The aim of this project was to record the Traditional Local Knowledge (TLK) concerning the traditional uses of wild food plants together with all the practices linked as gathering, processing, cooking, including the therapeutic uses, to re-discover plant species often under-utilized or neglected and to identify those with new or underestimated healthy effects for human people. This research was performed in two areas belonging to the province of the city of Bologna (Emilia-Romagna region, Northern Italy) and in the Middle Agri Valley (Potenza province, Basilicata region, Southern Italy). Up to now no research has been carried out on the use of wild edibles in these territories and, therefore, this study represents the first attempt to collect and save from oblivion an important part of the cultural heritage preserved by these populations. Using an ethnobotanical approach, people still retaining TLK about wild food plants were interviewed recording the edible species and related practices used. By means of the Relative Frequency of Citation index (RFC), it was possible to state that Crepis vesicaria subsp. taraxacifolia (Thuill) Thell and Cichorium intybus L. were the most recognised and important wild food species respectively in the area of Bologna and in the Middle Agri Valley. The results of the two investigated areas were compared. The ethnobotanical study was followed by a metabolic screening of 34 plants samples mentioned by the informants (13 collected in Bologna’s area and 21 in Middle Agri Valley). Sanguisorba minor Scop. showed the highest antioxidant activity and polyphenol content; Mentha spicata L. exhibited the highest flavonoid content; Sinapis arvensis L. revealed the highest content of chlorophylls and carotenoids and Clematis vitalba L. the highest protein content. Finally, the most considerable and cited species in the area of Bologna, Crepis vesicaria subsp. taraxacifolia (Thuill) Thell, was analysed by an untargeted metabolomics approach.

Gli elementi trasponibili (TEs) sono componenti universali dei genomi di tutti gli esseri viventi. L'attività ed il movimento degli TEs hanno importanti effetti sul genoma ospite e per questo sono considerati dei fattori importanti nell'evoluzione del genoma. Le relazioni che intercorrono tra TEs e il loro genoma ospite sono ancora ampiamente dibattute. Hickey già nel 1982 suggerì che queste relazioni potevano essere influenzate dalla modalità di riproduzione del genoma ospite. Organismi bisessuali, attraverso i meccanismi propri della meiosi, riuscirebbero a contrastare la proliferazione degli TEs, al contrario negli organismi unisessuali, proprio a causa della mancanza di questi meccanismi, gli TEs tendono a proliferare ed accumulare all'interno del genoma. Al fine di valutare queste ipotesi, ho isolato e caratterizzato gli TEs in organismi con strategie riproduttive non canoniche. Ho condotto le mie analisi in due organismi: gli insetti stecco del genere Bacillus e nel fossile vivente Triops cancriformis. Entrambi presentano strategie riproduttive che vanno dal gonocorismo alla partenogenesi, rendendoli un eccellente modello per lo studio e la caratterizzazione degli TEs. Nel genere Bacillus mi sono focalizzata sullo studio del retrotransposone non-LTR R2. Ho isolato e caratterizzato sette elementi R2 completi; sia in specie gonocoriche che partenogenetiche. Ho trovato un elemento R2 degenerato presente nel genoma di B. rossius da almeno 5 milioni di anni. In oltre, le mie analisi suggeriscono per la prima volta che anche gli elementi R2 possono trasferirsi attraverso eventi di trasferimento orizzontale. I miei dati mostrano una panoramica della composizione di TEs nel genoma di una popolazione partenogenetica di Triops cancriformis. Dalle mie analisi si evince che il 20% della library di triops è composta da TEs. / Transposable elements (TEs) are universal components of all living organisms. TEs activity and movement have profound effects on host genome and today many researchers agree to consider them as important actors in genome evolution. The relationships between TEs and their host genomes are still under debate. Different hypotheses were proposed to explain TEs dynamics in host genome; one of these propose that host reproductive strategies can influence TEs evolutionary dynamics (Hickey, 1982). In fact, bisexual organisms, through homologous chromosomes recombination and reassortment during meiosis and amphimixis, can control the spread and proliferation of mobile elements, while unisexual organisms would experience an increase of these elements density due to the inability to eliminate them through exclusive mechanisms of sexual reproduction. In order to evaluate these hypothesis, I isolated and characterized TEs in organisms with non-canonical reproductive strategies. I performed my analyses in two organisms: the stick insects of the Bacillus genus and in the tadpole shrimps T. cancriformis. In both instances reproductive strategies range from bisexual gonochoric reproduction, to unisexual parthenogenesis, making them an excellent model for the study and characterization of TEs. In the Bacillus genus I focused on the R2 non-LTR retrotranposon. I isolated and sequenced by primer walking seven R2 complete elements; both gonochoric and parthenogenetic Bacillus species. I found a R2 degenerate element present in the B. rossius genome since 5 Myr ago. In addition, my analyses for the first time suggest that also R2 retrotransposons can use horizontal transfer as a strategy to colonize a new genome. My data showed a TEs overview in a T. cancriformis parthenogenetic population. In contrast with the theoretical, my analyses highlighted that the 20% of the library is composed by TEs, in which both TEs classes are widely represented (class I, 11.4% and class II, 8.9%).

