Programa de Pós-Graduação em Ciências Farmacêuticas. CIPHARMA, Escola de Farmácia, Universidade Federal de Ouro Preto. / Submitted by Oliveira Flávia (flavia@sisbin.ufop.br) on 2014-08-20T16:55:32Z No. of bitstreams: 1 DISSERTAÇÃO_ AvaliaçãoAtividadeLeishmanicida.pdf: 1353741 bytes, checksum: d35a91a5ba75fa3d48e954528062cc3a (MD5) / Rejected by Gracilene Carvalho (gracilene@sisbin.ufop.br), reason: O PDF não é o da descrição. on 2014-08-25T13:06:19Z (GMT) / Submitted by Oliveira Flávia (flavia@sisbin.ufop.br) on 2014-08-28T19:19:41Z No. of bitstreams: 1 DISSERTAÇÃO_ AvaliaçãoAtividadeLeishmanicida.pdf: 2387079 bytes, checksum: 408539ffed7be7b6d46f7b881d7c3cc6 (MD5) / Approved for entry into archive by Gracilene Carvalho (gracilene@sisbin.ufop.br) on 2014-08-29T16:38:38Z (GMT) No. of bitstreams: 1 DISSERTAÇÃO_ AvaliaçãoAtividadeLeishmanicida.pdf: 2387079 bytes, checksum: 408539ffed7be7b6d46f7b881d7c3cc6 (MD5) / Made available in DSpace on 2014-08-29T16:43:32Z (GMT). No. of bitstreams: 1 DISSERTAÇÃO_ AvaliaçãoAtividadeLeishmanicida.pdf: 2387079 bytes, checksum: 408539ffed7be7b6d46f7b881d7c3cc6 (MD5) Previous issue date: 2013 / O presente estudo avaliou a utilização de lipossomas convencionais como veículos vetores do antimônio trivalente, na forma de tártaro emético, bem como sua associação ao ácido ascórbico, para o tratamento da leishmaniose visceral no modelo murino. Lipossomas constituídos por distearoilfosfatidilcolina/colesterol (5:4) foram obtidos pela metodologia de congelamento/descongelamento. A caracterização da formulação mostrou tamanho aproximado de 222,5 nm, índice de polidispersão de 0,214, eficiência de encapsulação de 15%, concentração final de 4 mg SbIII/mL e cinética de liberação bifásica avaliada in vitro. Lipossomas vazios, igualmente obtidos e hidratados com tampão fosfato serviram como controle. Camundongos BALB/c com idade entre 6 e 8 semanas foram inoculados com 1 x 107 promastigotas de Leishmania (Leishmania) infantum (cepa C43) por via endovenosa. Após seis semanas de infecção, os animais foram divididos em sete grupos (n=8) e tratados por via intraperitoneal, em dose única com: (1) tampão fosfato, (2) antimônio trivalente (SbIII) - 9 mgSb/Kg, (3) lipossomas vazios, (4) lipossomas de SbIII (9 mgSb/Kg), (5) ácido ascórbico (AA) - 300 mg/kg; (6) associação do AA (300 mg/kg) ao SbIII (9 mgSb/kg) e (7) associação de AA (300 mg/kg) aos lipossomas de SbIII (9 mgSb/kg). Dez dias após o tratamento, os animais foram eutanasiados e avaliou-se: a carga parasitária no fígado, baço e medula óssea pelo método de diluição limitante; o padrão de células do baço por citometria de fluxo; a função renal, hepática e cardíaca por análises bioquímicas e o padrão histopatológico do fígado, rins e coração. Observou-se redução significativa na carga parasitária após tratamento com antimônio trivalente lipossomal no fígado, baço e medula óssea de 47%, 33% e 47%, respectivamente, indicando a capacidade do sistema de vetorização de direcionar o medicamento para esses compartimentos. A administração concomitante do AA e SbIII livre ou em formulação lipossomal não interferiu na atividade leishmanicida do SbIII. Não foi observada diminuição na carga parasitária após tratamento com antimônio trivalente livre em nenhum dos órgãos avaliados. Os resultados mostraram nenhuma alteração no padrão fenotípico de células do baço por citometria de fluxo ou nas funções hepática, cardíaca e renal por análises bioquímicas. As análises histopatológicas mostraram que a coadministração dos lipossomas de SbIII e AA foi capaz de preservar os tecidos hepático e renal quando comparados os diferentes grupos experimentais. Os resultados desse trabalho permitiram concluir que a referida preparação lipossomal representa uma alternativa terapêutica capaz de reduzir a carga parasitária nos diferentes órgãos alvo, com potencial para a eliminação do parasito na medula óssea. A coadministração do AA aos lipossomas de SbIII acarretou redução na toxicidade do SbIII. Em conjunto, nossos dados apontam os benefícios do encapsulamento do SbIII em lipossomas unilamelares demonstrados pela redução das alterações histopatológicas para fígado e rins, particularmente em combinação com AA. Acreditamos que nosso trabalho será útil para a proposição de novos regimes terapêuticos para o tratamento da leishmaniose visceral, garantindo a adesão do paciente à terapia devido à redução dos efeitos colaterais. _______________________________________________________________________________ / ABSTRACT: The present study evaluated the utilization of conventional liposomes as carriers of trivalent antimony (SbIII), and its association with ascorbic acid, for the treatment of murine visceral leishmaniasis. Liposomes composed of diestearoylphosphatidylcholine and cholesterol (5:4) were obtained according to the freeze / thaw technique and hydrated in a solution of tartar emetic. Empty