V. Oliveira

Insights into the Role of Extracellular Polysaccharides in Burkholderia Adaptation to Different Environments

The genus Burkholderia comprises more than 60 species able to adapt to a wide range of environments such as soil and water, and also colonize and infect plants and animals. They have large genomes with multiple replicons and high gene number, allowing these bacteria to thrive in very different niches. Among the properties of bacteria from the genus Burkholderia is the ability to produce several types of exopolysaccharides (EPSs). The most common one, cepacian, is produced by the majority of the strains examined irrespective of whether or not they belong to the Burkholderia cepacia complex (Bcc). Cepacian biosynthesis proceeds by a Wzy-dependent mechanism, and some of the B. cepacia exopolysaccharide (Bce) proteins have been functionally characterized. In vitro studies showed that cepacian protects bacterial cells challenged with external stresses. Regarding virulence, bacterial cells with the ability to produce EPS are more virulent in several animal models of infection than their isogenic non-producing mutants. Although the production of EPS within the lungs of cystic fibrosis (CF) patients has not been demonstrated, the in vitro assessment of the mucoid phenotype in serial Bcc isolates from CF patients colonized for several years showed that mucoid to non-mucoid transitions are relatively frequent. This morphotype variation can be induced under laboratory conditions by exposing cells to stress such as high antibiotic concentration. Clonal isolates where mucoid to non-mucoid transition had occurred showed that during lung infection, genomic rearrangements, and mutations had taken place. Other phenotypic changes include variations in motility, chemotaxis, biofilm formation, bacterial survival rate under nutrient starvation and virulence. In this review, we summarize major findings related to EPS biosynthesis by Burkholderia and the implications in broader regulatory mechanisms important for cell adaptation to the different niches colonized by these bacteria.

Excimer laser ablation of Al2O3-TiC ceramics : laser induced modifications of surface topography and structure

Abstract Samples of an Al2O3–34 wt% TiC ceramic were processed with KrF excimer laser radiation (248 nm), using a fluence in the range 2 to 8 J/cm2. The roughness and the ablation rate vary considerably with increasing number of pulses, reaching a constant value at >200 pulses. In stationary regime, they both increase when fluence increases. Laser processing results on the formation of a globular topography recast layer by a selective ablation process, alumina being rapidly laser sputtered by an electronic mechanism, while titanium carbide is thermally ablated.

LASER MICROMACHINING OF AL2O3-TIC CERAMICS

Al 2 O 3 −34 wt.% TiC ceramics have been machined with a KrF (248 nm) excimer laser under normal atmosphere. In the initial steps of the irradiation process both the roughness and the removal rate present a strong variation with the number of pulses. After approximately 200 pulses the process reaches a stationary regime where the roughness and the removal rate become constant. Characterization of the machined areas by scanning electron microscopy showed that the variations in roughness and removal rate are related to the evolution of the surface topography of the samples. Also, as a consequence of laser irradiation, TiC and Al 2 O 3 are partially transformed into TiO 2 , TiC 0.7 N 0.3 , and an Al–Ti solid solution.

Comparative Transcriptomic Analysis of the Burkholderia cepacia Tyrosine Kinase bceF Mutant Reveals a Role in Tolerance to Stress, Biofilm Formation, and Virulence

ABSTRACT The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.

Femtosecond laser ablation of dentin

The surface morphology, structure and composition of human dentin treated with a femtosecond infrared laser (pulse duration 500?fs, wavelength 1030?nm, fluences ranging from 1 to 3?J?cm?2) was studied by scanning electron microscopy, x-ray diffraction, x-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The average dentin ablation threshold under these conditions was 0.6???0.2?J?cm?2 and the ablation rate achieved in the range 1 to 2??m/pulse for an average fluence of 3?J?cm?2. The ablation surfaces present an irregular and rugged appearance, with no significant traces of melting, deformation, cracking or carbonization. The smear layer was entirely removed by the laser treatment. For fluences only slightly higher than the ablation threshold the morphology of the laser-treated surfaces was very similar to the dentin fracture surfaces and the dentinal tubules remained open. For higher fluences, the surface was more porous and the dentin structure was partially concealed by ablation debris and a few resolidified droplets. Independently on the laser processing parameters and laser processing method used no sub-superficial cracking was observed. The dentin constitution and chemical composition was not significantly modified by the laser treatment in the processing parameter range used. In particular, the organic matter is not preferentially removed from the surface and no traces of high temperature phosphates, such as the ?-tricalcium phosphate, were observed. The achieved results are compatible with an electrostatic ablation mechanism. In conclusion, the high beam quality and short pulse duration of the ultrafast laser used should allow the accurate preparation of cavities, with negligible damage of the underlying material.

