La période de transition, c'est-à-dire de 3 semaines avant à 3 semaines après la parturition, est une période critique pour les vaches laitières puisqu'elles font face à divers changements métaboliques et immunologiques. Pendant cette période, les vaches sont en état de balance énergétique négative causée par l'augmentation de la demande en nutriments pour la production laitière. Pour répondre à cette demande, les vaches doivent mobiliser leurs réserves corporelles ce qui peut entraîner d'importants problèmes de santé. Ce projet avait pour but d'évaluer si la traite incomplète ou la tétée pendant les 5 premiers jours de lactation peut diminuer les problèmes de santé suite à la parturition sans affecter la production laitière subséquente. Quarante-sept vaches Holstein multipares ont été assignées à l'un des 3 traitements suivants. Les vaches du traitement Témoin ont été complètement traites [i.e. traitées] 2x par jour; celles du traitement Incomplet ont été partiellement traites [i.e. traitées] (environ 1/3 du lait a été récolté) 2x par jour jusqu'au jour 5 et celles du traitement Tétée ont été laissées avec le veau qui pouvait téter ad libitum jusqu'au jour 5 en plus d'être traites complètement 1x par jour du jour 3 au jour 5.La production laitière, la consommation alimentaire, certains métabolites sanguins et quelques fonctions immunitaires ont été mesurés sur chacune des vaches. Les traitements n'ont pas affecté la production laitière subséquente des vaches et aucune différence n'a été observée entre les traitements pour la consommation alimentaire. Les vaches des traitements Partiels (Incomplet + Tétée) ont montré des concentrations sanguines 16% plus élevées en glucose, 21% plus faibles en acides gras non estérifiés et 44% plus faibles en bêtahydroxybutyrate. Ces différences de concentrations ont persisté jusqu'au jour 5 pour les acides gras non estérifiés et jusqu'au jour 21 pour le glucose et les bêta-hydroxybutyrate. Bref, les traitements Partiels ont permis d'améliorer la glycémie et de diminuer les concentrations en métabolites sanguins néfastes. Par contre, les fonctions immunitaires évaluées n'ont pas permis de mettre en évidence que la diminution de la récolte en lait en début lactation améliore le statut immunitaire des vaches laitières. Néanmoins, ils ont montré une différence significative entre le jour 2 et le jour 61 pour la prolifération des lymphocytes et la production d'interleukine-4. De plus, une corrélation négative a été observée entre la teneur en acides gras non estérifiés du sérum et la prolifération des lymphocytes. En conclusion, la diminution de la récolte en lait pendant les 5 premiers jours de lactation permet de réduire les désordres métaboliques sans avoir de répercussion négative sur la production laitière subséquente des vaches.

Esse projeto visa a melhoria das propriedades mecânicas e de processabilidade do Poli(3-hidroxibutirato) (PHB) para futuras aplicações. Foram preparados filmes de blendas de PHB por dissolução em clorofórmio. Poli(etileno glicol), com massa molar de 300 (PEG) ou 4.000.000 g mol-1 (PEO), foi misturado em proporções de 5 a 30 % em massa e análises termogravimétricas (TG), de calorimetria diferencial exploratória (DSC) e ensaios dinâmico-mecânicos (DMTA) foram realizadas para avaliar a miscibilidade da blenda. Foram também analisados filmes recém-preparados e filmes envelhecidos, para avaliar o avanço da cristalização e consequente alteração das propriedades dos filmes com o passar do tempo. Para isso foram feitas comparações visuais de imagens microscópicas da morfologia das blendas, obtidas através de microscopia eletrônica de varredura (MEV). As blendas foram analisadas quanto ao grau de cristalinidade através de análise calorimétrica (DSC) e de difratometria de raio-X. As curvas de DSC mostram uma redução da Tm do PHB com a adição de PEG e PEO, o que indica uma forte interação entre os polímeros. As análises de TG mostram uma etapa principal de degradação, evidenciando a miscibilidade entre os polímeros. A TG também mostra uma sensível redução na temperatura de degradação do PHB para concentrações acima de 10 % em PEG, o que é indesejável. Através do MEV nota-se que existe também uma segregação de fases que aumenta com o tempo de estocagem do material, levando à recristalização do PHB e sua consequente fragilização. A difração de raios-X nos mostra que a adição de PEG e PEO ao PHB traz uma redução considerável para a cristalinidade do sistema, e que o aumento de 5 % para 30 % no teor de PEG é responsável por apenas uma pequena redução na cristalinidade, mas uma considerável redução na recristalização sofrida pela blenda com o tempo de estocagem. As análises de DMTA mostram que as blendas possuem módulo de armazenamento similar ao de polímeros flexíveis, evidenciando uma efetiva tenacificação do PHB. Amostras utilizando PEO apresentam maior