• The activities in the year 2022/23 of the Doctoral Program in Life Sciences, as is our tradition, were carried out according to the following structure:
The doctoral students receive high-level training, which includes the courses organized, also in collaboration with the BEMM school. Each doctoral student, in addition 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):
•Seminars in the form of “readings” by course teachers and seminars by Italian and foreign researchers organized by the course.
• monthly Journal Clubs: 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 2023, the presentation took place on 16 October 2023 for first and second year PhD students.
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 38th cycle, in the presence of the students of the three years of the course. 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 encouraged to participate, at the expense of the doctorate, in conferences / workshops / courses (see activities of individual doctoral students).
The doctoral students have obtained significant results both in the field of scientific publications and also with applicative implications. It should be noted that the scientific production of the doctoral students was certainly significant (141 publications in 2022-23). Several doctoral students played a role as first author to underline their significant contribution to the experimental work.
In addition, PhD students have won competitive grants or prizes such as Sapienza "Avvio alla Ricerca" (12 grants) as well as grants for mobility or research abroad (including EMBO) and for participation to international conferences or courses (FEBS grants and prizes for best oral presentation). We also underline other significant participations by doctoral students in joint initiatives with companies or research institutions, including for application purposes (3 patents).
Among third-year doctoral students (36th cycle), who have concluded or are about to conclude their doctoral studies, there is a high percentage (>70%) of those employed or about to be employed (with grants, post-doc positions or other contract) in the academic sector and one in a company, confirming the excellent post-doctoral positioning of the students of the course.
REPORT SCIENTIFIC ACTIVITY 22/23
BUTT USMAN AKHTAR
Diverse microbial communities are observed in both healthy individuals and those with bladder cancer. These communities consist of both aerobic and anaerobic bacteria, meticulously separated through advanced techniques. The impairment of the urobiome may lead to troublesome symptoms. Consequently, bacterial colonies are methodically isolated for in-depth analysis, aimed at formulating a comprehensive protocol for understanding the microbial factors influencing a healthy state versus indicators of pathology, particularly in women, and contributing to the development of innovative interventions and biomarkers for bladder cancer.
Air pollution is the most harmful environmental toxicant in the modern world and growing evidence demonstrates a link between particulate matter exposure and an increased risk of Amyotrophic Lateral Sclerosis, Alzheimer's and Parkinson's disease. Focusing attention on Manganese (Mn), it is a trace metal element vital for biological functions but chronic exposure to Mn2+ has been associated with toxic effects on the brain. The aim of the project is to generate a 2D and 3D brain model based on hiPSCs derived neurons and analyze the effect of the manganese overexposure on the formation and maturation of nervous circuits. In order to study synaptic structures at high resolution using confocal microscopy, tissue clarification will also be applied and the collaboration with CrestOptics S.p.A will be continued as part of the PON project on green issues.To date, cultures were untreated or treated with two doses of Manganese from day 40 until the end of differentiation. I collected preliminary data about cortical populations of both control and treated groups. I performed viability assays, real-time PCR analysis and immunofluorescence analysis at different time points. Moreover, functional calcium imaging studies and patch-clamp recording were performed to analyze spontaneous intracellular calcium dynamics at network level and measure the membrane potential or the amount of current passing across the neuronal cell membrane.
DI RUSSO SARA
The tumour microenvironment has a vital influence in the formation of brain metastases. Breast cancer cells that metastasise into the brain (231 BR) use nutrients and amino acids to migrate, and have an altered metabolism due to the absence of transporters of these amino acids. Furthermore, the 231-BR cells compared to their parental counterparts have an altered NF-KB pathway and show an increased production of different cytokines that are potentially necessary for opening of the blood-brain barrier and invading the brain parenchyma.
FLORIS ERICA The depletion of the myogenic long non-coding RNA Charme in the heart impairs the maturation and functionality of resident cardiac mesenchymal stromal cells. These cells show plastic mesenchymal features, a significant depletion of the pro-fibrotic fraction, and an impoverished paracrine profile. Cardiac mesenchymal stromal cells could contribute to the altered cardiac architecture of Charme-/- mice, and their ability to face cardiac injury in the adult tissue could be impaired.
