Role of Magnesium and its mitochondrial transporter MRS2 in the modulation of drug-induced apoptosis leading to multidrug resistance phenotype

Magnesium is an essential element for many biological processes crucial for cell life and proliferation. Growing evidences point out a role for this cation in the apoptotic process and in developing multi drug resistance (MDR) phenotype. The first part of this study aimed to highlight the involvement of the mitochondrial magnesium channel MRS2 in modulating drug-induced apoptosis. We generated an appropriate transgenic cellular system to regulate expression of MRS2 protein. The cells were then exposed to two different apoptotic agents commonly used in chemotherapy. The obtained results showed that cells overexpressing MRS2 channel are less responsiveness to pharmacological insults, looking more resistant to the induced apoptosis. Moreover, in normal condition, MRS2 overexpression induces higher magnesium uptake into isolated mitochondria respect to control cells correlating with an increment of total intracellular magnesium concentration. In the second part of this research we investigated whether magnesium intracellular content and compartmentalization could be used as a signature to discriminate MDR tumour cells from their sensitive counterparts. As MDR model we choose colon carcinoma cell line sensitive and resistant to doxorubicin. We exploited a standard-less approach providing a complete characterization of whole single-cells by combining X-Ray Fluorescence Microscopy , Atomic Force Microscopy and Scanning Transmission X-ray Microscopy. This method allows the quantification of the intracellular spatial distribution and total concentration of magnesium in whole dehydrated cells. The measurements, carried out in 27 single cells, revealed a different magnesium pattern for both concentration and distribution of the element in the two cellular strains. These results were then confirmed by quantifying the total amount of intracellular magnesium in a large populations of cells by using DCHQ5 probe and traditional fluorimetric technique.

Effect of season and cryopreservation on oxidative status of stallion spermatozoa

The aim of this study was to investigate 1) the effect of different ROS and lipid peroxidation on sperm quality, and 2) differences in ROS between non-breeding and breeding seasons. Eighteen ejaculates from six stallions were collected in January and July (N = 36), processed for freezing. After 90��� of cooling, some straws were not frozen (unfrozen), some were frozen (frozen). Rapid sperm (RAP, CASA), membrane-acrosome integrity (MAI), high mitochondrial membrane potential (Mpos), intracellular Ca2+ (Fneg), lipid peroxidation (BODIPY), ROS (DCFH, MitoSOX) and chromatin fragmentation (DFI%) were evaluated by flow cytometry during incubation at +37��C at T0 (after 90 min at +4��C and after thawing), 3, 6, 12 and 24h. In winter, ROS and BODIPY were higher and faster (P < 0.0001) in frozen than unfrozen; DFI% was similar at 0h (P > 0.05) but higher in frozen after 3h of incubation (P < 0.0001). RAP, PMAI, Mpos and Fneg were lower in frozen compared to unfrozen (P < 0.0001). Summer and winter data were compared. Overall, ROS concentrations and BODIPY were higher and faster (P < 0.001) in winter, DFI% was lower in winter (P < 0.001), but similar between the two groups within seasons after thawing. Differences were found at 3h and 12h for DFI%, and for DCFH and MitoSOX at 0h and 12h of incubation in winter and summer respectively. A moderate positive correlations was found between DFI% and MitoSOX, DCFH, BODIPY, whereas a negative correlation, stronger in winter, between RAP, PMAI, Mpos, Fneg and BODIPY, DCFH, MitoSOX. DFI was not different in unfrozen and frozen, despite a significant higher ROS level in winter, and incubation allowed to asses differences in DFI, suggesting that incubation should be included when evaluating stallion frozen semen. Higher level of ROS and BODIPY in winter was less detrimental than freezing-thawing.

Contribution of rare damaging variants in Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) is a heterogeneous and highly heritable neurodevelopmental disorder with a complex genetic architecture, consisting of a combination of common low-risk and more penetrant rare variants. This PhD project aimed to explore the contribution of rare variants in ASD susceptibility through NGS approaches in a cohort of 106 ASD families including 125 ASD individuals. Firstly, I explored the contribution of inherited rare variants towards the ASD phenotype in a girl with a maternally inherited pathogenic NRXN1 deletion. Whole exome sequencing of the trio family identified an increased burden of deleterious variants in the proband that could modulate the CNV penetrance and determine the disease development. In the second part of the project, I investigated the role of rare variants emerging from whole genome sequencing in ASD aetiology. To properly manage and analyse sequencing data, a robust and efficient variant filtering and prioritization pipeline was developed, and by its application a stringent set of rare recessive-acting and ultra-rare variants was obtained. As a first follow-up, I performed a preliminary analysis on de novo variants, identifying the most likely deleterious variants and highlighting candidate genes for further analyses. In the third part of the project, considering the well-established involvement of calcium signalling in the molecular bases of ASD, I investigated the role of rare variants in voltage-gated calcium channels genes, that mainly regulate intracellular calcium concentration, and whose alterations have been correlated with enhanced ASD risk. Specifically, I functionally tested the effect of rare damaging variants identified in CACNA1H, showing that CACNA1H variation may be involved in ASD development by additively combining with other high risk variants. This project highlights the challenges in the analysis and interpretation of variants from NGS analysis in ASD, and underlines the importance of a comprehensive assessment of the genomic landscape of ASD individuals.

Graphene-based bioelectronic interfaces and devices and interpretative models targeting glial cells

Bioelectronic interfaces have significantly advanced in recent years, offering potential treatments for vision impairments, spinal cord injuries, and neurodegenerative diseases. However, the classical neurocentric vision drives the technological development toward neurons. Emerging evidence highlights the critical role of glial cells in the nervous system. Among them, astrocytes significantly influence neuronal networks throughout life and are implicated in several neuropathological states. Although they are incapable to fire action potentials, astrocytes communicate through diverse calcium (Ca2+) signalling pathways, crucial for cognitive functions and brain blood flow regulation. Current bioelectronic devices are primarily designed to interface neurons and are unsuitable for studying astrocytes. Graphene, with its unique electrical, mechanical and biocompatibility properties, has emerged as a promising neural interface material. However, its use as electrode interface to modulate astrocyte functionality remains unexplored. The aim of this PhD work was to exploit Graphene-oxide (GO) and reduced GO (rGO)-coated electrodes to control Ca2+ signalling in astrocytes by electrical stimulation. We discovered that distinct Ca2+dynamics in astrocytes can be evoked, in vitro and in brain slices, depending on the conductive/insulating properties of rGO/GO electrodes. Stimulation by rGO electrodes induces intracellular Ca2+ response with sharp peaks of oscillations (���P-type���), exclusively due to Ca2+ release from intracellular stores. Conversely, astrocytes stimulated by GO electrodes show slower and sustained Ca2+ response (���S-type���), largely mediated by external Ca2+ influx through specific ion channels. Astrocytes respond faster than neurons and activate distinct G-Protein Coupled Receptor intracellular signalling pathways. We propose a resistive/insulating model, hypothesizing that the different conductivity of the substrate influences the electric field at the cell/electrolyte or cell/material interfaces, favouring, respectively, the Ca2+ release from intracellular stores or the extracellular Ca2+ influx. This research provides a simple tool to selectively control distinct Ca2+ signals in brain astrocytes in neuroscience and bioelectronic medicine.