Este trabalho teve como objetivo caracterizar e padronizar métodos para a tipagem molecular de marcadores genéticos, para futuro uso em estudos de genética populacional de Plasmodium vivax na Amazônia brasileira. As amostras sanguíneas utilizadas neste estudo foram colhidas no Brasil, Camboja, Sri Lanka e Estados Unidos. Foram selecionados polimorfismos de única base (SNPs) distribuídos ao longo de cromossomos distintos de P. vivax; para estes polimorfismos, sete ensaios de tipagem de SNPs foram padronizados com sucesso. Com a tipagem molecular, foi possível definir haplótipos que caracterizam cada amostra, assim como identificar infecções geneticamente mistas (co-ocorrência de clones distintos na mesma amostra). No entanto, com este conjunto de marcadores não foi possível agrupar amostras de acordo com sua localização geográfica, por estes marcadores não serem suficientemente informativos do ponto de vista genético. Os sete SNPs avaliados, quando comparados a 13 marcadores de DNA microssatélite, revelaram menor proporção de infecções geneticamente mistas e menor diversidade genética. / This study aimed to characterize and standardize methods for molecular typing of genetic markers to be used in the future in studies of population genetics of Plasmodium vivax population in the Brazilian Amazon. Blood samples used in this study were collected in Brazil, Cambodia, Sri Lanka and United States. Single nucleotide polymorphisms (SNPs), distributed over different P. vivax chromosomes were chosen and seven typing assays were sucessfully standardized. Molecular typing of polymorphisms allowed to define haplotypes that characterize each sample, as well as to identify genetically mixed infections (co-occurrence of different clones in the same sample). However, with this markers set, it was not possible to group samples according to their geographical location, because probably they are not sufficiently genetically informative. Compared to 13 microsatellite markers, these seven SNPs revealed a lower proportion of mixed-clone infections and lower genetic diversity.

O gênero Phoradendron é um dos mais diversos entre as plantas parasitas, agrupando cerca de 230 espécies, que apresentam grande variedade quanto à morfologia e padrões de infestação. A presente dissertação comparou os padrões de infestação de duas espécies de Phoradendron parasitando diferentes hospedeiras: Tapirira guianensis e Cedrela fissilis, além de analisar os efeitos causados por tais parasitas na funcionalidade e na anatomia da madeira destas hospedeiras. Foram realizadas análises tradicionais de anatomia da madeira, análises de microtomografia e experimentos de anatomia funcional com infiltração de corante através da madeira da hospedeira. Os resultados mostraram que, enquanto P. crassifolium forma uma galha concisa sobre os ramos de T. guianensis, Phoradendron sp. é mais agressivo ao espalhar seu sistema endofítico através da madeira de C. fissilis, causando maiores rupturas dos tecidos xilemático e floemático da hospedeira. Sugere-se que tais rupturas poderiam levar a uma alteração local do balanço auxina/citocinina e à liberação de etileno. Esta hipótese é reforçada pelas alterações anatômicas observadas em ambos os casos na interface parasita-hospedeira, tais como hiperplasia e/ou hipertrofia, maior densidade de vasos, alterações no agrupamento dos vasos e redução da espessura da parede celular das fibras. P. crassifolium também provocou severo aumento da densidade de vasos embolisados na madeira de T. guianensis, aumentando também a densidade de vasos e o tamanho de raios, além de reduzir o diâmetro transversal dos vasos e a espessura da parede celular de fibras. Tais efeitos também podem estar relacionados às altas taxas de transpiração e aos potenciais hídricos extremamente baixos apresentados por plantas parasitas, o que pode culminar no aumento da transpiração total da hospedeira, elevando a formação de embolismos, causando estresse hídrico e consequente baixa pressão de turgor nas células derivadas do câmbio. Conclui-se que cada uma das espécies de parasita aqui analisada estabeleceu uma relação única com sua hospedeira, formando diferentes padrões de infestação e alterando de modo particular a xilogênese da hospedeira / Phoradendron is one of the most diverse genera among parasitic plants, comprising ca. 230 species and showing a great variety of morphologies and infestation patterns. The present work compared the infestation patterns of two Phoradendron species on different hosts: Tapirira guianensis and Cedrela fissilis, and also analyzed their effects on the hosts\' functionality and wood anatomy. Traditional wood anatomy procedures were used, along with the technique of microtomography analysis and functional anatomy experiments using a dye ascent through host wood. Results showed that while P. crassifolium forms a defined gall in branches of T. guianensis, Phoradendron sp. is more aggressive when spreading its endophytic tissue trough the wood of C. fissilis, causing major disruptions on both xylem and phloem tissues of the host. Such disruptions could lead to a local change of the auxin/cytokinin balance and probably ethylene liberation. This hypothesis is reinforced by the anatomical alterations observed in both cases at the host-parasite interface, such as hyperplasia and/or hypertrophy, higher vessel density, alterations of vessel grouping and thin cell-walled fibres. P. crassifolium also provoked a severe increase in embolism density on the wood of T. guianensis, along with increases on vessel density and ray size, besides reductions in vessel lumen diameter and fibre cell-wall thickness. These effects could also be related to the high transpiration rates and extremely low water potential presented by parasitic plants, which may increase the host\'s total transpiration, enhancing embolism formation, causing water stress and consequent low turgor in differentiating cambium derivates. In conclusion, each parasitic plant species analyzed established a unique relationship with its host, forming different infestation patterns and differently altering its host xylogenesis

