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Identification and validation of salivary biomarkers for systemic and oral diseases
No full textAn established target of medicine and modern healthcare is early diagnosis of diseases and the evaluation of patient risk profile. Detect a disease at an early stage and modulate the risk factors may improve the success of treatment, prevent complications and enhance prognosis and quality of life of patients.
The possibility to obtain an accurate diagnosis and effective screenings through reliable and non-invasive tools, seems to be a very attractive.
Within such background, the concept of “precision medicine” is rapidly developing. It usually employs molecular profiles to adapt a therapeutic strategy to a patient, with specific stage of disease and/or to determine the predisposition for. “Precision medicine” is based on biomolecular disciplines usually indicated with the suffix “omics”. It encompasses genomic, epigenomic, transcriptomic, proteomic, metabolomic, and others.
The combination of the above-mentioned concepts is leading to the development of novel technologies, based on easy and non-invasive methods to collect diagnostic human specimens, possibly with a high specificity and sensitivity and customized for specific patient.
One of the human fluids which has gained importance within this context is saliva.
The aim of the present thesis is to present translational data about salivary biomarkers and their potential application in clinical setting.
An update of the current knowledge about salivary diagnostics is provided in Chapter 1. Latest
literature confirms the fluid high potential as a matrix for multipurpose analysis with several different technological platforms; the number of recently published studies confirms the increasing interest in saliva. However, as routinely reliable diagnostic tools, salivary diagnostics has still little spreading.
Chapter 2 reports preliminary data of a pilot study of nutriepigenomic, which aims to highlight the role of nutrition in epigenetic variations (induced and inducible), as cause of cancer risk increasing or decreasing. The study evaluated the effect of a nutritional and lifestyle program [Nutritional and Lifestyle Intervention (NLI)] on salivary miRNAs expression in women carriers of BRCA1/2 germ mutations. Preliminary statistically results seem to demonstrate that 5 salivary miRNAs have a statistical difference between their expression before nutritional intervention and after 18 months.
The progression from normal mucosa to different grades of dysplasia is explored in relation to salivary metabolome in an experimental case-control study (Chapter 3). Metabolomics was performed on saliva samples using high resolution 1H-NMR (Nuclear Magnetic Resonance). A cohort of voluntary patients with histologically confirmed oral leukoplakia and oral lichen planus was enrolled and salivary specimens were collected before surgical procedures (incisional/excisional biopsies). Preliminary results are focused on the differences between metabolomes of patients with dysplastic and non-dysplastic leukoplakia as well as healthy controls.
Chapter 4 reports data about the possible relationships between the salivary and serum metabolomes to gain a comprehensive view of the metabolic phenotype under physiological conditions. Using 1H-NMR spectroscopy, we obtained the serum metabolite profiles of 20 healthy young individuals and compared them with the metabolomes of parotid, submandibular/sublingual, and whole saliva samples collected concurrently from the same individuals. The salivary gland-specific metabolic composition and the effective saliva collection protocol is reported in the Annex n° 1. During the samples collection, the Full-Mouth Bleeding Score (FMBS) was employed as a classification parameter. We identified a panel of metabolites differentially expressed in healthy subjects with high but physiological FMBS scores compared to those with lower score, and this set of metabolites may be associated with early stages of gingival inflammation. This correlation between salivary metabolites and gingival bleeding score is described in Chapter 5. The salivary fluid has been widely studied and genomic, transcriptomic, and proteomic profiles have been reported. Conversely, its metabolic composition is still mainly unknown: metabolites in submandibular/sublingual saliva have never been analyzed systematically. These results could provide basis to a further identification of salivary biomarkers in oral and systemic diseases.
Results emerging from 2 systematic reviews are presented in chapter 6 and 7.
The first was developed to answer the question: “Are salivary biomarkers reliable for diagnosis of dental caries?” (Chapter 6), while the second aims to identify the salivary metabolites for the diagnosis of oral cancer (Chapter 7).
For both reviews, results and quality assessment of selected studies are presented, highlighting the potential risks of bias in current literature. Moreover, a summary of statistical data and reported evidence are given.
