Use of Biotechnology to increase the content of bioactive compounds in fermented foods of plant origin

The objectives of this PhD research were: i) to evaluate the use of bread making process to increase the content of β-glucans, resistant starch, fructans, dietary fibers and phenolic compounds of kamut khorasan and wheat breads made with flours obtained from kernels at different maturation stage (at milky stage and fully ripe) and ii) to study the impact of whole grains consumption in the human gut. The fermentation and the stages of kernel development or maturation had a great impact on the amount of resistant starch, fructans and β-glucans as well as their interactions resulted highly statistically significant. The amount of fructans was high in kamut bread (2.1g/100g) at the fully ripe stage compared to wheat during industrial fermentation (baker’s yeast). The sourdough increases the content of polyphenols more than industrial fermentation especially in bread made by flour at milky stage. From the analysis of volatile compounds it resulted that the sensors of electronic nose perceived more aromatic compound in kamut products, as well as the SPME-GC-MS, thus we can assume that kamut is more aromatic than wheat, so using it in sourdough process can be a successful approach to improve the bread taste and flavor. The determination of whole grain biormakers such as alkylresorcinols and others using FIE-MS AND GC-tof-MS is a valuable alternative for further metabolic investigations. The decrease of N-acetyl-glucosamine and 3-methyl-hexanedioic acid in kamut faecal samples suggests that kamut can have a role in modulating mucus production/degradation or even gut inflammation. This work gives a new approach to the innovation strategies in bakery functional foods, that can help to choose the right or best combination between stages of kernel maturation-fermentation process and baking temperature.

Microbiome and resitome and foods of animal origin

To ensure food safety and to prevent food-borne illnesses, rapid and accurate detection of pathogenic agents is essential. It has already been demonstrated that shotgun metagenomic sequencing can be used to detect pathogens and their antibiotic resistance genes in food. In the studies presented in this thesis, the application shotgun metagenomic sequencing has been applied to investigate both the microbiome and resistome of foods of animal origin in order to assess advantages and disadvantages of shotgun metagenomic sequencing in comparison to the cultural methods. In the first study, it has been shown that shotgun metagenomics can be applied to detect microorganisms experimentally spiked in cold-smoked salmon. Nevertheless, a direct correlation between cell concentration of each spiked microorganism and number of corresponding reads cannot be established yet. In the second and third studies, the microbiomes and resistomes characterizing caeca and the corresponding carcasses of the birds reared in the conventional and antibiotic free farms were compared. The results highlighted the need to reduce sources of microbial contamination and antimicrobial resistance not only at the farm level but also at the post-harvest one. In the fourth study, it has been demonstrated that testing a single aliquot of a food homogenate is representative of the whole homogenate because biological replicates displayed overlapping taxonomic and functional composition. All in all, the results obtained confirmed that the application of shotgun metagenomic sequencing represents a powerful tool that can be used in the identification of both spoilage and pathogenic microorganism, and their resistome in foods of animal origin. However, a robust relationship between sequence read abundance and concentration of colony-forming unit must be still established.

The integration of immigrant-origin youths in Japan

The steadily growing immigration phenomenon in today’s Japan is showing a tangible and expanding presence of immigrant-origin youths residing in the country. International research in the migration studies area has underlined the importance of focusing on immigrant-origin youths to shed light on the character of the way immigrant incorporate in countries of destinations. In-deed, immigrants’ offspring, the adults of tomorrow, embody the interlocutor between first-generation immigrants and the receiving societal context. The extent of the presence of immigrants’ children in countries of destination is also a reliable yardstick to assess the maturation of the migration process, transforming it from a temporary phenomenon to a long-term settlement. Within this framework, the school is a privileged site to observe and analyze immigrant-origin youths’ integration. Alongside their family and peers, school constitutes one of the main agents of socialization. Here, children learn norms and rules and acquire the necessary tools to eventually compete in the pursuit of an occupation, determining their future socioeconomic standing. This doctoral research aims to identify which theoretical model articulated in the area of migration studies best describes the adaptation process of immigrant-origin youths in Japan. In particular, it examines whether (and to what extent) any of the pre-existing frameworks can help explain the Japanese occurring circumstances, or whether further elaboration and adjustment are needed. Alternatively, it studies whether it is necessary to produce a new model based on the peculiarities of the Japanese social context. This study provides a theoretical-oriented contribution to the (mainly descriptive but maturing) literature on immigrant-origin youths’ integration in Japan. Considering past growth trends of Japanese immigration and its expanding prospective projections (Korekawa 2018c), this study might be considered pioneering for future development of the phenomenon.

