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La leucemia linfatica cronica (CLL), è una patologia incurabile caratterizzata da un intenso traffico di cellule leucemiche tra il sangue periferico e gli organi linfatici. Durante questo ricircolo le cellule di CLL si adattano alle condizioni ambientali riarrangiando il loro citoscheletro, tuttavia, le differenze fra le cellule di CLL e quelle sane non sono note. La nostra ipotesi prevede che il citoscheletro potrebbe essere coinvolto nell’adattamento meccanico delle cellule. In questo contesto abbiamo identificato la proteina HS1 come candidato. HS1 è l’omologo ematopoietico di cortactin, interagisce con il citoscheletro e inoltre nei pazienti di CLL è identificato come un fattore prognostico. Per studiarla abbiamo introdotto un’analisi basata su una tecnica di super risoluzione confocale (STED), ottimizzata allo scopo di visualizzare e quantificare la distribuzione di HS1 in cellule primarie di pazienti con la CLL e donatori sani (HD-B). Dalle analisi abbiamo rilevato che HS1 è organizzato in nanocluster in tutto il volume extra-nucleare della cellula, inoltre la sua organizzazione correla con la prognosi dei pazienti con CLL. Attraverso ricostruzioni 3D dell’intera cellula, abbiamo rilevato la presenza di grossi assembramenti di HS1 nella regione di adesione, condizione particolarmente evidente nelle cellule di pazienti con cattiva prognosi. Questa organizzazione suggerisce la necessità di reclutare HS1 nelle adesioni focali. A questo scopo abbiamo quantificato il livello di interazione tra HS1 e la vimentina, un filamento intermedio coinvolto nella formazione di queste strutture sfruttando la STED e la phasor-FLIM-FRET. Questi approcci ci hanno consentito di identificare un aumento nell’interazione delle due proteine nella regione basale, a conferma della nostra ipotesi. Nel loro insieme, questo studio ci ha consentito di dimostrare che i pazienti con CLL possono essere classificati in base all’organizzazione di una singola proteina e inoltre ci suggerisce, Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of leukemic cells between the peripheral blood and lymphoid tissues. CLL cells’ ability to adapt to external cues and reorganize their cytoskeleton enables them to recirculate between tissues, however little is known about the differences between CLL and healthy B cells (HD-BD) during these processes. Our study started from the unexplored hypothesis that the cytoskeleton might be a key actor possibly involved in CLL cells' mechanical adaptation. In this context we identify the hematopoietic lineage cell-specific protein 1 (HS1) as a perfect candidate for our studies. HS1 is the hematopoietic homologous of cortactin and is a central cytoskeletal interactor. In CLL patients its phosphorylation, and expression are putative prognostic factors. We here introduce a super-resolution approach based on single-cell 3D-STED microscopy optimized to reveal the distribution of endogenous HS1 in HD-B and CLL primary cells. Our study shows that endogenous HS1 in primary cells and in MEC1 cells (CLL cell line), is organized in a nanocluster-like structure all over the extranuclear body, with a correlation between HS1 clustering and CLL patients’ prognosis. Using 3D reconstruction, we observed that HS1 nanoclusters form bulky assemblies at the bottom of the cell (adhesive side), especially in those with adverse outcomes, suggesting the need for its recruitment for cell adhesion. This observation agrees with a phasor-FLIM-FRET, and STED colocalization analysis of HS1 and vimentin, an intermediate filament involved in focal adhesion assembly, which showed an increased interaction at the basal site of the cells. Altogether these results enable for the first time the discrimination of CLL patients based on a single protein organization and suggest the presence of an altered cytoskeleton in CLL cells as compared to HD-B cells. Based on this evidence, we questioned if the organization of CLL

