Made available in DSpace on 2014-06-11T19:26:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-26Bitstream added on 2014-06-13T20:33:42Z : No. of bitstreams: 1 cassiano_gc_me_sjrp.pdf: 5020075 bytes, checksum: 3cff0abab9c0ce6b6d89b1c811d80e65 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Responsável por 300 a 500 milhões de novas infecções e 1,5 a 3 milhões de mortes por ano no mundo inteiro, a malária continua sendo a principal doença parasitária. Se importante é o combate contra os parasitos, importante também é o conhecimento dos aspectos de incidência, distribuição, especificidade de hospedeiros, além do conhecimento de fatores biológicos e moleculares que determinam a distribuição destes. Neste cenário, um dos principais parâmetros analisados para o controle e monitoramento da malária é a detecção de espécies de Plasmodium nos vetores capazes de infectarem humanos. Embora existam diversas metodologias com este fim, a maioria delas apresenta algumas limitações. O objetivo do presente estudo foi desenvolver um método que permita a identificação do P. falciparum, do P. malariae e das variantes do P. vivax no vetor. Uma PCR foi padronizada, utilizando iniciadores contra regiões específicas do gene CS. A PCR-RFLP foi utilizada para distinguir as variantes do P. vivax. A eficiência da metodologia desenvolvida foi comparada com uma nested-PCR, utilizando Anopheles infectados experimentalmente. Um total de 90 mosquitos foram infectados artificialmente com P. vivax (n = 30), P. falciparum (n = 30) e P.malariae (n = 30). Estes mosquitos infectados, juntamente com outros 30 não infectados foram avaliados em relação a identificação do Plasmodium por nested PCR e com o CS-PCR. A nested PCR para o P. vivax, P. falciparum e P. malariae identificou 19, 16 e 21 mosquitos positivos, respectivamente; enquanto o CS-PCR detectou em 17, 14 e 16 mosquitos, respectivamente. A comparação entre os dois métodos revelou uma boa concordância (κ = 0,723, 0,867 e 0,657, respectivamente, para P. vivax, P. falciparum... / Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, causing 1.5-3 million deaths. Is important the fight against the parasites, but it is also important the knowledge of the incidence, host specificity, molecular and biological factors determining the distribution of these parasites. In this scenario, identification of the human-specific Plasmodium species in the mosquito host is an essential component for planning and monitoring of malaria control operations. However, most of the detection methods show some potential limitations. The objective of this study was development an effective assay for detecting Plasmodium falciparum, Plasmodium malariae and Plasmodium vivax variants in Anopheles mosquitoes. A PCR was development targeting the CS gene. A PCR-RFLP was used to distinguish the P. vivax variants VK210, VK247 and P. vivax-like. The new PCR assay was compared against a nested PCR using artificially infected Anopheles mosquitoes. A total of 90 mosquitoes were artificially infected with P. vivax (n = 30), P. falciparum (n = 30) and P. malariae (n = 30). These infected mosquitoes along with another 30 unfed mosquitoes were checked for the identification of Plasmodium by nested PCR and with the CS-PCR. Nested PCR for P. vivax, P. falciparum and P. malariae detected positive infection in 19, 16 and 21 mosquitoes respectively; whereas CS-PCR detected in 17, 14 and 16 mosquitoes, respectively. The comparison revealed a close agreement between the two assays (κ = 0.723, 0.867 and 0.657, respectively for P. vivax, P. falciparum and P. malariae groups). Subsequently, PCR-RFLP efficiently discriminate P. vivax variants... (Complete abstract click electronic access below)

Submitted by JOÃO LUCAS BRUNO PRATES null (jhonylprates@yahoo.com.br) on 2017-08-30T20:18:59Z No. of bitstreams: 1 Dissertação de mestrado - João Lucas.pdf: 6449742 bytes, checksum: 4da0e1cbddc21aa321ccfa9245fbb63b (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-01T13:02:50Z (GMT) No. of bitstreams: 1 prates_jlb_me_araiq.pdf: 6449742 bytes, checksum: 4da0e1cbddc21aa321ccfa9245fbb63b (MD5) / Made available in DSpace on 2017-09-01T13:02:50Z (GMT). No. of bitstreams: 1 prates_jlb_me_araiq.pdf: 6449742 bytes, checksum: 4da0e1cbddc21aa321ccfa9245fbb63b (MD5) Previous issue date: 2017-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Na presente pesquisa foram sintetizados vários derivados piridínicos objetivando a descoberta de constituintes com atividade antiparasitária potencial. Estes compostos foram planejados a partir do alcaloide (–)-espectalina isolado de Senna spectabilis (Fabaceae), empregando alguns processos usuais da química medicinal como a simplificação molecular, isosterismo clássico e funcional. Deste procedimento experimental, foram sintetizados 18 substâncias, sendo todas obtidas por meio de reações simples, a partir de compostos comerciais. Das substâncias sintéticas produzidas, 14 foram submetidas à avaliação para atividade em Trypanosoma cruzi, visando potenciais antichagásicos, e em Leishmania infantum para identificar potenciais leishmanicidas. Os compostos testados mostraram-se inativos na inibição destes protozoários. Contudo, dois análogos (48 e 52) apresentaram atividade inibitória das cepas de Plasmodium falciparum, com CI50 = 5,0 µM e índice de seletividade bastante interessante (IS >50). / In the present research, several pyridine derivatives were synthesized aiming at the discovery of new