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Integrative computational approaches to study protein-driven mechanisms in adaptation and disease
No full textLe proteine giocano un ruolo chiave sia nell’adattamento che nelle patologie, perciò collegare variazioni nella sequenza genetica ai meccanismi molecolari patofisiologici rimane una sfida fondamentale. Questa tesi sviluppa e applica strategie computazionali integrative per identificare come le proteine codifichino la funzione, come tale funzione venga rimodulata dall’evoluzione o dalla patologia e come queste conoscenze possano essere sfruttate per progettare strumenti terapeutici o meccanicistici selettivi. In contesti biologici eterogenei, combino modellistica molecolare, dinamica molecolare classica, simulazioni a campionamento avanzato, docking proteina-proteina e proteina-DNA, genomica evolutiva e bioinformatica quantitativa per derivare ipotesi sperimentalmente testabili e, ove possibile, rilevanti dal punto di vista traslazionale. Il framework computazionale qui sviluppato si fonda su tre pilastri. Primo, la modellistica strutturale e dinamica viene utilizzata per risolvere stati conformazionali transitori, interfacce di legame e accoppiamenti allosterici non accessibili alle sole strutture statiche. Secondo, analisi di sequenza comparative e su scala di popolazione sono integrate con l’informazione strutturale per identificare domini conservati, innovazioni specifiche di linea evolutiva e deviazioni patogeniche, consentendo interpretazioni delle relazioni genotipo–fenotipo. Terzo, la progettazione guidata dalla struttura che comprende peptidi, miniproteine e piccole molecole, è impiegata per perturbare interfacce selezionate con specificità di isoforma o di bersaglio, fornendo dissezioni funzionali e, in alcuni casi, prototipi per lo sviluppo terapeutico. Nel complesso, questo lavoro mostra come il calcolo multi-scala possa colmare il divario tra sequenza, meccanismo e intervento. Unificando simulazione fisica, logica evolutiva e design razionale, questa tesi propone una roadmap generalizzabile per lo studio dei processi proteina-mediati nell’adattamento e nella malattia, e mette in evidenza il ruolo degli approcci computazionali non come accessorio, ma come motore primario della scoperta e dell’innovazione in biologia. Proteins sit at the core of biological adaptation and disease, yet connecting genetic sequence variation to pathophysiological molecular mechanisms remains a major challenge. This thesis develops and applies integrative computational strategies to identify how proteins encode function, how that function is rewired by evolution or pathology, and how such knowledge can be leveraged to design selective therapeutic or mechanistic tools. Across diverse biological contexts, I combine molecular modeling, classical molecular dynamics, enhanced-sampling simulations, protein-protein and protein-DNA docking, evolutionary genomics, as well as quantitative bioinformatics to derive hypotheses that are experimentally testable and, where possible, translationally relevant. The computational framework established here emphasizes three pillars. First, structural and dynamical modeling is used to resolve transient conformational states, binding interfaces, and allosteric couplings that are inaccessible to static structures alone. Second, comparative and population-scale sequence analyses are integrated with structure to identify conserved domains, lineage-specific innovations, and pathogenic deviations, enabling interpretations of genotype-phenotype relationships. Third, structure-guided design, spanning peptide, miniprotein, and small-molecule modalities, is employed to perturb selected interfaces with isoform or target specificity, providing functional dissection and, in some cases, prototypes for therapeutic development. Collectively, the work shows how multi-scale computation can bridge the gap from sequence to mechanism to intervention. By unifying physical simulation with evolutionary logic and rational design, this thesis offers a generalized roadmap for studying protein-driven processes in both adaptation and disease, and highlights the role of computational approaches not as an accessory, but as a primary engine of biological discovery and innovation. 