In conclusion, the researches described in this thesis highlight that there are many advantages to use saliva as a diagnostic fluid for monitoring health and diseases
Clinical management and differential diagnoses in the geriatric dogs with suspected idiopathic vestibular syndrome
No full textIn geriatric dogs, most neurological disorders often manifest with dramatic clinical signs. Nonetheless, certain conditions, such as idiopathic vestibular syndrome (IVS), can be effectively managed with symptomatic therapy and are associated with a favorable prognosis. Idiopathic vestibular syndrome is a neurological disorder frequently observed in geriatric dogs. Its etiology and pathogenesis remain unknown. The condition is believed to involve the peripheral components of the vestibular system and is typically characterized by an acute onset of clinical signs, followed by a rapid and often spontaneous improvement, with resolution generally occurring within a few weeks of onset. Despite this, residual clinical signs such as ataxia, head tilt, or facial hemiparesis may persist long-term without significantly compromising the patient’s quality of life. A meticulous diagnostic approach is essential, as IVS is a diagnosis of exclusion. Relying only on medical history and neurological examination findings may lead to a diagnostic risk, particularly given that geriatric patients are predisposed to other neurological conditions presenting with similar clinical features. The main objective in case of suspected IVS is to rule out peripheral vestibular disorders, like otitis media/interna. It is even more important to exclude central vestibular diseases, like intracranial neoplasms, meningoencephalitis, that may lead to a more unfavorable prognosis. Advanced imaging modalities, such as magnetic resonance imaging (MRI) or computed tomography (CT), are considered the gold standard in achieving a definitive diagnosis. However, due to the older age of the patients, some owners may decline advanced diagnostic procedures. In such cases, clinicians must rely on the inte
gration of alternative diagnostic tools to establish the most likely presumptive diagnosis and an appropriate therapeutic management. Follow-up evaluations are essential to reevaluate the initial diagnosis, particularly in patients where advanced imaging and other medical tests could not be performed. Constant monitoring also helps to identify any clinical evolution suggestive of an alternative or progressive underlying pathology among the differential diagnoses initially considered
Riconoscimento di metaboliti batterici, batteri Gram-negativi e della loro parete cellulare tramite recettori calix[4]arenici
No full textThis thesis deals with the study of calix[4]arenes receptors, in particular their synthesis and first interaction studies with bacterial metabolites, Gram-negative bacteria and their artificial models of cell wall.
The first chapter of this thesis is a general introduction of the topic of bacteria recognition and how can be made with supramolecular receptors, such as calix[4]arenes, or other multivalent ligands. Among the techniques that can be used to study this interaction, a focus is set on the quartz crystal microbalance, a mechanical system that is able to assess the presence of an interaction by detecting a variation in the mass that is set on the sensor surface.
Chapter 2 is about the interaction of two negatively charged calix[4]arenes with four biogenic polyamines (1,4-diamino butane, 1,6-diamino hexane, spermidine and spermine), and the study of this interaction has been carried out via 1H NMR titrations, resulting in a complex system with multiple equilibria occurring. To clarify the system fluorescence titrations were performed, consenting to determine the association constants of the complexes formed.
The third chapter is about the synthesis of derivatives of two calix[4]arenes already studied in this research group, bearing four methyl-L-proline and four methyl-L-phenylalanine at the upper rim respectively. These receptors have proven to be selective ligands for Gram-negative bacteria, opening the possibility to be exploited for multiple applications. Two derivatives that have been synthetized possess alkyl chains at the lower rim, that will consent to study if the ligand properties of the macrocycle changed upon alkylation of the phenolic groups. The other two derivatives possess alkyl chains that terminate with an azide, allowing the attachment of the calixarene to the surface. The synthetic strategy employed has not led to the products, and for time reasons I was not able to find a suitable strategy. Moreover, two models of the calixarenes with a monovalent amino acid moiety have been synthetized, with an azide on the other end to allow the linkage to the surface and study another type of multivalent surface.
Chapter 4 concerns about the work carried out in a six-months research period in University of Twente, the Netherlands, where quartz crystal microbalance was used to study the multivalent interactions between zwitterionic surfaces and Gram-negative bacterial membrane models. In the first model the supported lipid bilayers were used as models for the Gram-negative bacteria cell wall and on the surface solutions of methyl-L-proline and methyl-L-phenylalanine calix[4]arenes were flowed: the interaction was not detected with this system. Given that only the two monovalent calixarene models were successfully obtained, these were the sole molecules immobilized on a surface in the second approach. By subsequently introducing a vesicle solution over the modified surface, the interaction between the vesicles and the calixarene-functionalized surface was examined.