Casos de malária importada, contraídos em região endêmica, mas diagnosticados em um país não-endêmico, são um evento raro mas com desfecho potencialmente fatal. Nosso objetivo foi investigar se a análise de genomas mitocondriais permite inferir a origem geográfica de casos importados de malária vivax diagnosticados nos EUA, comparando os resultados com aqueles obtidos por análise de DNA microssatélite. Foi sequenciado o genoma mitocondrial completo de 63 amostras de P. vivax provenientes de infecções importadas dos EUA, além de 7 amostras do Brasil e 6 do Panamá. A rede de haplótipos com DNA mitocondrial foi construída com 412 sequências e foi possível classificar com precisão a origem geográfica presumida dos isolados da América do Sul, Coréia, Sudeste Asiático e Melanésia, porém os isolados do Sul da Ásia, América Central e África não puderam ser classificados geograficamente. A análise bayesiana realizada com a tipagem de marcadores de microssatélites não apresentou sucesso quanto à classificação geográfica dos isolados de P. vivax. / Cases of imported malaria, infection acquired in an endemic region, but diagnosed in a non-endemic country, are rare but can lead to a fatal outcome. Our objectives were investigate whether the analysis of mitochondrial genomes allows inferring the geographic origin of isolates of P. vivax derived from cases of imported malaria diagnosed in the USA, and compare the performance of mitochondrial genome and DNA microsatellite analysis. We sequenced full mitochondrial genomes from 63 P. vivax isolates collected at the USA from imported infections, and 7 samples from Brazil and 6 Panama. A network of mitochondrial DNA haplotypes was built with 412 genomic sequences and were able to classify accurately isolates from South America, Korea, Southeast Asia and Melanesia according to their presumed geographic origin, but failed to do so with samples from South Asia, Central America and Africa. The Bayesian analysis performed by typing microsatellite markers showed no success on the classification of geographical isolates of P. vivax.

A proteína de ligação em Duffy (PvDBP) dos merozoítos de P. vivax se liga a glicoproteínas de membrana, chamadas de grupo sanguíneo Duffy, conhecidas como DARC. Indivíduos DARC negativos são normalmente resistentes a infecção estágio sanguínea por P. vivax; portanto, PvDBP é um forte antígeno canditato a vacina. Aqui, investigamos respostas de anticorpos contra três proteínas derivadas de PvDBP e MSP119, em 343 indivíduos de uma região Amazônica brasileira. Anticorpos contra Sal III-His, OII-His, Sal III-IV-GST e MSP119-GST foram encontrados em 43,7%, 39,0%, 14,3% e 38,8% dos indivíduos. Os indivíduos FY*BESFY*BES foram os que menos apresentaram anticorpos contra PvDBP, porém encontramos proporções similares de respondedores entre indivíduos com outros genótipos. A análise de sequências revelou muitas variantes da região II de PvDBP em 41 isolados. A mais comum (encontrada em 28,8% dos isolados), difere de Sal I e PNG- O em seis codóns de aminoácidos. Nenhuma variante tipo Sal I, cujo protótipo de vacina está em teste clínico, foi encontrada nos parasitas locais. / The Duffy binding protein (PvDBP) of the P. vivax merozoites, which binds to a erythrocyte membrane glycoprotein known as Duffy blood group antigen for chemokines (DARC). DARC-negative individuals are usually resistant to blood-stage infection with P. vivax; therefore, PvDBP is a major vaccine candidate antigen. Here, we investigated antibody responses to three proteins derived from PvDBP and MSP119, in 343 subjects in the Amazon of Brazil. Antibodies to Sal III-His, OII-His, Sal III-IV-GST and MSP119-GST were found in 43,7 %, 39,0%, 14,3% and 38,8% of the subjects. FY*BESFY*BES individuals were less likely to have antibodies to PvDBP, but we found similar proportions of seropositive subjects with other genotypes. Sequence analysis revealed several region II PvDBP variants in 41 local P. vivax isolates. The most common of them (found in 28,8% isolates) differs from Sal I and PNG-O alleles in six amino acid codons. No Sal I-type variant, from which a PvDBP-based vaccine prototype currently under clinical testing has been derived, was found in local parasites.

