Differential gene expression during Trypanosoma cruzi metacyclogenesis

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the refered kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.

Effects of dietary protein type on oxidized cholesterol-induced alteration in age-related modulation of lipid metabolism and indices of immune function in rats.

Exogenous oxidized cholesterol disturbs both lipid metabolism and immune functions. Therefore, it may perturb these modulations with ageing. Effects of the dietary protein type on oxidized cholesterol-induced modulations of age-related changes in lipid metabolism and immune function was examined using differently aged (4 weeks versus 8 months) male Sprague-Dawley rats when casein, soybean protein or milk whey protein isolate (WPI) was the dietary protein source, respectively. The rats were given one of the three proteins in diet containing 0.2% oxidized cholesterols mixture. Soybean protein, as compared with the other two proteins, significantly lowered both the serum thiobarbituric acid reactive substances value and cholesterol, whereas it elevated the ratio of high density lipoprotein-cholesterol/cholesterol in young rats, but not in adult. Moreover, soybean protein, but not casein and WPI, suppressed the elevation of Delta6 desaturation indices of phospholipids in both liver and spleen, particularly in young. On the other hand, WPI, compared to the other two proteins, inhibited the leukotriene B4 production of spleen, irrespective of age. Soybean protein reduced the ratio of CD4(+)/CD8(+) T-cells in splenic lymphocytes. Therefore, the levels of immunoglobulin (Ig)A, IgE and IgG in serum were lowered in rats given soybean protein in both age groups except for IgA in adult, although these observations were not shown in rats given other proteins. Thus, various perturbations of lipid metabolism and immune function caused by oxidized cholesterol were modified depending on the type of dietary protein. The moderation by soybean protein on the change of lipid metabolism seems to be susceptible in young rats whose homeostatic ability is immature. These observations may be exerted through both the promotion of oxidized cholesterol excretion to feces and the change of hormonal release, while WPI may suppress the disturbance of immune function by oxidized cholesterol in both ages. This alleviation may be associated with a large amount of lactoglobulin in WPI. These results thus showed a possibility that oxidized cholesterol-induced perturbations of age-related changes of lipid metabolism and immune function can be moderated by both the selection and combination of dietary protein.

Effect of oxidized cholesterol on age-associated changes to immune parameters in spleen lymphocytes and peritoneal exudate cells derived from rats

The effects of oxidized cholesterol on immune parameters were examined by using spleen lymphocytes and peritoneal exudate cells (PEC) derived from 5-week- (Young) and 9-month-old (Adult) rats. The immunoglobulin (Ig) G and IgM production was inhibited by oxidized cholesterol in the rats of both ages when lymphocytes were exposed to 30 micrograms/ml of oxidized cholesterol for 24 hr. The intracellular IgA level was also lowered by 30 micrograms/ml of oxidized cholesterol, irrespective of age. In contrast, IgE production was significantly increased by the addition of 30 micrograms/ml of oxidized cholesterol in only young lymphocytes. Moreover, oxidized cholesterol enhanced the intracellular histamine accumulation in only adult PEC, although the total histamine level produced by PEC was similar in the rats of both ages. These results thus suggest the possibility that oxidized cholesterol can have different effects on the age-related modulation of immune functions such as Igs production and histamine release.

Construção e validação do Índice de Qualidade de Vida de Ferrans & Powers: versão feridas

O artigo visa a apresentar o processo de construção e validação do Índice de Qualidade de Vida de Ferrans & Powers - Versão Feridas (IQVFP-VF) para emprego em pessoas com feridas agudas e crônicas de diferentes etiologias. O estudo metodológico desenvolveu-se por meio de procedimentos teóricos, empíricos e analíticos. Os resultados indicaram que o instrumento possui consistência interna e estabilidade satisfatórias; confirmaram a validade de conteúdo, concorrente (com o item sua satisfação) e convergente (com o WHOQOL-breve), bem como sua capacidade de discriminar os indivíduos conforme o número e duração das feridas, intensidade de dor e idade. A análise fatorial confirmatória mostrou que o instrumento manteve-se razoavelmente ajustado ao modelo original. Em conclusão, pode-se considerar que o IQVFP-VF é válido e tem confiabilidade atestada nos aspectos mais importantes da Qualidade de Vida para a população com feridas - geral, saúde e aspectos psicológicos e espirituais.

Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein.

Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.

On the role of kerato-epithelin in the pathogenesis of 5q31-linked corneal dystrophies.

PURPOSE: Recently, the authors identified a gene, BIGH3, in which different mutations cause a group of hereditary corneal dystrophies: lattice type I and IIIA (CDLI and CDLIIIA), granular Groenouw type I (CDGGI), Avellino (CDA), and Reis-Bücklers' (CDRB). All these disorders are characterized by the progressive accumulation of corneal deposits with different structural organization. Experiments were conducted to determine the role of kerato-epithelin (KE), the product of BIGH3, in the pathogenesis of the diseases. METHODS: KE-15 and KE-2, two rabbit antisera raised against peptides from the 69-364 and 426 - 682 amino acid regions of KE respectively, were used for immunohistology of the corneas obtained after keratoplasty in six CDLI patients, three CDGGI patients, and one CDA patient. RESULTS: The nonamyloid deposits observed in CDGGI stained intensively with KE-15 and KE-2, whereas the amyloid deposits in all analyzed CDLI corneas reacted to KE-2 but not to KE-15. In the CDA cornea, where amyloid and nonamyloid inclusions were present, positive staining with both antisera was observed. CONCLUSIONS: Pathologic amyloid and nonamyloid deposits observed in CDLI, CDGGI-, and CDA-affected corneas are caused by KE accumulation. Different staining patterns of amyloid and nonamyloid deposits observed with antibodies against the amino and carboxyl termini of KE suggest that two mechanisms of KE misfolding are implicated in the pathogenesis of 5q31-linked corneal dystrophies.

ppGpp controlled by the Gac/Rsm regulatory pathway sustains biocontrol activity in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.

Aplicação dos conceitos de modelagem e de redes de Petri na análise do processo produtivo da indústria sucroalcooleira

A modelagem, como meio de análise de processos do agronegócio, bem como de cadeias produtivas, atende à demanda por métodos apropriados. O presente trabalho objetivou avaliar a aderência dos conceitos de modelagem e de rede de Petri aplicados ao complexo agroindustrial da cana-de-açúcar. Utilizou-se uma empresa real para a aquisição das informações. Os modelos das cadeias de atividades foram validados com o uso da rede de Petri, por meio de um método de análise em que são simulados todos os caminhos possíveis e os estados que o sistema modelado pode atingir. O estudo resultou em 14 conjuntos de etapas de produção, englobando todas as operações unitárias das cadeias produtivas do açúcar e do álcool, desde o plantio da cana até o armazenamento do açúcar e do álcool.

Dietary protein modifies oxidized cholesterol-induced alterations of linoleic acid and cholesterol metabolism in rats

Effects of dietary protein on oxidized cholesterol-induced alterations in linoleic acid and cholesterol metabolism were studied in 4-wk-old male Sprague-Dawley rats, using casein and soybean protein as dietary protein sources. The rats were fed one of the two proteins in cholesterol-free, 0.3% cholesterol or 0.3% oxidized cholesterol mixture diets using a pair-feeding protocol for 3 wk. In the soybean protein-fed group, rats fed oxidized cholesterol did not have lower activity of liver microsomal delta6 desaturase, the rate-limiting enzyme in the metabolism of linoleic acid to arachidonic acid, compared with rats fed cholesterol-free diet, whereas in the casein-fed group the desaturase activity was significantly greater in rats fed oxidized cholesterol than in those fed cholesterol-free diet. This was in contrast to a significant reduction in liver microsomal delta6 desaturase activity by cholesterol, irrespective of protein source. In general, these changes were reflected in the desaturation indices of liver phospholipids. Furthermore, soybean protein significantly increased the fecal excretion of neutral and acidic steroids and tended to reduce (P = 0.082) the accumulation of oxidized cholesterols in the liver. Thus, soybean protein partly modified some of the undesirable effects of oxidized cholesterol through its hypocholesterolemic effect and possibly through the modulation of hepatic delta6 desaturase activity.

