Bei überkritisch großen Läsionen des peripheren Nervensystems, die zum Verlust von Motorik bzw. Sensibilität an Extremitäten führen und damit eine erhebliche Beeinträchtigung der Lebensqualität des Patienten bedingen, ist der Einsatz von Überbrückungsstrukturen bzw. Nervenleitkanälen notwendig. Da weder autologe Transplantate noch künstliche Konstrukte im klinischen Alltag bislang zufriedenstellende Ergebnisse lieferten, ist die Nachfrage nach alternativen Materialien und Konzepten des Tissue Engineering hoch. Im Rahmen dieser Dissertation ist es gelungen zwei thermoplastische Methoden zu etablieren, mit denen aus dem im medizinischen Sinne interessanten und relativ neuartigen Material Poly(3-hydroxybuttersäure) in Kombination mit Polycaprolacton poröse resorbierbare Hohlstrukturen bzw. Hohlfasern erzeugt werden können, die den hohen aktuellen Anforderungen an eine Nervenleitschiene gerecht werden. Neben der Entwicklung und Charakterisierung sowie Modifizierung der erzeugten Leitkanäle bezüglich Porosität, Permeationsverhalten, mechanische Eigenschaften und Oberflächenfunktionalisierung, konnten strukturelle als auch biochemische Reize in diese integriert werden, die in einer Reihe von ex-vivo Studien mit neuronalen Primärzellen hinsichtlich Adhäsion, Vitalität und Ausbreitungsverhalten untersucht werden konnten. Es konnte eine Art „Toolbox“ aus PHB basierten Strukturen erstellt werden, die es erlaubt hierarchische Strukturen zusammenzustellen, die entsprechend des peripheren Defektes zusammengesetzt und biomolekular maßgeschneidert werden könnten, um die native Struktur bestmöglich temporär bis zur vollständigen Regeneration zu imitieren und damit die Therapie größerer Defekte zu ermöglichen bzw. die als Plattform für weitere Konzepte der Grundlagenforschung im Bereich des Neuro-Tissue Engineering dienen.

Devido ao crescente interesse em polímeros biodegradáveis, muitos estudos têm sido realizados a fim de se obter polímeros biodegradáveis com melhores propriedades mecânicas e de processamento. O poli(3-hidroxibutirato) (PHB) tem sido apresentado como um substituto para polímeros não-biodegradáveis em aplicações comerciais. Porém, sua elevada cristalinidade, instabilidade térmica e alto custo de produção são problemas que dificultam sua comercialização. Uma alternativa para modificá-lo e melhorar suas propriedades mecânicas é através de reações de transesterificação com poli(?-caprolactona) (PCL), um poliéster sintético, também biodegradável e com alto potencial para uso como biomaterial. Neste trabalho, realizou-se a extrusão reativa de blendas de PHB e PCL e o copolímero obtido foi caracterizado por ensaios de solubilidade, calorimetria exploratória diferencial (DSC), espectroscopia no infravermelho por transformada de Fourier (FTIR), espectrometria de ressonância magnética nuclear de hidrogênio (RNM-1H) e de carbono (RNM-13C). Os resultados indicaram que houve modificação do PHB resultando no copolímero PHB-co-PCL, que apresentou menor cristalinidade que os homopolímeros de partida. Com o PHB-co-PCL, foram feitas medidas de viscosidade e ensaios de eletrofiação, variando-se os parâmetros do processo e da solução. As análises por microscopia eletrônica de varredura (MEV) apontaram formação de fibras lisas e uniformes com diâmetros médios entre 900 a 1200 nm. Análise de viabilidade celular confirmou que o material não é citotóxico, favorecendo sua aplicabilidade em mantas porosas na engenharia de tecidos. / Due to the increasing interest in biodegradable polymers, many studies have been conducted in order to obtain biodegradable polymers with improved mechanical and processing properties. The poly(3-hydroxybutyrate) (PHB) has been proposed as an ideal substitute for non-biodegradable polymers in commercial applications. However, its high crystallinity, thermal instability and high production costs are problems that have limited its commercialization. An alternative to modify it and improve its mechanical properties is through transesterification reactions with poly(?-caprolactone) (PCL), a synthetic polyester, also biodegradable and with high potential for use as a biomaterial. In this work, reactive extrusion of PHB/PCL blend was carried out, and the copolymer was characterized by solubility tests, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), hydrogen (1H-NMR) and carbon (13C-NMR) nuclear magnetic resonance spectroscopies. The results indicated that PHB was modified, resulting in the copolymer PHB-co-PCL, showing lower crystallinity than the homopolymers. Viscosity measurements of PHB-co-PCL solutions were performed and electrospinning tests were carried out under different conditions. The scanning electron microscopy analysis (SEM) indicated the formation of smooth and uniform fibers, with average diameter between 900-1200 nm. Cell viability analysis confirmed that the material is not cytotoxic, favoring its applicability for the production of porous fiber mats in tissue engineering.

