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Supporting Scientific Research Through Machine and Deep Learning: Fluorescence Microscopy and Operational Intelligence Use Cases
Get PDFAlthough the debate of what data science is has a long history and has not reached a complete consensus yet, Data Science can be summarized as the process of learning from data.
Guided by the above vision, this thesis presents two independent data science projects developed in the scope of multidisciplinary applied research.
The first part analyzes fluorescence microscopy images typically produced in life science experiments, where the objective is to count how many marked neuronal cells are present in each image.
Aiming to automate the task for supporting research in the area, we propose a neural network architecture tuned specifically for this use case, cell ResUnet (c-ResUnet), and discuss the impact of alternative training strategies in overcoming particular challenges of our data.
The approach provides good results in terms of both detection and counting, showing performance comparable to the interpretation of human operators.
As a meaningful addition, we release the pre-trained model and the Fluorescent Neuronal Cells dataset collecting pixel-level annotations of where neuronal cells are located.
In this way, we hope to help future research in the area and foster innovative methodologies for tackling similar problems.
The second part deals with the problem of distributed data management in the context of LHC experiments, with a focus on supporting ATLAS operations concerning data transfer failures.
In particular, we analyze error messages produced by failed transfers and propose a Machine Learning pipeline that leverages the word2vec language model and K-means clustering.
This provides groups of similar errors that are presented to human operators as suggestions of potential issues to investigate.
The approach is demonstrated on one full day of data, showing promising ability in understanding the message content and providing meaningful groupings, in line with previously reported incidents by human operators
Insights on epidemiology and control of Marek’s disease through traditional and novel molecular tools
Get PDFMarek's disease (MD) is a contagious, lymphoproliferative and neuropathic disease of poultry caused by a ubiquitous lymphotropic and oncogenic virus, Gallid alphaherpesvirus 2 (GaHV-2).
MD has been reported in all poultry-rearing countries and is among the viral diseases with the highest economic impact in the poultry industry worldwide, including Italy. MD has been also recognized as one of the leading causes of mortality in backyard poultry.
The present doctoral thesis aimed at exploring Marek's disease virus molecular epidemiology in Italian commercial and backyard chicken flocks and, for the first time, in commercial turkeys affected by clinical MD. Molecular biology techniques targeting the full-length meq gene, the major GaHV-2 oncogene, were used to detect and characterize the circulating GaHV-2 strains searching for genetic markers of virulence.
A final study focused on the development of rapid, sensitive, and species-specific loop-mediated isothermal amplification assays coupled with a lateral flow device readout for the detection of conventional and recombinant HVT-based vaccines is included in the thesis. HVT vaccines, currently used to protect chickens from MD, are referred to as "leaky", as they do not impede the infection, replication, and shedding of field GaHV-2: vaccinal and field viruses can coexist in the vaccinated host and molecular tests able to discriminate between GaHV-2 and HVT are required. These new simple, fast, and accurate tests for the monitoring of MD vaccination success in the field could be greatly beneficial for field veterinarians, small laboratories, and more broadly for resource-limited settings
Whole Exome Sequencing widens the genetic landscape of Hereditary Optic Neuropathies
Get PDFHereditary optic neuropathies (HON) are a genetic cause of visual impairment characterized by degeneration of retinal ganglion cells. The majority of HON are caused by pathogenic variants in mtDNA genes and in gene OPA1. However, several other genes can cause optic atrophy and can only be identified by high throughput genetic analysis. Whole Exome Sequencing (WES) is becoming the primary choice in rare disease molecular diagnosis, being both cost effective and informative.
We performed WES on a cohort of 106 cases, of which 74 isolated ON patients (ON) and 32 syndromic ON patients (sON). The total diagnostic yield amounts to 27%, slightly higher for syndromic ON (31%) than for isolated ON (26%).
