On the respiratory metabolism of marine plankton: its application in assessing carbon fluxes and the role of substrates in its biochemical control

Programa de doctorado en Oceanografía. La fecha de publicación es la fecha de lectura

Researches on berry composition in red grape: agronomical, biochemical and molecular approaches

It is well known that the best grape quality can occur only through the achievement of optimal source/sink ratio. Vine balance is in fact a key parameter in controlling berry sugar, acidity and secondary metabolites content (Howell, 2001; Vanden Heuvel et al., 2004). Despite yield reduction and quality improvement are not always strictly related, cluster thinning is considered a technique which could lead to improvement in grape sugar and anthocyanin composition (Dokoozlian and Hirschfelt, 1995; Guidoni et al., 2002). Among several microclimatic variables which may impact grape composition, the effect of cluster light exposure and temperature, which probably act in synergistic and complex way, has been widely explored showing positive even sometimes contradictory results (Spayd et al., 2001; Tarara et al., 2008). Pre-bloom and véraison defoliation are very efficient techniques in inducing cluster microclimatic modification. Furthermore pre-bloom defoliation inducing a lower berry set percentage On these basis the aim of the first experiment of the thesis was to verify in cv Sangiovese the effects on ripening and berry composition of management techniques which may increase source/sink ratio and /or promote light incidence on berries throughout grape ripening. An integrated agronomic, biochemical and microarray approach, aims to understand which mechanisms are involved in berry composition and may be conditioned in the berries during ripening in vines submitted to three treatments. In particular the treatments compared were: a) cluster thinning (increasing in source/sink ratio) b) leaf removal at véraison (increasing cluster light exposure) c) pre-bloom defoliation (increasing source sink ratio and cluster light exposure). Vine response to leaf removal at véraison was further evaluated in the second experiment on three different varieties (Cabernet Sauvignon, Nero d’Avola, Raboso Piave) chosen for their different genetic traits in terms of anthocyanin amount and composition. The integrated agronomic, biochemical and microarray approach, employed in order to understand those mechanisms involved in berry composition of Sangiovese vines submitted to management techniques which may increase source/sink ratio and induce microclimatic changes, bring to interesting results. This research confirmed the main role of source/sink ratio in conditioning sugars metabolism and revealed also that carbohydrates availability is a crucial issue in triggering anthocyanin biosynthesis. More complex is the situation of pre-bloom defoliation, where source/sink and cluster light increase effects are associated to determine final berry composition. It results that the application of pre-bloom defoliation may be risky, as too much dependent on seasonal conditions (rain and temperature) and physiological vine response (leaf area recovery, photosynthetic compensation, laterals regrowth). Early induced stress conditions could bring cluster at véraison in disadvantage to trigger optimal berry ripening processes compared to untreated vines. This conditions could be maintained until harvest, if no previously described physiological recovery occurs. Certainly, light exposure increase linked to defoliation treatments, showed a positive and solid effect on flavonol biosynthesis, as in our conditions temperature was not so different among treatments. Except the last aspects, that could be confirmed also for véraison defoliation, microclimatic changes by themselves seemed not able to induce any modification in berry composition. Further studies are necessary to understand if the peculiar anthocyanic and flavonols composition detected in véraison defoliation could play important role in both color intensity and stability of wines.

