Non-invasive strategy in assessing asthma through biofluids metabolomics exploration: exhaled breath and urine potentialities

Asthma is a significant health issue in the pediatric population with a noteworthy growth over the years. The proposed challenge for this PhD thesis was the development of advanced methodologies to establish metabolomic patterns in urine and exhaled breath associated with asthma whose applicability was subsequently exploited to evaluate the disease state, the therapy adhesion and effect and for diagnostic purposes. The volatile composition of exhaled breath was studied combining headspace solid phase microextraction (HS-SPME) with gas chromatography coupled to mass spectrometry or with comprehensive two-dimensional gas chromatography coupled to mass spectrometry with a high resolution time of flight analyzer (GC×GC–ToFMS). These methodologies allowed the identification of several hundred compounds from different chemical families. Multivariate analysis (MVA) led to the conclusion that the metabolomic profile of asthma individuals is characterized by higher levels of compounds associated with lipid peroxidation, possibly linked to oxidative stress and inflammation (alkanes and aldehydes) known to play an important role in asthma. For future applications in clinical settings a set of nine compounds was defined and the clinical applicability was proven in monitoring the disease status and in the evaluation of the effect and / or adherence to therapy. The global volatile metabolome of urine was also explored using an HSSPME/GC×GC–ToFMS method and c.a. 200 compounds were identified. A targeted analysis was performed, with 78 compounds related with lipid peroxidation and consequently to oxidative stress levels and inflammation. The urinary non-volatile metabolomic pattern of asthma was established using proton nuclear magnetic resonance (1H NMR). This analysis allowed identifying central metabolic pathways such as oxidative stress, amino acid and lipid metabolism, gut microflora alterations, alterations in the tricarboxylic acid (TCA) cycle, histidine metabolism, lactic acidosis, and modification of free tyrosine residues after eosinophil stimulation. The obtained results allowed exploring and demonstrating the potential of analyzing the metabolomic profile of exhaled air and urine in asthma. Besides the successful development of analysis methodologies, it was possible to explore through exhaled air and urine biochemical pathways affected by asthma, observing complementarity between matrices, as well as, verify the clinical applicability.

HPLC-DAD methodology for the quantification of organic acids, furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo-diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250 4.6 mm, 5mm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan-3-ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans-resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n55) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rose ´ and fortified wines.

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the layer-by-layer technique

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.