Cellular and Molecular Biology

Brief description
Photodynamic therapy (PDT) is an emerging therapeutic modality, clinically approved for a wide variety of medical conditions as it is minimally invasive and versatile. The procedure involves the activation of photosensitive molecules, called photosensitizers (PS), used to promote cell death based on the interaction between visible or infrared light and molecular oxygen available in cellular environment. After irradiation, the excited PS reacts with molecular oxygen, triggering both reactive oxygen species (ROS) and single oxygen
formation. Signalling cascades, also involving calcium signals, are activated in target tissue, resulting in cell death.
The goal of the internship is to dissect the intracellular signalling pathway due to PS photoactivation.
Live cell fluorescent microscopy will be used to evaluate the temporal and spatial release of ROS and their interplay with intracellular Ca2+ signals. In particular dynamic ROS release will be monitored by means of Cell Rox (Thermo Fisher), a green fluorescent probe used for monitoring oxidative stress (and thus ROS production) in living cells. The ROS species released by the photoactivated PS will be defined using specific ROS scavengers.
Ca2+ signals dynamics will be monitored by ion imaging using different Ca2+ probes aiming at monitoring free cytosolic Ca2+ (Fura-2-AM or Fluo-4, Thermo Fisher) or intraorganellar Ca2+ dynamics by means of protein-based Ca2+ sensors. In particular GEM-CEPIAer will be used to measure the variation in ER Ca2+ concentration, and GEM-GECO1 to measure the dynamics in mitochondria Ca2+ concentration.
This thesis is in close collaboration with the laboratory guided by Prof Barbero, Department of Chemistry,

Knowledge, skills and competences to be acquired by the end of the traineeship (expected Learning Outcomes):
The student is expected to acquire advanced competences in live-cell imaging and ion imaging by means of fluorescence microscopy. The student will also acquire competences in image analyses and quantitative data analyses of the acquired data. Moreover, the student will also acquire basic cell biology procedures (cell culture maintenance, cell transfection).