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.

Neisseria meningitidis, the leading cause of bacterial meningitis, can adapt to different host niches during human infection. Both transcriptional and post-transcriptional regulatory networks have been identified as playing a crucial role for bacterial stress responses and virulence. We investigated the N. meningitidis transcriptional landscape both by microarray and by RNA sequencing (RNAseq). Microarray analysis of N. meningitidis grown in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. In particular, we identified a glucose-responsive hexR-like transcriptional regulator in N. meningitidis. Deletion analysis showed that the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of meningococcus, by targeting a DNA region overlapping putative regulatory sequences. Our results indicate that HexR coordinates the central metabolism of meningococcus in response to the availability of glucose, and N. meningitidis strains lacking the hexR gene are also deficient in establishing successful bacteremia in a mouse model of infection. In parallel, RNAseq analysis of N. meningitidis cultured under standard or iron-limiting in vitro growth conditions allowed us to identify novel small non-coding RNAs (sRNAs) potentially involved in N. meningitidis regulatory networks. Manual curation of the RNAseq data generated a list of 51 sRNAs, 8 of which were validated by Northern blotting. Deletion of selected sRNAs caused attenuation of N. meningitidis infection in a murine model, leading to the identification of the first sRNAs influencing meningococcal bacteraemia. Furthermore, we describe the identification and initial characterization of a novel sRNA unique to meningococcus, closely associated to genes relevant for the intracellular survival of pathogenic Neisseriae. Taken together, our findings could help unravel the regulation of N. meningitidis adaptation to the host environment and its implications for pathogenesis.

L’insorgenza di fenomeni coinvolti nello sviluppo della farmacoresistenza costituisce al momento la principale causa di mancata risposta al trattamento chemioterapico nell’osteosarcoma. Questo è in parte dovuto ad una sovraespressione di diversi trasportatori ABC nelle cellule tumorali che causano un aumento dell’efflusso extracellulare del chemioterapico e pertanto una ridotta risposta al trattamento farmacologico. L'oncogene C-MYC è coinvolto nella resistenza al metothrexate, alla doxorubicina e al cisplatino ed è un fattore prognostico avverso, se sovraespresso al momento della diagnosi, in pazienti affetti da osteosarcoma. C-MYC è in grado di regolare l'espressione di diversi trasportatori ABC, probabilmente coinvolti nella resistenza ai farmaci nell’osteosarcoma, e questo potrebbe spiegare l’impatto prognostico avverso dell’oncogene in questo tumore. L’espressione genica di C-MYC e di 16 trasportatori ABC, regolati da C-MYC e / o responsabili dell'efflusso di diversi chemioterapici, è stata valutata su due diverse casistiche cliniche e su un pannello di linee cellulari di osteosarcoma umano mediante real-time PCR. L'espressione della proteina è stata valutata per i 9 trasportatori ABC risultati più rilevanti.Infine l'efficacia in vitro di un inibitore, specifico per ABCB1 e ABCC1, è stata valutata su linee cellulari di osteosarcoma. ABCB1 e ABCC1 sono i trasportatori più espressi nelle linee cellulari di osteosarcoma. ABCB1 è sovraespresso al momento della diagnosi in circa il 40-45% dei pazienti affetti da osteosarcoma e si conferma essere un fattore prognostico avverso se sovraespresso al momento della diagnosi. Pertanto ABCB1 diventa il bersaglio di elezione per lo sviluppo di strategie terapeutiche alternative, nel trattamento dell’osteosarcoma, atte al superamento della farmacoresistenza. L’inibizione dell'attività di tale trasportatore causa un aumento della sensibilità al trattamento chemioterapico nelle linee cellulari di osteosarcoma farmacoresistenti, indicando questo approccio come una possibile strategia per superare il problema della mancata risposta al trattamento farmacologico nei pazienti con osteosarcoma che sovraesprimono ABCB1. / Resistance to chemoterapeutic agents is a major obstacle for successful treatment of high grade osteosarcoma. This is in part caused by disregulation of the activity of ATP-binding cassette transporters (ABC) responsible for the efflux of chemotherapeutic agents in cancer cells. Studying how these transporters are regulated at transcription level is a crucial step to understanding multidrug resistance in cancer. The C-MYC oncogene has been identified to be related to resistance against methotrexate, doxorubicin and cisplatin and to be a prognostic factor for high grade osteosarcoma. C-MYC can regulate the expression of several ABC transporter genes, possibly related to drug resistance in osteosarcoma, and this may explain its adverse prognostic impact in this tumor. We have analyzed C-MYC and 16 ABC transporters, which are regulated by C-MYC and/or have been found to be responsible for the efflux of conventional drugs used in the treatment of human tumors on two different series of clinical samples and in a panel of human osteosarcoma cell lines by real-time PCR. The protein expression was evaluated for the 9 ABC transporters resulted as the most relevant from gene espression experiments. Finally the efficacy of a specific ABCB1 and ABCC1 inhibitor, was evaluated on osteosarcoma cell lines. ABCB1 and ABCC1 were the most expressed transporters in osteosarcoma cell lines. ABCB1 overexpression at diagnosis was found in about 40-45% of osteosarcoma patients and it was confirmed to be associated with adverse prognosis, being involved in drug resistance of osteosarcoma cells. Because of this, ABCB1 can be considered as a candidate therapeutic target for improving the treatment response of high-grade osteosarcoma patients. Inhibition of ABCB1 activity resulted to increase chemotherapeutics sensitivity of drug resistant human osteosarcoma cell lines, indicating this approach as a possible strategy to overcome treatment unresponsiveness in ABCB1-overexpressing osteosarcoma patients.

