This thesis is inspired by a persistent limitation in the use of composite biomaterials for orthopaedic applications, namely the agglomeration of reinforcing particles in these composites, which has resulted in poor mechanical properties. The use of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and hydroxyapatite (HA) nanoparticles to produce biodegradable nanocomposites is investigated. More specifically, the thesis investigates different methods of composite processing, and interfacial modifying agents and the effect that these have on the nano- and micro- scale structure of composites and their mechanical properties. PHBV and HA were chosen because PHBV is a biodegradable/biocompatible polymer and it has a relatively high stiffness when compared to other biodegradable polymers frequently used in orthopaedic applications. HA is chemically similar to ceramic phase found in bones and hard tissues and the inclusion of HA into biomedical materials has been shown to enhance the rate of osteoconduction. HA/PHBV composites were produced using different dispersing agents including poly(acrylic acid) (PAA), a model dispersing agent, polyethyleneimine (PEI) which allowed for the development of a single solvent system for composite preparation, and heparin (Hep), a macromolecule which is produced in vivo. Additionally, HA/PAA/PHBV composites were prepared from both sonicated and non-sonicated HA/PAA suspensions up to approximately 17% by weight (wt %) of HA content. Attempts to prepare composites with higher HA loadings led to inhomogeneous composite mixtures, which were caused by the dual solvent system used for the composite preparation. The HA/PEI/PHBV and HA/Hep/PHBV composites were produced up to approximately 75 wt % of HA content. It was found that the HA/PEI/PHBV and HA/Hep/PHBV composites could be prepared at higher loadings than HA/PAA/PHBV composites due to the single solvent system used for the preparation of the HA/PEI/PHBV composites and the better dispersion of HA/Hep particles in precursor suspensions. Finally, selected HA/PEI/PHBV composites were further processed using a twin screw extruder. All of the composites were characterised in terms of their dispersion levels as well as their compressive mechanical properties. In addition, HA/PEI/PHBV composite reinforced with 20 wt % of HA content was also tested for its mechanical properties using three different test types; compression, three-point bending, and tensile tests. Finally, the HA/PAA/PHBV, HA/PEI/PHBV, and HA/Hep/PHBV composites were tested their compressive mechanical properties in wet state. It was found that the sonicated HA/PAA suspensions in general had better colloidal stability than non-sonicated ones and that this yielded composites with superior compressive moduli than those prepared from non-sonicated suspensions. In addition, the better dispersion of the particles in the composites prepared from the sonicated suspensions, as confirmed by transmission electron microscopic (TEM) images, led to higher percentage crystallinities when compared to the composites prepared from non-sonicated suspensions. It is likely that the greater number of individual HA particles and smaller HA agglomerates observed in the composites prepared from sonication treatment are acting as nuclei for crystal growth more effectively than large HA agglomerates. The largest modulus and yield strength that could be achieved with this system were approximately 1.45 GPa and 80 MPa, respectively. Composites of HA/PEI/PHBV and HA/Hep/PHBV with approximately 55 wt % of HA content were found to exhibit the largest compressive moduli of approximately 2.5 and 2.8 GPa, respectively. Moreover, the yield strengths for the same materials were found to be approximately 123 and 120 MPa, respectively. This was found to correlate with the better levels of dispersion within the nanocomposites that could be achieved using these stabilisers. The extruded samples were found to have an even greater degree of particle dispersion when compared to the unextruded ones. This improved degree of particle dispersion of the extruded samples resulted in higher moduli in comparison to unextruded samples. The largest compressive modulus and yield strength of the extruded samples were found to be approximately 3.2 GPa and 125 MPa, respectively. The compressive moduli of the composites produced in this thesis are significantly greater than that of cancellous bone (0.4 GPa), but significantly lower than that of cortical bone (12.8–17.7 GPa). However, maximum yield strengths of the HA/PEI/PHBV and HA/Hep/PHBV composites match to cortical bone (120–180 MPa), which is a noteworthy finding in this thesis. The wet mechanical results of all composites as well as pure PHBV polymer showed a reduction in both moduli and yield strengths when compared to dry state. In addition, after 2 weeks in wet state both moduli and yield strengths of the composites and pure polymer converged to approximately the same values. Finally, the HA/PEI/PHBV composite samples tested by tensile testing showed the highest Young’s modulus and those tested by compression testing possessed the lowest Young’s modulus. This resulted from the difference in periods of time for heating exposure and void contents of the tested samples, which were prepared by different methods. However, toughness values obtained from the samples tested using three-point bending and tensile tests, was not significantly different.

