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Urban water services in Sub Saharan Africa: access, private sector involvement and technical paradigm.
Get PDFThe thesis analyses the water supply sector in the Sub Saharan African region, focusing on the challenges experienced by the water utilities to fulfil their mandates, in a context of rapid urbanization.
In September 2000, building upon a decade of major United Nations (UN) conferences and summits, world leaders came together at UN Headquarters in New York to adopt the Resolution A/RES/55/2, committing their nations to a new global partnership to reduce extreme poverty and setting out a series of time-bound targets - with a deadline in 2015 - that have become known as the Millennium Development Goals (MDG). The goal number 7 was “Ensure environmental sustainability” and it included the Target 7.C which is to “halve, by 2015, the proportion of the population without sustainable access to safe drinking water and basic sanitation”.
The water MDG is dramatically off track in Sub Saharan Africa, with only 64% of the population covered in 2012 instead of the expected 77.5% (WHO and UNICEF 2014). These poor performances are driven by urban areas, where the water supply coverage through household connections declined while the access through other improved sources, like public taps, private hand pumps and protected wells, hardly compensated for that.
This calls for a reconsideration of the policies implemented in the sector following the prescriptions of the neoliberal agenda for the sector. In the ‘80s and ‘90s policymakers from International Financial Institutions (IFIs) and donors agencies designed a set of recipes to address poor performances of the urban water services in the developing world. This happened in the context of structural adjustment policies, such as trade liberalization, labor market reforms, financial deregulation and privatization of State Owned Enterprises (SOE). In the water sector, these orientations were translated into decentralization, private sector participation, commercialization and corporatization of water utilities, with the shift of governments from providers to regulators.
The thesis studies some of the devices and solutions typically adopted by the reformed utilities and justified by the expectation of positive outcomes for the access to water by the poor
The work shows however that some of these devices gained a certain degree of autonomy from access goals and became a priority as such, to be pursued by water utilities regardless their impacts and interaction with key social dimensions. This phenomenon was in some cases favoured by a biased attitude of water sector practitioners, benchmarking regulation and donors.
The work highlights that in some cases the reform solutions do not contribute to the achievement of the declared objectives, while their implementation can divert scarce resources and attention from key sector priorities.
Cost recovery, Private Sector Participation and household level metering issues are analysed.
The work is organized in three parts.
The first part proposes a review of the main notions and issues addressed by water economics (chapter 1), with particular attention to developing countries.
The second part is divided into three chapters, closely linked together for their arguments but conceived as autonomous papers and characterized by different methodological approaches: the first is quantitative, the second and the third are more qualitative in nature and they include original findings from interviews on the Lilongwe Water Board, a water utility from Malawi.
The second chapter focuses on the problems of cost recovery and access to drinking water in Sub Saharan Africa. A model explaining the dynamics in water coverage which accounts for financial performances of utilities is proposed. The data set covers 25 countries in the Sub Saharan region from 1995 to 2012. The results suggest that the access to water depends upon financial results, but this relationship is not linear: with increasing returns for relatively low levels of cost recovery and decreasing returns beyond a certain threshold. The results are consistent with the literature about the risks associated with corporatization and neoliberal reforms in the water sector, and they provide some supporting quantitative evidence and recommendations for sector policies in the region.
The third chapter refers to the Light Private Sector Involvement initiatives in the Sub Saharan Africa water supply sector, considering in particular efficiency improvements, aid effectiveness and related policy implications. The study analyses the determinants that can incentivize or discourage the partners of light forms of Private Sector Involvement (PSI) initiatives to achieve the expected results in the water supply sector in the Sub Saharan Africa region. This is done through a review of case studies involving management and service contracts, which are the lightest and lower risk forms of public-private partnership. While five cases are taken from the available literature, the sixth includes contributions from original research on Lilongwe Water Board (Malawi). The chapter considers the incentives to perform for both the private and the public partner, as determined by the contracts and by the wider context. The incentives necessary for both parties to engage in the partnership are also considered, jointly with the costs of creating these preconditions. The study concludes that the allocation of risks and decision making power are among the drivers of poor performances by light PSI initiatives. Moreover, as most partnerships are financed by development projects, the study discusses the policy implications of promoting these PSI initiatives.