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2019-03-13T00:48:12Z No. of bitstreams: 1 Camila_Hinterholz_2015.pdf: 2996844 bytes, checksum: f54562c9458e8a2460e3978c1be500c2 (MD5) / Made available in DSpace on 2019-03-13T00:48:12Z (GMT). No. of bitstreams: 1 Camila_Hinterholz_2015.pdf: 2996844 bytes, checksum: f54562c9458e8a2460e3978c1be500c2 (MD5) Previous issue date: 2015-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Biopolymers, especially Poly(3-hydroxybutyrate) (PHB), have been receiving big attention in order to minimize environmental damage caused by plastics from petrochemical industries. In this context, the aim of this paper was to formulate a mathematical model to describe the PHB production by Cupriavidus necator, from a detailed theoretical study. To this end, 6 models were evaluated, being 5 for cell growth and 1 for product formation, all from the literature. The ordinary differential equations system was solved numerically by Rosenbrock method. To the parameters estimation, an algorithm based on the Genetic Algorithms was developed and implemented in the software Maple®. To validate the models, experimental data at 30, 32,5, 35 and 37,5 °C were obtained from the literature. From the data analysis, it was observed that the best temperature, for both cell growth and product formation was 32,5 °C, and that the PHB production in partially associated with cell growth. To the parameters estimation, the ordinary differential equations system, obtained from the phenomenological modelling of non-structured and non-segregated models, was evaluated together with the models from the literature. The results for the objective function and correlation coefficient indicated that all the studied models adjusted well to the experimental data at all temperatures. Thus, some statistical tests were applied in order to better evaluate the models fitting, and the results indicated the Andrews’s (1968) model as the one that best represents the data from 32,5 °C, and Heinzle e Lafferty’s (1980) model for 35 °C. For the data at 30 and 37,5 °C there was no statistically valid models found. In conclusion, the statistical methodology applied for the models discrimination and fitting evaluation made it possible to say which model best represents data at each temperature. / Os biopolímeros, em especial o Poli(3-hidroxibutirato) (PHB), têm recebido grande atenção na tentativa de minimizar danos ambientais causados pelo acúmulo de plásticos de origem petroquímica. Neste âmbito, o objetivo deste trabalho foi formular um modelo matemático para o processo de produção do PHB por Cupriavidus necator, a partir de um estudo teórico detalhado. Para tanto, foram avalliados 5 modelos de crescimento celular e 1 modelo para a formação do produto, todos obtidos da literatura. O sistema de equações diferenciais ordinárias foi resolvido numericamente pelo método de Rosenbrock. Para a estimação dos parâmetros dos modelos foi desenvolvido um algoritmo baseado nos Algoritmos Genéticos, o qual foi implementado no software Maple®. Para a validação dos modelos, dados experimentais de ensaios a 30, 32,5, 35 e 37,5 °C foram obtidos da literatura. A partir da análise dos dados foi verificado que a melhor temperatura, tanto para o crescimento da biomassa quanto para a formação do produto, foi de 32,5 °C, e que a produção de PHB está parcialmente associada ao crescimento celular. Para a estimação dos parâmetros, o sistema de equações diferenciais ordinárias obtidas a partir da modelagem fenomenológica de modelos não estruturados e não seguregados foi avaliado juntamente com os modelos da literatura. Os resultados obtidos para a função objetivo e coeficiente de correlação indicaram que todos os modelos estudados se ajustaram bem aos dados experimentais em todas as temperaturas. Assim, para uma melhor avaliação dos ajustes foram aplicados alguns testes estatísticos, cujos resultados indicaram o modelo de Andrews (1968) como sendo o que melhor representa os dados à temperatura de 32,5 °C, e o modelo de Heinzle e Lafferty (1980) para os dados à 35 °C. Para as temperaturas de 30 e 37,5 °C não foi encontrado um modelo estatisticamente válido. Logo, a metodologia estatística aplicada para a discriminação de modelos e avaliação da qualidade dos ajustes tornou possível a identificação do modelo que melhor representa os dados em cada temperatura.

