Annual report

• The activities in the year 2021/22 of the Doctoral Program in Life Sciences and are, as is our tradition, were carried out according to the following structure:

• The first year doctoral students received the training proposal which includes the courses organized in collaboration with the BEMM school, mainly carried out electronically.
Each doctoral student, in addition of course to carrying out experimental work aimed at preparing his thesis at the facility to which he is assigned, participated in (all activities are held in English, with signature to stimulate participation):
• - monthly Journal Club activity: every month, with the exception of July and August, 4 recently published scientific articles and topics of general interest and of great impact are discussed. According to an established calendar, each PhD student presented an article within the topic chosen for each day.
• - annual report of their research activity and participation in the report days of all the other students. In 2022, the presentation took place on 17 October 2022 for first and second year PhD students.
• -participation in the seminars of the Pasteur Italia Institute.
• Seminars in the form of “readings” by course teachers and seminars by Italian and foreign researchers organized by the course, as per individual cards. In particular, online seminars were held during 2022.

The heterogeneity and complexity of the themes of the research carried out in the laboratories of the teachers and tutors of the doctoral school constitute the intrinsic added value but also the critical factor of the course.
Particular emphasis is therefore given, in all the activities carried out, to the attempt to maintain and improve a common knowledge and vocabulary among the various disciplines.
Also for this purpose, the PhD school organized a Kick-off meeting for the 37th cycle, in the presence of the students of the three years of the course, connected electronically, held in January 2022. The day was an opportunity for knowledge and involvement, also in the presence of the Coordinator and members of the teaching Board.

Each doctoral student was also asked to participate, at the expense of the doctorate, in a conference / workshops / course (see activities of individual doctoral students). Obviously, many activities were carried out online and the mobility was relatively limited, due to objective limitations.


35 cycle (third year)

