Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-01-10T11:15:29Z No. of bitstreams: 1 lilypaolamartinezabad.pdf: 2623487 bytes, checksum: bd4d6d3010f0286720ab5bcfb393b5ea (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2017-01-31T11:20:52Z (GMT) No. of bitstreams: 1 lilypaolamartinezabad.pdf: 2623487 bytes, checksum: bd4d6d3010f0286720ab5bcfb393b5ea (MD5) / Made available in DSpace on 2017-01-31T11:20:52Z (GMT). No. of bitstreams: 1 lilypaolamartinezabad.pdf: 2623487 bytes, checksum: bd4d6d3010f0286720ab5bcfb393b5ea (MD5) Previous issue date: 2016-09-30 / A leishmaniose visceral canina (LVC) representa um grave problema de saúde pública. No Brasil, a prevalência da infecção nos cães é bastante variável, podendo atingir níveis superiores a 60% em alguns surtos. O teste rápido Dual Path Platform (TRDPP®-Bio-Manguinhos), como teste de triagem, seguido por ELISA (EIE-BioManguinhos), como teste confirmatório, tornaram-se parte do protocolo de diagnóstico da LVC, credenciado no Brasil desde 2011. No entanto, o diagnóstico da LVC ainda precisa ser melhorado para alcançar uma taxa de detecção mais precisa. Recentemente, rKLO8, uma nova proteína antigênica de L. donovani do Sudão foi clonada e purificada, e mostrou alta reatividade para diagnosticar leishmaniose visceral em humanos. O presente estudo teve como objetivo avaliar a reatividade de soros de cães frente ao antígeno rKL08 e o antígeno de referência rK26, comparando ambas as proteínas, utilizadas como antígenos em testes de ELISA, com os testes DPP® e EIE, usados como testes de diagnóstico da LVC. Amostras de soros de cães de Governador Valadares, uma área endêmica para leishmaniose em Minas Gerais, Brasil, foram agrupadas da seguinte forma: (I) DPP®/EIE negativo (n = 100), (II) DPP® positivo / EIE negativo e (III) DPP® / EIE positivo (n = 100). Níveis séricos elevados de IgM e IgG para ambos os antígenos, rKLO8 e rK26, foram encontrados no grupo III (p <0,0001). Interessantemente, foram detectados níveis elevados de IgG2 e baixos níveis de IgG1 contra ambos os antígenos no grupo de cães DPP®/EIE positivo, sugerindo a ocorrência de um fenótipo predominantemente do tipo Th1 associado com infecção subclínica. O ELISA-rKLO8 (IgG) e o ELISA-rK26 (IgG) mostraram uma sensibilidade de 68% e 77%, e especificidade de 92% e 91%, respectivamente, determinado através da análise da curva ROC. Além disso, o coeficiente Kappa indicou boa concordância (0,739) entre o ELISA-rKLO8 versus o ELISA-rK26. Ainda, a combinação de antígenos rKLO8 e rK26 (rKLO8+rK26) em um mesmo teste exibiu maior sensibilidade (85%) e especificidade (93%). A análise kappa mostrou que o ELISA-rKLO8 + rK26 (IgG) teve melhor concordância com ambos os testes, DPP® e EIE, com valores de kappa igual a 0,700. Estes dados indicaram que a combinação dos antígenos rKLO8 e rK26 gera uma melhor acurácia no diagnóstico da LVC que os antígenos rKLO8 e rK26 usados em separado na detecção de IgG. Estes resultados demonstraram, pela primeira vez, a utilidade do antígeno rKLO8 no diagnóstico da LVC, e que ELISA-rKLO8, pode representar uma potencial ferramenta adicional para o diagnóstico de LVC. / Canine Visceral Leishmaniasis (CVL) represents a serious public health issue. In Brazil, the prevalence of infection in dogs is quite variable and may reach levels above 60% in some outbreaks. The dual Path Platform (DPP®-Bio-Manguinhos) as quick screening test followed by ELISA (EIE-Bio-Manguinhos) as a confirmatory test became part of the diagnostic protocol of CVL, nationally accreditated in Brazil since 2011. However, CVL diagnosis still needs to be improved to achieve a more accurate detection rate. Recently, rKLO8, a new antigenic protein of Sudanese L. donovani, was cloned and purified and had high reactivity to diagnose human VL. The present study aimed to evaluate serum reactivity to rKL08 and to the reference antigen rK26, and to compare both diagnostic proteins used in ELISA with the combined DPP® and EIE as diagnostic tests of CVL. Dog sera samples from Governador Valadares, an area endemic for leishmaniasis in Minas Gerais, Brazil, were grouped in the following way: (I) DPP®/EIE negative (n = 100), (II) DPP® positive/EIE negative and (III) DPP®/EIE positive dog sera (n = 100). Enhanced serum levels of IgM and IgG to both rKLO8 and rK26 were found in group III (p<0.0001). Interestingly, high IgG2 and low IgG1 levels against both antigens were detected in DPP®/EIE positive dogs, suggesting the occurrence of a predominant Th1 phenotype associated with subclinical infection. The rKLO8-ELISA (IgG) and the rK26-ELISA (IgG) showed a sensitivity of 68% and 77% and specificity of 92% and 91%, respectively, determined by ROC curve analysis. In addition, Kappa coefficient indicated good agreement (0.739) between rKLO8-ELISA and rK26-ELISA. Moreover, the combination of rKLO8 and rK26 antigens (rKLO8+rK26) exhibited higher sensitivity (85%) and specificity (93%). Kappa analysis established that rKLO8+rK26-ELISA (IgG) had better agreement with both DPP® and EIE, with kappa values of 0.700. These data indicate that the combination of rKLO8 and rK26 antigens has better accuracy in the diagnosis of CVL than rKLO8 and rK26 used separately at detecting IgG. These results showed for the first time the usefulness of rKLO8 antigen in the diagnosis of CVL, and that rKLO8-ELISA may represent a potential additional tool for the diagnosis of CVL.