Novel insights into hákarl: A deep dive into the microbiological and physico-chemical features of Iceland's traditional fermented shark
No full textHakarl, the traditional Icelandic product obtained from the fermentation of Greenland shark (Somniosus microcephalus), represents a unique food item from both cultural and microbiological perspectives. This study investigated commercial samples of h ́akarl using an integrated approach, combining physico-chemical analyses, lipid and volatile profiling, metataxonomic sequencing, and microbial isolation. Results confirmed the alkaline nature of the product (pH ~8) and a water activity (aw) of 0.96, sufficient to sustain an active and diverse microbial community. Lipid profiling revealed the predominance of monounsaturated fatty acids, with differences in PUFA and DHA levels between the analyzed producers, consistent with nutritionally favorable characteristics. Microbiological analysis highlighted bacterial communities dominated by Firmicutes, particularly Bacilli and Clostridia, with significant abundances of Tissierella creatinini and Atopostipes suicloacalis. Culturedependent methods led to the isolation of Carnobacterium antarcticum cultures, which were subsequently characterized for their enzymatic activities. These findings suggest potential biotechnological applications of the isolates, especially in fermentation and aroma development. Volatile compound analysis identified thirteen VOCs including alcohols, aldehydes, ketones, phenols, sulfur- and nitrogen-containing compounds. Trimethylamine was the predominant metabolite responsible for the strong ammonia-like odor, followed by phenol and sulfurcontaining compounds, which also contributed to the sensory profile. Overall, the results provide novel insights into the microbial ecology, physico-chemical traits, and volatile characteristics of h ́akarl, confirming its variability linked to artisanal production methods. The study emphasizes h ́akarl's role as a reservoir of protechnological microorganisms and advances current understanding of the factors influencing its safety, quality, and identity as traditional fermented food
Insights into the separation, enantioseparation and recognition mechanisms of amphetamine and methamphetamine isotopologues on achiral and polysaccharide-based chiral columns in high-performance liquid chromatography. First Baseline Separation of Isotopomers.
No full textThe different behavior of enantiomers of chiral compounds in non-isotropic environments (among them in living organism) is well known. On the other hand, the importance of a kinetic isotope effect in the biomedical field has become evident during past few decades. Thus, separation of both, enantiomers and isotopologues is now critical.
Only very few published studies have attempted the simultaneous separation of enantioisotopologues. In this study we report baseline separation of partially deuterated isotopologues of a few amphetamine derivatives in high-performance liquid chromatography (HPLC) using achiral columns. In addition, the simultaneous separations of enantiomers and isotopologues (i.e. enantioisotopologues) were attempted on polysaccharide-based chiral columns. For several compounds the isotope effect was tunable and could be switched from a “normal” to “inverse” by making changes to the mobile-phase composition. A stronger isotope effect was observed in acetonitrile-containing mobile phases compared to methanol-containing ones with both chiral and achiral columns. In a separation system where both “normal” and “inverse” isotope effects were observed the “normal”
isotope effect was favored in polar organic solvents while increasing content of the aqueous component in the reversed-phase (RP) mobile phase favored an “inverse” isotope effect. This observation indicates that polar, hydrogen bonding-type noncovalent interactions are involved in the “normal” isotope effect, while apolar hydrophobic-type interactions are mostly responsible for the “inverse” isotope effect.
In this study, the feasibility of very challenging separation of isotopomers by high-performance liquid chromatography in a common setup is successfully demonstrated with the examples of deuterated amphetamine and methamphetamine. Understanding the effect of various factors, such as analyte structure, location of heavy atoms within the structure, separation mode (i.e.,surface chemistry of the stationary phase), mobile phase components and their additives, pH of the mobile phase, and separation temperature, that enabled baseline separation of
Isotopomers with high selectivity is discussed. The results reported
here are significant for both the analytical separation of isotopomers and their preparative-scale separation. The understanding and fine-tuning of the mechanisms underpinning isotopomer retention and discrimination enabled the reversal of isotopomer elution order on specific chiral columns. The simultaneous separation of isotopomers and their enantiomers was observed on polysaccharide-based chiral columns. Molecular modeling was used to provide a reasonable explanation of the separation mechanism of the isotopomers
Effects of the seismic vertical component on base-isolated structures
