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.

Caratterizzazione clinica, eziopatogenetica e ricerca di marker diagnostici nella poliradicoloneurite acuta idiopatica del cane

La poliradicoloneurite acuta idiopatica (ACIP) è una patologia infiammatoria che interessa le radici di più nervi spinali, descritta soprattutto nel cane, più raramente nel gatto, caratterizzata da insorgenza acuta di paresi/paralisi flaccida. L’ACIP mostra notevoli similitudini con la sindrome di Guillan-Barrè dell’uomo (GBS), in cui la patogenesi è su base autoimmunitaria ed è stata correlata con la presenza di alcuni fattori scatenanti (trigger). Lo scopo di questo lavoro è stato quello di caratterizzare l’ACIP in 26 cani, descrivendone la sintomatologia, l’evoluzione clinica, i risultati degli esami diagnostici. La diagnosi si è basata sui riscontri dell’anamnesi, della visita neurologica e del decorso confermata, quando possibile, dai rilievi elettrodiagnostici. Su tutti i cani è stata valutata l’esposizione a specifici agenti infettivi (Toxoplasma gondii, Neospora canunim, Ehrlichia canis, Leishmania infantum), o altri fattori (come vaccinazioni) che potrebbero aver agito da “trigger” per l’instaurarsi della patologia; sull’intera popolazione e su 19 cani non neurologici (gruppo di controllo), si è proceduto alla ricerca degli anticorpi anti-gangliosidi. La sintomatologia di più frequente riscontro (25/26) ha coinvolto la funzione motoria (paresi/plegia) con prevalente interessamento dei 4 arti (24/25) . Sei cani hanno ricevuto una terapia farmacologica, che non ne ha influenzato il decorso, favorevole in 24/26 casi. In 9 pazienti è stata rilevata una precedente esposizione a potenziali trigger; in 10 casi si è riscontrato un titolo anticorpale positivo ad almeno un agente infettivo testato. In 17/26 cani si è ottenuto un titolo anticorpale anti-GM2 e anti-GA1; nella popolazione di controllo solo un caso è risultato positivo. Questi risultati hanno contribuito a consolidare le conoscenze di questa patologia, validando l’utilità della ricerca anticorpale anti-gangliosidica per la diagnosi di ACIP e facendo intravedere la possibilità che l’ACIP possa essere assimilate alla GBS anche dal punto di vista patogenetico, per la quale potrebbe essere considerata come modello animale spontaneo.

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

Structural and biochemical studies of Sporosarcina pasteurii UreE: a nickel-chaperone involved in the urease activation process

Urease is a nickel-dependent enzyme that catalyzes hydrolysis of urea in the last step of organic nitrogen mineralization. Its active site contains a dinuclear center for Ni(II) ions that must be inserted into the apo-enzyme through the action of four accessory proteins (UreD, UreE, UreF, UreG) leading to activation of urease. UreE, acting as a metallo-chaperone, delivers Ni(II) to the preformed complex of apo-urease-UreDFG and has the capability to enhance the GTPase activity of UreG. This study, focused on characterization of UreE from Sporosarcina pasteurii (SpUreE), represents a piece of information on the structure/mobility-function relationships that control nickel binding by SpUreE and its interaction with SpUreG. A calorimetric analysis revealed the occurrence of a binding event between these proteins with positive cooperativity and a stoichiometry consistent with the formation of the (UreE)2-(UreG)2 hetero-oligomer complex. Chemical Shift Perturbations induced by the protein-protein interaction were analyzed using high-resolution NMR spectroscopy, which allowed to characterize the molecular details of the protein surface of SpUreE involved in the complex formation with SpUreG. Moreover, backbone dynamic properties of SpUreE, determined using 15N relaxation analysis, revealed a general mobility in the nanoseconds time-scale, with the fastest motions observed at the C-termini. The latter analysis made it possible for the first time to characterize of the C-terminal portions, known to contain key residues for metal ion binding, that were not observed in the crystal structure of UreE because of disorder. The residues belonging to this portion of SpUreE feature large CSPs upon addition of SpUreG, showing that their chemical environment is directly affected by protein-protein interaction. Metal ion selectivity and affinity of SpUreE for cognate Ni(II) and non cognate Zn(II) metal ions were determined, and the ability of the protein to select Ni(II) over Zn(II), in consistency with the proposed role in Ni(II) cations transport, was established.

