Annual report

• The activities in the year 2020/21 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 2021, the presentation took place on 12 October 2021 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 2021, for reasons related to the pandemic.

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. Despite the limitations due to the meeting in online mode, the meeting met with a largely positive opinion from the students.

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.

36 cycle (I year)

As regards specifically the research activity of 1st year doctoral students, the main results achieved are summarized below, which led to the publication in 2021-22 of 12 scientific articles for students of the 36th cycle and to the presentation of the results by PhD students in national and international conferences, also in this case partially limited by reduced mobility. More details on scientific production can be found in the individual cards.

Benedetti Maria Cristina
The doctoral project focuses on the study of encephalopathy related to the GNAO1 gene, a rare disease that affects the development of the psychomotor system. Lines of iPS characterized by four different mutations in the GNAO1 gene have been generated using the CRISPR-Cas9 system and screening of individual clones is underway. The future goal is to generate 2D and 3D cultures to highlight alterations during development and subsequently carry out drug tests. Cortical and neuromuscular organoids were generated by adapting previously published protocols and subsequently characterized from a morphological and functional point of view showing their ability to recapitulate the complexity of the cortical system and the neuromuscular junction over time.

Bontempi Giulio
This year the mechanism of the antifibrotic response promoted by the treatment of primary fibrotic mesothelial cells (MCs) with the pharmacological inhibitor HDAC1 / 2 (HDACi) MS-275 was analyzed, focusing on the role of miR-769-5p among a series of HDACi-induced antifibrotic miRNAs. Gene silencing and ectopic expression experiments revealed that miR-769-5p controls the antifibrotic response induced by MS-275. In analyzing the molecular mechanisms of this process, we focused on the role of WT1 (Wilm's Tumor 1), a transcription factor that controls the differentiation of MCs, and validated a regulatory axis, including HDAC1-WT1-miR-769 -5p, potentially relevant for cell therapies aimed at counteracting organ fibrosis.

D'Antoni Chiara
The doctoral project focuses on the study of Fragile X, a neurodevelopmental disease associated with autism and epilepsy spectra. To better identify the effect of this mutation on neurodevelopment, cortical organoids were generated starting from wild type and patient-derived iPSCs cells. These organoids were subsequently characterized using immunofluorescence and real time-PCR techniques.

D'Ambrosio Luca
Preclinical evidence has shown that alterations in mitochondrial dynamics processes are associated with the development and progression of cardiovascular diseases. However, how deregulation of mitochondrial fusion leads to endothelial dysfunction, a common cause of cardiovascular disease, is not yet known. The levels of the OPA1 protein, the main effector of mitochondrial fusion, were analyzed in HUVEC subjected to different conditions: nutrient deficiency, hyperglycemic, oxidative and metabolic stress and condensed cigarette smoke. The first data collected show that OPA1 is differently modulated by the various treatments, in association with changes in the mitochondrial network and function after the treatments. It will be evaluated in the future whether the deregulation of OPA1 leads to a reduction in angiogenesis, both in vitro and in vivo.

De Simone Assia
The aim of the doctoral project is to analyze the toxic effect of repeated PACL and QAGR polypeptides, generated by RAN translation in DM2, using Drosophila melanogaster as a model organism. It has been observed that the expression of repeated tetrapeptides causes a strong neurodegeneration which affects the viability of Drosophila melanogaster and the development of the CNS. Furthermore, differences in subcellular localization and solubility specific to each repeated polypeptide were highlighted suggesting that there may be different molecular mechanisms at the base.

Ialongo Davide
The research project concerns the design and synthesis of new organic molecules with antitumor activity. During the first year, benzimidazoles and benzoxazoles were synthesized as new inhibitors of heparanase and of new molecules with a pyrimidine structure capable of inhibiting Aurora Kinase A. The first molecules were found to be active with IC50 values ​​in the order of submicromolar in in vitro assays. while the Aurora kinase A inhibitors have been shown to have an activity in the nanomolar order, preventing the interaction of the ATP molecule with the enzyme.

