Variation in peach fruit susceptibility to Monilinia laxa and gene expression

Brown rot caused by Monilinia laxa and Monilinia fructigena is considered one of the most important diseases affecting Prunus species. Although some losses can result from the rotten fruits in the orchard, most of the damage is caused to fruits during the post-harvest phase. Several studies reported that brown rot incidence during fruit development highly varies; it was found that at a period corresponding to the the pit hardening stage, fruit susceptibility drastically decreases, to be quickly restored afterwards. However the molecular basis of this phenomenon is still not well understood. Furthermore, no difference in the rot incidence was found between wound and un-wound fruits, suggesting that resistance associated more to a specifc biochemical response of the fruit, rather than to a higher mechanical resistance. So far, the interaction Monilinia-peach was analyzed through chemical approaches. In this study, a bio-molecular approach was undertaken in order to reveal alteration in gene expression associated to the variation of susceptibility. In this thesis three different methods for gene expression analysis were used to analyze the alterations in gene expression occurring in peach fruits during the pit hardening stage, in a period encompassing the temporary change in Monilinia susceptibility: real time PCR, microarray and cDNA AFLP techniques. In 2005, peach fruits (cv.K2) were weekly harvested during a 19-week long-period, starting from the fourth week after full bloom, until full maturity. At each sampling time, three replicates of 5 fruits each were dipped in the M.laxa conidial suspension or in distilled water, as negative control. The fruits were maintained at room temperature for 3 hours; afterwards, they were peeled with a scalpel; the peel was immediately frozen in liquid nitrogen and transferred to -80 °C until use. The degree of susceptibility of peach fruit to the pathogen was determined on 3 replicates of 20 fruits each, as percentage of infected fruits, after one week at 20 °C. Real time PCR analysis was performed to study the variation in expression of those genes encoding for the enzymes of the phenylpropanoid pathway (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), cinnamate 4-hydroxylase (C4H), leucoanthocyanidine reductase (LAR), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and of the jasmonate pathway, such as lipoxygenase (LOX), both involved in the production of important defense compounds. Alteration in gene expression was monitored on fruit samples of a period encompassing the pit hardening stage and the corresponding temporary resistance to M.laxa infections, weekly, from the 6thto the 12th week after full bloom (AFB) inoculated with M. laxa or mock-inoculated. The data suggest a critical change in the expression level of the phenylpropanoid pathway from the 7th to the 8th week AFB; such change could be directly physiologically associated to the peach growth and it could indirectly determine the decrease of susceptibility of peach fruit to Monilinia rot during the subsequent weeks. To investigate on the transcriptome variation underneath the temporary loss of susceptibility of peach fruits to Monilinia rot, the microarray and the cDNA AFLP techniques were used. The samples harvested on the 8th week AFB (named S, for susceptible ones) and on the 12th week AFB (named R, for resistant ones) were compared, both inoculated or mock-inoculated. The microarray experiments were carried out at the University of Padua (Dept. of Environmental Agronomy and Crop Science), using the μPEACH1.0 microarray together with the suited protocols. The analysis showed that 30 genes (corresponding to the 0.6% of the total sequences (4806) contained in the μPeach1.0 microarray) were found up-regulated and 31 ( 0.6%) down regulated in RH vs. SH fruits. On the other hand, 20 genes (0.4%) were shown to be up-regulated and 13 (0.3%) down-regulated in the RI vs. SI fruit. No genes were found differentially expressed in the mock-inoculated resistant fruits (RH) vs. the inoculated resistant ones (RI). Among the up-regulated genes an ATP sulfurylase, an heat shock protein 70, the major allergen Pru P1, an harpin inducing protein and S-adenosylmethionine decarboxylase were found, conversely among the down-regulated ones, cinnamyl alcohol dehydrogenase, an histidine- containing phosphotransfer protein and the ferritin were found. The microarray experimental results and the data indirectly derived, were tested by Real Time PCR analysis. cDNA AFLP analysis was also performed on the same samples. 339 transcript derived fragments considered significant for Monilinia resistance, were selected, sequenced and classified. Genes potentially involved in cell rescue and defence were well represented (8%); several genes (12.1%) involved in the protein folding, post-transductional modification and genes (9.2%) involved in cellular transport were also found. A further 10.3% of genes were classified as involved in the metabolism of aminoacid, carbohydrate and fatty acid. On the other hand, genes involved in the protein synthesis (5.7%) and in signal transduction and communication (5.7%) were found. Among the most interesting genes found differentially expressed between susceptible and resistant fruits, genes encoding for pathogenesis related (PR) proteins were found. To investigate on the association of Monilinia resistance and PR biological function, the major allergen Pru P1 (GenBank accession AM493970) and its isoform (here named Pru P2), were expressed in heterologous system and in vitro assayed for their anti-microbial activity. The ribonuclease activity of the recombinant Pru P1 and Pru P2 proteins was assayed against peach total RNA. As the other PR10 proteins, they showed a ribonucleolytic activity, that could be important to contrast pathogen penetration. Moreover Pru P1 and Pru P2 recombinant proteins were checked for direct antimicrobial activity. No inhibitory effect of Pru P1 or Pru P2 was detected against the selected fungi.

