Tesi di Dottorato
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Item Green biomaterials and advanced technologies for road pavements(Università della Calabria, 2021-04-08) Ashimova, Saltanat Zhandarbekovna; Cerra, Maria Carmela; Rossi, Cesare Oliverio; Burkhanbaevich, Teltayev BagdatThe main objective aims at developing methodologies and new biomaterials for the sustainability assessment of “green bituminous mixtures”. “Green bituminous mixtures” is used in the context as a general term for all types of bituminous mixtures in which specific materials or technologies are used with the aim of reducing the environmental impact and of reducing health risks of workers. In the present research new bioadditives were developed and tested to improve the adhesion between bitumen and stones, to modify the rheological characteristic of the bitumen as well as rejuvenating properties of the bitumen.Item Production of new bituminous materials (ecobinders)by recycling waste oil and rubber from end-of-life tire ("Crumb Rubber")(Università della Calabria, 2021) Porto, Michele; Cerra, Maria Carmela; Rossi, CesareNowadays the main problems faced by applied research and industry are towards environmental issues such as global warming, carbon footprint, reduction of greenhouse gas emission amongst others. The Kyoto Treaty was developed as a result in 1997 to setting the objective for European countries to develop policies and technologies in order to meet greenhouse gas reduction requirements. In this light, the asphalt industry accordingly implemented measures to facilitate eco-friendly operations. Examples of some of these measures include the introduction of the use of Warm Mix Asphalt (WMA) and Reclaimed Asphalt Pavement (RAP) which reduce production costs and reduce CO2 emissions. Another initiative used to address environmental problems is resource conservation which has made the use, recovery and recycling of resources more practical and effective in not only reducing production costs but also in making a regenerative circular economy. An example of this initiative is an innovation called End of Waste (EoW) product through which waste is converted into a new product and it is proving very practical and effective in recent times. The End of Waste (EoW) criteria means recovery and treatment processes under which waste could be converted in a new potential product. In particular, according to the European normative the main requirements for a given waste - possibly treated by industrial processes - to satisfy the EoW qualification are the following: a) the substance or object is intended to be used for specific purposes; b) there is a market or demand for this substance or object; c) the substance or object meets the technical requirements for the specific purposes and complies with the existing legislation and standards applicable to the products; d) the use of the substance or object will not lead to overall negative impacts on the environment or human health (in accordance with the Substance of Very High Concern (SVHC) list) From this perspective, the reuse of opportunely re-refined exhausted oils from automobile and industrial hydraulic applications - that have become unfit for the use for which they were originally intended - completely fulfil the circular economy goals. This PhD thesis deals with the conversion of End of Waste Product (EoW) in the form of Re-refined Engine Oil Bottom (REOB) obtained from exhausted oil regeneration processes – through the addition of recycled industrial waste materials - to high performing bituminous binder suitable for use in asphalt mix production. This can save the asphalt industry an even larger amount of resources if it is normalized to practice using this new potentially revolutionary binder together with Reclaimed Asphalt Pavement (RAP) which reduces the need for virgin aggregates in asphalt mix production. Indeed, in the present work asphalt concrete samples have been obtained by using 100 % Reclaimed Asphalt Pavement (RAP) aggregates. The asphalt concrete samples thus obtained underwent testing in order to study their behaviour with the goal to substitute virgin aggregates that are commonly employed in asphalt pavement. This would allow to reach the standards of most of the European countries where RAP is already used in the range of 70-90%, while up to now in Italy a maximum of 30% of RAP is used in the Hot Mix Asphalt (HMA) and Warm Mix Asphalt (WMA). The aged and more brittle bituminous binder that coats the aggregates limits the use of RAP material as it stiffens the resulting asphalt mixtures making the pavements more brittle and sometimes more prone to cracking especially at low temperatures. To overcome this problem a rejuvenating agent could be used.Item Edible oleogels: a rheological and microstructural analysis for structured emulsions design(Università della Calabria, 2022-06-01) Salvino, Samuele; Cerra, Maria Carmela; Gabriele, Domenico; Lupi, Francesca RomanaItem Rheological analysis of complex interfaces and bulk behaviour of starches and pea protein(Università della Calabria, 2021-06-24) Paleologo, Mario Florio Oraldo; Cerra, Maria Carmela; Baldino, Noemi; De Cindio, BrunoItem Development of new bio-materials and technologies for the green regeneration of RAP(Università della Calabria, 2021) Loise, Valeria; Cerra, Maria Carmela; Oliverio Rossi, CesareIl presente lavoro di ricerca ha avuto come scopo lo sviluppo di nuovi rigeneranti green per il fresato d’asfalto, noto anche come reclaimed asphalt pavement (RAP). Questa ricerca nasce dalla necessità di aumentare l’uso del RAP nelle pavimentazioni stradali. L’uso del RAP ha sia un impatto