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Big data analysis for drug discovery: from molecular simulations to lipidomics
No full textThis work highlights the growing significance of computational tools in drug discovery, from atomistic simulations that uncover molecular mechanisms behind therapeutic actions to multivariate analysis for pattern recognition in large datasets.
In Chapter 2, two metadynamics-based protocols were set-up and applied to classify M3 receptor antagonists by residence time, contributing to structure-kinetic relationship (SKR) analysis and demonstrating how chemical modifications influence the (un)binding kinetics.
Chapter 3 introduces a multiscale computational protocol based on enhanced sampling and QM/MM simulations to characterize the mechanism of action of known azole urea inhibitors of MGL. This approach was successfully applied to drive the synthesis of a new compound, endowed with nanomolar activity.
In Chapter 4 a similar computational protocol was applied to investigate the mechanism of action of new tyrosine kinase inhibitors (TKIs) targeting the catalytic lysine of EGFR, designed to overcome the resistance to osimertinib, the front-line treatment for non-small cell lung cancer (NSCLC).
Chapter 5 explores alternative strategies to prevent the insurgence of osimertinib resistance in NSCLC investigating the role played by lipid metabolism in cancer cells. Multivariate analysis of lipidomic data obtained from LC-MS experiments on osimertinib-sensitive and -resistant cells revealed that the combination of osimertinib with a glucosylceramide synthase inhibitor (e.g., venglustat) is a promising strategy to delay the onset of resistance, potentially extending the progression-free survival of NSCLC patients
The aesthetic and functional potential of reusing waste materials: innovation and critical awarenes.
No full textQuesta ricerca esplora il potenziale creativo e funzionale del riutilizzo dei ma
teriali di scarto nel contesto dell’arte e del design. Il primo obiettivo generale
della ricerca è evidenziare come approcci artistici abbiano contribuito nella storia a una maggiore consapevolezza critica nei confronti dei rifiuti, a elevarne il
loro valore funzionale e a generare in essi potenziale creativo, promuovendo un
cambiamento culturale e sociale.
L’obiettivo specifico è fornire un esempio progettuale che esplori le possibilità
estetiche e funzionali del riuso dei materiali, illustrando come tali principi possano tradursi in soluzioni innovative e sostenibili in contesti contemporanei. Attraverso un progetto di design innovativo, la ricerca dimostrerà le possibilità estetiche e funzionali del riuso dei materiali, riflettendo sul loro impatto sia a livello
pratico che simbolico. Questo approccio non solo valorizza il potenziale estetico dei materiali di scarto, ma stimola anche una riflessione più profonda sul ruolo
dell’arte e del design nella promozione di una cultura della sostenibilità e della
responsabilità ambientale. Il contributo della tesi si inserisce in un dibattito più
ampio sull’economia circolare e il ruolo del design nella creazione di soluzioni
sostenibili, proponendo nuove modalità di intervento che coniugano innovazione creativa e consapevolezza ambientale.This research explores the creative and functional potential of reusing waste materials
in the context of art and design. The first general objective
of the research is to highlight how artistic approaches have contributed throughout history to greater critical awareness of waste, to raising its
functional value, and to generating creative potential in it, promoting
cultural and social change.
The specific objective is to provide a design example that explores the aesthetic and functional possibilities
of reusing materials, illustrating how these principles can be translated into innovative and sustainable solutions in contemporary contexts. Through an innovative design project, the research will demonstrate the aesthetic and functional possibilities of reusing materials, reflecting on their impact at both a practical and symbolic level.
This approach not only enhances the aesthetic potential of waste materials, but also stimulates a deeper reflection on the role This approach not only enhances the aesthetic potential of waste materials, but also stimulates a deeper reflection on the role
of art and design in promoting a culture of sustainability and
environmental responsibility. The thesis's contribution is part of a broader debate
on the circular economy and the role of design in creating sustainable solutions ,
proposing new ways of working that combine creative innovation and environmental awareness
Realization and investigation of the properties of biocomposites based on biopolymers for novel application in agri-food and biomedical fields
No full textQuesta ricerca si concentra sullo sviluppo di biocompositi a base di poli(butilene adipato-co-tereftalato) (PBAT) e poliidrossialcanoati (PHA) per migliorare le proprietà meccaniche e garantire la biodegradabilità per applicazioni di imballaggio e biomediche.
