## 3 Methodology
### Experimental Studies
**Ex vivo studies on _lymphocytes_ from human donors:_Lymphocytes_ from human donors will be exposed to 5G signals (3.5 GHz), alone and in combination with other agents, to analyze the expression of a selection of cancer-related genes. The exposure system and dosimetry is similar to the one adopted for in vitro studies on keratinocytes. Ex vivo exposure will be performed on _lymphocytes_ collected from healthy human donors, with the same exposure conditions adopted for the short-term in vitro studies. Combined exposures will be carried out on lymphocytes with ethyl methanesulfonate (EMS)/menadione genotoxicants. The comet assay will be applied to evaluate the induction of DNA damage. Gene expression and epigenetic tests will be applied to selected conditions based on the Comet assay results. **Biological effects of RF-EMF exposure in humans:** Buccal cells from self-reported heavy and light mobile phone users will be used to investigate the expression of genes identified in in vitro and ex vivo studies. Self-reported mobile phone users will be selected  
Figure 3: Overall strategy for biological investigations. based on the amount of time calling with the mobile phone at the ear level. Information will be obtained related, e.g., to the type of phone used in the last 10 years and the behavior when calling, to estimate SAR values at the inner cheek level (dosimetry modeling). Moreover, actual measurements are envisaged to confirm the classification in both groups. Buccal cells will be taken from heavy and light mobile phone users following established methods for cell collection [33]. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) will be performed on a selection of genes, as defined in in vitro studies with the genomics tool. **Effects of RF-EMF exposure on human RBCs:** In a first study, blood samples will be taken from healthy volunteers following exposures based on the conditions defined elsewhere in the project (5G carrier frequencies (3G/4G/5G) and modulation envelope). The system will be an evaluation board based on software defined radio (SDR) technology with a maximum frequency of 6 GHz and a minimum quadrature sampling of 8 bits. The output frequency of the multiplier will be emitted in the FR2 band with a modulation scheme as defined in an earlier task. The available output emission will be amplified via a high-frequency amplifier and connected to a directional antenna whose gain will be set according to the required level. A second SDR will be used in reception to ensure the monitoring of the experiment. In a second exploratory study, healthy volunteers (15 males and 15 females aged 18-25 years) will participate in two double-blind sessions, one real and one sham (random) exposure (45 min). In samples taken after exposure, blood count, RBC indices, redox state, and NO level, CO-oximetry, RBC aggregability and morphology will be assessed. **Biophysical and Biochemical mechanisms of RF-EMF exposure in blood and RBC:** The RF-EMF (4G and 5G) interaction with human RBCs will be studied, in which the possible molecular, sub-cellular/cellular targets of the exposure, and the RF-EMF parameters (repetition frequency, slopes, on-off cycles, etc.) will be identified. A vector signal generator interconnected to an exposure setup equipped with a temperature controller and pumps to introduce flow conditions will be used to expose RBC suspensions. A microfluidic unit will be developed by using a radiofrequency generator and a directional plasmonic phase modulator adapted to 26 GHz. Both systems will allow multiple exposures to RF-EMF signals. A special system will be developed by combining a TEM line for the RF-EMF exposure with a high-performance network analyzer for real-time assessment of dielectric parameters. The responses of cellular hemoglobin (Hb) and membranes will be evaluated using dielectric spectroscopy to monitor changes in protein structure and its surface charge through the changes in free and bound water dynamics.