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-11T18:26:05Z No. of bitstreams: 1 patriciadealmeidamachado.pdf: 4548121 bytes, checksum: f773fa1c31a3db1b43310dcfaf89e879 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T15:22:16Z (GMT) No. of bitstreams: 1 patriciadealmeidamachado.pdf: 4548121 bytes, checksum: f773fa1c31a3db1b43310dcfaf89e879 (MD5) / Made available in DSpace on 2017-05-17T15:22:16Z (GMT). No. of bitstreams: 1 patriciadealmeidamachado.pdf: 4548121 bytes, checksum: f773fa1c31a3db1b43310dcfaf89e879 (MD5) Previous issue date: 2017-02-20 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As leishmanioses são doenças causadas por protozoários do gênero Leishmania, causando grande impacto na saúde pública mundial. O tratamento é limitado por uma série de fatores e a necessidade de alternativas para a quimioterapia é urgente. Assim, este trabalho teve como objetivo avaliar a atividade leishmanicida de bioisósteros do resveratrol e seus complexos metálicos e estudar o mecanismo de morte do parasito associado ao tratamento. Foram testados doze biosósteros do resveratrol, sendo cinco não complexados a metais, quatro complexados a ouro e três complexados a vanádio, em formas promastigotas e amastigotas de L. amazonensis, L. braziliensis e L. major e em macrófagos peritoneais. Para o teste em promastigotas e em macrófagos peritoneais, a viabilidade celular foi avaliada pelo método colorimétrico do MTT, enquanto no teste em amastigotas, o efeito dos compostos foi avaliado pela contagem das formas intracelulares. Foi ainda avaliada a seletividade e a especificidade dos compostos, através do cálculo do índice de seletividade e do índice de especificidade de cada composto. Os compostos complexados a metais, comparados aos não complexados, apresentaram significante efeito em promastigotas e amastigotas de Leishmania sp., e ainda foram seletivos ao parasito, em comparação com a célula hospedeira. Posteriormente, baseado na síntese, efetividade e seletividade, o composto 11 (VOSalophen), um complexo de vanádio, foi selecionado para estudos sobre mecanismos de morte e atividade em modelo de leishmaniose cutânea murina. Promastigotas de L. amazonensis tratadas com VOSalophen apresentam alterações mitocondriais, observados por meio das marcações com JC-1, Mitotracker® Red CM-H2XROS e rodamina 123 e da microscopia eletrônica de transmissão (MET); aumentam a produção de EROs (utilizando H2DCFDA), acumulam corpos lipídicos (marcação com Nile Red), apresentam alterações morfológicas, mas não alteram a integridade da membrana plasmática, visto não haver marcação com iodeto de propídeo. Além disto, os parasitos apresentam redução no tamanho celular e volume celular; externalização de fosfatidilserina na face externa da membrana plasmática do parasito (usando marcação com anexina V-FITC) e utilizando a técnica do TUNEL foi observada a fragmentação do DNA. Em amastigotas intracelulares de L. amazonensis, também foi verificada fragmentação do DNA (técnica TUNEL). O conjunto destes resultados sugere morte por apoptose-like. Além disso, foi verificado um aumento de formação de vacúolos autofágicos em promastigotas de L. amazonensis tratados com o VOSalophen (marcação com MDC e MET), sugerindo a ocorrência de autofagia. Em macrófagos infectados com L. amazonensis, VOSalophen induz um aumento de produção de ON e EROs, indicando modulação celular do composto em células infectadas. Em modelo de leishmaniose cutânea murina, o tratamento com o VOSalophen causou significativa redução da carga parasitária nas patas de camundongos BALB/c infectados com L. amazonensis, quando comparado ao controle e além disso, não mostrou toxicidade renal e hepática aos animais tratados, o que foi avaliado através das dosagens de AST, ALT, GGT e creatinina. Esses resultados, em conjunto, mostram o significante efeito in vitro e in vivo de bioisósteros do resveratrol complexados a metais. / Leishmaniasis is diseases caused by protozoa of the genus Leishmania, causing significant impact on global public health. The treatment is limited by a number of factors, and the need for alternatives to chemotherapy is urgent. Thus, this study aimed to evaluate the leishmanicidal activity of bioisosters of resveratrol and its metal complexes and to study the mechanism of parasite death associated with the treatment. Twelve biosósteros of resveratrol were tested, five not complexed to metals, four complexed to gold and three complexed to vanadium, in promastigotes and amastigotes of L. amazonensis, L. braziliensis and L. major and in peritoneal macrophages. In promastigotes and peritoneal macrophages, the cell viability was assessed by the MTT colorimetric method, while in amastigotes, the effect of the compounds was evaluated by counting the intracellular forms. It was also assessed the selectivity and specificity of the compounds, by calculating the selectivity index and the specificity index of each compound. The compounds complexed to metals, compared to non-complexed, showed a significant effect on promastigotes and amastigotes of Leishmania sp., and were further selective to the parasite compared to the host cell. Subsequently, based on the synthesis, effectiveness and selectivity, the compound 11 (VOSalophen), a vanadium