Trasduzione del segnale e poliamine nell'apoptosi di cellule cardiache

Introduction: Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, thus representing a potential therapeutic target. Apoptosis of cardiac cells can be induced experimentally by several stimuli including hypoxia, serum withdrawal or combination of both. Several lines of research suggest that neurohormonal mechanisms play a central role in the progression of heart failure. In particular, excessive activation of the sympathetic nervous system or the renin-angiotensin-aldosterone system is known to have deleterious effects on the heart. Recent studies report that norepinephrine (NE), the primary transmitter of sympathetic nervous system, and aldosterone (ALD), which is actively produced in failing human heart, are able to induce apoptosis of rat cardiomyocytes. Polyamines are biogenic amines involved in many cellular processes, including apoptosis. Actually it appears that these molecules can act as promoting, modulating or protective agents in apoptosis depending on apoptotic stimulus and cellular model. We have studied the involvement of polyamines in the apoptosis of cardiac cells induced in a model of simulated ischemia and following treatment with NE or ALD. Methods: H9c2 cardiomyoblasts were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation. Cardiomyocyte cultures were prepared from 1-3 day-old neonatal Wistar rat hearts. Polyamine depletion was obtained by culturing the cells in the presence of α-difluoromethylornithine (DFMO). Polyamines were separated and quantified in acidic cellular extracts by HPLC after derivatization with dansyl chloride. Caspase activity was measured by the cleavage of the fluorogenic peptide substrate. Ornithine decarboxylase (ODC) activity was measured by estimation of the release of 14C-CO2 from 14C-ornithine. DNA fragmentation was visualized by the method of terminal transferase-mediated dUTP nick end-labeling (TUNEL), and DNA laddering on agarose gel electophoresis. Cytochrome c was detected by immunoflorescent staining. Activation of signal transduction pathways was investigated by western blotting. Results: The results indicate that simulated ischemia, NE and ALD cause an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of caspase activity, a family of proteases that execute the death program and induce cell death. This effect was prevented in the presence of DFMO, an irreversible inhibitor of ODC, thus suggesting that polyamines are involved in the execution of the death program activated by these stimuli. In H9c2 cells DFMO inhibits several molecular events related to apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, down-regulation of Bcl-xL, and DNA fragmentation. The anti-apoptotic protein survivin is down-regulated after ALD or NE treatement and polyamine depletion obtained by DFMO partially opposes survivin decrease. Moreover, a study of key signal transduction pathways governing cell death and survival, revealed an involvement of AMP activated protein kinase (AMPK) and AKT kinase, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact polyamine depleted cells show an altered pattern of AMPK and AKT activation that may contrast apoptosis and appears to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. Conclusions: These results indicate that polyamines are involved in the execution of the death program activated in cardiac cells by heart failure-related stimuli, like ischemia, ALD and NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.

