Zugl.: Giessen, Univ., Diss., 2008

Marburg, Univ., Diss., 2009

Univ., Diss., 2010--Würzburg.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (Ph.D.)--Duquesne University, 2005. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references and abstracts.

Made available in DSpace on 2016-03-28T12:42:11Z (GMT). No. of bitstreams: 2 janaiara_cunha_ioc_mest_2014.pdf: 5340118 bytes, checksum: d30f5606e8d7731758e5708a036d73ec (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os primatas neotropicais dos gêneros Saimiri e Aotus são modelos recomendados pela OMS para estudos experimentais da malária humana, pois são susceptíveis à infecção por plasmódios humanos e reproduzem de forma relativamente confiável a patologia e a imunidade observadas em humanos. Eles apresentam, entretanto, uma importante limitação: a necessidade de se esplenectomizar o animal para que as parasitemias sejam elevadas e consistentes. O objetivo desse estudo foi avaliar a influência da administração de lipossomo-clodronato (LC), utilizado para depletar monócitos/macrófagos em vários modelos experimentais, na infecção por Plasmodium falciparum em Saimiri sciureus. Realizou-se um experimento in vitro utilizando cultivo de esplenócitos de Saimiri, incubados em presença ou não de LC, quantificando-se a depleção de macrófagos por citometria de fluxo. Primeiro experimento: foram utilizados seis animais não esplenectomizados divididos em dois grupos que receberam 1mL PBS ou de LC (5mg/mL) a partir do dia 0 de infecção com inóculo contendo 106 hemácias parasitadas com P. falciparum (cepa FUP). Segundo experimento: foram utilizados 14 animais não esplenectomizados divididos em seis grupos - três grupos não infectados, com dois animais cada, que receberam 1mL PBS, ou 0,5mL ou 1mL de LC e três grupos infectados no dia 0 que receberam as mesmas administrações; sendo que o grupo que recebeu PBS tinha dois animais e os grupos que receberam LC tinham três animais cada. Em ambos os experimentos as administrações foram por via intravenosa duas vezes por semana a partir de dia 0. Após eutanásia, foram realizados exames histopatológicos e ensaio de expressão de citocinas em células esplênicas No ensaio in vitro o LC induziu uma citotoxicidade dose-dependente de monócitos/macrófagos. No primeiro experimento o grupo tratado com LC apresentou aumento na parasitemia alcançando valores superiores a 20% no d11 e requerendo tratamento. O grupo tratado com PBS apresentou parasitemia de 0,029% a 8,15% e foi capaz de controlar espontaneamente a infecção no d18. No segundo experimento os animais infectados que receberam 0,5mL LC apresentaram parasitemias mais elevadas (pico entre 16,1% e 26,7% entre d10 e d14) do que os outros grupos (grupo PBS - picos entre 6,3% e 17,7% entre d12 e d13, e grupo 1mL LC \2013 pico entre 4,8% e 8,8% entre d11 e d15). Em todos os animais infectados, a temperatura foi relacionada com a presença de parasitemia e a hemoglobina e o hematócrito diminuíram alcançando valores mínimos quando parasitos já não são mais detectáveis na circulação. Os animais toleraram clinicamente a administração de LC, mas apresentaram sinais histopatológicos de toxicidade hepática. Em conclusão, o LC é capaz de promover parasitemias mais altas em infecções de P. falciparum em primatas S. sciureus. A infecção esteve associada a evidências de depleção parcial de macrófagos como diferenças no tamanho dos baços e menor presença de ferro nos baços e fígados dos animais que receberam LC. Ensaios ainda precisam ser realizados para se estabelecer volumes mínimos funcionais e superar o problema da aparente toxicidade hepática / The WHO recommends the Neotropical primates of the genus Saimiri and Aotus as models for experimental studies of human malaria. The se monkeys are susceptible to infection by human P lasmodi a and reproduce relatively reliably the pathology and immunity observed in man . However, the model has a s limitation the need of splenectomy for the obtention of high and consistent parasitemias . The aim was to evaluate the influence of administration of clo dronate - liposome (CL), used to deplete monocytes/macrophages in several experimental models, in Plasmodium falciparum infected Saimir i sciureus . We conducted an experiment using in vitro culture of spleen cells from Saimiri incubated in the presence or abs ence of C L and quantifying the depletion of macrophages by flow cytometry. Other experiment s were: Exp. 1 : six non splenectomized animals were divided into two groups receiv ing 1 mL of PBS or 1 mL of C L (5mg/mL) from the d ay 0 of infection with 10 6 P. falc iparum (FUP strain) parasitized erythrocytes . Exp. 2 : 14 non - splenectomized animals were divided in six groups - three non - infected groups , with two animals each, receiv ing 1mL PBS, 0.5 mL or 1 mL CL ; and three infected groups receiving the same injections . The group that received PBS had two animals each; and the groups that received CL had three animals each . In both experiments the injections were intravenous, two times a week from d0. After euthanasia, histopathological examination and testing of cytoki ne expression in splenic cells were performed. In vitro assay : the CL induced a dose - dependent monocyte / macrophage cytotoxicity. In Exp 1, the CL treated group showed an increase in parasitemia reaching values higher than 20% at d11 and requir ed treatm ent. T he group treated with PBS showed parasitemia from 0.029 % to 8.15% and was able to spontaneously control the infection by d18. In Exp 2, the infected animals receiv ing 0.5 ml CL showed higher parasitaemia (peak s between 16.1% and 26.7% between d10 and d14) than the other groups (PBS group - peaks between 6.3% and 17 7% between d12 and d13, and 1mL CL group - peak between 4.8% and 8.8% between d11 and d15). In all infected animals, the temperature was related to the presence of parasitaemia and hemoglob in and hematocrit decreased , reaching minimum values during or after clearance of the parasite. The animals tolerated clinically the CL administration but showed histopahological signs of liver toxicity. Animals receiving CL showed less iron in the spl ee n , suggesting a decreased erythrophagocitosis . In conclusion, the CL is capable of promoting higher parasitemia in P. falciparum infect ed S. sciureus primates. The infection was associated with evidence s of partial depletion of macrophages , such as differe nces in the spleen size s and decreased presence of iron in the spleens and livers of animals receiv ing CL. However , more tests still need to be conducted to define the lower volum es of LC that are still functional , to overcome the problem of the apparent l iver toxicity.

