2,598 research outputs found
Molecular techniques for the identification and detection of microorganisms relevant for the food industry
No full textThe research described in this thesis concerns the development and application in food microbiology of molecular identification and detection techniques based on 16S rRNA sequences. The technologies developed were applied to study the microbial ecology of two groups of bacteria, namely starter cultures and sporeforming spoilage bacteria, that are of importance to the food industry and in particular the dairy industry.In this Chapter the results are summarized and discussed in relation to recent developments. Moreover, the impact of the results obtained on quality control systems and risk assessment of the use of genetically modified microorganisms in food will be discussed.Role of ribosomal RNA sequences in the identification and detection of microorganismsIn Chapter 1 several identification and detection techniques based upon rRNA sequences are described. They are based upon the structural and sequence differences within the ribosomal operon, like ribotyping, analysis of the spacer regions, or restriction legnth polymorphism of amplified parts. Some of the techniques strictly involve specific sequence differences within the rRNA genes, which consist of alternating conserved and more variable regions. The rRNA genes are uniquely suited for the reliable identification of microorganisms and the rational probe design for the specific detection of microorganisms. In particular 16S rRNA sequences have become to play an important role in microbial taxonomy. Many microorganisms have been reclassified or renamed based on new insights in their phylogenetic relationships based on 16S rRNA sequence homology. Within the constraints of the current data-bases it is now possible to identify new isolates quickly and reliably by sequencing their 16S rRNA. The use of rRNA sequencing in the identification and detection of microorganisms has indicated that many organisms are still unknown and not characterized, comprising both culturable and nonculturable microorganisms.16S rRNA-derived DNA probes can either be based on conserved regions containing groupand/or genus-specific sequences or more variable regions containing speciesand/or subspecies- specific sequences. These probes can be applied in hybridizations to DNA or RNA fixed to a membrane or present in in situ fixed cells. In order to increase the specificity and the sensitivity of a detection method, such probes can also be applied in specific DNA amplifications. Direct amplification of specific 16S rRNA sequences from, for instance, a food matrix like cheese, is the most straightforward and sensitive detection method. Unfortunately, most of these food matrices, and also other environmental samples like soil and faecal material, contain components that may inhibit the PCR reaction. This necessitates complex DNA extraction protocols or the use of enrichment cultures in order to obtain nucleic acid extracts that can be applied in PCR amplifications successfully.It is possible to identify an isolate reliably at the species or subspecies level based on ribosomal sequences (Chapters 2 and 5). However, it is possible to distinguish isolates at the strain level with other techniques, like the frequently applied Random Amplified Polymorphic DNA (RAPD) technique, in which strain specific patterns are formed by the amplification with arbitrarily chosen primers of approximately ten nucleotides . This technique is often used in addition to 16S rRNA-based identification methods, as has been shown recently by the discrimination of different serotypes of Listeria monocytogenes. The combination of both approaches can be very useful, in particular in epidemiologic studies. Moreover, RAPD patterns can be used for the development of strain-specific DNA probes as well. This can also be a very successful approach for obtaining species-specific DNA probes for microorganisms, such as Campylobacter jejuni, Campylobacter coli and Campylobacter lari, that are difficult to design on the basis of ribosomal sequences. It has been described that some genera, like Rhizobium, include species that contain an almost identical 16S rRNA but share almost no DNA-DNA homology. In order to discriminate between these species, other techniques have to be applied, of which RAPD or other chromosomal fingerprinting methods are used most frequently.Development and application of molecular, 16S rRNA based, identification methods for Lactococcus speciesIn the food industry in general, but specifically in the dairy industry, lactic acid bacteria are used as starter cultures to initiate specific fermentations. Both for basic research on lactic acid bacteria and for their application in industrial food fermentations, reliable and simple methods for the identification of such bacteria are required. Because little difference exists in phenotypic properties of especially the mesophilic lactic acid bacteria, reliable identification and detection techniques have been developed based on specific sequences in variable regions of 16S rRNA (Chapter 1). Species-specific DNA probes, based on the first variable region (VI) of the 16S rRNA , were designed for Lactococcus garvieae, L. plantarum and L. raffinolactis and subspecies-specific DNA probes for L. lactis subsp. lactis and L . lactis subsp. cremoris. The third subspecies L. lactis subsp. hordnieae was not distinguishable from L lactis subsp. lactis based on 16S rRNA sequences because these differed in only one base pair. In addition, speciesspecific probes for Leuconostoc were developed based on the third variable region (V3) of the 16S rRNA .There is a growing need for new production strains for the innovation of dairy products. These can be obtained either by genetic modification