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.

Development of machine learning methods for multi-modal biomarkers detection and integration

In medicine, innovation depends on a better knowledge of the human body mechanism, which represents a complex system of multi-scale constituents. Unraveling the complexity underneath diseases proves to be challenging. A deep understanding of the inner workings comes with dealing with many heterogeneous information. Exploring the molecular status and the organization of genes, proteins, metabolites provides insights on what is driving a disease, from aggressiveness to curability. Molecular constituents, however, are only the building blocks of the human body and cannot currently tell the whole story of diseases. This is why nowadays attention is growing towards the contemporary exploitation of multi-scale information. Holistic methods are then drawing interest to address the problem of integrating heterogeneous data. The heterogeneity may derive from the diversity across data types and from the diversity within diseases. Here, four studies conducted data integration using customly designed workflows that implement novel methods and views to tackle the heterogeneous characterization of diseases. The first study devoted to determine shared gene regulatory signatures for onco-hematology and it showed partial co-regulation across blood-related diseases. The second study focused on Acute Myeloid Leukemia and refined the unsupervised integration of genomic alterations, which turned out to better resemble clinical practice. In the third study, network integration for artherosclerosis demonstrated, as a proof of concept, the impact of network intelligibility when it comes to model heterogeneous data, which showed to accelerate the identification of new potential pharmaceutical targets. Lastly, the fourth study introduced a new method to integrate multiple data types in a unique latent heterogeneous-representation that facilitated the selection of important data types to predict the tumour stage of invasive ductal carcinoma. The results of these four studies laid the groundwork to ease the detection of new biomarkers ultimately beneficial to medical practice and to the ever-growing field of Personalized Medicine.

Dynamic food systems and climate-induced risks transmission for food and nutritional security

Agriculture market instability impedes achieving the global goal of sustainable and resilient food systems. Currently, the support to producers reaches the mammoth USD 540 billion a year and is projected to reach USD 1.8 trillion by 2030. This gigantic increase requires a repurposing agricultural support strategy (RASS), considering the market country-specific circumstances. These circumstances may vary with geographic locations, marketing structures, and product value chains. The fruit production system is crucial for health-conscious consumers and profit-oriented producers for food and nutritional security. Export is one of the main driving forces behind the expansion of the fruit sector, and during the year 2010-2018, trade significantly outpaced production increases. The previous literature states that irregular and unpredictable behaviour — Chaos — can arise from entirely rational economic decision-making within markets. Different markets' direct/indirect linkages through trade create trade hubs, and uncertainty may function as an avenue to transmit adverse shocks and increase vulnerability rather than contribute to resilience. Therefore, distinguishing Chaos into an endogenous and exogenous pattern of behaviour is cradled to formulate an effective RASS for resilient food systems and to understand global food crises. The present research is aimed at studying the market dynamics of three regional trade hubs, i.e., Brazil (South America), Italy (Europe), and Pakistan (Asia), each representing advanced to traditional value chains to control uncertainty (risks). The present research encompasses 1) a systematic review to highlight the research dynamism and identify grey-areas of research. Based on the findings, we have investigated the 2) nonlinear impacts of climate-induced price responsiveness in monopsony markets. Once we highlighted the importance of marketing structures/arrangements, 3) we developed a risk transmission framework to address the co-evolving impacts in complex dynamic interactions.

Investigation of plant genetic-microbiome interactions in different plant species

The rhizosphere, i.e. the soil surrounding the plant roots, and endosphere, i.e. the microbial communities within the plant organs harbors microbes known to influence root and plant physiological processes. An important question is to what extent plant species, genotypes and environmental conditions affect bacterial and fungal communities. The objectives of the first research study were to unravel and compare the rhizospheric microbiota of grape in two independent vineyards using 16S and ITS amplicon sequencing, evaluate location and varietal effects, and test the correlation between bioavailable copper levels and other soil parameters with microbiota composition and diversity. Our results showed that the microbial alpha diversity based on Shannon index differed significantly between vineyards while it did not differ between two grape cultivars. In the second study, we were focusing on different wheat species and genotypes such as Bread Wheat, Wild Emmer Wheat, Domesticated Emmer Wheat, Durum Wheat Landraces, Durum Wheat cultivars, T. monococcum and triticale in two fields located in Bologna and Foggia. The objectives of this research experiment were to elucidate and compare the rhizospheric and endophytic microbiota of 30 diverse wheat genotypes in two different fields using 16S amplicon sequencing. Our results showed that the microbial alpha diversity based on Shannon index differed significantly between fields of Bologna and Foggia, in which Bologna had a higher diversity in respect to Foggia for both rhizospheric and endophytic communities. Using Shannon index there was significant differences, for instance, between Durum Emmer Wheat and Wild Emmer Wheat in Bologna, and between Bread Wheat and Durum Wheat Landraces in Foggia. Our results contribute to understand the role of wheat species and genotype and the filed management on the root-microbe-soil interactions in the perspective of understanding their impact on crop systems sustainability.

