Study of Italian isolates of Alternaria spp.: molecular and morphological characterization and pathogenesis on apple tree

It was decided to carry out a morphological and molecular characterization of the Italian Alternaria isolatescollected from apple , and evaluate their pathogenicity and subsequently combining the data collected. The strain collection (174 isolates) was constructed by collecting material (received from extension service personnel) between June and August of 2007, 2008, and 2009. A Preliminary bioassays were performed on detached plant materials (fruit and leaf wounded and unwounded), belonging to the Golden cultivar, with two different kind of inoculation (conidial suspension and conidial filtrate). Symptoms were monitored daily and a value of pathogenicity score (P.S.) was assigned on the basis of the diameter of the necrotic area that developed. On the basis of the bioassays, the number of isolates to undergo further molecular analysis was restricted to a representative set of single spore strains (44 strains). Morphological characteristics of the colony and sporulation pattern were determined according to previous systematic work on small-spored Alternaria spp. (Pryor and Michaelides, 2002 and Hong et al., 2006). Reference strains (Alternaria alternata, Alternaria tenuissima, Alternaria arborescens and four Japanese strains of Alternaria alternata mali pathotype), used in the study were kindly provided by Prof. Barry Pryor, who allows a open access to his own fungal collection. Molecular characterization was performed combining and comparing different data sets obtained from distinct molecular approach: 1) investigation of specific loci and 2) fingerprinting based on diverse randomly selected polymorphic sites of the genome. As concern the single locus analysis, it was chosen to sequence the EndoPG partial gene and three anonymous region (OPA1-3, OPA2- and OPa10-2). These markers has revealed a powerful tool in the latter systematic works on small-spored Alternaria spp. In fact, as reported in literature small-spored Alternaria taxonomy is complicated due to the inability to resolve evolutionary relationships among the taxa because of the lack of variability in the markers commonly used in fungi systematic. The three data set together provided the necessary variation to establish the phylogenetic relationships among the Italian isolates of Alternaria spp. On Italian strains these markers showed a variable number of informative sites (ranging from 7 for EndoPg to 85 for OPA1-3) and the parsimony analysis produced different tree topologies all concordant to define A. arborescens as a mophyletic clade. Fingerprinting analysis (nine ISSR primers and eight AFLP primers combination) led to the same result: a monophyleic A. arborescens clade and one clade containing both A. tenuissima and the A. alternata strains. This first attempt to characterize Italian Alternaria species recovered from apple produced concordant results with what was already described in a similar phylogenetic study on pistachio (Pryor and Michaelides, 2002), on walnut and hazelnut (Hong et al., 2006), apple (Kang et al., 2002) and citurus (Peever et al., 2004). Together with these studies, this research demonstrates that the three morphological groups are widely distributed and occupy similar ecological niches. Furthermore, this research suggest that these Alternaria species exhibit a similar infection pattern despite the taxonomic and pathogenic differences. The molecular characterization of the pathogens is a fundamental step to understanding the disease that is spreading in the apple orchards of the north Italy. At the beginning the causal agent was considered as Alteraria alternata (Marshall and Bertagnoll, 2006). Their preliminary studies purposed a pathogenic system related to the synthesis of toxins. Experimental data of our bioassays suggest an analogous hypothesis, considering that symptoms could be induced after inoculating plant material with solely the filtrate from pathogenic strains. Moreover, positive PCR reactions using AM-toxin gene specific primers, designed for identification of apple infecting Alternaria pathovar, led to a hypothesis that a host specific toxin (toxins) were involved. It remains an intriguing challenge to discover or not if the agent of the “Italian disease” is the same of the one previously typified as Alternaria mali, casual agent of the apple blotch disease.

Development of bionanotechnological strategies for signal enhancement in nucleic acids biosensors

