Archivio Istituzionale della Ricerca - Università degli Studi di Pavia
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Insights into Monoamine Oxidases: Biocatalytic Potential and Evolutionary Diversification
No full textMonoamine oxidases (MAOs) are flavin-dependent enzymes that catalyze oxidative deamination of primary and secondary amines to the corresponding aldehydes or ketones with the release of ammonia and hydrogen peroxide. In mammals, MAOs are part of the outer mitochondrial membrane and occur in two isoforms, MAO A and MAO B, which are pivotal in neurotransmitter metabolism and are directly implicated in neurological events and age-related disorders. Besides their biological significance, MAOs and their homologues have also seen growing interest as multi- functional biocatalysts. Their capacity for selective amine oxidations has found applications in the preparation of enantiopure drug intermediates, in biosensors, and in green chemistry.
The first part of this thesis (Chapter 1) introduces the structural and biochemical features of MAOs within the frame of the broader family of FAD-dependent amine oxidases. It points out the distribution and the physiological function of mammalian isoforms, analyzes homologous enzymes in fungi and bacteria, and considers their possible applications in biocatalysis and biosensing. This introduction prepares the reader for the subsequent experimental chapters, which deal with both the applied and evolutionary sides of the MAO family.
One of the primary focuses of this thesis has been the characterization of a novel thermostable bacterial enzyme, MAOTb, in a thermophilic Thermoanaerobacterales isolate (Chapter 3). MAOTb is distinguished by high expression levels in Escherichia coli and high thermostability, with retention of activity at temperatures greater than 70 °C. Biochemical characterization revealed an unequivocal preference for long- chain aliphatic amines, n-heptylamine being its most active substrate. Pre-steady- state kinetics experiments verified that the reduced enzyme reacts rapidly with molecular oxygen, making it a bona fide oxidase. Structural comparison at 1.5 Å resolution revealed close similarity with human MAOs, e.g., conserved aromatic cage and covalent FAD ligation, but also distinguishing features such as absence of membrane-anchoring helix, which accounts for its solubility. Such features make MAOTb a potential candidate for industrial and synthetic applications.
To investigate its engineering potential, structure-based mutagenesis was used (Chapter 4). Targeting critical residues that determine the active site from the substrate-bound crystallographic structure, variants with altered activity against aromatic monoamines were realized, demonstrating the feasibility of substrate specificity adjustment in this enzyme. These discoveries emphasize the suitability of MAOTb as a robust scaffold for tailoring oxidase activity toward drugs of pharmaceutical value.
Concurrently with this, the thesis also explores the evolutionary diversification of the MAO family (Chapter 5). Phylogenetic analysis revealed that the duplication event resulting in MAO A and MAO B occurred in the tetrapod ancestor, while non- tetrapod jawed vertebrates retained the ancestral gene (MAO A/B) along with a new paralog, MAO F. Functional investigation of recombinant MAO A/B and MAO F in representative species revealed that both have human MAO A-like features but with distinct enzymatic properties. These findings contribute to our understanding of vertebrate MAO evolutionary history and uncover a novel lineage of putative physiological importance.
In general, this thesis presents new insights into the monoamine oxidase family from complementary perspectives. The discovery and engineering of MAOTb introduce an intriguing new biocatalyst with industrial application, while the evolutionary analysis leading to the identification of MAO F further illuminates vertebrate molecular evolution. By combining different approaches, this work identifies a path spanning basic and applied research and illustrates both the ancient diversification and the modern utility of monoamine oxidases.Monoamine oxidases (MAOs) are flavin-dependent enzymes that catalyze oxidative deamination of primary and secondary amines to the corresponding aldehydes or ketones with the release of ammonia and hydrogen peroxide. In mammals, MAOs are part of the outer mitochondrial membrane and occur in two isoforms, MAO A and MAO B, which are pivotal in neurotransmitter metabolism and are directly implicated in neurological events and age-related disorders. Besides their biological significance, MAOs and their homologues have also seen growing interest as multi- functional biocatalysts. Their capacity for selective amine oxidations has found applications in the preparation of enantiopure drug intermediates, in biosensors, and in green chemistry.
