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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Analysis of polyhydroxyalkaloids in plant extracts by chromatography and mass spectrometry : including sequential tandem MS

Egan, Martin Joseph January 2000 (has links)
No description available.
12

Functional Characterization of Rainbow Trout (<em>Oncorhynchus mykiss</em>) Chemokine 2 (CK-2)

Eshaque, Shathi January 2006 (has links)
Chemokines are cytokines with chemoattractant ability, and comprise one of the major groups of molecules in immune system. These are small, secreted proteins cause the migration of leukocytes to the sites of injury. Over 40 mammalian chemokines have been identified to date, and they have been implicated in a number of immune mediated processes, including regulation of inflammation, antigen presentation, blood cell development, metastasis, viral infection and wound healing. In rainbow trout, there have been fewer chemokines reported and only one functional study has been published. Rainbow trout chemokine 2 (CK-2) is the only known CC chemokine with a mucin stalk, which has the potential for extensive <em>O</em>-glycosylation. However, no functional characterization has been performed on this molecule yet. CK-2 shares the presence of a mucin stalk with the mammalian chemokines, fractalkine (CX<sub>3</sub>CL1), lymphotactin (XCL1), and CXCL16. Another related trout CC chemokine sequence, CK-2. 1, has been discovered recently, which has 98% nucleotide sequence identity with CK-2. CK-2. 1 was believed to be a separate gene due to its apparent differential regulation in challenged rainbow trout. The question remained, however, whether or not CK-2. 1 was a separate gene or an allele of CK-2. The goal of this project was to further characterize both CK-2 and CK-2. 1. <br /><br /> Through genomic PCR on several outbred individuals it was shown that CK-2. 1 is an allele of CK-2 but not a separate gene. Reverse transcriptase (RT) PCR analysis revealed an increased level of transcript both CK-2 and CK-2. 1 in response to phytohaemagglutinin (PHA) stimulation of head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL) collected from fish with different allelic distributions. Similar results were also observed in the rainbow trout macrophage/monocyte cell line, RTS11. Moreover, an anti-CK-2 antiserum was developed in rabbits, which cross-reacted with CK-2. 1. This newly produced antibody was used to determine the protein expression levels in PHA stimulated rainbow trout tissues. RT-PCR was also performed on the same tissues in order to examine the transcript expression. Rainbow trout with both CK-2 and CK-2. 1 were used for this experiment. An overall decreasing pattern of transcript (both CK-2 and CK-2. 1) was observed in brain and HK over 24 hours, while protein was still detected at 24 hours post stimulation. However, in spleen the CK-2 transcript showed a slight upregulation at 4 hours post stimulation along with a very little or no CK-2. 1 expression, although no protein was detected in spleen. Liver showed a very low level of CK-2 and CK-2. 1 transcript at 8 hours post stimulation; while protein was again detected at 24 hours post stimulation. In addition, the sizes of the proteins found in different tissues were larger than expected (&le;30 kDa for CK-2 or &le;35 for CK-2. 1), perhaps due to the presence of extensive <em>O</em>-glycosylation at the mucin stalk of the protein. <br /><br /> A chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5??m pores toward CK-2 at an optimal concentration of 500ng/ml (17nm). Moreover, by pre-treating the recombinant chemokine with the polyclonal antisera, it was shown that the chemokine was actually causing the chemotactic activity. Pre-treatment of the cells with pertussis toxin, an inhibitor of G-protein signalling inhibited the migration of PBLs, established the fact that CK-2 caused chemotaxis by binding to a 7 transmembrane, G-coupled receptor just like all other known chemokines. Interestingly, CK-2 was also shown to attract RTS-11 cells. <br /><br /> Overall, the above findings indicate that CK-2 is functionally a chemokine with two very different alleles in rainbow trout. It is probably heavily <em>O</em>-glycosylated and different tissues express different sizes of the protein. This is only the second functional study of a fish chemokine.
13

Avaliação do sistema de mobilização de poli-3-hidroxibutirato em Burkholderia sacchari. / Evaluation of poly-3-hydroxybutyrate (P3HB) mobilization system in Burkholderia sacchari.

