InsubriaSPACE
Not a member yet
702 research outputs found
Sort by
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
Evolutionary processes in an environmental challenging site: the soda-lake Natron (Tanzania).
No full textLakes Natron and Magadi are neighbouring small soda localised in the Eastern Rift Valley, between Kenya and Tanzania, and are the remnants of the Paleolake Orolonga after a split that occurred around 9,000 years BP. These basins represent a really harsh environment, characterized by a complex geo-morphological structure, which may increase, by habitat fragmentation and isolation, the effects of evolutionary forces (like genetic drift and selection) on the native species and populations. These lakes harbour a small species flock of quite recent origin, the Alcolapia flock, endemic of this region and not found in any other place around the world. This flock is characterized by four different morphotypes: A. alcalicus, A. latilabris, A. ndalalani, endemic of Lake Natron, and A. grahami, found only in the Lake Magadi.
With the aim to understand the evolutionary processes that are shaping genetic diversity of this species, we have analysed by means of molecular genetics tools (mtDNA and nuclear DNA markers), 310 specimens of Alcolapia collected from eight populations placed around the Lake Natron and from one population located in the north-east part of Lake Magadi.
Phylogenetic analyses based on D-loop sequences of a subset of 69 Alcolapia have shown a monophyletic structure of the flock, as suggested by the more frequent haplotype (2lat) shared by all the morphotypes and corresponding to the Orolonga haplotype identified in previous studies (Seegers et al, 1999; Wilson et al., 2000 and 2004). Besides, the main starburst radiation occurred in Lake Natron Alcolapia, which evolved in relatively recent times
Functional characterization of the tumor antagonizing gene RNASET2.
No full textSeveral types of cancer including solid tumors and blood malignancies are associated with alterations in different regions of chromosome 6. In particular, the 6q region has been frequently found to be deleted in a wide range of solid tumors, including ovarian carcinoma. Therefore, this chromosomal region has been extensively investigated in order to identifying putative tumor suppressor genes. In specific, a minimal region of deletion for ovarian cancer has been defined in the peritelomeric 6q27 region, and our group has recently isolated from this region the RNASET2 gene, coding for a secreted ribonuclease belonging to the Rh/T2/S acid ribonuclease family.
The RNASET2 gene was found to be down-regulated at the transcriptional level in several primary ovarian tumors and ovarian cancer cell lines and its potential for tumor growth inhibition has been subsequently tested in vivo in a highly tumorigenic and metastatic Hey3Met2 ovarian cancer cell line.
To this aim, athymic mice were injected with Hey3Met2 cell clones stably transfected with expression vectors encoding wild-type or a catalytically inactive mutant RNASET2 protein. Strikingly, wild-type RNASET2-expressing cells formed smaller tumors in mice and gave rise to a lower number of metastasis when compared to control cells transfected with the vector only. Moreover, mutant RNASET2-expressing cells also displayed a similar or even more increased inhibition of tumorigenicity when compared to wild-type RNASET2, suggesting that catalytic activity is not involved in control of tumor suppression and metastasis.
With the purpose of further defining the biological role of RNASET2, we have undertaken a functional characterization of this gene, by comparing RNASET2-overespressing cells behavior both in vitro and in vivo. In sharp contrast with previous in vivo observations, several in vitro tests such as proliferation, clonogenicity, growth in soft agar and apoptosis assays failed to reveal any change in cancer-related phenotypes in the same RNASET2-overexpressing cell line, thus suggesting a non-cell autonomous activity for RNASET2 as a tumor suppressor.
This “asymmetric” in vivo/in vitro property has been recently ascribed to a novel class of tumor suppressor genes, called tumor antagonizing genes (TAGs), whose oncosuppressive activity is carried out only in vivo by induction of microenvironmental remodeling.
To confirm this hypothesis we turned to a xenograft assay and, as expected, Hey3Met2 cells expressing wild-type or mutant RNASET2 were not able to support tumor growth when compared to control mice.
Strikingly, a detailed histological analysis of tumor sections revealed a different architectural pattern between control mice and RNASET2-expressing tumors. The former showed a solid and diffuse growth with strong component of human tumor cells, whereas in the latter a more organized structure and a 2 massive infiltration of host stromal cells mainly derived from monocyte/macrophage lineage has been found.
In order to establish whether these cells could be functionally responsible for RNASET2-mediated tumor suppression, a further in vivo xenograft assay with Rag-2-/- γc-/- mice was performed, since these mice lack both lymphocytes and NK cells, thus retaining innate immunological response only through macrophages. As previously observed in nude mice, RNASET2-expressing cells were again clearly suppressed in tumorigenicity, further suggesting a functional role for macrophages recruitment in tumor suppression mediated by RNASET2.
To definitively confirm the involvement of this cell lineage, in an additional in vivo experiment Rag-2-/- γc-/- mice were pretreated with the macrophages-depleting agent clodronate before inoculation of Hey3Met2 cells. Untreated mice showed once again a clear effect of tumor suppression exerted by RNASET2 whereas in clodronate-treated mice a very similar tumor development was noticed in both control and RNASET2-expressing cells. Finally, immunohistochemical analysis showed that recruited immune cells mainly belong to M1 subtype of anti-tumorigenic macrophages, suggesting a possible mechanism of tumor suppression exerted by this secreted ribonuclease.
