Дисертації з теми "Infections à Burkholderia cepacia"
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1
Lameignère, Émilie. "Etudes structurales et fonctionnelles des lectines solubles de Burkholderia cenocepacia." Grenoble 1, 2009. http://www.theses.fr/2009GRE10024.
Анотація:
La colonisation des poumons par des germes comme Pseudomonas aeruginosa et Burkholderia cenocepacia représente la première cause de maladie et de mortalité chez les patients atteints de la mucoviscidose. Quatre gènes de séquence apparentée à celui codant pour la lectine soluble PA-IIL (LecB) de P. Aeruginosa ont été identifies dans le génome de B. Cenocepacia. Ces lectines pourraient être impliquées dans la reconnaissance des cellules epitheliales de l'hOte au stade précoce de l'infection, ou encore dans la formation du biofilm responsable de la résistance aux antibiotiques. Les travaux développés dans cette thèse portent sur l'étude structurale et fonctionnelle de deux de ces lectines : Bc1A et Bc1B, ainsi que sur la localisation et la fonction des protéines chez la bactérie. L'utilisation de glycan arrays associée à la microcalorimétrie de titration ont permis de déterminer la spécificité des lectines et leur affinity pour les meilleurs ligands. Les études de localisation ont montré ces lectines cytoplasmiques sont aussi détectées à la surface de la membrane externe. Enfin, l'obtention de cristaux de lectine complexée à son ligand permet de mieux comprendre l'interaction l'échelle moléculaireOpportunistic infection by pathogens such as Pseudomonas aeruginosa and Burkholderia cenocepacia is the first cause of morbidity and mortality in cystic fibrosis patients. The opportunistic pathogen Burkholderia cenocepacia contains three soluble carbohydratebinding proteins, related to the fucose-binding lectin PA-IIL from Pseudomanas aeruginosa. These lectins could play a role in early stage of infection through specific binding to epithelial cells of hosts. They could also be involved in the building of biofilm that is responsible for resistance to antibiotics. The thesis is focused on structure-function studies of two B. Cenocepacia lectins, Bc1A and Bc1B with the aim to correlate the data to the localization and function in the bacteria. Glycan array data associated with titration microcalorimetry allowed to determine the specificity of the lectins and the affinity towards the best ligands. Localization studies demonstrate the presence of the lectins in the bacteria cytoplasm but also on the outer membrane. Finally, crystal structures of lectin complexed with carbohydrate give the molecular basis of the interaction
2
Mesureur, Jennifer. "Réponse de l'hôte et virulence bactérienne durant une infection aiguë ou persistante causée par le complexe Burkholderia cepacia chez l'embryon de poisson-zèbre (Danio rerio)." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTT024.
Анотація:
Les bactéries appartenant au complexe Burkholderia cepacia (Bcc) provoquent des infections sévères chez les personnes atteintes de mucoviscidose. L'infection peut varier d'une forme asymptomatique à une forme plus aiguë pouvant entraîner une pneumonie nécrosante et une septicémie, connue sous le nom de syndrome cepacia. Afin d'étudier les infections causées par le Bcc, nous avons développé un nouveau modèle in vivo, l'embryon de poisson zèbre. Nous avons montré que B. cenocepacia K56-2 pouvait se répliquer dans les macrophages et causer une infection aiguë mortelle pour les embryons. En revanche, B. stabilis LMG14294 induit une infection persistante chez les embryons. Dans cette étude, nous avons montré que les macrophages jouaient un rôle-clé dans la multiplication de K56-2 et dans l'induction d'une réponse inflammatoire MyD88-dépendante, caractérisée par la surexpression des gènes codant pour Cxcl8 (ou IL-8) et l'IL-1b. En l'absence de macrophages, les bactéries sont incapables de se multiplier durant les premières 24h de l'infection, ce qui donne un avantage pour la survie des embryons. L'absence de MyD88 induit aussi l'augmentation de la survie des embryons infectés par K56-2. Mais de manière paradoxale, les bactéries se multiplient mieux chez les embryons myd88-/- mutants que chez les embryons sauvages. Ceci suggère que ce n'est pas le nombre de bactéries qui est important pour l'infection, mais que c'est la réponse inflammatoire excessive causée par cette infection qui entraîne la mort des embryons. Afin d'avoir une vision globale des changements d'expression des gènes de l'hôte durant l'infection, nous avons effectué une expérience de RNAseq. Comme attendu, l'infection aiguë se caractérise par une importante modulation du transcriptome de l'hôte qui augmente avec le temps. A l'opposé, l'infection persistante n'induit que très peu de changements. La réponse immunitaire innée, et en particulier la voie des TLR, ainsi que l'apoptose sont très fortement activées durant une infection aiguë. Pour sa part, B. stabilis module essentiellement les gènes codant pour le système du complément.Le rôle critique des macrophages lors d'une infection par Bcc chez les poissons zèbre est en accord avec les récentes observations cliniques. Ceci suggère que le stade intracellulaire de B. cenocepacia et la réponse inflammatoire qui s'ensuit peuvent être des cibles pour le développement de nouvelles thérapies permettant de lutter contre cette infectionBacteria belonging to the Burkholderia cepacia complex (Bcc) can cause chronic infection with periods of acute exacerbation and sometimes fatal necrotizing pneumonia (“cepacia syndrome”) in individuals with cystic fibrosis (CF), and are associated with poor prognosis. Here, we exploited the exciting possibilities for in vivo non-invasive imaging of Bcc infection in transparent zebrafish embryos, with an innate immune system with remarkable similarity to that of humans, and numerous genetic and genomic tools to study the role of host phagocytes and the innate immune response in the pro-inflammatory character of the infection.We show that macrophages play a critical role in intracellular multiplication of B. cenocepacia K56-2 and induction of a MyD88-dependent fatal inflammatory response, characterised by high levels of cxcl8 and il1b expression. Surprisingly, in sharp contrast to the situation found for infections with other pathogens including Mycobacterium marinum and Staphylococcus aureus, in the absence of macrophages, K56-2 survived but was unable to replicate in the first 24 h, which resulted in a significant pro-survival advantage to the host compared to wild type embryos that died within 2 to 3 days. The Toll-like receptor (TLR) pathway is a major arm of the cell-mediated innate immune response with MyD88 as a key adaptor protein involved in the production of pro-inflammatory cytokines. We found that the absence of MyD88 also provided a pro-survival effect to the embryos after infection with K56-2. Paradoxically, the bacteria replicated better in myd88-/- mutant than wild type embryos, suggesting that it is not bacterial burden per se, but the inflammatory response that kills the embryos. Interestingly, cxcl8 and il1b expression were not significantly induced during the first 7 hours in the myd88-/- mutant while a strong induction was seen in control embryos, suggesting that a Myd88-dependent inflammatory response during early macrophage stages significantly contributes to fatal infection.Next, we performed RNAseq to analyse global changes in host gene expression during acute and persistent infection induced by K56-2 and B. stabilis LMG14294 respectively. Whereas acute infection was characterised by strong modulation of host gene expression increasing over time, persistent infection showed modulation of only a small set of genes. TLR and apoptosis signaling pathways were amongst the strongly activated groups during acute infection, in line with the strong inflammatory character of K56-2. During persistent infection, the major differentially expressed gene set concerned genes encoding complement proteins. The critical role for macrophages in Bcc infection in zebrafish is in agreement with recent clinical observations. We suggest that the intracellular stages of B. cenocepacia and the ensuing inflammatory response are essential targets to explore for the development of new therapies to combat this infection
3
Coutinho, Carla Patrícia da Silva. "Differentiation of clonal variants of the burkholderia cepacia complex isolated during chronic respiratory infection in cystic fibrosis patients with FTIR spectroscopy." Tese, Faculdade de Farmácia da Universidade do Porto, 2010. http://hdl.handle.net/10216/63817.
4
Coutinho, Carla Patrícia da Silva. "Differentiation of clonal variants of the burkholderia cepacia complex isolated during chronic respiratory infection in cystic fibrosis patients with FTIR spectroscopy." Doctoral thesis, Faculdade de Farmácia da Universidade do Porto, 2010. http://hdl.handle.net/10216/63817.
5
Lowe, Carolyn Ann. "Iron regulation in Burkholderia cepacia and Burkholderia pseudomallei." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246990.
6
Lewenza, William Shawn. "Quorum sensing in Burkholderia cepacia." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/NQ54796.pdf.
7
Mykrantz, Hallie B. "Investigation of Burkholderia cepacia Virulence." Miami University / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=miami1114116170.
8
Maxwell, Alison Irene. "Antimicrobial strategies against Burkholderia cepacia." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/22461.
Анотація:
The aim of this project was to investigate novel antimicrobial strategies against B. cepacia, based on natural antimicrobial compounds present in plants and human airways as protection against bacterial disease. The project focused on a panel of 20 strains of B. cepacia complex. Isolates included strains representing major epidemic clones of B. cepacia and exhibiting a range of susceptibilities to all classes of conventional antibiotics. The project is focused on two major themes. First, a study of antimicrobials from plants and second, examination of the antimicrobial activities of cationic peptides present in human airway secretions. The susceptibility of the B. cepacia strain panel to plant extracts was investigated, in particular the activity of aqueous garlic extract (AGE) and thyme oil. The MIC of thyme oil for all 20 strains was found to be 0.01%. MICs of AGE ranged from 0.25%-3%. Killing curves suggesting that AGE produces a slow killing effect over a twenty four hour period, whereas thyme oil kills bacterial cells in less than 20 minutes. By electron microscopy, no intact bacteria were observed after three minutes incubation with thyme oil. In contrast, two hours incubation with AGE produced morphological changes in the cellular structure of B. cepacia consistent with much slower damage to the bacterial cell membrane. Attempts were then made to purify and identify the chemical nature of the antimicrobial agents using reverse phase HPLC. AGE was shown to contain allicin along with other anti-cepacia compounds. Thyme oil was shown to comprise the phenols thymol and its isomer, carvacrol. Human (hBD-1) and murine (mBD-1) b-defensins were examined for activity against B. cepacia. In contrast to the salt-sensitive antimicrobial activity observed with Pseudomonas aeruginosa, no antimicrobial activity was observed against strains of B. cepacia.
9
McNeely, Damian. "Biocontrol of multidrug resistant Burkholderia cepacia." Thesis, University of Ulster, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413831.
10
Detsika, Maria G. "Genetic variation amongst isolates of Burkholderia cepacia." Thesis, University of Liverpool, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269724.
11
Pope, Cassie Francesca. "Evolution of fluoroquinolone resistance in Burkholderia cepacia." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1445281/.
Анотація:
This study investigates the evolution of fluoroquinolone resistance in Burkholderia cepacia and assesses fitness of clinical isolates of the B. cepacia complex. B. cepacia was chosen as a clinically relevant model of antibiotic resistance because these bacteria cause chronic infections in cystic fibrosis patients, are highly resistant to killing by many antimicrobials and consequently require long term antibiotic treatment. Fluoroquinolones are a widely used class of antimicrobials, increasingly used in medical and veterinary practice. A method was optimised and used to determine the rate of mutation occurring in topoisomerase genes that confer resistance to fluoroquinolones. The fitness cost associated with fluoroquinolone resistance mutations was assessed as a measure of the stability of resistance in the bacterial population. Clinical isolates were assessed for hypermutability using mutation to fluoroquinolone resistance as a selective tool. In Gram-negative bacteria resistance to fluoroquinolones occurs via three major mechanisms drug efflux, reduced permeability and target alteration. The spectrum of fluoroquinolone resistance mutations occurring in vitro, the rate at which they arise, and the fitness costs of characterised topoisomerase mutations was investigated, using models relevant to transmission of the Burkholderia cepacia complex. Previous studies have shown that single point mutations in DNA gyrase, conferring resistance, have no or low cost. Only double mutations in gyrA and parC conferred a fitness cost. Second step mutations occur at a faster rate than first step mutations. Mutation in gyrA, therefore, may predispose the genome to mutation in topoisomerase genes.
12
Boaisha, Othman. "Burkholderia cepacia complex bacteria and their antimicrobial activity." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/23048/.