Artisanal food productions of animal origin: exploring food safety in the age of Whole Genome Sequencing

The artisanal food chain is enriched by a wide diversity of local food productions with delightful organoleptic characteristics and valuable nutritional properties. Despite their increasing worldwide popularity and appeal, several food safety challenges are addressed in artisanal facilities context suffering from less standardized processing conditions. In such scenario, recent advances in molecular typing and genomic surveillance (e.g., Whole Genome Sequencing [WGS]) represent an unprecedent solution capable of inferring sources of contamination as well as contributing to food safety along the artisanal food continuum. The overall objective of this PhD thesis was to explore potential microbial hazards among different artisanal food productions of animal origins (dairy and meat-derived) typical of the food culture and heritage landscape belonging to Mediterranean countries. Three different studies were then carried out, specifically focussing on: 1) compare the seasonal variability of microbiological quality and potential occurrence of microbial hazards in two batches of Italian artisanal fermented dairy and meat productions; 2) Investigate genetic relationships as well as virulome and resistome of foodborne pathogens isolated within dairy and meat-derived productions located in Italy, Spain, Portugal and Morocco; 3) investigate the population structure, virulome, resistome and mobilome of Klebsiella spp. isolates collected from study 1, including an extended range of public sequences.

Bioimaging of animal embryogenesis: mathematical methods and computational algorithms

Some fundamental biological processes such as embryonic development have been preserved during evolution and are common to species belonging to different phylogenetic positions, but are nowadays largely unknown. The understanding of cell morphodynamics leading to the formation of organized spatial distribution of cells such as tissues and organs can be achieved through the reconstruction of cells shape and position during the development of a live animal embryo. We design in this work a chain of image processing methods to automatically segment and track cells nuclei and membranes during the development of a zebrafish embryo, which has been largely validates as model organism to understand vertebrate development, gene function and healingrepair mechanisms in vertebrates. The embryo is previously labeled through the ubiquitous expression of fluorescent proteins addressed to cells nuclei and membranes, and temporal sequences of volumetric images are acquired with laser scanning microscopy. Cells position is detected by processing nuclei images either through the generalized form of the Hough transform or identifying nuclei position with local maxima after a smoothing preprocessing step. Membranes and nuclei shapes are reconstructed by using PDEs based variational techniques such as the Subjective Surfaces and the Chan Vese method. Cells tracking is performed by combining informations previously detected on cells shape and position with biological regularization constraints. Our results are manually validated and reconstruct the formation of zebrafish brain at 7-8 somite stage with all the cells tracked starting from late sphere stage with less than 2% error for at least 6 hours. Our reconstruction opens the way to a systematic investigation of cellular behaviors, of clonal origin and clonal complexity of brain organs, as well as the contribution of cell proliferation modes and cell movements to the formation of local patterns and morphogenetic fields.

B-type cytochromes of the DOMON domain superfamily (Novel redox elements of plant plasma membrane)

