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Please use this identifier to cite or link to this item: http://hdl.handle.net/2108/385

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contributor.advisorFederici, Giorgio-
contributor.advisorUrbani, Andrea-
contributor.authorD'Alessandro, Annamaria-
date.accessioned2008-01-03T09:41:45Z-
date.available2008-01-03T09:41:45Z-
date.issued2008-01-03T09:41:45Z-
identifier.urihttp://hdl.handle.net/2108/385-
description20. cicloen
description.abstractIl mantenimento dell’omeostasi e la capacità delle cellule di rispondere all’ambiente esterno dipendono dalla degradazione di proteine regolatorie. I due principali sistemi di degradazione delle proteine nelle cellule eucariotiche sono rappresentati dal sistema proteasoma-ubiquitina (UPS) e dall’autofagia (ALP). Pur essendo l’UPS più efficiente nella degradazione rispetto all’autofagia, in particolari condizioni (inibizione del proteasoma), l’autofagia diviene il sistema più adottato. Molte sostanze, sintetiche e naturali (per es. Epoxomicina) sono state descritte in modelli animali come reminescenze delle patologie neurodegenerative. Tali risultati ci hanno indotto ad effettuare una caratterizzazione molecolare degli effetti provocati dall’Epoxomicina su cellule di neuroblastoma (NB). Obiettivo della ricerca è stato esaminare gli effetti biologici di tale farmaco lesionante su SH-SY5Y, linea cellulare di NB umano, (cambiamenti morfologici, apoptosi, accumulo delle proteine poliubiquitinate ed attivazione dell’autofagia), chiarire attraverso proteomica funzionale l’impatto del farmaco sul proteoma di NB e caratterizzare le informazioni ottenute tramite lo studio dei networks proteici. In particolare, la caratterizzazione fenotipica, strutturale e funzionale dell’Epoxomicina su cellule di NB è stata effettuata usando diversi approcci di proteomica (elettroforesi bidimensionale associata a “Peptide Mass Fingerprinting”, Cromatografia in fase liquida associata alla Spettrometria di Massa, sia in Tandem che Esponenziale). Tutte le proteine differenzialmente espresse identificate (ctrl vs. trattato) sono state studiate e raggruppate nelle rispettive categorie funzionali. Alcune di esse sono state validate tramite western-blotting su differenti linee cellulari umane di NB, oltre che su neuroni primari murini, caratterizzati da differente background fenotipico e genetico. I risultati di proteomica sono stati poi analizzati tramite bioinformatica. Sulla base del “Knowledge based Database approach” abbiamo costruito dei networks funzionali, comprendenti le proteine identificate, e scoperto che molte di esse sono connesse con il beta-estradiolo, noto per il suo ruolo neuroprotettivo. Per confermare tali evidenze abbiamo trattato il nostro modello cellulare di NB con beta-estradiolo prima di esporre le cellule stesse ad Epoxomicina. I risultati ottenuti hanno evidenziato una riduzione dell’apoptosi e la ripresa del ciclo cellulare, associate ad una marcata riduzione delle inclusioni ubiquitinate ed induzione dell’autofagia. Questi dati sembrano quindi suggerire il ruolo protettivo dell’estradiolo nella rimozione degli aggregati proteici. Ulteriori studi saranno effettuati per definire in modelli animali i meccanismi tramite cui le proteine identificate sono coinvolte nella risposta all’Epoxomicina.en
description.abstractMaintenance of cellular homeostasis and ability of cells to respond to their environment depend on orderly degradation of key regulatory proteins. The two main routes of protein clearance in eukaryotic cells are the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathways (ALP). Even if UPS is more efficient than macroautophagy, in particular conditions (i.e. inhibition of proteasome), autophagy becomes the major clearance route. A variety of compounds, both synthetic analogs and natural products (i.e. Epoxomicin), have described in animal models as reminescence of neurodegenerative syndromes. These evidences have suggested us the need for a better characterization of the molecular insight induced by Epoxomicin. Our investigation sought to examine the biological effect of this injuring drug on SH-SY5Y cells, a human neuroblastoma (NB) cell line (cell morphological changes, induction of apoptosis, accumulation of polyubiquitinated proteins and activation of autophagy), to clarify by functional proteomics its impact on NB cells proteome and characterize the obtained informations flow through protein networks. The characterization of phenotypical, structural and functional impact of Epoxomicin on NB cells proteome was carried out by using different functional proteomic approaches (2DE combined to Peptide Mass Fingerprinting, Liquid Chromatography-Tandem Mass Spectrometry and nano-LC/MSE). All the distinct differentially expressed proteins (ctrl vs. treated) were examined for