Physiological and Biochemical Aspects of Iron Nutrition in Grapevine

The objective of this thesis was to study the response mechanisms of grapevine to Fe-deficiency and to potential Fe chlorosis prevention strategies. The results show that the presence of bicarbonate in the nutrient solution shifted the activity of PEPC and TCA cycle enzymes and the accumulation/translocation of organic acids in roots of Fe-deprived plants. The rootstock 140 Ruggeri displayed a typical behavior of calcicole plants under bicarbonate stress. The Fe chlorosis susceptible rootstock 101-14 reacted to a prolonged Fe-deficiency reducing the root activity of PEPC and MDH. Noteworthy, it accumulates high levels of citric acid in roots, indicating a low capacity to utilizing, transporting and/or exudating organic acids into the rhizosfere. In contrast, 110 Richter rootstock is capable to maintain an active metabolism of organic acids in roots, accumulating them to a lesser extent than 101-14. Similarly to 101-14, SO4 genotype displays a strong decrease of mechanisms associated to Fe chlorosis tolerance (PEPC and MDH enzymes). Nevertheless it is able to avoid excessive accumulation of citric acid in roots, similar as 110 Richter rootstock. Intercropping with Festuca rubra increased leaf chlorophyll content and net photosynthesis. In addition, intercropping reduces the activity of PEPC in roots, similary to Fe-chelate supply. Applications of NH4+ with nitrification inhibitor prevents efficiently Fe-deficiency, increases chlorophyll content, and induces similar root biochemical responses as Fe-EDDHA. Without the addition of nitrification inhibitors, the effectiveness of NH4+ supply on Fe chlorosis prevention resulted significantly lower. The aspects intertwined in this investigation highlight the complexity of Fe physiology and the fine metabolic tuning of grapevine genotypes to Fe availability and soil-related environmental factors. The experimental evidences reveal the need to carry out future researches on Fe nutrition maintaining a continous flow of knowledge between theoretical and agronomical perspectives for fully supporting the efforts devoted to convert science into practice.

The Domon Family of Plant Plasma Membrane B-Type Cytochromes: Heterologous Expression, Biochemical Characterization and Physiological Roles in Vivo

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

BioTuCSoN: biochemical extension of TuCSoN to support self-organising coordination

Starting from pervasive computing paradigm, we want to face the new system's requirements, concerning, mainly, self-organisation, situatedness and adaptivity, through the definition and execution of nature-inspired patterns. They are extracted by the study of dynamics in biological systems and we consider for their implementation the biochemical tuple spaces model. In particular, the aim of the thesis is to design and realize a first biochemical extension of TuCSoN (technology based on tuple spaces model) and, then, to verify its capabilities by means of a proper case study, that deals with local self-organisation and competition of services in an open and highly-dynamic environment.

Understanding The Physiological, Biochemical, and Molecular Mechanisms of Salinity Tolerance in Strawberry Cultivars and in HvTPK1-Overexpressed Barley

The research was carried out to investigate of main elements of salt stress response in two strawberry cultivars, Elsanta and Elsinore. Plants were grown under 0, 10, 20 and 40 mM NaCl for 80 days. Salinity dramatically affected growth in both cultivars, although Elsinore appeared to be more impaired than Elsanta. Moreover a significant reduction of leaf photosynthesis, evaporation, and stomatal conductance was recorded 24 hrs after the stress was applied in both cultivars, whereas physiological functions were differentially restored after acclimation. However, cv. Elsanta had more efficient leaf gas exchange and water status than cv. Elsinore. In general, Fruit yield reduced upon salinization, wheares fruit quality concerning fruit taste, aroma, appearance, total soluble solids and titratable acidity, did not change but rather was enhanced under moderate salinity. On the other hand fruit quality was impaired at severe salt stress. Fruit antioxidant content and antioxidant capacity were enhanced significantly by increasing salt concentration in both cultivars. The oxidative effects of the stress were defined by the measures of some enzymatic activities and lipid peroxidation. Consistently, an increase in superoxide dismutase (SOD), catalase (CAT), peroxide dismutase (POD) enzymes and higher content of proline and soluble proteins were observed in cv. Elsinore than in cv. Elsanta. The increase coincided with a decrease in lipid peroxidation. The research confirmed that although strawberry cultivars were sensitive to salinity, difference between cultivars exist; The experiment revealed that cv. Elsanta could stand severe salt stress, which was lethal to cv. Elsinore. The parameters measured in the previous experiment were proposed as early screening tools for the salt stress response in nine strawberry genotypes. The results showed that, wheares Elsanta and Elsinore cultivars had a lower dry weight reduction at 40 mM NaCl among cultivars, Naiad, Kamila, and Camarosa were the least salt-sensitive cultivars among the screened.