of known production strains or by isolation of new strains from natural ecological niches. Both for the application of genetically modified starter strains and to allow for an efficient search for strains from natural ecosystems, it is important to know if and where a Lactococcus strain survives outside the dairy environment. Lactose-utilizing Lactococcus isolates from environmental samples taken on cattle farms and in the waste flow of a cheese production plant were identified up to the species level, using amplified variable regions (VI) of the 16S rRNA in combination with species-specific DNA probes (Chapter 3). These isolates were further characterized by using specific PCR amplification of sequences related to cit P , prt P and nis A, coding for citrate permease, protease and prenisin, respectively. It was possible to isolate Lactococcus spp. from various environments, indicating that lactococci can survive outside the dairy plant and that some are able to persist in soil, effluent water, on vegetation and on cattle. During the characterization of the environmental lactococcal isolates discrepancies were observed between the 16S rRNA based identification of L . lactis strains and identification based on their phenotypical properties. Similar findings were obtained with isolates from spontaneous milk fermentations, collected from several european countries . The classical differentiation between L. lactis subsp. lactis and L. lactis subsp. cremoris is based on phenotypical differences. Llactis subsp. cremoris strains are characterized by their inability to hydrolyse arginine, to metabolize a number of sugars, and to grow at 37 °C and in the presence 4% NaCI . Based on SDS-PAGE of whole-cell proteins both phenotypes are distinguishable and form two seperate clusters. However, within the group of environmental isolates, identified as L.lactis subsp. lactis on the basis of their phenotype, 16S rRNA sequences belonging to both L. lactis subsp. lactis and L lactis subsp. cremoris were encountered. Detailed characterization of a large collection of lactococcal isolates has led to the conclusion that within the species L. lactis two ribotypes are present, each having a specific 16S rRNA sequence. However, for each ribotype different phenotypes can be found. The ribotype of the strain NCDO 712, the parental strain of MG1363, which is frequently used in genetic studies of L. lactis , shows that it belongs to L. lactis subsp. cremoris , confirming the conclusion based on the mapping of its chromosome. It is quite remarkable that this strain shows the L. lactis subsp. cremoris ribotype, while phenotypically it resembles L. lactis subsp. lactis .The phenotype described for L. lactis subsp. cremoris is only observed with isolates that have been obtained from industrial starter cultures or traditional fermented milk products like villi. Such strains may belong to either ribotype. This may suggest that the continuous culturing in milk has resulted in a differentiation of phenotypic properties between "starter" strains and strains originating from environmental sources. Detailed characterisation of L. lactis isolates from natural environments indicated that they differ from strains commonly present in starter cultures. This does not only suggest that starter isolates do not survive outside the dairy environment, but also that L. lactis strains present within the natural population have potential to be applied in product innovation and differentiation. This applies in particular to isolates that were obtained from spontaneous milk fermentations, which are still used in the southern part of Europe for the production of artisanel cheeses. An initial inventory of the biodiversity of such isolates illustrates the large diversity of properties, like aroma formation, acidification and bacteriocin production, which can be exploited to obtain differentiation in fermented milk products.Molecular detection techniques were not only used for the identification of environmental isolates but also for the monitoring of the survival of L lactis in the human gastrointestinal tract. For the potential application of L lactis as probiotic, as genetically modified starter culture, or as live vaccine, it is important to determine whether these bacteria survive in the gastrointestinal tract after consumption by humans. In Chapter 4, a human feeding study is described in which the fate was monitored of L. lactis strain TC165.5, which was genetically marked by insertion of the naturally occurring sucrose-nisin conjugative transposon Tn 5276 and spontaneous chromosomal resistance to rifampicin and streptomycin. A method was developed for the efficient extraction of microbial DNA from human faeces. The passage of strain TC165.5 through the intestinal tract was monitored by selective plating and specific detection of the nis A gene by PCR amplification. The study showed that up to 0.1-1% of L. lactis cells, consumed in a dairy product, may survive passage of the gastrointestinal tract, provided that they pass within 3 days after consumption. The partial survival of lactococci provides a positive prospective for the use of Lactococcus strains as probiotic or in the development of live vaccines.Biosafety assessment of the use of genetically modified Lactococcus spp. infermented food productsThe studies presented in Chapters 3 and 4 were part of an assessment of the biosafety aspects of the use of genetically modified starter cultures, specifically addressing general ecological parameters like survival, dissemination and transfer of genetic information. In order to quantify the survival of starter lactococci, careful monitoring of the waste flow of a cheese production plant was performed. This indicated the absence of typical industrial strains (Chapter 3) suggesting that most starter organisms