Studi ed approfondimenti sulla composizione in acidi grassi del latte bovino di razza Reggiana con diverso fenotipo per k-caseina e Beta-lactoglobulina

The aim of the present study was to examine the association between milk protein polymorphism and fatty acids profiles of bovine milk. Milk samples were collected from each of 55 Reggiana cows during early, mid and late lactation, respectively, in two farms within the production area of Parmigiano Reggiano cheese. Identification and quantification of fatty acids were performed by gas chromatography. Milk fatty acid composition using cows of differing κ-casein (κ-Cn) and β-lactoglobulin (β-Lg) phenotypes was investigated. Statistically significant results regarding the associations between milk fatty acid composition and κ-Cn phenotype were found, in particular, κ-Cn BB seems to influence de novo fatty acid synthesis in the mammary gland. Also κ-Cn AB seems to have the same effect. Proportions of C10:0 (2,29a AA; 2,53b AB; 2,59b BB), C12:0 (2,77a AA; 3,17b AB; 3,20b BB) and C14:0 (9,22a AA; 10,25b AB; 10,27b BB) were higher in the milk from cows with κ-Cn phenotype AB and BB vs κ-Cn phenotype AA (p<0,05). Conversely C18:0 (7,84b AA; 7,20a,b AB; 6,94a BB) and C18:1 (19,19b AA; 16,81a AB; 16,79a BB) were lower in the milk from cows with κ-Cn phenotype AB and BB vs κ-Cn phenotype AA. The association between milk fatty acid composition and β-Lg phenotype was not statistically significant, except for some fatty acids. In particular, C12:0 (3,05a AA; 3,04a AB; 3,33b BB) was higher in the milk from cows with β-Lg phenotype BB vs β-Lg phenotype AA and AB (p<0,05). Concentrations of C18:0 (6,93a AA; 7,86b AB; 6,59a BB) and C18:1 (16,74a,b AA; 18,24b AB; 16,07a BB) were lower in the milk from cows with β-Lg phenotype AA and BB vs β-Lg phenotype AB (p<0,05). Moreover this research, carried out in farms within the Parmigiano Reggiano cheese district, analysed also the size distribution of fat globules in bulk milk of Reggiana and Frisona breed cows. In particular, the size distribution of individual milk fat globules of Reggiana cows with differing κ-Cn phenotypes was considered. From first observations, no statistically significant differences were observed.

Validazione e applicazioni cliniche della metodica NIRA (Near Infrared Reflectance Analysis) per la valutazione del contenuto nutrizionale del latte materno in terapia intensiva neonatale

Phase 1: To validate Near-Infrared Reflectance Analysis (NIRA) as a fast, reliable and suitable method for routine evaluation of human milk’s nitrogen and fat content. Phase 2: To determine whether fat content, protein content and osmolality of HM before and after fortification may affect gastroesophageal reflux (GER) in symptomatic preterm infants. Patients and Methods: Phase 1: 124 samples of expressed human milk (55 from preterm mothers and 69 from term mothers) were used to validate NIRA against traditional methods (Gerber method for fat and Kjeldhal method for nitrogen). Phase 2: GER was evaluated in 17 symptomatic preterm newborns fed naïve and fortified HM by combined pH/intraluminal-impedance monitoring (pH-MII). HM fat and protein content was analysed by a Near-Infrared-Reflectance-Analysis (NIRA). HM osmolality was tested before and after fortification. GER indexes measured before and after fortification were compared, and were also related with HM fat and protein content and osmolality before and after fortification. Results: Phase 1: · A strong agreement was found between traditional methods’ and NIRA’s results (expressed as g/100 g of milk), both for fat and nitrogen content in term (mean fat content: NIRA=2.76; Gerber=2.76; mean nitrogen content: NIRA=1.88; Kjeldhal =1.92) and preterm (mean fat content: NIRA=3.56; Kjeldhal=3.52; mean nitrogen content: NIRA=1.91; Kjeldhal =1.89) mother’s milk. · Nitrogen content of the milk samples, measured by NIRA, ranged from 1.18 to 2.71 g/100 g of milk in preterm milk and from 1.48 to 2.47 in term milk; fat content ranged from 1.27 to 6.23 g/100 g of milk in preterm milk and from 1.01 to 6.01 g/100 g of milk in term milk. Phase 2: · An inverse correlation was found between naïve HM protein content and acid reflux index (RIpH: p=0.041, rho=-0.501). · After fortification, osmolality often exceeded the values recommended for infant feeds; furthermore, a statistically significant (p<.05) increase in non acid reflux indexes was observed. Conclusions: NIRA can be used as a fast, reliable and suitable tool for routine monitoring of macronutrient content of human milk. Protein content of naïve HM may influence acid GER in preterm infants. A standard fortification of HM may worsen non acid GER indexes and, due to the extreme variability in HM composition, may overcome both recommended protein intake and HM osmolality. Thus, an individualized fortification, based on the analysis of the composition of naïve HM, could optimize both nutrient intake and feeding tolerance.

