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PRE-NUCLEATION IN MOLECULAR LIQUIDS: A MOLECULAR DYNAMICS PERSPECTIVE
No full textI cristalli sono molto diffusi nella vita quotidiana e svolgono un ruolo centrale nei settori farmaceutico, alimentare e della scienza dei materiali, consentendo un’ampia gamma di applicazioni tecnologiche avanzate. Nonostante la loro importanza, la fase iniziale della cristallizzazione, la nucleazione, rimane ancora poco chiara. Le tecniche sperimentali sono in gran parte cieche alla nucleazione a causa delle elevate richieste in termini di risoluzione spaziale, temporale e sensibilità, mentre gli approcci computazionali sono messi alla prova dal fatto che la nucleazione è un evento raro, che richiede simulazioni di lunga durata su sistemi di grandi dimensioni. In questo lavoro, i fenomeni di pre-nucleazione, tra cui il parziale ordinamento e l’aggregazione nei liquidi molecolari, vengono investigati mediante una combinazione di simulazioni computazionali ed esperimenti di cristallizzazione.
Questa ricerca ha tre obiettivi principali: innanzitutto, studiare l’effetto del confinamento sul parziale ordinamento dei liquidi molecolari; in secondo luogo, investigare la formazione e le proprietà dei cluster subcritici durante la fase di pre-nucleazione; infine, esplorare il ruolo delle interazioni di stacking aromatico nei fenomeni di pre-nucleazione.
L’acido benzoico è stato selezionato come composto modello data la sua limitata flessibilità molecolare, il numero di atomi ridotto e la sua capacità di partecipare a diverse interazioni intermolecolari, inclusi il legame a idrogeno e lo stacking aromatico. Alcuni suoi derivati sono stati inoltre esaminati per fare un confronto. Le principali metodologie impiegate includono simulazioni di dinamica molecolare (MD) utilizzando il software MiCMoS, calcoli di energia statica mediante il metodo PIXEL, calcoli DFT-D ed esperimenti di cristallizzazione condotti con il cristallizzatore multi-reattore Crystal16.
Per affrontare il primo obiettivo, è stato sviluppato e implementato un algoritmo di confinamento in MiCMoS. Sono state eseguite simulazioni esplorative di MD su sistemi contenenti fino a otto molecole di acido benzoico confinate all’interno di piccole nanocavità cubiche per analizzare il comportamento di aggregazione e i pattern di interazione, mentre sono stati utilizzati calcoli DFT-D per valutare la stabilità degli aggregati ciclici. Successivamente, sono state condotte simulazioni di liquidi confinati in diverse geometrie, da nanostrati a nanotubi e nanocavità. Rispetto al liquido bulk non confinato, il confinamento determina un aumento della viscosità e un’intensificazione delle interazioni di stacking, guidate dal parziale ordinamento in prossimità delle barriere di confinamento, dove si formano strati di molecole adsorbite. L’aumento della rigidità del confinamento induce una transizione liquido-liquido tra stati a bassa e ad alta densità nelle nanocavità. Il confinamento è stato spinto al limite adottando una geometria a nanostrato che consente la presenza di un solo strato molecolare, nella quale è stato osservato un aggregato catemerico costituito da 26 molecole. Questo aggregato ricorda un pattern identificato in precedenti studi di predizione della struttura cristallina e suggerisce che, in queste condizioni, sia possibile accedere a un nuovo polimorfo dell’acido benzoico, che allo stato attuale è monomorfo.
Il secondo obiettivo è incentrato sulle disomogeneità su scala nanometrica nei liquidi bulk. Le fluttuazioni di densità risultano significativamente amplificate nel liquido surraffreddato, probabilmente a causa della formazione di aggregati più grandi e persistenti, identificati come cluster subcritici. A tal fine, è stato sviluppato un programma di analisi per caratterizzare la formazione degli aggregati ed è stata introdotta una nuova definizione di cluster subcritici basata sull’energia di interazione, sulla persistenza temporale e sull’energia in eccesso, una grandezza che misura se l’energia coesiva dell’aggregato supera le interazioni con il liquido circostante. Le distribuzioni delle dimensioni degli aggregati mostrano i trimeri come specie più abbondante, mentre i dimeri, che corrispondono al pattern dominante nella struttura cristallina, risultano essere i secondi più comuni. Tuttavia, tali aggregati sono troppo piccoli per essere qualificati come cluster subcritici. Il cluster subcritico più grande e persistente è osservato nel liquido surraffreddato ed è costituito da 17 molecole, con un tempo di vita superiore a 130 ps. Questo cluster adotta una forma globulare costituita da una catena ripiegata di molecole legate da legami a idrogeno, con interazioni di stacking aromatico intermittenti che contribuiscono alla sua stabilizzazione.