The majority of genes found are related to mitochondrial function and already reported for harbouring HON pathogenic variants: ACO2, AFG3L2, C19orf12, DNAJC30, FDXR, MECR, MTFMT, NDUFAF2, NDUFB11, NDUFV2, OPA1, PDSS1, SDHA, SSBP1, and WFS1. Among these OPA1, ACO2, and WFS1 were confirmed as the most relevant genetic causes of ON. Moreover, several genes were identified, especially in sON patients, with direct impairment of non-mitochondrial molecular pathways: from autophagy and ubiquitin system (LYST, SNF8, WDR45, UCHL1), to neural cells development and function (KIF1A, GFAP, EPHB2, CACNA1A, CACNA1F), but also vitamin metabolism (SLC52A2, BTD), cilia structure (USH2A), and nuclear pore shuttling (NUTF2).
Functional validation on yeast model was performed for pathogenic variants detected in MECR, MTFMT, SDHA, and UCHL1 genes. For SDHA and UCHL1 also muscle biopsy and fibroblast cell lines from patients were analysed, pointing to possible pathogenic mechanisms that will be investigated in further studies.
In conclusion, WES proved to be an efficient tool when applied to our ON cohort, for both common disease-genes identification and novel genes discovery. It is therefore recommended to consider WES in ON molecular diagnostic pipeline, as for other rare genetic diseases
Development of new chemical entities to target neuroinflammatory pathways.
Get PDFNeuroinflammatory pathways are main culprits of neurodegenerative diseases' onset and progression, including Alzheimer’s disease (AD). On this basis, several anti-inflammatory drugs were repurposed in clinical trials. However, they have failed, probably because neuroinflammation is a complex network, still not fully understood.
From these evidences, this thesis focused on the design and synthesis of new chemical entities as potential neuroinflammatory drugs or chemical probes. Projects 1 and 2 aimed to multi-target-directed ligand (MTDL) development to target neuroinflammation in AD. Polypharmacology by MTDLs is considered one of the most promising strategies to face the multifactorial nature of neurodegenerative diseases. Particularly, Project 1 took inspiration from a cromolyn-ibuprofen drug combination polypharmacological approach, which was recently investigated in AD clinical trials. Based on that, two cromolyn-(S)-ibuprofen codrug series were designed and synthesized. Parent drugs were combined via linking or fusing strategies in 1:2 or 1:1 ratio, by means of hydrolyzable bonds.
Project 2 started from a still ongoing AD clinical trial on investigational drug neflamapimod. It is a selective inhibitor of p38α-MAPK, a kinase strictly involved in neuroinflammatory pathways. On the other side, rasagiline, an anti-Parkinson drug, was also repurposed as AD treatment. Indeed, rasagiline’s propargylamine fragment demonstrated to be responsible not only for the MAO-B selective inhibition, but also for the neuroprotective activity. Thus, to synergistically combine these two effects into single-molecules, a small set of neflamapimod-rasagiline hybrids was developed. In the end BMX, a poorly investigated kinase, which seems to be involved in pro-inflammatory mediator production, was explored for the development of new chemical probes. High-quality chemical probes are a powerful tool in target validation and starting points for the development of new drug candidates. Thus, Project 3 focused on the design and synthesis of two series of optimized BMX covalent inhibitors as selective chemical probes
<Pare un assurdo, e pure e esattamente vero>. Imagination and Imaginary in Leopardi
Get PDFQuesta ricerca è volta all’analisi dell’immaginazione nell’opera di Giacomo Leopardi secondo una prospettiva critico-interpretativa, cercando, da una parte, di comprenderne la funzione all’interno delle dinamiche complessive della teoria della conoscenza leopardiana e, dall’altra, di indagare le modalità attraverso le quali Leopardi si accosta alle figure dell’immaginario. In quest’ottica si è deciso di dividere in due parti equivalenti il lavoro, destinando la prima, all’esame della facoltà immaginativa in una prospettiva prettamente gnoseologica, intesa nell’economia generale degli atti mentali, mentre la seconda, alle sue applicazioni pragmatiche, alla ricerca, da una parte, di una categorizzazione utile all’interpretazione dell’immaginario leopardiano, dall’altra, dell’utilità pratica e morale dell’immaginazione.La présente recherche vise à analyser le concept du imagination dans l’œuvre de Giacomo Leopardi selon une