Innovative approaches for the diagnosis of Parkinson’s disease and multiple system atrophy based on the analysis of the olfactory mucosa

Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases whose diagnosis is particularly complex, especially in the early stages, because the symptoms are similar to each other and to those of other diseases, including dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). All these disorders share a similar pathological process: the change in the structure of some proteins normally present in the brain which thus lose their function, begin to aggregate and deposit in specific brain areas, causing irreparable damage. In particular, PD, MSA and DLB are called α-synucleinopathies because they present aggregates of the α-synuclein protein (αSynD), which however are localized in different brain structures. PSP and CBD are instead called tauopathies because they are characterized by the presence of aggregates of the tau protein. These protein aggregates are considered disease-specific biomarkers because their detection and distribution (which can only be determined post-mortem on the patient's brain tissue) are used to formulate a definite diagnosis. As long as the patient is alive the diagnosis is only probable and does not have absolute accuracy. Consequently, some diagnoses made in life may change after the neuropathological assessments. Several evidence suggests that misfolded proteins can also appear in peripheral tissues such as the olfactory mucosa (OM, easily and periodically collectible with a nasal swab), but in such small quantities as not to be detectable with common diagnostic techniques. The recent advances in molecular and structural biology have provided insights into the processes involved in the pathogenesis of neurodegenerative diseases and have made it possible to recapitulate the protein misfolding process in vitro in a limited period of time through the development of innovative techniques, called seed amplification assays (SAAs), among which the real-time quaking-induced conversion (RT-QuIC). This new methodology exploits the ability of misfolded proteins to transmit their abnormal conformation to normal monomers, which are used as substrate of the reaction. Abnormally folded proteins are able to interact with these substrates and induce monomers to change conformation and subsequently aggregate. Therefore, the addition of misfolded proteins (considered “seeds”) to the substrate is able to trigger an aggregation phenomenon, known as “seeding effect” that might be exploited for diagnostic and therapeutic purposes. In my Ph.D. project I have firstly optimized the RT-QuIC assay, with the aim of analyzing OM samples collected from patients with PD, MSA, CBD and PSP, and evaluating the efficacy of the test in detecting traces of misfolded αSynD in α-synucleinopathy derived samples. The results of our study showed that most OM samples from patients with PD and MSA induced aggregation of the recombinant substrate protein, suggesting the presence of traces of αSynD. In contrast, the PSP and CBD samples had no effect on the substrate (since they do not contain abnormal αSyn). Interestingly, the RT-QuIC reaction products acquired biochemical and biophysical characteristics useful to discriminate, with a good degree of accuracy, patients with PD from patients with MSA. Moreover, by exposing neuronal-like differentiated SH-SY5Y cells to these products, we observed the induction of different inflammatory pathways. These findings suggested the existence of a link between the morphology of the aggregates and their inflammatory properties. To deepen this aspect, we have produced three different recombinant aggregates of αSyn, in order to generate, in a controlled environment, artificial αSyn seeds resembling to some extent the αSynD strains present in OM, and test their behavior by RT-QuIC without the presence of other tissue factors. Although capable to efficiently seed the aggregation of the substrate, αSv1, αSv2, and αSv3 did not transmit their seed-specific properties to the reaction products which showed comparable biochemical properties, instead. Probably, our experimental setting was too artificial to properly recapitulate the phenomenon of the seeding effect exerted by αSynD in RT-QuIC. However, when used to stimulate SH-SY5Y cells, αSv1, αSv2, and αSv3 acted on different activators of inflammatory pathways, thus strengthening the existence of a correlation between morphological and inflammatory properties of αSyn fibrils. In the last part of my project, we decided to evaluate how much the RT-QuIC assay could be used for diagnostic purposes in the field of α-synucleinopathies, by studying its reproducibility in other laboratories. Together with an American lab we have so analyzed a group of OM samples with the same experimental protocol and we obtained a 96% concordance of results. Furthermore, we observed that the OM of MSA behaved differently according to the pathological subtype. In fact, we know that this disease can manifest itself in a cerebellar form (MSA-C) or associated with parkinsonism (MSA-P). In our test, only MSA-P samples induced a seeding effect, allowing us to discriminate between the two pathological subtypes. These preliminary studies provide evidence that RT-QuIC of OM samples represents a reliable assay for supporting the diagnosis of α-synucleinopathy and may limit the negative effects that misdiagnosis produces in terms of costs for the healthcare system and improve overall patient care, treatment, and possible enrollment in future clinical trials

LA LEALT\uc0 DELLE INFORMAZIONI FORNITE SUGLI ALIMENTI AI CONSUMATORI NEL QUADRO DELLA DISCIPLINA GENERALE IN MATERIA DI PRATICHE COMMERCIALI SLEALI BUSINESS TO CONSUMER, FRA INCERTEZZE INTERPRETATIVE E QUESTIONI IRRISOLTE SUL PIANO SANZIONATORIO E RIMEDIALE