Castellanos, Nuri Andrea Merchan 19 October 2010 (has links)
O sistema de mobilização intracelular de poli-3-hidroxibutirato (P3HB) em Burkholderia sacchari foi analisado. A busca em genomas de Burkholderia spp. identificou duas oligômero hidrolases (PhaY1 e PhaY2) e pelo menos três P3HB despolimerases intracelulares (PhaZa1, PhaZa2 e PhaZd1). Mutantes de B. sacchari afetados na mobilização de P3HB e complementados com genes de Ralstonia eutropha apresentaram um aumento expressivo nas taxas de mobilização de P3HB, especialmente quando o gene phaZa1 foi superexpresso. A superexpressão dos genes phaZa2 ou phaZa3 também conduziu a aumentos nas taxas de mobilização embora em um grau menor que os valores obtidos com phaZa1. Dois mutantes afetados na mobilização de P3HB foram obtidos utilizando o transposon mini-Tn5 (NAM03 e NAM04). NAM03 apresentou interrupção em gene que codifica uma P3HB despolimerase intracelular (PhaZa1). NAM04 apresentou interrupção em gene anotado como serino peptidase LonA. Este pode representar um ativador da mobilização ou uma nova P3HB despolimerase intracelular. / The intracellular poly-3-hydroxybutyrate (P3HB) mobilization system in Burkholderia sacchari was analyzed. A search in Burkholderia spp. genomes identified two oligomer hydrolases (PhaY1 and PhaY2) and at least three intracellular P3HB depolymerase (PhaZa1, PhaZa2 e PhaZd1). B. sacchari mutants affected on P3HB mobilization and complemented by Ralstonia eutropha genes showed an expressive increase on P3HB mobilization rates, especially when phaZa1 was overexpressed. The overexpression of phaZa2 or phaZa3 also increased the mobilization rates though to a lesser extent than phaZa1. Two mutants affected on P3HB mobilization were obtained using the transposon mini-Tn5 (NAM03 and NAM04) .NAM03 was disrupted in a gene encoding an intracellular P3HB depolymerase (PhaZa1). NAM04 was disrupted in a gene annotated as a serine peptidase LonA. This could be a mobilization activator or a new intracellular P3HB depolymerase.
14

Functional Characterization of Rainbow Trout (<em>Oncorhynchus mykiss</em>) Chemokine 2 (CK-2)