perspective critique-interprétative, en essayant, d’une part, de comprendre sa fonction dans la dynamique globale de la théorie de la connaissance de Leopardi et, de l’autre, d’étudier les modalités à travers lesquelles Leopardi aborde les figures de l’imaginaire. Dans cette perspective, on a décidé de diviser l’œuvre en deux parties équivalentes et d’allouer la première, à l’examen de la faculté imaginative dans une perspective purement gnoséologique, considérée dans l’économie générale des actes mentaux, tandis que la seconde, est dédiée à ses applications pragmatiques, à la recherche, d’une part, d’une catégorisation utile pour l’interprétation de l’imaginaire léopardien, d’autre part, de l’utilité pratique et morale de l’imagination.This research analyses the concept of imagination in Giacomo Leopardi's works through a critical-interpretative perspective, trying, on one hand, to understand its function into Leopardi's global theory of knowledge and, on the other, to study how Leopardi approaches imaginary objects. In this perspective, the work has been divided into two parts: the first one is dedicated to the analysis of imagination through a gnoseological point of view, considered into the general scheme of mental acts. The second part is dedicated to its pragmatic applications, searching, on one side, for a category able to interpret Leopardi's imaginary and, on the other, for the practical and moral utility of imagination
Judicial Specialisation in Intellectual Property: the Unified Patent Court as a Model of Specialised Court
Get PDFThe aim of this thesis is to discuss and develop the Unified Patent Court project to account for the role it could play in implementing judicial specialisation in the Intellectual Property field. To provide an original contribution to the existing literature on the topic, this work addresses the issue of how the Unified Patent Court could relate to the other forms of judicial specialisation already operating in the European Union context. This study presents a systematic assessment of the not-yet-operational Unified Patent Court within the EU judicial system, which has recently shown a trend towards being developed outside the institutional framework of the European Union Court of Justice.
The objective is to understand to what extent the planned implementation of the Unified Patent Court could succeed in responding to the need for specialisation and in being compliant with the EU legal and constitutional framework. Using the Unified Patent Court as a case study, it is argued that specialised courts in the field of Intellectual Property have a significant role to play in the European judicial system and offer an adequate response to the growing complexity of business operations and relations.
The significance of this study is to analyse whether the UPC can still be considered as an appropriate solution to unify the European patent litigation system. The research considers the significant deficiencies, which risks having a negative effect on the European Union institutional procedures. In this perspective, this work aims to make a contribution in identifying the potential negative consequences of this reform. It also focuses on considering different alternatives for a European patent system, which could effectively promote innovation in Europe
S-nitrosylation homeostasis in plants: key enzymes and regulatory pathways
Get PDFThe nitrosylated form of glutathione (GSNO) has been acknowledged to be the most important nitrosylating agent of the plant cell, and the tuning of its intracellular concentration is of pivotal importance for photosynthetic life. During my time as a PhD student, I focused my attention on the enzymatic systems involved in the degradation of GSNO. Hence, we decided to study the structural and catalytic features of alcohol dehydrogenases (GSNOR and ADH1) from the model land plant Arabidopsis thaliana (At). These enzymes displayed a very similar 3D structure except for their active site which might explain the extreme catalytic specialization of the two enzymes. They share NAD(H) as a cofactor, but only AtGSNOR was able to catalyze the reduction of GSNO whilst being ineffective in oxidizing ethanol. Moreover, our study on the enzyme from the unicellular green alga Chlamydomonas reinhardtii (Cr) revealed how this S-nitrosoglutathione reductase (GSNOR) specifically use NADH to catalyze GSNO reduction and how its activity responds to thiol-based post-translational modifications. Contextually, the