are not able to persist in non-dairy environments, although natural niches are available, In addition, the numbers of lactococci found in the non-dairy environments were considerably lower than those that are daily released into the environment via the industrial production of fermented milk products. It was therefore concluded that there is no environmental release of viable starter bacteria resulting from the waste flow of the production process of fermented foods.Another avenue for the release of lactic acid bacteria into the environment is by means of the consumption of fermented milk products by humans. The results of the human feeding trial (Chapter 4) showed that only a small fraction of viable cells of the marked L.lactis strain survived passage through the human gastrointestinal tract.It is well known that L. lactis strains possess efficient systems for transfer of genes via plasmids, transposons and phages. A number of studies have been published on gene transfer in lactococci under natural conditions, including large-scale fermentation, cheese manufacturing, and passage through the gastro-intestinal tract of mice. The results showed that the transfer rates decreased rapidly under natural conditions where cell-to-cell contact and growth are limited. On the other hand, under conditions favourable for cell-to-cell contact, such as on agar plates and intestinal mucosal surfaces, the transfer frequencies are relatively high, up to 10 -4.The transfer of genetic information from one strain to another per se is not to be regarded as a potential hazard, since it was demonstrated that lactococci are already capable of transferring genetic elements. In some cases, however, the properties encoded by new genetic traits may be potentially hazardous in combination with specific strains or in specific ecosystems. If the encoded properties are already present in the ecosystem, no specific new hazard may be expected, since in the past it has not resulted in hazardous biological consequences. By relating the potential hazards of the application of genetically modified lactic acid bacteria to the regular hazards associated with the, consumption of fermented dairy products, the potential hazard may be normalized. So both for legislation purposes and for the acceptance by the consumers it is important to identify and normalize the biosafety aspects by relating them to current practice. In this way the potential hazards are more comprehensible and acceptable.Development and application of molecular detection and identification methods for Clostridium tyrobutyricumMajor spoilage problems in the food industry are caused by sporeforming bacteria, belonging to the genera Clostridium and Bacillus, which survive heat-treatments that are applied to prolong the shelf-life of the food product. Butyric acid fermentation in cheese (late blowing), caused by the germination of clostridial spores which survive the heat treatment of cheese milk, is still causing considerable loss in the cheese producing industry. For the production of semi- hard cheeses, like Gouda cheese, it is very important to limit the number of spores in the cheese milk of bacteria capable of causing late blowing. Although Clostridium tyrobutyricum is regarded as the causative agent of butyric acid fermentation, also spores of other clostridia, such as C. beijerinckii and C.sporogenes are frequently isolated from late-blown cheeses. The current detection method for C.tyrobutyricum is not specific, since also other clostridial species are able to form butyric acid and hydrogen. In Chapter 5 and 6 the development of specific detection methods is described for the Clostridium spp. most frequently encountered in dairy environments. Based on specific sequences in the V2 and V6 region of the 16S rRNA, species- specific DNA probes were developed for C. tyrobutyricum, C. acetobutylicum, C. beijerinckii, C. butyricum and C.sporogenes (Chapter 5).In Chapter 6 the causative relationship between C.tyrobutyricum and late blowing in cheese is demonstrated. Cheese experiments were performed to provoke this defect by using spores from different strains of several dairy-related clostridia. To overcome problems associated with isolation of Clostridium spp., specific clostridial sequences were directly detected in DNA isolated from cheese by a two-step specific PCR amplification. Only specific sequences of C.tyrobutyricum were detected in both the experimental and in commercially obtained cheeses showing signs of late blowing. This clearly identified C.tyrobutyricum as the causative agent of late blowing in cheese.In order to prevent late blowing in cheese, the number of spores has to be limited to less than 1 per 10 ml. of cheese milk. To improve the currently used methods for the routine detection of clostridial spores in milk, a specific and very sensitive method has to be developed. Although the current methods are very aspecific and often give false positive results mainly due to the presence of C.beijerinckii spores, it is possible to detect up to 1 spore per 100 nil by MPN-methods. Recently, Herman et al. have shown that it should be possible to detect one C.tyrobutyricum spore per 100 nil raw milk by concentrating the spores by centrifugation, followed by DNA extraction and a nested PCR amplification. However, all reports on DNA extraction from spores describe experiments with artificially obtained spore- suspensions. It is not clear if these methods can also be applied on natural spore populations. Since it is known that during the production of artificial spore suspensions residues of cell material, including DNA, can adhere to the outside of the spores, proper control experiments should be included. Such control experiments have not been described, so far making it impossible to evaluate the efficiency of the