This PhD Thesis includes five main parts on diverse topics. The first two parts deal with the trophic ecology of wolves in Italy consequently to a recent increase of wild ungulates abundance. Data on wolf diet across time highlighted how wild ungulates are important food resource for wolves in Italy. Increasing wolf population, increasing numbers of wild ungulates and decreasing livestock consume are mitigating wolf-man conflicts in Italy in the near future. In the third part, non-invasive genetic sampling techniques were used to obtain genotypes and genders of about 400 wolves. Thus, wolf packs were genetically reconstructed using diverse population genetic and parentage software. Combining the results on pack structure and genetic relatedness with sampling locations, home ranges of wolf packs and dispersal patterns were identified. These results, particularly important for the conservation management of wolves in Italy, illustrated detailed information that can be retrieved from genetic identification of individuals. In the fourth part, wolf locations were combined with environmental information obtained as GIS-layers. Modern species distribution models (niche models) were applied to infer potential wolf distribution and predation risk. From the resulting distribution maps, information pastures with the highest risk of depredation were derived. This is particularly relevant as it allows identifying those areas under danger of carnivore attack on livestock. Finally, in the fifth part, habitat suitability models were combined with landscape genetic analysis. On one side landscape genetic analyses on the Italian wolves provided new information on the dynamics and connectivity of the population and, on the other side, a profound analysis of the effects that habitat suitability methods had on the parameterization of landscape genetic analyses was carried out to contributed significantly to landscape genetic theory.