Lo studio esamina biocompositi di PBAT rinforzati con fosfato di calcio vetroso (CPG) e un complesso zeina-biossido di titanio (ZTC). Queste cariche migliorano la resistenza meccanica e la biodegradabilità senza ridurre la bioattività.
Inoltre, il poliidrossibutirrato (PHB) plastificato con glicerolo trilevulinato (GT) è stato valutato in termini di flessibilità e la stabilità della stampa 3D. la processabilità dei compositi PHB-GT è stata valutata, ottenendo miglioramenti nella flessibilità. L'incorporazione di vetro fosfato riassorbibile nel PHB plastificato ha permesso di ottenere compositi bioattivi per applicazioni biomedicali volte alla rigenerazione dei tessuti.
La biodegradabilità del PBAT-CPG e del PHB plastificato è stata testata in varie condiThis research focuses on the development of biocomposites based on poly(butylene adipate-co-terephthalate) (PBAT) and polyhydroxyalkanoates (PHA) to improve mechanical properties and ensure biodegradability for packaging and biomedical applications.
The study examines PBAT biocomposites reinforced with glassy calcium phosphate (CPG) and a zein-titanium dioxide (ZTC) complex. These fillers improve mechanical strength and biodegradability without reducing bioactivity.
In addition, polyhydroxybutyrate (PHB) plasticized with glycerol trilevulinate (GT) was evaluated for flexibility and 3D printing stability, and the processability of PHB-GT composites was evaluated, resulting in improvements in flexibility. The incorporation of resorbable phosphate glass into plasticized PHB enabled bioactive composites for biomedical applications aimed at tissue regeneration.
The biodegradability of PBAT-CPG and plasticized PHB was tested under various conditions, highlighting their potential for sustainable applications
Studio degli acidi biliari sierici e fecali pre e post trapianto del microbiota fecale in cani enteropatici cronici
No full textCanine chronic enteropathies (CE) can be characterized by intestinal dysbiosis and
reduced colonization by Clostridium (Peptacetobacter) hiranonis, resulting in impaired
conversion of primary to secondary bile acids (BAs). Such perturbations can adversely
affect intestinal and hepatic homeostasis through disturbance of the gut3liver axis. Fecal
microbiota transplantation (FMT) is a promising therapeutic approach aimed at restoring
microbial composition and metabolic functions by re-establishing C. hiranonis and
reactivating BA-transforming pathways. This prospective observational trial enrolled ten
dogs with diet non-responsive or partially responsive CE, evaluating the impact of FMT
on fecal microbiota (dysbiosis index, DI), fecal and serum BA profiles, and liver enzyme
activities (ALT, AST, SAP). Assessments were performed at T0 (day of FMT), T7 days,
and T30 days. Clinical improvement was observed (CIBDAI decreased from 3.5 to 1,
p=0.02), accompanied by a decline in DI (from 32.3 to 34.2, p=0.02), while no significant
changes were detected in serum or fecal BA levels or liver enzyme activities. At baseline,
three dogs showed low abundance of C. hiranonis and two of them demonstrated
evidence of recolonization after FMT. Secondary BA production was reduced in two dogs,
consistent with the lack of C. hiranonis and characterized by impaired conversion of
primary to secondary BAs. However, overall, BA profiles in CE dogs did not differ
substantially over time or when compared with healthy controls. In summary, FMT led to
clinical improvement and partial microbiota normalization without influencing BA
metabolism or liver function in the short term. These findings suggest that the therapeutic
efficacy of FMT in modulating the gut3liver axis may depend on the degree of baseline
dysbiosis and that combined fecal and serum BA quantification may provide a useful tool to evaluate its metabolic impact, although further studies are warranted
Action in the social brain : a fMRI study on the involvement of the mirror system and mentalization circuits
