Proteomics studies of protein homeostasis and aggregation in ageing and neurodegeneration

Upon ageing, a progressive disruption of protein homeostasis often leads to extensive protein aggregation and neurodegeneration. It is therefore important to study at the proteome level the origins and consequences of such disruption, which so far have remained elusive. Addressing this problem has recently become possible by major advances in mass spectrometry-based (MS) proteomics, which allows the identifications and quantification of thousands of proteins in a variety of biological samples. In the first part of this thesis, I analyse proteome-wide MS data for the nematode worm C. elegans upon ageing, in wild type (WT), long-lived and short-lived mutant strains. By comparing the total abundance and the soluble abundance for nearly 4000 proteins, I provide extensive evidence that proteins are expressed in adult worms at levels close to their solubility limits. With the use of sequence-based prediction tools, I then identify specific physico-chemical properties associated with this age-related protein homeostasis impairment. The results that I obtained reveal that the total intracellular protein content remains constant, in spite of the fact that the proteome undergoes wide remodeling upon ageing, resulting into severe protein homeostasis disruption and widespread protein aggregation. These results suggest a protein-dependent decrease in solubility associated with the protein homeostasis failure. In the second part of the thesis, I determine and classify potential interactions of misfolded protein oligomers with other proteins. This phenomenon is widely believed to give rise to cytotoxicity, although the mechanisms by which this happens are not fully understood. To address this question, I process and analyse MS data from structurally different oligomers (toxic type A and nontoxic type B) of the protein HypF-N, incubated in vitro with proteins extracted from murine cell cultures. I find that more than 2500 proteins are pulled down with the misfolded oligomers. These results indicate that the two types of oligomers interact with the same pool of proteins and differ only in the degree of binding. Functional annotation analysis on the groups reveals a preference of the oligomers to bind proteins in specific biological pathways and categories, including in particular mitochondrial membrane proteins, RNA-binding proteins and molecular chaperones. Overall, in this study I complement the powerful and high-throughput experimental approach of MS proteomics with bioinformatics analyses and prediction algorithms to define the physical, chemical and biological features of protein homeostasis disruption upon ageing and the interactome of misfolded oligomers

Machine learning approach to the safety assessment of a prestressed concrete railway bridge

Early structural anomalies identification allows to hold maintenance activities that avoid loss of both economic resources and human life. This is extremely important for crucial infrastructures like railway bridges. This paper illustrates the structural health monitoring approach applied to a simply supported prestressed concrete railway bridge. In the framework of long-term monitoring, both static quantities (displacements, strains, and rotations) and environmental measurements (temperatures) have been recorded. Machine learning techniques, Extreme Gradient boosting machine and Multi-Layer Perceptron, have been exploited to build regression correlation models associated with the undamaged structural condition after adequate pre-processing operations. In this way, alarm thresholds based on the expected residuals between the predicted structural quantities and the measured ones, have been defined. The thresholds turned out to be able to catch early-stage anomalies not pointed out by traditional damage thresholds based on the design values. The proposed damage index is chosen as the moving median of the residuals, allowing a significant reduction of false alarms. The used correlation models and the obtained results represent a starting point for the generalization of this approach to the bridges belonging to the same static typology

High time-resolved measurements of fine aerosol (PM2.5) in a hot-spot area during wintertime: multi-wavelength optical absorption properties and source apportionment

Black Carbon (BC) is the main absorber of solar radiation among the aerosol components, it influences cloud processes, and alters the melting of snow and ice cover. Although it is one of the most important individual climate-warming components, uncertainties on the radiative forcing related to BC-radiation interaction still cover more than one order of magnitude. Moreover, weakly absorbing organic material (brown carbon, BrC) in the form of particle coating or as particle as-is can be considered a further important contributor to aerosol absorption. The peculiarity of BrC is that it is very effective in the absorption of short-\u3bb radiation whereas its contribution to aerosol absorption is negligible in the red or near-IR bands. It is noteworthy that BC and BrC can also be used for source apportionment purposes (e.g. they can be helpful for the discrimination between fossil fuels combustion vs. biomass burning). Thus, aerosol absorption properties possibly related to mixing and/or size information, and BC content are currently of great interest. Moving in this frame, a multi-\u3bb polar photometer (PP_UniMI) has been developed at the Department of Physics of the University of Milan in the last years. The instrument is based on the measurement on the scattering plane of the light transmitted and scattered in the forward and back hemispheres by unloaded and loaded samples using a rotating photodiode. Data reduction aiming at the determination of the sample absorbance follows Petzold et al. (2004) and therein cited literature. Currently, PP_UNIMI allows performing 4-\u3bb measurements (870, 633, 532, 405 nm) on aerosol collected on different substrates, including aerosol collected with high-time resolution using a streaker sampler. Such sampler collects aerosol segregated in two size-classes (fine and coarse) on a rotating frame with hourly resolution. The set-up of the instrument was validated against independent measurements carried out using a Multi-Angle Absorption Photometer for what concerns the red-light results, considering possible artefact effects shown in Vecchi et al. (2013). The results presented here are related to the analysis of the high time-resolved trends of multiwavelength aerosol absorption properties measured on the fine aerosol fraction during a field campaign performed in Milan (Italy) in November 2015 (see an example in Figure 1). Such data will be used to test the possibility of applying source apportionment models based on optical properties (es. Aethalometer model) using off-line hightime resolved data. It is also noteworthy that equivalent BC can be quantified from the polar photometer measurements at 635 nm using a suitable mass absorption coefficient. Such information will be joined to the elemental components (Na-Pb) detected by Particle-Induced X-ray Emission technique carried out at the INFN-LABEC in Florence to perform receptor modelling analysis (e.g. Positive Matrix Factorization). The results of the source apportionment using such data will be also presented