Анотація:
Burkholderia cepacia complex (Bcc) are well known for their ability to produce antifungal agents. However, the majority of past studies have screened only a limited number of isolates, which were not taxonomically well characterized. In addition, while the activity of Bcc bacteria against plant pathogenic fungal species has been characterized, very few studies have examined the interaction between Bcc and saprotrophic wood decay basidiomycete species. The overall aim of this study was to begin to the characterization of the interactions of Bcc and other Burkholderia with the model ascomycete Candida albicans and woodland basidiomycete fungi. Methods. The study made use of a large collection (397 isolates) of taxonomically well identified Burkholderia species. A systematic screening of the antifungal activity of this collection was made against C. albicans and various wood decay basidiomycetes: Bjerkandera adusta, Trametes versicolor, Hypholoma fasciculare, Resinicium bicolor and Phanerochaete velutina. A wide range of media were evaluated for Burkholderia growth, antifungal metabolite production and the ability to support the mycelial growth of basidiomycetes. An agar overlay assay was used to examine the anti-candidal activity of Burkholderia isolates, while a novel overlay assay was developed using homogenised mycelial material for the basidiomycete species. Novel and existing extraction techniques were evaluated for their ability to partially purify active Burkholderia metabolites. The purification and identification of the active Burkholderia antibiotics were performed using both preparative thin layer chromatography (TLC) and liquid chromatography combined with mass spectrometry (LC-MS). A novel TLC-bioautography assay was also developed to reveal the presence of active Burkholderia metabolites in the antimicrobial extracts. The minimum inhibitory concentration (MIC) of the semi-purified Burkholderia antifungals was also determined. The natural diversity of Burkholderia and other bacteria associated with different rhizosphere environments was evaluated by cultivating isolates from local maize crops and examining a collection of Burkholderia isolates obtained from the rainforest in Sabah, Malaysia. The genetic basis for the secretion of antifungal agents by B. ambifaria was examined using transposon mutagenesis and screening for the mutants which had lost their anti-candidal activity. Additional transposon mutants that had lost their anti-bacterial activity and had been obtained in a previous screen were also examined for alterations in antifungal activity. Finally, the Burkholderia genome database and bioinformatic techniques were used to genetically characterize these mutants and other genetic loci that had been previously implicated in antibiotic production. Results. The majority of Burkholderia isolates possessed the same antagonistic activity towards C. albicans as they did towards B. adusta. Of the 397 isolates examined, antifungal activity was the greatest in the following Burkholderia species: B. ambifaria, B. cepacia, B. cenocepacia and B. contaminans. Interestingly, no antifungal activity was observed for B. multivorans (27 isolates) and B. stabilis (18 isolates) under the experimental conditions tested. The type of medium used to study the interaction between Burkholderia and the fungi considerably effected antifungal activity, with the most suitable media for studying antagonistic activity being Sabouraud agar and an acidic minimal salts medium (BSM; pH 6) supplemented with 0.4% glycerol. Of the 397 isolates screened, 47.85% were anticandidal (190 isolates; 89.47% of these were Bcc species and 10.53% were other Burkholderia species), while 48.10% were active against B. adusta (191 isolates; 93.19% of these were Bcc species and 6.81% were other Burkholderia). Extraction using Amberlite XAD16 resin binding followed by elution with methanol was an excellent means to isolate active Burkholderia antifungals. Fractionation of these extracts using silica gel TLC and ethyl-acetate:methanol:water (20:1:0.5) as the elution solvent was optimal for the separation of the majority of the active metabolites. A TLC-bioautography assay was developed in this current study as a preliminary screening technique to detect anti-microbial components of novel Burkholderia extracts. The assay was very useful for detecting anti-candidal and anti-bacterial compounds and was adapted for use for the first time with basidiomycetes. Antifungal Burkholderia isolates produced between 1 and 10 antifungal metabolites. Several antifungal agents were identified as enacyloxin, pyrrolnitrin, bactobolin and quinolines. Novel compounds and novel bactobolin derivatives were also present. For B. ambifaria strain AMMD a number of chromosomal loci were found to be involved in the production of antifungal metabolites. Quorum sensing was essential for the expression of antifungal activity as transposon mutations in the CepI synthase-encoding gene prevented the biosynthesis of all B. ambifaria anti-candidal compounds. B. ambifaria AMMD extracts containing enacyloxin IIa also produced an inhibitory effect at low concentration against E. coli, B. multivorans and A. baumannii. Conclusions. Burkholderia are potentially a very large reservoir of known and novel antimicrobial agents, especially with nearly 50% of isolates screened exhibiting antifungal activity. Antimicrobial compounds such as enacyloxin IIa were discovered for the first time to be produced by Burkholderi and novel extraction and bioassay methods for anti-fungals were developed in this study. Overall, we now have the tools to considerably advance the isolation and characterization of antifungal Burkholderia metabolites.
13
Hughes, Jayne. "The pathogenicity of Burkholderia cepacia in cystic fibrosis." Thesis, University of Edinburgh, 1998. http://hdl.handle.net/1842/21310.
Анотація:
This project has focused on the interaction of B. cepacia with neutrophils, the predominant immune cell in CF. As B. cepacia is resistant to neutrophil non-oxidative killing mechanisms, respiratory burst induction by B. cepacia was investigated using another CF pathogen, Pseudomonas aeruginosa, as a comparison. In general, non-opsonised CF isolates of B. cepacia induced little respiratory burst activity. By contrast, P. aeruginosa strains induced a greater range of responses, with a subset of strains inducing considerable activity. Opsonisation with specific immune sera increased neutrophil responses to both B. cepacia and P. aeruginosa. However, in view of the cleavage of opsonins and opsonin receptors within the CF lung, opsonisation may have little impact on host defences to B. cepacia within the respiratory tract. Lipopolysaccharide (LPS) extracted from B. cepacia was shown to upregulate neutrophil expression of complement receptor 3 (CR3) and to prime respiratory burst responses to fMet-Leu-Phe and to whole bacteria. Significantly LPS from the major epidemic strain of B. cepacia, ET12, increased neutrophil respiratory burst responses to P. aeruginosa but not to the ET12 strain itself. Thus it was speculated that B. cepacia may establish a foothold in the P. aeruginosa-infected lung by selectively upregulating immune responses to P. aeruginosa. In view of the development of environmental B. cepacia as biocontrol agents, environmental and clinical isolates were compared for potential virulence determinants. Few obvious differences were found between CF, non-CF clinical and environmental strains. Significantly LPS from two environmental strains of B. cepacia primed neutrophil responses to a similar degree as LPS from CF strains of B. cepacia. A surprising result was that an environmental B. cepacia strain was less effectively cleared in both CF and non-CF mice than the ET12 strain. This project has provided evidence to support the hypothesis that B. cepacia colonisation is associated with a marked and damaging immune response in CF patients.
14
Sood, Vandana. "Characterization of the 40 kDa protease of Burkholderia cepacia." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq24700.pdf.
15
Morgan, Michelle Marie. "Interaction of Burkholderia cepacia complex organisms and antimicrobial peptides." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.540768.
16
Bloodworth, Ruhullah. "Essential genes and genomes of the Burkholderia cepacia complex." Wiley, 2013. http://hdl.handle.net/1993/30912.
Анотація:
The Burkholderia cepacia complex (Bcc) are a group of closely related species known for their intrinsic multidrug resistance, large multipart genomes and ability to infect people with cystic fibrosis. The clinical relevance of the Bcc and their large multipart genomes make the study of their essential genes of broad interest. Essential genes are those required for survival in standard laboratory conditions this makes them potential targets for novel antibiotics against a group of species where few existing antibiotics are effective. Furthermore, while essential gene studies have been carried out in a number of bacterial species, only one of these species had multiple chromosomes and none had a genome as large as the Bcc. In my research I identified essential genes in B. cenocepacia K56-2, a member of the Bcc, by using transposon mutagenesis to deliver a rhamnose inducible promoter randomly into the genome and screening for a conditional growth (CG) phenotype. The utility of the CG mutant library was confirmed by showing that, when grown in suboptimal concentrations of rhamnose, only mutants that under-expressed the target of the antibiotic were hypersensitive. The CG mutant library included transposon insertions upstream from widely conserved, well-characterized essential genes suggesting that the system is capable of recovering essential gene mutants. A number of genes with either no or mixed records of essentiality in other microorganisms were also recovered. Among these was one of the three electron transfer flavoproteins (ETFs) in B. cenocepacia. The ETFs are a family of proteins found in a large number of eukaryotic, archaeal and bacterial species, which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions, high throughput screens have identified ETFs as putatively essential in several species. I showed that ETF expression is required for both viability and growth both on complex media and on media containing a variety of single carbon sources. Furthermore, cells depleted of ETF were determined to be nonviable and the morphologic shape of the cells changed from short rods to small spheres. In depth studies of essential genes are only possible for organisms with sequenced genomes. Of the 18 named species that currently comprise the Bcc, only 7 have been sequenced limiting the possibility of cross species comparative genomics. Therefore, I have assembled the first draft genomes of B. contaminans isolates, a species that has emerged as the dominant Bcc species recovered from the CF populations of Argentina and Spain. Identifying and characterizing essential genes in the Bcc, and sequencing additional Bcc species for comparative genomics are important first steps in understanding these clinically important bacteria.February 2016
17
Wigley, Paul. "Characterisation of Burkholderia cepacia from clinical and environmental origins." Thesis, Cardiff University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298072.
18
Padilha, Giovana da Silva 1976. "Caracterização, purificação e encapsulamento de lipase de Burkholderia cepacia." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/266999.