complex, was selected for studies on the mechanisms of death and activity in the murine model of cutaneous leishmaniasis. L. amazonensis promastigotes treated with VOSalophen exhibit mitochondrial alterations, which was observed by JC-1, Mitotracker® Red CMH2XROS e rhodamina 123 staining and transmission electronic microscopy (TEM); increased ROS production (using H2DCFDA); accumulate lipid bodies (Nile Red staining), exhibit morphological changes, but did not alter the integrity of the plasma membrane since no staining with propidium iodide. In addition, the parasites had a reduction in cell volume and size; phosphatidylserine externalization on the outer face of parasite's plasma membrane (annexin V-FITC staining) and using the TUNEL technique, DNA fragmentation was observed. In L. amazonensis intracellular amastigotes, DNA fragmentation was also observed (TUNEL technique). This set of results suggests death by apoptosis-like. Furthermore, there was an increase of autophagic vacuole formation in L. amazonensis promastigotes treated with VOSalophen (MDC staining and TEM), suggesting the occurrence of autophagy. In macrophages infected with L. amazonensis, VOSalophen induces an increase of nitric oxide and reactive oxygen species production, indicating a modulatory effect in infected cells. In the murine model of cutaneous leishmaniasis, the treatment with VOSalophen caused a significant reduction in the parasite load in the paws of BALB/c mice infected with L. amazonensis, when compared to control and furthermore not showed renal and hepatic toxicity to the treated animals, which was evaluated by the AST, ALT, GGT and creatinine dosages. These results, taken together, show the significant effect of bioisosters of resveratrol complexed to metals.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-18T13:19:42Z No. of bitstreams: 1 josegeraldodecastrojunior.pdf: 7182456 bytes, checksum: 31d33bc1bedcad933b465924da4098e3 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-18T13:40:17Z (GMT) No. of bitstreams: 1 josegeraldodecastrojunior.pdf: 7182456 bytes, checksum: 31d33bc1bedcad933b465924da4098e3 (MD5) / Made available in DSpace on 2017-05-18T13:40:17Z (GMT). No. of bitstreams: 1 josegeraldodecastrojunior.pdf: 7182456 bytes, checksum: 31d33bc1bedcad933b465924da4098e3 (MD5) Previous issue date: 2013-12-04 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / No Brasil, a leishmaniose visceral (LV), também conhecida como calazar, é uma zoonose, de transmissão vetorial, causada pelo protozoário Leishmania chagasi. Esta doença, anteriormente descrita como rural vem passando por um processo de urbanização. Em 2008, foram diagnosticados no município de Juiz de Fora, os primeiros casos considerados autóctones de leishmaniose visceral canina (LVC) e estudos sobre epidemias urbanas da LV têm indicado que a LVC vem precedendo a infecção humana, visto que os cães são os principais reservatórios domésticos. Assim, o presente estudo teve como objetivo pesquisar a infecção de LVC no município de Juiz de Fora, bem como avaliar os fatores de risco associados à doença. O trabalho foi realizado com animais do canil municipal e ONGs e a partir do soro destes foram realizadas três técnicas sorológicas: o imunocromatográfico “TR DPP® e ELISA (“EIE-Leishmaniose visceral canina®), ambos fornecidos pela FIOCRUZ/Bio-Manguinhos e ELISA in house. A amostra totalizou 781 animais e a prevalência da LVC variou de acordo com a técnica empregada: o teste DPP apresentou soropositividade de 4,87% (IC 95% de 3,5-6,7%); o ELISA Bio-Manguinhos de 2,18% (IC 95% de 1,3-3,5%) e ELISA in house de 13,73% (IC 10,8-17,3%). Em relação à variabilidade observada entre as técnicas, vale a pena destacar que as mesmas apresentam antígenos diferentes e isto pode refletir nos resultados. A amostra foi composta, em sua maioria por fêmeas adultas, sem raça definida, porte médio e pelo curto. Na análise univariada, utilizando-se o ELISA Bio-Manguinhos como confirmatório para a LVC, foi observada associação estatística (p< 0,05; IC 95%) com sintomatologia clínica segundo Quinnell et al. 2003, origem dos animais (canil municipal) e grupo racial; além disto, houve sugestão de associação com sexo masculino e porte médio/grande dos animais. Na análise multivariada, o fato de ser procedente do canil e sintomatologia clínica mantiveram associadas ao desfecho, sendo também sugestiva o sexo masculino. Este foi o primeiro inquérito da LVC no município de Juiz de Fora e a presença da doença relatada neste trabalho, reforça a idéia de que a leishmaniose está em processo de expansão e urbanização no Brasil, apontando a necessidade de vigilância epidemiológica ativa. / In Brazil, the visceral leishmaniasis (LV), also known as calazar, is a zoonotic disease, with vector transmission caused by the Leishmania chagasi protozoan. This disease, before described as rural is going through a process of urbanization. In 2008, were diagnosed in Juiz de Fora municipality, the first cases considered autochthones of canine visceral leishmaniasis (CVL) and research about urban epidemic of LV has showed that the CVL is preceding the human infection, since that the dogs are the main domestic reservoirs. Then, the present study aimed to investigate the CVL infection in Juiz de Fora, as well as to value the risk factors associated to the