Search

Natural products from mushrooms, plants, microalgae, and cyanobacteria have been intensively explored and studied for their preventive or therapeutic potential. Among age-related pathologies, neurodegenerative diseases (such as Alzheimer's and Parkinson's diseases) represent a worldwide health and social problem. Since several pathological mechanisms are associated with neurodegeneration, promising strategies against neurodegenerative diseases are aimed to target multiple processes. These approaches usually avoid premature cell death and the loss of function of damaged neurons. This review focuses attention on the preventive and therapeutic potential of several compounds derived from natural sources, which could be exploited for their neuroprotective effect. Curcumin, resveratrol, ergothioneine, and phycocyanin are presented as examples of successful approaches, with a special focus on possible strategies to improve their delivery to the brain.

AL amyloidosis is a life-threatening disease caused by deposition of immunoglobulin light chains. While the mechanisms underlying light chains amyloidogenesis in vivo remain unclear, several studies have highlighted the role that tissue environment and structural amyloidogenicity of individual light chains have in the disease pathogenesis. AL natural deposits contain both full-length light chains and fragments encompassing the variable domain (VL) as well as different length segments of the constant region (CL), thus highlighting the relevance that proteolysis may have in the fibrillogenesis pathway. Here, we investigate the role of major truncated species of the disease-associated AL55 light chain that were previously identified in natural deposits. Specifically, we study structure, molecular dynamics, thermal stability, and capacity to form fibrils of a fragment containing both the VL and part of the CL (133-AL55), in comparison with the full-length protein and its variable domain alone, under shear stress and physiological conditions. Whereas the full-length light chain forms exclusively amorphous aggregates, both fragments generate fibrils, although, with different kinetics, aggregate structure, and interplay with the unfragmented protein. More specifically, the VL-CL 133-AL55 fragment entirely converts into amyloid fibrils microscopically and spectroscopically similar to their ex vivo counterpart and increases the amorphous aggregation of full-length AL55. Overall, our data support the idea that light chain structure and proteolysis are both relevant for amyloidogenesis in vivo and provide a novel biocompatible model of light chain fibrillogenesis suitable for future mechanistic studies.

The AMPK/SNF1 pathway governs energy balance in eukaryotic cells, notably influencing glucose de-repression. In S. cerevisiae, Snf1 is phosphorylated and hence activated upon glucose depletion. This activation is required but is not sufficient for mediating glucose de-repression, indicating further glucose-dependent regulation mechanisms. Employing fluorescence recovery after photobleaching (FRAP) in conjunction with non-linear mixed effects modelling, we explore the spatial dynamics of Snf1 as well as the relationship between Snf1 phosphorylation and its target Mig1 controlled by hexose sugars. Our results suggest that inactivation of Snf1 modulates Mig1 localization and that the kinetic of Snf1 localization to the nucleus is modulated by the presence of non-fermentable carbon sources. Our data offer insight into the true complexity of regulation of this central signaling pathway in orchestrating cellular responses to fluctuating environmental cues. These insights not only expand our understanding of glucose homeostasis but also pave the way for further studies evaluating the importance of Snf1 localization in relation to its phosphorylation state and regulation of downstream targets.

The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.

Breast and pancreatic cancer remain difficult adversaries in the landscape of oncology, characterised by their aggressive behaviour, high metastatic potential, and limited treatment efficacy. These cancer cells are continuously challenged by limited oxygen, nutrient supply, and elevated protein synthesis that may cause induction of the Unfolding Protein Response (UPR), a complex signalling network activated by Endoplasmic Reticulum (ER) stress to restore cellular homeostasis. Accumulating evidence highlights the vital role of the UPR as a critical process that enables tumour cells to sustain malignancy. On the other hand, prolonged pharmacological activation of the UPR has been demonstrated to induce cell death in cancer. Considering this, we decided to use for our experiments, FR054, a competitive inhibitor of PGM3 enzyme. By inhibiting the Hexosamine Biosynthetic Pathway (HBP), FR054 causes a decrease in the N-glycosylation (N-GlcNAc) protein level and, consequently, an accumulation of misfolded proteins into the ER. This stress condition triggers a prolonged activation of UPR that induces ROS accumulation and subsequent cell death in breast cancer (BC) cells. Otherwise, as demonstrated by the non-complete restoration after N-Acetyl Cysteine (NAC) co-treatment, in pancreatic cancer (PC) cells this ROS-dependent cell death mechanism is somewhat inhibited. Our transcriptional data suggests a potential key player in this inhibition: the xCT/SLC7A11 antiporter. This antiporter indeeed is responsible for mediating the uptake of extracellular cystine in exchange for glutamate thereby participating in glutathione biosynthesis. To confirm this hypothesis, we observed how the inhibition of xCT/SLC7A11 by erastin, a recognized ferroptosis inducer, significantly enhanced the FR054 effect causing a noteworthy increase in cancer cell proliferation arrest and death. In conclusion, HBP inhibition triggers UPR in both breast and pancreatic cancer cells. Notably, pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable clinical challenge due to its aggressiveness and resistance to treatment. Notably, PDAC exhibits a pronounced resistance to various therapeutic modalities, often attributed to a wide metabolic reprogramming involving also the Hexosamine Biosynthetic Pathway (HBP). Transcriptional and bioinformatics analyses, performed under inhibition of HBP in PDAC cell models, indicated that activation of ER stress and Unfolded Protein Response (UPR), due to HBP inhibition, activates an NRF2-dependent antioxidative response, characterized by activation of several genes involved in glutathione (GSH) metabolism. Indeed, inhibition of both HBP and the xCT protein, the cystine/glutamate antiporter involved in GSH synthesis, in PDAC cells significantly reduced cell proliferation, enhanced cell death, altered cellular redox status, heightened sensitivity to oxidative stress, increased dependence on glutamine metabolism, and induced ferroptosis. These findings underscore the potential of HBP inhibition to synergize with ERA, offering a promising therapeutic strategy for managing PDAC.

The overlap between the geographic distribution of COVID-19 outbreaks and pollution levels confirmed a correlation between exposure to atmospheric particulate matter (PM) and the SARS-CoV-2 pandemic. The RAS system is essential in the pathogenesis of inflammatory diseases caused by pollution: the ACE/AngII/AT1 axis activates a pro-inflammatory pathway, which is counteracted by the ACE2/Ang(1-7)/MAS axis, which activates an anti-inflammatory and protective pathway. However, ACE2 is also known to act as a receptor through which SARS-CoV-2 enters host cells to replicate. Furthermore, in vivo systems have demonstrated that exposure to PM increases ACE2 expression. In this study, the effects of acute and sub-acute exposure to ultrafine particles (UFP), originating from different anthropogenic sources (DEP and BB), on the levels of ACE2, ACE, COX-2, HO-1, and iNOS in the lungs and other organs implicated in the pathogenesis of COVID-19 were analyzed in the in vivo BALB/c male mice model. Exposure to UFP alters the levels of ACE2 and/or ACE in all examined organs, and exposure to sub-acute DEP also results in the release of s-ACE2. Furthermore, as evidenced in this and our previous works, COX-2, HO-1, and iNOS levels also demonstrated organ-specific alterations. These proteins play a pivotal role in the UFP-induced inflammatory and oxidative stress responses, and their dysregulation is linked to the development of severe symptoms in individuals infected with SARS-CoV-2, suggesting a heightened vulnerability or a more severe clinical course of the disease. UFP and SARS-CoV-2 share common pathways; therefore, in a “risk stratification” concept, daily exposure to air pollution may significantly increase the likelihood of developing a severe form of COVID-19, explaining, at least in part, the greater lethality of the virus observed in highly polluted areas.