Obiettivo del lavoro è stato lo sviluppo e la validazione di nuovi bioassay e biomarker quali strumenti da utilizzare in un approccio ecotossicologico integrato per il biomonitoraggio di ambienti marino-costieri interessati da impatto antropico negli organismi che vivono in tali ambienti. L’ambiente reale impiegato per l’applicazione in campo è la Rada di Augusta (Siracusa, Italia). Una batteria di bioassay in vivo e in vitro è stata indagata quale strumento di screening per la misura della tossicità dei sedimenti. La batteria selezionata ha dimostrato di possedere i requisiti necessari ad un applicazione di routine nel monitoraggio di ambienti marino costieri. L’approccio multimarker basato sull’impiego dell’organismo bioindicatore Mytilus galloprovincialis in esperimenti di traslocazione ha consentito di valutare il potenziale applicativo di nuovi biomarker citologici e molecolari di stress chimico parallelamente a biomarker standardizzati di danno genotossico ed esposizione a metalli pesanti. I mitili sono stati traslocati per 45 giorni nei siti di Brucoli (SR) e Rada di Augusta, rispettivamente sito di controllo e sito impattato. I risultati ottenuti supportano l’applicabilità delle alterazioni morfometriche dei granulociti quale biomarker di effetto, direttamente correlato allo stato di salute degli organismi che vivono in un dato ambiente. Il significativo incremento dell’area dei lisosomi osservato contestualmente potrebbe riflettere un incremento dei processi degradativi e dei processi autofagici. I dati sulla sensibilità in campo suggeriscono una valida applicazione della misura dell’attività di anidrasi carbonica in ghiandola digestiva come biomarker di stress in ambiente marino costiero. L’utilizzo delle due metodologie d’indagine (bioassay e biomarker) in un approccio ecotossicologico integrato al biomonitoraggio di ambienti marino-costieri offre uno strumento sensibile e specifico per la valutazione dell’esposizione ad inquinanti e del danno potenziale esercitato dagli inquinanti sugli organismi che vivono in un dato ambiente, permettendo interventi a breve termine e la messa a punto di adeguati programmi di gestione sostenibile dell’ambiente. / The aim of the work was the development and validation of new bioassays and biomarkers as tools in an integrated ecotoxicological approach for the biomonitoring of impacted coastal marine environment environments. The Rada of Agusta (Syracuse, Sicily) was used as real environment for the field application of the proposed integrated approach. A battery of in vivo and in vitro bioassays was investigated as screening tool of the assessment of marine sediment toxicity. The battery has proven to have the necessary requirement for a routine application in marine coastal environment biomonitoring. The multimarker approach based on the use of bioindicator organism Mytilus galloprovincialis in translocation experiments allowed to evaluate the field application potential of new cytological and molecular biomarkers in parallel to standardized biomarkers of genotoxicity and heavy metal exposure. Mussels were caged for 45 days in Brucoli (SR) and Rada di Augusta, reference site and impacted site respectively. Results support the applicability of granulocytes morphometric alterations as effect biomarker, directly correlated to the health of the organism. Morphometric alterations were accompanied by a significative increase of the lysosomal compartment, which in turn could reflect the pollutant induced increase of the degradative and autophagic processes. Carbonic anhydrase activity in digestive gland proved to be a valuable biomarker of chemical stress in marine coastal environment. The functional role of carbonic anhydrase in the lysosomal compartment functioning was evaluated. The combined use of the two methodologies (bioassays and biomarkers) in an integrated ecotoxicological approach provides a sensitive and specific tool for the assessment of pollutant exposure and pollutant effects in biomonitoring of coastal marine environment, facilitating the application of monitoring data in risk-based decision making

Naphthenic acids (NAs) are an important group of organic pollutants mainly found in hydrocarbon deposits. Although these compounds are toxic, recalcitrant, and persistent in the environment, we are just learning the diversity of microbial communities involved in NAs- degradation and the mechanisms by which NAs are biodegraded. Studies have shown that naphthenic acids are susceptible to biodegradation, which decreases their concentration and reduces toxicity. Nevertheless, little is still known about their biodegradability. The present PhD Thesis’s work is aimed to study the biodegradation of simple model NAs using bacteria strains belonging to the Rhodococcus genus. In particular, Rh. sp. BCP1 and Rh. opacus R7 were able to utilize NAs such as cyclohexane carboxylic acid and cyclopentane carboxylic acid as the sole carbon and energy sources, even at concentrations up to 1000 mg/L. The presence of either substituents or longer carboxylic acid chains attached to the cyclohexane ring negatively affected the growth by pure bacterial cultures. Moreover, BCP1 and R7 cells incubated in the presence of CHCA or CPCA show a general increase of saturated and methyl-substituted fatty acids in their membrane, while the cis-mono-unsaturated ones decrease, as compared to glucose-grown cells. The observed lipid molecules modification during the growth in the presence of NAs is suggested as a possible mechanism to decrease the fluidity of the cell membrane to counteract NAs toxicity. In order to further evaluate this toxic effect on cell features, the morphological changes of BCP1 and R7 cells were also assessed through Transmission Electron Microscopy (TEM), revealing interesting ultrastructural changes. The induction of putative genes, and the construction of a random transposon mutagenesis library were also carried out to reveal the mechanisms by which these Rhodococcus strains can degrade toxic compounds such as NAs.

