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A framework in support of emergency management for specified and unspecified emergencies.
No full textmissin
Ecotoxicological assessment of freshwater ecosystems.
No full textL’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)
Analisi palinologiche e geofisiche per la ricostruzione delle trasformazioni ambientali nella regione lariana tra Tardiglaciale e Olocene: nuovi dati dal sondaggio di Piazza Verdi a Como.
No full textThe analyses of the present research were performed on lacustrine sediments sampled through a continuous drilling conducted in Como-Piazza Verdi (Lombardy, Southern Alps), about 200 m far from the shore of Lake Como. A multidisciplinary approach (mainly stratigraphy, palaeobotany, geophysics) was adopted to reconstruct the palaeoenvironmental evolution in Como basin.
The oldest phase of the Lateglacial was characterized by the melting of the Abduan ice tongue covering the present-day Como town area. Later, the slopes facing the proglacial and post-glacial Lake Como were colonized by pioneer vegetation. During the Bölling-Allerød the temperatures rose and a continental forest spread. At the end of the Lateglacial the role of this phytocoenosys gradually became less relevant and some mesophilous species appeared. Thanks to the climate amelioration during the Holocene these species formed a mesophilous mixed forest. During Copper Age the human factor prevailed over the climate: 5100 cal yr BP the use of fire, adopted by men to deforest, caused the disappearance of silver fir, a species very sensitive to fire; the cool-wet oscillation in progress at that time would naturally encourage its development.
The present research has innovative features from the methodological point of view and presents the first extensive pollen record for the Como subsurface. The results emphasize the need for further improving the knowledge about the palaeoenvironmental evolution in the Lake Como area
Correlative analysis of putative molecular predictive factors in patients with curatively resected stage III colon cancer, treated with adjuvant oxaliplatin-based chemotherapy.
No full textColon cancer is the second cause of death for neoplasm worldwide. In most cases it is diagnosed when still localized to the intestinal wall or in regional lymph nodes. Adjuvant therapy with 5-Fluorouracil (5FU) and folinic acid (FA), in combination with oxaliplatin (FOLFOX) are the standard options for patients with radically resected stage III disease. However, a proportion of patients will develop recurrence due to drug resistance and oxaliplatin-based chemotherapy is a regimen that may cause potentially disabling sensory neuropathy. Therefore there is an increase needing for a better selection of patients to be addressed to the most appropriate chemotherapeutic treatment, also in the adjuvant setting.
Several proteins and genetic markers have been described in an attempt to refine prognostic information and predict the benefit derived from systemic treatment. In particular TS protein expression, MSI, p53 expression, BRAF and TP53 mutations, have been described in several reports in relation to 5FU treatment, whereas ERCC1 polymorphism, ERCC1 expression and KRAS mutations, seem to be related to oxaliplatin efficacy in advanced colon cancer patients.
At this purpose we enrolled 230 patients from Argentina and Switzerland who underwent surgical resection, followed by 6-months adjuvant treatment: 106 were treated with 5FU alone and 124 with FOLFOX. In all the cases we investigated the MSI status by fragment analysis, we analyzed BRAF, KRAS, TP53 mutations and ERCC1 codon 118 polymorphism by direct sequencing and we performed ERCC1 expression analysis at protein and mRNA levels by immunohistochemistry and real-time PCR, respectively. Finally, we correlated the molecular and immunohistochemical results with the clinical data.
Above all, a little advantage in survival was observed for patients treated with FOLFOX regimen if compared to those treated with 5FU (51.3 and 41.6 months, respectively, for DFS; 55.4 and 49.3 months, respectively, for OS), although the difference was not statistically significant, probably due to the low number of analyzed cases.
We found MSI in 12% of cases, BRAF mutations in 9% of cases, KRAS mutations in 28% of cases and ERCC1 resulted over-expressed in 40% of cases detected by IHC and in 49% of cases detected by real-time PCR. These percentages, as well as the types of alterations, are in line with those published in the literature.
Concerning the correlations among markers, we observed a significant association between MSI and BRAF mutations (in agreement with the literature) and absence of association between KRAS mutations and ERCC1 expression (at odds with the hypothesis proposed in a recent preclinical study).
