## 3 Results and Discussion
### Analysis of Amide I Band (t\({}_{0}\) and t\({}_{24}\) Cells)  
The analysis of the different peaks constituting the Amide I band can offer precise information on the secondary structure of proteins and their conformational changes as a result of different doses of ionizing radiation. In the present case, the Amide I band of control samples and irradiated samples was  
Figure 7: Comparison of the absorbance ratios P/D1 (**A**), P/D2 (**B**), and DM (**C**) with dose, for cells fixed immediately after (**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\({}_{0}\); 0 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 \(p\leq 0.05\).  
analyzed using eight subcomponents related to parallel \(\beta\)-sheet (1626, 1635 cm\({}^{-1}\)), anti-parallel \(\beta\)-sheet (1617, 1697 cm\({}^{-1}\)), \(\alpha\)-helix (1661 cm\({}^{-1}\)), and \(\beta\)-turn (1674 cm\({}^{-1}\)); unordered structures are attributed to the band at 1646 cm\({}^{-1}\). In Figure 8A,B, the ratios between the secondary structures peak area, grouped according to the different structures, and the area of the entire Amide I peak, as a percentage, are reported for both t\({}_{0}\) and t\({}_{24}\) cells, respectively. In particular, the \(\beta\)-sheet bar is related to the contributions of different \(\beta\)-sheet subcomponents that are reported in Tables S2 and S3 (Supplementary Material) in terms of percentage of the areas together with the other subcomponent bands for t\({}_{0}\) and t\({}_{24}\) samples. As already indicated by the PR1, PR2, and PR3 analysis reported in Section 3.4, the exposure to X-rays induces several modifications in protein secondary structure. More specifically, a significant increase (\(p\leq 0.05\)) for the \(\beta\)-sheet structure is visible for all the t\({}_{0}\) irradiated samples combined with a decrease in the signal coming from the \(\alpha\)-helix structure; for both, the structure the maximum/minimum, respectively, occurs at 6 Gy. Moreover, for the t\({}_{0}\) cells, the \(\beta\)-turn structure shows modification at 2, 4, 6, and 8 Gy, with values higher than the control sample while the unordered component has lower values than the control for all doses. The ratios for t\({}_{24}\) cells show a stable course for all the sub-structures with significant variations (\(p\leq 0.05\)) only at the maximum dose of 10 Gy; in fact, a shift towards lower values of the \(\beta\)-sheet structure band is accompanied by an increase in the percentage linked to the \(\alpha\)-helix band and of the unordered structure.  
Figure 8: Variations of secondary protein structure contributions to Amide I band (1590–1710 cm\({}^{-1}\)) with dose, for cells fixed immediately (**A**) and 24 h after irradiation (**B**); the ratios between the secondary structures peaks area and the area of the entire Amide I peak are reported; asterisks of different colors indicate when a significant difference in respect to the corresponding control value occurred at \(p\leq 0.05\).