Sindrome da Asfissia Perinatale nel puledro neonato: protocolli diagnostico-terapeutici

La Sindrome da Asfissia Perinatale (PAS) è una delle più comuni patologie che colpiscono il puledro neonato nelle prime 72 h di vita. È una patologia difficile da diagnosticare in quanto non esistono parametri o segni clinici specifici, la sintomatologia è molto variabile in base alla durata e all’intensità dell’insulto ipossico ischemico e al tipo di organo maggiormente colpito. Lo scopo di questo studio è la ricerca e la valutazione di alcuni parametri biochimico-clinici e di alcuni biomarkers per la diagnosi precoce e il corretto trattamento dei puledri affetti da PAS. Nei puledri neonati che presentano questa patologia è stata riscontrata un’ipermagnesiemia al momento del ricovero associata a prognosi infausta, probabilmente causata da un grave danno cellulare con rilascio in circolo del magnesio intracellulare. La PAS potrebbe essere un’ulteriore causa di Euthyroid Sick Syndrome, in quanto abbiamo riscontrato una diminuzione delle concentrazioni di T3 e T4 nei puledri malati rispetto ai sani della stessa età, come avviene in altre malattie sistemiche. Lo studio del profilo proteomico ha permesso di separare le più importanti frazioni proteiche del liquido amniotico di cavalla, mettendo in evidenza similitudini e differenze qualitative e quantitative nei ferogrammi dei puledri sani e di quelli affetti da PAS ed una maggiore variabilità è stata riscontrata nei profili dei liquidi amniotici dei puledri malati. Il glutatione è risultato poco espresso nel puledro neonato, i puledri sani presentano concentrazioni più basse sia rispetto ai malati della stessa età sia agli adulti ma con una tendenza all’aumento nelle prime 24 ore di vita per i sani ed un calo nei malati. La somministrazione della terapia antiradicalica non influisce sulle concentrazioni di glutatione totale ed i puledri deceduti presentano concentrazioni più alte.

Innovative analytical methods for Central Nervous System Drug analysis in biological fluids

During recent years a consistent number of central nervous system (CNS) drugs have been approved and introduced on the market for the treatment of many psychiatric and neurological disorders, including psychosis, depression, Parkinson disease and epilepsy. Despite the great advancements obtained in the treatment of CNS diseases/disorders, partial response to therapy or treatment failure are frequent, at least in part due to poor compliance, but also genetic variability in the metabolism of psychotropic agents or polypharmacy, which may lead to sub-therapeutic or toxic plasma levels of the drugs, and finally inefficacy of the treatment or adverse/toxic effects. With the aim of improving the treatment, reducing toxic/side effects and patient hospitalisation, Therapeutic Drug Monitoring (TDM) is certainly useful, allowing for a personalisation of the therapy. Reliable analytical methods are required to determine the plasma levels of psychotropic drugs, which are often present at low concentrations (tens or hundreds of nanograms per millilitre). The present PhD Thesis has focused on the development of analytical methods for the determination of CNS drugs in biological fluids, including antidepressants (sertraline and duloxetine), antipsychotics (aripiprazole), antiepileptics (vigabatrin and topiramate) and antiparkinsons (pramipexole). Innovative methods based on liquid chromatography or capillary electrophoresis coupled to diode-array or laser-induced fluorescence detectors have been developed, together with the suitable sample pre-treatment for interference removal and fluorescent labelling in case of LIF detection. All methods have been validated according to official guidelines and applied to the analysis of real samples obtained from patients, resulting suitable for the TDM of psychotropic drugs.

Role of nuclear redox control, intra-population heterogeneity and oxygen tension in human amniotic fluid stem cells aging

Amniotic fluid stem cells (hAFSC) are emerging as a potential therapeutic approach for various disorders. The low number of available hAFSC requires their ex vivo expansion prior to clinical use, however, during their in vitro culture, hAFSC quickly reach replicative senescence. The principal aim of this study was to investigate the aging process occurring during in vitro expansion of hAFSC, focusing on the redox control that has been reported to be affected in premature and physiological aging. My results show that a strong heterogeneity is present among samples that reflects their different behaviour in culture. I identified three proteins, namely Nox4, prelamin A and PML, which expression increases during hAFSC aging process and could be used as new biomarkers to screen the samples. Furthermore, I found that Nox4 degradation is regulated by sumoylation via proteasome and involves interactions with PML bodies and prelamin A. Since various studies revealed that donor-dependent differences could be explained by cell-to-cell variation within each patient, I studied in deep this phenomenon. I showed that the heterogeneity among samples is also accompanied by a strong intra-population heterogeneity. Separation of hAFSC subpopulations from the same donor, using Celector® technology, showed that an enrichment in the last eluted fraction could improve hAFSC application in regenerative medicine. One of the other problems is that nowadays hAFSC are expanded under atmospheric O2 concentration, which is higher than the O2 tension in their natural niches. This higher O2 concentration might cause environmental stress to the in vitro cultured hAFSCs and accelerate their aging process. Here, I showed that prolonged low oxygen tension exposure preserves different hAFSC stemness properties. In conclusion, my study pointed different approaches to improve in vitro hAFSC expansion and manipulation with the purpose to land at stem cell therapy.

Flow of Complex Fluids in Geological Fractures

In this study, the lubrication theory is used to model flow in geological fractures and analyse the compound effect of medium heterogeneity and complex fluid rheology. Such studies are warranted as the Newtonian rheology is adopted in most numerical models because of its ease of use, despite non-Newtonian fluids being ubiquitous in subsurface applications. Past studies on Newtonian and non-Newtonian flow in single rock fractures are summarized in Chapter 1. Chapter 2 presents analytical and semi-analytical conceptual models for flow of a shear-thinning fluid in rock fractures having a simplified geometry, providing a first insight on their permeability. in Chapter 3, a lubrication-based 2-D numerical model is first implemented to solve flow of an Ellis fluid in rough fractures; the finite-volumes model developed is more computationally effective than conducting full 3-D simulations, and introduces an acceptable approximation as long as the flow is laminar and the fracture walls relatively smooth. The compound effect of shear-thinning fluid nature and fracture heterogeneity promotes flow localization, which in turn affects the performance of industrial activities and remediation techniques. In Chapter 4, a Monte Carlo framework is adopted to produce multiple realizations of synthetic fractures, and analyze their ensemble statistics pertaining flow for a variety of real non-Newtonian fluids; the Newtonian case is used as a benchmark. In Chapter 5 and Chapter 6, a conceptual model of the hydro-mechanical aspects of backflow occurring in the last phase of hydraulic fracturing is proposed and experimentally validated, quantifying the effects of the relaxation induced by the flow.