Neisseria meningitidis is a strictly human pathogen and is a major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a surface-exposed lipoprotein that binds human factor H (hfH) allowing the bacterium to evade the host innate immunity response. Of note, fHbp is a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, its level of expression varies among isolates and may influence strain susceptibility to anti-fHbp antisera. The aim of the study was to understand the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of a panel of 105 invasive strains and we identified nine fIR sequence types which represent 77% of the isolates. By mass spectrometry we obtained an absolute quantification of fHbp in the same panel of strains and found a correlation between the fIR type and fHbp amounts. By the generation of a series of isogenic recombinant strains, where fHbp expression was under the control of each of the nine fIR types, we were able to confirm that the fIR sequence determines a specific level of expression and investigate the major determinants involved. The quantity of fHbp on the surface of the bacteria correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies in immune sera. The influence of fHbp to mediate the evasion from generic complement-mediated killing presumably through binding of hfH was assessed and survival in human non-immune serum was less correlated with protein amounts measured from an in vitro growth culture. Overall, we demonstrated that the expression level of this antigen can be inferred by the DNA sequence of the fHbp intergenic region. Therefore, our findings can contribute to understand and predict vaccine coverage mediated by fHbp.

Il carcinoma del colon-retto (CCR) comprende tutte le neoplasie con sede tra il cieco ed il retto. E’ il tumore più frequente nella popolazione italiana. Tra le cause che ne influenzano il rischio sono noti sia fattori esogeni che endogeni. La componente genetica riveste un ruolo chiave, infatti le cellule tumorali presentano mutazioni ereditate o acquisite a carico di numerosi geni coinvolti nelle principali vie di segnalazione, replicazione e riparazione. I nostri studi si sono rivolti all’identificazione di modificazioni nell’ambito degli acidi nucleici, mediante analisi di un semplice campione di sangue periferico. Abbiamo voluto indagare sia sul rischio genetico di contrarre la malattia e dunque sulla predittività, che sul riscontro della patologia e dunque sulla diagnosi e sulla prognosi. Studi molecolari hanno difatti evidenziato che alcuni polimorfismi a carico di determinati geni possono contribuire allo sviluppo di patologie tumorali. Il mio lavoro ha mirato, da una parte, a verificare se alcune varianti alleliche dei geni CDH1, MDR1 (ABCB1), ROCK1 e ROCK2 potessero essere considerate fattori di suscettibilità alla patologia, per un possibile impiego in qualità di marcatori predittivi e, dall'altra, ad identificare mRNA marcatori specifici, selezionati tramite un programma bioinformatico utilizzando, per la validazione, campioni di sangue provenienti da pazienti affetti da cancro del colon-retto e controlli sani. L’indagine condotta sui polimorfismi scelti per i geni indagati, ha evidenziato, per alcuni, un’associazione significativa, che mi ha permesso di raccogliere dati interessanti sull’eziologia del CCR. Lo studio di mRNA candidati marcatori, mi ha permesso di identificare TSPAN8, LGALS4 e COL1A2 quali promettenti strumenti di diagnosi e prognosi per il carcinoma del colon-retto. Ulteriori studi saranno necessari per testare i candidati biomarcatori in test di predittività, screening e di follow-up. / The colorectal cancer (CCR) includes all cancers located between the caecum and the rectum. It is the most common cancer in the Italian population. Among the causes that influence the risk they are known both exogenous and endogenous factors. The genetic component plays a key role, in fact, cancer cells have inherited or acquired mutations of many genes involved in major signaling, replication and repair pathways. Our studies were directed to the identification of modifications in the context of nucleic acids, by analyzing a simple peripheral blood sample. We investigated both the genetic risk of contracting the disease and thus the prediction, and on the detection of the disease and thus on diagnosis and prognosis. Molecular studies have indeed shown that some polymorphisms of certain genes may contribute to the development of cancer. My work has focused, on the one hand, to see if some allelic variants of CDH1, MDR1 (ABCB1), ROCK1 and ROCK2 genes could be regarded as factors of susceptibility to disease, for possible use as predictive markers and, on the other hand, to identify specific mRNA markers, selected through a bioinformatics program using, for the validation, blood samples from patients with colorectal cancer and healthy controls. The investigation conducted on polymorphisms chosen for the investigated genes, showed, for some of them, a significant association, which allowed me to gather interesting data on the etiology of the CRC. The study of candidated mRNA markers, allowed me to identify TSPAN8, LGALS4 and COL1A2 such promising diagnostic and prognosis tools for colorectal cancer. Further studies will be needed to test the candidated biomarkers for predictive testing, screening and follow-up.

