Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-18T12:20:57Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeAntiplasmodialModelagem.pdf: 2404946 bytes, checksum: f50c8e09794716ea2356ae20dc9b113b (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-11-03T14:56:45Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeAntiplasmodialModelagem.pdf: 2404946 bytes, checksum: f50c8e09794716ea2356ae20dc9b113b (MD5) / Made available in DSpace on 2017-11-03T14:56:45Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeAntiplasmodialModelagem.pdf: 2404946 bytes, checksum: f50c8e09794716ea2356ae20dc9b113b (MD5) Previous issue date: 2008 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A malária é uma infecção causada pelo Plasmodium sp. e pode ser grave, se não tratada precocemente. Ela acomete importante fração da humanidade e tem profundo impacto sanitário mundial. Estima-se que 3,3 bilhões de pessoas estejam expostas ao risco de transmissão. Um dos problemas inerentes à infecção é o progressivo aumento da resistência do parasito aos antimaláricos. Nesse contexto, fazem-se necessários estudos para o desenvolvimento de novas alternativas quimioterápicas. Muitas substâncias têm atividade antiplasmodial comprovada, como as chalconas. Entretanto, as propriedades fisico-químicas dessas moléculas, que são importantes para suas ações biológicas, não estão bem estabelecidas. Neste trabalho, foi realizada a modelagem molecular e avaliada a atividade antiplasmodial de duas chalconas (HBR1 e LH2) e quatro derivados de chalconas (GH3, IV4, LCH1 e LCH3). Para isso, foram determinadas: as concentrações inibitórias em 50% do crescimento do P. falciparum in vitro e as propriedades físico-químicas das substâncias, como HOMO, LUMO, potencial eletrostático, C log P, energia de hidratação, polarizabilidade e volume molecular, através de cálculos virtuais. Os resultados dos valores calculados foram correlacionados com a atividade biológica, a fim de se identificar parâmetros químicos que possam influenciar a ação antiplasmodial. As concentrações inibitórias em 50% do crescimento do P. falciparum variaram de 0,2 a 1,7 μM, sendo que estes valores foram menores do que os descritos na literatura. O estudo da correlação entre as atividades biológicas e as propriedades fisico-químicas mostraram parâmetros determinantes de atividade biológica, como LUMO, potencial eletrostático, C log P e energia de hidratação, que podem auxiliar na seleção de moléculas mais ativas contra o P. falciparum. Assim, essas propriedades moleculares podem ser utilizadas no planejamento racional de novas chalconas e/ou derivados com atividade antiplasmodial. / Malaria is an infection caused by Plasmodium sp. and It can be serious, if not treated precociously. It affects significant fraction of humanity and has profound health impact worldwide. It is estimated that 3.3 billion people are exposed to the risk of transmission. One of the problems of the infection is the growing emergence of parasite resistance to antimalarial drugs. In this context, studies are needed to develop new alternative chemotherapy. Many substances, such as the chalcones, have had their antiplasmodial activity proven. However, the physicochemical properties of these molecules, which are important for biological actions, are not well established. In this work, molecular modeling was performed and the antiplasmodial activity was evaluated of two chalcones (HBR1, and LH2) and four derivatives of chalcones (GH3, IV4, LCH1, and LCH3). For that, we determined the drug concentration inhibitory of 50% of the growth of P. falciparum in vitro as well as the physicochemical properties of derivatives of chalcones as HOMO, LUMO, electrostatic potential, C log P, hydration energy, polarizability and molecular volume through virtual calculations. The results of the calculated values were correlated with the biological activity in order to identify chemical parameters that can influence the antiplasmodial action. The inhibitory concentrations in 50% of the growth of P. falciparum ranged from 0,2 to 1,7 M, and these values were smaller than described them in the literature. The study of the correlation between the biological activities and the physicochemical properties showed determinating parameters for the biological activity, as LUMO, electrostatic potential, C log P and hydration energy, which may help in the selection of molecules more active against P. falciparum. Thus, these molecular properties can be used in the rational planning of new chalcones and/or derivatives with antiplasmodial activity.

