A preclinical study of spinal cord injury focused on cellular and molecular modifications as potential targets for innovative therapies

Spinal Cord Injury (SCI) is a devastating condition for human and animal health. In SCI particularly, neurons, oligodendrocytes precursor cells, and mature oligodendrocytes are highly vulnerable to the toxic microenvironment after the lesion and susceptible to the elevated levels of noxious stimuli. Thus the regenerative response of the organism in case of SCI is significantly reduced, and only little spontaneous amelioration is observed in lesioned patients during the early phases. This work mainly focuses on studying and characterizing the modification induced by the SCI in a preclinical animal model. We investigated the ECM composition in the spinal cord segments surrounding the primary lesion site at a gene expression level. We found Timp1 and CD44 as a crucial hub in the secondary cascade of SCI in both spinal cord segments surrounding the lesion site. Interestingly, a temporal and anatomical difference in gene expression, indicating a complex regulation of ECM genes after SCI that could be used as a tool for regenerative medicine. We also investigated the modification in synaptic plasticity-related gene expression in spinal and supraspinal areas involved in motor control. We confirmed the anatomical and temporal difference in gene expression in spinal cord tissue. This analysis suggests that a molecular mapping of the lesion-induced modification could be a useful tool for regenerative medicine. In the last part, we evaluated the efficacy of an implantable biopolymer loaded with an anti-inflammatory drug and a pro-myelinating agent on the acute phase of SCI in our preclinical model. We found a consistent reduction of the inflammatory state in the spinal lesion site and the cord's surrounding segments. Moreover, we found increased preservation of the spinal cord tissue with a related upregulation of neuronal and oligodendroglial markers after lesion. Our treatment showed effective ameliorating functional outcome and reducing the lesion extension in the chronic phase.

Development of an innovative strategy to enhance the efficacy of gene therapy for CDKL5 deficiency disorder

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental disease caused by mutations in the CDKL5 gene, characterized by early-onset epileptic seizures, intellectual disability, motor and visual impairment and respiratory dysregulation. Although pharmacological treatments are used to control seizures, there is currently no cure to ameliorate symptoms for CDD. Albeit delivery of a wild-type copy of the mutated gene to cells represents the most curative approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for brain gene therapy. The major one regards the low efficiency of gene delivery to the CNS by viral vectors. We used a secretable Igk-TATk-CDKL5 protein to enhance the efficiency of a gene therapy for CDD. In view of the properties of the Igk-chain leader sequence, the TATk-CDKL5 protein produced by infected cells is secreted via constitutive secretory pathways. Importantly, due to the transduction property of the TATk peptide, the secreted CDKL5 protein is internalized by cells. We compared the effects of a CDKL5 gene therapy with an IgK-TATk-CDKL5 gene therapy in a Cdkl5 KO mouse model to validate whether the Igk-TATk-CDKL5 approach significantly improve the therapeutic efficacy. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to a higher CDKL5 protein replacement and Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with Cdkl5 KO mice treated with the AAVPHP.B_CDKL5 vector.

Investigation of innovative combustion systems for reduced fuel consumption and increased specific power production

This work resumes a wide variety of research activities carried out with the main objective of increasing the efficiency and reducing the fuel consumption of Gasoline Direct Injection engines, especially under high loads. For this purpose, two main innovative technologies have been studied, Water Injection and Low-Pressure Exhaust Gas Recirculation, which help to reduce the temperature of the gases inside the combustion chamber and thus mitigate knock, being this one of the main limiting factors for the efficiency of modern downsized engines that operate at high specific power. A prototypal Port Water Injection system was developed and extensive experimental work has been carried out, initially to identify the benefits and limitations of this technology. This led to the subsequent development and testing of a combustion controller, which has been implemented on a Rapid Control Prototyping environment, capable of managing water injection to achieve knock mitigation and a more efficient combustion phase. Regarding Low-Pressure Exhaust Gas Recirculation, a commercial engine that was already equipped with this technology was used to carry out experimental work in a similar fashion to that of water injection. Another prototypal water injection system has been mounted to this second engine, to be able to test both technologies, at first separately to compare them on equal conditions, and secondly together in the search of a possible synergy. Additionally, based on experimental data from several engines that have been tested during this study, including both GDI and GCI engines, a real-time model (or virtual sensor) for the estimation of the maximum in-cylinder pressure has been developed and validated. This parameter is of vital importance to determine the speed at which damage occurs on the engine components, and therefore to extract the maximum performance without inducing permanent damages.