When we matched the clinical data of the whole patients cohort with molecular alterations, we found a trend towards a better prognosis for patients with MSI than for those with a MSS status (p=0.17); we observed that KRAS mutations confer a worse prognosis to advanced colon cancer patients, borderline for the DFS (p=0.07) and statistically significant for the OS (p=0.004); finally we found a trend towards a better DFS (p=0.11) for patients showing low levels of ERCC1 mRNA expression.
When we subdivided the patients on the basis of the received treatment (5FU versus oxaliplatin-based chemotherapy), we observed similar percentages of alterations of all the markers between the two groups. By correlating the molecular alterations with clinical data, we found a trend towards a better survival for MSI patients treated 5FU (p=0.16 and p=0.37 for DFS and OS, respectively), while for FOLFOX patients no clinical differences were found between MSI and MSS cases.
As for KRAS mutations, in 5FU group we observed a statistical significant worse DFS (p=0.04) and a trend towards a worse OS (p=0.07) in KRAS mutated patients if compared to wild-type patients. In FOLFOX group, no statistical differences were identified between KRAS mutated and wild-type cases. Stratifying the population on the basis of KRAS mutational status, we noticed that in wild-type patients there was no difference in the clinical outcome in the two treatment modalities. On the contrary, in mutated cases a trend towards a better DFS (p=0.28) and OS (p=0.20) was observed in FOLFOX treated patients if compared to 5FU group.
As regards ERCC1 expression, we found only a trend toward a better DFS (p=0.17) in patients characterized by low ERCC1 mRNA levels when treated with FOLFOX.
As for the last markers, ERCC1 codon 118 polymorphism (AAT/AAC) and TP53 mutations, we found percentages of alterations in line with the literature (for ERCC1 polymorphism: TT genotype in 31% of cases, CC genotype in 21% of patients; for TP53: 44% of cases showed at least one mutation). The correlations between these two markers and the clinical outcome are now under evaluation.
In conclusion, looking at the whole cohort, we can confirm a better clinical outcome for adjuvant colon cancer patients treated with FOLFOX regimen with respect to 5FU treatment. MSI could be a useful tool indicating a better prognosis also for advanced colon cancer but its role in predicting 5FU or FOLFOX efficacy remains controversial. In addition, we propose to assess ERCC1 mRNA expression analysis before the administration of oxaliplatin-based chemotherapy, in order to early identify the patients who may benefit the most from this treatment.
Finally, we suggest that KRAS mutational status could help clinicians in selecting the best chemotherapeutic treatment in the adjuvant setting: only KRAS mutant patients should be treated with a platinum-based chemotherapy, while patients whose tumour is KRAS wild-type can be treated with 5FU alone, thus preventing adverse side effect in a consistent number of cases. Our results, of course, deserve confirmations
New approaches to cancer vaccination and immunotherapy, based on the optimization of the presentation of tumor antigens and stimulation of CD4 positive T helper cells.
No full textRecently we have shown that a number of murine tumors arising from mice of the Balb/c (MHC H-2d) strain, induced to express MHC class II molecule after transfection with CIITA (MHC class II transactivator) ( Accolla R.S. et al. 1986) can be rejected effectively, generating tumor specific T helper cell (TH) triggering, immunological memory and protection even against parental tumor (Meazza R. et al. 2003 ; Frangione V. et al. 2010; Mortara L. et al. 2006) . The aims of my PhD thesis were: a) - to assess whether the same results can be extended to tumors of different MHC background (H-2b); b) - to investigate, using knock-out C57Bl mice for dendritic cells, whether CIITA transfected tumor cells can act as “surrogate APC” for their tumor antigens in vivo. To this end, LLC (Lewis lung carcinoma, H-2b) cells were stably transfected with CIITA and selected for expression of MHC class II molecules. Parental tumor cells and CIITA-transfected cells (LLC-CIITA) were injected subcutaneously into C57/BL6 mice and tumor take and growth kinetics were assessed. Mice injected with LLC-CIITA cells were tumor-free for longer time than mice injected with parental tumor cells. The growth kinetics and the size of CIITAexpressing tumors were significantly reduced compared to the parental tumor.
