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Endocrine disrupters are strictly associated to cancer and several cardiovascular risk factors. Bisphenol A (BPA) is an endocrine disrupter commonly used in the manufacturing of plastics based on polycarbonate, polyvinyl chloride and resins. Our study aims to investigate whether BPA may cause pro-oxidative and pro-inflammatory effects on cardiomyoblasts, thus exacerbating the Doxorubicin (DOXO)-induced cardiotoxicity phenomena. We tested the metabolic effects of BPA at low doses analyzing its affections on the intracellular calcium uptake, oxidative stress, lipid peroxidation and production of nitric oxide and interleukins. Co-incubation of BPA and DOXO significantly reduced the cardiomyoblast viability, compared to only DOXO exposure cells. The mechanisms underlying these effects are based on the stimulation of the intracellular calcium accumulation and lipid peroxidation. Notably, BPA increase the production of pro-inflammatory interleukins involved in cardiovascular diseases as well as in DOXO-Induced cardiotoxicity phenomena. This study provides a rationale for translational studies in the field of cardio-oncology.

A histological study was conducted in red mullet, Mullus barbatus, collected from two sites characterized by different anthropogenic impacts. The aim of the study was to assess sex-, size-, season- and site-related variation in gonadal macrophage aggregate (MA) size, number, and relative area. Gonadal MAs were most abundant in males than in females. The number of MA was significantly higher in males from the most impacted site in October, with larger individuals showing more MA than smaller ones. MAs were always found in ripe testes, whereas they occurred only in regressing ovaries. These preliminary findings suggest that the presence of ovarian MA in red mullet is most likely related to ovary regression after spawning, whereas the presence of testicular MA is not necessarily associated to gonad regression, and may vary with season, size, and water quality.

An investigation on the removal of 17-a-Ethinyl Estradiol (EE2) from aqueous solutions using Polyacrylonitrile (PAN) beads has been carried out under closed conditions.

The protective effects of ethanolic olive fruit extract (OFE) and purified oleuropein (Ole) have been evaluated against H2O2-induced oxidative damage and apoptosis in human embryonic kidney (HEK-293) cells. Olive compounds demonstrated a protective effect against H2O2-induced cell death resulting in total rescue of cell viability. This effect was accompanied by a marked decrease in intracellular reactive oxygen species (ROS) and lipid peroxidation as evidenced by malondialdehyde (MDA) production. Consequently, olive compounds reduced the apoptotic cell death. This was confirmed by flow cytometry analysis for multicaspases and by western blot and real-time PCR analyses for the expression of key effectors involved in the apoptotic process. Our data suggest that the anti-apoptotic effect could be due to the activation of the Erk/p38 signaling pathway and to the modulation of Bcl-2/Bax ratio. These findings provide new insight into the mechanisms underlying the protective effects of OFE and Ole against oxidative damage-induced apoptosis.

In recent years, impaired fertility and endometrium related diseases are increased. Many evidences suggest that environmental pollution might be considered a risk factor for endometrial physiopathology. Among environmental pollutants, endocrine disrupting chemicals (EDCs) act on endocrine system, causing hormonal imbalance which, in turn, leads to female and male reproductive dysfunctions. In this work, we studied the effects of triclosan (TCL) and bisphenol A (BPA), two widespread EDCs, on human endometrial stromal cells (ESCs), derived from endometrial biopsies from woman not affected by endometriosis. Cell proliferation, cell cycle, migration and decidualization mechanisms were investigated. Treatments have been performed with both the EDCs separately or in presence and in absence of progesterone used as decidualization stimulus. Both TCL and BPA did not affect cell proliferation, but they arrested ESCs at G2/M phase of cell cycle enhancing cell migration. TCL and BPA also increased gene expression and protein levels of some decidualization markers, such as insulin growth factor binding protein 1 (IGFBP1) and prolactin (PRL), amplifying the effect of progesterone alone. All together, our data strongly suggest that TCL and BPA might alter human endometrium physiology so affecting fertility and pregnancy outcome.