The fourth chapter analyses the priorities and tools for Water Demand Management in urban Africa, focusing on household level water metering. The study presents an analysis of the issues associated with water metering at household level by utilities in low income areas or informal settlements of Sub Saharan African cities. Metering is considered a key tool for water demand management and recommended as a good practice in the water supply sector but, while its benefits are clearly spelled out by donors and development agencies, its costs and shortcomings are seldom considered. The chapter analyses such challenges, based on the available literature and on an original case study on Lilongwe Water Board (Malawi). It is argued that the technical paradigm of metered household level connection can be in some cases a constraint to the connection of low income households, due to the high cost and complexity of the practices associated to this paradigm, while the benefits in terms of demand management are not straightforward. Some alternatives to universal household level metering are also identified.
Finally, in the last part of the work the findings from the studies presented are summarized and some conclusions and recommendations are drawn about the importance of better focusing on the priority of water access, encompassing a wider set of operational solutions
Molecular and cellular processes of the innate immune response in insects: investigation on the immune modulation induced by entomopathogenic nematodes
No full textAim of this project was to investigate relationships between Rhynchophorus ferrugineus or Galleria mellonella and the entomopathogenic nematode Steinernema carpocapsae. In particular, the work was focused on the immune response of the insect host either in naïve larvae or in larvae infected with entomoparasites. We analyzed different immunological processes: the activity and modulation of prophenoloxidase-phenoloxidase (proPO) system, the cell-mediated encapsulation, the antimicrobial peptides (AMPs) inducible response and finally the phagocytosis activity of the host hemocytes. Furthermore, we investigated the immune depressive and immune evasive strategies of the parasite. Our results indicated that R. ferrugineus has an efficient immune system; however, in the early phase of infection, the presence of S. carpocapsae induces a strong inhibition of the host proPO system. In addition, the parasite does not seem to be susceptible to the encapsulation by host hemocytes; the parasite mimetic properties seem to be related to the structure of its body surface. S. carpocapsae, before the release of its symbiotic bacteria (X. nematophila), depresses and elude the host immune defenses, with the aim to create a favorable environment for its symbionts responsible of the septicemic death of the insect host. Besides, our results have demonstrated that X. nematophila is able to inhibit the synthesis and the activity of antimicrobial peptides. X .nematophila elude the recognition by hemocytes since it is not engulfed by the host cells. It is evident that the nematode and its symbiotic bacteria cooperate to elude and inhibit immune responses of the insect host. This study provides data that can help to a better understand of the relationships between parasites and their hosts
Enzyme promiscuity in amino acid oxidases: a tool for sustainable processes.
Get PDFEnzymatic promiscuity is the ability of enzymes to catalyze additional reactions different from those for which they have evolved. This phenomenon plays a key role in the divergent evolution of novel enzymes. Amino acid oxidases (AAOs) are a group of FAD containing enzymes that catalyze the oxidative deamination of amino acids. AAOs evolved to fulfill very different physiological roles, by reshaping of their functional and structural features. Thus these enzymes represent an ideal model to understand the mechanisms that originated molecular biodiversity in modern enzyme families. In addition, their strict enantioselectivity renders AAOs interesting biocatalysts for the production of optically pure amino acids, valuable compounds widely used in the pharmaceutical and food industries. Through extensive literature and database search we identified two novel L-amino acid oxidases (LAAOs).
Detailed structural and functional characterization showed that the first one, the aminoacetone oxidase from S. oligofermentans (SoAAO), is not a canonical LAAO, since it does no possess the typical features of these enzymes, and has only a low promiscuous activity on L-amino acids. Its preferred substrate is aminoacetone that is converted to 2,5-dimethylpyrazine. Thus we propose that SoAAO could act as a scavenger of aminoacetone (a prooxidant metabolite), protecting the cell from oxidative damage.
The second one, L-amino acid deaminase from P. myxofaciens (PmaLAAD), resembles more closely the typical LAAOs: it is a membrane associated protein active on large hydrophobic L-amino acids. PmaLAAD does not use molecular oxygen, but a cytochrome b-like protein, as a direct electron acceptor. We propose that PmaLAAD is involved in catabolic utilization of L-amino acids to fuel the electron-transfer chain of Proteus membranes.
Comparison of the 3D structure of the two proteins with other LAAOs reveals that SoAAO diverged very early from this group of flavoenzymes, originating a novel structural group, while PmaLAAD has a clear evolutionary link with LAAOs.
