Essential oils and hydrolates as potential tools for integrated prevention of bacterial diseases in plants

The present work aims to investigate the potential use of natural substances against bacterial plant pathogens. Microdilution tests were therefore carried out in vitro to identify the minimum inhibitory and bactericidal concentrations (MIC and MBC) of several EOs and Hys against selected bacterial pathogens. Commercial products based on a mixture of EOs were in addition assayed with macrodilution experiments against Erwinia amylovora (Ea-causal agent of fire blight). Subsequently, using selected EOs, Hys, and commercial products, ex vivo tests on disease incidence and Ea population dynamics were carried out; the latter experiment was followed by SEM observations. In addition, in vivo resistance induction test was carried out against bacterial leaf of tomato, caused by Xanthomonas vesicatoria (Xv). EOs and Hys showed high bactericidal activity in vitro (MBC <0.1 and <10% for the most active EOs and Hys: Origanum compactum and Thymus vulgaris EOs and Citrus aurantium var. amara Hy, respectively), but they were not effective ex vivo, while resulted very active when used in vivo as resistance inducers in the tomato-Xv pathosystem (relative protection >40%). Differently, commercial products resulted active in all tests, but not as resistance inducers against Xv. An open field trial with commercial products was carried out on strawberry plants naturally infected with Xanthomonas fragariae; the results showed discrete relative protection, concerning that provided by the conventional products based on copper; mostly, the disease severity reduction on those plants treated with EOs commercial products was significant when disease severity resulted high. The papers already published described in the present work investigate (1)the activity of Hys in comparison to EOs with respect to their active volatile content; (2) the potential use of EOs and Hys in cultural heritage; for the restoration of paintings; (3) the induction of resistance caused by plasma-activated water-based root treatments.

Characterisation of probiotic strains for the control and prevention of enteropathogens in the food chain

60 strains (belonging to the genera Lactobacillus, Bifidobacterium, Leuconostoc and Enterococcus) were tested for their capacity to inhibit the growth of 3 strains of Campylobacter jejuni: Lactobacilli and bifidobacteria were left to grow in MRS or TPY broth at 37°C overnight in anaerobic conditions; Campylobacter jejuni was inoculated in blood agar plates at 37°C for 24-48 hours in microaerophilic conditions. The inhibition experiments were carried out in vitro using ”Spot agar test” and “Well diffusion assay” techniques testing both cellular activity and that of the surnatant. 11 strains proved to inhibit the growth of Campylobacter jejuni. These strains were subsequently analised analised in order to evaluate the resistance to particular situations of stress which are found in the gastrointestinal tract and during the industrial transformation processes (Starvation stress, osmotic stress, heat stress, resistance to pH and to bile salts). Resistance to starvation stress: all strains seemed to resist the stress (except one strain). Resistance to osmotic stress: all strains were relatively resistant to the concentrations of 6% w/v of NaCl (except one strain). Resistance to heat stress: only one strain showed little resistance to the 55°C temperature. Resistance to pH: In the presence of a low pH (2.5), many strains rapidly lost their viability after approximately 1 hour. Resistance to bile salts: Except for one strain, all strains seemed to be relatively resistant to the 2% w/v concentration of bile salts. Afterward, strains were identified by using phenotipic and molecular techniques. Phenotipic identification was carried out by using API 50 CHL (bioMérieux) and API 20 STREP identification system (bioMérieux); molecular identification with species-specific PCR: the molecular techniques confirmed the results by phenotipic identification. For testing the antibiotic resistance profile, bacterial strains were subcultured in MRS or TPY broth and incubated for 18 h at 37°C under anaerobic conditions. Antibiotics tested (Tetracycline, Trimethoprim, Cefuroxime, Kanamycin, Chloramphenicol, Vancomycin, Ampycillin, Sterptomycin, Erythromycin) were diluted to the final concentrations of: 2,4,8,16,32,64,128,256 mg/ml. Then, 20 μl fresh bacterial culture (final concentration in the plates approximately 106 cfu/ml) were added to 160 μl MRS or TPY broth and 20 μl antibiotic solution. As positive control the bacterial culture (20 ul) was added to broth (160 ul) and water (20 ul). Test was performed on plates P96, that after the inoculum were incubated for 24 h at 37oC, then the antibiotic resistance was determined by measuring the Optical Density (OD) at 620 nm with Multiscan EX. All strains showed a similar behaviour: resistance to all antibiotic tested. Further studies are needed.