constituents with potential antiparasitic activity. All compounds were planned from the (-)-spectaline alkaloid isolated from Senna spectabilis (Fabaceae), employing some usual medicinal chemistry procedures, such as molecular simplification, classical and functional isosterism. From this experimental procedure, 18 compounds were synthesized, and all of these were obtained by means of simple reactions, from commercial compounds. Of the synthetic substances produced, 14 were evaluated for activity in Trypanosoma cruzi, aimed at the search for potential antichagasics, and against Leishmania infantum to identify potential leishmanicidal derivatives. All compounds tested were inactive to the inhibition of these protozoa. However, two analogues (48 and 52) showed inhibitory activity against Plasmodium falciparum strains, with IC50 = 5.0 μM and an interesting selectivity index (IS> 50).

Conserved residues in protein are crucial for maintaining structure and function, either by direct involvement in chemistry or indirectly, by being essential for folding, stability and oligomerisation and are mostly clustered near active sites. The variability of sequences of the same protein from diverse organisms is a reflection of the selective pressures of evolution. Sequence conservation analysis with 3397 bacterial triosephosphate isomerase (TIM) sequences using Plasmodium falciparum (Pf) TIM as template, showed full conservation of ten residues, K12, T75, H95, E97, C126, E165, P166, G209, G210 and G228. The integrity of the enzyme active site, which lies near the dimer interface, makes TIM an obligatory dimer. Attempts to engineer active monomeric TIM have not been successful. The present study assesses the effects of mutations at fully conserved position 75 (Thr) and the highly conserved position 64 (Q: 3011, E: 383) near the dimer interface, using the recombinant Plasmodial enzyme. Residue 64, Gln in Pf, and T75 interact with the catalytic E97 and K12, respectively. Preliminary analysis of available crystal structures showed that Gln 64 takes part in a single intersubunit interaction and maintains the obligatory strained backbone angles of the catalytic K12 residue, while Thr 75 is involved in four intersusunit hydrogen bond interactions. This led to the hypothesis that mostly, Gln at position 64 is crucial for enzyme activity and Thr at position 75 for the integrity of the dimer. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The major findings revealed that the mutations at position 64 have a significant effect on dimer integrity with a 1000 fold increase in the dimer dissociation constant compared to the wild type enzyme, while dimer stability was unimpaired for the T75 mutants. Concentration dependence of activity yielded an estimate of dimer dissociation constant (Kd) values (Q64N 73.7±9.2 nM and Q64E 44.6±8.4 nM). Enzyme activity values of the T75 mutants are comparable to the wild type, except for T75N which shows a 4-fold drop in activity. All four T75 mutants show a dramatic fall in activity between 35 °-45 °C. Crystal structure determination of the T75S/V/N mutant offers insights into the variation in local interactions with T75N showing the largest changes. These results were unanticipated emphasising the uncertainties involved in inferring functional and structural role for individual residues based only on analysis of interactions observed in crystal structures. Nanospray ionisation mass spectrometric studies has also been used to probe the oligomeric properties of the three mutant proteins Q64N, Q64E and T75S and the wild type enzyme in the gas phase. The gas phase distributions of dimeric and monomeric species have been examined under a wild range of collision energies (40 – 160 eV). The order in the gas phase, PfTIM wild type > T75S > Q64E ~ Q64N, together with the solution phase experiments described above establish the importance of Q64 and T75 in influencing stability and activity. Inhibition studies with a 27 residue synthetic dimer interface peptide and the Q64 mutants establish that the interaction between the protein and the peptide was facilitated in the case of monomeric species.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-05-23T20:00:00Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Karla L. Nogueira.pdf: 2581121 bytes, checksum: 51f4a19124d973da05a54ea9d7bc1132 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-05-23T20:04:15Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Karla L. Nogueira.pdf: 2581121 bytes, checksum: 51f4a19124d973da05a54ea9d7bc1132 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-05-23T20:04:36Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Karla L. Nogueira.pdf: 2581121 bytes, checksum: 51f4a19124d973da05a54ea9d7bc1132 (MD5) / Made available in DSpace on 2017-05-23T20:04:36Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Karla L. Nogueira.pdf: 2581121 bytes, checksum: 51f4a19124d973da05a54ea9d7bc1132 (MD5) Previous issue date: 2016-12-19 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / alaria is among the most important infectious diseases prevalent in the tropical and subtropical regions of the world that can lead to a serious and longstanding morbidity and is therefore considered a serious public health problem. The disease is possible to be transmitted by 5 species of the genus Plasmodium spp. Infecting humans that are inoculated into the human vertebrate host by biting the vector species of the genus Anopheles