Joints and innovative components for automotive applications

The research activities described in this thesis were focused on two main topics: the study of shaft-hub joint performance, with particular regard to interference-fitted and adhesively bonded connection, and the fatigue characterization of additively processed metal alloys. The research on interference-fitted shaft-hub joints dealt with some studies in the field of fretting fatigue. Rotating bending fatigue tests were performed on different materials by not conventional specimens to determine the fatigue properties of interference-fitted joints and to investigate the fretting fatigue phenomenon, which led to novel and original results. In adhesively bonded and interference-fitted shaft-hub connections (called hybrid joints) the synergic effect of anaerobic adhesive and interference has the capability of improving the joint strength. However, the adhesive contribution depends on several factors. Therefore, its behavior was investigated for different coupling pressure, coupling procedure, operating temperature and joint design. The study on additively manufactured metal alloy deals with rotating banding fatigue tests. AlSi10Mg and Maraging Stainless Steel CX were involved in the campaign for their wide applicability in Automotive. Build direction, heat and surface treatments were considered as input parameters. Fatigue results were interpreted by statistical method and microscopy analyses in order to determine the effectiveness and the beneficial or detrimental effects of the considered factors. Fracture mode and microstructure were investigated by fractographic and micrographic analyses

Analisi del tempo di reazione dei conducenti dei veicoli con metodologie innovative: l'influenza del sistema Adaptive Cruise Control

La presente tesi si pone l’obiettivo di studiare e comprendere l’influenza che i sistemi di assistenza alla guida (ADAS – Advanced Driver Assistance Systems), installati negli autoveicoli di nuova generazione, hanno sulla condotta di guida degli automobilisti, con particolare attenzione alla distrazione ed al workload che essi provocano. Punto centrale dell’analisi è il sistema Adaptive Cruise Control (ACC) che permette al guidatore del veicolo sia di mantenere una velocità di marcia costante sia di rilevare, tramite sensoristica, i veicoli che lo precedono, intervenendo sul sistema frenante e sulla centralina di controllo del motore, così da garantire il mantenimento della distanza di sicurezza selezionata. Lo studio, attraverso l’utilizzo di tecniche innovative, si sofferma, in particolare, sull’analisi del comportamento di guida dei conducenti a bordo di un veicolo dotato di Adaptive Cruise Control. Il fine della ricerca è quello di determinare se e quanto il sistema ACC influisca sul conducente in termini di carico di lavoro cognitivo e fisico e di livelli d’attenzione, concentrandosi sulla valutazione del tempo di reazione con sistema acceso o spento.

Development and optimization of fibre-shaped dye-sensitized solar cells employing an innovative fully organic sensitizer

The quality of human life depends to a large degree on the availability of energy. In recent years, photovoltaic technology has been growing extraordinarily as a suitable source of energy, as a consequence of the increasing concern over the impact of fossil fuels on climate change. Developing affordable and highly efficiently photovoltaic technologies is the ultimate goal in this direction. Dye-sensitized solar cells (DSSCs) offer an efficient and easily implementing technology for future energy supply. Compared to conventional silicon solar cells, they provide comparable power conversion efficiency at low material and manufacturing costs. In addition, DSSCs are able to harvest low-intensity light in diffuse illumination conditions and then represent one of the most promising alternatives to the traditional photovoltaic technology, even more when trying to move towards flexible and transparent portable devices. Among these, considering the increasing demand of modern electronics for small, portable and wearable integrated optoelectronic devices, Fibre Dye-Sensitized Solar Cells (FDSSCs) have gained increasing interest as suitable energy provision systems for the development of the next-generation of smart products, namely “electronic textiles” or “e-textiles”. In this thesis, several key parameters towards the optimization of FDSSCs based on inexpensive and abundant TiO2 as photoanode and a new innovative fully organic sensitizer were studied. In particular, the effect of various FDSSCs components on the device properties pertaining to the cell architecture in terms of photoanode oxide layer thickness, electrolytic system, cell length and electrodes substrates were examined. The photovoltaic performances of the as obtained FDSSCs were fully characterized. Finally, the metal part of the devices (wire substrate) was substituted with substrates suitable for the textile industry as a fundamental step towards commercial exploitation.