La tesi si propone di approfondire i rapporti fra il reg. Ue n. 1169/2011, relativo alla fornitura di informazioni sugli alimenti ai consumatori, e la dir. 2005/29/Ce, in materia di pratiche commerciali sleali business to consumer. Al riguardo, muovendo dalla disamina dei due plessi normativi, sembra possibile ritenere che fra essi intercorra un rapporto di species a genus, tale per cui, come affermato dal 5\ub0 considerando del reg. Ue n. 1169/2011, quest\u2019ultimo \ue8 destinato a integrare i precetti generali contenuti nella direttiva, disciplinando, in modo specifico, le informazioni che gli operatori del settore alimentare sono tenuti a comunicare ai consumatori, nonch\ue9 le modalit\ue0 per la loro presentazione. Tuttavia, non \ue8 stato chiarito dal legislatore europeo come il coordinamento fra le due discipline operi nel concreto. In particolare, posto che le violazioni delle norme contenute nel regolamento sembrano configurare tipologie specifiche di pratiche commerciali ingannevoli, ci si chiede se, ai fini del giudizio di slealt\ue0, tali violazioni integrino l\u2019elenco di pratiche commerciali in ogni caso sleali, di cui all\u2019allegato I della dir. 2005/29/Ce, oppure se sia necessario dimostrare la sussistenza dei requisiti generali di cui agli artt. 6 e 7 della direttiva, con particolare riferimento all\u2019idoneit\ue0 della pratica a falsare il comportamento economico del consumatore, tenuto conto delle circostanze del caso concreto. La questione assume rilevanza anche perch\ue9 si presta a incidere sul livello di tutela effettivamente accordato ai consumatori finali di alimenti. Con riferimento a questi ultimi, soprattutto nel caso in cui si opti per la seconda opzione interpretativa, si pone, peraltro, il problema di definire le loro caratteristiche percettive. Se, infatti, la figura del consumatore finale di alimenti, cui fa riferimento il reg. Ue n. 1169/2011, si fonda sul modello giurisprudenziale del consumatore medio, normalmente informato e ragionevolmente attento e avveduto, non \ue8 chiaro se la tutela predisposta dal regolamento possa, se del caso, estendersi anche alla figura del c.d. consumatore vulnerabile, introdotta dalla dir. 2005/29/Ce. Il problema dei rapporti fra la disciplina in materia di informazioni alimentari e la disciplina in materia di pratiche commerciali sleali business to consumer \ue8 destinato a ripercuotersi anche sul profilo sanzionatorio. Volgendo lo sguardo al nostro ordinamento giuridico, la disamina del d.lgs. n. 231/2017, che prevede le sanzioni amministrative pecuniarie applicabili nei confronti degli operatori del settore alimentare che violino le prescrizioni del reg. Ue n. 1169/2011, rivela un quadro normativo per nulla confortante. Il legislatore nazionale non si \ue8 preoccupato di delineare un raccordo fra il d.lgs. n. 231/2017 e la disciplina sanzionatoria in materia di pratiche commerciali scorrette, di cui al Codice del consumo; per contro, ha affidato all\u2019ICQRF, istituito presso il Ministero per le Politiche Agricole, Alimentari e Forestali, la competenza ad applicare le sanzioni previste dal d.lgs. n. 231/2017, determinando il pericolo di una sovrapposizione rispetto alla competenza sanzionatoria esercitata dall\u2019AGCM in materia di pratiche commerciali scorrette business to consumer. Una considerevole deminutio della tutela accordata ai consumatori di prodotti alimentari e una possibile violazione del principio del ne bis in idem sono solo alcune delle conseguenze che possono derivare da tale approccio. Sotto questo profilo, l\u2019analisi dei rapporti fra il reg. Ue n. 1169/2011 e la dir. 2005/29/Ce si conferma uno strumento utile a saggiare l\u2019adeguatezza delle scelte normative, adottate sul piano europeo e su quello nazionale, rispetto all\u2019obbiettivo di garantire un\u2019efficace tutela agli interessi dei consumatori di prodotti alimentari. Da qui, le ragioni dell\u2019indagine che segue

Decellularization and Delipidation Protocols of Bovine Bone and Pericardium for Bone Grafting and Guided Bone Regeneration Procedures