The development of human society, which is highly dependent on fossil fuels, is now facing a range of global issues, such as rising energy prices, energy security and climate changes. To successfully tackle the resultant issues, the energy transition from fossil fuels to renewable energy sources, such as solar energy, tide energy, hydroelectric power, geothermal heat and biofuels, is under way. Biodiesel, as an important type of biofuels, has been increasingly produced from vegetable oil or used cooking oil, especially in Europe. Nevertheless, considering the high production cost of biodiesel, there is still much to be done to improve the economics of biodiesel industry. Utilisation of crude glycerol, the main by-product of the biodiesel industry, to produce value-added products appears to be a promising solution. Poly(3-hydroxybutyric acid) (PHB), a biodegradable plastic, can be converted from glycerol by Cupriavidus necator DSM 545 under unbalanced growth conditions, such as nitrogen limitation. One way to enhance the batch production of PHB is to genetically engineer the strain of C. necator, which requires insights of the dynamic impact of extracellular environment on cell phenotypes. Hence in this thesis, we aim to perform metabolic modelling based on experimental measurements to gain a better understanding of the behaviour of the metabolic network of Cupriavidus necator DSM 545 and identify potential bottlenecks of the process. Initially, C. necator DSM 545 is a strain that hardly grows on glycerol, so in a preliminary study, we investigate the process by which the strain was adapted to consume glycerol through serial subcultivation. It is found that the adaptation can be achieved within 15 cell generations over three passages in basal mineral medium, and the acquired phenotype is sufficiently stable upon further passage. The study of metabolism started with the reconstruction of the cell's metabolic network, followed by a thermodynamic analysis to check the feasibility and reversibility of all the biochemical reactions included. Then the static flux balance analysis was extended and applied to analyse the shift of metabolic states during the microbial fermentation in different batch conditions. The resulting patterns of flux distribution reveal the TCA cycle to be the major competitor for PHB synthesis at the ACCoA node. Cells have the potential to enter an efficient PHB-production phase that features minimal TCA/PHB flux split ratio, and the length of the phase can be manipulated by aeration. Although low aeration rate favours optimal flux split ratio, such condition that limits respiration also limits nutrient uptake, leading to low PHB productivity overall. To identify the actual limiting factors of PHB synthesis in the system, we further performed metabolic control analysis based on the calculated flux distributions. The analysis demonstrated how the distribution of the metabolic control can vary widely, depending on the aeration conditions used and the flux split ratios. Glycerolipid pathway, glycolysis, PHB metabolism, as well as the electron transport chain are revealed to be potential engineering targets as they contribute to the great majority of the positive control of PHB flux.