Effects of Global Warming on Berry Composition of cv. Sangiovese: Biochemical and Molecular Aspects and Agronomical Adaptation Approaches

Wine grape must deal with serious problems due to the unfavorable climatic conditions resulted from global warming. High temperatures result in oxidative damages to grape vines. The excessive elevated temperatures are critical for grapevine productivity and survival and contribute to degradation of grape and wine quality and yield. Elevated temperature can negatively affect anthocyanin accumulation in red grape. Particularly, cv. Sangiovese was identified to be very sensitive to such condition. The quantitative real-time PCR analysis showed that flavonoid biosynthetic genes were slightly repressed by high temperature. Also, the heat stress repressed the expression of the transcription factor “VvMYBA1” that activates the expression of UFGT. Moreover, high temperatures had repressing effects on the activity of the flavonoids biosynthetic enzymes “PAL” and “UFGT”.Anthocyanin accumulation in berry skin is due to the balance between its synthesis and oxidation. In grape cv. Sangiovese, the gene transcription and activity of peroxidases enzyme was elevated by heat stress as a defensive mechanism of ROS-scavenging. Among many isoforms of peroxidases genes, one gene (POD 1) was induced in Sangiovese under thermal stress condition. This gene was isolated and evaluated via the technique of genes transformation from grape to Petunia. Reduction in anthocyanins concentration and higher enzymatic activity of peroxidase was observed in POD 1 transformed Petunia after heat shock compared to untrasformed control. Moreover, in wine producing regions, it is inevitable for the grape growers to adopt some adaptive strategies to alleviate grape damages to abiotic stresses. Therefore, in this thesis, the technique of post veraison trimming was done to improve the coupling of phenolic and sugar ripening in Vitis vinifera L. cultivar Sangiovese. Trimming after veraison showed to be executable to slow down the rate of sugar accumulation in grape (to decrease the alcohol potential in wines) without evolution of the main berry flavonoids compounds.

Effects of pesticides on honey bees (Apis mellifera L.): study of a specific route of exposure and evaluation of biochemical-physiological changes in the assessment of the pesticides toxicity

In this study, some important aspects of the relationship between honey bees (Apis mellifera L.) and pesticides have been investigated. In the first part of the research, the effects of the exposure of honey bees to neonicotinoids and fipronil contaminated dusts were analyzed. In fact, considerable amounts of these pesticides, employed for maize seed dressing treatments, may be dispersed during the sowing operations, thus representing a way of intoxication for honey bees. In particular, a specific way of exposure to this pesticides formulation, the indirect contact, was taken into account. To this aim, we conducted different experimentations, in laboratory, in semi-field and in open field conditions in order to assess the effects on mortality, foraging behaviour, colony development and capacity of orientation. The real dispersal of contaminated dusts was previously assessed in specific filed trials. In the second part, the impact of various pesticides (chemical and biological) on honey bee biochemical-physiological changes, was evaluated. Different ways and durations of exposure to the tested products were also employed. Three experimentations were performed, combining Bt spores and deltamethrin, Bt spores and fipronil, difenoconazole and deltamethrin. Several important enzymes (GST, ALP, SOD, CAT, G6PDH, GAPDH) were selected in order to test the pesticides induced variations in their activity. In particular, these enzymes are involved in different pathways of detoxification, oxidative stress defence and energetic metabolism. The results showed a significant effect on mortality of neonicotinoids and fipronil contaminated dusts, both in laboratory and in semi-field trials. However, no effects were evidenced in honey bees orientation capacity. The analysis of different biochemical indicators highlighted some interesting physiological variations that can be linked to the pesticide exposure. We therefore stress the attention on the possibility of using such a methodology as a novel toxicity endpoint in environmental risk assessment.