RNA interference technology as a potential control method for fruit and horticultural crops pathogens Botrytis cinerea and Plasmopara viticola

Chapter 1, a general introduction on Botrytis cinerea and its threat to crop production is presented. What Botrytis looks like, its life cycle, why it is a threat to agricultural production, its worldwide pest status, and its current state of management is further elaborated on. Chapter 2, a general introduction on Plasmopara viticola, its threat to grape production and management strategies presented. Chapter 3, titled " RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges ", presents the rapid improvement and extensive implementation of RNA interference (RNAi) technology for the management of fungal pathogens. In this chapter, we describe the application of exogenous RNAi involved in plant pathogenic fungi and discuss dsRNA production, formulation, and RNAi delivery methods. Chapter 4, titled " Exogenous dsRNAs against chitin synthase and glucan synthase genes suppress the growth of the pathogenic fungus Botrytis cinerea " addresses two important questions: Is RNAi technology functional for B. cinerea control ? And which target genes can be exploited for RNAi-based B.cinerea disease control ? Upon target genes selections, an exogenous RNAi protocol was set up and we could effectively deliver a known dose of bacterially produced double stranded RNA (dsRNA) to induce RNAi in B. cinerea. Chapter 5, titled " Double-Stranded RNA Targeting Dicer-Like Genes Compromises the Pathogenicity of Plasmopara viticola on Grapevine “, which deals mainly on RNAi induction against Plasmopara viticola. This chapter addresses two main questions: Is RNAi technology functional in contrasting Plasmopara viticola? And which target genes can be exploited for RNAi-based disease control in Plasmopara viticola?. In the last Chapter (Chapter 6) titled “General discussions and perspectives for future research”, the major research findings from this thesis are discussed together with perspectives for future research.

Consumer behaviour: conceptualisation, scale development and validation of agro-food chain fairness

Although there is broad agreement on the need to transition to a fairer agro-food system, consumer potential in shaping a fair food system has often been overlooked. There is no unique definition of the concept of fairness from the consumer’s perspective. In addition, there are no scales in the academic literature that address fairness in its broad sense, as the existing scales focus on specific and limited aspects that provide a partial picture of the concept. Lack of a true and trustworthy measurement of the notion has been a significant barrier to the knowledge of fairness in agro-food systems from the individual-differences perspective. The individual-differences perspective helps explain why some individuals are more likely than others to put emphasis on the extent to which agro-food chains are fair. Individual consumer perception of an ethical problem is followed by the perception of various alternatives that might lead to a solution. Therefore, the current research intends to make two significant contributions by resolving these constraints. First, advance the literature by providing a new viewpoint to understand fairness in the agro-food chain. Indeed, the research provides a comprehensive conceptualisation of fairness that embraces different aspects of fairness and describes the concept in all its facets and nuances. Second, the research provides a valid, reliable, and invariant measurement of the individual disposition toward fairness in agro-food chains by rooting the items in the theoretical underpinnings of the fairness literature. Overall, this research provides a comprehensive suite of approaches and tools to enhance the resilience, integrity and sustainability of agro-food chains.

Exploring mediterranean durum wheat wild relatives and elite diversity panel for future breeding