Анотація:
Orientadoesr: Elias Basile Tambourgi, Ranulfo Monte AlegreTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo : O presente trabalho foi dividido em três etapas e teve como objetivo a caracterização, purificação e encapsulamento da lípase a partir da cepa de Burkholderia cepacia. Para produção da enzima, o microorganismo foi cultivado em meio contendo sais, extrato de levedura, peptona bacteriológica e 6% de óleo de soja, por fermentação líquida em biorreator do tipo Bioflo III. As fermentações foram conduzidas a 150 rpm em 30ºC. Após 96 horas de cultivo, o sobrenadante foi separado das células por centrifugação e foi utilizado como extrato enzimático, uma vez que a Burkholderia cepacia produz lípase extracelular. O extrato bruto apresentou atividade usando azeite de oliva como substrato. Na primeira etapa do trabalho, analisou-se a condições ótimas de trabalho e estabilidade sob condições diversas e na presença de diferentes íons. A temperatura ótima foi a 37ºC, no entanto a 50ºC a enzima perdeu 10% da atividade em relação à temperatura em 37ºC. O cálculo de energia de ativação, seguindo a equação de ARRHENIUS, foi de 10,1 kJ/mol. O efeito do pH sobre a hidrólise do azeite de oliva pela lípase foi investigado com diferentes tampões na faixa de pH entre 3 e 11. Foram mantidas aproximadamente 80% da atividade entre os pHs 5 e 7, com atividade máxima no pH 8. Atividades razoáveis foram obtidas mesmo nos valores mais ácidos de pH e menores atividades nos valores entre 9 e 11. No entanto, no pH 11 observou-se queda de 80% de atividade em relação à atividade ótima da lípase. A estabilidade da atividade enzimática em diferentes tampões e valores de pH 5, 8 e 11 também foi investigada, onde nas 4 horas de incubação a diferentes valores de pH, a enzima mostrou-se bastante estável. Um estudo de estabilidade com diferentes íons por incubação da lípase durante 30 dias também foi realizado. Íons como Mn2+, Co2+, I- e Ca2+ mantiveram ou aumentaram a atividade enzimática, mas a enzima foi inibida na presença de Fe2+, Hg2+ e Al3+. À temperatura de 37 ºC e pH 8 foi feita a determinação dos parâmetros cinéticos. Os dados obtidos experimentalmente permitiram a obtenção da curva cinética, que mostrou a influência da emulsão óleo e água contendo diferentes proporções de azeite de oliva (0,1 a 50% v/v) na velocidade de hidrólise pela lípase. De acordo com a metodologia de LINEWEAVER-BURK, calculou-se os valores de Km e Vmáx de 43,90 mg/mL e 0,0258 U/mg, respectivamente. A segunda etapa foi purificar a lípase de Burkholderia cepacia em sistema bifásico aquoso polietileno glicol (PEG)/fosfato. Foram preparadas soluções estoques de PEG com massas molares de 1500, 4000 e 6000 Da (50% m/m) e tampão fosfato nos pHs 6, 7 e 8 (20% m/m de KH2PO4/K2HPO4). Um planejamento fatorial 23 foi feito para avaliar a massa molecular do PEG, pH e comprimento da linha de amarração (tie-line) no coeficiente de partição da atividade específica da lípase. Os resultados mostraram que maiores valores de atividade específica foram obtidos ao usar a menor massa molecular de PEG (1500 Da) em pH 6 e 8 e maior comprimento da linha de amarração. Após a purificação, determinou-se 33 kDa a massa molecular e 6,0 o ponto isoelétrico da lípase de Burkholderia cepacia. Na terceira etapa do trabalho, a enzima foi encapsulada por gelificação iônica usando alginato de sódio e cloreto de cálcio. Para a encapsulação foi feito um planejamento fatorial 22 variando o tamanho do bico atomizador (2 e 0,5 mm) e a concentração de CaCl2 (2 e 4% m/v). A enzima imobilizada foi caracterizada quanto à eficiência de encapsulamento, estabilidade, tamanho médio e sua distribuição e morfologia. No estudo de estabilidade verificou-se, em todas as condições de processo, que a enzima imobilizada manteve maior estabilidade em relação ao extrato bruto durante os 28 dias analisados. Nas análises de microscopia óptica, as microcápsulas formadas apresentaram formas esféricas, com média de tamanho de 400 e 100 ?m para os bicos de 2 e 0,5 mm, respectivamente. Nas análises por microscopia eletrônica de varredura (MEV), as microcápsulas foram secas em estufa e mantiveram as formas esféricas, com redução de tamanho, mesmo após esse processo de secagem. Usando o bico atomizador de 2 mm nas concentrações de 2 e 4% (m/v) de CaCl2, a microcápsula seca apresentou um tamanho de 139 e 150 µm, respectivamente, contra a média de 400 µm das hidratadas. Para o bico atomizador de 0,5 mm nas concentrações de 2 e 4% (m/v) de CaCl2, as microcápsulas apresentaram tamanhos de 15 e 6 µm, respectivamente
Abstract: This work was divided into three stages and aimed the characterization, purification and encapsulation of the lipase from a strain of Burkholderia cepacia. For enzyme production, the microorganism was grown in medium containing salts, yeast extract, bacteriological peptone and 6% soybean oil, liquid fermentation in bioreactor type Bioflo III. The fermentations were performed at 150 rpm and 30ºC. After 96 hours, the supernatant was separated from the cells by centrifugation and it was used as enzyme extract, since the Burkholderia cepacia produces extracellular lipase. In the first stage of this work, the enzymatic activity and stability were analyzed under different conditions and in the presence of different ions. The crude extract showed activity using olive oil as substrate. The optimum temperature was 37ºC, however at 50ºC the enzyme remained activity, with loss of activity of 10% compared to activity at 37°C. The calculation of activation energy, ARRHENIUS equation, was 10.1 kJ/mol. The effect of pH on hydrolysis of olive oil by lipase was investigated in the pH range between 3 and 11. About 80% of the activity was kept in the pH range between 5 and 7, with maximum activity at pH 8. Reasonable activities were obtained even in the more acidic pH values and lower activities in the values between 9 and 11. At pH 11 we observed a decrease of 80% of activity relative to the optimal activity of the enzyme. The stability of the enzyme activity of the crude extract in different buffers and pH values of 5, 8 and 11 was also investigated, where the 4 hour incubation at different pH values, the enzyme was stable. A stability study with different ions for incubation the lipase for 30 days was also realized. Ions such as Mn2+, Co2+, I- and Ca2+ remained stable or increased enzyme activity but the enzyme was inhibited in the presence of Fe2+, Hg2+ and Al3+. The determination of kinetic parameters was performed at 37°C and pH 8. The data obtained experimentally allowed obtaining the kinetic curve, which showed the influence of oil-water emulsion containing different proportions of olive oil (0.1 to 50% v/v) at the rate of hydrolysis by the lipase. According to the method of LINEWEAVER-BURK, it was calculated Km and Vmax values of 43.90 mg/mL and 0.0258 U/mg, respectively. The second step was to purify the lipase from Burkholderia cepacia in a polyethylene glycol (PEG)/phosphate aqueous two-phase system. Stock solutions were prepared with PEG molecular mass of 1500, 4000 and 6000 Da (50% w/w) and phosphate buffer in pHs 6, 7 and 8 (20% w/w KH2PO4/K2HPO4). A factorial design 23 was made to evaluate the molecular mass of PEG, pH and length of the tie-line in the partition coefficient of the enzyme specific activity. The results showed that higher values of specific activity of the enzyme were obtained when using the lower molecular mass PEG (1500 Da) at pH 6 and 8 and greater tie-line length. After purification, 33 kDa was determined for the molecular mass and 6.0 for the isoelectric point of the lipase from Burkholderia cepacia. In the third stage, the enzyme was encapsuled by ionic gelation using sodium alginate and calcium chloride. For the encapsulation, a factorial design 22 was made with 2 and 0.5 mm atomizer sizes and CaCl2 (2 and 4% w/v) concentration. The immobilized enzyme was characterized as to its encapsulation efficiency, stability, size and distribution size, and morphology. In the 28 day stability study, it was found in all process conditions that the immobilized enzyme remained more stable compared to the crude extract. In optical microscopy, the formed microspheres were spherical with average size of 400 and 100 µm for the 2 and 0.5 mm nozzles respectively. In the scanning electron microscope (SEM) analyses, the microspheres were dried in oven and maintained their spherical shapes with size reduction, even after the drying process. Using a 2 mm atomizer in the 2 and 4% (w/v) CaCl2 concentrations, the dry microcapsule presented a size of 139 and 150 µm respectively, against the average of hydrated 400 ?m. For the 0.5 mm atomizer in 2 and 4% (w/v) concentrations the dry microcapsules were 15 and 6 ?m, respectively
Doutorado
Sistemas de Processos Quimicos e Informatica
Doutor em Engenharia Química
19
Nzula, Sazini. "The Burkholderia cepacia complex : a clinical and biotechnological paradox." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/22530.
Анотація:
The aims of this thesis were to investigate the biological properties of both clinical and environmental isolates of the B. cepacia complex, with particular relevance to their pathogenicity and potential biotechnological exploitation. B. vietnamiensis were more sensitive to ceftazidime and chloramphenicol than strains from the other genomovars, and environmental B. cepacia genomovar III strains were more sensitive to ciprofloxacin and chloramphenicol than clinical isolates of the same genomovar. Although resistance to antibiotics is not uniform across all the subgroups of the B. cepacia complex, the antibiotic-sensitive strains can readily mutate to high levels of resistance. With the exception of catalase and melanin that were only produced by clinical strains, other putative virulence factors were detected in both clinical and environmental isolates. Certain factors including genetic markers for epidemic spread, were also detected in candidate biopesticide strains. The phytopathogenicity of clinical and environmental isolates was also found to be similar. Lack of knowledge regarding the fate of the B. cepacia complex strains introduced to the environment is a major obstruction to the organisms' commercial exploitation. Of major concern is the possibility of genetic exchange between different B. cepacia complex strains had between the B. cepacia complex and other soil microflora. Natural transformation of B. cepacia complex strains was demonstrated with DNA from the well-characterised epidemic lineage ET12, represented by the Edinburgh isolate J2315. The transformed bacteria included candidate biopesticide strains. The similarity of putative B. cepacia complex virulence factors produced by clinical, environmental and candidate biopesticide strains, as well as the natural exchange of genes between all subgroups suggests that caution should be exercised on the commercial application of members of the B. cepacia as biopesticides.
20
Verdier, Philippe. "Septicémies à pseudomonas cepacia en réanimation : étude clinique." Clermont-Ferrand 1, 1987. http://www.theses.fr/1987CLF13026.
Анотація:
Etude clinique sur des infevtions graves à Pseudomonas cepacia. A partir d' une série continue de 62 patients ayant présenté une hémoculture positive à P. Cepacia de mars 1983 à août 1986 dans les services cliniques de Clermont-Ferrand, l' auteur a réalisé une enquête clinique et une étude thérapeutique et pronostique de ces infections. L' étude des observations cliniques a permis d' identifier les principaux paramètres des infections graves à P. Cepacia. La résistance de ce germe à de nombreux antibiotiques pose un difficile problème thérapeutique. Le travail a essayé de préciser les différents traitements antibiotiques qui restent actifs sur ce germe. Enfin l' auteur a essayé de définir à partir d' une enquête épidémiologiques une attitude préventive qui devrait permettre une réduction de la fréquence de ces infections.
21
Shalaby, Moustafa El-Sayed Abd El-Hameid. "Biological degradation of substrate mixtures composed of phenol, benzoate and acetate by Burkholderia cepacia G4 Biologischer Abbau von Substratgemischen aus Phenol, Benzoat und Acetat durch Burkholderia cepacia G4 /." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=967643740.
22
彭志明 and Chi-ming Pang. "Molecular analysis of the dehalogenase IVa of Burkholderia cepacia MBA4." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1999. http://hub.hku.hk/bib/B31240872.
23
Farmer, Kate Louise. "Isolation and characterisation of exoproduct deficient mutants of Burkholderia cepacia." Thesis, University of Sheffield, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301450.
24
Sam, Laiju. "Molecular biology of a cryptic dehalogenase from Burkholderia cepacia MBA4." Thesis, Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B21827187.
25
Pang, Chi-ming. "Molecular analysis of the dehalogenase IVa of Burkholderia cepacia MBA4 /." Hong Kong : University of Hong Kong, 1999. http://sunzi.lib.hku.hk/hkuto/record.jsp?B21827230.
26
Horiuchi, Kenichi. "Biotransformation of Organic Residues to Useful Materials by Burkholderia cepacia." Kyoto Universtiy, 2001. http://hdl.handle.net/2433/108359.
27
Baxter, Ian A. "Studies of the nature of Burkholderia cepacia in cystic fibrosis." Thesis, Aston University, 1996. http://publications.aston.ac.uk/11039/.
Анотація:
37 B. cepacia isolates from clinical and botanical sources were characterised via metabolic capabilities, antibiotic sensitivity, fatty acid methyl ester (FAME) profiles restriction digest analysis of chromosomal DNA by pulsed-gel electrophoresis (PFGE) (with the use of two separate restriction enzymes) and outer membrane protein (OMP) profiles. This revealed isolates of the UK CF epidemic strain to form a distinct group with a specific OMP profile. Cluster analysis of PFGE and FAME profiles revealed the species Burkholderia gladioli and Burkholderia vietnamiensis to be more closely related to each other and to laboratory strains of B. cepacia than to the CF epidemic strain considered a member of the latter species. The epidemic strain of B. cepacia may therefore be worthy of species definition in its own right. All the strains studied showed a high level of resistance to antibiotics, including the carbapenems. Considering this, carbapenemase production by isolates of B. cepacia was investigated. A metallo--lactamase from a clinical strain of B. cepacia was isolated and partially purified of using Cibacron blue F3GA-coupled agarose. The resulting preparation showed a single band of -lactamase activity (pI 8.45) after analytical isoelectric focusing. The enzyme was particularly effective in the hydrolysis of imipenem. Meropenem, biapenem, cephaloridine, ceftazidime, benzylpenicillin, ampicillin and carbenicillin were hydrolysed at a lower rate. An unusual inhibition profile was noted. Inhibition by the metal ion chelators ethylene diamine tetra acetic acid and o-phenanthroline was reversed by addition of zinc, indicating a metallo-enzyme, whilst >90% inhibition was attainable with 0.1mM concentrations of tazobactam and clavulanic acid. A study of 8 other clinical isolates showed an enzyme of pI 8.45 to be present and inducible by imipenem in each case. This enzyme was assigned PCM-I (Pseudomonas cepacia metalloenzyme I).