Devido ao crescente interesse em polímeros biodegradáveis, muitos estudos têm sido realizados a fim de se obter polímeros biodegradáveis com melhores propriedades mecânicas e de processamento. O poli(3-hidroxibutirato) (PHB) tem sido apresentado como um substituto para polímeros não-biodegradáveis em aplicações comerciais. Porém, sua elevada cristalinidade, instabilidade térmica e alto custo de produção são problemas que dificultam sua comercialização. Uma alternativa para modificá-lo e melhorar suas propriedades mecânicas é através de reações de transesterificação com poli(?-caprolactona) (PCL), um poliéster sintético, também biodegradável e com alto potencial para uso como biomaterial. Neste trabalho, realizou-se a extrusão reativa de blendas de PHB e PCL e o copolímero obtido foi caracterizado por ensaios de solubilidade, calorimetria exploratória diferencial (DSC), espectroscopia no infravermelho por transformada de Fourier (FTIR), espectrometria de ressonância magnética nuclear de hidrogênio (RNM-1H) e de carbono (RNM-13C). Os resultados indicaram que houve modificação do PHB resultando no copolímero PHB-co-PCL, que apresentou menor cristalinidade que os homopolímeros de partida. Com o PHB-co-PCL, foram feitas medidas de viscosidade e ensaios de eletrofiação, variando-se os parâmetros do processo e da solução. As análises por microscopia eletrônica de varredura (MEV) apontaram formação de fibras lisas e uniformes com diâmetros médios entre 900 a 1200 nm. Análise de viabilidade celular confirmou que o material não é citotóxico, favorecendo sua aplicabilidade em mantas porosas na engenharia de tecidos. / Due to the increasing interest in biodegradable polymers, many studies have been conducted in order to obtain biodegradable polymers with improved mechanical and processing properties. The poly(3-hydroxybutyrate) (PHB) has been proposed as an ideal substitute for non-biodegradable polymers in commercial applications. However, its high crystallinity, thermal instability and high production costs are problems that have limited its commercialization. An alternative to modify it and improve its mechanical properties is through transesterification reactions with poly(?-caprolactone) (PCL), a synthetic polyester, also biodegradable and with high potential for use as a biomaterial. In this work, reactive extrusion of PHB/PCL blend was carried out, and the copolymer was characterized by solubility tests, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), hydrogen (1H-NMR) and carbon (13C-NMR) nuclear magnetic resonance spectroscopies. The results indicated that PHB was modified, resulting in the copolymer PHB-co-PCL, showing lower crystallinity than the homopolymers. Viscosity measurements of PHB-co-PCL solutions were performed and electrospinning tests were carried out under different conditions. The scanning electron microscopy analysis (SEM) indicated the formation of smooth and uniform fibers, with average diameter between 900-1200 nm. Cell viability analysis confirmed that the material is not cytotoxic, favoring its applicability for the production of porous fiber mats in tissue engineering.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2015-05-21T17:56:37Z No. of bitstreams: 1 Dissertação de Grasielly Karine Martins de Souza.pdf: 2433825 bytes, checksum: 1a67453fb90b15dc21744d94950f544d (MD5) / Made available in DSpace on 2015-05-21T17:56:37Z (GMT). No. of bitstreams: 1 Dissertação de Grasielly Karine Martins de Souza.pdf: 2433825 bytes, checksum: 1a67453fb90b15dc21744d94950f544d (MD5) Previous issue date: 2015-03-05 / A crescente utilização de produtos fabricados a partir de polímeros não-biodegradáveis, provenientes de rota petroquímica, como copos descartáveis, garrafas e sacolas plásticas, têm contribuído para o acúmulo de resíduos plásticos no planeta. Uma alternativa viável para solução deste problema é a utilização de polímeros biodegradáveis para produção destes produtos. Além disso, alguns polímeros biodegradáveis, como o poli(3-hidroxibutirato) (PHB) e a quitosana, têm aplicações médico-hospitalares e alimentícias, por apresentarem propriedades, como atoxicidade e biocompatibilidade. No entanto, nestes setores há a necessidade da esterilização dos seus produtos. Portanto, o presente trabalho teve como principal objetivo a avaliação das propriedades morfológicas, mecânicas e térmicas de blendas de PHB/quitosana após processos de esterilização térmica ou radiolítica. As blendas foram preparadas pelo método casting solution (0,7% m/m de quitosana) e suas propriedades foram avaliadas a partir de diversas técnicas, como espectroscopia do infravermelho com transformada