extraction methods described.Relevance of molecular identification and detection techniques based on ribosomalsequences for the food microbiology in general and in particular for the dairyindustryThe availability of molecular methods for the detection and identification of microorganisms has several advantages. In many cases the identity of both desired and unwanted microorganisms present in food products is of major importance. Enormous efforts have been invested in the reproducible identification of isolates based upon phenotypic properties such as carbohydrate fermentation, formation of specific metabolites, enzyme activities, and lipid composition. Unfortunately, most of these methods appeared to be irreproducible or not sufficiently discriminative for proper identification. The 16S rRNA-based methods are far more suited for the quick and reliable identification of isolates. However, the 16S rRNA sequencing share one particular limitation with many other identification methods, like those of API or Biolog, namely that it is only possible to obtain a correct identification within the limitations of the database. As many other databases, the RPD-database is not complete and contains only the 16S rRNA sequences of approximately 3000 bacteria. Most of the widely distributed species are present, but the 16S rRNA sequences of many bacteria frequently encountered in foods have not yet been determined or deposited. The absence of most Brevibacterium spp. and many other relatives of the Arthrobacter group, including Corynebacterium spp., is very problematic since they are important in the dairy industry as part of the surface microflora of surface-ripened cheeses and as spoilage organisms of pasteurized milk and cream. It appears that the biodiversity within this group is enormous, complicating clear systematic descriptions for these bacteria.Molecular identification methods based on ribosomal sequences have revolutionized the classification and systematics for many bacteria, but also the ability to specifically detect microorganisms has made a great impact on food microbiology. This is particularly the case with respect to the detection and specific quantification of those bacteria that are unculturable, like Candidatus arthromitus , or those for which no suitable selective media are available, like Bifidobacterium spp.. In addition, molecular techniques will allow to obtain more detailed information on critical points in the production processes of foods. Such information is essential for safeguarding the product quality. For instance, by the use of strain- specific RAPD patterns to monitor the population dynamics of mixed-strain starter bacteria during the fermentation process, better insights could be obtained in which factors are important for the quality of the resulting fermented food product.Although the potential sensitivity of molecular methods should make them suitable for the reliable detection of pathogens and spoilage organisms in food products and raw materials, there are some major practical problems that still have to be solved. One of the largest problems is the presence of PCR-inhibiting components in several food products, like cheese and meat. This inhibition can be circumvented by applying enrichment procedures before the actual detection with a specific PCR reaction. Even if these procedures result in a quicker and more reliable detection of the target organism there are some important limitations to such procedures. It is known that a significant problem with the detection of some pathogens, like Salmonella, is the unreliability of a successful pre-enrichment in buffered peptone. This failure to obtain growth in the pre- enrichment step can be caused by competing flora or the physiological state of the target organisms. Still, the detection kits that are currently available, based on DNADNA hybridization, can be successfully used as verification methods for Listeria and Salmonella after a successful pre-enrichment.Besides the practical problems, the implementation of molecular methods is also complicated by legislation, codes of practice, and regulations for processing and product control. Each adapted detection protocol for bacteria like Salmonella, Listeria and other important food pathogens requires extensive tests and validations before it is accepted by regulatory authorities. In addition, the routi
BRCA1 and BRCA2 mutations in a population-based study of male breast cancer
No full textBackground: The contribution of BRCA1 and BRCA2 to the incidence of male breast cancer (MBC)
in the United Kingdom is not known, and the importance of these genes in the increased risk of female
breast cancer associated with a family history of breast cancer in a male first-degree relative is unclear.
Methods: We have carried out a population-based study of 94 MBC cases collected in the UK. We
screened genomic DNA for mutations in BRCA1 and BRCA2 and used family history data from these
cases to calculate the risk of breast cancer to female relatives of MBC cases. We also estimated the
contribution of BRCA1 and BRCA2 to this risk.
Results: Nineteen cases (20%) reported a first-degree relative with breast cancer, of whom seven also
had an affected second-degree relative. The breast cancer risk in female first-degree relatives was 2.4
times (95% confidence interval [CI] = 1.4–4.0) the risk in the general population. No BRCA1 mutation
carriers were identified and five cases were found to carry a mutation in BRCA2. Allowing for a
mutation detection sensitivity frequency of 70%, the carrier frequency for BRCA2 mutations was 8%
(95% CI = 3–19). All the mutation carriers had a family history of breast, ovarian, prostate or
pancreatic cancer. However, BRCA2 accounted for only 15% of the excess familial risk of breast
cancer in female first-degree relatives.
