Made available in DSpace on 2016-08-11T14:41:23Z (GMT). No. of bitstreams: 1 CamilaFAC_DISSERT.pdf: 3171290 bytes, checksum: 6f7ce9f3dcccd165d00b2a266f0a28ef (MD5) Previous issue date: 2014-03-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Visceral Leishmaniasis (VL) is a anthropozoonosis whose occurrence depends on the presence of species involved in the transmission chain and favorable environmental conditions. In the city of Mossoró, Rio Grande do Norte, VL is endemic, and the disease vector, the sandfly Lutzomyia longipalpis species present in significant numbers throughout the year, and its wetlands to development coupled with the presence of organic matter and animals. These factors justify the choice of the areas of this study, near the River Mossoró, being wet and biodiversity areas that may favor the development of the vector. Based on the foregoing, this study aimed to characterize the risk of transmission of VL near the River Mossoró areas in the city of Mossoró, Rio Grande do Norte. The research took place in the districts of Alto da Conceição Ilha de Santa Luzia and Paredões, with 478 residences that are nearby the bed of the Rio Therefore, an entomological survey was conducted with CDC light traps using the methodology employed by the Ministry of Health Brazil and analyzed the influence of temperature, humidity, rainfall and winds over the sandflies captured. Questionnaires with 478 residents of the area was also conducted, addressing the population's knowledge about the LV as well as routine activities of residents and environmental characteristics that could favor the maintenance of the vector. It was observed that sand flies are present in the area throughout the year, and in greater quantity peridomiciles 71.3% (p<0.05). The female L. longipalpis predominate over other species, 92.2% (p<0.05) and the number of males than females was 62.4% (p<0.05). With the questionnaires, it was observed that with respect to social profile, the majority of respondents were female, with ages between 18 and 40 years in education and 53.8% had completed elementary (p<0,05). Still on the responses, 61.5% raised dogs, 95.9% had little knowledge about the characteristics inherent to sandflies and 85.3% were unaware of the environments preferably this vector (p<0.05). The characteristics of the environment, sewage were dumped mainly in the River (44.6%) and 76.6% of the respondents complained about the presence of garbage in the streets accumulation (p<0.05). Associations analyzed, statistical significance between education, knowledge about the transmission of VL and local preference vector (p<0.05), demonstrating that the level of education may influence the population's knowledge about disease transmission. Thus, comparing the vector density to environmental factors, it can be seen that the high temperature and humidity, rainfall and low wind speeds, make the environment conducive for the development of the vector. Regarding the lack of knowledge of the population about the LV and the environment where these people reside, these facts reflect the risk of maintaining the sandfly and consequently in disease transmission. May conclude that the lack of knowledge about the disease and ways to vector control and leave the residents of these areas dogs at risk of infection / A Leishmaniose Visceral (LV) é uma antropozoonose cuja ocorrência depende da presença de espécies envolvidas na cadeia de transmissão e condições ambientais favoráveis. Na cidade de Mossoró, Rio Grande do Norte, a LV é endêmica, e o vetor da doença, flebotomíneo da espécie Lutzomyia longipalpis, apresenta-se em número significativo ao longo de todo o ano, tendo seu desenvolvimento atrelado a áreas úmidas, com presença de matéria orgânica e animais. Esses fatores justificam a escolha das áreas do presente estudo, próximas ao Rio Mossoró, por serem áreas úmidas e com biodiversidade que pode favorecer o desenvolvimento do vetor. Baseado no exposto, este estudo teve como objetivo principal caracterizar o risco de transmissão da LV em áreas próximas ao Rio Mossoró, na cidade de Mossoró, Rio Grande do Norte. A pesquisa ocorreu nos bairros de Alto da Conceição, Ilha de Santa Luzia e Paredões, com 478 residências que estão nas proximidades do leito do Rio. Para tanto, foi realizado levantamento entomológico com armadilhas luminosas tipo CDC utilizando a metodologia empregada pelo Ministério da Saúde do Brasil e analisado a influência de temperatura, umidade, pluviosidade e ventos sobre os flebotomíneos capturados. Também foi realizada aplicação de questionários com 478 moradores da área, abordando o conhecimento da população sobre a LV, bem como atividades de rotina dos moradores e características ambientais que poderiam favorecer a manutenção do vetor. Observou-se que os flebotomíneos estão presentes na área durante todo o ano, sendo em maior quantidade no peridomicílio 71,3% (p<0,05). As fêmeas de L. longipalpis predominaram sobre outras espécies, 92,2% (p<0,05) e a quantidade de machos foi superior a de fêmeas 62,4% (p<0,05). Com aplicação dos questionários, observou-se que com relação ao perfil social, a maioria dos entrevistados era do sexo feminino, com a faixa etária entre 18 a 40 anos e na escolaridade 53,8% tinham até o fundamental completo (p<0,05). Ainda sobre as respostas, 61,5% criavam cães, 95,9% demonstrava pouco conhecimento sobre características inerentes aos flebotomíneos e 85,3% desconheciam os ambientes de preferência desse vetor (p<0,05). Nas características do ambiente, os esgotos eram despejados principalmente no Rio (44,6%) e 76,6% dos entrevistados se queixaram da presença de acúmulo lixo nas ruas (p<0,05). Das associações analisadas, houve significância estatística entre a escolaridade, o conhecimento sobre a transmissão da LV e locais de preferência do vetor (p<0,05), demonstrando que o nível de escolaridade pode influenciar no conhecimento da população sobre a transmissão da doença. Deste modo, comparando a densidade vetorial aos fatores ambientais, percebe-se que a alta temperatura e umidade, as chuvas e a baixa velocidade dos ventos, tornam o ambiente propicio para o desenvolvimento do vetor. Com relação a falta de conhecimento da população sobre a LV e o ambiente onde essas pessoas residem, esses fatos refletem no risco de manutenção do flebotomíneo e consequentemente na transmissão da doença. Podendo concluir que, o desconhecimento sobre a doença e as formas de controle vetorial deixam os moradores e os cães dessas áreas expostos ao risco de infecção.

Leishmaniasis are infectious diseases caused by protozoa of the genus Leishmania, that are digenetic parasites alternating between an extracellular promastigote stage (in sand fly vector) and an intracellular amastigote stage (in mammalian host). Promastigote phagocytosis results in a macrophage respiratory burst in vitro, leading to the generation of reactive oxygen species (ROS) such as superoxide anion (O2 -), hydrogen peroxide (H2O2), singlete oxygen (1O2) and hydroxyl radical (OH). Thus, we aimed to evaluate in vitro ROS toxicity for L. L. chagasi promastigotes, as well as for their amastigote counterparts. To evaluate ROS toxicity for promastigotes, we selected 16 L. L. chagasi isolates (in log phase growth) and incubated them under increasing concentrations of menadione (0-750ìM) for 4 hours, after which we determined ROS viability for these parasites by quantifying the number of mobile forms. For ROS toxicity for amastigote L. L. chagasi, we infected J774.16, a murine macrophage cell line, with ROS susceptible and resistant L. L. chagasi (in stationary phase of growth) in cultures treated by LMNA, an iNOS inhibitor (to block the synthesis of nitric oxide), treatment with diethyldithiocarbamate (DETC), a SOD-1 inhibitor (to increase superoxide anion synthesis) as well as incubation with N-acetylcysteine, NAC ( an antioxidant).These infection experiments were conducted in 8 well plates in a 5:1 ratio (parasites/cell). Subsequently, we incubated these plates for 4, 24, 48 and 72 hours at 37oC and 5% CO2. After that, the plates were stained and the number of amastigotes (parasite burden) determined. We observed that 14 out 16 isolates treated with menadione showed 50% or more loss of their viability at concentrations varying from 15 to 750 ìM, whereas two of them exhibited even 70% or more viability at 750 mM. From ROS toxicity evaluation for L. L. chagasi amastigotes, we observed in J774.16 cultures infected by ROS susceptible and resistant L. L. chagasi a decrease in parasitic load for both isolates. This decrease in parasite burden was observed also in cultures treated by LMNA, an iNOS inhibitor. Inhibition of SOD by DETC also promoted a decrease in the number of amastigotes for both of these J774.16 cultures from 24 hour infection. The addition of NAC to these cultures increased the number of amastigotes for ROS resistant infected J774.16 cells but nor for those ROS susceptible infected cultures. These data indicate that damage on these amastigotes induced by DETC is oxidative. Thus, it is possible to conclude that reactive oxygen species are crucial toxic agents for L. L. chagasi as in promastigote form, as well as in amastigote form. / As leishmanioses sao doencas infecciosas causadas por parasitos do genero Leishmania. Leishmania sao protozoarios digeneticos, alternando entre as formas promastigota (flebotomineo) e amastigota (hospedeiro mamifero). A fagocitose de promastigotas desencadeia em macrofagos um gburst oxidativo h, gerando especies reativas do oxigenio (ROS) como o anion superoxido (O2 -), peroxido de hidrogenio (H202), o oxigenio singlete (1O2) e o radical hidroxila (OH). Assim, objetivamos avaliar in vitro a atividade leishmanicida de ROS para promastigotas de L.(L.) chagasi, bem como para suas respectivas formas amastigotas. Para a avaliacao da toxicidade de ROS para promastigotas, selecionamos 16 isolados de L.(L.) chagasi (em fase log de crescimento), incubando-os sob concentracoes crescentes de menadiona (0-750 ÊM) por um periodo de 4h, ao fim do qual determinamos a viabilidade desses parasitos, quantificando o numero de formas moveis. Na avaliacao da acao leishmanicida de ROS para as formas amastigotas de L. L. chagasi, infectamos uma linhagem celular de macrofagos murinos J774.16 com um isolado resistente e dois isolados susceptiveis a ROS (na proporcao 5:1 parasitos/celulas) em culturas com LMNA, inibidor de iNOS, (para bloquear a sintese de oxido nitrico), incubadas com o inibidor da enzima SOD-1, dietilditiocarbamato, DETC (para aumentar a producao de O2 -) e com N-acetilcisteina, NAC (um antioxidante) em placas de 8 pocos e incubadas por 4, 24, 48 e 72 horas. Ao termino de cada incubacao, coramos as placas com panotipo, para determinacao do numero de amastigotas (carga parasitaria). A partir da exposicao de promastigotas a concentracoes crescentes de menadiona, observamos que dos 16 isolados avaliados, 14 deles apresentaram perdas de 50% ou mais de suas viabilidades entre concentracoes de 15 a 750 ÊM de menadiona (formas susceptiveis a ROS), enquanto apenas dois deles apresentaram 70% ou mais de viabilidade a 750 ÊM de menadiona (formas resistentes a ROS). Na avaliacao da toxicidade de ROS para as formas amastigotas, observamos nas culturas de celulas J774.16 infectadas por L. L. chagasi susceptiveis e resistente a ROS diminuicao na carga parasitaria de ambos os isolados a partir do periodo de 48 horas. Para as culturas incubadas com DETC, observamos a reducao dos numeros de amastigotas para essas culturas, a partir do tempo de 24 horas de infeccao. A adicao de NAC a essas culturas reverteu a carga parasitaria das culturas infectadas pelo isolado resistente, mas nao as que foram infectadas pelos isolados susceptiveis a ROS, indicando que o dano induzido por DETC sobre essas amastigotas e oxidativo. Assim, e possivel afirmar que as especies reativas do oxigenio sao importantes agentes toxicos para promastigotas e amastigotas de L. L. chagasi.

Visceral Leishmaniasis (VL) is a severe desease, endemic in the Brazil, with a tendency to be more prevalent in urban areas. The pathology of VL is associated with a reduced production of IFN-. by T-cells and the increased production of IL-10, which suppresses the activation of macrophages and promotes development of the disease. Activated T cells can express CD40L interacting with CD40 expressed on antigen presenting cells (APC), this interaction is important for the activation of APCs and production of inflammatory cytokines, chemokines, nitric oxide (NO) and metalloproteinases, triggering a protective response in experimental models of LV. Data from our group showed that VL patients have low serum levels of sCD40L before treatment and that these levels increase over the same, titles reaching close to those found in endemic control, suggesting a protective effect of this molecule. In this work we evaluated the participation of sCD40L in modulating the immune response of macrophages infected with Leishmania chagasi. In this work we evaluated the participation of sCD40L in modulating the immune response of macrophages infected with Leishmania chagasi. For this, human macrophages were infected with promastigotes of Leishmania chagasi in the presence of serum containing high titers of sCD40L with and without addition of blocking antibody, anti-CD40L. After 72 hours, the microbicidal response was evaluated by number of infected macrophages and the number of intracellular parasites We observed that there was a 43% reduction in the percentage of infected macrophages and 58% of the number of intracellular parasites when compared to the medium (P = 0.01) and this effect was reversed by the blockade of sCD40L, because there was a 24% increase in the percentage of infected macrophages and 33% of the number of amastigotes. From the results we can conclude that the sCD40Lestá related to development of a protective immune response by activating microbicidal mechanisms of macrophages to control infection by L. chagasi. / A Leishmaniose Visceral (LV) é uma doença grave e endêmica no Brasil, sendo mais prevalente em áreas urbanas. A patologia da LV está associada a uma menor produção de IFN-. por células T e ao aumento da produção de IL-10, que suprime a ativação dos macrófagos e promove o desenvolvimento da doença. Células T ativadas podem expressar CD40L que interagem com o CD40 expresso em células apresentadoras de antígeno (APC), essa interação é importante para a ativação de APCs e produção de citocinas inflamatórias, quimiocinas, óxido nítrico (NO) e metaloproteinases, desencadeando uma resposta protetora em modelos experimentais de LV. Dados do nosso grupo demostraram que pacientes com LV apresentam baixos níveis séricos de sCD40L antes do tratamento e que esses níveis aumentam no decorrer do mesmo, atingindo títulos próximos aos valores encontrados no controle endêmico, sugerindo um efeito protetor dessa molécula. Neste trabalho avaliamos a participação do sCD40L presente no soro na resposta microbicida de macrófagos infectados por Leishmania chagasi. Para isso, macrófagos humanos obtidos a partir de PBMC foram infectados com promastigotas de Leishmania chagasi na presença de soro contendo altos títulos de sCD40L, com e sem adição de anticorpo de bloqueio, anti-CD40L. Avaliamos a resposta microbicida a partir do número de macrófagos infectados e a quantidade de parasitos intracelulares. Observamos que houve uma redução de 43% do percentual de macrófagos infectados e de 58% do número de parasitos intracelulares quando comparado ao meio (p=0.01), sendo esse efeito foi revertido com o bloqueio do sCD40L, pois ocorreu um aumento de 24% do percentual de macrófagos infectados e de 33% do número de amastigotas. A partir dos resultados obtidos concluimos que o sCD40L está relacionado ao desenvolvimento de uma resposta imunológica protetora por ativar mecanismos microbicidas dos macrófagos para o controle da infecção por L. chagasi.

In Brazil leishmaniasis reach 19 states, and more than 90% of human cases of the disease are concentrated in Northeast region, there is still important regions foci in the Midwest, North and Southeast regions. Studies point for the occurrence of about 20.000 new cases annually of the disease. The need for new drugs more effective, safe and accessible for the treatment of leishmaniasis, visceral in particular, later on, affects liver, spleen, reticuloendothelial system, bone marrow and lymph nodes becomes relevant. Furthermore, according to the World Health Organization, the plant species are the best and major source of drugs for humanity and Brazil has 60.7% of its territory of natural and planted forests, representing the second largest forest area in the world, only behind from Russia. Thus, this study aimed investigated the activities of the essential oil of Cymbopogon citratus and Citral against Leishmania (L.) chagasi. In order for this to happen, oil from fresh leaves were harvested on the Mother Earth farm, located in Santana do Sao Francisco/SE. The Citral was acquired from Sigma-Aldrich (Darmstadt, Germany). Gaseous Chromatography and Detections by Flame Ionization were carried to identify its chemical constituents, just like an evaluation of inhibitory concentrations 50% in promastigotes and amastigotes in Leishmania mentioned beforehand, cytotoxicity assays, nitric oxide production and fluorescence to detect possible increase of membrane permeability in the presence of Citral were developed. Pharmacological activities were found in promastigotes (CI50/48 hours 25 Êg/mL for oil and 30 Êg/mL for Citral), however, activities were not found in amastigotes. The cytotoxicity (CI50 was 26.25 Êg/mL for oil and 33.96 Êg/mL for Citral). As for the increased production of nitric oxide, this did not occur, however, pore formation was observed in cell membranes of promastigotes in the presence of Citral. Therefore, the essential oil of Cymbopogon citratus and Citral may come to be used as preventive measure against visceral leishmaniasis. Since the promastigotes are the ways that infect vertebrates, including man. / No Brasil as leishmanioses atingem 19 estados, sendo que mais de 90% dos casos humanos da doenca concentram-se na regiao Nordeste, havendo ainda focos importantes nas regioes Centro-Oeste, Norte e Sudeste. Estudos sinalizam para a ocorrencia de cerca de 20.000 casos anuais da doenca. A necessidade de novos farmacos mais eficazes, seguros e acessivel para o tratamento das leishmanioses, em especial a visceral, posto, acomete figado, baco, sistema reticuloendotelial, medula ossea e linfonodos torna-se relevante. De acordo com a Organizacao Mundial de Saude, as especies vegetais sao fontes de farmacos importantes para humanidade e o Brasil possui 60,7% do seu territorio de florestas naturais e plantadas, representando a segunda maior area florestal do mundo, atras apenas da Russia. Assim, esse estudo objetivou investigar as atividades do oleo essencial de Cymbopogon citratus e Citral contra Leishmania (L.) chagasi. Para tal, obteve-se o oleo das folhas fresca colhidas na fazenda Mae Terra, localizada em Santana do Sao Francisco/SE, ja o Citral foi adquirido da Sigma-Aldrich (Darmstadt, Germany). Foram realizadas suas Cromatografias Gasosas e Deteccoes por Ionizacao de Chama para identificar seus constituintes quimicos, bem como avaliacao das concentracoes inibitorias 50% nas promastigotas e amastigotas na Leishmania supracitada, ensaios de citotoxicidade, producao de oxido nitrico e fluorescencia para detectar possivel aumento da permeabilidade de membrana em presenca do Citral foram desenvolvidos. Atividades farmacologicas foram constatadas em promastigotas (CI50/48 horas 25 Êg/mL para oleo e 30 Êg/mL para Citral), entretanto, nao foi encontrada acao nas amastigotas. A citotoxicidade (CI50 foi de 26,25 Êg/mL para oleo e 33,96 Êg/mL para Citral). Quanto ao aumento na producao de oxido nitrico, essa nao ocorreu, todavia, foi observada formacao de poros nas membranas celulares nos promastigotas em presenca de Citral. Portanto, o oleo essencial de Cymbopogon citratus e Citral pode vir a ser usados como medida preventiva contra leishmaniose visceral. Uma vez que as promastigotas sao as formas que infectam os animais vertebrados, inclusive o homem.

La protéine découplante mitochondriale UCP2 est exprimée basalement dans différents tissus et le rôle de régulateur négatif de la production des espèces actives oxygénées (ROS) mitochondriales a été avancé. Les travaux présentés se sont intéressés à l'implication potentielle d'UCP2 dans le déroulement de la réponse immunitaire. Une induction de l'expression d'UCP2 est démontrée dans un modèle murin d'encéphalite par le virus Herpès simplex de type 1 (HSV-1) tout d'abord, puis dans un modèle parasitaire de leishmaniose causée par Leishmania donovani. Cette expression d'UCP2 a été observée dans les cellules immunitaires, colocalisée avec différents marqueurs inflammatoires exprimés à des niveaux élevés. Le modèle d'infection virale a montré un délai entre la mise en place de la réponse immunitaire innée et l'apparition d'UCP2 dans le cerveau, impliquant UCP2 dans les étapes tardives de la défense antivirale. A cette étape, la réplication du virus et la neuroinflammation sont au maximum, expliquant la susceptibilité des souris déficientes en UCP2. De plus, l'invalidation des gènes codant pour TNF-a et/ou IL-ip sensibilise au virus HSV-1 les souris naturellement résistantes et modifie le profil d'expression d'UCP2. Dans le modèle parasitaire, l'invalidation du gène codant UCP2 n'a pas permis de diminuer la croissance des parasites dans la première phase de la leishmaniose. Ainsi la protéine UCP2 ne serait pas impliquée dans les étapes précoces de mise en place de la défense antiparasitaire; les tactiques des parasites pour inhiber la réponse de l'hôte pourraient expliquer l'absence de différences nettes liées à la perte d'UCP2. La dernière étude portait sur la chronologie d'apparition d'UCP2 par rapport aux marqueurs neuroinflammatoires suite à l'injection intrastriatale de LPS. Dans ce modèle, une forte expression transitoire de TNF-a, bcBa et TLR2 est repérée dans la zone adjacente au site d'injection dès 6 h suivant l'injection puis diminue. UCP2 est seulement observable 48 h post injection dans les mêmes régions cérébrales. En conclusion, nous avons démontré dans 3 types de stimulations infectieuses de la réponse immunitaire que l'apparition d'UCP2 était liée avec un délai à l'expression de facteurs inflammatoires et UCP2 n'a semblée être qu'un marqueur. En effet, la fonction antioxydante tenue par UCP2 dans les étapes tardives de l'immunité semble modeste à la vue des résultats des souris déficientes en UCP2, mais ce rôle reste encore à préciser. / Mitochondrial uncoupling protein 2 (UCP2) is basally expressed in diverse tissues and a function of negative regulator of reactive oxygen species (ROS) production has been proposed. Studies presented in this thesis emphasize the potential role of UCP2 in immunity. Induction of UCP2 expression was demonstrated first in a murine model of encephalitis caused by Herpes simplex virus type 1 (HSV-1), then in a parasitic leishmaniasis model. UCP2 mRNA expression was observed in immune cells, colocalized with different inflammatory factors at high levels. Models of viral infection demonstrated that the transcriptional activation of UCP2 was delayed compared with the inflammatory response, involving UCP2 in the later stages of the antiviral response. At those later steps, viral replication and neuroinflammation were maximal, which could explain susceptibility in UCP2-deficient mice. Moreover, invalidation of the genes encoding TNFa and/or IL1B in resistant mice allowed HSV-1 for replicating in neurons and modified the distribution pattern of UCP2m RNA in the brain. In parasitic models, growth of parasites in UCP2- deficient mice was not impaired in the first phase of pathogenesis. Therefore UCP2 did not appear to be implicated in early steps of the antiparasitic response; parasites could develop strategies for inhibiting the host response, which would explain the similarity in the immune responses between UCP2-deficient and wild type mice. The last study addressed the chronology of UCP2 expression in relation with neuroinflammation following intrastriatal injection of LPS. In this model, strong and transitory expressions of TNFa, IkB and TLR2 were shown in ipsilateral region at 6h after injection. UCP2 was only expressed 48h post injection in the same cerebral region. In conclusion, we have demonstrated, in three models of infection triggering an immune response that the induction of UCP2 expression occurs in a coordinated but delayed manner with that of common inflammatory factors. UCP2 appears to be a marker of the severity of the immune response. It does not appear, based on the results obtained in UCP2-deficient mice, to play an essential role in the models of infection that we used. The role of UCP2 in those models remains to be folly elucidated.

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.