The combination of bone grafting materials with guided bone regeneration (GBR) membranes seems to provide promising results to restore bone defects in dental clinical practice. In the first part of this work, a novel protocol for decellularization and delipidation of bovine bone, based on multiple steps of thermal shock, washes with detergent and dehydration with alcohol, is described. This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study. Furthermore, histological and morphological analyses confirm the maintenance of an intact bone extracellular matrix (ECM). In vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively. In the second part of this study, two methods of bovine pericardium decellularization are compared. The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method. Overall, the results of this study demonstrate the characterization of a novel protocol for the decellularization of bovine bone to be used as bone graft, and the acquisition of a method to produce a pericardium membrane suitable for GBR applications

CMB Polarization Systematics, Cosmological Birefringence and the Gravitational Waves Background

Cosmic Microwave Background experiments must achieve very accurate calibration of their polarization reference frame to avoid biasing the cosmological parameters. In particular, a wrong or inaccurate calibration might mimic the presence of a gravitational wave background, or a signal from cosmological birefringence, a phenomenon characteristic of several non-standard, symmetry breaking theories of electrodynamics that allow for \textit{in vacuo} rotation if the polarization direction of the photon. Noteworthly, several authors have claimed that the BOOMERanG 2003 (B2K) published polarized power spectra of the CMB may hint at cosmological birefringence. Such analyses, however, do not take into account the reported calibration uncertainties of the BOOMERanG focal plane. We develop a formalism to include this effect and apply it to the BOOMERanG dataset, finding a cosmological rotation angle $\alpha=-4.3^\circ\pm4.1^\circ$. We also investigate the expected performances of future space borne experiment, finding that an overall miscalibration larger then $1^\circ$ for Planck and $0.2\circ$ for EPIC, if not properly taken into account, will produce a bias on the constraints on the cosmological parameters and could misleadingly suggest the presence of a GW background.Comment: 10 pages, 3 figure

A highly pathogenic porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) strongly modulates cellular innate and adaptive immune subsets upon experimental infection

Highly pathogenic (HP) PRRSV isolates have been discovered within both PRRSV-1 and PRRSV-2 genotypes and investigated in recent years especially for their ability to cause extremely severe disease in conventional pig herds. The exacerbation of general and respiratory clinical signs has been attributed not only to an efficient replication (virulence) but also to the ability to dysregulate viral recognition and induce mechanisms of immune evasion or immune enhancement of humoral and cellular anti-viral responses differently from non-HP PRRSV isolates in terms of intensity and temporal onset. Thus, the understanding of the immunopathogenesis of HP PRRSV is a major concern for the study of virus biology and development of efficacious vaccines. The present study aims at addressing the modulation of relevant immune cell subsets by flow cytometry in the blood of 4- week-old pigs experimentally infected with the recently discovered PR40/2014 HP PRRSV-1.1 strain phenotypically characterized in Canelli et al. (2017) compared to pigs infected with a non-HP PRRSV isolate (PR11/ 2014) and uninfected controls. PR40 infected animals showed an early and marked reduction of pro-inflammatory CD172α+ CD14+CD16+ and CD14+CD163+ monocytes and TCRγδ+CD8α+/CD8α- lymphocytes when pigs were most infected, possibly due to a recruitment sustaining an acute inflammatory response in target tissues. The prolonged increased CD3+CD16+ NKT cell levels may sustain peripheral inflammation and/ or the anti-viral response. The late reduction (potential depletion) of γ/δ T lymphocytes and CD3+CD4+CD8α- naïve Th lymphocytes paralleled with the delayed increase of CD3+CD4+CD8α+ memory and CD3+CD4- CD8α/β+cytotoxic T lymphocytes. In addition, PR40 infection showed an early depletion of activated CD4+CD25+ T lymphocytes and Tregs together with an intense and lasting depletion of CD21+ B lymphocytes. Overall, these features demonstrate that the more severe clinical signs observed upon infection with the HP PR40 strain are sustained by remarkable changes in the peripheral blood distribution of immune cells and provide further insights into the immune regulation/immunopathogenesis induced by PRRSV-1 subtype 1 European isolates

Effects of opioids on proximal renal tubular cells undergoing ATP depletion.

Abstract This study investigated the effect of morphine, fentanyl, butorphanol and buprenorphine on viability and caspase-3 activity in renal proximal tubular cells exposed to opioids for 2 h before or 12 h after chemical anoxia. Cell viability decreased regardless the treatment although intracellular ATP content was elevated in morphine and fentanyl pre-treated cells at 12 h. Anoxia increased caspase activity but this effect was significantly reduced in cells treated before or after with morphine, fentanyl and in cell treated with butorphanol for 12 h. No influence of buprenorphine was detected. Morphine, fentanyl and butorphanol might have protective effects during kidney ischemia

Preliminary Results on a "Real" Iris Recognition System under Challenging Operational Conditions

