Estudos espectroscopicos de hidrogenases bacterianas: papel do níquel no metabolismo de hidrogenio

Tese apresentada para obtenção do grau de Doutor em QuímicaForam isoladas e caracterizadas hidrogenases de bactérias redutoras de sulfato do género Desulfovibrio (DI) (Dlqiqas, (NClB 9332), D=salexiqens (estirpe British Guiana, NClB 8403), Dlbaculatus(estirpe 9974, D5M 1743) e D.desulfuricans (ATCC 27774» e de uma bactéria metanogénica (Methanosarcina (Ns.) barkeri, D5M 800). Utilizou-se como técnica básica de caracterização a espectroscopia de Ressonância Paramagnética Electrónica (RPE), complementada, para a hidrogenase de D.qiqas, com estudos de espectroscopia de Mõssbauer. Todas as enzimas estudadas contêm niqueI e centros Fe/S pertencendo ao grupo das hidrogenases [NiFe]. A hidrogenase de D.qiqas (NClB 9932), tem uma massa molecular de 89,5 kDa, é composta de duas subunidades não idênticas de 62 e 26 kDa, res~ectivamente e contém 11 átomo-g de ferro e 1 átomo-g de niqueI por mole de enzima. Os àtomos de ferro encontram-se agrupados em centros Fe/5: um centro [3Fe-x5] e dois centros [4Fe-45]2+/1+. No estado nativo todos os centros Fe/5 estão no estado oxidado. O centro de niqueI contém um ião niqueI, provavelmente no estado de oxidação Ni 3+. Os estudos espectroscópicos de RPE e M~ssbauer revelaram a ausência de interaccções magnéticas entre estes quatro centros metálicos no estado nativo da enzima. O espectro de RPE do estado nativo apresenta essencialmente dois sinais de RPE: um sinal praticamente isotropico,centrado a g=2,01, detectável a temperaturas até cerca qe 30 K,atribuido, através de estudos de M~ssbauer, a um centro [3Fe-x5]oxidado (5=1/2); um sinal rõmbico, saturado com a potência da radiação de microondas a baixas temperaturas, detectado a temperaturas elevadas (facilmente observável a 77 K), com valores de g a 2,31, 2,23 e 2,02 (Qingl Ni-A). Em algumas preparações da hidrogenase é detectàvel outro sinal r6mbico, de menor intensidade,com valores de g a 2,33 2,16 e 2,02 (§ingl tli-B) a intensidade relativa destes dois sinais rdmbicos pode ser modificada por ciclos de redução/reoxidação anaer6bica da hidrogenase. Por substituição isot6pica com 61Ni foi possivel atribuir inequivocamente estes sinais de RPE rômbicos a espécies paramagneticas de niqueI, possivelmente NiCIII). A hidrogenase de D.gigas foi também isolada a partir de células crescidas em meio enriquecido em 57 Fe, o que permitiu realizar estudos detalhados de espectroscopia de Mõssbauer nos estados oxidado e reduzidos desta enzima.JNICT, INIC, NATO e AI

Heterogeneous malaria transmission in long-term Afghan refugee populations: a cross-sectional study in five refugee camps in northern Pakistan.

BACKGROUND: Afghan refugees in northern Pakistan have been resident for over 30 years and current information on malaria in this population is sparse. Understanding malaria risk and distribution in refugee camps is important for effective management both in camps and on return to Afghanistan. METHODS: Cross-sectional malariometric surveys were conducted in five Afghan refugee camps to determine infection and exposure to both Plasmodium falciparum and Plasmodium vivax. Factors associated with malaria infection and exposure were analysed using logistic regression, and spatial heterogeneity within camps was investigated with SatScan. RESULTS: In this low-transmission setting, prevalence of infection in the five camps ranged from 0-0.2 to 0.4-9 % by rapid diagnostic test and 0-1.39 and 5-15 % by polymerase chain reaction for P. falciparum and P. vivax, respectively. Prevalence of anti-malarial antibodies to P. falciparum antigens was 3-11 and 17-45 % for P. vivax antigens. Significant foci of P. vivax infection and exposure were detected in three of the five camps. Hotspots of P. falciparum were also detected in three camps, only one of which also showed evidence of P. vivax hotspots. CONCLUSIONS: There is low and spatially heterogeneous malaria transmission in the refugee camps in northern Pakistan. Understanding malaria risk in refugee camps is important so the malaria risk faced by these populations in the camps and upon their return to Afghanistan can be effectively managed

Effectiveness of a serological tool to predict malaria transmission intensity in an elimination setting.