Eshaque, Shathi January 2006 (has links)
Chemokines are cytokines with chemoattractant ability, and comprise one of the major groups of molecules in immune system. These are small, secreted proteins cause the migration of leukocytes to the sites of injury. Over 40 mammalian chemokines have been identified to date, and they have been implicated in a number of immune mediated processes, including regulation of inflammation, antigen presentation, blood cell development, metastasis, viral infection and wound healing. In rainbow trout, there have been fewer chemokines reported and only one functional study has been published. Rainbow trout chemokine 2 (CK-2) is the only known CC chemokine with a mucin stalk, which has the potential for extensive <em>O</em>-glycosylation. However, no functional characterization has been performed on this molecule yet. CK-2 shares the presence of a mucin stalk with the mammalian chemokines, fractalkine (CX<sub>3</sub>CL1), lymphotactin (XCL1), and CXCL16. Another related trout CC chemokine sequence, CK-2. 1, has been discovered recently, which has 98% nucleotide sequence identity with CK-2. CK-2. 1 was believed to be a separate gene due to its apparent differential regulation in challenged rainbow trout. The question remained, however, whether or not CK-2. 1 was a separate gene or an allele of CK-2. The goal of this project was to further characterize both CK-2 and CK-2. 1. <br /><br /> Through genomic PCR on several outbred individuals it was shown that CK-2. 1 is an allele of CK-2 but not a separate gene. Reverse transcriptase (RT) PCR analysis revealed an increased level of transcript both CK-2 and CK-2. 1 in response to phytohaemagglutinin (PHA) stimulation of head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL) collected from fish with different allelic distributions. Similar results were also observed in the rainbow trout macrophage/monocyte cell line, RTS11. Moreover, an anti-CK-2 antiserum was developed in rabbits, which cross-reacted with CK-2. 1. This newly produced antibody was used to determine the protein expression levels in PHA stimulated rainbow trout tissues. RT-PCR was also performed on the same tissues in order to examine the transcript expression. Rainbow trout with both CK-2 and CK-2. 1 were used for this experiment. An overall decreasing pattern of transcript (both CK-2 and CK-2. 1) was observed in brain and HK over 24 hours, while protein was still detected at 24 hours post stimulation. However, in spleen the CK-2 transcript showed a slight upregulation at 4 hours post stimulation along with a very little or no CK-2. 1 expression, although no protein was detected in spleen. Liver showed a very low level of CK-2 and CK-2. 1 transcript at 8 hours post stimulation; while protein was again detected at 24 hours post stimulation. In addition, the sizes of the proteins found in different tissues were larger than expected (&le;30 kDa for CK-2 or &le;35 for CK-2. 1), perhaps due to the presence of extensive <em>O</em>-glycosylation at the mucin stalk of the protein. <br /><br /> A chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5µm pores toward CK-2 at an optimal concentration of 500ng/ml (17nm). Moreover, by pre-treating the recombinant chemokine with the polyclonal antisera, it was shown that the chemokine was actually causing the chemotactic activity. Pre-treatment of the cells with pertussis toxin, an inhibitor of G-protein signalling inhibited the migration of PBLs, established the fact that CK-2 caused chemotaxis by binding to a 7 transmembrane, G-coupled receptor just like all other known chemokines. Interestingly, CK-2 was also shown to attract RTS-11 cells. <br /><br /> Overall, the above findings indicate that CK-2 is functionally a chemokine with two very different alleles in rainbow trout. It is probably heavily <em>O</em>-glycosylated and different tissues express different sizes of the protein. This is only the second functional study of a fish chemokine.
15

Ackumuleringspotential av PHA  från restströmmar inom pappersbruk : En studie om PHA från Gruvöns, Bäckhammars och Skoghalls bruk