Chemical, biochemical and microbiological indicators to assess soil quality in temperate agro-ecosystems

Soil is a critically important component of the earth’s biosphere. Developing agricultural production systems able to conserve soil quality is essential to guarantee the current and future capacity of soil to provide goods and services. This study investigates the potential of microbial and biochemical parameters to be used as early and sensitive soil quality indicators. Their ability to differentiate plots under contrasting fertilization regimes is evaluated based also on their sensitivity to seasonal fluctuations of environmental conditions and on their relationship with soil chemical parameters. Further, the study addresses some of the critical methodological aspects of microplate-based fluorimetric enzyme assays, in order to optimize assay conditions and evaluate their suitability to be used as a toll to asses soil quality. The study was based on a long-term field experiment established in 1966 in the Po valley (Italy). The soil was cropped with maize (Z. mays L.) and winter wheat (T. aestivum L.) and received no organic fertilization, crop residue or manure, in combination with increasing levels of mineral N fertilizer. The soil microbiota responded to manure amendment increasing it biomass and activity and changing its community composition. Crop residue effect was much more limited. Mineral N fertilization stimulated crop residue mineralization, shifted microbial community composition and influenced N and P cycling enzyme activities. Seasonal fluctuations of environmental factors affected the soil microbiota. However microbial and biochemical parameters seasonality did not hamper the identification of fertilization-induced effects. Soil microbial community abundance, function and composition appeared to be strongly related to soil organic matter content and composition, confirming the close link existing between these soil quality indicators. Microplate-based fluorimetric enzyme assays showed potential to be used as fast and throughput toll to asses soil quality, but required proper optimization of the assay conditions for a precise estimation of enzymes maximum potential activity.

Oxidative Stress and Friedreich’s Ataxia

Friedreich’s Ataxia (FRDA) is a neurodegenerative disorder caused by a deficiency of the protein frataxin and characterized by oxidative stress. The first aim of my research project was to analyze the effects of tocotrienol in FRDA patients. Patients received for 2 months a low dose of tocotrienol. A number of biochemical parameters related to oxidative stress were studied. We consistently showed that taking for 2 months a low dose of tocotrienol led to the decrease of oxidative stress indexes in FRDA patients. Also, this study provides a suitable model to investigate the efficacy of natural compounds to counteract the oxidative stress in FRDA. Furthermore, we investigated whether the tocotrienol was able to modulate the expression of the frataxin isoforms (FXN-1, FXN -2, FXN-3) in FRDA patients. We demonstrated that tocotrienol leads to a specific and significant increase of FXN-3 expression. As no structural and functional details were available for FNX-2 and FXN-3, 3D-models were built. FXN-1, the canonical isoform, was then docked on the human iron-sulphur complex and functional interactions were computed; when FXN-1 was replaced by FXN-2 or FNX-3, we found that the interactions were maintained, thus suggesting a possible biological role for both isoforms. The second aim of my research project was to investigate the role of a single nucleotide polymorphism (SNP) in the protein Sirtuin 6 in FRDA patients. In fact, it was known that those who harbour a SNP (Asn46/Ser46) in the gene enconding Sirt6 show a better outcome those individuals who are homozygous for the Asn 46 allele. We found that fibroblasts and iPSC-derived neurons from FRDA patients harboring the SNP (Asn46/Ser46) have a reduced amount of Sirt6 protein compared to cells from individuals who are homozygous for the prevalent Asn allele. Our studies provide new information on the role of Sirtuins in FRDA pathogenesis.

Biochemical analysis of the phosphatase domain of the human soluble epoxide hydrolase (sEH)

