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Pop bioaccumulation in aquatic trophic webs.
No full textPersistent Organic Pollutants (POPs) are chemical substances that persist in the environment for long time since they are resistant to chemical, biological, and photolytic degradation, are long-range transported because they are semi-volatile, bioaccumulate in organisms due to their lipophilicity and have potential adverse effects on wildlife and human health.
Starting from the first half of the last century, POPs have been developed and used in a wide range of products and they have been released in the environment through combustion. In the present PhD thesis, attention was posed to some “old generation POPs”: commercial mixtures of PCBs (PolyChlorinated Biphenyls), used as dielectric and hydraulic fluids, and some pesticides and their isomers/metabolites: DDTs (DichloroDiphenylTrichloroethane), HCHs (HexaChlorocycloHexanes) and HCB (HexaChloroBenzene). In the past century, million of tons of these chemicals were released into the environment. Nowadays, their use is banned in many areas of the world, including Italy, even if in certain countries, like in Africa for example, DDT is still widely used to fight against malaria vectors.
Lake and marine sediments or the ice of remote areas are important POPs reservoirs which can be considered “chemical time-bombs” since they are not watertight environment and unexpected events can cause POP release with a boomerang effect.
The present work was aimed at clarifying some aspects of POP bioaccumulation in the aquatic trophic webs. Although these compounds have been studied a lot in the last decades, there are still some open topics to understand the fate of these pollutants in real ecosystems.
Many empirical and mechanistic models of different complexity have been developed since the ‘60s, in order to predict the fate of POPs and their potential for bioaccumulation in the aquatic food webs. However, some problems arise when filed data are used to verify these theoretical models.
In this thesis, a bioenergetic steady-state model was applied to the pelagic trophic web of Lake Maggiore, one of the largest and deepest Italian lakes characterized by DDT contamination of industrial origin until 1996. In particular, the seasonal and spatial variability of water concentration and the role of zooplankton in the transfer of POPs were investigated. With this aim, water, zooplankton and fishes of Lake Maggiore were analysed for the determination of DDT content.
Although DDT contamination along the water column became rather homogeneous starting from 2007, the hypothesis of a steady-state condition was rejected, probably due to the algal production. An apparent thermodynamic paradox was then found since zooplankton showed DDT concentrations and bioaccumulation factors on lipid basis (BAFL) higher than fishes. Beyond the uncertainty of the Log Kow values, the underestimation of bacteria and phytoplankton capability to bioconcentrate POPs and/or the underestimation of the trophic level of zooplankton, food consumption rates and other kinetic parameters were taken into account to explain the findings of this study. Moreover, the zooplankton species might be more endangered by POPs than predicted by the steady-state model.
Concerning Italian deep subalpine lakes, other factors can obstacle the occurrence of a steady-state condition. The influence of secondary pollution sources was tested on two glacier-fed lakes (Como and Iseo), starting from the monitoring of a littoral filter-feeding mollusc (Dreissena polymorpha) and a pelagic fish species (Alosa fallax lacustris). In spite of the Italian DDT ban in 1978, in 2005 a large DDT contamination caused very high concentration in both monitored species. A plausible explanation for this phenomenon was that DDT, widely used from 1950s to 1970s for fruit treatment in valleys just below the glaciers, was trapped in the ice and then was released in the last 2 years as a consequence of the ice melting during the recent glacier retreat. In mussels, levels 150 times higher than those recorded before caused a degeneration of the reproductive apparatus, while concentrations in fish were of concern for human consumption.
With the aim of confirming high-altitude glacier melting as the source of the secondary pollution by DDT, past and present contamination was reconstructed by the analyses of POPs stored in a sediment core collected in 2008 in an undisturbed deposition area of Lake Iseo. As expected, it was observed that DDT concentrations sharply increased starting from the mid ninety with a contamination peak in 2005, probably ascribed to the complete overturn of the lake. On the contrary of DDTs, PCBs showed different accumulation profile indicating that these pollutants reached the lake from different pollution sources. Since sediments can not be considered a watertight compartment their role as secondary pollution source can not be ignored.
Within the national PRIN project “An Integrated Approach to the Conservation and Management of the European Eel in the Mediterranean Region”, POP contamination in European eel (Anguilla anguilla), a critically endangered species prone to POP bioaccumulation, was evaluated in order to understand the possible causes of its decline. POP bioaccumulation in eel poses concern even for human health because of its commercial importance and its wide distribution in Europe.