28
Gallagher, Malcolm. "Epidemiology and resistance of Burkholderia cepacia complex in cystic fibrosis." Thesis, University of Liverpool, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399191.
29
Butler, Sarah Louise. "Pulmonary colonisation of patients with cystic fibrosis by Burkholderia cepacia." Thesis, University of Edinburgh, 1994. http://hdl.handle.net/1842/27646.
Анотація:
This thesis describes studies investigating the biological properties of B. cepacia, in particular those which may contribute to the organism's role as an opportunist pathogen and considers the host-bacterium interaction in CF including factors involved in early colonisation and the host immune response to B. cepacia colonisation. Emphasis is placed on studies of a highly transmissible or epidemic strain isolated in Edinburgh in 1989 and responsible for colonisation of CF patients in other regional CF centres. B. cepacia adhesion to respiratory mucin was measured in an ELISA-based mucin adherence assay. The majority of B. cepacia strains did not adhere to purified respiratory mucin. Interestingly, the epidemic strain exhibited the greatest degree of mucin adherence. Adhesion of B. cepacia to buccal epithelial cells studied by fluorescent labelling and flow cytometry also showed that the epidemic strain demonstrated greatest adhesion. The humoral immune response in CF patients colonised with B. cepacia was investigated by ELISA, incorporating B. cepacia LPS and by immunoblotting against LPS, flagella and outer membrane protein antigens. Elevated levels of specific anti-B. cepacia IgG, IgA and IgM were observed in patients chronically colonised by B. cepacia, especially in those patients colonised by the epidemic strain. Detection of anti-B. cepacia antibodies may aid in early diagnosis of B. cepacia colonisation, but as yet does not appear to have prognostic value. Evidence presented in this thesis indicates that B. cepacia persists in the CF respiratory tract despite a specific humoral immune response and causes bacteraemia despite being serum sensitive. These factors together with the intractability of B. cepacia to antimicrobial therapy and the close taxonomic relationship with the highly virulent Burkholderia pseudomallei, allows speculation that the association B. cepacia with CF may involve a stage of intracellular growth for survival.
30
Langley, Ross John. "Novel agents with inhibitory activity against the Burkholderia cepacia complex." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/29213.
Анотація:
The purpose of these studies was to investigate novel antimicrobial agents against the Bcc, in particular, but also other problematic and emerging CF pathogens, including multiresistant epidemic P. aeruginosa strains, methicillin resistant S. aureus (MRSA), and Sten. maltophilia. The studies were based on three themes: First, the natural antimicrobial properties of honey: second, the lytic properties of bacteriophage and their associated lytic enzymes; third, the antimicrobial properties of novel mammalian cationic β-defensins. The studies were able to use a large collection of bacterial isolates held in the Edinburgh strain repository, including clinical, environmental and epidemic strains. In the case of the Bcc, resistance to the major families was shown to vary across the Bcc. In general, clinical isolates were more resistant to conventional antibiotics than environmental isolates. The results show that resistance against antimicrobial agents varies within the Bcc. NZMh exerts a promising bactericidal effect on members of the Bcc, including B. cenocepacia J2315. The novel Bcc bacteriophages may prove to be a useful panel for further study, either as vectors for horizontal gene transfer or as therapeutic agents. The results conform previous reports showing that Bcc isolates are inherently resistant to CAMPs, including the novel 5-cysteine defensin, Defrl. However, synthetic Defr1 was shown to be active against a panel of multiresistant pathogens associated with infections in CF. Further research is required to optimise the recombinant expression of Bcep781 endolysin, D3 lysin, and Defr1, to allow investigation of their antimicrobial properties and their potential as therapeutics agents against multiresistant CF pathogens.
31
Lopes, Andreia Rodrigues Josefino. "Avaliação de mecanismo de escape imunológico do complexo Burkholderia cepacia." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/8527.
Анотація:
Dissertação para a obtenção do Grau de Mestre em Genética Molecular e BiomediciaO Complexo Burkholderia cepacia (BCC) tem vindo a ter cada vez mais atenção por parte da comunidade científica, principalmente devido ao perigo que representa para os doentes com fibrose quística (FQ). Os mecanismos de infecção e invasão estão a ser cada vez mais estudados, e a versatilidade deste grupo de bactérias tem-se provado excepcional. Por outro lado, também se tem começado a estudar a sua interacção com o hospedeiro. Assim com o objectivo de contribuir para o esclarecimento desta questão são estudados, neste trabalho, de forma comparativa 4 variantes clonais de B. cenocepacia isoladas de um mesmo doente, sendo este o primeiro trabalho a abordar questões imunológicas em isolados clonais. O objectivo deste trabalho foi verificar se existiam diferenças entre os isolados ao nível de: internalização por células dendríticas (DCs) e macrófagos derivados de monócitos humanos, capacidade para induzir morte celular nas DCs e macrófagos, indução de maturação das DCs e no tipo e capacidade de produção de citocinas pelas DCs. Este trabalho sugere que parte do sucesso na adaptação da B. cenocepacia ao ambiente pulmonar dos doentes de FQ e na infecção do mesmo passa também pela evolução nos mecanismos de escape imunológico desta bactéria. Demonstrou-se que os isolados mais virulentos são os menos internalizados, estimulam maior expressão de citocinas pró-inflamatórias e maior supressão da sua maturação. Os nossos resultados sugerem ainda que a morte celular induzida por estas bactérias é provocada maioritariamente por apoptose. Estes factores poderão explicar as diferenças de virulências entre os vários isolados clonais, através da subversão da resposta imune para uma melhor instalação da infecção.
32
Mirambell, Viñas Antonio. "Aspectos microbiológicos de Burkholderia cepacia complex en pacientes con fibrosis quística." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/329293.
Анотація:
La fibrosis quística (FQ) es la enfermedad hereditaria autosómica recesiva más frecuente entre la población caucásica. La principal causa de morbilidad y mortalidad en estos pacientes es por afectación pulmonar; mayoritariamente se producen infecciones respiratorias por Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia y algunas especies del complejo Burkholderia cepacia (Bcc). Cerca del 5% de los pacientes con FQ, se encuentran colonizados o infectados por Bcc, aunque éstas también son conocidas por infectar a pacientes con enfermedad granulomatosa crónica y por ser patógenas nosocomiales. Las especies del Bcc son bacilos gramnegativos fenotípicamente similares y con una relación genética muy estrecha; por ello la identificación y diferenciación mediante técnicas de microbiología convencionales es de elevada complejidad. Actualmente se han descrito 20 especies diferentes dentro Bcc. Debido a su elevada resistencia natural a numerosos antimicrobianos, su erradicación en los pacientes con FQ es dificultosa. Además el Bcc se caracteriza por poseer múltiples factores de virulencia, así como por su capacidad de formar biofilm. Entre las especies descritas del Bcc, B. multivorans y B. cenocepacia son las más prevalentes; no obstante esta distribución no se ha mantenido igual en el tiempo, ni en todos los países. Los objetivos del presente trabajo fueron; estudiar la epidemiolgía-molecular de los aislados bacterianos pertenecientes a Bcc obtenidos de muestras respiratorias de pacientes con FQ del Hospital Vall d’Hebron de Barcelona durante los años 2010 y 2012, determinar la sensibilidad a antimicrobianos y establecer la capacidad para formar biofilm de estos aislados. Por ello se identificaron mediante secuenciación del gen recA los aislados. También se comprobó la utilidad de MALDI-toF (VITEK MS y Daltonics MicroflexLT) para su identificación. La relación clonal se realizó por electroforesis en campo pulsante (PFGE) mediante la enzima SpeI. A su vez se determinaron los secuenciotipos (MLST) de los aislados y la relación clonal de éstos. Se estudió la sensibilidad a diferentes antimicrobianos utilizando la técnica disco-difusión y mediante la técnica del cristal violeta la capacidad de formar biofilm. El 5,3% de pacientes con FQ estuvo colonizado o infectado por Bcc. B. multivorans fue la especie más prevalente (43,8%), seguida de B. contaminans (37,5%), B. cepacia (18,8%) y B. stabilis (6,3%). Ambos MALDI-ToF ensayados determinaron el género del 100% de aislados, no obstante VITEK MS no determinó la especie de los aislados que pertenecían a B. stabilis y B. contaminans. Igualmente Daltonics MicroflexLT no identificó a nivel de especie los aislados de B. contaminans y un aislado de B. cepacia. En seis pacientes se aisló un mismo clon de B. contaminans (ST482), por lo que se constató la diseminación clonal de secuentiotipo. Se describieron los secuenciotipos ST17, ST450, ST723, ST724, ST725 y ST814 de B. multivorans; ST9 y ST726 de B. cepacia y ST51 de B. stabilis. De estos, ST723, ST724, ST725, ST726 y ST814 se describieron por primera vez en este estudio. El 68,2% de los aislados fueron sensibles a meropenem, 65,1% a piperacilina-tazobactam, 60,3% a ceftazidima, 54,6% a doxiciclina, 47,6% a trimetoprim-sulfametoxazol y 26,9% a ciprofloxacina. B. multivorans y B. cepacia fueron las especies más resistentes a antibióticos β-lactámicos, trimetoprim-sulfametoxazol y ciprofloxacina. B. contaminans y B. stabilis fueron los más sensibles a antibióticos β-lactámicos. Doxiciclina fue el antimicrobiano más activo frente a los aislados de B. multivorans y meropenem a los aislados de B. cepacia. Prácticamente todos los aislados tuvieron capacidad de formar biofilm. La capacidad de formar biofilm es diferente según la especie, siendo B. multivorans la que mostró mayor capacidad, aunque los aislados de un mismo clon mostraron cuantificaciones diferentes esgrimiendo la existencia de diferentes subpoblaciones de un mismo clon, con diferente capacidad de formar biofilm.Cystic fibrosis (CF) is the most common autosomal recessive hereditary disease among caucasian population. The main cause of morbidity and mortality in patients with cystic fibrosis are the current and chronic lung infections. Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and some of the Burkholderia cepacia complex (Bcc) species are linked to the CF lung infections. About 5% of CF patients, mainly adults, are colonized or infected by Bcc, although this bacterial complex is also known to infect patients with chronic granulomatous disease and also as nosocomial pathogen. Bcc gram-negative bacilli are phenotypically very similar and genetically related, consequently the identification and differentiation using conventional microbiological techniques is difficult. Due to its high natural resistance to many antimicrobials, the eradication of Bcc in CF patients is complicated. Bcc is characterized by multiple virulence factors, including the ability to form biofilm. Among the Bcc species, B. multivorans and B. cenocepacia are the most prevalent among CF patients; however the distribution of Bcc species among the CF patients it’s changing over the years and countries. The aim of this study was to reveal the distribution of the Bcc species among the isolates obtained through respiratory samples from CF patients attended at the CF unit of Vall d’Hebron University Hospital (Barcelona) between 2010 and 2012; the susceptibility to antimicrobial agents and the ability to form biofilm were studied. 63 Bcc isolates were identified from 16 CF patients by sequencing the recA gene. Also the identification of these isolates by MALDI-TOF (VITEK MS and Daltonics Microflex LT) was determined. The clonal relatedness was performed by the PFGE employing the SpeI enzyme and the ST of all the clonal isolates by MLST. Finally the susceptibility to different antimicrobial and the biofilm quantification were analyzed by the disc-diffusion method and crystal violet technique respectively. 5,3% of CF patients were colonized or infected by Bcc. The most prevalent specie was B. multivorans (43,8%) followed by B. contaminans (37,5%), B. cepacia (18,8%) and B. stabilis (6,3%). Both MALDI-ToF analyzed in this study determined the genus of all isolates. However VITEK MS misidentified the all the B. stabilis and B. contaminans isolates. Daltonics Microflex also misidentified the B. contaminans isolates and one B. cepacia isolate. From six patients the same B. contaminans (ST482) clone was isolated, therefore the spread capacity of this clone is proven. The ST723, ST724, ST725, ST726 and ST814 have been described for the first time in this study. From all B. multivorans isolates, ST17, ST450, ST723, ST724, ST725 and ST814 were determined, from B. cepacia, ST726, ST9 and from B. stabilis, ST51 was found. By the antimicrobial susceptibility the 68,2% of the isolates were susceptible to meropenem, 65,1% to piperacillin-tazobactam, 60,3% to ceftazidime, 54,6% to doxycycline, 47,6% to trimethoprim-sulfamethoxazole and 26,9% to ciprofloxacin. B. multivorans and B. cepacia isolates were the most resistant to the betalactams, trimethoprim-sulfamethoxazole and ciprofloxacin antimicrobials. B. contaminans and B. stabilis were the most susceptible to betalactam. Doxycycline was the most effective antimicrobial agent against B. multivorans isolates and meropenem to B. cepacia isolates. Finally most of the Bcc isolates had the capacity to produce biofilm, nevertheless significant differences were determined among all the species, being B. multivorans the greatest. However the same clonal isolates showed significant differences at biofilm quantifications suggesting the presence of different subpopulations of the same clone, with different biofilm quantifications.
