Application of chromatographic and spectroscopic techniques in the evaluation of the lipid fraction of animal products

Lipolysis and oxidation of lipids in foods are the major biochemical and chemical processes that cause food quality deterioration, leading to the characteristic, unpalatable odour and flavour called rancidity. In addition to unpalatability, rancidity may give rise to toxic levels of certain compounds like aldehydes, hydroperoxides, epoxides and cholesterol oxidation products. In this PhD study chromatographic and spectroscopic techniques were employed to determine the degree of rancidity in different animal products and its relationship with technological parameters like feeding fat sources, packaging, processing and storage conditions. To achieve this goal capillary gas chromatography (CGC) was employed not only to determine the fatty acids profile but also, after solid phase extraction, the amount of free fatty acids (FFA), diglycerides (DG), sterols (cholesterol and phytosterols) and cholesterol oxidation products (COPs). To determine hydroperoxides, primary products of oxidation and quantify secondary products UV/VIS absorbance spectroscopy was applied. Most of the foods analysed in this study were meat products. In actual fact, lipid oxidation is a major deterioration reaction in meat and meat products and results in adverse changes in the colour, flavour and texture of meat. The development of rancidity has long recognized as a serious problem during meat handling, storage and processing. On a dairy product, a vegetal cream, a study of lipid fraction and development of rancidity during storage was carried out to evaluate its shelf-life and some nutritional features life saturated/unsaturated fatty acids ratio and phytosterols content. Then, according to the interest that has been growing around functional food in the last years, a new electrophoretic method was optimized and compared with HPLC to check the quality of a beehive product like royal jelly. This manuscript reports the main results obtained in the five activities briefly summarized as follows: 1) comparison between HPLC and a new electrophoretic method in the evaluation of authenticity of royal jelly; 2) study of the lipid fraction of a vegetal cream under different storage conditions; 3) study of lipid oxidation in minced beef during storage under a modified atmosphere packaging, before and after cooking; 4) evaluation of the influence of dietary fat and processing on the lipid fraction of chicken patties; 5) study of the lipid fraction of typical Italian and Spanish pork dry sausages and cured hams.

Genomic and transcriptional analysis of allergen genes in apple (Malus x domestica)

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Heterosis in maize (Zea mays, L.): characterization of heterotic quantitative trait loci (QTL) for agronomic traits in near isogenic lines (NILs) and their testcrosses

In a previous study on maize (Zea mays, L.) several quantitative trait loci (QTL) showing high dominance-additive ratio for agronomic traits were identified in a population of recombinant inbred lines derived from B73 × H99. For four of these mapped QTL, namely 3.05, 4.10, 7.03 and 10.03 according to their chromosome and bin position, families of near-isogenic lines (NILs) were developed, i.e., couples of homozygous lines nearly identical except for the QTL region that is homozygote either for the allele provided by B73 or by H99. For two of these QTL (3.05 and 4.10) the NILs families were produced in two different genetic backgrounds. The present research was conducted in order to: (i) characterize these QTL by estimating additive and dominance effects; (ii) investigate if these effects can be affected by genetic background, inbreeding level and environmental growing conditions (low vs. high plant density). The six NILs’ families were tested across three years and in three Experiments at different inbreeding levels as NILs per se and their reciprocal crosses (Experiment 1), NILs crossed to related inbreds B73 and H99 (Experiment 2) and NILs crossed to four unrelated inbreds (Experiment 3). Experiment 2 was conducted at two plant densities (4.5 and 9.0 plants m-2). Results of Experiments 1 and 2 confirmed previous findings as to QTL effects, with dominance-additive ratio superior to 1 for several traits, especially for grain yield per plant and its component traits; as a tendency, dominance effects were more pronounced in Experiment 1. The QTL effects were also confirmed in Experiment 3. The interactions involving QTL effects, families and plant density were generally negligible, suggesting a certain stability of the QTL. Results emphasize the importance of dominance effects for these QTL, suggesting that they might deserve further studies, using NILs’ families and their crosses as base materials.

A moment symbolic representation of Lévy processes with applications

By using a symbolic method, known in the literature as the classical umbral calculus, a symbolic representation of Lévy processes is given and a new family of time-space harmonic polynomials with respect to such processes, which includes and generalizes the exponential complete Bell polynomials, is introduced. The usefulness of time-space harmonic polynomials with respect to Lévy processes is that it is a martingale the stochastic process obtained by replacing the indeterminate x of the polynomials with a Lévy process, whereas the Lévy process does not necessarily have this property. Therefore to find such polynomials could be particularly meaningful for applications. This new family includes Hermite polynomials, time-space harmonic with respect to Brownian motion, Poisson-Charlier polynomials with respect to Poisson processes, Laguerre and actuarial polynomials with respect to Gamma processes , Meixner polynomials of the first kind with respect to Pascal processes, Euler, Bernoulli, Krawtchuk, and pseudo-Narumi polynomials with respect to suitable random walks. The role played by cumulants is stressed and brought to the light, either in the symbolic representation of Lévy processes and their infinite divisibility property, either in the generalization, via umbral Kailath-Segall formula, of the well-known formulae giving elementary symmetric polynomials in terms of power sum symmetric polynomials. The expression of the family of time-space harmonic polynomials here introduced has some connections with the so-called moment representation of various families of multivariate polynomials. Such moment representation has been studied here for the first time in connection with the time-space harmonic property with respect to suitable symbolic multivariate Lévy processes. In particular, multivariate Hermite polynomials and their properties have been studied in connection with a symbolic version of the multivariate Brownian motion, while multivariate Bernoulli and Euler polynomials are represented as powers of multivariate polynomials which are time-space harmonic with respect to suitable multivariate Lévy processes.