spp. Of each endemic region in the world. In South America, Brazil has an important representation in malaria indices also attributed to the endemic Amazon region. The search for new antimalarial drugs is an urgent necessity, as cases of resistance to most of the drugs used in restricted antimalarial therapy worldwide are increasing. Historically quinine and artemisinin have been used as classic antimalarial drugs whose isolation was from plants and which served as a model for the development of synthetic and / or semisynthetic antimalarials with improved physicochemical and pharmacological characteristics. The objective of this study was to reproduce by means of semisynthesis some derivatives of a larger mass scale of the natural substance 4-nerolidylcatechol (4-NC), isolated from Piper peltata, in order to evaluate them for their antimalarial activity in vitro, By inhibition of Plasmodium falciparum, cytotoxic activity in normal fibroblastic cell line (MRC-5) and to determine their selectivity indices (IS), in addition to the in vivo evaluation to verify the suppression of Plasmodium berghei (malaria transmitting species for murine model) and evaluation Of acute toxicity after oral administration of the derivatives to the healthy animal model. Nine 4-NC derivatives were synthesized, two of them unpublished, as well as two new derivatives of peltatol A, a substance also isolated from P. peltata. The chemical and reactional improvements led to the achievement of derivatives with higher yields, a reduction in reaction time in a day scale, and the possibility of substitution of chlorinated solvents by cleaner solvents, which lead to yields equivalent to those obtained with chlorinated solventes. Regarding the results obtained in vitro, only the new propionylated derivative of 4-NC was considered partially active against P. falciparum (IC50 = 10.5 μM). In the in vivo analysis, the percentage of parasite suppression of the derivatives against the plasmodial species causing malaria in rodents and that is widely used for this type of evaluation, it was found that the new dipropionylated, dibutyrylated and dipentylated derivatives, as well as the mixture of the monomethylated and epoxidized derivatives and peltatol A, all at a dose of 200 mg / kg / day, showed suppression of parasitemia against P. berghei of 41, 33, 47, 36, 40 and 51%, respectively, compared to controls treated with the vehicle alone (blank). The derivatives were not considered to be toxic to the MRC-5 lineage nor did they stimulate any signs of acute toxicity in healthy mice after oral administration and still showed satisfactory selectivity indices. Finally, although the inhibition levels of the derivatives studied were not considered significant at a dose of 200 mg / kg / day, their mechanism of antimalarial action - inhibition of isoprenoid biosynthesis and inhibition of hemozoin formation - still makes them competitive and which may be better investigated for their exploitation as antimalarials and / or other biological activities / A malária está entre as mais importantes doenças infecciosas prevalentes nas regiões tropicais e subtropicais no mundo que pode conduzir a um grave e duradouro estado de morbidade, sendo portanto considerada um grave problema de saúde pública. A doença é possível de ser transmitida por 5 espécies do gênero Plasmodium spp. infectantes de humano que são inoculadas no hospedeiro vertebrado humano mediante a picada das espécies vetoras do gênero Anopheles spp. próprias de cada região endêmica no mundo. Na América do Sul, o Brasil tem importante representação nos índices de malária também atribuída à endêmica região amazônica. A busca por novas drogas antimaláricas é uma necessidade urgente visto que em todo o mundo são crescentes os casos de resistência à maioria das drogas utilizadas na restrita terapia antimalárica. Historicamente tem-se quinina e artemisinina como antimaláricos clássicos cujo isolamento deu-se a partir de plantas e os quais serviram como modelo para o desenvolvimento de antimaláricos sintéticos e/ou semissintéticos com características físico-químicas e farmacológicas mais aprimoradas. Este estudo teve como objetivo reproduzir por meio de semissíntese alguns derivados, em maior escala maior de massa, da substância natural 4-nerolidilcatecol (4-NC), isolado de Piper peltata, afim de avaliá-los quanto a sua atividade antimalárica in vitro, mediante inibição de Plasmodium falciparum, atividade citotóxica em linhagem celular fibroblástica normal (MRC-5) e determinar seus índices de seletividade (IS) além da avaliação in vivo para verificação da supressão de Plasmodium berghei (espécie transmissora de malária para modelo murino) e avaliação da toxicidade aguda após administração oral dos derivados ao modelo animal sadio. Foram sintetizados nove derivados de 4-NC, sendo dois deles inéditos, bem como dois novos derivados de peltatol A, substância também isolada de P. peltata. Os melhoramentos químicos e reacionais conduziram a obtenção de derivados com maiores rendimentos, diminuição de tempo reacional em escala de dias além da confirmada possibilidade da substituição de solventes clorados por solventes mais limpos que conduzem a obtenção de rendimentos equivalentes aos obtidos por meio de solventes clorados. Em relação aos resultados obtidos in vitro, apenas o novo derivado propanoilado de 4-NC foi considerado parcialmente ativo contra P. falciparum (CI50 = 10,5 μM). Já na análise in