A wide range of endocrine disrupter chemicals can mimic steroid hormones causing adverse health effects. Nonylphenol (NP) and t-octhylphenol (t-OP) are man-made alkylphenolic environmental contaminants possessing controversial endocrine disruption properties. This study has investigated the effects of NP and t-OP enriched diets on hepatic tissue and biotransformation activities in the liver. To this aim, sea bream juveniles were fed with commercial diet enriched with three different doses of NP (NP1: 5mg/kg bw, NP2: 50mg/kg bw and NP3: 100mg/kg bw) or t-OP (t-OP1: 5mg/kg bw, t-OP2: 50mg/kg bw and t-OP3: 100mg/kg bw) for 21 days. A significant increase of the hepatosomatic index was observed in NP1 and t-OP1. Alteration of liver morphology was observed in both NP and t-OP exposed juveniles although the most altered endpoints were observed in t-OP2 with 100% of tissue degeneration. Ethoxyresorufin-O-deethylase activity was significantly inhibited by NP and t-OP (p0.05), while catalase activity was significantly induced, at both doses. A different pattern of protein expression of different isoforms of both vitellogenin and zona radiata protein was evidenced within the treatments. In addition, a significant increase in the abundance of the stress induced heat shock protein 70 gene in the liver of t-OP2 fish and a significant increase in the abundance of the estrogen induced cathepsin D gene in the liver of NP1 and t-OP2 fish, were observed. Finally, estradiol-17β (E2) and testosterone (T) plasma levels and E2/T showed significantly different patterns in NP and t-OP exposed against control fish.

The potential of Raman spectroscopy with excitation in the visible as a tool for quantitative determina- tion of single components in food industry products was investigated by focusing the attention on glu- cose content in commercial sport drinks. At this aim, micro-Raman spectra in the 600-1600 cm À1 wavenumber shift region of four sport drinks were recorded, showing well defined and separated vibra- tional fingerprints of the various contained sugars (glucose, fructose and sucrose). By profiting of the spectral separation of some peculiar peaks, glucose content was quantified by using a multivariate statis- tical analysis based on the interval Partial Least Square (iPLS) approach. The iPLS model needed for data analysis procedure was built by using glucose aqueous solutions at known sugar concentrations as cali- bration data. This model was then applied to sport drink spectra and gave predicted glucose concentra- tions in good agreement with the values obtained by using a biochemical assay. These results represent a significant step towards the development of a fast and simple method for the on-line glucose quantifica- tion in products of food and beverage industry.

A monolithic silica gel matrix with entrapped glucose oxidase (GOD) was constructed as a bioactive element in an optical biosensor for glucose determination. Intrinsic fluorescence of free and immobilised GOD was investigated in the visible range in presence of different glucose concentrations by time-resolved spectroscopy with time-correlated single-photon counting detector. A three-exponential model was used for analysing the fluorescence transients. Fractional intensities and mean lifetime were shown to be sensitive to the enzymatic reaction and were used for obtaining calibration curve for glucose concentration determination. The sensing system proposed achieved high resolution (up to 0.17 mM) glucose determination with a detection range from 0.4 mM to 5 mM.

Micro-Attenuated Total Reflection (ATR) Fourier Transform Infrared spectroscopy was used to investigate sol–gel layers for biosensing applications prior and after glucose oxidase (GOD) immobilization. The changes occurring in sol–gel infrared spectrum after GOD immobilization were clearly evidenced confirming the retaining of the enzyme activity. Moreover, micro-ATR experimental technique allowed us to investigate the spatial distribution of enzyme concentration. The non-destructive nature of our approach also enabled to monitor the time stability of sol–gel layers and of embedded GOD. The temporal evolution of some peaks in infrared spectra of these sol–gel layers was compared with absorption and steady-state fluorescence measurements. The results reported here confirm that micro-ATR infrared spectroscopy can be usefully employed for a non- or minimally invasive detailed characterization of supports for enzyme immobilization.