disease. This work was done with the animals of the public kennel and ONGs and with the serum were realized three serological techniques: the immunochomatographic “TR DPP® and ELISA (EIE-leishmaniose visceral canine®), both given by FIOCRUZ/BIO-MANGUINHOS and ELISA in house. The samples totalized 781 animals and the prevalence of CVL varied according with the technique used: the test DPP showed soropositivity of 4.87% (IC 95% of 3.5-6.7%); the ELISA Bio-Manguinhos of 2.18% (IC 95% of 1.3-3.5%) and ELISA in house of 13.73% (IC 10.8-17.3%). In relation to variability observed among the techniques, as worth pointing out that same showed different antigens and this can reflect in the results. The sampIes were compound in majority by adult females, without definite race, medium-sized and short hair. In the univarious analysis using the ELISA Bio-Manguinhos as confirmatory for the CVL, was observed statistic association (p<0.05; IC 95%) with clinic sintomatology according by Quinnell et al. (2003), origin of animals (public kennel) and racial group; and also, there was suggestion of the association with masculine sex and medium/big port animals. In the multivarious analysis, the fact of being precedent from the kennell and clinic sintomatology kept associated to the result, being suggestive the masculine sex. This was the first enquiry of CVL in Juiz de Fora municipality and presence of the disease showed in this research, reinforce the idea that the leishmaniasis is in the process of expansion and urbanization in Brazil, pointing out necessity of an active epidemiologic vigilance.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-17T11:16:52Z No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 16756474 bytes, checksum: 2304892df326fd4997c78bc7f0870bae (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-17T11:34:54Z (GMT) No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 16756474 bytes, checksum: 2304892df326fd4997c78bc7f0870bae (MD5) / Made available in DSpace on 2017-08-17T11:34:54Z (GMT). No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 16756474 bytes, checksum: 2304892df326fd4997c78bc7f0870bae (MD5) Previous issue date: 2017-02-15 / As leishmanioses representam um grande problema de saúde pública mundial com sérias limitações na quimioterapia atual, como: número limitado de fármacos, baixa eficácia, custo elevado, crescente resistência parasitária e toxicidade. O objetivo do presente trabalho foi avaliar a atividade de uma série de dez derivados de 4- aminoquinolinas em espécies de Leishmania relacionadas a leishmaniose tegumentar. Avaliar os possíveis mecanismos de ação do composto com atividade antileishmanial promissora, identificando as organelas alvo e os processos de morte celular desencadeados no parasito, bem como a sua eficácia in vivo. Dentre os compostos avaliados, o derivado AMQ-j foi o mais ativo, com atividade expressiva em L. amazonensis e L. braziliensis (CI50 menor que 6.0 μg/mL e 2,5 μg/mL em promastigotas e amastigotas intracelulares, respectivamente) para ambas as espécies avaliadas. O composto AMQ-j apresentou baixa toxicidade para macrófagos murinos (CC50> 40.0 μg/mL), sendo mais tóxico para amastigotas intracelulares (Índice de Seletividade>12,0). Os resultados preliminares acerca do modo de ação apontam que o composto AMQ-j induziu drásticos efeitos na mitocôndria do parasito e caracterizado pelo colapso do potencial de membrana mitocondrial (ΔΨm), inchaço da organela, aumento na produção de Espécies Reativas do Oxigênio (EROS) e acúmulo de corpúsculos lipídicos (CLs) no citoplasma. O tratamento com AMQ-j induziu nas formas promastigotas uma série de alterações bioquímicas e celulares sugestivas de morte por apoptose-like como redução do volume celular, exposição de fosfatidilserina no folheto externo da membrana plasmática, manutenção da integridade da membrana plasmática e alterações drásticas no núcleo celular evidenciadas por meio da desorganização da cromatina e fragmentação do DNA. O efeito do composto em promastigotas está também associado à indução de morte por autofagia evidenciada pelo aumento de vacúolos autofágicos, presença de corpos multivesiculares dentro de vacúolos, vesículas citoplasmáticas e acúmulo de compartimentos acídicos no citoplasma dos promastigotas. O composto também induziu fragmentação do DNA dos amastigotas intracelulares de modo seletivo, sem induzir fragmentação da célula hospedeira. Estudos in silico sugerem que AMQ-j é um potencial inibidor da tripanotiona redutase (TryR), enzima fundamental na defesa antioxidante do parasito. Estudos in vivo em modelo murino de infecção com L. amazonensis demonstraram a eficácia do AMQ-j pela via intralesional na redução do tamanho da lesão e da carga parasitária, sem indução de toxicidade hepática, cardíaca e renal. Os estudos de predição in silico relacionados a propriedades farmacocinéticas (ADMET) e características físico-químicas (regra de Lipinsky) sugerem que AMQ-j pode ser utilizado pela via oral. O efeito leishmanicida do composto está associado a múltiplos alvos, desencadeando a morte do parasito por diferentes vias, como apoptose e autofagia. O efeito in vivo do composto aponta para a necessidade da continuidade dos estudos no intuito de melhor estabelecer o seu efeito leishmanicida. / Leishmaniasis represents a major global public health