Malaria affects the daily lives of more than 2 billion people worldwide and has been estimated to result in 300-500 million clinical cases annually leading to approximately 2 million deaths, mainly caused by the most virulent malaria species, Plasmodium falciparum. The lack of a vaccine and the rapid emergence and spread of drug resistant strains of P. falciparum, necessitate the development of new antimalarials and the identification and validation of new parasite-specific therapeutic targets. Numerous studies directed at interfering with the polyamine biosynthetic pathway in P. falciparum have shown its potential as a target for the development of a new class of antimalarials. The essential nature of P. falciparum spermidine synthase (PfSpdSyn), an enzyme in the polyamine pathway of the parasite warranted the further investigation to find novel lead compounds. The high cost and attrition rate of drug discovery has resulted in the implementation of smart drug discovery platforms in both academia and industry. The strategy implemented in this study involved the development of a dynamic receptor-based pharmacophore model (DPM) of PfSpdSyn complemented by a knowledge-based rational design strategy. The use of pharmacophore models to identify lead compounds has become increasingly popular over the last decade and has been shown to be a reliable method in the drug discovery process. The development of a DPM allows for the incorporation of protein exibility within the drug design process. This methodology results in a wealth of information of the chemical space of the active site and was incorporated in designing new inhibitors against PfSpdSyn using a knowledge-based rational design strategy. The active site of PfSpdSyn was subdivided into four binding regions (DPM1-DPM4) to allow for the identi cation of fragments binding within these speci c binding regions. DPMs representative of the chemical characteristics of each binding region were constructed and subsequently screened against the drug-like subset of the ZINC database. From the screens a total of nine compounds were selected for in vitro testing, complementing each other in exploring specific active site binding characteristics. From these compounds a new lead compound N-(3-aminopropyl)-cyclohexylamine (NAC; Ki 2.8 μM) was identified for PfSpdSyn. NAC was specifically designed to bind in both the putrescine and decarboxylated adenosylmethionine cavities by chemically bridging the catalytic center and was confirmed by kinetic studies. NAC shows great potential for lead optimization to increase its binding affinity. This study then paves the way for lead optimization and possibly the development of a novel antimalarial. The development of a DPM for PfSpdSyn has seen the establishment of this methodology in the Bioinformatics and Computational Biology Unit, Department of Biochemistry at the University of Pretoria. It can be concluded that the development of a DPM complemented by a knowledge-based rational design strategy is an effective approach for the identification of novel lead compounds in the presence of a 3D target structure. This paves the way for more studies on both malaria and other drug targets using DPMs. Copyright / Thesis (PhD)--University of Pretoria, 2009. / Biochemistry / unrestricted

