Ecosustainable and phytobased alternative methods for the conservation of biodeteriorated stone materials

Il presente lavoro di tesi propone di sviluppare un metodo finalizzato al trattamento di materiali lapidei biocolonizzati, tramite l’impiego di sostanze chimiche di origine naturale (i.e. fitochimici), caratterizzati da un basso impatto ambientale, una bassa tossicità per l’uomo e che non compromettano lo stato conservativo dei beni culturali. In questa prospettiva, sono state sfruttate le già note proprietà biocide di tre Oli Essenziali (EOs) già largamente studiati e impiegati in diversi ambiti, tra cui quello della conservazione dei beni culturali. Tali sostanze sono ottenute a partire da piante molto comuni della flora mediterranea, ed in particolare si tratta degli oli di Origanum vulgare, Thymus vulgaris e Clinopodium nepeta. Ogni olio essenziale ha una composizione chimica molto eterogenea, sebbene siano sempre presenti uno o due componenti chimici maggioritari, anche definiti Principi Attivi (APs), che ne definiscono il chemiotipo e sembrerebbero condizionarne l’azione biocida, sebbene non sia ancora stato sistematicamente dimostrato. In virtù di quanto detto, l’effetto biocida dei tre oli essenziali è stato confrontato con quello dei loro rispettivi principi attivi ovvero: carvacrolo per O. vulgare, timolo per T. vulgaris e pulegone per C. nepeta. Per valutarne un possibile incremento dell’azione biocida, ogni olio è stato applicato singolarmente e in combinazione con gli altri due, in modo da creare diverse miscele contenenti rispettivamente, uno, due e tre composti, e lo stesso è stato fatto per i principi attivi. L’applicazione sulle superfici lapidee biocolonizzate ha previsto l’incapsulamento delle sostanze attive all’interno di una matrice idrogel (HG) di nuova formulazione, composta dalla miscela di sostanze polimeriche e tensioattivi, i cui più grandi vantaggi sono legati all’atossicità nella composizione chimica e alla capacità di formare un film omogeneo, pelabile, facile da rimuovere dalle superfici una volta asciutto. I composti sono stati testati su diversi biofilm presenti su substrati lapidei in granito e travertino. In particolare, le sperimentazioni hanno previsto l’applicazione su campioni di granito (in laboratorio) e su due superfici murarie, una in granito e una in travertino, esposte a diverse condizioni ambientali. Per la valutazione dell’azione biocida e pulente dei composti fitochimici, sono state effettuate misure non invasive di colore e di fluorescenza che potranno essere replicate anche sui beni culturali. Il sistema ottenuto dalla combinazione dell’idrogel e dei fitochimici (phyto-HG) ha mostrato buoni risultati per quanto riguarda l’azione pulente e biocida nei confronti di differenti biofilm, anche rispetto a quelle dimostrate da un sistema classicamente utilizzato per la pulitura meccanica dei beni culturali (spazzolatura) e di un comune biocida (Preventol® RI80). Le prove sperimentali hanno messo in evidenza l’efficacia dei principi attivi i quali, possono essere considerati, nella maggior parte dei casi, più efficienti rispetto ai corrispondenti oli essenziali. Questo risultato è molto incoraggiante per le future sperimentazioni riguardanti lo sviluppo del metodo, che potrebbe prevedere l’impiego dei soli principi attivi per la realizzazione delle formulazioni biocide, con i notevoli vantaggi legati ai minori costi di produzione e alla possibilità di ridurre i limiti legati all’impossibilità di controllare le composizioni chimiche degli estratti naturali. L’azione ottenuta dalla combinazione di più sostanze necessita ulteriori approfondimenti, in quanto sembrerebbe che la loro azione sia condizionata dalla sinergia che si ottiene da più sostanze nei confronti di specifici microorganismi presenti. Il monitoraggio a medio e a breve termine effettuato sui campioni sperimentali ha evidenziato un’azione prolungata delle sostanze nel tempo. Sebbene questo non possa essere considerato sempre vantaggioso, in quanto ci si potrebbero aspettare delle reazioni secondarie che potrebbero verificarsi tra i residui e la matrice lapidea, è importante comunque sottolineare i vantaggi legati all’inibizione a lungo termine nei confronti di una possibile ricolonizzazione della superficie. Per queste ragioni, risulta importante analizzare più approfonditamente la stabilità dei fitochimici nel tempo, tramite l’osservazione di possibili modifiche delle proprietà chimiche ed estetiche dei materiali.The present PhD thesis aims to develop a valid strategy for the treatment of the biocolonization of stone materials, by employing natural chemical substances (i.e. phytochemicals) harmless towards ecosystems, human health, and cultural heritage materials. To achieve this goal, the well know biological properties of the Essential Oils (EOs) have been exploited, by employing three of these plant extracts that have already demonstrated inhibitory effects towards different microorganisms. These oils are obtained from Mediterranean plants of the Lamiaceae family, in particular: Origanum vulgare, Thymus vulgaris and Clinopodium nepeta. Each EO is characterized by the presence of one or two mayor chemical components, also defined Active Principles (APs). It seems that the biocidal action of each EO is established by the presence of specific APs, although this was not systematically proven. To demonstrate what has just been said, in this study, the effect of the EOs has been compared to the one of the respective APs, which are carvacrol for O. vulgare, thymol for T. vulgaris and pulegone for C. nepeta. Each EO has been applied alone and combined with the two others, in mixtures containing two and three EOs. The same process occurred with the APs. This was done to assess a possible empowerment of the biocidal action when more substances are combined. For their application on biodegraded stone surfaces, the phytochemicals were incorporated inside an innovative hydrogel (HG) matrix based on a mixture of polymeric substances and surfactants; whose main advantage is linked to its ability of forming a peelable layer when applied on surfaces. The compounds were tested on biofilms, colonizing granite and travertine surfaces. The experimentations were performed on granite samples and on two biocolonized walls, one in granite and the other in travertine. The biocidal and cleaning properties of the systems composed by the phytochemical substances and the hydrogel have been assessed through non-invasive colorimetric and fluorescence measurements. The phyto-HG systems demonstrated good cleaning and large spectrum biocidal properties against different biofilms, also compared to the ones of a commonly employed mechanical cleaning tool (brushing) and a common biocide (Preventol ® RI80). The APs also showed their broad-spectrum biocidal action, which can be considered, in many cases, higher than the one of the respective EOs. This is an encouraging result, in sight of possible future developments of the method, that will provide the employment of the only APs for the creation of the formulations, with notable advantages linked to the reduction of the production costs and the possibility of reducing the limits linked to the impossibility of controlling the chemical composition of the natural extracts (i.e. the EOs). The action of the combined substances compared to biofilms needs to be further analysed, because it seems that their action is strongly linked to the synergism occurring between two substances and the specific microorganisms present. The short-term and medium-term monitoring carried out on the experimental samples evidenced a prolonged action of the substances in time. Even though this is not always encouraged, since possible secondary reactions may occur between the chemical residuals and the mineralogical matrix, it is important to stress the advantage related to the long-term inhibition towards a secondary biological growth on the surfaces. For these reasons, it will be important to further analyse the stability of the substances in time, by observing possible modifications in the properties of the stones during the time

