Un approcio integrato sperimentale/teorico per lo studio delle transizioni del ciclo cellulare in fibroblasti umani irraggiati con fotoni

This thesis work is based on the development of a mathematical model to describe the perturbations of cell cycle induced by irradiation with X-rays. Flow cytometry experimental measurements have been performed on the human fibroblast IMR90 cell line, providing data to validate the model without perturbation and to test its descriptive and predictive power after irradiation. Cell cycle is divided in four phases: G1 phase (in which cell gets ready to the DNA synthesis), S phase (where the DNA is replicated), G2 phase (where the cell begins the preparation for mitosis), M phase (cell division), and each phase is characterized by a different length. Various proteins, cyclins and cyclin-dependent kinases, regulate transitions between two phases and block progression in the cycle in specific checkpoints if the system detects errors. Radiation can delay or arrest cell cycle, mainly through the activation of DNA repair mechanisms, and if there is to much damage, the stop in the cycle can cause cell death. The thesis is focused on two activities: experimental data collection and development of a mathematical model. For the experimental part, cells were irradiated with X-rays at IRCCS Istituti Clinici Maugeri (6 MV LINAC, Varian) with two different doses: 2 Gy and 5 Gy. Cell samples were analyzed at different time points after irradiation (0, 6 ,16, 24, 48, 72 h) to study the temporal dynamic of the cell distribution in the cell cycle and its perturbation induced by radiation. This analysis was performed with the flow cytometer (Attune NxT AFC, ThermoFisher) available in the Radiobiology and Radiation Biophysics Laboratory at the Physics Department of University of Pavia, in order to discriminate cells by DNA content (through FxCycle Violet staining), to recognize cell that are replicating the DNA (through EdU staining) and to differentiate G2 and M (through H3 histone phosphorylation). This information allows to discern the four phases and obtain the percentage of cell in each phase. Starting from the work of Basse et al. (2003), a compartmental model was developed, in which each compartment represents a phase of the cell cycle as a function of time and DNA content, so that the model can reproduce experimental data and obtain information on transitions between phases. The temporal evolution is described by a system of partial differential equations, whose parameters represent the transition rates, division rate and rate of DNA synthesis. During exponential growth, the total number of cells increases in time, but their distribution between the phases remains stationary in absence of external perturbations. Therefore, experimental data obtained from cell population in pure exponential growth were used as input of the model to estimate its initial parameters. Perturbation is implemented as an alteration of the value of the model parameters: the variation of the parameters that best reproduce the cell cycle profile of the irradiated conditions as a function of dose and time gives information on the mechanism behind radiation effects. The model can be extended to experimental data from literature (other cell lines or different radiation qualities), or can be used to study new potential perturbations. The model can e.g. be a tool for predictions in radiotherapy on the variation of the dynamics of cell cycle with different fractionation schemes or with the possible synergistic combination with phase-dependent chemotherapeutic drugs.