Conclusion: These data suggest that other genes that confer an increased risk for both female and
male breast cancer have yet to be found
Music for classical guitar by South African composers : a historical survey, notes on selected works and a general catalogue
No full textIncludes abstract.Includes bibliographical references (leaves 296-309).This is the first comprehensive investigation of music for, or including, the classical guitar by South African composers. The focus of this research has been, firstly, to uncover as much of the repertoire as possible, and, secondly, to collate, study, catalogue and report on the information. A brief historical survey of the guitar in South Africa provides the context within which this study was conducted. The primary sources of quantitative data collection were through the archival catalogues of the South African Music Rights Organisation and through personal contact with guitarists, composers and guitar teachers. Other sources consulted were publishers, broadcasting corporations, recording companies, libraries and the internet. The body of the dissertation comprises biographical sketches, background notes, analyses and technical notes on 17 selected solo and chamber works dating from 1947 to 2007 by some of South Africa's most prominent composers and guitaristcomposers. The repertoire ranges in style from the traditional and ethnically inspired to the experimental and abstract. As this is an empirical survey, each selected entry includes details on instrumentation, duration, level of difficulty, number of pages, scordatura, commissions or requests, sources or publishers, premières and recordings. A biography of each composer is provided as well as background notes which offer an overview of the selected work. The notes discuss historical, cultural, musical and extra-musical influences, and frequently include references to interview material. The commentaries on the selected works, with musical examples, include an analytical component describing structure, form, stylistic and compositional elements, while the technical observations include performance suggestions and a grading for each work
Variáveis associadas ao desempenho cognitivo tardio de pacientes com traumatismo crânio-encefálico grave
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Ciências MédicasObjetivos: O trauma cranioencefalico (TCE) e uma das principais causas de mortalidade e morbidade. Ha raros estudos prospectivos que investigam a associacao de variaveis clinicas e laboratoriais da fase aguda do TCE e o prognostico cognitivo tardio dos pacientes vitimas de TCE. Este estudo tem como objetivo identificar variaveis clinicas, laboratoriais e biomarcadores de lesao tecidual associados ao prognostico cognitivo em pacientes vitimas de TCE. Metodos: Foram coletadas prospectivamente as variaveis da internacao hospitalar de 234 pacientes consecutivos com TCE grave (GCS admissao . 8). Dos 172 sobreviventes, uma amostra representada de 46 pacientes realizaram avaliacao cognitiva (composta de 15 testes neuropsicologicos) em media 3 (+ - 1,8) anos apos a hospitalizacao. Um sub-grupo de 22 pacientes que foram avaliados cognitivamente realizaram analise dos niveis plasmaticos de TBARS (indicativo de dano por estresse oxidativo a lipideos) e Carbonil (indicador de dano por estresse oxidativo a proteinas) em amostras de sangue coletadas na fase aguda de TCE (mediana de 10, 30 e 70 horas apos o impacto do TCE). Um grupo controle (n=23) pareado por sexo, idade e nivel socio-educacional foi avaliado cognitivamente para comparacao com os pacientes. Resultados: A media de idade dos pacientes foi 34 (+ - 13) anos sendo 85% do sexo masculino, com escolaridade media de 9 (+ - 4,7) anos. Os pacientes apresentaram um desempenho inferior em todos os testes neuropsicologicos. A analise por regressao linear evidenciou uma forte associacao independente (R coeficiente = 0,6 a 0,8) entre maior escolaridade e menor idade e o desempenho cognitivo em 14 dos 15 testes neuropsicologicos avaliados. O desempenho nos testes cognitivos nao esteve associado ao genero, escore de admissao na Escala de Coma de Glasgow (ECG), exame das pupilas, presenca de trauma em outros orgaos, e classificacao da escala de Marshall na tomografia computadorizada na admissao (TC). Niveis elevados de glicose e presenca de hemorragia sub-aracnoide na TC mostraram-se independentemente associados a um menor desempenho no teste de Retencao de Aprendizagem de Rey e de Memoria Logica respectivamente. Embora os niveis plasmaticos de TBARS e Carbonil tenham sido significativamente elevados na fase aguda do TCE, estes biomarcadores nao se mostraram associados ao desempenho cognitivo dos pacientes. Conclusoes: Baixa escolaridade e idade mais avancada sao preditores independentes de pior desempenho cognitivo tardio apos o TCE grave. O exame de TC e glicemia mostraram limitada capacidade de predicao do desempenho cognitivo enquanto que o exame das pupilas, ECG na admissao, presenca de trauma associado nao foram preditores do desempenho em nenhum dos testes neuropsicologicos avaliados. A medida dos niveis plasmaticos de TBARS e Carbonil tambem nao se mostrou associada com o desempenho cognitivo dos pacientes. A identificacao de variaveis clinicas e laboratoriais associadas ao prognostico cognitivo apos o TCE grave permanece um desafio para a area de neuropsicologia clinica
Avaliação das alterações da saturação periférica de oxigênio, da tensão arterial e do pulso em pacientes submetidos à sedação consciente para endoscopia digestiva alta
No full textTrabalho de Conclusão de Curso - Universidade Federal de Santa Catarina. Curso de Medicina. Dapartamento de Clínica Cirúrgica
Will the thyroglobulin assay with lower functional sensitivity whilst the patients are on L-T4 treatment replace the TSH-stimulated thyroglobulin assay in the follow-up of patients with differentiated thyroid cancer?