The aim of the present study is understanding the properties of a new group of redox proteins having in common a DOMON-type domain with characteristics of cytochromes b. The superfamily of proteins containing a DOMON of this type includes a few protein families. With the aim of better characterizing this new protein family, the present work addresses both a CyDOM protein (a cytochrome b561) and a protein only comprised of DOMON(AIR12), both of plant origin. Apoplastic ascorbate can be regenerated from monodehydroascorbate by a trans-plasma membrane redox system which uses cytosolic ascorbate as a reductant and comprises a high potential cytochrome b. We identified the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis auxin-responsive gene air12. The protein, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol-modification signal, and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, to be fully reduced by ascorbate and fully oxidized by monodehydroascorbate. Redox potentiometry suggests that AIR12 binds two high-potential hemes (Em,7 +135 and +236 mV). Phylogenetic analyses reveal that the auxin-responsive genes AIR12 constitute a new family of plasma membrane b-type cytochromes specific to flowering plants. Although AIR12 is one of the few redox proteins of the PM characterized to date, the role of AIR12 in trans-PM electron transfer would imply interaction with other partners which are still to be identified. Another part of the present project was aimed at understanding of a soybean protein comprised of a DOMON fused with a well-defined b561 cytochrome domain (CyDOM). Various bioinformatic approaches show this protein to be composed of an N-terminal DOMON followed by b561 domain. The latter contains five transmembrane helices featuring highly conserved histidines, which might bind haem groups. The CyDOM has been cloned and expressed in the yeast Pichia pastoris, and spectroscopic analyses have been accomplished on solubilized yeast membranes. CyDOM clearly reveal the properties of b-type cytochrome. The results highlight the fact that CyDOM is clearly able to lead an electron flux through the plasmamembrane. Voltage clamp experiments demonstrate that Xenopus laevis oocytes transformed with CyDOM of soybean exhibit negative electrical currents in presence of an external electron acceptor. Analogous investigations were carried out with SDR2, a CyDOM of Drosophila melanogaster which shows an electron transport capacity even higher than plant CyDOM. As quoted above, these data reinforce those obtained in plant CyDOM on the one hand, and on the other hand allow to attribute to SDR2-like proteins the properties assigned to CyDOM. Was expressed in Regenerated tobacco roots, transiently transformed with infected a with chimeral construct GFP: CyDOM (by A. rhizogenes infection) reveals a plasmamembrane localization of CyDOM both in epidermal cells of the elongation zone of roots and in root hairs. In conclusion. Although the data presented here await to be expanded and in part clarified, it is safe to say they open a new perspective about the role of this group of proteins. The biological relevance of the functional and physiological implications of DOMON redox domains seems noteworthy, and it can but increase with future advances in research. Beyond the very finding, however interesting in itself, of DOMON domains as extracellular cytochromes, the present study testifies to the fact that cytochrome proteins containing DOMON domains of the type of “CyDOM” can transfer electrons through membranes and may represent the most important redox component of the plasmamembrane as yet discovered.

Study of the building and decorative materials and techniques used in Hungarian Roman sites

The aims of this research were: - To identify the characteristics, properties and provenance of the building and decorative material found in three Hungarian Roman sites: Nagyharsány, Nemesvámos-Balácapuszta and Aquincum - To provide a database of information on the different sites - To have an overview of main conservation strategies applied in Hungary. Geological studies, macroscopical and microscopical observations, XRD investigations, physical and chemical analyses allowed us to define the characteristics and properties of the different kinds of collected materials. Building stones sampled from Nagyharsány site showed two different kinds of massive limestone belonging to the areas surrounding the villa. Also Building stones sampled from Nemesvámos-Balácapuszta Roman villa proved to be compatible with limestone belonging to local sources. Mural painting fragments show that all samples are units composed of multilayered structures. Mosaic tesserae can be classified as following: -Pale yellow , blackish and pink tesserae are comparable with local limestone; -White tessera, composed of marble, was probably imported from distant regions of the Empire, as the usual practice of Romans. Mortars present different characteristics according to the age, the site and the functions: -Building mortars are generally lime based, white or pale yellow in colour, present a high percentage of aggregates represented by fine sand; -Supporting mortars from both mosaics and mural paintings are reddish or pinkish in colour, due to the presence of high percentage of brick dust and tiles fragments, and present a higher content of MgO. Although the condition of the sites, there is an insignificant content of soluble salts. Database The whole study has allowed us to provide work sheets for each samples, including all characteristics and properties. Furthermore, all sites included in the frame of the research have been described and illustrated on the base of their floor plans, material and construction methodologies. It can be concluded that: 1. In Nagyharsány Archaeological site, it is possible to define a sequence of different construction phases on the base of the study of building material and mortars. The results are comparable with the chronology of the site provided by the archaeologists 2. The material used for construction was of local origin while the more precious ones, used for decorative elements, were probably imported from long distance 3. Construction techniques in Hungary mainly refer to the usual Roman knowledge and practice (Vitruvius); few differences have been found 4. The database will represent an archive for Archaeologists, Historians and Conservators dealing with Roman period in Hungary.

