Littérature scientifique sur le sujet « Analisi trascrittomica »

2011/2012
Il tema principale di questo lavoro di tesi è la discussione dei metodi che, mediante l’utilizzo di strumenti creati ad-hoc e di software di terze parti, hanno permesso analizzare sequenze trascritte di 5 organismi non-modello: Mytilus galloprovincialis, Ruditapes philippinarum, Latimeria menadoensis, Astacus leptodactylus e Procambarus clarkii.
XXV Ciclo
1981

Agli ellagitannini (ET), tannini condensati tipici di molti tipi di frutta, tra cui frutti rossi e melograno, sono attribuite svariate attività biologiche positive per la salute umana ancora poco sostanziate da chiare evidenze scientifiche. La chiave per comprendere questa variabilità potrebbe risiedere nella biotrasformazione che gli ET subiscono dopo l’ingestione: nell’intestino vengono trasformati nei metaboliti derivati, le urolitine. In relazione alla tipologia di urolitine prodotte dall’uomo sono stati individuati tre fenotipi metabolici: metabotipo A, tipico di soggetti che producono in prevalenza urolitina A; metabotipo B, di soggetti che producono anche isourolitina A ed urolitina B; metabotipo 0, di coloro che non producono urolitine. Nel presente elaborato, che si inserisce nel più ampio progetto di ricerca NIKE, sono stati testati gli effetti putativi dei metabotipi in colture primarie di macrofagi, una tipologia cellulare plastica e ubiquitaria che riveste un ruolo centrale nella risposta immunitaria. I macrofagi sono stati attivati in senso classico (M1) o alternativo (M2) e supplementati per sei giorni con due diverse combinazioni di urolitine, per ricalcare i metabotipi A e B, in concentrazioni coerenti con quelle plasmatiche date dal consumo di alimenti ricchi in ET. Parallelamente sono stati coltivati anche macrofagi non attivati come controllo. Al termine del trattamento, si è proceduto all’analisi del profilo di espressione genica con tecnologia microarray a genoma intero per indagare le basi molecolari del supposto effetto antinfiammatorio dei metaboliti testati. Il trascrittoma dei macrofagi è risultato essere influenzato in maniera differente dai due metabotipi: il trascrittoma degli M1 è stato minimamente influenzato dalla supplementazione, mentre negli M2 il metabotipo A ha indotto cambiamenti significativi in un considerevole numero di trascritti, mostrando un miglioramento delle proprietà antinfiammatorie macrofagiche.

The research project has been developed on the equine inflammatory respiratory diseases, which can be divided in Recurrent Airway Obstruction (RAO) and Inflammatory Airway Disease (IAD). The aim of this study was to investigate immune-related genes expression in the respiratory tract of IAD and RAO-affected horses. Clinical examination and endoscopy were performed. On the Broncho-Alveolar Lavage (BAL) fluid, obtained during endoscopy, cytological and microbiological analysis were performed to evaluate correlations between the gene expressions values and the clinical parameters. A first analysis was developed by real time RT-PCR comparing the gene expression profile of 10 immune-related target genes (IL-1ß, IL-6, IL-8, IL-13, IL-17, TNFa, INF?, TGF-ß1, NF?-ß and TRL 4) in the BAL of healthy horses and RAO-affected ones, on which sampling was performed twice within 15 days. The aim was to deepen the effects of the respiratory disease on the equine immune system and to assess a potential temporal evolution of the gene expression values and of the other parameters considered. In addition, biopsies of the bronchial tissue were obtained and subsequent, histological evaluation and gene expression analyses were performed. Six of the target genes showed a significant expression values increase in the RAO group compared to the control one. A positive statistical correlation between the amount of mucus in the airways and the expression of some genes investigated was found. Regarding inflammatory mediators expression in the biopsy tissue, neither of target genes was significantly differentially expressed between the RAO horses and the control group. The second part of the research project included also the study of IAD. On all horses, clinical investigations and assessments of gene expression profiles were carried out twice within 15 days, at the diagnosis moment and at the end of the pharmacological treatment. Considering the results of the first study, no biopsies of the respiratory tissue were performed. The development of a microarray platform specific for equine immune-related genes, provide a global view of the pathways involved in the IAD and RAO inflammatory response. The statistical analyses showed that 379 transcripts (55 up-regulated and 324 down-regulated) were significantly differentially expressed between the IAD group and control horses and 1763 genes (903 up-regulated and 860 down-regulated) between the RAO-affected horses and the healthy animals. Between IAD-affected