75 research outputs found
Fiscal decentralization and urban public transport
Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Urban Studies and Planning, 2006.Includes bibliographical references (leaves 163-168).Financing public transport through public funds is a common practice that can be justified on different grounds: equity, natural monopoly and, particularly with the increasing motorization rate, externalities produced by private transport (congestion, pollution, road accidents) especially in urban areas. In addition, there is a belief that transport investments support economic growth, in particular transit investments because they help fostering the agglomeration effect. Whether local or national tax sources should be used for subsidizing public transport is a fairly recent question, at least in Europe where, historically, countries used to be very centralized. Several national policy reviews as well as academic papers suggest that the reforms aiming at decentralizing power and responsibility for urban public transport management lead to successful outcomes. Yet, there is no literature on the effects of decentralization of tax raising on public transport provision although an increasing number of subnational governments reports a mismatch between transferred resources and devolved responsibilities and the public finance literature indicates that decentralization of finance authority can improve the results of decentralization reforms .(cont.) The objective of this thesis, thus, is to test if the theoretical benefits of fiscal decentralization (i.e. decentralization of taxing power in addition to management responsibilities) apply to the urban public transport sector and to what extent. Using a sample of five European cities (Madrid, Barcelona, Paris, Milan and Stockholm) in decentralized countries as case studies, the thesis identifies three major outcomes of fiscal decentralization. First it increases the expenditure in public transport infrastructure. Second it increases the entrepreneurship of the local policy-makers. Third it generally improves the predictability of the revenues and therefore helps planning in the medium to long term. On the other hand, the research also shows that there is no increased willingness to tax at the local level therefore the stability of funding and its adequacy to the needs is not guaranteed by local fiscal autonomy. The thesis moreover suggests that a mixed system of national dedicated taxation and local capacity to incrementally adapt the tax rates is an optimal scheme for financing local public transport. Finally, in light of the case studies, the thesis provides some recommendations to Transport for London (TfL), in order to guide the ongoing debate on the necessity of increased fiscal autonomy for the local authorities in the UK to solutions that are likely to improve TfL's situation.by Giorgia Favero.S.M
Characterization of the risk of palytoxin and analogues as seafood contaminants
2010/2011The present thesis was developed for the characterization of the risk associated to palytoxins as seafood contaminants. To this aim, an integrated approach between in vitro and in vivo studies was chosen. Palytoxin and its analogues are known seafood contaminants that can accumulate in several edible species of shellfish, fish, crustaceans and echinoderms. Generally, primary symptoms associated to the ingestion of contaminated food involve the gastro-intestinal apparatus and later develop with the involvement of the muscular tissue. For a better comprehension of the mechanism of action of this family of biotoxins, the effects of PLTX have been studied on primary culture of mouse skeletal muscle cells. The myotoxic insult triggered by PLTX was described in detail with the definition of the cytotoxicity together with the description of the morphologic alterations and functional impairment caused by the toxin. Moreover, the influence of the ionic composition of the extracellular medium on the effects of the toxin was elucidated. Primary cultures of skeletal muscle cells, that presents in vitro many of the peculiarities of the adult muscle fiber, allowed the investigation of the membrane mechanisms that regulate the intracellular calcium increase triggered by the toxin. It was possible to discriminate the difference between calcium release from intracellular stores and the calcium entrance from extracellular compartment both elicited by the toxin, and to understand the importance of the latter in relation to the toxic event. Moreover, the involvement of the main membrane channels and transporters that may be related to the entrance of calcium was investigated and the crucial role of stretch-activated channels in the mechanism of toxicity was demonstrated. Once defined the crucial molecular mechanisms of action of PLTX, experiments were also performed with two of its analogue: the 42-hydroxyl-palytoxin and the ostreocin-D.
