Dinamica molecolare delle rodopsine con modelli multi-scala

In questa tesi viene presentato un modello multi-scala per le rodopsine. La funzione di queste proteine è di tradurre stimoli luminosi (nel visibile) in segnali chimici per comunicazione trans cellulare. Specificamente sono state considerate due ropsine, prototipiche di due sottoclassi funzionali: la rodopsina batterica e la rodopsina dei mammiferi. La prima è una pompa di protoni localizzata nella membrana purpurea del batterio Halobacterium salinarum; la seconda si trova nelle cellule fotorecettrici della retina dei mammiferi ed ha lo scopo di generare il segnale che avvia il processo della visione. Il loro funzionamento è molto simile: entrambe contengono al loro interno un retinale che, con l'assorbimento di un fotone, cambia conformazione; questa transizione dà il via ad una serie di cambiamenti strutturali delle proteine che portano al rilascio del segnale. Il “fotociclo” si conclude con il ritorno delle proteine nello stato iniziale, ed ogni suo stato è identificabile da un diverso assorbimento ottica della proteina. Le due rodopsine hanno anche una struttura terziaria molto simile, ma le strutture dei vari stati della batterica sono note sperimentalmente con maggiore accuratezza, mentre per la rodopsina dei mammiferi i dati strutturali sono più frammentari e meno accurati. Si è dunque scelta la rodopsina batterica come prototipo per il modello che è poi stato trasferito all’altra. Il modello sviluppato rappresenta la proteina a due diversi livelli di risoluzione: per il retinale e le molecole di acqua interne alla proteina si è usata una rappresentazione quasi atomistica, in cui i gradi di libertà espliciti sono le coordinate degli atomi “pesanti” (C, N e O) mentre gli idrogeni ad essi legati sono trattati implicitamente; per tutto il resto della proteina si è usato un modello “minimalista” in cui un amminoacido è rappresentato da un singolo atomo, il carbonio alfa, (Cα). In questo modo la parte peptidica della proteina è rappresentata da una catena lineare di centri interattivi. Questa rappresentazione mista con parte peptidica a bassa risoluzione e sito attivo (retinale + acqua) ad alta risoluzione consente di rappresentare con buona accuratezza le fasi iniziali veloci del fotociclo che coinvolgono la transizione strutturale del retinale, e contemporaneamente mantiene un basso costo computazionale, che consente la rappresentazione dell’intero fotociclo su scale di tempi macroscopici. La parametrizzazione delle interazioni nel modello (il campo di forze) è la parte principale di questo lavoro di tesi. Il campo di forze del retinale ha costituito un problema minore, in quanto i campi per le interazioni atomistiche o quasi atomistiche sono stati sviluppati e ottimizzati nel corso degli ultimi quattro-cinque decenni, e quindi esistono molti dati di letteratura per i parametri. Invece i modelli a bassa risoluzione sono relativamente nuovi nel panorama della modellistica biomolecolare, e non standardizzati. Si è quindi proceduto a ottimizzare i parametri di ciascun termine del campo di forze, fittandoli in modo da riprodurre set di dati strutturali sperimentali e dinamici da simulazioni atomistiche. Si sono ottenuti così diversi set di parametri, ciascuno ottimizzato per un diverso stato del fotociclo. Infine si è proceduto a combinare i diversi campi di forze tra loro, in modo da poter rappresentare (in simulazioni di dinamica molecolare classica) le transizioni tra uno stato e l’altro. In questa fase sono stati inclusi dati sperimentali sulla stabilità relativa dei diversi stati. Si è così ottenuto un campo multi-stabile, in grado di rappresentare nella simulazione l’intero fotociclo. Poiché il campo include al livello empirico dati di varia natura di varia origine, è in grado di riprodurre accuratamente aspetti sia strutturali che cinetici che energetici, oltre a consentire la simulazione del fotociclo completo su scala macroscopica. Entrambe le cose non sono possibili in modelli atomistici (accurati strutturalmente ma non in grado di riprodurre transizioni, energie relative e cinetica su larga scala temporale), o interamente a bassa risoluzione (non in grado di riprodurre accuratamente le fasi veloci del fotociclo)