Chapter 5 is a preliminary study on a new project, the SAMBA project, where the aim is to obtain nano-objects made of polymers with different shapes, bearing units of calix[4]arenes with properties of Gram-negative bacteria ligands. This work represents a preliminary investigation into the feasibility of conducting RAFT polymerization by anchoring the RAFT agent to the lower rim of calix[4]arene. After synthesizing calixarenes functionalized with between one and four RAFT agent units, polymerization was carried out using the calixarene with two RAFT agents, yielding the polymers subsequently analyzed
Myocardial injury in patients with septic shock echocardiographic and laboratory evaluation of troponins within the first 24 hours and after one week in patients with septic shock
No full textSeptic shock is a severe, potentially life-threatening clinical condition characterized by an excessive and dysregulated systemic inflammatory response to infection, leading to hypoperfusion, organ dysfunction, and hemodynamic instability. Its management represents a complex challenge in emergency medicine, both in human and veterinary practice.
Over the years, sepsis has been the subject of numerous studies and updates aimed at improving early recognition and therapeutic strategies. In this regard, in 2021 the Surviving Sepsis Campaign (SSC) published new guidelines for the treatment of sepsis in adult human patients, with the objective of improving prognosis and reducing mortality through a timely and standardized diagnostic and therapeutic approach.
In veterinary medicine, sepsis and septic shock are relatively frequent conditions associated with high morbidity and mortality, particularly in dogs and cats. The causes are multiple: among the most common are infectious diseases, such as parvovirosis, as well as pathologies secondary to severe infectious processes, including pyometra, pyelonephritis, pneumonia, and septic peritonitis due to gastrointestinal perforations.
The heterogeneity of clinical presentations and the rapid progression of the disease require veterinarians to adopt a timely, aggressive, and highly targeted diagnostic and therapeutic approach, despite the absence of species-specific guidelines. In clinical practice, this implies the need to adapt recommendations developed for human medicine to canine and feline patients, while facing several challenges, such as drug selection and dosage, the applicability of intensive monitoring techniques, and the economic sustainability of care.
Among the target organs of septic shock, the heart plays a pivotal role. Myocardial injury is widely described in human medicine as a transient myocardial dysfunction. In veterinary medicine, however, myocardial injury or dysfunction is a recognized complication, but one that remains poorly investigated. Available literature is limited, and most of the current knowledge, both in terms of diagnosis and therapeutic management, derives from the translation of human data.
In this context, the use of specific biomarkers, such as cardiac troponins, and echocardiography plays a fundamental role in the assessment of myocardial injury, allowing evaluation of ventricular function and contractile performance. The integration of laboratory and echocardiographic data provides a valuable tool to better characterize myocardial damage, monitor its temporal evolution, and assess its prognostic impact on clinical outcomes.
The main objective of this thesis is to provide an updated review of the literature regarding myocardial injury associated with septic shock, with particular attention to its relevance in veterinary medicine. This is complemented by a case series analysing patients with septic shock, evaluated through echocardiography and cardiac troponin measurements within the first 24 hours and, when possible, after one week.Lo shock settico è una condizione clinica grave, potenzialmente letale, caratterizzata da
una risposta infiammatoria sistemica eccessiva e disfunzionale ad un9infezione, che porta
ad ipoperfusione, disfunzione d9organo e instabilità emodinamica. La sua gestione
rappresenta una sfida complessa nella medicina d9urgenza, sia in ambito umano che
veterinario.
Nel corso degli anni, la sepsi è stato oggetto di numerosi studi e aggiornamenti, con
l9obiettivo di migliorare il riconoscimento precoce e l9approccio terapeutico. A questo
proposito, nel 2021, la Surviving Sepsis Campaign (SSC) ha pubblicato nuove linee guida per
il trattamento della sepsi negli adulti in medicina umana, con l9obiettivo di migliorare la
prognosi e ridurre la mortalità attraverso un approccio diagnostico e terapeutico tempestivo
e standardizzato.