The POP analysis of eels caught in three Italian coastal waters (Tevere River, Lesina Lagoon and Caprolace Lake) showed a high pollution variability within each subpopulation. Nevertheless, it was clear that Tevere eels were much more polluted by POPs with an industrial origin than the others collected in two coastal lagoons. As concern the risk for eel, levels of dioxine-like PCBs that could cause embryo fish mortality, were found in the river eels, also infested by the exotic swimbladder nematode Anguillicoloides crassus, which could jeopardize their swimming ability.
Moreover, in eels collected in the Tevere River gonad alterations not directly related to POP contamination or to parasites were observed. The very low lipid contents measured in some Caprolace eels, probably due to the scarce food availability, could be the cause of an unsuccessful migration to the spawning site.
The main conclusions of this study were that eel contamination by POPs seems to be of great concern for the survival both of this species and of its predators and, therefore, for the human consumption.
The suitability of Anguilla anguilla as bioaccumulator species for POPs monitoring in European water bodies was also considered indicating the suitability of eels as bioindicators in brackish environments and rivers, with particular attention to the physiological condition of the fish.
In the end the occurrence of DDT in equatorial African lakes, notable for the conservation of the endangered lesser flamingos (Phoeniconaias minor), was investigated. The monitoring of DDT pollution was carried out on sediments to evaluate the potential exposition. In spite of the residual usage of DDT in equatorial Africa to fight malaria, the remote areas of lakes Natron and Bogoria in the Rift Valley region were poorly affected by this kind of pollution
Synthesis of fused-ring heterocycles from intramolecular reactions on alkene derivatives.
No full textHeterocycles form by far the largest of the classical divisions of organic chemistry and are of immense importance biologically, industrially, and indeed to the functioning of any developed human society. The majority of pharmaceuticals and biologically active agrochemicals are heterocyclic, as are countless additives and modifiers used in industries as varied as cosmetics, reprography, information storage, and plastics.
The work accomplished in this thesis concerns three different projects to achieve fused-ring heteropolicyclic systems having potential pharmacological properties. The synthetic procedures employed to reach this goal involve well known methodologies based on the functionalization of carbon-carbon double bonds.
The first project deals with diversity-oriented synthesis to access complex structures containing a piperidine unit starting from enantiopure 2-allyl-piperidine.
The second one concerns the study of a synthetic protocol for a new class of enantiopure tetracyclic 1,4-benzodiazepin-5-ones, containing an imidazole and a pyrazole ring.
Finally, third one is related to the obtainment of bicyclic oxazolidinone derivatives by Pd(II)-CuCl2 catalyzed intramolecular reactions on alkoxycarbonyl-protected amino alkenes
The proteomic approach to investigate Parkinson's desease: pathogenetic mechanisms and biomarkers discovery.
No full textThe overall goals of basic research in improving the clinical approach to a disease are, on the one hand, to unravel its natural history, finding its cause and developing better treatments and, on the other hand, to find biomarkers that would permit to ameliorate the diagnosis, to distinguish among patient subtypes and to set up clinical trials of new therapeutic agents. The common threads of the two projects developed in this thesis are the technique used, proteomics, and the main object, Parkinson‟s disease (PD). Proteomics is a powerful methodology to investigate how protein expression is affected in the pathogenesis of a disease process and should be exploited both to investigate PD pathogenetic mechanisms and to discover peripheral biomarkers.
In the first part of this thesis the project “A SH-SY5Y model to investigate PD pathogenetic mechanisms” is discussed. We investigated the expression pattern of cellular proteins following dopamine exposure in catecholaminergic SH-SY5Y human neuroblastoma cells overexpressing α-synuclein by a proteomic approach. Using 2D gel image analysis and ANOVA statistics we identified proteins that correlate with the experimental conditions in our model. Significant changes in specific cellular processes were observed, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis and neuronal plasticity. Proteins were analyzed by a network enrichment tool that automatically enriched the network model in terms of the most relevant proteins that seem to be missing. Moreover, the bioinformatic approach found out the enriched Gene Ontology (GO) categories, allowing us to draw attention to the NF-κB pathway. To validate experimentally the hypothesis of the NF-κB involvement, we performed a luciferase gene reporter assay that revealed a downregulation of the transcription factor activation by α-synuclein increased expression and its complete quenching by dopamine exposure.
