Analisi morfologica e molecolare di cellule da colture primarie umane esposte a resine biocompatibili

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized adhesive systems due to degradation processes or the incomplete polymerization of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental adhesives. Cytotoxic effects due to high concentrations of HEMA have already been investigated, but the influence of minor toxic concentrations for long-term exposition on specific proteins such as type I collagen and tenascin has not been studied in depth. The objective of this project was to study the effect of minor toxic concentrations of HEMA on human gingival fibroblasts (HGFs) and human pulp fibroblasts (HPFs), investigating modification in cell morphology, cell viability, and the influence on type I collagen and tenascin proteins. Different concentrations of the resin monomer and different times of exposition were tested on both cell lines. The cell vitality was determined by MTT assay, and high-resolution scanning electron microscopy analysis was performed to evaluate differences in cell morphology before and after treatment. To evaluate the variability in the expression and synthesis of procollagen α1 type I and tenascin proteins on HGFs and HPFs treated with HEMA at different concentrations immunofluorescence, RT-PCR and western blot analysis, were carried out. The treatments on HGFs with 3mmol/L HEMA, showed a strong reduction of procollagen α1 type I protein at 72h and 96h, demonstrating that HEMA interferes both with the synthesis of the procollagen α1 type I protein and its mRNA expression. The results obtained on HPFs treated with different concentrations of HEMA ranging from 0,5mmol/L to 3mmol/L and for different exposition times showed a strong reduction in cell viability in specimens treated for 96h and 168h, while immunofluorescence and western blotting analysis demonstrated a reduction of procollagen α1 type I and an overexpression of tenascin protein. In conclusion, our results showed that the concentrations of HEMA we tested, effect the normal cell production and activity, such as the synthesis of some dental extracellular matrix proteins.

Stereoselective synthesis of heterocycles as intermediates of biologically active compounds

The aim of this thesis was to investigate the synthesis of enantiomerically enriched heterocycles and dehydro-β-amino acid derivatives which can be used as scaffolds or intermediates of biologically active compounds, in particular as novel αvβ3 and α5β1 integrin ligands. The starting materials of all the compounds here synthesized are alkylideneacetoacetates. Alkylidene derivates are very usefull compounds, they are usually used as unsaturated electrophiles and they have the advantage of introducing different kind of functionality that may be further elaborated. In chapter 1, regio- and stereoselective allylic amination of pure carbonates is presented. The reaction proceeds via uncatalyzed or palladium-catalyzed conditions and affords enantiopure dehydro-β-amino esters that are useful precursor of biologically active compounds. Chapter 2 illustrates the synthesis of substituted isoxazolidines and isoxazolines via Michael addition followed by intramolecular hemiketalisation. The investigation on the effect of the Lewis acid catalysis on the regioselectivity of the addition it also reported. Isoxazolidines and isoxazolines are interesting heterocyclic compounds that may be regarded as unusual constrained -amino acids or as furanose mimetics. The synthesis of unusual cyclic amino acids precursors, that may be envisaged as proline analogues, as scaffolds for the design of bioactive peptidomimetics is presented in chapter 3. The synthesis of 2-substituted-3,4-dehydropyrrole derivatives starting from allylic carbonates via a two step allylic amination/ring closing metathesis (RCM) protocol is carried out. The reaction was optimized by testing different Grubbs’ catalysts and carbamate nitrogen protecting groups. Moreover, in view of a future application of these dehydro-β-amino acids as central core of peptidomimetics , the malonate chain was also used to protect nitrogen prior to RCM. Finally, chapter 4 presents the synthesis of two novel different classes of integrin antagonists, one derived from dehydro-β-amino acid prepared as described in chapter 1 and the other one has isoxazolidines synthesized in chapter 2 as rigid constrained core. Since that these compounds are promising RGD mimetics for αvβ3 and α5β1 integrins, they have been submitted to biological assay. and to interpret on a molecular basis their different affinities for the αvβ3 receptor, docking studies were performed using Glide program.