33
Thomas, Laura. "The molecular basis for preservative resistance in Burkholderia cepacia complex bacteria." Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/14475/.
Анотація:
Burkholderia cepacia complex bacteria can contaminate and survive in a variety of antimicrobial and preserved industrial products. Contamination may lead to economic loss for manufacturers and also potentially pose a risk to the health of vulnerable consumers. Understanding the interaction between Bcc bacteria and preservatives, and the molecular basis for their resistance, is essential in order to better target these organisms and to facilitate the implementation of improved preservative strategies which target resistance mechanisms. In the present study, multi-locus sequence typing analysis of a collection of 67 Bcc isolates from environmentalindustrial sources was used to expand the current knowledge of Bcc species diversity within this niche and identified B. lata (n=17) and B. cenocepacia (n=11) as predominant species groups. The relationship between Bcc species diversity, isolation source and preservative susceptibility was investigated using a collection of 83 genetically diverse Bcc strains from clinical, environmental and environmental-industrial isolation sources. Susceptibility to eight preservatives was not related to Bcc taxonomy, as susceptibility profiles varied both between and within species groups. However, Bcc isolates from environmental-industrial sources had a significantly higher minimum inhibitory and minimum bactericidal concentration (MIC and MBC) for the formaldehyde releasing agent DMDM hydantoin. This suggests that for this preservative agent, susceptibility was related to source and that the selection of highly tolerant Bcc bacteria had occurred within the niche. Isothiazolone, DMDM hydantoin, phenoxyethanol and methyl paraben preservatives were observed to be highly efficacious against Bcc bacteria when evaluated in growth medium at the maximum concentration regulated for use in rinse-off personal care products in EU-regulated countries. Benzethonium chloride and sodium benzoate preservatives had the weakest anti-Bcc activity at these levels but were effective against several strains. Combinations of preservatives, and preservatives with potentiating agents, were evaluated for synergistic anti- Bcc activity. The greatest anti-Bcc activity was observed when isothiazolone preservatives were combined with EDTA or phenoxyethanol, with each combination resulting in an additive effect. The competency of Bcc bacteria to adapt to preservatives was explored via the progressive sub-culture of B. lata strain 383 in subinhibitory preservative concentrations. This genome sequence strain represented a Bcc species commonly encountered in the environmental-industrial niche. Stable adaptive-resistance to isothiazolone and benzethonium chloride preservatives was developed. Phenoxyethanol, DMDM hydantoin and methyl paraben preservatives were recalcitrant to B. lata strain 383 adaptation. The preservative and antibiotic susceptibility profiles of the adapted B. lata strain 383 derivatives differed, suggesting the induction of agent-specific adaptive-resistance mechanisms had occurred. The B. lata 383-CMIT,-BIT, derivatives (adapted respectively to chloromethylisothiazolinone and benzisothiazolinone), demonstrated cross-resistance to isothiazolone preservatives and fluoroquinolone antibiotics. Sequence analysis of the topoisomerase genes in these derivatives revealed fluoroquinolone resistance was not mediated by target modification. Preservative-induced adaptive resistance was not associated with overall increased multi-drug resistance. The molecular basis for resistance to DMDM hydantoin and isothiazolone preservatives was investigated via the random transposon mutagenesis of B. lata strain 383 using pTnModOTp’. Several genetic pathways were identified as putative preservative resistance determinants, suggesting that resistance is multi-factorial. These included the detoxification of formaldehyde via a glutathione-dependent pathway; a type II general secretory system (A3244_A3233 genes); a homologue of an ABC-type efflux system involved in resistance to organic solvents (A3512_A3517 genes); homologues of multi-drug RND-type efflux systems EmrB/QacA-Emr-TolC; and bacterial defence mechanisms against oxidative stress. A transcriptomic microarray-based approach was used to profile global gene expression of B. lata strain 383 in response to sub-MIC of 0.00162% DMDM hydantoin and 0.00001498% of a methylisothiazolinone and CMIT blend, as well as isothiazolone-induced adaptive resistance. With a 1.5-fold change and P < 0.05 confidence level criterion applied, few significant changes were observed after a single sub-MIC exposure, and the differential expression of putative resistance determinants identified by transposon mutagenesis was not induced at these concentrations. Isothiazolone-induced adaptive-resistance involved a greater number of significant gene expression changes that were stable irrespective of the presence of the priming agent, with 126 up-regulated and 90 down-regulated genes. Transcriptomic analysis suggested that isothiazolone-induced adaptive resistance was multi-factorial in nature, and identified active efflux as a putative key resistance mechanism. A novel role for a RND-type efflux system (B1004_B1006 genes) was identified, and the up-regulation of the ABC-efflux system (A3512_A3517 genes) and bacterial defence mechanisms against oxidative stress corroborated the transposon mutagenesis findings. B. lata strain 383-CMIT demonstrated a four-fold reduction in MIC for the priming preservative (2.81E-04%) in the presence of 512 mg/L of the efflux inhibitor PAβN. Resistance mechanism targeted preservative strategies such as using efflux inhibitors may work to improve preservation.
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Chung, Yiu-kay Wilson, and 鍾堯基. "Identification of the regulatory element of dehalogenase IVa of Burkholderia cepacia MBA4." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B29517291.
35
Bartholdson, Sara Josefin. "Putative virulence factors and novel antimicrobial targets of the Burkholderia cepacia complex." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/16939.
Анотація:
Members of the Bcc, previously described as non-mucoid, produced copious amounts of EPS when onion extract was provided as a sole nutrient. When investigating the onion extract content, it was found that fructose, sucrose and alcohol sugars were able to induce this change in phenotype. Interestingly, none of the virulent B. cenocepacia ET-12 isolates was able to produce EPS on any medium tested. This loss of EPS phenotype correlated with an 11 bp deletion in bceB, which is part of the bce gene cluster associated with the biosynthesis of cepacian, a previously characterised Burkholderia EPS. It has been proposed that instead of a non-mucoid to mucoid conversion, which is strikingly characteristic of P. aeruginosa infections in CF patients, members of the Bcc revert from a mucoid to a non-mucoid phenotype, suggesting a role for EPS in initial persistence, and that a loss of mucoidy may lead to increased virulence. Bcc LPS has previously been shown to be constitutively modified with 4-amino-4-deoxy-β-L-arbinoase (L-Ara4N). The enzymes involve din L-Ara4N biosynthesis were recently shown to be essential for B. cenocepacia viability. In this study, UDP-glucose dehydrogenase (Ugd) and 4-amino-4-deoxy-β-L-arbinose transferase (ArnT), the first and last enzymes of the biosynthetic pathway, were investigated. The L-Ara4N biosynthetic enzymes may be an Achilles’ heel of B. cenocepacia. Inhibitors to any of the enzymes in the L-Ara4N biosynthetic pathway could potentially be used alone, or in combination with other antimicrobial agents that would, under normal conditions, not be able to penetrate the outer membrane.
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Marques, Petrus Pires 1987. "Estudo da imobilização de lipase de Burkholderia cepacia em alginato de sódio." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/266917.
Анотація:
Orientador: Elias Basile TambourgiDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: A lipase é uma enzima de alto interesse comercial, com aplicabilidades diversas na indústria, desde aditivo de detergentes até ferramenta de síntese da indústria farmacêutica. O uso da enzima é limitado, no entanto, pelo alto custo de obtenção em nível de pureza adequado, de acordo com a finalidade. O objetivo desse trabalho foi imobilizar a lipase diretamente de seu extrato bruto, excluindo no processo outras proteínas, promovendo uma imobilização seletiva, atuando como extração, ou purificação de baixa resolução. A enzima foi produzida utilizando uma cepa de Burkholderia cepacia com óleo de oliva como indutor. O fermentado produzido pela bactéria foi caracterizado quanto à sua atividade lipolítica utilizando o p-npp como substrato. O extrato apresentou temperatura ótima de 50°C e pH ótimo 9,0. A enzima foi avaliada ainda quanto à sua afinidade pelo substrato utilizado, revelando um Km=18,4 mM, com uma velocidade máxima de atividade de 5,56 mM.min-1. Através dos dados da cinética foi possível calcular a energia de ativação da enzima em 38 kJ.mol-1.K-1. O extrato enzimático foi utilizado em procedimento de imobilização utilizando alginato de sódio em solução. As já documentadas interações entre o alginato e lipases permitiram realizar um processo de imobilização seletiva por precipitação com cloreto de cálcio. Foram estudadas, com o auxílio de planejamento estatístico de experimentos, as seguintes variáveis atuantes no processo: pH e concentração da solução de alginato, concentração da solução de cloreto de cálcio, tamanho das esferas produzidas e relação volume alginato/volume solução enzimática. As melhores condições encontradas para o processo foram concentração de alginato de 2%, pH da solução 3,68 e concentração de cloreto de cálcio 200mM, com uma relação de 40% de volume alginato/60% solução enzimática, produzindo esferas de 2 mm de diâmetro. Com essas condições obteve-se uma taxa de imobilização de 168,07%, que representa uma concentração da proteína alvo em 1,68 vezes, e recuperação superior a 99% em um procedimento de etapa única
Abstract: Lipase is an enzyme of high commercial value, with several applications in industry, from detergent additive to pharmaceutical synthesis tool. Nevertheless, the lipase use is limited by the high cost of its production in the necessary purity grade according to its use. The objective of this work was to entrap lipase directly from its crude extract excluding contaminant proteins in the process, promoting a selective entrapment, acting as a pre-purification or extraction. The enzyme was produced using a strain of Burkholderia cepacia and olive oil as inducer. The crude extract was characterized according to its lipolytic activity using p-npp as substrate. The crude extract presented optimal temperature of 50 °C and optimal pH of 9.0. Lipase was also evaluated according to its affinity for the substrate, revealing a Km of 18.4 mM and maximum velocity of 5.56 mM.min-1. From the data obtained it was also possible to calculate the activation energy of the enzyme: 38 kJ.mol-1.K-1. This crude extract was used in the immobilization procedure using sodium alginate solution. The already published data about the affinity of lipases and alginate allowed a performance of a selective entrapment by precipitation with calcium chloride. Using statistical design of experiments the following variables were studied in the process: concentration and pH of alginate solution, concentration of calcium chloride solution, diameter of the produced beads and relation alginate volume/enzyme solution volume. The best conditions found were: alginate concentration 2% at pH 3.68, 200 mM calcium chloride concentration and a relation 40% alginate solution volume/60% enzyme solution, producing 2 mm diameter beads. In these conditions, it was possible to obtain a immobilization yield of 168.07%, which represents a concentration of 1.68 times of the target protein and an enzyme recovery superior to 99% in a single step procedure
Mestrado
Sistemas de Processos Quimicos e Informatica
Mestre em Engenharia Química
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Thickett, Kathleen Mary. "Towards developing a multilocus sequence typing (MLST) scheme for Burkholderia cepacia complex." Thesis, University of Warwick, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403134.