Evidences for direct magnetic patterning via diffusive transformations using femtosecond laser interferometry

The application of femtosecond laser interferometry to direct patterning of thin-film magnetic alloys is demonstrated. The formation of stripe gratings with submicron periodicities is achieved in Fe1−xVx (x = 18–34 wt. %) layers, with a difference in magnetic moments up to Δμ/μ ∼ 20 between adjacent stripes but without any significant development of the topographical relief (<1% of the film thickness). The produced gratings exhibit a robust effect of their anisotropy shape on magnetization curves in the film plane. The obtained data witness ultrafast diffusive transformations associated with the process of spinodal decomposition and demonstrate an opportunity for producing magnetic nanostructures with engineered properties upon this basis.

XeCl laser ablation of Al2O3–TiC ceramics

Al2O3–TiC ceramic composites are promising materials for micromechanical components. However, due to their hardness, brittleness and contraction during sintering, they are difficult to shape into high-accuracy parts using conventional forming techniques. Laser micromachining can be an alternative technique to shape this type of materials. Previous work using KrF (λ=248 nm) excimer lasers showed that micromachining was accompanied by the formation of a thin melted layer of resolidified material and, in certain conditions, a cone-like surface topography. This was explained by the different ablation behaviour of Al2O3 and TiC. To verify this assumption, Al2O3–TiC, Al2O3 and TiC ceramics were processed with a XeCl (λ=308 nm) excimer laser and their ablation behaviour was investigated. It was found that TiC has a higher ablation rate than Al2O3. The experimental results suggest that chemical reactions occur during processing of Al2O3–TiC in air and that cone formation can be due to the shielding of laser radiation by the resulting compounds.

A mathematical description of surface texture development in laser-ablated dentin

A cone-shaped texture is observed when a transverse cross section of human dentin cut perpendicularly to the tooth axis is processed with KrF excimer laser radiation using appropriate processing parameters. In the present paper, a mathematical model describing the kinetics of cone growth as a function of the number of laser pulses is presented. It is shown that the fractional area occupied by cones is adequately described by a modified Johnson-Mehl-Avrami-Kolmogorov equation and that the boundary lines between cones define a Voronoi tessellation generated from the corresponding initial tubule location. This model allows the surface texture evolution in dentin to be predicted from the initial tubule distribution.

Ultrafast laser texturing of Ti-6Al-4V surfaces for biomedical applications

By controlling processing parameters such as the average fluence, number of laser pulses and beam polarization direction, different types of multiscale surface textures were produced on Ti-6Al-4V surfaces by ultrafast laser processing. The samples were textured in ambient atmosphere using an Yb: KYW chirped-pulse-regenerative amplification laser with a wavelength of 1030 nm and pulse duration of 500 fs. The wetting of simulated biological fluids as well as the human mesenchymal stem cells (hMSCs) behavior were assessed. Three types of textured surfaces were tested, consisting of: (i) Laser-Induced Periodic Surface Structures-LIPSS; (ii) nanopillars-like structures; and (iii) LIPSS overlapped to microcolumns. The laser textured surfaces present hydrophilic behavior and high affinity for HBSS (Hank’s balanced salt solution). Cell spreading and adhesion strength is reduced by the laser nanotextures as compared to a polished control surface. Cytoskeleton stretching and stress fibers were clearly observed on LIPSS while significant filopodia formation was verified on nanopillars. There was no cell proliferation on the laser nanotextured surfaces. Ultrafast laser texturing of Ti-6Al-4V surfaces is an efficient technique for increasing surface wettability, and is potentially useful as a technique to control the behavior of hMSCs by changing the cytoskeleton shape, FAPs distribution and area, and proliferation.By controlling processing parameters such as the average fluence, number of laser pulses and beam polarization direction, different types of multiscale surface textures were produced on Ti-6Al-4V surfaces by ultrafast laser processing. The samples were textured in ambient atmosphere using an Yb: KYW chirped-pulse-regenerative amplification laser with a wavelength of 1030 nm and pulse duration of 500 fs. The wetting of simulated biological fluids as well as the human mesenchymal stem cells (hMSCs) behavior were assessed. Three types of textured surfaces were tested, consisting of: (i) Laser-Induced Periodic Surface Structures-LIPSS; (ii) nanopillars-like structures; and (iii) LIPSS overlapped to microcolumns. The laser textured surfaces present hydrophilic behavior and high affinity for HBSS (Hank’s balanced salt solution). Cell spreading and adhesion strength is reduced by the laser nanotextures as compared to a polished control surface. Cytoskeleton stretching and stress fibers were clearly observed on L...

Influence of tubule orientation on cone-shaped texture development in laser-ablated dentin

In the present paper, the influence of tubule orientation on surface texture development was studied. Specimens of dentin with a wide range of tubule orientations were extracted from caries-free human teeth, processed using KrF laser radiation, and analyzed by scanning electron microscopy. When a transverse cross section of dentin cut perpendicularly to the tooth axis is processed with KrF laser radiation, a cone-like topography develops in the inner dentin where tubules are parallel to the laser beam. When laser processing is carried out in the outer dentin, because tubules are significantly tilted with respect to the laser beam, flat surfaces are achieved. The surface texture after laser processing depends effectively on the angle between the tubules and the laser beam. The dependency of cone growth on tubule orientation was confirmed using a simple differential ablation model.