No full textIl seguente studio, svolto nell’ambito delle neuroscienze cognitive, indaga il concetto di cervello sociale e le strutture cerebrali coinvolte nell’elaborazione delle interazioni sociali. Partendo da una prospettiva evolutiva del “cervello sociale” viene evidenziato come la socialità sia stata fondamentale nel modellare le capacità
cognitive avanzate nei primati. La presente tesi pone l’attenzione sui correlati neurali della cosiddetta “Teoria
della mente” - la capacità di comprendere stati mentali altrui - tradizionalmente associata a strutture come la corteccia prefrontale mediale, il solco temporale superiore e la giunzione temporo-parietale, e sui correlati neurali relativi al “Sistema Specchio” - responsabili della comprensione delle azioni altrui attraverso un meccanismo di rispecchiamento motorio -, quali il giro frontale inferiore, il lobulo parietale inferiore, il solco temporale superiore e la corteccia motoria primaria. A partire da un’analisi della letteratura più recente
su questi circuiti, è stato ipotizzato che entrambi siano coinvolti e interconnessi nella codifica di azioni socialmente rilevanti. Questo è stato verificato tramite uno studio di risonanza magnetica funzionale (fMRI) nel quale i partecipanti osservavano altre persone compiere azioni di afferramento. Manipolando variabili come lo scopo finale dell’azione, la prospettiva, e il contatto visivo, lo studio esplora attraverso un’analisi di similarità
rappresentazionale come il livello di ‘coinvolgimento’ (‘engagement’) sociale espresso dall’azione osservata moduli l’attivazione di questi network, valutando l’ipotesi di una possibile connettività rappresentazionale, di contro alla visione “classica” di una distinzione funzionale netta delle rispettive proprietà funzionali.The following study, carried out in the field of cognitive neuroscience, investigates the concept of the social brain and the brain structures involved in processing of social interactions. Starting from an evolutionary perspective of the “social brain,” it is highlighted how sociality has been fundamental in shaping the capacities
advanced cognition in primates. This thesis focuses attention on the neural correlates of the so-called “Theory
of mind"- the ability to understand mental states of others -, traditionally associated with structures such as the medial prefrontal cortex, the superior temporal sulcus and the temporo-parietal junction, and on neural correlates related to the “Mirror System”- responsible for understanding of others' actions through a motor mirroring mechanism - such as the inferior frontal gyrus, inferior parietal lobule, superior temporal sulcus
and the primary motor cortex. From an analysis of the most recent literature on these circuits, it has been hypothesized that both are involved and interconnected in the encoding of socially relevant actions. This has been verified through a functional magnetic resonance imaging (fMRI) study in which participants observed other people performing grasping actions. Manipulating variables such as the end goal of the action, perspective taking, and eye contact, the study explores through a similarity analysis representational how the level of social 'involvement' ('engagement') expressed by the observed action modules the activation of these networks, evaluating the hypothesis of a possible representational connectivity, as opposed to the 'classical' view of a sharp functional distinction of the respective functional properties
Dinamiche di formazione e trasformazione del bacino documentario altomedievale più ricco d’Europa: il Diplomatico Arcivescovile e le Biblioteche dell’Archivio storico diocesano di Lucca
No full textIl saggio propone un approfondimento sulla formazione del patrimonio custodito presso l’Archivio Storico Diocesano di Lucca, uno dei bacini documentari più ricchi per l’Alto Medioevo europeo e, in particolare, sui suoi due nuclei principali: le oltre 1800 pergamene del Diplomatico Arcivescovile e gli 11 codici dei secoli VIII e IX conservati nelle Biblioteche Capitolare Feliniana e Arcivescovile
Development of innovative Oxygen Transport Membranes materials for application in Catalytic Membrane Reactors.