Die lösliche Epoxidhydrolase (sEH) gehört zur Familie der Epoxidhydrolase-Enzyme. Die Rolle der sEH besteht klassischerweise in der Detoxifikation, durch Umwandlung potenziell schädlicher Epoxide in deren unschädliche Diol-Form. Hauptsächlich setzt die sEH endogene, der Arachidonsäure verwandte Signalmoleküle, wie beispielsweise die Epoxyeicosatrienoic acid, zu den entsprechenden Diolen um. Daher könnte die sEH als ein Zielenzym in der Therapie von Bluthochdruck und Entzündungen sowie diverser anderer Erkrankungen eingesetzt werden. rnDie sEH ist ein Homodimer, in dem jede Untereinheit aus zwei Domänen aufgebaut ist. Das katalytische Zentrum der Epoxidhydrolaseaktivität befindet sich in der 35 kD großen C-terminalen Domäne. Dieser Bereich der sEH s wurde bereits im Detail untersucht und nahezu alle katalytischen Eigenschaften des Enzyms sowie deren dazugehörige Funktionen sind in Zusammenhang mit dieser Domäne bekannt. Im Gegensatz dazu ist über die 25 kD große N-terminale Domäne wenig bekannt. Die N-terminale Domäne der sEH wird zur Haloacid Dehalogenase (HAD) Superfamilie von Hydrolasen gezählt, jedoch war die Funktion dieses N-terminal Domäne lange ungeklärt. Wir haben in unserer Arbeitsgruppe zum ersten Mal zeigen können, dass die sEH in Säugern ein bifunktionelles Enzym ist, welches zusätzlich zur allgemein bekannten Enzymaktivität im C-terminalen Bereich eine weitere enzymatische Funktion mit Mg2+-abhängiger Phosphataseaktivität in der N-terminalen Domäne aufweist. Aufgrund der Homologie der N-terminalen Domäne mit anderen Enzymen der HAD Familie wird für die Ausübung der Phosphatasefunktion (Dephosphorylierung) eine Reaktion in zwei Schritten angenommen.rnUm den katalytischen Mechanismus der Dephosphorylierung weiter aufzuklären, wurden biochemische Analysen der humanen sEH Phosphatase durch Generierung von Mutationen im aktiven Zentrum mittels ortsspezifischer Mutagenese durchgeführt. Hiermit sollten die an der katalytischen Aktivität beteiligten Aminosäurereste im aktiven Zentrum identifiziert und deren Rolle bei der Dephosphorylierung spezifiziert werden. rnrnAuf Basis der strukturellen und möglichen funktionellen Ähnlichkeiten der sEH und anderen Mitgliedern der HAD Superfamilie wurden Aminosäuren (konservierte und teilweise konservierte Aminosäuren) im aktiven Zentrum der sEH Phosphatase-Domäne als Kandidaten ausgewählt.rnVon den Phosphatase-Domäne bildenden Aminosäuren wurden acht ausgewählt (Asp9 (D9), Asp11 (D11), Thr123 (T123), Asn124 (N124), Lys160 (K160), Asp184 (D184), Asp185 (D185), Asn189 (N189)), die mittels ortsspezifischer Mutagenese durch nicht funktionelle Aminosäuren ausgetauscht werden sollten. Dazu wurde jede der ausgewählten Aminosäuren durch mindestens zwei alternative Aminosäuren ersetzt: entweder durch Alanin oder durch eine Aminosäure ähnlich der im Wildtyp-Enzym. Insgesamt wurden 18 verschiedene rekombinante Klone generiert, die für eine mutante sEH Phosphatase Domäne kodieren, in dem lediglich eine Aminosäure gegenüber dem Wildtyp-Enzym ersetzt wurde. Die 18 Mutanten sowie das Wildtyp (Sequenz der N-terminalen Domäne ohne Mutation) wurden in einem Expressionsvektor in E.coli kloniert und die Nukleotidsequenz durch Restriktionsverdau sowie Sequenzierung bestätigt. Die so generierte N-terminale Domäne der sEH (25kD Untereinheit) wurde dann mittels Metallaffinitätschromatographie erfolgreich aufgereinigt und auf Phosphataseaktivität gegenüber des allgemeinen Substrats 