Polyhydroxyalkanoates (PHAs) are polyesters, produced by many bacteria and some archaea. The most commonly characterised is polyhydroxybutyrate (PHB). Produced when nutrients are growth limiting and carbon available in excess, PHA serves as a carbon-energy storage material and forms generally spherical insoluble inclusions between 50-500nm in diameter in the cytoplasm. The key enzyme for PHA synthesis is the PHA synthase and this enzyme catalyses the polymerisation of (R)-3-hydroxy fatty acids into PHA. PHA synthase remains covalently attached to the growing polyester chain and is displayed on the surface of the PHA granule. Other proteins associated with PHA granules include depolymerases for mobilisation or degradation of granules, regulatory proteins and phasins, proteins that aid in PHA granule stability. PHA bio-beads displaying an IgG binding protein were produced and used to purify IgG from serum demonstrating that the PHA synthase can be engineered to display functional synthase fusion proteins at the PHA granule surface. Correctly folded eukaryotic proteins were also produced and displayed at the PHA granule surface as phasin fusion proteins. Multiple-functionality was also achievable by co-expression of various hybrid genes suggesting that this biotechnological bead production strategy might represent a versatile platform technology. The production of functional eukaryotic proteins at the PHA bead surface represents a novel in vivo matrix-assisted protein folding system. Protein engineering of PHA granule surface proteins provides a novel molecular tool for the display of antigens for FACS based analysis and offers promising possibilities in the development of future biotechnological production processes. Overall, the results obtained in this study strongly enhance the applied potential of these polyester beads in biotechnology and medicine.

This research is directed toward understanding the effect of nanoparticle attributes and polymer morphology on the properties of the nanocomposites with analogous nanoparticle chemistry. In order to develop this understanding, polymer nanocomposites containing calcium phosphate nanoparticles of different specific surface areas and shapes were fabricated and characterized through thermal and thermomechanical techniques. Nanoparticles were synthesized using reverse microemulsion technique. For nanocomposites with different surface area particles, the mobility of amorphous polymer chains was restricted significantly by the presence of particles with an interphase network morphology at higher loadings. Composites fabricated with different crystallinity matrices showed that the dispersion characteristics and reinforcement behavior of nanoparticles were governed by the amount of amorphous polymer fraction available. The study conducted on the effect of nanoparticle shape with near-spherical and nanofiber nanoparticles illustrated that the crystallization kinetics and the final microstructure of the composites was a function of shape of the nanoparticles. The results of this research indicate that nanoparticle geometry and matrix morphology are important parameters to be considered in designing and characterizing the structure-property relationship in polymer nanocomposites.