vivo, o percentual de supressão parasitária dos derivados contra a espécie plasmodial causadora de malária em roedores e que é amplamente utilizada para este tipo de avaliação, constatou-se que os novos derivados dipropanoilado, dibutirilado e dipentanoilado, e ainda a mistura dos derivados monometilados e epoxidados e peltatol A, todos à dose de 200 mg/kg/dia, exibiram supressão da parasitemia contra P. berghei de 41, 33, 47, 36, 40 e 51%, respectivamente, em comparação aos controles tratados apenas com o veículo (branco). Os derivados não foram considerados tóxicos para a linhagem MRC-5, tampouco estimularam quaisquer sinais de toxicidade aguda em camundongos sadios após administração oral e ainda apresentaram satisfatórios índices de seletividade. Por fim, ainda que os níveis de inibição dos derivados estudados não tenham sido considerados significativos à dose de 200 mg/kg/dia, seu mecanismo de ação antimalárica – inibição da biossíntese de isoprenóides e inibição da formação de hemozoína – ainda os torna competitivos e passíveis de melhores estudos acerca de sua exploração como antimaláricos e/ou outras atividades biológicas.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-08-28T15:50:17Z No. of bitstreams: 2 Dissertação - Elizabeth N. Andrade.pdf: 1864684 bytes, checksum: c599ae6a377eaefe4189fdca36efee53 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-08-28T15:50:44Z (GMT) No. of bitstreams: 2 Dissertação - Elizabeth N. Andrade.pdf: 1864684 bytes, checksum: c599ae6a377eaefe4189fdca36efee53 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-28T15:50:44Z (GMT). No. of bitstreams: 2 Dissertação - Elizabeth N. Andrade.pdf: 1864684 bytes, checksum: c599ae6a377eaefe4189fdca36efee53 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2010-03-17 / Introduction: Malaria is one of the most serious health problems affecting Brazilians, and it is in the Amazon area that one can find the largest number of people affected by the disease. In that region, the State of Amazonas is by far, the place that presents the highest incidence of the illness. Malaria brings a strong impact on personal and collective grounds and is considered the most debilitating disease affecting residents of that large area of land. Such epidemiological importance plus an outstanding harm to the hematopoietic sector imposes the need for studies that enhance a better understanding of the problem as proposed by this research. Methods: A study of a 29 patient group with falciparum malaria, primo-infected and no previous treatment, all older 15 years of age, admitted to the Tropical Medicine Foundation of Amazonas from 1998 to 1999. Clinical and laboratory data (hematology, biochemistry), myelogram and bone marrow biopsy collected from patients were analyzed to identify changes resulting from bone marrow impairment in acute malaria caused by Plasmodium falciparum. Results: The patients studied had low levels of parasitemia (52%), thrombocytopenia with platelet counts in peripheral blood, varying inversely to peripheral parasitemia (p value 0.016), hemolysis presumed positive by Coombs test in 16 patients; dyserithropoiesis, dysmegakariopoiesis and leukocyte phagocytosis in a total of 152 dyspoietics events involving the three medullary series and presence of lymphoid aggregates (17.24%) Conclusions: Acute malaria caused by Plasmodium falciparum in this study provided important changes that affect cell maturation in bone marrow, primarily in erythroid and megakaryocytic lineage, besides causing the appearance of uncertain evolutionary potential lymphoid aggregates. / Introdução: A malária é um dos mais graves problemas da saúde que afeta os brasileiros, sendo que se encontra na Região Amazônica a maior quantidade de pessoas afetadas pela doença. Naquela região, o Estado do Amazonas é aquele que apresenta a maior casuística da doença. A malária é uma doença com forte impacto pessoal e coletivo, sendo considerada a doença mais debilitante a afetar os moradores daquela região. A esta importância epidemiológica agrega-se o notório comprometimento do setor hematopoiético pela malária, impondo a necessidade de estudos que aumentem o entendimento sobre a questão como se propõe esta pesquisa. Metodologia: Foram estudados 29 pacientes portadores de malária por Plasmodium falciparum, primo infectados, virgens de tratamento, maiores de 15 anos, atendidos na Fundação de Medicina Tropical do Amazonas no período de 1998 a 1999. Dados clínicos, laboratoriais (hematológicos, bioquímicos), mielograma e biópsia de medula óssea coletados dos pacientes foram analisados visando identificar alterações decorrentes do comprometimento da medula óssea na malária aguda por Plasmodium falciparum. Resultados: Os pacientes estudados apresentaram nível de parasitemia baixo (52%); plaquetopenia com a contagem de plaquetas no sangue periférico variando negativamente com a parasitemia periférica (p valor 0,016); hemólise, presumida pelo teste de Coombs positivo, em 16 pacientes; diseritropoiese, dismegacariopoiese e fagocitose de leucócitos totalizando 152 eventos dispoiéticos acometendo as três séries medulares e presença de agregados linfóides (17,24%) Conclusões: A malária aguda por Plasmodium falciparum proporcionou neste estudo alterações medulares importantes que afetam a maturação celular na medula óssea, principalmente na linhagem eritróide e megacariocítica, além de ocasionar o aparecimento de agregados linfóides de potencial evolutivo incerto.