Nucleic acid biosensors represent a powerful tool for clinical and environmental pathogens detection. For applications such as point-of-care biosensing, it is fundamental to develop sensors that should be automatic, inexpensive, portable and require a professional skill of the user that should be as low as possible. With the goal of determining the presence of pathogens when present in very small amount, such as for the screening of pathogens in drinking water, an amplification step must be implemented. Often this type of determinations should be performed with simple, automatic and inexpensive hardware: the use of a chemical (or nanotechnological) isothermal solution would be desirable. My Ph.D. project focused on the study and on the testing of four isothermal reactions which can be used to amplify the nucleic acid analyte before the binding event on the surface sensor or to amplify the signal after that the hybridization event with the probe. Recombinase polymerase amplification (RPA) and ligation-mediated rolling circle amplification (L-RCA) were investigated as methods for DNA and RNA amplification. Hybridization chain reaction (HCR) and Terminal deoxynucleotidil transferase-mediated amplification were investigated as strategies to achieve the enhancement of the signal after the surface hybridization event between target and probe. In conclusion, it can be said that only a small subset of the biochemical strategies that are proved to work in solution towards the amplification of nucleic acids does truly work in the context of amplifying the signal of a detection system for pathogens. Amongst those tested during my Ph.D. activity, recombinase polymerase amplification seems the best candidate for a useful implementation in diagnostic or environmental applications.

MetaVaccinology: a new vaccine discovery tool

In the last decade, the reverse vaccinology approach shifted the paradigm of vaccine discovery from conventional culture-based methods to high-throughput genome-based approaches for the development of recombinant protein-based vaccines against pathogenic bacteria. Besides reaching its main goal of identifying new vaccine candidates, this new procedure produced also a huge amount of molecular knowledge related to them. In the present work, we explored this knowledge in a species-independent way and we performed a systematic in silico molecular analysis of more than 100 protective antigens, looking at their sequence similarity, domain composition and protein architecture in order to identify possible common molecular features. This meta-analysis revealed that, beside a low sequence similarity, most of the known bacterial protective antigens shared structural/functional Pfam domains as well as specific protein architectures. Based on this, we formulated the hypothesis that the occurrence of these molecular signatures can be predictive of possible protective properties of other proteins in different bacterial species. We tested this hypothesis in Streptococcus agalactiae and identified four new protective antigens. Moreover, in order to provide a second proof of the concept for our approach, we used Staphyloccus aureus as a second pathogen and identified five new protective antigens. This new knowledge-driven selection process, named MetaVaccinology, represents the first in silico vaccine discovery tool based on conserved and predictive molecular and structural features of bacterial protective antigens and not dependent upon the prediction of their sub-cellular localization.

Intensificazione di processi biologici per la Bioremediation aerobica di suoli contaminati

Enzyveba, a partially characterized complex consortium of not-adapted microorganisms developed through prolonged stabilization of organic wastes, was found to markedly intensify the aerobic remediation of aged PAH- and PCB-contaminated soil by acting as a source of exogenous specialized microorganisms and nutrients. Thus, Enzyveba was tested in the bioremediation of Diesel (G1) and HiQ Diesel (G2) contaminated soils under aerobic slurry-phase conditions by means of a chemical, microbiological, ecotoxicological integrated analytical procedure. The addition of Enzyveba resulted in a higher availability of cultivable specialized bacteria and fungi but this resulted in a slight intensification of soil remediation, probably because of the high content of nutrients and specialized microorganisms of the soil. In many cases, the biotreatability of soils impacted by diesel fuel is limited by their poor content of autochthonous pollutant-degrading microorganisms. Thus, bioaugmentation with stable and reproducible cultures with the required broad substrate specificity might be the solution for a successful remediation. Two microbial consortia, ENZ-G1 and ENZ-G2, were enriched from Enzyveba on G1 and G2. Both consortia consist of a similar composition of bacterial and fungal species. They exhibited a comparable and significant biodegradation capability by removing about 90% of 1 g/l of diesel fuel under liquid culture conditions. Given their remarkable biodegradation potential, richness of quite diverse microbes, stability and resistance after cryopreservation at -20 °C for several months, both consortia appear very interesting candidates for bioaugmentation on site. The mycoflora of a soil historically contaminated by high concentration of PCBs was characterised before, at the beginning and at the end of the biotreatment mentioned above. Several mitosporic fungi isolated from soil grew in presence of a mixture of three PCBs congeners when also glucose was provided. This is the first study in which 5 strains of mitosporic species able to biodegrade PCB are reported in the literature.

Biometanazione della frazione organica dei rifiuti solidi urbani attraverso codigestione con letame bovino

An anaerobic consortium, capable of efficiently converting into methane the organic fraction of mechanically sorted municipal solid waste (MS-OFMSW), was obtained through a dedicated enrichment procedure in a 0.36 L up-flow anaerobic recirculated reactor. This result was obtained after several micro-reactor fed-batch procedures that allowed to obtain only a few methanization of the MS-OFMSW.