Il terzo obiettivo affronta il ruolo delle interazioni di stacking aromatico nella nucleazione. Circa 200 strutture dimeriche di derivati dell’acido benzoico sono state ottenute dal Cambridge Structural Database (CSD) e le loro energie di stacking sono state stimate mediante il metodo PIXEL. Dopo l’applicazione di una serie di criteri, sono stati individuati quattro composti per l’analisi sperimentale. La solubilità cinetica è stata esaminata in diversi solventi ed è stato selezionato l’isopropanolo come il più idoneo. Dopo un’accurata determinazione della solubilità termodinamica, un composto è stato escluso perché insolubile e un altro per ragioni di tempo. I due composti rimanenti sono stati impiegati in esperimenti di cristallizzazione utilizzando il Crystal16 per determinare le velocità di nucleazione. A causa della natura intrinsecamente stocastica della nucleazione, sono state eseguite 80 ripetizioni usando cinque diversi livelli di sovrasaturazione, per un totale di 400 esperimenti per ciascun composto. Le velocità di nucleazione sono state determinate attraverso un fitting della probabilità cumulativa di nucleazione in funzione del tempo di induzione. Sono state condotte simulazioni complementari di MD per correlare la propensione alla nucleazione con le interazioni di stacking aromatico, supportate dallo sviluppo di un nuovo programma di analisi specificamente progettato per caratterizzare lo stacking. I composti che mostrano una maggiore propensione alla nucleazione presentano anche energie di stacking più favorevoli, un numero maggiore di interazioni di stacking con tempi di vita più lunghi e la formazione di aggregati stacked di dimensioni maggiori. Questi aggregati adottano disposizioni di tipo colonnare che richiamano i motivi di impaccamento cristallino, pur mantenendo un elevato grado di disordine strutturale.Crystals are ubiquitous in everyday life and play a central role in pharmaceuticals, the food industry, and materials science, enabling a wide range of advanced technological applications. Despite their importance, the initial stage of crystal formation, i.e. nucleation, remains poorly understood. Experimental techniques are largely insensitive to nucleation due to the extremely high spatial and temporal resolution and sensitivity required, while computational approaches are challenged by the rare-event nature of nucleation, which demands long simulations of large systems. In this work, pre-nucleation phenomena such as partial ordering and aggregation in molecular liquids are investigated through a combination of computer simulations and crystallization experiments.
This research pursues three main objectives: first, to study the effect of confinement on the partial ordering of molecular liquids; second, to investigate the formation and properties of subcritical clusters during the pre-nucleation stage; and third, to explore the role of aromatic stacking interactions in pre-nucleation phenomena.
Benzoic acid was selected as the model compound owing to its limited molecular flexibility, small number of atoms, and ability to engage in a variety of intermolecular interactions, including hydrogen bonding and aromatic stacking. Some derivatives were also examined for comparison. The primary methodologies employed include molecular dynamics (MD) simulations using the MiCMoS software, static energy calculations with the PIXEL method, DFT-D calculations, and crystallization experiments performed with the Crystal16 multi-reactor crystallizer.