This thesis is inspired by a persistent limitation in the use of composite biomaterials for orthopaedic applications, namely the agglomeration of reinforcing particles in these composites, which has resulted in poor mechanical properties. The use of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and hydroxyapatite (HA) nanoparticles to produce biodegradable nanocomposites is investigated. More specifically, the thesis investigates different methods of composite processing, and interfacial modifying agents and the effect that these have on the nano- and micro- scale structure of composites and their mechanical properties. PHBV and HA were chosen because PHBV is a biodegradable/biocompatible polymer and it has a relatively high stiffness when compared to other biodegradable polymers frequently used in orthopaedic applications. HA is chemically similar to ceramic phase found in bones and hard tissues and the inclusion of HA into biomedical materials has been shown to enhance the rate of osteoconduction. HA/PHBV composites were produced using different dispersing agents including poly(acrylic acid) (PAA), a model dispersing agent, polyethyleneimine (PEI) which allowed for the development of a single solvent system for composite preparation, and heparin (Hep), a macromolecule which is produced in vivo. Additionally, HA/PAA/PHBV composites were prepared from both sonicated and non-sonicated HA/PAA suspensions up to approximately 17% by weight (wt %) of HA content. Attempts to prepare composites with higher HA loadings led to inhomogeneous composite mixtures, which were caused by the dual solvent system used for the composite preparation. The HA/PEI/PHBV and HA/Hep/PHBV composites were produced up to approximately 75 wt % of HA content. It was found that the HA/PEI/PHBV and HA/Hep/PHBV composites could be prepared at higher loadings than HA/PAA/PHBV composites due to the single solvent system used for the preparation of the HA/PEI/PHBV composites and the better dispersion of HA/Hep particles in precursor suspensions. Finally, selected HA/PEI/PHBV composites were further processed using a twin screw extruder. All of the composites were characterised in terms of their dispersion levels as well as their compressive mechanical properties. In addition, HA/PEI/PHBV composite reinforced with 20 wt % of HA content was also tested for its mechanical properties using three different test types; compression, three-point bending, and tensile tests. Finally, the HA/PAA/PHBV, HA/PEI/PHBV, and HA/Hep/PHBV composites were tested their compressive mechanical properties in wet state. It was found that the sonicated HA/PAA suspensions in general had better colloidal stability than non-sonicated ones and that this yielded composites with superior compressive moduli than those prepared from non-sonicated suspensions. In addition, the better dispersion of the particles in the composites prepared from the sonicated suspensions, as confirmed by transmission electron microscopic (TEM) images, led to higher percentage crystallinities when compared to the composites prepared from non-sonicated suspensions. It is likely that the greater number of individual HA particles and smaller HA agglomerates observed in the composites prepared from sonication treatment are acting as nuclei for crystal growth more effectively than large HA agglomerates. The largest modulus and yield strength that could be achieved with this system were approximately 1.45 GPa and 80 MPa, respectively. Composites of HA/PEI/PHBV and HA/Hep/PHBV with approximately 55 wt % of HA content were found to exhibit the largest compressive moduli of approximately 2.5 and 2.8 GPa, respectively. Moreover, the yield strengths for the same materials were found to be approximately 123 and 120 MPa, respectively. This was found to correlate with the better levels of dispersion within the nanocomposites that could be achieved using these stabilisers. The extruded samples were found to have an even greater degree of particle dispersion when compared to the unextruded ones. This improved degree of particle dispersion of the extruded samples resulted in higher moduli in comparison to unextruded samples. The largest compressive modulus and yield strength of the extruded samples were found to be approximately 3.2 GPa and 125 MPa, respectively. The compressive moduli of the composites