Benedetti, Rosaria (2011) UV Laser crosslink induction: an innovative tool for eigenetic studies. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Date Deposited:||06 Dec 2011 11:10|
|Last Modified:||30 Apr 2014 19:47|
SUMMARY: Establishing a stable covalent bond between proteins and nucleic acids, usually referred to as crosslinking is a powerful tool of molecular biology, which affords access to study otherwise transient interactions between bio-molecules. Crosslinking with fs- UV lasers has been presented in the literature as a revolutionary technique to increase the otherwise low process yield of conventional methods based on chemical catalysts, conventional UV sources, or longer UV pulses. It is known that crosslinking induced in cells by ultrashort laser pulses has a twofold advantage over conventional methods. (i) It binds only species that are in proximity (“zero length” covalent bond) of the absorbed photons rather than favoring unspecific bonds amongst many possible species in the cell. (ii) It should only operate until the radiation is incident on the sample, thus paving the way for time-resolved studies of transient interactions. Moreover, when combined with biochemical techniques such as Chromatin Immunoprecipitation (ChIP) to analyze the produced bonds, the UV laser method will make feasible the characterization of the dynamics of the transcription factors bindings on chromatin in living cells, thus overcoming the technical limitations of the current technologies. The aim of this research project was to find the best operating condition for the induction of crosslink between DNA and proteins in living human cells mediated by an UV laser source, by the exploration of ultra short laser characteristics. Particular attention was focalized on the role of laser repetition rate and on the total energy parameter. To screen in a easy and rapid way the possible combinations of both parameters a cellular model was construct, in which the crosslink yield is analyzed as readout of fluorescence signals. On the bases of so obtained informations, ChIP experiments were carried out against a specific epigenetic mark (Ch3K4H3) on different genetic promoter regions, coding for apoptosis involved protein (TRAIL), structural protein (H2B) and cellular trafficking involved one (SCAMP5). The response of human cells to UV laser radiation was also kept in consideration, looking at cellular mortality, cell phases distribution and DNA damages induction, in order to clarify the principles underpinning the crosslink induction processes. It was found that in some laser operating conditions, although the crosslink induction was still present, the DNA damages (and the consequent cell death) were too high to allow the ChIP experiment itself. Instead the conditions in which the cell viability was maximized, and so the apoptotic and pro-death cellular pathways minimized, fitted with effectively crosslinked experimental points (in terms of protein stability and DNA amplificability). The set of data displayed and discussed in the thesis, suggests that as each in vivo technique, for the establishment of UV Laser mediated crosslink many physical and biological parameters need to be consider. First of all, the UV Laser light characteristics assume key role in the setting of ChIP experiments by the definition of the better balance between total energy and repetition rate. Also some structural features are important: among them the apparatus’s geometry used for the irradiation, the material of the cuvette, the size of Laser beam, the speed of stirring. The most important thing to consider, however, is still the cellular behavior in response to the UV incident radiation and the way by which the crosslink could be formed. To be sure that the creation of stable bonds between DNA and proteins is obtained, the cells must be hit with sufficient energy and the energy reaching the biological model does not disrupt the proteins or DNA itself.
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