Genomic characterization of the italian wolf (Canis lupus): the genes involved in black coat colour determination and application of microarray technique for snps detection.

This study provides a comprehensive genetic overview on the endangered Italian wolf population. In particular, it focuses on two research lines. On one hand, we focalised on melanism in wolf in order to isolate a mutation related with black coat colour in canids. With several reported black individuals (an exception at European level), the Italian wolf population constituted a challenging research field posing many unanswered questions. As found in North American wolf, we reported that melanism in the Italian population is caused by a different melanocortin pathway component, the K locus, in which a beta-defensin protein acts as an alternative ligand for the Mc1r. This research project was conducted in collaboration with Prof. Gregory Barsh, Department of Genetics and Paediatrics, Stanford University. On the other hand, we performed analysis on a high number of SNPs thanks to a customized Canine microarray useful to integrate or substitute the STR markers for genotyping individuals and detecting wolf-dog hybrids. Thanks to DNA microchip technology, we obtained an impressive amount of genetic data which provides a solid base for future functional genomic studies. This study was undertaken in collaboration with Prof. Robert K. Wayne, Department of Ecology and Evolutionary Biology, University of California, Los Angeles (UCLA).

A candidate gene approach to identify DNA markers associated with meat quality and production traits: investigation of several cathepsin genes in Italian heavy pigs.

The cathepsin enzymes represent an important family of lysosomal proteinases with a broad spectrum of functions in many, if not in all, tissues and cell types. In addition to their primary role during the normal protein turnover, they possess highly specific proteolytic activities, including antigen processing in the immune response and a direct role in the development of obesity and tumours. In pigs, the involvement of cathepsin enzymes in proteolytic processes have important effects during the conversion of muscle to meat, due to their influence on meat texture and sensory characteristics, mainly in seasoned products. Their contribution is fundamental in flavour development of dry-curing hams. However, several authors have demonstrated that high cathepsin activity, in particular of cathepsin B, is correlated to defects of these products, such as an excessive meat softness together with abnormal free tyrosine content, astringent or metallic aftertastes and formation of a white film on the cut surface. Thus, investigation of their genetic variability could be useful to identify DNA markers associated with these dry cured hams parameters, but also with meat quality, production and carcass traits in Italian heavy pigs. Unfortunately, no association has been found between cathepsin markers and meat quality traits so far, in particular with cathepsin B activity, suggesting that other genes, besides these, affect meat quality parameters. Nevertheless, significant associations were observed with several carcass and production traits in pigs. A recent study has demonstrated that different single nucleotide polymorphisms (SNPs) localized in cathepsin D (CTSD), F (CTSF), H and Z genes were highly associated with growth, fat deposition and production traits in an Italian Large White pig population. The aim of this thesis was to confirm some of these results in other pig populations and identify new cathepsin markers in order to evaluate their effects on cathepsin activity and other production traits. Furthermore, starting from the data obtained in previous studies on CTSD gene, we also analyzed the known polymorphism located in the insulin-like growth factor 2 gene (IGF2 intron3-g.3072G>A). This marker is considered the causative mutation for the quantitative trait loci (QTL) affecting muscle mass and fat deposition in pigs. Since IGF2 maps very close to CTSD on porcine chromosome (SSC) 2, we wanted to clarify if the effects of the CTSD marker were due to linkage disequilibrium with the IGF2 intron3-g.3072G>A mutation or not. In the first chapter, we reported the results from these two SSC2 gene markers. First of all, we evaluated the effects of the IGF2 intron3-g.3072G>A polymorphism in the Italian Large White breed, for which no previous studies have analysed this marker. Highly significant associations were identified with all estimated breeding values for production and carcass traits (P<0.00001), while no effects were observed for meat quality traits. Instead, the IGF2 intron3-g.3072G>A mutation did not show any associations with the analyzed traits in the Italian Duroc pigs, probably due to the low level of variability at this polymorphic site for this breed. In the same Duroc pig population, significant associations were obtained for the CTSD marker for all production and carcass traits (P < 0.001), after excluding possible confounding effects of the IGF2 mutation. The effects of the CTSD g.70G>A polymorphism were also confirmed in a group of Italian Large White pigs homozygous for the IGF2 intron3-g.3072G allele G (IGF2 intron3-g.3072GG) and by haplotype analysis between the markers of the two considered genes. Taken together, all these data indicated that the IGF2 intron3-g.3072G>A mutation is not the only polymorphism affecting fatness and muscle deposition in pigs. In the second chapter, we reported the analysis of two new SNPs identified in cathepsin L (CTSL) and cathepsin S (CTSS) genes and the association results with meat quality parameters (including cathepsin B activity) and several production traits in an Italian Large White pig population. Allele frequencies of these two markers were evaluated in 7 different pig breeds. Furthermore, we mapped using a radiation hybrid panel the CTSS gene on SSC4. Association studies with several production traits, carried out in 268 Italian Large White pigs, indicated positive effects of the CTSL polymorphism on average daily gain, weight of lean cuts and backfat thickness (P<0.05). The results for these latter traits were also confirmed using a selective genotype approach in other Italian Large White pigs (P<0.01). In the 268 pig group, the CTSS polymorphism was associated with feed:gain ratio and average daily gain (P<0.05). Instead, no association was observed between the analysed markers and meat quality parameters. Finally, we wanted to verify if the positive results obtained for the cathepsin L and S markers and for other previous identified SNPs (cathepsin F, cathepsin Z and their inhibitor cystatin B) were confirmed in the Italian Duroc pig breed (third chapter). We analysed them in two groups of Duroc pigs: the first group was made of 218 performance-tested pigs not selected by any phenotypic criteria, the second group was made of 100 Italian Duroc pigs extreme and divergent for visible intermuscular fat trait. In the first group, the CTSL polymorphism was associated with weight of lean cuts (P<0.05), while suggestive associations were obtained for average daily gain and backfat thickness (P<0.10). Allele frequencies of the CTSL gene marker also differed positively among the visible intermuscular extreme tails. Instead, no positive effects were observed for the other DNA markers on the analysed traits. In conclusion, in agreement with the present data and for the biological role of these enzymes, the porcine CTSD and CTSL markers: a) may have a direct effect in the biological mechanisms involved in determining fat and lean meat content in pigs, or b) these markers could be very close to the putative functional mutation(s) present in other genes. These findings have important practical applications, in particular the CTSD and CTSL mutations could be applied in a marker assisted selection (MAS) both in the Italian Large White and Italian Duroc breeds. Marker assisted selection could also increase in efficiency by adding information from the cathepsin S genotype, but only in the Italian Large White breed.