No full textL'urgenza di aumentare la sostenibilità ambientale nelle attività industriali per limitare i cambiamenti irreversibili, a cui assistiamo quotidianamente nel clima e nella natura, è diventata una tra le maggiori sfide a livello mondiale. Tra i metodi green per la produzione di energia proposti dalla comunità scientifica hanno guadagnato molta attenzione le membrane per il trasporto dell'ossigeno (OTM), poiché possono essere impiegate in diversi ambiti, apportando un contributo significativo alla decarbonizzazione del sistema energetico. Le OTM permettono di separare l'ossigeno da miscele di gas, come l'aria, e possono quindi essere utilizzate per produrre ossigeno puro per i processi industriali di ossidazione. Inoltre, studi recenti hanno applicato le OTM in reattori catalitici a membrana (CMR), che sfruttano le proprietà del trasporto di ossigeno per eseguire processi di conversione chimica. I CMR possono potenzialmente essere impiegati per produrre diversi prodotti chimici e vettori energetici, tra cui l'idrogeno da una fonte abbondante e priva di carbonio come l'acqua.
Per funzionare, gli OTM devono condurre sia elettroni che ioni; pertanto, sono costituiti principalmente da conduttori misti di elettroni e ioni (MIEC). Sebbene gli OTM abbiano applicazioni promettenti, funzionano a temperature elevate e in ambienti altamente riducenti. Il limite più significativo per l’applicazione degli OTM nei processi reali è che presentano una bassa stabilità nelle condizioni operative o una bassa permeabilità. La sfida è quindi quella di progettare nuovi materiali OTM che presentino un buon equilibrio tra stabilità e permeabilità.
L'obiettivo di questo progetto di dottorato è stato quello di identificare, sintetizzare e caratterizzare materiali MIEC innovativi con proprietà promettenti per l'applicazione in OTM operanti in condizioni difficili, come atmosfere riducenti e contenenti CO2 . Le attività si sono concentrate sugli ossidi MIEC appartenenti alla classe delle perovskiti SrTi1-xFexO3- , che ha recentemente attirato l'attenzione grazie alla sua maggiore stabilità chimica rispetto ai materiali MIEC più studiati. In particolare, è stato studiato l'effetto della sostituzione del sito A sulla perovskite SrTi0,8Fe0,2O3- (STF82). Pertanto, inizialmente si è scelto di sostituire lo stronzio con il calcio, che potrebbe aumentare la tolleranza del materiale alla CO2 ma, avendo un raggio ionico più piccolo, potrebbe avere un impatto negativo sulle prestazioni di permeazione dell'ossigeno. Per studiare in via preliminare una possibile gamma di sostituzioni, sono state scelte due diverse frazioni di Ca, il 10% e il 40%, e sono state così preparate per reazione allo stato solido Sr0,9Ca0,1Ti0,8Fe0,2O3- (SCTF9182) e Sr0,6Ca0,4Ti0,8Fe0,2O3- (SCTF6482). La stabilità di queste polveri nei confronti della CO2 è stata studiata con l'analisi termogravimetrica (TGA) e sono stati eseguiti test di permeabilità su membrane dense sinterizzate nell'intervallo 600°-1000°C in gradiente di pressione parziale con aria e argon come gas di alimentazione e sweep. Inoltre, sono stati eseguiti test di permeazione a lungo termine sulle membrane SCTF9182 in atmosfera di CO2. È stato osservato che la stabilità del reticolo di perovskite in ambiente contenente CO2 è stata migliorata dalla sostituzione del Ca, mitigando la formazione indesiderata di carbonati. Al contrario, la permeabilità non è stata ridotta dall'introduzione del Ca, il che rende questa sostituzione promettente per lo sviluppo di nuovi materiali per gli OTM. Inoltre, la membrana SCTF9182 ha mostrato un flusso costante di separazione dell'ossigeno e nessuna degradazione di fase dopo aver permeato per 340 ore a 900 °C con elio come gas di sweep e per 170 ore a 900 °C con un flusso gassoso di sweep contenente il 40% di CO2.