Berglund, Alfred January 2018 (has links)
Plastic is one of the most universal materials used today. With a good future view, with new implementations and applications, it makes a lot of time to look at the production and management of the plastic materials. Plastic materials that have been used in our daily lives cause serious environmental problems. Millions of tons of these non-degradable plastics accumulate in the environment every year. The basic problem is that plastic is not naturally occurring in nature since containers are usually made of polyethylene terephthalate. This means that microorganisms do not have the ability to break it down to the current cycle. It takes hundreds of years for plastic containers to break down, not biologically but only degenerate into smaller and smaller pieces. Plastic breaks down into smaller pieces that become smaller and smaller until we cannot see them with the naked eye, mainly through heat and UV light. Although we cannot see them, they are still present and become part of our nature forever. Bioplastics is the plastic industry's tool to try to reduce these little pieces of our nature that will remain forever so that they do not grow more. With today's plastic packaging, which is said to be bioplastic, additives of, for example, cobalt and nickel, which are said to make it easier for the polymers to break down over time, have proven to be not as effective as they thought. Polyhydroxyalkanoates (PHA) are polymers which are biodegradable as based on their composition have different physical properties. PHA is a family of natural polyesters synthesized from various microorganisms discovered in 1926. Once discovered, interest has been high due to their biodegradability and its production from renewable resources. The polymers can be described generally as production from microorganisms under controlled conditions, where they occur naturally in organisms that classify them as biopolymers. Some of these polymers are already industrially produced on a large scale today. However, many still apply to several new areas but must be optimized for commercial production. Biopolymers can be classified into four groups. Amino-acid-based polysaccharides from bacteria, polyphenol-based and polyesters that this study is looking at. Depending on what the microorganisms possess for character traits and what they give to the substrate to break down, it gives polyesters with different physical properties. This case is a short-chain polyester to be formed, more specifically P3HB which is a three-carbon PHB polyester in its polymer which can be up to 5-7 units long. To avoid ongoing problems, a solution is needed. A solution that has received much attention to reduce plastic residues in nature is the use of biodegradable plastics and among them polyhydroxyalkanoates. Polyhydroxyalkanoates (PHAs) are common intracellular compounds found in bacteria, archaea and in few eukaryotes such as yeast and fungi. PHA acts as an energy storage polymer that is produced in some microorganisms when the carbon source is abundant and other nutrients such as nitrogen, phosphorus, oxygen or sulfur are limited. These polymers accumulate intracellularly up to 90% of the dry weight of the cell under nutritional conditions and act as energy saving materials. It has resembled mechanical properties like the traditional oil-based plastic such as polypropylene or polyethylene that can be formed with other synthetic polymers. PHA plastics possess many more applications, in agriculture, packaging and in the medical industry. It is biodegradable and also immunologically compatible. What the PHAs plaster can cause is an ultimate decomposition from a non-fossil source, which is exactly why it is very attractive. The purpose of this study was that from a hypothesis see within a limited time frame of ten hours of bio sludge from Gruvön, Skoghall and Bäckhammar's use could accumulate PHA with the aid of added readily degradable substrate. The process of the study will be a small part of a current research project together with Paper Province, Promiko, Pöyry and RISE. The aim of their study is to use residues from the forest industry to make hydrogen as well as bioplastics. This study will help to look at a subprocess of their cascading process. The aim of the study is to be able to measure the amount of PHA that could accumulate and rank the potential of the different uses. Using chemical analysis methods and extractions, it will provide opportunities to measure the accumulation of PHA in the various bacterial cultures of biomass from the use. The methods involve soxhlet extraction to successfully extract PHA from the bacteria. Dosage of substrate is sodium acetate piped from egg-diluted solution at 600 mg per dosage. In order for the dosage to be added at the right time, DO and the pH of the reactors were measured and logged throughout the course. FT-IR is used to view the course of events during the experimental period, linked to known features that may indicate that PHA is present in the bio sludge. Nutrients like phosphorus and nitrogen are measured, along with SÄ, SS, TOC, several before and after the experiment to compile discussion of the results. The conclusion was based on the analysis methods that the bio sludge that yielded the best yield was from Gruvöns use. This also relates best to the hypothesis of celebration and starvation, the relationship to which the bio sludge is exposed. The mine has a slurry in its five-step process which causes the bio sludge to return from step five where there is a shortage of food for bacteria to step three where there is a lot of food to consume. The rankings of the different uses relate to the hypothesis that the use of mining was best and the worst was the use of Bäckhammar. Based on the analysis methods included in the study, it can be concluded that the bio sludge that yielded the best yield was Gruvöns use with 13.6% of PHA / VS from the soxhlet extraction, the practice was best matched to the hypothesis. The ranking of the different bio sludge of the use is based on the hypothesis that Skoghall's use was second best followed by Bäckhammar's use which was the worst in accumulating PHA in the bacterial culture.
16

Identificação de bactérias da rizosfera de inhame, macaxeira e batata-doce potencialmente produtoras de polihidroxialcanoatos

Pereira, Juliana de Castro Nunes 28 February 2013 (has links)
Submitted by Nathália Neves (nathalia.neves@ufpe.br) on 2015-03-04T18:33:43Z No. of bitstreams: 2 Dissertação Formato digital - Juliana de Castro 2013.1.pdf: 985270 bytes, checksum: 14f3a13c691b9d8853fcfbe2c0c26fe2 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-04T18:33:43Z (GMT). No. of bitstreams: 2 Dissertação Formato digital - Juliana de Castro 2013.1.pdf: 985270 bytes, checksum: 14f3a13c691b9d8853fcfbe2c0c26fe2 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-02-28 / Inhame, macaxeira e batata-doce são vegetais ricos em amido e importantes sócio economicamente para o Nordeste brasileiro. O solo da rizosfera desses tubérculos e raízes é ambiente rico em bactérias que produzem metabólitos com diversas aplicações biotecnológicas, a exemplo de Polihidroxialcanoatos (PHAs), polímeros biodegradáveis produzidos a partir dos produtos da hidrolise do amido e utilizados na produção de plásticos. O objetivo do trabalho foi isolar e identificar do solo da rizosfera e do aderido à superfície desses três vegetais, bactérias potencialmente produtoras de PHAs, selecionando-as por PCR de colônia e avaliando a produção de PHAs por coloração de Sudan Black. Colônias bacterianas isoladas com características morfológicas distintas foram selecionadas para a PCR de colônia. Das 214 bactérias selecionadas, 11 amostras foram positivas para o gene phaC da via de síntese de PHA. Nove bactérias apresentaram resultado satisfatório para produção de grânulos de PHAs após coloração de Sudan Black. Dentre as bactérias positivas na PCR e Sudan Black, oito foram identificadas por seqüenciamento ao nível de gênero ou espécie, tais como: Pseudomonas, Bacillus, Agrobacterium, Stenotrophomonas, Cupriavidus e Microvirga flocculans, sendo esta última descrita pela primeira vez como produtora de PHA. A aplicação da PCR de colônia com coloração por Sudan Black mostrou-se eficiente e rápida para seleção de bactérias potencialmente produtoras de PHAs a partir de amostras de solo.
17

