CHARACTERIZATION OF MOLECULAR MECHANISMS DRIVING EPIGENETIC CONVERSION AND PHENOTYPE SWITCH OF FIBROBLASTS INTO INSULIN SECRETING CELLS

La conversione epigenetica \ue8 una tecnica promettente che consente ad una cellula somatica matura di passare ad un fenotipo funzionale diverso ed alternativo rispetto a quello di origine. Questo risultato viene perseguito senza alcuna modificazione transgenica e senza l'acquisizione di uno stato di pluripotenza stabile e irreversibile, caratteristiche che rendono questo approccio molto prezioso per la medicina rigenerativa. Il protocollo di conversione epigenetica \ue8 robusto, riproducibile e assicura una buona efficienza ed il conseguimento di un fenotipo funzionale. Tuttavia, le cellule ottenute non sono completamente mature e differenziate ed \ue8 necessario identificare le migliori condizioni per realizzare uno \u201cscale-up\u201d che permetta l\u2019applicazione in studi preclinici. Lo scopo del presente progetto di dottorato \ue8 stato quello di individuare condizioni di coltura fisiologiche, limitando le differenze tra l\u2019ambiente in vitro e quello in vivo, al fine di aumentare l'efficienza del processo di differenziamento. Pi\uf9 precisamente, sono state testate concentrazioni fisiologiche di ossigeno e di glucosio per poter valutare l\u2019efficienza di conversione cellulare nei diversi ambienti. Parallelamente, \ue8 stato valutato l'uso di sistemi di coltura tridimensionale (3D), con lo scopo di studiare il loro impatto sull\u2019efficienza di conversione e sull'acquisizione di un fenotipo funzionale e maturo. I dati ottenuti suggeriscono che il \u201cbackground genetico\u201d ha un effetto significativo sulla risposta cellulare alle diverse condizioni di ossigeno durante il processo di differenziamento. D\u2019altro canto, l'efficienza di conversione \ue8 risultata strettamente dipendente dalle concentrazioni di glucosio utilizzate durante l\u2019isolamento delle cellule dal tessuto di origine. Inoltre, l\u2019utilizzo di sistemi di coltura 3D, che riflettono la rigidit\ue0 e l\u2019elasticit\ue0 proprie dell'organo in vivo, ha dimostrato un effetto positivo per l'acquisizione di un fenotipo pancreatico maturo, tipico delle cellule terminalmente differenziate. Infine, le informazioni molecolari ottenute dagli esperimenti di \u201cgenome editing\u201d (ancora in corso) dovrebbero ulteriormente chiarire e corroborare i dati ottenuti. Complessivamente, i risultati di questa tesi possono fornire informazioni utili sia per la comprensione dei meccanismi di base che regolano la crescita e il differenziamento cellulare, cos\uec come per la messa a punto di un protocollo di \u201cscale-up\u201d da utilizzare nella realizzazione di studi preclinici finalizzati alla medicina rigenerativa del diabete.Epigenetic conversion is a powerful technique that allows a mature somatic cell to switch into a different and alternative functional phenotype. The result is acquired without any transgenic modification, nor the acquisition of a stable and irreversible pluripotent state, making this approach very valuable for regenerative medicine. The protocol is robust, reproducible and ensures good functional efficiency, however, cells obtained are not completely mature and the optimal scale up conditions are needed for clinical translation. Aim of the present PhD project was to investigate whether the use of ambient conditions that try to closely mimic the physiological milieu, and limit the differences between in vitro and in vivo situations, may generate terminally differentiated cells and boost efficiency. To this purpose, physiological oxygen and different glucose concentrations were tested in order to assess cell responses and conversion ability in the different environments. In parallel, the use of three-dimensional (3D) culture systems was investigated, with the specific aim to study the impact of stiffness on epigenetic conversion and the acquisition of a functional, mature phenotype. The data obtained suggest that genetic background has a profound effect on the response to oxygen during the differentiation process and that conversion efficiency is strictly dependent on the glucose concentrations applied at cell isolation from the original tissue. 3D culture systems that match the stiffness typical of the original organ were able to increase differentiation and favored the acquisition of a mature pancreatic phenotype, distinctive of terminally differentiated cells. Last but not least, key molecular informations deriving from the ongoing gene editing experiments are expected to further clarify and substantiate the data obtained. Altogether, the information derived in this PhD project may find useful applications in order to design the best in vitro conditions and obtain a powerful scale-up protocol for pre-clinical studies and regenerative medicine of diabetes