Adoptive cell transfer of purified CD4+ TH cells from mice injected with LLCCIITA into naïve mice demonstrated that these cells were able to protect from LLC parental tumor growth. Taken together these results strongly suggest that, similarly to H-2d strain, also H-2b tumors can be rejected if expressing CIITAmediated MHC class II molecules, confirming the general applicability of our tumor vaccination model. To achieve the second goal we performed in vivo experiments in a novel transgenic mouse model named CD11.c DOG, in which it is possible to induce a conditional depletion of dendritic cells (DC) by diphtheria toxin (DT) treatment. These mice express the diphtheria toxin receptor (DTR) under the control of the promoter of the CD11c molecule, expressed preferentially in DC. Once injected with DT, dendritic cells are eliminated for the period of treatment up to 12 days. DT-treated CD11c.DOG mice where injected with LLC-CIITA tumor cells and tumor take and tumor growth was followed during time. We found that LLC-CIITA cells can be recognized and rejected better than parental tumor even in CD11c-DOG mice.
These results suggest that CIITA-tumor cells may act in vivo as surrogate APCs for their own tumor antigens and trigger and adaptive immune response mediated by CD4+ TH cells
Development of engineered nanoparticles for biomedical and industrial applications.
No full textIn this work four nanoparticle systems were designed: two for medical and two for industrial applications.
The purpose of the first part of my study concerns the manufacture of a NPs-enzyme system for cancer therapy: Fe3O4 NPs@APTES-DAAO. This system combine the magnetic properties of iron oxide nanoparticles (Fe3O4 NPs) with the ROS generating enzyme D-amino acid oxidase (DAAO) in order to efficiently direct the enzyme into the tumor and kill tumor cells.
The second medical system designed consists in magnetic iron oxide nanoparticles conjugated with the antibiotics teicoplanin. Similar to the previous system, Fe3O4 NPs@Teicoplanin wants to takes advantage of the magnetic character of NPs for a specific targeting of the antibiotic.
The two systems for industrial application were composed of Fe3O4 NPs conjugated to a cephalosporin C acylase (VAC) and a L-aspartate oxidase (LASPO) respectively. In industrial enzymatic application processes, immobilization of enzymes can offer several advantages, including the ability to be used repeatedly, improvement of enzyme stability and broadening the optimum pH range of enzyme. The first system Fe3O4 NPs@APTES-VAC allows the one-step conversion of cephalosporin C in 7-aminocephalosporanic acid, which is the precursor of in semisynthetic cephalosporins antibiotics. The second system Fe3O4 NPs@APTES-LASPO allows the resolution of a racemic mixture of D, L-aspartate
Ruolo del processo autofagico in cellule tumorali umane mantenute in differenti condizioni di ossigenazione a seguito del trattamento chemioterapico.
No full textIl carcinoma del colon retto (CRC) rappresenta uno dei tumori maligni più comuni nei Paesi occidentali e presenta un tasso di incidenza in aumento in tutto il mondo. Come la maggior parte dei tumori solidi, anche i carcinomi del colon presentano spesso al loro interno regioni ipossiche in cui si assiste all’attivazione del fattore trascrizionale HIF-1, che svolge un ruolo chiave nell’insorgenza di fenomeni di chemioresistenza. Alla risposta adattativa attuata dalle cellule nei confronti dell'ipossia partecipa anche il processo autofagico, il cui ruolo nella risposta delle cellule tumorali al trattamento chemioterapico e nei fenomeni di farmacoresistenza indotta dall'ipossia risulta molto controverso. Cellule mantenute in condizioni ipossiche presentano in genere livelli di autofagia più elevati rispetto a cellule normossiche. Questo fenomeno potrebbe rappresentare una misura difensiva nei confronti dello stress ipossico, contribuendo alla resistenza terapeutica, tuttavia una attività autofagica particolarmente intensa e/o persistente sembrerebbe in grado di indurre morte cellulare.
Su queste premesse è stata formulata un'ipotesi di lavoro in base alla quale cellule trattate con 5-Fluorouracile (5-FU), in grado di indurre autofagia citoprotettiva, e sottoposte a condizioni di ipossia (in cui il processo autofagico dovrebbe essere iperattivo) potrebbero essere indotte ad attivare un programma di morte cellulare in seguito all'esposizione a composti in grado di aumentare ulteriormente la risposta autofagica. Tale ipostesi è stata testata in un modello sperimentale basato sull'uso della linea cellulare HCT116, derivata da adenocarcinoma del colon umano, esponendo le cellule a 5-FU in combinazione con Luteolina (LUT), un flavonoide di origine naturale dotato di un ampio spettro di azioni potenzialmente antiproliferative/citotossiche, o Obatoclax (OBX), composto dotato di attività BH3-mimetica grazie alla quale è in grado di attivare la fase mitocondriale del processo apoptotico. Per entrambi i composti è stata riportata una attività pro-autofagica.
