Development of functional MRI protocols in the study of neurodegenerative diseases: MRI study in patients with REM sleep behavior disorder, idiopathic Parkinson's disease and multisystem atrophy

In the central nervous system, iron in several proteins is involved in many important processes: oxygen transportation, oxidative phosphorylation, mitochondrial respiration, myelin production, the synthesis and metabolism of neurotransmitters. Abnormal iron homoeostasis can induce cellular damage through hydroxyl radical production, which can cause the oxidation, modification of lipids, proteins, carbohydrates, and DNA, lead to neurotoxicity. Moreover increased levels of iron are harmful and iron accumulations are typical hallmarks of brain ageing and several neurodegenerative disorders particularly PD. Numerous studies on post mortem tissue report on an increased amount of total iron in the substantia nigra in patients with PD also supported by large body of in vivo findings from Magnetic Resonance Imaging (MRI) studies. The importance and approaches for in vivo brain iron assessment using multiparametric MRI is increased over last years. Quantitative MRI may provide useful biomarkers for brain integrity assessment in iron-related neurodegeneration. Particularly, a prominent change in iron- sensitive T2* MRI contrast within the sub areas of the SN overlapping with nigrosome 1 were shown to be a hallmark of Parkinson's Disease with high diagnostic accuracy. Moreover, differential diagnosis between Parkinson's Disease (PD) and atypical parkinsonian syndromes (APS) remains challenging, mainly in the early phases of the disease. Advanced brain MR imaging enables to detect the pathological changes of nigral and extranigral structures at the onset of clinical manifestations and during the course of the disease. The Nigrosome-1 (N1) is a substructure of the healthy Substantia Nigra pars compacta enriched by dopaminergic neurons; their loss in Parkinson’s disease and atypical parkinsonian syndromes is related to the iron accumulation. N1 changes are supportive MR biomarkers for diagnosis of these neurodegenerative disorders, but its detection is hard with conventional sequences, also using high field (3T) scanner. Quantitative susceptibility mapping (QSM), an iron-sensitive technique, enables the direct detection of Neurodegeneration

Neuro-ophthalmological and electrophysiological characterization of mitochondrial diseases.

Aims and methods: 1) characterization of patients with Dominant Optic Atrophy (DOA) associated with mutations in AFG3L2 and ACO2 genes in comparison with classical OPA1-DOA; 2) characterization of patients with mtDNA mutations causing MELAS and MERRF syndromes and correlation with heteroplasmy; 3) longitudinal evaluation of subacute m.11778G>A/MTND4 Leber’s Hereditary Optic Neuropathy (LHON) patients co-treated with rAAV2/2-ND4 gene therapy and idebenone. We performed a comprehensive neuro-ophthalmological assessment coupled with electrophysiological examination. Results: 1) We described and compared 23 ACO2 and 13 AFG3L2 patients with 72 OPA1 patients. All patients presented temporally predominant optic atrophy, with ACO2 showing higher RNFL and GCL thicknesses at OCT, while AFG3L2 was virtually-indistinguishable from OPA1. 2) Retinopathy was the most common manifestation in 17/33 MELAS patients, conversely, optic atrophy was the most common finding in 7/8 MERRF patients. Correlation of heteroplasmy with neuro-ophthalmological parameters failed to disclose any significance in MELAS, while it negatively correlated with OCT parameters in MERRF. 3) We compared modifications in visual acuity, OCT and electrophysiological parameters at 3 timepoints in 9 LHON patients. We observed significant decrease of RNFL thickness and reduction of PhNR amplitude. Visual acuity improved of about -0.37 LogMAR, correlating significantly with time from onset and from injection, but not with idebenone therapy duration. Discussion: 1) ACO2 seems associated to better preservation of retinal ganglion cells, depending on a different pathogenic mechanism involving mtDNA maintenance, as opposed to AFG3L2 which is involved in OPA1 processing. 2) MELAS and MERRF patients presented with a clearly distinct ocular phenotype, possibly reflecting a selective susceptibility of different retinal cell types to global energy defect or oxidative stress. 3) Follow up of LHON patients treated with gene therapy confirmed the deterioration in OCT and electrophysiological parameters, while the amount of visual improvement was similar to the one observed in recent clinical trials.