38
Huber, Birgit Alexandra. "Identifizierung von Genen in Burkholderia cepacia, die für die Formation von Biofilmen auf abiotischen Oberflächen von Bedeutung sind." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964907321.
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Pencreac'h, Gaëlle. "Biocatalyse en milieu organique : étude comparative des propriétés de la lipase de Pseudomonas cepacia en milieu aqueux et en milieu organique." Aix-Marseille 1, 1996. http://www.theses.fr/1996AIX11014.
Анотація:
L'objectif de cette etude a ete de comprendre les differents parametres qui gouvernent l'activite lipasique en milieu organique. Dans un premier temps une nouvelle methode de mesure de l'activite lipasique en milieu organique a ete developpee. La reaction est l'hydrolyse d'un ester d'acide gras et de p-nitrophenol. Le p-nitrophenol forme est dose par colorimetrie a 410 nm apres extraction par une phase aqueuse alcaline. La comparaison des proprietes en milieu aqueux et organique de la lipase de pseudomonas cepacia purifiee a revele que le fonctionnement en milieu organique est caracterise par: une augmentation de la stabilite thermique, de la temperature optimale et de l'energie d'activation de la reaction, une faible modification de la specificite d'acide gras et une tres forte diminution de l'activite. Les precautions experimentales necessaires (elimination des limitations diffusionnelles, controle de l'activite thermodynamique de l'eau en milieu organique) ont ete prises. La diminution de l'activite en milieu organique par rapport au milieu aqueux est caracteristique des lipases relativement a d'autres classes d'enzymes. Pour 24 autres lipases, le rapport de l'activite en milieu organique et aqueux varie fortement. Cinq lipases sont plus actives en milieu organique qu'en milieu aqueux. La plupart des preparations enzymatiques contiennent des additifs qui influencent l'activite en milieu organique. Nous avons egalement etudie les proprietes de la lipase de p. Cepacia immobilisee sur polypropylene poreux (accurel ep-100). Globalement, les proprietes de la lipase ne sont pas modifiees apres immobilisation. Toutefois, l'activite specifique en milieu organique augmente d'un facteur 3. De meme, pour 18 autres lipases on observe une augmentation (entre 10 et 4300 fois) de l'activite apres immobilisation
40
Edler, Carola [Verfasser], and Ralf Matthias [Akademischer Betreuer] Hagen. "Comparison of Mast Burkholderia cepacia, Ashdown + gentamycin and Burkholderia pseudomallei selective agar for the selective growth of Burkholderia spp. / Carola Edler ; Betreuer: Ralf Matthias Hagen." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2017. http://d-nb.info/1139492764/34.
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Bamford, Sarah. "Lipopolysaccharide as a major virulence factor in the pathogenesis of Burkholderia cepacia syndrome." Thesis, Cardiff University, 2008. http://orca.cf.ac.uk/55761/.
Анотація:
In cystic fibrosis (CF), bacteria of the Burkholderia cepacia complex (Bcc) can induce a fulminant inflammation with pneumonitis and sepsis. Lipopolysaccharide (LPS) may be an important virulence factor associated with the increased morbidity and mortality seen in Bcc infection but little is known about the molecular pathogenesis of Bcc LPS. In this study, the inflammatory response to highly purified LPS from different Bcc clinical isolates and the cellular signalling pathways employed was investigated. It has been suggested that the large inflammatory response from BcLPS may be due to contaminants in the LPS preparation. Phenol-chloroform petroleum ether (PCP) purified BcLPS preparations were compared to more highly purified, enzyme treated preparations of BcLPS. There were no significant differences in the levels of IL-6 and TNF-a induced from monocytes (MM6) and in levels of IL-8 from epithelial cells (A549), which indicates that there were no contaminants present that could cause an inflammatory response from these cells. The inflammatory response elicited by LPS from different Bcc species that were tested was seen to be varied. LPS from different clinical isolates of the same clonal ET12 strain of Burkholderia cenocepacia were found to induce a varied inflammatory response. Some isolate's LPS produced as much cytokine as prototypical Escherichia coli LPS and all Bcc isolates tested with the exception of environmental samples produced higher levels of inflammatory cytokine than LPS from the CF pathogen Pseudomonas aeruginosa. It was shown that passaging over time under laboratory conditions was not responsible for this variation. Bcc LPS samples were tested in their ability to prime MM6 cells for respiratory burst, samples that had previously produced high levels of cytokine from direct stimulation of MM6 cells were found to not be the most efficient primers of respiratory burst. It was found that the inflammatory response from Bcc LPS stimulated monocytes was separate and in some cases opposite to the ability to prime for respiratory burst. Inhibition experiments were used to investigate the Toll-like receptors and associated adaptor molecules and pathways utilized when monocytes were stimulated by Bcc LPS. The use of anti-CD14, anti-TLR4 and anti-TLR2 antibodies in Bcc LPS stimulated monocytes showed that all Bcc LPS samples tested signalled via CD 14 and TLR4 and not via TLR2. Inhibition of MyD88 using an inhibitor peptide proved that all but one sample required MyD88 to signal. LPS from clinical isolate of Burkholderia multivorans was found to activate the inflammatory response via MyD88-independent pathways. Using MAP kinase inhibitors to test for reduction of cytokine response from stimulated MM6 cells and direct analysis of activation through western blotting, LPS from all clinical Bcc isolates were seen to activate all three major MAPKs p42/44, p38 and JNK. Degradation of the NF-kB inhibitory protein IicB-a was tested using anti-lKB-a antibodies and activation of the transcription factor NF-kB was tested using an electrophoretic mobility shift assay (EMSA). IicB-a was degraded and NF-kB was activated in all the BcLPS samples tested. This study suggests that LPS alone from clinical isolates of Bcc is major virulence factor in CF that it can cause a massive inflammatory response from cells, and that the LPS induced signalling cascade is via classical TLR4-mediated signalling pathways similar to highly inflammatory LPS purified from E.coli.
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施國雄 and Johnny Sze. "Heterologous expression and localization of cryptic haloacid dehalogenase Chd1 of Burkholderia cepacia MBA4." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B3122670X.
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Lin, Qiaoyi. "Physiological and genetic studies of 2,4-dichlorophenoxyacetate dissimilation by Burkholderia cepacia strain 2a." Thesis, University of Kent, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.655200.
Анотація:
A chemo-heterotrophic bacterium Burkholderia cepacia strain 2a was isolated from garden soil in Wales by Dr. A. R. W. Smith (personal communication), and it has been the subject for the biochemical and genetic studies on 2,4- dichlorophenoxyacetate (2,4-D) metabolism. The genes involved in 2,4-D metabolism were located on the plasmid pIJB1 in a composite transposon Tn5530. This plasmid was sequenced in this project for the evolutionary study of this group of plasmid. The in-silico analysis of the 99,001 bp DNA sequence of pIJB1 revealed 93 open reading frames. Over 52 % of the sequence was comprised of well conserved IncPI backbone genes; the rest of the plasmid was composed of accessory genes for 2,4-D and malonate degradation on the Tn5530 transposon, and for mercury resistance on a Tn5058-like transposon. Phylogenetic analysis against twenty-two IncP-I plasmids revealed that pIJBI has the highest evolutionary relationship with the IncPI-1) plasmids pAKD4 (from Pseudomonas putida UWCI), and pEST40II (from Achromobacter xylosoxidans ssp. denitrificans EST4002). The loss of the 2,4-D metabolising phenotype during growth in non-selective (succinate-minimal salts) medium was confirmed to be due to homologous recombination of two identical ISI071 ::IS1471 elements flanking the Tn5530 transposon. Loss of the ability of strain 2a to utilise 2,4-D was followed through seven sequential subcultures in succinateMS liquid medium at different phases of growth. A cumulative 9 % of the viable cells in the seventh sub-culture was observed to have lost the 2,4-D phenotype. The functionality of the mer resistance gene operon on pIJB 1 was investigated by observing the level of inhibition different mercury compounds have on the growth of strain 2a on 4 mM succinate-MSM agar. Preliminary observation revealed that strain 2a possesses broad-spectrum resistance to both organic and inorganic mercury compounds. Strain 2a was tested for its ability to utilise a panel of the chlorinated phenoxyalkanoates to elucidate the specificity of the 2,4-D degradation pathway. Strain 2a was unable to grow on a range of substituted phenoxyalkanoates. Preliminary evidence showed that the organism cleaved the ether linkage in several of these compounds to produce the corresponding phenols, and strain 2a was observed to utilise glyoxylate, the other product of the ether-cleavage reaction; but was unable to utilise most of the phenols, due to the narrower substrate specificity of the second enzyme of the 2,4-D pathway, 2,4-dichlorophenol monooxygenase. Succinate exerted carbon catabolite repression on 2,4-D metabolism when both carbon substrates were presented in minimal salts medium (MSM) to strain 2a together. In this medium, the duration of the lag period between succinate exhaustion and the onset of 2,4-D metabolism was influenced by the previous history of the inoculum, grown in 2,4-D MSM, provided for the culture. Cells taken at mid-log phase for inoculation reproducibly produced longer lag periods (5 h) than those taken at early or late stationary phase (3 h and 3.7 h respectively). This was attributed to the different levels of poly-β-hydroxybutyrate (Hill) accumulated during growth in succinate; the greater the accumulation inside cells, the longer the lag was sustained. However, the causal link between PHB accumulation during succinate metabolism in diauxic growth and the state of the cells taken from different growth phases in 2,4-D-MSM for inoculation was unclear. The constancy of ATP levels around the period of the inter-growth lag phase suggested that PHB utilisation may have sustained cellular energy demands and thereby delayed the onset of2,4-D utilisation. The presence of the tfdK gene within the 2,4-D dissimilatory operon on pIJBl prompted the investigation into involvement of the encoded product in 2,4-D metabolism in strain 2a In-silico comparison of the tfdK amino acid sequence with its analogue in Achromobacter xylosoxidans ssp denitrificans strain EST4002 (pEST40II) and in Cupriavidus necator strain JMP134 (pJP4) revealed 100 % and 76 % identity respectively. The chemotactic role of tfdK was implicated by testing for swarming on soft-agar containing 2,4-D in JMP134 and EST4002, but little sign of swarming was observed with strain 2a The tfdK gene expression level in the three organisms, determined using qPCR, revealed that this phenotypic difference was possibly due to a diminished level of expression of the gene in stain 2a compared with the levels in the other two strains.
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Sze, Johnny. "Heterologous expression and localization of cryptic haloacid dehalogenase Chd1 of Burkholderia cepacia MBA4 /." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23735892.
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Caulkins, Juliana Carvalho de Arruda. "Identificação de genes envolvidos na síntese de polihidroxialcanoatos em Burkholderia cepacia linhagem IPT64." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-06082009-111247/.
Анотація:
Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado.The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.
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MONTEIRO, Josineide Neri. "Identificação de bactérias do complexo Burkholderia cepacia através de utilização de ferramentas computacionais." Universidade Federal de Pernambuco, 2016. https://repositorio.ufpe.br/handle/123456789/25984.