No full textI sistemi di isolamento sismico sono ampiamente utilizzati per ridurre le azioni sismiche orizzontali trasmesse alle strutture, grazie al principio di disaccoppiamento tra edificio e terreno. Tali sistemi aumentano il periodo fondamentale della costruzione garantendo una marcata riduzione delle accelerazioni sismiche. Tuttavia, essi non garantiscono la stessa efficacia nei confronti della componente verticale del sisma, che può rivelarsi significativa in condizioni near-fault, con accelerazioni verticali comparabili o superiori a quelle orizzontali. Nonostante i progressi nella ricerca, molte normative, tra cui quella italiana, forniscono ancora indicazioni limitate sulla valutazione delle componenti tridimensionali del sisma in condizioni near-fault. Il presente lavoro analizza una delle possibili soluzioni progettuali da adottare nella ricostruzione del centro storico di Castelluccio di Norcia, gravemente colpito dall’evento del 30 ottobre 2016. Il progetto si basa sulla tecnologia dell’“Artificial Ground Isolation”, che consente di isolare intere porzioni di territorio mediante un’unica piastra isolata. Lo studio si propone di valutare gli effetti di input sismici tridimensionali in contesti near-fault sul sistema di isolamento usato nella ricostruzione di Castelluccio di Norcia. Inizialmente viene valutata l’influenza della rigidezza verticale degli isolatori e dello smorzamento verticale della piastra e degli edifici sulla risposta dinamica del sistema strutturale. Successivamente vengono investigati gli effetti della componente verticale sul modello numerico dell’intervento, proponendo inoltre una metodologia semplificata per la modellazione degli edifici, utile a studiare l’interazione dinamica degli stessi con la piastra riducendo gli oneri computazionali. Infine, una volta definito il modello “finale”, vengono individuati i principali parametri progettuali tenendo conto della variabilità del coefficiente di attrito dei dispositivi di isolamento.Seismic isolation systems are widely used to reduce the horizontal seismic actions transmitted to structures, thanks to the decoupling principle between the building and the ground. These systems increase the fundamental period of the structure, ensuring a marked reduction of seismic accelerations. However, they do not guarantee the same effectiveness with respect to the vertical seismic component, which may become significant in near-fault conditions, where vertical accelerations can be comparable to or even higher than horizontal ones. Despite advances in research, many design codes, including the Italian one, still provide limited guidance on the assessment of the three-dimensional seismic components under near-fault conditions. The present work analyzes one of the possible design solutions to be adopted in the reconstruction of the historical center of Castelluccio di Norcia, severely damaged by the event of October 30, 2016. The project is based on the “Artificial Ground Isolation” technology, which allows entire portions of territory to be isolated through a single isolated slab. This study aims to evaluate the effects of three-dimensional seismic inputs in near-fault contexts on the isolation system adopted for the reconstruction of Castelluccio di Norcia. Initially, the influence of the vertical stiffness of the isolators and the vertical damping of the slab and buildings on the dynamic response of the structural system is assessed. Subsequently, the effects of the vertical component are investigated through the numerical model of the intervention, also proposing a simplified methodology for building modeling, useful for studying their dynamic interaction with the slab while reducing computational effort. Finally, once the “definitive” model is developed, the main design parameters are identified, taking into account the variability of the friction coefficient of the isolation devices
Exploring the biodiversity of acclimation: effects of nutrient limitation and light regime in marine microalgae
No full textWidespread across diverse environments from fresh and marine waters to symbiotes with other organisms, microalgae have adapted to numerous habitats where they form the base of marine food webs and are responsible for roughly half of global carbon fixation. Microalgae derive from a complex evolutionary history characterized by multiple endosymbiotic events through which they acquired their photosynthetic plastids, giving rise to many distinct lineages. As a result, they display broad diversity in the structure and composition of their photosynthetic apparatus, as well as in their physiological adaptations to ever-changing environments, shaped by habitat and phylogeny. In this
thesis, we investigate long-term acclimation strategies to different nutrient limitations and light regimes in microalgae belonging to distinct phylogenetic groups and ecological niches.
The first introductory chapter covers the state of the art on: (i) the structure and function of the photosynthetic apparatus, (ii) sulphur and iron uptake and assimilatory pathways, (iii) environmental variations in nutrient and light availability and their effects on microalgal physiology, (iv) microalgal species under investigation in the following chapters.
In chapter 2 examined the responses to long-term sulphur limitation in three marine microalgae: the chlorophytes Tetraselmis suecica and Dunaliella salina, and the model diatom Phaeodactylum tricornutum. Sulphur is an essential component of proteins, lipids, and other metabolites, and it plays a direct role in photosynthesis through both electron transport and carbon fixation pathways. The three species were grown under sulphur-replete and sulphur-deficient conditions, and we analysed
their growth, elemental and pigment composition, in vivo photosynthesis, and the accumulation of proteins related to photosynthesis and sulphur metabolism. Under low sulphate conditions, all species prioritized the allocation of resources to photosynthesis: through modulation of pigment content and stoichiometry of their photosynthetic apparatus, S-limited cells maintained in vivo photosynthetic activity close to that of control cultures while adjusting their growth and cellular composition in species-specific ways. These results were interpreted in an evolutionary framework, considering how changes in oceanic sulphate availability throughout Earth’s history paralleled by shifts in the ecological dominance of different algal groups may have shaped the responses of the species studied.