4-Nitophenylphosphat getestet. Diejenigen Mutanten, die Phosphataseaktivität zeigten, wurden anschließend kinetischen Tests unterzogen. Basiered auf den Ergebnissen dieser Untersuchungen wurden kinetische Parameter mittels vier gut etablierter Methoden berechnet und die Ergebnisse mit der „direct linear blot“ Methode interpretiert. rnDie Ergebnisse zeigten, dass die meisten der 18 generierten Mutanten inaktiv waren oder einen Großteil der Enzymaktivität (Vmax) gegenüber dem Wildtyp verloren (WT: Vmax=77.34 nmol-1 mg-1 min). Dieser Verlust an Enzymaktivität ließ sich nicht durch einen Verlust an struktureller Integrität erklären, da der Wildtyp und die mutanten Proteine in der Chromatographie das gleiche Verhalten zeigten. Alle Aminosäureaustausche Asp9 (D9), Lys160 (K160), Asp184 (D184) und Asn189 (N189) führten zum kompletten Verlust der Phosphataseaktivität, was auf deren katalytische Funktion im N-terminalen Bereich der sEH hindeutet. Bei einem Teil der Aminosäureaustausche die für Asp11 (D11), Thr123 (T123), Asn124 (N124) und Asn185 (D185) durchgeführt wurden, kam es, verglichen mit dem Wildtyp, zu einer starken Reduktion der Phosphataseaktivität, die aber dennoch für die einzelnen Proteinmutanten in unterschiedlichem Ausmaß zu messen war (2 -10% and 40% of the WT enzyme activity). Zudem zeigten die Mutanten dieser Gruppe veränderte kinetische Eigenschaften (Vmax allein oder Vmax und Km). Dabei war die kinetische Analyse des Mutanten Asp11  Asn aufgrund der nur bei dieser Mutanten detektierbaren starken Vmax Reduktion (8.1 nmol-1 mg-1 min) und einer signifikanten Reduktion der Km (Asp11: Km=0.54 mM, WT: Km=1.3 mM), von besonderem Interesse und impliziert eine Rolle von Asp11 (D11) im zweiten Schritt der Hydrolyse des katalytischen Zyklus.rnZusammenfassend zeigen die Ergebnisse, dass alle in dieser Arbeit untersuchten Aminosäuren für die Phosphataseaktivität der sEH nötig sind und das aktive Zentrum der sEH Phosphatase im N-terminalen Bereich des Enzyms bilden. Weiterhin tragen diese Ergebnisse zur Aufklärung der potenziellen Rolle der untersuchten Aminosäuren bei und unterstützen die Hypothese, dass die Dephosphorylierungsreaktion in zwei Schritten abläuft. Somit ist ein kombinierter Reaktionsmechanismus, ähnlich denen anderer Enzyme der HAD Familie, für die Ausübung der Dephosphorylierungsfunktion denkbar. Diese Annahme wird gestützt durch die 3D-Struktur der N-terminalen Domäne, den Ergebnissen dieser Arbeit sowie Resultaten weiterer biochemischer Analysen. Der zweistufige Mechanismus der Dephosphorylierung beinhaltet einen nukleophilen Angriff des Substratphosphors durch das Nukleophil Asp9 (D9) des aktiven Zentrums unter Bildung eines Acylphosphat-Enzym-Zwischenprodukts, gefolgt von der anschließenden Freisetzung des dephosphorylierten Substrats. Im zweiten Schritt erfolgt die Hydrolyse des Enzym-Phosphat-Zwischenprodukts unterstützt durch Asp11 (D11), und die Freisetzung der Phosphatgruppe findet statt. Die anderen untersuchten Aminosäuren sind an der Bindung von Mg 2+ und/oder Substrat beteiligt. rnMit Hilfe dieser Arbeit konnte der katalytischen Mechanismus der sEH Phosphatase weiter aufgeklärt werden und wichtige noch zu untersuchende Fragestellungen, wie die physiologische Rolle der sEH Phosphatase, deren endogene physiologische Substrate und der genaue Funktionsmechanismus als bifunktionelles Enzym (die Kommunikation der zwei katalytischen Einheiten des Enzyms) wurden aufgezeigt und diskutiert.rn