Parent selection for cocoa resistance to witches'-broom

The objective of this work was to identify genotypes with high general combining ability for resistance to witches'-broom (Moniliophthora perniciosa) in populations formed from a first cycle of recurrent selection. Highly productive and resistant clones from different origins were interbred using the North Carolina II design. The clones SCA 6, CSUL 7, RB 39, CEPEC 89, OC 67, BE 4, EEG 29 and ICS 98 were used as paternal parents, while the maternal ones were NA 33, CCN 10, IMC 67, P 4B, CCN 51, CEPEC 86, SGU 54 and ICS 9. Twenty days after germination, 56 seedlings of each cross (four replicates of 14 seedlings) received the inoculation of a 1-mL suspension with 7.5x10(4 ) basidiospores mL-1. Symptoms were evaluated 60 days after inoculation. Significant differences were observed among paternal and among maternal parents, for resistance to witches'-broom assessed according to the proportion of progeny seedlings with the disease symptoms. Differences were also observed between groups of mothers or fathers previously defined as resistant, and groups previously defined as susceptible. It is possible to obtain a combination of genes that can increase the level of resistance to witches'-broom directly from the first cycle of recurrent selection.

Wavelet Analysis in Turbulence

The objective of this thesis is to study wavelets and their role in turbulence applications. Under scrutiny in the thesis is the intermittency in turbulence models. Wavelets are used as a mathematical tool to study the intermittent activities that turbulence models produce. The first section generally introduces wavelets and wavelet transforms as a mathematical tool. Moreover, the basic properties of turbulence are discussed and classical methods for modeling turbulent flows are explained. Wavelets are implemented to model the turbulence as well as to analyze turbulent signals. The model studied here is the GOY (Gledzer 1973, Ohkitani & Yamada 1989) shell model of turbulence, which is a popular model for explaining intermittency based on the cascade of kinetic energy. The goal is to introduce better quantification method for intermittency obtained in a shell model. Wavelets are localized in both space (time) and scale, therefore, they are suitable candidates for the study of singular bursts, that interrupt the calm periods of an energy flow through various scales. The study concerns two questions, namely the frequency of the occurrence as well as the intensity of the singular bursts at various Reynolds numbers. The results gave an insight that singularities become more local as Reynolds number increases. The singularities become more local also when the shell number is increased at certain Reynolds number. The study revealed that the singular bursts are more frequent at Re ~ 107 than other cases with lower Re. The intermittency of bursts for the cases with Re ~ 106 and Re ~ 105 was similar, but for the case with Re ~ 104 bursts occured after long waiting time in a different fashion so that it could not be scaled with higher Re.

Avaliação da reprodutibilidade da tomografia computadorizada no estadiamento da pancreatite aguda