Анотація:
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CAPES
O gênero Burkholderia compreende bactérias gran-negativas, aeróbicas pertencentesà classe β-proteobacteria. Estudos de 16S rDNA revelaram que o gênero Burkholderia é composto por bactérias que, apesar de intimamente relacionadas e fenotipicamente muito similares, possuem múltiplas diferenças genéticas, suficientes para permitir subdivisões em espécies ou variantes genômicas, que formam o complexo B. cepacia. Dados biológicos, especialmente os de sequenciamento genômico, vêm sendo gerados em ritmo acelerado nas últimas décadas. Com o surgimento da Bioinformática, podemos aplicar técnicas computacionais para manipular dados biológicos. O alinhamento múltiplo de sequências (MAS) é um conjunto de técnicas utilizadas para entender informações biológicas de um conjunto de sequências sendo considerada a tarefa mais comum e mais importante da bioinformática, visto que pode fornecer consideráveis informações sobre estrutura e função de genes. Os algoritmos genéticos (AGs) permitem uma simplificação na formulação e solução de problemas de otimização visto que incorporam uma solução potencial para um problema específico numa estrutura semelhante à de um cromossomo e aplicam operadores de seleção e cruzamento a essas estruturas de forma a preservar informações críticas relativas à solução do problema. O presente trabalho objetivou aplicar técnicas computacionais visando solucionar o problema de alinhamento genético de sequências biológicas de DNA de bactérias do complexo Burkholderia cepacia. As sequências analisadas (586) foram obtidas através do banco de dados GenBank do National Center for Biotechnology Information (NCBI). Para alinhamento das sequências, utilizou-se as seguintes ferramentas: Clustal ômega e Kalign. Das ferramentas utilizadas, nenhuma conseguiu gerar dados de boa acurácia. Desse modo, conclui-se que existe a necessidade de desenvolvimento de novos algoritmos/ferramentas de alinhamento genético visando trabalhar com grande quantidade de dados para obtenção de uma otimização. Para o caso de várias sequências, o problema do alinhamento múltiplo é considerado NP-difícil. Desse modo, foi observado que é necessário desenvolver novos algoritmos, para sua resolução em tempo hábil buscando sempre soluções bem aproximadas da solução ótima.
The genus Burkholderia comprises gran-negative bacteria, aerobic belonging to β-proteobacteria class. 16S rDNA analyzes have revealed that the genus Burkholderia is composed of bacteria which, although closely related and phenotypically very similar, have multiple genetic enough differences to allow subdivisions species or genomic variants that constitute the B. cepacia complex. Biological data, especially the genomic sequencing, are being generated at a rapid pace in recent decades. With the emergence of bioinformatics, we can apply computational techniques to manipulate biological data. The multiple sequence alignment (MAS) is a set of techniques used to understand biological information from a set of sequences is considered the most common and most important task of bioinformatics, since it can provide considerable information about the structure and function of genes. AGs allow a simplification in the design and optimization of troubleshooting as incorporate a potential solution to a specific problem in a structure similar to a chromosome and apply selection and crossover operators such critical information to preserve the form of structures for the solution problem. This study aimed to apply computational techniques aimed at solving the genetic alignment problem of biological DNA sequences of bacteria Burkholderia cepacia complex. The sequences analyzed (586) were obtained from the GenBank database of the National Center for Biotechnology Information (NCBI). For aligning the sequences, the following tools were used: Clustal omega and Kalign. The tools used, none was able to generate good data accuracy. Thus, it is concluded that there is a need to develop new algorithms / alignment tools genetic targeting working with large amounts of data to obtain an optimization. In the case of multiple sequences, the problem of multiple alignment is considered to be NP-hard. Thus, it was observed that it is necessary to develop new algorithms for its resolution in a timely manner and always seeking approximate solutions of the optimal solution.
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Neto, Orlando Carlos da Conceição. "Complexo Burkholderia cepacia em pacientes com fibrose cística: caracterização das espécies, avaliação do perfil de susceptibilidade aos antimicrobianos e da diversidade genética." Universidade do Estado do Rio de Janeiro, 2013. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=9298.
Анотація:
O Complexo Burkholderia cepacia (CBc) é um grupo de 17 espécies intimamente relacionadas que estão associadas à deterioração pulmonar e aumento da mortalidade em pacientes com Fibrose Cística (FC). Essas espécies variam entre si em relação à prevalência, quadros clínicos e virulência. Pouco é conhecido em relação ao perfil de resistência aos antimicrobianos. Uma vez estabelecida a infecção, a abordagem terapêutica e as medidas de controle atualmente adotadas são baseadas no CBc, sem considerar cada espécie em particular. O objetivo deste estudo foi determinar a prevalência das espécies do CBc em pacientes atendidos em dois centros de referência no Rio de Janeiro, bem como estabelecer perfis de resistência a antimicrobianos e avaliar a diversidade molecular entre as espécies. Cem amostras do CBc isoladas de 38 pacientes com FC no período de janeiro de 2010 a fevereiro de 2012 foram identificadas por métodos fenotípicos e pelo sequenciamento do gene recA. As CIMs para amicacina, aztreonam, ceftazidima, trimetoprim/sulfametoxazol e tobramicina foram determinadas por microdiluição e a genotipagem das espécies foi realizada por PFGE com a enzima SpeI. B. vietnamiensis (44%) foi a espécie mais prevalente, seguida de B. cenocepacia IIIA (36%), B. multivorans (10%), B. cenocepacia IIIB (1%) e B. stabilis (1%). Cinco por cento das amostras não foram identificadas. B. vietnamiensis foi identificada em mais da metade dos pacientes (58,3%). Foram observadas diferenças no perfil de susceptibilidade entre as espécies do CBc. B. cenocepacia IIIA foi a espécie que apresentou as maiores taxas de resistência aos antimicrobianos, sobretudo para trimetoprim/ sulfametoxazol (80,5%), principal antimicrobiano utilizado no tratamento de infecções causadas pelo CBc. Amostras com perfis MDR ocorreram em todas as espécies, destacando-se o perfil A, resistente simultaneamente aos cinco antimicrobianos, observado em 58,8% das amostras de B.cenocepacia IIIA. A análise do polimorfismo genético mostrou que, apesar de B. vietnamiensis ter sido a espécie mais prevalente, a ocorrência de nove grupos clonais sugere que a aquisição dessas cepas tenha se dado a partir de uma fonte ambiental comum. Para B. cenocepacia IIIA, 52,9% das amostras foram atribuídas a um mesmo grupo clonal (BcA), compartilhado entre nove pacientes atendidos em um mesmo centro de referência. Oitenta por cento dessas amostras apresentaram ainda resistência a todos os antimicrobianos testados. Os dados mostram que, mesmo com o emprego de técnicas moleculares, é difícil a identificação do CBc em nível de espécie; que B. cenocepacia IIIA é caracterizada por índices de resistência superiores às outras espécies e que a transmissão cruzada entre os indivíduos aponta para a necessidade do estabelecimento de medidas de vigilância do CBc nos centros de referência.The Burkholderia cepacia complex (BCC) is a group of 17 closely related species that are associated with pulmonary deterioration and increased mortality in patients with Cystic Fibrosis (CF). These species differ from each other in prevalence, clinical status and virulence. Little is known about the profile of antimicrobial resistance. Once the infection, the therapeutic approach and the control measures currently adopted are based on BcC, without considering each particular species. The aim of this study was to determine the prevalence of BcC species in patients from two reference centers in Rio de Janeiro, as well as establishing antimicrobial resistance profiles and assess the molecular diversity among them. One hundred samples of BcC isolates from 38 CF patients from January 2010 to February 2012 were identified by phenotypic methods and by sequencing the recA gene. The MIC for amikacin, aztreonam, ceftazidime, trimethoprim /sulfamethoxazole and tobramycin were determined by microdilution species and genotyping was carried out by PFGE with the enzyme SpeI. B. vietnamiensis (44%) was the most prevalent species, followed by B. cenocepacia IIIA (36%), B. multivorans (10%), B. cenocepacia IIIB (1%) and B. stabilis (1%). Five percent of the samples were not identified. B. vietnamiensis was identified in over half of patients (58.3%). There were differences in susceptibility profiles among BcC species. B. cenocepacia IIIA showed the highest rates of antimicrobial resistance, particularly to trimethoprim/ sulfamethoxazole (80.5%), primary antimicrobial used to treat infections caused by BcC. Samples with MDR profiles were observed for all species, highlighting the profile A, simultaneously resistant to five antibiotics, observed in 58.8% of B.cenocepacia IIIA samples. The analysis of genetic polymorphism showed that despite B. vietnamiensis was the most prevalent species, the occurrence of nine clonal groups suggests that these strains acquisition has taken place from a common environmental source. For B. cenocepacia IIIA, 52.9% of the samples were assigned to the same clonal group (BcA), shared among nine patients treated at a single referral center. Eighty percent of these samples also showed resistance to all antimicrobials tested. The data show that, even with the use of molecular techniques, the identification of BcC on species level is difficult; that B. cenocepacia IIIA is characterized by higher levels of resistance to other species and that the cross transmission between individuals points to the need for the establishment of BcC surveillance in reference centers.
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Capizzani, Carolina Paulino da Costa. "Epidemiologia das infecções bacterianas em pacientes com fibrose cística envolvendo Achromobacter e bactérias do complexo Burkholderia cepacia." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-23112017-100715/.
Анотація:
Achromobacter sp. e Burkholderia sp. são considerados patógenos problemáticos em pacientes com fibrose cística (FC), principalmente por apresentarem linhagens que podem ser transmissíveis e multidroga resistentes. Este trabalho teve como objetivo analisar isolados de Achromobacter e do complexo Burkholderia cepacia (CBc) de pacientes com FC atendidos no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto (HCFMRP-USP) e no Hospital das Clínicas da Faculdade de Ciências Médicas de Campinas (HCFCM-UNICAMP): identificar gênero/espécies; avaliar a sensibilidade a antimicrobianos; investigar relações genéticas entre os isolados por Pulsed-field Gel Electrophoresis (PFGE); elucidar a taxonomia e epidemiologia molecular dos isolados por Multilocus Sequence Typing (MLST) e correlacionar os resultados com dados clínicos. Entre julho/2011 a setembro/2014, nos dois hospitais, as espécies mais prevalentes de Achromobacter e CBc foram A. xylosoxidans e B. vietnamiensis, respectivamente. Os antibióticos mais efetivos contra isolados de Achromobacter sp. de pacientes do HCFMRP-USP foram imipenem e meropenem e do HCFCM-UNICAMP foram meropenem e ceftazidima. Os antibióticos mais efetivos contra CBc de pacientes do HCFMRP-USP foram sulfametoxazol-trimetoprim e meropenem e do HCFCM-UNICAMP foram ceftazidima e meropenem. Houve suspeita de contaminação cruzada entre alguns pacientes que apresentaram isolados com o mesmo perfil de PFGE. No HCFMRP-USP, isolados de B. vietnamiensis de pacientes diferentes tiveram o mesmo perfil de PFGE e apenas 2 pacientes tinham infecção crônica. No HCFCM-UNICAMP, isolados de B. cenocepacia IIIB de 4 pacientes apresentaram o mesmo pulsotipo, porém nenhum dos pacientes tinha infecção crônica. Isolados de B. vietnamiensis e B. multivorans de pacientes diferentes no HCFCM-UNICAMP também apresentaram o mesmo pulsotipo, e apenas um paciente colonizado por B. multivorans tinha infecção crônica. No HCFCM-UNICAMP, isolados de Achromobacter apresentaram perfis únicos de PFGE, enquanto que no HCFMRP-USP houve suspeita de contaminação cruzada somente entre pacientes colonizados por A. xylosoxidans, sendo que 3 destes pacientes estavam com infecção crônica. Nos dois hospitais, 17 STs foram identificados em isolados do CBc, 14 deles pela primeira vez e 3 STs (ST17, ST369 e ST911) apresentaram distribuição intercontinental. Em isolados de pacientes dos dois hospitais foram identificados alguns STs em comum (STs 1056, 1057, 369 e 911), o que pode sugerir ancestral comum. No total, 6 STs diferentes foram identificados em isolados de A. xylosoxidans de pacientes do HCFMRP-USP, dos quais 3 STs apareceram pela primeira vez e os outros 3 STs apresentaram distribuição intercontinental. Nenhuma das espécies apresentou linhagens epidêmicas descritas. Os pacientes colonizados cronicamente por A. xylosoxidans apresentaram valores de escore de Shwachman, índice de massa corporal (IMC) e função pulmonar menos preservados e exacerbações ligeiramente mais frequentes do que pacientes colonizados por bactérias do CBc. Este estudo possibilitou a correta identificação dos patógenos proporcionando a adoção de medidas de controle mais efetivas e tratamentos mais adequados, além de atualização do banco de dados epidemiológicos, o que facilita a análise colaborativa multicêntrica e auxilia no controle de infecção global destes patógenos.Achromobacter sp. and Burkholderia sp. are troublesome pathogens in cystic fibrosis (CF) patients, mainly because they may have transmissible and multidrug resistant strains. The aim of this study was to analyze the Achromobacter and Burkholderia cepacia complex (Bcc) isolates from CF patients treated at the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto (HCFMRP-USP) and Hospital das Clínicas da Faculdade de Ciências Médicas de Campinas (HCFCM-UNICAMP); to identify genus/species; to evaluate antimicrobial susceptibility; to investigate clonal relatedness among isolates by Pulsed-field Gel Electrophoresis (PFGE); to elucidate taxonomy and molecular epidemiology of the isolates by Multilocus Sequence Typing (MLST), and to relate the results to clinical data. Between July/2011 and September/2014, in both hospitals, the most prevalent species of Achromobacter and Bcc were A. xylosoxidans and B. vietnamiensis, respectively. The most effective antibiotics against Achromobacter sp. isolates of patients from HCFMRP-USP were imipenem and meropenem, and from HCFCM-UNICAMP were meropenem and ceftazidime. The most effective antibiotics against Bcc isolates of patients from HCFMRP-USP were sulfamethoxazole-trimethoprim and meropenem, and from HCFCM-UNICAMP were ceftazidime and meropenem. Cross-contamination was suspected among some patients who presented isolates with the same PFGE profile. In HCFMRP-USP, isolates of B. vietnamiensis from different patients showed the same PFGE profile, and only 2 patients had chronic infection. In HCFCM-UNICAMP, isolates of B. cenocepacia IIIB of 4 patients showed the same pulsetype, but none of the patients had chronic infection. Isolates of B. vietnamiensis and B. multivorans from different patients from HCFCM-UNICAMP also showed the same pulsetype, and only one patient colonized by B. multivorans had chronic infection. In HCFCM-UNICAMP, Achromobacter isolates showed unique profiles of PFGE, whereas in HCFMRP-USP cross-contamination was only suspected among patients colonized by A. xylosoxidans, and 3 of these patients had chronic infection. In both hospitals, 17 STs were identified in Bcc isolates, 14 of them for the first time and 3 STs (ST17, ST369 and ST911) presented intercontinental distribution. In both hospitals, some common STs (STs 1056, 1057, 369 and 911) were identified, which may suggest a common ancestor. In total, 6 different STs were identified in A. xylosoxidans isolates of patients from HCFMRP-USP, of which 3 STs were identified for the first time, and the other 3 STs presented intercontinental distribution. None of the species presented described epidemic strains. Patients chronically colonized by A. xylosoxidans showed less preserved Shwachman score, body mass index (BMI) and lung function, and slightly more frequent exacerbations than patients colonized by Bcc bacteria. This study provided the correct identification of the pathogens, allowing the adoption of more effective control measures and adequate treatments, besides updating the epidemiological database, which facilitates the multicentric collaborative analysis and assists in the control of global infection of these pathogens
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Silva, André Leonardo Patrício. "Preparação de sílicas organofuncionalizadas para imobilização da lipase de Burkholderia capacia." Universidade Federal da Paraíba, 2012. http://tede.biblioteca.ufpb.br:8080/handle/tede/7123.