liposomes, hydrated in phosphate buffer, served as control vehicles. Characterization of the liposomal formulation revealed size, polydispersity index and encapsulation efficiency of 222.5 nm, 0.214 and 15%, respectively, for a formulation containing 4 mg SbIII/mL. It also displayed biphasic release kinetics in vitro. BALB/c mice, aged 6 to 8 weeks, were intravenously inoculated with 1 x 107 Leishmania (Leishmania) infantum promastigotes (C43 strain). Six weeks post infection, the animals were divided into seven groups (n=8) and treated intraperitoneally, as a single dose, with either (1) phosphate buffer, (2) SbIII at 9 mg Sb/kg, (3) empty liposomes, (4) SbIII-entrapped liposomes at 9 mg Sb/kg, (5) ascorbic acid (AA) at 300 mg/kg, (6) association of AA (300 mg/kg) with SbIII at 9 mg Sb/kg or (7) association of AA (300 mg/kg) with SbIII-entrapped liposomes at 9 mg Sb/kg. Ten days after treatment, animals were euthanized and the parasite load associated to the liver, spleen and bone marrow evaluated through the limiting dilution technique. Immunophenotyping of spleen cells was performed using flow cytometry. Enzyme activities in the serum were used to monitor hepatic, kidney and cardiac functions. Histopathological examinations of the liver, kidney and heart were also conducted. After treatment with SbIII-entrapped liposomes it was observed 47%, 33% and 47%, reductions in parasite load in the liver, spleen and bone marrow, respectively, indicating the ability of such delivery system to target the active principle to these compartments. Parallel administration of ascorbic acid with either free SbIII or SbIII-entrapped liposomes did not interfere with SbIII´s leishmanicide activity. No reduction in parasite load was observed after treatment with free SbIII for any of the investigated organs. Our results did not reveal any significant alteration in the profile of spleen cells. In addition, no impairment in the hepatic, renal and cardiac functions were observed. Histopathological analyses demonstrated that the co-administration of ascorbic acid with SbIII-entrapped liposomes was able to preserve the integrity of the hepatic and kidney tissues, in comparison to those from other experimental groups. The obtained results allowed us to conclude that our proposed SbIII liposomal preparation represents a therapeutic alternative able to reduce parasite burden with the potential to eliminate parasitism at the bone marrow. Co-administration of AA and SbIII-entrapped liposomes promoted reduced toxicity of SbIII. Collectively, our data extended the proposed benefits of unilamellar liposomes carrying SbIII by demonstrating their reduced toxicity, particularly in combination with ascorbic acid to the liver and kidney tissues. We believe our investigation can be useful for the proposal of novel therapeutic regimens to successfully treat visceral leishmaniasis aiming to guarantee patient compliance whilst minimizing potential side effects.

Made available in DSpace on 2014-06-11T19:23:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-03-05Bitstream added on 2014-06-13T19:09:01Z : No. of bitstreams: 1 braga_et_me_araca.pdf: 626001 bytes, checksum: 6e857013fc0dc12bd561b1176533abb8 (MD5) / O presente estudo teve por objetivos determinar a prevalência de hipertensão arterial sistêmica, bem como caracterizar, por meio de exames laboratoriais e de histopatologia, o tipo de lesão renal em cães naturalmente acometidos por leishmaniose visceral. Foram avaliados 68 cães divididos em dois grupos; G1 constituído por dez cães com síndrome urêmica, e G2 composto por 58 cães com valores séricos de creatinina e uréia dentro da normalidade ou apenas um pouco elevados, sem alterações significativas ao exame físico e ao exame de urina que pudessem caracterizar uma insuficiência renal. Verificou-se uma prevalência de 8,8% de hipertensão arterial sistêmica na população estudada, com 5/10 (50%) cães hipertensos no primeiro grupo e 1/58 (1,7%) no segundo. O estadiamento da doença renal nos animais hipertensos identificou 66,7% dos cães em estágio avançados a doença. Uma proteinúria, evidenciada por meio da P/C U, esteve presente em 77,94% dos cães. Foram observadas alterações glomerulares em 66/68 (97%), intersticiais em 48/68 (70,6%) e tubulares em 27/68 (39,7%) animais. A alteração glomerular mais frequente foi a glomerulonefritemembranoproliferativa, seguida de fibrose e glomerulonefrite membranosa. Dos seis cães em que se identificou hipertensão arterial sistêmica, quatro (66,7%) possuíam glomerulonefrite membranoproliferativa associada à fibrose glomerular em graus variados de lesão, um (16,7%) apresentava glomerulonefrite membranosa de grau intenso, e em outro animal verificou-se uma fibrose glomerular associada a glomerulonefrite proliferativa de grau leve / The aim of the present study was to determe the prevalence of hypertension and to characterize, by