problem with serious limitations in current chemotherapy, such as limited number of drugs, low efficacy, high cost, increasing parasitic resistance and toxicity. The objective of the present study was to evaluate a series of ten 4-aminoquinolines derivatives (AMQs) on Leishmania species related to tegumentary leishmaniasis. It was also evaluated the possible mechanisms of action of a compound with promising leishmanicidal activity, identifying the target organelles and the type of death triggered in the parasite, as well as to its leishmanicidal effect in vivo. Among the evaluated compounds, the AMQ-j derivative was the most active, with expressive activity on L. amazonensis and L. braziliensis (IC50 less than 6.0 μg/mL and 2.5 μg/mL against intracellular promastigotes and amastigotes, respectively) for both evaluated species. Furthermore, AMQ-j showed low toxicity for murine macrophages (CC50> 40.0 μg/mL) being more destructive to the intracellular parasites (selectivity index > 12.0). Preliminary studies about the mode of action showed that AMQ-j compound induced marked effects on the parasite mitochondria, characterized by mitochondrial membrane potential collapse (ΔΨm), organelle swelling, increased of Reactive Oxygen Species (ROS) production and lipidic bodies accumulation in the cytoplasm. The treatment with AMQ-j induced in the promastigote forms a series of biochemical and cellular alterations which suggest apoptosis-like death, including reduction of cellular volume, phosphatidylserine exposure on the outer leaflet of the plasma membrane, maintenance of plasma membrane integrity and drastic changes in the cell nucleus evidenced by chromatin disorganization and DNA fragmentation. The effect of AMQ-j in promastigote forms is also associated with the induction of death by autophagy evidenced by the increase of autophagic vacuoles, presence of multivesicular bodies inside vacuoles, cytoplasmic vesicles and accumulation of acidic compartments in the promastigote cytoplasm. The compound also induced DNA fragmentation of intracellular amastigotes selectively, without inducing host cell fragmentation. In silico studies suggest that this compound is a potential inhibitor of key redox enzyme trypanothione reductase (TryR). In vivo studies in murine infection model of L. amazonensis demonstrated the efficacy of AMQ-j by the intralesional route, reducing lesion size and parasite load, without induction of hepatic, cardiac and renal toxicity. The in silico prediction studies on pharmacokinetic properties (ADMET) and physico-chemical characteristics (Lipinsky's rule) suggest that AMQ-j can be used orally. Taken together, the results suggest that the leishmanicidal effect of the compound is associated with multiple targets and triggers parasite death through different pathways, including apoptosis and autophagy. The in vivo effect indicates that studies with this compound should be continued in order to better establish its leishmanicidal effect.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-04-04T18:08:27Z No. of bitstreams: 1 anacarolinaribeirogomesmaia.pdf: 687971 bytes, checksum: 19edf64a9349768df07f14662c3d6df6 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-04-05T11:27:48Z (GMT) No. of bitstreams: 1 anacarolinaribeirogomesmaia.pdf: 687971 bytes, checksum: 19edf64a9349768df07f14662c3d6df6 (MD5) / Made available in DSpace on 2018-04-05T11:27:48Z (GMT). No. of bitstreams: 1 anacarolinaribeirogomesmaia.pdf: 687971 bytes, checksum: 19edf64a9349768df07f14662c3d6df6 (MD5) Previous issue date: 2015-05-04 / PROQUALI (UFJF) / A NTPDase 1 (50 kDa) de promastigotas de Leishmania infantum foi purificada por eletroforese em gel não-desnaturante. A identidade desta proteína ou de seu domínio B conservado (r83-122) foi confirmada por “Western blots” utilizando soros imunes produzidos contra a apirase de batata (SA), r-potDomínioB (SB), um polipeptídeo recombinante derivado do domínio B desta proteína vegetal, e LbB1LJ (SC; r82-103) e LbB2LJ (SD; r102-121), peptídeos sintéticos derivados do domínio B da NTPDase 1 de L. braziliensis. Sua antigenicidade foi evidenciada em “Western blots” pela reatividade com soros de cães com leishmaniose visceral. Os soros imunes SC ou SD inibiram sua atividade (87-99%) em preparação de promastigotas, sugerindo um efeito direto sobre o domínio B. Por ELISA, 45-50% de 38 cães infectados foram soropositivos para LbB1LJ e LbB2LJ confirmando a antigenicidade deste domínio. / The NTPDase 1 (50 kDa) of Leishmania infantum promastigotes was purified by nondenaturing gel electrophoresis. The identity of this protein or of its conserved domain B (r83-122) was confirmed by Western blots using immune sera raised against potato apyrase (SA); r-potDomainB (SB), a recombinant polypeptide derived from the B domain of this vegetable protein, and LbB1LJ (SC; r82-103) and LbB2LJ (SD; r102-121), synthetic peptides derived from the B domain of the L. braziliensis NTPDase 1. Its antigenicity was evidenced in Western blots by the reactivity with serum samples from dogs with visceral leishmaniasis. The immune sera SC and SD inhibited its activity (87- 99%) in promastigotes preparation, suggesting a direct effect on the B domain. By ELISA, 45-50% of 38 infected dogs were seropositive for LbB1LJ and LbB2LJ confirming the antigenicity of this domain.