produced in this thesis are significantly greater than that of cancellous bone (0.4 GPa), but significantly lower than that of cortical bone (12.8–17.7 GPa). However, maximum yield strengths of the HA/PEI/PHBV and HA/Hep/PHBV composites match to cortical bone (120–180 MPa), which is a noteworthy finding in this thesis. The wet mechanical results of all composites as well as pure PHBV polymer showed a reduction in both moduli and yield strengths when compared to dry state. In addition, after 2 weeks in wet state both moduli and yield strengths of the composites and pure polymer converged to approximately the same values. Finally, the HA/PEI/PHBV composite samples tested by tensile testing showed the highest Young’s modulus and those tested by compression testing possessed the lowest Young’s modulus. This resulted from the difference in periods of time for heating exposure and void contents of the tested samples, which were prepared by different methods. However, toughness values obtained from the samples tested using three-point bending and tensile tests, was not significantly different.

Disponibiliza??o em conte?do parcial do trabalho, conforme Termo de Autoriza??o. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-02-14T19:00:46Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lays_fernanda_nunes_dourado_parcial.pdf: 389665 bytes, checksum: 2289d8ac4ca389d5cdaf516878ddae78 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-03-06T12:24:50Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lays_fernanda_nunes_dourado_parcial.pdf: 389665 bytes, checksum: 2289d8ac4ca389d5cdaf516878ddae78 (MD5) / Made available in DSpace on 2017-03-06T12:24:50Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lays_fernanda_nunes_dourado_parcial.pdf: 389665 bytes, checksum: 2289d8ac4ca389d5cdaf516878ddae78 (MD5) Previous issue date: 2016 / Na ind?stria farmac?utica, os pol?meros s?o capazes de desempenhar a fun??o de matriz para libera??o controlada de f?rmacos. Isto tem como vantagem a minimiza??o dos efeitos adversos dos medicamentos e a otimiza??o do tratamento. Entre os pol?meros utilizados para tal finalidade, tem-se o poli(3-hidroxibutirato) (PHB) conhecido por sua origem natural, um material que se degrada a produtos n?o t?xicos para o organismo, o que garante o seu emprego como biomaterial. Quando utilizado em associa??o a outros pol?meros, s?o denominados Blendas, essas misturas modificam as caracter?sticas f?sico-qu?micas dos pol?meros para serem empregados em finalidades diversas. Desta forma este trabalho teve como objetivo o desenvolvimento de uma matriz polim?rica capaz de ser utilizada para libera??o controlada de f?rmacos. Para tal, foram produzidas duas membranas diferentes uma de PHB e outra de Blenda Poli (3-hidroxibutirato)/Polipropilenoglicol, PHB/PPG (90:10), com concentra??es diferentes de f?rmaco, contendo 5% e 25% em massa. O f?rmaco modelo foi a Sinvastatina. Estes dispositivos foram produzidos por dissolu??o seguida de evapora??o do solvente, formando filmes de 0,5 mm de di?metro que foram analisados quanto a sua degrada??o in vitro e in vivo, em implantes subcut?neos. Os materiais foram analisados utilizando espectroscopia na regi?o do infravermelho, termogravimetria, microscopia eletr?nica de varredura e por testes histol?gicos. Os filmes ? base de PHB, apresentaram degrada??o in vitro e in vivo mais lenta quando comparados ?s blendas, no entanto em ambos os casos, os materiais que continham maior percentual de Sinvastatina apresentaram maior degrada??o e libera??o do f?rmaco. As l?minas histol?gicas revelam a presen?a do tecido em torno dos dispositivos e aus?ncia de inflama??o o que comprova a biocompatibilidade dos materiais estudados. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / In the pharmaceutical industry, the polymers are able to drive the controlled release of drugs. Its brings the advantage of minimizing adverse effects of medicines and optimization of treatment. Among the polymers used for this purpose the poly (3-hydroxybutyrate) has known for their natural origin, its degradation to non-toxic effects to the life tissue which ensures their use as subcutaneous implants. A blend is a mixture of different polymers, these mixtures are able to modify the physicochemical characteristics of the polymers for several uses. This study aimed to develop a polymer matrix can be used as a way for controlled release of drugs. For this purpose, two different membranes were produced, the PHB and a Poly(3-hydroxybutyrate)/Polypropylene glycol blends, PHB/PPG (90:10), with different concentrations of drug with 5% and another 25%. The simvastatin was chosen as a model. These devices were manufactured by casting to form films of 0.5 mm diameter that were analyzed for in vitro and in vivo degradation tests using subcutaneous implants. The materials were analyzed using infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy and histological tests. The films based on PHB presented a slower degradation in vitro and in vivo degradation tests when compared to the blends, however in both cases those materials containing a higher percentage of simvastatin has shown the higher degradation and release of the drug. The histological sections showed a tissue colonization and absence of inflammation which demonstrates the biocompatibility of the implants.

This master’s thesis deals with plasticization of poly(3-hydroxybutyrate), polylactid acid and their blend. It explores effect of chemical structure of plasticizer on mechanical properties of this polymer blend and on its diffusion from the polymer blend. Syntheses of plasticizers based on oligomeric polyadipates, citrates, lactate and esters of 2 ethylhexanoic acid with poly(ethyleneglycol) were carried out. Molecular weight distribution of synthesized plasticizers was determined using gel permeation chromatography. Poly(3-hydroxybutyrate), polylactid acid and their blend were plasticized with synthesized and commercial plasticizers. From commercial, chosed plasticizers were based on citrates and ester of 2-ethylhexanoic acid with poly(ethyleneglycol). Thermal stability of selected commercial plasticizers in polylactid acid was studied using thermogravimetry. Diffusion of plasticizers from poly(3-hydroxybutyrate), polylactid acid and their blend during exposure to 110 °C was also investigated. Mechanical properties of prepared blends were tested by tensile test. Almost all used plasticizers showed positive softening effect in blend. The highest elongation at break was detected for the blend with commercial acetyltributylcitrate, where elongation at break reached 328 % relative to 21 % for neat non-plasticized blend.

O objetivo deste trabalho foi o estudo das propriedades mecânicas e térmicas do PHB e de compósitos de PHB/pó de madeira processados, reprocessados e irradiados. Foram preparados compósitos com PHB e pó de madeira com concentrações de PHB/pó de madeira de 90/10, 80/20 e 70/30 (m/m). Foram realizados ensaios de propriedades mecânicas, HDT, ponto de amolecimento Vicat, TGA e DSC. A incorporação do pó de madeira aumentou o grau de cristalinidade e a temperatura de cristalização do polímero, e nos compósitos PHB/pó de madeira 80/20 e 70/30 a rigidez do material aumentou em relação ao polímero puro. O compósito PHB/pó de madeira 70/30 apresentou os melhores resultados em termos econômicos, de processamento e de estabilidade em relação ao reprocessamento. A irradiação após o processamento, em dose de 30 kGy, provocou aumento da rigidez do PHB puro e dos compósitos PHB/pó de madeira 90/10 e 80/20, embora as outras propriedades tenham decrescido. O compósito PHB/pó de madeira 70/30 apresentou queda de todas as propriedades com a irradiação. / The purpose of this work was the study of the mechanical and thermal properties of processed, reprocessed and irradiated PHB, as well as PHB/wood flour composites. PHB/wood flour composites were prepared with PHB/wood relation of 90/10, 80/20 e 70/30 (w/w). Mechanical properties, HDT, Vicat melting point and thermo analyses (TGA and DSC) tests were carried out. The introduction of the wood flour increased both polymer crystallinity and crystallization temperature. The material stiffness increased in PHB/wood flour composites (80/20 and 70/30) resulting in higher values in some properties when compared with pure PHB. The PHB/wood flour composite 70/30 showed the best results in terms of economy, processing and stability during reprocessing. The irradiation after processing in 30kGy doses led to a stiffness increasing of pure PHB and PHB/wood flour composites (90/10 and 80/20) while other properties have decreased. The PHB/wood flour 70/30 showed decreasing in all properties after irradiation.