The transcribed ultraconserved regions (T-UCRs) are a group of long non-coding RNAs involved in human carcinogenesis. The factors regulating the expression of T-UCRs and their mechanism of action in human cancers are unknown. In this work it was shown that high expression of uc.339 associates with lower survival in 204 non-small cell lung cancer (NSCLC) patients. Moreover, it was shown that uc.339 found up-regulated in archival NSCLC samples, acts as a decoy RNA for miR-339-3p, -663-3p and -95-5p. So, Cyclin E2, a direct target of three microRNAs is up-regulated, inducing cancer growth and migration. Evidence of this mechanism was provided from cell lines and primary samples confirming that TP53 directly regulates uc.339. These results support a key role for uc.339 in lung cancer.

The study of optic flow on postural control may explain how self-motion perception contributes to postural stability in young males and females and how such function changes in the old falls risk population. Study I: The aim was to examine the optic flow effect on postural control in young people (n=24), using stabilometry and surface-electromyography. Subjects viewed expansion and contraction optic flow stimuli which were presented full field, in the foveral or in the peripheral visual field. Results showed that optic flow stimulation causes an asymmetry in postural balance and a different lateralization of postural control in men and women. Gender differences evoked by optic flow were found both in the muscle activity and in the prevalent direction of oscillation. The COP spatial variability was reduced during the view of peripheral stimuli which evoked a clustered prevalent direction of oscillation, while foveal and random stimuli induced non-distributed directions. Study II was aimed at investigating the age-related mechanisms of postural stability during the view of optic flow stimuli in young (n=17) and old (n=19) people, using stabilometry and kinematic. Results showed that old people showed a greater effort to maintain posture during the view of optic flow stimuli than the young. Elderly seems to use the head stabilization on trunk strategy. Visual stimuli evoke an excitatory input on postural muscles, but the stimulus structure produces different postural effects. Peripheral optic flow stabilizes postural sway, while random and foveal stimuli provoke larger sway variability similar to those evoked in baseline. Postural control uses different mechanisms within each leg to produce the appropriate postural response to interact with extrapersonal environment. Ageing reduce the effortlessness to stabilize posture during optic flow, suggesting a neuronal processing decline associated with difficulty integrating multi-sensory information of self-motion perception and increasing risk of falls.

