Dottorato in SCIENZE DELLA VITA

Ricerca: Spettroscopia ed imaging di aggregati proteici alla nanoscala

TITLE OF PhD RESEARCH PROJECT
Infrared nanospectroscopy and nanoimaging of protein aggregates

PhD THEMATIC AREA
Biophysics; Infrared spectroscopy; Nanoscale spectroscopy and imaging; Protein-RNA interaction

BRIEF DESCRIPTION OF PhD PROJECT
α-Synuclein is a non-nucleic acid-binding protein in the monomeric state and its aggregation is linked to neurodegeneration. Recently, it has been shown that α-synuclein acquires the ability to sequester total RNA when aggregating and this protein-RNA interaction impacts on the aggregation rate of α-synuclein. In the framework of my PhD, I investigate the morphological and structural properties of α-synuclein fibrils forming in-vitro in the presence and absence of total RNA by using the atomic force microscopy-assisted infrared spectroscopy technique (AFM-IR). Specifically, in the group I am a part of, we track the changes of the amide-I infrared absorption band at wavelengths around 6 μm, which is related to C=O stretching mode of each peptide unit in the protein polypeptide chain and is sensitive to protein secondary structure. The in-vitro analysis of aggregates is performed at different time points by casting a drop of solution on an ultraflat gold surface suitable for AFM-IR analysis. As α-synuclein aggregation proceeds, we observe a faster increase of the parallel β-sheet component (a spectral marker of amyloid fibril formation) upon total RNA addition. The simultaneous AFM nanoimaging of the forming α-synuclein fibrils verifies the earlier formation of micrometer-long amyloid fibrils when total RNA is present in the aggregating solution. AFM analysis also reveals significant differences in the morphology and packing density of the α-synuclein fibrils forming with and without total RNA addition. In parallel, we also characterize the aggregation process with circular dichroism spectroscopy and FTIR micro-spectroscopy, finding the results consistent with AFM-IR. Finally, persistence length measurements on individual fibrils and analysis of parallel β-sheet central frequencies measured by AFM-IR show that total RNA increases the rigidity of the forming α-synuclein fibrils. These results show that AFM-IR allows a unique structural approach into studying the effect of total RNA on the in-vitro protein aggregation.

EDUCATION
1 Nov 2021 – Current: PhD student in Life Sciences program
Department of Biochemical Sciences, La Sapienza University of Rome in collaboration with the Center for Life Nano- & Neuro-Science (CLN2S), Istituto Italiano di Tecnologia
Project: Infrared nanospectroscopy and nanoimaging of protein aggregates
Supervisor: Prof. Michele Ortolani

1 Oct 2018 – 11 Dec 2020: Master Degree in Chemistry (with honours)
Department of Chemistry, University of Crete, Greece
MSc Thesis: Biomolecular residue analysis in archaeological potsherds by means of UV-vis absorption and fluorescence spectroscopy
Supervisor: Prof. Demetrios Anglos

1 Oct 2014 – 30 Sep 2018: Bachelor Degree in Chemistry (with merit)
Department of Chemistry, University of Crete, Greece
BSc Thesis: Atmospheric processes, climatic change and air quality: UV and infrared absorption cross sections determination for critical volatile organic compounds and heterogeneous interaction of gaseous sulfur dioxide with dust surfaces
Supervisor: Dr. Vassileios C. Papadimitriou


WORKING EXPERIENCE / FELLOWSHIP
1 Feb 2021 – 30 Oct 2021: Early stage researcher internship
Center for Life Nano- & Neuro-Science (CLN2S), Istituto Italiano di Tecnologia
Project: Infrared spectroscopy of photoactive proteins
Supervisor: Prof. Michele Ortolani

1 Mar 2020 – 30 Jun 2020: Horizon 2020 Erasmus+ trainee
Università di Pisa, Department of Chemistry and Industrial Chemistry, Italy
Project: Analysis of collagen in samples from archaeological bone using analytical pyrolysis
Supervisors: Prof. Ilaria Bonaduce and Prof. Erika Ribechini

1 Jul 2017 – 30 Sep 2017: Horizon 2020 Erasmus+ trainee
Institut de Combustion Aérothermique Réactivité Environnement, ICARE, CNRS, Orléans, France
Project: Investigation of temperature dependence of UV-vis cross-sections for several ketones
Supervisor: Dr. Abdelwahid Mellouki

CONFERENCE / SCHOOL / WORKSHOP / MEETING PARTECIPATION
3 − 6 September 2023: Annual retreat of Center for Life Nano-& Neuro-Science (CLN2S), IIT, Preci, Italy
1 −8 September 2023: FEBS Advanced Lecture Course in Protein Folding, Aggregation and Compartmentalization, (Poster presentation) Spetses island, Greece
27 August−1 September 2023: 12th International Conference on Advanced Vibrational Spectroscopy, ICAVS12, (Poster presentation) Krakow, Poland
22 – 23 June 2023: 15th Bessy@HZB User Meeting, (Poster presentation) Berlin, Germany
12 – 14 June 2023: International Summer School on Vibrational Spectroscopy VISPEC, Perugia, Italy
11 – 18 December 2022: Workshop and Winter School in Biophotonics, Bioelectronics, and Biomechanics, Biocube meeting, (Poster presentation) Sestriere, Italy
20 − 23 September 2022: Annual retreat of Center for Life Nano-& Neuro-Science (CLN2S), IIT, Preci, Italy
28 August − 2 September 2022: 47th International Conference on Infrared, Millimeter, and Terahertz Waves (Poster presentation) Delft, Netherlands
4 − 8 July 2022: Plasmonica Summer School and Workshop (Poster presentation), Torino, Italy

BEAMTIME
19 − 25 June 2023: Conduct experiments at Bessy II synchrotron facility, Helmholtz-Zentrum Berlin, Berlin, Germany
Research proposal: Infrared micro-spectroscopy of pure and RNA-bound alpha-synuclein aggregates

25 April − 1 May 2022: Conduct experiments at Bessy II synchrotron facility, Helmholtz-Zentrum Berlin, Berlin, Germany
Research proposal: Mid-infrared microspectroscopy and nanospectroscopy of the aggregation pathway of TDP-43

OTHER ACTIVITIES
1 Nov 2023 – 31 Oct 2024: Student representative in the PhD Board
1 Nov 2023 – 31 Oct 2024: Tutor in Biochemistry Master degree course (Bando BT-B2 1/2023)
1 Feb 2023 – 15 Jul 2023: Tutor in Biochemistry Master degree course (Bando BT-B2 2/2022)
1 Oct 2022 – 31 Jan 2023: Tutor in Biochemistry Master degree course (Bando BT-B2 1/2022)