horses and RAO animals, were showed significant differences of the respiratory rate at rest and of the amount of mucus in the airways. Some transcripts involved in the genesis, length and motility of the respiratory epithelium cilia, were down-regulated both in IAD and in RAO horses. In the IAD population, has been demonstrated the over-expression of genes coding for inflammatory mediators. Some of the transcripts up-regulated in the RAO group, are involved in the inflammatory response, bronchoconstriction, apoptosis and hypoxia pathway. In the same disease, some genes involved in the genesis of the protective muco-protein film of the respiratory epithelium were under-expressed. The analyses carried out by the software Gene Sets Enrichment Analysis (GSEA) showed that the pathway activated during human asthma, is also enriched in equine RAO, albeit marginally significant (False Discovery Rate <25%, p value 0.08 ). The low quality of the RNA extracted from the BAL of some samples, did not allow to reach a significant number of horses, considered before and after the pharmacological treatment, to assess the effect of the therapy on gene expression profiles. In conclusion, the present studies provided information about the immunological mechanisms activated during the most important equine respiratory diseases. In the future, the information obtained could lead to the development of new therapies for IAD and RAO, by the inhibition of molecules involved in the pathogenesis of these diseases, as is already done in human medicine. The involvement of the same pathway in human asthma and equine RAO, could suggest a possible role of horses as animal model for the study of human chronic respiratory diseases.
Il lavoro di ricerca svolto nell’arco dei tre anni di dottorato, è stato articolato in due progetti sviluppati nell’ambito delle malattie respiratorie su base infiammatoria che colpiscono gli equini. Tali patologie possono essere distinte in due grandi gruppi: Recurrent Airway Obstruction (RAO) ed Inflammatory Airway Disease (IAD). Lo scopo dei progetti di ricerca si è basato sull’indagine dei profili di espressione di geni immuno-correlati nell’albero respiratorio di cavalli affetti da IAD e RAO, in relazione ad un gruppo di controllo. Su tutti i soggetti, sono stati eseguiti gli esami clinici mirati alla valutazione dell’apparato respiratorio, l’esame endoscopico e l’esame citologico e microbiologico da Broncho-Alveolar Lavage (BAL), per valutare le potenziali correlazioni esistenti tra i profili di espressione genica ed i parametri clinici. Il primo progetto è stato sviluppato comparando cavalli sani con soggetti affetti da RAO, su cui i campionamenti sono stati ripetuti due volte nell’arco di 15 giorni, al fine valutare una potenziale evoluzione temporale dell’espressione genica e degli altri parametri considerati nella ricerca. Inoltre, sono state eseguite biopsie del tessuto bronchiale, sottoposto sia a valutazione istologica che ad analisi di espressione genica. Mediante real time RT-PCR, sono stati indagati i profili di espressione di 10 geni target immuno-correlati (IL-1ß, IL-6, IL-8, IL-13, IL-17, TNFa, INF?, TGF-ß1, NF?-ß e TRL 4), sei dei quali hanno dimostrato una aumento statisticamente significativo dei livelli di espressione nel gruppo RAO rispetto al gruppo di controllo. Le analisi statistiche condotte, hanno riscontrato una correlazione positiva tra la quantità di muco nelle vie aeree e l’ espressione di alcuni dei geni indagati. Non sono state evidenziate differenze di espressione, dei geni inclusi nello studio, tra i tessuti bioptici prelevati dai soggetti affetti da RAO e quelli ottenuti dal gruppo di controllo. Il secondo progetto di ricerca, è stato sviluppato ampliando la casistica dei cavalli affetti da RAO ed introducendo lo studio della IAD. Su tutti i soggetti, le indagini cliniche e le valutazioni dei profili di espressione genica sono state condotte sia al momento della diagnosi che al termine del trattamento farmacologico della durata di 15 giorni. Valutati i risultati del primo lavoro, non sono state eseguite biopsie del tessuto respiratorio. Lo sviluppo di una piattaforma microarray specifica per i geni immuno-correlati di cavallo ha permesso di ottenere una visione globale dei pathway coinvolti nella risposta infiammatoria delle due patologie. Le analisi statistiche effettuate hanno evidenziato una differenza di espressione significativa per 379 trascritti (di cui 55 sovra-espressi e 324 sotto-espressi) tra il gruppo IAD ed il gruppo di controllo e per 1763 geni (di cui 903 sovra-espressi e 860 sotto-espressi) tra i pazienti affetti da RAO ed i soggetti sani. Da un punto di vista clinico, sono state riscontrate differenze statisticamente significative sia della frequenza respiratoria a riposo che della quantità di muco presente nelle vie aeree dei cavalli affetti da IAD rispetto ai soggetti RAO. Tra i geni sotto-espressi nei