BACKGROUND: Sri Lanka achieved the WHO certificate as a malaria free country in September 2016, thus monitoring of malaria transmission using sensitive and effective tools is an important need. Use of age-specific antibody prevalence as a serological tool to predict transmission intensity is proven to be a cost effective and reliable method under elimination settings. This paper discusses the correlation of four anti-malarial antibodies against vivax and falciparum malaria with the declining transmission intensities in two previously high malaria endemic districts i.e. Kurunegala and Moneragala of Sri Lanka. METHODS: Sera was collected from 1,186 individuals from the two districts and were subjected to standard ELISA together with control sera from non-immune individuals to obtain Optical Density (OD) values for four anti-malarial antibodies i.e. anti-MSP1 and anti-AMA1 for both Plasmodium vivax and Plasmodium falciparum. The sero-positive samples were determined as mean OD + 3SD of the negative controls. The sero-prevalence was analyzed against the demographic characteristics of the population. A simple reversible catalytic model was fitted into sero-prevalence data to predict the sero-conversion and sero-reversion rates. RESULTS: Over 60% of the population was sero-positive for one or more antibodies except young children (<10 years). The sero-prevalence was zero in young children and very low in young adults when compared to the older age groups. The model developed for falciparum malaria that assumed the presence of a change in transmission was not significant in the Kurunegala district although significant reduction in transmission was observed when the model was used for P. vivax antibody data in that district. In Moneragala district however, all the serological markers indicated a change in transmission that has occurred approximately 15 years ago. CONCLUSIONS: Assessment of MSP1 and AMA1 anti-malarial antibodies of P. vivax and P. falciparum proved to be useful indicators in predicting transmission under elimination settings as prevailed in Sri Lanka. The sero-conversion rates for the two districts studied are shown to be very low or zero indicating the absence of active and/or hidden transmission confirming a "true" state of elimination at least, in the two study districts in Sri Lanka

USP38, FREM3, SDC1, DDC, and LOC727982 Gene Polymorphisms and Differential Susceptibility to Severe Malaria in Tanzania

Populations exposed to Plasmodium falciparum infection develop genetic mechanisms of protection against severe malarial disease. Despite decades of genetic epidemiological research, the sickle cell trait (HbAS) sickle cell polymorphism, ABO blood group, and other hemoglobinopathies remain the few major determinants in severe malaria to be replicated across different African populations and study designs. Within a case-control study in a region of high transmission in Tanzania (n = 983), we investigated the role of 40 new loci identified in recent genome-wide studies. In 32 loci passing quality control procedures, we found polymorphisms in USP38, FREM3, SDC1, DDC, and LOC727982 genes to be putatively associated with differential susceptibility to severe malaria. Established candidates explained 7.4% of variation in severe malaria risk (HbAS polymorphism, 6.3%; α-thalassemia, 0.3%; ABO group, 0.3%; and glucose-6-phosphate dehydrogenase deficiency, 0.5%) and the new polymorphisms, another 4.3%. The regions encompassing the loci identified are promising targets for the design of future treatment and control interventions

African glucose-6-phosphate dehydrogenase alleles associated with protection from severe malaria in heterozygous females in Tanzania.

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers

Novel Insights Into the Protective Role of Hemoglobin S and C Against Plasmodium falciparum Parasitemia.

Although hemoglobin S (HbS) and hemoglobin C (HbC) are well known to protect against severe Plasmodium falciparum malaria, conclusive evidence on their role against infection has not yet been obtained. Here we show, in 2 populations from Burkina Faso (2007-2008), that HbS is associated with a 70% reduction of harboring P. falciparum parasitemia at the heterozygous state (odds ratio [OR] for AS vs AA, 0.27; 95% confidence interval [CI], .11-.66; P = .004). There is no evidence of protection for HbC in the heterozygous state (OR for AC vs AA, 1.49; 95% CI, .69-3.21; P = .31), whereas protection even higher than that observed with AS is observed in the homozygous and double heterozygous states (OR for CC + SC vs AA, 0.04; 95% CI, .01-.29; P = .002). The abnormal display of parasite-adhesive molecules on the surface of HbS and HbC infected erythrocytes, disrupting the pathogenic process of sequestration, might displace the parasite from the deep to the peripheral circulation, promoting its elimination at the spleen level