To address the first objective, a confining algorithm was developed and implemented in MiCMoS. Exploratory MD simulations of up to eight benzoic acid molecules confined within small cubic nanocavities were carried out to analyze aggregation behavior and interaction motifs, while DFT-D calculations were used to assess the stability of cyclic aggregates. Subsequently, simulations of confined liquids were performed in different geometries, ranging from nanolayers to nanotubes and nanocavities. Compared to the unconfined bulk liquid, confinement leads to an increase in viscosity and enhanced stacking interactions, driven by partial ordering near the confining barriers, where adsorbed molecular layers form. Increasing the stiffness of confinement induces a liquid-liquid transition between low-density and high-density states in nanocavities. Confinement was further pushed to the limit by adopting a nanolayer geometry allowing only a single molecular layer, in which a catemeric aggregate composed of 26 molecules was observed. This aggregate closely resembles a motif previously identified in crystal structure prediction studies and suggests that, under these conditions, access to a previously unobserved polymorphic form of benzoic acid, reported as monomorphic to date, may be possible.
The second objective focuses on nanoscale inhomogeneities in bulk liquids. Density fluctuations are significantly enhanced in the undercooled liquid, likely due to the formation of larger and more persistent aggregates, identified as subcritical clusters. To investigate these features, a dedicated analysis tool was developed to characterize aggregate formation, and a new definition of subcritical clusters was introduced based on interaction energy, time persistence, and excess energy, which measures whether the cohesive energy of an aggregate exceeds its interactions with the surrounding liquid. Aggregate size distributions reveal trimers as the most abundant species, while dimers, corresponding to the dominant motif in the crystal structure, are the second most common. However, these aggregates are too small to qualify as subcritical clusters. The largest and most persistent subcritical cluster is observed in the undercooled liquid and consists of 17 molecules with a lifetime exceeding 130 ps. This cluster adopts a globular shape formed by a folded chain of hydrogen-bonded molecules, with intermittent aromatic stacking interactions contributing to its stabilization.
The third objective addresses the role of aromatic stacking interactions in nucleation. Approximately 200 dimeric structures of benzoic acid derivatives were retrieved from the Cambridge Structural Database (CSD), and their stacking energies were estimated using the PIXEL method. After applying a series of selection criteria, four candidate compounds were identified for experimental investigation. Kinetic solubility was screened in several solvents, and isopropanol was selected as the most suitable. Following an accurate determination of thermodynamic solubility, one compound was excluded due to insolubility and another due to time reasons. The remaining two compounds were subjected to crystallization experiments using Crystal16 to determine nucleation rates. Owing to the stochastic nature of nucleation, 80 repetitions were performed at five different supersaturation levels, yielding a total of 400 experiments per compound. Nucleation rates were obtained by fitting the cumulative probability of nucleation as a function of induction time. Complementary MD simulations were conducted to correlate nucleation propensity with aromatic stacking interactions, supported by the development of a new analysis tool specifically designed to characterize stacking behavior. Compounds exhibiting higher nucleation propensities also display stronger stacking energies, a greater number of stacking interactions with longer lifetimes, and the formation of larger stacked aggregates. These aggregates adopt pillar-like arrangements reminiscent of crystal packing motifs, while still retaining a significant degree of structural disorder
Phenotypic and genomic characterization of the Maltese hunting dog (Kelb tal-Kaċċa ta' Malta)
No full textThe traditional Maltese Hunting Dog (Kelb tal-Kaċċa ta’ Malta, KTKM) is deeply rooted in Maltese historical and cultural identity, being valued for its versatility in traditional hunting, where it usually flushes out and retrieves small migratory bird species. This study aims to characterise the KTKM phenotypically and genomically, to support its official recognition as a breed and the drafting of a breed standard. Morphological and visual examination of 36 adult KTKM, underline their similarity to European Braque-type dogs, presenting medium-sized and meso-dolicomorphic body, mesocephalic head, large drop ears, thick hanging lips, and a short haired orange-and-white coat. Genomic data of 33 KTKM were compared with 41 dog breeds, including Italian and European breeds belonging to hunting dogs, primitive dogs, terriers, and shepherds. Genomic structure analyses placed the KTKM within the pointing dog cluster, phenotypically and functionally consistent, but also highlighted possible historical links to the Kelb tal-Fenek (indigenous to Malta and known internationally as the Pharaoh Hound) and spaniels. Admixture analysis showed high genetic uniformity and uniqueness within the KTKM sampled population, while runs of homozygosity indicated a relatively low inbreeding coefficient. In conclusion, the KTKM can be considered as being a distinct, genetically uniform breed that is aligned with pointing dogs, showing moderate levels of inbreeding and good heterozygosity. Breed recognition and conservation strategies are crucial to safeguard its gene pool and cultural significance
DIETARY BIOACTIVES IN THE MODULATION OF METABOLIC, FUNCTIONAL AND AGING BIOMARKERS
No full textBioactive compounds and bioactive-rich foods play a crucial role in modulating key physiological pathways related to human health, including cardiometabolic regulation, intestinal function, and healthy aging. In particular, dietary (poly)phenols and xanthines have gained attention for their potential benefits on several metabolic and functional biomarkers. However, the mechanisms are not completely understood and the evidence in humans are still scarce. The present Ph.D. thesis investigates the role of dietary bioactives and bioactives-rich foods in modulating functional biomarkers of human health, with a focus on the mechanisms involving lipid metabolism, oxidative stress and inflammation, intestinal permeability, and telomere integrity. The research was structured into three main chapters, combining both in vitro and in vivo approaches.
