## 3 Results and Discussion
### Analysis of Relative Absorbance Ratios (t\({}_{0}\) and t\({}_{24}\) Cells)
In Figure 6A-E, the ratio values LS, PP1, PP2, P/L1, and P/L2 are reported, respectively, for the different doses and times of fixation. In particular, the LS ratio gives an indication about lipid saturation; PP1 and PP2 ratios are related to protein phosphorylation, while P/L1 and P/L2 ratios report on the relative protein and lipid content (see Table 1). The ratio between the absorbance bands of CH\({}_{3}\) and CH\({}_{2}\) asymmetric stretching (LS - Figure 6A) evidences a significantly increasing trend with dose, for both t\({}_{0}\) (for all doses, \(p\leq 0.05\)) and t\({}_{24}\) cells (for doses higher than 4 Gy, \(p\leq 0.05\)) indicating an increasing lipid saturation effect [20; 50]. These modifications can suggest a cell apoptosis process that has been observed to occur together with several membrane changes, such as phosphatidylserine exposure, membrane blebbing, and vesicle formation [46]. Moreover, some researchers noticed an increase in saturated fatty acids containing phospholipids during apoptosis in neuronal cells [45]. Phospholipids are the most important structural components of membranes delimiting many intracellular organelles (e.g., lysosomes, endoplasmic reticulum, and nuclei) and a salient change in their composition could cause deformation and porosity of such membranes. As a consequence, during apoptosis, a characteristic cleavage and laddering of chromosomal DNA occur caused by a deoxyribonuclease flow entering into the nucleus. [PERSON] et al. [51] also suggested that the occurrence of a significant increase in saturated fatty acids only in the apoptotic cells can produce an increase by 10%-20% in saturated fatty acids in a mixed population of apoptotic and non-apoptotic cells. This indication of a possible occurrence of apoptosis is also in agreement with the results of a Raman micro-spectroscopy investigation already reported ([52] and references therein). Figure 5: Comparison of the absorbance ratios PR1 (**A**), PR2 (**B**), and PR3 (**C**) with dose, for cells fixed immediately (blue) and 24 h after irradiation (red); the ratio variations (Mean \(\pm\) SD) are reported, as a value normalized with respect to the corresponding ratio value of the non-irradiated samples (0 Gy, t\({}_{0}\): O Gy, t\({}_{24}\)); blue and red asterisks indicate when a significant difference in respect to the control value for the t\({}_{0}\) and t\({}_{24}\) samples, respectively, occurred at \(p\leq 0.05\). The ratios PP1 (Figure 6B) and PP2 (Figure 6C), which are both linked to protein phosphorylation [53], show significant (\(p\leq 0.05\)) modifications only at 10 Gy for t\({}_{0}\) cells. For t\({}_{24}\) cells, the ratio PP1 shows an increasing behavior also at 6, 8, and 10 Gy doses which became significant (\(p<0.05\)) at 10 Gy while the ratio PP2 does not show a significant trend in the occurring modifications. These modifications confirm the occurrence of changes in the DNA conformation, from the B-DNA form to the A-DNA form already noticed in Sections 3.2 and 3.3[44; 45]. The ratios P/L1 (Figure 6D) and P/L2 (Figure 6E), both linked to cell protein/lipid content, show an increasing trend with the dose for t\({}_{0}\) cells, which is significant (\(p<0.05\)) at all doses and at 10 Gy dose, respectively; on the contrary, t\({}_{24}\) samples show a significant shift to a higher value (\(p\leq 0.05\)) only for the P/L2 ratio at 10 Gy. The analysis of the P/L1 and P/L2 changes indicates modifications in membrane fluidity and their protein content [43].