No full textThe author reviews the literature on the new assays for serum thyroglobulin (sTg) presenting lower functional sensitivity and demonstrates that its use, whilst the patients are taking L-T4, presents better results than sTg following TSH stimulation in the follow-up of patients with differentiated thyroid carcinoma. Therefore, he suggests a revision on the guidelines for the follow-up of these patients (developed when the available assays present a sensitivity of 1 ng/mL), proposing the use of sTg assays with functional sensitivity of 0.1-0.2 ng/mL with the patients on L-T4 treatment instead of sTg stimulated by TSH.O autor apresenta evidências recentes da literatura que mostram que ensaios de tiroglobulina sérica (sTg) com maior sensibilidade funcional apresentam a mesma qualidade que a obtenção da sTg estimulada por rhTSH ou hipotiroidismo, no seguimento de pacientes com câncer diferenciado de tiróide (CDT). Desta forma, propõe modificar a prática recomendada pelas diretrizes de sociedades internacionais para o seguimento desses pacientes (desenvolvidas enquanto os ensaios disponíveis apresentavam sensibilidade de 1 ng/mL), substituindo-se a obtenção da sTg estimulada por rhTSH ou hipotiroidismo pelo acompanhamento dos pacientes na vigência da terapia com L-T4 com a medida da sTg desde que se empreguem técnicas com sensibilidade funcional da ordem de 0,1-0,2 ng/mL.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaFleuryUNIFESP, EPM, Depto. de MedicinaSciEL
Avaliação da atividade tripanocida e leishmanicida de produtos naturais da flora mato-grossense
No full textTCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.O arsenal quimioterápico disponível para o tratamento da Doença de Chagas e das Leishmanioses é restrito a poucos fármacos, os quais apresentam eficácia limitada e efeitos colaterais indesejáveis. A biodiversidade de plantas brasileiras representa uma rica fonte para a busca por novos compostos com potencial antiparasitário. Neste estudo, 20 extratos, 8 frações e 2 compostos isolados da flora mato-grossense foram avaliados. As amostras foram solubilizadas em DMSO (50mg/ml) e mantidas a 4o C até o uso. Promastigotas de Leishmania amazonensis (cepa 575) e L. chagasi (cepa PP75), e epimastigotas de Trypanosoma cruzi (cepa Y) (5x106 parasitos/ml), mantidos respectivamente em meio Schneider e LIT, forma incubados em triplicata em placas de 96 orifícios a 26o C com diferentes concentrações dos produtos naturais (1,6 a 1000 μg/ml). Após 72h de incubação, a atividade contra T. cruzi foi determinada pela contagem do número de parasitos vivos em câmara de Neubauer, e a atividade leishmanicida foi determinada pelo método de MTT. Anfotericina B (0,1μM) e Benzonidazol (10μg/ml), e DMSO 1% foram usados como controles positivo e negativo, respectivamente. Os produtos ativos contra epimastigotas de T. cruzi foram incubados com tripomastigotas sanguíneos (1x106 parasitos/ml) a 4o C, usando cristal violeta e DMSO 1% como controles positivo e negativo, e a sobrevivência dos parasitos foi determinada após 48h. A avaliação in vitro da atividade leishmanicida contra amastigotas intracelulares foi realizada com células J774-A1 infectadas com L. amazonensis em placas de 96 orifícios na presença de diferentes concentrações dos produtos naturais por 48 horas. As monocamadas foram fixadas com metanol e coradas com Giemsa, e o número de amastigotas intracelulares foi determinado pela avaliação randômica de 200 células. A citotoxicidade das amostras ativas foi avaliada usando células J774-A1 (2x104 células/orifício) pelo método de MTT. Todos os ensaios forma realizados duas vezes, em triplicata. Dois extratos hexânicos (Xilopia aromatica e Aspidosperma cuspa) e uma fração (“Ipê Pimenta”) apresentaram atividade tripanocida contra epimastigotas (CI50 = 87,07; 66,41 e 100,46 μg/ml, respectivamente), e o extrato hexânico de A. cuspa foi ativo contra tripomastigotas sangüíneos (CI50 = 150,62 μg/ml). Quatro extratos hexânicos (X. aromatica, Bowdichia virgiloides, A. cuspa e Acosmium dasycarpum), duas frações de Zanthoxylum hasslerianum e o composto isolado Cumarina foram ativos contra promatigotas de L. amazonensis (CI50 = 15,52 – 120,9 μg/ml) e L. chagasi (CI50 = 3,976 – 166,40 μg/ml). Desses, apenas o extrato hexânico de A. dasycarpum não apresentou atividade em testes com amastigotas intracelulares de L. amazonensis, sendo que os demais produtos testados apresentaram inibição acima de 40% nas concentrações de 1,6, 8 e 40 μg/ml. Nenhum