Prevention of binge drinking in young adolescents through the promotion of a balanced level of psychological well-being: be-dry

This doctoral thesis presents a project carried out in secondary schools located in the city of Ferrara with the primary objective of demonstrating the effectiveness of an intervention based on Well-Being Therapy (Fava, 2016) in reducing alcohol use and improving lifestyles. In the first part (chapters 1-3), an introduction on risky behaviors and unhealthy lifestyle in adolescence is presented, followed by an examination of the phenomenon of binge drinking and of the concept of psychological well-being. In the second part (chapters 4-6), the experimental study is presented. A three-arm cluster randomized controlled trial including three test periods was implemented. The study involved eleven classes that were randomly assigned to receive well-being intervention (WBI), lifestyle intervention (LI) or not receive intervention (NI). Results were analyzed by linear mixed model and mixed-effects logistic regression with the aim to test the efficacy of WBI in comparison with LI and NI. AUDIT-C total score increased more in NI in comparison with WBI (p=0.008) and LI (p=0.003) at 6-month. The odds to be classified as at-risk drinker was lower in WBI (OR 0.01; 95%CI 0.01–0.14) and LI (OR 0.01; 95%CI 0.01–0.03) than NI at 6-month. The odds to use e-cigarettes at 6-month (OR 0.01; 95%CI 0.01–0.35) and cannabis at post-test (OR 0.01; 95%CI 0.01–0.18) were less in WBI than NI. Sleep hours at night decreased more in NI than in WBI (p = 0.029) and LI (p = 0.006) at 6-month. Internet addiction scores decreased more in WBI (p = 0.003) and LI (p = 0.004) at post-test in comparison with NI. Conclusions about the obtained results, limitations of the study, and future implications are discussed. In the seventh chapter, the data of the project collected during the pandemic are presented and compared with those from recent literature.

Synergies among safety and security in the prevention of major accidents related to dangerous substances

Historical evidence shows that chemical, process, and Oil&Gas facilities where dangerous substances are stored or handled are target of deliberate malicious attacks (security attacks) aiming at interfering with normal operations. Physical attacks and cyber-attacks may generate events with consequences on people, property, and the surrounding environment that are comparable to those of major accidents caused by safety-related causes. The security aspects of these facilities are commonly addressed using Security Vulnerability/Risk Assessment (SVA/SRA) methodologies. Most of these methodologies are semi-quantitative and non-systematic approaches that strongly rely on expert judgment, leading to security assessments that are not reproducible. Moreover, they do not consider the synergies with the safety domain. The present 3-year research is aimed at filling the gap outlined by providing knowledge on security attacks, as well as rigorous and systematic methods supporting existing SVA/SRA studies suitable for the chemical, process, and Oil&Gas industry. The different nature of cyber and physical attacks resulted in the development of different methods for the two domains. The first part of the research was devoted to the development and statistical analysis of security databases that allowed to develop new knowledge and lessons learnt on security threats. Based on the obtained background, a Bow-Tie based procedure and two reverse-HazOp based methodologies were developed as hazard identification approaches for physical and cyber threats respectively. To support the quantitative estimation of the security risk, a quantitative procedure based on the Bayesian Network was developed allowing to calculate the probability of success of physical security attacks. All the developed methods have been applied to case studies addressing chemical, process and Oil&Gas facilities (offshore and onshore) proving the quality of the results that can be achieved in improving site security. Furthermore, the outcomes achieved allow to step forward in developing synergies and promoting integration among safety and security management.

N-3 fatty acids and cardiovascular prevention

In this study we elucidate the role of polyunsaturated fatty acids (PUFAs) in the prevention of cardiovascular diseases, focusing the attention on their role in the modulation of acyl composition of cell lipids and of gene expression. Regarding this latter mechanism, the effectiveness of PUFAs as activators of two transcriptional factors, SREBPs and PPARs, have been considered. Two different model system have been used: primary cultures of neonatal rat cardiomyocytes and an human hepatoma cell line (HepG2). Cells have been supplemented with different PUFAs at physiological concentration, and special attention has been devoted to the main n-3 PUFAs, EPA and DHA. PUFAs influence on global gene expression in cardiomyocytes has been evaluated using microarray technique. Furthermore, since it is not fully elucidated which transcription factors are involved in this modulation in the heart, expression and activation of the three different PPAR isoforms have been investigated. Hepatocytes have been used as experimental model system in the evaluation of PUFAs effect on SREBP activity. SREBPs are considered the main regulator of cholesterol and triglyceride synthesis, which occur mainly in the liver. In both experimental models the modification of cell lipid fatty acid composition subsequent to PUFAs supplementation has been evaluated, and related to the effects observed at molecular level. The global vision given by the obtained results may be important for addressing new researches and be useful to educators and policy makers in setting recommendations for reaching optimal health through good nutrition.