In medicina veterinaria la sepsi e lo shock settico rappresentano condizioni relativamente
frequenti e gravate da un elevato tasso di morbilità e mortalità, soprattutto nel cane e nel
gatto. Le cause possono essere molteplici: tra le più comuni si annoverano infezioni, come
la parvovirosi, ma anche patologie secondarie a processi infettivi gravi, come piometra,
pielonefrite, polmonite e peritonite settica da perforazioni gastrointestinali.
L9eterogeneità dei quadri clinici e la rapidità di progressione della patologia, impongono al
medico veterinario un approccio diagnostico-terapeutico tempestivo, aggressivo e altamente mirato, nonostante l9assenza di linee guida specifiche in medicina veterinaria.
Nella pratica clinica, questo comporta la necessità di adattare le raccomandazioni date in
medicina umana, al cane e al gatto, affrontando numerose difficoltà, tra cui la scelta dei
farmaci e i relativi dosaggi, l9applicabilità delle tecniche di monitoraggio intensivo e la
sostenibilità economica delle cure.
Tra gli organi bersaglio dello shock settico, il cuore riveste un ruolo di primaria importanza.
In medicina veterinaria il danno miocardico o la disfunzione miocardica è una complicanza
riconosciuta, ma ancora poco approfondita, a differenza di quello che avviene in ambito
umano. La letteratura disponibile infatti risulta essere limitata e la maggior parte delle
conoscenze, sia per quanto riguarda la diagnosi sia per quanto riguarda l9approccio
terapeutico, derivano dalla traslazione dei dati presenti in medicina umana. In questo contesto, un ruolo fondamentale nella valutazione del danno miocardico è dato
dall9impiego di biomarcatori specifici, come le troponine cardiache, e dall9esame
ecocardiografico, che permette di studiare la funzione ventricolare e la performance
contrattile. L9integrazione dei dati laboratoristici ed ecocardiografici rappresenta uno
strumento utile per caratterizzare meglio il danno, monitorare l9evoluzione temporale e
valutare l9impatto prognostico sul decorso clinico dei pazienti.
L9obiettivo principale di questo elaborato è offrire una revisione aggiornata della letteratura
relativa al danno miocardico associato a shock settico, con particolare attenzione alla sua
rilevanza in medicina veterinaria, integrata da una Case Series in cui sono stati analizzati
pazienti con shock settico, sottoposti a valutazione ecocardiografica e al dosaggio delle
troponine cardiache entro le prime 24 ore dalla diagnosi e, quando possibile, a distanza di
una settimana
Correlation between haematological markers and graphic test indices with the PMOH method : a retrospective study of 20 clinical cases
No full textIl PANS (Sindrome Neuropsichiatrica Pediatrica ad Esordio Acuto) è una sindrome neuropsichiatrica
ad insorgenza acuta, in cui, nella maggior parte dei casi, è presente un’infezione da parte di un agente
esterno, quali virus, batteri, ecc. Nel paziente affetto da tale sindrome insorgono una serie di sintomi
neuropsichiatrici, che affliggono sia la componente fisiologica che quella psicologica. Il presente studio prende in considerazioni questi due aspetti della PANS, andando ad analizzare, su un campione di pazienti, marker ematologici ed indici psicodiagnostici. L’obiettivo di questa analisi è quello di indagare una possibile relazione tra gli indici delle analisi del sangue, legati agli aspetti infettivi della malattia, e gli indici del test proiettivo del disegno della figura umana (DFU) È stato riscontrato che, così come alcuni valori ematici risultano alterati in quasi tutto il campione, allo stesso modo alcuni indici del test proiettivo risultano più frequenti nel nostro campione. Inoltre, sono state evidenziate delle correlazioni significative tra alcuni marker ematici e alcuni indici
del test DFU
Untargeted/targeted methods for quality assessment in hazelnuts, cocoa, and finished products of the confectionery industry
No full textThis Ph.D. thesis explores the development and application of targeted and untargeted analytical methods for quality assessment in hazelnuts, cocoa, and finished confectionery products. Using chromatographic techniques such as UHPLC-HRMS and GC-IMS, the research focuses on the analysis of defects in hazelnuts, the discrimination of cocoa origins, and the monitoring of phenolic compound stability during shelf life, within the context of the food industry. These approaches are supported by advanced chemometric tools, enabling the management and reduction of data complexity, providing effective solutions for optimizing business strategies and quality control.