In Chapter 1, the mechanisms of action of selected bioactives, including (poly)phenols and xanthine-derived metabolites, were explored in different cellular models. In particular, 3T3-L1 adipocytes and Caco-2 intestinal epithelial cells were used to investigate processes such as lipid accumulation, glucose metabolism, and intestinal barrier integrity. Additionally, in 3T3-L1 adipocytes, a systematic review highlighted the modulatory role of dietary (poly)phenols on adipocyte metabolism, showing that different classes of (poly)phenols can influence key markers involved in lipid and glucose metabolism (e.g., AMPK, SIRT1). Experimental studies further revealed that quercetin-derived metabolites can reduce glucose-stimulated lipid accumulation, while blueberry and raspberry extracts protect intestinal barrier function under pro-inflammatory conditions by preserving tight junction integrity and reducing oxidative stress. Additionally, xanthine metabolites demonstrated protective effects on age-associated intestinal barrier dysfunction, suggesting their potential contribution to gut health during aging.
Chapter 2 translated these mechanistic insights into the clinical setting, evaluating the effects of blueberry consumption, as source of dietary bioactives such (poly)phenols, in a human dietary intervention study. The human trial (the BLUMET study) assessed the impact of an 8-week blueberry intervention on biomarkers related to cardiometabolic health, oxidative stress, inflammation and intestinal permeability in individuals with metabolic syndrome. Preliminary findings suggest that blueberry intake may modulate some biomarkers of adipose tissue metabolism such as adiponectin and slightly reduce total cholesterol, supporting its potential role in the protection of human health. Conversely, no significant findings were documented for the rest of the biomarkers analyzed suggesting the need for further investigations.
In Chapter 3, the focus was placed on telomere biology, a well-established hallmark of aging. The work was carried out as part of an Erasmus+ traineeship program at the University of Copenhagen (Denmark). The experiments investigated the role of xanthine and (poly)phenol metabolites in human samples (PBMCs) from the BLUMET study, as well as in cell culture models (THP-1). All activities were conducted under the supervision of Professor Peter Møller at the Department of Public Health, Section of Environmental Health, University of Copenhagen. In particular, cellular experiments, consisting in a dysmetabolism-induced monocytic cell line, demonstrated the potential of (poly)phenol metabolites to prevent telomere shortening suggesting their ability to modulate key pathways involved in cellular senescence. In addition, the findings from the BLUMET study suggested a possible protective effect of wild blueberry consumption on telomere length in individuals with metabolic syndrome, linking nutritional interventions to the preservation of genomic stability.