ERAP1, a crucial aminopeptidase in peptide generation for major histocompatibility complex class I (MHC-I) binding, and it has shown potential as a target for enhancing antitumor immunity since low levels of this enzyme have been associated with increased antitumor response. Our research focuses on investigating the role of ERAP1 in the tumor immune microenvironment of mouse models and exploring its modulation to improve tumor responsiveness to immunotherapy. In ex vivo assays involving ERAP1 genetic or pharmacological inhibition in neuroblastoma and melanoma murine models, we observed significant increases in apoptosis and tumor cell death, along with enhanced activation markers in certain immune cell populations.
The formalism based on Zernike polynomials can measure the level of shape and electrostatic complementarity in protein-protein complexes, distinguishing between interacting and non-interacting proteins, as well as between transient and permanent interactions (as recently demonstrated for the SARS-CoV-2 ACE2 protein complex). Moreover, it is extendable to other properties, both of a physicochemical nature, such as hydrophilic and hydrophobic contributions, and of an evolutionary nature, such as the coevolutionary process.
This method can thus be employed to develop a protocol for recognizing binding regions in interacting proteins, which could serve for a docking algorithm
Ferritin, an iron storage protein, thanks to its biocompatibility, biodegradability, and size uniformity has a greeting attraction in nanomedicine. Its capability to bind TfR1 (Transferrin Receptor 1), overexpressed by several cancer cells, renders that protein an excellent tool for the targeted delivery of small therapeutic molecules.
We selected and mutated polar/charged amino acids through genetic engineering into hydrophobic ones. This new hydrophobicity enhanced-ferritin mutant has been fully characterized and used to encapsulate small hydrophobic molecules, thus increasing their solubility and action selectivity towards cancer cells.
The modifications of the morphology and of the secondary structure of fibrils of wild type α-synuclein and of its C-terminally truncated isoform (αS103) forming in vitro in the presence and absence of total RNA have been studied by using the atomic force microscopy-assisted infrared spectroscopy (AFM-IR) technique. When total RNA is present in the aggregating solution, a faster increase of the parallel β-sheet component (a spectral marker of amyloid fibril formation) is observed accompanied by a higher rigidity of the forming α-synuclein fibrils. Furthermore, attenuated total reflectance (ATR) Fourier-transform infrared (ATR-FTIR) spectroscopy has been used to show that the new “green'' solvent Cyrene does not denaturate or modify during solubilization the native conformation of a range of proteins and enzymes: BSA, ferritin, hemoglobin, RNase, and D-amino acid oxidase for a Cyrene concentration up to 60 wt %.
KROL KAMILA JULIA
Natural killer (NK) cells are among the most powerful immune cells with anticancer activity. Their cytotoxic activity is regulated by signals from inhibitory receptors such as killer immunoglobulin-like receptors (KIRs) whose main ligands are the major histocompatibility complex (MHC) class I molecules. Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) is a protein that processes peptides by trimming them to the most favorable length to bind MHC class I. Depletion of ERAP1 in cancer cells causes a change in the repertoire of peptides presented by MHC class I molecules which enhance immunogenicity.
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by upper and lower motor neurons (MNs) degeneration, causing paralysis and death. The onset of disease can occur as subtle cramping or weakness in the limb or bulbar muscles, and then it progresses to paralysis of nearly all skeletal muscles. Some subsets of motor neurons, such as those that innervate extraocular muscles or sphincters, are spared until late in disease. However, the disease is fatal, and it usually leads to death within 3-5 years.
The first goal of my PhD project has been the generation of 3D NMOs carrying FUS P525L mutation. Since there is high variability among different lines of iPSC in the generation of organoids,
he first step was to produce a new FUS P525L line from iPSCs that have been proved suitable for the generation of NMOs. To this aim, I took advantage of a TALEN construct for FUS gene editing
to generate a new iPSC line carrying the P525L mutation from the WTSIi004-A line (EbiSC).
In the second year of my PhD, I focused on the development FUS P525L and isogenic WT NMOs and their characterization.
PELLEGRINI FRANCESCA ROMANA
Silencing of ATAT1 induces the ATG7 -dependent pathway of autophagy. In addition, ATAT1 silencing renders cancer cells more sensitive to oxidative stress and provokes an alteration of cellular metabolism. The effect on these processes is directly dependent on the absence of the enzyme and independent from the acetylation of lysine 40 residue on the alpha tubulin chain.