OBJETIVO: Medir a reprodutibilidade da tomografia computadorizada sem e com contraste na avaliação da gravidade da pancreatite aguda. MATERIAIS E MÉTODOS: Cinqüenta e um exames de tomografia computadorizada abdominal sem e com contraste de pacientes com pancreatite aguda foram analisados por dois radiologistas (observadores 1 e 2). Calculamos o índice morfológico pela tomografia computadorizada sem e com contraste, separadamente, e o índice de gravidade da tomografia computadorizada para pancreatite aguda. Medimos a reprodutibilidade intra- e interobservador da tomografia computadorizada através do índice kappa (kapa). RESULTADOS: Para a concordância interobservador obtivemos kapa de 0,666, 0,705, 0,648, 0,547 e 0,631 para índice morfológico sem e com contraste, presença de necrose pancreática, extensão da necrose pancreática e índice de gravidade da tomografia computadorizada, respectivamente. Para a concordância intra-observador dos observadores 1 e 2 obtivemos, respectivamente, kapa de 0,796 e 0,732 para o índice morfológico sem contraste; 0,725 e 0,802 para o índice morfológico com contraste; 0,674 e 0,849 para a presença de necrose pancreática; 0,606 e 0,770 para a extensão da necrose pancreática; e 0,801 e 0,687 para o índice de gravidade da tomografia computadorizada. CONCLUSÃO: O estadiamento da pancreatite aguda pela tomografia computadorizada por meio do índice morfológico e do índice de gravidade da tomografia computadorizada é um método bastante reprodutível. O não-uso do contraste não afeta a reprodutibilidade da tomografia computadorizada para o cálculo do índice morfológico.

Linfonodo sentinela após mamoplastia de aumento pela via transaxilar: estudo prospectivo controlado por meio de linfocintilografia em 43 pacientes

OBJETIVO: Verificar se a mamoplastia de aumento pela via transaxilar apresenta potencial de prejudicar a identificação futura do linfonodo sentinela. MATERIAIS E MÉTODOS: Estudo prospectivo controlado em que foram selecionadas 22 pacientes divididas em grupo pós-mamoplastia e grupo controle, totalizando 43 mamas (22 no grupo pós-mamoplastia e 21 no grupo controle) avaliadas por meio de linfocintilografia imediatamente após injeções periareolares de fitato-99mTc. Os testes estatísticos consideraram como diferenças significativas valores de p < 0,05. RESULTADOS: Todas as mamas do grupo pós-mamoplastia apresentaram drenagem linfática para a cadeia axilar, sem diferença com o grupo controle (p = 0,488). A média de linfonodos captantes foi de 1,27 ± 0,46 no grupo pós-mamoplastia e 1,33 ± 0,58 no grupo controle (p = 0,895). A média de tempo para visualização do primeiro linfonodo foi de 3,14 ± 4,42 minutos no grupo pós-mamoplastia e 5,48 ± 5,06 minutos no grupo controle, novamente sem diferença significativa (p = 0,136). CONCLUSÃO: A mamoplastia de aumento pela via transaxilar não acarretou prejuízo na identificação futura do linfonodo sentinela.

Avaliação dos custos para realização de controles de qualidade de radiofármacos marcados com [99mTc]tecnécio em serviços de medicina nuclear no Brasil

OBJETIVO: Estabelecer os custos dos controles de qualidade para os radiofármacos marcados com [99mTc]tecnécio nos serviços de medicina nuclear do Brasil, em atenção às resoluções RDC nº 38/2008 e RDC nº 63/2009 editadas pela Agência Nacional de Vigilância Sanitária. MATERIAIS E MÉTODOS: Foram apurados preços de materiais de consumo, equipamentos e de mão-de-obra para a realização dos controles de qualidade. Os valores foram convertidos para unidades de volume, tempo e outras unidades cabíveis para a determinação do preço unitário. RESULTADOS: O investimento para aquisição de materiais de consumo e equipamentos foi estimado ser de R$ 35.500,00. O custo final para o controle de cada kit variou entre R$ 6,44 e R$ 7,80, dependendo do produto a ser analisado e do profissional selecionado para execução do procedimento. Esses valores podem representar de 0,5% a 10% do valor recebido pelas instituições pela realização dos exames. Na prática, o custo efetivo pode ser menor, uma vez que o produto de um kit pode ser utilizado em diversos pacientes. CONCLUSÃO: Em face do ganho de qualidade e segurança dos pacientes, concluímos que os custos da implantação do programa de controle de qualidade podem ser absorvidos no planejamento financeiro dos serviços de medicina nuclear.