dos extratos ativos foi citotóxico para células J774-A1 acima de 90 μg/ml.The chemotherapy arsenal available for treatment of Chagas disease and leishmaniasis is restricted to few drugs which presented limited efficacy and show undesirable side effects. The Brazilian plant biodiversity represents a rich source of new potential antiparasitic compounds. In this study the trypanocidal and leishmanicidal activity of 20 extracts, 8 fractions and 2 isolated compounds from Mato Grosso State plants was evaluated. Samples were solubilized in DMSO (50mg/ml) and maintained at 4ºC until use. Promastigotes of Leishmania amazonensis (575 strain) and L. chagasi (PP75 strain), and epimastigotes of Trypanosoma cruzi (Y strain) (5x106 cells/ml), maintained respectively in Schneider’s and LIT medium, were incubated in triplicate in 96-well microplates at 26ºC with different concentrations of natural products (1.6 to 1,000μg/ml). After 72h of incubation, the activity was determined by counting the number of live parasites in Neubauer chambers (T. cruzi) and by MTT method (Leishmania sp). Amphotericin B (0.1μM) and Benzonidazol (10μg/ml), and DMSO 1% were used as positive and negative controls, respectively. Products active against epimastigotes were incubated with blood trypomastigotes (1x106 cells/ml) at 4oC, using violet cristal and DMSO 1% as positive and negative controls, and survival parasites determined after 48h. The in vitro evaluation of leishmanicidal activity against intracellular amastigotes was realized by using J774.A1 cultures infected with L. amazonensis in 96-well plates in the presence of different natural products dilutions for 48 hours. The monolayers were fixed with methanol and Giemsa stained, and intracellular amastigote numbers were determined in 200 randomly chosen cells. Citotoxicity of active extracts was evaluated using J774.A1 cells (2x104 cells/well) by the MTT method. All assays were performed two times in triplicate. Two tested hexanic extracts (Xilopia aromatica and Aspidosperma cuspa) and a fraction (“Ipê Pimenta”) showed trypanocidal activity for epimastigotes (IC50 = 87.07; 66.41 e 100.46 μg/ml, respectively) and hexanic extract of A. cuspa was active against blood trypomastigotes (IC50 = 150.62 μg/ml).. Four hexanic extracts (X. aromatica, Bowdichia virgiloides, A. cuspa and Acosmium dasycarpum), two fractions of Zanthoxylum hasslerianum and the isolated compound Coumarin were active against L. amazonensis (IC50 = 15.52 – 120.9 μg/ml) and L. chagasi (IC50 = 3.976 – 166.40 μg/ml) promastigotes. From this, only the hexanic extract of A. dasycarpum didn’t show activity on tests with L. amazonensis intracelular amastigotes, and the other tested products showed inhibition up to 40% on 1.6, 8 and 40 μg/ml. The extracts, fractions and isolated compounds apresent low citotoxicity for J774.A1 cells up to 90 μg/ml
Active insulation:computational assessment of the performance of a forced convective dynamic insulation system
No full textClinical relevance of soluble c-erbB-2 for patients with metastatic breast cancer predicting the response to second-line hormone or chemotherapy
No full textConcentrations of soluble c-erbB-2 were determined in the sera of 64 patients with distant metastasis from advanced breast cancer receiving second-line hormone or chemotherapy in comparison to 35 breast cancer patients without detectable recurrent disease and 17 healthy blood donors. The sera of non-metastatic breast cancer patients contained s-erbB-2 concentrations similar to those of healthy blood donors. Patients with distant metastasis from advanced breast cancer had significantly higher values of s-erbB-2 in comparison to patients with non-disseminated disease (mean: 59.6 vs. 11.6 U/ml; p = 0.022). A significant correlation was observed between s-erbB-2 serum levels and serum LDH concentrations (p < 0.001), levels of alkaline phosphatase (p < 0.001), and the presence of hepatic metastasis (p = 0.001). Time to tumor progression was significantly shorter in patients with s-erbB-2 levels above 40 U/ml (mean: 23.4 vs. 56.7 months; p = 0.002). Furthermore, breast cancer patients with hepatic metastasis and those with elevated s-erbB-2 serum levels above 40 U/ml had limited response to hormone or chemotherapy. Non-responders had significantly higher s-erbB-2 levels (mean: 270.3, range: 42-500 U/ml;) compared with the responder group (mean: 23.1, range: 0-149 U/ml; p < 0.001). Logistic regression analysis indicated that elevated s-erbB-2 serum levels above 40 U/ml independently predicted an unfavorable response to second-line hormone or chemotherapy in patients with advanced metastatic breast cancer. Copyright (C) 2002 S. KargerAG, Basel