Adapter proteins are a class of proteins that play a fundamental role in the context of regulating cellular homeostasis, acting as a dock for different signaling molecules, allowing them to form supramolecular complexes and become functional. In particular, the FRS2 (fibroblast growth factor receptor substrate 2) protein plays a crucial role in signaling pathways by being phosphorylated by the fibroblast growth factor and nerve growth factor receptor families. FRS2 consists of the PTB domain (phosphotyrosine-binding domain) and a long disordered tail. The characterization of the folding mechanism of the PTB domain allowed to observe how the presence or absence of a disulfide bridge probably determines a conformational variation of the protein. Furthermore, this variation does not particularly influence the interaction with the two physiological ligands.
PINZON GRIMALDOS ALESSANDRA
Familial Combined Hypolipidemia (FHBL2, OMIM #605019) is a genetic disorder characterized by very low levels of plasmatic lipoproteins and lower susceptibility to cardiovascular disease caused by loss-of-function mutations in the gene encoding for Angiopoietin-like 3 (ANGPTL3).
In this context, we aimed to determine whether ANGPTL3 deficiency-induced hypolipidemia impacts on monocyte/macrophage metabolism, ER membrane stability, and inflammasome and IFN response. Then, to recapitulate in vitro the effects of poor lipid exposure on monocyte response, we have set up a system of monocyte culture in conditions of LDL deprivation or supplementation: this system allows obtaining monocytes with varying degrees of intracellular lipid accumulation. Also, in this setting, we analyzed ISG15 expression, and we detected higher ISG15 expression in lipid-deprived compared to the normal serum condition. In addition, we evaluated the presence of ER stress response through flow cytometry and quantitative Real Time PCR. Recently, we are analyzing ROS production and mitochondrial stability through flow cytometry.
The field of structural bioinformatics is currently witnessing substantial growth in available algorithms, particularly those driven by artificial intelligence. Despite the expansive opportunities created, a critical focus on the application of these algorithms is imperative. Recognizing the paramount importance of user-friendly tools, the effort is directed towards developing tools featuring graphical interfaces to facilitate both educational and research endeavors in the realm of structural bioinformatics. Simultaneously, the projects in software development extend beyond theoretical constructs to find practical applications in everyday research, specifically in the domains of protein engineering and the rational design of pharmaceuticals.
Antigen presentation and cytotoxicity assays on HEK cells for fibrous meshes with ERAP compounds encapsulated showed that ERAP_A decreases antigen presentation and increases toxicity in PVA fibers, and ERAP_E compound decreases antigen presentation in PCL fibers. Permeability assays using mass spectroscopy for ERAP_A samples showed that the compound has a good release with PCL fiber and was detected for PVA fibers in lower amounts. Electrospun fibrous meshes (PCL fibrous meshes) were a promising method to encapsulate ERAP inhibitors.
With the hope of providing a wide-spectrum treatment option for SARS-CoV-2, a non-structural protein, namely nsp13 with 99.8% conservation ratio among variants was chosen as a target. Accordingly, a novel set of eighteen different analogues of indolyl diketo acids were designed, synthesized, purified, and characterized. To evaluate the biological potency, nsp13 associated unwinding and ATPase enzymatic activities were further assessed, whose findings suggests that all the analogues, excluding one, were active at low micromolar range reaching to 4.73 ±0.5 and 1.8 ± 0.19 µM for unwinding and ATPase activity, respectively.
Silencing SEMA5A in human A375 and murine B16/F10 melanoma cells reduced their proliferation and colony formation. Elevated levels of specific semaphorins (SEMA3A, SEMA4D, SEMA3E, SEMA3F, and SEMA5A) in melanoma patients were linked to metastasis and BRAF mutations. Additionally, Bcl-2 depletion in A375 cells altered multiple semaphorin mRNA expressions, including SEMA5A. Modulation of SEMA6A and SEMA7A expression by Bcl-2 levels was confirmed in human melanoma M14 cells via western blotting.
Early diagnosis of prion diseases on easily accessible fluids is crucial for patients, physicians and caregivers. We showed that RT-QuIC test is effective on biological fluids spiked with CNS prions.
We will further develop this test with biological fluids that we are collecting from prion-affected subjects.
