Development of new collagen cross-linkers for adhesive bonding stabilization

Aims: This thesis aimed to investigate the influence of different collagen cross-linkers, as separate primers or contained within desensitizing agents, on the longevity of dental restorations and on the dentinal enzymatic activity immediately, or after aging in vitro. Methods: A series of studies was conducted using several different cross-linking molecules and several adhesive systems. Four studies investigated the longevity of the hybrid layer by means of microtensile bond strength test, and the enzymatic activity using gelatin and in situ zymography, immediately or after 1 year of aging in the artificial saliva. The first study tested samples bonded with or without a cross-linking agent, that were previously aged for 5 years. The degradation of the hybrid layer was observed using transmission electron microscopy, the enzymatic activity in the hybrid layer using in situ zymography. Raman spectroscopy was used to investigate whether the active substance was still within the hybrid layer after 5 years. Results: The results of the studies showed that collagen cross-linkers were efficient in preserving bond strength after aging in vitro when used as separate primers on demineralized or partially demineralized dentin. In the cases when the cross-linker was utilized on mineralized dentin, bond strength results were higher than in the control groups immediately and after aging, however, no difference in enzymatic activity was detected after aging. Conclusions: The tested cross-linker molecules used as separate primers in etch-and-rinse and self-etch adhesives seem to be clinically applicable, since the procedure is not overly time-consuming and seems to preserve the hybrid layer over time. As for the cross-linkers contained in the desensitizing agent, when utilized before the adhesive procedures, it has shown to increase the bond strength of self-etch adhesives, but further studies are needed to better understand its effect on the enzymatic activity and crosslinking effects on mineralized dentin.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

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.

Relação entre resistência ao cisalhamento no plano e parâmetros processuais de co-cura de juntas em compósitos auto-reforçados de polietileno de matriz reprocessada

Este estudo investiga a otimização da resistência ao cisalhamento no plano de juntas de sobreposição co-curadas do compósito termoplástico unidirecional auto-reforçado de polietileno de baixa densidade reciclado reforçado por fibras de polietileno de ultra alto peso molecular através da relação desta resistência com os parâmetros processuais de prensagem a quente para a conformação da junta (pressão, temperatura, tempo e comprimento). A matriz teve sua estrutura química analisada para verificar potenciais degradações devidas à sua origem de reciclagem. Matriz e reforço foram caracterizados termicamente para definir a janela de temperatura de processamento de junta a ser estudada. A elaboração das condições de cura dos corpos de prova foi feita de acordo com a metodologia de Projeto de Experimento de Superfície de Resposta e a relação entre a resistência ao cisalhamento das juntas e os respectivos parâmetros de cura foi obtida através de equação de regressão gerada pelo método dos Mínimos Quadrados Ordinários. A caracterização mecânica em tração do material foi analisada micro e macromecanicamente. A análise química da matriz não demonstrou a presença de grupos carboxílicos que evidenciassem degradação por ramificações de cadeia e reticulação advindos da reciclagem do material. As metodologias de ensaio propostas demonstraram ser eficazes, podendo servir como base para a constituição de normas técnicas. Demonstrou-se que é possível obter juntas com resistência ótima ao cisalhamento de 6,88 MPa quando processadas a 1 bar, 115°C, 5 min e com 12 mm. A análise da fratura revelou que a ruptura por cisalhamento das juntas foi precedida por múltiplas fissuras longitudinais induzidas por sucessivos debondings, tanto dentro quanto fora da junta, devido à tensão transversal acumulada na mesma, proporcional a seu comprimento. A temperatura demonstrou ser o parâmetro de processamento mais relevante para a performance da junta, a qual é pouco afetada por variações na pressão e tempo de cura.

Study of new Catalysts for the Selective Oxidation of n-Butane to Maleic Anhydride: The Role of Catalysts Thermal Treatment

Maleic anhydride (MA) is a very versatile molecule, indeed, with three functional groups (two carbonyl groups and one double bond C=C) it is an excellent joining and cross-linking material. It is obtained via selective oxidation of n-butane, using vanadyl pyrophosphate as a catalyst. The catalytic system has been largely studied over the years and it is normally used in the industrial production of MA, but the main open problem is to completely control its preparation. This thesis reports the effect of different preparation parameters employed during the calcination procedure for the transformation of precursor into the active catalyst. The thermal treatment is already known to be favoured in the presence of water, hence the first study was on the role of different amount of water co-fed with air, leading to obtain catalysts with an higher crystallinity. This is not the only parameter to control: the molar ratio of oxygen has also an important role, to obtain an active and selective catalyst. Some tests decreasing the “oxidizing power” of the mixture were carried out and it was observed a progressive development of VPP phase instead of oxidized V/P/O systems. Established the role of water and oxygen, the optimal conditions have been found when a mixture composed of air, water and nitrogen was used for the calcination, in the molar ratio of 30:10:60% respectively. Also at the lower temperature tested, i.e. 400°C, the catalyst presents the higher conversion of n-butane and MA yield compared to all other samples. The important conclusion we have reached is that not higher amount of water is necessary to obtain the most performing catalyst, thus leading to economic savings. Performing the same experiments on two different precursors, give catalysts with different activity but the mixture previously descripted is always the one that leads to the best performance.

Inequality of income and poverty persistence in the Municipality of Bologna: a cross-sectional and longitudinal analysis through administrative data

This work is part of a project promoted by Emilia-Romagna that aims at encouraging research activities in order to support the innovation strategies of the regional economic system through the exploitation of new data sources. To gain this scope, a database containing administrative data is provided by the Municipality of Bologna. This is achieved by linking data from the Register Office of the Municipality and fiscal data coming from the tax returns submitted to the Revenue Agency and released by the Ministry of Economy and Finance for the period 2002-2017. The main purpose of the project is the analysis of the medium term financial and distributional trends of income of the citizens residing in the Municipality of Bologna. Exploiting this innovative source of data allow us to analyse the dynamics of income at municipal level, overcoming the lack of information in official survey-based statistic. We investigate these trends by building inequality indicators and by examining the persistence of in-work poverty. Our results represent an important informative element to improve the effectiveness and equity of welfare policies at the local level, and to guide the distribution of economic and social support and urban redevelopment interventions in different areas of the Municipality.