Plasmodium falciparum est un parasite protozoaire responsable de la forme la plus sévère de la malaria. Depuis quelques années, les cas de résistance aux antipaludiques sont devenus de plus en plus fréquents et de plus en plus répandus. En plus de sa résistance aux drogues actuellement disponibles, ce parasite reste jusqu' à aujourd'hui réfractaire aux vaccinations. L’identification de nouvelles approches basées sur l`inhibition spécifique de certaines de ses cibles moléculaires vitales est devenue une nécessité. La recombinase à site spécifique de P. falciparum (Pf-Int) est un enzyme qui a été récemment identifié dans le laboratoire à partir de PlasmoDB. Cette recombinase à site spécifique joue potentiellement un rôle clé dans le système de recombinaison nécessaire à la viabilité du parasite. Cette protéine de 490 acides aminés, soit ~57 kDa, contient une région C-terminale qui porte les résidus conservés du site catalytique des recombinases à tyrosine R-H-K-R-(H/W)-Y. La prédiction montre une région N-terminale qui ressemble à celle de l’intégrase du phage lambda avec un mélange de structures secondaires α et β.Lors de ces travaux, nous avons d’abord montré par RT-PCR que le gène (MAL13P1.42) qui code pour PF-Int est transcrit pendant le cycle intra-érythrocytaire avec un maximum pendant la phase schizont. Nous avons ensuite essayé de montrer l`implication de Pf-Int dans le cycle parasitaire. Ceci a été réalisé grâce à un parasite (KO: knock-out) dont le gène Pf-Int a été invalidé. Ces analyses montrent que Pf-Int n'a aucun impact apparent sur le cycle de développement intra-érythrocytaire du parasite, en particulier sur la durée du cycle et le taux de croissance. Au niveau moléculaire, nous avons également procédé à la production d'anticorps anti-Pf-Int en utilisant le fragment C-162 (Résidus 162-490). La comparaison des profils de marquage, par cet anticorps, des extraits protéiques du KO et du parasite sauvage par la technique de Western blot n'a pas permis d'identifier la protéine endogène dans le parasite sauvage. Dans le but de déterminer la localisation sub-cellulaire de Pf-Int, nous avons réalisé des essais de sur-expression de différentes protéines de fusion dans le parasite. Nous avons essayé de déterminer l’impact de trois codons d’initiation différents ainsi que l’impact de la présence de la région N-terminale (1-190aa) de Pf-Int sur sa localisation subcellulaire en utilisant une chimère entre la partie N-terminale et la protéine GFP. Lors de ces travaux, nous avons réussi à sur-exprimer différentes régions de Pf-Int sous forme recombinante dans E. coli. Nous l’avons d’abord caractérisé par des études biophysiques. Ainsi nous avons pu déterminer, par dichroïsme circulaire (CD), le contenu en structures secondaires de Pf-Int, qui est proche de celui des autres membres de la même famille. Nous avons également démontré sa stabilité par CD couplé à la dénaturation thermique. Le spectre RMN-1D a aussi pu être enregistré. La troisième partie de nos travaux a concerné l’identification des cibles ADN de Pf-Int. Deux stratégies de recherche de cibles par affinité ont été utilisées au laboratoire en utilisant une première bibliothèque de séquences synthétisées chimiquement et une deuxième bibliothèque formée de fragments d’ADN génomique de P. falciparum. Ces deux approches ont permis l’identification de deux séries de cibles ADN. Grace aux cibles ADN identifiées, nous avons pu démontrer l’interaction de différents fragments de Pf-Int avec ces cibles par des expériences de retard sur gel natif (EMSA). Nous avons aussi pu démontrer que les protéines recombinantes sont actives in vitro. En effet, ces dernières sont capables de former des complexes covalents en présence de l’ADN cible. La conservation de la protéine, ainsi que son expression différentielle nous laisse à penser que son rôle est certes loin d’être élucidé, mais que Pf-Int reste une cible potentielle pour P. falciparum. / Plasmodium falciparum is a protozoan parasite responsible for the most severe form of malaria. In recent years, cases of resistance to antimalarial drugs have become increasingly frequent and common. In addition to its resistance to drugs currently available, there is no vaccine available against this parasite till now. The identification of new approaches based on the specific inhibition of some of its molecular targets has become vital.The identification of the Pf-Int site specific recombinase in Plasmodium falciparum by analysis of PlasmoDB is a new opportunity to study the role of genetic variation in this parasite as it needs to adapt to its hosts. This ~ 57 kDa protein contains a C-terminal domain carrying the putative tyrosine recombinase conserved active site residues R-H-K-R-(H/W)-Y, an N-terminus with a predicted alpha-helical bundle and a mixed alpha-beta domain resembling Lambda-Int. Here, we show that the sequence is highly conserved among members of the Plasmodia. It is expressed differentially during distinct life stages as estimated by RT-PCR, namely with a peak in the schizont phase. We then tried to show the