L’anguilla europea, è una specie eurialina catadroma con un complesso ciclo biologico: l’area di riproduzione, unica, si trova molto distante da quella di distribuzione. La specie necessita di una gestione dello stock a fini conservazionistici. Il problema è europeo: lo stock è unico, distribuito in Europa e nell’Africa settentrionale, si riproduce in Atlantico ed è panmittico. C’è preoccupazione per il declino del reclutamento e delle catture di adulti. Lo scopo del progetto è di individuare possibili unità di stock nella penisola italiana. La ricerca è basata sullo studio degli otoliti mediante analisi morfometrica e microchimica. I contorni degli otoliti sono sottoposti ad analisi ellittica di Fourier per individuare eventuali gruppi. Gli otoliti sono stati levigati per effettuare: letture d’età, indagini microstrutturali al SEM delle fasi larvali, analisi microchimiche LA-ICP-MS del nucleo, studiarne l’origine e valutare l’ambiente di sviluppo. Le indagini morfometriche mostrano evidenti pattern ontogenetici, ma non legati ocorrelati alla località, sesso o anno di nascita. Le indagini microstrutturali hanno evidenziano l’alto contenuto organico nucleare, un pattern comune di crescita ed eventi chiave delle fasi larvali, con una media di 212 anelli giornalieri. La microchimica rivela che le larve si sviluppano in acque salate fino alla metamorfosi, poi migrano verso acque meno salate. Le analisi su campioni nati nello stesso anno, evidenziano due gruppi: individui di rimonta naturale e individui di ripopolamento. I profili nucleo bordo evidenziano la permanenza a salinità intermedie degli adulti. L’attività di ricerca si è dimostrata proficua dal punto di vista tecnico con la messa a punto di protocolli innovativi e con forti ricadute sulla riduzione dei tempi e costi d’analisi. Il debole segnale di possibili unità di stock andrà verificato in futuro mediante analisi più dettagliate discriminando meglio la storia di ogni singolo individuo. / The European eel is euryhaline catadromous with a complex life cycle: spawning area, unique, is far from that of distribution. The species needs a stock management and its conservation. The problem is European: the stock is unique, distributed in Europe and North Africa, spawning in Atlantic Sea and is panmictic. There is concern about the decline of recruitment and catches of adults. The purpose of the project is to identify possible units of stock in the Italian peninsula. The research is based on the study of otoliths by morphometric and microchemical analyses . The otoliths contours are analyzed by elliptic Fourier function to discover any groups. Otoliths were polished to make: ageing, SEM microstructural investigations of larval stages, LA-ICP-MS microchemical analysis of the core to study the origin and along a transect core to edge to evaluate the development environment. Morphometric investigations show obvious ontogenetic pattern, but no location, gender or year of birth correlation. The microstructural investigations showed high organic content in the core, a common pattern of growth and key events of the larval stages, with an average of 212 daily rings. The microchemical reveals that the larvae develop into salt water until metamorphosis, then migrate to waters less salty. Analyses of samples born in the same year, showed two groups: wild and restocked. The profiles confirm that the adult stay in brackishwater. The development of innovative protocols and improved technics have guaranteed a strong reduction of time and cost of analysis. The weak signal of possible stock units will be further tested through more detailed analysis discriminating about the life history of each individual.

Mutations in OPA1 gene have been identified in the majority of patients with Dominant Optic Atrophy (DOA), a blinding disease, and the syndromic form DOA-plus. OPA1 protein is a mitochondrial GTPase involved in various mitochondrial functions, present in humans in eight isoforms, resulting from alternative splicing and proteolytic processing. In this study we have investigated the specific role of each isoform through expression in OPA-/- MEFs, by evaluating their ability to improve the defective mitochondrial phenotypes. All isoforms were able to rescue the energetic efficiency, mitochondrial DNA (mtDNA) content and cristae integrity, but only the presence of both long and short forms could recover the mitochondrial morphology. In order to identify the OPA1 protein domains crucial for its functions, we selected and modified the isoform 1, shown to be one of the most efficient in preserving mitochondrial phenotype, to express three specific OPA1 variants, namely: one with a different N-terminus portion, one unable to generate short form owing to deletion of S1 cleavage site and one with a defective GTPase domain. We demonstrated that the simultaneous presence of the N- and C-terminus of OPA1 was essential for the mtDNA maintenance; a cleavable isoform generating s-forms was necessary to completely rescue the energetic competence and the presence of the C-terminus was sufficient to partially recover the cristae ultrastructure. Lastly, several pathogenic OPA1 mutations were inserted in MEF clones and the biochemical features investigated, to correlate the defective phenotypes with the clinical severity of patients. Our results clearly indicate that this cell model reflects very well the clinical characteristics of the patients, and therefore can be proposed as an useful tool to shed light on the pathomechanism underlying DOA.