means of laboratory tests and histopathology, kidney lesions in dogs naturally affected by visceral leishmaniasis. Sixty eight dogs were divided into two groups: G1 consisting of ten dogs with uremic syndrome, and G2 consisting of 58 dogs with normal or slightly elevated serum creatinine and urea, without significant changes on physical examination and on urinalysis that could characterize renal failure. There was a 8.8% prevalence of hypertension in the population, with 5/10 (50%) hypertensive dogs in the first group and 1/58 (1.7%) in the second. The staging of renal disease in hypertensive animals identified 66.7% of the dogs in an advanced stage of the disease. Proteinuria, evidenced by U P/C, was present in 77.94% of the dogs. Glomerular lesions were observed in 66/68 (97%), interstitial lesions in 48/68 (70.6%) and tubular lesions in 27/68 (39.7%) dogs. Membranoproliferative glomerulonephritis was the glomerular lesion most frequently observed, followed by fibrosis and membranous glomerulonephritis. Four out of six (66.7%) dogs with hypertension had membranoproliferative glomerulonephritis associated with glomerular fibrosis in varying degrees of injury, one (16.7%) had intense membranous glomerulonephritis, and in another (16.7%) dog we identified a glomerular fibrosis associated with a mild proliferative glomerulonephritis

Made available in DSpace on 2016-09-27T13:39:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-04. Added 1 bitstream(s) on 2016-09-27T13:45:08Z : No. of bitstreams: 1 000870645_20170104.pdf: 242400 bytes, checksum: 9d69e8bb5f77471c03c024ed5ca56917 (MD5) Bitstreams deleted on 2017-01-06T13:21:19Z: 000870645_20170104.pdf,. Added 1 bitstream(s) on 2017-01-06T13:22:02Z : No. of bitstreams: 1 000870645.pdf: 525092 bytes, checksum: 20064957cf347be120a7f3cbbb1f7137 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Dogs infected with Leishmania infantum showed a reduction in the number of lymphocytes T. PD-1 (Programmed cell death 1) a new member of the B7-CD28 family, it is expressed by cells of the immune system and its binding to PD-L1 (CD274) or PD-L2 (CD273) induces deactivation of T cells or apoptosis. This study aimed to evaluate the PD-1 expression and its ligands as well as their role in T lymphocyte induction of apoptosis, TNF-α and IL-4 secretion, nitric oxide production and parasite load in dogs with visceral leishmaniasis. We observed that in canine visceral leishmaniasis PD-1 and its ligands involved in induction of apoptosis of T lymphocytes, are regulating the nitric oxide production, TNF-α, IL-4, as well as the parasitic load. This study helps to clarify the mechanism of immune response and immunotherapeutic drugs such as blocking monoclonal antibodies that can influence the LVC / FAPESP: 2013/066849

Resumo:Cães infectados com Leishmania infantum, apresentam uma redução do número de linfócitos T. PD-1(programmed cell death 1) um novo membro da família B7-CD28, é expresso por células do sistema imune e sua ligação a PD-L1 (CD274) ou PD-L2 (CD273) induz à desativação dos linfócitos T ou à apoptose. O presente estudo teve como objetivo avaliar a expressão de PD-1 e seus ligantes bem como sua função na indução de apoptose de linfócitos T, secreção de TNF-α e IL-4, óxido nítrico e na carga parasitária em cães com leishmaniose visceral. Observamos que na leishmaniose visceral canina PD-1 e seus ligantes participam na indução da apoptose celular de linfócitos T, estão regulando a produção de óxido nítrico, TNF-α, IL-4, além da carga parasitária. Esse estudo ajuda a esclarecer o mecanismo da resposta imunológica e drogas imunoterapeuticas como anticorpos monoclonais bloqueadores que podem influenciar na LVC / Abstract:Dogs infected with Leishmania infantum showed a reduction in the number of lymphocytes T. PD-1 (Programmed cell death 1) a new member of the B7-CD28 family, it is expressed by cells of the immune system and its binding to PD-L1 (CD274) or PD-L2 (CD273) induces deactivation of T cells or apoptosis. This study aimed to evaluate the PD-1 expression and its ligands as well as their role in T lymphocyte induction of apoptosis, TNF-α and IL-4 secretion, nitric oxide production and parasite load in dogs with visceral leishmaniasis. We observed that in canine visceral leishmaniasis PD-1 and its ligands involved in induction of apoptosis of T lymphocytes, are regulating the nitric oxide production, TNF-α, IL-4, as well as the parasitic load. This study helps to clarify the mechanism of immune response and immunotherapeutic drugs such as blocking monoclonal antibodies that can influence the LVC / Orientador:Valéria Marçal Felix de Lima / Banca:Alexandra Ivo de Medeiros / Banca:Caris Maroni Nunes / Mestre