Characterization of the Caspr2 and NLGN2 ligands: a proteomic and biochemical approach

Autism Spectrum Disorder (ASD) is a range of early-onset conditions classified as neurodevelopmental disorders, characterized by deficits in social interactions and communication, as well as by restricted interest and repetitive behaviors. Among the proteins associated with this spectrum of disease there are Caspr2, α-NRXN1, NLGN1-4. Caspr2 is involved in the clustering of K+ channels at the juxtaparanodes, where it is proposed to bind TAG-1. Recent works reported a synaptic localization of Caspr2, but little is know on its role in this compartment. NRXNs and their ligand NLGNs, instead, have a well-defined role in the formation and maintenance of synapses. Among the neuroligins, NLGN2 binds NRXNs with the lowest affinity, suggesting that it could have other not yet characterized ligands. The aim of this work was to better characterize the binding of Caspr2 to TAG-1 and to identify new potential binding partner for Caspr2 and NLGN2. Unexpectedly, using Isothermal Titration Calorimetry and co-immunoprecipitation experiments the direct association of the first two proteins could not be verified and the results indicate that the first evidences reporting it were biased by false-positive artifacts. These findings, together with the uncharacterized synaptic localization of Caspr2, made the identification of new potential binding partners for this protein necessary. To find new proteins that associate with Caspr2 and NLGN2, affinity chromatography in tandem with mass spectrometry experiments were performed. Interestingly, about 25 new potential partners were found for these two proteins and NLGN1, that was originally included as a control: 5 of those, namely SFRP1, CLU, APOE, CNTN1 and TNR, were selected for further investigations. Only the association of CLU to NLGN2 was confirmed. In the future, screenings of the remaining candidates have to be carried out and the functional role for the proposed NLGN2-CLU complex has to be studied.

Genetic and morphological variation and differentiation of Raja clavata populations in the European seas: marker development and preliminary data

In this study the population structure and connectivity of the Mediterranean and Atlantic Raja clavata (L., 1758) were investigated by analyzing the genetic variation of six population samples (N = 144) at seven nuclear microsatellite loci. The genetic dataset was generated by selecting population samples available in the tissue databases of the GenoDREAM laboratory (University of Bologna) and of the Department of Life Sciences and Environment (University of Cagliari), all collected during past scientific surveys (MEDITS, GRUND) from different geographical locations in the Mediterranean basin and North-east Atlantic sea, as North Sea, Sardinian coasts, Tuscany coasts and Cyprus Island. This thesis deals with to estimate the genetic diversity and differentiation among 6 geographical samples, in particular, to assess the presence of any barrier (geographic, hydrogeological or biological) to gene flow evaluating both the genetic diversity (nucleotide diversity, observed and expected heterozygosity, Hardy- Weinberg equilibrium analysis) and population differentiation (Fst estimates, population structure analysis). In addition to molecular analysis, quantitative representation and statistical analysis of morphological individuals shape are performed using geometric morphometrics methods and statistical tests. Geometric coordinates call landmarks are fixed in 158 individuals belonging to two population samples of Raja clavata and in population samples of closely related species, Raja straeleni (cryptic sibling) and Raja asterias, to assess significant morphological differences at multiple taxonomic levels. The results obtained from the analysis of the microsatellite dataset suggested a geographic and genetic separation between populations from Central-Western and Eastern Mediterranean basins. Furthermore, the analysis also showed that there was no separation between geographic samples from North Atlantic Ocean and central-Western Mediterranean, grouping them to a panmictic population. The Landmark-based geometric morphometry method results showed significant differences of body shape able to discriminate taxa at tested levels (from species to populations).

Valutazione di marker diagnostici, epidemiologia e nuove prospettive terapeutiche nell' avvelenamento da rodenticidi anticoagulanti nel cane.