I materiali identificati sono stati impiegati per preparare membrane dual-phase composte da una miscela del noto conduttore ionico ossido di cerio dopato con gadolinio (CGO) e delle perovskiti precedentemente sintetizzate STF82, SCTF9182 e SCTF6482. Sono state identificate le condizioni di sinterizzazione più adatte per ottenere membrane dense a tenuta di gas ed evitare qualsiasi interazione di fase tra CGO e STF/CSFT. Sono stati eseguiti test di permeabilità sia su membrane dense che su membrane dense con uno strato di attivazione di LSCF sulla superficie, che supera le limitazioni legate alle reazioni di scambio superficiale. Le membrane dual-phase non hanno migliorato la permeabilità rispetto alle membrane single-phase e, aggiungendo lo strato di attivazione, tutte le membrane hanno mostrato la stessa permeabilità, evidenziando che il fattore limitante, in questo caso, è la conduzione ionica attraverso la fase CGO.
Infine, è stata studiata la sostituzione del bario. In questo caso, il bario ha un raggio ionico più alto dello stronzio, che potrebbe facilitare la formazione di vacancies di ossigeno e migliorare la permeazione degli ioni di ossigeno. Il bario è stato sostituito nelle stesse percentuali del calcio, quindi le perovskiti Sr0,9Ba0,1Ti0,8Fe0,2O3- (SBTF9182) e Sr0,6Ba0,4Ti0,8Fe0,2O3- (SBTF6482) sono state sintetizzate per reazione allo stato solido e il trattamento di sinterizzazione è stato ottimizzato per entrambe le composizioni, al fine di evitare la formazione di cricche e ottenere membrane bulk a tenuta di gas. I test di permeabilità hanno rivelato una permeabilità comparabile per le tre perovskiti STF82, SBTF9182 e SBTF6482 per l'intervallo di temperatura 750-1000 °C. Come nel caso del Ca, la sostituzione di Sr con Ba in frazioni fino al 40% sembra avere solo un effetto minore sulle prestazioni di permeabilità all'ossigeno.The urgency of increasing the environmental sustainability of the human activities, to limit the irreversible changes we see daily in the climate and nature, has become one of the biggest problems worldwide. Amongst the new methods proposed by the scientific community, Oxygen transport Membranes (OTMs) have gained a lot of attention, since they can be employed in different applications, potentially bringing a significant contribution for the decarbonization of the energy system. OTMs allow oxygen separation from gas mixtures like or ambient air, and therefore they can be used to produce pure oxygen for oxy-fired industrial processes. Moreover, recent studies have applied OTMs in Catalytic Membrane Reactors (CMRs), which exploit the properties of oxygen transport to perform processes of chemical conversion. CMRs can potentially be employed to produce several chemical commodities and energy vectors, including hydrogen from and earth-abundant and carbon-free source likewater.
To work, OTMs need to conduct both electrons and ions; therefore, they are mainly made of Mixed Electron and Ion Conductors (MIEC). Although OTMs have promising applications, they work at high temperatures and in highly reducing environments. The most significant limit for the diffusion of OTMs in real processes is that they exhibit either low stability in operating conditions, or low permeability. The challenge is then to design new OTMs materials that exhibit a good balance between stability and permeability.