presence of NADPH-dependent GSNO-degrading systems in algal protein extract was highlighted and resulted to be relatively efficient in this model organism. This activity could be ascribed to several proteins whose contribution has not been defined yet. Intriguingly, protein extract from GSNOR null mutants of Arabidopsis displayed an increased NADPH-dependent ability to degrade GSNO and our quantitative proteome profiling on the gsnor mutant revealed the overexpression of two class 4 aldo-keto reductases (AKR), specifically AtAKR4C8 and AtAKR4C9. Later, all four class 4 AKRs showed to possess a NADPH-dependent GSNO-degrading activity. Finally, we initiated a preliminary analysis to determine the kinetic parameters of several plant proteins, including GSNOR, AKR4Cs, and thioredoxins. These data suggested GSNOR to be the most effective enzyme in catalyzing GSNO reduction because of its extremely high catalytic proficiency compared to NADPH-dependent systems
Efficacy of Tocilizumab for treament of chronic active antibody mediated rejection in kidney transplant recipient
Get PDFIn the last decades significant improvements has been reached in short term graft survival, conversely long-term graft survival in still an open challenge for the scientific community. One of the major causes of long term graft loss is represented by chronic- active antibody mediated rejection (cAMR), a recently identified entity whose diagnosis is based on laboratoristic and histologic elements: the presence of DSA associated to specific morphological lesions as inflammation and microvascular damage associated or not to C4d deposition. Treatment of cAMR is an open field of debate. Tocilizumab, an anti-IL6 monoclonal antibody has been recently proposed as a first line treatment for cAMR, showing encouranging results.
We describe our monocentric experience using Tocilizumab as first-line therapy for cAMR.
Graft function (eGFR), proteinuria and DSA have been evaluated every 6 month for 24 months; histology have been performed after 12 months of treatment.
No adverse events have been observed during study period.
12 patients completed the study with a follow-up of 24 months. Kidney function showed a worsening during follow-up that reaches statistical significance at 12 and 24 months (eGFR from 32.2±13.9 ml/min to 26.9±13 ml/min), but far less than expected for these kind of patients. 4 patients (30%) reached ESRD during follow-up, 3 requiring renal replacement therapy.
We did not observed any statically significant variation in proteinuria and in DSA MFI levels.
From a histological point of view, we observed a significant improvement in active cAMR lesions (C4d deposition and Acute tissue injury (MTA, g>0/ptc>0, v>0) and no progression among chronic lesions (Transplant glomerulopathy, PTC multilayering and aterial intimal fibrosis)
Tocilizumab shown good results, with a stabilization of graft function, a reduction in kidney inflammation and active lesions in kidney biopsy and not allowing progression of chronic lesions.In the last decades significant improvements has been reached in short term graft survival, conversely long-term graft survival in still an open challenge for the scientific community. One of the major causes of long term graft loss is represented by chronic- active antibody mediated rejection (cAMR), a recently identified entity whose diagnosis is based on laboratoristic and histologic elements: the presence of DSA associated to specific morphological lesions as inflammation and microvascular damage associated or not to C4d deposition. Treatment of cAMR is an open field of debate. Tocilizumab, an anti-IL6 monoclonal antibody has been recently proposed as a first line treatment for cAMR, showing encouranging results.
We describe our monocentric experience using Tocilizumab as first-line therapy for cAMR.
Graft function (eGFR), proteinuria and DSA have been evaluated every 6 month for 24 months; histology have been performed after 12 months of treatment.
No adverse events have been observed during study period.
12 patients completed the study with a follow-up of 24 months. Kidney function showed a worsening during follow-up that reaches statistical significance at 12 and 24 months (eGFR from 32.2±13.9 ml/min to 26.9±13 ml/min), but far less than expected for these kind of patients. 4 patients (30%) reached ESRD during follow-up, 3 requiring renal replacement therapy.
We did not observed any statically significant variation in proteinuria and in DSA MFI levels.
