Halogenated aliphatic and aromatic aerobic biodegradation via direct metabolism and cometabolism: batch and continuous tests

In this thesis the application of biotechnological processes based on microbial metabolic degradation of halogenated compound has been investigated. Several studies showed that most of these pollutants can be biodegraded by single bacterial strains or mixed microbial population via aerobic direct metabolism or cometabolism using as a growth substrates aromatic or aliphatic hydrocarbons. The enhancement of two specific processes has been here object of study in relation with its own respective scenario described as follow: 1st) the bioremediation via aerobic cometabolism of soil contaminated by a high chlorinated compound using a mixed microbial population and the selection and isolation of consortium specific for the compound. 2nd) the implementation of a treatment technology based on direct metabolism of two pure strains at the exact point source of emission, preventing dilution and contamination of large volumes of waste fluids polluted by several halogenated compound minimizing the environmental impact. In order to verify the effect of these two new biotechnological application to remove halogenated compound and purpose them as a more efficient alternative continuous and batch tests have been set up in the experimental part of this thesis. Results obtained from the continuous tests in the second scenario have been supported by microbial analysis via Fluorescence in situ Hybridisation (FISH) and by a mathematical model of the system. The results showed that both process in its own respective scenario offer an effective solutions for the biological treatment of chlorinate compound pollution.

Uremic Neuropathy: Epiemiological Study in Hemodialysis Patients

Background/Aims. Uremic Neuropathy (UN) highly limits the individual self-sufficiency causing near-continuous pain. An estimation of the actual UN prevalence among hemodialysis patients was the aim of the present work. Methods. We studied 225 prevalent dialysis patients from two Italian Centres. The Michigan Neuropathy Score Instrument (MNSI), already validated in diabetic neuropathy, was used for the diagnosis of UN. It consisted of a questionnaire (MNSI_Q) and a physical-clinical evaluation (MNSI_P). Patients without any disease possibly inducing secondary neuropathy and with MNSI score  3 have been diagnosed as affected by UN. Electroneurographic (ENG) lower limbs examination was performed in these patients to compare sensory conduction velocities (SCV) and sensory nerve action potentials (SNAP) with the MNSI results. Results. Thirtyseven patients (16.4%) were identified as being affected by UN, while 9 (4%) presented a score <3 in spite of neuropathic symptoms. In the 37 UN patients a significant correlation was found between MNSI_P and SCV (r2 = 0.1959; p<0.034) as well as SNAP (r2 = 0.3454; p=0.027) both measured by ENG. Conclusions. UN is an underestimated disease among the dialysis population even though it represents a huge problem in terms of pain and quality of life. MNSI could represent a valid and simple clinical-instrumental screening test for the early diagnosis of UN in view of an early therapeutic approach.

Continuous geochemical monitoring by mass-spectometer in the Campi Flegrei geothermal area. An application at Pisciarelli-Solfatara (diffuse and fumarolic gases) and at the mud gases during drilling of the CFDDP pilot hole

During this work has been developed an innovative methodology for continuous and in situ gas monitoring (24/24 h) of fumarolic and soil diffusive emissions applied to the geothermal and volcanic area of Pisciarelli near Agnano inside the Campi Flegrei caldera (CFc). In literature there are only scattered and in discrete data of the geochemical gas composition of fumarole at Campi Flegrei; it is only since the early ’80 that exist a systematic record of fumaroles with discrete sampling at Solfatara (Bocca Grande and Bocca Nuova fumaroles) and since 1999, even at the degassing areas of Pisciarelli. This type of sampling has resulted in a time series of geochemical analysis with discontinuous periods of time set (in average 2-3 measurements per month) completely inadequate for the purposes of Civil Defence in such high volcanic risk and densely populated areas. For this purpose, and to remedy this lack of data, during this study was introduced a new methodology of continuous and in situ sampling able to continuously detect data related and from its soil diffusive degassing. Due to its high sampling density (about one measurement per minute therefore producing 1440 data daily) and numerous species detected (CO2, Ar, 36Ar, CH4, He, H2S, N2, O2) allowing a good statistic record and the reconstruction of the gas composition evolution of the investigated area. This methodology is based on continuous sampling of fumaroles gases and soil degassing using an extraction line, which after undergoing a series of condensation processes of the water vapour content - better described hereinafter - is analyzed through using a quadrupole mass spectrometer