A two-wire, low component count soil temperature sensor was developed. The sensor uses one wire for ground and the other wire is used for both power and communication. Pulse width modulation is used to send temperature measurements to the master, where the duty cycle is proportional to the temperature. The sensor parasitically powers itself from the bidirectional data line. In order to reduce the number of components necessary, a microcontroller with an internal temperature sensor is used. Finally, the sensor can receive data from the master on the bidirectional communication line, which is used for calibrating the sensor.

The rapid development of DNA sequencing technologies has opened up new avenues of research, including the investigation of population structure within infectious diseases (both within patient and between populations). In order to take advantage of these advances in technologies and the generation of new types of data, novel bioinformatics tools are needed that won’t succumb to artifacts introduced by the data generation, and thus provide accurate and precise results. To achieve this goal I have create several tools. First, SeekDeep, a pipeline for analyzing targeted amplicon sequencing datasets from various technologies, is able to achieve 1-base resolution even at low frequencies and read depths allowing for accurate comparison between samples and the detection of important SNPs. Next, PathWeaver, a local haplotype assembler designed for complex infections and highly variable genomic regions with poor reference mapping. PathWeaver is able to create highly accurate haplotypes without generating chimeric assemblies. PathWeaver was used on the key protein in pregnancy-associated malaria Plasmodium falciparum VAR2CSA which revealed population sub-structuring within the key binding domain of the protein observed to be present globally along with confirming copy number variation. Finally, the program Carmen is able to utilize PathWeaver to augment the results from targeted amplicon approaches by reporting where and when local haplotypes have been found previously. These rigorously tested tools allow the analysis of local haplotype data from various technologies and approaches to provide accurate, precise and easily accessible results.

Thin-film solar cells based on an absorber layer of chalcogenide compounds (CIGS, CdTe) are today among the most promising photovoltaic technologies due to their long-term ability to gain a foothold in mass commercial production as an alternative to conventional Si solar cells. Despite this success, the physical origin of the defects present in the thin films are still insufficiently elucidated, especially in the compounds of the chalcopyrite family Cu(In_{1x},Ga_{x})(S_{y},Se_{1y})_{2}. The research focuses on the identification and analysis of microstructural defects responsible for the electrical instability of chalcopyrite-based thin-film solar cells with a typical heterostructure arrangement ZnO:Al/i-ZnO/CdS/Cu(In,Ga)Se_{2}/Mo. The non-uniform polycrystalline nature of semiconductor materials in this complex multilayer structure requires a comprehensive analysis of electro-optical, structural and compositional properties associated with the actual morphology at the macroscopic, microscopic or even nanoscopic level. The observed predominant ohmic or non-ohmic current conduction in the dark transport characteristics was also reflected in the slope deviations of the excessive noise fluctuations, which were in the spectral domain exclusively in the form of flicker noise with dependency S_{i} ~ f^{1}. Spatially resolved electroluminescence based on stimulated photon emission by charge carriers injecting into the depletion region, not only showed a significantly inhomogeneous distribution of intensity in planar heterojunction under forward bias, but also revealed light emitting local spots in reverse bias due to a trap-assisted radiative recombination through the high density of defect states. Microscopic examination of the defect-related light emitting spots revealed rather extensive defective complexes with many interruptions through the layers, especially at the heterojunction CdS/Cu(In,Ga)Se_{2} interface. Besides, the high leakage current via these defective complexes subsequently led to a considerable local overheating, which caused a clearly observable structural and morphological changes, such as deviations in absorber layer stoichiometry due to Cu–In–Ga–Se segregation, Cu-rich and Ga-rich grains formation with an occurrence of Se-poor or Cu_{x}Se_{y} secondary phases regions, material redeposition accompanied by evaporation of ZnO:Al/i-ZnO/CdS layers together with the formation of Se structures on the surface around the defects. Within the research, analytical modelling of transport characteristics was implemented with parameters extraction of individual transport mechanisms to understand the non-ohmic shunt behaviour due to leakage current. In addition to the proper current path along the main heterojunction, the proposed model contains parasitic current pathways as a consequence of recombination-dominated charge transport or current conduction facilitated by multi-step tunnelling via high density of mid-gap defect states in the depletion region, ohmic leakage current caused by pinholes or low-resistance paths along grain boundaries in Cu(In,Ga)Se_{2}, or space-charge limited current due to metals diffusion from the ZnO:Al layer and grid Ag contacts through disruptions in i-ZnO/CdS layers.

Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its ability to provide resistance to the drug methotrexate (MTX). The DNA and amino acid sequences are known, and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmaniaare folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis. PTR1 catalyzes the transfer of hydride from NADPH to the 2-amino-4-oxo-pteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of 7, 8-dihydropteridines yielding 5,6, 7, 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates. The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme family. Although this is typical for members of this enzyme family, it does not allow for unambiguous classification in this family. In fact, when the DNA sequence of PTR1was first determined, PTR1 was classified as an aldoketo reductase. To classify PTR1 definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determined; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTR1 were developed. The stereochemistry of hydride transfer The use of [3H]-folate, showed that the ultimate product of PTR1 was 5, 6, 7, 8-tetrahydrofolate. 4R-[3H]-NADPH and 4S-[3H]-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTR1 was coupled to thymidylate synthase (TS), and tritium from 4S-[3H]-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure I-1). The transfer of the pro-Shydride indicates that PTR1 is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase (SDR) family. The kinetic mechanism of PTR1 When NADPH was varied at several fixed concentrations of folate (and vice-versa) V/K (Vmax/KM) showed a dependence upon concentration of the fixed substrate. This is consistent with a ternary complex mechanism, in contrast to a substituted enzyme mechanism that exhibits no dependence of V/K on fixed substrate. Product inhibition patterns using NADP+ and 5-deazatetrahydrofolate (5dTHF, a stable product analog) were consistent with an ordered ternary complex mechanism in which NADPH binds first and NADP+ dissociates last. However, an enzyme-DHF binary complex was detected by fluorescence. Isotope partitioning experiments showed that the enzyme-DHF binary complex was not catalytically competent whereas the enzyme-NADPH complex was. Measurement of the tritium isotope effect on V/K (T(V/K)) at high and low dihydrofolate confirmed that PTR1 proceeds via a steady state ordered mechanism. Rapid quench analysis showed that dihydrofolate was a transient intermediate during the reduction of folate to tetrahydrofolate and that folate reduction is biphasic. Catalytic Residues of PTR1 The amino acid sequences of dihydropteridine reductase and 3-α, 20-β, hydroxy steroid dehydrogenase were aligned to that of PTR1. Based on the results of the alignment, site directed mutagenesis was used to investigate the role of specific residues in the catalytic cycle of PTR1. Variant enzymes were screened based on their ability to rescue a dihydrofolate reductase (DHFR) deficient strain of E. coli. Selected PTR1 variants (some complementing and some non-complementing) were purified and further characterized. Tyrosine 193 of the wild type enzyme was found to be involved in the reduction of pteridines, but not in the reduction of 7, 8-dihydropteridines, and eliminated the substrate inhibition of 7, 8-dihydropteridines observed with the wild type enzyme. Both PTR1(K197Q) and PTR1(Y193F/K197Q) had decreased activity for all substrates and low affinity for NADPH. In contrast to the wild type enzyme, NADPH displayed substrate inhibition towards PTR1(K197Q). All PTR1(D180) variants that were purified were inactive except for PTR1(D180C), which showed 2.5% of wild type activity with DHF. The binary complexes of PTR1(D180A) and PTR1(D180S) with NADPH showed a decrease in affinity for folate. Based on the kinetic properties of the PTR1 variants, roles for Y193, K197, and D180 are proposed. In conjunction with D180, Y193 acts as a proton donor to N8 of folate. K197 forms hydrogen bonds with NADPH in the active site and lowers the pKaof Y193. D180 participates in the protonation of N8 of folate and N5 of DHF. Crystallization of PTR1 and PTR1-ligand complexes The crystallization of PTR1 from L. major and L. tarentolea as unliganded and as binary and ternary complexes was attempted. Several crystal forms were obtained including L. major PTR1-NADPH-MTX crystals that diffracted to ~ 3.2 Å resolution. It was not possible to collect a full data set of any of the crystals. At their current stage, none of the crystal forms is suitable for structural work.