Анотація:
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Previous issue date: 2012-09-19Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The preparation of stable immobilized lipases is one of the great challenges for modern biotechnology that involves the use of these enzymes in biocatalyzed processes. The commercial application of these biocatalysts depends of an efficient immobilization and the appropriate use of supports to ensure stability to enzymes. This work focused on the study of the preparation of chemically modified supports derived from silica gel. The organofunctionalized silicas were prepared by silylation through of the heteregeneous route using the compounds 3-aminepropyl- and 3-chloropropyl trimethoxysilane resulting in the solids named Sil-propil-NH2 and Sil-propil-Cl, respectively. The solids Sil-propil-NH2 and Sil-propil-Cl reacted subsequently with the cyanuric chloride and 1,6 diaminehexane as spacer, followed by covalent attack of cyanuric chloride resulting in the matrices Sil-propil-N-CC and Sil-propil-Hex-CC. Silica gel and the modified solid were characterized measures of adsorption / desorption of nitrogen, CHN elemental analysis, thermogravimetry (TG), Si29 and C13 NMR and FTIR spectroscopy. The modified support were used in the immobilization of the Burkholderia cepacia lipase, and the catalytic performance and stability of the immobilized enzyme derivatives were investigated in the hydrolysis reaction of p-nitrophenylpalmitate (pNPP) in five consecutive reaction cycles. The results of the preparation of matrizes showed the anchoring of the triazine molecule onto silanized surface. For material Sil-propil-N-CC was observed the increasing of the nitrogen content as indicated CNH elemental analysis, that corresponded to 1.82% of N due the introduction of amino group and 3.08% of N after the reaction of the triazine molecule. For the material Sil-propil-Cl, it was observed the increasing in the nitrogen percentage for the support with spacer (2.13% of N) and after the reaction with cyanuric chloride (3.6% of N). The tests of catalytic activity operational stability of the immobilized enzymes onto supports were 2910, 3000 e 3430 U/g, respectively, for supports with aminopropyl and triazine molecule, chloropropyl with the spacer and for the surface with spacer and triazine molecule. For catalytic test were observed a higher tendency for loss of stability for support without spacer. The obtained results showed that the chemical modification reactions of silica gel enabled the covalent anchoring of the cyanuric chloride and the use of spacer resulted in higher catalytic activity and stability for the immobilized bio catalysts.
A obtenção de lipases imobilizadas estáveis é um dos maiores desafios para a biotecnologia moderna que envolve o emprego destas enzimas em processos biocatalisados. A aplicação comercial desses biocatalisadores depende de métodos eficientes de imobilização e da utilização de um suporte apropriado para assegurar estabilidade às enzimas. Nessa direção, o escopo do presente trabalho envolveu a preparação de suportes quimicamente modificados a partir da sílica gel. As sílicas organofuncionalizadas foram preparadas por silanização pela rota heterogênea, utilizando os compostos 3-aminopropiltrimetoxissilano e 3-cloropropiltrimetoxissilano originando os sólidos denominados Sil-propil-NH2 e Sil-propil-Cl, respectivamente. Os sólidos Sil-propil-NH2 e Sil-propil-Cl reagiram subsequentemente com cloreto cianúrico e o com o espaçador 1,6 diaminohexano, seguido do ataque covalente do cloreto cianúrico resultando nas matrizes Sil-propil-N-CC e Sil-propil-Hex-CC. A sílica gel e os sólidos modificados foram caracterizados pelas técnicas de termogravimetria, medidas de adsorção/dessorção de nitrogênio, análises elementar de CHN, ressonância magnética nuclear de Si29 e C13 e espectroscopia de absorção na região do infravermelho. Os suportes modificados foram utilizados na imobilização da lipase de Burkholderia cepacia, sendo que o desempenho catalítico e estabilidade dos derivados enzimáticos imobilizados foram investigados em reações de hidrólise do éster palmitato de p-nitrofenila (p-NPP) em cinco ciclos reacionais consecutivos. Os resultados das preparações dos suportes mostraram o ancoramento da molécula triazínica nas superfícies silanizadas. No material Sil-propil-N-CC foi observado um aumento do teor de nitrogênio obtido da análise elementar, que correspondeu a 1,82% de N referente à introdução do grupo amino e 3,08% de N após a reação com o composto triazínico. No caso do material Sil-propil-Cl, observou-se um aumento na porcentagem de nitrogênio do suporte contendo espaçador (2,13% N) e após a reação com o cloreto cianúrico (3,6% N). Os testes de atividade hidrolítica e estabilidade das lipases imobilizadas mostraram 2910, 3000 e 3430 U/g, respectivamente para os suportes contendo aminopropil e o anel triazínico, cloropropil e o espaçador e a superfície com espaçador e o anel triazínico. Nos ensaios catalíticos foi observada uma maior tendência de perda de estabilidade na ausência do espaçador. Os resultados obtidos mostraram que as reações de modificação química da superfície da sílica possibilitaram o ancoramento covalente do cloreto cianúrico e a presença de um espaçador possibilitou maior atividade e estabilidade aos biocatalisadores imobilizados.
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SILVA, Walkíria Alves da. "Caracterização epidemiológica da podridão em escama da cebola." Universidade Federal Rural de Pernambuco, 2016. http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/5987.
Анотація:
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The onion is the third vegetable in economic importance on the world, with emphasis on Brazil as one of the most economically important vegetable, both by the volume and the income produced. This culture can be affected by various diseases, especially the scale rot, caused by bacteria of the Burkholderia cepacia complex. In an attempt to determine the favorable conditions for the development of epidemics, the knowledge of host-pathogen-environment interaction is essential. Thus, the appropriate inoculum concentration, the temperature range, the period of humidification exposure and the age, which the plant host becomes more susceptible to the establishment of high levels of disease, should be identified for each host-pathogen association. Although these epidemiological characteristics are the key factors for infection and subsequent development of rot in scale, there is no consistent information about the influence of these parameters on the behavior of the disease. Therefore, this study aimed to select and identify six isolates of B. cepacia complex, and determine the in vitro temperature, evaluate the effect of the inoculum concentration, temperature, presence and exposure to moisture chamber and the age of the bulbs in the severity of scale rot onion. Through Bayesian inference, the isolates CRMB31, and CRMB109 CRMB259 were identified as B. cenocepacia, while the isolates CRMB76, and CRMB199 CRMB222 were identified as B. arboris. The optimum temperature for in vitro growth of the isolates B. cenocepacia was 30°C, while for the isolates of B. arboris was 28°C. The conditions that predispose the occurrence of rot severity scale at higher inoculum were load of 108 CFU/mL, together on a wetness of 48 h, temperature between 35 and 40°C and more young tissues. To our knowledge, this is the first study to determine the environmental factors favorable to the development of rot in onion scale. In addition, the information obtained in this study will be useful for understanding the epidemics of the rot in scale and will assist in the implementation of strategies to control the disease.
A cebola é a terceira hortaliça em importância econômica no mundo, tendo destaque no Brasil como uma das hortaliças economicamente mais importantes, tanto pelo volume produzido como pela renda gerada. Esta cultura pode ser acometida por várias doenças, destacando-se a podridão em escama causada por bactérias do complexo Burkholderia cepacia. Na tentativa de determinar as condições favoráveis ao desenvolvimento de epidemias, o conhecimento da interação patógeno-hospedeiro-ambiente é imprescindível. Assim sendo, a concentração de inóculo adequada, a faixa de temperatura, o período de exposição à umidificação e a idade em que a planta hospedeira se torna mais suscetível para o estabelecimento de altos níveis de doença devem ser definidos para cada associação patógeno-hospedeiro. Embora essas características epidemiológicas sejam fatores primordiais para infecção e posterior desenvolvimento da podridão em escama, não existem informações consistentes a respeito da influência desses parâmetros sobre o comportamento da doença. Portanto, o presente trabalho teve como objetivos selecionar e identificar seis isolados do complexo B. cepacia e determinar a temperatura in vitro, avaliar o efeito da concentração de inóculo, temperatura, presença e tempo de exposição à câmara úmida e idade dos bulbos na severidade da podridão em escama da cebola. Por meio de Inferência Bayesiana, os isolados CRMB31, CRMB109 e CRMB259 foram identificados como B. cenocepacia, enquanto os isolados CRMB76, CRMB199 e CRMB222 foram identificados como B. arboris. A temperatura ideal de crescimento in vitro para os isolados de B. cenocepacia foi de 30°C, enquanto para os isolados de B. arboris foi de 28°C. As condições que predispuseram a ocorrência de severidade da podridão em escama mais elevadas foram carga de inóculo de 108 UFC/mL, em conjunto com um período de molhamento de 48 h, temperaturas entre 35 e 40°C e tecidos mais novos. Para o nosso conhecimento, este é o primeiro estudo realizado para determinação dos fatores ambientais favoráveis ao desenvolvimento da podridão em escama da cebola. Além disso, as informações obtidas neste estudo serão úteis para o entendimento de epidemias da podridão em escama e auxiliarão a implementação de estratégias para o controle da doença.