Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management Bull’s eye rot (BER) caused by Neofabraea alba is one of the most frequent and damaging latent infection occurring in stored pome fruits worldwide. Fruit infection occurs in the orchard, but disease symptoms appear only 3 months after harvest, during refrigerated storage. In Italy BER is particularly serious for late harvest apple cultivar as ‘Pink Lady™’. The purposes of this thesis were: i) Evaluate the influence of ‘Pink Lady™’ apple primary metabolites in N. alba quiescence ii) Evaluate the influence of pH in five different apple cultivars on BER susceptibility iii) To find out not chemical method to control N. alba infection iv) Identify some fungal volatile compounds in order to use them as N. alba infections markers. Results regarding the role of primary metabolites showed that chlorogenic, quinic and malic acid inhibit N. alba development. The study based on the evaluation of cultivar susceptibility, showed that Granny Smith was the most resistant apple cultivar among the varieties analyzed. Moreover, Granny Smith showed the lowest pH value from harvest until the end of storage, supporting the thesis that ambient pH could be involved in the interaction between N. alba and apple. In order to find out new technologies able to improve lenticel rot management, the application of a non-destructive device for the determination of chlorophyll content was applied. Results showed that fruit with higher chlorophyll content are less susceptible to BER, and molecular analyses comforted this result. Fruits with higher chlorophyll content showed up-regulation of PGIP and HCT, genes involved in plant defence. Through the application of PTR-MS and SPME GC-MS, 25 volatile organic compounds emitted by N. alba were identified. Among them, 16 molecules were identified as potential biomarkers.

Il cancro batterico dell’actinidia causato da Pseudomonas syringae pv.actinidiae (Psa) suscita grande interesse a livello globale a partire dal 2008. La malattia è comparsa in Giappone e in due anni ha avuto una diffusione epidemica in tutte le aree di coltivazione mondiale di actinidia. Gravi perdite economiche hanno attirato l’attenzione internazionale su questa problematica e grandi sforzi sono stati rivolti allo studio di questo patosistema ancora poco conosciuto. E’ emerso infatti che il patogeno può rimanere in fase latente per lunghi periodi senza causare sintomi caratteristici nelle piante infette, e che dalla comparsa dei sintomi la pianta muore nell’arco di un paio d’anni. Il monitoraggio ed il controllo della situazione è perciò di fondamentale importanza ed è ancora più importante prevenire la comparsa di nuovi focolai di infezione. A questo proposito sarebbe opportuno l’impiego di materiale vegetale di propagazione non infetto, ma in molti casi questo diventa difficile, dal momento che il materiale impiegato è generalmente quello asintomatico, non analizzato precedentemente per la presenza del patogeno. Negli ultimi anni sono state perciò messe a punto molte tecniche molecolari per l’identificazione di Psa direttamente da materiale vegetale. L’obiettivo di questo lavoro è stato quello di studiare l’epidemiologia di Psa in piante adulte infette e di verificare l’efficacia di metodi di diagnosi precoce per prevenire la malattia. A tale scopo il lavoro sperimentale è stato suddiviso in diverse fasi: i) studio della localizzazione, traslocazione e sopravvivenza di Psa nelle piante, a seguito di inoculazione in piante adulte di actinidia di ceppi marcati Psa::gfp; ii) studio della capacità di Psa di essere mantenuto in germogli di actinidia attraverso sette generazioni di micropropagazione dopo l’inoculazione delle piante madri con lo stesso ceppo marcato Psa::gfp; iii) studio ed applicazioni di un nuovo metodo di diagnosi precoce di Psa basato sull’analisi molecolare del “pianto”. / Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) involved all global interest since 2008. The disease started from Japan and in two year it was causing epidemic outbreaks around the world, in every country that has an actinidia’s cultivations. Huge amount of economical losses brought the international attention on this problem and on strong efforts were devoted study this relatively unknown pathosystem. It appears in fact that pathogen can be maintained in latent form for long periods, without show any characteristic symptom on the affected plants, when it suddenly induce symptoms the plant die in one or two years. Monitor and control the real situation on symptoms are fundamental, but more important is to prevent the appearance of new infection events. This could be supported by the use of pathogen free propagation materials, but in several cases this is only theoretically achieved since materials employed are just asymptomatic but not tested to be pathogen-free. In the recent years a lot of molecular techniques were developed for Psa detection and diagnosis, directly from plant material. The objectives of this work were to clarify the spreading of Psa into the infected adult plant, and to verify the effectiveness of Psa early detection methods on disease prevention. Toward these aims the following experimental steps were carried out: i) study the localization, movement and survival ability of Psa into the plant after inoculation with a reference marked strains of Psa::gfp several actinidia adult plants; ii) study the Psa ability to be maintained in shoots during seven micropropagated generations after inoculation with the same marked strain of Psa::gfp mother plants; iii) study and application of a novel Psa detection method based on bleeding sap molecular testing.