Glioblastoma (GB) is the most common and aggressive brain tumor. The treatment for newly diagnosed glioblastoma is surgical resection of the primary tumor mass, followed by radiotherapy and chemotherapy. However, recurrences frequently occur in proximity to the surgical resection area. In these cases, none of the current therapies is effective. Recently, implantable biomaterials seem to be a promising strategy against GB recurrence. Here, for the first time we combined the tailorable properties of soy-protein hydrogels with the versatility of drug-loaded liposomes to realize a hybrid biomaterial for controlled and sustained nanoparticles release. Hydrogel consisting of 18–20 % w/v soy-protein isolated were fabricated in absence of chemical cross-linkers. They were biodegradable (−10 % and −30 % of weight by hydrolytic and enzymatic degradation, respectively in 3 days), biocompatible (>95 % of cell viability after treatment), and capable of sustained release of intact doxorubicin-loaded liposomes (diffusion coefficient between 10−18 and 10 −19 m2 s−1). A proof-of-concept in a “donut-like” 3D-bioprinted model shows that liposomes released by hydrogels were able to diffuse in a model with a complex extracellular matrix-like network and a 3D structural organization, targeting glioblastoma cells. The combination of nanoparticles' encapsulation capabilities with the hydrogels' structural support and controlled release properties will provide a powerful tool with high clinical relevance that could be applicable for the treatment of other cancers, realizing patient-specific interventions.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Nonostante gli enormi passi avanti sulla comprensione dei meccanismi d’azione del cancro e nello sviluppo di farmaci antitumorali, la maggior parte delle forme di cancro è ancora incurabile. La ragione di tale insuccesso è radicata nella complessità di questa malattia, che è ancora poco conosciuta sia in fase di insorgenza, dovuta alla misregolazione di oncogeni, sia in fase di metastatizzazione delle cellule cancerose. Lo studio di tali caratteristiche da un punto di vista meccanico può fornire una visione differente dei meccanismi di azione del cancro ed aiutare a contestualizzarli all’interno di processi estremamente complessi. L’insorgenza del cancro è dovuta ad una mancata regolazione del ciclo cellulare, che a sua volta dipende spesso da un’alterata espressione dei cosiddetti oncogeni. Ne è un esempio il proto-oncogene c-KIT, che codifica per un fattore di crescita delle cellule staminali. La regolazione della sua espressione è controllata dal suo promotore prossimale, la cui struttura è caratterizzata dalla presenza di 3 superstrutture tridimensionali complesse che si formano su uno dei due filamenti della sua sequenza, chiamate G-quadruplexes. Tali strutture sembrerebbero avere un ruolo nel riconoscimento del promotore da parte della polimerasi e nell’assemblaggio della macchina trascrizionale, ma la loro funzione non è ancora chiara. Lo studio della nanomeccanica di tali strutture a livello di singola molecola (grazie al Magnetic Tweezers) può fornire importanti informazioni sul loro ruolo e sulle modalità con cui regolano l’espressione dell’oncogene. In seguito all’insorgenza del cancro, le cellule coinvolte subiscono forti cambiamenti strutturali: iniziano a dividersi velocemente e a migrare incontrollatamente. Entrambi questi processi richiedono un grande cambiamento nella struttura cellulare ed in particolare nella rigidità della cellula. La leucemia linfoide cronica (CLL) rappresenta un caso esemplare di tali capacità migratorie, non solo, With the term cancer are intended many species of diseases having quite different properties from each other. Despite such vast differences, the mechanisms beyond the onset of any kind of cancer are very similar and can be classified in two main groups depending on their stage. The first is related to the dysregulation of particular genes (oncogenes), that results in an impairment of the cell cycle. The second concerns the ability of cancer cells to continuously divide and migrate through tissues, that results in a highly invasive potential. From a mechanical point of view, the investigation of such features can be crucial for a deeper understanding of cancer onset and progression as well as for the study of novel pharmacological treatments. The outbreak of cancer is caused by a deficiency in the regulation of the cell cycle which, in turn, often depends on an abnormal expression of oncogenes. It is the case of the proto-oncogene c-KIT, that encodes for a mast/stem cell growth factor receptor. Its regulation relies mainly on its promoter, which is constituted by 3 distinct three-dimensional DNA structures called G-quadruplexes (G4s). Those structures can be studied by means of nanomechanical tools such as Magnetic Tweezers, which can recognize folded G4s at single-molecule level, thus enabling to study their role in the regulation of the oncogene. After the onset of cancer, a generic cell undergoes mechanical changes: it divides quickly, and it starts migrating. Both phenomena require a modification in the cell structural phenotype, eventually modifying its rigidity. Chronic lymphocytic leukemia is a case in point: malignant B lymphocytes continuously traffic between peripheral blood and lymphoid tissues. Such frequent migrations require a change in the rigidity of cells. In this case, Atomic Force Microscopy can provide a nanomechanical approach allowing to measure the stiffness of single cells from patients with leukemia, which is slightly decreased if compared to

Two‐photon laser writing is used here to fabricate 3D proteinaceous microstructures with photothermal functionality in the near‐infrared spectral region and tunable elasticity. The photo‐cross‐linking is initiated in bovine serum albumin (BSA) by rose bengal or methylene blue and the photo‐thermal effect arises from gold non‐spherically symmetric nanoparticles dispersed in the ink. Massive energy transfer of the plasmonic resonances of the gold nanoparticles to methylene blue prevents effective photo‐crosslinking of BSA. However, stable microstructures with photo‐thermal functionality can be fabricated in the rose bengal proteinaceous inks. On these microstructures, with a gold atom concentration as low as 1% w/w, a highly localized temperature increase can be quickly (≅1 s) reached and maintained under continuous wave laser irradiation at 800 nm. The photothermal efficiency under continuous wave laser irradiation depends on the thickness of the microstructure and can reach 12.2 ± 0.4 °C W−1 These proteinaceous microstructures represent therefore a promising platform for future applications in the fields like physical stimulation of cells for regenerative nanomedicine.

By functionalizing the surface of PEG-liposomes with linkers bearing quaternary ammonium compounds (QACs), we generated novel bacteria disruptors with anti-adhesive properties and reduced cytotoxicity compared to free QACs. Furthermore, QAC-functionalized liposomes are a promising platform for future drug encapsulation. The QAC (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide (MTAB) was attached to maleimide-functionalized liposomes (DSPE-PEG) via thiol linker. The MTAB-functionalized liposomes were physicochemically characterized and their biological activity, in terms of anti-adherence activity and biofilm prevention in Escherichia coli were assessed. The results showed that MTAB-functionalized liposomes inhibit bacterial adherence and biofilm formation while reducing MTAB toxicity.