Made available in DSpace on 2015-04-11T13:54:37Z (GMT). No. of bitstreams: 1 Camila Fabbri.pdf: 1702364 bytes, checksum: aad2c6cd689b654fb570e596447becf9 (MD5) Previous issue date: 2012-01-19 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The human malaria is an infectious disease, febrile, acute or chronic, caused by protozoa transmitted by vectors, being the disease more widely distributed in the world and one of the most prevalent parasitic diseases today. About 300 to 500 million cases occur annually and about 1 to 2 million people - mostly children - die from malaria. In the state of Amazonas, between january 2007 and march 2009, 22,081 cases of malaria were diagnosed and reported, with the largest record (14,249) in 2007. Although Plasmodium vivax malaria is considered a benign form of the disease with a low mortality rate in relation to Plasmodium falciparum, it can cause a serious disease where complications such as jaundice, severe anemia, renal failure and pulmonary complications are described. Oxidative stress occurs when there is an imbalance in the concentration of oxidants, free radicals, and antioxidants, such as enzymes, vitamins C and E, β-carotene. During the malaria disease, oxidative stress may be developed by two mechanisms: by the parasite, when it is to reproduce, generates reactive species and the host immune system, which makes use of these reactive species to try to combat the parasite. In order to study oxidative stress in patients with severe vivax malaria who developed jaundice in the course of the disease in plasma and erythrocytes, and also to check the influence of this high concentration of bilirubin in oxidative stress, the following groups of patients was studied: non-severe malaria , severe malaria presenting jaundice, concurrent malaria and dengue, healthy patients with no history of malaria (control group) and one patient with deficiency in the enzyme glucose-6-phosphate dehydrogenase which contracted malaria. In all patients were measured the levels of antioxidant enzymes and lipid peroxidation marker malondialdehyde on day zero (when diagnosed with malaria) and on day fourteen (free of symptoms and parasitemia). The levels of malondialdehyde and the enzymes celuroplasmin, lactate dehydrogenase and glutathione reductase were increased (p <0.02) in plasma of patients with severe malaria compared with the control group, unlike the enzymes catalase, superoxide dismutase and thioredoxin reductase where levels were decreased (p <0.03) compared to the control group. The levels of glutathione reductase and malondialdehyde marker also showed significantly higher levels in severe malaria groups when compared with non-severe malaria group. The correlation between glutathione reductase and total bilirubin was significant (p < 0.0001) in all patients of each group. Patients who developed malaria and dengue fever showed levels of oxidative stress similar to patients with severe malaria. With all these results, we conclude the patients with severe vivax malaria who developed jaundice had a higher oxidative stress than the patients who contracted the disease in milder form. Thus, these high concentrations of bilirubin may develop the role of signaling to oxidation in patients with malaria / A malária humana é uma doença infecciosa, febril, aguda ou crônica, causada por protozoários transmitidos por vetores, sendo a doença mais amplamente distribuída no mundo e uma das doenças parasitárias mais prevalentes da atualidade. Cerca de 300 a 500 milhões de casos ocorrem anualmente e cerca de 1 a 2 milhões de pessoas - na maioria crianças - morrem de malária. No estado do Amazonas, entre janeiro de 2007 a março de 2009, foram diagnosticados e notificados 22.081 casos de malária, com maior registro (14.249) em 2007. Embora a malária por Plasmodium vivax seja considerada a forma benigna da doença, com uma taxa de letalidade baixa em relação ao Plasmodium falciparum, ela pode causar uma doença grave, onde complicações como icterícia, anemia grave, insuficiência renal e complicações pulmonares são descritas. O estresse oxidativo ocorre quando há um desequilíbrio na concentração de substâncias oxidantes, os radicais livres, e antioxidantes, como enzimas, vitaminas C e E, β-caroteno. Na malária, o estresse oxidativo pode ser desenvolvido por dois mecanismos: através do parasita quando este ao se reproduzir gera espécies reativas e o sistema imunológico do hospedeiro, que lança mão dessas espécies reativas para tentar combater o parasita. Com o intuito de estudar o estresse oxidativo em pacientes com malária vivax grave que desenvolveram icterícia ao decorrer doença no plasma e eritrócitos, além de verificar a influência desta alta