involvement of Pf-Int in the parasitic cycle. We were able to create a parasite where the Pf-Int gene was knocked-out. The comparison test showed that Pf-Int has apparently no impact on the intraerythrocytic developmental cycle of the parasite, particularly in the cycle length and the growth rate.At the molecular level, we produced two sets of anti-Pf-Int antibodies using the purified recombinant fragment C-162 (residues 162-490). Comparison of protein extracts from KO and wild parasite by Western blot technique using our antibody has failed to identify the endogenous protein in the wild type parasite.We also tried to determine the subcellular localization of Pf-Int and the role of possible alternate initiation codons by over-expressing different constructs in the parasite Plasmodium falciparum. In order to determine the impact of the N-terminal region (1-190aa) of Pf-Int on its subcellular localization, we also created a chimeric protein using a fusion of Pf-Int(1-190aa) with the GFP. We successfully expressed a variety of the recombinant form of Pf-Int in E. coli. We have first determined its secondary structure content by circular dichroism (CD) and its solution stability by thermal denaturation-CD. An 1-D NMR spectrum was also recorded. The third part of our work has involved the identification of the DNA targets of Pf-Int. Two search strategies conducted in the laboratory using a library of chemically synthesized sequences and a second library made of fragments of genomic DNA of P. falciparum. Both approaches have allowed the identification of two sets of target DNA. Secondly, electrophoretic mobility shift assays (EMSA) were used to show its affinity and specificity for DNA. The recombinant proteins were shown to be functional as they form a covalent complex with DNA. Thus Pf-Int could be a potential agent that binds to and alters DNA, either in a specific or in random fashion. Its conservation and differential expression leads us to conclude that although its role is far from being understood, Pf-Int remains a key target for P. falciparum.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-03-15T22:01:31Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNiveisSericos.pdf: 48252101 bytes, checksum: 6d60c3aff86212a2a89095b9b67f48bf (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-03-18T12:22:17Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNiveisSericos.pdf: 48252101 bytes, checksum: 6d60c3aff86212a2a89095b9b67f48bf (MD5) / Made available in DSpace on 2013-03-18T12:22:17Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNiveisSericos.pdf: 48252101 bytes, checksum: 6d60c3aff86212a2a89095b9b67f48bf (MD5) Previous issue date: 1997 / O objetivo desta pesquisa foi avaliar o comportamento dos níveis séricos de cortisol e dehidroepiandrosterona (DHEA) em pacientes com malária por Plasmodium falciparum. Como o cortisol apresenta um efeito imunossupressor e o DHEA um efeito imunoestimulador, estudou- se a correlação entre os níveis destes esteróides e a condição clínica do paciente de malária. A amostra constou de 24 pacientes com malária por P. falciparum não-complicada, sendo 18 do sexo masculino e 6 do sexo feminino, com idade variando de 15 a 47 anos, 12 primoinfectados e 12 multi-infectados, provenientes de área endêmica de malária da Amazônia. Coletaram-se amostras diárias de sangue de 20 em 20 minutos no pré-tratamento (D0), 24 horas após o início da medicação (D1) e no 8º dia de acompanhamento (D7), quando o paciente já se encontrava assintomático. Todos os pacientes apresentavam parasitemia negativa em D7. Dosaram-se: os níveis séricos de cortisol em D0, D1 e D7; DHEA em D0 e D7; os níveis de anticorpos totais IgG anti-P. falciparum, anti-P. vivax, e anticorpos IgM anti-P. falciparum em D0. Comparam-se os níveis séricos de cortisol dos três dias, concluindo-se que os níveis de cortisol eram significativamente mais elevados em D0 do que nos outros dias. Foram correlacionados os níveis de cortisol com a parasitemia, obtendo-se como significativas as correlações entre cortisol D0 e parasitemia D1, assim como cortisol D1 com parasitemia D1, levando-se a deduzir que o cortisol pode interferir na resposta inicial à terapêutica de pacientes com malária por P. falciparum. O cortisol foi correlacionado com a temperatura, tempo de evolução da doença, níveis de anticorpos IgG anti-P. falciparum, não se obtendo resultados estatisticamente significativos, levando a inferir que a temperatura não interfere nos níveis de cortisol e o mesmo não interfere nos níveis de anticorpos, e não apresenta variações importantes com o tempo de evolução da doença. Os níveis de DHEA em D0, foram significativamente mais elevados do que em D7, apesar dos pacientes estarem sintomáticos há mais de um dia, já que um estímulo mantido do eixo hipotálamo-hipófise-adrenal (HPA) leva a uma diminuição deste esteróide. O DHEA foi correlacionado com a parasitemia obtendo-se um resultado significativo na correlação DHEA D0 com parasitemia D1. A correlação entre cortisol e DHEA em D0 não foi significativa (p = 0,057), porém este resultado