The objective of this PhD project was to identify, synthesize, and characterize innovative MIEC materials with promising properties for application in OTM operating under challenging conditions, like reducing and CO2-containing atmospheres. , The activities were focused on MIEC oxides belonging to the class of SrTi1-xFex-O3- perovskites, which had recently attracted attention thanks to its higher chemical stability with respect to the most studied MIEC materials. In particular, it was investigated the effect of A-site substitution on the perovskite SrTi0,8Fe0,2O3- (STF82). . Therefore, initially, it was chosen to substitute strontium with calcium, which might increase the material tolerance to CO2 but, having a smaller ionic radius, potentially have a negative impact on the oxygen permeation performance. Two different Ca fractions were chosen to preliminary investigate a possible range of substitutions, 10% and 40%, and so Sr0,9Ca0,1Ti0,8Fe0,2O3- (SCTF9182) and Sr0,6Ca0,4Ti0,8Fe0,2O3- (SCTF6482) were prepared through solid-state reaction. The stability of these powders against CO2 was studied with Thermogravimetric Analysis (TGA), and permeability tests were carried out on sintered dense membranes in the range of 600°-1000°C under partial pressure gradient with air and argon as feed and sweep gases. In addition, long-term permeation tests on SCTF9182 membranes were performed in CO2 atmosphere. It was observed that the stability of the perovskite lattice in CO2-containing environment was improved by Ca substitution, with mitigate the undesired formation of carbonates. On the contrary, the permeability was not reduced by Ca introduction, which makes this substitution promising for the development of new materials for OTMs. Moreover, SCTF9182 membrane showed constant oxygen separation flux and no phase degradation after permeating for 340 h at 900 °C when swept with He and for 170 h at 900 °C when swept with a gaseous stream containing 40% CO2.
The identified materials were employed to prepare dual-phase membranes composed by a mixture of the well-known ionic conductor gadolinium doped ceria (CGO) and the perovskites previously synthesised STF82, SCTF9182 and SCTF6482. Appropriate sintering conditions to obtain dense gas-tight membranes and avoid any phase interaction between CGO and STF/CSFT were identified. Permeability tests were carried out on both dense membranes and dense membranes with an activation layer of LSCF on the surface, which overcomes the limitations related to surface exchange reactions. Dual-phase membranes did not improve the permeability compared to single-phase membranes, and by adding the activated layer, all membranes exhibited the same permeance, highlighting that the limiting factor, in this case, is the ionic conduction through CGO phase.
Finally, the substitution of barium was investigated. In this case, it has a higher ionic radius than strontium, which could facilitate the formation of oxygen vacancies and improve the permeation of oxygen ions. Barium was substituted in the same percentages as calcium, and so the perovskites Sr0,9Ba0,1Ti0,8Fe0,2O3-(SBTF9182) and Sr0,6Ba0,4Ti0,8Fe0,2O3- (SBTF6482) were synthesised by solid state reaction and the sintering treatment was optimized for both compositions, in order to avoid cracks formation and obtain gas-tight bulk membranes. Permeability tests revealed comparable permeance for the three perovskites STF82, SBTF9182 and SBTF6482 for in the temperature range 750 – 1000 °C. Like in the case of Ca, the substitution of Sr with Ba at fractions up to 40 % seems to have only a minor effect on the oxygen permeability performance
L'utilizzo dei segnali sociali per l'autoregolazione nei macachi : un' indagine etologica preliminare
No full textSocial interactions are profoundly influenced by contextual information and group characteristics, which shape individual behavior. In the field of social neuroscience, numerous neurophysiological studies have highlighted the neural mechanisms underlying the encoding of self- and other-related information. However, studying social behavior in ecologically valid conditions remains a challenge, as the complexity of these phenomena is often reduced in highly controlled experimental settings. This study presents a preliminary ethological investigation aimed at exploring social behavior in a group of four macaques, allowing the subjects to spontaneously express their interactive strategies in an unrestricted experimental context. The employed paradigm is based on a turn-taking task, structured around the use of sticks of different color, each corresponding to a specific individual, administered in a freely moving setting. This setup, designed to enhance the likelihood of observing spontaneous interactions, aims to assess whether individuals demonstrate awareness of the association between a specific stimulus and a given group member. Additionally, the study investigates the monkeys’ strategies of social monitoring and self-regulation during the task. The results reveal that, during others’ trials, monkeys primarily direct their gaze toward the individual associated with the presented color stick. Moreover, an overall inhibition of gaze exchanges follows the presentation of stimuli associated with specific subjects, depending on their hierarchical rank. At the same time, during their own trials, monkeys show increased