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-01-26T21:18:13Z No. of bitstreams: 1 TalytaDelfinoRolimDeAlbuquerque_DISSERT.pdf: 2284989 bytes, checksum: 6b25b44bf6d63685506ea96437eef00b (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-02-01T23:27:08Z (GMT) No. of bitstreams: 1 TalytaDelfinoRolimDeAlbuquerque_DISSERT.pdf: 2284989 bytes, checksum: 6b25b44bf6d63685506ea96437eef00b (MD5) / Made available in DSpace on 2016-02-01T23:27:08Z (GMT). No. of bitstreams: 1 TalytaDelfinoRolimDeAlbuquerque_DISSERT.pdf: 2284989 bytes, checksum: 6b25b44bf6d63685506ea96437eef00b (MD5) Previous issue date: 2013-03-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / C?es s?o os reservat?rios prim?rios dos parasitos do g?nero Leishmania. A resposta imune induzida pela infec??o por Leishmania infantum nestes animais n?o est? completamente elucidada, e poucos estudos tem investigado a correla??o entre a express?o de citocinas e quimiocinas e as manifesta??es cl?nicas dos animais portadores de leishmaniose visceral canina (LVC). O objetivo deste estudo foi correlacionar os achados cl?nico em c?es naturalmente infectados por L. infantum (das ?reas rurais do munic?pio de Mossor?, Estado do Rio Grande do Norte, Brasil) com os n?veis de express?o de citocinas (IFN-?, TNF-?, IL-10 e IL-17), iNOS, quimiocinas (CCL1, CCL2, CCL3, CCL4 , CCL5, CCL17, CCL20, CCL24, CCL26, CXCL9, CXCL10), receptores de quimiocinas (CXCR3, CCR3, CCR4, CCR5, CCR6, CCR8), e parasitismo no f?gado e no ba?o. Inicialmente, foi realizado levantamento soroepidemiol?gico de c?es de 12 assentamentos rurais de Mossor?-RN, totalizando 398 c?es examinados. A positividade dos c?es para o parasito foi determinada pela reatividade em dois testes sorol?gicos (RIFI e ELISA). Todas as localidades rurais apresentaram c?es positivos para L. infantum, com positividade variando entre 6,6% a 49% dos animais examinados por localidade, e um total de 27% de c?es de todas as localidades. Posteriormente, vinte e um c?es foram clinicamente avaliados e classificados como assintom?ticos (n = 11) ou sintom?ticos (n = 10). A infec??o foi confirmada pela realiza??o de imprint no ba?o, e observa??o de formas amastigotas. Esplenomegalia, perda de peso e onicogrifose foram os sinais cl?nicos mais frequentes. No f?gado, os n?veis de express?o de RNAm de citocinas (IFN-?, TNF-?, IL-10 e IL-17), iNOS e quimiocinas (CCL1, CCL17, CCL26, CCR3, CCR4, CCR5, CCR6, e CCR8) foram mais baixos nos animais sintom?ticos do que nos animais assintom?ticos. Em compara??o com os animais n?o infectados, c?es sintom?ticos obtiveram n?veis mais baixos de express?o de quase todas as mol?culas analisadas. Al?m disso, foi observada alta carga parasit?ria e baixa inflama??o hep?tica em animais sintom?ticos, quando comparado aos assintom?ticos. Conclui-se que a diminui??o da express?o de citocinas, quimiocinas e receptores de quimiocinas resultaria na migra??o e ativa??o deficiente de leuc?citos, dificultando o controle do parasitismo e conduzindo ao desenvolvimento da doen?a. / Dogs are the primary reservoirs of the parasites of the genus Leishmania. The immune response induced by infection with Leishmania infantum in these animals is not completely understood, and few studies have investigated the correlation between the expression of cytokines and chemokines and clinical manifestations of animals with canine visceral leishmaniasis (CVL). The aim of this study was to correlate the clinical status of dogs naturally infected with L. infantum (rural municipality of Mossoro, Rio Grande do Norte, Brazil) with the expression levels of cytokines (IFN-?, TNF-?, IL-10 and IL-17), iNOS, chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, CCL17, CCL20, CCL24, CCL26, CXCL9, CXCL10), chemokine receptors (CXCR3, CCR3, CCR4, CCR5, CCR6, CCR8), and parasitism in the liver and spleen using real-time PCR. Initially, we conducted seroepidemiological survey of dogs from 12 rural settlements from Mossor?-RN, totaling 398 dogs examined. The positivity of dogs for the parasite was determined by reativity in two serological tests (ELISA and IFA). All rural localities showed dogs positive for L. infantum, with positivity varying between 6,6% and 49% of the animals examined by location, totalizing 27% of dogs from all settlements. Subsequently, Twenty-one dogs were clinically evaluated and classified as asymptomatic (n = 11) or symptomatic (n = 10). The infection was confirmed by performing imprint in the spleen, and observation of amastigotes. Splenomegaly, weight loss and onychogryphosis were the most frequent clinical signs. In the liver, levels of mRNA expression of cytokines (IFN-?, TNF-?, IL-10 and IL-17), iNOS and chemokines (CCL1, CCL17, CCL26, CCR3, CCR4, CCR5, CCR6, CCR8 and) were lower in symptomatic animals than in asymptomatic animals. In comparison with uninfected animals, symptomatic dogs had lower levels of expression of almost all molecules tested. In addition, the parasitic load was observed high and low symptomatic hepatic inflammation in animals when compared to asymptomatic. In conclusion, the decreased expression of cytokines, chemokines and chemokine receptors result in poor migration and activation of leukocytes, hindering control of parasitism and leading to the development of the disease.