LC-MS based methods for phytosterols determination in oenological matrices. Targeted metabolomic study of Rioja grape varieties

221 p.+ anexos

Development of new applications of inductively coupled plasma mass spectrometry (ICP-MS) hyphenated with different sample introduction systems

225 p. : il. Texto en español con conclusiones en inglés

Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

Background -- N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model. Methods -- CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling. Results -- No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells. Conclusions -- In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

Determination of drugs used in combined cardiovascular therapy

Cardiovascular diseases are nowadays the first cause of mortality worldwide, causing around the 30% of global deaths each year. The risk of suffering from cardiovascular illnesses is strongly related to some factors such as hypertension, high cholesterol levels, diabetes, obesity The combination of these different risk factors is known as metabolic syndrome and it is considered a pandemic due to the high prevalence worldwide. The pathology of the disorders implies a combined cardiovascular therapy with drugs which have different targets and mechanisms of action, to regulate each factor separately. The simultaneous analysis of these drugs turns interesting but it is a complex task since the determination of multiple substances with different physicochemical properties and physiological behavior is always a challenge for the analytical chemist. The complexity of the biological matrices and the difference in the expected concentrations of some analytes require the development of extremely sensitive and selective determination methods. The aim of this work is to fill the gap existing in this field of the drug analysis, developing analytical methods capable of quantifying the different drugs prescribed in combined cardiovascular therapy simultaneously. Liquid chromatography andem mass spectrometry (LCMS/MS) has been the technique of choice throughout the main part of this work, due to the high sensitivity and selectivity requirements.

Mass spectrometry coupled to imaging techniques: The better the view the greater the challenge

These are definitively exciting times for membrane lipid researchers. Once considered just as the cell membrane building blocks, the important role these lipids play is steadily being acknowledged. The improvement occurred in mass spectrometry techniques (MS) allows the establishment of the precise lipid composition of biological extracts. However, to fully understand the biological function of each individual lipid species, we need to know its spatial distribution and dynamics. In the past 10 years, the field has experienced a profound revolution thanks to the development of MS-based techniques allowing lipid imaging (MSI). Images reveal and verify what many lipid researchers had already shown by different means, but none as convincing as an image: each cell type presents a specific lipid composition, which is highly sensitive to its physiological and pathological state. While these techniques will help to place membrane lipids in the position they deserve, they also open the black box containing all the unknown regulatory mechanisms accounting for such tailored lipid composition. Thus, these results urges to different disciplines to redefine their paradigm of study by including the complexity revealed by the MSI techniques.

Development of procedures for the triazole fungicides determination in fruits and liquid samples using microextraction techniques and chromatographic separation

Agricultural crops can be damaged by funguses, insects, worms and other organisms that cause diseases and decrease the yield of production. The effect of these damaging agents can be reduced using pesticides. Among them, triazole compounds are effective substances against fungus; for example, Oidium. Nevertheless, it has been detected that the residues of these fungicides in foods as well as in derivate products can affect the health of the consumers. Therefore, the European Union has established several regulations fixing the maximum residue of pesticide levels in a wide range of foods trying to assure the consumer safety. Hence, it is very important to develop adequate methods to determine these pesticide compounds. In most cases, gas or liquid chromatographic (GC, LC) separations are used in the analysis of the samples. But firstly, it is necessary to use proper sample treatments in order to preconcentrate and isolate the target analytes. To reach this aim, microextraction techniques are very effective tools; because allow to do both preconcentration and extraction of the analytes in one simple step that considerably reduces the source of errors. With these objectives, two remarkable techniques have been widely used during the last years: solid phase microextraction (SPME) and liquid phase microextraction (LPME) with its different options. Both techniques that avoid the use or reduce the amount of toxic solvents are convenient coupled to chromatographic equipments providing good quantitative results in a wide number of matrices and compounds. In this work simple and reliable methods have been developed using SPME and ultrasound assisted emulsification microextraction (USAEME) coupled to GC or LC for triazole fungicides determination. The proposed methods allow confidently determine triazole concentrations of μg L‐1 order in different fruit samples. Chemometric tools have been used to accomplish successful determinations. Firstly, in the selection and optimization of the variables involved in the microextraction processes; and secondly, to overcome the problems related to the overlapping peaks. Different fractional factorial designs have been used for the screening of the experimental variables; and central composite designs have been carried out to get the best experimental conditions. Trying to solve the overlapping peak problems multivariate calibration methods have been used. Parallel Factor Analysis 2 (PARAFAC2), Multivariate Curve Resolution (MCR) and Parallel Factor Analysis with Linear Dependencies (PARALIND) have been proposed, the adequate algorithms have been used according to data characteristics, and the results have been compared. Because its occurrence in Basque Country and its relevance in the production of cider and txakoli regional wines the grape and apple samples were selected. These crops are often treated with triazole compounds trying to solve the problems caused by the funguses. The peel and pulp from grape and apple, their juices and some commercial products such as musts, juice and cider have been analysed showing the adequacy of the developed methods for the triazole determination in this kind of fruit samples.

La cerámica de la Segunda Edad del Hierro en el País Vasco: Estudio tecnológico, funcional y social

670 p. Capítulos de introducción, metodología, discusión y conclusiones en castellano e inglés.

Systematic Studies on 3d- and 4f-Metal Containing Polyoxometalates Suitable for Organic Derivatization

232 p.