Around 100 kya, H. sapiens started its colonization of the world, gradually adapting to new environmental conditions and to extremely different alimentary resources. Interaction between the human genome and nutritional/climatic conditions is regulated by physiological mechanisms that modulate body temperature and the energetic balance. Genes involved in these processes are thus considered as one of the principal targets of biomedical research focused on chronic metabolic diseases. The aim of this project is to investigate micro-evolutionary processes that shaped metabolic variability among human populations through the description of patterns of genetic diversity and their potential association with phenotypes linked to nutrition and thermoregulation. Particularly the identification of potential adaptation processes was carried on through three major research projects. The first one focused on European populations, which have experienced several migration and adaptation processes during their recent evolutionary history. Through an in silico approach, it was possible to analyse the pattern of genetic variability in Europe for genes involved in different metabolic processes and to find multiple signals of local adaptation to climate. In the second study, thanking advantage from previous researches carried on by our research group on Italian samples and by means of genome-wide approaches, we investigate patterns of genetic variability at three genes associated to metabolic phenotypes along Italy. A targeted analysis has been conducted on samples from several Italian provinces, highlighting a different pattern of variability among distinct macro-areas of the peninsula thus indicating that different adaptation processes may have acted on such loci. Finally, in the last project, taking advantage from data generated for a probiotic treatment case study on individual affected by celiac disease, it was possible to evaluate the influence of this pathologic condition on gut microbiota composition and thus to get evolutionary insights into the complex interplay between gut microbiota and celiac disease susceptibility.

One of the main problems in reconstructing the biological profile of unidentified human remains is the reliability of the methods used. These methods are normally developed on modern identificated human skeletal collections, but human variability can introduce problematic biases when applied on osteological collections from different geografical and cronological contexts. This study test the reliability of selected macroscopic methods for sex determination: the index of sexualization of Acsadi and Nemeskeri, the Phenice method and for the age estimation: the synostosis of cranial sutures (Meindl and Lovejoy), the morphological variations at the pubic symphysis (Suchey and Brooks), the morphological variations at ileum surface (Lovejoy, et al.), the modifications of the sternal end of the fourth rib (Iscan et al.) and the variations of the degree of sacral vertebral body fusion (Belcastro et al.) on a large sample (n.489) of know sex and age from Certosa Cemetery of Bologna and “Colecção Esqueletos Identificados" of Coimbra. Regarding age, for each method was calculated the standard values of bias and inaccuracy. The method of pubic symphysis might be the most reliable for age estimation but we must consider the width of age ranges considered by the method. The values of inaccuracy are over 10 years in individuals older than 50 and 27 years in individuals older than 60. The index of sexualization confirm in more than 99% of cases the known sex and the Phenice method gave good results with the 87% of confirm. The reliability of the tested methods in the two european populations was confirmed, with the pelvis as the most diagnostic district, in particular the pubic symphysis. Nevertheless, the dramatic increase in values of bias and inaccuracy with increasing age confirms the need for new or revised age estimation methods that better suits the study of the remains of older individuals.

Yellowfin tuna (Thunnus albacares; YFT) represents one of the most important seafood commodities in the world. The rationale of this Ph.D. project was identified by prioritizing key issues as objectives for contributing to the conservation of YFT and helping to develop a more realistic stock assessment and sustainable management of this species. Specifically, in this work the current mismatch between the biology and/or ecology and the realized management strategies was highlighted, putting particular emphasis on YFT population structure, which is currently characterized by a high degree of uncertainty at both local and global scale. This general pattern was confirmed by the results obtained using a panel of microsatellite loci, which cannot reject the null hypothesis of the existence of only one panmictic population at the global scale. On the contrary, the access to more powerful and cost effective genetic tools would represent the first step for resolving YFT population structure at both global and local scale. After having evaluated the efficiency and usefulness of 2b-RAD genotyping technique for investigating population genetic structure in highly migratory fish species, a panel of 972 SNPs (Single Nucleotide Polymorphisms) was generated. Using this panel, three distinct populations were identified in the Atlantic, Indian and Pacific Oceans. Additionally, it was possible to define a subset of 33 outlier loci putatively under selection to delineate and separate sub-populations within both the Atlantic and the Pacific Oceans (following an east-west division). Finally, it was emphasized for the first time that in the Atlantic Ocean larger YFT females allocate a greater fraction of surplus energy to egg production than smaller ones, improving noticeably the spawning quality. This result sheds light on the important contribution that larger and most experienced spawners have for the YFT productivity.