A series of electrospun mats of styrene–maleic anhydride copolymers has been functionalized by modification with two types of spacers – a polymer with a flexible hydrophilic polyether chain (Jeffamine® ED) and a rigid low-molecular-weight spacer (р-phenylenediamine). The successful modification of the microfibrous mats with these bifunctional compounds has been proved by ATR-FTIR and X-ray photoelectron spectroscopy (XPS) analyses. In a further step, acetylcholinesterase (AChE) has been covalently immobilized onto the modified mats using glutaraldehyde as a binding agent. The amount of bound protein and the specific activity of the immobilized AChE have been determined. The highest activity has been displayed by AChE covalently bound to Jeffamine-modified microfibrous mats. Moreover, the immobilized AChE is characterized by enhanced thermal and storage stability as compared to the free enzyme.

A monolithic silica gel matrix with entrapped glucose oxidase was constructed as a bioactive element in an optical biosensor for glucose determination. Physicochemical and biochemical characterizations of the catalytic matrix were performed, and the intrinsic fluorescence of immobilised glucose oxidase (GOD) was investigated in the UV and visible range by performing steady state and time course measurements. In all cases, the silica gel matrix proved to be a suitable support for optical biosensing owing to its superior optical properties (e.g., high transmittance and reliable fluorescence and GOD absorption spectra after immobilisation). From steady state measurements, calibration curves were obtained as a function of glucose concentration. When time course measurements were performed, the silica gel support displayed a larger linear calibration range and higher sensitivity than other immobilisation systems. In addition, a glucose optical biosensor was developed and characterised using as catalytic element GOD immobilised on a gel disk bound to a bundle of optical fibres.

Glucose oxidase (GOD) immobilized into sol–gel matrices was studied by using Micro-Attenuated Total Reflection Fourier Transform Infrared (micro-ATR FT-IR) spectroscopy in order to characterize enzyme distribution and secondary structure in systems with valuable potentialities in amperometric and optical biosensing. Spectra were acquired in the 4000–600 cm− 1 frequency region and the analysis of specific fingerprints in the FT-IR spectra evidenced that the enzyme was actually immobilized in the matrix. The enzyme spatial distribution was obtained by examining the amide I and amide II band region of spectra from defined sample positions. The deconvolution of the amide I band in terms of lorentzian functions provided information on the secondary structure of the immobilized GOD. By this approach a macroscopic preservation of GOD activity upon immobilization was evidenced along with the existence of some matrix sites with locally inactivated GOD. To our knowledge this is the first example of point-by-point characterization of conformational changes of immobilized enzyme by means of micro-ATR infrared spectroscopy, thus confirming that this technique can be usefully employed for a non- or minimally-invasive detailed micro-characterization of catalytic supports in order to improve their functionality.

Laccases are cuproproteins belonging to the group of oxidoreductases that are able to catalyze the oxidation of various aromatic compounds (particularly phenols) with the concomitant reduction of oxygen to water. They are characterized by low substrate specificity and have a catalytic competence which widely varies depending on the source. Additionally, laccases have also very peculiar optical properties due to their copper active sites which participate to the reduction processes. All these characteristics make laccases very flexible biotic element for biotechnological applications. During the years, a number of studies have been devoted at exploiting catalytic properties of laccases and very few at profiting of their optical properties. Some preliminary studies by absorption, fluorescence FT-IR and Raman spectroscopies of commercial laccases have evidenced their potential usefulness for optical biosensing of phenol compounds as cathecol. Moreover the sol-gel process offers a convenient and versatile method for preparing optically transparent matrices at room temperature that can represent a very convenient support for laccase immobilization. Also for immobilised enzymes the above-mentioned techniques have allowed a detailed characterization of their optical properties that confirmed the potentials of laccases in optical biosensors and represented a fundamental step in the designing of an optimised optical biosensing scheme.