Towards a FrameNet Resource for the Legal Domain

In the AI&Law community, the importance of frame-based ontologies has been acknowledged since the early 90\u27s with the Van Kralingen\u27s proposal of a frame language for legal knowledge representation. This still appears to be a strongly felt need within the community. In this paper, we propose to face this need by developing a FrameNet resource for the legal domain based on Fillmore\u27s Frame Semantics, whose final outocme will include a frame-based lexical ontology and a legal corpus annotated with frame information. In particular, the paper focuses on methodological and design issues, ranging from the customization and extension of the general FrameNet for the legal domain to the linking of the developed resource with already existing Legal Ontologies

Active fixation coronary sinus lead in a patient with persistence of left superior vena cava

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Proteome-wide observation of the phenomenon of life on the edge of solubility.

To function effectively proteins must avoid aberrant aggregation, and hence they are expected to be expressed at concentrations safely below their solubility limits. By analyzing proteome-wide mass spectrometry data of Caenorhabditis elegans, however, we show that the levels of about three-quarters of the nearly 4,000 proteins analyzed in adult animals are close to their intrinsic solubility limits, indeed exceeding them by about 10% on average. We next asked how aging and functional self-assembly influence these solubility limits. We found that despite the fact that the total quantity of proteins within the cellular environment remains approximately constant during aging, protein aggregation sharply increases between days 6 and 12 of adulthood, after the worms have reproduced, as individual proteins lose their stoichiometric balances and the cellular machinery that maintains solubility undergoes functional decline. These findings reveal that these proteins are highly prone to undergoing concentration-dependent phase separation, which on aging is rationalized in a decrease of their effective solubilities, in particular for proteins associated with translation, growth, reproduction, and the chaperone system

Saharan dust impact in central Italy: An overview on three years elemental data records

In southern European countries, Saharan dust may episodically produce significant increases of PM10, which may also cause the exceedance of the PM10 daily limit value established by the European Directive (2008/50/EC). The detection with very high sensitivity of all the elements that constitute mineral dust makes PIXE technique a very effective tool to assess the actual impact of these episodes. In this work, a review of long-term series of elemental concentrations obtained by PIXE has been accomplished with the aim of identifying the occurrence of Saharan dust transport episodes over long periods in Tuscany and characterising them in terms of composition and impact on PM concentration, tracing back their contribution to the exceedances of the PM10 limit value. The impact of the different Saharan intrusions on PM10 showed a very high variability. During the most intense episodes (which occurred with a frequency of few times per year) the calculated soil dust concentration reached values as high as 25\u201330 \u3bcg m 123, to be compared with background values of the order of 5 \u3bcg m 123. The Saharan dust contribution was decisive to cause the exceedance of the PM10 daily limit value in the 1\u20132% of the days considered in the present work

The relevance of molecular genotyping to allocate cases in a suspected outbreak of Legionella pneumonia in patients with prolonged immunosuppressive therapy

Three cases of pneumonia caused by Legionella pneumophila serogroup 1 (Lp1) in immunosuppressed patients with repeated hospitalization were suspected as a healthcare-associated cluster. The environmental investigation did not reveal the presence of legionellae in the hospital patient rooms. Water samples collected from the homes of two patients were also negative for Legionella spp. In the absence of environmental strains potentially involved in the infections, we proceeded to genotype environmental Lp1 strains isolated in the hospital during routine water sampling during the decade 2009–2019 and recovered after long-term storage at −20 °C. These 'historical' strains exhibited a high grade of similarity and stability over time, regardless of the disinfection systems. The different molecular profiles shown among the clinical and environmental strains excluded a nosocomial outbreak. The study suggests that the application of molecular typing may be a useful tool to discriminate hospital vs community-acquired cases, mostly for severely immunosuppressed patients in whom the symptomatology could be insidious and the incubation period could be prolonged. Moreover, the genotyping allowed us to exclude any link between the cases. Keywords: Legionnaires' disease, Immunosuppressed patients, Sequence-based typing, Cluster, Environmental strains, Clinical strain

Assessing spleen stiffness by point shear‐wave elastography: Is it feasible and reproducible in patients with chronic liver disease? Is it useful to predict portal hypertension?

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