Sara Andreone
The PhD project aim is to explore the anti-tumor mechanisms of IL-33 and eosinophils, focusing the attention on immune checkpoint (I.C.) modulation. These granulocuytes can acquire tumor-expressed I.C. through trogocytosis and this process is strongly enhanced by activation stimuli (i.e., IL-33) that promote CD11b/CD18-dependent cell adhesion and increase the expression of specific ligands
Marianna Bufano
A Virtual Screening protocol was applied on several targets for the identification of small molecules that could inhibits the proliferation of some type of cancers. Molecular dynamics simulations were applied in order to study the stability of the proposed binding mode. Also, using implemented approach of classical molecular dynamics simulations the behavior of some scaffold proteins after ligand or protein binding was investigated to know more about their possible conformational landscape.
Elena Gugole
Functional and structural characterization of proteins involved in lignin degradation by the fungus Pycnoporus Cinnabarinus with emphasis on understanding the mechanism of action of the auxiliary protein PcODH.
Soyanni Holness
The final year activities comprised but not limited to studying the priming effect of one stress (HL ) on the plants response to a subsequent stress (Drought). The sequential stress experiments were established and the priming effect was analysed at the molecular and physiological level by qPCR gene expression analysis and assessing photosynthesis efficiency, water status and carbon assimilation. Additionally, H3K4me3 as a potential epigenetic mark that could be associated with this priming/stress memory was investigated.
Karimpour Ghavianeh Angela
The experiments concerned the study of the association of GOLPH3 with mTOR. Tor is an evolutionarily conserved serine/threonine protein kinase that controls cell growth, proliferation, metabolism, and survival as the central component of two multiprotein complexes named mTORC1 and mTORC2 (mTOR complexes 1 and 2). It has been shown that dGOLPH3 could regulate the formation of Rheb/Tctp complexes by recruiting Rheb to the Golgi apparatus. Collectively, my data provide the first in vivo demonstration that GOLPH3 regulates organ growth by directly associating with mTOR signaling proteins.
Francesca Romana Liberati
The PhD project is focused on the study of the riboregulation of SHMT, it has a key role in the One Carbon Metabolism (OCM) for the control of the redox balance of the cells and production of nucleotides. SHMTs can bind RNA; the RNA-SHMT1 (the cytosolic isoform) interaction not only control RNA translation but also the catalytic activity of the enzyme. Due to the role of SHMT proteins in OCM and the importance of this pathway for highly proliferating cells, the lack of effective inhibitors that block the activity of these enzymes represents a significant problem.
During the PhD project it has been demonstrated that riboregulation of the mitochondrial SHMT2 isoform also occurs, and that the RNA-binding feature of SHMT2 might be utilized to target this protein (in vitro, in cellulo, in vivo) through the production of an RNA-based inhibitor (iRNA).
This research represents a starting point for understanding how metabolic enzymes are riboregulated and provides the proof-of-concept for the development of a unique RNA-based approach to inhibit cancer cell growth, a novel potentially effective tool for patient’s chemotherapy.
Sara Petillo
MLN4924 treatment induces the upregulation of the NKG2D ligand MICA and MICB on the plasma membrane of Multiple Myeloma cells and this triggers Natural Killer (NK) cell activation and cytotoxicity resulting in an increased lysis of Myeloma cells. Moreover MLN4924, alone and even more in combination with the monoclonal antibodies Daratumumab/Elotuzumab, directly enhances NK cell degranulation and killing of Myeloma cells as the result of the stabilization of the CRL substrates Rac1 and RhoA, Rho GTPases involved in cytoskeletal rearrangements and cytotoxic granule release after target recognition. Furthermore, neddylation inhibition partially counteracts the TGFβ-mediated impairment of NK cell effector functions by preventing the activation of the CRL4-AMBRA1 complex that mediates non-proteolytic polyubiquitylation of Smad4 to enhance its transcriptional activity.
Caterina Sanchini
The PhD research activity was focused on the characterization of the microglia microtubule cytoskeleton rearrangement entailed by microglia reactivity, using murine primary microglia cells and validating the in vitro results with an in vivo mouse model of microglia retinal activation. The findings indicated that microtubules play a crucial role in the cytokine release and morphological changes that take place during microglia activation. The study suggested that manipulating microtubules could present innovative therapeutic strategies to counteract neuroinflammation.
Adriano Setti
The role of RNA interactions has been explored in two areas related to muscle and motor neuron pathology: in the field of muscle pathology, it has been studied how the expression of circRNAs is altered in rhabdomyosarcoma and the role of the RNA helicase DDX5 and the m6A epitranscriptomic reader YTHDC1 in supporting their expression in this tumor; in the field of motor neuron pathology, it has been studied how over-expression of the FUS RNA binding protein with wild type sequence or with mutation associated with Amyotrophic Lateral Sclerosis (P525L) influences the RNA composition of Stress Granules, cytoplasmic molecular condensates produced in response to oxidative stress. In particular, the properties of RNAs showing altered recruitment in Stress Granules in the presence of the mutated protein and the role of m6A epitranscriptomic modification in this process have been characterized.
Beatrice Silvestri
The research project concerns the study of neuromuscular junction defects and cell death in FUSP525L and sporadic ALS, using human induced pluripotent stem cells (hiPSCs) to recapitulate a co-culture neural-muscle model system.
Flavia Trionfetti
During this year we focus on the evaluation of the effects of HDAC1/2 (using specific pharmacological inhibitors such as Entinostat, Mocetinostat, and genetic silencing) in mesenchymal-like mesothelial cells (MCs) in two distinct experimental systems developed in the past two years: 1) use of Poly(I:C) to mimic a viral dsRNA infection; 2) infection of MCs with SARS-CoV-2.
Gloria Tucci In vitro and in vivo experiments to study the ability to suppress TNFR2+ and TNRF2- Tregs.

36 cycle (second year)