leva a crer que o DHEA acompanha o aumento dos níveis de cortisol. Obteve-se uma correlação negativa entre DHEA e tempo de evolução de doença, apesar destes níveis estarem aumentados no pré-tratamento. Calculou-se a correlação parcial entre cortisol, DHEA e temperatura, concluindo-se que a temperatura interfere positivamente na correlação cortisol e DHEA. Uma vez que a febre reflete o momento em que ocorre a lise das hemácias secundária a esquizogonia, provavelmente esta lise com conseqüente liberação de citocinas serve como um fator agudizador da estimulação do eixo HPA, sugerindo que a liberação dos dois hormônios apresenta mecanismo comum. A correlação entre DHEA e anticorpos não foi significativa, portanto o DHEA não deve interferir na produção de anticorpos de pacientes com malária por P. falciparum. / The main purpuse of our study was to determine the levels of both cortisol and dehydroepiandrosterone (DHEA) in serum samples from patients suffering from Plamodium falciparum malaria. Since cortisol is potentially immunesupressive, and, conversely, DHEA is inherently immunopotentiating, we sought to assess the possible association between serum levels of these steroids and patient's clinical conditions. We enrolled to participate in this study 24 patients aged 12 to 47 years, of whom 18 were male and 6 female, suffering from uncomplicated P. falciparum malaria. All patients lived in areas of the Amazon were malaria is endemic. Half of them were found to be primo-infected, whereas the others were being reinfected by P. falciparum when recruited for this investigation. Blood samples were obtained from each patients as follows: at 20-minutes intervals during the pre-treatment phase (i. e. on day 0, D0), 24 hours after starting drug therapy (D1) and at the 8th day of follow-up (D7), when patients were asymptomatic. All patients at D7 presented with negative parasitemia. Serum levels of cortisol and DHEA were measured on D0, Dl and D7 and D0 and D7, respectively. In addition, the determination of IgG antibodies to both P. falciparum and P. vivax was performed only on D0. Our results indicated that levels of cortisol in serum samples collected on D0 were significantly higher than those of D1 and D7. High levels of cortisol on D0/D1 and significant parasitemia on D1 led us to postulated that this corticosteroid may interfere with the initial response of P. falciparum-infected patients to treatment. The cortisol levels did not correlate with the intensity of fever, duration of illness and the levels of IgG antibories to P. falciparum. These findings suggest that temperature does not interfere with the cortisol levels, and these, on the other land, do not significantly ralate to either antibody response or the duration of illness. The DHEA levels were found to be significantly more elevated on D0 than on D7, even though patients were already symptomatic for more than one day when first serum samples was taken. The progressive decrease in the DHEA levels is therefore likely to be mediated by a continuous stimulus from the hypothalamic-pituitary-adrenal (HPA) axis. Similarly to cortisol, the DHEA levels on D0 correlated significantly with D1 parasitemia. Thus, it is suggested that in cortisol levels paralels that for DHEA. Of interest, the DHEA serum levels seem to inversely correlate with the duration of illness, in spite of high levels of this steroid detected at the pre-treatment phase. A not significant correlation has been noted if cortisol and DHEA serum levels are compared with temperature. This clinical parameter, however, was found to directly interfere with the correlation that exist between both cortisol and DHEA levels. It is known that fever reflects the occasion when erythrocytes disrupt from the schizogony, with release of cytokines , which act as an acute stimulating factor for the HPA axis. It would therefore be proposed that liberation of both hormones has a commom mechanism. The lack of significant interrelationships between DHEA levels and IgG antibodies indicates that this hormone does not seem to interfere with the production of antibodies by P. falciparum infected patients.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-07T16:03:10Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNivelConcordancia.pdf: 1258819 bytes, checksum: 6d1ffa3e55c412538882010cdc01808f (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-13T16:58:07Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNivelConcordancia.pdf: 1258819 bytes, checksum: 6d1ffa3e55c412538882010cdc01808f (MD5) / Made available in DSpace on 2014-02-13T16:58:07Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNivelConcordancia.pdf: 1258819 bytes, checksum: 6d1ffa3e55c412538882010cdc01808f (MD5) Previous issue date: 2007 / O diagnóstico precoce e o tratamento adequado dos casos de malária é a principal estratégia para o controle da doença. Várias alternativas para o diagnóstico microscópico tradicional foram propostas nos últimos anos, os testes imunocromatográficos que capturam antígenos alvos dos parasitos da malária estão sendo