New materials for electrochemiluminescence

The study of electrochemiluminescence (ECL) involves photophysical and electrochemical aspects. Excited states are populated by an electrical stimulus. The most important applications are in the diagnostic field where a number of different biologically-relevant molecules (e.g. proteins and nucleic acids) can be recognized and quantified with a sensitivity and specificity previously not reachable. As a matter of fact the electrochemistry, differently to the classic techniques as fluorescence and chemiluminescence, allows to control the excited state generation spatially and temporally. The two research visits into A. J. Bard electrochemistry laboratories were priceless. Dr. Bard has been one of ECL pioneers, the first to introduce the technique and the one who discovered in 1972 the surprising emission of Ru(bpy)3 2+. I consider necessary to thank by now my supervisors Massimo and Francesco for their help and for giving me the great opportunity to know this unique science man that made me feel enthusiastic. I will never be grateful enough… Considering that the experimental techniques of ECL did not changed significantly in these last years the most convenient research direction has been the developing of materials with new or improved properties. In Chapter I the basics concepts and mechanisms of ECL are introduced so that the successive experiments can be easily understood. In the final paragraph the scopes of the thesis are briefly described. In Chapter II by starting from ECL experimental apparatus of Dr. Bard’s laboratories the design, assembly and preliminary tests of the new Bologna instrument are carefully described. The instrument assembly required to work hard but resulted in the introduction of the new technique in our labs by allowing the continuation of the ECL studies began in Texas. In Chapter III are described the results of electrochemical and ECL studies performed on new synthesized Ru(II) complexes containing tetrazolate based ligands. ECL emission has been investigated in solution and in solid thin films. The effect of the chemical protonation of the tetrazolate ring on ECL emission has been also investigated evidencing the possibility of a catalytic effect (generation of molecular hydrogen) of one of the complexes in organic media. Finally, after a series of preliminary studies on ECL emission in acqueous buffers, the direct interaction with calf thymus DNA of some complexes has been tested by ECL and photoluminescence (PL) titration. In Chapter IV different Ir(III) complexes have been characterized electrochemically and photophysically (ECL and PL). Some complexes were already well-known in literature for their high quantum efficiency whereas the remaining were new synthesized compounds containing tetrazolate based ligands analogous to those investigated in Chapt. III. During the tests on a halogenated complex was unexpectedly evidenced the possibility to follow the kinetics of an electro-induced chemical reaction by using ECL signal. In the last chapter (V) the possibility to use mono-use silicon chips electrodes as ECL analitycal devices is under investigation. The chapter begins by describing the chip structure and materials then a signal reproducibility study and geometry optimization is carried on by using two different complexes. In the following paragraphs is reported in detail the synthesis of an ECL label based on Ru(bpy)3 2+ and the chip functionalization by using a lipoic acid SAM and the same label. After some preliminary characterizations (mass spectroscopy TOF) has been demonstrated that by mean of a simple and fast ECL measurement it’s possible to confirm the presence of the coupling product SAM-label into the chip with a very high sensitivity. No signal was detected from the same system by using photoluminescence.

Toward a 3D in vitro model based on decellularized thymus to maintain adult thymic ephitelial cells functionality

During my PhD,I have been develop an innovative technique to reproduce in vitro the 3D thymic microenvironment, to be used for growth and differentiation of thymocytes, and possible transplantation replacement in conditions of depressed thymic immune regulation. The work has been developed in the laboratory of Tissue Engineering at the University Hospital in Basel, Switzerland, under the tutorship of Prof.Ivan Martin. Since a number of studies have suggested that the 3D structure of the thymic microenvironment might play a key role in regulating the survival and functional competence of thymocytes, I’ve focused my effort on the isolation and purification of the extracellular matrix of the mouse thymus. Specifically, based on the assumption that TEC can favour the differentiation of pre-T lymphocytes, I’ve developed a specific decellularization protocol to obtain the intact, DNA-free extracellular matrix of the adult mouse thymus. Two different protocols satisfied the main characteristics of a decellularized matrix, according to qualitative and quantitative assays. In particular, the quantity of DNA was less than 10% in absolute value, no positive staining for cells was found and the 3D structure and composition of the ECM were maintained. In addition, I was able to prove that the decellularized matrixes were not cytotoxic for the cells themselves, and were able to increase expression of MHC II antigens compared to control cells grown in standard conditions. I was able to prove that TECs grow and proliferate up to ten days on top the decellularized matrix. After a complete characterization of the culture system, these innovative natural scaffolds could be used to improve the standard culture conditions of TEC, to study in vitro the action of different factors on their differentiation genes, and to test the ability of TECs to induce in vitro maturation of seeded T lymphocytes.