Longo Chiara
The project investigates the involvement of Polycomb Repressive Complex 2 (PRC2) in the response to cold stress in plants, using both a genetic and pharmacological strategy, to analyze the epigenome and transcriptome in response to cold stress, in the presence of an inhibitor. of PRC2. During the first year, adequate set-ups for cold stress tests (chilling and freezing) were designed and confirmed, considering the concentration and duration of treatment with the inhibitor. The toxicity of the inhibitor in the plant and the uptake kinetics from the medium were also evaluated.

Toscanelli Walter
Oxidative stress plays a crucial role in the replication of the influenza virus and in triggering the inflammatory response to infection.
Two of the main factors involved in redox-sensitive pathways, NRF2 and G6PD, are under-regulated following influenza virus infection. This thesis project aims to study the role of another redox factor, APE1, related to NRF2, in the inflammatory response and viral replication during influenza virus infection.

35 Cycle (II year)

Andreone Sara
The goal of the doctoral project is to increase the antitumor activity of interleukin -33 and eosinophils against melanoma, paying particular attention to the modulation of immune checkpoints and the process called "trogocytosis". In particular, it has been observed that the eosinophils activated by IL-33 have the ability to acquire foreign molecules from surrounding cells, such as PD-1 and TIGIT.

Bufano Marianna
Through virtual screening, a new inhibitor of the DVL1 protein was identified through binding to its PDZ domain. To obtain more information on the binding mode, molecular dynamics was carried out and the analysis of the trajectories helped to identify the interactions to have a stable bond. In particular, the molecular dynamics analysis showed more stable interactions for the S enantiomer. Biological assays confirmed a greater activity of the same and a selectivity towards the PDZ of DVL1 rather than NHERF1.

Gugole Elena
The PhD project focuses on the structural and functional characterization of the dehydrogenases involved in the degradation of lignin, by the fungus Picnoporus Cinnabarinus. We obtained single crystals of PcODH in complex with coumaric acid, a secondary electron acceptor of the enzyme. Data analysis showed the presence of the electron density of cinnamic acid in the active site and a conformational rearrangement following its binding.

Holness Soyanni
Single abiotic stress reliever treatments with drought or high light intensities can trigger a molecular response that can last from several days to weeks and can help plants cope with recurring stresses. However, it is often unclear whether this is due to acclimatization or epigenetic memory (priming) and the underlying mechanisms that help plants cope with subsequent stresses. In this PhD project, the potential priming effects in plants subjected to subsequent stresses are investigated, with particular attention to two specific abiotic stresses: drought and bright light.

Karimpour Ghahnavieh Angela
Affinity purification coupled with mass spectrometry (AP-MS) was used this year to identify the GOLPH3 oncoprotein interactome in Drosophila melanogaster. Using co-immunoprecipitation (Co-IP) and glutathione S-transferase (GST) pull-down experiments, some selected interactors, including endocytic and secretory trafficking proteins, were validated. In parallel, interactions with different proteins involved in Akt / TOR signaling, such as TCTP, 14-3-3ζ and LST8, were validated. To further demonstrate the involvement of dGOLPH3 in the TOR pathway, the genetic interaction with Tctp and Rheb in the imaginal wing disc was analyzed. Overall the results indicate a strong genetic interaction of dGOLPH3 with the TOR signaling pathway.

Liberati Francesca Romana
The PhD project focuses on the study of the reboregulation of SHMT, a key enzyme of the one-carbon metabolism and often over-expressed in many types of cancer cells. It has been shown that when SHMT binds RNA, its enzymatic activity changes: in the presence of RNA, the enzyme is unable to reversibly convert serine into glycine, but only glycine into serine. To better clarify the molecular details of this mechanism, plasmids were produced for the expression of specific nucleic acid sequences under the control of an inducible tetracycline system to finely regulate their production: by transfecting cells with these plasmids, their metabolism was studied. , the survival and proliferation rate. The results showed that SHMT's RNA binding ability could be used as a Trojan horse to control the monocarbon metabolism of cancer cells and to inhibit cancer growth. At the moment, few small molecule inhibitors of SHMT have been identified and the in vivo efficiency is poor, and therefore the possibility of using RNA inhibitors is being explored, which appears to be a promising strategy.