An atomic-level look at the structure-property relationship of cerium-doped glasses using classical molecular dynamics

Ce-containing bioactive glasses are of great interest in biomedical field since they exert antioxidant properties associated with low toxicity and a broad spectrum of bacteriostatic activities. The results obtained by classical molecular dynamics simulations allow the elucidation of the correlations between the effect of the inclusion of cerium doping ions into the structure of phosphosilicate and silicate bioactive glasses and their properties. The addition of small quantities of Ce to the silicate bioglass favours the depolymerisation of the silicate network with a positive effect on the ability to dissolve in body fluid. Moreover, the under coordination of both the Ce3+ and Ce4+ species in these glasses enhances their catalytic activity towards hydrogen peroxide. Conversely, the formation of cerium phosphate domains in the phosphosilicate glasses leads to detrimental effects for both the solubility and the catalytic activity of the glasses. Finally, a new quantitative view of the structure-activity relationships governing the macroscopic properties of these glasses has been obtained by means of structural descriptor that takes into account the fragmentation of the Si network and the consequent rearrangement of the modifier ions and the network destruction per cerium unit descriptor

From the Amelioration of a NADP+-dependent Formate Dehydrogenase to the Discovery of a New Enzyme: Round Trip from Theory to Practice

NADP+-dependent formate dehydrogenases (FDHs) are biotechnologically relevant enzymes for cofactors regeneration in industrial processes employing redox biocatalysts. Their effective applicability is however hampered by the low cofactor and substrate affinities of the few enzymes described so far. After different efforts to ameliorate the previously studied GraFDH from the acidobacterium Granulicella mallensis MP5ACTX8, an enzyme having double (NAD+ and NADP+) cofactor specificity, we started over our search with the advantage of hindsight. We identified and characterized GraFDH2, a novel highly active FDH, which proved to be a good NAD+-dependent catalyst. A rational engineering approach permitted to switch its cofactor specificity, producing an enzyme variant that displays a 10-fold activity improvement over the wild-type enzyme with NADP+. Such variant resulted to be one of the best performing enzyme among the NADP+-dependent FDHs reported so far in terms of catalytic performance

Sweat analysis with a wearable sensing platform based on laser-induced graphene

The scientific community has shown increasing interest in laser scribing for the direct fabrication of conductive graphene-based tracks on different substrates. This can enable novel routes for the noninvasive analysis of biofluids (such as sweat or other noninvasive matrices), whose results can provide the rapid evaluation of a person's health status. Here, we present a wearable sensing platform based on laser induced graphene (LIG) porous electrodes scribed on a flexible polyimide sheet, which samples sweat through a paper sampler. The device is fully laser manufactured and features a two layer design with LIG-based vertical interconnect accesses. A detailed characterization of the LIG electrodes including pore size, surface groups, surface area in comparison to electroactive surface area, and the reduction behavior of different LIG types was performed. The bare LIG electrodes can detect the electrochemical oxidation of both uric acid and tyrosine. Further modification of the surface of the LIG working electrode with an indoaniline derivative [4-((4-aminophenyl)imino)-2,6-dimethoxycyclohexa-2,5-dien-1-one] enables the voltammetric measurement of pH with an almost ideal sensitivity and without interference from other analytes. Finally, electrochemical impedance spectroscopy was used to measure the concentrations of ions through the analysis of the sweat impedance. The device was successfully tested in a real case scenario, worn on the skin during a sports session. In vitro tests proved the non-cytotoxic effect of the device on the A549 cell line

Device‐to‐Materials Pathway for Electron Traps Detection in Amorphous GeSe‐Based Selectors