Atomic Force Microscopy Studies of the Amyloidogenic Processes of Intrinsically Unstructured Proteins related to Neurodegenerative Diseases

Protein aggregation and formation of insoluble aggregates in central nervous system is the main cause of neurodegenerative disease. Parkinson’s disease is associated with the appearance of spherical masses of aggregated proteins inside nerve cells called Lewy bodies. α-Synuclein is the main component of Lewy bodies. In addition to α-synuclein, there are more than a hundred of other proteins co-localized in Lewy bodies: 14-3-3η protein is one of them. In order to increase our understanding on the aggregation mechanism of α-synuclein and to study the effect of 14-3-3η on it, I addressed the following questions. (i) How α-synuclein monomers pack each other during aggregation? (ii) Which is the role of 14-3-3η on α-synuclein packing during its aggregation? (iii) Which is the role of 14-3-3η on an aggregation of α-synuclein “seeded” by fragments of its fibrils? In order to answer these questions, I used different biophysical techniques (e.g., Atomic force microscope (AFM), Nuclear magnetic resonance (NMR), Surface plasmon resonance (SPR) and Fluorescence spectroscopy (FS)).

Analysis of the memory B cell response against glycoconjugate vaccines

The development of vaccines directed against polysaccharide capsules of S. pneumoniae, H. influenzae and N. meningitidis have been of great importance in preventing potentially fatal infections. Bacterial capsular polysaccharides are T-cell-independent antigens that induce specific antibody response characterized by IgM immunoglobulins, with a very low IgG class switched response and lack of capability of inducing a booster response. The inability of pure polysaccharides to induce sustained immune responses has required the development of vaccines containing polysaccharides conjugated to a carrier protein, with the aim to generate T cell help. It is clear that the immunogenicity of glycoconjugate vaccines can vary depending on different factors, e.g. chemical nature of the linked polysaccharide, carrier protein, age of the target population, adjuvant used. The present study analyzes the memory B cell (MBC) response to the polysaccharide and to the carrier protein following vaccination with a glycoconjugate vaccine for the prevention of Group B streptococcus (GBS) infection. Not much is known about the role of adjuvants in the development of immunological memory raised against GBS polysaccharides, as well as about the influence of having a pre-existing immunity against the carrier protein on the B cell response raised against the polysaccharide component of the vaccine. We demonstrate in the mouse model that adjuvants can increase the antibody and memory B cell response to the carrier protein and to the conjugated polysaccharide. We also demonstrate that a pre-existing immunity to the carrier protein favors the development of the antibody and memory B cell response to subsequent vaccinations with a glycoconjugate, even in absence of adjuvants. These data provide a useful insight for a better understanding of the mechanism of action of this class of vaccines and for designing the best vaccine that could result in a productive and long lasting memory response.

Sviluppo di un'applicazione bioinformatica per la gestione dei dati di antibiotico sensibilità di isolati clinici

Il problema dell'antibiotico-resistenza è un problema di sanità pubblica per affrontare il quale è necessario un sistema di sorveglianza basato sulla raccolta e l'analisi dei dati epidemiologici di laboratorio. Il progetto di dottorato è consistito nello sviluppo di una applicazione web per la gestione di tali dati di antibiotico sensibilità di isolati clinici utilizzabile a livello di ospedale. Si è creata una piattaforma web associata a un database relazionale per avere un’applicazione dinamica che potesse essere aggiornata facilmente inserendo nuovi dati senza dover manualmente modificare le pagine HTML che compongono l’applicazione stessa. E’ stato utilizzato il database open-source MySQL in quanto presenta numerosi vantaggi: estremamente stabile, elevate prestazioni, supportato da una grande comunità online ed inoltre gratuito. Il contenuto dinamico dell’applicazione web deve essere generato da un linguaggio di programmazione tipo “scripting” che automatizzi operazioni di inserimento, modifica, cancellazione, visualizzazione di larghe quantità di dati. E’ stato scelto il PHP, linguaggio open-source sviluppato appositamente per la realizzazione di pagine web dinamiche, perfettamente utilizzabile con il database MySQL. E’ stata definita l’architettura del database creando le tabelle contenenti i dati e le relazioni tra di esse: le anagrafiche, i dati relativi ai campioni, microrganismi isolati e agli antibiogrammi con le categorie interpretative relative al dato antibiotico. Definite tabelle e relazioni del database è stato scritto il codice associato alle funzioni principali: inserimento manuale di antibiogrammi, importazione di antibiogrammi multipli provenienti da file esportati da strumenti automatizzati, modifica/eliminazione degli antibiogrammi precedenti inseriti nel sistema, analisi dei dati presenti nel database con tendenze e andamenti relativi alla prevalenza di specie microbiche e alla chemioresistenza degli stessi, corredate da grafici. Lo sviluppo ha incluso continui test delle funzioni via via implementate usando reali dati clinici e sono stati introdotti appositi controlli e l’introduzione di una semplice e pulita veste grafica.