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-09T23:35:51Z No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-15T22:34:21Z (GMT) No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) / Made available in DSpace on 2016-08-15T22:34:21Z (GMT). No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) Previous issue date: 2015-04-23 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Nas Am?ricas, a infec??o por Leishmania tem como principal agente etiol?gico Leishmania infantum. Nos ?ltimos 30 anos o padr?o de distribui??o das leishmanioses tem mudado substancialmente e a doen?a tem apresentado um perfil emergente na periferia dos grandes centros urbanos. A infec??o por Leishmania pode evoluir com um amplo espectro cl?nico desde o acometimento da pele, mucosas e v?sceras. Dos indiv?duos infectados por L. infantum apenas 10% desenvolvem a doen?a, sabe-se que 90% da infec??o humana ? assintom?tica e diversos fatores est?o envolvidos no curso da infec??o. Os principais fatores envolvidos no desenvolvimento da doen?a s?o a resposta imune do hospedeiro, a esp?cie e o conte?do g?nico do parasita. O sequenciamento dos isolados de Leishmania poderia aumentar a compreens?o acerca da sintomatologia dos indiv?duos. Dessa forma, o objetivo desse estudo foi avaliar a diversidade gen?tica de cepas de Leishmania circulantes entre humanos, sintom?ticos e assintom?ticos e c?es de ?reas end?micas do Rio Grande do Norte. A variabilidade gen?tica de um grupo de amostras de humanos e caninos com a doen?a visceral, doen?a cut?nea e infec??o assintom?tica foi avaliado com os marcadores moleculares hsp70 e ITS1. O genoma completo de 20 isolados de Leishmania oriundos de humanos, sintom?ticos e assintom?ticos, e c?es foram sequenciados para avaliar a diversidade dessas amostras. Os fragmentos amplificados de hsp70 e ITS1 das amostras e foram analisados e montadas utilizando o pacote Phred/Phrap. Os dendogramas foram constru?dos aplicando o m?todo neighbor joining com 500 bootstraps, seguido das infer?ncias sobre as rela??es entre as variantes de Leishmania. As sequ?ncias dos 20 isolados brasileiros foram mapeadas com o genoma de refer?ncia Leishmania infantum JPCM5, usando o programa Bowtie2, com identifica??o de 36 contigs. As informa??es dos SNPs v?lidos foram utilizadas na PCA. Os SNPs foram visualizados pelo Geneious 7.1 e IGV. As anota??es do genoma foram ent?o transferidas para seus respectivos cromossomos e visualizadas utilizando o Geneious. As sequ?ncias consenso de todos os cromossomos (com m?nimo de 75% das reads com a mesma base) foram alinhadas usando Mauve. As ?rvores filogen?ticas foram calculadas de acordo com c?lculos de m?xima verossimilhan?a e modelos JTT. Como resultados obtivemos que hsp70 e ITS1 n?o foram capazes de definir variabilidade gen?tica entre os isolados de humanos e c?es; nem para os isolados de cultura e de sangue perif?rico, oriundos de um mesmo paciente.O sequenciamento gen?mico dos 20 isolados brasileiros revelou uma forte rela??o entre as cepas de Leishmania circulantes em no Rio Grande do Norte. Os isolados da Grande Natal de humanos e caninos permaneceram agrupados em todas as an?lises, sugerindo que existe proximidade genot?pica e geogr?fica entre os isolados. As amostras isoladas na d?cada de 1990 apresentaram uma maior diversidade genot?pica quando comparadas as amostras recentemente isoladas. De forma geral, n?o encontramos correla??o entre as formas cl?nicas sintom?ticas e assintom?ticas e o conte?do g?nico dos isolados brasileiros de Leishmania. / Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector?s species and the parasite?s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-12-20T15:50:22Z No. of bitstreams: 1 texto completo.pdf: 1478810 bytes, checksum: be9e97e7629b09268a428ed4728be5c8 (MD5) / Made available in DSpace on 2016-12-20T15:50:22Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1478810 bytes, checksum: be9e97e7629b09268a428ed4728be5c8 (MD5) Previous issue date: 2016-08-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A leishmaniose visceral (LV) é uma doença causada por protozoários parasitas intracelulares da espécie Leishmania infantum chagasi nas Américas. Esses parasitos possuem forte tropismo por órgãos viscerais, especialmente baço, fígado e medula óssea, onde se proliferam e provocam lesões podendo ser letal caso não sejam tratadas. O parasito possui moléculas consideradas fatores de virulência que vem sendo intensamente investigadas. Entre elas destacamos as proteínas ligante de heparina (PLH), glicoproteínas relacionadas em diversos trabalhos na literatura com o processo de adesão e internalização do parasito à célula hospedeira. Nesse trabalho