Durum wheat (Triticum durum) is an important crop that has been used for millennia for human consumption, and modern breeding can take advantage of the wide variability useful for the adaptation to new challenges. Novel beneficial alleles can be found in wild relatives and landraces thus enhancing crop adaptation to many biotic and abiotic stresses. This dissertation considers the source of variability from both before and after wheat domestication, that caused a loss of potentially useful alleles. Chapter 1. is the thesis introduction, which outlines the importance of wheat in the world, providing an historical overview of the domestication, the evolution mechanisms that led to the current forms of durum wheat and the use of wild relatives as a source of germplasm for future breeding programs is crucial. Moreover, the emergence of Z. tritici has been considered as the main pathogen of wheat since it contains extremely high levels of genetic variability and is thus difficult to control. Chapter 2. Considers the contribution of the phenotypic diversity of 242 accessions of Aegilops tauschii from the Open Wild Wheat Consortium, involved in wheat domestication, provided with whole-genome resequencing. The accessions were phenotyped both in the field and in controlled conditions and A k-mer-based GWAS was performed to identify genomic regions involved in useful traits. Chapter 3. Describes the genetic basis of resistance to Z. tritici in a durum wheat elite diversity panel representative of the germplasm bred in Mediterranean. Quantitative trait loci (QTL) analysis results revealed several loci involved in the STB response that were found in several chromosome regions with a high infection rate. The genomic regions associated with STB resistance identified in this study could be of interest for marker assisted selection (MAS) in durum wheat breeding programs.

Adapting fruit production to climate change: how to increase intrinsic fruit quality and save water through deficit irrigation scheduling.

Fruit crops are an important resource for food security, since more than being nutrient they are also a source of natural antioxidant compounds, such as polyphenols and vitamins. However, fruit crops are also among the cultivations threatened by the harmful effects of climate change This study had the objective of investigating the physiological effects of deficit irrigation on apple (2020-2021), sour cherry (2020-2021-2022) and apricot (2021-2022) trees, with a special focus on fruit nutraceutical quality. On each trial, the main physiological parameters were monitored along the growing season: i) stem and leaf water potentials; ii) leaf gas exchanges; iii) fruit and shoot growth. At harvest, fruit quality was evaluated especially in terms of fruit size, flesh firmness and soluble solids content. Moreover, it was performed: i) total phenolic content determination; ii) anthocyanidin concentration evaluation; and iii) untargeted metabolomic study. Irrigation scheduling in apricot, apple and sour cherry is surely overestimated by the decision support system available in Emilia-Romagna region. The water stress imposed on different fruit crops, each during two years of study, showed as a general conclusion that the decrease in the irrigation water did not show a straightforward decrease in plant physiological performance. This can be due to the miscalculation of the real water needs of the considered fruit crops. For this reason, there is the need to improve this important tool for an appropriate water irrigation management. Furthermore, there is also the need to study the behaviour of fruit crops under more severe deficit irrigations. In fact, it is likely that the application of lower water amounts will enhance the synthesis of specialized metabolites, with positive repercussion on human health. These hypotheses must be verified.

Investigating the causes of late fruit drop in ‘Regina’ sweet cherry (Prunus avium)

The presented study aimed to correctly describe the (late) fruit drop pattern of sweet cherry cv. Regina grafted on ‘Gisela 5’ and investigate its internal causes. In the first season, a method to describe the fruit drop pattern was defined and validated. The second season was devoted to a province-based screening of the phenomenon to identify potential influences of environmental, physiological and management factors. The multisite trial involved 6 commercial orchards located at different elevations, from 225 up to 1175m a.s.l. The third season was dedicated to find confirmation of the hypothesis formulated during the previous year. The multisite comparison was maintained but reduced to only two orchards to allow more frequent samplings. It emerged that late fruit drop is a complex phenomenon showing variable intensity: the percentage of late fruit drop ranged from 7 to 76% of the fruitlets set, depending on the orchard and on the year considered. Two main waves of fruitlets drop have been observed: the first one was composed by unfertilized parthenocarpic fruitlets, probably caused by late or missing fertilization, that immediately after bloom already showed smaller diameters and symptoms of senescence; the second one (the focus of this study) was composed by fully developed fruits that at a certain point decreased their growth rate and got senescent. All the late dropped cherries showed an aborted embryo. This sudden change has been observed to be concomitant both with prolonged periods of low temperatures (or sudden severe decreases in the daily Growing Degree Hours accumulation) and with extraordinary high temperatures close to or above 30°C. Other factors, such as the position of the limb within the canopy, its orientation (sunny vs. shady side) or nutrition played only a marginal role. Excessive vigor can increase late fruit drop intensity but is not its main cause.