Micro-Raman spectroscopy and interval Partial Least Square (iPLS) multivariate analysis have been used for determining glucose concentration in various commercial sport drinks. By employing a visible excitation light (633 nm), micro- Raman spectra in the 600-1600 cm -1 wavenumber shift region have been recorded, showing well defined and separated vibrational fingerprints of the various contained sugars (glucose, fructose and sucrose). Glucose content was quantified by using the iPLS analysis based on a model built by employing aqueous glucose solutions as reference samples. The estimated glucose concentrations are in good agreement with the values obtained by using a biochemical assay. These results represent a significant step towards the development of a fast, simple, cost-effective Raman-based method for glucose quantification in products of food and beverage industry, alternative to expensive, time-, sample- and chemicals-consuming biochemical assays currently used in production and quality control processes.

The investigation of materials suitable for enzyme immobilization in biosensing applications has a widespread interest. There are many studies on physico-chemical properties of these materials at macroscopic level but few studies have been devoted to examine and correlate these properties at microscopic level. FT-IR spectroscopy with Micro-Attenuated Total Reflection (Micro-ATR) approach can be extremely useful for understanding a variety of aspects of materials which can be used for optimising immobilization procedures. Moreover, this experimental approach is particularly simple to use (no sample preparation is required) and minimally invasive. Using a Perkin Elmer Spectrum One FT-IR spectrometer equipped with a mercury-cadmium-telluride detector and a micro-ATR element we investigated different materials used for immobilization procedures with various enzymes widely used for biosensing in environmental and clinical applications. In particular, composite membranes constituted by a chemically modified poly-acrylonitrile (PAN) membrane plus layers of tethered chitosan of different molecular weight have been examined. Also silica gel matrices without and with glucose oxidase have been investigated. Spectra have been analysed and the contribution of principal functional groups has been evidenced.

Liquid samples of clarified apple and apricot juices at different production stages were investigated using visible light micro-Raman spectroscopy in order to assess its potential in monitoring fruit juice production. As is well-known, pectin plays a strategic role in the production of clarified juice and the possibility of using Raman for its detection during production was therefore evaluated. The data analysis has enabled the clear identification of pectin. In particular, Raman spectra of apple juice samples from washed and crushed fruits revealed a peak at 845 cm-1 (typical of pectin) which disappears in the Raman spectra of depectinised samples. The fructose content was also revealed by the presence of four peaks at 823 cm(-1), 872 cm(-1), 918 cm(-1) and 975 cm(-1). In the case of apricot juice, several Raman fingerprints of beta-carotene at 1008, 1159 and 1520 cm-1 were also highlighted. Present results resulted interesting for the exclusive use of optical methods for the quantitative determination of the above-mentioned substances in place of the biochemical assays generally used for this purpose, which are time consuming and require different chemical reagents for each of them.

Immobilised enzymes can be reused, easily separated from the reaction medium, and are more stable in most of the cases. Despite of these advantages, there are still some problems facing the usage of the immobilised enzyme in industry. One of those problems is diffusion-limitation of both the reactants and the products. This problem becomes even more serious when the products are inhibitors of the enzymes. Different strategies for overcoming this problem have been discussed in this thesis.A new solution to overcome diffusion limitation is based on processing the enzymatic reaction under non-isothermal conditions. In such a bioreactor the enzymes have to be immobilised on a hydrophobic membrane.In this thesis, two enzymes,β-galactosidase and penicillin G acylase have been immobilised onto teflon and nylon membranes. Two grafting techniques have been used to modify the membranes in order to be able to bind the enzyme. These grafting techniques were based on using high-energy radiation as well as chemical modification. Both the grafting technique and enzyme immobilisation method has been optimised. The performance of the membranes has been tested in the non-isothermal bioreactor. A higher activity under non-isothermal conditions was found in comparison to isothermal operation. Furthermore, the application of the non-isothermal conditions is promising for solving the diffusion-limitation problems.