utilizamos a PLH de L. infantum chagasi (PLHLc) em experimentos de imunização de camundongos BALB/c para avaliar a proteção conferida a esses animais após desafio com as formas promastigotas do parasito. Foram avaliadas a carga de parasitos no baço e no fígado, produção de citocinas (IFN- , TNF, IL-2, IL-17, IL-6, IL-10 e IL-4) e de óxido nítrico (NO) após imunização com PLHLc isolada ou associada ao Adjuvante Incompleto de Freund (AIF) e Adjuvante Saponina (SAP), quatro semana após o desafio. Os camundongos foram imunizados por via intraperitoneal ou subcutânea, sendo submetidos a duas doses de reforço utilizando o mesmo protocolo da primeira imunização. Quinze dias após a última dose os camundongos foram infectados com 1 x 10 7 promastigotas de L. infantum chagasi em fase final logarítmica de crescimento. Quatro semanas após a infecção os animais foram eutanasiados e o baço e o fígado foram coletados para ensaio da quantificação da carga parasitária e avaliação da produção de citocinas e NO por citometria de fluxo e pelo método de Griess, respectivamente. Nossos resultados mostraram que o grupo imunizado apenas com PLHLc não apresentou redução da carga parasitária e os esplenócitos estimulados in vitro com antígeno particulado de L. infantum chagasi (AgLc) produziram citocinas IL-6 e IL-17, ambas de padrão de resposta imune Th17 e a citocina IL-10. O grupo imunizado com PLHLc + AIF apresentou redução da carga de parasitos apenas no baço e produziram citocinas IFN- e IL-2 (Th1), IL-6 e IL-17 (Th17) e IL-10. O grupo imunizado com PLHLc + SAP apresentou redução da carga parasitária no baço e no fígado. Os esplenócitos desse grupo estimulados in vitro com AgLc produziram maiores médias de citocinas de padrão de resposta imune Th1 e Th17 e a citocina reguladora IL-10 comparado com os grupos anteriores. Em relação a produção de NO por esplenócitos estimulados in vitro com AgLc níveis basais foram detectados pelo método de Griess em todos os grupos avaliados. Os resultados alcançados nos mostra que a PLHLc associada ao adjuvante Saponina representa uma forte candidata a vacina contra LV e nos direciona para uma nova etapa de investigação, a proteção conferida pela PLHLc recombinante. / Visceral leishmaniasis (VL) is a disease caused by the protozoan intracellular parasite Leishmania infantum chagasi specie in the Americas. These parasites exhibit a strong tendency to invade visceral organs, especially the spleen, liver and bone marrow, where they proliferate and cause injuries and can be lethal if not treated. The parasite exhibit molecules considered as virulence factors that have been intensively searched. Among then we highlight the heparin-binding proteins (HBP), glycoproteins related in several studies in the literature with the process of adhesion and internalization of the parasite to the host cell. In this work, we used L. infantum chagasi HBP (HBPLc) in immunization experiments using BALB/c mice to evaluate the protection given to the animals after challenge with promastigote forms of the parasite. Were evaluate the parasite load in the spleen and liver, cytokine production (IFN- , TNF, IL-2, IL-17, IL-6, IL-10 e IL-4) and nitric oxide (NO) after immunization with isolated HBPLc or associated with Incomplete Freund’s Adjvant (IFA) and Saponin Adjuvant (SAP), four weeks after challenge. The mice were immunized by intraperitoneal or subcutaneous route and submitted two times to booster doses using the same protocol of the first immunization. Fifteen days after the last dose the mice were infected with 1 x 10 7 L. infantum chagasi promastigotes end logarithmic phase of growth. Four weeks after infection the animals were euthanized and spleen and liver were collected for assay quantification of the parasite load and evaluation of the cytokine and NO production by flow cytometry and by Griess method, respectively. Our results showed that the group immunized with only HBPLc showed no reduction in parasite load and spleen cells stimulated in vitro with crude extract of L. infantum chagasi (AgLc) produced cytokines IL-6 and IL-17, both standard immune response Th17 and IL-10 cytokine. The group immunized with HBPLc + IFA decreased the parasite load only in the spleen and produced cytokines IFN- and IL-2 (Th1), IL-6 and IL-17 (Th17) and IL-10 cytokine. The group immunized with HBPLc + SAP decreased the parasite load in the spleen and liver. The spleen cells this group in vitro stimulated with AgLc generated pattern cytokines of Th1 and Th17 and regulatory cytokine IL-10 compared to previous groups. About NO production by spleen cells stimulated in vitro with AgLc baseline levels were detected by Griess method in all groups evaluated. The results achieved shows that the HBPLc associated with Saponin adjuvant is a strong candidate against VL vaccine and directs us to a new stage of research, the protection given by recombinant HBPLc.