The choice of the ideal material employed in selector devices is a tough task both from the theoretical and experimental side, especially due to the lack of a synergistic approach between techniques able to correlate specific material properties with device characteristics. Using a material-to-device multiscale technique, we propose a reliable protocol for an efficient characterization of the active traps in amorphous GeSe chalcogenide. The resulting trap maps trace back the specific features of materials responsible for the measured findings, and connect them to an atomistic description of the sample. Our metrological approach can be straightforwardly extended to other materials and devices, which is very beneficial for an efficient material-device co-design and the optimization of novel technologies

Pirfenidone for Idiopathic Pulmonary Fibrosis and Beyond

Pirfenidone (PFD) slows the progression of idiopathic pulmonary fibrosis (IPF) by inhibiting the exaggerated fibrotic response and possibly through additional mechanisms, such as anti-inflammatory effects. PFD has also been evaluated in other fibrosing lung diseases. Myocardial fibrosis is a common feature of several heart diseases and the progressive deposition of extracellular matrix due to a persistent injury to cardiomyocytes may trigger a vicious cycle that leads to persistent structural and functional alterations of the myocardium. No primarily antifibrotic medications are used to treat patients with heart failure. There is some evidence that PFD has antifibrotic actions in various animal models of cardiac disease and a phase II trial on patients with heart failure and preserved ejection fraction has yielded positive results. This review summarises the evidence about the possible mechanisms of IPF and modulation by PFD, the main results about IPF or non-IPF interstitial pneumonias and also data about PFD as a potential protective cardiac drug

A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis- Associated Strains

Vulvovaginal candidiasis (VVC) affects nearly 3/4 of women during their lifetime, and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a switch from a commensal to pathogenic state, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e., multilocus sequence type [MLST] profile, rate of growth, and filamen- tation), but they show strikingly different behaviors during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal prolif- eration and shedding of fungi and epithelial cells. Transcriptome sequencing (RNA-seq) analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis, and type I interferon pathways; the latter has been implicated in damage protection. Strikingly, inhibition of type I interferon signaling selectively increases fungal shedding of strains in the colonizing cohort, suggesting that increased shedding correlates with lower interferon pathway activation. These data suggest that VVC strains may intrinsically have enhanced pathogenic potential via differential elicitation of epithelial responses, including the type I interferon pathway. Therefore, it may eventually be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis

Disclosing the Interaction of Gold Nanoparticles with Aβ(1–40) Monomers through Replica Exchange Molecular Dynamics Simulations

Amyloid-β aggregation is one of the principal causes of amyloidogenic diseases that lead to the loss of neuronal cells and to cognitive impairments. The use of gold nanoparticles treating amyloidogenic diseases is a promising approach, because the chemistry of the gold surface can be tuned in order to have a specific binding, obtaining effective tools to control the aggregation. In this paper, we show, by means of Replica Exchange Solute Tempering Molecular Simulations, how electrostatic interactions drive the absorption of Amyloid-β monomers onto citrates-capped gold nanoparticles. Importantly, upon binding, amyloid monomers show a reduced propensity in forming β-sheets secondary structures that are characteristics of mature amyloid fibrils

A Multi-Scale–Multi-Stable Model for the Rhodopsin Photocycle

We report a multi-scale simulation study of the photocycle of the rhodopsins. The quasi-atomistic representation (“united atoms” UA) of retinal is combined with a minimalist coarse grained (CG, one-bead-per amino acid) representation of the protein, in a hybrid UA/CG approach, which is the homolog of QM/MM, but at lower resolution. An accurate multi-stable parameterization of the model allows simulating each state and transition among them, and the combination of different scale representation allows addressing the entire photocycle. We test the model on bacterial rhodopsin, for which more experimental data are available, and then also report results for mammalian rhodopsins. In particular, the analysis of simulations reveals the spontaneous appearance of meta-stable states in quantitative agreement with experimental data