Osteoarthritis (OA) is a highly disabling pathology, characterized by synovial inflammation and cartilage degeneration. Orthobiologics have shown promising results in OA treatment thanks to their ability to influence articular cells and modulate the inflammatory OA environment. Considering their complex mechanism of action, the development of reliable and relevant joint models appears as crucial to select the best orthobiologics for each patient. The aim of this study was to establish a microfluidic OA model to test therapies in a personalized human setting. The joint-on-a-chip model included cartilage and synovial compartments, containing hydrogel-embedded chondrocytes and synovial fibroblasts, separated by a channel for synovial fluid. For the cartilage compartment, a Hyaluronic Acid-based matrix was selected to preserve chondrocyte phenotype. Adding OA synovial fluid induced the production of inflammatory cytokines and degradative enzymes, generating an OA microenvironment. Personalized models were generated using patient-matched cells and synovial fluid to test the efficacy of mesenchymal stem cells on OA signatures. The patient-specific models allowed monitoring changes induced by cell injection, highlighting different individual responses to the treatment. Altogether, these results support the use of this joint-on-a-chip model as a prognostic tool to screen the patient-specific efficacy of orthobiologics.

Neuronal differentiation is regulated by nerve growth factor (NGF) and other neurotrophins. We explored the impact of NGF on mitochondrial dynamics and metabolism through time-lapse imaging, metabolomics profiling, and computer modeling studies. We show that NGF may direct differentiation by stimulating fission, thereby causing selective mitochondrial network fragmentation and mitophagy, ultimately leading to increased mitochondrial quality and respiration. Then, we reconstructed the dynamic fusion–fission–mitophagy cycling of mitochondria in a computer model, integrating these processes into a single network mechanism. Both the computational model and the simulations are able to reproduce the proposed mechanism in terms of mitochondrial dynamics, levels of reactive oxygen species (ROS), mitophagy, and mitochondrial quality, thus providing a computational tool for the interpretation of the experimental data and for future studies aiming to detail further the action of NGF on mitochondrial processes. We also show that changes in these mitochondrial processes are intertwined with a metabolic function of NGF in differentiation: NGF directs a profound metabolic rearrangement involving glycolysis, TCA cycle, and the pentose phosphate pathway, altering the redox balance. This metabolic rewiring may ensure: (a) supply of both energy and building blocks for the anabolic processes needed for morphological reorganization, as well as (b) redox homeostasis.

Liquid-liquid phase separation (LLPS) is pivotal in forming biomolecular condensates, which are crucial in several biological processes. Intrinsically disordered regions (IDRs) are typically responsible for driving LLPS due to their multivalency and high content of charged residues that enable the establishment of electrostatic interactions. In our study, we examined the role of charge distribution in the condensation of the disordered N-terminal domain of human topoisomerase I (hNTD). hNTD is densely charged with oppositely charged residues evenly distributed along the sequence. Its LLPS behavior was compared with that of charge permutants exhibiting varying degrees of charge segregation. At low salt concentrations, hNTD undergoes LLPS. However, LLPS is inhibited by high concentrations of salt and RNA, disrupting electrostatic interactions. Our findings show that, in hNTD, moderate charge segregation promotes the formation of liquid condensates that are sensitive to salt and RNA, whereas marked charge segregation results in the formation of aberrant condensates. Although our study is based on a limited set of protein variants, it supports the applicability of the “stickers-and-spacers” model to biomolecular condensates involving highly charged IDRs. These results may help generate reliable models of the overall LLPS behavior of supercharged polypeptides.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by ataxia and other neurological manifestations, with a poor prognosis and a lack of effective therapies. The amyloid aggregation of the ataxin-3 protein is a hallmark of SCA3 and one of the main biochemical events prompting its onset, making it a prominent target for the development of preventive and therapeutic interventions. Here, we tested the efficacy of an aqueous Lavado cocoa extract and its polyphenolic components against ataxin-3 aggregation and neurotoxicity. The combination of biochemical assays and atomic force microscopy morphological analysis provided clear evidence of cocoa flavanols’ ability to hinder ATX3 amyloid aggregation through direct physical interaction, as assessed by NMR spectroscopy. The chemical identity of the flavanols was investigated by ultraperformance liquid chromatography-high-resolution mass spectrometry. The use of the preclinical model Caenorhabditis elegans allowed us to demonstrate cocoa flavanols’ ability to ameliorate ataxic phenotypes in vivo. To the best of our knowledge, Lavado cocoa is the first natural source whose extract is able to directly interfere with ATX3 aggregation, leading to the formation of off-pathway species.

Microglial cells play a key role in protecting the central nervous system from pathogens and toxic compounds and are involved in the pathogenesis of different neurodegenerative diseases. Cadmium is a widespread toxic heavy metal, released into the environment at a rate of 30,000 tons/year by anthropogenic activities; it is easily uptaken by the human body through diet and cigarette smoke, as well as by occupational exposure. Once inside the body, cadmium enters the cells and substitutes to zinc and other divalent cations altering many biological functions. Its extremely long half-life makes it a serious health threat. Recent data suggest a role for heavy metals in many neurodegenerative diseases; however, the role of cadmium is still to be elucidated. In this work we report the investigation of cadmium toxicity towards murine BV2 microglial cells, a widely used model for the study of neurodegeneration. Results show that increasing cadmium concentrations increase oxidative stress, a proposed mechanism of neurodegeneration, but also that BV2 cells can keep oxidative stress under control by increasing glutathione reduction. Moreover, cadmium induces alterations of cell morphology and metabolism leading to mitochondrial impairment, without switching the cells to Warburg effect. Finally cadmium induces the release of proinflammatory cytokines, but does not markedly switch BV2 cells to M1 phenotype.