I risultati ottenuti in una prima fase dello studio hanno evidenziato che LUT ed OBX potenziano l'effetto citotossico del 5-FU, in tutte le condizioni sperimentali adottate. Con entrambe le combinazioni si osserva in effetti un aumento dei livelli di autofagia; tuttavia l'osservazione che, a seguito dell'inibizione del processo autofagico, l'effetto citotossico sinergico risulta ulteriormente potenziato porta ad escludere che l'effetto chemiosensibilizzante di LUT e OBX possa essere dovuto all'induzione di autofagia letale.
Al fine di identificare uno fra i possibili meccanismi alla base dell’azione chemiosensibilizzante mediata da LUT ed OBX al 5-FU, è stata valutatata la possibilità che i due composti fossero in grado di modulare negativamente i livelli di espressione della proteina HIF-1α e/o l'attività trascrizionale del fattore HIF-1. I dati ottenuti hanno evidenziato che entrambi i composti sono in grado di inibire l'attività di HIF-1, ma verosimilmente in base a meccanismi diversi. Infatti, mentre nel caso della LUT a fronte di una ridotta attività trascrizionale si assiste ad un aumento dei livelli proteici di HIF-1α apparentemente paradossale, nel caso di OBX la downregolazione dell'attività di HIF-1 si accompagna, ed è verosimilmente dovuta, a una diminuzione dei livelli di espressione della subunità inducibile.
Esperimenti volti a chiarire se OBX fosse in grado di modulare i livelli di HIF-1α agendo sul trascritto, sulla sintesi della proteina o sulla sua degradazione suggeriscono che il meccanismo principale sia rappresentato da una ridotta stabilità della proteina, la cui degradazione da parte del proteasoma risulta facilitata, indipendentemente dalla via "canonica", attiva in normossia, che prevede l’idrossilazione O2-dipendente di residui critici di prolina e l'interazione con il complesso ubiquitina-ligasico coordinato dalla proteina di von Hippel-Lindau.
In base ai dati ottenuti possiamo concludere che, contrariamente all’ipotesi inizialmente formulata, l'esacerbazione dello stimolo pro-autofagico da parte dai trattamenti utilizzati per questo studio, non è stata tale da permettere il superamento di un livello soglia, oltrepassato il quale l'induzione di autofagia perde il suo ruolo citoprotettivo per trasformarsi in un evento letale. Nelle nostre condizioni sperimentali, quindi, non solo il fenomeno non rende ragione dell'effetto sinergico delle combinazioni utilizzate, ma potrebbe anzi contribuire alla resistenza terapeutica.
Tuttavia, i dati ottenuti fino a questo momento chiariscono, almeno parzialmente, il meccanismo alla base dell'azione chemiosensibilizzante di LUT ed OBX nelle condizioni in cui HIF-1 risulta attivato, permettendoci di ipotizzare un loro utilizzo in combinazioni polichemioterapiche per il controllo farmacologico della crescita e della progressione di carcinomi colorettali e di altri tumori solidi in cui spesso si assiste all’insorgenza di fenomeni di chemioresistenza indotta da ipossia
Multi-level approaCh to the study of exposure to nanoparticles.