their known biological function and grouped in the respective functional categories. Some of them were also validated by western-blotting on different human NB cell lines and also on primary murine neurons, characterized by different genetic and phenotipical background. A more comprehensive analysis of the proteomic results was performed by a bioinformatic approach. Applying a Knowledge based Database approach we have drawn functional networks including the identified proteins and found that several of them are directed towards beta-estradiol, known for its neuroprotective properties. To confirm the central role played by estradiol we have treated our NB cell model with beta-estradiol, before the exposure to Epoxomicin. Results showed apoptosis reduction and cell cycle resumption associated to strong reduction of the ubiquitinated inclusions and autophagy induction. These data seem to suggest a protective role played directly by beta-estradiol in protein aggregates removing. Further investigation will be necessary to define the in vivo mechanism by which the identified proteins can be involved in responce to Epoxomicin.en
description.tableofcontentsIntroduction - 1 Protein degradation 1.1-Lysosomes‘ degradation - 1.2 Autophagy (Proteins degradation and Cell Death) - 1.3 Autophagy in Cancer and Neurodegenerative disorders - 1.4 Proteolysis by proteasomes - 1.5 Tables and Figures - 2 Ubiquitin Proteasome System - 2.1 Background - 2.2 Description - 2.3 Structure and organization - 2.4 Assemblation and degradation process - 2.5 Biological implication of Proteasome degradation - 2.6 The role of Ubiquitin in Protein Degradation - 2.7 Ubiquitin family - 2.8 Figures - 3 Diseases linked to UPS impairment - 3.1 Inherited disorders - 3.2 Acquired disorders - 3.3 Neurodegenerative diseases - 3.4 Protein-degradation pathways in neurodegeneration - 3.4 Figures - 4 Proteasome Inhibitors (PIs) - 4.1 Synthetic Inhibitors of Proteasome - 4.2 Natural Product Inhibitors of Proteasome - 4.3 Proteasome Inhibitors as Therapeutic Agents - 4.4 Figures - 5 Models of Proteasome dysfunction - 5.1 Pharmacological models of Proteasome dysfunction - 5.2 Genetic models of Proteasome dysfunction - 5.3 Figures and Tables - 6 Proteomics analysis of biological model - 6.1 Tools of Proteomics - 6.2 Mass Spectrometry tecniques and instruments - 6.3 Applications of Proteomics - 6.4 Determinants of the Proteome - 6.5 Figures - 7 Bioinformatic analysis - 7.1 Classical bioinformatics: prediction of fingerprinting patterns - 7.2 Computational genomics and functional association - 7.3 Gene Ontologies - 7.4 Beyond the classical view: New methods and Possibilities - 7.5 Bioinformatics methods for the prediction of protein interactions - 7.6 Future direction - 7.7 Figures and Tables . - Objectives of the research - Results - 1 Biological effect of Epoxomicin on NB cell model - 2 Evaluation of Epoxomicin impact on NB proteome - 2.1 Differential expression and protein identification - 2.2 Expression Analysis - 3 Immunoblotting validation - 4 Protein networks involved in response to Epoxomicin - 5 Protective role of beta-estradiol on Epoxomicin cell model - 6 Figures and Tables. - Discussion - 1 Biological effects of Epoxomicin - 2 Proteins involved in response to Epoxomicin treatment - 3 Protein networks: β-estradiol in neurodegenerative disease - 4 Figures . - Concluding remarks - Materials and Methods - 1 Reagents - 2 Cell Lines - 3 Neuronal Cultures - 4 Viability Assays - 5 Flow cytometric analysis - 5.1 Analysis of cells volume and morphological complexity - 5.2 Fluorescence-activated cell sorting of SH-SY5Y cells - 5.3 Assays of apoptosis and necrosis induction - 5.4 Propidium Iodide-Based Cell Cycle Analysis - 6 Immunoblot Analysis - 7 Two-dimensional gel electrophoresis - 8 Image analysis - 9 In gel trypsin digestion - 10 Protein identification by Peptide Mass Fingerprinting (PMF) - 11 Protein identification by LC-MS/MS - 12 Protein identification and quantitation by LC-MSE - 13 Bioinformatic Analysis of Gene Ontologies - 14 Figures - Bibliography - Curriculum vitae et studiorum - Publications - Copy of publicationsen
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language.isoenen
subjectEpoxomicinen
subjectNeuroblastoma (NB)en
subjectneurodegenerative diseaseen
subjectautophagyen
subjectproteomicsen
subjectfunctional network analysisen
subjectbeta-estradiolen
titleCharacterization of protein degradation arrest inducted by Epoxomicin in a neuroblastoma cell line modelen
typeThesisen
degree.nameDottorato in biochimica e biologia molecolareen
degree.levelDottoratoen
degree.disciplineFacoltà di medicina e chirurgiaen
degree.grantorUniversità degli studi di Roma Tor Vergataen
date.dateofdefenseA.A. 2007/2008en
Appears in Collections:Tesi di dottorato in medicina

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