Ce travail de thèse a eu pour but la synthèse de nouveaux composés hétérocycliques (rhodanines et pyrazoles) potentiellement actifs sur des protéines kinases, des lignées cellulaires tumorales, les influx SOCE (Store Operated Calcium Entry) et a ciblé principalement comme pathologies la malaria, la leishmaniose et le cancer. La première partie de cette étude a permis la synthèse d'une nouvelle famille de dérivés 5-arylidène rhodanines dissymétriques comportant un bras espaceur diaminé et ce par l'intermédiaire de la technologie micro-onde. Sur les 7 protéines kinases testées avec ces composés, CK1δ/ε et CDK5/p25 ont été inhibées spécifiquement avec des CI50 comprises entre 1,1 et 10 µM. L'activité anticancéreuse enregistrée est moyenne avec des CI50 variant de 8 à 23 µM. Les travaux réalisés au cours de la seconde partie de cette étude se sont appuyés sur le SKF-96365 comme modèle structural et ont permis d'accéder à 3 librairies inédites d'analogues comportant les plateformes pyrazole et rhodanine. L'activité pharmacologique visée ici était une modulation des influx SOCE et diverses variations structurales ont été effectuées en vue de réaliser une étude Relation Structure-Activité (RSA). Plusieurs analogues "pyrazoles" ont montré une activité supérieure à celle du SKF-96365 et de la GSK-7975A sur les influx SOCE de la lignée HEK-293. Les 2 composés montrant la meilleure activité (30f et 30h), sont également plus actifs que la Synta 66 aux faibles concentrations. Ces analogues ont également une activité sélective des canaux SOCE concernés car totalement inactifs sur l'ensemble des protéines kinases testées. Les CI50 les plus significatives pour l'activité anticancéreuse varient entre 3 et 8 µM. / This thesis work has been aimed the synthesis of new heterocyclic compounds (rhodanines and pyrazoles) potentially active on kinase proteins, tumor cell lines, SOCE impulses (Store Operated Calcium Entry) and has mainly targeted pathologies such as malaria, leishmaniasis and cancer. The first part of this study allowed the synthesis of a new family of 5-arylidene rhodanine derivatives asymmetric having a diamino spacer arm and via microwave technology. Of the 7 protein kinases tested with these compounds, CK1δ/ε and CDK5/p25 have been specifically inhibited with IC50 between 1.1 and 10 µM. The recorded anticancer activity is average with IC50 ranging from 8 to 23 µM. The work carried out during the second part of this study was based on SKF-96365 as structural model and provided access to 3 unpublished libraries of analogs containing pyrazole and rhodanine platforms. The desired pharmacological activity was SOCE modulating and various structural changes were made to undertake a study Structure-Activity Relationship (SAR). Several "pyrazole" analogs have shown a higher activity than SKF-96365 and GSK-7975A on SOCE of HEK-293 line. The two compounds showing the best activity (30f and 30h) are also more active than Synta 66 at low concentrations. These analogs are completely inactive on all protein kinases tested, indicating selectivity for SOCE channels concerned. The most significant IC50 for the anticancer activity vary between 3 and 8 µM.

La leishmaniose viscérale (LV) est la forme la plus sévère de la leishmaniose humaine. Elle est transmise par la piqûre d'un phlébme. La leishmaniose viscérale est mortelle en l'absence de traitement. Les options thérapeutiques courantes contre la leishmaniose viscérale sont limitées. En région méditerranéenne, la LV est due à Leishmania infantum, zoonose dont le chien est le principal réservoir. A côté de quelques cas d'infection systémique, un nombre important d'humains porteurs asymptomatiques a été mis en evidence. En première partie, nous avons étudié l'intérêt du MultiLocus Microsatellite Genotyping (MLMT) pour l'identification des souches de Leishmania du sud de la France. Par MLMT nous avons étudié la variabilité génétique de différentes souches et recherché une association avec les différentes formes cliniques, la résistance aux médicaments et les phénomènes de rechutes. Nous avons observé une hétérogénéité génétique entre les différentes souches de L. infantum MON-1. Si l'association de certains génotypes avec les différentes expressions cliniques de la leishmaniose n'a pu être démontrée, nous avons par contre observé une répartition préférentielle géographique de certains génotypes. En deuxième partie, nous avons mis au point un protocole expérimental destiné au criblage de nouveaux agents anti-Leishmania infantum ayant pour cible la machinerie cellulaire mise en route par la cellule hôte pour l'élimination du parasite intracellulaire. Nos résultats ont montré que les altérations du système de trafic intracellulaire de la cellule-hôte induites par certains composés étaient corrélées à la mort du parasite et à son élimination. / Visceral leishmaniasis (VL) is the most severe form of Human Leishmaniases, which occurs when protozoan parasites Leishmania donovani or L. infantum, given by phlebotomine sandfly bites. The disease is fatal when untreated. Current treatment options against VL are very limited with few drug molecules, often expensive, not always safe and able to induce resistance phenomenon.In this report, we have characterized on one hand, different genetic variants of Leishmania infantum strains isolated in different geographical areas from southern France and on the other hand have identified new potential anti-Leishmania infantum compounds and characterized their molecular mechanism of action.In the first part, we studied the interest of Multilocus Microsatellite Genotyping (MLMT) for the identification of Leishmania strains from southern France. By genotyping technique MLMT, we studied the genetic variability of different strains and sought an association with different clinical forms of leishmaniasis, resistance to drugs and relapse. We observed genetic heterogeneity among different strains of L. infantum-MON-1. we observed a preferential geographic distribution of certain genotypes.In the second part, we have developed an experimental protocol for the screening of new anti-Leishmania infantum compounds that target the host cell machinery responsible for the intracellular parasite killing, we studied the different steps of endocytic pathways potentially targeted by these compounds. Our results showed that with some compounds, modifications of the intracellular trafficking of the host cell were correlated with parasite death and its elimination.