In conclusion, the characterization of two novel microbial LAAOs allowed us to define their structure/function relationships, to clarify their physiological role and to obtain new insights on the underlying mechanisms of the molecular evolution of AAOs
L’analisi del modello di sviluppo albanese nel periodo post-comunismo: il cambiamento economico e la specializzazione.
Get PDFThis dissertation examines the model of economic development in Albania during the political-economic transition of 90-ies, mainly with regard to the economic change, trading specialization and restructuring of economy.
The main hypothesis of this study is the possibility to enable the development of the country through the export orientation as well as the potential restructuring of the actual manufacturing structure, focusing especially on new sectors such as industry, and also not overlooking those in which Albania has already paved its way of development, like agriculture. Also, by a thorough analysis of conditions, on a national and international scale, it is expounded on the reality that characterized Albania after the fall of socialist system, and highlighting strong points and lost possibilities in the realm of economic development process. With the analysis of the characteristics of this process, we have intended to highlight the problems and difficulties that have been evident during the transition period in Albania.
We have mainly carried out a thorough analysis of primary problems which have hindered the economic development of the country for a long time, from the beginning of ’90-ies and onwards. In addition, we have evaluated the policies and instruments of economic development, which on one hand, may be important for the economic policy in the following years, but on the other side, they serve to comprehend the conditions that favor investments. These investments indicate the role that a good investment plays in the civil society, with reference to the aspect of employment and increase of people welfare.
Concerning the employment in question, it is noted that the undertaken reforms during the beginning of `90-ies, have not produced the expected effects. Economic transformation in Albania was more difficult than being considered. Or rather, first monetary reforms that their primary aim was to stabilize the inflation, licensing inhibition (staff dismissing) by the public enterprises and public enterprises privatization, did not build a solid base in order to enhance the economic development of the country.
The economy and price liberalization initiating since 1992 along with massive privatization of public enterprises induced in a paradoxical way a paralyzation of the country’s production and a substantial deterioration of the trade balance. Albania was transformed from an isolated country that had “embraced” the motto: to produce everything by ourselves, had to abandon this philosophy, by becoming day by day dependent on import, with a high unemployment rate and with a social context of extreme economic poverty, a thing which stimulated the domestic population to emigrate towards industrial and developed countries. Consequently, these reforms have generated effects, which are nowadays being reflected in trade exchanges. Trade liberalization and the interference lack of the state in the economy, after 40 years of protectionism, brought about the restructuring of domestic production, in which the industrial sector was the most damaged. This phenomenon was at that period called the deindustrialization of the country. In fact, it is detected that all production activities were redimensioning or completely disappearing in the field of heavy, chemical, textile and food industry. With a primitive technology, the enterprises were not able to produce qualitative products that competed in the market, and under such circumstances, there occurred the shrinkage in the production activities towards their final closure. A part of light industry continued in the years to come to produce less than the production capacities at their disposal. Whereas, in the sector of agriculture, agrarian reform fragmented the enterprises and this led to direct management of rural areas families and consequently losing the quality and production technology. The lack of technology caused the decline of productivity and market competition, which later would rise to auto-consumption production.
In the following years, after the fall of communist regime, home policy-makers demanded a rapid polarization economy in those sectors reflecting comparative advantages for Albania that derived from the raw material availability such as: chrome, iron-nickel, petroleum and copper, geographical position and the level of a very low pay.
The main objective of this study is precisely the analysis of the typical economic aspects and the decisions made by the governance in the aforementioned period with the aim and hope to present a detailed and clear panorama of the economic transition and with its progress in Albania. In the first phases, Albania experienced what was later defined by the World Bank as a growth without development.
The study of the economic development model in Albania for a long-term period, allows on one hand the clear designation of different approaches followed by the governments in order to manage the economic policy, while on the other side, it highlights the choices and “errors” made by the economic policy. Based on these data, we have attempted to define the instruments and strategies that a certain state applies to generate a substantial change of the development model, from the labor intensive production, remittances, financings or aids by the international institutions and the raw material export, to a new development model which focuses on raw material transformation, production growth in various sectors of economy, the import decline and export increase.
In this study, it is demonstrated that through raw material transformation such as petroleum processing, is guaranteed the home market demand, the export and consequently the improvement of trade balance.