Valorization of pomaces from the mechanical extraction of virgin olive oils in dairy animal feeding

This research work is aimed at the valorization of two types of pomace deriving from the extra virgin olive oil mechanical extraction process, such as olive pomace and a new by-product named “paté”, in the livestock sector as important sources of antioxidants and unsaturated fatty acids. In the first research the suitability of dried stoned olive pomace as a dietary supplement for dairy buffaloes was evaluated. The effectiveness of this utilization in modifying fatty acid composition and improving the oxidative stability of buffalo milk and mozzarella cheese have been proven by means of the analysis of qualitative and quantitative parameters. In the second research the use of paté as a new by-product in dietary feed supplementation for dairy ewes, already fed with a source of unsaturated fatty acids such as extruded linseed, was studied in order to assess the effect of this combination on the dairy products obtained. The characterization of paté as a new by-product was also carried out, studying the optimal conditions of its stabilization and preservation at the same time. The main results, common to both researches, have been the detection and the characterization of hydrophilic phenols in the milk. The analytical detection of hydroxytyrosol and tyrosol in the ewes’ milk fed with the paté and hydroxytyrosol in buffalo fed with pomace showed for the first time the presence in the milk of hydroxytyrosol, which is one of the most important bioactive compounds of the oil industry products; the transfer of these antioxidants and the proven improvement of the quality of milk fat could positively interact in the prevention of some human cardiovascular diseases and some tumours, increasing in this manner the quality of dairy products, also improving their shelf-life. These results also provide important information on the bioavailability of these phenolic compounds.

Neurodegenerative diseases: molecular mechanisms and new strategies for neuroprotection

The term neurodegeneration defines numerous conditions that modify neuron’s normal functions in the human brain where is possible to observe a progressive and consistent neuronal loss. The mechanisms involved in neurodegenerative chronic and acute diseases evolution are not completely understood yet, however they share common characteristics such as misfolded proteins, oxidative stress, inflammation, excitotoxicity, and neuronal loss. Many studies have shown the frequency to develop neurodegenerative chronic diseases several years after an acute brain injury. In addition, many patients show, after a traumatic brain injury, motor and cognitive manifestations that are close to which are observed in neurodegenerative chronic patients. For this reason it is evident how is fundamental the concept of neuroprotection as a way to modulate the neurodegenerative processes evolution. Neuroinflammation, oxidative stress and the apoptotic process may be functional targets where operate to this end. Taking into account these considerations, the aim of the present study is to identify potential common pathogenetic pathways in neurodegenerative diseases using an integrated approach of preclinical studies. The goal is to delineate therapeutic strategies for the prevention of neuroinflammation, neurodegeneration and dysfunctions associated to Parkinson’s disease (PD) and cerebral ischemia. In the present study we used a murine model of PD treated with an isothiocyanate, 6-MSITC, able to quench ROS formation, restore the antioxidant GSH system, slow down the apoptotic neuronal death and counteract motor dysfunction induced by 6-OHDA. In the second study we utilized a transgenic mouse model knockout for CD36 receptor to investigate the inflammation involvement in a long term study of MCAo, which shows a better outcome after the damage induced. In conclusion, results in this study allow underlying the connection among these pathologies, and the importance of a neuroprotective strategy able to restore neurons activity where current drugs therapies have shown palliative but not healing abilities.

Influence of the natural environment on health: evaluation of different environmental contexts (urban, green spaces) and intervention proposals

Urbanization has grown during the last decades, with an increase in population concentrated in cities. Cities are usually relatively nature-poor, and the loss of green urban space likely leads to less contact with the natural world for urban dwellers. It is known that the natural environment could provide important advantages, and the loss of contact with this type of environment has potential negative impacts on the quality of life. The use of green urban space demonstrated stronger benefits for mental health and stress reduction. In general, exposure to green urban space is linked to a reduction in mortality rates, due to the promotion of a healthy lifestyle. Green urban space could be an optimal environment in which to perform physical activity. Undertaking regular physical activity is one of the major determinants of health. The benefits of exercise have been widely demonstrated through a wide range of studies. Benefits are linked to the treatment and prevention of most chronic and non-communicable diseases, that are not contagious, but they are usually long-lasting. Regular physical activity could reduce mental health problems, such as anxiety. The World Health Organization proposed to improve physical activity programs through the implementation of interventions in green urban spaces. Green urban space provides a safe, accessible, and attractive place to perform physical activity. All the interventions aimed to promote the practice of physical activity and to reduce sedentary behavior are important. It is well known that physical activity has several positive effects, a great amount of the population remains inactive. A good strategy could be to show people how integrated physical activity into their all-day life, for example through the use of green urban space or active commuting. The results in the present thesis showed the effectiveness of performing physical activity in a natural environment and of active commuting.