Iris recognition algorithms have recently demonstrated excellent performance in the authentication task. In this paper, we present a technology transfer project for the development and testing of a biometric recognition system under challenging operational conditions. Due to the stringent operational requirements, the design and implementation of the system included a phase of selecting technologically advanced hardware. The lack of corresponding data sets implied a novel acquisition step. The evaluation phase is preliminary as the data set is being expanded for the acquisition of new samples capable of highlighting the system’s critical issues. Current samples were acquired in very different lighting conditions and in the presence of glasses, which was not yet done in the literature. In addition to the selected hardware, such data allowed us to simulate a realistic environmental context for the project’s final prototype

Gene expression in mdx mouse muscle in relation to age and exercise: aberrant mechanical-metabolic coupling and implications for pre-clinical studies in duchenne muscular dystrophy

Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. Mechanical activity modulates gene expression and muscle plasticity. Here, we investigated the outcome of 4 (T4, 8 weeks of age) and 12 (T12, 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in the gastrocnemius muscle of mdx and wild-type (WT) mice using quantitative real-time PCR. Basal expression of the exercise-sensitive genes peroxisome-proliferator receptor γ coactivator 1α (Pgc-1α) and Sirtuin1 (Sirt1) was higher in mdx versus WT mice at both ages. Exercise increased Pgc-1α expression in WT mice; Pgc-1α was downregulated by T12 exercise in mdx muscles, along with Sirt1, Pparγ and the autophagy marker Bnip3. Sixteen weeks old mdx mice showed a basal overexpression of the slow Mhc1 isoform and Serca2; T12 exercise fully contrasted this basal adaptation as well as the high expression of follistatin and myogenin. Conversely, T12 exercise was ineffective in WT mice. Damage-related genes such as gp91-phox (NADPH-oxidase2), Tgfβ, Tnfα and c-Src tyrosine kinase were overexpressed in mdx muscles and not affected by exercise. Likewise, the anti-inflammatory adiponectin was lower in T12-exercised mdx muscles. Chronic exercise with minor adaptive effects in WT muscles leads to maladaptation in mdx muscles with a disequilibrium between protective and damaging signals. Increased understanding of the pathways involved in the altered mechanical-metabolic coupling may help guide appropriate physical therapies while better addressing pharmacological interventions in translational researchWeakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. Mechanical activity modulates gene expression and muscle plasticity. Here, we investigated the outcome of 4 (T4, 8 weeks of age) and 12 (T12, 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in the gastrocnemius muscle of mdx and wildtype (WT) mice using quantitative real-time PCR. Basal expression of the exercise-sensitive genes peroxisome-proliferator receptor g coactivator 1α (Pgc-1α) and Sirtuin1 (Sirt1) was higher in mdx versus WT mice at both ages. Exercise increased Pgc-1α expression in WT mice; Pgc-1α was downregulted by T12 exercise in mdx muscles, along with Sirt1, Pparγ and the autophagy marker Bnip3. Sixteen weeks old mdx mice showed a basal over expression of the slowMhc1 isoform and Serca2; T12 exercise fully contrasted this basal adaptation as well as the high expression of follistatin and myogenin. Conversely, T12 exercise was ineffective in WT mice. Damage-related genes such as gp91-phox (NADPH-oxidase2), Tgfβ, Tnfα and c-Src tyrosine kinase were overexpressed in mdx muscles and not affected by exercise. Likewise, the anti-inflammatory adiponectin was lower in T12-exercised mdx muscles. Chronic exercise with minor adaptive effects in WT muscles leads to maladaptation in mdx muscles with a disequilibrium between protective and damaging signals. Increased understanding of the pathways involved in the altered mechanical-metabolic coupling may help guide appropriate physical therapies while better addressing pharmacological interventions in translational research

Evaluation of the effects of cyclododecane on oil paintings

The solubility of oil paint components during the application of cyclododecane in solvent mixtures was evaluated in order to predict if the application of cyclododecane during restoration may significantly alter the chemical state of the paint layer in oil paintings. The chemical affinity between some of the oil binder components and non-polar cyclododecane could potentially lead to interactions or leaching during the application. In order to investigate these effects a set of samples taken from oil paintings from the early 1900s and 2008, were treated with cyclododecane in a solution, melted, and sprayed as aerosol. The samples were also submitted to a comparative extractive treatment with cyclododecane and organic solvents of different polarities. After the treatments, the extracted components were analysed by gas chromatography-mass spectrometry (GC/MS), which provided detailed molecular information on the composition of the extracts, together with a quantitative profile of fatty acids in extracted triglycerides, after saponification and derivatisation. The results show that applications of cyclododecane both as a spray and in a saturated solution in a hydrocarbon solvent determine the extraction of a low amount of lipids from the paint. On the other hand, when cyclododecane is applied in the melted form, there is an extraction of lipid components of the paint into the treatment solution