Petillo Sara
The doctoral project aims to understand whether the administration of the Neddylation inhibitor, MLN4924, is able to promote the recognition and elimination of Multiple Myeloma (MM) cells by Natural Killer (NK) cells. MLN4924 induces the upregulation of MICA and MICB, ligands of the NK activating receptor NKG2D, on the membrane of MM cells by promoting the cytotoxic activity of NK which results in greater lysis of MM cells . The role of Neddylation in the regulation of NK function and activity in the context of the MM tumor microenvironment is also currently being studied.

Sanchini Caterina
The purpose of the doctoral project is to analyze the rearrangements of microtubules that occur during microglial activation, in physiological and neurodegenerative contexts. By in vitro and ex vivo analyses, the dynamics and nucleation of microtubules in the microglia were characterized, identifying a pathway mediated by the NLRP3 inflammasome complex that connects the recruitment of nucleation material in the perinuclear zone in the pro-inflammatory microglia with the release by il1b.

Setti Adriano
During the second year of the PhD, two projects were launched: the first concerning the characterization of the role of long non-coding MN2 in the development of motor neurons; the second concerns the study of "stress granules", cytoplasmic molecular condensates that are formed following oxidative stress. Regarding the first project: the abolition of the lncMN2 locus leads to a reduced production of motor neurons. To understand its function in motor neuron biogenesis, single cell RNA-Seq data of locus mutants produced in “Embryoid bodies”, deriving from the differentiation of mESC to motor neurons, were analyzed, characterizing the molecular circuits involved in this function. Regarding the second project: through the analysis of RNA-Seq of precipitates of "stress granules" their RNA composition was clarified. In particular, it is studied how the pathogenic mutation of the FUS protein (P525L), known to be associated with amyotrophic lateral sclerosis (ALS), can alter its molecular composition.

Silvestri Beatrice
The aim of the PhD project is to evaluate whether the alteration of the molecular circuits involving the FUS, HuD and GAP43 proteins, in addition to the alteration of neurites, can lead to the disruption of the neuromuscular junction observed in the pathology of ALS. To recapitulate the neuromuscular circuit in vitro, human induced pluripotent stem cells are used to obtain a 2D model system consisting of the neural and muscle component.

Trionfetti Flavia
During this year, the role of TLR ligands was analyzed, focusing in particular on PolyI: C (a TLR3 ligand that mimics viral RNA) in the genesis of the mesenchymal / fibrotic phenotype in mesothelial cells. Based on the information obtained using the synthetic TLR3 ligand, the role of SARS-CoV-2 infection in mesothelial cells was studied. Mesothelial cells have been shown to support SARS-CoV-2 infection and modulate the immune / inflammatory response.

Tucci Gloria
During this second year of the doctorate, the role of TNFα in the expansion of regulatory T cells (Treg) was investigated more thoroughly. It had already been shown that TNF is fundamental for the proliferation of Tregs, that these up-regulate TNF itself and TNFR2 (especially in the tumor microenvironment), and that the trend of receptor 2 is opposite to the production of TNF in the two subsets of Treg TNFR2 + and -. This year, with a gene expression analysis, it was confirmed that TNFR2 + Tregs are more active and proliferating, with a more defined regulatory identity, while TNFR2-, probably due to their sensitivity and responsiveness to the IFNa pathway , have a more “tissue-repair” and unstable phenotype. Finally, to fully understand the causes that lead TNFR2 + Tregs to transcribe the same mRNA but translate less TNF protein than TNFR2- it was discovered that this translation block is probably due to the greater expression of miR146a by Treg TNFR2 +, hypothesis confirmed thanks to the use of antagomiRs.