Furthermore, this study shows the geography of trade exchanges where Italy was the primary trade partner of Albania: exports towards Italy and Greece made up to 83% of the total export and 44% of total imports in 2006. 72% of exports and 26% of imports went and came from Italy. After 2006, with the membership of Albania in CEFTA (Central European Free Trade Agreement), it is observed a redimensioning of import-exports in favor of the countries of CEFTA. Generally speaking or either in particular, it is observed an increase of imports from China and exports towards Spain and Turkey. As far as the construction of trade is concerned, due to the membership of Albania in CEFTA, this is relevant for the “meat” product and no other.
From the qualitative and quantitative analysis of the Albanian economy, it is noticed that during transition, with the pass of time, Albania has made a good performance of the economic growth (with the exception of last years that growth has been retarded). Anyhow, it is clear that this growth is probably not stable with time and it does not possess the accurate capacities to generate incomes and enough employment for the domestic population. For this reason, we can say that the Albanian economy is facing a crossroad: it should go ahead with a “low” development direction which enables (as it occurs nowadays) sufficient rise in employment with a low pay and being concentrated in the sector which does not control autonomously in the future. Otherwise, it should choose a direction with a high development, by promoting the hidden capacities which are expected to give a hop in sectoral as the only ones, by ensuring simultaneous stability of the future incomes and the possibility to itself govern the progress of the country development.
In this dissertation, we have noted that the policies and strategies implemented by the Albanian governments in the years of transition have not fully worked out. This has occurred due to an increase in the gross production, real incomes in economy and employment, but on the other side these have not been sufficient in order to narrow the existing gap in developed countries. The employment and incomes are very low and as a result, they do not develop important complementarities and sectoral interdependencies. Most of all, they do not stimulate consumption and investments in order to consolidate production and promote domestic product export.
This study concludes in an analysis of the economic policy implemented in Albania during the recent years, in which the state shows a low protagonism in the economic development. Also, several strategies for development have been compiled and their application has encountered several problems. The economic and industrial policy in the country does not intend to create other new sectors, but rather the consolidation of the existing ones, considered as significant for the economic development of the country.
Whereas, with the investigation of the structural problems of the economy, the government has acknowledged that free market has not fully guaranteed investments and capital in new and existing activities, so as to engender a rise in productivity, i.e. development.
Governments have avoided their intervention in the economy and have regarded the market as the best mechanism for the income distribution, an issue that is questioned by our analysis.
As a conclusion, we can say that structural reforms should be coupled with horizontal policies that reinforce the institutions, technical competencies and above all policies of structural transformation. Accordingly, good governance on one hand should fight corruption and on the other hand, revitalize the economy of the country
Analysis of the role of liprins protein in breast cancer cell invasion.
No full textThe metastatic process requires the ability of cancer cells to break the basement mem¬brane and migrate through a complex three-dimensional environment. The laboratory has recently identified the protein liprin-α1 as an important regulator of integrin me¬diated focal adhesion dynamics and cell motility in non-neuronal cells (Asperti et al., 2009, Asperti et al., 2010). Liprins are a family of cytosolic scaffold proteins including the liprin-α and liprin-β subfamilies based on sequence similarities (Serra Pagés et al., 1998). The human genome encodes four liprin-α (liprin-α1-4) and two liprin-β proteins (liprin-β1 and liprin-β2). Interestingly, the gene PPFIA1 for liprin-α1 is frequently am-plified in tumors. Moreover, the levels of expression of the liprin-α1 protein are often increased in human breast cancers (Astro et al., 2011). Functional analysis has revealed that liprin-α1 is specifically required for migration and invasion in vitro of highly invasi¬ve MDA-MB-231 human breast cancer cells. The analysis of lamellipodia dynamics has revealed a decrease of the stability of these protrusions in cells depleted of endogenous liprin-α1, which are defective in cell motility. Furthermore, liprin-α1 silencing causes a reduction of tumor cell invasion through Matrigel. The examination of the invasive po¬tential has demonstrated that liprin-α1 is important also for the degradation of the extra¬cellular matrix (ECM) (Astro et al., 2011). Starting from these observations, the first aim of my project has been to investigate the function of liprin-α1 in vivo. I have generated MDA-MB-231-derived cell lines with either stable overexpression or stable depletion of liprin-α1, and I have used these cells for injection or transplantation in mice, to determine their invasive potential. The characterization of these cell lines in vitro has confirmed that liprin-α1 overexpression causes an increase of both cell migration on FN and invasion through Matrigel, by promoting the stability of the lamellipodia. On the other hand, li¬prin-α1-depleted cells have reduced ability to both migrate and invade in vitro. However, all the cell lines with altered liprin-α1 levels have shown similar proliferation rates and viability compared to the control MDA-MB-231 cells. To investigate the involvement of liprin-α1 in invasion in vivo, experimental metastasis assays and spontaneous metastasis assays were performed. In both assays, the formation of lung metastases by the modified and control breast cancer cell lines has been evaluated. The results indicated that liprin-α1 overexpression did not affect lung colonization. Considering the high invasive ability of MDA-MB-231 wild type cells, increase in lung colonization by liprin-α1 overexpression may be irrelevant in vivo. On the contrary, injection of liprin-α1-depleted cells resulted in the reduction of the formation of lung metastases compared to control cells. This is the first evidence that liprin-α1 is not involved in primary tumor growth, while it is important for tumor cell invasion.
