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.

Design and Performance Evaluation of Network-on-Chip Communication Protocols and Architectures

The scale down of transistor technology allows microelectronics manufacturers such as Intel and IBM to build always more sophisticated systems on a single microchip. The classical interconnection solutions based on shared buses or direct connections between the modules of the chip are becoming obsolete as they struggle to sustain the increasing tight bandwidth and latency constraints that these systems demand. The most promising solution for the future chip interconnects are the Networks on Chip (NoC). NoCs are network composed by routers and channels used to inter- connect the different components installed on the single microchip. Examples of advanced processors based on NoC interconnects are the IBM Cell processor, composed by eight CPUs that is installed on the Sony Playstation III and the Intel Teraflops pro ject composed by 80 independent (simple) microprocessors. On chip integration is becoming popular not only in the Chip Multi Processor (CMP) research area but also in the wider and more heterogeneous world of Systems on Chip (SoC). SoC comprehend all the electronic devices that surround us such as cell-phones, smart-phones, house embedded systems, automotive systems, set-top boxes etc... SoC manufacturers such as ST Microelectronics , Samsung, Philips and also Universities such as Bologna University, M.I.T., Berkeley and more are all proposing proprietary frameworks based on NoC interconnects. These frameworks help engineers in the switch of design methodology and speed up the development of new NoC-based systems on chip. In this Thesis we propose an introduction of CMP and SoC interconnection networks. Then focusing on SoC systems we propose: • a detailed analysis based on simulation of the Spidergon NoC, a ST Microelectronics solution for SoC interconnects. The Spidergon NoC differs from many classical solutions inherited from the parallel computing world. Here we propose a detailed analysis of this NoC topology and routing algorithms. Furthermore we propose aEqualized a new routing algorithm designed to optimize the use of the resources of the network while also increasing its performance; • a methodology flow based on modified publicly available tools that combined can be used to design, model and analyze any kind of System on Chip; • a detailed analysis of a ST Microelectronics-proprietary transport-level protocol that the author of this Thesis helped developing; • a simulation-based comprehensive comparison of different network interface designs proposed by the author and the researchers at AST lab, in order to integrate shared-memory and message-passing based components on a single System on Chip; • a powerful and flexible solution to address the time closure exception issue in the design of synchronous Networks on Chip. Our solution is based on relay stations repeaters and allows to reduce the power and area demands of NoC interconnects while also reducing its buffer needs; • a solution to simplify the design of the NoC by also increasing their performance and reducing their power and area consumption. We propose to replace complex and slow virtual channel-based routers with multiple and flexible small Multi Plane ones. This solution allows us to reduce the area and power dissipation of any NoC while also increasing its performance especially when the resources are reduced. This Thesis has been written in collaboration with the Advanced System Technology laboratory in Grenoble France, and the Computer Science Department at Columbia University in the city of New York.

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).

Studio della trasduzione del Segnale Nucleare Inositide-Dipendente: identificazione di eEF1A2 come nuovo Fosfosubstrato di PKC βI

Introduction Phospholipase Cb1 (PLC-β1) is a key player in the regulation of nuclear inositol lipid signaling and of a wide range of cellular functions, such as proliferation and differentiation (1,2,3). PLCb1 signaling depends on the cleavage of phosphatidylinositol 4,5-bisphosphate and the formation of the second messengers diacylglycerol and Inositol tris-phosphate which activate canonical protein kinase C (cPKC) isoforms. Here we describe a proteomic approach to find out a potential effector of nuclear PLC-b1 dependent signaling during insulin stimulated myogenic differentiation. Methods Nuclear lysates obtained from insulin induced C2C12 myoblasts were immunoprecipitated with anti-phospho-substrate cPKC antibody. Proteins, stained with Comassie blue, were excised, digested and subsequently analysed in LC-MS/MS. For peptide sequence searching, the mass spectra were processed and analyzed using the Mascot MS/MS ion search program with the NCBI database. Western blotting, GST-pull down and co-immunoprecipitation were performed to study the interaction between eEF1A2 and cPKCs. Site direct mutagenesis was performed to confirm the phosphorylated motif recognized by the antibody. Immunofluorescence analysis, GFP-tagged eEF1A2 vector and subcellular fractionation were performed to study nuclear localization and relative distribution of eEF1A2. Results We have previously shown that PLC-β1 is greatly increased at the nuclear level during insulin-induced myoblasts differentiation and that this nuclear localization is essential for induction of differentiation. Thus, nuclear proteins of insulin stimulated C2C12 myoblasts, were immunoprecipitated with an anti-phospho-substrate cPKC antibody. After Electrophoretic gel separation of proteins immunoprecipitated, several molecules were identified by LC-MS/MS. Among these most relevant and unexpected was eukaryotic elongation factor 1 alpha 2 (eEF1A2). We found that eEF1A2 is phosphorylated by PKCb1 and that these two molecules coimmunolocalized at the nucleolar level. eEF1A2 could be phosphorylated in many sites among which both threonine and serine residues. By site direct mutagenesis we demonstrated that it is the serine residue of the motif recognized by the antibody that is specifically phosphorylated by PKCb1. The silencing of PLCb1 gives rise to a reduction of expression and phosphorylation levels of eEF1A2 indicating this molecule as a target of nuclear PLCb1 regulatory network during myoblasts differentiation.