economico positivo, infatti non è necessario usare inerti e bitume fresco, ma soprattutto ha un alto un impatto in termini di ecosostenibilità, il RAP da rifiuto da smaltire diventa di fatto materia prima, implementando l’economia circolare e l’end waste. Infatti il riuso del fresato d’asfalto consentirebbe di non trattare più questo materiale come uno scarto, con tutti le relative problematiche ambientali derivabili, ma bensì come una risorsa. Inoltre sono state sviluppate ed implementate tecniche di indagine chimico-fisiche e nuove metodologie per determinare i meccanismi di azione di un rigenerante sulla struttura del bitume e distinguere in modo univoco un rigenerante da un semplice flussante. Tale tesi è stata la base scientifica che ha permesso di definire una norma europea e nazionale da seguire per le aziende che commercializzano rigeneranti bituminosi. Spesso infatti il fresato d’asfalto viene trattato con agenti che non modificano la struttura colloidale del bitume riportandola alla loro struttura originaria, ma che semplicemente hanno una mera attività flussante (abbassare la viscosità del sistema). Le “classiche” metodologie di indagine generalmente basate su parametri fisici-meccanici ed ingegneristici, non sono sufficienti per discriminare l’azione di un rigenerante da quella di un flussante. Tale approccio finora comunemente usato ha avuto un forte impatto sulla durata e qualità delle pavimentazioni stradali, effetto devastante dal punto di vista economico, sociale e soprattutto ambientale. The present research aimed to develop a procedure and methodologies that could uniquely distinguish between a fluxing agent and a rejuvenating agent. Recently, it has been realized that the reclaimed asphalt pavement (RAP) was a resource of considerable importance. In fact, it contains an appreciable amount of binder, which under certain conditions can be recycled for the construction of new pavements, thus reducing not only costs but also the use of raw materials. Therefore, this allows to consider the RAP more as a resource than a waste. Most of the time, the bitumen obtained from the RAP is treated with agents that have a mere flux function (lowering the viscosity of the system). These agents do not modify the colloidal structure of the bitumen. Therefore, they are not able to restore the structure of the aged bitumen to that of the neat bitumen. Furthermore, the investigation methodologies generally used to counter the regenerating effect of an additive are based on physical-mechanical and engineering parameters. These parameters are almost always insufficient to discriminate the action of a regenerant from that of a flux. For this reason, chemical-physical investigation techniques have been developed and implemented to determine the action mechanisms of a rejuvenator on the inner structure of the bitumen. In this way, it was possible to uniquely discriminate between a real rejuvenator and a fluxing agent. Finally, this thesis was the scientific basis that allowed the definition of a new European and National standard to be followed for companies that market bituminous regenerants.Item Cardiac adaptation to hypoxia: goldfish as a natural animal model(Università della Calabria, 2020-11-29) Leo, Serena; Cerra, Maria Carmela; Imbrogno, Sandra; Filice, MariacristinaItem In search of genetic and epigenetic markers of human aging and longevity: a study in the Calabrian population(Università della Calabria, 2021-02-01) Iannone, Francesca; Cerra, Maria Carmela; Rose, GiuseppinaItem Torsin a acting as a mechanosensor modulates nuclear transport in pathophysiological conditions(Università della Calabria, 2020-11-25) Galli, Giulia Raffaella; Cerra, Maria Carmela; Maggiolini, MarcelloGli esseri umani e l’ambiente circostante comunicano anche attraverso forze meccaniche. Gli input meccanici provenienti dal mondo che ci circonda svolgono un ruolo cruciale nei processi fisiologici e nel mantenimento dell’omeostasi dell’organismo umano. D'altra parte, difetti nelle proprietà meccaniche delle cellule o alterazioni delle forze meccaniche sono coinvolti nelle cause di diverse patologie tra cui il cancro. Le cellule del nostro corpo rispondono attivamente alle forze meccaniche, traducendo tali stimoli in segnali biochimici e nella regolazione dell'espressione genica. Il nucleo, costituito da due membrane separate che controllano il trasporto delle molecole, svolge un ruolo centrale nelle vie della meccano- trasduzione. Il trasporto nucleare delle molecole è meccanicamente sensibile ed è costituito da varie proteine coinvolte nel trasferimento di forze tra il citoscheletro e l'interno del nucleo. La ricerca condotta ha riguardato il ruolo dell'ATP-ase Torsin A come potenziale meccano-sensore in grado di rispondere ai cambiamenti della rigidità del substrato e modulare la traslocazione di proteine attraverso i pori nucleari. Sono stati pertanto studiati i cambiamenti nell'accumulo nucleare di MRTFA e YAP, poiché tali fattori di trascrizione sono fortemente sensibili ai segnali meccanici e risultano coinvolti nella progressione tumorale. Gli esperimenti sono stati eseguiti piastrando le cellule su gel di poliacrilammide con rigidità variabile e modulando i livelli di espressione di Torsin A attraverso tecniche di silenziamento genico ed over-espressione. In seguito ai cambiamenti di espressione di Torsin A è stata osservata un’alterazione nella traslocazione nucleare dei fattori di trascrizione summenzionati, supportando l'ipotesi 5 che Torsin A può agire nella meccano-sensibilità per il trasporto nucleare. L’utilizzo di costrutti mutanti di Torsin A ha ulteriormente consentito di dimostrare che Torsin A è