Being a scaffold protein, liprin-α1 is unlikely to act alone as a regulator of tumor cell invasion. Previous studies have described the interaction between liprin-α1 and liprin-β1 (Serra-Pages et al., 1998), and have suggested a possible role of liprin-β2 in migration and invasion (von Thun et al., 2012). However, the available data on the functions of li¬prin-β proteins and their relationship with liprin-α1 are not exhaustive. As the second aim of my PhD, I have addressed the biochemical interaction of liprin-α1 with either liprin-β1 or liprin-β2, and I have tried to elucidate the role of the two proteins in cell motility and invasion. While liprin-α1 interacts with liprin-β1, it does not interact with liprin-β2. This is the first evidence of the different ability of the two liprin-β proteins to interact with liprin−α1. The biochemical analysis has shown that the interaction between liprin-α1 and liprin-β1 occurs via the C-terminus of liprin-α1, and that two of the three SAM domains of liprin-α1 are sufficient to mediate this interaction.
The study of the subcellular localization has indicated that liprin-β1 colocalizes with liprin-α1 at the cell edge, whereas liprin-β2 partially colocalizes with cortactin-positive invadopodia. Functional analysis has shown that liprin-β1 silencing did not affect cell invasion through matrigel, whereas liprin-β2 silencing led to an increase of cell invasion, and enhanced ECM degradation, supporting the hypothesis of the different role of this protein in regulating the function of invadopodia with respect to liprin-α1 and liprin-β1. Analysis of the involvement of liprin-β1 and liprin-β2 in cell migration underlined the different effects of the two proteins. As previously observed for liprin-α1 (Astro et al, 2011), silencing of liprin-β1 led to a decrease of the speed of the cells in random migra¬tion assays. On the contrary, liprin-β2 silencing did not significantly affect cell motility. These data support the hypothesis of a cooperation between liprin-α1 and liprin-β1 in regulating cell motility, while they indicate that liprin-β2 does not have a relevant role in this process.
Altogether the work presented in my thesis sustains a key role of liprin-α1 as a positive regulator of the invasive apparatus of tumour cells in vivo, and has highlighted for the first time first time distinct roles of liprin-β1 and liprin-β2 in tumor cell motility
Alterazioni epigenetiche reversibili associate alla transizione epitelio mesenchimale (EMT) in cellule prostatiche tumorali.
No full textLa transizione epitelio mesenchimale (EMT) è un processo dinamico e regolato, fondamentale per lo sviluppo embrionale, per il mantenimento dell’omeostasi dei tessuti, per la fibrosi tissutale e per il cancro (Sanchez-Tillo et al, 2014). L’EMT induce un cambiamento reversibile dello stato fenotipico delle cellule che perdono le caratteristiche epiteliali e acquisiscono un fenotipo mesenchimale. Queste stesse cellule inoltre, possono revertire il loro stato fenotipico mesenchimale in epiteliale, durante il processo inverso di EMT, la transizione mesenchimale- epiteliale (MET) (Tam WL. & Weinberg RA., 2013).
