Interazione del poli(etilenglicol) con il sistema cesio-perfluoro-ottanoato/H2O: indagine spettroscopica tramite 19F NMR e 133Cs NMR

Il presente lavoro di tesi si inserisce in un filone di ricerca che, presso il nostro Dipartimento, ha visto la collaborazione dei gruppi di termodinamica e spettroscopia NMR. Questi studi mirano alla comprensione della natura delle interazioni che si instaurano fra le micelle, per esempio di cesio-perfluoro-ottanoato (CsPFO), e polimeri idrosolubili quali il poli(etilenglicol), PEG, in acqua. Studi analoghi sono già stati effettuati, con risultati incoraggianti, su un altro surfattante, il Sodio-Dodecil-Solfato. In questa tesi viene presentato lo studio del sistema CsPFO/H2O, senza e con aggiunta di PEG di diverso peso molecolare, attraverso 19F NMR e 133Cs NMR. La registrazione degli spettri del cesio è stata suggerita dall’idea che sia proprio il controione il tramite essenziale dell’interazione, molto studiata ma ancora poco chiara, fra polimero e micelle. Le pagine seguenti si propongono, dunque, di riassumere gli studi effettuati fino ad ora, presentare i risultati del nostro lavoro e suscitare curiosità ed interrogativi sulle cui tracce potrebbero essere condotti successivi approfondimenti

Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) 7> 256 mu g/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 mu g/ml] and rifampin (MBBC and MBEC = 128 mu g/ml). After the treatment of MRSA biofilm with 1024 mu g/ml vancomycin, similar to 5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells

The HCN domain couples voltage gating andcAMP response in hyperpolarization-activatedcyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms

The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltagedependent gating in three HCN isoforms

Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) > 256 μg/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 μg/ml] and rifampin (MBBC and MBEC = 128 μg/ml). After the treatment of MRSA biofilm with 1024 μg/ml vancomycin, ∼5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells

Studio delle proprieta molecolari di elastomeri liquido-cristallini mediante spettroscopia 2HNMR

L'elaborato è costituito da due sezioni, la prima delle quali dà il titolo alla tesi: - studio delle proprietà molecolari di elastomeri liquido-cristallini; - studio delle interazioni in un sistema micellare perfluorurato. Nella prima parte, dopo un'accurata descrizione delle teorie (Landau de Gennes) e delle tecniche (2HNMR, DSC, microscopia) alla base dell'analisi, sono esposti e analizzati i risultati riguardanti lo studio dell'ordine molecolare di sistemi elastomerici nematici. La novità di tale studio, che lo differenzia dai lavori riportati in letteratura, consiste nell'aver indagato direttamente una componente strutturale dell'elastomero liquido cristallino (il crosslinker selettivamente deuterato), senza dover ricorrere all'introduzione di molecole probe (che avrebbero alterato la struttura e la composizione del sistema). Dagli splitting quadrupolari del nucleo di deuterio sono quindi stati ottenuti gli andamenti del parametri d'ordine, analizzati in base alle previsioni della teoria di Landau-de Gennes. Viene sviluppato il confronto con il crosslinker disciolto in un solvente liquido-cristallino convenzionale e con dati di letteratura. La seconda parte indaga i sistemi micellari in soluzione, costituiti dai sali di Litio, Sodio e Cesio dell'acido perfluoroottanoico. Sono stati monitorati gli andamenti di chemical shift dei nuclei 19F, molto sensibili al processo di micellizzazione. Lavori di letteratura segnalano "anomalie" nelle curve calorimetriche nel momento in cui in questi sistemi vengono aggiunti poli(etilenglicol) di diverso peso molecolare. Sulla scia di questi lavori, l'analisi NMR è stata condotta su sistemi costituiti dal solo sale, dal sale più il polimero, dal sale più eteri corona (quest'ultimi mirati ad valutare l'azione sia del controione sia, per confronto, del polimero). I risultati ottenuti e la validità delle ipotesi formulate hanno trovato conferma in calcoli teorici

2H NMR orientational study of a probe dissolved in nematic solution and, used as crosslinker, in a liquid crystalline elastomer

The orientational order parameters, Szz and Biax, of UB-d4 [1,4-bis(undec-10-en-1-yloxy)benzene-d4] in the ZLI1167 nematic mixture are studied by means of 2H NMR at variable temperature. The Szz(T) trend fairly follows the Maier–Saupe theoretical curve; therefore, UB-d4 can be considered an excellent probe to monitor the ordering of the nematic phase itself, provided that Biax is not neglected and the complete analysis of quadrupolar and dipolar splittings is performed. UB-d4 is a typical crosslinker in liquid crystal elastomers (LCEs): the order parameters here collected help us to discuss the peculiarities of the crosslinker as a probe for the study of internal order in LCEs. From the analysis of our data and of data in the literature, we infer that the order parameter Biax of the crosslinker in the LCE matrix is low enough to be neglected without severely perturbing the determination of Szz. The crosslinker UB-d4 is a reliable probe to monitor the nematic order degree inside the LSCE-UB-d4 sample