The first part of this thesis (Chapter 1) introduces the structural and biochemical features of MAOs within the frame of the broader family of FAD-dependent amine oxidases. It points out the distribution and the physiological function of mammalian isoforms, analyzes homologous enzymes in fungi and bacteria, and considers their possible applications in biocatalysis and biosensing. This introduction prepares the reader for the subsequent experimental chapters, which deal with both the applied and evolutionary sides of the MAO family.
One of the primary focuses of this thesis has been the characterization of a novel thermostable bacterial enzyme, MAOTb, in a thermophilic Thermoanaerobacterales isolate (Chapter 3). MAOTb is distinguished by high expression levels in Escherichia coli and high thermostability, with retention of activity at temperatures greater than 70 °C. Biochemical characterization revealed an unequivocal preference for long- chain aliphatic amines, n-heptylamine being its most active substrate. Pre-steady- state kinetics experiments verified that the reduced enzyme reacts rapidly with molecular oxygen, making it a bona fide oxidase. Structural comparison at 1.5 Å resolution revealed close similarity with human MAOs, e.g., conserved aromatic cage and covalent FAD ligation, but also distinguishing features such as absence of membrane-anchoring helix, which accounts for its solubility. Such features make MAOTb a potential candidate for industrial and synthetic applications.
To investigate its engineering potential, structure-based mutagenesis was used (Chapter 4). Targeting critical residues that determine the active site from the substrate-bound crystallographic structure, variants with altered activity against aromatic monoamines were realized, demonstrating the feasibility of substrate specificity adjustment in this enzyme. These discoveries emphasize the suitability of MAOTb as a robust scaffold for tailoring oxidase activity toward drugs of pharmaceutical value.
Concurrently with this, the thesis also explores the evolutionary diversification of the MAO family (Chapter 5). Phylogenetic analysis revealed that the duplication event resulting in MAO A and MAO B occurred in the tetrapod ancestor, while non- tetrapod jawed vertebrates retained the ancestral gene (MAO A/B) along with a new paralog, MAO F. Functional investigation of recombinant MAO A/B and MAO F in representative species revealed that both have human MAO A-like features but with distinct enzymatic properties. These findings contribute to our understanding of vertebrate MAO evolutionary history and uncover a novel lineage of putative physiological importance.
In general, this thesis presents new insights into the monoamine oxidase family from complementary perspectives. The discovery and engineering of MAOTb introduce an intriguing new biocatalyst with industrial application, while the evolutionary analysis leading to the identification of MAO F further illuminates vertebrate molecular evolution. By combining different approaches, this work identifies a path spanning basic and applied research and illustrates both the ancient diversification and the modern utility of monoamine oxidases
BIOCOMPATIBLE HYDROGELS BASED ON POLYSACCHARIDES AND DERIVATIVES AS VERSATILE PLATFORMS FOR LOCAL APPLICATIONS
No full textSynthesis, structural characterization, and biological evaluation of cinnamaldehyde–azomethine hybrids: In Vitro and In Silico approaches
No full textWe herein reported the synthesis and investigation of biologically active novel compounds, which integrate cinnamaldehydes and azomethine moieties into one molecule. Seven cinnamaldehyde-based azomethines with polyamine backbones were synthesized by condensation reaction in a non-catalyst medium, and their structures were studied by NMR, FTIR, mass and elemental analysis. Antibacterial activity was investigated against Gram-positive and Gram-negative bacterial strains by a two-fold microdilution method, and results were compared with the known antibiotics. The obtained results revealed that compound 5 exhibited promising activity against a clinical isolate of S. aureus, with a MIC