monitoring toward higher-ranking individuals, and the subjects' task performance is negatively affected by being observed or engaged in an interaction with another individual, with this effect depending on their relative positions within the group’s hierarchy. These data suggest that learned correspondences between environmental stimuli and specific group members enable monkeys to predict other’s behavior and allow them to use this information for self-regulated social interaction strategies. This approach, which preserves the natural behavioral expression of the subjects, provides a foundation for future studies investigating the neural bases of social affordances, contributing to a more ecologically valid understanding of the processes underlying social interactions.Le interazioni sociali sono profondamente influenzate dalle informazioni contestuali e dalle caratteristiche del gruppo, che modellano il comportamento individuale. Nell’ambito delle neuroscienze sociali, numerosi studi neurofisiologici hanno evidenziato i meccanismi neurali alla base della codifica delle informazioni relative a sé e agli altri. Tuttavia, lo studio del comportamento sociale in condizioni ecologicamente valide rappresenta ancora una sfida, poiché la complessità di questi fenomeni viene spesso ridotta in contesti sperimentali
altamente controllati. Il presente studio propone un'indagine etologica preliminare volta a esplorare il
comportamento sociale in un gruppo di quattro macachi, consentendo ai soggetti di esprimere spontaneamente le proprie strategie interattive in un contesto sperimentale privo di restrizioni. Il paradigma adottato si basa su un task di turn-taking, strutturato sull’uso di bastoncini di diverso colore, ciascuno associato a un individuo specifico, somministrati in un contesto di libero movimento. Questo disegno sperimentale, concepito per favorire l’espressione di interazioni spontanee, mira a valutare se gli individui dimostrino consapevolezza della specifica associazione tra gli stimoli e i membri del gruppo. Inoltre, lo studio indaga le strategie di monitoraggio sociale e autoregolazione adottate dai macachi durante il task. I risultati mostrano che, durante i turni altrui, i macachi dirigono prevalentemente lo sguardo verso l’individuo associato al bastoncino del colore presentato. Inoltre, in seguito alla presentazione di stimoli associati a soggetti specifici, si osserva una generale inibizione del monitoraggio sociale, che varia in funzione del rango gerarchico degli individui coinvolti. Allo stesso tempo, durante i propri turni, i macachi manifestano un incremento del monitoraggio nei confronti degli individui di rango superiore, e la loro prestazione nelcompito risulta negativamente influenzata dall’essere osservati o dall’essere coinvolti in un’interazione da un altro individuo, con un effetto modulato dalla posizione relativa all’interno della gerarchia del gruppo. Questi dati suggeriscono che le corrispondenze apprese tra stimoli ambientali e membri specifici del gruppo consentono ai macachi di prevedere il comportamento altrui e di utilizzare tali informazioni per strategie di interazione sociale autoregolata. Questo approccio, che preserva l’espressione naturale del comportamento dei soggetti, fornisce una base per studi futuri volti a indagare le basi neurali delle affordance sociali, contribuendo a una comprensione ecologicamente più valida dei processi alla base delle interazioni sociali
Unlocking the potential of Nanobodies: Immunotagging for targeted therapy
No full textNanobodies, or VHHs, are single-domain antibodies derived from camelid heavy-chain antibodies, have emerged for their stability, specificity, and small size, which allows for superior tissue penetration and access to epitopes inaccessible to conventional antibodies. However, their lack of an Fc region limits their immune effector function and reduces circulation time. To address these limitations, this research introduces the ImmunoJunction (IJ) platform, which combines VHHs with an engineered ImmunoTag—a fragment of tetanus toxin capable of recruiting anti-tetanus antibodies present in over 85% of the global population. ImmunoTag facilitates immune engagement through opsonization and cellular activation, effectively redirecting pre-existing
immunity to target various pathogens or tumor antigens. The modularity of the ImmunoJunction platform offers significant potential for both therapeutic and diagnostic applications.
This thesis explores the development and characterization of nanobodies targeting critical proteins associated with viral and bacterial pathogenesis, specifically the Spike protein of SARS-CoV-2 and the virulence factors PhtD, PnrA, and pneumolysin of Streptococcus pneumoniae.