Functional and Genetic characterization of new genomic islands from an E. coli strain associated with neonatal meningitis.

Enterobacteriaceae genomes evolve through mutations, rearrangements and horizontal gene transfer (HGT). The latter evolutionary pathway works through the acquisition DNA (GEI) modules of foreign origin that enhances fitness of the host to a given environment. The genome of E. coli IHE3034, a strain isolated from a case of neonatal meningitis, has recently been sequenced and its subsequent sequence analysis has predicted 18 possible GEIs, of which: 8 have not been previously described, 5 fully meet the pathogenic island definition and at least 10 that seem to be of prophagic origin. In order to study the GEI distribution of our reference strain, we screened for the presence 18 GEIs a panel of 132 strains, representative of E. coli diversity. Also, using an inverse nested PCR approach we identified 9 GEI that can form an extrachromosomal circular intermediate (CI) and their respective attachment sites (att). Further, we set up a qPCR approach that allowed us to determine the excision rates of 5 genomic islands in different growth conditions. Four islands, specific for strains appertaining to the sequence type complex 95 (STC95), have been deleted in order to assess their function in a Dictyostelium discoideum grazing assays. Overall, the distribution data presented here indicate that 16 IHE3034 GEIs are more associated to the STC95 strains. Also the functional and genetic characterization has uncovered that GEI 13, 17 and 19 are involved in the resistance to phagocitation by Dictyostelium d thus suggesting a possible role in the adaptation of the pathogen during certain stages of infection.

Ricerca di Escherichia coli produttori di verocitotossine e definizione di obiettivi di performance nella produzione primaria di alimenti di origine animale

La presenza di Escherichia coli produttori di verocitotossine (VTEC o STEC) rappresenta una tra le più importanti cause di malattia alimentare attualmente presenti in Europa. La sua presenza negli allevamenti di animali destinati alla produzione di alimenti rappresenta un importante rischio per la salute del consumatore. In conseguenza di comuni contaminazioni che si realizzano nel corso della macellazione, della mungitura i VTEC possono essere presenti nelle carni e nel latte e rappresentano un grave rischio se la preparazione per il consumo o i processi di lavorazione non comportano trattamenti in grado d’inattivarli (es. carni crude o poco cotte, latte non pastorizzato, formaggi freschi a latte crudo). La contaminazione dei campi coltivati conseguente alla dispersione di letame o attraverso acque contaminate può veicolare questi stipiti che sono normalmente albergati nell’intestino di ruminanti (domestici e selvatici) e anche prodotti vegetali consumati crudi, succhi e perfino sementi sono stati implicati in gravi episodi di malattia con gravi manifestazioni enteriche e complicazioni in grado di causare quadri patologici gravi e anche la morte. Stipiti di VTEC patogeni ingeriti con gli alimenti possono causare sintomi gastroenterici, con diarrea acquosa o emorragica (nel 50% dei casi), crampi addominali, febbre lieve e in una percentuale più bassa nausea e vomito. In alcuni casi (circa 5-10%) l’infezione gastroenterica si complica con manifestazioni tossiemiche caratterizzate da Sindrome Emolitico Uremica (SEU o HUS) con anemia emolitica, insufficienza renale grave e coinvolgimento neurologico o con una porpora trombotica trombocitopenica. Il tasso di mortalità dei pazienti che presentano l’infezione da E. coli è inferiore all’1%. I dati forniti dall’ECDC sulle infezioni alimentari nel periodo 2006-2010 hanno evidenziato un trend in leggero aumento del numero di infezioni a partire dal 2007. L’obiettivo degli studi condotti è quello di valutare la prevalenza ed il comportamento dei VTEC per una analisi del rischio più approfondita.