Regarding hazelnuts, a targeted UHPLC-HRMS approach was used to quantify bitter compounds associated with the "cimiciato" defect (such as Carpinontriol-B), providing an alternative to visual analysis for quality control. Additionally, the untargeted analysis identified molecular markers linked to the "rotten" defect, caused by bedbug attack, which were detectable even at very low concentrations (up to 1%).
The thesis also focused on cocoa, aiming to develop effective tools for geographic origin discrimination. Through an untargeted approach, key molecular compounds were identified for geographic discrimination, allowing for a successful correlation of origin with specific sensory attributes using a sensomics approach. Subsequently, the GC-IMS analysis of volatile compounds, integrated with automated chemometric tools developed in Python, proposed a new rapid tool for cocoa origin verification.
Lastly, the thesis addressed the stability of phenolic compounds during the shelf life of sugar-free candies enriched with botanical extracts to ensure the product’s nutritional quality during storage. In particular, the study focused on the persistence of phenolic compounds from grape seed and green tea extracts, using both targeted and untargeted UHPLC-HRMS approaches. The untargeted approach, based on phenolic fingerprinting, proved most effective for monitoring these compounds throughout the storage period
Bioactive compounds in hop leaves: from synthesis factors to practical applications
No full textIn recent years, consumer preference for organic and sustainably produced products has changed significantly. Growing demand has prompted both farmers and industry to rethink and adapt their production methods. This change in consumer behavior is having a profound impact on the way the industry approaches production and is driving innovation in the area of sustainability. Another consequence is that research is also focusing on new strategies for obtaining these biomolecules. In this context, recycling and reuse agri-food waste seems to be the best solution from an economic and ecological point of view.
Based on these considerations, the doctoral project focused on the use of hop plants as a source of biomolecules, in line with the 2030 Green Agenda. The hop plant, known for its cones used in beer brewing, has much more to offer and my research aimed to highlight this untapped potential.
In fact, while the cones are the main product harvested for their economic value in beer production, the rest of the plant, including the leaves and stems, is usually discarded as waste and represents a burden for the farmers. Over the last three years, this research has characterized the hop vegetative biomass in terms of bioactive compounds, in order to individuate several potential applications and increase the sustainability of the hop culture.
The research has been carried out in three research lines: i) Open field - Hop leaves: a source of bio-compounds ii) Hop biomass -from waste to resource; iii) Bioactive compounds from vitro- derived hop plantlets: a green opportunity.
On closer inspection, the first research line focused on the biochemical and molecular characterization of leaves collected from plants of ten hop varieties at the time of cone harvesting; moreover, only for the cv. Cascade, one of the most widely grown hop varieties, the development of the biochemical leaf profile during the growing cycle was investigated. In particular, the bioactivity of the leaf extracts against food-borne and additionally the possible interaction with proteins such as human insulin, amyloid beta peptide, mucin and bovine serum albumin (BSA) was evaluated, indicating their potential to counteract inflammatory processes and protect against Alzheimer’s disease.
Thanks to the collaboration with Packtin srl, a company partner of this research program, that uses innovative techniques to process plant and food waste, hop vegetative biomass was dried and pulverized to obtain a powder that was characterized and employed in different application fields. Specifically, after a biochemical characterization, hop biomass powder was used, in collaboration with University of Palermo, as ingredient in bakery product and, in collaboration with University of Ferrara, its extracts were evaluated for crop protection. Hop biomass powder confirmed its high content bioactive compounds; in detail, when added to bread, hop biomass powder improved its organoleptical and nutritional quality, extending, also, its shelf life. Extracts obtained with different solvents were tested in vitro and in vivo against the bacterium Xanthomonas campestris pv. campestris and showed promising antibacterial activity. The results demonstrated the effective antimicrobial activity of these matrices. In addition, the high efficacy, environmental compatibility and associated sustainability as well as the low cost and low toxicity make these extracts an ideal means of controlling black rot in cruciferous crops. The possibility of converting hop waste into valuable by-products opens up new opportunities for the industry and offers environmentally friendly alternatives that can reduce the amount of waste and increase the sustainability of production processes.