In chapter 3 following the physiological characterization of S limitation, we performed a
transcriptomic analysis of D. salina grown under control and growth-limiting sulphur concentrations to obtain a broader view of the metabolic pathways affected during acclimation. We focused on genes related to photosynthesis, including those encoding antenna proteins, as well as genes involved in sulphur metabolism and protein turnover, guided by insights from the physiological data. Although our de novo transcriptome assembly provided good coverage of the targeted pathways, relatively few differentially expressed genes previously reported in the literature were identified. This may reflect
differences in gene expression patterns between cells undergoing commonly studied acute stress responses and those acclimated to long-term nutrient limitation. Nevertheless, our results indicated an increase in protein-related metabolic activity, consistent with studies suggesting elevated protein mistranslation under sulphur limitation.
In chapter 4 we then assessed the effects of iron limitation on the chlorophytes T. suecica and D. salina. Iron is essential for redox reactions and photosynthesis, particularly as a component of Fe–S clusters in many photosynthetic complexes, and it is one of the major factors limiting marine primary production, with its availability varying across habitats. The two species differed in their ability to reduce cellular Fe requirements: T. suecica exhibited stronger reductions in growth and photosynthesis, while D. salina, adapted to survive in low-Fe environments, maintained its relative Fe content through regulation of cell size and the activation of Fe-sparing strategies.
In chapter 5 differences in photosynthetic characteristics identified in earlier experiments prompted a detailed analysis of the acclimation of T. suecica and D. salina to different light regimes. Cultures were acclimated to low light, high light, and fluctuating light conditions. T. suecica displayed relatively low Chl a/b ratios (for chlorophytes), consistent with species possessing large antenna complexes. In line with this, T. suecica showed minimal changes in the Chl a/b under low light. In contrast, D. salina increased its pigment content under low light. The species also differed in their responses to fluctuating light and in their photoprotective mechanisms: T. suecica exhibited NPQ
kinetics similar to those of the model species Chlamydomonas reinhardtii, whereas D. salina showed a distinctive pattern characterized by strong NPQ induction at the onset of light, followed by relaxation, and a broader NPQ amplitude across growth conditions, suggesting a fundamentally different mode of NPQ regulation.
In chapter 6 in light of the importance of photosynthetic acclimation to light availability, we examined the effects of far-red light on in-hospite coral symbionts, a topic that remains poorly explored. Far red wavelengths may act as cues for regulatory or acclimatory responses in symbiotic algae. We grew the hard coral Cladocora caespitosa and the soft-bodied anemone Anemonia viridis for two months under white light and white light supplemented with infrared light, monitoring growth, symbiont density, and photosynthetic activity. Although no major differences in coral health or growth were
detected, some differences in photophysiology emerged. Symbionts of C. caespitosa exhibited higher Chl a/c2 ratios than those of A. viridis, suggesting that the two hosts harbor distinct Symbiodiniaceae lineages. Under IR-enriched light, C. caespitosa symbionts showed increased activation of photoprotective mechanisms, while A. viridis symbionts did not exhibit significant changes in photosynthetic regulation. Although the duration of the experiment may have been too short to reveal effects on host coral growth, the observed differences in symbiont responses suggest that in some algal species, far-red light may serve as an environmental indicator of proximity to the water surface
From Smart Assistants to Smart Spaces: AI in Fashion Retail and the Emerging Need for In-Store Behavioral Intelligence
No full textIn the context of the digital transformation of the retail sector, physical fashion stores are increasingly seeking intelligent systems that can provide real-time insights into shopper behaviour. This paper presents an AI-powered monitoring system that has been deployed in a large fashion retail store. It integrates a dense network of Xovis top-view sensors with a real-time analytics. The system uses a temporal proximity-based clustering algorithm to perform fine-grained analysis of customer flow, gender composition, and group segmentation. Over the course of one year, the system recorded and analysed more than 479,000 shopper interactions, revealing temporal patterns, spatial preferences and behavioural trends. The results demonstrate a consistent predominance of female shoppers, a high frequency of individual shopping behaviour and distinct peak hours that align with daily routines and promotional campaigns. The analytics are designed to support operational decision-making and enables managers to optimise store layout, staffing and marketing strategies. The proposed system is a scalable, privacy-conscious solution for behavioural intelligence in physical retail. It offers a foundation for predictive modelling, adaptive merchandising and data-driven retail design. Future developments will explore extended behavioural profiling, automated alerts and integration with business KPIs to enhance strategic decision-making
Neuromorphic Intelligence in Practice: Applications of Spiking Neural Networks in Awake Neurosurgery, Eye-Tracking, and Autonomous Systems
No full textLe Spiking Neural Networks (SNN) rappresentano un paradigma computazionale ispirato al cervello, apprezzato per l’elevata efficienza energetica e la naturale capacità di elaborare dati temporali. Tuttavia, la loro diffusione pratica è stata limitata dalla complessità dell’addestramento e da una prevalente applicazione a compiti di classificazione. Questa tesi mira a colmare il divario tra il potenziale teorico delle SNN e il loro impiego reale, attraverso un’indagine articolata su più livelli.