LTA4H expression in canine oral melanomas: methodological set up and preliminary results

Leukotriene A4 hydrolase (LTA4H) is a hydrolytic enzyme which converts leukotriene A4 intoleukotriene B4 inside the arachidonic acid cascade. Besides playing a well-known role in inflammation,it has also been investigated for possible implications in different types of tumors, including canine uvealmelanoma (Chen et al., 2004; Malho et al., 2013). In the present study, we set up RT-PCR andimmunohistochemical protocols to investigate the expression of LTA4H gene and protein, respectively,in formalin fixed paraffin embedded (FFPE) specimens of canine melanomas. 16 samples of canine oralmelanomas were histopathologically evaluated and divided in two subgroups based on morphologicalcriteria of malignancy. RT-PCR protocols for the target gene LTA4H were set up on frozen and FFPEsamples of the same tumor. Immunohistochemical investigation of LTA4H protein was performed witha mouse monoclonal antibody anti-LTA4H (clone 1E9). Preliminary tests were carried out to define theprotocol: with and without bleaching, with different unmaskings, with different serial dilutions of theprimary antibody. RT-PCR set up resulted in comparable good efficiency on both frozen and FFPEsamples. Final immunohistochemical protocol included: hydrogen peroxide bleaching, water bathunmasking and 1:100 dilution of the primary antibody. Positive immunostaining for LTA4H was presentin neoplastic cells of all melanoma samples, with variable localization and intensity. These preliminaryresults encourage future applications of the studied techniques to quantify differential expression ofLTA4H in canine melanomas both at molecular and histological level. Laboratory results will becompared with follow up data, in order to verify if LTA4H can be proposed as a valuable prognosticmarker