The in vitro antimalarial activity of clofazimine and seven of its analogues, all TMP(tetramethyl-piperidyl group)-derivatives except 8669, against the R8-1 and pfUP-1 laboratory strains of Plasmodium falciparum was investigated using a flow cytometric procedure. The flow cytometric method was compared with microscopy and radiometry for efficiency in quantitating the level of parasitemia in malaria cultures. The flow cytometric method compared well, as determined by the 81and and Altman measure of agreement, with both microscopy and radiometry and was chosen for use in this study due to its speed, precision and convenience (includes a fixing step that allows samples to be evaluated at anyone time). The riminophenazine agents were found to exhibit antimalarial action of varying degrees: B669, B4100, B4103, B4112 and B4158 showed the best activity followed by B4121 and B4169. Clofazimine did not exhibit any activity at concentrations up to 2µg/ml in this system. Their effective concentrations in vitro were comparable to that of standard antimalarial agents such as chloroquine. The agents B4103 and B4112 exhibited additive antimalarial activities when combined with chloroquine. The inclusion of the TMP group and extent of halogenation of six of the riminophenazines tested indicate that it is these structural properties which are the major determinants of the antiplasmodial activity. This is the first study to establish an antiplasmodial activity of riminophenazines and further tests are necessary to establish their antiparasitic mode of action and therapeutic potential in animal models of experimental chemotherapy. / Dissertation (MSc)--University of Pretoria, 2010. / Pharmacology / unrestricted

Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2004 / Malaria is the most prevalent parasitic disease in the world and the emergence of drug resistant strains of Plasmodium falciparum has made the search for new antimalarial drugs important. Protein kinases play an important role in cellular function and the phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in diverse cellular processes such as glucose transport, cell survival and proliferation. A homology based approach identified an open reading frame (ORF) coding for the catalytic region of part of the 6.4 Kb ORF of PFE0765w gene sequence found at plasmoDB. The ORF consisted of 1 758 base pairs which coded for a 586 amino acid protein with a molecular weight of 68.5 KDa. The PfPI3K ORF was amplified from P.falciparum DNA, subcloned into an expression vector and the sequence verified. Analysis of the expressed protein obtained by Western blotting and probing with anti-His monoclonal antibody showed a protein of 68.5 KDa as well as some smaller products. / IT2018

A Research Report Submitted to the School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the Degree of Master of Science in Medicine in Tropical Diseases (Epidemiology and Biostatistics Option). Johannesburg, January 2001 / Background: increasing spread of drug resistance among Plasmodium falciparum poses a serious threat to malaria treatment. The situation is complicated not only because new drugs are expensive and slow in development but also because they must be effective, preferably have a novel method of action, with an acceptable level of adverse effects, and be deployed in such a way as to prolong their use. / IT2018