In parallel to in vitro studies, the effects of repeated oral administration of PLTX in mice were also investigated. In fact, in vitro studies are not sufficient for the complete comprehension of the real hazard associated to a food contaminant, since molecules once in contact with the body may undergo adsorption, distribution and metabolism before reaching the target tissue. Short-term (7 days) administration of the toxin revealed toxicity at all the doses tested and lethality was recorded in the treated animals already from the dose of 30 µg/kg. Histological analysis highlighted alterations in several tissues: severe inflammatory processes and even foci of necrosis were observed in lungs. Alteration of the muscular tissues was visible as fiber separation and degeneration in the heart and increased cellularity between fibers in skeletal muscle. Moreover, depletion of glycogen content of hepatocytes and moderate alterations of the spleen were also observed. Data collected in the present project revealed, for the first time, toxicity of PLTX at doses much lower than that currently used by European Food Safety Authority (EFSA) for the estimation of limit values for presence of these compounds in seafood. For this reason, these results are likely to have a considerable impact at regulatory level and to have crucial importance for the protection of the consumers.Il presente lavoro di tesi è stato sviluppato con lo scopo di caratterizzare il rischio associato alla presenza delle palitossine quali contaminanti dei prodotti ittici destinati ad uso alimentare. A tal fine, è stato scelto un approccio integrato tra studi in vitro e in vivo. La palitossina e i suoi analoghi sono noti contaminanti dei prodotti ittici e possono accumularsi in diverse specie edibili di molluschi, pesci, crostacei ed echinodermi. Generalmente, i primi sintomi associati all’ingestione di cibo contaminato coinvolgono l’apparato gastro-intestinale e poi si sviluppano con l’interessamento del tessuto muscolare. Per una migliore comprensione del meccanismo d’azione di questa famiglia di biotossine, gli effetti della PLTX sono stati studiati mediante colture primarie murine di cellule muscolari scheletriche. L’insulto miotossico indotto dalla PLTX è stato descritto nel dettaglio con la definizione della citotossicità assieme alla descrizione delle modifiche morfologiche e delle alterazioni funzionali causate dalla tossina. Inoltre, è stata caratterizzata l’influenza della composizione ionica dell’ambiente extracellulare negli effetti della tossina. Le colture primarie di cellule muscolari scheletriche, che presentano molte delle caratteristiche peculiari della fibra muscolare adulta, hanno permesso l’indagine dei meccanismi che regolano l’aumento intracellulare di calcio indotto dalla tossina. E’ stato possibile discriminare la differenza tra il rilascio di calcio dai depositi intracellulari e l’entrata di calcio dai compartimenti extracellulari, entrambi effetti indotti dalla tossina, e comprendere l’importanza del secondo meccanismo in relazione agli eventi tossici. E’ stato indagato il coinvolgimento dei canali e trasportatori di membrana in relazione all’ingresso di calcio ed è stato dimostrato il ruolo cruciale dei canali attivati da stiramento nel meccanismo di tossicità . Una volta definiti i meccanismi d’azione cruciali per la PLTX, esperimenti sono stati condotti anche con due dei suoi analoghi, la 42-idrossi-palitossina e l’ostreocina-D.
Parallelamente agli studi in vitro, sono stati studiati gli effetti della somministrazione orale ripetuta della PLTX nel topo. Infatti, gli studi in vitro non sono sufficienti alla comprensione del reale pericolo associato ad un contaminate alimentare, dal momento che, le molecole, una volta a contatto con l’organismo, possono subire assorbimento, distribuzione e metabolismo prima di raggiungere il tessuto bersaglio. La somministrazione a breve termine della tossina (7 giorni) ha rivelato tossicità a tutte le dosi somministrate, e letalità è stata registrata negli animali trattati già alla dose di 30 µg/kg. Le analisi istologiche hanno evidenziato alterazioni a carico di diversi tessuti: a livello polmonare sono stati osservati severi processi infiammatori associati anche a focolai di necrosi. Le alterazioni del tessuto muscolare erano visibili come degenerazione e separazione delle fibre nel cuore e aumento degli elementi cellulari tra le fibre del muscolo scheletrico. Inoltre sono stai osservati, deplezione del contenuto di glicogeno negli epatociti e moderate alterazioni della milza. I dati raccolti nel presente elaborato hanno rivelato, per la prima volta, la tossicità della palitossina a dosi molto inferiori rispetto a quelle correntemente utilizzate della European Food Safety Authority (EFSA) per la stima di valori limite per la presenza di questi composti nei prodotti ittici destinati ad uso alimentare. Per questo motivo, i risultati avranno probabilmente un considerevole impatto a livello legislativo e un’importanza cruciale per la protezione dei consumatori.XXIV Ciclo198
A flow SPR immunosensor based on a sandwich direct method
