found to inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit the proliferation and activation of NK cells. In summary, we found that establishment of JEV infection and production of cytokines like IFN-β, TNF-α, IL-6 along with MMP-9 in human endothelial cells. These cytokines may also indirectly lead to the reported damage and leukocyte infiltration across infected and uninfected vicinal endothelial cells. The increased surface expression of HLA-E in fibroblast and release of sHLA and sHLA-E molecules from endothelial cells may have an important immunoregulatory role. HLA-E is an inhibitory ligand for NKG2A/CD94 positive CD8+ T and NK cells. Hence our finding that sHLA-E can inhibit NK cell proliferation suggests an immune evasive strategy by JEV.