Kiwifruit (genus Actinidia) is an important horticultural crop grown in the temperate regions. The four world’s largest producers are China, Italy, New Zealand and Chile. More than 50 species are recognized in the genus but the principal species in cultivation are A. deliciosa and A. chinensis. In Italy, as well as in many other countries, the kiwifruit crop has been considered to be relatively disease free and then no certification system for this species has been developed to regulate importation of propagation plant material in the European Union. During the last years a number of fungal and bacterial diseases have been recorded such as Botrytis cinerea and Pseudomonas syringae pv. actinidiae. Since 2003, several viruses and virus-like diseases have been identified and more recent studies demonstrated that Actinidia spp can be infected by a wide range of viral agents. In collaboration with the University of Auckland we have been detected thirteen different viral species on kiwifruit plants. During the three years of my PhD I worked on the characterization of Cucumber mosaic virus (CMV) and Pelargonium zonate spot virus (PZSV). The determination of causal agents has been based on host range, symptom expression in the test plant species and morphological properties of the virus particles using transmission electron microscopy (TEM) and using specific oligonucleotide primers in reverse transcription-polymerase chain reaction (RT-PCR). Both viruses induced several symptoms on kiwifruit plants. Moreover with new technologies such as high-throughput sequencing we detected additional viruses, a new member of the family Closteroviridae and a new member of the family Totiviridae. Taking together all results of my studies it is clear that, in order to minimize the risk of serious viral disease in kiwifruit, it is vital to use virus-free propagation material in order to prevent the spread of these viruses.

Physical inactivity is a hallmark of Chronic Obstructive Pulmonary Disease (COPD), which relates to outcomes relevant for patients and healthcare providers. Despite its importance, no patient reported outcome (PRO) instrument exists that captures physical activity that maximally reflects the experience of patients with COPD. The aim of the PROactive project is to develop, validate and use two PRO instruments (one for daily assessment, while the second as a clinical measure) to measure physical activity in COPD, researching and incorporating all dimensions of physical activity reported by patients. These tools are developed in accordance with the guidelines from the Food and Drug Administration (FDA) and European Medicines Agency (EMA). At the end of the first phase of the project, it has an initial version of the two PROs instruments validated. In the second phase, the PROs instruments are used and validated in several clinical trials that will determine, among other things, the utility, viability and sensitivity to change in different samples from patients with COPD / La inactivitat física és un aspecte clau en la MPOC que es relaciona amb esdeveniments de salut rellevants tant per als pacients com per als proveïdors de salut. Malgrat la importància de l’activitat física, no existeix cap instrument de resultats percebuts pels pacients (patient-reported outcomes – PRO) que capturi l'activitat física reflectint al màxim l'experiència dels pacients amb MPOC. L'objectiu del projecte PROactive és desenvolupar, validar i utilitzar dos instruments PRO (un per a l'avaluació diària, i un com a mesura clínica) per mesurar l'activitat física en la MPOC, investigant i incorporant totes les dimensions de l'activitat física reportades pels pacients. Aquestes eines es desenvolupen d'acord amb les directrius de l'Administració d'Aliments i Medicaments (FDA) i l'Agència Europea de Medicaments (EMA). Al final de la primera fase del projecte, que compta amb una versió inicial dels instruments PRO validats. A la segona fase, els qüestionaris PROs són utilitzats i validats en diversos assajos clínics que determinaran, entre altres coses, la utilitat, la viabilitat i sensibilitat al canvi en diferents mostres de pacients amb MPOC.