Each element arising from the present study could represent a valuable starting point for focused investigations aimed to better understand key issue of PD pathogenesis. For instance, a protein that completely disappeared after dopamine treatment was VDAC2, a porin of the outer mitochondrial membrane. Since the impairment of mitochondrial function is considered among the major pathogenetic factors of neurodegeneration in PD, we further investigated the regulation of VDAC2 degradation and the role of its phosphorylation.
Accumulating evidence supports a specific role of DJ-1 in protecting dopaminergic neurons from dopamine itself. Since this protein act as a pool of different forms possibly associated to different function and localization, we analyzed DJ-1 2D electrophoresis pattern in SH-SY5Y cells after exposure to dopamine; we observed a specific increase in the most acidic forms in the pattern together with a significant decrease of the most basic spot. Unlike cells exposed to generic oxidative conditions, no additional shift was observed. The results are corroborated by a meta-analysis of the literature showing that in the absence of dopamine treatment the specific acidic form is underrepresented.
The second part of the thesis is focused on the project “Dopamine response of dopaminergic circulating cells: the Jurkat cell model”. The functions of immune cells are regulated not only by cytokines, but also by several neurotransmitters. In particular, peripheral blood lymphocytes possess a complex dopaminergic regulatory system. Human T-lymphocytes express all five dopamine receptors (D1-D5), each of which exerts different actions on the regulation of T-cell functions. The Jurkat human leukemia CD4+ T-cell line has been thoroughly used and characterized as a suitable cell model to investigate T-cell signaling and apoptosis, nevertheless its characterization as a model of circulating dopaminergic cells was never reported before. In this regard, we characterized the dopaminergic system in Jurkat cells and through a proteomic approach we analyzed their response to a dopamine challenge in order to highlight metabolic pathways that could specifically reveal alterations linked to PD. A dopamine challenge, with no effect on cell viability (50 μM), induced quantitative changes in the protein expression pattern. The proteins that showed quantitative differences were identified by peptide mass fingerprinting and analyzed in terms of both interaction network and GO classification enrichment. We obtained a significant model for network enrichment and a functional association to GO classification (unfolded protein binding). Eventually, we observed a modification of β-actin and 14-3-3 two-dimensional pattern following dopamine exposure.
In both projects, by combining the experimental and the bioinformatic approach, we fulfilled the expectations for proteomics to generate new hypotheses. Of course, one must consider that models have limitations and that hypotheses need further validation, but they could be a good starting-point to unravel biochemical pathways altered in PD, a disease with a poorly understood etiology
Pharmacological properties of the phytocannabinoids Δ9-tetrahydrocannabivarin and cannabidiol.
No full textCannabis and its derivatives have been used for medicinal purpose for thousand of years. More recently, the main cannabis constituents, cannabinoids, have been found to act and target at cannabinoid as well as other receptors. This brought particular interest around the pharmacology of phytocannabinoids and their possible clinical applications. The research object of this study focused on two phytocannabinoids, Δ9-tetrahydrocannabivarin (Δ9-THCV) and cannabidiol (CBD), and it has been divided in three individual investigations:
In the first study, we investigated the pharmacology of Δ9-THCV at cannabinoid type 1 (CB1) and type 2 (CB2) receptors. We found and confirmed that Δ9-THCV acts as antagonist at CB1 receptors in experiments of [35S]GTPγS binding assay on human CB1-CHO (Chinese hamster ovary) cell membranes. Also, in the same set of experiments, Δ9-THCV displayed a slight inverse agonism at CB1 receptors, which was confirmed in experiments of cyclic AMP assay in hCB1-CHO cells. At CB2 receptors, we found that Δ9-THCV can behave as a partial agonist when the measured response is inhibition of forskolin-induced stimulation of cyclic AMP production in hCB2-CHO cells or stimulation of [35S]GTPγS binding to membranes obtained either from hCB2-CHO cells or from mouse spleen membranes. No such effect was displayed by Δ9-THCV in untransfected CHO cells, pertussis toxin (PTX)-treated hCB2-CHO cells or CB2 -/- mouse spleen membranes.