From a histological point of view, we observed a significant improvement in active cAMR lesions (C4d deposition and Acute tissue injury (MTA, g>0/ptc>0, v>0) and no progression among chronic lesions (Transplant glomerulopathy, PTC multilayering and aterial intimal fibrosis)
Tocilizumab shown good results, with a stabilization of graft function, a reduction in kidney inflammation and active lesions in kidney biopsy and not allowing progression of chronic lesions
Practical use and clinicopathological significance of the excreted fractions of electrolytes in the recognition of acute kidney injury (AKI) in different clinical settings
Get PDFRicerche nel campo del danno renale acuto e frazioni urinarie escrete.
La prima parte verte sull’analisi dei dati provenienti da una casistica di cani con leptospirosi, sono stati confrontati due gruppi di cani, il primo con AKI da leptospirosi e l’altro con AKI per eziologie differenti. In queste due popolazioni di pazienti abbiamo valutato alcuni analiti sierici ed urinari come ad esempio l’escrezione elettrolitica frazionata e biomarker di AKI come NGAL. I cani con leptospirosi, hanno mostrato maggiore kaliuresi e più grave glicosuria rispetto a quelli non affetti da leptospirosi, così come erano più frequentemente glicosurici rispetto agli altri. Questi dati sono in analogia con quanto è riportato nell’uomo e dimostrano un pattern di danno tubulare tipico in corso di questa malattia se paragonato appunto ad altre cause di danno tubulare acuto e AKI
La seconda parte riguarda la valutazione della funzionalità renale e del danno renale acuto in cani affetti da insufficienza valvolare mitralica. E’ stata incentrata sul danno renale in corso di cardiopatie e per questa ragione abbiamo pensato di valutare esclusivamente pazienti con MVD per varie ragioni: poiché sono pazienti che si presentano frequentemente nella pratica clinica; la malattia è tipicamente cronica e il paziente rimane stabile a lungo con un andamento progressivo della malattia; questi pazienti possono presentare frequenti episodi di AKI legati allo scompenso cardiaco e/o alla terapia con diuretico 3/o ace-i che questi animali ricevono. Abbiamo valutato prospetticamente l'impatto della terapia orale con furosemide sulla chimica urinaria, nei cani con malattia della valvola mitrale mixomatosa. Tali differenze sono state attribuite all'effetto della terapia con furosemide sugli elettroliti renali. La chimica urinaria è utile per stimare la risposta diuretica nei cani con malattie cardiache.
I dati suggeriscono differenze significative tra i diversi stadi ACVIM con particolare riferimento all’escrezione elettrolitica di Sodio, Potassio e Cloro.Research in the field of acute kidney injury and excreted urinary fractions.
The first part focuses on the analysis of data from a series of dogs with leptospirosis, two groups of dogs were compared, the first with AKI from leptospirosis and the other with AKI for different etiologies. In these two populations we evaluated some serum and urinary analytes such as fractional electrolyte excretion and biomarker of AKI such as NGAL. Dogs with leptospirosis showed greater kaliuresis and more severe glycosuria than those without leptospirosis, as well as were more frequently glycosuric than the others. These data are in analogy with what is reported in humans and demonstrate a typical tubular damage pattern in the course of this disease when compared to other causes of acute tubular damage and AKI.
The second part concerns the evaluation of renal function and AKI in dogs with mitral valve insufficiency. It focused on renal damage in the course of heart disease and for this reason we decided to exclusively evaluate patients with MVD for various reasons: they are patients who frequently present themselves in clinical practice; the disease is typically chronic and the patient remains stable for a long time with a progressive course of the disease; these patients may present with frequent episodes of AKI related to heart failure and / or to the 3 / or ace-i diuretic therapy these animals receive. We prospectively evaluated the impact of oral furosemide therapy on urinary chemistry in dogs with myxomatous mitral valve disease. These differences have been attributed to the effect of furosemide therapy on renal electrolytes. Urinary chemistry is useful for estimating the diuretic response in dogs with heart disease.
