Effective malaria vaccine might help improve control strategies against malaria, but the complexity of interactions between the parasite and its hosts poses challenges. The asexual blood stage P. falciparum antigen Pf332 has potentials as one of the proteins in understanding the complex host-parasite interactions. The interest in Pf332 as a target for parasite neutralizing antibodies, evolved from previous studies demonstrating that Pf332-reactive antibodies inhibits parasite growth in vitro. The presence of natural P. falciparum infection also indicated that Pf332 has the ability to induce protective antibodies. In paper I, we identified and characterized the immunogenicity of a C-terminal region of Pf332. Immunological analyses carried out with this fragment revealed that rabbit anti-C231 antibodies possess parasite in vitro inhibitory capabilities. In paper II, the functional activity of C231 specific antibodies was confirmed with human-affinity purified antibodies, where the antibodies inhibited late stage parasite development, by the presence of abnormal parasites and disintegrated red cell membranes. Epidemiological data from malaria endemic area of Senegal (Paper III & IV), showed that antibodies were reactive with two different fragments of Pf332 (C231 and DBL). Distribution of anti-C231 antibodies in the IgG subclasses, gave similar levels of IgG2 and IgG3. The levels of anti-C231 antibodies were associated with protection from clinical malaria, but with DBL reactive antibodies IgG3 was associated with protection from clinical malaria. We hereby conclude that antigen Pf332 contains immunogenic epitopes, and is a potential target for parasite neutralizing antibodies. The Pf332 protein should thus be considered as a candidate antigen for inclusion in a subunit P. falciparum malaria vaccine. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (M.Sc. (Biochemistry))--Mahidol University, 1982.

Thesis (Ph.D. (Tropical Medicine))--Mahidol University, 2006. / LICL has E-Thesis 0014 ; please contact computer services. LIRV has E-Thesis 0014 ; please contact circulation services.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 126-137).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 9, 2009). Includes bibliographical references.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2013-08-06T19:23:03Z No. of bitstreams: 1 SARAH_RESENDE_MBCM_Final.pdf: 6553798 bytes, checksum: cb7ea34b3f0a947f3d81c20d5448938f (MD5) / Made available in DSpace on 2013-08-06T19:23:03Z (GMT). No. of bitstreams: 1 SARAH_RESENDE_MBCM_Final.pdf: 6553798 bytes, checksum: cb7ea34b3f0a947f3d81c20d5448938f (MD5) Previous issue date: 2013 / Cepas de Plasmodiumresistentes a diferentes drogas têm sido descritasao redor do mundo. Embora os mecanismos de desenvolvimento de resistência não sejam bem conhecidos, sabe-se que defeitos nos sistemas de reparo do DNA podem estar envolvidos. Esses defeitos estão relacionados principalmente a mutações nas enzimas do sistema de reparo de mal pareamento do DNA ou mismatch repair (MMR) e já foram descritos em populações naturais de diversos organismos. Devido ao conhecimento limitado sobre o sistema MMR de Plasmodium, faz-se necessário um amplo estudo sobre os genes que codificam as proteínas envolvidas nesse sistema. Neste trabalho, foi realizado um estudo sobre as enzimas envolvidas no sistema de reparo do mal pareamento do DNA em Plasmodium: variabilidade intra e interespecífica em Plasmodium, principalmente nos domínios funcionais, e comparação entre níveis de expressão entre cepas/isolados de P. falciparum.Os parasitos foram também avaliados quanto ao número de cópias e expressão dos genes gch-1emdr1. Foram identificadas proteínas pertencentes às classes MSH2, MSH6, MLH1 e PMS1. As sequências de proteínas mostraram-se muito conservadas, tanto entre o gênero Plasmodium, quanto em relação a outros organismos distantes evolutivamente. Foi encontradaum proteína homóloga a MutS que possui os domínios I e V, mas ainda não identificada quanto à sua classificação. O gene codificador desta proteína teve sua expressão confirmada neste e em outros trabalhos. Alguns SNPs foram encontrados em cepas/isolados depositados no PlasmoDB, no entanto, o sequenciamento da região que compreende os principais domínios funcionais apontou apenas 1 SNP na proteína PMS1. Os genes estudados, em sua maioria, apresentaram-se mais expressos entre 10 e 30 horas após a sincronização. W2 e 3D7 apresentam 2 cópias do genes gch-1e mdr1. BHZ apresentou apenas 1 cópia do mdr1. Os resultados da análise de expressão desses genes ligados à resistência concordam com os resultados encontrados para o número de cópias gênicas. Este estudo fornece uma análise ampla das principais enzimas do MMR e será importante para estudos futuros do papel funcional destas enzimas e seu envolvimento no desenvolvimento de resistência às drogas. / Drug-resistant Plasmodiumstrains have been reported world wide. The mechanisms underlying resistance development are not well understood, but failure in DNA repair could be involved in this process. This failure is mainly related to mutations in the enzymes of the DNA mismatch repair (MMR). Because of the limited knowledge about the PlasmodiumMMR system, it is necessary a comprehensive study about the genes encoding proteins involved in this system. In this work, we studied the enzymes involved in the PlasmodiumMMR, considering the intraspecific and interspecific variability in Plasmodium, especially within the functional domains and comparing the expression levels between strains/isolates of P. falciparum.Parasites were also assessed for copy number and expression of the genes pfgch-1and pfmdr1. We identified proteins related to MSH2, MSH6, MLH1 and PMS1. The protein sequences were very conserved among the genus Plasmodium, as well in relation to other evolutionarily unrelated organisms. We found a putative protein homologous to MutS showing the domains I and V, butnot classified yet. The gene encoding this protein has its expression confirmed here and in other previous studies. SNPs were found in some strains/isolates deposited in PlasmoDB, however, the sequencing of the region comprising the main functional domains showed only one SNP in PMS1. The genes studied, mostly, were more expressed between 10 and 30 hours after synchronization. W2 and 3D7 showed 2 copies of the gene gch-1 and mdr1. In BHZ, only one copy of the mdr1 were founded. The results of expression of these genes related to the resistanceagree with the findings for the gene copy number. This study provides a comprehensive analysis of the major enzymes of the MMR and will be important to further functional studies of this enzymes and their role in drug resistance development.