concentração de bilirrubina no estresse oxidativo, os seguintes grupos de pacientes foram estudados: malária não grave, malária grave apresentando icterícia, malária e co-infeccão com dengue, pacientes saudáveis sem histórico de malária (grupo controle) e um paciente portador da deficiência na enzima glicose-6-fosfato desidrogenase que contraiu malária. Em todos os pacientes foram mensurados os níveis de enzimas antioxidantes e o marcador de lipoperoxidação malondialdeído no dia zero (quando diagnosticado com malária) e no dia quatorze (livre de sintomas e parasitemia). Os níveis de malondialdeído e das enzimas celuroplasmina, lactato desidrogenase e glutationa redutase estavam aumentados (p < 0,02) no plasma de pacientes com malária grave em comparação ao grupo controle, diferentemente das enzimas catalase, superóxido dismutase e tioredoxina redutase onde os níveis estavam diminuídos (p < 0,03) em comparação ao grupo controle. Os níveis da enzima glutationa redutase e do marcador malondialdeído também mostraram níveis significativamente mais elevados no grupos malária grave quando comparado com o grupo malária não grave. A correlação entre os níveis de bilirrubina e glutationa redutase foi significativa (p < 0.0001) em todos os pacientes de cada grupo. Pacientes que desenvolveram malária e dengue apresentaram níveis de estresse oxidativo semelhante aos pacientes com malária grave. Com todos estes resultados, podemos concluir que os pacientes com malária vivax grave que desenvolveram icterícia apresentaram um maior estresse oxidativo do que os pacientes que contrairam a doença na forma mais branda. Dessa forma, estas altas concentrações de bilirrubina podem estar desempenhando a função de sinalizadoras do processo oxidativo em pacientes com malária

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-03-02T15:45:37Z No. of bitstreams: 2 Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-03-02T15:45:50Z (GMT) No. of bitstreams: 2 Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-02T15:45:50Z (GMT). No. of bitstreams: 2 Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-10-04 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The steady increase in the number of current drug resistance cases used without malaria treatment has encouraged studies on a discovery of potentially active new drugs. In previous work by LAPAAM / INPA has revealed the in vitro and in vivo antimalarial activity of indole alkaloids, such as elipticine, olivacin and derivatives. The great challenge has been found other indolic structures, which are easier to obtain and which present similar or superior antimalarial activity as found in these structures. In this search, nucleic acid tricyclic derivatives obtained from carbazolic and -carbolinic skeletons were synthesized and attributes against in vitro assays. In total, twenty-three derivatives were synthesized, of which eighteen were evaluated in vitro biological assays against K1 P. falciparum strains and cytotoxicity in non-tumor cells (MRC-5). In addition were submitted to the tests five commercial substances harmine (24), harmane (25), harmaline (27) and carbazole (29). As the main reactions for the nitration, demethylation, formation of salts through hydrogen chloride bubbling, condensation reaction of Pictet-Splenger and alkylation, a quality formed by the unpublished compound in the 9-(2,3-dihydroxy)-harmano (25.1). Among the derivatives submitted antimalarial assays, 3-nitrocarbazole (29.4), O-acetyl-harmol (26.1) and the harmine salt HCl (24.4) were the most active with IC50 8,87 M, 12,2 M and IC50 19,31 M, respectively. The 8-nitroharmano (25.2) and the 9-(2,3-dihydropropyl)-harmane (25.1) had their activity potentiated with IC50 values lower than the harmane. And more active commercial substances harmaline with IC50 14,7 M and the norharman with IC50 17.67 M. According to the factory, there is no concentration of 50 g / mL, except for 6-nitro-harmano (25.3) and O-acetyl-harmol, whose lower viability is less than 25%. / O constante aumento no número de casos de resistências aos medicamentos atuais utilizados no tratamento da malária tem incentivado estudos sobre a descoberta de novas drogas potencialmente ativas. Trabalhos anteriores realizados pelo LAPAAM/INPA revelaram a atividade antimalárica in vitro e também in vivo de alcaloides indólicos, tais como elipticina, olivacina e derivados. O grande desafio tem sido encontrar outras estruturas indólicas, de mais fácil obtenção e que apresente atividade antimalárica similar ou superior às encontradas nestas estruturas. Nessa busca, derivados tricíclicos com núcleo indólicos obtidos a partir esqueletos carbazólicos e -carbolínicos foram sintetizados e avaliados