Design of an innovative electric vehicle simulator for Charging Systems' End-of-Line Testing

The trend related to the turnover of internal combustion engine vehicles with EVs goes by the name of electrification. The push electrification experienced in the last decade is linked to the still ongoing evolution in power electronics technology for charging systems. This is the reason why an evolution in testing strategies and testing equipment is crucial too. The project this dissertation is based on concerns the investigation of a new EV simulator design. that optimizes the structure of the testing equipment used by the company who commissioned this work. Project requirements can be summarized in the following two points: space occupation reduction and parallel charging implementation. Some components were completely redesigned, and others were substituted with equivalent ones that could perform the same tasks. In this way it was possible to reduce the space occupation of the simulator, as well as to increase the efficiency of the testing device. Moreover, the possibility of conjugating different charging simulations could be investigated by parallelly launching two testing procedures on a unique machine, properly predisposed for supporting the two charging protocols used. On the back of the results achieved in the body of this dissertation, a new design for the EV simulator was proposed. In this way, space reduction was obtained, and space occupation efficiency was improved with the proposed new design. The testing device thus resulted to be way more compact, enabling to gain in safety and productivity, along with a 25% cost reduction. Furthermore, parallel charging was implemented in the proposed new design since the conducted tests clearly showed the feasibility of parallel charging sessions. The results presented in this work can thus be implemented to build the first prototype of the new EV simulator.

Innovative Infrastructures for Sustainable Mobility in Urban Areas: Protective Asphalt to Prevent Vulnerable Road Users Injuries.

Nell’elaborato viene studiato il fenomeno dell’incidentalità singola delle utenze deboli sulla strada (perdita di controllo, scivolamento ecc..ecc..), con particolare enfasi nell’analizzare le conseguenze traumatiche dovute all’impatto con la superficie stradale. Una nuova tecnologia viene sviluppata per rendere le pavimentazioni di piste ciclabili, marciapiedi e zone pedonali con capacità di assorbimento degli impatti, prevenendo, o riducendo drasticamente, la probabilità e entità di eventi traumatici a seguito di una caduta. Attraverso una stesa di prova avvenuta ad Imola (BO), si sono analizzate e risolte le problematiche dovute alle discrepanze riscontrate fra “costruzione” in laboratorio e costruzione nella realtà urbana. La nuova tecnologia, infatti, è stata studiata come “construction-friendly”, permettendo alle società di costruzione e pavimentazione di stendere il nuovo manto protettivo con strumenti e macchinari tradizionali. Infine l’asfalto modificato è stato testato nei laboratori svedesi del KTH – Royal Institute of Technology per provarne l’efficacia in termini di assorbimento degli impatti. Nel “Drop Impact Test”, test specifico per l’approvazione dei caschi protettivi da bicicletta, l’asfalto modificato ha performato ben al di sotto della soglia di approvazione dei caschi da ciclista, e si colloca in un range di valore di accelerazione lineare perfino al di sotto della soglia di “Low Risk of Injuries”. La nuova tecnologia, inoltre, fa utilizzo di gomma granulata riciclata da pneumatici fuori uso. Tale dettaglio conferisce maggiore sostenibilità al progetto: oltre all’utilizzo di legante a freddo, che diminuisce le emissioni di CO2, e di incentivare l’utilizzo della mobilità dolce attraverso una più sicura rete infrastrutturale, l’utilizzo di gomma riciclata dà nuova vita al materiale che altrimenti andrebbe in discarica e prolunga, così, la vita utile del materiale.