Chronic pancreatitis is an established risk factor for pancreatic ductal adenocarcinoma (PDAC) development. Polymorphisms in the pro-inflammatory cytokine gene interleukin 1β (IL-1β), as well as high IL-1β or low IL-1 receptor antagonist (IL-1RA) serum levels, are associated to worse prognosis in PDAC patients. To characterize the role of IL-1β in pancreatic tumorigenesis, we generated a transgenic mouse model bearing KRASG12D mutation combined to chronic inflammation induced by pancreatic overexpression of human IL-1β (KC-IL-1β). We found that IL-1β overexpression induced PDAC onset in 6 out of 13 KRASG12D bearing animals (46%), with a median overall survival of 10.5 months, compared to only 1 out of 13 mice carrying KRASG12D mutation alone (KC)(7.7% p= 0.02). In primary pancreatic KRASG12D organoid cultures, IL-1β exposure increased the number of spheroids and induced gene expression changes consistent with epithelial to mesenchymal transition (EMT), as shown by increased expression of vimentin, Zeb1, Snail. All these changes were counteracted using a recombinant human IL-1receptor antagonist (IL1-RA). Consistently, immuno-histochemical analysis confirmed that in KC-IL-1β tumor epithelial cells and metastasis were strongly positive for vimentin. The relevance of these findings was confirmed in human PDAC, showing higher IL-1 receptor I (IL1-RI) and vimentin expression in tumor tissue compared with adjacent normal pancreas. Regarding the mechanism involved in EMT activation, IL-1β exposure was found to induce an up-regulation of ribosome biogenesis rate, with consequent down-regulation of p53 protein expression which has been shown to be responsible for EMT changes. The finding that IL-1β/IL1-RI inflammatory pathway stimulates acinar cell proliferation and promotes EMT provides the rationale for a therapeutic strategy based on IL-1β receptor blockade to counteract inflammation-induced pancreatic tumorigenesis

Strategies to promote active aging and counteract the development of age-related diseases are among the most challenging researches in the framework of Horizon 2020, accordingly with the World Health Organization's declaration that "increased longevity without quality of life is an empty prize”. The aim of this thesis is to investigate the role of blood circulating microRNAs (miRs) and their expression profile characterizing aging and longevity trajectories, and particularly to distinguish between healthy and unhealthy longevity. To this purpose two experimental designs were defined, the former applied the advanced technology of smallRNA-sequencing (Illumina platform) to screen circulating miRs in a small cohort of different aged people, the latter was based on selected miRs analyzed on a larger cohort. The protocol for sequencing analysis, including library preparation, was optimized and applied on 12 donors, i.e. 3 young healthy donors, 3 old healthy donors, 3 healthy centenarians and 3 unhealthy centenarians. Significant miRs identified by sequencing, i.e. miR-30a-5p, -766-3p, -598-3p, were measured on a larger cohort of 48 subjects. Aging-related miRs previously described, i.e. miR-133a-3p, -206, -16, were analyzed in the same cohort of 48 donors. Circulating miR-206 and miR-16 levels described significant trajectories of aging, while miR-598-3p and miR-133a-3p levels characterized longevity trajectories. All these miRs are involved in the PI3K-Akt signaling, a central pathway for aging process. Finally, blood circulating molecules able to distinguish between healthy and unhealthy were obtained by joining the identified miRs and hemato-biochemical parameters, opening the possibility for further studies on therapeutic approaches.

Urban airborne particulate matter (PM) is known to increase morbidity and mortality due to cardiopulmonary diseases related to inflammatory processes and genotoxic effects. The aim of this thesis is to highlight the toxic and carcinogenic potential of airborne particulate matters collected during different seasons at a site that is located in the northern area of the city of Bologna by using alternative in vitro tests, such as the cell transformation assay with BALB/c 3T3 A31-1-1 and Bhas 42 cells. The purpose is also to evaluate the lifetime cancer risks associated with air inhalation in different sites (rural and urban), by using the relative potency of compounds belonging to the same chemical class (PAHs and nitro-PAHs) and the specific unit of carcinogenic risk. None of the organic or inorganic extracts of PM2.5 and PM1 induced a significant increase in the average number of transformed foci/plate or in the transformation frequency of BALB/c 3T3 A311-1 cells, whereas the results obtained by Bhas 42 cell transformation showed a significant increase in the average number of transformed foci/plate. All the analyzed organic extracts showed promoting effects in Bhas 42 cells. The application of the UR cancer risk to the transformed value of B(a)P equivalents in the winter– autumn campaigns leads to estimate an increase in the cancer risk similar to that defined in the literature (1 x 10-4 for exposure to 1 ng/m3). The calculated cancer risk was about one order of magnitude lower in the summer campaigns. In conclusion, the proposed approach, based on the integration of the data derived from in vitro testing and cancer risk assessment, could represent a reliable model for investigating environmental mixtures and predicting their effects on toxicological relevant endpoints.