Avaliação do sistema de mobilização de poli-3-hidroxibutirato em Burkholderia sacchari. / Evaluation of poly-3-hydroxybutyrate (P3HB) mobilization system in Burkholderia sacchari.

Nuri Andrea Merchan Castellanos 19 October 2010 (has links)
O sistema de mobilização intracelular de poli-3-hidroxibutirato (P3HB) em Burkholderia sacchari foi analisado. A busca em genomas de Burkholderia spp. identificou duas oligômero hidrolases (PhaY1 e PhaY2) e pelo menos três P3HB despolimerases intracelulares (PhaZa1, PhaZa2 e PhaZd1). Mutantes de B. sacchari afetados na mobilização de P3HB e complementados com genes de Ralstonia eutropha apresentaram um aumento expressivo nas taxas de mobilização de P3HB, especialmente quando o gene phaZa1 foi superexpresso. A superexpressão dos genes phaZa2 ou phaZa3 também conduziu a aumentos nas taxas de mobilização embora em um grau menor que os valores obtidos com phaZa1. Dois mutantes afetados na mobilização de P3HB foram obtidos utilizando o transposon mini-Tn5 (NAM03 e NAM04). NAM03 apresentou interrupção em gene que codifica uma P3HB despolimerase intracelular (PhaZa1). NAM04 apresentou interrupção em gene anotado como serino peptidase LonA. Este pode representar um ativador da mobilização ou uma nova P3HB despolimerase intracelular. / The intracellular poly-3-hydroxybutyrate (P3HB) mobilization system in Burkholderia sacchari was analyzed. A search in Burkholderia spp. genomes identified two oligomer hydrolases (PhaY1 and PhaY2) and at least three intracellular P3HB depolymerase (PhaZa1, PhaZa2 e PhaZd1). B. sacchari mutants affected on P3HB mobilization and complemented by Ralstonia eutropha genes showed an expressive increase on P3HB mobilization rates, especially when phaZa1 was overexpressed. The overexpression of phaZa2 or phaZa3 also increased the mobilization rates though to a lesser extent than phaZa1. Two mutants affected on P3HB mobilization were obtained using the transposon mini-Tn5 (NAM03 and NAM04) .NAM03 was disrupted in a gene encoding an intracellular P3HB depolymerase (PhaZa1). NAM04 was disrupted in a gene annotated as a serine peptidase LonA. This could be a mobilization activator or a new intracellular P3HB depolymerase.
18

Elaboration et caractérisation de matériaux multiphasiques à base de polylactide (PLA) et de polyhydroxyalcanoates (PHA) / Elaboration and characterization of multiphasic polylactide (PLA) and polyhydroxyalkanoates (PHA) multiphasic blends