Loss of eff/UbcD1 (an E2 ubiquitin conjugating enzyme) gene, confers resistance to radiation-induced double-strands breaks in Drosophila melanogaster model. The overexpression of eff/UbcD1 confers radiosensitivity to DSBs, suggesting a negative role of the protein in this phenomenon. Moreover IP experiments show that eff/UbcD1 interacts with factors involved in DNA repair, like Nbs,Rad 50, Ku70, PCNA and Hp1, suggesting that is involved in the ubiquitination of these factors. A western blot- time point analysis of the expression level of g-H2Av (marker for DSBs) after irradiation, in both conditions of UbcD1 depletion/overexpression suggest that UbcD1 may alter g-H2av kinetics.
Monkeypox is a zoonotic infection endemic in both West and Central Africa, and in 2022 an unusual epidemic outbreak occurred in non-endemic countries and has generated a great concern. Despite cases of human MPXV infection occurs from many years, innate immunity against this virus, such as the Interferon (IFN) response, has not been extensively characterized. Investigation of the IFN-mediated response in samples from MPXV-infected patients reveals increased mRNA levels of type I IFNs in samples with higher MPXV DNA, while IFN-γ is highly expressed in samples with lower MPXV DNA. Pre-treatment of A549, HeLa, and VeroE6 cells with high concentrations of IFN-α and IFN-αn did not significantly inhibit MPXV replication, suggesting a possible resistant phenotype of MPXV to IFN-α. Sensitivity of MPXV to the antiviral activity of other types of IFNs needs to be investigated.
FOLLO FRANCESCA CARMEN
During the first months of the Ph.D., an automated tracking system was developed for the phenotypic analysis of Caenorhabditis elegans transgenic lines expressing human beta-amyloid at the neuronal level, GRU102 (gnaIs2) [myo-2::YFP + unc-119::Aβ1-42]), or at the muscle level, CL2120 (dvIs14) [(pCL12) unc-54::Aβ1-42 + (pCL26) mtl-2::GFP].
The data confirm that the mutant lines are characterized by locomotor and pharyngeal pumping defects, and reduced lifespan.
The analysis conducted at different time points in the animal's life showed that these defects (locomotion and pharyngeal function) are progressive, becoming more severe with aging.
project concerns the understanding of Mast Cell phenotype and function during Colorectal Cancer progression. Mast cells (MCs) are tissue-resident immune cells characterized by their high granularity and by the surface expression of the high-affinity receptor for IgE (FcεRI) and CD117/KIT, the receptor for stem cell factor (SCF) which triggers maturation, survival and activation of MCs. All together our results underscore the ability of SCF, in combination with IL-33, to shape phenotype and function of MCs that through the secretion of IL-6 and TNF-α may contribute to chronic inflammation and disease in the intestinal microenvironment.
Pathological alterations observed in tauopathies involve the development of harmful misfolded and aggregated proteins. Notably, studies have revealed the presence of Aβ plaques and hyperphosphorylated tau in the retinas of Alzheimer's Disease (AD) patients, even in early-stage cases. Additionally, the extent of tau pathology in the retina has been correlated with cognitive decline in Frontotemporal Dementia (FTD) patients. Despite ongoing research, the study of tauopathies in the retina remains an evolving field. Specifically, the project aims to investigate the impact of an intronic tau mutation related to FTD/AD on iPSC-derived retinal models. This intronic mutation leads to an increased expression of 4R tau isoforms. Given that accumulating evidence highlights the importance of maintaining an equimolar ratio of 3R and 4R tau isoforms for normal neuronal function, the research will analyze pathological alteration during retinal differentiation.
Recently, growing numbers of studies are showing how the agonists of Sigma-1 Receptor have neuroprotective effects in several diseases, like Huntington's disease. Nevertheless, the structural basis for agonism or antagonism on Sigma-1 is largely unknown. We decided to use computational methods to investigate new binding modes of Iloperidone-like structures, an antipsychotic drug which demonstrated a high binding affinity for this receptor. Starting to these data, the chemical structure of this drug will be further modified, and the proposed compounds will be synthesized and tested in vitro, to obtain a pronounced and selective agonist of the σ1R for the treatment of Huntington's disease.