Maria Cristina Benedetti
Creation of a collection of hiPSCs carrying different mutations linked to GNAO1 encephalopathies with their isogenic counterpart using CRISPR-Cas9 system. Differentiation and characterization a GNAO1 mutated iPSC lines toward a cortical fate, in order to highlight alterations that occur along human brain development both at a molecular and functional level compared to its isogenic control
Giulio Bontempi
Beginning characterization of the role of lncRNA H19 in the EMT process in primary mesothelial cells. Conclusion and publication of the article “Restoration of WT1/miR-769-5p axis by HDAC1 inhibition promotes MMT reversal in mesenchymal-like mesothelial cells”. Collaboration with Sergio Valente in the article “Novel pyridine-containing histone deacetylase inhibitors strongly arrest proliferation, induce apoptosis and modulate miRNAs in cancer cells” evaluating the markers of apoptosis and proliferation and evaluating the modulation of some miRNAs following pharmacological treatment.
Luca D'Ambrosio
Mitochondrial dynamics are modulators of endothelial cell functionality, their impairment can lead to mitochondrial dysfunction and the resulting damage. In vitro and ex vivo analyzes were performed to evaluate the modulations of OPA1 protein levels and function in Huvec cells treated with cardiovascular stressors and vascular reactivity in TgOPA1 and OPA1 +/- mice.
Assia De Simone
The expression of PACL and QAGR repeated tetrapeptides in flies’s motor neurons significantly affect both survival and locomotor abilities. At first, has been analyse locomotor activity, using a muscle-specific driver Mef2, this induces locomotory defects in both tetrapeptides that can be calculated by measuring the number of peristaltic contraction and also induces pupal lethality. The expression of peptides in cervical cancer cells (HeLa) showed a localization of PACL in the cytoplasm of HeLa cells while QAGR peptides expression in HeLa cells causes the formation of protein aggregates in the nucleolus. PACL could be involved in stress granules formation, so control cells were treated with arsenite which induces stress granules formation and when PACL is express in HeLa cells the granules present in PACL colocalize with stress granules, similarly at the cells induced by arsenite treatment.
Chiara D'Antoni
In the second year of PhD, the study of Fragile X Syndrome exploiting 2D and 3D cell cultures was pursued. These systems were characterized using Real time PCR, Immunoflurescence and Calcium imaging assays. Moreover, the effect of KW-60002, a drug currently used to teat Parkinson disease, was tested both on 2D and 3D cultures.
Davide Ialongo
While the current clinical scenario is increasingly dominated by the new biologics, a renewed interest has been experiencing for small molecules thanks to their pharmacokinetics and -dynamics characteristics and the possibility of being properly designed, making them an extraordinary starting point for the design of new drugs, especially as anticancer agents. In the light of the previous obtained data, the thesis consists in the design and synthesis of innovative Heparanase inhibitors, based on an ureidic or thioureidic core symmetrically functionalized by heterocycles, active in vitro and able to inhibit the cellular proliferation, the metastatic potential, and to induce autophagy in different tumor cell lines. In parallel, a series of newly synthesized functionalized bis-anilino- and phenoxy-pyrimidines are currently being evaluated as inhibitors of various involved-in-tumor-onset kinases (AURKA, VEGFR-A, PDGFR, EGFR, B-RAF, c-KIT, wt and mutated) by ATP competition, by covalent binding, or by protein-protein interaction inhibition.
Chiara Longo
As for the main project, the study of PRC2 role in response to plant cold stress by using also a human epidrug, RDS3434, after inconsistent results obtained with the first climatic chamber, I ran a new cycle of acclimation and freezing experiments with a Fitortron climate chamber, and by measuring phenotypical stress parameters of wild-type plants and prc2 mutants, with or without pharmacological treatment, I found that treatment with RDS3434 resulted in increased viability and higher chlorophyll content of acclimated plants compared to the untreated.
Moreover, after a preliminary expression kinetic analysis of cold and abiotic stress key genes, I designed and sent an RNA sequencing analysis, whose 48 samples were sent to an external service, passed the first quality control and are currently being analysed.
As for the minor project, in which I’m testing in plant human epidrugs against different epigenetic targets, such as Arabidopsis HAC protein family, P300/CBP homolog, following the phenotypic screening, I tested the effect of the most effective molecules, RDS3061 and RDS3029, on gene expression, I cloned and purified the catalytic domain of HAC1 and ran an in vitro inhibition assay of RDS3061 and measured its up-take by plants; the results are published at DOI 10.3390/IJMS231810446.
Walter Toscanelli
Study of redox-sensitive pathways involved in the regulation of the inflammatory response to respiratory virus infection, with a particular focus on the role of the APE1/REF-1 protein in the modulation of cellular antioxidant and immune response to influenza virus infection.

37 cycle (first year)