Characterization of optical transduction-based molecular systems and nanoparticles for the development of chemical sensors

With the increasing importance that nanotechnologies have in everyday life, it is not difficult to realize that also a single molecule, if properly designed, can be a device able to perform useful functions: such a chemical species is called chemosensor, that is a molecule of abiotic origin that signals the presence of matter or energy. Signal transduction is the mechanism by which an interaction of a sensor with an analyte yields a measurable form of energy. When dealing with the design of a chemosensor, we need to take into account a “communication requirement” between its three component: the receptor unit, responsible for the selective analyte binding, the spacer, which controls the geometry of the system and modulates the electronic interaction between the receptor and the signalling unit, whose physico-chemical properties change upon complexation. A luminescent chemosensor communicates a variation of the physico-chemical properties of the receptor unit with a luminescence output signal. This thesis work consists in the characterization of new molecular and nanoparticle-based system which can be used as sensitive materials for the construction of new optical transduction devices able to provide information about the concentration of analytes in solution. In particular two direction were taken. The first is to continue in the development of new chemosensors, that is the first step for the construction of reliable and efficient devices, and in particular the work will be focused on chemosensors for metal ions for biomedical and environmental applications. The second is to study more efficient and complex organized systems, such as derivatized silica nanoparticles. These system can potentially have higher sensitivity than molecular systems, and present many advantages, like the possibility to be ratiometric, higher Stokes shifts and lower signal-to-noise ratio.

Metagenomic trajectory of gut microbiome in the human lifespan

Co-evolving with the human host, gut microbiota establishes configurations, which vary under the pressure of inflammation, disease, ageing, diet and lifestyle. In order to describe the multi-stability of the microbiome-host relationship, we studied specific tracts of the bacterial trajectory during the human lifespan and we characterized peculiar deviations from the hypothetical development, caused by disease, using molecular techniques, such as phylogenetic microarray and next-generation sequencing. Firstly, we characterized the enterocyte-associated microbiota in breast-fed infants and adults, describing remarkable differences between the two groups of subjects. Successively, we investigated the impact of atopy on the development of the microbiome in Italian childrens, highlithing conspicuous deviations from the child-type microbiota of the Italian controls. To explore variation in the gut microbiota depending on geographical origins, which reflect different lifestyles, we compared the phylogenetic diversity of the intestinal microbiota of the Hadza hunter-gatherers of Tanzania and Italian adults. Additionally, we characterized the aged-type microbiome, describing the changes occurred in the metabolic potential of the gut microbiota of centenarians with respect to younger individuals, as a part of the pathophysiolology of the ageing process. Finally, we evaluated the impact of a probiotics intervention on the intestinal microbiota of elderly people, showing the repair of some age-related dysbioses. These studies contribute to elucidate several aspects of the intestinal microbiome development during the human lifespan, depicting the microbiota as an extremely plastic entity, capable of being reconfigured in response to different environmental factors and/or stressors of endogenous origin.

The genetic history of Italians: new-insights from uniparentally-inherited markers

Genetic differences among human groups can be ascribed both to the broad-scale extents of pre-historical and historical migrations and to the fine-scale impacts of socio-cultural and geographic heterogeneity. In this thesis, the genetic information provided by uniparental markers were exploited to address different aspects of the Italian population history, by combining macro- and micro-geographic investigations at different spatial and temporal scales. To firstly assess the overall Italian variability, Y-chromosome and mtDNA markers were deeply typed in ~900 individuals from continental Italy, Sicily and Sardinia. Sex-biased patterns and contrasting demographic histories were observed for males and females. Differential European and Mediterranean contributions were invoked to explain the paternal genetic sub-structure observed in peninsular Italy, compared to the homogeneous maternal genetic landscape. If Neolithic showed to be one principal determinant of the detected paternal structure, local insights into specific Italian regional contexts highlighted the importance of Post-Neolithic contributions. Among them, migrations from the Balkans (particularly Greece) during late Metal Ages, played a relevant role in the cultural and genetic transitions occurred in Sicily and Southern Italy. On a finer geographic and temporal perspective, the more recent layers of Italian genetic history and some aspects of the gene-culture interaction were assessed by exploring the genetic variability within two “marginal populations”: Arbereshe of Southern Italy and Partecipanza in Northern Italy. The Arbereshe are Albanian-speaking communities settled in Sicily and Calabria since the end of Middle Ages. Despite sharing common genetic and cultural backgrounds, these groups revealed diverging micro-evolutionary histories, implying different founding events and different patterns of cultural isolation and local admixture. Partecipanza is an idiosyncratic institution of Medieval origin aimed at sharing and devolving collective lands. This case-study exemplified that socio-economic stratification within the same population may induce sex-biased genetic structuring and the maintenance of otherwise hidden historical genetic traces.