La communauté Rurale de Mont-Rolland (région de Thiès, Sénégal) est un foyer endémique de leishmaniose canine décrit depuis 1970. Des études épidémiologiques récentes ont montré que l'agent étiologique est Leishmania (Leishmania) infantum et que le cycle de transmission est bien établi sur l'ensemble de la commune, avec plus de 30% des chiens et plus de 20% des hommes séropositifs. Néanmoins, malgré les différentes études épidémiologiques, le vecteur reste toujours inconnu. Jusqu'à présent, aucune espèce de Phlebotomus, connue pour être vecteur de L. infantum (sous genre Larroussius et secondairement Adlerius), n'a été identifiée au Sénégal. Dans ce contexte, l'objectif principal de cette thèse était d'identifier les vecteurs potentiels de la maladie et de caractériser le cycle de transmission de la leishmaniose canine par des études écologiques, parasitologiques et moléculaires des populations de phlébotomes. Ainsi, 7442 spécimens de phlébotomes ont été récoltés par piégeage adhésif, piégeage lumineux et par pulvérisations intradomiciliaires d'insecticides pyréthrinoïdes. Neuf espèces de phlébotomes ont été identifiées : deux appartiennent au genre Phlebotomus, P. duboscqi (vecteur de la leishmaniose cutanée au Sénégal) et P. rodhaini, les autres espèces appartiennent au genre Sergentomyia, S. adleri, S. clydei, S. antennata, S. buxtoni, S. dubia, S. schwetzi, et S. magna. La distribution spatiale des phlébotomes est hétérogène selon le village, selon l'écosystème et l'environnement de capture. Les études parasitologiques et les études moléculaires (PCR diagnostique de Leishmania) ont révélé trois espèces de Sergentomyia infectées par L. infantum, S. dubia (dissection et PCR), S. schwetzi (dissection et PCR) et S. magna (PCR). Il faut noter que plus de 2% des individus femelles à jeun testées étaient PCR positives, soulignant la survie des parasites chez ces espèces après la digestion. Toutes les autres espèces étaient négatives aussi bien en dissection qu'en PCR. Les analyses statistiques ont montré : que la distribution de S. dubia est significativement associée à la séroprévalence chez les chiens, que les individus de S. schwetzi positifs en PCR sont significativement associés à la séroprévalence chez le chien, qu'il y a un effet significatif du nombre de phlébotomes positifs en PCR (les 3 espèces confondues) sur la séroprévalence chez l'homme. Toutes ces données démontrent pour la première fois que 3 espèces du genre Sergentomyia, S. dubia, S. schwetzi et probablement S. magna, sont les vecteurs de la leishmaniose canine dans le foyer de Mont-Rolland. Ces résultats remettent en question le dogme qui stipule que seul le genre Phlebotomus serait capable de transmettre le parasite Leishmania dans l'Ancien-Monde. L'étude du comportement et de la distribution de ces trois espèces et de leurs spécimens infectés nous ont permis de proposer un modèle de transmission dans ce foyer. / AbstractThe rural community of Mont-Rolland (Thiès, Sénégal) is an endemic focus of canine leishmaniasis described since 1970. Recent epidemiological studies showed that the causative agent is Leishmania infantum with well established transmission cycle throughout the area. However, despite the several epidemiological studies, the vector species is still unknown. So far, no species of Phlebotomus belongs to Larroussius and Adlerius subgenera, known as vector of L. infantum, was identified in Senegal. In this context, the main objective of this thesis was to identify potential vectors of the disease and to characterize the cycle of canine leishmaniasis transmission by ecological, parasitological and molecular studies on sandfly populations. Thus, 7442 specimens were collected by sticky traps, light traps and indoor spraying with pyrethroid insecticide from different environments (intradomiciliary, peridomiciliary, culture area, isolated area) and different villages and ecosystems (sandy soils, lateritic gravel soils and sandy clay soils). Nine species of sand flies were identified; two belong to the Phlebotomus genus, P. duboscqi (vector of cutaneous leishmaniasis in Senegal) and P. rodhaini, the other species belong to the Sergentomyia genus, S. adleri, S. clydei, S. antennata, S. buxtoni, S. dubia, S. schwetzi, and S. magna. Two species of the Phlebotomus genus were significantly underrepresented (292 specimens) compared to species of the Sergentomyia genus (7150 specimens). The spatial distribution of sand flies is heterogeneous depending on the village, the ecosystem and environment, suggesting that several species are capable of transmitting the parasite. Parasitological and molecular studies (diagnostic PCR of Leishmania) revealed that three species of Sergentomyia were infected with L. infantum, S. dubia (dissection and PCR), S. schwetzi (dissection and PCR) and S. magna (PCR). It is worth noting that more than 2% of un-fed females of these species were PCR-positive, emphasizing the survival of parasites after digestion. All other species were found to be negative in both PCR and dissection. In addition, statistical analysis showed that 1) the distribution of S. dubia is significantly associated with seroprevalence in dogs, 2) PCR-positive individuals of S. schwetzi are significantly associated with seroprevalence in dogs, 3) there is a significant effect of PCR-positive sand flies (all species considered) on seroprevalence in humans. All these data demonstrated for the first time that three species of Sergentomyia, S. dubia, S. schwetzi and probably S. magna, are the vectors of the canine leishmaniasis in the Mont-Rolland commune. The results presented in this study challenge the dogma which states that only the Phlebotomus genus would be capable of transmitting Leishmania parasites in the Old World. The behaviour and distribution of these three species and their infected specimens allowed us to propose a model of transmission in the focus.