Modeling human neuronal properties in physiological and pathological conditions is essential to identify novel potential drugs and to explore pathological mechanisms of neurological diseases. For this purpose, we generated a three-dimensional (3D) neuronal culture, by employing the readily available human neuroblastoma SH-SY5Y cell line, and a new differentiation protocol. The entire differentiation process occurred in a matrix and lasted 47 days, with 7 days of pre-differentiation phase and 40 days of differentiation, and allowed the development of a 3D culture in conditions consistent with the physiological environment. Neurons in the culture were electrically active, were able to establish functional networks, and showed features of cholinergic neurons. Hence here we provide an easily accessible, reproducible, and suitable culture method that might empower studies on synaptic function, vesicle trafficking, and metabolism, which sustain neuronal activity and cerebral circuits. Moreover, this novel differentiation protocol could represent a promising cellular tool to study physiological cellular processes, such as migration, differentiation, maturation, and to develop novel therapeutic approaches.

Le nanotecnologie stanno rivoluzionando il settore medico grazie al potenziale terapeutico e diagnostico delle nanoparticelle (NP). Le NP proteiche hanno ottenuto particolare attenzione come veicoli per la somministrazione di farmaci. Tra di esse spicca la NP vault, la più grande particella ribonucleoproteica conosciuta e presente naturalmente nelle cellule degli eucarioti superiori. La vault è costituita da 78 copie della Major Vault Protein (MVP) che forma una struttura a barile, in grado di racchiudere altre proteine, come la poli (ADP-ribosio) polimerasi, la proteina 1 associata alla telomerasi e alcuni RNA non tradotti. I ruoli fisiologici della vault non sono del tutto chiari ma si riporta essere coinvolta in diverse funzioni cellulari per la sopravvivenza di citoprotezione. La vault presenta caratteristiche uniche: non è immunogenica, è biodegradabile e ha una struttura dinamica e una cavità spaziosa, che la rendono idonea per la somministrazione mirata di molecole terapeutiche, spesso destinate alle linee cellulari tumorali. Tuttavia, la produzione e la purificazione di vault rappresentano un processo complesso. Nel capitolo 1, è presentata una procedura semplificata che combina l'espressione di vault nel lievito Komagataella phaffii con una procedura di purificazione che coinvolge il pretrattamento con RNasi seguito da cromatografia a esclusione molecolare. Lo studio affronta anche il problema dell'aggregazione proteica, identificando le condizioni di conservazione ottimali. La NP vault è capace di incapsulare proteine cargo se fuse con il dominio INT, derivato dalla proteina vPARP. Nel capitolo 2, viene esplorata l’incapsulazione di GFP-INT come proteina cargo all'interno di vault, prodotte con una procedura semplificata. L'incapsulazione di GFP-INT sfrutta la proprietà di "respirazione" delle vault. Il lavoro ha anche valutato la stechiometria del carico di GFP-INT nella vault, evidenziando una saturazione con circa 20 copie di GFP-INT. Inoltre, e, In recent years, nanotechnology have made a significant impact in the field of medicine through the remarkable therapeutic and diagnostic potential of nanoparticles (NPs). Among these, protein-based NPs have gained considerable attention as drug delivery systems and vaults emerge as ideal candidates. Vaults are the largest known ribonucleoprotein particles naturally occurring in higher eukaryotic cells. 78 copies of the dominant component Major Vault Protein (MVP) assemble into a barrel-like "nano-capsule”, enclosing other proteins like poly (ADP-ribose) polymerase, telomerase-associated protein-1, and some small untranslated RNAs. While the precise physiological roles of this nanocomplex are not completely understood, it has been associated with numerous cellular functions that promote cell survival and provide cytoprotective effects. Vaults possess unique properties, including non-immunogenicity, biodegradability, dynamic structure, and a spacious cavity, making them highly attractive for delivery of therapeutic molecules, often targeted to cancer cell lines. However, existing production and purification protocols for vault are complex, partly due to their reliance on higher eukaryotes as expression systems. In Chapter 1, a newly published simplified procedure is presented, combining human vault expression in the yeast Komagataella phaffii and a purification process involving RNase pretreatment followed by size-exclusion chromatography, significantly streamlining the process. The study also addresses the challenge of protein aggregation and identifies optimal storage conditions. One remarkable vault feature is its ability to encapsulate cargo proteins by fusing them with the INT domain, derived from the vault-interacting vPARP protein. Chapter 2 explores the encapsulation of GFP-INT as a reporter cargo within vaults expressed and purified using the simplified procedure. Encapsulation of GFP-INT was achieved through a straightforward mixing process, harnessing vaul

Protein fibrillation is commonly associated with pathologic amyloidosis. However, under appropriate conditions several proteins form fibrillar structures in vitro that can be used for biotechnological applications. MNEI and its variants, firstly designed as single chain derivatives of the sweet protein monellin, are also useful models for protein fibrillary aggregation studies. In this work, we have drawn attention to a protein dubbed Mut9, already characterized as a super stable MNEI variant. Comparative analysis of the respective X-ray structures revealed how the substitutions present in Mut9 eliminate several unfavorable interactions and stabilize the global structure. Molecular dynamic predictions confirmed the presence of a hydrogen-bonds network in Mut9 which increases its stability, especially at neutral pH. Thioflavin-T (ThT) binding assays and Fourier transform infrared (FTIR) spectroscopy indicated that the aggregation process occurs both at acidic and neutral pH, with and without addition of NaCl, even if with a different kinetics. Accordingly, Transmission Electron Microscopy (TEM) showed a fibrillar organization of the aggregates in all the tested conditions, albeit with some differences in the quantity and in the morphology of the fibrils. Our data underline the great potential of Mut9, which combines great stability in solution with the versatile conversion into nanostructured biomaterials

Laccases are multicopper oxidases able to oxidize several phenolic compounds and find application in numerous industrial applications. Among laccase producers, white-rot fungi represent a valuable source of multiple isoforms and isoenzymes of these multicopper oxidases. Here we describe the identification, biochemical characterization, and application of laccase 2 from Trametes polyzona (TP-Lac2), a basidiomycete fungus emerged among others that have been screened by plate assay. This enzyme has an optimal temperature of 50 degrees C and in acidic conditions it is able to oxidize both phenolic and non-phenolic compounds. The ability of TP-Lac2 to decolorize textile dyes was tested in the presence of natural and synthetic mediators at 30 degrees C and 50 degrees C. Our results indicate that TP-Lac2 most efficiently decolorizes (decolorization rate > 75%) malachite green oxalate, orange G, amido black10B and bromocresol purple in the presence of acetosyringone and 2,2 '-azinobis (3-ethylbenzthiazoline-6-sulfonate)-ABTS. Overall, the laccase mediator system consisting of TP-Lac2 and the natural mediator acetosyringone has potential as an environmentally friendly alternative for wastewater treatment in the textile industry.