de Fourier (FTIR), ressonância magnética nuclear (RMN-1H), microscopia eletrônica de varredura (MEV), ensaio de tração, calorimetria exploratória diferencial (DSC) e análise termogravimétrica (TGA). Os filmes obtidos de PHB e PHB/quitosana apresentaram-se homogêneos e opacos, enquanto os filmes de quitosana ficaram homogêneos e transparentes. Através da análise por componentes principais dos espectros de infravermelho dos filmes de PHB puro, quitosana pura e das blendas PHB/quitosana foi possível observar a formação de dois agrupamentos distintos em relação às suas estruturas químicas. Além disso, também foram evidenciadas alterações nos espectros de infravermelho das blendas antes e após irradiação. No espectro de RMN-1H não foi observada nenhuma nova estrutura com a adição da quitosana, nem após irradiação. Com base no MEV, foi possível observar na blenda a presença de grânulos, referentes à quitosana, dispersos na matriz de PHB. Também foi observado que após esterilização térmica, a blenda apresentou uma superfície rugosa, com a presença de grânulos e alguns vazios na matriz. Entretanto, após a esterilização radiolítica, os filmes apresentaram superfície lisa e homogênea. As propriedades mecânicas (tensão máxima, deformação específica e módulo de elasticidade) da blenda se mantiveram aproximadamente constantes após processo de esterilização térmica, não sendo comprometidas com a presença da quitosana. Porém após esterilização radiolítica as blendas se apresentaram quebradiças. A temperatura e entalpia de cristalização, a temperatura de fusão e o grau de cristalinidade das blendas não foram alteradas com a adição de quitosana, nem após esterilização térmica, porém para as doses de 50 e 75 kGy, houve uma redução no valor da entalpia de cristalização e no grau de cristalinidade da blenda. Provavelmente, a radiação-γ, nessas doses, comprometeu a região cristalina da blenda. O comportamento térmico da blenda, antes e após processos de esterilização térmica ou radiolítica, observado na curva de TGA foi semelhante ao do filme de PHB puro. As blendas não apresentaram atividade antimicrobiana, provavelmente devido ao baixo percentual de quitosana presente na blenda.

Orientador: Maria Isabel Felisberti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-11T21:04:55Z (GMT). No. of bitstreams: 1 Alves_SabrinadeSousa_M.pdf: 1156094 bytes, checksum: 5b56bbbe834e47047d9d7c14d74333c1 (MD5) Previous issue date: 2008 / Resumo: Materiais poliméricos com propriedades como biodegradabilidade e biocompatibilidade, como o poli(hidroxibutirato) (PHB) e a poli(caprolactona), vêm sendo amplamente estudados para aplicações com alto valor agregado, como a área biomédica. A síntese de poliuretanas contendo blocos de PHB e PCL visa a obtenção de um material que mantenha as características de biodegradabilidade e biocompatibilidade e que possua propriedades mecânicas diferentes dos materiais de partida, o que viabiliza um aumento na gama de aplicações destes polímeros. Neste trabalho, polióis de PHB e PCL com massa molar inferior a 6000 g mol foram obtidos através de reações de glicólise. Diferentes rotas e condições experimentais, utilizando diferentes solventes, catalisadores, tempo e temperatura, foram testadas para esta reação. A caracterização destes oligômeros foi feita através de análise de RMN H e C, DSC, TGA, índice de hidroxila e GPC. Na segunda etapa do trabalho, foram realizadas reações dos oligômeros obtidos com hexametileno diisocianato (HDI), um isocianato de cadeia linear de baixo poder carcinogênico. As estruturas químicas das poliuretanas obtidas nesta reação foram caracterizadas por FTIR e RMN H e C, as propriedades térmicas caracterizadas por DSC, TGA e DMA, físicas através de GPC e teste de solubilidade para verificar existência de reticulação / Abstract:The synthesis of polyurethanes containing PHB and PCL blocks aims to obtain materials with controlled mechanical properties maintain biodegradable and biocompatible polymers such as poly(hidroxybutyrate) (PHB) and poly(caprolactone) (PCL) have been largely studied. The synthesis of polyurethanes containing blocks of PHB and PCL aims to obtain materials with controlled mechanical properties maintaing the biodegradability and biocompatibility. In this work, polyols of PHB and PCL with molar weight less than 6000 g mol were obtained from glicolise reactions with ethylene glycol. The polyols were characterizated by RMN H e C, DSC, TGA, hydroxyl index and GPC. Diferent routes and experimental conditions, using diferent solvents, catalysts, time and temperature were tested. In a second step of this research reactions between the polyols and hexamethylene diisocianate (HDI), an alifatic isocianate with low carcinogenic toxicity were conduced in order to obtin polyurethanes. The chemical structure of polyurethanes obtained was characterized by FTIR and RMN H e C, thermal properties were characterized by DSC, TGA and DMA em physical properties by GPC and solubility test / Mestrado / Físico-Química / Mestre em Química