Applied genomics in livestock: genome-wide association studies and population genomics analysis in pig and rabbit breeds

The domestication and selection processes in pigs and rabbits have resulted in the constitution of multiple breeds with broad phenotypic diversity. Population genomics analysis and Genome-wide association study analysis can be utilized to gain insights into the ancestral origins, genetic diversity, and the presence of lethal mutations across these diverse breeds. In this thesis, we analysed the dataset obtained from three Italian Pig breeds to detect deleterious alleles. We screened the dataset for genetic markers showing homozygous deficiency using two approaches single marker and haplotype-based approach. Moreover, Genome-wide association study analyses were performed to detect genetic markers associated with pigs' reproductive traits. In rabbits, we investigated the application of SNP bead chip for detection signatures of selection in rabbits using different methods. This analysis was implemented for the first time in different fancy and meet rabbit breeds. Multiple approaches were utilized for the detection of the selection of signatures including Fst analysis, ROH analysis, PCAdapt analysis, and haplotype-based analysis. The analysis in pigs was able to identify five putative deleterious SNPs and nine putative deleterious haplotypes in the analysed Italian Pig breeds. The genomic regions of the detected putative deleterious genomic markers harboring loss of function variants such as the Frameshift variant, start lost, and splice donor variant. Those variants are close to important candidate genes such as IGF2BP1, ADGRL4, and HGF. In rabbits, multiple genomic regions were detected to be under selection of signature. These genomic regions harbor candidate genes associated with coat color phenotype (MC1R, TYR, and ASIP), hair structure (LIPH), and body size (HMGA2 and COL2A1). The described results in rabbits and pigs could be used to improve breeding programs by excluding the deleterious genetic markers carriers and incorporating candidate genes for coat color, body size, and meat production in rabbit breeding programs to enhance desired traits

Metabolomics to investigate the effects of treatments on food and of food consumption on health

Metabolomics has established itself as a discipline that can offer a unique point of view on how a technological treatment could impact on the charactersitics of a food. Even more, the same analytical platforms necessary for the purpose can also effectively unravel intricate interactions between such food and human health upon consumption. This PhD thesis investigates the application of metabolomics in understanding the impact of technological treatments on food and their subsequent effects on human health, utilizing 1H-NMR as the analytical platform. The study involves the development of standard operating procedures (SOPs) to ensure a fast and stable preparation of seafood samples, incorporating novel algorithms to enhance the accuracy of metabolome profiles. To gain insight on how metabolomics can allow exploring the effects of a technological treatment on a food, we performed three sets of experiments to investigate the application of metabolomics in studying the impact of high hydrostatic pressure (HHP) treatment on seafood metabolome during storage. The first experiment employs untargeted metabolomic analysis on chill-stored rose shrimp, revealing significant post-HHP treatment metabolic alterations and mechanisms. The investigation is extended to grey mullet in the second experiment, utilizing both untargeted and targeted metabolomic analyses to account for matrix-related effects. The third experiment assesses the targeted metabolome of striped prawns, showing that HHP significantly influences metabolic pathways, positively impacting freshness and taste through alterations in related metabolites. Shifting focus to the effects of food on humans, the study explores the impact of multistrain probiotics on cirrhosis patients using 1H-NMR. The platform reveals notable alterations in glutamine/glutamate metabolism, enhancing the patients' ammonia detoxification capacity. This research underscores the potential of metabolomics in uncovering intricate interactions between technological treatments, food, and human health, providing valuable insights for both the food industry and healthcare interventions.

Applied genomics to monitor the genetic diversity and integrity of honey bee (Apis mellifera) populations and define sustainable strategies to benefit the beekeeping sector

Honey bees are considered keystone species in ecosystem, the effect of harmful pesticides for the honey bees, the action of extreme climatic waves and their consequence on honey bees health can cause the loss of many colonies which could contribute to the reduction of the effective population size and incentive the use of non-autochthonous queens to replace dead colonies. Over the last decades, the use of non-ligustica bee subspecies in Italy has increased and together with the mentioned phenomena exposed native honey bees to hybridization, laeding to a dramatic loss of genetic erosion and admixture. Healthy genetic diversity within honey bee populations is critical to provide tolerance and resistance to current and future threatening. Nowadays it is urgent to design strategies for the conservation of local subspecies and their valorisation on a productive scale. In this Thesis we applied genomics tool for the analysis of the genetic diversity and the genomic integrity of honey bee populations in Italy are described. In this work mtDNA based methods are presented using honey bee DNA or honey eDNA as source of information of the genetic diversity of A. mellifera at different level. Taken together, the results derived from these studies should enlarge the knowledge of the genetic diversity and integrity of the honey bee populations in Italy, filling the gap of information necessary to design efficient conservation programmes. Furthermore, the methods presented in these works will provide a tool for the honey authentication to sustain and valorise beekeeping products and sector against frauds.