Alzheimer's disease (AD) is a multifactorial and progressive form of dementia with a senile onset that affects specific areas of the brain. Recent genome wide association (GWA) studies reported that the allele 4 of apolipoprotein E (APOE) and single nucleotide polymorphisms (SNPs) in other genes that regulate inflammatory pathways, such as the gene coding for clusterin (CLU), are associated with AD. The hypothesis is that all of these genes may be involved in different mechanisms mediated by herpes viruses and we argued that the concomitant presence of SNPs in these genes in the same individual may represent a genetic signature predisposing to AD. The present study is focused on SNPs in CLU, interferon (IFN)-λ3/IL-28B, Med23 and the transcription factor IRF7, which are genes involved in antiviral responses and their association with AD and cognitive deterioration. Moreover, the effects of IL-28B, Med23 and IRF7 genotypes upon the presence of epstein-barr virus (EBV) and human herpes virus 6 (HHV-6) in the peripheral blood of AD and controls (CTR) have been also investigated. The activation of the innate immune system has a key role as a promoting factor for AD and in AD patients activated microglia release cytokines that induce neuro-inflammation. In this thesis gene variants and different expression of genes involved in the innate immune response in case-control population studies and in a mouse model of AD were investigated. Results from these experiments suggest that individuals with a particular genetic makeup in defensive mechanisms of the innate immunity may be at risk of defective immune responses. Impaired immunity against persistent viruses such as those of herpes family, might result in chronic and inappropriate activation of microglia, abnormal Aβ production and increased amyloid deposition. Cycles of virus latency and infections may therefore contribute to neurodegeneration associated with AD in genetically predisposed elderly.

Many oomycetes species are pathogens of plants, animals and humans; some of them are well studied because they cause significant economic losses in agriculture and acquaculture. The cell wall of oomycetes consists mainly of cellulose, β- (1,3) and β- (1,6)- glucan and in some species a small amount of chitin is also present. The biosynthesis of cellulose in oomycetes is still poorly studied and therefore misunderstood, although it’s the target of some fungicides that inhibit this process, such as carboxylic acid amides. Since 2010, single amino acid exchanges in CesA3 protein conferring CAA resistance in Plasmopara viticola have been identified. In this work, we initially provide the first evidence of the presence of mandipropamid resistant populations of Plasmopara viticola in commercial vineyards in Italy (paper I). We continue by studying the different activity of four CAAs fungicides (benthiavalicarb, dimethomorph, iprovalicarb, mandipropamid) toward P. viticola resistant strains (paper II). The results show that the G1105S mutation affects all four CAAs, but its impact is varied. These results confirm that they are cross resistant, although many gaps in the mode of action are still present. In order to confirm our previous findings, we performed a microscopical base method to assess the sensitivity of four CAAs, and preliminary microscopical data confirmed the different activity toward the CAA-resistant and CAA-sensitive populations (chapter ten). Furthermore, we present a study (chapter eleven) in which we randomly selected samples from paper I, in order to test them using a different approach, simulating a scenario more close to the field. With this test, we were able to confirm the data presented in the paper I.

Plant bacterial diseases are nowadays routinely managed with scheduled treatments based on heavy metal compounds or, in the worst cases, on antibiotics; to overcome the environmental consequences linked to the use of these chemical compounds, such as pollution or selection of antibiotic resistant pathogens, an integrated control management is required. The use of bacterial antagonists, biological agents, plant defence response elicitors or resistant host plant genotypes play an important role in the frame of sustainable agriculture. In this work, the activity of plasma activated water (PAW) and different bioagents aimed to the control of the two bacterial pathogens Xanthomonas vesicatoria and Pseudomonas syringae pv. actinidiae, causal agents of tomato bacterial leaf spot and kiwifruit bacterial canker, respectively, were studied. All these tools were assayed for their direct efficacy and for their ability, as elicitors, to trigger the plant immune system against these two bacterial pathogens. Moreover, a study on several Actinidia sp. accessions was carried out to evaluate their susceptibility against bacterial canker of kiwifruit. PAW resulted unable to direct inhibit X. vesicatoria growth in in vitro assays, however it showed the ability to trigger tomato plant immune system by reducing disease severity up to approx. 38% when tested in three experiments on two tomato genotypes conducted under greenhouse conditions. When tested in in vitro and in vivo experiments against X. vesicatoria and P. syringae pv. actidinidiae, the tested bioagents, based on natural extracted compounds or on different strains of Bacillus sp., showed a direct efficacy against both bacterial pathogens. Moreover, they were also able to elicit the plant defence response by significantly lowering the disease severity on tomato and kiwifruit leaves. In addition, the A. chinensis accession NPK3 resulted the less susceptible to the bacterial canker in comparison to more than 20 accessions tested.