Alimenti Funzionali e Componenti Nutraceutici come Biomodulatori

Recent knowledge supports the hypothesis that, beyond meeting nutrition needs, diet may modulate various functions in the body and play beneficial roles in some diseases. Research on functional foods is addressing the physiologic effects and health benefits of foods and food components, with the aim of authorizing specific health claims. The recognition that oxidative stress plays a major role in the pathophysiology of cardiac disorders has led to extensive investigations of the protective effects of exogenous antioxidants, but results are controversial. A promising strategy for protecting cardiac cells against oxidative damage may be through the induction of endogenous phase 2 enzymes with the enhancement of cellular antioxidant capacity. Sulforaphane (SF), a naturally occurring isothiocyanate abundant in Cruciferous vegetables, has gained attention as a potential chemopreventive compound thanks to its ability to induce several classes of genes implicated in reactive oxygen species (ROS) and electrophiles detoxification. Antioxidant responsive element (ARE)-mediated gene induction is a pivotal mechanism of cellular defence against the toxicity of electrophiles and ROS. The transcription factor NF-E2-related factor-2 (Nrf2), is essential for the up-regulation of these genes. We investigated whether SF could exert cardioprotective effects against oxidative stress and elucidated the mechanisms underpinning these effects. Accordingly, using cultured rat neonatal cardiomyocytes as a model system, we evaluated the time-dependent induction of gene transcription, the corresponding protein expression and activity of various antioxidant and phase 2 enzymes (catalase, superoxide dismutase, glutathione and related enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase, NAD(P)H: quinone oxidoreductase 1 and thioredoxine reductase) elicited by SF. The results were correlated to intracellular ROS production and cell viability after oxidative stress generated by H2O2, and confirmed the ability of SF to exert cytoprotective effects acting as an indirect antioxidant. Furthermore, to get better insight into SF mechanism of action, we investigated the effect of SF treatment on Nrf2 and the upstream signalling pathways MAPK ERK1/2 and PI3K/Akt, known to mediate a pro survival signal in the heart. The use of specific inhibitors of ERK1/2 and Akt phosphorylation demonstrated their involvement in phase 2 enzymes induction. The concentration of SF tested in this study is comparable to peak plasma concentration achieved after dietary exposure giving clear relevance to our data to support dietary intake of Cruciferous vegetables in cytoprotection against oxidative stress, a common determinant of many cardiovascular diseases.

Manipolazione del metabolismo degli xenobiotici da frutta convenzionale ed attività chemiopreventiva

A reduced cancer risk associated with fruit and vegetable phytochemicals initially dictated chemopreventive approaches focused on specific green variety consumption or even single nutrient supplementations. However, these strategies not only failed to provide any health benefits but gave rise to detrimental effects. In parallel, public-health chemoprevention programmes were developed in the USA and Europe to increase whole vegetable consumption. Among these, the National Cancer Institute (NCI) sponsored plan “5 to 9 a day for a better health” was one of the most popular. This campaign promoted wide food choice through the consumption of at least 5 to 9 servings a day of colourful fruits and vegetables. In this study the effects of the diet suggested by NCI on transcription, translation and catalytic activity of both xenobiotic metabolizing (XME) and antioxidant enzymes were studied in the animal model. In fact, the boost of both antioxidant defences and “good” phase-II together with down-regulation of “bad” phase-I XMEs is still considered one of the most widely-used strategies of cancer control. Six male Sprague Dawley rats for each treatment group were used. According to the Italian Society of Human Nutrition, a serving of fruit, vegetables and leafy greens corresponds to 150, 250 and 50 g, respectively, in a 70 kg man. Proportionally, rats received one or five servings of lyophilized onion, tomato, peach, black grape or lettuce – for white, red, yellow, violet or green diet, respectively - or five servings of each green (“5 a day” diet) by oral gavage daily for 10 consecutive days. Liver subcellular fractions were tested for various cytochrome P450 (CYP) linked-monooxygenases, phase-II supported XMEs such as glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UDPGT) as well as for some antioxidant enzymes. Hepatic transcriptional and translational effects were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. dROMs test was used to measure plasmatic oxidative stress. Routine haematochemical parameters were also monitored. While the five servings administration didn’t significantly vary XME catalytic activity, the lower dose caused a complex pattern of CYP inactivation with lettuce exerting particularly strong effects (a loss of up to 43% and 45% for CYP content and CYP2B1/2-linked XME, respectively; P<0.01). “5 a day” supplementation produced the most pronounced modulations (a loss of up to 60% for CYP2E1-linked XME and a reduction of CYP content of 54%; P<0.01). Testosterone hydroxylase activity confirmed these results. RT-PCR and Western blot analysis revealed that the “5 a day” diet XMEs inactivations were a result of both a transcriptional and a translational effect while lettuce didn’t exert such effects. All administrations brought out none or fewer modulation of phase-II supported XMEs. Apart from “5 a day” supplementation and the single serving of lettuce, which strongly induced DT- diaphorase (an increase of up to 141 and 171%, respectively; P<0.01), antioxidant enzymes were not significantly changed. RT-PCR analysis confirmed DT-diaphorase induction brought about by the administration of both “5 a day” diet and a single serving of lettuce. Furthermore, it unmasked a similar result for heme-oxygenase. dROMs test provided insight into a condition of high systemic oxidative stress as a consequence of animal diet supplementation with “5 a day” diet and a single serving of lettuce (an increase of up to 600% and 900%, respectively; P<0.01). Haematochemical parameters were mildly affected by such dietary manipulations. According to the classical chemopreventive theory, these results could be of particular relevance. In fact, even if antioxidant enzymes were only mildly affected, the phase-I inactivating ability of these vegetables would be a worthy strategy to cancer control. However, the recorded systemic considerable amount of reactive oxygen species and the complexity of these enzymes and their functions suggest caution in the widespread use of vegan/vegetarian diets as human chemopreventive strategies. In fact, recent literature rather suggests that only diets rich in fruits and vegetables and poor in certain types of fat, together with moderate caloric intake, could be associated with reduced cancer risk.