Gérard, Thibaut 29 August 2013 (has links)
L'objectif de ce travail de thèse est de fournir une étude systématique et détaillée des propriétés de mélanges de PLA et de PHA préparés à l'état fondu. En particulier, le comportement rhéologique, la miscibilité, la morphologie, les propriétés mécaniques et l'influence du vieillissement physique sur les propriétés en traction des mélanges PLA/PHBV réalisés en mélangeur interne et par extrusion bi-vis sur toute la gamme de composition ont été étudiés.La sensibilité du PHBV à la dégradation thermique a été mise en évidence, ainsi que l'importance des interfaces entre le PLA et le PHBV sur le comportement rhéologique aux basses fréquences. Les analyses de calorimétrie différentielle à balayage ont montré l'immiscibilité des deux polymères. Deux types de morphologies distinctes (nodulaire et co-continue) selon la composition de mélange ont été observées par microscopie électronique à balayage et microscopie optique en réflexion. La résistance au choc des mélanges PLA/PHBV avec et sans modifiant choc a été étudiée et les propriétés en traction de différents mélanges de PLA avec du PHBV, du Lotader GMA ou du PBAT ont été caractérisées. L'ajout d'une faible quantité de phase dispersée dans la matrice de PLA, initialement fragile, permet d'augmenter considérablement son allongement à la rupture. Cependant, les propriétés ductiles obtenues diminuent avec le temps écoulé entre la mise en forme des échantillons et leur test mécanique. Une étude de l'influence du vieillissement physique sur les propriétés des mélanges à base de PLA a donc été réalisée et un modèle qualitatif a été proposé. / The objective of this PhD work is to provide a systematic and detailed study of the properties of PLA and PHA blends prepared by melt mixing. In particular, the rheological behaviour, the miscibility, the morphology, the mechanical properties and the influence of physical aging on the tensile properties of PLA/PHBV blends prepared in an internal mixer and by twin screw extrusion over the entire composition range were studied.The sensibility of PHBV to thermal degradation was highlighted, as well as the importance of the interfaces between PLA and PHBV on the rheological behaviour at low frequencies. The differential scanning calorimetry showed that the two polymers are immiscible. Two different types of morphologies (nodular and co-continuous) depending on blend composition have been observed by scanning electron microscopy and optical microscopy in reflection. The impact resistance of the PLA/PHBV blends with or without impact modifier was studied and the tensile properties of different blends containing PLA blended with PHBV, Lotader GMA, or PBAT were characterised. The addition of a small amount of dispersed phase in the initially brittle PLA matrix increases considerably its elongation at break. However, the obtained ductile properties decrease as the time between preparation of samples and their mechanical testing increases. A study of the influence of physical aging on the properties of PLA based blends was conducted and a qualitative model was proposed.
19

Construção de mutantes de Pseudomonas abrigando diferentes PHA sintases em seu genoma, para produção de 3HB-co-3HAMCL. / Construction of recombinant Pseudomonas strains harboring different PHA synthases in its genome to produce 3HB-co-3HAMCL.