Agenti di encefalopatie spongiformi trasmissibili e zoonosi: tipizzazione molecolare

Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases caused by the conversion of the host-encoded cellular protein (PrPC) to a disease-associated isoform (PrPSc). The agent responsible for prion diseases may exist as different strains with specific biological and biochemical properties. According to the protein-only hypothesis, prion strain diversity is enciphered in PrPSc conformation. Molecular strain typing methods are based on the electrophoretic mobility of protease resistant core of PrPSc, on the susceptibility to protease digestion, on the glycosylation profile of PrPres and on the conformational stability of PrPSc. In this study a new conformational stability assay was developed based on the differential solubility of PrPC and PrPSc: CSSA (conformational stability and solubility assay). The conformational stability assay was performed by measuring PrPSc solubility in homogenates treated with increasing concentrations of GdnHCl, in the absence of proteinase K. Indeed, dose-response curves allowed estimation of the concentration of GdnHCl able to solubilise 50% of PrPSc. The results showed that this method is valuable for the biochemical typing of strains in bank voles and it is also a promising tool for molecular analysis of natural prion isolates. CSSA also revealed strain-specific PrPSc conformational stabilities of ovine natural isolates so that this feature, combined with the N-terminal PrPSc cleavage, allowed differentiation of classical scrapie, including CH1641-like, from natural goat BSE and experimental sheep BSE. In view of the implications concerning strain similarity between animal and human TSEs, the physico-chemical properties of the Nor98 with two human prion diseases (VPSPr and GSS) were compared in order to investigate the extent of the similarity between animal and human prion strains. The results showed an unexpected heterogeneity of the molecular features among human and sheep TSEs associated with internal PrPres fragments with the possible exception of Nor98 and a case of GSS P102L. These similarities and differences need further investigation by N- and C-terminal sequencing and biological characterization.

Immunoconiugati contenenti tossine vegetali o siRNA per la deplezione selettiva di cellule leucemiche. Preparazione, citotossicità e studi di mutagenesi sito-specifica.

CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.

Sindrome da Asfissia Perinatale nel puledro neonato: protocolli diagnostico-terapeutici

La Sindrome da Asfissia Perinatale (PAS) è una delle più comuni patologie che colpiscono il puledro neonato nelle prime 72 h di vita. È una patologia difficile da diagnosticare in quanto non esistono parametri o segni clinici specifici, la sintomatologia è molto variabile in base alla durata e all’intensità dell’insulto ipossico ischemico e al tipo di organo maggiormente colpito. Lo scopo di questo studio è la ricerca e la valutazione di alcuni parametri biochimico-clinici e di alcuni biomarkers per la diagnosi precoce e il corretto trattamento dei puledri affetti da PAS. Nei puledri neonati che presentano questa patologia è stata riscontrata un’ipermagnesiemia al momento del ricovero associata a prognosi infausta, probabilmente causata da un grave danno cellulare con rilascio in circolo del magnesio intracellulare. La PAS potrebbe essere un’ulteriore causa di Euthyroid Sick Syndrome, in quanto abbiamo riscontrato una diminuzione delle concentrazioni di T3 e T4 nei puledri malati rispetto ai sani della stessa età, come avviene in altre malattie sistemiche. Lo studio del profilo proteomico ha permesso di separare le più importanti frazioni proteiche del liquido amniotico di cavalla, mettendo in evidenza similitudini e differenze qualitative e quantitative nei ferogrammi dei puledri sani e di quelli affetti da PAS ed una maggiore variabilità è stata riscontrata nei profili dei liquidi amniotici dei puledri malati. Il glutatione è risultato poco espresso nel puledro neonato, i puledri sani presentano concentrazioni più basse sia rispetto ai malati della stessa età sia agli adulti ma con una tendenza all’aumento nelle prime 24 ore di vita per i sani ed un calo nei malati. La somministrazione della terapia antiradicalica non influisce sulle concentrazioni di glutatione totale ed i puledri deceduti presentano concentrazioni più alte.

Urine proteome in animals of veterinary interest: species comparison and new biomarkers of nephropathy

Urine is considered an ideal source of biomarkers, however in veterinary medicine a complete study on the urine proteome is still lacking. The present work aimed to apply proteomic techniques to the separation of the urine proteome in dogs, cats, horses, cows and some non-conventional species. High resolution electrophoresis (HRE) was also validated for the quantification of albuminuria in dogs and cats. In healthy cats, applying SDS-PAGE and 2DE coupled to mass spectrometry (MS), was produced a reference map of the urine proteome. Moreover, 13 differentially represented urine proteins were linked with CKD, suggesting uromodulin, cauxin, CFAD, Apo-H, RBP and CYSM as candidate biomarkers to be investigated further. In dogs, applying SDS-PAGE coupled to MS, was highlighted a specific pattern in healthy animals showing important differences in patients affected by leishmaniasis. In particular, uromodulin could be a putative biomarker of tubular damage while arginine esterase and low MW proteins needs to be investigated further. In cows, applying SDS-PAGE, were highlighted different patterns between heifers and cows showing some interesting changes during pregnancy. In particular, putative alpha-fetoprotein and b-PAP needs to be further investigated. In horses, applying SDS-PAGE, was produced a reference profile characterized by 13±4 protein bands and the most represented one was the putative uromodulin. Proteinuric horses showed the decrease of the putative uromodulin band and the appearance of 2 to 4 protein bands at higher MW and a greater variability in the range of MW between 49 and 17 kDa. In felids and giraffes was quantified proteinuria reporting the first data for UTP and UPC. Moreover, by means of SDS-PAGE, were highlighted species-specific electrophoretic patterns in big felids and giraffes.