In conclusion, this thesis contributes to the growing body of evidence supporting the beneficial role of dietary bioactives, in particular (poly)phenols, in modulating key markers of cardiometabolic health, gut function, and cellular aging. By integrating mechanistic, cellular, and clinical data, it highlights the importance of both individual metabolites and whole food matrices in modulating lipid metabolism, intestinal health, vascular function, and telomere maintenance. These findings underscore the need for future studies to elucidate the complex interactions between dietary bioactives and biological processes, with the ultimate purpose of defining nutritional strategies with the contribution of bioactive-rich foods able to enhance overall human health and well-being
MUSCLE-SPECIFIC MECP2 MISEXPRESSION INDUCES SKELETAL AND VISCERAL MUSCLE DEFECTS RESCUED BY BUTYRATE SUPPLEMENTATION IN DROSOPHILA
No full textSummary
Background
MeCP2 is a chromatin-associated protein whose dosage alterations cause two severe neurodevelopmental disorders: Rett syndrome (RTT), linked to loss-of-function mutations, and MECP2 duplication syndrome (MDS), linked to overexpression. Although widely studied for their neurological symptoms, these disorders also display prominent non-exclusively neurological features, including gastrointestinal dysmotility and muscle hypotonia - suggesting that MeCP2 dysfunction may also impact peripheral tissues. Since MECP2 is expressed in muscle, yet its role outside the nervous system remains poorly defined, this work explores the tissue-autonomous effects of MECP2 misexpression in muscle.
Aims
This project aims to establish a Drosophila model to investigate the impact of MECP2 misexpression in muscle tissue and its role in muscle development, assess whether the resulting phenotypes are autonomous or secondary to neuronal dysfunction, and test the therapeutic potential of short-chain fatty acids (SCFAs) - specifically butyrate - as chromatin-targeting interventions.
Results
Because Drosophila lacks an endogenous MECP2 gene, we used transgenic lines overexpressing either wild-type or mutant human MECP2. Using the Gal4/UAS system, tissue-specific expression revealed that muscle-targeted MECP2 overexpression led to the strongest phenotypes, such as reduced viability, disorganized skeletal and visceral muscle, impaired locomotion, and delayed gut transit. In addition, mitochondrial abnormalities - increased organelle number and altered cristae structure - were observed by electron microscopy in muscle fibers. Visceral muscle defects were most pronounced when MECP2 was expressed during gut development, suggesting interference with tissue maturation.
We also employed the model to functionally classify MECP2 variants. Alleles were scored based on their ability to enhance or suppress wild-type MECP2 phenotypes. R106W suppressed lethality and caused mild muscle disruption, consistent with loss of function. Δ166 enhanced lethality but caused minimal pathology, suggesting a benign effect. R294X produced strong muscle phenotypes and variable lethality across tissues, indicating partial function.
To determine whether these effects were secondary to neural dysfunction, we expressed MECP2 in neurons but observed no impact on muscle structure or gut function. Similarly, MECP2 expression in muscle did not disrupt synaptic architecture. These findings support a tissue-autonomous mechanism.
With a clear muscle-specific phenotype and considering their energetic and epigenetic functions, we next tested whether SCFAs supplementation could reverse these defects. High-dose propionate reduced survival. In contrast, acetate, sodium butyrate, and the butyrate-rich postbiotic Lalbaay® improved viability and development, with Lalbaay® showing the strongest effects.
Motor function was also enhanced, supporting the beneficial role of butyrate molecule. Indeed, supplementation with sodium butyrate, an HDAC inhibitor with energetic role, specifically restored muscle structure and gut motility. Valproic acid, a pan-HDAC inhibitor without metabolic properties, produced similar improvements, suggesting an epigenetic mechanism of action.
Finally, we began dissecting genetic interactions. Since Drosophila lacks endogenous MECP2, it provides a clean background to identify functional modifiers. Recent work implicated PHF14 - a MeCP2 interactor mutated in a Rett-like syndrome - as a candidate modifier. Given its conservation in flies, we initiated a mutagenesis screen via transposon excision. To date, 22 candidate mutant lines have been isolated and are being validated, paving the way for future genetic interaction studies.