Funzioni della subunità θ e del dominio PHP della subunità α nel core catalitico della DNA polimerasi III di Escherichia coli

Il core catalitico della DNA polimerasi III, composto dalle tre subunità α, ε e θ, è il complesso minimo responsabile della replicazione del DNA cromosomiale in Escherichia coli. Nell'oloenzima, α ed ε possiedono rispettivamente un'attività 5'-3' polimerasica ed un'attività 3'-5' esonucleasica, mentre θ non ha funzioni enzimatiche. Il presente studio si è concentrato sulle regioni del core che interagiscono direttamente con ε, ovvero θ (interagente all'estremità N-terminale di ε) e il dominio PHP di α (interagente all'estremità C-terminale di ε), delle quali non è stato sinora identificato il ruolo. Al fine di assegnare loro una funzione sono state seguite tre linee di ricerca parallele. Innanzitutto il ruolo di θ è stato studiato utilizzando approcci ex-vivo ed in vivo. I risultati presentati in questo studio mostrano che θ incrementa significativamente la stabilità della subunità ε, intrinsecamente labile. Durante gli esperimenti condotti è stata anche identificata una nuova forma dimerica di ε. Per quanto la funzione del dimero non sia definita, si è dimostrato che esso è attivamente dissociato da θ, che potrebbe quindi fungere da suo regolatore. Inoltre, è stato ritrovato e caratterizzato il primo fenotipo di θ associato alla crescita. Per quanto concerne il dominio PHP, si è dimostrato che esso possiede un'attività pirofosfatasica utilizzando un nuovo saggio, progettato per seguire le cinetiche di reazione catalizzate da enzimi rilascianti fosfato o pirofosfato. L'idrolisi del pirofosfato catalizzata dal PHP è stata dimostrata in grado di sostenere l'attività polimerasica di α in vitro, il che suggerisce il suo possibile ruolo in vivo durante la replicazione del DNA. Infine, è stata messa a punto una nuova procedura per la coespressione e purificazione del complesso α-ε-θ

Therapeutic strategies for modulation of the vaginal microbiota

The vaginal microbiota of healthy women consists of a wide variety of anaerobic and aerobic bacteria, dominated by the genus Lactobacillus. The activity of lactobacilli is essential to protect women from genital infections and to maintain the natural healthy balance of the vaginal ecosystem. This role is particularly important during pregnancy because vaginal infection is one of the most important mechanisms for preterm birth. The most common vaginal disorder is bacterial vaginosis (BV). BV is a polymicrobial disorder, characterized by a depletion of lactobacilli and an increase in the concentration of other bacteria, including Gardnerella vaginalis, anaerobic Gram-negative rods, anaerobic Gram-positive cocci, Mycoplasma hominis, and Mobiluncus spp. An integrated molecular approach based on real-time PCR and PCR-DGGE was used to investigate the effects of two different therapeutic approaches on the vaginal microbiota composition. (i) The impact of a dietary supplementation with the probiotic VSL#3, a mixture of Lactobacillus, Bifidobacterium and Streptococcus strains, on the vaginal microbial ecology and immunological profiles of healthy women during late pregnancy was investigated. The intake was associated to a slight modulation of the vaginal microbiota and cytokine secretion, with potential implications in preventing preterm birth. (ii) The efficacy of different doses of the antibiotic rifaximin (100 mg/day for 5 days, 25 mg/day for 5 days, 100 mg/day for 2 days) on the vaginal microbiota of patients with BV enrolled in a multicentre, double-blind, randomised, placebo-controlled study was also evaluated. The molecular analyses demonstrated the ability of rifaximin 25 mg/day for 5 days to induce an increase of lactobacilli and a decrease of the BV-associated bacteria after antibiotic treatment, and a reduction of the complexity of the vaginal microbial communities. Thus, confirming clinical results, it represents the most effective treatment to be used in future pivotal studies for the treatment of BV.