To further explore the potential of hop vegetative biomass, in vitro culture techniques have been applied to work independently from the seasonal constraints that normally affect hop production in the field and, moreover, guarantees a continuous and standardized supply of biomass to stakeholders.
Hop plant (cvs. Cascade, Columbus, Gianni and Magnum) in vitro multiplication was carried out at the University of Parma to obtain the vegetative biomass for further characterization. Specifically, biomass obtained in vitro from all four varieties was analysed in terms of biochemical and molecular properties, which also provides information on the potential of in vitro cultivated hop plants as a source of bioactive compounds.
In another study, four different cytokinins added to the culture medium were tested for the cvs. Cascade and Columbus at the Catholic University of Valencia to increase the production of biomolecules. The results of the growth parameters and biochemical tests showed a strong influence of the genotype on the response of the plant. Specifically, in cv. Columbus, the addition of cytokinins reduced plant growth, but triggered the synthesis of (poly)phenols and molecules with antioxidant activity.
In the last two studies, carried out in collaboration with the University of Palermo, the effects on growth parameters and the stimulation of secondary metabolism were investigated by modifying the culture system and the culture medium composition. In particular, the use of the temporary immersion system (TIS), in comparison with culture on solid medium, with or without sucrose, was tested on the cv. Cascade; while on the cv. Columbus only TIS was carried out evaluating different immersion times and the influence of sucrose in the culture medium.
In conclusion, research conducted during this PhD has demonstrated that hop plants are a valuable resource that goes far beyond their traditional use in brewing. The leaves, stems and even the vitro derived materials offer promising applications in a wide range of industrial applications, contributing to sustainability and reducing waste
Sistemi supramolecolari per nuove applicazioni: dai coloranti incapsulati in macrocicli calix[6]arenici agli studi elettrochimici di strutture interbloccate
No full textThe advancement of modern molecular nanotechnology presents increasing complexities and demands. Deep understanding and precise manipulation of properties at the molecular level are critical to designing materials with desired chemical and physical properties. This thesis introduces a novel supramolecular approach aimed at fabricating novel plasmonic nanostructures hybridized with organic dyes based on cyanines, styryl cyanines, and thiazo[5,4-d]thiazoles. The optical and electrochemical properties of these dyes can be modulated through their geometrically oriented inclusion in asymmetric calix[6]arene-based receptors and molecular cages derived from them.L'avanzamento delle moderne nanotecnologie molecolari presenta complessità e richieste crescenti. La comprensione profonda e la manipolazione precisa delle proprietà a livello molecolare sono fondamentali per progettare materiali con le caratteristiche chimiche e fisiche desiderate. Questa tesi introduce un nuovo approccio supramolecolare finalizzato alla realizzazione di nuove nanostrutture plasmoniche ibridate con coloranti organici a base di cianine, cianine stiriliche e tiazo[5,4-d]tiazoli. Le proprietà ottiche ed elettrochimiche di questi coloranti possono essere modulate attraverso la loro inclusione, geometricamente orientata, in recettori calix[6]areni asimmetrici e in gabbie molecolari da quest'ultimi derivate
I meccanismi molecolari alla base della Malattia di Darier: dai geni alle proteine
No full textDarier Disease (DD) (OMIM #124200) is a rare autosomal dominant genodermatosis affecting around 1 in 50,000 people. From a histopathological point of view, DD is characterized by loss of intercellular adhesion (acantholysis), premature and abnormal keratinization (dyskeratosis) and presence of rounded keratinocytes (corps ronds). Skin lesions typically appear in patients between the age of 6 and 20 years, predominantly in body areas more exposed to UV irradiation, heat and friction. DD is primarily attributed to missense, non-sense, deletion, insertion and altered splicing mutations within the ATP2A2 gene (12q23-24.1) encoding the Sarco/Endoplasmic Reticulum Calcium ATPase type 2 (SERCA2), a ubiquitously expressed cellular pump responsible for the translocation of Ca2+ from the cytosol to the Endoplasmic Reticulum. Altered SERCA2 function impairs the intracellular calcium homeostasis leading to ER stress response and cell apoptosis. The perturbation of calcium levels could be responsible for desmosomes assembly alteration, consequently impairing cell-to-cell adhesion. However, the molecular mechanism by which SERCA2 mutations cause disease abnormalities is not well clarified.