How to Deal with Weak Interactions in Noncovalent Complexes Analyzed by Electrospray Mass Spectrometry: Cyclopeptidic Inhibitors of the Nuclear Receptor Coactivator 1-STAT6

Mass spectrometry, and especially electrospray ionization, is now an efficient tool to study noncovalent interactions between proteins and inhibitors. It is used here to study the interaction of some weak inhibitors with the NCoA-1/STAT6 protein with KD values in the μM range. High signal intensities corresponding to some nonspecific electrostatic interactions between NCoA-1 and the oppositely charged inhibitors were observed by nanoelectrospray mass spectrometry, due to the use of high ligand concentrations. Diverse strategies have already been developed to deal with nonspecific interactions, such as controlled dissociation in the gas phase, mathematical modeling, or the use of a reference protein to monitor the appearance of nonspecific complexes. We demonstrate here that this last methodology, validated only in the case of neutral sugar–protein interactions, i.e., where dipole–dipole interactions are crucial, is not relevant in the case of strong electrostatic interactions. Thus, we developed a novel strategy based on half-maximal inhibitory concentration (IC50) measurements in a competitive assay with readout by nanoelectrospray mass spectrometry. IC50 values determined by MS were finally converted into dissociation constants that showed very good agreement with values determined in the liquid phase using a fluorescence polarization assay

Gauging circadian variation in ketamine metabolism by real-time breath analysis

The time-of-day of drug application is an important factor in maximizing efficacy and minimizing toxicity. Real-time in vivo mass spectrometric breath analysis of mice was deployed to investigate time-of-day variation in ketamine metabolism. Different production rates of ketamine metabolites, including the recently described anti-depressant hydroxynorketamine, were found in opposite circadian phases. Thus, breath analysis has potential as a rapid and 3Rs (Replacement, Reduction and Refinement) conforming screening method to estimate the time-dependence of drug metabolism

Tumor type M2-pyruvate-kinase levels in pleural fluid versus plasma in cancer patients: a further tool to define the need for invasive procedures

Pleural effusion is a common diagnostic problem and a challenge to the thoracic surgeon. The analysis of serum and body fluids for tumor markers is an established diagnostic procedure. Among various markers, tumors are linked to the overexpression of a glycolytic isoenzyme, M2-pyruvate-kinase (M2-PK). This preliminary study evaluated this enzyme as a tumor marker to differentiate malignant from benign pleural effusion

Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts

Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis

Changes in serum levels of TNF-α, IL-6, OPG, RANKL and their correlation with radiographic and clinical assessment in fragility fractures and high energy fractures

Stages of bone turnover during fracture repair can be assessed employing serum markers of osteoblastic and osteoclastic activity, inflammatory cytokines, clinical evaluation and imaging instruments. Our study compare the fracture healing process in fragility fractures and high energy fractures by evaluating serum changes of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), osteoprotegerin (OPG) and receptor activator of the nuclear factor-kB ligand (RANKL) in combination with radiographic (Radiographic Union Scale for Tibial fractures, RUST) and clinical (Lower extremity measure, LEM) assessments. We enrolled 56 patients divided into four corresponding groups: group A with high energy trauma fracture (tibial/femoral shaft); group B with low energy trauma fracture (femoral fractures); healthy (control A) and osteoporotic subjects (control B). Blood samples were collected before surgery (T0) and after 10 weeks (T10). Serum concentrations of IL-6, TNF-α, RANKL and OPG were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. Our results show that RANKL values are significantly higher at T10 than at T0 in low energy trauma fractures (group B). OPG is significantly lower in each control group than that of the respective fractured group and its concentration at T0 and at T10 is significantly lower in high than in low energy fractures. RANKL/OPG ratio is significantly higher in both controls than in fractured groups, and significantly increases after 10 weeks. IL-6 and TNF-α concentrations significantly decrease during fracture healing and are higher in high (group A) than in low energy fractures (group B). Significant differences were also found in both RUST score and LEM between groups A and B. Changes in TNF-α and IL-6 levels correlate with RUST and LEM in fragility and high energy fractures, while RANKL/OPG ratio is associated with these clinical parameters only in fragility fractures. These findings suggest that serum levels of IL-6, TNF-α, RANKL and OPG might be used to monitor the stages of fracture repair. Further studies will be needed to confirm the role of these cytokines in fracture repair

Atherosclerosis, dyslipidemia, and inflammation: the significant role of polyunsaturated fatty acids