Bifidobacterium - Human Host Interaction: Role of Human Plasminogen

Bifidobacterium is an important genus of the human gastrointestinal microbiota, affecting several host physiological features. Despite the numerous Bifidobacterium related health-promoting activities, there is still a dearth of information about the molecular mechanisms at the basis of the interaction between this microorganism and the host. Bacterial surface associated proteins may play an important role in this interaction because of their ability to intervene with host molecules, as recently reported for the host protein plasminogen. Plasminogen is the zymogen of the trypsin-like serine protease plasmin, an enzyme with a broad substrate specificity. Aim of this thesis is to deepen the knowledge about the interaction between Bifidobacterium and the human plasminogen system and its role in the Bifidobacterium-host interaction process. As a bifidobacterial model, B. animalis subsp. lactis BI07 has been used because of its large usage in dairy and pharmaceutical preparations. We started from the molecular characterization of the interaction between plasminogen and one bifidobacterial plasminogen receptor, DnaK, a cell wall protein showing high affinity for plasminogen, and went on with the study of the impact of intestinal environmental factors, such as bile salts and inflammation, on the plasminogen-mediated Bifidobacterium-host interaction. According to our in vitro findings, by enhancing the activation of the bifidobacterial bound plasminogen to plasmin, the host inflammatory response results in the decrease of the bifidobacterial adhesion to the host enterocytes, favouring bacterial migration to the luminal compartment. Conversely, in the absence of inflammation, plasminogen acts as a molecular bridge between host enterocytes and bifidobacteria, enhancing Bifidobacterium adhesion. Furthermore, adaptation to physiological concentrations of bile salts enhances the capability of this microorganism to interact with the host plasminogen system. The host plasminogen system thus represents an important and flexible tool used by bifidobacteria in the cross-talk with the host.

From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.

RNA non-codificanti indotti da danno al DNA regolano il fattore di trascrizione HIF-1α

Recenti analisi sull’intero trascrittoma hanno rivelato una estensiva trascrizione di RNA non codificanti (ncRNA), le quali funzioni sono tuttavia in gran parte sconosciute. In questo lavoro è stato dimostrato che alte dosi di camptotecina (CPT), un farmaco antitumorale inibitore della Top1, aumentano la trascrizione di due ncRNA antisenso in 5’ e 3’ (5'aHIF-1α e 3'aHIF-1α rispettivamente) al locus genico di HIF-1α e diminuiscono i livelli dell’mRNA di HIF-1α stesso. Gli effetti del trattamento sono Top1-dipendenti, mentre non dipendono dal danno al DNA alla forca di replicazione o dai checkpoint attivati dal danno al DNA. I ncRNA vengono attivati in risposta a diversi tipi di stress, il 5'aHIF-1α è lungo circa 10 kb e possiede sia il CAP in 5’ sia poliadenilazione in 3’ (in letteratura è noto che il 3'aHIF-1α è un trascritto di 1,7 kb, senza 5’CAP né poliadenilazione). Analisi di localizzazione intracellulare hanno dimostrato che entrambi sono trascritti nucleari. In particolare 5'aHIF-1α co-localizza con proteine del complesso del poro nucleare, suggerendo un suo possibile ruolo come mediatore degli scambi della membrana nucleare. È stata dimostrata inoltre la trascrizione dei due ncRNA in tessuti di tumore umano del rene, evidenziandone possibili ruoli nello sviluppo del cancro. È anche noto in letteratura che basse dosi di CPT in condizioni di ipossia diminuiscono i livelli di proteina di HIF-1α. Dopo aver dimostrato su diverse linee cellulari che i due ncRNA sopracitati non potessero essere implicati in tale effetto, abbiamo studiato le variazioni dell’intero miRnoma alle nuove condizioni sperimentali. In tal modo abbiamo scoperto che il miR-X sembra essere il mediatore molecolare dell’abbattimento di HIF-1α dopo trattamento con basse dosi di CPT in ipossia. Complessivamente, questi risultati suggeriscono che il fattore di trascrizione HIF-1α venga finemente regolato da RNA non-codificanti indotti da danno al DNA.