Considering the SERCA2 role in the modulation of the NOTCH1 pathway in several disease models, our hypothesis posits that SERCA2 loss-of-function variants could inactivate NOTCH1 activity and its signaling. The project's aims include the definition of the role of SERCA2 mutations on the NOTCH1 and other molecular pathways and the transcriptional effects of SERCA2 inhibition.
By combining in vitro and in vivo assays we identified novel signaling pathways deregulations. In particular, upregulated ones included immunological, inflammatory, and cell responses to external stimuli, while genes associated with metabolic functions such as fatty acid metabolism, ribosomal structural components, and mitochondrial activity resulted to be downregulated. Additionally, NOTCH1 signaling target proteins were found to be dysregulated, according to our starting hypothesis.
Our findings provide important insights into the molecular mechanisms underlying Darier disease, which may offer potential targets for therapeutic interventions.La malattia di Darier (OMIM #124200) è una rara genodermatosi autosomica dominante che colpisce circa 1 persona su 50.000. Da un punto di vista istopatologico, la DD è caratterizzata da perdita di adesione intercellulare (acantolisi), cheratinizzazione prematura e anomala (discheratosi) e presenza di cheratinociti tondeggianti (corps ronds). Le lesioni cutanee compaiono tipicamente in pazienti di età compresa tra i 6 e i 20 anni, prevalentemente nelle aree corporee più esposte ai raggi UV, al calore e allo sfregamento. La DD è principalmente attribuita a mutazioni missenso, non-senso, di delezione, di inserzione e di splicing alterato all'interno del gene ATP2A2 (12q23-24.1) che codifica per la Sarco/ATPasi del calcio del reticolo endoplasmatico di tipo 2 (SERCA2), una pompa cellulare ubiquitariamente espressa responsabile della traslocazione degli ioni Ca2+ dal citosol al lume del reticolo endoplasmatico. L'alterata funzione della proteina SERCA2 compromette l'omeostasi intracellulare del calcio portando all’attivazione della risposta allo stress ER e all'apoptosi cellulare. La perturbazione dei livelli di calcio potrebbe essere responsabile dell'alterazione dell'assemblaggio dei desmosomi, compromettendo di conseguenza l'adesione cellula-cellula. Tuttavia, il meccanismo molecolare con cui le mutazioni di SERCA2 causano anomalie della malattia non è ben chiarito. Considerando il ruolo di SERCA2 nella modulazione della via di segnalazione di NOTCH1 in diversi modelli di malattia, la nostra ipotesi postula che le varianti con perdita di funzione di SERCA2 potrebbero compromettere la maturazione NOTCH1 e di conseguenza la trascrizione dei suoi geni target. Gli obiettivi del progetto includono la definizione del ruolo delle mutazioni di SERCA2 sulla via di segnalazione di NOTCH1 e di altre vie molecolari e gli effetti trascrizionali dell'inibizione di SERCA2.
Combinando saggi in vitro ed in vivo abbiamo identificato la deregolazione di diverse vie di segnalazione. In particolare, quelle sovra regolate includono processi immunologici, infiammatori e risposte cellulari a stimoli esterni, mentre i geni associati alle funzioni metaboliche come il metabolismo degli acidi grassi, le componenti strutturali ribosomiali e l'attività mitocondriale risultano sotto regolati. Inoltre, è stato riscontrato che la segnalazione NOTCH1 è deregolata. I nostri risultati forniscono importanti informazioni sui meccanismi molecolari alla base della malattia di Darier, che possono offrire potenziali bersagli per interventi terapeutici
The importance of surgical margin in oncological surgeries
No full textI tumori cutanei sono comuni nel cane, e i più frequentemente riscontrati sono i mastocitomi, i sarcomi e i carcinomi. L’escissione chirurgica rappresenta il trattamento di prima scelta; tuttavia, ottenere un’escissione istologicamente completa può risultare complesso e il suo valore prognostico è ancora oggetto di dibattito.