Il tumore alla vescica è uno dei più diffusi a livello mondiale. Spesso non presenta sintomi e l’alta frequenza di tumori non muscolo invasivi ad alto grado è associata ad un elevato rischio di recidiva, con la necessità di controlli periodici, con impatti a livello economico. Le recidive portano alla progressione a tumori muscolo invasivi correlati ad una prognosi sfavorevole. I metodi di diagnosi più utilizzati sono la cistoscopia e la TURBT, quest’ultima utilizzata anche a scopo terapeutico. Tuttavia, spesso si verifica un’incompleta rimozione del tumore, dovuta ad una difficoltà nello stabilire l’entità della penetrazione, con aumento della probabilità di diagnosi errate e insorgenza di recidive. Diventa quindi cruciale identificare marcatori che permettano di assegnare, al momento della resezione, la corretta classificazione, migliorando la prognosi. La riprogrammazione del metabolismo è considerato un tratto distintivo della crescita tumorale, e lo studio dei processi metabolici alterati nella patologia può rappresentare un bersaglio clinico utilizzabile per nuove strategie terapeutiche. Questa tesi, nata dal progetto Horizon2020 AMPLITUDE (Advanced Multimodal Photonics Laser Imaging Tool for Urothelial Diagnosis in Endoscopy), riassume il percorso svoltosi nel corso di tre anni, volto a studiare le caratteristiche funzionali e metaboliche di un pannello di sei linee cellulari di tumore alla vescica, con lo scopo di trovare un marcatore indicativo della progressione tumorale. Una prima caratterizzazione delle linee cellulari cresciute in colture 2D ha rivelato un’eterogeneità intrinseca tra queste cellule. Le cellule muscolo invasive hanno mostrato un ampio spettro di proprietà morfo-funzionali che garantisce loro di manifestare un fenotipo più aggressivo rispetto alle linee non muscolo invasive. Allo stesso modo, anche il fenotipo metabolico si è rivelato altamente eterogeneo, senza correlazioni con la progressione tumorale. Tuttavia, è stato p, Bladder cancer (BC) is one of the most common malignancies worldwide. Symptoms are often absent and the frequent non muscle invasive (NMIBC) high grade tumors are associated to a high risk of relapses, resulting in high costs for patient follow-up. Frequent relapses often progress to muscle invasive tumors (MIBC), that result in a poor prognosis. Cystoscopy and transurethral resection of bladder tumor (TURBT) are the most used diagnostic methods. However, the frequent incomplete resections, related to the difficulty to establish the level of penetration of the tumor, cause most of the wrong diagnoses and relapse onsets. Therefore, identifying new markers is crucial to attribute the correct classification at the time of resection, improving patient’s prognosis. Energy metabolism reprogramming is an established cancer hallmark, and altered metabolic pathways can represent attractive clinical targets exploitable in new therapeutic strategies. This thesis, part of the Horizon2020 project AMPLITUDE (Advanced Multimodal Photonics Laser Imaging Tool for Urothelial Diagnosis in Endoscopy), represents the path followed these three years to study the functional and metabolic features of a panel of six bladder cancer cell lines, to find a marker for cancer progression. A first characterization of the cell lines grown in 2D models showed the intrinsic heterogeneity between these cells. MIBC cell lines showed a range of morpho-functional properties that allow them the manifestation of a more aggressive phenotype, compared to the two NMIBC cell lines. The metabolic phenotype as well showed high heterogeneity not relatable with the cancer progression, although some features correlated with the genetic background of the cell lines. From 2D models, we decided to grow cells forming spheroids, known to be more representative for the in vivo pathology, better simulating the architectural complexity of a tumor mass. The characterization of the 2D to 3D transition confirmed that the hete

Animal testing is the current standard for drug and chemicals safety assessment, but hazards translation to human is uncertain. Human in vitro models can address the species translation but might not replicate in vivo complexity. Herein, we propose a network-based method addressing these translational multiscale problems that derives in vivo liver injury biomarkers applicable to in vitro human early safety screening. We applied weighted correlation network analysis (WGCNA) to a large rat liver transcriptomic dataset to obtain co-regulated gene clusters (modules). We identified modules statistically associated with liver pathologies, including a module enriched for ATF4-regulated genes as associated with the occurrence of hepatocellular single-cell necrosis, and as preserved in human liver in vitro models. Within the module, we identified TRIB3 and MTHFD2 as a novel candidate stress biomarkers, and developed and used BAC-eGFPHepG2 reporters in a compound screening, identifying compounds showing ATF4-dependent stress response and potential early safety signals.

Since the mid-1960s, methylotrophic yeast Komagataella phaffii (previously described as Pichia pastoris) has received increasing scientific attention. The interest for the industrial production of proteins for different applications (e.g., feed, food additives, detergent, waste treatment processes, and textile) is a well-consolidated scientific topic, and the importance for this approach is rising in the current era of environmental transition in human societies. This review aims to summarize fundamental and specific information in this scientific field. Additionally, an updated description of the relevant products produced with K. phaffii at industrial levels by a variety of companies—describing how the industry has leveraged its key features, from products for the ingredients of meat-free burgers (e.g., IMPOSSIBLETM FOODS, USA) to diabetes therapeutics (e.g., Biocon, India)—is provided. Furthermore, active patents and the typical workflow for industrial protein production with this strain are reported.

Mitochondria have been the focus of extensive research for decades since their dysfunction is linked to more than 150 distinct human disorders. Despite considerable efforts, researchers have only been able to skim the surface of the mitochondrial social complexity and the impact of inter-organelle and inter-organ communication alterations on human health. While some progress has been made in deciphering connections among mitochondria and other cytoplasmic organelles through direct (i.e., contact sites) or indirect (i.e., inter-organelle trafficking) crosstalk, most of these efforts have been restricted to a limited number of proteins involved in specific physiological pathways or disease states. This research bottleneck is further narrowed by our incomplete understanding of the cellular alteration timeline in a specific pathology, which prevents the distinction between a primary organelle dysfunction and the defects occurring due to the disruption of the organelle’s interconnectivity. In this perspective, we will (i) summarize the current knowledge on the mitochondrial crosstalk within cell(s) or tissue(s) in health and disease, with a particular focus on neurodegenerative disorders, (ii) discuss how different large-scale and targeted approaches could be used to characterize the different levels of mitochondrial social complexity, and (iii) consider how investigating the different expression patterns of mitochondrial proteins in different cell types/tissues could represent an important step forward in depicting the distinctive architecture of inter-organelle communication.

The production of synthetic plastics, especially polyethylene, has reached a crucial level, making highly challenging plastic waste management. This paper aims to elucidate the role of two novel laccase-like multicopper oxidases in polyethylene oxidative degradation based on transcriptomic data from Rhodococcus opacus R7 grown on polyethylene. The purification of the recombinant proteins, LMCO2 and LMCO3 belonging respectively to the three- and two-domains laccase families, showed that they are endowed with different functional features. LMCO2 showed the highest enzyme activity at 65 °C with an optimal pH of 7.0, while LMCO3 exhibited the highest activity at 80 °C at acidic pH. LMCOs showed activity on both phenolic (i.e., 2,6-DMP) and non-phenolic (i.e., ABTS) compounds and they were thermostable. The oxidative activity of LMCO2 and LMCO3 on untreated low-density polyethylene was assessed by combining Fourier transform infrared spectroscopy and gas chromatography coupled with mass spectrometry analyses. Oxidation of PE was recorded within a short time range (24-48 h) and revealed previously not described patterns of alkyl compounds and oxygenated products including ketones, alcohols, and carboxylic acids. Structural analysis of LMCO2 and LMCO3 together with density functional theory calculations allowed to identify structural and electronic elements presumably involved in the oxidation of polyethylene.