due gruppi di cavalli affetti da malattia respiratoria, hanno acquistato importanza alcuni trascritti coinvolti nella genesi, lunghezza e motilità dell’apparato ciliare dell’epitelio respiratorio. Nella popolazione IAD, è stata dimostrata la sovra-espressione di geni codificanti per mediatori coinvolti nella risposta infiammatoria. I geni sovra-espressi nel gruppo RAO, caratterizzati da maggior rilievo, sono coinvolti nella risposta infiammatoria, nella broncocostrizione, nella via apoptotica e nel pathway dell’ipossia. Nella medesima patologia, si sono mostrati sotto-espressi anche alcuni geni coinvolti nella genesi del film muco-proteico di protezione dell’epitelio respiratorio. Lo studio condotto mediante Gene Set Enrichment Analysis (GSEA), ha evidenziato che il pathway attivato in corso di asma umano, viene arricchito anche nella patologia RAO equina, sebbene la significatività statistica sia marginale (False Discovery Rate < 25%, p value 0,08). Non è stato possibile valutare l’effetto della terapia farmacologica sui profili di espressione genica, poiché la bassa qualità dell’RNA estratto dal BAL di alcuni campioni non ha permesso di raggiungere un numero significativo di soggetti valutati prima e dopo il trattamento terapeutico. Gli studi effettuati hanno quindi permesso di far luce su alcuni dei meccanismi immunologici che stanno alla base delle patologie respiratorie equine di maggiore importanza veterinaria ed economica. In futuro, le informazioni ottenute, potrebbero condurre allo sviluppo di nuovi mezzi terapeutici per l’inibizione delle molecole coinvolte nello sviluppo di IAD e RAO, come già avviene in medicina umana. Infine, il coinvolgimento di un medesimo pathway nell’asma umano e nella RAO equina, potrebbe condurre all’utilizzo di tale specie come modello animale per lo studio delle patologie respiratorie croniche umane.

From the second half of past century to nowadays aquaculture keeps on being the fastest-growing animal-food-producing sector, so that it has provided the 46% of total food fish supply in 2010. However, we are faced with a few problems closely connected to some aspects that are still unknown in the biology of certain relevant species such as the gilthead seabream (Sparus aurata). Functional genomics can offer very well-grounded tools to get information about the molecular mechanisms which are involved in physiological processes whose consequences may be very high also from an economic point of view. The issue concerning how the marine organisms and populations react to climatic changes is a question of paramount importance which is still rather unsettled. The gilthead bream is very sensitive to low temperatures, so that it does not survive when temperature falls under 5°C. In fact, in winter time the breeding cause often huge economic losses to their owners since the mortality rate rises because of metabolic syndrome known as winter disease. In this study we considered the gene expression profiles of Sparus aurata individuals which have been exposed to low temperatures, in experimental conditions that could represent as realistic as possible the winter season. The gene expression profile can be used as a tool to link up the genotype to the physiology and to the phenotype. Moreover, the study looked into populations coming from regions with different climatic conditions (Veneto and Sicily), by assuming a different tolerance to cold exposition. Four groups of wild sea bream (120±16 g), coming in pairs from the two regions, were exposed for 21 days to two temperature treatments: 16 ± 0.3 °C (control groups) and 6.8 ± 0.3 °C (cold groups). Liver and gill samples were collected during acute (0, 6 and 24 hours) and chronic exposure (21 days). The gene expression profiles were analyzed using an oligo-nucleotide microarray technology, with about 19,715 ESTs. Results revealed a complex transcriptomic response to cold with many molecular pathways involved among which: lipid and carbohydrate metabolism, regulation of heat shock proteins (HSPs) and other protein chaperones, protein degradation and repair, regulation of cell death, RNA and DNA metabolism, immune response. The earliest transcriptional response is linked to oxidative stress and anti-oxidant/survival cell response, suggesting an immediate disturbance of systemic oxygen balance. The largest transcriptional difference between cold and control groups occurred during long-term exposure, involving primarily several genes of lipid metabolism with a role in the re-allocation of energy sources and immune-related genes indicating an immunosuppressive effect of cold exposure. The data on the liver and gill transcriptome of the gilthead sea bream exposed to cold provide a starting point to investigate physiological mechanisms underlying long term cold adaptation in fish and to address future research for the identification of cold tolerant S. aurata strain for aquaculture.