Usman Akhtar Butt
The project will focus on the characterization of the microbiota of the urinary trait. A literature search was performed, as well as techniques in the field of microbiology, molecular and microbial were practised. Started the collection of urine samples from the hospitals. Preliminary experiments were run to assess the better conditions.
Erica Debbi
Analysis of the impact of heavy metals present in atmospheric particulate on synaptic
development using a 2D brain model based on hiPSCs derived neurons and high-resolution
microscopy. Control and treated cell cultures for cell viability assays (MTS), RT-PCR, immunoflorescence analysis, calcium imaging recordings. iDisco Tissue Clearing experiments with cortical organoids.
Sara Di Russo
The PhD project studies the tumor microenvironment, in particular the process of breast cancer brain metastasis. It is used a complex model mimicking the brain microenvironment, using mouse brain extracellular fluid, Breast cancer cells able to metastasize with 100% of frequency to the brain, and BBB derived endothelial cells. We investigated the involvement of glutamate, present in significant quantity on the brain side of the BBB and the inflammatory environment in the formation of brain metastases by breast cancer cells.
Erica Floris
The project focuses on the characterization of primary cardiac stromal cells isolated from 5-week-old mouse models knock-out for the long non-coding RNA Charme. Cells are isolated through the explant culture method and characterized by their immunophenotype and 3D growth, as well as the paracrine function exerted on other cardiac cell types, and their ability to activate and differentiate to mediate cardiac repair processes.
Paula Gragera Alvarez
After the characterization of different cell lines, a melanoma and a neuroblastoma models were selected for the study. CRISPR/Cas9 technology was used as a method to stably inhibit the expression of ERAAP in the previously mentioned models. The ERAAP-KO neuroblastoma model was succesfully obtained and the protocol is being optimized for the melanoma model.
Greta Grassmann
Development of new computational methods for the investigation of the molecular mechanisms underlying the interaction among proteins, with the final aim of creating a new docking algorithm. Two novel methods have been designed, one to obtain the minimal geometrical representation (in terms of points) of a protein surface and one for the evaluation of electrostatic complementarity between partners. Both are going to be tested in the evaluation and prediction of proteins interaction when merged with other methods, like the Zernike-based shape complementarity evaluation (which in the mean time has been applied to systems of interest like the SARS-CoV-2 – ACE2 complex).
Kamila Julia Król
Investigating 13 cell lines in matter of ERAP1 activity and protein expression, MHC class I surface expression. Designing constructs to knockout exon 2 or 3 of ERAP1 gene with CRISPR-Cas9 system and obtaining ERAP1 silenced cell lines and their scrambled sgRNA equivalents. Testing already established silenced cell lines- proliferation and viability, cut efficiency, protein expression, MHC class I expression, ERAP1 induction by IFNg stimulation.
Alessio Incocciati
Research activity focused on the development, production, and purification of recombinant ferritins for Theragnostics purposes. In the last months, these protein chimeras have been characterized and tested on appropriate experimental models.
Antonia Intze
Atomic force microscopy-assisted infrared spectroscopy technique (AFM-IR) has been used to simultaneously observe the morphology and the modifications of the secondary structure of individual nanoscale protein aggregates of α-synuclein (αS) and TAR DNA binding protein 43 (TDP-43), down to 50 nm diameter and 5 nm thickness. The changes of the Amide-I infrared absorption band were tracked at wavelengths around 6 μm, which is related to C=O stretching mode of peptide bonds and is sensitive to the different protein secondary structure components (α-helix, parallel and antiparallel β-sheet, turn, and random coil). The co-aggregation of αS with RNA and its effect on the in vitro protein aggregation was also studied, finding statistically significant differences in the fibril morphology and packing density, in the percentage of parallel β-sheet component and in the aggregation timescales if compared to the RNA-free samples.
Michela Mochi
A gene editing strategy was set up to obtain a new line of iPSCs containing the P525L mutation of the FUS gene, with the aim of then using this line to obtain neuromuscular organoids. A plasmid allowing to overexpress a particular isoform of a protein (HuD) was developed to investigate the implications in the molecular and phenotypic alterations observed in motor neurons with the mutation of the FUS gene. Using the generated lines, a protocol is being set up to generate neuromuscular organoids expressing the P525L mutation of the FUS gene and wild type, previously used in the laboratory. We analyzed the effects of the overexpression of the HuD isoform at various time points of iPSC differentiation into motor neurons and the expression of the same isoform during differentiation in wild type lines and homozygous for the P525L mutation of FUS.
Francesca Romana Pellegrini
In this project I aim to investigate the role of tubulin acetylation, and in particular ATAT1 acetyltransferase, in the autophagic process and how its involvement may influence lung cancer progression and aggressiveness. To this end, I will study, in cultured lung cancer cells, the effect of absence or overexpression of ATAT1 enzyme on the autophagic process and on other hallmarks of cancer.
Valeria Pennacchietti
During the first year of the PhD, the folding mechanism of the PTB domain of the FRS2 protein was characterized and the interaction between the PTB domain and two ligands, FGFR1 and TRKB. In particular, kinetic experiments suggest that the PTB domain folds through a three-state mechanism with the formation of a low-energy intermediate. While, the study of the interaction between the PTB domain and the two ligands allowed to determine some of the residues involved in the binding.
Alessandra Pinzon Grimaldos
To determine whether ANGPTL3 deficiency-induced hypolipidemia impacts on monocyte/macrophage metabolism, ER membrane stability and IFN response.
Serena Rosignoli
The research activities for 2022 have been focused on the development of multiple GUI-based platforms for assisting molecular docking analyses, sequence and structures of proteins analyses and protein-DNA interactions. Such platforms vary from being plugins of the popular Molecular Graphics Viewer PyMOL, to being developed for a command-line usage, to being integrated as freely accessible web-servers. Moreover, the software were also presented at seminars and congresses, and started to be used in didactic courses.
Filipa Vasconcelos
The main objective of theresearch is the development of ERAP/IRAP modulators delivery systems for in vitro/in vivo proof of concept with extended release capable of tissue targeting and minimization of side effects for the treatment of cancer/autoimmune diseases using electrospinning and nanoparticle techniques.
The encapsulation of ERAP/IRAP modulators in nanofibers and/or nanoparticles and respective characterization was performed and achieved a prolonged release rate when tested for drug release + drug quantification.

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