In primo luogo, viene dimostrata l’efficacia delle SNN in un’applicazione medica critica: un sistema di classificazione in tempo reale degli stati del paziente durante neurochirurgia da sveglio, con prestazioni competitive rispetto a modelli ricorrenti allo stato dell’arte. Successivamente, viene esplorato il loro utilizzo in interazioni uomo-macchina a bassa latenza mediante un modello di eye-tracking addestrato con surrogate gradient.
Il contributo metodologico centrale è la proposta e validazione di un nuovo framework di regressione ispirato al population coding, che consente alle SNN di affrontare problemi complessi e multivariabili, superando il tradizionale ambito della classificazione. Infine, l’applicabilità pratica dei principi neuromorfici viene dimostrata implementando un controllore basato su SNN su un robot mobile, addestrato tramite imitation learning per replicare una politica di controllo classica.
Nel complesso, il lavoro evidenzia la maturazione delle SNN da modelli teorici a strumenti versatili per l’ingegneria reale, offrendo nuove applicazioni, un avanzamento metodologico per la regressione e una validazione concreta in ambito robotico.Spiking Neural Networks (SNNs) are a brain-inspired computational paradigm known for their energy efficiency and natural suitability for temporal data processing. However, their practical adoption has been limited by training challenges and a historical focus on classification tasks. This dissertation bridges the gap between the theoretical potential of SNNs and their real-world application through a comprehensive investigation.
First, it demonstrates the effectiveness of SNNs in a critical medical setting by developing a real-time patient state classification system for awake neurosurgery, achieving performance competitive with state-of-the-art recurrent models. It then explores low-latency human–computer interaction through a surrogate gradient-trained eye-tracking model.
The core methodological contribution is the proposal and validation of a novel regression framework inspired by population coding. This approach enables SNNs to perform complex, multi-variable regression tasks, significantly extending their capabilities beyond classification. Finally, the practical applicability of neuromorphic principles is shown by implementing an SNN-based controller on a mobile robot, trained via imitation learning to replicate a classical control policy.