The porcine animal model goes through the 3Rs: development of in vitro and ex vivo system to study vascular biology

Since the publication of the book of Russell and Burch in 1959, scientific research has never stopped improving itself with regard to the important issue of animal experimentation. The European Directive 2010/63/EU “On the protection of animals used for scientific purposes” focuses mainly on the animal welfare, fixing the Russell and Burch’s 3Rs principles as the foundations of the document. In particular, the legislator clearly states the responsibility of the scientific community to improve the number of alternative methods to animal experimentation. The swine is considered a species of relevant interest for translational research and medicine due to its biological similarities with humans. The surgical community has, in fact, recognized the swine as an excellent model replicating the human cardiovascular system. There have been several wild-type and transgenic porcine models which were produced for biomedicine and translational research. Among these, the cardiovascular ones are the most represented. The continuous involvement of the porcine animal model in the biomedical research, as the continuous advances achieved using swine in translational medicine, support the need for alternative methods to animal experimentation involving pigs. The main purpose of the present work was to develop and characterize novel porcine alternative methods for cardiovascular translational biology/medicine. The work was mainly based on two different models: the first consisted in an ex vivo culture of porcine aortic cylinders and the second consisted in an in vitro culture of porcine aortic derived progenitor cells. Both the models were properly characterized and results indicated that they could be useful to the study of vascular biology. Nevertheless, both the models aim to reduce the use of experimental animals and to refine animal based-trials. In conclusion, the present research aims to be a small, but significant, contribution to the important and necessary field of study of alternative methods to animal experimentation.

Demersal communities in the Mediterranean Sea: a case study of Triglidae (Osteichthyes, Scorpaeniformes) on the conservation and sustainable use of marine resources

An appropriate management of fisheries resources can only be achieved with the continuous supply of information on the structure and biology of populations, in order to predict the temporal fluctuations. This study supports the importance of investigating the bio-ecology of increasingly exploited and poorly known species, such as gurnards (Osteichthyes, Triglidae) from Adriatic Sea (Mediterranean), to quantify their ecological role into marine community. It also focuses on investigate inter and intra-specific structuring factor of Adriatic population. These objectives were achieved by: 1) investigating aspects of the population dynamics; 2) studying the feeding biology through the examination of stomach contents; 3) using sagittal otoliths as potential marker of species life cycle; 4) getting preliminary data on mDNA phylogeny. Gurnards showed a specie-specific “critical size” coinciding with the start of sexual maturity, the tendency to migrate to greater depths, a change of diet from crustaceans to fish and an increase of variety of food items eaten. Distribution of prey items, predator size range and depth distribution were the main dimensions that influence the breadth of trophic niche and the relative difference amongst Adriatic gurnards. Several feeding preferences were individuated and a possible impact among bigger-size gurnards and other commercial fishes (anchovy, gadoids) and Crustacea (such as mantis prawn and shrimps) were to be necessary considered. Otolith studies showed that gurnard species have a very fast growth despite other results in other areas; intra-specific differences and the increase in the variability of otolith shape, sulcus acusticus shape, S:O ratios, sulcus acusticus external crystals arrangement were shown between juveniles and adults and were linked to growth (individual genetic factors) and to environmental conditions (e.g. depth and trophic niche distribution). In order to facilitate correct biological interpretation of data, molecular data were obtained for comparing morphological distance to genetic ones.

