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FTIR Microspectroscopy in Studies of DNA Damage Induced by Proton Microbeam in Single PC-3 Cells

100%
Lipiec E., Kowalska J., Lekki J., Wiecheć A., Kwiatek W.
Acta Physica Polonica A
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2012
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vol. 121
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issue 2
506-509
EN
In recent years, the Fourier transformed infrared spectroscopy is often applied in studies of biological materials on cellular level. Undoubted advantage of this method is high sensitivity. In presented research the FTIR microspectroscopy was used to analyse the DNA damage in single PC-3 cells (prostate cancer cell line derived from bone metastases) irradiated by counted number of protons. Focused proton microbeam 2 MeV from the Van de Graaff accelerator at the Institute of Nuclear Physics, Polish Academy of Sciences, was used as an irradiation source. Four groups of single cells were irradiated with 1000, 2000, 4000, and 8000 protons per cell, respectively. Following irradiation cells were fixed in 70% ethanol and then analyzed by IR microspectroscopy. Bond analysis of IR spectra served as a base for result analysis. This research has focused on the detection of changes in DNA backbone spectral range (950-1240 cm^{-1}), which could be related to damages such as single and double strand breaks, DNA-DNA, and DNA-protein cross links. Switches and differences in intensity of DNA backbone bands (980-1149 cm^{-1}, 1151-1350 cm^{-1} - symmetric and asymmetric PO^{2-} stretching vibrations, as well as in 1110 cm^{-1} - symmetric stretching of P-O-C band) were observed. Experimental spectra of irradiated and control cells were compared with simulated spectra generated by HyperChem software. The multivariate statistical methods of principal component analysis and hierarchical cluster analysis (Ward's method) were also performed and are discussed.
2
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Comparison of Methods in Studies of Cell Death Mechanisms

86%
Borkowska A., Nowakowski M., Miszczyk J., Lipiec E., Wiltowska-Zuber J., Rawojć K., Kwiatek W.
Acta Physica Polonica A
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2018
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vol. 133
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issue 2
263-266
EN
While studying the influence of ionizing radiation or certain chemical agents on cells, it is crucial to not only determine cytotoxicity, but also to follow cell death mechanisms. There are different methods to screen processes of cell death and still very important question remains unanswered about differences in results that could be caused by various experimental steps in procedures. Based on literature review two protocols of cell death determination were compared. First protocol regarded collecting cells floating in medium before trypsinization and following centrifugation of them. In the second protocol floating cells were discarded and attached ones were stained and fixed. In all experiments three different untreated cell lines (A172, DU145 as cancer cell lines and in comparison, fibroblasts (FB CCL 110), as a non- cancerous cell line) were used to test applied protocols. Cells were cultured and death processes were examined at different time points up to 120 h. Compared protocols showed statistically significant differences, especially in terms of necrosis, which was higher when included floating cells from culture medium and then centrifuging them. Therefore, presented results show importance of choosing a valid experimental procedure in case of evaluating cells viability and types of cell death pathways quantitatively.
3
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Infrared Spectroscopy in Molecular Study of the Piezoelectric Effect in Pig's Shin Bone

86%
Lipiec E., Kowalska J., Wiecheć A., Zieliński P., Kwiatek W., Iwaniec M.
Acta Physica Polonica A
|
2012
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vol. 121
|
issue 2
539-542
EN
Despite growing number of literature demonstrating the piezoelectric effect in bones at macro level there's still a lack of papers describing this effect at molecular level. In order to examine this effect more specifically, a study has been conducted to show the possibility of using infrared spectroscopy on samples contained in the electric field. This is the first known research on applying infrared spectroscopy to study the influence of electric field and compressive stress on bones' structure represented by the molecular IR spectrum. The samples used in this experiment as a model were prepared in pellet form, made from powdered pig's shin bone mixed with KBr (mass ratio of about 1:100). The spectra were obtained in transmission mode. Three different types of experiments were performed at each sample, which were subjected to: (a) fixed electric field (E < 40 kV/m), (b) variable electric field (0-40 kV/m), (c) compressive stress (P < 100 MPa). The obtained data have shown that the electric field and compressive stress on sample modified infrared spectra of the bone. Qualitative as well as quantitative changes in the spectral range between 900 cm^{-1} and 1200 cm^{-1} and the band at 562 cm^{-1} (PO_4^{3-} asymmetric and symmetric stretching, respectively) were observed. The relative area obtained by decomposition of the PO_4^{3-} band reaches an extreme value in the external compressive stress (10 MPa), that occur under physiological conditions. Based on presented data, it was proved that the applied factors (electric field, compressive stress) can change the oscillation energy and the number of molecule's degrees of freedom.
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