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.

Molecular architecture of fur binding to iron-induced and - repressed genes in Helicobacter pylori

The ferric uptake regulator protein Fur regulates iron-dependent gene expression in bacteria. In the human pathogen Helicobacter pylori, Fur has been shown to regulate iron-induced and iron-repressed genes. Herein we investigate the molecular mechanisms that control this differential iron-responsive Fur regulation. Hydroxyl radical footprinting showed that Fur has different binding architectures, which characterize distinct operator typologies. On operators recognized with higher affinity by holo-Fur, the protein binds to a continuous AT-rich stretch of about 20 bp, displaying an extended protection pattern. This is indicative of protein wrapping around the DNA helix. DNA binding interference assays with the minor groove binding drug distamycin A, point out that the recognition of the holo-operators occurs through the minor groove of the DNA. By contrast, on the apo-operators, Fur binds primarily to thymine dimers within a newly identified TCATTn10TT consensus element, indicative of Fur binding to one side of the DNA, in the major groove of the double helix. Reconstitution of the TCATTn10TT motif within a holo-operator results in a feature binding swap from an holo-Fur- to an apo-Fur-recognized operator, affecting both affinity and binding architecture of Fur, and conferring apo-Fur repression features in vivo. Size exclusion chromatography indicated that Fur is a dimer in solution. However, in the presence of divalent metal ions the protein is able to multimerize. Accordingly, apo-Fur binds DNA as a dimer in gel shift assays, while in presence of iron, higher order complexes are formed. Stoichiometric Ferguson analysis indicates that these complexes correspond to one or two Fur tetramers, each bound to an operator element. Together these data suggest that the apo- and holo-Fur repression mechanisms apparently rely on two distinctive modes of operator-recognition, involving respectively the readout of a specific nucleotide consensus motif in the major groove for apo-operators, and the recognition of AT-rich stretches in the minor groove for holo-operators, whereas the iron-responsive binding affinity is controlled through metal-dependent shaping of the protein structure in order to match preferentially the major or the minor groove.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Candidate genes affecting fat deposition, carcass composition and meat quality traits in pigs