Marine environments, especially coral reefs, are the source of a wide range of bioactive natural products with structurally unique features. Secondary metabolites extracted from soft corals have been extensively studied for their potential pharmacological properties of interest to human health, especially concerning their possible cytotoxic effect against different cancer cell lines. Our recently published review related to this topic focuses on Alcyoniidae, one of the largest octocoral families. From this research, 344 cytotoxic compounds were found, most of which have been discovered in the last 20 years proving that this particular research field only recently exploded. To prove and underline the potential of this research, 21 of these secondary metabolites displayed comparable and even higher cytotoxic activity than the chemotherapy drug used as a positive control in the various studies against specific cancer lines. However, the important comparison between the viability study on the cancer cells and on the normal one of the same tissues remains to be further investigated. In the initial part of our research project, we extracted the Maldivian soft coral Sarcophyton sp. by using ethyl acetate and dichloromethane solvents. Moreover, further purification by column chromatography of the ethyl acetate extract led to three fractions and the metabolites profile was determined by applying a multi- analytical approach based on mass spectrometry and by comparison with reference data from the current literature. By MTT assay was found that ethyl acetate and dichloromethane crude extracts caused a reduction in viability of human colorectal cancer (CRC) cell lines SW480 and E705, while were only weakly active against human healthy mucosa cell line CCD841. Furthermore, fraction 2 and 3 displayed cytotoxicity against CRC cell lines while showing a lower effect toward CCD841. This study provides a better understanding of the chemical nature of Sarcophyton sp. and indicates a

Secondary metabolites extracted from soft corals have been extensively studied for their potential pharmacological properties of interest to human health especially in relation to their possible cytotoxic effect against different cancer cell lines. However, the comparison between the viability study on the cancer cell lines and on the healthy cells of the same tissue remains to be further investigated. In this work, the soft coral Sarcophyton sp. was extracted by using ethyl acetate and dichloromethane solvents. Moreover, further purification by column chromatography of the ethyl acetate extract led to three fractions and the metabolites profile was determined by applying a multi-analytical approach based on mass spectrometry and by comparison with reference data from the current literature. By MTT assay was found that ethyl acetate and dichloromethane crude extracts caused a reduction in viability of human colorectal cancer (CRC) cell lines SW480 and E705, while were only weakly active against human healthy mucosa cell line CCD841. Furthermore, fraction 2 and fraction 3 displayed cytotoxicity against CRC cell lines while showed a lower effect towards CCD841. This study provides a better understanding of the chemical nature of Sarcophyton sp. and indicates a promising fraction which represents an excellent starting point for further work on the isolation, structural characterization, and biochemical investigation of new compounds with a potential anticancer effect. In addition, the future aim will be to extend the study to other soft corals genera as well as other marine organisms.

Colorectal cancer (CRC) is the second-leading cause of cancer death, with a worldwide incidence rate constantly increasing; thus, new strategies for its prevention or treatment are needed. Here, we describe the adjuvant effect of the polyphenol-enriched fractions of cinnamon, from cinnamon bark and buds, when co-administered with a potent anticancer drug, cetuximab, used for CRC therapy. The co-administration significantly reduces the cetuximab dose required for the antiproliferative activity against colorectal cancer cell line E705, which is sensitive to EGFR-targeted therapy. The anticancer activity of these cinnamon-derived fractions, whose major components (as assessed by UPLC–HRMS analysis) are procyanidins and other flavonoids, strictly correlates with their ability to induce apoptosis in cancer cell lines through ERK activation and the mitochondrial membrane potential impairment. Due to the severe side effects of cetuximab administration, our results suggest the use of nutraceuticals based on the polyphenolic fractions of cinnamon extracts as adjuvants in the therapy of CRC.

Coronary artery disease (CAD) is the most common type of heart disease and represents the leading cause of death in both men and women worldwide. Early detection of CAD is crucial for decreasing mortality, prolonging survival, and improving patient quality of life. Herein, a non-invasive is described, nanoparticle-based diagnostic technology which takes advantages of proteomic changes in the nano-bio interface for CAD detection. Nanoparticles (NPs) exposed to biological fluids adsorb on their surface a layer of proteins, the “protein corona” (PC). Pathological changes that alter the plasma proteome can directly result in changes in the PC. By forming disease-specific PCs on six NPs with varying physicochemical properties, a PC-based sensor array is developed for detection of CAD using specific PC pattern recognition. While the PC of a single NP may not provide the required specificity, it is reasoned that multivariate PCs across NPs with different surface chemistries, can provide the desirable information to selectively discriminate the condition under investigation. The results suggest that such an approach can detect CAD with an accuracy of 92.84%, a sensitivity of 87.5%, and a specificity of 82.5%. These new findings demonstrate the potential of PC-based sensor array detection systems for clinical use.

Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. l-Serine is the precursor of the two main co-agonists of the N-methyl-d-aspartate receptor, glycine and d-serine. Strikingly, dysfunctions in both l- and d-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l-serine, glycine, threonine, l- and d-aspartate (which level is unexpectedly higher than that of d-serine) show the same biosynthetic time course. A significant utilization of l-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d-serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP). Methods: Transcriptional and bioinformatics analysis were performed to obtain information about the effect of the HBP inhibition in two cell models of PDAC. Cell count, western blot, HPLC and metabolomics analyses were used to determine the impact of the combined treatment between an HBP’s Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival. Results: Here we show that the combined treatment applied to different PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death. Conclusion: Our study discloses that HBP inhibition enhances, via UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy.

Heterogeneity describes the differences among cancer cells within and between tumors. It refers to cancer cells describing variations in morphology, transcriptional profiles, metabolism, and metastatic potential. More recently, the field has included the characterization of the tumor immune microenvironment and the depiction of the dynamics underlying the cellular interactions promoting the tumor ecosystem evolution. Heterogeneity has been found in most tumors representing one of the most challenging behaviors in cancer ecosystems. As one of the critical factors impairing the long-term efficacy of solid tumor therapy, heterogeneity leads to tumor resistance, more aggressive metastasizing, and recurrence. We review the role of the main models and the emerging single-cell and spatial genomic technologies in our understanding of tumor heterogeneity, its contribution to lethal cancer outcomes, and the physiological challenges to consider in designing cancer therapies. We highlight how tumor cells dynamically evolve because of the interactions within the tumor immune microenvironment and how to leverage this to unleash immune recognition through immunotherapy. A multidisciplinary approach grounded in novel bioinformatic and computational tools will allow reaching the integrated, multilayered knowledge of tumor heterogeneity required to implement personalized, more efficient therapies urgently required for cancer patients.