rigidez do que as amostras contendo PEG. / This project aims the enhancement of both processability and mechanical properties of poly(3-hydroxybutyrate) (PHB) for future applications. Films of blends of PHB and poly(ethylene oxide) were prepared by chloroform solution casting and evaporation. Poly(ethylene oxide) having molar mass of 4,000,000 (PEO) or 300 g.mol-1 (PEG) was blended in proportion of 5 to 30 wt %, Thermogravimetric Analysis (TG), Differential Scanning Calorimetry (DSC) and Dynamic-Mechanic Analysis (DMTA) were performed in order to evaluate the miscibility of the blends. As-cast and aged films were also compared in order to analyze the crystallization progress and the consequent changes in the blends properties due to the aging process. For this, visual comparison was drawn between microscopy pictures obtained through scanning electron microscopy (SEM). The blends have their crystallinity degree determined by analyzing their X-ray diffraction curves. The DSC curves show a reduction at the PHBs Tm as the mass percentage of PEG increases, indicating a strong interaction in between the polymers. The TG and DTG analyses reveal a single main degradation step, what denotes miscibility in between the polymers. The TG also shows a considerable reduction of the PHBs degrading temperature for PEG concentrations over 10 %, which is undesirable. Through SEM one can note a phase segregation that increases with storaging time, leading to further crystallization of PHB and its subsequent enbrittlement. The X-ray diffraction curves show that the PEG and PEO bring a considerable crystallinity reduction to the system, and that the increasing of PEG content from 5 to 30% has only a minor effect per se, though also a considerable reduction of the perfection undergone by the system with storage time. The DMTA shows that the blends have a storage modulus similar to the one of flexible polimers, hence showing an effective PHB toughening. Samples containing PEO are more rigid than those containing PEG.

Em animais, dietas cetogênicas induzem o Browning de tecidos adiposos brancos, fenômeno caracterizado pelo de aumento de adipócitos capazes de expressar a proteína desacopladora 1 (UCP1) e outros marcadores de gordura marrom em meio a gordura branca. Estudo anterior demonstrou que o β-hidroxibutirato (βHB), principal corpo cetônico, aumenta marcadores do processo de browning em adipócitos brancos (in vitro) após 24 horas de incubação. No entanto, as doses testadas foram suprafisiológicas (50 mM) ou apenas encontradas durante a cetoacidose (25 mM). As dietas cetogênicas aumentam a cetonemia em torno de 1-3 mM. O jejum prolongado pode aumentá-lo para 4-7 mM. Uma vez que poderia ser provocada in vivo através de intervenções dietéticas, estudamos o impacto de concentrações fisiológicas de βHB no metabolismo e marcadores de Browning em adipócitos brancos / tecido adiposo em diferentes modelos: adipócitos isolados de ratos Wistar, células 3T3 -L1 e in vivo, através da suplementação de sais de βHB em ratos Wistar. Demostramos que o βHB: não induz o aparecimento diferentes marcadores de Browning (tais como o incremento: da capacidade oxidativa, da atividade de citrato sintase e de genes relacionados ao Browning) em adipócitos isolados após 24 ou 48 horas de tratamento; não exerce efeito permissivo no browning induzido por agonismo β-adrenérgico. Além disso, os adipócitos 3T3-L1 diferenciados com βHB (4mM) tiveram diminuição de 52% na expressão de Ucp1, resultado que foi reproduzido no tecido adiposo inguinal subcutâneo de ratos Wistar após a ingestão de sais DL- βHB, onde a expressão gênica de Ucp1 foi indetectável. Em conclusão, embora as causas de browning do tecido adiposo branco induzido por dietas cetogênicas permaneçam inconclusivas, nosso estudo demonstra a incapacidade de, em concentrações fisiológicas, os corpos cetônicos serem, por si, responsáveis por esse fenômeno. Pelo contrário, em algumas situações, o βHB pode prejudicar a expressão da Ucp1. / In animals, ketogenic diets induce browning of white adipose tissue, a phenomenon characterized by the increase of adipocytes capable of expressing the uncoupling protein 1 (UCP1) and other markers of brown fat in among white fat. A previous study demonstrated that β-hydroxybutyrate (βHB), the major ketone body, increases markers of the browning process in white adipocytes (in vitro) after 24 hours of incubation. However, the doses tested were supraphysiological (50 mM) or only found during ketoacidosis (25 mM). Ketogenic diets increase ketonemia by about 1-3 mM. Prolonged fasting can increase it to 4-7 mM. Since it could be elicited in vivo through dietary interventions, we studied the impact of physiological concentrations of βHB on metabolism and browning markers on white adipocytes/adipose tissue in different models: adipocytes isolated from Wistar rats, 3T3-L1 