34 Cycle (III year)

Boi Dalila
The research project consisted in the study and identification of protein-protein interacting inhibitors of the kinase Aurora-A and the oncoprotein N-Myc complex in neuroblastoma cells. Using bioinformatic approaches, we have identified molecules able to disrupt the complex and peptides potentially acting as competitors of the Aurora-A/N-Myc interaction. After an in vitro characterization of the binding between the two proteins, experiments were performed in neuroblastoma cell lines, which revealed the efficacy of the identified molecules in decreasing the levels of N-Myc protein, disrupting the complex and inducing the expression of markers of cell death only in MYCN-dependent cells.
Cordella Federica
The PhD project is focused on the impact of a systemic antibiotic treatment on microglia and synaptic signaling within the hippocampus. Taking advantage of electrophysiological recordings, immunofluorescence analysis and real time PCR, the effects of an altered gut-brain axis on brain homeostasis were evaluated, in particular on microglia functionality, astrocytes reactivity and synaptic functions.
De Leo Alessandro
A new library of quinolinonyl-based compounds were designed and synthesized a as anti-HIV RHIs. They were active at μM/sub-μM concentrations both in enzymatic and cell-based assays with a good cytotoxic profile. These derivatives were selective vs RH over IN, they interacted with a highly conserved region of the enzyme, also present in drug-resistant strains.

Esposito Chiara
The interaction process with ligands and inhibitors of the Aurora A kinase has been studied.

Frasca Federica
The PhD project planned (i) to determine the prevalence of respiratory viruses and (ii) to evaluate the impact of microbial infections on the respiratory antiviral immunity of patients suffering from bronchiolitis and cystic fibrosis (CF). Molecular epidemiology analysis of respiratory viruses indicated that RSV (ON1 strain), and HRV were the most frequently detected viruses, in infants with bronchiolitis and in all-age CF patients, respectively. Nasal microbiota of RSV-A bronchiolitis patients presents significant perturbations of both the nasal microbiota structure and the microbial relationships with respect to RSV-B and virus-negative. Data obtained from RSV bronchiolitis infants, stratified according to RSV genotype (NA1, ON1 and BA) revealed a weaker control of RSV-A ON1 (and BA) replication and/or an inadequate host immune response which may impact the risk of respiratory sequelae. Concerning CF disease, the data pointed out that HRV infection is associated with a profound dysregulation of the antiviral defence. In addition, HRV can promote bacteria colonization resulting in detrimental effects in CF individuals.
Garone Maria Giovanna
The PhD project was focused on the characterization of the causative link between the alterations in RNA metabolism and Amyotrophic Lateral Sclerosis (ALS), taking advantage of an iPSC-based model system that allows the disease modeling in human motor neurons (MNs) carrying a pathogenic mutation in the RNA binding protein (RBP) FUS (FUSP525L).
Messina Elena
In this project the topic of whether miRNAs can be used to overcome therapeutic problems encountered in cancer and increase treatment efficiency. It was found that in Epstein Barr Virus-related lymphomas, in ovarian cancer and in adipocyte stem cells, by modulating different miRNAs, it is possible to regulate Immune-Checkpoints, oncogenes and senescence related genes, hold in check the adverse effects of conventional chemotherapy and increase its efficiency.
Musavizadeh Zahrasadat
A combination of molecular dynamics (MD) and essential dynamics (ED) analyses was carried out to fully map the effects of phosphorylation, ADP, and conformation disrupting (CD) inhibitors (i.e., CD532 and MLN8054) on the dynamics of the Aurora-A kinase protein. Additionally, the free energy landscape based on distance from MD simulation was done to gain an overall view of the conformational landscape accessible to Aurora-A.
Souvalidou Fani
Kinase activity assays for newly synthetized compounds, based on the structure of RPM1722 against the kinase Aurora-A were carried out. Aurora-A C290A/C393A was purified and used for crystallography in complex with the previously mentioned compounds.
Spizzichino Sharon
During the PhD, the biochemical and structural characterization of SHMT1 protein-protein and protein- R(D)NA transient interactions was carried out. It was demonstrated the formation of the SHMT1-TYMS-DHFR complex both ex vivo and in vitro. Furthermore, the cryo-EM structure of the SHMT1-RNA complex was solved.
Terri Michela
During the PhD, the impact of HDAC1-3 inhibition in the adhesion process of epithelial ovarian cancer cells (EOC) on mesenchymal-like mesothelial cells (MCs) was evaluated. Using MS-275 it was demonstrated that HDAC1-3 inhibition on mesenchymal-like MCs leads to a deregulation of molecular mechanisms involved in the activation of α5β1 Integrin and consequently to a reduction of EOC cells adhesion on MCs monolayer.

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