Progranulin is a pleiotropic growth factor with important physiological roles in embryogenesis and maintenance of adult tissue homeostasis. While-progranulin deficiency is associated with a broad range of pathological conditions affecting the brain, such as frontotemporal dementia and neuronal ceroid lipofuscinosis, progranulin upregulation characterizes many tumors, including brain tumors, multiple myeloma, leiomyosarcoma, mesothelioma and epithelial cancers such as ovarian, liver, breast, bladder, adrenal, prostate and kidney carcinomas. The increase of progranulin levels in tumors might have diagnostic and prognostic significance. In cancer, progranulin has a pro-tumorigenic role by promoting cancer cell proliferation, migration, invasiveness, anchorage-independent growth and resistance to chemotherapy. In addition, progranulin regulates the tumor microenvironment, affects the function of cancer-associated fibroblasts, and modulates tumor immune surveillance. However, the molecular mechanisms of progranulin oncogenic function are not fully elucidated. In bladder cancer, progranulin action relies on the activation of its functional signaling receptor EphA2. Notably, more recent data suggest that progranulin can also modulate a functional crosstalk between multiple receptor-tyrosine kinases, demonstrating a more complex and context-dependent role of progranulin in cancer. Here, we will review what is currently known about the function of progranulin in tumors, with a focus on its molecular mechanisms of action and regulation.

Artificial intelligence is getting a foothold in medicine for disease screening and diagnosis. While typical machine learning methods require large labeled datasets for training and validation, their application is limited in clinical fields since ground truth information can hardly be obtained on a sizeable cohort of patients. Unsupervised neural networks – such as Self-Organizing Maps (SOMs) – represent an alternative approach to identifying hidden patterns in biomedical data. Here we investigate the feasibility of SOMs for the identification of malignant and non-malignant regions in liquid biopsies of thyroid nodules, on a patient-specific basis. MALDI-ToF (Matrix Assisted Laser Desorption Ionization - Time of Flight) mass spectrometry-imaging (MSI) was used to measure the spectral profile of bioptic samples. SOMs were then applied for the analysis of MALDI-MSI data of individual patients’ samples, also testing various pre-processing and agglomerative clustering methods to investigate their impact on SOMs’ discrimination efficacy. The final clustering was compared against the sample's probability to be malignant, hyperplastic or related to Hashimoto thyroiditis as quantified by multinomial regression with LASSO. Our results show that SOMs are effective in separating the areas of a sample containing benign cells from those containing malignant cells. Moreover, they allow to overlap the different areas of cytological glass slides with the corresponding proteomic profile image, and inspect the specific weight of every cellular component in bioptic samples. We envision that this approach could represent an effective means to assist pathologists in diagnostic tasks, avoiding the need to manually annotate cytological images and the effort in creating labeled datasets.

Il cancro è una delle principali cause di morte nel mondo e glioblastoma e tumore al seno triplo negativo sono due tipologie di tumore caratterizzate da una prognosi sfavorevole e da un alto rischio di recidive. I trattamenti farmacologici oggi a disposizione non sono sufficienti all’ottenimento di una guarigione completa sia a causa della bassa selettività che impedisce l’utilizzo di dosaggi più elevati, sia a causa dello sviluppo di meccanismi di resistenza da parte delle cellule tumorali. In questo contesto le nanoparticelle possono essere sviluppate sia come nuovi trattamenti terapeutici sia per superare alcune limitazioni delle terapie convenzionali. In questo progetto di tesi, ho scelto di utilizzare nanoparticelle di H-ferritina (HFn), una forma ricombinante di ferritina che possiede alcune caratteristiche che la rendono un buon candidato per migliorare i trattamenti terapeutici già disponibili per questi due tumori. L’HFn è infatti biocompatibile, la sua cavità interna può essere utilizzata per il caricamento di farmaci e molecole di detection e la sua superficie può essere facilmente modificata sfruttando i numerosi gruppi funzionali esposti verso l’esterno. La HFn è, inoltre, in grado di legarsi al recettore della transferrina (TfR1), over-espresso dal 98% dei tumori solidi. Tale recettore è over espresso anche dalle cellule endoteliali che compongono la barriera ematoencefalica, rendendo così la HFn in grado di attraversarla. Il mio lavoro di tesi può essere suddiviso in due progetti. Nel primo progetto i gruppi funzionali esposti sulla superficie della HFn sono stati utilizzati per la coniugazione dell’anticorpo monoclonale cetuximab al fine di studiare il mantenimento della sua attività antitumorale in modelli tumorali 3D e di paragonarla a quella del cetuximab non coniugato. Come modelli tumorali 3D sono stati utilizzati sferoidi ottenuti da una linea cellulare di glioblastoma e da una di tumore al seno triplo negativo. Sulla base dei prom, Cancer is a leading cause of disease worldwide and glioblastoma and triple negative breast cancer represent two types of tumors with poor prognosis and a high risk of recurrence. The therapeutic options currently in use are still subject to failure, due to (1) the poor selectivity of drugs, which compromises dose escalation to more therapeutically active levels and (2) the development of resistance by different mechanisms that cells put in place to defend themselves from the action of drugs. In this scenario, nanoparticles can be developed as new therapeutic strategies or to overcome the limitations of the treatments currently in use. In my PhD project, I exploited the unique features of a nanoparticle-based on recombinant heavy-chain ferritin cages (HFn) to improve the therapeutic treatments already available for these tumors. HFn can be a promising candidate for this study because 1) it is biocompatible; 2) it can be loaded with drugs; 3) it can be easily modified by exploiting the functional groups on its surface; 4) it exhibits tumor targeting by recognition of TfR1, overexpressed in 98% of human cancers and directly correlated with tumor stage or cancer progression; 5) HFn is able to cross the blood-brain barrier (BBB) exploiting the same TfR1 receptor, also found overexpressed on the BBB endothelial wall. My work is divided into two main subprojects, all sharing the use of HFn. In the first subproject (chapter one), the functional groups present on the surface of HFn were exploited for the conjugation of the monoclonal antibody cetuximab, studying the maintenance of its anticancer activity against a 3D tumor model in comparison with unconjugated cetuximab. Cancer spheroids of glioblastoma and triple negative breast cancer were selected and produced for this study. Then we focused on the documented ability of cetuximab to promote an indirect anticancer effect (ADCC) through the activation of immune systems cells. ADCC activation was evaluated both in live imagi

