http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-06-19T15:18:16Z http://hdl.handle.net/2067/1848 Title: Comparative proteomics and transcriptomics analyses of livers from two different Bos taurus breeds: “Chianina and Holstein Friesian” Authors: Timperio, Anna Maria; D'Alessandro, Angelo; Pariset, Lorraine; D'Amici, Gian Maria; Valentini, Alessio; Zolla, Lello Abstract: The Holstein Friesian and Chianina cattle breeds are representative of extreme selection for milk and meat traits, respectively, with significant changes in metabolism resulting from human selection over the past centuries. In the present study, we wanted to assess whether selection for different purposes has had a measurable effect on liver metabolism through a comparison of the protein and gene expression profiles of the two breeds. We applied 2-DE in order to identify proteins which were differentially expressed in the livers of the two breeds and relate them to different liver functions. We expected to find that only a small number of proteins would be differentially expressed, due to the relatively short phylogenetic distance between these cattle breeds. Nonetheless, thirty nine differentially-expressed proteins were characterized between Chianina and Holstein Friesian, out of a total of 560 ± 57 spots that matched. Microarray analyses evidenced the differential expression of 167 genes (148 for the Holstein Friesian and 19 for the Chianina). Despite being closely related at the genetic level, the disparity of the proteomic and transcriptomic profiles of these two breeds allows us to perform pathway analysis thus to pinpoint proteins whose expression might render the latter capable of greater milk production, or proteins involved in altered thermoregulatory ability or hormone production. On the other hand, we found proteins and gene transcripts in Chianina, not expressed in Holstein, which, upon interaction pathway analysis, were mainly involved in anabolic pathways. In brief, our integrated study provides molecular evidences to support the physiological differences between Holstein and Chianina cattle breeds. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1843 Title: Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation Authors: D'Amici, Gian Maria; Timperio, Anna Maria; Gevi, Federica; Grazzini, Giuliano; Zolla, Lello Abstract: Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advates, Helixate NexGens and Refactos), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGens showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refactos showed glutathione S-transferase and b-lactamase, whereas Advates apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants. Description: L'artcoo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1887 Title: Comparative proteomics and transcriptomics analyses of livers from two different Bos taurus breeds: “Chianina and Holstein Friesian” Authors: Timperio, Anna Maria; D'Alessandro, Angelo; Pariset, Lorraine; D'Amici, Gian Maria; Valentini, Alessio; Zolla, Lello Abstract: The Holstein Friesian and Chianina cattle breeds are representative of extreme selection for milk and meat traits, respectively, with significant changes in metabolism resulting from human selection over the past centuries. In the present study, we wanted to assess whethe selection for different purposes has had a measurable effect on liver metabolism through a comparison of the protein and gene expression profiles of the two breeds. We applied 2-DE in order to identify proteins which were differentially expressed in the livers of the two breeds and relate them to different liver functions. We expected to find that only a small number of proteins would be differentially expressed, due to the relatively short phylogenetic distance between these cattle breeds. Nonetheless, thirty nine differentially expressed proteins were characterized between Chianina and Holstein Friesian, out of a total of 560 ± 57 spots that matched. Microarray analyses evidenced the differential expression of 167 genes (148 for the Holstein Friesian and 19 for the Chianina). Despite being closely related at the genetic level, the disparity of the proteomic and transcriptomic profiles of these two breeds allows us to perform pathway analysis thus to pinpoint proteins whose expression might render the latter capable of greater milk production, or proteins involved in altered thermoregulatory ability or hormone production. On the other hand, we found proteins and gene transcripts in Chianina, not expressed in Holstein, which, upon interaction pathway analysis, were mainly involved in anabolic pathways. In brief, our integrated study provides molecular evidences to support the physiological differences between Holstein and Chianina cattle breeds. 2011-04-05T22:00:00Z http://hdl.handle.net/2067/1970 Title: Metodi elettroforetici bidimensionali applicati allo studio di diversi sistemi membranali Authors: D'Amici, Gian Maria Abstract: Il progredire delle metodiche scientifiche e delle tecnologie nelle ultime decadi, ha catalizzato un’estensione degli scopi connessi agli studi in campo biologico spostando l’attenzione dalle riduttive analisi biochimiche a carico delle singole proteine ad un esame attento ed approfondito esteso all’intero proteoma. La proteomica nasce come naturale evoluzione della chimica delle proteine che rappresentava uno dei capisaldi nello studio della biologia durante gli anni ottanta. L’analisi proteomica implica la necessità di separare le proteine prima della loro caratterizzazione. Così tutta la qualità dell’intera analisi molto dipende dalle performance dei metodi di separazione adottati. Questa lavoro di tesi nasce dal condensamento di diverse pubblicazioni e vuole proporsi come un saggio delle diverse metodiche di elettroforesi bidimensionale applicabili allo studio di proteine di membrana. Utilizzando differenti tecniche elettroforetiche bidimensionali e di rivelazione sono state affrontate due tematiche di ricerca diverse su differenti sistemi biologici: nel capitolo 2 con una proteomica differenziale di tipo “classico” (2D IEF-SDS-PAGE) è stata studiata le degradazione delle proteine della membrana eritrocitaria durante la conservazione del sangue ad uso trasfusionale, nel capitolo 3 invece, prendendo come modello le proteine delle membrane fotosintetiche tilacoidali, è stato proposto un nuovo sistema elettroforetico bidimensionale nativo, sia in prima che in seconda dimensione (2D N-LP-IEF-SDS-PAGE), accoppiabile con una terza dimensione denaturante.; Developments in methods and technologies have resulted in an expansion of the purpose of biological studies from the reductionist biochemical analysis of single proteins to proteome-wide measurements. Proteomics and other complementary analysis methods are essential components of the emerging 'systems biology'. Proteome analysis implies the ability to separate proteins as a first step prior the characterization. Thus, the overall performance of the analysis strongly depends on the performance of the separation tool, usually two dimensional electrophoresis. This thesis gives an introduction to the proteomic technique and shows how twodimensional electrophoresis works with membrane proteins. Different subjects and biological systems were investigated using different separative two-dimensional electrophoresis techniques. In chapter one, two-dimensional gel electrophoresis (2D IEF-SDS-PAGE) and mass spectrometry were used to identify protein profile changes in red blood cell membranes stored over time under atmospheric oxygen, in the presence or absence of protease inhibitors. In chapter two, a new 3D native electrophoretic protocol is proposed for an exhaustive separation and identification of membrane proteins. This method is based on native liquid phase isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second dimension, where both the pI and the molecular masses of protein complexes (2D N-LP-IEF-BN) were used to separate them in their native form. Description: Dottorato di ricerca in Genetica e biologia cellulare 2008-04-03T22:00:00Z