Orientadora: Profa. Dra. Márcia Aparecida Sperança / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, São Bernardo do Campo, 2018. / A leishmaniose compreende um grupo de doenças zoonóticas causadas por protozoários da família Trypanosoma e do gênero Leishmania, sendo transmitida por vetores flebotomíneos. O género Leishmania compreende 30 espécies, incluindo 20 espécies capazes de causar doença em humanos, com diferentes manifestações clínicas, que vão desde lesões assintomáticas, cutânea e mucocutânea, até a forma visceral grave, que é letal se não tratada. No continente americano, a leishmaniose visceral (LV) é causada por Leishmania infantum chagasi e o Brasil concentra 90% dos casos registrados na América Latina. O diagnóstico da LV na América apresenta dificuldades devido a reações cruzadas com outras tripanossomíases endêmicas. Além disso, os sinais clínicos e as alterações hematológias da LV são encontrados em outras doenças infecciosas e em alterações malignas hematológicas. Com o intuito de buscar ferramentas e estratégias para o diagnóstico diferencial da LV, neste trabalho foram desenvolvidos métodos de isolamento e detecção molecular de L. infantum chagasi, além da busca por características demográficas específicas e avaliação do diagnóstico diferencial da LV em aspirados de medula óssea de pacientes com sintomas de doença hematológica da região de Marília-SP, no período de 2010 a 2017. O método de isolamento de L. infantum chagasi foi realizado a partir de amostras de medula óssea e sangue periférico de indivíduos sintomáticos. O diagnóstico molecular por reação em cadeia da polimerase, convencional, foi desenvolvido utilizando como alvo o gene de cópia única que codifica a enzima quitinase de L. infantum, o qual permite o diagnóstico específico e direto a partir de diferentes amostras clínicas. Os testes apresentaram elevada especificidade e mostraram sensibilidade aumentada quando comparados com métodos utilizados rotineiramente nos serviços de saúde. Os resultados da análise demográfica e do diagnóstico diferencial a partir de aspirados de medula óssea de pacientes com suspeita de doenças hematológicas indicaram que 19,4% apresentaram LV, e que a população com maior risco possui menos de 20 anos. O estudo realizado demonstra que a investigação de LV em pacientes com alterações hematológicas é fundamental não só para identificar casos de LV, mas também para tratar corretamente os casos de doenças infecciosas e hematológicas associadas. / Leishmaniasis covers a group of zoonotic diseases caused by protozoa parasites of the Trypanosoma Family and the genus Leishmania, being transmitted by phlebotomine insect vectors. The genus Leishmania comprises 30 species, including 20 species capable of causing disease in humans, with different clinical manifestations, ranging from asymptomatic, cutaneous and mucocutaneous lesions, to severe visceral form, which is lethal if untreated. In the American continent, visceral leishmaniasis (VL) is caused by Leishmania infantum chagasi and Brazil accounts for 90% of the Latin America registered cases. The diagnosis of VL in America presents difficulties due to cross-reactions with other endemic trypanosomiasis. In addition, clinical signs and VL hematological alterations are found in other infectious diseases and hematological malignancies. In order to search for tools and strategies for the differential VL diagnosis, in this work we have developed methods of isolation and molecular detection of L. infantum chagasi, as well as the search for specific demographic characteristics and evaluation of the differential diagnosis of VL in bone marrow biopsies of patients with symptoms of haematological diseases in the region of Marília-SP, from 2010 to 2017. The method of L. infantum chagasi isolation was performed from samples of bone marrow and peripheral blood of symptomatic individuals. Conventional polymerase chain reaction (PCR) was developed using the single copy gene encoding the chitinase enzyme of L. infantum, which allows specific and direct diagnosis from different clinical samples. Both diagnostic tests showed high specificity and increased sensitivity when compared with routinely used methods in health services. The results of the demographic analysis and the differential diagnosis from bone marrow biopsy of patients with supposed hematological diseases indicated that 19.4% had VL, and that the population with the highest risk is less than 20 years old. The present study demonstrates that the investigation of VL in patients with hematological alterations is fundamental not only to identify cases of VL but also to correctly treat cases of VL associated infectious and hematological diseases.

A Leishmaniose visceral é um problema que cresce no Estado de São Paulo afetando o homem e o cão. Os exoantígenos da membrana das leishmanias são liberados no meio de cultura. Os exoantígenos são importantes na indução da imunidade mediada pelas células T e B estimulando a produção elevada de anticorpos. Realizamos uma avaliação comparativa por ELISA e Immunoblotting de exoantígenos e antígenos totais de L. (L.) amazonensis, L. (V.) braziliensis e L. (L.) chagasi, no diagnóstico da leishmaniose visceral humana e canina. Obteve-se por ELISA sensibilidade de 100% para ambos os preparados antigênicos independente da espécie de Leishmania. A melhor especificidade em humanos e cães foi com os exoantígenos. O exoantígeno da L. (L.) chagasi teve a melhor especificidade e média de absorbâncias comparadas aos das outras espécies (p<0.005). Para o hospedeiro humano o ELISA com exoantígenos, não discriminou pacientes com leishmaniose cutânea e ou mucocutânea. O Immublotting dos exoantígenos de L. (L.) chagasi (IBleish) apresentou 100% de sensibilidade e especificidade para os cães. Os dados do IBleish-L. (L.) chagasi demonstraram a possibilidade de sua utilização como método confiável para a confirmação do diagnóstico da leishmaniose canina. / The visceral leishmaniasis is a new problem that grows in the State of São Paulo, affecting men and dogs. The exoantigens from the membrane of the Leishmania, are released out of culture medium. The exoantigens are important in the induction of immunity mediated by T and B cells, stimulating the production of antibodies. This work was carried out an evaluation of ELISA and Immunoblotting using comparatively exoantigens and total antigens of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) chagasi for the diagnosis of canine and human visceral leishmaniasis. ELISA sensitivity was 100% for all different antigens, independent of the species of Leishmania employed. The best specificity for both human and dogs were obtained with exoantigens. Exoantigen from L. (L.) chagasi was what showed the best specificity and mean absorbance compared to those of other species (p<0.05). The ELISA with exoantigens for the human host not discriminate individuals with leishmaniasis cutaneous or mucocutaneous. The Immunoblotting that used the exoantigens of L. (L.) chagasi (IBleish) showed 100% sensitivity and specificity for the dogs. The IBleish-L. (L.) chagasi showed the possibility of its use as a reliable method to confirm the diagnosis of canine leishmaniasis.