In collaboration with Dr. Barbara Costa and Dr. Dino Maione, we also showed that Δ9-THCV shares the ability of established selective CB2 receptor agonist to reduce signs of inflammation and inflammatory pain (Guindon, Hohmann 2008). These experiments were performed on in vivo models of λ-carrageenan-induced oedema and thermal hyperalgesia, and formalin-induced nociception.
In the second investigation we brought further evidences that Δ9-THCV is a partial agonist at CB2 receptors. In detail, we applied a protocol to hCB2-CHO cells in order to convert the selective CB2 receptor antagonist/inverse agonist, AM630, into an apparent neutral antagonist. In these experimental conditions, we found that Δ9-THCV still behaves as a CB2 receptor agonist and is antagonized by AM630 in experiments of cyclic AMP assay. Additional tests were also conducted to better understand the pharmacology of the ligand, AM630, at CB2 receptors.
In the third study, we investigated the effect of CBD at serotonin type 1A (5-HT1A) autoreceptors. This research was based on previous in vivo findings showing that CBD-induced anti-emetic and anti-nausea effects were mediated by somatodendritic 5-HT1A autoreceptors located in the raphe nuclei (Rock et al. 2009, Parker et al. 2010). Experiments of [35S]GTPγS binding conducted in rat brainstem membranes revealed that CBD, in a bell-shaped manner, is able to enhance the dose-response curve of the selective 5-HT1A receptor agonist, DPAT. In addition, our results suggest that CBD does not interact directly with 5-HT1A receptors, and that CBD-mediated DPAT dose-response curve enhancement might implicate the involvement of an other receptor
Molecular biomarkers for fish welfare and species authentication.
No full textAquaculture is currently contributing almost half of fish consumed by the human population and it keeps growing more rapidly than other animal food production sectors.
The introduction of molecular techniques, such as various genome projects, gene expression analysis and functional genomics, and the monitoring of stress levels through
very early indicators such as molecular biomarkers, can bring considerable benefits to the quantity and quality of production and improve welfare of reared animals. Moreover in recent years, some teleost species have become model organisms, such as zebrafish and pufferfish, and the knowledge about them could be transferred to further improve reared animals and husbandry. Therefore, species of interest in aquaculture could, in turn, become new animal models. In this context, we looked for a new molecular biomarker for stress in Dicentrarchus labrax.
Stress could involve alterations of brain functioning that may precipitate to mood disorders. The neurotrophin Brain Derived Neurotrophic Factor (BDNF) has recently been involved in stress-induced adaptation. BDNF is a key regulator of neuronal plasticity and adaptive processes. Regulation of BDNF is complex and may reflect not only stressspecific mechanisms, but also hormonal and emotional responses. For this reason, we used, as an animal model of stress, a fish, D. labrax, whose brain organization is very similar to that of higher vertebrates, but is generally considered free of emotional reactions. We provide, for the first time in a species of great interest in aquaculture, a comprehensive characterization of BDNF gene and its transcriptional, translational and post-translational regulation following acute stress. While total BDNF mRNA levels are unchanged, BDNF splicing variants 1c and 1d resulted down regulated after acute stress.
Acute stress induces also a significant increase in proBDNF levels and reduction in mature BDNF suggesting altered regulation of proBDNF proteolytic processing. Notably, we provide here the first evidence that fishes possess a simplified proteolytic regulation of BDNF since the pro28kDa form, generated by the SKI-1 protease in mammals, is absent in fishes. The cleavage site, in fact, has first emerged in reptilians. Finally, we show that the proBDNF/totBDNF ratio is a highly predictive novel quantitative biomarker to detect stress in fishes with sensitivity = 100%, specificity = 87%, and Negative Predictive Value = 100%.
The high predictivity of proBDNF/totBDNF ratio for stress in lower vertebrates indicates that processing of BDNF is a central mechanism in adaptation to stress and predicts that a similar regulation of pro/mature BDNF has likely been conserved throughout evolution of vertebrates from fish to man.
The second part of this thesis is focused on the problem of seafood and fish species authentication. This is an important issue within the seafood industry to protect consumers from fraudulent practices, like species substitution, resulting from the increasingly wide diversification of species and globalization of fish trade. DNA-based methods for species identification are by far the best: they are generally based on PCR amplification of a target sequence, followed by a post-PCR analysis of amplified products, which could consist in sequencing or obtaining species-specific patterns of restriction fragments.