The data suggest significant differences between the different ACVIM stages with particular reference to the electrolytic excretion of Sodium, Potassium and Chlorine
Nicolò dell’Abate en France: dessins et projets décoratifs. Proposition pour un catalogue
Get PDFLa tesi prende in esame l’attività artistica di Nicolò dell’Abate (Modena, 1509 – Parigi, 1571), con particolare riferimento alla sua grafica. L’artista lavorò circa sessanta anni suddivisi fra l’Emilia, in alcune delle sue piccole entità politiche autonome come Modena, Scandiano, Soragna, Busseto, Bologna, e la Francia, potente stato nazionale che, all’epoca qui presa in considerazione, era guidato dalla casata dei Valois: la carriera italiana di Nicolò dell’Abate si sviluppa in un arco temporale che si estende dal 1529 alla primavera del 1552, quando si trasferisce definitivamente oltralpe, morendo a Parigi alla fine di marzo del 1571. L’elaborato è diviso in quattro capitoli, dei quali il primo, introduttivo, ripercorre per sommi capi le principali tappe della sfaccettata attività italiana; il secondo e il terzo sono invece incentrati sull’esame dell’attività artistica francese del maestro, con particolare riferimento alla grafica, indagata sulla base della ricostruzione del contesto storico, culturale e artistico della Francia dell’epoca, con specifico riguardo alla corte reale della famiglia Valois nei periodi compresi fra l’arrivo di Nicolò, nel 1552, e la morte prematura del re Enrico II, nel 1559, e fra il 1560 e il 1571, durante il quale deteneva il potere de facto la regina madre Caterina de’ Medici, moglie di Enrico. Il quarto e ultimo capitolo costituisce il catalogo dei disegni italiani e francesi: è preceduto da un saggio introduttivo che esamina lo stile disegnativo di Nicolò dell’Abate in Italia e in Francia, le sue caratteristiche fondamentali e il suo mutamento, oltre alle tecniche grafiche impiegate dall’artista, la loro evoluzione e variazione e il loro ruolo nella sua poetica. Infine, in appendice, è presente il regesto delle fonti francesi sull’attività dell’artista e di alcuni dei maggiori esponenti della cosiddetta scuola di Fontainebleau.The thesis examines the artistic activity of Nicolò dell'Abate (Modena, 1509 - Paris, 1571), with a particular reference to his draughtsmanship. The thesis is divided into four chapters: the first, introductory, retraces in detail the main stages of Nicolò’s Italian activity; the second and third chapters focus on the examination of his French artistic activity, with a particular reference to his draughtsmanship, studied on the basis of the reconstruction of the historical, cultural and artistic context of France at the time, with a specific reference to the Royal Court of the Valois in the periods between the arrival of Nicolò, in 1552, and the premature death of King Henry II in 1559, and that of 1560 to 1571, during which the queen-mother Catherine de Medici held power. The fourth chapter is the catalogue of Italian and French drawings: it is preceded by an introductory essay which examines the style of drawing of Nicolò dell'Abate in Italy and France. Finally, in the appendix, the French sources on the activity of the artist and some of the principal representatives of the school of Fontainebleau are reported.La thèse examine l’activité artistique de Nicolò dell’Abate (Modène, 1509 - Paris, 1571), avec une référence particulière à sa production dessinée. La thèse est divisé en quatre chapitres : le premier, introductif, retrace en détail les principales étapes de l’activité italienne; le deuxième et le troisième chapitres sont centrés sur l’examen de l’activité artistique française du maître, avec une référence particulière à ses dessins, étudié sur la base de la reconstruction du contexte historique, culturel et artistique de la France de l’époque, avec une référence spécifique à la cour royale des Valois dans les périodes comprises entre l’arrivée de Nicolò, en 1552, et la mort prématurée du roi Henri II, en 1559, et celle de 1560 à 1571, durant laquelle la reine-mère Catherine de Médicis détenait le pouvoir. Le quatrième chapitre constitue le catalogue des dessins italiens et français : il est précédé d’un essai introductif qui examine le style de dessin de Nicolò dell’Abate en Italie et en France. Enfin, en annexe, les sources françaises sur l’activité de l’artiste et de quelques-uns des principaux représentants de l’école de Fontainebleau sont reprises