Dalla seconda metà del secolo scorso ad oggi l‟acquacoltura continua ad essere il settore delle produzioni animali in più rapida crescita, con il 46% di pesce fornito sul totale consumato nel 2010. Rimangono, tuttavia, problematiche strettamente legate ad aspetti ancora sconosciuti nell‟ambito della biologia di alcune specie d‟interesse come l‟orata comune (Sparus aurata). La genomica funzionale può fornire validi strumenti per ottenere informazioni sui meccanismi molecolari coinvolti nei processi fisiologici importanti anche da un punto di vista economico. Come le popolazioni e le specie marine reagiscono ai cambiamenti climatici è una questione di importanza centrale ancora non del tutto risolta. L‟orata comune risente fortemente del freddo, non sopravvivendo a temperature inferiori ai 5°C e spesso, durante l‟inverno, gli allevamenti subiscono ingenti danni economici per l‟elevata mortalità data dalla sindrome metabolica winter disease. In questo studio sono stati valutati i profili di espressione genica di individui di S. aurata esposti alle basse temperature, in condizioni sperimentali che fossero il più realistiche possibile con la stagione invernale. Il profilo di espressione genica può servire come strumento per legare il genotipo alla fisiologia e al fenotipo. Sono state, inoltre, esaminate popolazioni provenienti da regioni con condizioni climatiche diverse, Veneto e Sicilia, ipotizzando una differente tolleranza al freddo. Quattro gruppi di orate (120±16 g), provenienti a coppie dalle due regioni, sono state esposte per 21 giorni a due trattamenti di temperatura: 16 ± 0,3 °C (gruppi di controllo) e 6,8 ± 0,3 °C (gruppi dei trattati). Campioni di fegato e branchia sono stati raccolti durante esposizione acuta (0, 6 e 24 ore) e cronica (21 giorni). I profili di espressione sono stati analizzati usando un microarray a oligo-nucleotidi con circa 19.715 geni. I risultati hanno rivelato una risposta trascrizionale complessa per la risposta al freddo, con numerosi pathway coinvolti: metabolismo di lipidi e carboidrati, heat shock protein (HSP) e chaperoni, degradazione proteica, apoptosi, metabolismo di RNA e DNA, risposta immunitaria. La prima risposta è legata allo stress ossidativo, suggerendo un disturbo immediato del bilancio dell‟ossigeno a livello sistemico, mentre le più grandi differenze trascrizionali tra trattati e controlli si rilevano durante l‟esposizione a lungo termine, e coinvolgono principalmente geni del metabolismo lipidico per la ridistribuzione delle riserve energetiche e geni dell‟immunità per l‟importante effetto immuno-soppressivo del freddo. I dati del trascrittoma di branchia e fegato di orate esposte alle basse temperature forniscono un punto di partenza per indagare i meccanismi fisiologici sottostanti l‟adattamento al freddo a lungo termine nei pesci e per indirizzare ricerche future volte all‟identificazione di ceppi di S. aurata resistenti al freddo in acquacoltura.