cells and in vivo, through the supplementation of βHB salts in Wistar rats. We demonstrate that βHB does not increase any browning markers (such as: oxidative capacity, citrate synthase activity, and browning related genes expression) in isolated adipocytes after 24 or 48 hours of treatment; does not exert a permissive effect on browning induced by β-adrenergic agonism. In addition, 3T3-L1 adipocytes differentiated with βHB (4mM) had a 52% decrease in Ucp1 expression, a result that was reproduced in the subcutaneous inguinal adipose tissue of Wistar rats after ingestion of DL-βHB salts, where Ucp1 expression was undetectable. In conclusion, although the causes of browning of white adipose tissue during ketogenic diets remain inconclusive, our study demonstrates the inability, in physiological concentrations, of ketone bodies themselves to be responsible for this phenomenon. In contrast, in some situations, βHB may impair the expression of Ucp1.

This dissertation evaluated nutritional approaches such as the addition of betaine, prebiotics, probiotics, transitional metabolic substrates, and β-mercaptoacetate (MAA; a compound which inhibits β-oxidation) to the diet of lactating dairy cows to determine their impact on physiological, metabolic, hormonal and production responses during thermal stress. The first objective was to evaluate the use of an organic osmolyte, betaine to reduce the impact of heat stress (HS). Cows were fed either 0 (control; CON), 57 mg/kg BW (mid) or 114 mg/kg (high; HI) body weight (BW) betaine and subjected to thermoneutral (TN) and HS conditions. There was an increase in milk yield during TN with HI betaine over controls (P< 0.01), but the advantage was lost during HS. Plasma glucose increased during HS in HI dose cows compared to control (P < 0.01) as did plasma insulin (P = 0.01). Betaine increased milk production during TN and plasma glucose in HS, but did not improve the HS response. Objective two evaluated the use of a probiotic or direct fed microbial (DFM), Calsporin (Bacillus subtilus C-3102) to decrease the effects of HS in dairy cows. We hypothesized that feeding Calsporin prior to and during HS would reduce pathogenic strains of bacteria, maintain commensal microbes, and improve ruminal anaerobic fermentation resulting in improved milk yield (MY). Milk yield was numerically increased (1.26 kg, P = 0.11) in cows fed Calsporin during TN but was reduced under HS (-2.67 kg, P < 0.01) and milk protein content was decreased (P = 0.05). The DFM tended to decrease somatic cell count (SCC) across periods (P = 0.07). Calsporin addition to the diet did not affect respiration rates and was associated with higher rectal temperature at 1800 in HS (P = 0.02). The expression of heat shock protein 27 (HSP27) was decreased with Calsporin treatment (P = 0.03) and in both HS and TN. The fecal microbial count did not change with the exception of the Calsporin strain in treated animals (P < 0.01). The third objective was to feed OmniGen-AF (OG) to dairy cows before and during thermal stress. We hypothesized that feeding OG to HS dairy cows will improve the immune response, and decrease production losses associated with HS. Cows fed OG maintained lower SCC compared to control (P < 0.01) during the recovery period. We did not detect differences between groups in serum calcium while serum non-esterified fatty acid (NEFA) concentrations (P = 0.10) tended to be greater in OG fed cows across the Agricultural Research Center (ARC) portion including HS. Serum Adrenocorticotropic hormone (ACTH) levels were greater in OG cows (P<0.0001) across all sample days. Feeding OG reduced the HS response including serum Cortisol. The final study measured the effects of the metabolic substrate β-hydroxybutyrate (BHB) during HS on feed intake and metabolites. Under TN conditions the cows received a bolus dose of BHB and dry matter intake (DMI) and metabolites were measured. The second part of this study used a bolus of MAA to limit the up-stream production of acetyl-CoA available for ketogenesis by inhibiting ß-oxidation. We proposed that dosing lactating dairy cows with BHB would decrease DMI, increase plasma insulin, decrease NEFAs and increase skin temperature by vasodilatation. The same cows were then subjected to HS and dosed with saline and MAA on different test days. The infusion of BHB increased skin temperature (time 0.5, 1, 2, 3 and 4°C r² =0.98 with serum BHB) and decreased serum NEFA levels (P < 0.01). There was no change in mean DMI, glucose or insulin. The bolus of MAA decreased feed intake, vaginal temperature, and insulin. There was an increase in serum BHB with the initial dose of MAA and an initial decrease in serum glucose (P < 0.0001) with MAA. Serum glucose increased as insulin decreased with MAA. The infusion of BHB did not alter feed intake in this study despite high plasma levels of BHB.