Lo studio di un modello sperimentale di EMT- MET in cellule tumorali di prostata, mediante l’induzione con terreno condizionato da CAFs (fibroblasti associati al tumore), per 3 giorni, in cellule prostatiche PC3 e non in LN-CaP (linea cellulare di controllo) ha attivato l’EMT; mentre il trattamento con CM-HPFs (fibroblasti umani periferici) usato come controllo, non ha indotto il processo. La conferma dell’attivazione di EMT è stata ottenuta mediante l’analisi di espressione proteica e di RNA messaggero di marcatori chiave di tale processo, come E-caderina, N-caderina e Vimentina (Thiery J.P. et al, 2009). Inoltre, la riespressione di E-caderina e la diminuzione di N-caderina e Vimentina, nel trattamento di PC3 con terreno ottenuto da cellule PC3 mantenute in coltura in assenza di siero per 48 ore (CM-SS PC3 o recovery medium, RM), per ulteriori 3 giorni, ha dimostrato l’attivazione del processo di MET.
La riduzione di espressione delle sequenze LINE1, considerate come surrogato della metilazione globale del DNA, nelle cellule PC3 in EMT, è in accordo con la diminuzione dell’espressione di UHRF1 e di DNMT1, DNA metiltransferasi di mantenimento; inoltre il ripristino di espressione delle medesime, durante la MET, evidenzia lo stato dinamico e reversibile di tali processi.
L’aumento, seppur lieve, di espressione di DNMT3a (DNMTs “de novo) in EMT e il successivo ripristino di espressione della stessa, durante la MET, mostra la presenza di alterazioni degli enzimi implicati nella metilazione del DNA e riconferma la reversibilità del processo di EMT-MET.
La presenza del legame di DNMT3a e non di DNMT1, sul promotore del gene di CDH1, in cellule PC3 in EMT, mediante tecnica ChIP, e l’aumento dello stato di metilazione di una regione del promotore di CDH1, mediante pyrosequencing confermano una significativa variazione dello stato di metilazione di questo gene. Inoltre, i dati ottenuti mediante analisi di Illumina 450K su di oltre 485 mila isole CpGs distribuite all’interno dell’intero genoma, ha prodotto risultati importanti sulla reversibilità dello stato di metilazione di alcuni geni implicati nel processo di EMT, come CDH1, N-CAD, ZEB1 e VIMENTINA. Infatti, in questi geni lo stato di ipometilazione e/o di ipermetilazione durante il processo di EMT, è esattamente speculare al dato ottenuto, per gli stessi geni, durante il processo di MET. I risultati acquisiti da queste analisi hanno inoltre confermato lo stato di ipermetilazione di CDH1 durante la EMT che ne determina la sua repressione trascrizionale, come si osserva dalle analisi di qPCR e WB eseguite precedentemente.
Quindi, durante il processo di EMT-MET avvengono delle alterazioni epigenetiche reversibili a livello dello stato di metilazione del DNA di alcuni geni, in particolare di CDH1.
Inoltre, la presenza contemporanea di H3K4me3 e di H3K27me3 sul promotore di CDH1 in EMT, ha mostrato lo stato di gene ambiguo di E-caderina e l’acquisizione di uno stato “bivalente”, caratterizzato da plasticità e reversibilità (Ke X.S. et al, 2009).
Questi risultati ottenuti, però, non sono ancora in grado di spiegare se l’attivazione del processo di EMT richieda la presenza di variazioni dello stato di metilazione del DNA o se tali alterazioni sono solo la conseguenza del processo, tuttavia sono ancora in corso esperimenti ed analisi per rispondere a questa domanda
Evaluation of microarray technology and cell line models in modern toxicology
Get PDFModern toxicology puts together existing knowledge of classical biology with new technologies to study effects of perturbations and to predict adverse outcomes resulting from those perturbations.
Modern toxicology employs high throughput technology as microarray and cell lines as in vitro models to investigate the effect of potential toxic compounds.
In my thesis I evaluate the usefulness and reproducibility of microarray technology and cell line models. I have been mainly interested in the study of the effects on the regulation of gene transcription in cell lines. I used HepG2 cell line as a model for liver to study cadmium-induced cytotoxicity, Caco-2 as model for intestinal epithelial to study nanoparticles and HeLa cells from cervical cancer to evaluate genomic instability.