In Medicina Veterinaria l'avvelenamento da rodenticidi anticoagulanti è conosciuto e studiato ormai da anni, essendo una delle intossicazioni più comunemente riscontrate nelle specie non target. In letteratura si rinvengono numerose pubblicazioni ma alcuni aspetti sono rimasti ancora inesplorati.Questo studio si propone di valutare il processo infiammatorio, mediante le proteine di fase acuta (APPs), in corso di fenomeni emorragici, prendendo come modello reale un gruppo di soggetti accidentalmente avvelenati da rodenticidi anticoagulanti. I 102 soggetti avvelenati presentano un valore più elevato di proteina C reattiva (CRP)con una mediana di 4.77 mg/dl statisticamente significativo rispetto alla mediana delle due popolazioni di controllo di pari entità numerica create con cross match di sesso, razza ed età; rispettivamente 0.02 mg/dl dei soggetti sani e 0.37 mg/dl dei soggetti malati di altre patologie. Inoltre all'interno del gruppo dei soggetti avvelenati un valore di CRP elevato all'ammissione può predisporre al decesso. La proteina C reattiva assume quindi un ruolo diagnostico e prognostico in questo avvelenamento. Un'altra finalità, di non inferiore importanza, è quella di definire una linea guida terapeutica con l'ausilio di biomarker coagulativi e di valutare la sicurezza della vitamina K per via endovenosa: in 73 cani, non in terapia con vitamina k, intossicati da rodenticidi anticoagulanti, i tempi della coagulazione (PT ed aPTT) ritornano nel range di normalità dopo 4 ore dalla prima somministrazione di 5 mg/kg di vitamina k per via endovenosa e nessun soggetto durante e dopo il trattamento ha manifestato reazioni anafilattiche, nessuno dei pazienti ha necessitato trasfusione ematica e tutti sono sopravvissuti. Infine si è valutata l'epidemiologia dell'ingestione dei prodotti rodenticidi nella specie oggetto di studio e la determinazione dei principi attivi mediante cromatografia liquida abbinata a spettrofotometria di massa (UPLC-MS/MS).

Studio delle citochine nella distrofia muscolare di Emery-Dreifuss: Possibili marker patogenetici e bersagli di cura della malattia

La distrofia muscolare di Emery-Dreifuss (EDMD) è una miopatia degenerativa ereditaria caratterizzata da debolezza e atrofia dei muscoli senza coinvolgimento del sistema nervoso. Individui EDMD presentano, inoltre, cardiomiopatia con difetto di conduzione che provoca rischio di morte improvvisa. Diversi studi evidenziano un coinvolgimento di citochine in diverse distrofie muscolari causanti infiammazione cronica, riassorbimento osseo, necrosi cellulare. Abbiamo effettuato una valutazione simultanea della concentrazione di citochine, chemochine, fattori di crescita, presenti nel siero di un gruppo di 25 pazienti EDMD. L’analisi effettuata ha evidenziato un aumento di citochine quali IL-17, TGFβ2, INF-γ e del TGFβ1. Inoltre, una riduzione del fattore di crescita VEGF e della chemochina RANTES è stata rilevata nel siero dei pazienti EDMD rispetto ai pazienti controllo. Ulteriori analisi effettuate tramite saggio ELISA hanno evidenziato un aumento dei livelli di TGFβ2 e IL-6 nel terreno di coltura di fibroblasti EDMD2. Per testare l’effetto nei muscoli, di citochine alterate, abbiamo utilizzato terreno condizionante di fibroblasti EDMD per differenziare mioblasti murini C2C12. Una riduzione del grado di differenziamento è stata osservata nei mioblasti condizionati con terreno EDMD. Trattando queste cellule con anticorpi neutralizzanti contro TGFβ2 e IL-6 si è avuto un miglioramento del grado di differenziamento. In C2C12 che esprimevano la mutazione H222P del gene Lmna,non sono state osservate alterazioni di citochine e benefici di anticorpi neutralizzanti. I dati mostrano un effetto patogenetico delle citochine alterate come osservato in fibroblasti e siero di pazienti, suggerendo un effetto sul tessuto fibrotico di muscoli EDMD. Un effetto intrinseco alla mutazione della lamina A è stato rilevato sul espressione di caveolina 3 in mioblasti differenziati EDMD. I risultati si aggiungono a dati forniti sulla patogenesi dell' EDMD confermando che fattori intrinseci ed estrinseci contribuiscono alla malattia. Utilizzo di anticorpi neutralizzanti specifici contro fattori estrinseci potrebbe rappresentare un approccio terapeutico come mostrato in questo studio.

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.