This work was undertaken with the objective of isolating and characterising the bdh gene of P. aeruginosa PAOI. Isolation of the bdh gene was initially attempted by PCR amplification and then by heterologous complementation of E. coli (LS5218) and S. meliloti (Rm11107) strains unable to catabolise 3-hydroxybutyrate. Three classes of plasmids were isolated. Class I comprised two plasmids, p5218-02 and p5218-07, isolated via complementation of LS5218, which were capable of complementing both LS5218 and Rm11107 for growth on 3-hydroxybutyrate. 3-hydroxybutyrate dehydrogenase (BDH) activity was not detected in an extract of LS5218 (p5218-02). The sole 3.6-kb EcoRI fist was partially sequenced and found to have three putative open reading frames (ORF). ORF 1 is homologous to the fusE gene of E. coli. We hypothesised that p5218-02 encodes an enzyme capable of degrading 3 hydroxybutyrate, but does not encode the bdh gene. Plasmids of class II (p30065) and class III (p30066) were isolated via complementation of Rm11107. Significant BDH activity was detected in an extract of Rm11107 (p30066), but not in Rm11107, leading to the hypothesis that p30066 carries the bdh gene.

This study was undertaken to characterize the role and pathway for assimilation of the intracellular carbon storage compound, poly-beta-hydroxybutyrate (PHB), in Rhizobium (Sinorhizobium) meliloti. Mutants unable to utilize the degradation intermediates, 3-hydroxybutyrate (HB) and/or acetoacetate (AA) were characterized. A mutant unable to utilize HB (Hbu-) while retaining the ability to utilize AA was found to be deficient in 3-hydroxybutyrate dehydrogenase (Bdh) activity. The bdhA mutant showed no symbiotic defects in association with alfalfa plants. However, when co-inoculated with the wild type, the mutant showed significantly reduced competitiveness. A more severe competition defect was observed for a PHB synthesis mutant (phaC). Both these mutants also showed reduced competitiveness when subjected to multiple cycles of subculturing through alternating carbon-rich and carbon-poor media, with the phaC mutant showing a greater loss in competitiveness. The results indicate that the ability to efficiently deposit and utilize cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability. / The gene encoding Bdh (bdhA) was isolated and sequenced. Two transcription start sites, S1 and S2 were identified but no known consensus promoter sequences were identified upstream of either start site. A sigma 54 consensus binding sequence was found to be located between S1 and S2 but no corresponding transcript was detected. Transcriptional bdhA-lacZ fusion studies indicated that gene expression was growth-phase associated. The bdhA gene from Rhizobium sp. NGR234 was also isolated and characterized and found to be highly homologous to the R. meliloti bdhA sequence. Unlike R. meliloti , NGR234 is able to accumulate PHB during symbiosis. An NGR234 bdhA mutant showed symbiotic defects on Leucaena but not on Tephrosia, Macroptilium or Vigna host plants, indicating that the phenotype was host-dependent. / Mutations that suppress the Hbu- phenotype without restoring Bdh activity were identified, indicating the existence of a Bdh-independent pathway for HB utilization. These mutations mapped to the age-1 locus, which causes enhanced growth rate on HB and AA minimal media. Introduction of plasmid-borne multiple copies of a gene encoding acetoacetyl-CoA synthetase (acsA) into the bdhA mutant also results in suppression of the Hbu- phenotype. A possible mechanism of suppression involving direct activation of HB to 3-hydroxybutyryl-CoA, followed by reduction to acetoacetyl-CoA by the NADP-acetoacetyl-CoA reductase (encoded by phaB) was investigated. A strain carrying the triple mutations, age-1::Tn5-Tp, bdhA ::Tn5 and phaB::OSmSp retained the ability to utilize HB, indicating that the bypass mechanism does not involve NADP-acetoacetyl-CoA reductase. / The phaB mutant does not accumulate PHB or utilize HB or AA. Furthermore, colonies of the phaB and phaC mutants exhibit non-mucoid phenotype on yeast extract mannitol agar. The observation that a R. meliloti exoS null mutant is also Hbu- provides further support for a link between PHB and exopolysaccharide synthesis. Since ExoS is a positive regulator of succinoglycan biosynthesis it is hypothesized that regulation of succinoglycan synthesis by ExoS requires PHB synthesis.

