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Final Report Summary - TROJAN-LIPID-SENSOR (Trojan-Lipid-Sensor)

The aim of my project is to design and produce an RNA sensor capable of recognizing membrane phospholipids to study cellular dynamics and the molecular mechanisms that control cell adhesion and movement. These RNA molecules bind to conditionally fluorescent molecules that are structurally related to the GFP Chromophore and are called Spinach. Spinach can be used to create small molecule sensors. If a small molecule–binding aptamer and Spinach share the critical stem required for Spinach fluorescence, small-molecule can fold the aptamer and stabilize the stem, resulting in fluorescence. Spinach can therefore function as a sensor that becomes fluorescent only upon binding to specific target molecules, in my case lipid molecules.
Since the cell membrane is the first structure that connects the inside of the cell with the outside, it is clear that the proteins and lipids contained therein not only play a fundamental structural role, but also play a role as transductor of cellular messages and external stimuli.
During the outgoing phase I had the opportunity to work at Cornell Medicine in New York City, in the lab of prof. Jaffrey, a luminaire in the field of the development of RNA sensors. During my time at Prof Jaffrey's lab I achieved several experiential and training goals I had planned during the drafting of the project. First, I gained experience with SELEX technology that allows RNA to be selected based on its binding properties to a target molecule. Subsequently, I became an expert on Circular Dichroism and Thermo Isothermal Calorimetry, analytical techniques that allow the study of conformational changes occurring on macromolecules. Finally, I participated in numerous seminars and meetings, creating a network of knowledge that will help me build my career in the future.
In the second year of my experience at Prof Jaffrey's lab, I spent a collaborative time with Professor Foster, Hunter College, New York City. Prof Foster is a world expert in Phospholipase D, Phosphatidic Acid and Cancer biology. During the collaboration with Prof. Foster, under the supervision of Prof Jaffrey, I was able to study and develop a GFP-Based sensor for Phosphatidic Acid (GFP-PASS).

During the third and final year of my project, which was held in the laboratory of Prof. Alessandra Cambi, I could use the PASS-GFP phosphatidic acid sensor. This sensor was found to be very promising for the dynamics of Membrane Phosphatidic Acid. In fact, this sensor has helped me to obtain valuable data for the completion of my scientific work on the role of PLD1 and PLD2 in controlling podosomes in dendritic cells. Hence, I imaged phosphatidic acid dynamic in live cell imaging and I was able to image accumulation of Phosphatidic acid at membrane level
Actomyosin-mediated reorganization of the cells cytoskeleton is the primary mechanism of cell migration and invasion. Podosome are actin structures involved in extracellular matrix adhesion and invasion found in several cell types. For the first time using Total Internal Reflection (TIRF) microscopy, I was able to image the accumulation of Phosphatidic acid islet at the site of podosome formation.
Moreover, during the final part of my project, my research goals included broadening my understanding of a wide range of topics in the fields of molecular and chemical biology. Additionally, I expanded my repertoire of technical skills and I became familiar with the most cutting-edge research in microscopy and cell biology field. Finally, I build on my training in grant writing, public speaking, and paper drafting that expected my long-term goal of establishing a successful career as the principle investigator of a research lab. Overall my experience in the outgoing host institution was very prolific in terms of professional experience and acquired scientific data. I also managed to complete the paper I will submit shortly.