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.

Sintesi, modifica e caratterizzazione di polimeri da fonti rinnovabili

Gli argomenti trattati all’interno di questa tesi di dottorato riguardano la sintesi e la modifica di polimeri ottenuti a partire da fonti rinnovabili. L’acido polilattico (PLA) è stato modificato per ottenere film estensibili per uso alimentare. La scelta del materiale si è basata sull’analisi del suo ciclo di vita e perché è riconosciuto come sicuro per l’utilizzo nel campo alimentare. Le formulazioni testate, a base di PLA, sono state preparate con l’aggiunta di una serie di additivi utilizzati per migliorare le proprietà meccaniche del materiale. La lavorazione è stata eseguita mediante estrusione, ottenendo dei pellet a composizione omogenea successivamente lavorati nell’estrusore a bolla, modalità industriale di produzione dei film estensibili. È stata poi effettuata la sintesi diretta di nuovi poliesteri insaturi a base di dimetil succinato e 1,6-esandiolo. L’insaturazione della catena è stata ottenuta mediante l’uso, durante la sintesi, di derivati dell’olio di ricino, l’acido ricinoleico e il suo derivato insaturo metil undecenoato. Un’altra molecola insatura utilizzata è stata il citronellolo, scelto tra i terpeni. I polimeri così ottenuti sono stati modificati tramite reazioni radicaliche indotte con radiazioni UV utilizzando sostanze antibatteriche (sale 3-esadecil-1-vinilimidazolo bromuro) al fine di ottenere materiali con attività biocida a lungo termine e senza rilascio. Si è proceduto inoltre alla polimerizzazione reversibile di monomeri furanici con oli vegetali utilizzando una strategia di tipo double click. Si è partiti dalla sintesi di monomeri derivanti da oli vegetali contenenti eterocicli furanici attaccati mediante addizione tiol-enica (prima reazione click chemistry) e si è proseguito con la loro successiva polimerizzazione attraverso una reazione di tipo Diels-Alder con molecole con gruppi maleimmidici (seconda reazione click chemistry). I polimeri così ottenuti sono materiali potenzialmente auto-riparanti, grazie alla possibilità di spostare l’equilibrio verso i prodotti o i reagenti semplicemente variando le condizioni di temperatura.