Aerobic biodegradation of chlorinated solvents and plastics in batch and continuous-flow bioreactors: process development, and identification of suitable chemical pre-Treatments

The purpose of the first part of the research activity was to develop an aerobic cometabolic process in packed bed reactors (PBR) to treat real groundwater contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA). In an initial screening conducted in batch bioreactors, different groundwater samples from 5 wells of the contaminated site were fed with 5 growth substrates. The work led to the selection of butane as the best growth substrate, and to the development and characterization from the site’s indigenous biomass of a suspended-cell consortium capable to degrade TCE with a 90 % mineralization of the organic chlorine. A kinetic study conducted in batch and continuous flow PBRs and led to the identification of the best carrier. A kinetic study of butane and TCE biodegradation indicated that the attached-cell consortium is characterized by a lower TCE specific degredation rates and by a lower level of mutual butane-TCE inhibition. A 31 L bioreactor was designed and set up for upscaling the experiment. The second part of the research focused on the biodegradation of 4 polymers, with and with-out chemical pre-treatments: linear low density polyethylene (LLDPE), polyethylene (PP), polystyrene (PS) and polyvinyl chloride (PVC). Initially, the 4 polymers were subjected to different chemical pre-treatments: ozonation and UV/ozonation, in gaseous and aqueous phase. It was found that, for LLDPE and PP, the coupling UV and ozone in gas phase is the most effective way to oxidize the polymers and to generate carbonyl groups on the polymer surface. In further tests, the effect of chemical pretreatment on polyner biodegrability was studied. Gas-phase ozonated and virgin polymers were incubated aerobically with: (a) a pure strain, (b) a mixed culture of bacteria; and (c) a fungal culture, together with saccharose as a co-substrate.

Studio pancreatic cystic follow-up, an international collaboration (PACYFIC study). Valutazione prospettica della sorveglianza delle cisti pancreatiche basato sulla consensus Europea sui tumori cistici del pancreas.

Introduzione Nel 2014 è stato proposto un protocollo di studio riguardante la sorveglianza delle lesioni cistiche del pancreas (intese come IPMN) e denominato PACYFIC Study. Obiettivi Obiettivo primario era di stabilire l’impatto di un programma di sorveglianza in termini di pazienti arruolati e pazienti con indicazione chirurgica. Obiettivi secondari erano: 1) stabilire l’impatto dei fattori demografici, clinici, radiologici e della strategia di sorveglianza sull’indicazione chirurgica, sulla capacità individuare le lesioni maligne, sulla sopravvivenza. Materiali e Metodi Lo studio su cui si è basata la raccolta dei dati è uno studio di tipo prospettico, di coorte, multicentrico, internazionale. Lo studio ha incluso gli individui con una IPMN, di nuova o pregressa diagnosi, che giustifichi una sorveglianza o il trattamento chirurgico. I dati clinici, demografici, radiologici e chirurgici sono stati raccolti in un database prospettico. Le variabili discrete sono state espresse come frequenza e percentuale. Le continue come medie e deviazioni standard o mediane e range interquartile (IQR). Per l’analisi statistica sono stati utilizzati il test di Fischer, il test del Chi quadro, il test di Spearman, il test di Student. L’analisi multivariata è stata eseguita utilizzando la regressione logistica espressa come Odds Ratio e intervallo di confidenza al 95 %. Per la sopravvivenza è stato utilizzato il metodo di Kaplan-Meier. L’analisi multivariata sulle sopravvivenze è stata eseguita mediante la regressione di Cox. Risultati Il protocollo di sorveglianza ha permesso l'arruolamento di 516 pazienti. 53 pazienti hanno raggiunto l'indicazione chirurgica. La sopravvivenza globale della coorte è stata di 326.8± 9.1 mesi. I fattori predittivi la sopravvivenza sono risultati età (OR 1.07, P-value<0.001), sesso (OR 1.82, P-value=0.006), ittero, noduli murali (OR 4.84, P-value=0.018 e OR 2.19, P-value=0.016), chirurgia (OR 0.46, P-value 0.038). Conclusioni L'introduzione del protocollo di sorveglianza ha portato ad un aumento di identificazione di lesioni e ha avuto impatto sulla sopravvivenza