This research followed acetate carbon (C) uptake, metabolism, and fate through a typical modern Kraft pulp and paper mill AS system. Freshly collected mill biomass (AS floc suspensions) was placed under conditions representing four key phases of AS biotreatment: (1) initial acetate uptake by aerated starved AS; (2) ongoing acetate uptake; (3) aerobic metabolism of acetate-loaded AS in acetate-stripped effluent; and (4) anaerobic, settled biomass metabolism. Conditions mimicked the mill system as closely as possible. Acetate carbon uptake kinetics and conversion to CO2, growth products, PHB, and secreted metabolites in each of the four phases were measured. The role of PHB synthesis in the initial stripping of acetate from mill effluent and the PHB production potential of this mill AS were also investigated. / Results showed that acetate was rapidly taken up by high-affinity systems in the AS. During the initial exposure of mill-starved AS, acetate greatly stimulated AS-O2 uptake, and was quickly converted to PHB and CO 2. Upon depletion of available effluent acetate, as occurs in the downstream sections of the aeration tank, O2-uptake rates decreased and the acetate-C stored in AS-PHB was slowly released as CO2, and partly used for growth. Under secondary clarifier-like anaerobic conditions, the AS released virtually no CO2. However, substantial amounts of PHB were used for growth under anaerobic conditions and a small proportion of the original acetate C exited the cells as organic acids.

abstract: The production of monomer compounds for synthesizing plastics has to date been largely restricted to the petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economic feasibility. Cyanobacteria have, however, become attractive microbial factories to produce renewable fuels and chemicals directly from sunlight and CO2. To explore the feasibility of photosynthetic production of (S)- and (R)-3-hydroxybutyrate (3HB), building-block monomers for synthesizing the biodegradable plastics polyhydroxyalkanoates and precursors to fine chemicals, synthetic metabolic pathways have been constructed, characterized and optimized in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803). Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing PHB biosynthesis pathway further promoted the 3HB production. Analysis of the intracellular acetyl-CoA and anion concentrations in the culture media indicated that the phosphate consumption during the photoautotrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. Fine-tuning of the gene expression levels via strategies, including tuning gene copy numbers, promoter engineering and ribosome binding site optimization, proved critical to mitigating metabolic bottlenecks and thus improving the 3HB production. One of the engineered Synechocystis strains, namely R168, was able to produce (R)-3HB to a cumulative titer of ~1600 mg/L, with a peak daily productivity of ~200 mg/L, using light and CO2 as the sole energy and carbon sources, respectively. Additionally, in order to establish a high-efficiency transformation protocol in cyanobacterium Synechocystis 6803, methyltransferase-encoding genes were cloned and expressed to pre-methylate the exogenous DNA before Synechocystis transformation. Eventually, the transformation efficiency was increased by two orders of magnitude in Synechocystis. This research has demonstrated the use of cyanobacteria as cell factories to produce 3HB directly from light and CO2, and developed new synthetic biology tools for cyanobacteria. / Dissertation/Thesis / Ph.D. Biological Design 2014

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-25T13:55:38Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação CENTRAL FINAL.pdf: 2543250 bytes, checksum: b3adf9bd3291a6638da52621c387fab7 (MD5) / Made available in DSpace on 2017-04-25T13:55:38Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação CENTRAL FINAL.pdf: 2543250 bytes, checksum: b3adf9bd3291a6638da52621c387fab7 (MD5) Previous issue date: 2016-03-14 / PRH-ANP/MCT / Muitos trabalhos vem norteando a utilização do poli(3-hidroxibutirato)-PHB, polímero da família dos Poli(hidroxialcanoatos), que possui como principais características, biodegradabilidade e biocompatibilidade, o que lhe permite aplicações na área médicohospitalar, médico-farmacêutica, embalagens alimentícias, entre outras. O estudo sobre a degradação de polímeros quando exposto a qualquer tipo de fonte degradativa é tema de grande interesse científico e industrial, em razão das alterações químicas que podem ocorrer na cadeia, podendo ocasionar a cisão (ou quebra) de ligações na cadeia principal ou em grupos laterais, reticulação, eliminação ou substituição de cadeias laterais, reações intramoleculares, auto-oxidação e despolimerização. A avaliação das propriedades do PHB após um processo de esterilização, ou mesmo da estabilidade desse material quando exposto a alguma substância química e/ou radiação gama é um