propostos, como o teste OptiMAL-IT® que detecta a desidrogenase lática do Plasmodium sp.. O estudo teve como objetivo a avaliação do nível de concordância entre o teste imunocromatográfico (OptiMAL-IT®) e a gota espessa para o diagnóstico da malária no Município de Mazagão – Amapá. Foram analisados 413 indivíduos com sintomatologia de malária, que procuraram o serviço da Unidade Mista de Saúde de Mazagão, com idade entre 01-68 anos. Os resultados do teste OptiMAL-IT® foram comparados com os resultados obtidos (das amostras) através da gota espessa corada pelo Giemsa. Dos 413 pacientes suspeitos de apresentarem malária, 317(76.8%) eram positivos através da GE e 311 (75.3%) eram positivos pelo TDR. Das lâminas de GE positivas, foram encontrados 27.4% de P. falciparum e 72.6% de P. vivax. O teste OptiMAL-IT® detectou 27.7% de P. falciparum e 72.3% de P. vivax. A sensibilidade obtida com o TDR para o P. falciparum foi de 97.7% e para o P. vivax foi de 98.2%, a sensibilidade global do TDR foi de 98.1% e a especificidade global e para ambas as espécies foi de 100%. Foram encontrados valores preditivos positivos e negativos de 100% e 94.1%, respectivamente. O teste OptiMAL-IT®, teve uma alta concordância com a GE, foi específico e eficiente, podendo ser usado no diagnóstico de malária nas situações onde a microscopia não está disponível. / The precocious diagnosis and the opportune treatment of the cases of malaria is one of the main strategies for the control of the disease. Several alternatives for the traditional microscopic diagnosis were proposed in the last years, the Immunochromatographic tests that capture white antigens of the parasites of the malaria they are being proposed, as the test OptiMAL-IT® that captures the lactic desidrogenase of the Plasmodium sp.. The study had as objective the evaluation of the level of agreement between the Immunochromatographic test (OptiMAL-IT®) and the thick drop for the diagnosis of the malaria in the City of Mazagão – Amapá, Brazil. 413 individuals were analyzed with malaria sintomatology that had looked for the service of the unit of health service of the city, with age among 01-68 years. The results of the OptiMAL-IT® test were compared with the obtained results, of the same samples, through the thick drop red-faced by the Giemsa. Of the 413 patients suspicious to present malaria, 317(76.8%) were positive through GE and 311 (75.3%) were positive for TDR OptiMAL-IT®. Of the positive blades of GE, had been found 27.4% of P. falciparum and 72.6% of P. vivax . The OptiMAL-IT® test detected 27.7% of P. falciparum and 72.3% of P. vivax. The sensibility obtained with TDR for P. falciparum was of 97.7% and for P. vivax was of 98.2%, the global sensibility of TDR was of 98.1% and the global specificity for both the species was of 100%. They were found preditivos values positive and negative of 100% and 94.1%, respectively. The OptiMAL-IT® test had a high agreement with thick drop, it is specific and efficient. It can be used in the diagnosis of malaria in the situations where microscopy is not available.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-12T12:04:47Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_MonitoramentoConcentracaoPlasmatica.pdf: 1602384 bytes, checksum: d99ae72ff74a26891b27d49d62237a6a (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-22T14:14:11Z (GMT) No. of bitstreams: 2 Dissertacao_MonitoramentoConcentracaoPlasmatica.pdf: 1602384 bytes, checksum: d99ae72ff74a26891b27d49d62237a6a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-22T14:14:11Z (GMT). No. of bitstreams: 2 Dissertacao_MonitoramentoConcentracaoPlasmatica.pdf: 1602384 bytes, checksum: d99ae72ff74a26891b27d49d62237a6a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2006 / Governo do Estado do Amapá / A correlação entre resposta parasitológica e a concentração plasmática de quinina + doxiciclina e de mefloquina foi monitorada por 28 dias em trinta pacientes com malária por Plasmodium falciparum no Estado do Amapá. No grupo (A) 12 pacientes receberam o esquema terapêutico oral de quinina (3 dias) + doxiciclina (5 dias), e no grupo (B) 18 pacientes receberam mefloquina oral dose única (1º dia). No grupo (A) quatro pacientes (33,3%) apresentaram plasmódios resistentes do tipo RI, e (66,7%) foram sensíveis (S). A média da concentração plasmática de quinina em D1, D3 e D5 foi de 2,575μg/mL, 2,3334μg/mL e 0,532μg/mL para os (S) e de 2,4667μg/mL, 2,1784μg/mL e 0,450μg/mL para os (RI), não houve diferença estatisticamente significativa entre essas concentrações. No grupo (B), 100% dos pacientes apresentaram plasmódios sensíveis. A média da concentração plasmática da mefloquina foi: D0(0 μg/mL), D1 (0,709μg/mL), D3 (0,543 μg/mL), D5 (0,361 μg/mL), D7 (0,187 μg/mL), D14 (0,125 μg/mL), D21 (0,046 μg/mL) e D28 (0 μg/mL). A correlação entre resposta parasitológica e a concentração plasmática de quinina +doxiciclina foi de 94,23% para os (S) e de 76,70% para os (RI). Houve absorção adequada desses fármacos tanto para os (S), quanto, para os (RI), portanto , não foi a má absorção a causa da