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.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-09-12T20:11:13Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) regina_inez_souza.pdf: 2322505 bytes, checksum: fb22e286692f3f78b323a1aa5767c86b (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-09-18T13:03:41Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) regina_inez_souza.pdf: 2322505 bytes, checksum: fb22e286692f3f78b323a1aa5767c86b (MD5) / Made available in DSpace on 2017-09-18T13:03:41Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) regina_inez_souza.pdf: 2322505 bytes, checksum: fb22e286692f3f78b323a1aa5767c86b (MD5) Previous issue date: 2017 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Na ind?stria farmac?utica, os pol?meros s?o bastante utilizados em sistemas para libera??o controlada de f?rmacos, por serem capazes de liber?-los efetivamente no local alvo, aumentando os benef?cios terap?uticos e minimizando os efeitos colaterais. Entre os pol?meros utilizados para essa finalidade, tem-se o poli(3-hidroxibutirato) (PHB) pertencente a classe dos poli(hidroxialcanoatos), sendo um pol?mero de origem microbiana, que destacase por ser biorreabsorv?vel e biocompat?vel. Quando utilizado em misturas f?sicas com outros pol?meros, as denominadas blendas, modifica-se propriedades f?sicas, mec?nicas e biol?gicas do PHB para aumentar sua aplicabilidade. Nesse sentido, o propilenoglicol (PPG) foi utilizado para prepara??o de blendas PHBPPG (90/10). O objetivo do trabalho foi preparar e estudar filmes polim?ricos constitu?dos por PHB e PHBPPG (90/10) contendo 1% e 5% de Snitrosoglutationa (GSNO), um doador de ?xido n?trico (NO) bem como sintetizar o f?rmaco e avaliar sua estabilidade ap?s armazenamento a 23?C, 4?C e -18?C durante 90 dias por an?lise t?rmica e por espectroscopia na regi?o do infravermelho. Os filmes obtidos contendo GSNO foram caracterizados por an?lise t?rmica, espectroscopia no infravermelho e avaliou-se o perfil de libera??o in vitro dos mesmos. A citotoxicidade dos extratos das matrizes constitu?das somente por PHB e PHBPPG (90/10) foi avaliada sobre c?lulas mononucleares do sangue perif?rico (PBMC) e leuc?citos totais pelo m?todo de exclus?o com azul de Tripan e sobre hem?cias pela avalia??o da atividade hemol?tica. Os resultados da an?lise t?rmica e infravermelho mostraram que a melhor forma de armazenamento de GSNO ? em 4?C e -18?C. As matrizes polim?ricas de PHB e da blenda PHBPPG (90/10) contendo 1% e 5% de GSNO, avaliadas por an?lise t?rmica foram capazes de liberar NO e apresentaram-se mais est?veis termicamente quando comparados aos filmes de PHB e da blenda sem o f?rmaco. O perfil de libera??o dos filmes de PHB e da blenda PHBPPG (90/10) mostrou que h? uma libera??o r?pida inicial de GSNO nas 24 horas iniciais seguido por um padr?o de libera??o controlada no per?odo de 1 a 8 dias para os filmes contendo 5% de GSNO, sendo o filme da blenda PHBPPG (90/10) contendo 5% de f?rmaco, o que apresentou maior porcentagem de libera??o no per?odo avaliado. Extratos provenientes dos filmes de PHB e da blenda PHBPPG (90/10) n?o apresentaram car?ter citot?xico por n?o induzirem hem?lise das hem?cias e n?o reduzirem a viabilidade celular de PBMCs e leuc?citos totais. As blendas desenvolvidas neste trabalho possuem a capacidade de libera??o controlada e in situ de ?xido n?trico, e possuem aplica??o potencial, por exemplo, no revestimento de stents coronarianos para preven??o da restenose p?s-angioplastia. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / Polymers are widely used for controlled drug release systems improving the therapeutic benefits and minimizing side effects by the pharmaceutical industry due their capability of releasing it effectively at the target site. Among the polymers used for this purpose there is poly(3-hydroxybutyrate, PHB), a polymer from microbial origin of the poly(hydroxyalkanoate) class that is both bioresorbable and biocompatible. When used as a physical blends with other polymers several properties of PHB (physical, mechanical and biological) could be modified to increase their applicability. To produce blends with PHB the propyleneglycol (PPG) was used to prepare PHBPPG blends (90/10). The aim of this work was to prepare and study polymeric films composed by PHB and PHBPPG (90/10) containing 1% and 5% S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor. The GSNO was synthesized and had its stability evaluated after storage at 23 ? C, 4 ? C and -18 ? C for 90 days by thermal analysis and infrared spectroscopy. The polymer materials containing GSNO were characterized by thermal analysis, infrared spectroscopy and also their drug release capability was evaluated. The cytotoxicity of matrices constituted by PHB and PHBPPG (90/10) was evaluated on peripheral blood mononuclear cells (PBMC) and total leukocytes by the exclusion method with Tripan blue and on red blood cells by the evaluation of hemolytic activity. The thermal and infrared analysis showed that the best form of GSNO storage is at 4 ? C and -18 ? C. Polymeric matrices of PHB and PHBPPG (90/10) containing 1% and 5% of GSNO, were able to release NO and were more thermally stable when compared to PHB and blends without the drug. The release profile of the PHB and PHBPPG blends (90/10) showed an initial fast release of GSNO at the initial 24 hours followed by a controlled release pattern in the period of 1 to 8 days for materials containing 5% of GSNO. The PHBPPG (90/10) blends containing 5% of drug presented the highest percentage of release in the evaluated period. The PHB and PHBPPG (90/10) blends did not present cytotoxic effects. The studied materials did not induced hemolysis of the red blood cells and did not reduced the cellular viability of PBMCs and total leukocytes. The developed blends are capable to exert the in situ controlled-release of nitric oxide and can potentially be used for example to coat coronary stents and thus help to prevent post-angioplasty restenosis.

Made available in DSpace on 2017-07-10T18:08:19Z (GMT). No. of bitstreams: 1 Manoela E Boff Zortea.pdf: 4020211 bytes, checksum: 0f828faa5dff1470fa28c16252974f2d (MD5) Previous issue date: 2012-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The diversity of use of polymeric materials causes concern because the use of non-renewable source for its production, as well as the environmental problems caused by the accumulation of the volume of solid waste generated. Seeking to minimize this problem were sought alternative products similar to plastics of petrochemical origin, one of them is the biodegradable polymer. The polyhydroxyalkanoates (PHAs) are polyesters synthesized by microorganisms from natural substances such as carbon and energy reserves, and accumulated by microbial cell in the form of intracellular granules, which may represent up to 80 % of the dry cell. The most studied polymer of this class is the polyhydroxybutyrate (P(3HB)) a biodegradable polymer that has similar features to the plastics from petroleum. This study aimed to develop for growth of bacteria Cupriavidus necator for the production of poly (3-hydroxybutyrate) using medium supplemented with cassava bagasse, whey and soybean oil. The first crops were carried out using a 2³ factorial design, to obtain the best condition at this stage of growth with the supplements tested. Other experiments used medium limited in nitrogen and higher amounts of supplements. The results obtained from the experimental design showed that the highest biomass production during growth was obtained in experiments which showed through the three supplements