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2012-08-13T04:12:34Z No. of bitstreams: 1 bianca_e_gama_ioc_bp_0042_2011.pdf: 1968573 bytes, checksum: 439595e4d28df18cd5a083cc9b3673ad (MD5) / Made available in DSpace on 2012-08-13T04:12:34Z (GMT). No. of bitstreams: 1 bianca_e_gama_ioc_bp_0042_2011.pdf: 1968573 bytes, checksum: 439595e4d28df18cd5a083cc9b3673ad (MD5) Previous issue date: 2011 / Instituto Nacional de Câncer. Centro de Transplantes de Medula Óssea. Rio de Janeiro, RJ, Brasil. / A malária, doença infecciosa causada por parasitas do gênero Plasmodium, é um conhecido flagelo das populações humanas desde a antiguidade. Na falta de uma vacina, a política de controle baseia-se principalmente no diagnostico rápido e no tratamento dos casos. Devido ao impacto da quimiorresistência parasitária no controle, faz-se necessário o constante monitoramento da eficácia de drogas. Portanto, para contextualizar o Brasil no panorama mundial, nosso objetivo se constituiu em realizar um estudo descritivo sobre a ocorrência de mutações (SNPs) nos genes considerados marcadores moleculares associados à resistência como pfcrt, pfmdr1, pfdhfr, pfdhps e pvdhfr, assim como em genes potencialmente associados à quimiorresistência (pfatpase6 e pvmdr1) em isolados de P. falciparum e P. vivax coletados nas cidades Amazônicas de Porto Velho, Paragominas e Manaus. Para tal, utilizamos PCRs seguidas do sequenciamento de DNA. Ao investigarmos a ocorrência de mutações nos genes pfcrt, pfdhfr e pfdhps em amostras de P. falciparum provenientes de Porto Velho e Paragominas foi possível detectar 1 isolado apresentando um haplótipo ou perfil de sensibilidade CVMNK no gene pfcrt e ACNSVI no gene pfdhfr e, além disso, pudemos observar uma redução no numero de mutações no gene pfdhps, ao confrontarmos os nossos resultados com os de um estudo retrospectivo. Em relação ao P. vivax, a análise do gene pvdmr1 num conjunto de amostras provenientes de Paragominas revelou o predomínio de mutações no códon 976 do gene pvmdr1 (preliminarmente associado com a resistência à cloroquina) e a presença de haplótipos duplo-mutante FRTNI e triplo-mutante FRTNL para o gene pvdhfr. dando continuidade à caracterização de P. falciparum no Brasil, foi estabelecido um registro de base da ocorrência de mutações nos genes pfmdr1 e pfatpase6, em amostras procedentes de Porto Velho, Paragominas e Manaus. Tal análise permitiu a identificação de um haplótipo prevalente NEF/CDVY no pfmdr1, assim como identificação de mutantes em um único códon ou duplo-mutantes (630 e/ou 402) no caso do gene pfatpase6. também não foi encontrada a mutação 769N, associada com a diminuição da susceptibilidade ao arteméter. Concluímos que: (a) existem no Brasil popluações parasitárias de P. falcipoarum portando haplótipos sensíveis para o gene pfcrt e o pfdhfr; (b) a mutação no códon 976 no gene pfmdr1 pode não ser válida para o monitoramento da resistência à cloroquina em isolados brasileiros de P. vivax; (c) as populações de P. vivax avaliadas apresentaram mutações no gene pvdhfr, apesar de nunca ter sido instituído o tratamento com sulfadoxina-pirimetamina para malária vivax e; (d) as amostras de P. falciparum avaliadas não portavam todos os alelos do gene pfmdr1 associados a um potencial desenvolvimento de resistência à combinação arteméter-lumefantrina / Acknowledged as a febrile syndrome of human populat ions since ancient times, malaria is an infectious disease caused by parasites of the Plasmodium genus. In the absence of a reliable vaccine, the c ontrol policy is based mainly on diagnosis and case management. Since para site chemoresistance is a major factor hampering th e disease control, there is a need for drug efficacy surveillance. Therefore, to place Brazil into the w orld scenario, our goal was to conduct a descriptive study on the occurrence of mutations (SNPs) in genes acknowledge d as molecular markers to chemoresistance as pfcrt , pfmdr1 , pfdhfr , pfdhps and pvdhfr , as well as in genes potentially associated with chemoresistance ( pfatpase6 and pvmdr1 ) in isolates of P. falciparum and P. vivax collected in Porto