Starch is a polymer of D-glucose that plants accumulate as semi-crystalline and osmotically inert granules. Besides being the major energy storage in plants, starch is of primary importance also in human and animal diet, industry and biofuels production. Two kinds of starch, structurally indistinguishable, but different for location and rates of synthesis and degradation are found in plants: secondary starch (characterized by long term-accumulation and found in storage organs) and transitory starch (mainly located in chloroplasts, produced during the day and degraded the subsequent night to meet the energy demand of the plant). Due to its structure, several enzymes are required in starch biosynthesis and degradation, controlling distinct features of starch granules and conferring different physical-chemical properties. Here, the TILLING approach was used on the barley TILLMore population to identify new alleles in five genes related to secondary starch metabolism and known to be expressed in barley seeds. Moreover, the role in Arabidopsis development of the (phospho)glucan, water dikinase proteins (GWD1, GWD2, PWD), known to be involved in nighttime transitory starch degradation, was investigated. Other enzymes, such as β-amylase 1 (BAM1) and α-amylase 3 (AMY3), were demonstrated to be involved in diurnal transitory starch degradation in specialized cells or under stress conditions. Here, we demonstrated that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline, a compatible solute, required to face osmotic stress. Moreover, the behaviour of Arabidopsis BAM1 and AMY3 enzymes under oxidative treatments and the possible role of glutathionylation, a redox post-translational modification occurring mainly under stress conditions and promoted by ROS, were investigated. AtBAM1 and AtAMY3 were found to be sensitive to oxidants and glutathionylated, with a modulatory and protective effect on protein activity.

In this research the phenomenon of monoclonal antibodies (mAbs) cooperative bactericidal activity and the structure of the N.meningitidis antigen NadA var.3 are investigated using Transmission Electron Microscopy (TEM). The mAbs cooperativity is a mechanism that occurs when mAbs that individually show low or no bactericidal activity become bactericidal when coupled together. Revealing the structural bases of cooperative bactericidal activity is fundamental for a thorough understanding of antibody-based mechanisms of protection. Meningococcal factor H binding protein (fHbp), surface-exposed lipoprotein presents in the vaccine against serogroup B Neisseria meningitidis, Bexsero®. A comparison of the structure of the murine cooperative complex with the human cooperative one has been performed revealing a higher level of flexibility and instability of the first complex. Moreover we have been able to prove a simultaneous binding of factor H (fH) to the immune cooperative complex. The 3D structures of the cooperative complexes demonstrated that the angle formed between fHbp-antibodies is orientated only depending on the epitope location on the antigen. Non-cooperative human complexes show a structural dissimilarities compared with the cooperative couples mainly for the absence of the complex formation due to the partial epitopes overlapping, as proved by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX_MS). The NadA var. 3, the variant included in Bexsero® vaccine, structure was determined by the Cryo-Electron Microscopy (Cryo-EM) technique combined with Single Particle (SP) reconstruction method. The 3D reconstruction of NadA var.3, generated from Cryo-TEM data, shows an elongated and thin stalk decorated by a globular compact head characterized by a three-fold symmetry. Moreover the 3D EM map shows three evident points of interruption in the density of the stalk region presumably correlated with the three interruptions present in the coiled-coil periodicity of NadA var.3 the sequence. This result indicates a possible alternative mechanism of the antigen flexibility.

Natural systems face pressures exerted by natural physical-chemical forcings and a myriad of co-occurring human stressors that may interact to cause larger than expected effects, thereby presenting a challenge to ecosystem management. This thesis aimed to develop new information that can contribute to reduce the existing knowledge gaps hampering the holistic management of multiple stressors. I undertook a review of the state-of-the-art methods to detect, quantify and predict stressor interactions, identifying techniques that could be applied in this thesis research. Then, I conducted a systematic review of saltmarsh multiple stressor studies in conjunction with a multiple stressor mapping exercise for the study system in order to infer potential important synergistic stressor interactions. This analysis identified key stressors that are affecting the study system, but also pointed to data gaps in terms of driver and pressure data and raised issues for potentially overlooked stressors. Using field mesocosms, I explored how a local stressor (nutrient availability) affects the responses of saltmarsh vegetation to a global stressor (increased inundation) in different soil types. Results indicate that saltmarsh vegetation would be more drastically affected by increased inundation in low than in medium organic matter soils, and especially in estuaries already under high nutrient availability. In another field experiment, I examined the challenges of managing co-occurring and potentially interacting local stressors on saltmarsh vegetation: recreational trampling and smothering by deposition of excess macroalgal wrack due to high nutrient loads. Trampling and wrack prevention had interacting effects, causing non-linear responses of the vegetation to simulated management of these stressors, such that vegetation recovered only in those treatments simulating the combined prevention of both stressors. During this research I detected, using molecular genetic methods, a widespread presence of S. anglica (and to a lesser extent S. townsendii), two previously unrecorded non-native Spartinas in the study areas.