in their central levels, followed by testing supplements containing cheese whey and soybean oil combined or the presence of only one of these two components. Four experiments with increasing concentrations of supplement showed that waste and cheese whey hydrolyzed starch presented as good culture media for growth of C. necator, and the increased production of cell biomass (5,8 g.L-1) at the time of the nutrient nitrogen limitation occurred in the culture containing 100 % hydrolyzed starch conversion factor and the best substrate was obtained in cells in medium containing the highest amount supplement cheese whey (50 %). / A diversidade de utilização de materiais poliméricos acarreta preocupação devido a utilização de fonte não renovável para a sua produção, bem como os problemas ambientais causados pelo acúmulo do volume de lixo sólido gerado. Procurando minimizar este problema buscaram-se alternativas de produtos semelhantes aos plásticos de origem petroquímica, uma delas é o polímero biodegradável. Os polihidroxialcanoatos (PHAs) são poliésteres sintetizados por micro-organismos como substâncias naturais de reserva de carbono e energia, sendo acumulados pela célula microbiana na forma de grânulos intracelulares, podendo representar até 80% da massa seca celular. O polímero mais estudado desta classe é o polihidroxibutirato (P(3HB)) um polímero biodegradável que possui características semelhantes às dos plásticos provenientes de petróleo. O presente trabalho teve como objetivo desenvolver estratégias de crescimento da bactéria Cupriavidus necator para produção de poli (3-hidroxibutirato), utilizando meio suplementado com bagaço de mandioca, soro de queijo e óleo de soja. Os primeiros cultivos foram realizados seguindo um planejamento experimental 2³, visando obter nesta etapa a melhor condição de crescimento com os suplementos testados. Os demais experimentos utilizaram meio limitado em nitrogênio e maior quantidade de suplementos. Os resultados obtidos através do planejamento experimental mostraram que as maiores produções de biomassa na fase de crescimento foram obtidas nos experimentos cujo meio apresentava os três suplementos em seus níveis centrais, seguido pelos ensaios que continham os suplementos soro de queijo e óleo de soja combinados ou somente a presença de um destes dois componentes. Quatro experimentos com aumento nas concentrações de suplemento mostraram que os resíduos soro de queijo e hidrolisado amiláceo apresentaram-se como bons meios de cultivo para o crescimento de C. necator, sendo que a maior produção de biomassa celular (5,8 g.L-1) no momento da limitação do nutriente nitrogênio ocorreu no cultivo contendo 100 % de hidrolisado amiláceo e o melhor fator de conversão de substrato em células foi obtido no meio contendo maior quantidade do suplemento soro de queijo (50 %).

Abstract To date, plant endophytic bacteria have mainly been studied in roots of crop plants. However, shoot-associated endophytes are less diverse than root-associated ones. Hence, endophytic bacteria of plant shoots evolved different traits, than root colonizers, especially with types of host tissues infected and patterns of growth and development. This study found Methylobacterium extorquens colonized pine seedlings similarly to stem-colonizing rhizobia of other plants. M. extorquens DSM13060 was isolated from meristematic cells in shoot tip cultures of Scots pine (Pinus sylvestris L.). M. extorquens infected the plant stem through epidermis or stomatal apertures, forming infection pockets in the root and stem epidermis, or cortex. Post-infection, thread-like infection structures passed through the endoderm, invading vascular tissues. This led to systemic colonization of above and below ground-parts, observed in in vitro grown Scots pine. A novel mechanism enabling