Development and Detailed Characterization of Innovative, High-Performance Membrane Materials for CO2 Capture Processes

The scope of this dissertation is to study the transport phenomena of small molecules in polymers and membranes for gas separation applications, with particular attention to energy efficiency and environmental sustainability. This work seeks to contribute to the development of new competitive selective materials through the characterization of novel organic polymers such as CANALs and ROMPs, as well as through the combination of selective materials obtaining mixed matrix membranes (MMMs), to make membrane technologies competitive with the traditional ones. Kinetic and thermodynamic aspects of the transport properties were investigated in ideal and non-ideal scenarios, such as mixed-gas experiments. The information we gathered contributed to the development of the fundamental understanding related to phenomenon like CO2-induced plasticization and physical aging. Among the most significant results, ZIF-8/PPO MMMs provided materials whose permeability and selectivity were higher than those of the pure materials for He/CO2 separation. The CANALs featured norbornyl benzocyclobutene backbone and thereby introduced a third typology of ladder polymers in the gas separation field, expanding the structural diversity of microporous materials. CANALs have a completely hydrocarbon-based and non-polar rigid backbone, which makes them an ideal model system to investigate structure-property correlations. ROMPs were synthesized by means of the ring opening metathesis living polymerization, which allowed the formation of bottlebrush polymers. CF3-ROMP reveled to be ultrapermeable to CO2, with unprecedented plasticization resistance properties. Mixed-gas experiments in glassy polymer showed that solubility-selectivity controls the separation efficiency of materials in multicomponent conditions. Finally, it was determined that plasticization pressure in not an intrinsic property of a material and does not represent a state of the system, but rather comes from the contribution of solubility coefficient and diffusivity coefficient in the framework of the solution-diffusion model.

Innovative strategies for the mitigation of acrylamide content in different food products

Acrylamide (AA) is an undesirable food toxic compound, classified as 'probably carcinogenic to humans' by the International Agency for Research on Cancer due to its toxic effects, including neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. AA is mainly formed during the heat treatment of foods (> 120 °C) by the Maillard reaction, an essential reaction that also allows the desired levels of shelf-life and sensory properties of various food products to be achieved. Over the years, authorities and regulations have become more restrictive regarding the maximum levels of AA permitted in foods and beverages. The latest Commission Regulation (EU) 2017/2158 contains reference levels and measures to reduce AA in several food groups that contribute to the highest dietary intake, making necessary the study of promising AA mitigation strategies. The aim of this PhD research project was to identify, characterise and optimise some AA mitigation strategies in the most at-risk widely consumed foods such as potato, coffee and bakery products. Some AA control strategies were selected and investigated for each food category, also considering the main quality characteristics of the final products. The comprehensive results obtained during the three years of research activity have allowed a deeper knowledge of the traditional and innovative AA mitigation strategies, which can be extremely useful for both the food industry and international authorities. The most promising strategies studied in terms of reduction of AA while maintaining the main quality characteristics of the examined foods were: the application of pulsed electric fields and yeast immersion as pre-treatments of chips for frying; the selection of high roasting degrees for coffee products; the selection of static baking conditions for biscuits; the optimisation of alternative biscuit’ formulations by both the use of chickpea legume flour and of flour from bean with intact cotyledon cell walls.

Valorization of by-products from olive oil industry and their utilization for innovative food formulation

The purpose of this thesis work was the valorization of the main by-products obtained from olive oil production chain (wastewater and pomace) and their utilization in innovative food formulation. In the first part of the thesis, an olive mill wastewater extract rich in phenols were used in the formulation of 3 innovative meat products: beef hamburgers, cooked ham and würstels. These studies confirms that olive mill wastewaters extract rich in phenols could be an alternative for the reduction/total replacement of additives (i.e., nitrites) in ground and cooked meat preparations, which would promote the formulation of healthier clean label products and improve the sustainability of the olive oil industry with a circular economy approach, by further valorizing this olive by-product. In the second part of the thesis, the lipid composition and oxidative stability of a spreadable product obtained from a fermented and biologically de-bittered olive pomace, was assessed during a shelf-life study. This study confirmed that olive pomace represents an excellent ingredient for the formulation of functional foods In the third and last part of the thesis, carried out at the Universidad de Navarra (Pamplona, Spain), during a period abroad (3 months), three extracts obtained from purification of olive mill wastewaters, were subjected to in-vitro digestion and characterized. From the analysis of the three phenolic extracts, it emerged that the most promising extract to be used in the food field is the spry-dried one. Thanks to its formulation containing maltodextrins it manages to maintain its antioxidant capacity even after being underwent to in-vitro digestion. This thesis work is a part of the PRIN 2015 project (PROT: 20152LFKAT) "Olive phenols as multifunctional bioactives for healthier food: evaluation of simplified formulation to obtain safe meat products and new foods with higher functionality", coordinated by University of Perugia.