value of 0.016 μmol/mL (8 μg/mL); therefore, due to its lowest MIC, the time-kill kinetics and growth inhibitory effects were further evaluated at 2× MIC to assess its bactericidal potential. Cytotoxicity studies demonstrated that compound 5 exhibited minimal toxicity toward human keratinocyte cells, indicating low overall cytotoxicity on mammalian cells. To study the mechanism of action of compound 5, the effect of the investigated molecule on the ultrastructural organization of S. aureus was studied by using transmission electron microscopy, which showed that the investigated sample directly affects the process of bacterial replication by revealing nucleoid enlightenment, remnants of destructively altered fragments of the cell septum, disruption of cell wall structure. To elucidate the antibacterial mechanism of the targeted compound, molecular docking identified target proteins 3VSL and 5M18 with their catalytic and allosteric sites, and 100 ns MD simulations (RMSD, Rg, SASA, RMSF) confirmed the synthesized ligand's stable binding and interaction with these proteins
Preposterous Fake News, the Breach of Democratic Trust, and Intellectual Humility
No full textWhat normative relationship should citizens and political representatives establish with people voicing preposterous fake news, defying basic epistemic rules? To answer this pressing matter, we defend the view that the spread of preposterous fake information is best accounted for by an expressivist explanation, according to which agents have practical reasons, rather than genuinely epistemic, to share preposterous fake news, as expressive of their commitment toward a specific narrative about social reality. Then, we investigate the worrying effects of these dynamics on the democratic fabric and on mutual trust among citizens. The pars construens of our argument consists in a strategy for mending the breach of democratic trust based on the virtue of reasonableness. We argue that to restore minimal trust between citizens and government, reasonable citizens ought to embrace intellectual humility, therefore approaching disagreement showing respect for others, dispensing with a superior and dismissive attitude toward agents holding unreasonable beliefs
Characterizing tuberculosis and non-tuberculous mycobacterial enzymes to fight antibiotic resistance by targeting novel essential mechanisms and virulence factors.
No full textLo sviluppo diastematico della notazione Ravennate: l ́impatto della diastemazia sulla notazione delle indicazioni performative.
No full textIl presente progetto di dottorato si propone di indagare lo sviluppo della notazione musicale Ravennate tra XI e XII secolo. In particolare, la ricerca si focalizza sullo studio della graduale integrazione di indicazioni diastematiche (relative la precisa definizione visiva dell ́altezza dei singoli suoni) da parte degli scribi nel corso dei due secoli.
Dopo l ́introduzione della ricerca descritta nel primo capitolo, la tesi si concentra sul definire le metodologie analitiche utilizzate nell ́individuare specifiche tipologie notazionali. Le fonti Ravennate sopravvissute, databili dalla fine del X al XII secolo, presentano caratteristiche peculiari che consentono di identificare una pratica notazionale specifica di Ravenna. La scrittura adiastematica si distingue per l’organizzazione orizzontale dei neumi, l’inclinazione verso destra dell ́angolo di scrittura, specifiche morfologie dei segni. La compresenza di tali elementi permette di delimitare la scrittura musicale Ravennate. Tra l ́XI ed il XII secolo, il variegato vocabolario neumatico iniziale si ridusse progressivamente, abbandonando alcune morfologie utilizzate per indicare specificità di articolazione vocale della melodia. La scrittura divenne più rigida, con contrasti più marcati e prendendo sembianze simili alla notazione quadrata. Questa trasformazione interessò non solo il ductus scrittorio, ma altresì una diversa disposizione dei neumi nello spazio di scrittura musicale della notazione: i segni neumatici iniziarono ad essere posizionati secondo un ́asse verticalizzata, seguendo la gerarchia tonale intercorsa tra un suono e l ́altro.
Il terzo capitolo ha carattere prettamente analitico, indagando le tipologie notazionali di Ravenna attraverso lo studio dell ́organizzazione dello spazio di scrittura, del vocabolario neumatico, delle morfologie dei segni, dell ́uso di neumi specifici e di forme composte, segmenti neumatici e formule melodiche. A seguito dell ́analisi delle fonti, sono state individuate tre principali tipologie di notazione Ravennate:
• notazione adiastematica (fine X – XI secolo): caratterizzata da una disposizione orizzontale della scrittura in modo da non permettere di inferire visivamente relazioni di altezza tra i suoni; da un ductus scorrevole, e dall ́utilizzo di un ́ampia gamma neumatica comprendente altresì varie forme ad occhiello;
• notazione a tendenza diastematica (fine XI – XII secolo): caratterizzata da una intenzionalità diastematica più marcata attraverso la disposizione della scrittura seguendo un asse verticale; le morfologie neumatiche prendono sembianze più rigide, vicine alle forme dei neumi su rigo caratterizzati da un forte contrasto di scrittura; nonché inizia il processo di riduzione della varietà neumatica antecedente attraverso l ́assenza di segni quilismatici e forme ad occhiello;
• notazione su rigo (XII secolo): scrittura caratterizzata dall ́uso del rigo guidoniano con linee tracciate a secco, delle quali due colorate in rosso e giallo per i salti semitonali di Fa e Do, lettere chiave e custos a fine rigo per indicare il primo suono del sistema di rigo sottostante; i neumi presentano morfologie marcate, con un elevato contrasto di scrittura assimilabile alla successiva notazione quadrata.
L’analisi di queste fonti ha permesso di ipotizzare una graduale integrazione delle indicazioni diastematiche nella pratica notazionale Ravennate.
Il quarto capitolo si concentra sull ́esame comparativo delle notazioni, al fine di individuare affinità con scritture coeve e possibili legami. I confronti indicano forti legami con Bologna e, in maniera ancora più decisa, con l ́area Toscana. Influenze West-Frankish sono state inoltre individuate, con alcuni influissi delle scritture ispaniche nelle forme di neumi resupini e ad occhiello. Il quinto capitolo contiene un excursus sulle dinamiche storico-culturali sottostanti lo sviluppo della scrittura ravennate tra XI e XII secolo.The doctoral research regards the development of the Ravennate notation between the 11th and 12th centuries, with particular focus on the manner in which the indication of diastemacy (the pitch-relation between individual sounds) were integrated in the praxis of notating melodies.
The thesis begins with an introduction to the research, to which follows – in Chapter Two – the definition of the analytical methodologies used to identify specific notational types. The Ravennate surviving sources from the late 10th to the 12th centuries show specific features that allow identifying a notational practice distinctive of Ravenna. The adiastematic notation is characterized by the horizontal arrangement of the neumes, a writing axis that tends to the right, and specific neumatic morphologies. The coexistence of these elements allows to circumscribe the Ravennate notation. Between the 11th and 12th centuries, the neumatic variety was progressively reduced, eliminating some of the earlier peculiar morphologies. The notation became more rigid, with stronger writing contrasts in a similar manner of forms of the square notation. This transformation affected also a different spatial organization of the neumes on the musical writing surface: the signs began to be placed according to a vertical axis, following the tonal hierarchy between pitches.
The Third Chapter is analytic, examining the Ravenna notational typologies through the investigation of: spatial organization of the music writing, neumatic vocabulary, morphologies, the use of specific neumes and compound forms, neumatic segments, and melodic formulas. Based on the analysis of the sources, three main types of Ravennate notation have been identified:
• adiastematic notation (late 10th – 11th century): characterized by the horizontal disposition of the writing, which does not allow for a visual inference of pitch relationships between sounds; by a fluid ductus; and by the use of a wide neumatic vocabulary, including various looped forms.
• notation of diastematic tendency (late 11th – 12th century): characterized by a diastematic intention of the writing through the vertical arrangement of the neumes; the morphologies become more rigid, closer to the forms of the staff notation having more writing contrasts; initial reduction of the earlier neumatic variety (absence of quilisma signs and looped forms).
• staff notation (12th century): characterized by the use of the Guidonian staff with dry-lines, two colored red and yellow for the semitone-steps F and C, letter clefs, and custos at the end of the line to indicate the first pitch on the following staff; the neumes present morphologies with high contrast, comparable to the later square notation.
The analysis of these sources suggests a gradual integration of diastematic indications by the Ravennate scribes between the 11th and 12th centuries.