Cadmium is currently classified as carcinogen for human. In my first study, gene expression is used to explain the possible toxic mechanism of cadmium carcinogenity, using HepG2 cell line. A suggested mechanism of cadmium-induced carcinogenicity involves defects in the cell response to DNA damage and in the resistance to apoptosis. In this regard, I focused on the tumour suppressor protein P53, since its inactivation is a common feature found in human cancers and it is a crucial component of the cellular response to DNA damage. The results presented in this thesis demonstrate that in HepG2 cells exposed to cadmium, P53 was correctly moved and accumulated into the nucleus to accomplish its function as a transcription factor. However, in spite of this correct nuclear localization, the signals for the cell cycle arrest were not activated. In this context, the important mediator of cell cycle arrest P21, a P53 downstream protein, was upregulated at the gene level but not at the protein level. These results could be explained by the involvement of a post-transcriptional activity mediated by miRNA, as demonstrated by the upregulation of mir-372 in cadmium-treated HepG2 cells, which was able to affect p21 expression and to promote cell proliferation.
Recent advances in materials science have resulted in the creation of particles in the nano-scale range and their use is spreading. This creates the need to evaluate their possible toxic effect on human health. In the second study, gene expression is applied to evaluate nanoparticle effect on Caco-2 cells as in vitro model of intestinal epithelial cells. The main focus was to compare the effect of gold nanoparticles (AuNP) of two sizes (30 nm and 5 nm), using microarray technology. Smaller AuNPs (5 nm) inhibit Caco-2 cell growth. Gene expression analysis shows a broad range of responses induced by small size AuNPs. A bioinformatics reconstruction of the possible pathways regulated by smaller AuNPs indicates that Caco-2 cells activate defence responses even if it was not enough to prevent the observed toxic effect. The response at the transcriptome level upon exposure to larger AuNPs (30 nm) is very weak. This effect can be due to the low uptake of these larger AuNPs. The third study focuses on the issue concerning cell line variability using different batches of HeLa cells obtained from different laboratories. The microarray expression analysis was performed on different batches of HeLa cells exposed to hypoxic conditions. In response to a hypoxic stimulus, each cell line batch activated different pathways, although the regulation of genes related to hypoxia is conserved. A genetic analysis show the high level of extensive chromosome instability in HeLa clones obtained from different laboratories. Each clone accumulates genomic variability in a time-dependent manner. The large differences in gene expression profiles suggest that the use of uncharacterized clones may lead to faulty conclusions and to irreproducible results in studies of gene function and pathway analysis.
In the three papers presented in this thesis I have not only shown the reproducibility of microarray by comparison to real time PCR, the gold standard technique for mRNA quantification, but also the potential to create possible model for the mechanistic effects on biological pathways.
In my thesis I have used different cell lines as in vitro models. They have shown that they have many advantages and that can give strong support for modern toxicology and be used to study biological mechanism. However, a possible drawback, which should be taken into account, is highlighted in the third study showing a high variability in the responses of different batches of the same cell line
Fish health and fillet quality following substitution of fishmeal (FM) and fish oil (FO) with vegetable meal (VM) and oil (VO) in the modern aquaculture.
Get PDFPlant proteins in vegetable meals (VM) and oils are used in an increasing percentage in diets for carnivorous fish to replace fish meal (FM) and fish oil (FO) in order to reduce production costs and to face the limitation of marine resources and to be sustainable in respect to the protection of oceanic biodiversity. However, several studies report that fish respond to such replacements by reducing growth rate, feed conversion and fillet quality. It may be due to a deficiency or poor availability in VM ingredients of some essential amino acids, but also to the occurrence of anti-nutritionals and inflammatory compounds acting on the fish intestine.
This can lead to economic losses for fish farmers, increased fish diseases occurrence, and negative environmental impact. This study focuses on European sea bass and Rainbow trout, as two among the most commercially important fish species in Italy. Aims of the study are to improve feed formulation and optimize feed additives such taurine, as well as butyrate, salt of a short chain fatty acid (SCFA), to protect fish intestine and evaluate the effects on fish fillet quality of the modern commercial diets where FM is partially substitute with VM.
In that context, our experimental study investigated butyrate and taurine in the diet, as nutraceutics able to reduce or minimize the negative effects of VM inclusion in fish performances and health.
After improvement of analytical methods for butyrate titration in fecal samples, the possibility that a butyric fermentation occurs in fish intestine has been experimentally evaluated. The protective effect of butyrate on fish intestine has been studied by following approaches including histology with light and electronic microscopy, then a molecular approach to study inflammatory cytokines, as well as the epigenetic action of this SCFA.