CO2-EVAR: An innovative approach to Automated Carbon Dioxide Angiography during Endovascular Abdominal Aortic Aneurysm repair

Objectives CO2-EVAR was proposed for treatment of AAA especially in patients with CKD. Issues regarding standardization, such as visualization of lowest renal artery (LoRA) and quality image in angiographies performed from pigtail or introducer-sheath, are still unsolved. Aim of the study was to analyze different steps of CO2-EVAR to create an operative protocol to standardize the procedure. Methods Patients undergoing CO2-EVAR were prospectively enrolled in 5 European centers (2018-2021). CO2-EVAR was performed using an automated injector. LoRA visualization and image quality (1-4) were analyzed and compared at different procedure steps: preoperative CO2-angiography from Pigtail/Introducer-sheath (1st Step), angiographies from Pigtail at 0%,50%,100% main body (MB) deployment (2nd Step), contralateral hypogastric artery (CHA) visualization with CO2 injection from femoral Introducer-sheath (3rd Step) and completion angiogram from Pigtail/Introducer-sheath (4th Step). Intra-/postoperative adverse events were evaluated. Results Sixty-five patients undergoing CO2-EVAR were enrolled, 55/65(84.5%) male, median age 75(11.5) years. Median ICM was 20(54)cc; 19/65(29.2%) procedures were performed with 0-iodine. 1st Step: median image quality was significantly higher with CO2 injected from femoral introducer [Pigtail2(3)vs.3(3)Introducer,p=.008]. 2nd Step: LoRA was more frequently detected at 50% (93%vs.73.2%, p=.002) and 100% (94.1%vs.78.4%, p=.01) of MB deployment compared with first angiography from Pigtail; image quality was significantly higher at 50% [3(3)vs.2(3),p=<.001] and 100% [4(3) vs.2(3),p=.001] of MB deployment. CHA was detected in 93% cases (3rd Step). Mean image quality was significantly higher when final angiogram (4th Step) was performed from introducer (Pigtail2.6±1.1vs.3.1±0.9Introducer,p=<.001). Rates of intra-/postoperative adverse events (pain,vomit,diarrhea) were 7.7% and 12.5%. Conclusions Preimplant CO2-angiography should be performed from Introducer-sheath. MB steric bulk during its deployment should be used to improve image quality and LoRA visualization with CO2. CHA can be satisfactorily visualized with CO2. Completion CO2-angiogram should be performed from femoral Introducer-sheath. This operative protocol allows to perform CO2-EVAR with minimal ICM and low rate of mild complications.

Design and synthesis of E3 ligase RNF5 inhibitors as innovative strategy to trigger mutant CFTR rescue in Cystic Fibrosis

In Cystic Fibrosis (CF) the deletion of phenylalanine 508 (F508del) in the CFTR anion channel is associated to misfolding and defective gating of the mutant protein. Among the known proteins involved in CFTR processing, one of the most promising drug target is the ubiquitin ligase RNF5, which normally promotes F508del-CFTR degradation. In this context, a small molecule RNF5 inhibitor is expected to chemically mimic a condition of RNF5 silencing, thus preventing mutant CFTR degradation and causing its stabilization and plasma membrane trafficking. Hence, by exploiting a virtual screening (VS) campaign, the hit compound inh-2 was discovered as the first-in-class inhibitor of RNF5. Evaluation of inh-2 efficacy on CFTR rescue showed that it efficiently decreases ubiquitination of mutant CFTR and increases chloride current in human primary bronchial epithelia. Based on the promising biological results obtained with inh-2, this thesis reports the structure-based design of potential RNF5 inhibitors having improved potency and efficacy. The optimization of general synthetic strategies gave access to a library of analogues of the 1,2,4-thiadiazol-5-ylidene inh-2 for SAR investigation. The new analogues were tested for their corrector activity in CFBE41o- cells by using the microfluorimetric HS-YFP assay as a primary screen. Then, the effect of putative RNF5 inhibitors on proliferation, apoptosis and the formation of autophagic vacuoles was evaluated. Some of the new analogs significantly increased the basal level of autophagy, reproducing RNF5 silencing effect in cell. Among them, one compound also displayed a greater rescue of the F508del-CFTR trafficking defect than inh-2. Our preliminary results suggest that the 1,2,4-thiadiazolylidene could be a suitable scaffold for the discovery of potential RNF5 inhibitors able to rescue mutant CFTRs. Biological tests are still ongoing to acquire in-depth knowledge about the mechanism of action and therapeutic relevance of this unprecedented pharmacological strategy.