Studio degli effetti funzionali e tossici di derivati di Brassicaceae in modelli sperimentali

I vegetali appartenenti alla famiglia delle Brassicaceae, sono ricchi di molecole biologicamente attive note per le numerose proprietà salutari. L’effetto di un estratto di germogli di cavolo nero toscano (TBCSE) è stato investigato, in termini chemiopreventivi, sugli enzimi epatici del metabolismo degli xenobiotici e antiossidanti, in ratti trattati con TBCSE. I risultati hanno mostrato un complesso pattern di modulazione, con una prevalente inibizione, del sistema citocromo P450-dipendente, e induzioni significative degli enzimi di fase II (glutatione transferasi e glucuronosiltransferasi) e antiossidanti (catalasi, NAD(P)H:chinone reduttasi, glutatione reduttasi e perossidasi). Successivamente, l’effetto di TBCSE è stato studiato nei confronti delle alterazioni provocate da un’alimentazione iperlipidica nel ratto. Il trattamento si è dimostrato efficace nel contrastare gli effetti deleteri dei grassi presenti nella dieta, come l’iperlipidemia, l’aumento del peso corporeo e del fegato, l’indebolimento delle attività degli enzimi antiossidanti e del potenziale detossificante a livello epatico. Complessivamente, TBCSE emerge essere un promettente prodotto nutraceutico con potenziali effetti chemiopreventivi, e da impiegare come strategia alimentare per contrastare gli effetti correlati ad una dieta iperlipidica. Il consumo di dosi sovralimentari di molecole isolate dalle Brassicaceae, tramite per esempio integratori dietetici, come strategia alimentare preventiva, potrebbe tuttavia rappresentare un rischio per la salute. La potenziale tossicità del sulforafane, glucorafanina, indolo-3-carbinolo, e 3,3'-diindolimetano, è stata valutata in epatociti primari di ratto. La citotossicità e l’induzione di stress ossidativo, osservate a concentrazioni non lontane da quelle che potrebbero essere raggiunte in vivo, insieme ad una forte modulazione dell’espressione genica, riguardante principalmente il metabolismo degli xenobiotici, risposte ad alterazioni dello stato ossidoredutivo, eventi di riparazione del DNA e di proteine, induzione dell’apoptosi, e meccanismi (co)cancerogeni, sottolineano la potenzialità di queste molecole di determinare un rischio tossicologico, in seguito ad un’assunzione prolungata e ad alte dosi.

Use of bioassays and biomarkers in Daphnia magna to assess the effect of pharmaceutical residuals in freshwater ecosystems

Widespread occurrence of pharmaceuticals residues has been reported in aquatic ecosystems. However, their toxic effects on aquatic biota remain unclear. Generally, the acute toxicity has been assessed in laboratory experiments, while chronic toxicity studies have rarely been performed. Of importance appears also the assessment of mixture effects, since pharmaceuticals never occur in waters alone. The aim of the present work is to evaluate acute and chronic toxic response in the crustacean Daphnia magna exposed to single pharmaceuticals and mixtures. We tested fluoxetine, a SSRI widely prescribed as antidepressant, and propranolol, a non selective β-adrenergic receptor-blocking agent used to treat hypertension. Acute immobilization and chronic reproduction tests were performed according to OECD guidelines 202 and 211, respectively. Single chemicals were first tested separately. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design with concentrations based on Toxic Units. The conceptual model of Concentration Addition was adopted in this study, as we assumed that the mixture effect mirrors the sum of the single substances for compounds having similar mode of action. The MixTox statistical method was applied to analyze the experimental results. Results showed a significant deviation from CA model that indicated antagonism between chemicals in both the acute and the chronic mixture tests. The study was integrated assessing the effects of fluoxetine on a battery of biomarkers. We wanted to evaluate the organism biological vulnerability caused by low concentrations of pharmaceutical occurring in the aquatic environment. We assessed the acetylcholinesterase and glutathione s-transferase enzymatic activities and the malondialdehyde production. No treatment induced significant alteration of biomarkers with respect to the control. Biological assays and the MixTox model application proved to be useful tools for pharmaceutical risk assessment. Although promising, the application of biomarkers in Daphnia magna needs further elucidation.