In spite, dietary butyrate showed no significant differences in weight gain or SGR (specific growth rate) of sea bass, the protective effect on the intestine walls was highlighted, together with its epigenetic effects on hiperacetylation histone H4 at lysine 8, while no effects has been found on the histone H3 at Lys9. A butyrate effect on the liver, as an increased transcriptional expression of the genes Dicer1, ehmt2, and hdac11 and il-10 have been assessed, suggesting an anti-inflammatory as well as an antiviral role of butyrate in seabass receiving a 2% of Na-butyrate as diet supplement. The effect of a dietary supplementation of the sulphonic aminoacid taurine, almost absent in the VM, was studied on seabass growth performances and on its potential role as antioxidant. Seabass fed with 1.5% taurine addition showed an increase of the fish growth performance and a marked reduction of the ROS production under resting conditions that was enhanced following forced swimming performances.
The synergistic effect of butyrate and taurine as feed additives was also explored in seabass feeding diet containing an elevate concentration of a soy protein. Different cytological protective mechanisms have been observed, indicating the existence of a synergistic action of the two nutraceutical compounds.
The quality of fish fillet in trout fed with 6 different commercial feed where FM was differently substituted by VM, has been studied by assessing the fillet lipidograms. Trout was utilized in this study as is know for the capacity to elongate and desaturate the n-3 long chain fatty acid EPA in DHA. The EPA/DHA ratio in feed and in fillet were expected to be modified by the fish, with an increased restitution of DHA as consequence of a reduction in EPA. Actually, such a dynamic was observed in fish fed with only 4 of the challenged commercial feed and that was supposed to be due to an effect on the fatty acids metabolism, following an unbalancement of the essential aminoacids in the diets where higher substitution of FM with VM was operated.
In conclusion, a number of the expected negative effects due the FM substitution with VM have been actually assessed. On the same time, the possibility to mitigate such negative performances with diet manipulation have been documented as well. Beside some practical results immediately applicable by the fish farming industry, indications for further researches are emerging from this study
Ecotoxicological assessment of freshwater ecosystems.
Get PDFL’ecotossicologia è una scienza recente nata come filiazione dalla tossicologia dalla quale ha derivato principi, concetti e metodi, coniugati però con l’ecologia. L’ecotossicologia è dunque la scienza dei veleni per l’ambiente e il suo scopo finale risulta essere la protezione degli ecosistemi.
La conservazione dell’ambiente ha un’importanza sia per quanto riguarda la salvaguardia dell’integrità degli ecosistemi per il loro valore intrinseco, sia per quanto riguarda i servizi che essi rendono all’uomo. Tra le risorse utili, l’acqua è indubbiamente è una delle più importanti, particolarmente vulnerabile, essenziale per la vita e lo sviluppo di tutti gli esseri viventi.
La tutela e il suo uso razionale è un obiettivo molto impegnativo che non può essere raggiunto con un approccio settoriale e di emergenza, ma richiede una politica preventiva che parta dalla cognizione della situazione al tempo presente ed incida poi sulle cause del degrado con una correttagestione ambientale.
Da questo punto di vista, a livello normativo un’importante novità è senza dubbio rappresentata della Direttiva Quadro sulle Acque (WFD 2000/60/EC), che impone ai Paesi membri la tutela e il recupero degli ecosistemi acquatici fissando obiettivi di qualità non più basati esclusivamente su indicatori chimici e fisici ma, soprattutto di tipo biologico ed ecosistemico.
L’approccio ecotossicologico risulta essere un valido strumento per la valutazione della qualità degli ecosistemi acquatici (sia comparto acqua che comparto sedimenti) sia attraverso il suo approccio classico con l’utilizzo dei test di ecotossicità in laboratorio su organismi target con lo scopo di identificare l’effetto che le sostanze pure possono avere sull’ambiente. Sia attraverso un approccio più oolistico che prevede l’utilizzo dei test di ecotossicità come strumenti di monitoraggio per identificare eventuali effetti negativi derivanti dalla presenza di miscele in ambiente (strumenti di monitoraggio effect-based). Quest’ultimo approccio risulta essere perfettamente in linea con quanto previsto dalle WFD portando i test di ecotossicità ad ricoprire un ruolo fondamentale all’interno di quelli che sono i classici metodi di monitoraggio basati esclusivamente sulla ricerca analitica delle singole sostanze in grado di provocare effetti avversi sull’ambiente (strumenti di monitoraggio stressors-based)