La poli(ADP-ribosilazione) è una modifica post-traduzionale del proteoma, che svolge un ruolo importante nell ambito di diverse funzioni cellulari (e.g., la regolazione della struttura della cromatina a steady state e durante lo sviluppo ed il differenziamento, la modulazione della espressione genica, i meccanismi di riparazione del DNA, l attivazione e la regolazione del processo apoptotico, la necrosi). La reazione è catalizzata dalle proteine PARP [Poly(ADP-ribose)Polymerases], una famiglia di enzimi che catalizzano l aggiunta di polimeri di poli(ADP-ribosio) alle proteine bersaglio, le cui attività molecolari e funzioni biologiche vengono di conseguenza modulate. Nell ambito di questo progetto di ricerca, abbiamo determinato la struttura genomica dei geni della famiglia PARP in Homo sapiens e in Mus musculus ed abbiamo analizzato la loro espressione in cellule alfa e beta del pancreas di topo, sia a steady state che dopo trattamento con citochine con la conseguente induzione di apoptosi: secondo un consenso generale, questo modello sperimentale consente di riprodurre in vitro alcuni dei fenomeni che sono correlati all insorgenza del Diabete Mellito (DM). I nostri risultati dimostrano una notevole attivazione della espressione di diverse PARP, soprattutto nelle cellule beta, e suggeriscono quindi un loro possibile ruolo nella patogenesi del DM. Per converso, questi stessi dati dovrebbero consentire anche una ulteriore definizione delle funzioni biologiche e del ruolo fisiologico di queste proteine, Infine, questi dati suggeriscono il possibile utilizzo di inibitori delle PARP per applicazioni di Medicina Traslazionale al trattamento clinico del Diabete Mellito.

Plants are sessile organisms and often they have to cope with environmental stresses (abiotic factors) such as drought, cold, heat, extreme light, excessive soil salinity, or several combinations of them. The genotype x environment (GxE) interaction is the source of the main variability in the responses to these constrains. Among the abiotic factors that can influence plant physiology, drought is the most relevant because it can influence plant growth and yield, and affects fruits composition. Secondary metabolism contributes to the adaptation of a plant to its environment. In fruit crops such as grapevine (Vitis vinifera L.), secondary metabolism also largely determines fruit quality. Grapevine is considered a drought-tolerant plant and traditionally is not irrigated, especially in Europe. Mediterranean climate, with warm and dry summers and cold and wet winters, is considered optimal for viticulture. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the accumulation of secondary metabolites in the grapes, thus affecting wine quality. We adopted a multidisciplinary approach that considered a two-years field trial, high-throughput transcripts profiling (RNA-sequencing) and large-scale targeted metabolite analyses to investigate the effect of drought events on the berry metabolism during fruit development and ripening in white and red grape varieties. An open field experiment was therefore conducted on Tocai friulano (white grape variety) and Merlot (red grape variety) vines in 2011 and 2012, in a North Italian viticultural area characterized by transient drought events during the summer. Two irrigation treatments were applied to the vines: (i) control vines were weekly irrigated, in order to keep their stem water potential (a sensitive indicator of grapevine water status) between -0.4 and -0.6 MPa; (ii) deficit irrigated vines were not irrigated from fruit set to harvest except in case of acute water deficit. Merlot vines were sheltered with an open-sided transparent cover at the beginning of the seasons, while Tocai friulano vines were cultivated without any cover and hence subjected to the natural precipitations. In Merlot, deficit irrigated vines experienced water deficit from 40-50 days after anthesis in both seasons. In Tocai friulano, deficit irrigated vines manifested a late deficit (from 80-90 days after anthesis) in 2011, and a prolonged water deficit from early stages of fruit development (from 30-40 days after anthesis) to harvest in 2012. For both varieties, berries were sampled for transcript and metabolite analyses during berry development and ripening. Furthermore, at harvest, grapes were microvinificated and wines composition was evaluated focusing on the secondary metabolites that largely contribute