Dipartimento di Chimica e Tecnologie Chimiche - Tesi di Dottorato

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Questa collezione raccoglie le Tesi di Dottorato afferenti al Dipartimento di Chimica e Tecnologie Chimiche dell'Università della Calabria.

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Author: search.filters.author.Gabriele, BartoloSubject: search.filters.subject.Spettrometria di massa

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Now showing 1 - 6 of 6
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    Advanced mass spectrometry-based strategies for the isolation and characterization of G protein-coupled estrogen receptor 1(GPR)
    (2014-11-28) Thangavel, Hariprasad; Gabriele, Bartolo; Sindona, Giovanni; Napoli, Anna
    Estrogen signaling plays a vital role in breast, ovarian and endometrial cancers. The actions of estrogen are mainly mediated by classical estrogen receptors, ERα and ERβ that belongs to the nuclear receptor superfamily. In recent years, a class of membrane-associated estrogen receptors are found to mimic the functions of classical ERs, including genomic as well as non-genomic signaling. These non-genomic signaling events include pathways that are usually thought of as arising from transmembrane growth factor receptors and G protein-coupled receptors (GPCRs). GPCRs belong to a superfamily of cell surface signaling proteins. GPCRs represent the most significant family of validated pharmacological targets in medical biology. A member of the GPCR family, named GPER, mediates rapid biological responses to estrogen in diverse normal and cancer cells, as well as transformed cell types. The identification and characterization of GPER will lead to understand the mechanisms underlying complex biological pathways and identify potentially new drug targets. Here, we proposed a novel gel-free method to isolate and enrich GPER from crude lysate using home-made hydroxyapatite column (HTP). The HTP eluate was subjected to cellulose acetate (CA) filteration, followed by on-membrane protein digestion with different proteases and analyzed by MALDI MS. GPER was identified by peptide mass fingerprinting (PMF) after intensive data analysis. Sequence analysis reports 3 potential N-glycosylation in GPER. We manually validated 2 out of 3 glycosylation sites in GPER from the obtained MS/MS data and also validated the glycan moieties predicted by Glycomod. This approach is the first of its kind to identify GPER and characterize post-translational modifications (PTMs) by MS-based proteomic analysis. The proposed method is simple, robust and unique with great reproducibility. Finally, we designed and synthesized polymer nanoparticles (NPs) in an effort to capture GPER with high affinity and selectivity from crude lysate. PNIPAm-based NPs were synthesized by a free radical precipitation polymerization method with no control over the functional monomer sequence. The NP binding affinity was evaluated against both truncated-GPER (short peptide epitopes) and GPER (whole protein). As the NPs were designed with complementary functionality against the peptides/protein, the NPspeptide/ protein binding will be through multipoint interactions. The initial qualitative results obtained by immunoblotting analysis revealed interesting hints on GPER’s competitive affinity towards NPs when probed against multiple antibodies. We anticipate to use this strategy as a sample purification step prior to MS-based proteomic analysis
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    Traceability of foodstuffs by high tech methodologies of mass spectrometry
    (2011-10-26) Naccarato, Attilio; Bartolino, Roberto; Gabriele, Bartolo; Sindona, Giovanni
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    Modern mass spectrometric applications in the structure and function evaluation of active principles
    (2011-10-26) Malaj, Naim; Gabriele, Bartolo; Sindona, Giovanni; Bartolino, Roberto
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    Synthesis and application of new solid phase techniques in quantitative proteomics using MALDI and ESI mass spectrometry
    (2008-10-07) Moschidis, Petros; Giovanni Sindona, Giovanni; Gabriele, Bartolo
    Proteomics, the analysis of the protein complement of a cell or an organism has grown rapidly as a category of the life sciences. Mass spectrometry (MS) is one of the central detection techniques in proteome analysis, yet it has to rely on prior sample preparation steps that reduce the enormous complexity of the protein mixtures obtained from biological systems. For that reason, a number of tagging (or labeling) strategies have been developed that target specific amino acid residues or post-translational modifications, enabling the enrichment of subfractions via affinity clean up, resulting in the identification of an ever-increasing number of proteins. In addition, the attachment of stable isotope-labeled tags now allow the relative quantitation of protein levels of two samples, e.g. those representing different cell states, which is of great significance for drug discovery and molecular biology. This work presents the research for new stable isotope-labeled (as well as stable-isotope free) solid phase strategies for the identification and relative quantitation of complex protein mixtures using MALDI and ESI-MS, MS/MS technology.
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    Food control quality by isotope dilution liquid chromatography-tandem mass spectrometry
    (2014-06-13) Benabdelkamel, Hicham; Tagarelli, Antonio; Gabriele, Bartolo
    The late part of the 20th century the advancement of knowledge regarding nutrition and disease prevention provided an opportunity for individuals to affect their own health. This expanding body of information helped people to understand how the environment and their own behaviour affected their body. People now had powerful tools to help maintaining and protecting their health. The understanding of how our diet affects our well being has dramatically changed the lifestyles and attitudes of people, who began to make menu and purchasing decisions based on how foods would affect their heath. A shift toward healthier lifestyles and healthier diets began. Food processors and marketers had to refocus their efforts from promoting foods for pleasure to promoting foods that fit in to a healthy diet. Primarily, the focus was on reducing fat and cholesterol in the diet and supplementing vitamins and minerals. Research began to demonstrate the presence of various phytochemicals in wine and juice make it from fruits and vegetables( such as stilbenes, falvoniods, polyphenols…) and specially in olive oil as ( polyphenol in dialdehyed form: oleochanthal, hydroxyoleocanthal..) These compounds have come to be known as nutraceuticals. The list of nutraceuticals present in wine, fruit drink and olive oil that are believed to have positive biological properties has been expanding. Food processors and developers have become very interested in exploiting these nutraceuticals for the production of foods that are not only part of a healthy diet but also improve the consumer’s health in another specific way. These foods have become known as “functional foods” means quality marker. The aim of this thesis has been to develop a analytical methods to determine the concentration of a group of these nutraceuticals in food , such as, quantitative determination of resveratrol in wine, pterostilbene in blueberry juice and dialdehyde form in olive oil using a sensitive high-performance liquid chromatographic separation method coupling with tandem-mass and isotope dilution to order to optimal the conditions for the analysis method, such as extraction procedure, matrices, column, quality controls, wavelength, mobile phases, run time, optimal separation (gradient, retention times), temperature, capillary voltage, cone voltages, vacuum and labelled internal standards, resulting in the best sensitivity and selectivity,The goodness and satisfactory of the method was performed according to, containing linear measuring range, quantification, lower limit of quantification (LLOQ), lower limit of detection (LLOD), quality controls, precision(RSD %), accuracy, recovery, stability and matrix effects. In conclusion, the described high-performance liquid chromatographic separation method with tandem-mass spectrometry detection and isotope dilution showed a satisfactory overall analytical performance well suited for applications in food quality control.