recrudescência. Os resultados sugerem que houve resistência do P. falciparum ao esquema administrado. Para os pacientes que receberam mefloquina a correlação foi de 91,82%. Houve absorção e manutenção da concentração desse fármaco durante o período de tempo necessário para completa eliminação dos parasitas. / The correlation between the plasma concentration of quinine and doxycycline and of mefloquine with the parasitological answer was monitored by 28 days in thirty patients with malaria by Plasmodium falciparum in the State of the Amapá. In the group (A) twelve patient received the oral scheme of quinine (3 days) + doxycycline (5 days), and in the group (B) eighteen patients received mefloquine oral unique dose (1º day). In the group (A) four sick (33,3%) presented Plasmodium resistant of the type RI, and (66,7%) were sensible (S). On average of the plasma concentration of the quinine in D1, D3 and D5 was of 2,5750μg/mL, 2,3334μg/mL and 0,532μg/mL for the (S) and of 2,4667μg/mL, 2,1784μg/mL and 0,450μg/mL for the (RI), had not difference statistical significant between those concentrations. In the group (B), patients 100% presented sensible Plasmodium. The average of the concentration plasma of the mefloquine was: D0 (0μg/mL), D1 (0,709μg/mL), D3 (0,543 μg/mL), D5 (0,361 μg/mL), D7 (0,187 μg/mL), D14 (0,125 μg/mL), D21 (0,046 μg/mL) and D28 (0μg/mL). The correlation between parasitological answer and the plasma concentration of quinine + doxicyclyne was of 94,23% for the (S) and of 76,70% for the (RI), the quinine and doxycycline adequately was absorption so much for the (S), as well as, for the (RI), therefore, was not to bad absorption of the drug the cause of the recrudescence. The results suggest that there was resistance of the P. falciparum to the administered treatment. For the patients that received mefloquine the correlation was of 91,82%, having the absorption and maintenance of the drug concentration during the period of necessary time for complete elimination of the parasites.

Cerebral malaria is characterised by an accumulation of infected erythrocytes in the microvasculature of the brain. Plasmodium falciparum infected erythrocytes have been shown to bind to a Human Brain Endothelial Cell line (HBEC-5i) in vitro. This provides a model for the investigation of interactions between P. falcuparum and human brain endothelium. Currently neither the parasite adhesion ligands on infected erythrocytes, nor the host endothelial cell receptors necessary for this interaction have been identified. In this work, the identity of the host receptor on brain endothelial cells was addressed by binding assays of selected and unselected parasites on a wide range of malaria-associated host molecules. The identity of the parasite ligand was investigated by microarray analysis of parasites after selection for cytoadherence to HBEC-5i. The hypothesis being tested was that the gene encoding the parasite cytoadherence ligand would show significant upregulation in selected compared to unselected paarasites. The P. falciparum laboratory strains 3D7, HB3 and IT/FCR3 were selected for binding to HBEC-5i using a panning assay. Compared to unselected parasites, HBEC-5i selected parasites showed a distinct phenotype with reduced platelet-mediated clumping. There was no significant increase in binding of parasites to any of the known endothelial cytoadherence receptors for P. falciparum after selection on HBEC-5i. Binding inhibition assays with various antibodies and soluble receptors did not greatly block the adhesion of parasites to HBEC-5i except for heparin. Altogether, the receptor(s) mediating the interation with HBEC-5i remains unknown. In order to carry out transcriptional analysis of selected and unselected paarasites form all three parasite strains, it was necessary to update the existing microarray chip which is based on the 3D7 genome. This is because each parasite train has a unique repertoire of variant surface antigens (VSAs) including var, rif and stevor genes. Therefore, to fully analysis HB3 and IT genomes. Unique oligonnucleotide probes were then designed for each new sequence and the 3D7-based microarray chip was updated. Transcriptional analysis was then carried out on selected and unselected parasites of all strains. Microarray data clearly indicated that the most highly upregulated genes after selection were group A or group A-like var genes (HB3var3, 3D7_PFDOO2Oc, ITvar7 and ITvar19), showing 11 to over 100 fold upregulation in selected parasites. The rif gene adjacent to the upregulated var gene was also highly expressed. To a lesser extent some exported proteins like RESA-1, PfEMP3 or PHIST family members also showed increased transcription in HBEC-selected parasites (2-3 fold upregulation). Reverse transcriptase-PCR confirmed the upregulation of group A var genes in selected parasites, suggessted that the group A PfEMP1 variants are major candidate ligands for parasite binding to HBEC-5i. These findings are consistent with previous work showing an association between Group A var genes and cerebral malaria.

Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-09-21T19:14:04Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInVitro.pdf: 1439306 bytes, checksum: 7f71a06de5db3f1824c5314ed19c43b4 (MD5) / Rejected by Edisangela Bastos (edisangela@ufpa.br), reason: on 2017-10-10T17:00:49Z (GMT) / Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-10-17T18:38:23Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInVitro.pdf: 1439306 bytes, checksum: 7f71a06de5db3f1824c5314ed19c43b4 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-11-24T15:00:16Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInVitro.pdf: 1439306 bytes, checksum: 7f71a06de5db3f1824c5314ed19c43b4 (MD5) / Made available in DSpace on 2017-11-24T15:00:16Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInVitro.pdf: 1439306 bytes, checksum: 7f71a06de5db3f1824c5314ed19c43b4 (MD5) Previous issue date: 2011-08-12 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O artesunato representa uma das principais drogas utilizadas como antimaláricos em diversos países. É um composto semi-sintético derivado de artemisinina, substancia extraída da planta chinesa Artemisia annua L. Apesar da ampla utilização do artesunato na terapêutica antimalárica, estudos demonstrando seus efeitos genotóxicos e citotóxicos em cultura de linfócitos humanos são ainda hoje quase inexistentes. Portanto, no presente trabalho, avaliamos os efeitos genotóxicos e citotóxicos do artesunato em cultura de linfócitos humanos. Nossos resultados demonstraram aumento significativo (p<0,05) no número de células apoptóticas dos linfócitos, tanto em 24 quanto em 48 h de tratamento. Desta forma, demonstrou-se em nosso trabalho, que o artesunato é uma droga genotóxica e citotóxica em cultura de linfócitos humanos, nas condições avaliadas. / Artesunate is one of the main drugs used as antimalarials in various countries. It is a semi-synthetic compound from artemisinin, a substance extracted from the Chinese plant Artemisia annua L. Despite the widespread use of antimalarial artesunato in malaria treatment, studies demonstrating its cytotoxic and genotoxic effects in human lymphocyte cultures are almost nonexistent. Therefore, in this study, we evaluated possible cytotoxic and genotoxic effects of artesunate on cultured human lymphocytes. A significant increase (p <0.05) in the rate of DNA damage and micronucleus frequency was observed after artesunato treatment. We also observed that artesunato induces a statically significant increase (p <0,05) in the number of apoptotic cells in both 24 and 48 h of treatment. Thus, we conclude in our work that artesunate is a highly cytotoxic and genotoxic drug in cultured human lymphocytes.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-03-22T12:53:35Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoInVitro.pdf: 4204396 bytes, checksum: a66ea9273f20dfc9a3cd37f2eab74163 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-03-27T12:50:35Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoInVitro.pdf: 4204396 bytes, checksum: a66ea9273f20dfc9a3cd37f2eab74163 (MD5) / Made available in DSpace on 2017-03-27T12:50:35Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoInVitro.pdf: 4204396 bytes, checksum: a66ea9273f20dfc9a3cd37f2eab74163 (MD5) Previous issue date: 2015-10-23 / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / A malária é uma das patologias infecto-contagiosas mais graves no mundo, apresentando distribuição geográfica bastante extensa em zonas tropicais. Seu tratamento é baseado na administração de drogas específicas, como, a artemisinina e seus derivados: artesunato, o qual será objeto deste estudo, e artemeter. O artesunato, é um composto semi-sintético derivado da artemisinina, substância extraída da planta chinesa Artemisia annua L. Apesar da ampla utilização do artesunato na terapia antimalárica, e de haver fortes evidências de que outros antimaláricos como, a partenina e a cloroquina, apresentem efeitos genotóxicos in vitro; ainda hoje são escassos os trabalhos que demonstrem seus efeitos genotóxicos em linfócitos humanos. Em estudos prévios realizados no laboratório de citogenética humana, foi demonstrado que o artesunato induz danos genotóxicos e citotóxicos ao DNA de linfócitos humanos em