In order to extend RNASET2 functional characterization, beside the experimental model described above a complementary research approach based on gene silencing was carried out, aimed at studying how the lack of RNASET2 could affect tumor development and providing a powerful tool for further investigations.
Our working hypothesis considers RNASET2 inactivation as a critical step in ovarian cancer progression, therefore cancer cell lines endowed with a low in vivo tumorigenic potential and expressing high endogenous RNASET2 levels might represent an ideal model system to challenge this hypothesis. In such a context, RNAi-mediated gene knockdown could provide additional information on this topic.
RNAi-mediated silencing of the RNASET2 gene has therefore been attempted in the human ovarian carcinoma cell line OVCAR3, which displays the above mentioned features.
Complete knockdown of RNASET2 in OVCAR3 cell line was successfully achieved and preliminary in vitro tests are in keeping with our previous data, showing no significant differences in proliferation and clonogenic potential in vitro in RNASET2-silenced when compared to control clones, and further supporting a “non-cell autonomous” behavior of RNASET2.
Gene silencing of RNASET2 also allowed us to studying both the intracellular routing and likely the mechanism(s) by which extracellular RNASET2 affects in vivo cell behavior, demonstrating that providing an external source of RNASET2 to silenced OVCAR3 cells resulted in the binding of the ribonuclease to the cell surface and its subsequently internalization
Terremoti crostali e zonazione sismica dell’Ecuador attraverso l’integrazione dei dati geologici, sismologici e morfostrutturali.
No full textmissin
Validazione del protocollo analitico di equilibrazione competitiva dei leganti accoppiata a stripping catodico per la speciazione di elementi in traccia.
No full textThe analytical performances of Competitive Ligand Equilibration with Cathodic Stripping Voltammetric detection of the labile fraction (CLE-CSV) were assessed and optimized. This speciation method enables the concentration of natural ligand(s) and their conditional stability constants for the complexation of the investigated metal to be determined through thermodynamic considerations.
The CLE-CSV procedure requires three working steps: firstly, total metal determination has to be performed with a good accuracy and precision; secondly, the calibration of the alfa coefficient of the added ligand with the metal considered is required; finally, titration of the samples with increasing metal additions provides the speciation results mentioned above.
To allow an accurate and precise measurement of the total metal concentration at trace levels (nM), the entire analytical procedure was assessed in term of effect of acidification of the samples and/or reagents required for the analysis and in term of influence of the UV-photooxidation used as pretreatment step for the total metal quantification with voltammetric techniques. The purity grade of nitric and hydrochloric acids produced by subboiling distillation was evaluated with the aim to avoid metal contamination during acidification processes. UV-photooxidation is commonly accepted as a sample pretreatment for voltammetric measurements to remove organic matter. Starting from the critical evaluation of the performances and costs of the commercially available systems, we decided to design and develop a prototype UV-digester, using only commonly commercialised parts (e.g. low cost, standard connection UV lamps). As a result, the target of coupling low investment and low maintenance costs to high performances is completely fulfilled. Its suitability for the treatment of natural waters prior to cathodic stripping voltammetry was assessed: the total concentration of copper, uranium and platinum in natural waters could be reliably determined applying a 30 minute UV treatment.
The final aim of this doctorate thesis is the complete evaluation of the speciation procedure CLE-CSV in term of accuracy and precision
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
Molecular and genetic approaches to identify the role of CDKL5 in the nervous system.
No full textRett syndrome (RS) is a progressive neurodevelopmental disorder with a large impact on society due to its high incidence in the female population. After a period of apparently normal development, RS girls lose acquired skills and start manifesting stereotypic hand movements, epilepsy, mental retardation, respiratory disturbances, motor deterioration and cardiac troubles. In 1999, mutations in the X-linked gene coding for methyl-CpG binding protein 2 (MeCP2) were identified as the molecular cause of RS. MeCP2 is a transcriptional repressor that, by binding methylated promoters, down-regulates transcription of its target genes. According to the fact the RS is an exclusively neurological disorder, MeCP2 regulates the transcription of specific neuronal genes such as Bdnf. In particular, Bdnf expression is modulated by an activity dependent phosphorylation of MeCP2.
In addition to classical RS, which is strongly associated with MECP2 mutations, RS variants have been described presenting some general features of the classic form but with different onset and severity. Less than half of the patients with these variants display MECP2 mutations, indicating that other genetic loci are involved opening the way to unravel the relationships between MeCP2 and other
genes causing RS variants. In recent years, mutations in the X-linked cyclin-dependent kinase like 5 (CDKL5) were identified in the RS Hanefeld variant, which is characterized by the absence of a normal period of development and the early onset of intractable seizures. CDKL5 is a serine/threonine kinase containing a catalytic domain in the N-terminal region and a large COOH-terminal tail. CDKL5 is a rather uncharacterized protein, however, its involvement in RS pathogenesis can be explained by the fact that the kinase interacts with MeCP2 in vivo mediating its phosphorylation in vitro.