The gene coding for the 5S ribosomal RNA is a suitable target for fish species identification because, for its particular sequence features, it does not require any further treatment after PCR. This gene consists of a small coding conserved region and a variable region of noncoding DNA, which is termed not transcribed spacer (NTS). Both regions are tandem repeated in the genome. The NTS, which is species-specific for length and sequence, has been used, here, to discriminate species subjected to substitution in the Italian fish market. Although preliminary, our results have demonstrated the value of this approach
Valutazione del danno da ischemia e riperfusione epatica in corso di laparoscopia mediante microdialisi intra-parenchimale: studio sperimentale e clinico.
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Plant DNA analysis for forensic and environmental purposes.
No full textAmong the disciplines included by forensic science, forensic biology is one of the most applied, particularly for human identity testing.
But other biological evidence can be found at a crime scene, which can possibly be helpful to solve the case and among the possibilities a very frequent one is the presence of plant material. Since the same molecular techniques used for human DNA are suited for plant DNA, they can be used to establish links between the victim, the suspect and the crime scene.
In the last decade a good amount of knowledge about the genetic structure of many plant species has been made available, whose provenance is mainly from population and conservation genetics studies. In fact, the assessment of the amount and distribution of genetic diversity and of the kind of differentiation in a given species are useful tool to understand evolutionary issues and to plan sound conservation strategies.
The aim of this project has been therefore to investigate the limits and potentials of forensic botany, still under-utilized in forensic casework, and investigate the use of the same genetic data for the floristic heritage management. The main species chosen was Turkey Oak (Quercus cerris L.), because of its wide distribution on the Italian territory and its ecological importance. A total of 509 trees from 16 populations have been sampled and genotyped by 8 heterologous microsatellites or SSRs (Single Sequence Repeats).
The amount of variability and genetic differentiation within and among populations was estimated, and the genetically homogenous groups were identified by the Bayesian clustering method. The species appears derived from no more than two homogeneous gene pools of origin and displays low differentiation (FST = 0.057), this indicating that the colonization of the Italian peninsula after the last glaciation is still under way.
Analysis of molecular variance (AMOVA), performed to partition the total genetic variation among the regions indicated by the Bayesian analysis revealed a significant amount of variability between the populations derived from the two putative gene pools.
The low differentiation observed between the Turkey oak populations unfortunately prevents this species from being a good candidate to be used as forensic tool. However, a population assignment test indicated that some welldifferentiated populations can be easily recognized by a multilocus genotyping.
Finally, after necessary authorization, we applied the knowledge acquired till now to a murder casework analyzing, by means of 4 SSRs, leaves of Hophornbeam tree (Ostrya carpinifolia Scop.), found near the corpse of a young woman. In this second case, albeit the area under study was small and the genetic information non-existent in this species, we were able to pinpoint the provenance of the leaves to a specific tree present on the crime scene
Chemometrics for complex data handling and process optimization in environmental sciences.
No full textScientists involved in environmental studies are called to investigate the effects of pollutants in environment compartments and on human beings. On one hand they are required to develop adequate environmental analytical methods, on the other hand they are charged with collecting and evaluating complex and often inexplicit data characterized by an extremely varying character. In this thesis successful applications of chemometric techniques in these field of environmental analysis are described.
Chapter one focuses on the study of a complex groundwater system situated on the Lake Como area. Geochemical methods and exploratory Principal Component Analysis (PCA) were applied on the chemical data and their results were finally compared. In the second chapter the study concerning some of the main tropospheric pollutants in the Lake Como area from 1992 to 2007 is presented. The pros and cons of the use of unfolding PCA and multiway methods (Tucker3) were discussed. Chapter three and four show how PCA and PLS (Partial Least Square Discriminans Analysis) could be used in classification matters characterized by a limited samples number.
Chapter three shows a new strategy for the unambiguous establishment of the link between the chemical and isotope fingerprint of historical copper mines and ancient copper artifacts. In chapter four multivariate analysis analysis was employed for the volatile profile discrimination of different Italian Pecorino cheese samples. Chapter five and chapter six show how multivariate regression could be employed in the process optimization using the “Experimental Design" strategy. The chapter five with the study of a new low cost analytical method for the platinum determination by Anodic Stripping Voltammetry (ASV) while the chapter six illustrates the optimization of a prevention strategy against Candida Albicans biofilm based on a molecule with antifouling effect