Thesis (Ph.D. (Tropical Medicine))--Mahidol University, 1999.

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 2003.

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

Introduction Malaria retinopathy is a set of visible changes in the retina which are specific to falciparum malaria. Studies to date have been mostly limited to comatose African children. Retinal changes in adults with severe malaria and severely unwell patients without malaria have been less well studied and the specificity, pathogenesis, diagnostic and prognostic value of malarial retinopathy in adults are not known. Methods A series of observational studies of retinopathy in Bangladesh, India and Malaysia were done from 2008-2012. The aims were to describe the spectrum of retinal changes in falciparum and knowlesi malaria in adults, determine their specificity for severe falciparum malaria, quantify the impact of malaria retinopathy on visual function, understand its pathogenesis and assess the potential contribution of retinopathy to confirming diagnosis of malarial coma, predicting prognosis and understanding pathogenesis of cerebral malaria. Results 495 patients were enrolled and underwent retinal photography (305 with P. falciparum malaria (112 cerebral, 68 noncerebral severe, 125 uncomplicated), 44 P. knowlesi, 43 sepsis, 41 encephalopathy and 62 healthy). Retinal whitening and white-centred haemorrhages were common and specific to severe falciparum malaria. Retinopathy was most common and severe in cerebral (88%) and fatal (91%) falciparum malaria. Moderate-severe retinopathy was 95% specific for cerebral malaria in comatose patients, and its severity correlated with depth of coma. Vessel whitening was not seen and papilloedema was rare. In noncerebral severe falciparum malaria, retinopathy predicted increased likelihood of later development of coma and death. Retinal findings in Bangladeshi children were similar to those in adults. Optic nerve sheath diameter was mildly increased and brain swelling minimal on MRI. Severity of retinopathy correlated with plasma lactate, serum bicarbonate, sequestered parasite load and red cell stiffness suggesting a central role for microvascular obstruction in the pathogenesis. Severity of retinal whitening correlated with decreased visual acuity. Conclusions Retinal changes seen in severe P. falciparum malaria in Asian adults is similar, but not identical, to that seen in African children. They have potential to help with diagnosis and prognosis of Asian adults with severe falciparum malaria. Microvascular obstruction is prominent in the pathogenesis of retinopathy and coma in adults whereas raised intracranial pressure is not.

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.

Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.

Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.