development of endophyte-host symbiosis is discovered within the M. extorquens – Scots pine model. This mechanism involves ability of M. extorquens to produce polyhydroxybutyrates (PHB) to protect itself from host-induced oxidative stress during infection. Upon initial colonization on the host surface, M. extorquens DSM13060 consumes methanol as a carbon source, using it to biosynthesize PHB. PHB are then degraded, upon host infection, by PHB depolymerases (PhaZ) to yield methyl-esterified 3-hydroxybutyrate oligomers. These oligomers have substantial antioxidant activity towards host-induced oxidative stress, enabling the bacterium to bypass host defenses and colonize further tissues. The bacteria can also store PHBs for future protection. The capacity for PHB production and, thus, protection from oxidative stress, is discovered in a wide taxonomic range of bacteria. This study also shows meristematic endophytes are important in growth and development of their hosts. Unlike many bacterial root endophytes, M. extorquens DSM13060 does not induce plant growth through hormones. However, this bacterium can colonize the interior of living host cells, where it aggregates around the nucleus of the host plant. M. extorquens DSM13060 genome encodes nucleomodulins, eukaryotic-like transcription factors, which may intervene in host transcription and metabolism. / Tiivistelmä Kasvin sisällä elävien endofyyttisten bakteerien tutkimus on perinteisesti keskittynyt viljelykasveihin ja niiden juuristoon. Kasvien maanpäällisissä versoissa elävät endofyytit eroavat merkittävästi juuriston bakteereista lajirikkauden suhteen. Versoissa eläville bakteereille on todennäköisesti kehittynyt erilaisia sopeumia kuin juuriston endofyyttilajeille. Endofyyttinen Methylobacterium extorquens DSM13060 elää männyn silmujen kasvusolukossa lisäten isäntäkasvin kasvua. Tässä tutkimuksessa M. extorquens –bakteerin todettiin siirtyvän männyn taimiin samoja mekanismeja käyttäen kuin Rhizobium –suvun typensitojabakteerit. Metylobakteeri tunkeutui isäntäkasviin aktiivisesti soluseinien läpi tai varren ilmarakojen kautta muodostaen mikropesäkkeitä juuren ja varren pinnoille, sekä infektiotaskuja kuorisolukkoon. Bakteeri eteni infektiolankojen avulla endodermin ohi johtosolukoihin, mikä mahdollisti bakteerin siirtymisen muualle taimeen. M. extorquens käytti kasvin pinnalla runsaana olevaa metanolia hiilenlähteenään, varastoiden sen solujen sisäiseksi polyhydroksibutyraatti (PHB) polymeeriksi. Infektion myöhemmissä vaiheissa bakteeri hajotti varastoidun polymeerin PHB-depolymeraasientsyymien (PhaZ) avulla lyhyiksi rasvahappoketjuiksi. Nämä metyloidut 3-hydroksibutyraatin oligomeerit suojasivat bakteeria isäntäkasvin puolustuksen tuottamilta happiradikaaleilta mahdollistaen infektion etenemisen. Tutkimuksessa saatujen tulosten perusteella endofyytin solunsisäinen energiavarasto, PHB, toimii pelkistävänä varastona ympäristön hapettavaa stressiä vastaan. Löytö osoitti uudenlaisen antioksidatiivisen puolustumekanismin, joka on levinnyt laajalle bakteerikunnassa ja liittyy yleisesti bakteerien kykyyn sietää vaikeita olosuhteita. Toisin kuin useat juurissa elävät bakteeriendofyytit, M. extorquens ei lisää isäntäkasvin kasvua tuottamalla kasvihormoneja. Bakteeri kykenee elämään männyn elävien solujen sisällä tumien läheisyydessä. M. extorquens DSM13060 genomi sisältääkin useita geenejä, jotka koodaavat nukleomoduliineja, eukaryoottisolujen säätylytekijöiden kaltaisia entsyymejä, joiden avulla bakteeri todennäköisesti vaikuttaa isäntäkasvin aineenvaihduntaan. Vastaavaa vaikutusmekanismia ei ole aikaisemmin kuvattu endofyyteillä. Tutkimus korostaa aiemmin tuntemattomien meristemaattisten bakteeriendofyyttien merkitystä isäntäkasvin kasvussa ja erilaistumisessa.

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.