Orientador: Maria do Carmo Gonçalves / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T02:02:44Z (GMT). No. of bitstreams: 1 Gomes_AlvaroLuiz_M.pdf: 3109264 bytes, checksum: b83bd9f308a60b69ae93e1dbd94dea75 (MD5) Previous issue date: 2011 / Resumo: O poli(3-hidroxibutirato), ou PHB, é um poliéster de grande importância tecnológica por ser biodegradável, proveniente de fontes renováveis e possuir muitas das propriedades típicas de poliésteres derivados do petróleo. No entanto, sua baixa disponibilidade e seu perfil de cristalização fazem com que o polímero ainda apresente muitos desafios para aplicações usuais. A preparação de blendas do PHB com outros polímeros é uma das soluções propostas por muitos pesquisadores. Neste estudo foram preparadas blendas do PHB com três distintos derivados de celulose e foram observados o comportamento térmico e de cristalização destas blendas. Foi constatado que o PHB apresenta miscibilidade com o acetato butirato de celulose (CAB) mas apresenta imiscibilidade com o acetato de celulose (CA) e com o acetato butirato de carboximetil celulose (CMCAB). A influência do método de preparação foi investigada e foi observado que o método de gelificação do PHB e posterior secagem produziu misturas com maior uniformidade microscópica, quando comparado a métodos como solvent casting e precipitação seguida de termomoldagem, principalmente para uma blenda considerada imiscível. A cinética de cristalização do PHB nas blendas foi também influenciada pelo método de preparação. A blenda PHB/CMCAB preparada por gelificação mostrou maior energia necessária para a formação de um núcleo de tamanho crítico para cristalização. Diferentes massas molares do PHB foram utilizadas na preparação das blendas e foi verificado que a massa molar influiu nas propriedades térmicas apenas quando a blenda era miscível, contribuindo com uma diminuição mais intensa da Tg da blenda para o PHB de menor massa molar nas blendas PHB/CAB. Polímeros de PHB de menor massa molar apresentaram, conforme esperado no estudo da cinética de cristalização com a utilização das equações de Lauritzen-Hoffman, constantes de nucleação menores e este mesmo comportamento foi observado também para PHB de diferentes massas molares na blenda com o CMCAB / Abstract: The poly(3-hydroxybutyrate), or PHB, is a polyester with very important technological characteristics. The polymer is biodegradable, made from renewable sources and has many of the typical properties of regular polyesters derived from petroleum. However, its current availability and crystallization profile introduces many challenges to more usual applications. The preparation of blends of PHB with other polymers is one of the solutions proposed by many researchers. In this study PHB blends were prepared with three different cellulose derivatives and the thermal behavior and crystallization of these blends were observed. It was found that PHB exhibits miscibility with cellulose acetate butyrate (CAB) but it shows to be not miscible with cellulose acetate (CA) and the carboxymethyl cellulose acetate butyrate (CMCAB). The influence of the preparation method was investigated and it was observed that the method of gel formation with PHB and subsequent drying produced blends with better microscopic uniformity when compared to traditional methods such as solvent casting or precipitation followed by hot pressing, mainly for an immiscible blend. The crystallization kinetics of PHB in the blends was also influenced by the method of preparation. The PHB/CMCAB blends prepared by gel formation showed higher energy required to form a nucleus of critical size for crystallization. Different molecular weights of PHB were used in the preparation of the blends, and it was observed that the molecular weight affected the thermal properties only when the blend was miscible. In this case, a decrease in the Tg value of the PHB/CAB blend was observed when PHB with lower molecular weight was used. PHB polymers with lower molecular weight showed, as expected for the study of crystallization kinetics with the Lauritzen-Hoffman equation, lower nucleating constants and this same trend was also observed for different molecular weights of PHB in the blend with CMCAB / Mestrado / Físico-Química / Mestre em Química