Les maladies tropicales négligées, telle que la leishmaniose, constituent un problème majeur de santé publique, en particulier dans les pays à faible revenu où cette maladie sévit. Le nombre limité des médicaments disponibles, leurs coûts, leurs effets indésirables et l’émergence de résistances, exige la recherche de nouveaux agents thérapeutiques. Les champignons endophytes constituent une source prometteuse de nouvelles molécules bioactives. Dans ce contexte, une étude mycochimique a été menée sur une souche endophyte de Botryosphaeria mamane, un micromycète relativement peu étudié, isolée des feuilles de Bixa orellana. Cette souche a été sélectionnée suite à un criblage préliminaire mené contre un modèle d’amastigotes axéniques de Leishmania infantum. Les études chimiques sur ce champignon ont permis d’isoler 9 molécules pures (BM1-BM9), dont 6 de structures nouvelles (BM1-BM4, BM8, BM9) et deux mélanges BM10 et BM11. Les molécules BM1-BM4 appartiennent à la famille des cyclopeptides de type thiodicétopipérazines que nous avons nommés botryosulfuranols A-D. Ils possèdent un squelette original caractérisé par deux centres spirocycliques. BM5-BM7 se sont révélés être des isocoumarines dérivés de la mélleine (cis-4-hydroxymélleine, trans-4-hydroxymélleine et 5-hydroxymélleine). BM8 et BM9 sont deux polycétides de structure nouvelle. Enfin, BM10 et BM11, sont des mélanges de cyclopentapeptides. L’évaluation de leur activité biologique sur amastigotes axéniques de Leishmania infantum a révélé que les thiodicétopipérazines avaient les meilleures activités. Le composé BM4 est le plus actif (CI50 = 0.03 µM) mais également le plus sélectif (IS = 190) de L. infantum par comparaison avec sa cytotoxicité sur des macrophages. BM1 et BM3 sont aussi actifs mais peu sélectifs (CI50 = 0.69 et 0.44 µM, IS = 3 et 15, respectivement) en raison de leur toxicité sur macrophages, et BM2 (CI50 = 3.88 µM) n’a montré aucune sélectivité. Les composés BM8 et BM9, bien que non cytotoxiques sur macrophages, ont montré une activité plus modérée contre L. infantum. Les composés BM5-BM7, et les mélanges BM10, BM11 n’ont présenté aucune activité leishmanicide. L'ensemble de ce travail souligne ainsi l'intérêt des endophytes fongiques comme sources de nouveaux composés leishmanicides. / Neglected tropical diseases, such as leishmaniasis, are a major public health problem, especially in the poorest countries of the world. The limited number of drugs available, their costs, adverse effects and the emergence of resistances, require the search for new therapeutic agents. Endophytic fungi are a promising source of new bioactive molecules. In this context, a mycochemical study was carried out on an endophytic strain of Botryosphaeria mamane, a relatively poorly studied micromycete, isolated from Bixa orellana leaves. This strain was selected from a preliminary screening for its good activity against Leishmania infantum axenic amastigotes. Chemical investigations on this fungus led to the isolation of 9 pure molecules (BM1-BM9), 6 of which are new structures (BM1-BM4, and BM8, BM9) and two mixtures BM10 and BM11. BM1-BM4 belong to the family of cyclodipeptides and are thiodiketopiperazines derivatives that we called botryosulfuranols A-D. The latter possess an unprecedent skeleton characterized by two spirocyclic centers. BM5-BM7 are isocoumarins derived from mellein (cis-4-hydroxymellein, trans-4-hydroxymellein and 5-hydroxymellein). BM8 and BM9 are two new polyketides. Finally, BM10 and BM11 are mixtures of cyclopentapeptides. Evaluation of leishmanicidal activities of these 9 compounds and the two mixtures on axenic amastigotes of Leishmania infantum showed that thiodiketopiperazines possessed the highest activities. BM4 was the most active (IC50 = 0.03 μM) and the most selective (IS = 190) when compared to its cytotoxicity on macrophages. The other thiodiketopiperazines BM1 and BM3 were active but less selective (IC50 = 0.69 and 0.44 μM, SI = 3 and 15, respectively), whereas BM2 was moderately active (IC50 = 3.88 μM) and not selective. BM8 and BM9, despite their absence of cytotoxicity against macrophages, were also moderately active against L. infantum (IC50 = 13.35 and 4.53 μM respectively). Compounds BM5-BM7, and the mixtures BM10 and BM11 did not show any leishmanicidal activities. This work highlights the interest of fungal endophytes as sources of new leishmanicidal compounds.