In this study, we report the development of an SPR (Surface Plasmon Resonance) immunosensor for the detection of ampicillin, operating under flow conditions. SPR sensors based on both direct (with the immobilization of the antibody) and competitive (with the immobilization of the antigen) methods did not allow the detection of ampicillin. Therefore, a sandwich-based sensor was developed which showed a good linear response towards ampicillin between 10-3 and 10-1 M, a measurement time of ≤20 min and a high selectivity both towardsβ-lactam antibiotics and antibiotics of different classes. © 2016 by the author
Alternaria toxins as casein kinase 2 inhibitors and possible consequences for estrogenicity: a hybrid in silico/in vitro study
Emerging mycotoxins produced by Alternaria spp. were previously reported to exert cytotoxic, genotoxic, but also estrogenic effects in human cells. The involved mechanisms are very complex and not fully elucidated yet. Thus, we followed an in silico target fishing approach to extend knowledge on the possible biological targets underlying the activity of alternariol, taken as the signature compound of Alternaria toxins. Combining ligand-based screening and structure-based modeling, the ubiquitous casein kinase 2 (CK2) was identified as a potential target for the compound. This result was validated in a cell-free in vitro CK2 activity assay, where alternariol inhibited CK2 with an IC50 of 707 nM. As CK2 was recently discussed to influence estrogen receptor (ER) transcription and DNA-binding affinity, we assessed a potential impact on the mRNA levels of ERα or ERβ by qRT-PCR and on nuclear localization of the receptors by confocal microscopy, using estrogen-sensitive Ishikawa cells as a model. While AOH did not affect the transcription of ERα or ERβ, an increase in nuclear localization of ERα after incubation with 10 µM AOH was observed. However, this effect might be due to ER binding affinity and therefore estrogenicity of AOH. Furthermore, in silico docking simulation revealed not only AOH, but also a number of other Alternaria toxins as potential inhibitors of CK2, including alternariol monomethyl ether and the perylene quinone derivative altertoxin II (ATX-II). These findings were representatively confirmed in vitro for the perylene quinone derivative altertoxin II, which was found to inhibit the kinase with an IC50 of 5.1 µM. Taken together, we propose CK2 inhibition as an additional mechanism to consider in future studies for alternariol and several other Alternaria toxins
Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean Sea: a multidisciplinary scientific approach
The increased presence of potentially toxic microalgae in the Mediterranean area is a matter of great concern. Since the end of the last century, microalgae of the genus Ostreopsis have been detected more and more frequently in the Italian coastal waters. The presence of Ostreopsis spp. has been accompanied by the presence of previously undetected marine biotoxins (palytoxins) into the ecosystem with the increased possibility of human exposure. In response to the urgent need for toxicity characterization of palytoxin and its congeners, an integrated study encompassing both in vitro and in vivo methods was performed
Persistence of the antagonistic effects of a natural mixture of Alternaria mycotoxins on the estrogen-like activity of human feces after anaerobic incubation
Several Alternaria mycotoxins are believed to act as endocrine disruptive chemicals (EDCs), since they are reported to bind estrogen receptors in several experimental models. After ingestion of contaminated food commodities, the mycotoxins reach the intestine, where they come into direct contact with food constituents as well as the gut microbiota. Thus, the aim of the present work was to evaluate the modulatory potential of a complex extract of cultured Alternaria fungi (CE; containing eleven chemically characterized compounds) on the estrogenic signaling cascade of mammalian cells before and after anaerobic incubation with fecal slurries, in order to simulate an in vivo-like condition in the gut. Assessing alkaline phosphatase expression in Ishikawa cells as a measure for estrogenicity, we found the CE to partially quench the intrinsic estrogenic properties of fecal slurries and fecal waters, even after 3 h of fecal incubation. Investigation of the mechanisms underlying the effects observed carried out through an in vitro/in silico approach revealed the ability of the extract to decrease the ERα/ERβ nuclear ratio, while a possible action of the mycotoxins as ER-antagonists was excluded. Our results suggest that Alternaria mycotoxins might act as EDCs in vivo, and warrant further investigation in animal models
Response of intestinal HT-29 cells to the trichothecene mycotoxin deoxynivalenol and its sulfated conjugates