cultura. Apesar destes achados, os mecanismos indutores de tais efeitos não foram devidamente caracterizados devido a limitações das técnicas utilizadas. Assim, o presente estudo teve como objetivo caracterizar in vitro os efeitos genotóxicos e citotóxicos do artesunato em linfócitos de sangue periférico humano utilizando técnicas como FISHMN, ensaios de estresse oxidativo e imunocitoquímica por imunofluorescência. Pretendeu-se através do uso de tais técnicas, elucidar os mecanismos responsáveis pelos efeitos do artesunato no DNA de linfócitos humanos. A partir dos resultados encontrados no presente estudo foi possível inferir que o artesunato induz a formação de ROS e outros radicais livres e que estas substâncias estão causando danos no DNA dos linfócitos humanos em cultura. Assim, as células com o DNA danificado, não sendo capazes de reverter tal condição, ativam a apoptose pelas vias extrínseca e intrínseca. / Malaria is one of the most serious infectious disease in the world, with quite extensive geographic distribution in tropical areas. Its treatment is based on administration of specific drugs, as artemisinin and its derivatives: artesunate, which will be the subject of this study, and artemether. The artesunate is a semi-synthetic compound derived from artemisinin, a substance extracted from the Chinese plant Artemisia annua L. Despite the widespread use of artesunate in antimalarial therapy and the strong evidences that other antimalarials such as partenin and chloroquine present genotoxic effects in vitro; there are few studies that demonstrate artesunate genotoxic effects in human lymphocytes. In previous studies carried out in laboratory human cytogenetics, it was shown that artesunate induces cytotoxic and genotoxic effects in human lymphocytes in vitro. Despite these findings, the mechanisms of these effects have not been adequately characterized due to limitations of the techniques used. This study aimed to assess in vitro the cytotoxic and genotoxic effects of artesunate on human peripheral blood lymphocytes using assays such as FISHMN, oxidative stress and immunocytochemistry by immunofluorescence. We aimed through these tools elucidate the mechanisms responsible for the effects of artesunate in DNA of human lymphocytes. The results found in this study suggest that the artesunate induces the formation of ROS and other free radicals and that these substances are causing DNA damage in human lymphocytes in culture. Thus cells with damaged DNA, not being able to reverse this condition, activate apoptosis through the extrinsic and intrinsic pathways.

Abstract not available

Le développement érythrocytaire du parasite Plasmodium falciparum est composé de deux phases successives : une prolifération intense responsable de la maladie et une différenciation en gamétocytes responsable de la dissémination du parasite. Ce changement de statut de la cellule serait en partie dû à une expression différentielle des gènes, notamment à une régulation transcriptionnelle. Cette régulation nécessite l'interaction de deux acteurs dont la caractérisation devrait aboutir à une meilleure connaissance du développement du parasite et permettre de trouver de nouvelles voies pour combattre la maladie.<br />Après identification des promoteurs de gènes, des éléments connus chez les autres eucaryotes ainsi que des éléments dits spécifiques de Plasmodium ont été recherchés à l'aide de différents programmes bioinformatiques, puis regroupés en modules. Les différentes familles de gènes dont l'expression est cordonnée ou altérée par l'expression diminuée d'un facteur de transcription, devraient partager dans leurs promoteurs des éléments de régulation leur permettant d'être exprimées à un moment précis du développement.<br />Des facteurs se liant à l'ADN et impliqués dans la régulation transcriptionnelle ont été recherchés dans le génome du parasite. Des phases ouvertes de lecture codant des facteurs appartenant aux familles de protéines à domaine Myb, à doigt de zinc ou encore avec une architecture beta ont été identifiées et les protéines correspondantes modélisées. Le clonage et la caractérisation biochimique de trois de ces facteurs ont confirmé la pertinence de la mise en évidence informatique de ces protéines.