Pig meat quality is determined by several parameters, such as lipid content, tenderness, water-holding capacity, pH, color and flavor, that affect consumers’ acceptance and technological properties of meat. Carcass quality parameters are important for the production of fresh and dry-cure high-quality products, in particular the fat deposition and the lean cut yield. The identification of genes and markers associated with meat and carcass quality traits is of prime interest, for the possibility of improving the traits by marker-assisted selection (MAS) schemes. Therefore, the aim of this thesis was to investigate seven candidate genes for meat and carcass quality traits in pigs. In particular, we focused on genes belonging to the family of the lipid droplet coat proteins perilipins (PLIN1 and PLIN2) and to the calpain/calpastatin system (CAST, CAPN1, CAPN3, CAPNS1) and on the gene encoding for PPARg-coactivator 1A (PPARGC1A). In general, the candidate genes investigation included the protein localization, the detection of polymorphisms, the association analysis with meat and carcass traits and the analysis of the expression level, in order to assess the involvement of the gene in pork quality. Some of the analyzed genes showed effects on various pork traits that are subject to selection in genetic improvement programs, suggesting a possible involvement of the genes in controlling the traits variability. In particular, significant association results have been obtained for PLIN2, CAST and PPARGC1A genes, that are worthwhile of further validation. The obtained results contribute to a better understanding of biological mechanisms important for pig production as well as for a possible use of pig as animal model for studies regarding obesity in humans.

A molecular phylogeny of bivalve mollusks: ancient radiations and divergences as revealed by mitochondrial genes

The main scope of my PhD is the reconstruction of the large-scale bivalve phylogeny on the basis of four mitochondrial genes, with samples taken from all major groups of the class. To my knowledge, it is the first attempt of such a breadth in Bivalvia. I decided to focus on both ribosomal and protein coding DNA sequences (two ribosomal encoding genes -12s and 16s -, and two protein coding ones - cytochrome c oxidase I and cytochrome b), since either bibliography and my preliminary results confirmed the importance of combined gene signals in improving evolutionary pathways of the group. Moreover, I wanted to propose a methodological pipeline that proved to be useful to obtain robust results in bivalves phylogeny. Actually, best-performing taxon sampling and alignment strategies were tested, and several data partitioning and molecular evolution models were analyzed, thus demonstrating the importance of molding and implementing non-trivial evolutionary models. In the line of a more rigorous approach to data analysis, I also proposed a new method to assess taxon sampling, by developing Clarke and Warwick statistics: taxon sampling is a major concern in phylogenetic studies, and incomplete, biased, or improper taxon assemblies can lead to misleading results in reconstructing evolutionary trees. Theoretical methods are already available to optimize taxon choice in phylogenetic analyses, but most involve some knowledge about genetic relationships of the group of interest, or even a well-established phylogeny itself; these data are not always available in general phylogenetic applications. The method I proposed measures the "phylogenetic representativeness" of a given sample or set of samples and it is based entirely on the pre-existing available taxonomy of the ingroup, which is commonly known to investigators. Moreover, it also accounts for instability and discordance in taxonomies. A Python-based script suite, called PhyRe, has been developed to implement all analyses.

Analysis of Copy Number Variants identifies new candidate genes for Autism Spectrum Disorder and Intellectual Disability

Autism spectrum disorder (ASD) and Intellectual Disability (ID) are complex neuropsychiatric disorders characterized by extensive clinical and genetic heterogeneity and with overlapping risk factors. The aim of my project was to further investigate the role of Copy Numbers Variants (CNVs), identified through genome-wide studies performed by the Autism Geome Project (AGP) and the CHERISH consortium in large cohorts of ASD and ID cases, respectively. Specifically, I focused on four rare genic CNVs, selected on the basis of their impact on interesting ASD/ID candidate genes: a) a compound heterozygous deletion involving CTNNA3, predicted to cause the lack of functional protein; b) a 15q13.3 duplication containing CHRNA7; c) a 2q31.1 microdeletion encompassing KLHL23, SSB and METTL5; d) Lastly, I investigated the putative imprinting regulation of the CADPS2 gene, disrupted by a maternal deletion in two siblings with ASD and ID. This study provides further evidence for the role of CTNNA3, CHRNA7, KLHL23 and CADPS2 as ASD and/or ID susceptibility genes, and highlights that rare genetic variation contributes to disease risk in different ways: some rare mutations, such as those impacting CTNNA3, act in a recessive mode of inheritance, while other CNVs, such as those occurring in the 15q13.3 region, are implicated in multiple developmental and/or neurological disorders possibly interacting with other susceptibility variants elsewhere in the genome. On the other hand, the discovery of a tissue-specific monoallelic expression for the CADPS2 gene, implicates the involvement of epigenetic regulatory mechanisms as risk factors conferring susceptibility to ASD/ID.