frente a ensaios in vitro. No total foram sintetizados vinte e três derivados, dos quais dezoito foram avaliados em ensaios biológicos in vitro contra cepas K1 de P. falciparum e citotoxidade em células não tumorais (MRC-5). Também foram submetidos aos ensaios, as cinco substâncias comerciais norhamano (23), harmina (24), harmano (25), harmalina (27) e carbazol (29). As principais reações utilizadas foram nitração, desmetilação, formação de sais através do borbulhamento de cloreto de hidrogênio, reação de condensação de Pictet-Splenger e alquilação, a qual formou o composto inédito na literatura 9-(2,3-diidroxi)-harmano (25.1). Entre os derivados submetidos aos ensaios antimaláricos, o 3-nitro-carbazol (29.2), O-acetil-harmol (26.1) e o sal de harmina.HCl (24.4) foram os mais ativos com CI50 8,87 M , 12,2 M e CI50 19,31 M, respectivamente. O 8-nitroharmano (25.2) e o 9-(2,3-diidropropil)-harmano (25.1) tiveram sua atividade potencializada com valores de IC50 inferiores ao harmano. E entre as substâncias comerciais, as mais ativas foram a harmalina com CI50 14,7 M e o norharmano com CI50 17,67 M. De maneira geral todos as estruturas estudas neste trabalho apresentaram baixa toxicidade em células normais com viablidade maior que 50 %, na concentração de 50 g/mL, com exceção do 6-nitro-harmano (25.3) e do O-acetil-harmol, cujas viablidades foram inferiores a 25%.

Plasmodium falciparum (Pf) est un parasite intracellulaire capable d’infecter l’Homme. Dans les 48h après l’invasion des érythrocytes, il passe par le stade d’une cellule géante multinucléée qui se divise en 16 à 32 parasites. Cette multiplication rapide nécessite des mécanismes spécifiques de régulation très subtilement orchestrés. Parmi les modifications post-traductionnelles décrites chez les cellules eucaryotes, la phosphorylation réversible des protéines par les kinases/phosphatases est une des voies majeures dans la transduction des signaux cellulaires. Chez Plasmodium, des études de génétique inverse ont démontré que PfPP1, phosphatase majeure du parasite, est essentielle pour sa survie. L’activité de PP1 est connue pour être contrôlée par divers régulateurs endogènes. Cependant, malgré leur importance dans le ciblage de l’holoenzyme à un compartiment subcellulaire spécifique et/ou dans la régulation de son activité, peu de recherches ont été consacrées à l’identification de partenaires de PP1 chez Pf.Dans le but d’approfondir nos connaissances sur la régulation de PfPP1 et son impact sur la biologie de Pf, nous étudions les partenaires de cette enzyme qui seraient impliqués dans le contrôle de la phosphatase. Nos recherches récentes, basées sur des études de génomique comparative, ont permis d’identifier 4 régulateurs de PfPP1 : PfLRR1, PfI3, PfI2 et PfeiF2β. Au-delà de la capacité de ces protéines à contrôler la fonction de PP1 in vitro, nous avons montré par génétique inverse que leur rôle est vital pour Pf. En parallèle, nous avons entrepris une démarche plus globale pour la recherche de nouveaux partenaires/régulateurs de PfPP1. Nous avons notamment réalisé un criblage par double hybride de levure (Y2H) où PfPP1 est utilisé comme appât.Dans la 1ère partie de ma thèse, nous avons analysé les clones obtenus en criblage Y2H et initié la caractérisation de plusieurs d’entre eux. Notre choix d’étude s’est porté par la suite sur PF3D7_091900 et PF3D7_1202600, retrouvées 8 et 10 fois lors du criblage et présentant une interaction forte avec PP1. Mon projet de thèse avait pour but de caractériser au niveau moléculaire et fonctionnel le rôle de ces protéines sur l’activité de PP1 et de déterminer les régions/motifs de ces potentiels régulateurs pouvant intervenir au niveau de la relation structure/fonction du complexe qu’ils forment avec PfPP1.Dans une 2ème partie, nous avons étudié la séquence de ces protéines. PF3D7_0919900, protéine spécifique du parasite, possède le motif RVxF, souvent impliqué dans l’interaction avec PP1 et présente des motifs RCC1 connus pour interagir avec des protéines. Ainsi elle sera nommée RCC-PIP pour Regulator of Chromosome Condensation - Phosphatase Interacting Protein. PF3D7_1202600 est, quant à elle, un orthologue de Caliban chez la drosophile, et sera nommée CLP pour Caliban-Like Protein. Elle présente 17 motifs RVxF dont 7 se situent dans le fragment obtenu suite au criblage. Différentes approches ont confirmé que ces 2 protéines sont des partenaires de PfPP1. Cependant, la réalisation d’un test pNPP in vitro a mis en évidence une fonction activatrice de RCC-PIP, alors que CLP ne présente pas d’effet.Dans une 3ème partie, l’objectif était d’étudier plus en détails RCC-PIP. Nous avons démontré que le motif RVxF est impliqué dans l’interaction avec PfPP1. Puis