to the final color, taste, and aromatic features. A large effect of water deficit on fruit secondary metabolism of the white grape variety Tocai friulano was observed in the season when the deficit occurred from early stages of berry development to harvest. In this particularly season, increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were observed, while carotenoid and flavonoid accumulations were differentially modulated by water deficit accordingly to the berry developmental stage. In parallel, RNA sequencing analysis revealed that many key genes of the phenylpropanoid, flavonoid, and terpenoid pathways were modulated by water deficit indicating a transcriptional regulation of these specific pathways in the berry under drought. II The higher co-regulation of several terpenoid transcripts with monoterpene accumulation under water deficit and the enrichment of drought-responsive elements in the promoter region of many terpenoid genes highlight that drought can enhance the production of these flavour components in grapes with potential effects on wine composition and sensory features. The wines produced from grapes subjected to water deficit revealed a more pronounced accumulation of monoterpenes and C13-norisoprenoids, and especially glycosidically-bound compounds. However, differences in the metabolic response between seasons suggest that the endurance of water deficit and the timing of application strongly impact this response. In Merlot berries, the drought stress response encompassed both ABA-dependent and ABA-independent signal transduction pathways with several VviAREB/ABFs, VvibZIP, and VviAP2/ERF-DREB transcription factors that were up-regulated by water deficit at one or more developmental stages. These transcription factors can play critical roles in the drought response by modulating a large suite of genes. Analyses of the central and specialized berry metabolism was conducted both at the transcript and metabolite levels by investigating several metabolic pathways (glycolysis and sugar accumulation, tricarboxylic acid cycle and amino acid biosynthesis, phenylpropanoid, flavonoid, terpenes, carotenoids, and fatty acid degradation pathways). The study revealed that water deficit enhanced the accumulation of several osmoprotectants (proline, sucrose, and raffinose) and of secondary metabolites such as anthocyanins and C5, C7, C8, and C9 volatile organic compounds. Furthermore, a weighted gene co-expression network analysis clustered in a module several genes involved in the branched chain amino acids biosynthesis, phenylpropanoid and flavonoid pathways, and sugar derivative metabolism together with the transcription factors mentioned above involved in the drought-stress signal, indicating a putative role of these transcription factors on the regulation of the response of the fruit metabolism to drought in Merlot berries. The wines produced from grapes subjected to water deficit revealed a higher concentration of anthocyanins that determined higher color intensity and a bluer coloration of the wines. A comparison between the two genotypes can be done only for the 2012 season, when levels and endurances of water deficit were similar between Tocai friulano and Merlot deficit irrigated vines. The data indicate that water deficit modulated the accumulation of several secondary compounds; however, the modulation of the secondary metabolism varied between cultivars indicating a genotype x environment interaction. In the Tocai friulano berry, water deficit specifically stimulated the synthesis of phenolic acids, such as gallic acid, ellagic acid, and caftaric acid, tocopherols, and monoterpenes, such as linalool, α-terpineol, nerol, and hotrienol. In Merlot, the response included an overproduction of anthocyanins, such as the tri-substituted delphinidin, petunidin, and malvidin both in the glycosylated and acylated form, and C5, C7, C8 and C9 volatile organic, such as 1-penten-3-ol, (E)-2-heptenal, (E)-2-octenal, 1-octen-3-ol, and nonanol. A common response between the two varieties included a higher accumulation of gallic acid, zeaxanthin, (E)-2-heptenal, (E)-2-octenal, 1-octen-3-ol, and nonanol in the berry. Furthermore, these results indicate that drought events can affect the composition and sensory features of white and red wines by increasing the accumulation of benzoic and cinnamic acids, pigments in the red grape variety and monoterpenes in the white grape variety.

