Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Grafting of adipic anhydride to carbon nanotubes through a Diels-Alder cycloaddition/oxidation cascade reaction

Accepted Manuscript

Microencapsulation of ellagic acid from pomegranate husk and karaya gum by spray drying

Objective: The aim of this study was to obtain and characterize microcapsules with Ellagic Acid (EA) from pomegranate as core material and Karaya Gum (KG) as wall material. Methods: EA was obtained from dry pomegranate peel powder via methanolysis and quantified by HPLC. Microcapsules were obtained preparing a dispersion containing KG and EA in phosphate buffer pH 8. The dispersion was processed in a spray dryer under specific conditions (inlet temperature at 150 °C, feed flow at 30% and aspirator at 100 %) for obtaining of microcapsules. Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) were used for characterization. Results: Obtained material contains 98.03±2.82 mg EA/g of pomegranate peel. FTIR showed that there were changes in the molecular structure of microcapsules referred to raw materials. SEM confirmed that particles obtained had micron-size (1-5 µm). DSC analysis showed that raw materials had glass transition temperatures of 79.58 and 83.41 °C and for microcapsules the value was67.25 °C. Conclusion: Methanolysis is a viable technique for the obtaining of EA from the peel of pomegranate. KG shows good potential for be used as wall material for EA microencapsulation.

Poly lactic acid based biodegradable stents and functionalization techniques: brief review

Commercial stents, especially metallic ones, present several disadvantages, and this gives rise to the necessity of producing or coating stents with different materials, like natural polymers, in order to improve their biocompatibility and minimize the disadvantages of metallic ones. This review paper discusses some applications of natural-based polymers in stents, namely polylactic acid (PLA) for stent development and chitosan for biocompatible coatings of stents . Furthermore, some effective stent functionalization techniques will be discussed, namely Layer by Layer (LBL) technique.

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r=0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.