The aim of this PhD thesis was to elucidate the role of CDKL5 in the nervous system both in physiological and in pathological conditions. The expression profile of the kinase in embryonic and postnatal brains from normal mice was compared to that of MeCP2 in order to better understand where and when the two proteins might enter in contact. Using an immunohistochemistry approach, we found that, despite an expression profile generally overlapping that of MeCP2, CDKL5 peaks later in development and, in some cases, its levels appear to be modulated in an opposite manner to that of MeCP2. A similar distribution was observed in adult human brain tissues reinforcing the relevance of the kinase in the nervous system.
By a series of immunofluoresence analyses on cultured primary hippocampal neurons we found that CDKL5 is a neuronal protein detectable in glutamatergic as well in GABAergic neurons but not in astrocytes. In mature neurons, the kinase is distributed both in the nucleus, with a particular dotted organization, and in the cytoplasm where it localizes in the cell soma and in the dendritic branches. Regarding the modulation of the subcellular distribution of CDKL5 in different stages of in vitro neuronal differentiation and in cultured non-neuronal cells, we demonstrated that an active nuclear export mechanism is involved in localizing the protein to the cytoplasm. Taken together, our results indicate the importance of CDKL5 for postnatal brain functioning. Eventually, we identified CDKL5 also in non-neuronal murine tissues, including kidney and liver. However, the neurological phenotype observed in patients with CDKL5 mutations leads to hypothesize a crucial role of the kinase in the nervous system where it is expressed at higher levels.
Since animal model represents an optimal tool to better understand the biological role of a protein and the mechanisms underlying a pathogenesis, we decided to generate a mouse possibly manifesting some pathological features observed in patients with CDKL5 defects. We therefore planned to conditionally inactivate the endogenous Cdkl5 gene in mice by a Cre-recombinase strategy. An ES-cell clone containing the correctly targeted Cdkl5 locus was obtained and injected into blastocysts of pseudopregnant females and the resultant chimeric animals were breeded for germline transmission. Unfortunately, no mice have so far been obtained carrying the targeted Cdkl5 locus in germline. As an important by-product of this project, however, an ES-cell line devoid of CDKL5 that can be differentiated into the neuronal lineage has been generated. This provides an important tool for studying neuronal differentiation and maturation in the absence of CDKL5 thereby helping answering some questions regarding the onset of RS and related variants in patients mutated in CDKL5
Quantificazione plasmatica del DNA libero circolante come marcatore diagnostico e prognostico nei pazienti affetti da neoplasia colorettale.
No full textmissin
Muscle development and differentiation in the urodele Ambystoma mexicanum.
No full textSkeletal muscle in vertebrates is the most representative tissue and it is composed by different types of fibers which differ for anatomical and physiological features. These traits permit to distinguish them basing on the speed of twitch and the load resistance. The characteristics of a fiber depend on the amount of glycogen and mitochondria in their cytoplasm, as well as by the presence of glycolitic and oxidative enzymes.
In particular, slow fibers are specific for a long-lasting contraction, they express a MyHC isoform having an ATPase activity at acidic pH (pH 4.0) and an aerobic oxidative metabolism. In contrast, fast fibers are larger in size, with few mitochondria. They are specialized in rapid and short-lasting contractions and express a MyHC isoform, having an ATPase activity at basic pH (pH 10.0) and an anaerobic glycolitic metabolism.
These different types of fibers, both in amniotes and in lower vertebrates such as fish and anuran amphibians s come from distinct populations of myoblasts which appear in the somites during the embryonic development.
Even if substantial data concerning the muscle differentiation and the characterization of the different types of fibers in zebrafish and Xenopus are available, nothing is still known about the mechanisms regulating these processes in amphibian urodeles. Since these animals present some anatomical features and life style between fish and anurans, the study of myogenic processes in these animals could be useful to clarify the evolutionary changes which lead to the formation of skeletal muscle in the trunk of land vertebrates.
To shed light on the myogenic processes in urodele amphibians we focused our studies on the axolotl Ambystoma mexicanum and we analysed several embryonic stages of this amphibian in order to identify the different types of fibers and their pattern of distribution during the myogenic process. Using morphological analysis, enzymatic hystochemistry and immunohystochemistry we showed that in A. mexicanum, as in zebrafish, the first differentiating fibers are the slow ones deriving from myoblasts localized close to the notochord.
These fibers then migrate towards the somitic surface and here they give rise to a uperficial layer of slow fibers, while the myoblasts forming the medial part of the somite differentiate into fast fibers.
Further, in order to evaluate the possible involvement of muscle-specific transcriptional factors and protein signalling in the regulation of myogenic process of this amphibian, we used molecular biological approaches to identify and clone the muscle-specific transcriptional factor (Myf5) and the Sonic hedgehog signalling protein, known to regulate the muscle development and differentiation both in amniotes and lower vertebrates