Glucose-6-phosphate dehydrogenase polymorphisms and susceptibility to mild malaria in Dogon and Fulani, Mali.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with protection from severe malaria, and potentially uncomplicated malaria phenotypes. It has been documented that G6PD deficiency in sub-Saharan Africa is due to the 202A/376G G6PD A-allele, and association studies have used genotyping as a convenient technique for epidemiological studies. However, recent studies have shown discrepancies in G6PD202/376 associations with severe malaria. There is evidence to suggest that other G6PD deficiency alleles may be common in some regions of West Africa, and that allelic heterogeneity could explain these discrepancies. METHODS: A cross-sectional epidemiological study of malaria susceptibility was conducted during 2006 and 2007 in the Sahel meso-endemic malaria zone of Mali. The study included Dogon (n = 375) and Fulani (n = 337) sympatric ethnic groups, where the latter group is characterized by lower susceptibility to Plasmodium falciparum malaria. Fifty-three G6PD polymorphisms, including 202/376, were genotyped across the 712 samples. Evidence of association of these G6PD polymorphisms and mild malaria was assessed in both ethnic groups using genotypic and haplotypic statistical tests. RESULTS: It was confirmed that the Fulani are less susceptible to malaria, and the 202A mutation is rare in this group (<1% versus Dogon 7.9%). The Betica-Selma 968C/376G (~11% enzymatic activity) was more common in Fulani (6.1% vs Dogon 0.0%). There are differences in haplotype frequencies between Dogon and Fulani, and association analysis did not reveal strong evidence of protective G6PD genetic effects against uncomplicated malaria in both ethnic groups and gender. However, there was some evidence of increased risk of mild malaria in Dogon with the 202A mutation, attaining borderline statistical significance in females. The rs915942 polymorphism was found to be associated with asymptomatic malaria in Dogon females, and the rs61042368 polymorphism was associated with clinical malaria in Fulani males. CONCLUSIONS: The results highlight the need to consider markers in addition to G6PD202 in studies of deficiency. Further, large genetic epidemiological studies of multi-ethnic groups in West Africa across a spectrum of malaria severity phenotypes are required to establish who receives protection from G6PD deficiency

Recommendations for Epidemiological Research in ME/CFS from the EUROMENE Epidemiology Working Group

The European Network on Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (EUROMENE) was established after a successful grant application to the European Cooperation is Science and Technology (COST). This network aimed to assess the existing knowledge and/or experience on health care delivery for people with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) in the European countries and worldwide, and to enhance coordinated research and health care provision in this field. The EUROMENE proposal, was based on the establishment of interrelated working groups (WGs), where the participants contributed with specific knowledge and viewpoints according to their specialties and/or areas of interest. In this paper we outline the work of a multidisciplinary team of researchers, including epidemiologists, clinicians, statisticians, biomedical scientist and heath economists, who set out their recommendations to guide data acquisition for ME/CFS research, aiming to standardise data collection and improve epidemiological research.</jats:p

Characterizing the impact of sustained sulfadoxine/pyrimethamine use upon the Plasmodium falciparum population in Malawi

Background Malawi experienced prolonged use of sulfadoxine/pyrimethamine (SP) as the front-line anti-malarial drug, with early replacement of chloroquine and delayed introduction of artemisinin-based combination therapy. Extended use of SP, and its continued application in pregnancy is impacting the genomic variation of the Plasmodium falciparum population. Methods Whole genome sequence data of P. falciparum isolates covering 2 years of transmission within Malawi, alongside global datasets, were used. More than 745,000 SNPs were identified, and differences in allele frequencies between countries assessed, as well as genetic regions under positive selection determined. Results Positive selection signals were identified within dhps, dhfr and gch1, all components of the parasite folate pathway associated with SP resistance. Sitting predominantly on a dhfr triple mutation background, a novel copy number increase of ~twofold was identified in the gch1 promoter. This copy number was almost fixed (96.8% frequency) in Malawi samples, but found at less than 45% frequency in other African populations, and distinct from a whole gene duplication previously reported in Southeast Asian parasites. Conclusions SP resistance selection pressures have been retained in the Malawian population, with known resistance dhfr mutations at fixation, complemented by a novel gch1 promoter duplication. The effects of the duplication on the fitness costs of SP variants and resistance need to be elucidated