Plants are sessile organisms and often they have to cope with environmental stresses (abiotic factors) such as drought, cold, heat, extreme light, excessive soil salinity, or several combinations of them. The genotype x environment (GxE) interaction is the source of the main variability in the responses to these constrains. Among the abiotic factors that can influence plant physiology, drought is the most relevant because it can influence plant growth and yield, and affects fruits composition. Secondary metabolism contributes to the adaptation of a plant to its environment. In fruit crops such as grapevine (Vitis vinifera L.), secondary metabolism also largely determines fruit quality. Grapevine is considered a drought-tolerant plant and traditionally is not irrigated, especially in Europe. Mediterranean climate, with warm and dry summers and cold and wet winters, is considered optimal for viticulture. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the accumulation of secondary metabolites in the grapes, thus affecting wine quality. We adopted a multidisciplinary approach that considered a two-years field trial, high-throughput transcripts profiling (RNA-sequencing) and large-scale targeted metabolite analyses to investigate the effect of drought events on the berry metabolism during fruit development and ripening in white and red grape varieties. An open field experiment was therefore conducted on Tocai friulano (white grape variety) and Merlot (red grape variety) vines in 2011 and 2012, in a North Italian viticultural area characterized by transient drought events during the summer. Two irrigation treatments were applied to the vines: (i) control vines were weekly irrigated, in order to keep their stem water potential (a sensitive indicator of grapevine water status) between -0.4 and -0.6 MPa; (ii) deficit irrigated vines were not irrigated from fruit set to harvest except in case of acute water deficit. Merlot vines were sheltered with an open-sided transparent cover at the beginning of the seasons, while Tocai friulano vines were cultivated without any cover and hence subjected to the natural precipitations. In Merlot, deficit irrigated vines experienced water deficit from 40-50 days after anthesis in both seasons. In Tocai friulano, deficit irrigated vines manifested a late deficit (from 80-90 days after anthesis) in 2011, and a prolonged water deficit from early stages of fruit development (from 30-40 days after anthesis) to harvest in 2012. For both varieties, berries were sampled for transcript and metabolite analyses during berry development and ripening. Furthermore, at harvest, grapes were microvinificated and wines composition was evaluated focusing on the secondary metabolites that largely contribute to the final color, taste, and aromatic features. A large effect of water deficit on fruit secondary metabolism of the white grape variety Tocai friulano was observed in the season when the deficit occurred from early stages of berry development to harvest. In this particularly season, increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were observed, while carotenoid and flavonoid accumulations were differentially modulated by water deficit accordingly to the berry developmental stage. In parallel, RNA sequencing analysis revealed that many key genes of the phenylpropanoid, flavonoid, and terpenoid pathways were modulated by water deficit indicating a transcriptional regulation of these specific pathways in the berry under drought. The higher co-regulation of several terpenoid transcripts with monoterpene accumulation under water deficit and the enrichment of drought-responsive elements in the promoter region of many terpenoid genes highlight that drought can enhance the production of these flavour components in grapes with potential effects on wine composition and sensory features. The wines produced from grapes subjected to water deficit revealed a more pronounced accumulation of monoterpenes and C13-norisoprenoids, and especially glycosidically-bound compounds. However, differences in the metabolic response between seasons suggest that the endurance of water deficit and the timing of application strongly impact this response. In Merlot berries, the drought stress response encompassed both ABA-dependent and ABA-independent signal transduction pathways with several VviAREB/ABFs, VvibZIP, and VviAP2/ERF-DREB transcription factors that were up-regulated by water deficit at one or more developmental stages. These transcription factors can play critical roles in the drought response by modulating a large suite of genes. Analyses of the central and specialized berry metabolism was conducted both at the transcript and metabolite levels by investigating several metabolic pathways (glycolysis and sugar accumulation, tricarboxylic acid cycle and amino acid biosynthesis, phenylpropanoid, flavonoid, terpenes, carotenoids, and fatty acid degradation pathways). The study revealed that water deficit enhanced the accumulation of several osmoprotectants (proline, sucrose, and raffinose) and of secondary metabolites such as anthocyanins and C5, C7, C8, and C9 volatile organic compounds. Furthermore, a weighted gene co-expression network analysis clustered in a module several genes involved in the branched chain amino acids biosynthesis, phenylpropanoid and flavonoid pathways, and sugar derivative metabolism together with the transcription factors mentioned above involved in the drought-stress signal, indicating a putative role of these transcription factors on the regulation of the response of the fruit metabolism to drought in Merlot berries. The wines produced from grapes subjected to water deficit revealed a higher concentration of anthocyanins that determined higher color intensity and a bluer coloration of the wines. A comparison between the two genotypes can be done only for the 2012 season, when levels and endurances of water deficit were similar between Tocai friulano and Merlot deficit irrigated vines. The data indicate that water deficit modulated the accumulation of several secondary compounds; however, the modulation of the secondary metabolism varied between cultivars indicating a genotype x environment interaction. In the Tocai friulano berry, water deficit specifically stimulated the synthesis of phenolic acids, such as gallic acid, ellagic acid, and caftaric acid, tocopherols, and monoterpenes, such as linalool, α-terpineol, nerol, and hotrienol. In Merlot, the response included an overproduction of anthocyanins, such as the tri-substituted delphinidin, petunidin, and malvidin both in the glycosylated and acylated form, and C5, C7, C8 and C9 volatile organic, such as 1-penten-3-ol, (E)-2-heptenal, (E)-2-octenal, 1-octen-3-ol, and nonanol. A common response between the two varieties included a higher accumulation of gallic acid, zeaxanthin, (E)-2-heptenal, (E)-2-octenal, 1-octen-3-ol, and nonanol in the berry. Furthermore, these results indicate that drought events can affect the composition and sensory features of white and red wines by increasing the accumulation of benzoic and cinnamic acids, pigments in the red grape variety and monoterpenes in the white grape variety.