Bioética em discurso: efeitos sobre os processos de constituição do sujeito enfermeira/o na terapia intensiva
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-graduação em EnfermagemInvestigação qualitativa, balizada na analítica foucaultiana com aproximações ao referencial pós-estruturalista, tendo como objetivos: analisar a constituição histórica do discurso da bioética em sua articulação com o discurso da tecnobiomedicina e o modo como os desdobramentos estratégicos e tecnológicos deste discurso atravessam o processo de produção do sujeito enfermeiro/a gerando determinados modos de conceber e intervir deste sujeito no contexto da terapia intensiva; conhecer as tensões no fazer/saber enfermagem que podem ser discursivamente articuladas à bioética, ou problematizadas a partir da emergência deste discurso. A tese utilizou fontes documentais e de entrevistas com enfermeiros/as intensivistas e teve sua proposição aprovada por Comitê de Ética (Parecer n° 186/07/CEP/UFSC). O corpus documental foi composto por 113 artigos publicados no período de 1984-2007, nos periódicos nacionais de enfermagem: Revista Latino-Americana de Enfermagem, Revista Acta Paulista, Revista Texto & Contexto Enfermagem, Revista Escola de Enfermagem USP), Revista Brasileira de Enfermagem, além da Revista o Mundo da Saúde (multidisciplinar). A busca foi orientada pelos descritores bioética e UTI e enfermagem, ética e UTI, bioética e enfermagem e ampliada para artigos com temas relacionais com a bio/ética. Foram entrevistados/as 20 (vinte) enfermeiros/as inseridos/as no contexto da terapia intensiva, utilizando-se o critério de saturação de dados. Com baliza ao corpus de análise, foram exploradas as temáticas das iatrogenias, autonomia e responsabilidade no cuidar. Na temática iatrogenias, ativou-se uma reflexão sobre o agir do/a enfermeiro/a em um contexto permeado pela possibilidade sempre latente de falhar tanto no procedimento como na conduta; e do encontro deste profissional com a obrigação de corrigir esta falha, não tanto ou não apenas no conhecimento ou na lei, mas na prática de si mesmo. O tema da autonomia foi analisado a partir do conceito de cuidado de si, desdobrando-se em categorias que expressaram o privilegiamento da moral como obediência à Lei; da conduta e da moral sobre o conhecimento técnico; e da governabilidade de si no confronto com a técnica. Estas se configuraram como possibilidades éticas do sujeito enfermeira/o intensivista, não como etapas seqüenciais ou concorrentes, mas coligadas e confluentes na experiência atual. A questão da responsabilidade do cuidar desdobrou-se em categorias que expressaram a responsabilidade frente às novas linguagens e a enfermagem como guardiã de certos atributos da UTI. Autonomia e responsabilidade no cuidar, de um lado, evidenciaram algumas possibilidades éticas do sujeito enfermeira/o, constituídas e desfeitas estrategicamente na contingência histórica; de outro lado, permitiram mapear um dos desdobramentos estratégicos e tecnológicos do discurso da bio/ética, gerando modos de conceber e intervir do sujeito enfermeiro/a na UTI. Uma autonomia como prática de si; uma responsabilidade do cuidar que evidencia uma #sutileza# na obrigação do dever ser, em um tempo de viver a enfermagem, também, como perita, conselheira, autoridade patente. Ao contrário de um apagamento da relação paradoxal entre a responsabilidade e a autonomia dos/as enfermeiros/as, sinalizou-se as múltiplas possibilidades de combinações de graus de autonomia e de responsabilidade, nem sempre ideais ou caminhando no mesmo sentido. This qualitative investigation, landmarked in Foucaltian analysis with approximationg to post-structuralist theory, explores iatrogenias as one of the tensions of nursing performance/knowledge which can be discursively articulated to bioethics and to techno biomedicine. Documental sources and interviews with intensive care nurses permitted an activation of reflection upon a nurse#s actions in a context permeated by the always latent possibility of failing, in both procedure and conduct. Based on such possibility, intensive care nurses find themselves obligated to correct their failure, not as much or merely in knowledge, nor law, but in self practice
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