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.

Studio molecolare dei meccanismi dell'autoincompatibilità gametofitica in pero europeo (Pyrus communis)

Self-incompatibility (SI) systems have evolved in many flowering plants to prevent self-fertilization and thus promote outbreeding. Pear and apple, as many of the species belonging to the Rosaceae, exhibit RNase-mediated gametophytic self-incompatibility, a widespread system carried also by the Solanaceae and Plantaginaceae. Pear orchards must for this reason contain at least two different cultivars that pollenize each other; to guarantee an efficient cross-pollination, they should have overlapping flowering periods and must be genetically compatible. This compatibility is determined by the S-locus, containing at least two genes encoding for a female (pistil) and a male (pollen) determinant. The female determinant in the Rosaceae, Solanaceae and Plantaginaceae system is a stylar glycoprotein with ribonuclease activity (S-RNase), that acts as a specific cytotoxin in incompatible pollen tubes degrading cellular RNAs. Since its identification, the S-RNase gene has been intensively studied and the sequences of a large number of alleles are available in online databases. On the contrary, the male determinant has been only recently identified as a pollen-expressed protein containing a F-box motif, called S-Locus F-box (abbreviated SLF or SFB). Since F-box proteins are best known for their participation to the SCF (Skp1 - Cullin - F-box) E3 ubiquitine ligase enzymatic complex, that is involved in protein degradation through the 26S proteasome pathway, the male determinant is supposed to act mediating the ubiquitination of the S-RNases, targeting them for the degradation in compatible pollen tubes. Attempts to clone SLF/SFB genes in the Pyrinae produced no results until very recently; in apple, the use of genomic libraries allowed the detection of two F-box genes linked to each S haplotype, called SFBB (S-locus F-Box Brothers). In Japanese pear, three SFBB genes linked to each haplotype were cloned from pollen cDNA. The SFBB genes exhibit S haplotype-specific sequence divergence and pollen-specific expression; their multiplicity is a feature whose interpretation is unclear: it has been hypothesized that all of them participate in the S-specific interaction with the RNase, but it is also possible that only one of them is involved in this function. Moreover, even if the S locus male and female determinants are the only responsible for the specificity of the pollen-pistil recognition, many other factors are supposed to play a role in GSI; these are not linked to the S locus and act in a S-haplotype independent manner. They can have a function in regulating the expression of S determinants (group 1 factors), modulating their activity (group 2) or acting downstream, in the accomplishment of the reaction of acceptance or rejection of the pollen tube (group 3). This study was aimed to the elucidation of the molecular mechanism of GSI in European pear (Pyrus communis) as well as in the other Pyrinae; it was divided in two parts, the first focusing on the characterization of male determinants, and the second on factors external to the S locus. The research of S locus F-box genes was primarily aimed to the identification of such genes in European pear, for which sequence data are still not available; moreover, it allowed also to investigate about the S locus structure in the Pyrinae. The analysis was carried out on a pool of varieties of the three species Pyrus communis (European pear), Pyrus pyrifolia (Japanese pear), and Malus × domestica (apple); varieties carrying S haplotypes whose RNases are highly similar were chosen, in order to check whether or not the same level of similarity is maintained also between the male determinants. A total of 82 sequences was obtained, 47 of which represent the first S-locus F-box genes sequenced from European pear. The sequence data strongly support the hypothesis that the S locus structure is conserved among the three species, and presumably among all the Pyrinae; at least five genes have homologs in the analysed S haplotypes, but the number of F-box genes surrounding the S-RNase could be even greater. The high level of sequence divergence and the similarity between alleles linked to highly conserved RNases, suggest a shared ancestral polymorphism also for the F-box genes. The F-box genes identified in European pear were mapped on a segregating population of 91 individuals from the cross 'Abbé Fétel' × 'Max Red Bartlett'. All the genes were placed on the linkage group 17, where the S locus has been placed both in pear and apple maps, and resulted strongly associated to the S-RNase gene. The linkage with the RNase was perfect for some of the F-box genes, while for others very rare single recombination events were identified. The second part of this study was focused on the research of other genes involved in the SI response in pear; it was aimed on one side to the identification of genes differentially expressed in compatible and incompatible crosses, and on the other to the cloning and characterization of the transglutaminase (TGase) gene, whose role may be crucial in pollen rejection. For the identification of differentially expressed genes, controlled pollinations were carried out in four combinations (self pollination, incompatible, half-compatible and fully compatible cross-pollination); expression profiles were compared through cDNA-AFLP. 28 fragments displaying an expression pattern related to compatibility or incompatibility were identified, cloned and sequenced; the sequence analysis allowed to assign a putative annotation to a part of them. The identified genes are involved in very different cellular processes or in defense mechanisms, suggesting a very complex change in gene expression following the pollen/pistil recognition. The pool of genes identified with this technique offers a good basis for further study toward a better understanding of how the SI response is carried out. Among the factors involved in SI response, moreover, an important role may be played by transglutaminase (TGase), an enzyme involved both in post-translational protein modification and in protein cross-linking. The TGase activity detected in pear styles was significantly higher when pollinated in incompatible combinations than in compatible ones, suggesting a role of this enzyme in the abnormal cytoskeletal reorganization observed during pollen rejection reaction. The aim of this part of the work was thus to identify and clone the pear TGase gene; the PCR amplification of fragments of this gene was achieved using primers realized on the alignment between the Arabidopsis TGase gene sequence and several apple EST fragments; the full-length coding sequence of the pear TGase gene was then cloned from cDNA, and provided a precious tool for further study of the in vitro and in vivo action of this enzyme.