Conclusions
This thesis demonstrates that MECP2 misexpression in Drosophila muscle leads to autonomous morphological and functional defects, which can be partially reversed by SCFAs such as butyrate and Lalbaay®, likely via chromatin remodeling and possibly metabolic support. Disease-associated MeCP2 variants reproduce the pathogenicity reported in patients, showing distinct tissue-specific behaviours and underscoring the validity of this model for functional interpretation. These findings highlight the systemic nature of MECP2 disorders and advocate for therapeutic strategies that extend beyond the nervous system to include peripheral tissues such as muscle
Review of the book by Elisa Segnini & Michael Subialka (eds.). (2023). Gabriele D’Annunzio and World Literature. Multilingualism, Translation, Reception
No full textConventional pulsed-field ablation versus pulsed-field ablation with non-integrated three-dimensional mapping for paroxysmal and persistent atrial fibrillation ablation
No full textBackground: There is limited evidence on efficiency, effectiveness, and safety outcomes in the context of pulsed-field ablation (PFA) of atrial fibrillation (AF) when a 3D mapping system is used. Purpose: To assess the impact of non-integrated 3D mapping systems in PFA for the treatment of AF. Methods: Consecutive patients undergoing PFA (FARAPULSE system) at 17 centers were included. Procedures were stratified according to the use of 3D mapping system (MAP vs standard; STD). Results: A total of 1804 patients were included, 484 (26.8%) with a 3D mapping system. MAP procedures had longer skin-to-skin (90 [75–120] min vs. 60 [50–70] min, p < 0.0001), cath lab utilization (120 [100–165] min vs. 70 [60–94] min, p < 0.0001), and fluoroscopy time (22 [17–28] min vs. 15 [11–21] min, p < 0.0001). The use of 3D mapping was not associated with a better long-term clinical outcome in the overall population (freedom from AF/atrial tachycardia (AT) of 82.5% in MAP procedures vs. 77.4% in STD procedures, HR = 0.737, 95% CI 0.53–1.03, p = 0.0746) as well as in PVI-only paroxysmal AF (86.5% vs. 80.3%, 0.67, 0.38–1.19, p = 0.172), in PVI plus additional lesion sets paroxysmal AF (72.7% vs. 77.8%, 1.08, 0.44–2.65, p = 0.869), or in PVI only persistent AF (76.0% vs. 68.0%, 0.86, 0.36–2.04, p = 0.727). A significantly higher arrhythmia recurrence-free rate was noticed in patients with persistent AF undergoing additional lesion set ablation (83.7% vs 70.3%, 0.45, 0.24 to 0.86, p = 0.016). Three major complications (0.2%) occurred, all in the STD group. Conclusion: The use of a non-integrated 3D mapping system for PFA of AF did not significantly affect long-term success rates in paroxysmal AF or in patients undergoing PVI-only procedures
The role of the PPAR system in diabetic cardiovascular risk and beyond
No full textPurpose of review: Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate metabolic homeostasis and play a key role in the management of a number of metabolic disorders (e.g. diabetes and liver steatosis). This review aims to provide an overview on the impact of the three isoforms, PPAR-α, PPAR-β/δ and PPAR-γ, on diabetic-driven metabolic diseases. Recent findings: The lack of clinical benefit observed in the PROMINENT trial with pemafibrate (a selective PPAR-α agonist) has raised questions regarding the therapeutic potential of PPAR-α activation in the prevention of major cardiovascular events. Conversely, evidence suggests a possible therapeutic role in peripheral artery disease. To reduce the adverse effects occurring consequently to PPAR-γ activation, partial agonists or selective PPAR-γ modulators (SPPARγMs) have been developed. In the context of metabolic dysfunction associated steatohepatitis, pan-PPAR agonism appears necessary to achieve significant improvements in histological endpoints. Summary: These diversified effects, albeit with a limited risk of significant side effects, make PPAR agonists an area of growing interest and with an expanding range of potential applications
Transforming jet flavour tagging at ATLAS
No full textJet flavour tagging enables the identification of jets originating from heavy-flavour quarks in proton–proton collisions at the Large Hadron Collider, playing a critical role in its physics programmes. This paper presents GN2, a transformer-based flavour tagging algorithm deployed by the ATLAS Collaboration that represents a different methodology compared to previous approaches. Designed to classify jets based on the flavour of their constituent particles, GN2 processes low-level tracking information in an end-to-end architecture and incorporates physics-informed auxiliary training objectives to enhance both interpretability and performance. Its performance is validated in both simulation and collision data. The measured c-jet (light-jet) rejection in data is improved by a factor of 3.5 (1.8) for a 70% b-jet tagging efficiency, compared to the previous algorithm. GN2 provides substantial benefits for physics analyses involving heavy-flavour jets, such as measurements of Higgs boson pair production and the couplings of bottom and charm quarks to the Higgs boson, and demonstrates the impact of advanced machine learning methods in experimental particle physics