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, has recently emerged in the foxhound population in the United States and parts of Canada. Leishmania infections are usually spread to mammals by infected sand flies, however epidemiological data do not support a role for sand fly transmission in North America. The purpose of this work was to isolate and characterize L. infantum from a naturally infected foxhound from Virginia (LIVT-1 isolate). A mouse model of North American leishmaniasis was developed using immunocompetent and genetically immunodeficient mouse strains infected with LIVT-1 promastigotes by different inoculation routes. The intravenous route of infection was superior to the subcutaneous route for inducing consistent experimental infections and mice lacking interferon gamma, inducible nitric oxide synthase, or B-cells were resistant to clinical disease. Experimental infections in dogs were performed to examine the infectivity, immune responses, and pathogenicity of LIVT-1. Experimentally infected dogs developed parasitologically proven infections and a range of clinical manifestations that were similar to those observed in naturally occurring disease. Diagnostic tests including culture and cytologic evaluation of bone marrow and lymph node aspirates, polymerase chain reaction, and serology by indirect fluorescent antibody test, and recombinant K39 (rK39) immunoassay were evaluated. Kappa statistics revealed that PCR had the highest level of agreement with culture and cytology results although the rK39 dipstick assay consistently identified more experimentally infected dogs. Flow cytometry revealed no significant differences (p>0.05) in CD4+ or CD8+ expression on peripheral blood lymphocytes. Alternate transmission mechanisms in experimentally inoculated mice and dogs were investigated. PCR revealed a low level of vertical and direct transmission of LIVT-1 in inoculated BALB/c mice. Leishmania DNA was detectable by PCR in tissues from puppies from a LIVT-1 infected beagle. Although the strain of L. infantum infecting foxhounds in North America appears to predominantly use a non-vector transmission mode, the disease it produces is similar to canine leishmaniasis in other parts of the world. Non-sand fly transmission may be responsible for maintaining infections in the foxhound population. Results from this work will lead to improvement in diagnosis, clinical management, and control of canine leishmaniasis in North America. / Ph. D.

Le parasite protozoaire Leishmania possède un cycle de vie dimorphique; le stade promastigote présent chez l'insecte vecteur et le stade amastigote présent dans les phagolysosomes des macrophages de l'hôte vertébré. L'étude des gènes exprimés préférentiellement ou spécifiquement au stade intracellulaire pourrait nous en apprendre d'avantage sur les mécanismes de survie du parasite à l'intérieur des macrophages. Parmi les gènes exprimés préférentiellement au stade amastigote nous avons identifié la famille des amastines qui codent pour des protéines de surface. Cette famille est présente chez plusieurs espèces de Leishmania où on retrouve en moyenne 50 membres. Toutes les amastines possèdent une séquence signature de 11 acides aminés qui leur est unique. La majorité des amastines sont plus abondantes au stade amastigote et cette régulation implique des séquences situées en 3'UTR. La comparaison des génomes de deux espèces de Leishmania causant des pathologies distinctes, soit L. major et L. infantum a révélé une grande conservation au niveau de la synténie du génome. Cela suggère que ce qui est responsable de chaque pathologie se situe au niveau des quelques gènes uniques à chaque espèce ou encore au niveau de l'expression différentielle des gènes communs. Pour identifier de nouveaux gènes exprimés au stade intracellulaire et également identifier des gènes impliqués dans le tropisme, une analyse du transcriptome entier a été effectuée à l'aide de la technologie de puces à ADN. Lors de cette étude, où des parasites intracellulaires ont été utilisés, les résultats ont montré que chez L. infantum, 7.1 % des gènes étaient différemment exprimés entre les deux stades tandis que 9.3% l'étaient chez L. major. Parmi ces gènes modulés très peu (=10%) sont communs aux deux espèces. Une expérience indépendante avec les puces a été réalisée avec des parasites axéniques de L. infantum, pour comparer les deux stades de développement mais aussi pour évaluer la pertinence du système de culture axénique pour les amastigotes par rapport aux amastigotes obtenus de macrophages ou d'animaux infectés. Cette expérience a révélé que 12.8% des gènes étaient modulés entre les deux stades et que parmi ces gènes très peu étaient en commun avec ceux identifiés chez les amastigotes intracellulaires.