coinvolta nel trasporto nucleare. Successivamente, sono stati anche valutati cambiamenti nella posizione di Torsin A in cellule piastrate su gel a diversa rigidità. Poiché il comportamento di Torsin A è fortemente dipendente dal cofattore noto come Lull1, che permette la traslocazione di Torsin A dal reticolo plasmatico all'involucro nucleare, la posizione di Torsin A in presenza e in assenza di tale cofattore è stata dunque valutata in cellule piastrate su gel a diversa rigidità. I risultati ottenuti hanno suggerito che la posizione di Torsin A è dipendente dalla presenza del cofattore Lull1. Inoltre, è stato osservato che l'accumulo di Torsin A nell'involucro nucleare diminuisce inibendo l'attività di Importin ß, indicando dunque che Importin ß è coinvolta nella localizzazione di Torsin A. È stato infine osservato che l’accumulo perinucleare di Torsina A è inversamente associato alla polimerizzazione dell'actina. Tali risultati indicano che Torsin A è un meccano-sensore in grado di rispondere ai cambiamenti della rigidità del substrato e di modulare la traslocazione nucleare di proteine coinvolte in importanti processi biologici.Item The effect of cholesterol on the function of eukaryotic membrane transporters for amino acids(Università della Calabria, 2020-11-25) Cosco, Jessica; Cerra, Maria Carmela; Indiveri, CesareAmino acid transport in mammalian cells is mediated by different amino acid transporters. Amino acid flow, which is important under physiological conditions, becomes particularly relevant under pathological conditions such as in cancer cells where high demand for these nutrients is required to satisfy the uncontrolled growth and proliferation. Therefore, to guarantee a sufficient supply of nutrients a lot of amino acid transporters are highly expressed in cancer cells. In this context, the amino acid transporters hLAT1 and hASCT2 are widely studied for their role as potential targets for drug development. hASCT2 belongs to SLC1 family and assembles at the plasma membrane as a trimeric complex. Studies conducted using the recombinant protein showed that this transporter is strongly stimulated by cholesterol. The stimulation is due to an improvement of protein insertion in the phospholipid bilayer and direct interaction with the protein. In fact, cholesterol increased the Vmax of the transport, without affecting the external Km, indicating that it increases the rate of conformational changes. Thanks to docking analysis, 6 putative cholesterol binding sites were predicted, some of these matched with the electron densities identified on the cryo-EM structure of ASCT2. Two poses are on the TM6, where a CRAC and a CARC motif has been identified. Experimental demonstrated the direct binding of cholesterol to the protein. In particular, Koshland’s reagent and SH-reagents have been used for the target of tryptophan and cysteine residues close to the cholesterol poses. hLAT1 belongs to SLC7 family and it forms a heterodimer complex (HAT) with the glycoprotein 4F2hc (also known as CD98 in mice), a member of SLC3 family. hLAT1 is the sole component involved in the transport of essential amino acids, as previously demonstrated (Napolitano, Scalise et al. 2015). In this work, the influence of cholesterol has been evaluated on the recombinant protein hLAT1. Moreover, putative regulators involved in energy metabolism have been tested on the transport. The transport activity increased up 75 μg cholesterol/ mg phospholipids. Moreover, the internal substrate affinity increased in the presence of cholesterol suggesting a stabilization of the inward conformation of hLAT1. The transporter is also stimulated by ATP at physiological concentration. This effect occurs only in the presence of cholesterol and was seen also on the native protein. This finding suggested that cholesterol and ATP binding sites are close to each other. The computational analysis confirmed this hypothesis. In fact, a hydrophobic region between the TMs 1, 5 and 7 was found to be close to a hydrophilic one. Docking results for ATP suggested an electrostatic interaction of the ϒ-phosphate of ATP with Lys 204, which was confirmed by site-directed mutagenesis. This residue is conserved in the other SLC7 proteins and for a serendipity event, it has been seen that Lys204 is also important in the substrate binding and pH-sensitive. In this work, the attention was focused also on another amino acid transporter CAT2, from Solanum lycopersicum. The specific interest in tomato resides in the well-recognized role for this species in biotechnology. In fact, tomato has been used as the primary model for the study of climacteric fruit ripening. SlCAT2 belongs to APC superfamily, as LAT1, and it is involved in the transport of cationic amino acids like arginine, lysine and the non-proteogenic amino acid ornithine. The experimental data on CAT2 highlighted an asymmetric regulation by cations and osmotic pressure, in line with the localization of the transporter in vacuoles. Like the other human transporters, CAT2 is also stimulated by cholesterol. On the basis of the 3D structure of the amino acid transporter GkApcT, the homology model of SlCAT2 was built and putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of membrane transporters to metabolic and membrane changes. Moreover the identification of hydrophobic or hydrophilic sites interacting with cholesterol or physiological effectors, respectively, could be important for applications in human pathology.Item Effetto del bisfenolo A sulla vascolatura uterina materna e sull'unità feto-placentare(Università della Calabria, 2021-12-01) Barberio, Laura; Cerra, Maria Carmela; Mandalà, Maurizio