Velho, Manaus and Paragominas ci ties in Brazilian Amazon. With this aim, we used PC R technique followed by DNA sequencing. The analysis of mutations in pfcrt , pfdhfr and pfdhps genes in P. falciparum samples from Porto Velho and Paragominas allowed us to detect a single isolate presenting a sensitivity profile CVMNK in the pfcrt gene and ACNCSVI in the pfdhfr gene. Additionally, we could observe a decrease in the mutations number for the pfdhps gene, when comparing our results with those from a retrospective study. Concerning P. vivax , the analysis from Paragominas samples revealed th e full predominance of mutations at codon 976 in the pvmdr1 gene (preliminarily associated with chloroquine resistance), and the presence of double-and triple- mutant haplotypes (FRTNI and FRTNL) for the pvdhfr gene. Lastly, to establish a genotypic baseline for pfmdr1 and pfatpase6 genes, P. falciparum isolates from Porto Velho, Manaus and Paragominas were also evaluated. This analysis allowed us to identify a major haplot ype NEF/CDVY in pfmdr1 gene, as well as to detect a single-mutant (in 630 or 402 codons) and double-mutant (in 630 and 402 codons) when pfatpase6 gene was surveyed. The 769N mutation, associated w ith decreased susceptibility to artemether, was not detected. We conclude that: (a) there exist P. falciparum populations presenting sensitive alleles for the gene pfcrt and pfdhfr in Brazil; (b) the 976 mutation in the pvmdr1 gene may not be valuable for monitoring chloroquine resistan ce in Brazilian P. vivax isolates; (c) P. vivax populations herein investigated presented mutations in the pvdhfr gene, although sulphadoxine-pyrimethamine therapy has never been preconized for malaria vivax and; (d ) the P. falciparum samples analyzed did not carry all alleles in the pfmdr1 gene that were associated with a potential develop ment of resistance to artemether- lumefantrine

Artemisinin-resistant Plasmodium falciparum parasites have been reported in the Greater Mekong Subregion since 2007. Artemisinin combination therapy (ACT) is the mainstay of antimalarial treatment and is responsible for decreases in malaria-related morbidity and mortality over the past fifteen years. The slowed parasite clearance rates following ACT indicates resistance to artemisinin derivatives. This resistance places increasing selective pressure for variants or traits that confer resistance to the partner drug used in combination and has led to the rapid failure of several partner drugs. While a single nucleotide polymorphism (SNP) in kelch13 has been shown to mediate some resistance phenotypes, the complete mechanism of artemisinin resistance is poorly understood. The known mechanisms of resistance hint at a connection to autophagy, an intracellular pathway that cells use to degrade waste molecules or organelles in response to stress and starvation, which is poorly characterized in Plasmodium. In this doctoral thesis project, I investigated the role of an autophagy-like mechanism in P. falciparum in the mechanism of artemisinin resistance. I found a SNP in autophagy-related gene 18 (atg18) that was associated with clinical delayed parasite clearance half-life following ACT. This gene encodes PfAtg18, a protein that I characterized as being similar to mammalian/yeast homologues in terms of structure, binding abilities, and ability to form puncta in response to stress. In order to investigate the contribution of the mutation in this protein, I edited the atg18 gene using CRISPR/Cas9 and screened the mutant and parent parasites against a drug library of over 6000 unique compounds. I discovered that while the SNP did not change the mutant parasite's susceptibility to any of the antimalarial compounds using a 72-hour drug pulse, it did alter the susceptibility to 227 other compounds. Further, I found that the SNP offers parasites a fitness advantage by allowing them to grow better in nutrient-limited settings. Finally, I determined that neither this atg18 SNP nor several polymorphisms in kelch13 modulate a dormancy phenotype that appears to be involved in the artemisinin-resistance mechanism.