Validation of a new method to assess the long-term Hypothalamic-Pituitary-Adrenal Activity using hair glucocorticoids as biomarkers. - Studies on pigs and laboratory Sprague-Dawley Rats

The evaluation of chronic activity of the hypothalamic-pituitary-adrenal (HPA) axis is critical for determining the impact of chronic stressful situations. The potential use of hair glucocorticoids as a non-invasive, retrospective, biomarker of long term HPA activity is of great interest, and it is gaining acceptance in humans and animals. However, there are still no studies in literature examining hair cortisol concentration in pigs and corticosterone concentration in laboratory rodents. Therefore, we developed and validated, for the first time, a method for measuring hair glucocorticoids concentration in commercial sows and in Sprague-Dawley rats. Our preliminary data demonstrated: 1) a validated and specific washing protocol and extraction assay method with a good sensitivity in both species; 2) the effect of the reproductive phase, housing conditions and seasonality on hair cortisol concentration in sows; 3) similar hair corticosterone concentration in male and female rats; 4) elevated hair corticosterone concentration in response to chronic stress manipulations and chronic ACTH administration, demonstrating that hair provides a good direct index of HPA activity over long periods than other indirect parameters, such adrenal or thymus weight. From these results we believe that this new non-invasive tool needs to be applied to better characterize the overall impact in livestock animals and in laboratory rodents of chronic stressful situations that negatively affect animals welfare. Nevertheless, further studies are needed to improve this methodology and maybe to develop animal models for chronic stress of high interest and translational value in human medicine.