importante tema de estudo pois é necessário adequar os materiais às normas, principalmente em aplicações como embalagem para uso médico hospitalar. Esse trabalho teve como objetivo avaliar a estabilidade à radiação gama do PHB, bem como investigar a estabilidade desse polímero quando imerso nas soluções de glutaraldeído 2% e etanol 70%. Os filmes de PHB foram preparados pela técnica de solution casting. Analisou-se amostras de filmes de PHB puro; PHB irradiados a 25 kGy; PHB imerso em glutaraldeído 2% irradiado 25 kGy e não irradiados com tempo de imersão na solução por 2, 4, 6 e 8 horas; PHB imerso em etanol 70% (EtOH) irradiado 25 kGy e não irradiados, com tempo de imersão na solução por 2, 4, 6 e 8 horas. As propriedades destes foram avaliadas a partir de diversas técnicas, tais como: infravermelho com transformada de Fourier (FTIR), ensaio de tração, calorimetria exploratória diferencial (DSC), termogravimetria (TGA). A partir das análises por componentes principais realizadas através dos espectros de infravermelho das amostras dos filmes de PHB puro, PHB imersos em glutaraldeído 2%, etanol 70% e, após exposição à radiação gama, apresentaram alterações químicas na sua estrutura, após exposição à radiação gama ou imersão nos agentes químicos estudados. Verificou-se, portanto, que o PHB é sensível à radiação gama, apresentando como efeito fundamental perdas de suas propriedades mecânicas, o que pode ser resultante do processo de cisão molecular. Contudo, em relação a esterilização do polímero com glutaraldeído e etanol, não se evidenciou perdas nas propriedades mecânicas das amostras imersas em ambas as soluções. Quanto às propriedades térmicas, amostras de PHB imersas em glutaraldeído 2% e etanol 70% apresentaram comportamento semelhante ao PHB puro. A degradação térmica dos filmes de PHB ocorreu em um único estágio e em uma faixa de temperatura que variou para a amostra PHB puro de 225,4 ºC a 295,1 ºC. O comportamento térmico das amostras de PHB imersas em glutaraldeído e etanol observado pelas curvas de TGA, antes e após processos de esterilização por radiação gama, foi semelhante ao comportamento térmico do filme de PHB puro. / A lot of study have conducted the use of poly (3-hydroxybutyrate)-PHB, a polymer from the poly (hydroxyalkanoates) family, which has main characteristics such as biodegradability and biocompatibility that allows applications in the medical-hospital, medical-pharmaceutical and food packaging areas. A study on polymer degradation when exposed to any type of degradative source is a subject of great scientific and industrial interest due to the chemical changes that may occur in the chain such as scission, crosslinking, elimination or substitution of side chains, intramolecular reactions, auto-oxidation and depolymerization. Sterilized PHB or even its stability when exposed to some chemical substance or gamma radiation, becomes important topics of study to fit the standards, especially in applications such as packaging for hospital-medical use. The objective of this work was to evaluate the PHB gamma radiation stability and to investigate the stability of this polymer when immersed in 2% glutaraldehyde and 70% ethanol solutions. PHB films were prepared by the technique of solution casting. The analyzed samples were: films of pure PHB; PHB irradiated at 25 kGy; PHB immersed in 2% glutaraldehyde irradiated 25 kGy and non-irradiated, with immersion time in the solution for 2, 4, 6 and 8 hours; PHB immersed in ethanol 70% (EtOH) irradiated 25 kGy and nonirradiated, with immersion time in the solution for 2, 4, 6 and 8 hours. Its properties were evaluated from several techniques such as: Fourier transform infrared (FTIR), tensile test, differential scanning calorimetry (DSC), thermogravimetry (TGA). Based on the analysis of the main components of different types of PHB samples (PHB immersed in 2% glutaraldehyde, 70% ethanol and after exposure to gamma radiation) showed chemical changes in structure, after exposure to gamma radiation or immersion in the chemical agents studied. It was verified that the PHB is sensitive to gamma radiation, presenting as fundamental effect losses of its mechanical properties, which may be the result of the molecular scission process. However, in relation to the sterilization of the polymer with glutaraldehyde and ethanol, no losses were observed in the mechanical properties of the samples immersed in both solutions. Regarding to the thermal properties PHB samples immersed in 2% glutaraldehyde and 70% ethanol presented similar behavior in comparison to pure PHB. The thermal degradation of PHB films occurred in a single stage and in a temperature range, which varied for the pure PHB sample from 225.4 ° C to 295.1 ° C. The thermal behavior of PHB samples immersed in glutaraldehyde and ethanol prior to and after gamma ray sterilization procedures observed by the TGA curves were similar to the thermal behavior of the pure PHB film.