Prodotti dell'alveare: la propoli e le sue proprietà benefiche

La propoli è una sostanza naturale prodotta dalle api operaie in seguito alla raccolta di resine ed estratti vegetali; è impiegata nell’alveare come materiale di costruzione con funzione cementante e protettiva. Essendo un prodotto di origine naturale, la sua composizione è estremamente complessa ed allo stesso tempo peculiare: molteplici sono le sostanze chimiche che ne fanno parte, la loro presenza e concentrazione dipende dal luogo d’origine, dall’ape, dalla stagione, dalla specie vegetale bottinata e da numerose altre variabili. È importante considerare la variabilità compositiva della propoli per poterne valutare le caratteristiche qualitative, che ancora oggi non sono ufficialmente definite a livello Europeo, ma che fanno riferimento principalmente all’intensità con cui essa è in grado di determinare effetti benefici nell’uomo. Dalla peculiarità delle molecole che la compongono emergono le sue potenzialità come sostanza antimicrobica, antiossidante, antiinfiammatoria ed antitumorale, caratteristiche che la rendono sempre più spesso oggetto di studi e ricerche scientifiche. In questo studio vengono messi a confronto i risultati ed i metodi analitici degli studi effettuati su tale matrice: ne deriva una forte difformità, data dall’origine dei campioni e dalla impossibilità, ad oggi, di ottenere un prodotto standardizzato.

Modelli PLI per la pianificazione di attività di satelliti

Questo lavoro è incentrato sulla pianificazione delle acquizioni e scaricamento del satellite PLATiNO. Le operazioni permesse a questo satellite sono vincolate da varie attività tra cui acquisizioni, manovre e scaricamento di dati. L'obiettivo finale è quello di soddisfare più richieste possibili massimizzando le operazioni del satellite senza però violare i vincoli imposti. A questo scopo, è stato sviluppato un modello in formulazione MILP per una versione rilassata del problema. In questa Tesi vengono innanzitutto trattati i principali argomenti di programmazione lineare e intera in modo da poter affrontare il modello matematico inerente al problema di downlink di PLATiNO. Successivamente viene descritto nel dettaglio il problema da modellizzare, con particolare attenzione alla strategia di downlink, che costituisce l'aspetto più problematico nella costruzione del modello. Si opta, infatti, per una formulazione mista rilassando i vincoli inerenti allo scaricamento dei dati. Infine, vengono valutate le prestazioni del modello rilassato confrontandolo con la sua versione esatta.

Metodi e modelli per l’analisi della domanda di un prodotto industriale

Il presente lavoro di tesi è stato svolto presso Marr S.p.A.: azienda italiana leader nella distribuzione di prodotti alimentari alla ristorazione extra domestica. Il fulcro dell'attività di MARR S.p.A risiede nell'approvvigionamento dei prodotti ai clientii. Per assicurare un servizio efficiente e redditizio è necessaria un'attenta pianificazione logistica, perciò, il lavoro di questa tesi si è concentrato su due aspetti centrali dell'operazione di riordino merce: la predizione della domanda futura dei prodotti, sulla base di dati riferiti al passato e l'individuazione della quantità effettiva da riordinare all'interno dei magazzini dell'azienda. Si è quindi svolta un'attenta analisi dell'attuale gestione dell'approvvigionamento da parte dell'azienda, quindi, ne sono state messe in luce le principali criticità, le quali risiedono nel fatto che il processo di riordino viene eseguito in maniera manuale sulla base dell'esperienza del Responsabile Acquisti. quindi, sono state sviluppate delle proposte di miglioramento delle attività di riordino: attraverso l’applicazione di modelli previsionali, è stato calcolato, per un campione ristretto di prodotti, il loro fabbisogno mensile, successivamente, questi risultati sono stati integrati con i vincoli di tempo di copertura e minimo d’ordine. Sono stati definiti e calcolati alcuni indici che potessero esprimere il grado di miglioramento della performance aziendale. I risultati avuti attraverso il lavoro di tesi cosa dimostrano quanto l’utilizzo di metodi basati sulle serie storiche forniscano una stima dei volumi di vendita futura più affidabili confronto a quelli ottenuti utilizzando i dati di vendita del mese corrente come stima delle vendite future. Attraverso l’utilizzo dei metodi di Media Mobile, Smorzamento Esponenziale e di Holt-Winters si può arrivare ad un livello di precisione maggiore di quello in uso in MARR S.p.A. ed è possibile giungere ad un’ottimizzazione dei magazzini, riducendone la giacenza del 10%.