Overall, this work shows that SNNs are evolving from theoretical constructs into versatile engineering tools, providing new applications, a key methodological advancement for regression, and practical validation in robotics
Root topography in high-density olive orchards
No full textRoot distribution and growth in high-density olive orchards require more detailed investigation. Proper root development can promote vegetative growth during the early years after planting and postpone physiological ageing in adult olive groves. The topography of the root was evaluated in the ‘Maurino’ cultivar in 2- and 10-year-old high-density olive orchards (4×2 m, 1,250 trees ha-1), located in Rome and Ancona (central Italy), respectively. Soil samples were taken at predetermined distances and depths on 8 trees of similar size in each orchard. The soil samples were dried, weighed and the olive roots were separated using a sieve and then scanned. Images were processed with WinRHIZOTM software to obtain the total root length, the mean root diameter and the root length density (RLD). In the young olive orchard, roots had larger diameter in the most superficial layers of soil and in the areas closest to the stem, while the RLD was greater in the first centimetres of soil along the row. Root also explored the inter-row without limitations due to the presence of the natural mulching. In the adult orchard, the results were consistent with those of the young one even though the root density was minimal at about 1-m distance between the trees along the row and the diameter of the roots was very small in the area of the inter-row subjected to compression by the passage of the machines. In conclusion, in the considered high-density orchards, the root system grew both along the row and in the inter-row. In adult trees, limitations to the root development may be due to autopathic phenomena between contiguous olive trees along the row or by portions of soil subjected to excessive compaction due to the periodical passage of machines in the inter-row
How much do users matter? An integrated method for building flood vulnerability and exposure assessment in Historic Urban Areas
Full text linkThe definition of sustainable strategies for risk mitigation in Urban Built Environments (UBEs) prone to flooding should be based on holistic yet quick approaches that comprise hazard, physical vulnerability, and user factors. In particular, the ways users occupy, live and behave in UBEs introduce significant spatiotemporal dynamics in the final risk due to their user exposure (how many?) and vulnerability (of which type?). These effects can be relevant in Historic UBEs (HUBEs), due to building heritage features and the related need to balance mitigation strategies with conservation and preservation. Therefore, quickly applicable analyses, exploiting available databases, should be developed, and the reliability of methods that incorporate user-related, dynamic parameters should be demonstrated in comparison to established “static” analyses of hazards and physical elements. This work proposes a building-scale approach for vulnerability and exposure assessment to floods, aimed at identifying “hot-spots” in HUBEs, by combining “static” and “dynamic” assessment methods. “Static” (e.g. material degradation, construction typology, urban morphological features) and “dynamic” (e.g. daily occupancy schedules, occupant densities/typologies) are combined within a GIS database, using single and multi-factor metrics. The method is demonstrated using a relevant Italian case study. Results remark that considering user exposure and vulnerability over time introduces significant differences in flood risk metrics and HUBEs hotspots, for both public buildings, due to daytime occupancy schedules, and residential buildings, where risk levels increase up to 80%, considering the possible low physical vulnerability of these buildings. This work therefore provides robust approaches to support informed decision-making in the prioritisation of targeted mitigation strategies within HUBEs
Multicenter study of invasive pulmonary aspergillosis in intensive care units: facing the new challenge
No full textInvasive pulmonary aspergillosis (IPA) is a life-threatening conditions which typically develops in immunosuppressed patients. However admission to intensive care units (ICU) has emerged as a risk factor for the development of IPA, even in those patients without classical risk factors.
As the clinical presentation is commonly atypical in non-neutropenic critically ill patients and the isolation of Aspergillus spp. is not always a sign of infection, the diagnosis of this condition often poses a considerable challenge to clinicians.
In this retrospective, observational study, all critically ill patients admitted in the ICUs of three hospitals in the Marche region with an Aspergillus spp. isolate obtained from a lower respiratory tract specimen over a six-year period (2019-2024) were classified as infected or colonized according to the main diagnostic criteria, to evaluate differences between colonized and infected patients and factors associated with mortality.
A total of 186 patients were identified: in 73 of them IPA was diagnosed with at least one diagnostic algorithm, while 113 were deemed as colonized. Four proven cases were diagnosed by histopathology.
Compared to colonized patients, those with IPA more frequently presented with structural lung abnormalities, chronic cardiovascular comorbidity, history of receipt of corticosteroid or other immunosuppressive therapies, severe respiratory viral infection, ARDS, pneumonia, respiratory failure, acute kidney injury, their previous hospitalization was longer, history of receipt antibiotic or antifungal therapy and growth of A. fumigatus; furthermore, galactomannan testing and chest CT were more frequently performed and an antifungal therapy was more frequently prescribed. On the contrary, colonized patients presented higher SOFA score and were more frequently admitted because of hemorrage, stroke or trauma.
Thirty-day mortality were higher in the infected group compared to colonized patients (p = 0.11). Factors shown to be independently associated with 30-day mortality were septic shock (OR 4.446, CI 1.022 – 19.339, p 0.047) and infection caused by A. fumigatus (OR 4.486, CI 1.239 – 16.245, p 0.022).
Our study confirmed the high mortality of IPA in critically ill patients. Early diagnosis is essential to promptly start an appropriate antifungal treatment, underscoring the potential role of non-culture-based methods and CT-scan in achieving an earlier diagnosis