The AMP-activated protein kinase (AMPK) and the target of rapamycin complex 1 (TORC1) are central kinase modules of two opposing signaling pathways that control eukaryotic cell growth and metabolism in response to the availability of energy and nutrients. Accordingly, energy depletion activates AMPK to inhibit growth, while nutrients and high energy levels acti-vate TORC1 to promote growth. Both in mammals and lower eukaryotes such as yeast, the AMPK and TORC1 pathways are wired to each other at different levels, which ensures homeostatic control of growth and metabolism. In this context, a previous study (Hughes Hallett et al., 2015) reported that AMPK in yeast, that is Snf1, prevents the transient TORC1 reactivation during the early phase following acute glucose starvation, but the underlying mechanism has remained elusive. Using a combination of unbiased mass spectrometry (MS)-based phosphoproteomics, genetic, biochemical, and physiological experiments, we show here that Snf1 temporally maintains TORC1 inactive in glucose-starved cells primarily through the TORC1-regulatory protein Pib2. Our data, therefore, extend the function of Pib2 to a hub that integrates both glucose and, as reported earlier, glutamine signals to control TORC1. We further demonstrate that Snf1 phos-phorylates the TORC1 effector kinase Sch9 within its N-terminal region and thereby antagonizes the phosphorylation of a C-terminal TORC1-target residue within Sch9 itself that is critical for its activity. The consequences of Snf1-mediated phosphorylation of Pib2 and Sch9 are physio-logically additive and sufficient to explain the role of Snf1 in short-term inhibition of TORC1 in acutely glucose-starved cells.

Liquid–liquid phase separation (LLPS) is responsible for the formation of so-called membrane-less organelles (MLOs) that are essential for the spatio-temporal organization of the cell. Intrinsically disordered proteins (IDPs) or regions (IDRs), either alone or in conjunction with nucleic acids, are involved in the formation of these intracellular condensates. Notably, viruses exploit LLPS at their own benefit to form viral replication compartments. Beyond giving rise to biomolecular condensates, viral proteins are also known to partition into cellular MLOs, thus raising the question as to whether these cellular phase-separating proteins are drivers of LLPS or behave as clients/regulators. Here, we focus on a set of eukaryotic proteins that are either sequestered in viral factories or colocalize with viral proteins within cellular MLOs, with the primary goal of gathering organized, predicted, and experimental information on these proteins, which constitute promising targets for innovative antiviral strategies. Using various computational approaches, we thoroughly investigated their disorder content and inherent propensity to undergo LLPS, along with their biological functions and interactivity networks. Results show that these proteins are on average, though to varying degrees, enriched in disorder, with their propensity for phase separation being correlated, as expected, with their disorder content. A trend, which awaits further validation, tends to emerge whereby the most disordered proteins serve as drivers, while more ordered cellular proteins tend instead to be clients of viral factories. In light of their high disorder content and their annotated LLPS behavior, most proteins in our data set are drivers or co-drivers of molecular condensation, foreshadowing a key role of these cellular proteins in the scaffolding of viral infection-related MLOs.

Particulate matter (PM) is a harmful component of urban air pollution and PM2.5, in particular, can settle in the deep airways. The RAS system plays a crucial role in the pathogenesis of pollution-induced inflammatory diseases: the ACE/AngII/AT1 axis activates a pro-inflammatory pathway counteracted by the ACE2/Ang(1-7)/MAS axis, which in turn triggers an anti-inflammatory and protective pathway. However, ACE2 acts also as a receptor through which SARS-CoV-2 penetrates host cells to replicate. COX-2, HO-1, and iNOS are other crucial proteins involved in ultrafine particles (UFP)-induced inflammation and oxidative stress, but closely related to the course of the COVID-19 disease. BALB/c male mice were subjected to PM2.5 sub-acute exposure to study its effects on ACE2 and ACE, COX-2, HO-1 and iNOS proteins levels, in the main organs concerned with the pathogenesis of COVID-19. The results obtained show that sub-acute exposure to PM2.5 induces organ-specific modifications which might predispose to greater susceptibility to severe symptomatology in the case of SARS-CoV-2 infection. The novelty of this work consists in using a molecular study, carried out in the lung but also in the main organs involved in the disease, to analyze the close relationship between exposure to pollution and the pathogenesis of COVID-19.

The growing use of photosynthetic microorganisms for food and food-related applications is driving related biotechnology research forward. Increasing consumer acceptance, high sustainability, demand of eco-friendly sources for food, and considerable global economic concern are among the main factors to enhance the focus on the novel foods. In the cases of not toxic strains, photosynthetic microorganisms not only provide a source of sustainable nutrients but are also potentially healthy. Several published studies showed that microalgae are sources of accessible protein and fatty acids. More than 400 manuscripts were published per year in the last 4 years. Furthermore, industrial approaches utilizing these microorganisms are resulting in new jobs and services. This is in line with the global strategy for bioeconomy that aims to support sustainable development of bio-based sectors. Despite the recognized potential of the microalgal biomass value chain, significant knowledge gaps still exist especially regarding their optimized production and utilization. This review highlights the potential of microalgae and cyanobacteria for food and food-related applications as well as their market size. The chosen topics also include advanced production as mixed microbial communities, production of high-value biomolecules, photoproduction of terpenoid flavoring compounds, their utilization for sustainable agriculture, application as source of nutrients in space, and a comparison with heterotrophic microorganisms like yeast to better evaluate their advantages over existing nutrient sources. This comprehensive assessment should stimulate further interest in this highly relevant research topic.

Le proteine prive di una struttura terziaria stabile sono definite proteine intrinsecamente disordinate (IDP). La risultante plasticità conformazionale conferisce alle IDP l’abilità di interagire con numerosi partner. Di conseguenza, le IDP sono coinvolte nelle cascate di trasduzione del segnale e nei fenomeni di condensazione proteica (detti separazione di fase liquido-liquido), i quali originano i cosiddetti “organelli privi di membrana” (MLO). Gli MLO includono svariati condensati citoplasmatici e nucleari che svolgono funzioni biologiche cruciali nella cellula. Le IDP mantengono il proprio network di interazioni principalmente attraverso contatti elettrostatici, essendo le loro catene particolarmente ricche di residui cationici ed anionici. La distribuzione delle cariche opposte (o patterning delle cariche) è emersa come caratteristica chiave responsabile delle dimensioni complessive e della propensione alla condensazione delle IDP. Un parametro di sequenza, k, è stato introdotto per descrivere in maniera quantitativa il patterning delle cariche nelle catene polipeptidiche. Investigare l’importanza del patterning delle cariche in relazione alla conformazione delle IDP e alla separazione di fase è l’obiettivo principale di questa tesi, che si organizza in due sezioni. Nella prima sezione, è stato studiato l’effetto della distribuzione delle cariche sull’ensemble conformazionale di IDP modello, determinando parallelamente l’influenza del contenuto di proline e degli elementi di struttura secondaria. Tre regioni intrinsecamente disordinate modello (IDR), ovvero NTAIL, PNT4 e NFM, similmente cariche ma con differente contenuto di proline, sono state permutate così da ottenere, a partire da ciascuna sequenza wild-type (wt), due varianti di k che presentano una differente distribuzione dei residui carichi: un costrutto “Low k”, che mostra un’alternanza più regolare rispetto alla proteina wt dei residui dotati di carica opposta; una variante “High k” con una se, Proteins lacking a stable tertiary structure are defined as intrinsically disordered proteins (IDPs). The resulting conformational plasticity gives IDPs the ability to rapidly and reversibly respond to environmental changes and to interact with multiple partners, acquiring an ordered structure upon binding or maintaining fuzzy conformations. As a consequence, IDPs are largely involved in signal transduction cascades and protein condensation phenomena (designated as liquid-liquid phase separation), which give rise to the so-called “membraneless organelles” (MLOs). MLOs include many cytoplasmatic and nuclear condensates, such as stress granules and the nucleolus, which exert crucial biological functions in the cell. IDPs maintain this interaction network primarily through electrostatic contacts, being their chains particularly enriched in cationic and anionic residues, together with prolines and glycines. Besides the net charge of the proteins, the distribution of opposite charges (or charge patterning) has emerged as a key feature dictating the overall size and condensation propensity of IDPs. A sequence parameter, k, has been introduced to quantitatively describe charge patterning in polypeptide chains. Investigating the relevance of charge patterning on IDP conformation and phase separation is the main objective of this thesis, which is organized into two sections. In the first section, the effect of charge distribution on the conformational ensemble of model IDPs was studied, in parallel assessing the influence of other sequence features such as proline content and secondary structure elements. Three model intrinsically disordered regions (IDRs), namely NTAIL, PNT4 and NFM, similarly charged yet with different proline fractions, were permutated in order to obtain from each wild-type (wt) sequence two k -variants with different distributions of charged residues: a “Low-k" variant, with a more regular alternation of oppositely charged residues than in the wt protei