Made available in DSpace on 2014-12-17T14:07:07Z (GMT). No. of bitstreams: 1 LuizFMC_TESE.pdf: 3268756 bytes, checksum: e846724b8b5adbbee4ec9a2ecaf76796 (MD5) Previous issue date: 2011-06-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The use of raw materials from renewable sources for production of materials has been the subject of several studies and researches, because of its potential to substitute petrochemical-based materials. The addition of natural fibers to polymers represents an alternative in the partial or total replacement of glass fibers in composites. In this work, carnauba leaf fibers were used in the production of biodegradable composites with polyhydroxybutyrate (PHB) matrix. To improve the interfacial properties fiber / matrix were studied four chemical treatments to the fibers..The effect of the different chemical treatments on the morphological, physical, chemical and mechanical properties of the fibers and composites were investigated by scanning electron microscopy (SEM), infrared spectroscopy, X-ray diffraction, tensile and flexural tests, dynamic mechanical analysis (DMA), thermogravimetry (TGA) and diferential scanning calorimetry (DSC). The results of tensile tests indicated an increase in tensile strength of the composites after the chemical treatment of the fibers, with best results for the hydrogen peroxide treated fibers, even though the tensile strength of fibers was slightly reduced. This suggests a better interaction fiber/matrix which was also observed by SEM fractographs. The glass transition temperature (Tg) was reduced for all composites compared to the pure polymer which can be attributed to the absorption of solvents, moisture and other low molecular weight molecules by the fibers / O uso de mat?rias-primas de fontes renov?veis para a produ??o de materiais vem sendo objeto de diversos estudos e pesquisas, devido ao seu potencial na substitui??o de derivados petroqu?micos. A adi??o de fibras vegetais a pol?meros representa uma alternativa na substitui??o parcial ou total de fibras de vidro em comp?sitos. Neste trabalho, fibras da folha de carna?ba (Copernicia prun?fera) foram utilizadas na produ??o de comp?sitos biodegrad?veis com matriz de polihidroxibutirato (PHB). Para melhorar as propriedades interfaciais fibra/matriz foram estudados quatro tratamentos qu?micos, para as fibras. O efeito dos diferentes tratamentos qu?micos nas propriedades morfol?gicas, f?sicas, qu?micas e mec?nicas das fibras e dos comp?sitos foram investigados por microscopia eletr?nica de varredura (MEV), espectroscopia no infravermelho, difra??o de raios-X, ensaios mec?nicos de tra??o e flex?o, an?lise din?mico-mec?nica (DMA) e an?lises t?rmicas por termogravimetria (TG) e calorimetria explorat?ria diferencial (DSC). Os resultados dos ensaios de tra??o dos comp?sitos indicaram um acr?scimo de resist?ncia ? tra??o ap?s modifica??o qu?mica das fibras, sendo o melhor resultado obtido com o tratamento de per?xido de hidrog?nio, mesmo havendo uma pequena redu??o na resist?ncia ? tra??o das fibras tratadas. Isto indica uma melhor intera??o entre as fibras e a matriz polim?rica que foi tamb?m observada nas imagens de microscopia eletr?nica de varredura. A temperatura de transi??o v?trea (Tg) foi reduzida para todos os comp?sitos em rela??o ao pol?mero puro o que foi atribu?do a absor??o de solventes, umidade e outras mol?culas de baixo peso molecular pelas fibras

The thesis was focused on the preparation and optimization of the preparation of wound dressing from materials with bioactive ingredients. In this work were prepared nanofiber dressings based on polyhydroxybutyrate and non-fibrous dressings from alginate and chitosan. Nanofibers were prepared by electrospinning and forcespinning methods. The bioactive components, which were used to functionalize the prepared dressings, were plant extracts, clotrimazole, ampicillin, lysozyme, and proteolytic enzymes. The theoretical part is focused on the description of the use of nanofibrous and non-fibrous materials in medicine, characterization of materials for the production of wound dressings and bioactive components. Furthermore, this section describes the methods used to prepare and characterize wound dressings. In the practical part were prepared aqueous and oil extracts from selected plants. Extracts were characterized for polyphenols content and antioxidant activity. PHB-based nanofibers were prepared using electrospinning and forcepinning methods. Nanofibers were enriched with selected plant oil extracts and clotrimazol. Modified nanofibres were detemined for antioxidant activity, short-term and long-term stability. Non-fibrous wound dressings were prepared from alginate and chitosan. These dressings were functionalized by the addition of selected aqueous extracts, ampicillin, lysozyme, papain, bromelain, and collagenase. Non-fibrous wound dressings were determined for antioxidant activity, short-term stability and proteolytic activity. The prepared wound dressings were tested for their antimicrobial effects on cultures of Micrococcus luteus, Serratia marcescens, Staphylococcus epidermidis and Escherichia coli. In conclusion, successfully prepared bioactive wound dressings with antioxidant and antimicrobial agents were tested for safety on human cells. The determination was performed using the MTT cytotoxicity test on human keratinocytes.

This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.

First artificial PHB granules were prepared under the terms of this thesis. The effect of used PHB solvent, ultrasonic bath temperature and time, solvent evaporation temperature and stabilizing agent nature was investigated using dynamic and electrophoretic light scattering. The most proper parameters were demonstrated at samples which were prepared by dissolving of PHB in chloroform, stabilizing with CTAB or lecithin, ultrasonifying at 35 °C followed by chloroform evaporating at 60 °C. Based on ATR-FTIR and Raman spectroscopy results, it was found out that PHB within the artificial granules were in crystalline form. The native PHB granules were isolated from Cupriavidus necator using either lysosyme, deoxyribonuclease and cell disruption by ultrasonification or digestion with alcalase, SDS and EDTA. Granules obtained by both isolation procedures were characterized by ATR-FTIR, Raman spectroscopy, light scattering techniques and DSC. According to the analyses results, the second mentioned procedure turned out to be more effective for obtaining the polymer in amorphous state. Furthermore, the polymer within granules recovered by using this procedure was thermally more stable. Last but not least, the native PHB granules samples were exposed to effect of acetone, lipase and sodium hypochlorite, assuming that polymer crystallinity should be increased by these chemicals in varying degrees. The highest degree of crystallinity was achieved after their treatment with lipase.