The fourth chapter focuses on the comparative analysis, identifying affinities with contemporary notations and potential links. The comparisons indicate strong affinities with Bologna and, more specific, the Tuscan area. West-Frankish influences are also identified, along with some elements from Iberian notation, particularly in the resupine and looped forms.
The fifth chapter provides an excursus on the historical-cultural dynamics underlying the development of Ravennate notation between the 11th and 12th centuries. This section describes the roles of the Ravennate archbishops (as Gerbert of Aurillac and Gebeardo), the impact of the Camaldolese reform of Romuald, the relationships between Teodaldo of Canossa and Guido of Arezzo, and the period of the conflicts between Ravenna and Rome
Artificial intelligence use and performance in detecting and predicting healthcare-associated infections: A systematic review
No full textObjectives
The increasing digitisation of healthcare data and the rapid development of Artificial Intelligence (AI) pave the way for innovative strategies for infectious disease management. This study aimed to systematically retrieve and summarize current evidence on the use and performance of AI-based models for healthcare-associated infection (HAI) detection (i.e., identifying infections already present in available data) and prediction (i.e., estimating future risk based on earlier patient information).
Methods
PubMed, Embase, Scopus and Web of Science were searched for experimental and observational studies published between 1 July 2018 and 12 February 2024. Primary outcomes included technical performance metrics for HAI detection and prediction (e.g. recall, precision, AUROC). Any reported clinical, organisational or economic impacts were evaluated as secondary outcomes.
Results
Of 4489 records initially identified, 121 studies were included. Twenty-five studies (20.6 %) focused on HAI detection, with more than half achieving an AUROC above 0.90. In contrast, studies on HAI prediction (n = 93, 76.9 %) reported more heterogeneous performance. Among studies comparing AI with traditional methods (n = 32), AI models outperformed conventional approaches in 81.3 % of cases (n = 26).
Conclusions
A growing body of evidence suggests that AI models are equal to or superior to traditional methods for HAI detection and prediction, but challenges remain in evaluating performance, with many studies lacking comparators, few prospective evaluations, and limited assessment of organisational impact
Molecular signatures across Alzheimer’s disease and Andean populations
No full textThe rapid advancement of omics technologies and ultrasensitive biomarkers-detection platforms has profoundly reshaped research in both medical and evolutionary biology, supporting the concept of molecular signatures as a new strategy for characterizing pathological processes and adaptive traits. This thesis explores different molecular signatures in two separated contexts: the pathophysiology of Alzheimer's disease (AD) and the high-altitude adaptation of Andean populations. Accordingly, the discussion is structured in two main sections.
The first one examines the AD clinical continuum across multiple molecular levels. It begins with a locus-specific methylation analysis of two regions within the APOE 5’UTR, performed on both peripheral blood mononuclear cells (PBMCs) and hippocampal (HIC) samples to identify differentially methylated CpG sites and assess their association with clinical parameters. The following two chapters focus on transcriptomic signatures. The first explores AD-related changes in both mRNA and lncRNA through a whole-transcriptome analysis carried out using a newly developed Compi pipeline. The second examines the small extracellular vesicles (SEVs)-derived miRNome from patients with AD and frontotemporal dementia (FTD). This section ends with a study measuring established AD-related fluid biomarkers in plasma and cerebrospinal fluid (CSF) across the AD continuum, aimed at evaluating their relationships with clinical parameters, disease phenotypes and patients’ profiles.
Conversely, the second section addresses the identification of epigenetic and molecular signatures underlying high-altitude adaptation in two Andean groups. Specifically, it presents a comparative analysis of the whole methylome of the Kichwa, who inhabit high-altitude regions of the Ecuadorian Andes, and the Ashaninka, residing at low-altitude areas of the Peruvian Amazon. The aim is to provide insights into altitude-related adaptations and achieve an unprecedented resolution of epigenetic variation within these populations.
Taken together, these studies highlight the importance of molecular signatures to unravel both disease mechanisms and adaptive processes, bridging clinical and evolutionary perspectives