Outcome a lungo termine dei pazienti pediatrici con rene singolo. Valutazione di marker laboratoristici e strumentali di danno renale

Obiettivi: valutare in pazienti con rene singolo congenito la correlazione tra il filtrato glomerulare misurato con il DTPA (DTPA-VFG) e 1) marker laboratoristici di danno renale (creatinina, cistatinaC, proteinuria) 2) formule per stimare il filtrato glomerulare 3) parametri di valutazione della crescita renale ecografica. Materiali e metodi: Sono stati arruolati 118 pazienti con rene singolo congenito tra 0 e 18 anni. Sono stati valutati a ogni visita altezza, creatinina, cistatinaC, proteinuria e lunghezza ecografica renale. E’ stato calcolato il filtrato stimato con formule basate sulla creatinina (Schwartz), sulla cistatina C (Zappitelli, Filler, Grubb e Bokenkamp) e su entrambe (equazione di Zappitelli). La crescita renale è stata valutata come rapporto lunghezza ecografica/altezza corporea (USL/H), differenza percentuale tra lunghezza renale misurata e attesa per età (delta%) e presenza o meno d’ipertrofia compensatoria. In 74 bambini è stata misurata la DTPA-VFG. Risultati: Il follow-up è di 2.1 ± 0.9 anni. Il 65% sono maschi. Nessun paziente ha sviluppato danno renale cronico. La media del DTPA-VFG era di 135±44ml/min/1.73m², il valore medio della creatinina 0.47±0.17mg/dl e di cistatinaC di 1±0.4mg/L. La lunghezza ecografica renale media era di 100±17 mm, il rapporto USL/H medio di 0.8±0,1 e il delta% di 1,13±11,4, il 66% presentava ipertrofia renale. Le uniche correlazioni significative con DTPA-VFG sono inversa con la creatinina (p=<.001) e lineare con USL/H (p=<.001). Discussione: Lo studio ha mostrato che come per altre nefrouropatie, la creatina e l’ecografia renale siano due strumenti validi per il follow-up dei pazienti con rene singolo congenito. Il limite principale è dovuto al fatto che nessuno dei pazienti ha sviluppato danno renale cronico e pertanto non è stato possibile stabilire dei cutt-off di rischio per parametri quali USL/H.

A novel marker for heart failure: Cross sectional area assessment in a large vein by conductance catheter.

The present study investigates the feasibility of a new application able to check the heart failure status in a patient through the estimation of the venous distension. In this way it would be possible to follow up patients, avoiding invasive or expensive exams such as cardiac catheterization and echocardiography. Moreover, the devices would also be able to diagnose the decline of the disease, in order to allow a new adaptation to therapy, and vice versa to check the improvement in the patient’s conditions after the CRT device implant. This thesis is essentially divided into three parts: an analytical model was used to obtain an estimation of the error committed for the calculation of the CSA and to understand how the accuracy and sensitivity depend on the different configurations of the electrodes and the catheter position inside the vein; secondly, an in-vitro experiment was carried out in order to verify the practical feasibility for these kinds of measurements, in a very simplified model; in the end, several animal experiments were done to test the in-vivo practicability of the proposed method. The obtained results showed the feasibility of this approach. In fact, the error committed in the estimation of CSA, during the animal experiments, can be considered acceptable (CSAerror_max ≈ -14%). Moreover, it has been demonstrated that the conductance catheter allows assessing, not only the vein CSA, but also the breathing of the animal.

Lysyl oxidase expression is an independent marker of prognosis and a predictor of lymph node metastasis in oral and oropharyngeal squamous cell carcinoma (OSCC)

Proteins of the lysyl oxidase (LOX) family are important modulators of the extracellular matrix. However, they have an important role in the tumour development as well as in tumour progression. To evaluate the diagnostic and prognostic value of the LOX protein in oral and oropharyngeal squamous cell carcinoma (OSCC) we performed QRT-PCR and immunohistochemical analysis on two tissue microarrays (622 tissue samples in total). Significantly higher LOX expression was detected in high grade dysplastic oral mucosa as well as in OSCC when compared to normal oral mucosa (P < 0.001). High LOX expression was correlated with clinical TNM stage (P = 0.020), lymph node metastases for the entire cohort (P < 0.001), as well as in the subgroup of small primary tumours (T1/T2, P < 0.001). Moreover, high LOX expression was correlated with poor overall survival (P = 0.004) and disease specific survival (P = 0.037). In a multivariate analysis, high LOX expression was an independent prognostic factor, predicting unfavourable overall survival. In summary, LOX expression is an independent prognostic biomarker and a predictor of lymph node metastasis in OSCC. Moreover, LOX overexpression may be an early phenomenon in the pathogenesis of OSCC and thus an attractive novel target for chemopreventive and therapeutic strategies.