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Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-04-11T13:11:41Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Neuza B. Barros.pdf: 2710465 bytes, checksum: 4f522332291442b7edb66a372b6259ce (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-04-11T13:11:55Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Neuza B. Barros.pdf: 2710465 bytes, checksum: 4f522332291442b7edb66a372b6259ce (MD5) / Made available in DSpace on 2018-04-11T13:11:55Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Neuza B. Barros.pdf: 2710465 bytes, checksum: 4f522332291442b7edb66a372b6259ce (MD5) Previous issue date: 2017-12-04 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cutaneous leishmaniasis (CL) is endemic in several regions of the world. In Brazil, despite the wide variety of clinical manifestations, 75% of patients with CL display a typical clinical picture, characterized by one or more ulcers and high round edges with a central necrosis, usually in the lower extremities. Leishmania amazonensis is a major etiologic agent of a wide spectrum of clinical forms of leishmaniasis. The objective of this study was to investigate the in vitro and in vivo antileishmania amazonensis activity of phospholipase A2 (Asp49) isolated from Bothrops jararacussu venom encapsulated in liposomal system. The cytotoxicity of PLA2-Asp49-liposomal was determined by MTT method in J774 macrophages after 48h of incubation. Macrophages treated with PLA2-Asp49-liposomes had cell viability around 82% at the highest concentration (100 μg/mL), while promastigotes treated with the same concentration resulted 78% of inhibition. On the other hand, peritoneal macrophages from Balb/c mice infected and treated with PLA2-Asp49-liposomes showed a 28% reduction in phagocytic index and a 55% reduction (p <0.05) in the number of intracellular amastigotes after 48h of treatment compared to the control group. After the treatments, the supernatants of peritoneal macrophages were collected, and the production of TNF-α, IL-10 and nitric oxide was evaluated. It was observed that there was no IL-10 production by macrophages treated with PLA2-Asp49-liposomes when compared to the untreated control. However, this group presented significant levels of TNF-α and nitric oxide. After the physicochemical characterization of the liposomes it was observed that the size of the vesicles remained close to the diameter of the membrane used in the extrusion, with an average diameter of 205.2 nm for PLA2-Asp49-liposomes and 241.9 nm for the control liposomes. PLA2-Asp49 were also efficiently encapsulated in liposomes (69.6%). The molecular docking showed that lipids strongly interact with the active site of the enzyme PLA2-Asp49. The activity of the toxin-containing liposomes was evaluated in BALB/c mice, previously infected with 1x105 of the parasite's promastigotes. After six weeks of infection, treatments in different groups of animals were initiated and observed for 21 days. The size of the paw lesion in PLA2-Asp49-liposomal treated animals was observed as decreasing by approximately 18% relative to the untreated control group and the Glucantime®-treated animals, which was used as a reference drug. At the end of the treatment, the animals were sacrificed and the paw and lymph node tissues were collected. Part of the collection was used to recover amastigotes and another to quantify cytokines and nitrites. In the group treated with PLA2-Asp49-liposomes the parasitic load was observed to be reduced by 73.5% in the macerated lymph node, compared to the control group. Comparatively, in the paw tissue was observed a reduction of 57.12%. The infected groups treated with PLA2-Asp49-liposomes showed significant production in TNF-α (45 and 80 pg/mL) and nitrite levels (32 and 36 μM), respectively, in lymph node and paw tissues, compared to infected untreated groups. The results indicate that the liposomal system containing PLA2-Asp49 is a promising biotechnological tool to confer an antileishmania activity in infected macrophages. / A leishmaniose cutânea é uma doença endêmica em várias regiões do mundo. No Brasil, 75% dos pacientes com a doença exibem uma forma clínica típica, caracterizada por uma ou mais úlceras de bordas redondas e elevadas com um centro necrótico. Leishmania amazonensis é um dos principais agentes etiológicos de formas clínicas da leishmaniose. O objetivo do presente trabalho foi o de investigar os efeitos in vitro e in vivo