Evolutionary genetics of lactase persistence in Eurasian human populations

Although ability to digest lactose generally declines after weaning in all mammals, in some human populations it persists also in adult individuals, a condition named lactase persistence (LP). Studies on the prevalence of the LP phenotype in worldwide human populations have shown that the frequency of this trait is highly variable in different ethnic groups, appearing to be positively correlated with the importance of milk in the diet. In particular, several single-nucleotide polymorphisms (SNPs) in the proximity of the LCT gene have been proved to be associated with LP. Nevertheless, few studies have till now analyzed genetic variation underlying LP in a wide set of Eurasian populations and, especially, in the Italian one. In the present study, we thus typed 40 SNPs surrounding the LCT gene in more than 1,000 samples from Italian and Arabic peninsulas to investigate patterns of LP-related genetic diversity in two regions which have played a pivotal role in the recent human evolutionary history according to their geographical position and historical/archaeological records. Our results underline a high and complex variability of the explored genomic region in both studied populations. In particular, a clear diversification of Northern Italian groups from the rest of the peninsula, was observed, with the formers being genetically more similar to Northern European populations than to Southern Italians. These observation are consistent with known decreasing pattern of LP from Northern to Southern Italy and suggest the possibility of an independent evolution of LP-associated genotypes in Northern Italy. A similar scenario was observed in the Arabian peninsula, with Dhofari Arabs from Southern Oman and Yemeni clustering together with respect to Arabs from Northern Oman and the subgroup of Omanis of Asian origin which appeared instead to be genetically closer to Europeans than to the rest of Arabic groups.

Evaluation of the photosynthetic efficiency of sweet sorghum under drought and cold conditions

Sweet sorghum, a C4 crop of tropical origin, is gaining momentum as a multipurpose feedstock to tackle the growing environmental, food and energy security demands. Under temperate climates sweet sorghum is considered as a potential bioethanol feedstock, however, being a relatively new crop in such areas its physiological and metabolic adaptability has to be evaluated; especially to the more frequent and severe drought spells occurring throughout the growing season and to the cold temperatures during the establishment period of the crop. The objective of this thesis was to evaluate some adaptive photosynthetic traits of sweet sorghum to drought and cold stress, both under field and controlled conditions. To meet such goal, a series of experiments were carried out. A new cold-tolerant sweet sorghum genotype was sown in rhizotrons of 1 m3 in order to evaluate its tolerance to progressive drought until plant death at young and mature stages. Young plants were able to retain high photosynthetic rate for 10 days longer than mature plants. Such response was associated to the efficient PSII down-regulation capacity mediated by light energy dissipation, closure of reaction centers (JIP-test parameters), and accumulation of glucose and sucrose. On the other hand, when sweet sorghum plants went into blooming stage, neither energy dissipation nor sugar accumulation counteracted the negative effect of drought. Two hybrids with contrastable cold tolerance, selected from an early sowing field trial were subjected to chilling temperatures under controlled growth conditions to evaluate in deep their physiological and metabolic cold adaptation mechanisms. The hybrid which poorly performed under field conditions (ICSSH31), showed earlier metabolic changes (Chl a + b, xanthophyll cycle) and greater inhibition of enzymatic activity (Rubisco and PEPcase activity) than the cold tolerant hybrid (Bulldozer). Important insights on the potential adaptability of sweet sorghum to temperate climates are given.