Benedetti, Rosaria
(2011)
UV Laser crosslink induction: an innovative tool for eigenetic studies.
[Tesi di dottorato]
(Unpublished)
| Item Type: |
Tesi di dottorato
|
| Lingua: |
English |
| Title: |
UV Laser crosslink induction: an innovative tool for eigenetic studies |
| Creators: |
| Creators | Email |
|---|
| Benedetti, Rosaria | rosaria.benedetti@gmail.com |
|
| Date: |
28 November 2011 |
| Number of Pages: |
100 |
| Institution: |
Università degli Studi di Napoli Federico II |
| Department: |
Chimica organica e biochimica |
| Scuola di dottorato: |
Biotecnologie |
| Dottorato: |
Scienze biotecnologiche |
| Ciclo di dottorato: |
24 |
| Coordinatore del Corso di dottorato: |
| nome | email |
|---|
| Sannia, Giovanni | UNSPECIFIED |
|
| Tutor: |
| nome | email |
|---|
| Marino, Gennaro | UNSPECIFIED | | Velotta, Raffaele | UNSPECIFIED |
|
| Date: |
28 November 2011 |
| Number of Pages: |
100 |
| Uncontrolled Keywords: |
Crosslinking,lasers |
| Settori scientifico-disciplinari del MIUR: |
Area 03 - Scienze chimiche > CHIM/11 - Chimica e biotecnologia delle fermentazioni |
[error in script]
[error in script]
| Date Deposited: |
06 Dec 2011 11:10 |
| Last Modified: |
15 Jul 2015 01:00 |
| URI: |
http://www.fedoa.unina.it/id/eprint/8596 |
Abstract
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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