Oliveira Filho, Edmar Ramos de 02 February 2017 (has links)
Polihidroxialcanoatos (PHA) são biopolímeros naturalmente produzidos e acumulados por diversos organismos, como bactérias, archaeas e alguns eucariontes, como fungos e leveduras. São materiais termoplásticos, biodegradáveis, biocompatíveis e podem ser produzidos a partir de fontes renováveis, exibindo grande potencial para substituir plásticos produzidos a partir de recursos não renováveis. Copolímeros híbridos de PHA, que podem ser formados por monômeros de cadeia curta e média, como P(3HASCL-co-3HAMCL), apresentam características físico-químicas diferenciadas, semelhantes às dos plásticos derivados de petróleo, sendo por isso interessantes para a indústria de materiais. A PHA sintase é considerada a enzima chave na síntese de PHA, responsável por catalisar a polimerização de diferentes monômeros de (R)-hidroxiacil-CoA, influenciando a composição monomérica do polímero formado. Sistemas de recombinação baseados em transposons bacterianos são explorados como ferramentas moleculares para inserção de sequências gênicas no cromossomo de bactérias Gram-negativas. Por exemplo, elementos mini-Tn7 podem ser prontamente transferidos para a construção de cepas recombinantes. No presente trabalho, é apresentada a construção de diferentes linhagens recombinantes a partir de Pseudomonas sp. LFM 046 e LFM 461, portando em seus cromossomos genes de PHA sintase de Ralstonia eutropha, Aeromonas hydrophila ou Aeromonas sp. TSM 81. Clones candidatos foram triados quanto a inserção das sequências de interesse em seu cromossomo, sendo os positivos avaliados em relação à capacidade de produção de PHA em ensaios em agitador rotativo, com glicose como única fonte de carbono. Um dos recombinantes obtidos se mostrou produtor do copolímero P(3HB-co-3HO-co-3HD), acumulando aproximadamente 2 % de sua massa seca celular na forma de PHA. / Polyhydroxyalkanoates (PHA) are biopolymers naturally produced and accumulated by many organisms such as bacteria, archaeas and some eukaryotes, such as fungi and yeasts. As thermoplastics, biodegradable, biocompatible and possibly made from renewable resources, they exhibit great potential to replace oil-derived plastics. Hybrid PHA copolymers can be formed by short and medium-chain monomers, P(3HASCL-co-3HAMCL), and present industry desired physicochemical properties, becoming similar to conventional oil-based plastics. PHA synthase is the key enzyme in PHA biosynthesis, responsible for catalyzing the polymerization of (R)-hydroxyacyl-CoA molecules, influencing polymer monomeric composition. Tn7-based recombination strategies represent powerful molecular tolls designed for gene delivery in Gram-negative bacteria, as mini-Tn7 elements can be readily transferred to recombinants production. In this work, its presented the constructions of recombinant Pseudomonas strains harboring PHA synthase genes from Ralstonia eutropha and Aeromonas strains in specific sites of its chromosome, and the production of P(3HB-co-3HO-co-3HD). Obtained clones were screened to confirm chromosomic insertion of the phaC sequences. Positive clones PHA production and composition were evaluated in shaken-flasks assays using glucose as the only carbon source. One of the constructed recombinants accumulated P(3HB-co-3HO-co-3HD), corresponding about to 2 % of its Cell Dry Weight as PHA.
20

Caracterização de linhagens bacterianas isoladas da biodiversidade brasileira quanto à produção de biopolímeros. / Characterization of brazilian biodiversity isolated bacterial strains on the production of biopolymer.

Matias, Fernanda 29 January 2009 (has links)
O lixo urbano tem sido apontado como um dos maiores poluentes ambientais. O lixo plástico chega a representar 20% do volume do lixo doméstico. Como alternativa aos plásticos petroquímicos, produtos plásticos menos agressivos ao meio ambiente e mais biodegradáveis têm sido estudados, entre eles os polihidroxialcanoatos (PHA). Os PHA são poliésteres biodegradáveis acumulados como material de reserva por inúmeras bactérias e que possuem aplicabilidade comercial bastante abrangente. Recentemente, os actinomicetos passaram a ser estudados para a produção destes polímeros. Em trabalho prévio, 53 novas linhagens de actinomicetos produtoras de biopolímeros foram isoladas de solo. Neste trabalho foi feita a seleção das bactérias quanto aos polímeros produzidos em diferentes de carbono. Das quatro linhagens selecionadas, duas foram analisadas quanto à produção de um novo polímero. Nas outras duas linhagens foram amplificados e estudados os genes sintetizadores dos polímeros. Em todas as linhagens foram feitas análises taxonômicas e cultivos em rejeitos industriais. / The urban waste has been described as one of the largest environmental pollutants. The plastic garbage can represent up to 20% of the volume of household waste. As an alternative to petrochemical plastics, plastic products less damaging to the environment and more biodegradable have been studied, among them polyhydroxyalkanoates (PHA). The PHA is a biodegradable polyester material accumulated as a reserve material by many bacteria and they have very broad commercial applicability. Recently, the actinomycetes have been studied for the production of polymers. In previous work, 53 new strains of actinomycetes producers of polymers were isolated from soil. In this work the bacterial the selection of bacteria was made concerning the polymers production on different carbon. Of the four strains selected, two were analyzed for the production of a new polymer. In the other two strains were amplified and studied the genes of polymers synthases. In all lineages were analyzed taxonomically and in cultivation on industrial waste.

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