Orientador: Virgínia Bodelão Richini Pereira / Resumo: As infecções intestinais parasitárias são um problema mundial. Parasitas veiculados em água e alimentos como podem ser provenientes da falta de higiene durante o manuseio dos alimentos, contaminação ambiental por material fecal, irrigação de cultivos agrícolas com águas poluídas ou fossas sépticas precárias, situações comuns em países como o Brasil. Tais questões, colaboram para ocorrência de surtos por água e alimentos na população. Assim, o objetivo deste trabalho foi investigar a presença de parasitas importantes em saúde pública em hortaliças e água de irrigação de propriedades do município de Bauru, São Paulo; bem como, nas fezes e nas mãos de manipuladores dos cultivos. As amostras foram coletadas de cinco propriedades do município de Bauru, sendo uma localizada em área urbana e quatro em área rural. Foram obtidas 33 amostras de água de irrigação, 62 de hortaliças, 31 das mãos e dez fecais dos manipuladores. Todas as amostras foram submetidas a análise molecular e as águas de irrigação submetidas ainda a análise microbiológica. Na análise microbiológica foi detectado coliformes totais e E. coli em três propriedades. Na análise molecular, o parasita mais prevalente em água de irrigação e hortaliça foi Cyclospora cayetanensis. Taenia spp. foi detectada em uma hortaliça e Giardia spp. foi mais prevalente nas amostras humanas. Não foi detectado Toxoplasma gondii. As amostras de água de irrigação apresentaram maior quantidade de amostras positivas. Atividades de educação em ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Parasitic intestinal infections are a worldwide problem. Parasites carried in water and food as they may come from the lack of hygiene during food handling, environmental contamination by fecal material, irrigation of agricultural crops with polluted water or precarious septic tanks, common situations in countries like Brazil. Such issues contribute to the occurrence of outbreaks by water and food in the population. Thus, the objective of this work was to investigate the presence of important parasites in public health in vegetables and irrigation water on rural properties in the municipality of Bauru, São Paulo; as well as in the feces and in the hands of crop handlers. The samples were collected from five rural properties in the municipality of Bauru, one of them located in an urban area and another four in a rural area. Were collected 33 samples of irrigation water, 62 of vegetables, 31 of hands and 10 fecal samples from handlers. All samples were subjected to molecular analysis and irrigation water was also subjected to microbiological analysis. In this microbiological analysis, total coliforms were detected at high rates and E. coli in three of the properties. In molecular analysis, the parasite most prevalent in the analysis and most common in irrigation water and vegetables was Cyclospora cayetanensis. Taenia spp. was detected in one greenery. Giardia spp. was most detected in human samples. Toxoplasma gondii was not detected. The samples of irrigation water had a grea... (Complete abstract click electronic access below) / Mestre

In this thesis, the design of passive networks with the aid of full-wave simulation software and geometry-based models of lumped elements is investigated. This is done by examining the results of a number of simulation examples, as well as measured data from manufactured designs to compare against simulated equivalents. One such example is a chip antenna evaluation board design, in which the PCB, antenna, matching components and connector are all modeled. When measured, the simulation agreed with the board’s best matched frequency of 5.5 GHz to within 20 MHz. In another, a new antenna layout is generated from an existing evaluation design which, produced a match of about -15 dB at the design frequency with a similar bandwidth to that shown on the antenna datasheet on the first attempt at manufacture. Additionally, a statistical experiment was conducted in order to provide insight into the phenomenon of coupling between lumped components, and to define clearly when it starts to become an important effect to consider. For both chip capacitors and inductors, a behavioral model of how much crosstalk is present in a prospective circuit was developed which takes into account angle and distance between components, as well as case size. Finally, a simple discrete gradient descent was implemented in a commercial full-wave simulation software in order to assist in the refinement of designs containing 3-D geometry-defined component models.