Development of new molecular methods for the diagnosis and the study of viral diseases of fish

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

Probiotics for the prevention/treatment of human diseases and ecological study of the intestinal microbiota

The interest in human intestinal microbiota has increased in the last 20 years and significant advances have been achieved with regard to its composition and functions. The gut microbiota contributes to the maintenance of the host health status and, since alterations in the gut microbiota have been involved in the pathogenesis/progression of some diseases, several studies have focused on the manipulation of its composition. Probiotics are a strategy to maintain/restore the correct balance of gut microbial population and to prevent/treat diseases. The aim of this thesis was to explore the possibility of probiotic supplementation for the prevention/treatment of human diseases and the related study of the intestinal microbial environment. After reviewing studies concerning the use of Bifidobacterium breve as probiotic in paediatric diseases, the effectiveness of a probiotic formulation consisting of two strains of B. breve was assessed in paediatric subjects for the prevention or alleviation of gastrointestinal disorders, including coeliac disease and paediatric obesity. As the emerging role of gut microbiota in neurological diseases, the intestinal microbial environment in amyotrophic lateral sclerosis patients compared to healthy controls and the effects of a probiotic administration were examined. Considering the role of viruses in shaping gut microbiota, gut bacteriophages and bacterial community of preterm infants were investigated. The results evidenced differences in gut microbial composition of healthy controls and diseased subjects in coeliac and amyotrophic lateral sclerosis patients. The probiotic approach was effective in restoring the microbial composition in the former, whereas, in the latter, the influence was focused only on some microbial groups. The probiotic intervention was effective in improving the glyco-insulinemic profile in obese children and in preventing gastrointestinal disorders in healthy newborns. The study of the bacterial and phage composition in preterm infants suggested a transkingdom interplay between bacteria and viruses with a reciprocal influence on their composition.

Design and synthesis of (pro)electrophilic compounds for investigating the multifactorial nature of neurodegenerative diseases: focus on inflammation-driven events

Neuroinflammation constitutes a major player in the etiopathology of neurodegenerative diseases (NDDs), by orchestrating several neurotoxic pathways which in concert lead to neurodegeneration. A positive feedback loop occurs between inflammation, microglia activation and misfolding processes that, alongside excitotoxicity and oxidative events, represent crucial features of this intricate scenario. The multi-layered nature of NDDs requires a deepen investigation on how these vicious cycles work. This could further help in the search for effective treatments. Electrophiles are critically involved in the modulation of a variety of neuroprotective responses. Thus, we envisioned their peculiar ability to switch on/off biological activities as a powerful tool for investigating the neurotoxic scenario driven by inflammation in NDDs. In particular, in this thesis project, we wanted to dissect at a molecular level the functional role of (pro)electrophilic moieties of previously synthesized thioesters of variously substituted trans-cinnamic acids, to identify crucial features which could interfere with amyloid aggregation as well as modulate Nrf2 and/or NF-κB activation. To this aim, we first synthesized new compounds to identify bioactive cores which could specifically modulate the intended target. Then, we systematically modified their structure to reach additional pathogenic pathways which could in tandem contribute to the inflammatory process. In particular, following the investigation of the mechanistic underpinnings involving the catechol feature in amyloid binding through the synthesis of new dihydroxyl derivatives, we incorporated the identified antiaggregating nucleus into constrained frames which could contrast neuroinflammation also through the modulation of CB2Rs. In parallel, Nrf2 and/or NF-κB antinflammatory structural requirements were combined with the neuroprotective cores of pioglitazone, an antidiabetic drug endowed with MAO-B inhibitory properties, and memantine, which notably contrasts excitotoxicity. By acting as Swiss army knives, the new set of molecules emerge as promising tools to deepen our insights into the complex scenario regulating NDDs.

Essential oils and hydrolates as potential tools for integrated prevention of bacterial diseases in plants

The present work aims to investigate the potential use of natural substances against bacterial plant pathogens. Microdilution tests were therefore carried out in vitro to identify the minimum inhibitory and bactericidal concentrations (MIC and MBC) of several EOs and Hys against selected bacterial pathogens. Commercial products based on a mixture of EOs were in addition assayed with macrodilution experiments against Erwinia amylovora (Ea-causal agent of fire blight). Subsequently, using selected EOs, Hys, and commercial products, ex vivo tests on disease incidence and Ea population dynamics were carried out; the latter experiment was followed by SEM observations. In addition, in vivo resistance induction test was carried out against bacterial leaf of tomato, caused by Xanthomonas vesicatoria (Xv). EOs and Hys showed high bactericidal activity in vitro (MBC <0.1 and <10% for the most active EOs and Hys: Origanum compactum and Thymus vulgaris EOs and Citrus aurantium var. amara Hy, respectively), but they were not effective ex vivo, while resulted very active when used in vivo as resistance inducers in the tomato-Xv pathosystem (relative protection >40%). Differently, commercial products resulted active in all tests, but not as resistance inducers against Xv. An open field trial with commercial products was carried out on strawberry plants naturally infected with Xanthomonas fragariae; the results showed discrete relative protection, concerning that provided by the conventional products based on copper; mostly, the disease severity reduction on those plants treated with EOs commercial products was significant when disease severity resulted high. The papers already published described in the present work investigate (1)the activity of Hys in comparison to EOs with respect to their active volatile content; (2) the potential use of EOs and Hys in cultural heritage; for the restoration of paintings; (3) the induction of resistance caused by plasma-activated water-based root treatments.