## 3 Results and Discussion
### Analysis of Relative Absorbance Ratios  
The intensity of an infrared band is considered to be proportional to the concentration of the species that are associated with the band. However, the use of band absorbance values, themselves for quantitative analysis, can be influenced by experimental artifacts such as variations in sample thickness. For this reason, it is preferable to use their ratios [41; 49; 50]. In Table 1, the ratios between the absorbance of chosen bands used in this work are listed, the biochemical processes to which they are related are also indicated. In the panels of Figure 4, the values of the different ratios mentioned in Table 1 are \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{4}{c}{**t\({}_{\textbf{0}}\) Cells**} \\ \hline
**Control** & **Assignments** & **0.5 Gy** & **2 Gy** & **4 Gy** \\ \hline Peak (cm\({}^{-1}\)) & & & & \\ \hline
1180–1240 & \(\beta\)-sheets & - & - & - \\ \(\%\)A = \(86\pm 8\) & & \(\%\)A = 74 \(\pm\) 15 & \(\%\)A = 74 \(\pm\) 20 & \(\%\)A = 76 \(\pm\) 15 \\ \hline
1240–1270 & \(\%\)A = 4 \(\pm\) 2 & Random coil & - & - & - \\ \(\%\)A = \(5\pm 2\) & \(\beta\)-turn & \(\%\)A = 7 \(\pm\) 3 & \(\%\)A = 7 \(\pm\) 5 & \(\%\)A = 7 \(\pm\) 3 \\ \hline
1290–1330 & \(\%\)A = 5 \(\pm\) 2 & \(\alpha\)-helix & - & - & - \\ \(\%\)A = \(5\pm 2\) & \(\%\)A = \(10\pm 4\) & \(\%\)A = \(10\pm 4\) & \(\%\)A = \(13\pm 5\) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Amide III deconvolution results for control and irradiated sample fixed immediately after irradiation, with assignments in accordance with the data reported in the literature [39; 48]. The ratios between the area of the different subcomponents and the area of the entire Amide III band are reported as a mean value of percentage (A\(\%\)) \(\pm\) SD. Figure 3: Variations of secondary protein structure contributions to Amide III band (1350–1180 cm\({}^{-1}\)) with dose, for cells fixed immediately after irradiation; the ratios between the secondary structure peak areas and the area of the entire Amide III peak are reported as Mean \(\pm\) SD. Asterisks indicate when a significant difference with respect to the corresponding control value occurred at \(p\leq 0.05\). reported as a function of the dose. As is evident, only some of these ratios show statistically significant variations. Some of the ratios related to DNA modifications (DNA1 and DM) are among these. DNA1, DNA2, and DM indicate changes in the antisymmetric and symmetric O-P-O stretching modes that are related to double-strand breaks (DBS) [12; 42]. DNA1 and DNA2 show an increase when proton dose increases, while DM shows a decrease when proton dose raises. The modifications evidenced by DNA1, DNA2, and DM are in agreement with the results obtained in other cell systems exposed to ionizing radiation that indicate the occurrence of damage in the primary, secondary, and tertiary structure of nucleic acid [42]. Figure 4: Comparison of the absorbance area ratios for different proton doses. See Table 1 for details on the reported ratios (Mean \(\pm\) SD) that are normalized to the corresponding ratio value the non-irradiated sample. Asterisks indicate when a significant difference occurred at \(p<0.05\). The LS ratio gives an indication about lipid saturation and shows a statistically significant increasing trend with proton dose in agreement with results obtained when human cells are exposed to X-ray doses [6; 47]. These modifications are suggestive of apoptosis, for which several membrane changes, such as phosphatidylserine exposure, membrane blebbing, and vesicle formation, are known to represent early events [51]. In addition, an increase in saturated fatty acids containing phospholipids during apoptosis in neuronal cells was reported in [40]. Phospholipids are the most relevant structural components of membranes bounding many intracellular organelles (e.g., lysosomes, endoplasmic reticulum, and nuclei) and a relevant change in their composition could induce deformation and porosity of such membranes.