The aim of this work was study of the detoxification of lignocellulose material hydrolysates and to investigate sawdust suitability as a substrate for microbial production of PHA by bacteria Burkholderia cepacia and Burkholderia sacchari. In the experimental part of the work the most suitable way of detoxification of model and real hydrolysate was studied. After that, detoxification methods used were evaluated. Criteria for evaluation were concentration of polyphenols as the most important microbial inhibitors and reduction saccharides as the main carbon substrate. Furthermore, fermentability of the hydrolysates was also tested by cultivation of two bacteria capable of PHA accumulation. Burkholderia sacchari demonstrated higher ability to accumulate PHA then Burkholderia cepacia. Then in the summary – most effective way for detoxification was ‚overliming‘. Major increase of PHB in biomass was obtained when Burkholderia sacchari was cultivated on media gained by application of overliming of real lignocellulose hydrolysate. However, total gains of PHB were more likely low and then sawdust can not be considered as a substrate for PHB production at industrial scale.

In drei inhaltlich abgeschlossen Teilen werden Fragestellungen bearbeitet, die sich mit dem Einsatz von Polyhydroxybutyraten als Scaffoldmaterialien für das Tissue Engioneering von Knochen beschäftigen. Zunächst wird ein Prozess optimiert, in dem mittels Verpressen und Auslösen von Platzhaltern (Porogen) poröse Träger (Scaffolds) aus Poly-3-hydroxybuttersäure (P3HB) sowie aus P3co4HB hergestellt werden. Diese Scaffolds werden in der Folge mechanisch und strukturell charakterisiert, wobei Druckfestigkeit, Dauerfestigkeit und Viskoelastizität untersucht werden. Im Ergebnis finden sich mehrere Kandidaten, die für die weitere Testung im Tierversuch in Frage kommen. Weiter wird das Abbauverhalten von schmelzgeponnenen P3HB-Fäden untersucht. Dabei wird ein beschleunigtes Modellsystem gewählt, das noch möglichst nahe am physiologischen Fall aber ohne biologisch aktive Komponente (zB. Enzyme) definiert wurde. Die Charakterisierung bedient sich hier der Gelpermeationschromatographie (GPC), des gasgestützten Elektronenrastermikroskops (ESEM), der differentiellen Thermoanalyse (DSC) und der Rasterkraftmikroskopie. Als Ergebnis zeichnete sich ab, dass neben der hydrolytischen Degradation im Gegensatz zu PHB mit kleinerer spezifischer Oberfläche bei den Fäden auch Erosion zum Abbau beiträgt. Eine partikuläre Freisetzung wird nicht beobachtet. Im dritten Teil werden textile Scaffolds aus P3HB mit einer künstlichen extrazellulären Matrix aus Chondroitinsulfaten (CS) und Kollagen versehen. Dem CS kann hier ein positiver Einfluss auf die osteogene Differenzierung von humanen mesenchymalen Stammzellen (hMSC) nachgewiesen werden. Dies wird zum einen durch die verstärkte Expression der alkalischen Phosphatase (ALP) sowie durch die Hochregulation von Proteinen ersichtlich, die bei der osteogenen Differenzierung essentiell sind. In wenigen Gene-Arrays lässt sich ebenfalls erkennen, dass die osteogene Differenzierung durch CS positiv beeinflusst wird. Insbesondere frühe Marker wie ZBTB16 und IGFBPs werden hier identifiziert. Basierend auf den Teilergebnissen wird am Ende ein Beitrag geliefert, der das Tissue Engineering insbesondere für überkritische Röhrenknochendefekte als Methode interessant erscheinen lässt. Dabei werden mechanische Lasten durch konventionelle Fixateure aufgenommen und der Defektraum durch den mehrfachen Einsatz von bio-funktionalisierten flachen Scaffolds gefüllt.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Materials based on polyhydroxybutyrate (PHB), chitosan (CHI) and fibroin (SF) are biocompatible and attractive for applications in bone tissue engineering. In this work, 3D scaffolds of PHB/CHI and PHB/CHI/SF in different proportions was prepared, characterized and evaluated the in vitro behavior: group I: PHB/CHI (50:50 wt.%), group II: PHB/CHI/SF (50:45:5 wt.%) and group III: PHB/CHI/SF (50:35:15 wt.%). The scaffolds were produced by the lyophilization method of the components mixtures. The physical-chemical characterization of the scaffolds was performed by X-ray diffraction, infrared spectroscopy, scanning electron microscopy and thermogravimetric analysis. A highly porous nature was revealed by SEM analysis of the scaffolds. The FTIR analysis revealed that the constituents used in the preparation of the scaffolds interacted chemically with each other. Thermal analysis showed that fibroin increases the thermal stability of the scaffolds. The viability and cell proliferation were assessed by the MTT method and the cytotoxicity results showed that all scaffolds are non- cytotoxic. In addition, the scaffolds stimulated cell proliferation and are promising for tissue engineering applications. / Materiais à base de polihidroxibutirato (PHB), quitosana (QUI) e fibroína (SF) são biocompatíveis e atrativos para aplicações na engenharia tecidual óssea. Neste trabalho foi preparado, caracterizado e avaliado o comportamento in vitro de arcabouços 3D de PHB/QUI/SF em diferentes proporções: grupo I: PHB/QUI (50:50 % em massa), grupo II-PHB/QUI/SF (50:45:5 % em massa) e grupo III- PHB/QUI/SF (50: 35:15 % em massa). Os arcabouços foram produzidos pelo método da liofilização das misturas dos componentes dos três grupos. A caracterização físico-química dos arcabouços foi realizada por difração de raios X, espectroscopia no infravermelho, microscopia eletrônica de varredura e análise termogravimétrica. A análise de MEV mostrou que os arcabouços 3D apresentam uma boa porosidade. Por FTIR observou- se que os componentes utilizados na preparação dos arcabouços interagiram quimicamente entre si. As análises térmicas indicam que a fibroína aumenta a estabilidade térmica dos arcabouços. A viabilidade e a proliferação celular foram avaliadas pelo método do MTT e os resultados de citotoxicidade mostraram que ambos os arcabouços não são citotóxicos. Além disso, os arcabouços estimularam a proliferação celular, sendo promissores para aplicações em engenharia tecidual.