Evaluation of the cleaning effect of natural-based biocides. Application on different phototropic biofilms colonizing the same granite wall

Natural derivatives, such as essential oils, are presented as an alternative to classical biocides to the treatment of biocolonization. Thus, in this work, the cleaning and biocidal potential of some natural derivatives towards two natural biofilms' growth on the same granite wall, with different microbial composition, was evaluated. For this purpose, three essential oils (EOs) (from Origanum vulgare, Thymus vulgaris and Calamintha nepeta) and their main active principles (APs) (carvacrol, thymol and R-(+)-pulegone, respectively) were embedded in a hydrogel matrix, with different combinations of EOs and APs, in order to evaluate the synergistic action of different actives. For comparative purposes, pure hydrogel and a mechanical method (brushing) were also used. Colorimetric measurements and chlorophyll a fluorescence analyses were performed to evaluate the cleaning action of the treatments on the biofilms. Overall, the EOs and APs present in the hydrogel proved to be reliable treatments to limit natural biocolonization, with O. vulgare being one of the most effective treatments in combination with other compounds, due to the majority presence of carvacrol. Moreover, the effect of the different treatments strictly depended on the biofilm in question, as well as its ability to adhere to the substrate

Phytochemical Compounds as Cleaning Agents on Granite Colonized by Phototrophic Subaerial Biofilms

The society has become increasingly interested in using natural products over chemicals for cleaning activities. In this study, the cleaning potential of formulations embedded in a hydrogel matrix and composed respectively of essential oils (EOs) of Origanum vulgare, Thymus vulgaris, and Calamintha nepeta, and their respective main active components (EO-ACs), viz., Carvacrol, Thymol, and Pulegone, on a phototropic biofilm growing on granite was investigated. In addition, and for comparative purposes, analysis with the combination of the three EOs, the combination of the three EO-ACs, and Preventol RI-80® (one of the most effective commercial cleaning agents based on quaternary ammonium salts) in all three cases embedded in a hydrogel matrix, as well as only the hydrogel matrix, distilled water, and Preventol RI-80®, in both latter cases applied with brush, were also studied. The cleaning effect of the treatments was assessed immediately after the treatment and after one and two weeks by color spectrophotometry, a reliable tool to evaluate the presence and vitality of the phototrophs and the cleaning effectiveness in granite. C. nepeta and its active component Pulegone proved to be the most effective and yielded similar results, comparable to those of uncolonized granite, and better than those obtained with Preventol RI-80® applied with brush (most common way), especially at the end of the experiment. These promising first results support the suitable use of the phytochemical compounds used on phototrophs field where there are still few published studies and encourage further investigation toward the evaluation of their exhibited biocidal activityThis study was partly funded by projects ED431C 2018/32 and CGL2016-79778-R AEI/FEDER, UE. E. Fuentes is supported by a PhD Fellowship-Contract MICINN-FPI (BES-2017-079927)S

Spatial abilities and the gender gap in mathematics (Matabì)