nous avons étudié les motifs RCC1 et leur interactome par la réalisation d’un criblage Y2H en utilisant ces motifs comme appât. Une kinase a été trouvée (PfCDPK7) suggérant que RCC-PIP aurait un rôle de plateforme capable d’interagir avec 2 enzymes antagonistes. L’étude du rôle de RCC-PIP chez le parasite est actuellement en cours. La réalisation d’un knock-in a démontré l’accessibilité du locus. Un knock-out a également été effectué, mais l’absence d’intégration du plasmide indique que RCC-PIP serait essentielle au parasite. Pour confirmer cette observation, un knock-out conditionnel chez P. berghei est en cours de réalisation. / Plasmodium falciparum is an intracellular parasite that evolves in several stages of development in the vertebrate host. Within 48 hours after the invasion of erythrocytes, it goes through the stage of a multinucleated giant cell which divides into as many parasites as nuclei (16-32 parasites). This growth/fast division requires specific regulatory mechanisms subtly orchestrated. Among the post-translational modifications described in eukaryotic cells, the reversible phosphorylation of proteins by kinases/phosphatases is one of the major pathways in the cellular signal transduction. In Plasmodium, PP1 is predicted to catalyze the majority of protein dephosphorylation events, and has been shown to be essential in its survival using reverse genetic approaches. The activity of PP1 is known to be tightly controlled by various endogenous regulators. However, despite their importance in targeting the holoenzyme to a specific subcellular compartment and/or regulating its activity, little has been devoted to identify PP1 partners in the parasite.In order to deepen our understanding of the regulation of PfPP1 and its impact on the biology of Plasmodium, we study the partners of this enzyme that may be involved in the control of its location, its specificity and activity. Our recent research, based on comparative genomic studies, have identified 4 regulators of PfPP1: PfLRR1, PfI3, PfI2 and PfeiF2β. In parallel, since Plasmodium has a particular cell cycle and the function of PP1 should be adapted, we have undertaken a more global approach to the search for new partners/regulators of PfPP1 using different approaches including a yeast two-hybrid screening where PfPP1 was used as bait.In the first part of my thesis, the clones obtained in Y2H screening were analyzed and 2 clones were selected for further characterization. These clones, corresponding to PF3D7_091900 and PF3D7_1202600 were identified 8 and 10 times during the screening, a good indication about their expression in blood stages and their interactions with PfPP1 can be still detectable under high stringency conditions. Hence, my thesis project aimed to characterize molecularly and functionally of the role of these proteins on PfPP1. _x000D_In the second part, we have analysed the sequence of these two proteins. PF3D7_0919900, a parasite-specific protein, shows the canonical binding motif « RVxF », known to be involved in the interaction with PP1, and present on the fragment obtained following the screening. The sequence also has RCC1 motifs known to interact with proteins. Thereafter, this protein was designated as RCC-PIP for Regulator of Chromosome Condensation - Phosphatase Interacting Protein. As far as PF3D7_1202600 is concerned, it seems to be an ortholog of Caliban expressed by Drosophila, and designated Pf Caliban CLP-Like Protein. It has 17 potential RVXF binding motif, of which 7 are located in the fragment obtained following the screening. Different approaches such as GST pull-down or ELISA, identified these two proteins as partners of PfPP1. Using pNPP in vitro assay, we showed a slight activation of PfPP1 by RCC-PIP, while CLP had no effect.In the third part, the objective was to study in more detail the RCC-PIP. We showed that the RVxF motif is involved in the interaction with PfPP1. We then tied to identify the partners of RCC1 by screening a cDNA library of P. falciparum using RCC1 containing protein as bait. We showed that RCC-PIP is able to interact with a kinase (PfCDPK7) suggesting that RCC-PIP may act as a platform since it is able to interact with 2 enzymes with opposed activities. Analysis of the role of RCC-PIP in the parasite is currently underway. The production of a knock-in demonstrated the accessibility of the locus. A knock-out was also carried out, but the lack of integration of the plasmid suggests that RCC-PIP is essential to the parasite. To confirm this observation, a conditional knock-out in P. berghei is in progress.

Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease

The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.