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Identifier 000421611
Title Study of lymphoma cell lines before and after Nutlin-3a induced wild type (wt) p53-activation by Raman spectroscopy
Alternative Title Μελέτη λεμφωματικών κυτταρικών σειρών με φασματοσκοπία Raman πριν και μετά την επαγόμενη από την Νατλίνη-3α ενεργοποίηση του αγρίου τύπου πρωτεΐνης p53
Author Κατσαρά, Κλυταιμνήστρα Μ.
Thesis advisor Γανωτάκης, Δημήτριος
Reviewer Αίβαλιώτης, Μιχάλης
Τσιώτης, Γεώργιος
Abstract The p53 tumor suppressor gene is commonly mutated in many human cancers. However, the majority of hematological malignancies express wild type (wt) p53, which is somehow inactivated. MDM2 (HDM2 in humans) binds to wt TP53, negatively modulating its transcriptional activity and stability. Nutlin-3a (N3a) inhibits the p53-MDM2 interaction, resulting in stabilization and non-genotoxic reactivation of the wt TP53 signaling pathway in lymphoma cells, followed by cell cycle arrest, and in most of the cases apoptosis. Raman spectroscopy allows the detection of chemical bonds vibration, the recognition of the four building blocks of biomolecules: amino acids, lipids, nucleic acids, carbohydrates and finally the analysis of the molecular profile of specimens without the need for labels or selective probes, and most importantly in a non-invasive manner. Our research had two main objectives: a) The analysis and comparison of three different lymphoma Hodgkin and non-Hodgkin model cell lines, before and after N3a-induced (wt) TP53-activation, through Raman spectroscopy. b) The detection and observation of N3a into lymphoma cells, in order to follow its course and determine its localization and site of action within the cell. Moreover, this analysis revealed possible difficulties regarding the entry of N3a in the cell. After testing and optimization of different experimental procedures, mainly concerning sample preparation and analysis, we developed an experimental procedure yielding satisfactory results. Briefly, cell suspension from different lymphoma cell types, at the appropriate cell growth stage, before and after N3a treatment, was placed directly on CaF2 microscope slide, for Raman imaging in different temperatures and focal conditions. Cell observations were made with an Olympus 60x water immersion lens in direct contact with the sample. With Raman spectroscopy we managed to detect distinct “signature” signals, which can differentiate the two cell models used, by receiving signals from three intracellular areas (nucleus, membrane, cytoplasm) and one extracellular, at different temperatures. However, the comparison of the two cell models was based on Raman signals from the nucleus area, because the corresponding spectrums presented higher repeatability, compared to those received from the cytoplasm and the membrane. Using PCA we managed to confirm the differentiation of the two cell models. Furthermore, with Raman spectroscopy and analysis we managed to determine the signature peaks of N3a that do not correspond to biological compounds. Those were used for the observation and detection of N3a intracellularly and extracellularly. Based on our results, in N3a-effected (possibly apoptotic) cells, N3a is present inside and outside of the cell, in contrast to live cells where N3a is not detected inside, but only outside of the cell. In conclusion, our study supports Raman spectroscopy as a potential promising, fast and non-invasive strategy, for the early, accurate diagnosis, categorization and assessment of putative drug efficacy against lymphoma and other human diseases.
Language English
Subject Cancer
Hodgkin lymphoma
Hodgkin λέμφωμα
Imaging
MDM2
PCA
Signal
TP53
Απεικόνιση
Καρκίνος
Σήμα
Issue date 2019-03-27
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Chemistry--Post-graduate theses
  Type of Work--Post-graduate theses
Permanent Link https://elocus.lib.uoc.gr//dlib/e/c/2/metadata-dlib-1552495781-537366-23749.tkl Bookmark and Share
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