Learning mathematics is a general problem in Italy, but it is more relevant for females. Male students typically outperform their female classmates in maths test scores from the earliest years of schooling, and the gap worsens as school grades progress. The different experiences and expectations about potential abilities of boys and girls are key to understanding the possible causes of the observed relative differences in education performance. Early life experiences are essential for developing a child’s cognitive capacities, and environmentally induced gender differences may arise when children are exposed to heterogeneous sources of development opportunities. One of the sources of gender inequalities in mathematics is related to differences in the acquisition of visuospatial abilities between girls and boys from a very young age. Existing works find that playing with specific toys facilitates learning in maths and science. Boys usually gain more experience than girls because of different parental and educators’ beliefs and behaviour regarding the gender-specific suitability of toys. Moreover, self-confidence and anxiety are not gender-neutral and can affect educational outcomes. The Matabì project aims to enhance spatial abilities and reduce the gender gap via construction play (i.e., by using building toys and Lego Duplo brick sets). Using the bricks should help students process abstract concepts, while the playful approach should reduce maths anxiety. Girls should benefit more from Matabì than males, who are (on average) able to develop these skills early on in life. The project started in October 2022, so we would present the overall structure and what has been accomplished so far. This should be engaging in three respects: the teachers' reinforcement of their spatial abilities, the instruments used to collect the outcomes, and the impact evaluation design conducted with Randomized Control Trial (RCT). Teachers’ training includes a pre- and post-standardised test, the mental rotation section of the Revised Purdue Spatial Visualization Test. The RCT involves around 60 classes in five schools in Torino (60 teachers and 1200 third and fourth grade pupils). Within each school, we randomly assigned a third of the teachers to the control group and two-thirds to the treatment group. The study will provide evidence of the effects of teaching methods exploiting construction play. The primary research questions are: 1- What is the impact on the spatial abilities of teachers? 2- What is the impact on the spatial abilities of pupils? 3- How does the impact differ by gender? 4- How does the treatment affect the gender gap in mathematics

Corylus avellana L. Aroma Blueprint: Potent Odorants Signatures in the Volatilome of High Quality Hazelnuts

The volatilome of hazelnuts (Corylus avellana L.) encrypts information about phenotype expression as a function of cultivar/origin, post-harvest practices, and their impact on primary metabolome, storage conditions and shelf-life, spoilage, and quality deterioration. Moreover, within the bulk of detectable volatiles, just a few of them play a key role in defining distinctive aroma (i.e., aroma blueprint) and conferring characteristic hedonic profile. In particular, in raw hazelnuts, key-odorants as defined by sensomics are: 2,3-diethyl-5-methylpyrazine (musty and nutty); 2-acetyl-1,4,5,6-tetrahydropyridine (caramel); 2-acetyl-1-pyrroline (popcorn-like); 2-acetyl-3,4,5,6-tetrahydropyridine (roasted, caramel); 3-(methylthio)-propanal (cooked potato); 3-(methylthio)propionaldehyde (musty, earthy); 3,7-dimethylocta-1,6-dien-3-ol/linalool (citrus, floral); 3-methyl-4-heptanone (fruity, nutty); and 5-methyl-(E)-2-hepten-4-one (nutty, fruity). Dry-roasting on hazelnut kernels triggers the formation of additional potent odorants, likely contributing to the pleasant aroma of roasted nuts. Whiting the newly formed aromas, 2,3-pentanedione (buttery); 2-propionyl-1-pyrroline (popcorn-like); 3-methylbutanal; (malty); 4-hydroxy-2,5-dimethyl-3(2H)-furanone (caramel); dimethyl trisulfide (sulfurous, cabbage) are worthy to be mentioned. The review focuses on high-quality hazelnuts adopted as premium primary material by the confectionery industry. Information on primary and secondary/specialized metabolites distribution introduces more specialized sections focused on volatilome chemical dimensions and their correlation to cultivar/origin, post-harvest practices and storage, and spoilage phenomena. Sensory-driven studies, based on sensomic principles, provide insights on the aroma blueprint of raw and roasted hazelnuts while robust correlations between non-volatile precursors and key-aroma compounds pose solid foundations to the conceptualization of aroma potential

Endothelial Cells Promote Osteogenesis by Establishing a Functional and Metabolic Coupling With Human Mesenchymal Stem Cells

Bone formation involves a complex crosstalk between endothelial cells (EC) and osteodifferentiating stem cells. This functional interplay is greatly mediated by the paracrine and autocrine action of soluble factors released at the vasculature-bone interface. This study elucidates the molecular and functional responses triggered by this intimate interaction. In this study, we showed that human dermal microvascular endothelial cells (HMEC) induced the expression of pro-angiogenic factors in stem cells from human exfoliated deciduous teeth (SHED) and sustain their osteo-differentiation at the same time. In contrast, osteodifferentiating SHED increased EC recruitment and promoted the formation of complex vascular networks. Moreover, HMEC enhanced anaerobic glycolysis in proliferating SHED without compromising their ability to undergo the oxidative metabolic shift required for adequate osteo-differentiation. Taken together, these findings provide novel insights into the molecular mechanism underlying the synergistic cooperation between EC and stem cells during bone tissue renewal