Il glioblastoma multiforme (GBM) è un astrocitoma di IV grado ed è il tumore più comune e aggressivo del sistema nervoso centrale (SNC). Il GBM è caratterizzato da una natura infiltrante, un’alta eterogeneità, aggressività e recidiva. Attualmente non ci sono terapie risolutive e il trattamento standard è basato sul protocollo di Stupp, che prevede chirurgia, radioterapia e cicli di chemioterapia con temozolomide (TMZ). Tuttavia, la resistenza alla terapia e il cosiddetto ‘cancer immune escape’ sono delle sfide molto importanti per i medici e gli scienziati. La nanomedicina ha ricevuto molta attenzione grazie alla sua versatilità di applicazione, che include l’attraversamento della barriera emato-encefalica (BBB) e la veicolazione di farmaci al sito bersaglio. Inoltre, la nanomedicina è stata applicata con successo nel campo dell’immunoterapia tumorale, per esempio utilizzando nanoparticelle (NPs) per il trasporto di farmaci, anticorpi monoclonali o acidi nucleici. Il razionale dietro l’immunoterapia è la stimolazione del sistema immunitario affinché reagisca contro il cancro, anche in tumori con scarsa risposta immunitaria come il GBM. Nel campo della terapia delle malattie cerebrali, la nanomedicina si è rivelata uno strumento eccellente per la veicolazione di farmaci, specialmente considerando che le cellule tumorali di GBM possono sfruttare delle strutture cellulari come i Tunneling nanotubes (TnTs) per scambiare organelli, materiale cellulare, fattori solubili, farmaci e persino NPs. Data questa premessa, i TnTs potrebbero essere sfruttati per impedire questa comunicazione cellulare (inibendo la loro formazione) oppure per diffondere i farmaci chemioterapici tra cellule diverse. Infine, negli ultimi anni gli inibitori delle istone deacetilasi (HDAC, HDACis) sono stati studiati come farmaci antitumorali in diversi tumori, compreso il GBM. Gli HDACis sono suddivisi in pan-HDACis (che diminuiscono l’attività di tutte le HDAC zinco-dipendenti) e in HDAC, Glioblastoma multiforme (GBM) is a IV grade astrocytoma and it is the most common and aggressive tumor of the central nervous system (CNS). GBM is characterized by an infiltrative nature, high heterogeneity, aggressiveness, and recurrence. There are no curative treatments and the standard care is based on Stupp’s protocol, with surgery, radiotherapy and temozolomide (TMZ) chemotherapy. However, therapy resistance and cancer immune escape are important challenges for physicians and scientists. Nanomedicine gained a lot of attention given the versatility of its application, including the crossing of blood-brain barrier (BBB), specific targeted therapy and drug delivery. In addition, nanomedicine has been successfully applied in the field of cancer immunotherapy, for example using nanoparticles (NPs) for the delivery of drugs, monoclonal antibodies (mAbs) or nucleic acids. The rationale behind immunotherapy is to stimulate the immune system to react against cancer, even in tumors with scarce immune response like GBM. In the context of brain disease therapy, nanomedicine turned out to be an excellent tool also for drug delivery, especially considering that GBM cancer cells can exploit cellular structures like Tunneling nanotubes (TnTs) to exchange organelles, cellular material, soluble factors, drugs and even NPs. Given this information, TnTs could be exploited to prevent cellular communication (by inhibiting their formation) or to spread anticancer drugs among different cells. Finally, in the last years histone deacetylase (HDAC) inhibitors (HDACis) were investigated as anticancer drugs in many tumors, GBM included. HDACis are divided into pan-HDACi (that decrease the activity of all Zn-dependent HDACs) and selective-HDACis (specific for one or a few HDACs) and they were proven to be effective in inducing apoptosis, modifying protein expression and acting on therapy resistance. Here, we synthetized 150 nm-sized liposomes loaded with pan-HDACi Givinostat, both unfunctio

A key aspect of adaptation to cold environments is the production of cold-active enzymes by psychrophilic organisms. These enzymes not only have high activity at low temperatures, but also exhibit remarkable structural flexibility and thermolability. In this context, the role of metal ions has been little explored, and the few available studies seem to suggest that metal binding counteracts structural flexibility. This article reports an investigation into the role of the binding of manganese ion (Mn2+) in the thermal adaptation of an esterase (M-Est) of the GDSx family, identified in the genome of the Antarctic bacterium Marinomonas sp. ef1. M-Est is specific for esters containing acetate groups and turned out to be a highly thermolabile cold-active enzyme, with a catalysis optimum temperature of 5 °C and a melting temperature of 31.7 °C. A combination of biochemical and computational analyses, including molecular dynamics simulations, revealed that M-Est binds Mn2+ ions via a single binding site located on the surface of the enzyme, close to the active site. Although the interaction between M-Est and Mn2+ induces only local conformational changes involving the active site, quite surprisingly they trigger an improvement in both thermal stability and catalytic efficiency under mild temperature conditions. These results, together with the conservation of the Mn2+ binding site among psychrophilic and psychrotolerant homologues, suggest that Mn2+ binding may be a useful, albeit atypical, strategy to mitigate the detrimental effects of temperature on true cold-active enzymes.