The first part of the study centers on SARS-CoV-2, targeting its Spike protein as a key antigen.
Initially, well-characterized nanobodies from the literature were expressed as recombinant proteins to assess their neutralizing capacity both independently and in conjugation with ImmunoTag. In vitro neutralization assays demonstrated enhanced viral neutralization with ImmunoJunction constructs, validating this approach. In collaboration with Preclinics, the study further advanced by generating novel anti-Spike and anti-nucleocapsid protein (N) nanobodies via a llama-derived immune cDNA library. Screening these libraries through phage display led to the isolation of promising nanobodies, which were then expressed in Escherichia coli and evaluated for
their potential neutralizing capacity. The neutralization assays revealed significant differences in efficacy among the candidates, with nanobody H8 exhibiting the strongest neutralization activity against the Wuhan variant, and nanobodies A12 and G3 demonstrating effective neutralization against Omicron BA.1 and BA.4/5. These three most effective candidates were then expressed in Escherichia coli conjugated with ImmunoTag, but unfortunately only A12-IJ was expressed and purified to appreciable levels. The second part of the research focuses on Streptococcus pneumoniae, a clinically significant pathogen, particularly in the context of rising antibiotic resistance. This segment focused on creating immune libraries to identify nanobodies targeting surface proteins PhtD, PnrA, and Pneumolysin, which play critical roles in bacterial virulence and host colonization. VHH libraries against these antigens were constructed and underwent selection by phage display to identify potent binders. For selected candidates targeting PhtD and PnrA, E. coli expression protocols were optimized to increase VHH yield and solubility. Although solubility issues initially posed challenges,
the use of autoinducing media and alternative E. coli strains improved the protein yield of four VHHs: A11 (PhtD), H2 (PhtD), H5 (PhtD), and D4 (PnrA). The purified proteins were then analyzed using a peptide ELISA assay to verify binding to their respective target proteins, with results indicated that H5 (PhtD) and D4 (PnrA) produced higher signals, suggesting a greater binding affinity to their respective targets.
For pneumolysin, nearly 30 high-affinity nanobody candidates were isolated, with ongoing efforts directed toward sequencing, expression, and evaluation to assess their potential.
In conclusion, this work exemplifies the versatility of nanobodies in targeted therapy, emphasizing
their promise in tackling antibiotic-resistant bacteria and complex viral pathogens. The findings underscores the need to evaluate not only binding affinity but also functional efficacy, indicating the necessity for further studies into the neutralization mechanisms and interactions of these nanobodies. The introduction of the ImmunoJunction platform represents a novel strategy with its modular approach that harnesses widespread tetanus immunity to enhance nanobody potency and immune engagement. This innovation opens new possibilities for nanobody-based therapeutics and diagnostics
Role of KCNQ1 Gene in Mental and Metabolic Disturbances Associated with Insulin Signalling Dysregulation
No full textMetabolic imbalances (such as diabetes, obesity, and metabolic syndrome) significantly increase the risk of cognitive disturbances, including Alzheimer’s disease, dementia, obsessive compulsive disorders, autism spectrum disorders, and attention deficit hyperactivity disorders. In this context, KCNQ1, a voltage-gated potassium channel, is a key connecting metabolism and cognition. Different scholars, including preclinical and clinical scientists, and geneticists, have identified a potential contribution of the KCNQ1 gene as a key mediator of insulin signalling pathways. My project aimed to identify mechanisms involving peripheral and central dysregulation of insulin signalling, assessing KCNQ1-related effects in the brain. To achieve this aim, I conducted behavioural, physiological, and metabolic tests in a conditional kcnq1 knockout mouse model. In those experimental subjects, kcnq1 is downregulated in a time- (during adolescence and adulthood) and spatial- (only in the brain) specific manner. My findings showed that kcnq1 deletion in the brain significantly impairs cognitive abilities in rodents; specifically, executive functions.My experimental findings disclose innovative potential therapeutic avenues revolving around KCNQ1 as a candidate target to mitigate insulin-mediated cognitive decline