Phospholipids play an essential role in cell membrane structure and function. The length and number of double bonds of fatty acids in membrane phospholipids are main determinants of fluidity, transport systems, activity of membrane-bound enzymes, and susceptibility to lipid peroxidation. The fatty acid profile of serum lipids, especially the phospholipids, reflects the fatty acid composition of cell membranes. Moreover, long-chain n-3 polyunsatured fatty acids decrease very-low-density lipoprotein assembly and secretion reducing triacylglycerol production. N-6 and n-3 polyunsatured fatty acids are the precursors of signalling molecules, termed “eicosanoids,” which play an important role in the regulation of inflammation. Eicosanoids derived from n-6 polyunsatured fatty acids have proinflammatory actions, while eicosanoids derived from n-3 polyunsatured fatty acids have antiinflammatory ones. Previous studies showed that inflammation contributes to both the onset and progression of atherosclerosis: actually, atherosclerosis is predominantly a chronic low-grade inflammatory disease of the vessel wall. Several studies suggested the relationship between long-chain n-3 polyunsaturated fatty acids and inflammation, showing that fatty acids may decrease endothelial activation and affect eicosanoid metabolis

Mountain high and valley deep: epigenetic controls of pluripotency and cell fate

All the somatic cells composing a mammalian organism are genetically identical and contain the same DNA sequence. Nevertheless, they are able to adopt a distinct commitment, differentiate in a tissue specific way and respond to developmental cues, acquiring a terminal phenotype. At the end of the differentiation process, each cell is highly specialized and committed to a distinct determined fate. This is possible thanks to tissue-specific gene expression, timely regulated by epigenetic modifications, that gradually limit cell potency to a more restricted phenotype-related expression pattern. Complex chemical modifications of DNA, RNA and associated proteins, that determine activation or silencing of certain genes are responsible for the 'epigenetic control' that triggers the restriction of cell pluripotency, with the acquisition of the phenotypic definition and the preservation of its stability during subsequent cell divisions. The process is however reversible and may be modified by biochemical and biological manipulation, leading to the reactivation of hypermethylated pluripotency genes and inducing cells to transit from a terminally committed state to a higher plasticity one. These epigenetic regulatory mechanisms play a key role in embryonic development since they drive phenotype definition and tissue differentiation. At the same time, they are crucial for a better understanding of pluripotency regulation and restriction, stem cell biology and tissue repair process

Severe Obstructive Sleep Apnea Disrupts Vigilance-State-Dependent Metabolism

The direct pathophysiological effects of obstructive sleep apnea (OSA) have been well described. However, the systemic and metabolic consequences of OSA are less well understood. The aim of this secondary analysis was to translate recent findings in healthy subjects on vigilance-state-dependent metabolism into the context of OSA patients and answer the question of how symptomatic OSA influences metabolism and whether these changes might explain metabolic and cardiovascular consequences of OSA. Patients with suspected OSA were assigned according to their oxygen desaturation index (ODI) and Epworth Sleepiness Scale (ESS) score into symptomatic OSA and controls. Vigilance-state-dependent breath metabolites assessed by high-resolution mass spectrometry were used to test for a difference in both groups. In total, 44 patients were eligible, of whom 18 (40.9%) were assigned to the symptomatic OSA group. Symptomatic OSA patients with a median [25%, 75% quartiles] ODI of 40.5 [35.0, 58.8] events/h and an ESS of 14.0 [11.2, 15.8] showed moderate to strong evidence for differences in 18 vigilance-state-dependent breath compounds compared to controls. These identified metabolites are part of major metabolic pathways in carbohydrate, amino acid, and lipid metabolism. Thus, beyond hypoxia per se, we hypothesize that disturbed sleep in OSA patients persists as disturbed sleep-dependent metabolite levels during daytime

NLRC5 promotes transcription of BTN3A1-3 genes and Vγ9Vδ2 T cell-mediated killing

BTN3A molecules-BTN3A1 in particular-emerged as important mediators of Vγ9Vδ2 T cell activation by phosphoantigens. These metabolites can originate from infections, e.g. with Mycobacterium tuberculosis, or by alterations in cellular metabolism. Despite the growing interest in the BTN3A genes and their high expression in immune cells and various cancers, little is known about their transcriptional regulation. Here we show that these genes are induced by NLRC5, a regulator of MHC class I gene transcription, through an atypical regulatory motif found in their promoters. Accordingly, a robust correlation between NLRC5 and BTN3A gene expression was found in healthy, in M. tuberculosis-infected donors' blood cells, and in primary tumors. Moreover, forcing NLRC5 expression promoted Vγ9Vδ2 T-cell-mediated killing of tumor cells in a BTN3A-dependent manner. Altogether, these findings indicate that NLRC5 regulates the expression of BTN3A genes and hence open opportunities to modulate antimicrobial and anticancer immunity