In a comprehensive attempt to understand the molecular and cellular processes driving seedlessness in grapevine, a seeded variety (wild-type) and its seedless somatic variant (mutant) were characterized at the morphological, genomic and transcriptomic levels in relation to berry development and seed content. The overall importance of clonal variability and the application of Next Generation Sequencing technology in highlighting the molecular events during seed formation within a developing berry have been clearly demonstrated. In this thesis three hypothesis were formulated, tested and confirmed. First it was hypothesized that the mutant has a gross morphology identical to the wild-type except for berry size and seed content. In testing this hypothesis quantitative and qualitative traits that relate to berry development and seed content were compared in the two clones. Here traits that were significantly different in the two lines are those that relate only to berry size and seed content. This evaluation was performed both in control conditions (self-pollination) and after anther/stigma removal which allowed the investigation of a possible role for parthenocarpy, stenospermocarpy or other mechanisms in promoting the phenotype of the seedless somatic variant. The second hypothesis states that the mutant is sterile or partly sterile hence cannot produce viable seeds. In order to verify this hypothesis pollen germination and viability assays were carried out in both clones. The tests confirmed pollen germination and vitality percentage of the mutant was significantly lower than that of the wild-type. The third hypothesis concerned the existence of genomic/transcriptomic differences between the two lines and could be tested through the power of the Next generation Sequencing technology. In particular, we raised the following questions. Are there somatic mutations that can allow the wild-type and mutant to be distinguished? What are the temporal and spatial changes that could occur in their respective transcriptomes? Especially how does expression levels of key regulatory genes change before, during and after fertilization in the two clones? These key questions were addressed with the aid of Molecular marker analysis, Array based SNP genotyping method and RNA-Seq approach. Using 58 microsatellites, the analyzed loci showed identical profile in the wild-type and the mutant. The 20K grapevine Illumina Chip revealed 16333 identical SNP loci in the two clones, thus a further confirmation of the true identity of the seedless line. Conversely variant calling from RNA-Seq enabled the identification of several somatic mutations at the whole-genome level in the two lines. At the same time, RNA-Seq allowed the creation of inventories of gene expression at successive stages of seed formation. i.e. stages E-L 15 (single flowers in compact groups), E-L 27 (young berries enlarging) and E-L 38 (berries harvest-ripe). Here the transcriptomes revealed by Illumina mRNA-Seq technology had approximately 98% of grapevine annotated transcripts and about 80% of them were commonly expressed in the two lines. Differential gene expression analysis revealed a total of 1075 differentially expressed genes (DE) in the pairwise comparison of developmental stages, which included DE genes specific to the wild-type background, DE genes specific to the mutant background and DE genes commonly shared in both backgrounds. The analysis of differential expression patterns and functional category enrichment of wild-type and mutant DE genes highlighted significant coordination and enrichment of pollen and ovule developmental pathways. The expression of some selected DE genes was further confirmed by real-time RT-PCR analysis. To the best of our knowledge the work presented in this thesis represents the most comprehensive attempt to characterize the genetic bases of seed formation in grapevine. We have shown that a seeded wine grape and its seedless somatic variant are similar in several biological processes except for berry size and seed content. With a high throughput method we could identify an inventory of genes with altered expression in the mutant compared to the wild-type, which may be responsible for the seedless phenotype. The genes located within known genomic regions regulating seed content may be used for the development of molecular tools to assist table grape breeding. Therefore the data reported here have provided a rich genomic resource for practical use and functional characterization of the genes that potentially underpin seedlessness in grapevine.