This diploma thesis is focused on the study of bioactive wound dressings. During the thesis, hydrogel, lyophilized and nanofiber wound dressings were prepared. Hydrogel and lyophilized wound dressings were prepared on basis of two polysaccharides – alginate and chitosan. Nanofiber wound dressings were prepared by spinning polyhydroxybutyrate. All prepared wound dressings were enriched with bioactive substances, which represented analgesics (ibuprofen), antibiotics (ampicillin) and enzymes (collagenase). Into hydrogel and lyophilized wound dressings were all the mentioned active substances incorporated, whereas nanofiber wound dressings were only with ibuprofen and ampicillin prepared. The theoretical part deals with the anatomy and function of human skin. There was explained the process of wound healing and also there were introduced available modern wound dressings. The next chapter of the theoretical part deals with materials for preparing wound dressings (alginate, chitosan, polyhydroxybutyrate) and with active substances, which were used during the experimental part of this thesis. In the theoretical part, the methods of preparation of nanofiber wound dressings and also the methods of cytotoxicity testing used in this work were presented. The first part of the experimental part of this thesis was focused on preparing already mentioned wound dressings. Then, their morphological changes over time and also the gradual release of incorporated active substances into the model environment were monitored. The gradual release of ampicillin was monitored not only spectrophotometrically, but also by ultra-high-performance chromatography. In wound dressings, in which collagenase was incorporated, was also the final proteolytic activity of this enzyme monitored. The effect of the active substances was observed on three selected microorganisms: Escherichia coli, Staphylococcus epidermidis and Candida glabrata. The cytotoxic effect of the active substances on the human keratinocyte cell line was monitored by MTT test and LDH test. A test for monitoring the rate of wound healing – a scratch test – was also performed.