da potencial atividade antileishmania amazonensis da Fosfolipase A2 (PLA2) da peçonha de Bothrops jararacussu encapsulada em sistema lipossomal. O método de MTT foi utilizado para avaliação da citotoxicidade da PLA2-Asp49-lipossomal em macrófagos J774 após 48h de incubação. Os macrófagos tratados com a PLA2-Asp49-lipossomal apresentaram viabilidade celular em cerca de 82% na maior concentração utilizada (100 μg/mL). No ensaio com as formas promastigotas tratadas com a mesma concentração da PLA2-Asp49-lipossomal ocorreu uma inibição de 78% dos parasitos. Para o teste de infecção macrófagos peritoneais de camundongos Balb/c, infectados e incubados com PLA2-Asp49-lipossomal apresentaram redução de 28% no índice fagocítico e de 55% (p < 0.05) na quantidade de amastigotas intracelulares, após 48h de tratamento, comparado ao grupo controle. Os sobrenadantes de cultura dos macrófagos peritoneais foram coletados para a avaliação da produção de citocinas (TNF-α e IL-10) e de óxido nítrico. Não houve produção de IL-10 pelos macrófagos tratados com o PLA2-Asp49-lipossomal in vitro. Entretanto, o grupo tratado com PLA2-Asp49-liposomal produziu níveis significativos de TNF-α e óxido nítrico. Após a caracterização físico-química dos lipossomas foi observado que o tamanho das vesículas permaneceu próximo ao tamanho da membrana utilizada na extrusão, com diâmetro médio entre 205,2 nm para PLA2-Asp49-liposomal e 241,9 nm para os lipossomas controle. A PLA2-Asp49 foi eficientemente encapsulada nos lipossomas (69,6%). A atividade dos lipossomas contendo a toxina foi avaliada em camundongos BALB/c, previamente infectados com 1x105 promastigotas do parasito. Após seis semanas foi iniciado o tratamento com as preparações dos diferentes grupos de animais, acompanhados por 21 dias. Foi observado nos animais tratados com PLA2-Asp49-lipossomal diminuição de aproximadamente 18% do tamanho da lesão das patas, em relação ao grupo controle sem tratamento e aos animais tratados com o fármaco referência (Glucantime®). Ao término do tratamento, os animais foram mortos, os tecidos das patas e linfonodos foram coletados para recuperação de amastigotas e quantificação de citocinas e nitritos nos sobrenadantes destes tecidos. No macerado do linfonodo, no grupo tratado com PLA2-asp49-liposomal observou-se redução da carga parasitária de 73,5%, comparado com o grupo controle. Ainda nessa comparação, no macerado do tecido da pata houve redução de 57,12% dos números de amatigotas transfomadas para promastigotas. Os grupos infectados e tratados com PLA2-Asp49-lipossomal apresentaram aumentos significativos, respectivamente, nos tecidos dos linfonodos e patas, nos níveis de IL-12 (1.200 e 400 pg/mL), fator de necrose tumoral (TNF-α) (50 e 100 pg/mL), e de nitritos (32 e 36 μM), quando comparados aos grupos infectados e sem tratamento. Os resultados sugerem que o sistema lipossomal contendo PLA2-Asp-49 é uma ferramenta biotecnológica promissora para conferir atividade antileishmania a macrófagos infectados, aumentando seus mecanismos celulares microbicidas e auxiliando para o estabelecimento de uma resposta imunológica mais eficiente.

1 ABSTRACT B-cell non-Hodgkin lymphomas (B-NHL) represent the most common mature lymphoproliferative diseases. B-NHL arise at different stages of B-cell development and represent their malignant counterpart. Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are aggressive types of B-NHLs. Deregulation of cell cycle control, inhibition of apoptosis or abnormal DNA damage response play a key role in the pathogenesis of DLBCL and MCL. Aberrant activation of several signaling pathways that further promote survival, cell proliferation or affect the tumor microenvironment have been recently recognized. Increased understanding of the oncogenic mechanisms implicated in pathogenesis of B-NHL lead to development of novel agents that target the oncogenic drivers of distinct lymphoma subtypes. MCL is an aggressive subtype of B-NHL associated with poor prognosis. In vivo models of human MCL for experimental therapy are however scarce. We established and characterized several mouse models of human MCL by xenotransplantation of either primary cells or established cell lines into immunodeficient mice (publication no 1). We demonstrated that engrafted MCL cells displayed complex changes of gene expression profile, phenotype and sensitivity to cytotoxic agents compared to the original in vitro growing...

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