Active Control of the Acoustic Field in a Vehicle Cabin

In this thesis, a thorough investigation on acoustic noise control systems for realistic automotive scenarios is presented. The thesis is organized in two parts dealing with the main topics treated: Active Noise Control (ANC) systems and Virtual Microphone Technique (VMT), respectively. The technology of ANC allows to increase the driver's/passenger's comfort and safety exploiting the principle of mitigating the disturbing acoustic noise by the superposition of a secondary sound wave of equal amplitude but opposite phase. Performance analyses of both FeedForwrd (FF) and FeedBack (FB) ANC systems, in experimental scenarios, are presented. Since, environmental vibration noises within a car cabin are time-varying, most of the ANC solutions are adaptive. However, in this work, an effective fixed FB ANC system is proposed. Various ANC schemes are considered and compared with each other. In order to find the best possible ANC configuration which optimizes the performance in terms of disturbing noise attenuation, a thorough research of \gls{KPI}, system parameters and experimental setups design, is carried out. In the second part of this thesis, VMT, based on the estimation of specific acoustic channels, is investigated with the aim of generating a quiet acoustic zone around a confined area, e.g., the driver's ears. Performance analysis and comparison of various estimation approaches is presented. Several measurement campaigns were performed in order to acquire a sufficient duration and number of microphone signals in a significant variety of driving scenarios and employed cars. To do this, different experimental setups were designed and their performance compared. Design guidelines are given to obtain good trade-off between accuracy performance and equipment costs. Finally, a preliminary analysis with an innovative approach based on Neural Networks (NNs) to improve the current state of the art in microphone virtualization is proposed.

Sinus rhythm restoration with electrical cardioversion: acute effect of shock configuration and subsequent modifications in peripheral flow and sleep.

Atrial fibrillation (AF) is a widespread arrhythmia, associated with higher risk of stroke, sleep disorders and dementia. In some conditions, electrical cardioversion (ECV) represents the best choice for rhythm control. Nowadays, there is a growing interest in developing new devices for screening and monitoring of AF patients. We aimed to improve acute efficacy of ECV procedure and to explore the feasibility of the use of new wearable devices for monitoring in candidates to AF ECV. We compared antero-apical pads vs antero-posterior patches approach for AF ECV, and we elaborated a decision algorithm to improve acute efficacy. After, we evaluated the feasibility of the use of new wearable devices for monitoring of candidates to AF ECV. In particular, we analysed the effect of AF ECV on heart rate variability and vascular age parameters derived from PPG signals registered with Empatica (CE 1876/MDD 93/42/EEC), and on EEG pattern registered with Neurosteer (Israel). From December 2005 to September 2019, 492 patients were enrolled. We evaluated acute efficacy of the two approaches for AF ECV and we elaborated a decision algorithm based on body surface area, weight, and height. The decision algorithm improved first shock efficacy (93.2% vs. 87.2%, p=0.025). From 1st November 2021 to 1st April 2022, 24 patients were enrolled in PPEEG-AF pilot study. Considering vascular age parameters, a significant reduction in TPR and a wave was observed (p<0.001). Considering sleep patterns, a tendency to higher coherence was observed in registrations acquired during AF, or considering signals registered for each patient independently from AF. The new decision algorithm improved acute efficacy and reduced costs associated with adhesive patches. Significant modifications were observed on vascular age parameters measured before and after ECV, and a possible AF effect on sleep pattern was noticed. More data are necessary to confirm these preliminary results.