di Pasquale, Pamela (2014) DNA repair and protection mechanisms in Escherichia coli and Mycobacterium tuberculosis. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: DNA repair and protection mechanisms in Escherichia coli and Mycobacterium tuberculosis
Creators:
CreatorsEmail
di Pasquale, Pamelapamela.dipasquale@unina.it
Date: 31 March 2014
Number of Pages: 114
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Scuola di dottorato: Scienze chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 26
Coordinatore del Corso di dottorato:
nomeemail
Paduano, Luigiluigi.paduano@unina.it
Tutor:
nomeemail
Pucci, PieroUNSPECIFIED
Duilio, AngelaUNSPECIFIED
Date: 31 March 2014
Number of Pages: 114
Uncontrolled Keywords: DNA repair, DNA methylation, tuberculosis
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Area 03 - Scienze chimiche > CHIM/01 - Chimica analitica
Date Deposited: 10 Apr 2014 08:11
Last Modified: 26 Jan 2015 11:35
URI: http://www.fedoa.unina.it/id/eprint/9924

Abstract

Alkylating molecules are exogenous or endogenous chemicals commonly capable to modify nucleic acids in all living organisms causing dangerous and toxic effects. Several repair systems were developed by all organisms to avoid DNA alkylating modifications. In Escherichia coli AidB protein is involved in the adaptive response to alkylation stress in a still obscure fashion [1]. The same mechanism is also present in Mycobacterium tuberculosis (Mtb), the causing agent of tuberculosis. Human immune response usually targets bacterial systems which are essential for cells survival like DNA alkylating response [2]. All the Information concerning these mechanisms are deeply interesting objects in order to understand molecular mechanism of MTB infection and to lead the identification of new putative therapeutic drugs. In this biological contest, the study of DNA alkylation response processes in E.coli and MTB was the main interest in this PhD thesis. The first part was principally addressed to obtain a comprehensive description of E.coli response to alkylation stress while the second part was entirely focused on the Mtb response to methylating molecules. Finally in the third part a new tandem MS approach was set up to detect and quantify methylating DNA extracted from complex matrices. A complete elucidation of DNA response to alkylation damages in E.coli, a model organism for all bacteria was performed. Proteomic approaches were designed in order to identify and functionally analyze all the proteins differentially expressed in the presence and in the absence of methylating molecules. Subsequently the project focused on the exploration of MTB response to alkylation stress. Different clinical tubercular and not tubercular strains were treated with alkylating agents in order to analyze sensitivity and morphological alterations. The results obtained showed the occurrence of a DNA response mechanism in MTB similar to that found in E.coli. In silico investigations revealed the presence of FadE8, a putative DNA protection protein homologous to E.coli AidB. The corresponding gene was cloned in several plasmidic vectors and expressed in E.coli. The recombinant FadE8 protein was structurally and functionally characterized. A Multiple Reaction Monitoring (MRM) tandem MS procedure was used to qualitative and quantitative analyze DNA modifications. Through this approach E.coli and MTB systems were eventually compared and the functional roles of homologue proteins involved were highlighted.

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