Questo studio clinico prospettico ha valutato 72 tumori asportati in 61 cani, con analisi istologica dei margini in tutti i casi e dati di sopravvivenza disponibili per 45 soggetti (follow-up ≥ 6 mesi). La popolazione era costituita prevalentemente da cani adulti e anziani, con una distribuzione delle razze che rifletteva sia l’andamento demografico sia le predisposizioni note.
Complessivamente, l’88% dei tumori è stato asportato con margini indenni da neoplasia, mentre il 12% ha presentato margini infiltrati. Una recidiva locale si è verificata in un solo cane (2%), affetto da carcinoma squamocellulare auricolare escisso con margini incompleti. L’analisi di sopravvivenza non ha evidenziato differenze significative tra le escissioni complete e incomplete, suggerendo che fattori quali la biologia del tumore, il ruolo del chirurgo nell’orientamento del campione chirurgico e la tecnica istologica impiegata nella valutazione del pezzo rivestano un’importanza cruciale nell’esito clinico.
I mastocitomi hanno rappresentato il 77% dei casi, erano per lo più di basso grado e di dimensioni inferiori a 3 cm, caratteristiche che hanno facilitato l’escissione e contribuito all’elevata percentuale di margini completi. I sarcomi (15%) erano prevalentemente di basso grado; nonostante alcuni fossero voluminosi, non si sono registrate recidive. I carcinomi hanno rappresentato l’8% dei tumori, con recidiva documentata solo in un carcinoma squamocellulare di grandi dimensioni e infiltrante.
La tomografia computerizzata ha supportato la pianificazione chirurgica in casi selezionati, soprattutto in regioni anatomiche complesse, mentre la tecnica del cross-sectioning è risultata la più utilizzata per la valutazione istologica dei margini. Le metastasi linfonodali, riscontrate nel 62% dei linfonodi asportati, non hanno avuto un impatto significativo sulla sopravvivenza a breve termine.
Questi risultati indicano che, per molti mastocitomi di basso grado e di dimensioni inferiori a 2 cm, un’escissione proporzionale può essere sufficiente, mentre per le neoplasie di alto grado o localizzate in sedi anatomiche difficili risultano più sfidanti. Una pianificazione chirurgica accurata può comunque contribuire a un buon esito oncologico. Sarà necessario uno standard nella valutazione dei margini e un follow-up più prolungato per affinare le linee guida chirurgiche basate sull’evidenza.Cutaneous tumors are common in dogs, with mast cell tumors, sarcomas and carcinomas being the most prevalent. Surgical excision is the primary treatment, yet, the histologically complete excision of the lesion may be difficult to achieve, and its prognostic role is still debated.
This clinical prospective study evaluated 72 tumors excised from 61 dogs, with histological margin assessment performed in all cases and survival data available for 45 dogs (≥ 6 months follow-up). The population was mainly composed of middle-aged to senior dogs, with breed distribution reflecting both demographic trends and known predispositions.
Overall, 88% of tumors were excised with tumor-free margins, while 12% were incomplete. Despite this, local recurrence occurred in only one dog (2%), affected by an auricular squamous cell carcinoma excised with incomplete margins. Survival analysis did not show significant differences between complete and incomplete excisions, suggesting that factors such as tumor biology, role of the surgeon in adequately orient the surgical sample and histological technique in evaluating the specimen may play a key role in the clinical outcome.
Mast cell tumors represented 77% of cases, they were mostly low-grade and smaller than 3 cm, which facilitated wide excision and contributed to the high rate of complete margins. Sarcomas (15%) were predominantly low-grade; despite some being large, no recurrence was recorded. Carcinomas accounted for 8% of tumors, with recurrence documented only in one large, infiltrating squamous cell carcinoma.
Computed tomography supported surgical planning in selected cases, particularly in complex anatomical regions, while cross-sectioning was the most common histological method for margin evaluation. Lymph node metastasis, detected in 62% of excised nodes, did not significantly impact short-term survival.
These findings indicate that proportional excision may be sufficient for many low-grade mast cell tumors smaller than 2 cm, while high-grade and anatomically constrained neoplasms are more challenging. Nonetheless, accurate surgical planning may aid in achieving a good oncologic outcome. Standardized margin assessment and longer-term follow-up are needed to refine evidence-based surgical guidelines