This project is based on Whole Genome Sequencing (WGS) techniques, performed by rapid methods like the ONT (Oxford Nanopore Technologies), in part complemented by Illumina sequencing, bioinformatic analyses, extraction and characterization of plasmids. WGS is applied on key bacterial strains, isolated from hospitalized patients, with the aim to identify endemic clones circulating in hospitals, animals, and the environment and to describe their evolution in a global perspective. Since WGS allows the identification of microbial markers predicting and orienting empiric therapy of sepsis in critical patients, information obtained with these studies will be used to build rapid bacterial typing methods and to follow colonization and/or infection in critical patients. Due to its capacity to collect multiple antimicrobial-resistance determinants, Klebsiella pneumoniaeis is one of the biggest threats to public health. In Italy the dissemination of multidrug-resistant K. pneumoniaeis is driven by few high-risk clones expressing the KPC carbapenemase enzyme, such as those belonging to the Clonal Groups (CGs) 101, 147, 258 and 307.
Some RNA binding proteins play an important role in intercellular communication by loading RNA into the extracellular vesicles, regulating several processes such as cancer development and progression. The RBP SYNCRIP, known to play a role in loading specific microRNAs within the EVs, was silenced in hepatocarcinoma cells in order to analyze the EVs microRNA cargo and, as a consequence, the effect of the same EVs on many types of receiving cells. The microRNAs carried by the EVs derived from the silenced cancer cells were thus sequenced and analyzed through a bioinformatics pipeline developed by me.
SHMT1 and SHMT2 are metabolic proteins often found overexpressed in different types of tumors, that are able to bind specific RNA sequences and this bond inhibits their enzymatic function. Direct transfection of the above-mentioned RNA sequences on adenocarcinoma tumor cell lines causes a significant reduction in tumor cells viability. Cytotoxic effects of smaller RNA sequences have been evaluated for the implementation of a new RNA- based strategy for cancer therapy.
In order to compare the expression of genes of tissues selectively exposed to xenobiotics (in particular micro and nano plastics), an automated bioinformatic analysis pipeline was developed. This pipeline, divided into several modules and highly parallelized, performs preprocessing, then performs alignments and finally creates statistics and differential expression graphs.
Now the pipeline is in a pre-alpha state, and as such it is already able to produce analyses, albeit crude ones; in an attempt to answer the project's question, however, we also want to take the opportunity to create a tool that can draw as much information as possible from the
single data and make it as user friendly as possible for the novice, possibly integrating it with a visual data aggregation system, for use in the web app.
Allosteric inhibitors of bacterial DNA-gyrase represent a promising class of novel antibacterial agents albeit their effectiveness is hindered by a hERG mediated cardiotoxicity. Chemo-informatics techniques were applied to identify new putative inhibitors endowed with the allosteric mechanism of action and an improved safety profile. Nearly 15 millions compounds were evaluated by a hybrid virtual screening procedure using a pharmacophore model of ligand interactions (nearly 80 holo and apo crystal structures considered) and a new in-house python code to parallelize docking calculations. A group of nearly 20 molecules was selected for biological testing.
VALENTI VALENTINA FPR1 inhibition coupled with the restoration of Toll-like receptor 3 activity
protects the heart from Doxorubicin -induced cardiomyopathy. Our HYPOTHESIS: 1. Inhibition of FPR1 can protect DOX-treated mice from DOXCM. 2. FPR1 inhibition combined with pIC administration protects the heart from DOXCM and restoring the chemotherapeutic effect on tumor cells.
VENTURINO MARIA ROSARIA
The PhD project aims to develop a three-dimensional tumor-chip system to evaluate the interactions between immune and cancer cells thus contributing to develop precise and effective immunotherapies.
During the first year, the research activity was focused on: i) design and development of a three-dimensional microfluidic platform able to evaluate the interactions between immune and tumor cells, in collaboration with Dr. Luca Businaro (CNR, Rome); ii) protocol set-up for cell cultures, fluorescent dye labeling and cell loading on the 3D device to establish standard operating procedures (SOP); iii) migration analysis of immune cells, isolated from peripheral blood (PBMCs), towards cancer cells by confocal laser scanning microscopy (CLSM) and ImageJ software. In the first experiments, the Sk-Mel-28 melanoma cell line treated with an epigenetic drug combined with interferon type I (IFNa2b) was used and preferential migration of PBMCs to drug-treated cancer cells was observed, with respect to untreated control.
Moreover, in parallel experiments, the pro-apoptotic effect of drugs was evaluated, by testing caspase 3/7 directly in the tumor-on-chip, by detecting the fluorescent signal of caspase 3/7 added in both immune and tumor chambers of tumor-on-chip. These results suggest that the combined treatment of melanoma cells can induce cancer cell death by apoptosis and attract immune cells to the tumor site.