Abstract The sulfated forms of the Fusarium toxin deoxynivalenol (DON), deoxynivalenol-3-sulfate (DON-3-Sulf) and deoxynivalenol-15-sulfate (DON-15-Sulf) were recently described, however little is known about their mechanism of action in mammalian cells. DON-3-Sulf and DON-15-Sulf were taken up by HT-29 colon carcinoma cells, although to a lesser extent compared to DON. All three compounds were found to enhance the intracellular ROS level in the dichlorofluorescein assay (≥ 1μM), even though substantial differences were observed in their cytotoxic potential. In silico modelling highlighted that DON-sulfates do not share the classical mechanism of action of DON, being unable to fit into the ribosomal pocket and trigger the classical ribotoxic stress response. However, DON-3-Sulf and DON-15-Sulf sustained a distinctive proliferative stimulus in HT-29 and activated autophagy. The mechanisms of action of DON-3-Sulf and DON-15-Sulf suggest a potential interplay between the onset of ribosomal inhibition and autophagy activation as an alternative and/or complementary mode of action for DON and its sulfated analogues
Cellular Biomechanic Impairment in Cardiomyocytes Carrying the Progeria Mutation: An Atomic Force Microscopy Investigation
Given the clinical effect of progeria syndrome, understanding the cell mechanical behavior of this pathology could benefit the patient's treatment. Progeria patients show a point mutation in the lamin A/C gene (LMNA), which could change the cell's biomechanical properties. This paper reports a mechano-dynamic analysis of a progeria mutation (c.1824 C > T, p.Gly608Gly) in neonatal rat ventricular myocytes (NRVMs) using cell indentation by atomic force microscopy to measure alterations in beating force, frequency, and contractile amplitude of selected cells within cell clusters. Furthermore, we examined the beating rate variability using a time-domain method that produces a Poincaré plot because beat-to-beat changes can shed light on the causes of arrhythmias. Our data have been further related to our cell phenotype findings, using immunofluorescence and calcium transient analysis, showing that mutant NRVMs display changes in both beating force and frequency. These changes were associated with a decreased gap junction localization (Connexin 43) in the mutant NRVMs even in the presence of a stable cytoskeletal structure (microtubules and actin filaments) when compared with controls (wild type and non-treated cells). These data emphasize the kindred between nucleoskeleton (LMNA), cytoskeleton, and the sarcolemmal structures in NRVM with the progeria Gly608Gly mutation, prompting future mechanistic and therapeutic investigations
Deoxynivalenol induces structural alterations in epidermoid carcinoma cells A431 and impairs the response to biomechanical stimulation
Morphology together with the capability to respond to surrounding stimuli are key elements governing the spatial interaction of living cells with the environment. In this respect, biomechanical stimulation can trigger significant physiological cascades that can potentially modulate toxicity. Deoxynivalenol (DON, vomitoxin) is one of the most prevalent mycotoxins produced by Fusarium spp. and it was used to explore the delicate interaction between biomechanical stimulation and cytotoxicity in A431 cells. In fact, in addition of being a food contaminant, DON is a relevant toxin for several organ systems. The combination between biomechanical stimulation and the mycotoxin revealed how DON can impair crucial functions affecting cellular morphology, tubulin and lysosomes at concentrations even below those known to be cytotoxic in routine toxicity studies. Sub-toxic concentrations of DON (0.1\u20131 \u3bcM) impaired the capability of A431 cells to respond to a biomechanical stimulation that normally sustains trophic effects in these cells. Moreover, the effects of DON (0.1\u201310 \u3bcM) were partially modulated by the application of uniaxial stretching (0.5 Hz, 24 h, 15% deformation). Ultimately, proteomic analysis revealed the potential of DON to alter several proteins necessary for cell adhesion and cytoskeletal modulation suggesting a molecular link between biomechanics and the cytotoxic potential of the mycotoxin
The Cardiomyopathy Lamin A/C D192G Mutation Disrupts Whole-Cell Biomechanics in Cardiomyocytes as Measured by Atomic Force Microscopy Loading-Unloading Curve Analysis
Atomic force microscopy (AFM) cell loading/unloading curves were used to provide comprehensive insights into biomechanical behavior of cardiomyocytes carrying the lamin A/C (LMNA) D192G mutation known to cause defective nuclear wall, myopathy and severe cardiomyopathy. Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls. Furthermore, there seems to be a connection between this lamin nuclear mutation and cell adhesion behavior since LMNA D192G cardiomyocytes displayed loss of AFM probe-to-cell membrane adhesion. We believe that this loss of adhesion involves the cytoskeletal architecture since our microscopic analyses highlighted that mutant LMNA may also lead to a morphological alteration in the cytoskeleton. Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein. These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties
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