No full textBackground and aims
Exposure to nanoaerosols (particles with diameter below 100 nm) is an important topic in epidemiological and toxicological studies and is deemed to be a major risk affecting human health, both for general population (exposed to “ultrafine particles” - UFP) and for workers (involved in the production or application of manufactured nanomaterials -MNM- and nanoparticles - NP). In fact, high concentrations of airborne nano-sized particles are associated with increased pulmonary and cardiovascular mortality. Further, recent studies assesse that UFP and NP can reach the deeper region of the respiratory system and overcome the alveolar barrier and enter the bloodstream, contributing to increased risk of cancer, thrombosis, and cardiovascular diseases. An increasing number of studies are indicating that the health risk deriving from exposure to airborne nanoaerosols is not adequately addressed by conventional exposure evaluation methods and strategies capable of measuring exposure against these attributes. In fact, in recent years, society has become increasingly sensitive to individual risks, and thus data on the exposure needs to be personalized. Therefore, airborne particle studies were performed in the recent years to identify the main UFP and NP sources and to characterize population exposure. In this regard, personal monitoring is considered as the only way to obtain accurate exposure data, which are critical to further reduce exposure misclassification in epidemiological studies. The drawback of such method, however, is the high cost of implementation and the associated small number of observations that tends to produce sample biases. For this reason, personal monitoring is often used as a complement in exposure models to assess air pollution exposures in health studies. These models use personal or household exposure monitoring, and appear well-suited to overcome the problem of achieving population representative samples while understanding the role of exposure variation at the individual level. The monitoring and characterization techniques discussed in this thesis aims to evaluate nanoaerosols exposure in terms of mass, surface-area and/or number concentration. These methods were developed and used to exposure characterization both in environmental and occupational settings. The goal of this PhD project is firstly to perform an exposure assessment of UFP using innovative techniques and strategies. Secondly, it will be shown that microenvironmental models are effective tool, capable to model exposure to UFP in populations and sub populations. Finally, newly developed strategies and techniques are applied in occupational settings, in order to perform an occupational exposure assessment for workers involved in the production or application of MNM and NP.
Materials and methods
Exposure to airborne pollutants can be determined using indirect exposure models or through a direct approach (i.e. air quality measurements). In this study, UFP concentrations in different urban microenvironments (ME) were firstly measured by personal monitoring in repeated sampling campaigns, along fixed routes in two Italian cities, in order to measure personal exposure in
transport MEs. Measurements followed a multi-parametric and multi-metric approach, including on-line monitoring of UFP Particle Number Concentration (PNC), mean diameter (mean d) and lung-deposited surface area (LDSA). Secondly, average daily UFP exposure of adult Milan subpopulations (defined on the basis of gender and then for age, employment or educational status), in different exposure scenarios (typical working day in summer and winter) were simulated using a microenvironmental stochastic simulation model. The basic concept of this kind of model is that time-weighted average exposure is defined as the sum of partial microenvironmental exposures, which are determined by the product of UFP concentration and time spent in each microenvironment. Furthermore, this thesis describes the development of an instrumental approach for measurement of NP exposures in occupational settings, which takes into account the major potential route of exposure and factors that may influence biological activity and potential toxicity of nanomaterials and incorporates a risk management approach: different methods were used to measure and assess occupational exposures to engineered nanoparticles (NP) with a multi-parametric approach: the first method involved off-line gravimetric analysis of filter samples collected with Low Pressure Impactor. The second method used different, hand-held, direct-reading instruments to obtain a time series of particle number concentrations (PNC), mean diameter and surface-area concentration for NP.
Results and conclusions
This thesis provides important insights into UFP exposure in urban environments, that should be considered in developing additional and larger studies on population’s exposure. First, continuous real-time monitoring provided the information necessary to define the influence of local sources or changes in local circumstances on UFP concentrations. In addition, continuous monitoring allowed for the evaluation of short-term particle concentrations and demonstrated temporal and spatial variability for the studied urban microenvironments. Further, the simulation model, used to estimate the average daily UFP exposure of adult subpopulations in a major Italian urban area and in different exposure scenarios, have defined that demographic and socio-demographic factors (e.g., gender, age, profession, instruction level), as well as environmental patterns, have to be considered as major determinants of pollutant exposure in urban environments. Furthermore, this research detected UFP levels and average particle sizes and their seasonal variability, as well as comprehensive information on average particle number and mass concentrations, sizes and surface area in various microenvironments within urban areas, which is fundamental to evaluate the variability of human exposure in urban environments and to support the relevance of traffic-related exposure for health. Thus, findings derived from this study may represent an important tool in the definition of health and social implication of UFP exposure for general population and to provide complete and accurate exposure assessment data for risk assessors, including exposure metrics, mostly relevant as health effects indicators. Finally, regarding occupational settings, this study defined an experimental protocol, which was intended to be useful in determining potential exposure to engineered nanomaterials and nanoparticles in the workplace with complementary approaches. These information may also be used to determine whether engineering controls are effective in preventing release of the engineered nanomaterials to the workplace atmosphere