Orientador: Paulo Pinto Joazeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T01:42:36Z (GMT). No. of bitstreams: 1 Lucchesi_Carolina_M.pdf: 3174282 bytes, checksum: b63549c83e46027b51da37ffd45ad2d7 (MD5) Previous issue date: 2006 / Resumo: O copolímero Poli(hidroxibutirato-co-hidroxivalerato) (PHBV) tem sido intensamente estudado como substrato para a engenharia de tecidos, sendo conhecido como um poliéster hidrofóbico. A modificação da superfície por plasma é uma técnica efetiva e econômica para os materiais e tem ganhado crescente interesse da engenharia biomédica, por melhorar a biocompatibilidade da superfície. Neste estudo, avaliou-se as vantagens da modificação da superfície de membranas de PHBV tratadas por plasma de Oxigênio e Nitrogênio a fim de acelerar o processo de adesão e proliferação celular. O PHBV foi dissolvido em c1oreto de metileno à temperatura ambiente. Membranas de PHBV foram submetidas ao tratamento de plasma de Oxigênio e Nitrogênio, através de um gerador de plasma. As membranas foram esterilizadas por radiação UV por 30 min e colocadas em placas de 96 poços. Células Vero foram semeadas sobre as membranas, sendo determinada a proliferação celular sobre as matrizes, a citotoxicidade e adesão celular. Após 2, 24,48 e 120h de incubação, o crescimento e proliferação dos fibroblastos foram observados por microscopia eletrônica de varredura (MEV). As análises das membranas indicaram que o tratamento por plasma aumentou o ângulo de contato e a rugosidade, alterando a morfologia da superfície, e conseqüentemente, melhorou ocomportamento hidrofílico do polímero. A microscopia eletrônica de varredura das células Vero mostrou que as modificações da superfície proporcionaram melhor adesão, espalhamento e proliferação celular. O tratamento da superfície do polímero somado às suas propriedades químicas é um caminho para obtenção de estruturas aplicáveis a engenharia de tecido / Abstract: The copolymers poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) are being intensely studied as a tissue engineering substrate. It is know that Poly(hydroxybutic) (PHB) and their copolymers are quite hydrophobic polyesters. Plasma-surface modification is an effective and economical surface treatment technique for many materiaIs and of growing interest in biomedical engineering. In this study we investigate the advantages of oxygen and nitrogen plasma treatment to modify the PHBV surface to enable the acceleration of Vero cell adhesion and proliferation. PHBV was dissolved in methylene chloride at room temperature. The PHBV membranes were modified by oxygen or nitrogen-plasma treatments using a plasma generator. The membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates. Vero cells were seeded onto the membranes and their proliferation onto the matrices was also determined by cytotoxicity and cell adhesion assay. After 2, 24, 48 and 120h of incubation, growth of fibroblasts on matrices was observed by scanning electron microscopy (SEM). The analyses of the membranes indicated that the plasma treatment increased the contact angle and their roughness, it also changed the surface morphology, and consequently, enhanced the hydrophilic behavior of PHBV polymers. Scanning electron microscopy analysis of Vero cell adhered to plasma treated PHBV showed that the modified surface had allowed better cell attachment, spreading and growth than the untreated membrane. This combination of surface treatment and polymer chemistry is a valuable guide to prepare appropriated surface for tissue engineering application / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural