Probabilistic Envelope Curves for Extreme Rainfall Events - Curve Inviluppo Probabilistiche per Precipitazioni Estreme

A regional envelope curve (REC) of flood flows summarises the current bound on our experience of extreme floods in a region. RECs are available for most regions of the world. Recent scientific papers introduced a probabilistic interpretation of these curves and formulated an empirical estimator of the recurrence interval T associated with a REC, which, in principle, enables us to use RECs for design purposes in ungauged basins. The main aim of this work is twofold. First, it extends the REC concept to extreme rainstorm events by introducing the Depth-Duration Envelope Curves (DDEC), which are defined as the regional upper bound on all the record rainfall depths at present for various rainfall duration. Second, it adapts the probabilistic interpretation proposed for RECs to DDECs and it assesses the suitability of these curves for estimating the T-year rainfall event associated with a given duration and large T values. Probabilistic DDECs are complementary to regional frequency analysis of rainstorms and their utilization in combination with a suitable rainfall-runoff model can provide useful indications on the magnitude of extreme floods for gauged and ungauged basins. The study focuses on two different national datasets, the peak over threshold (POT) series of rainfall depths with duration 30 min., 1, 3, 9 and 24 hrs. obtained for 700 Austrian raingauges and the Annual Maximum Series (AMS) of rainfall depths with duration spanning from 5 min. to 24 hrs. collected at 220 raingauges located in northern-central Italy. The estimation of the recurrence interval of DDEC requires the quantification of the equivalent number of independent data which, in turn, is a function of the cross-correlation among sequences. While the quantification and modelling of intersite dependence is a straightforward task for AMS series, it may be cumbersome for POT series. This paper proposes a possible approach to address this problem.

Probabilistic approaches in civil engineering: generation of random fields and structural identification with genetic algorithms

The inherent stochastic character of most of the physical quantities involved in engineering models has led to an always increasing interest for probabilistic analysis. Many approaches to stochastic analysis have been proposed. However, it is widely acknowledged that the only universal method available to solve accurately any kind of stochastic mechanics problem is Monte Carlo Simulation. One of the key parts in the implementation of this technique is the accurate and efficient generation of samples of the random processes and fields involved in the problem at hand. In the present thesis an original method for the simulation of homogeneous, multi-dimensional, multi-variate, non-Gaussian random fields is proposed. The algorithm has proved to be very accurate in matching both the target spectrum and the marginal probability. The computational efficiency and robustness are very good too, even when dealing with strongly non-Gaussian distributions. What is more, the resulting samples posses all the relevant, welldefined and desired properties of “translation fields”, including crossing rates and distributions of extremes. The topic of the second part of the thesis lies in the field of non-destructive parametric structural identification. Its objective is to evaluate the mechanical characteristics of constituent bars in existing truss structures, using static loads and strain measurements. In the cases of missing data and of damages that interest only a small portion of the bar, Genetic Algorithm have proved to be an effective tool to solve the problem.

Insulin and TOR pathways regulate cellular and organismal growth through the myc oncogene in Drosophila

A large body of literature documents in both mice and Drosophila the involvement of Insulin pathway in growth regulation, probably due to its role in glucose and lipid import, nutrient storage, and translation of RNAs implicated in ribosome biogenesis (Vanhaesebroeck et al. 2001). Moreover several lines of evidence implicate this pathway as a causal factor in cancer (Sale, 2008; Zeng and Yee 2007; Hursting et al., 2007; Chan et al., 2008). With regards to Myc, studies in cell culture have implied this family of transcription factors as regulators of the cell cycle that are rapidly induced in response to growth factors. Myc is a potent oncogene, rearranged and overexpressed in a wide range of human tumors and necessary during development. Its conditional knock-out in mice results in reduction of body weight due to defect in cell proliferation (Trumpp et al. 2001). Evidence from in vivo studies in Drosophila and mammals suggests a critical function for myc in cell growth regulation (Iritani and Eisenman 1999; Johnston et al. 1999; Kim et al. 2000; de Alboran et al. 2001; Douglas et al. 2001). This role is supported by our analysis of Myc target genes in Drosophila, which include genes involved in RNA binding, processing, ribosome biogenesis and nucleolar function (Orain et al 2003, Bellosta et al., 2005, Hulf et al, 2005). The fact that Insulin signaling and Myc have both been associated with growth control suggests that they may interact with each other. However, genetic evidence suggesting that Insulin signaling regulates Myc in vivo is lacking. In this work we were able to show, for the first time, a direct modulation of dMyc in response to Insulin stimulation/silencing both in vitro and in vivo. Our results suggest that dMyc up-regulation in response to DILPs signaling occurs both at the mRNA and potein level. We believe dMyc protein accumulation after Insulin signaling activation is conditioned to AKT-dependent GSK3β/sgg inactivation. In fact, we were able to demonstate that dMyc protein stabilization through phosphorylation is a conserved feature between Drosophila and vertebrates and requires multiple events. The final phosphorylation step, that results in a non-stable form of dMyc protein, ready to be degraded by the proteasome, is performed by GSK3β/sgg kinase (Sears, 2004). At the same time we demonstrated that CKI family of protein kinase are required to prime dMyc phosphorylation. DILPs and TOR/Nutrient signalings are known to communicate at several levels (Neufeld, 2003). For this reason we further investigated TOR contribution to dMyc-dependent growth regulation. dMyc protein accumulates in S2 cells after aminoacid stimulation, while its mRNA does not seem to be affected upon TORC1 inhibition, suggesting that the Nutrient pathway regulates dMyc mostly post-transcriptionally. In support to this hypothesis, we observed a TORC1-dependent GSK3β/sgg inactivation, further confirming a synergic effect of DILPs and Nutrients on dMyc protein stability. On the other hand, our data show that Rheb but not S6K, both downstream of the TOR kinase, contributes to the dMyc-induced growth of the eye tissue, suggesting that Rheb controls growth independently of S6K.. Moreover, Rheb seems to be able to regulate organ size during development inducing cell death, a mechanism no longer occurring in absence of dmyc. These observations suggest that Rheb might control growth through a new pathway independent of TOR/S6K but still dependent on dMyc. In order to dissect the mechanism of dMyc regulation in response to these events, we analyzed the relative contribution of Rheb, TOR and S6K to dMyc expression, biochemically in S2 cells and in vivo in morphogenetic clones and we further confirmed an interplay between Rheb and Myc that seems to be indipendent from TOR. In this work we clarified the mechanisms that stabilize dMyc protein in vitro and in vivo and we observed for the first time dMyc responsiveness to DILPs and TOR. At the same time, we discovered a new branch of the Nutrient pathway that appears to drive growth through dMyc but indipendently from TOR. We believe our work shed light on the mechanisms cells use to grow or restrain growth in presence/absence of growth promoting cues and for this reason it contributes to understand the physiology of growth control.

Expression and cellular localization of Copper transporter 2 (Ctr2) in Mus musculus

The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.

Neuroprotective strategies against neurodegeneration in cellular model systems

In the present study we analyzed new neuroprotective therapeutical strategies in PD (Parkinson’s disease) and AD (Alzheimer’s disease). Current therapeutic strategies for treating PD and AD offer mainly transient symptomatic relief but it is still impossible to block the loss of neuron and then the progression of PD and AD. There is considerable consensus that the increased production and/or aggregation of α- synuclein (α-syn) and β-amyloid peptide (Aβ), plays a central role in the pathogenesis of PD, related synucleinopathies and AD. Therefore, we identified antiamyloidogenic compounds and we tested their effect as neuroprotective drug-like molecules against α-syn and β-amyloid cytotoxicity in PC12. Herein, we show that two nitro-catechol compounds (entacapone and tolcapone) and 5 cathecol-containing compounds (dopamine, pyrogallol, gallic acid, caffeic acid and quercetin) with antioxidant and anti-inflammatory properties, are potent inhibitors of α-syn and β-amyloid oligomerization and fibrillization. Subsequently, we show that the inhibition of α-syn and β-amyloid oligomerization and fibrillization is correlated with the neuroprotection of these compounds against the α-syn and β-amyloid-induced cytotoxicity in PC12. Finally, we focused on the study of the neuroprotective role of microglia and on the possibility that the neuroprotection properties of these cells could be use as therapeutical strategy in PD and AD. Here, we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine (6-OHDA), which induces a Parkinson-like neurodegeneration, with Aβ42, which induces a Alzheimer-like neurodegeneration, and glutamate, involved in the major neurodegenerative diseases. We show that MCM nearly completely protects CGNs from 6-OHDA neurotoxicity, partially from glutamate excitotoxicity but not from Aβ42 toxin.

Probabilistic tomography of atmospheric parameters from GNSS data

The objective of the work is the evaluation of the potential capabilities of navigation satellite signals to retrieve basic atmospheric parameters. A capillary study have been performed on the assumptions more or less explicitly contained in the common processing steps of navigation signals. A probabilistic procedure has been designed for measuring vertical discretised profiles of pressure, temperature and water vapour and their associated errors. Numerical experiments on a synthetic dataset have been performed with the main objective of quantifying the information that could be gained from such approach, using entropy and relative entropy as testing parameters. A simulator of phase delay and bending of a GNSS signal travelling across the atmosphere has been developed to this aim.

Cellular and Molecular Biomarkers for Risk Assessment of Colorectal cancer

The detection of Colorectal Cancer (CRC), at early stages, is one of the proven strategies resulting in a higher cure rate. In recent years, several studies have appeared identifying potential cancer markers in serum, plasma and stool in an attempt to improve actual screening procedures. Thus, the aim of the study was (1) Evaluate MN frequency, (2) Evaluate plasma ultrafiltrate capacity to induce MN formation, (3) Evaluate SEPT9 and NOTCH3 promoter methylation profile in peripheral blood lymphocytes from subjects resulted positive to fecal occult blood test and examined by colonoscopy. MN frequency was significantly higher in subjects with histological diagnosis of CRC and adenoma than control (p ≤ 0.001 and p ≤ 0.01, respectively). About, CF-MN analysis, a statistically significant difference was observed between CRC and control (p ≤ 0.05). On the other hand, SEPT9 and NOTCH3 promoter methylation status was significantly lower in CRC subjects than controls; additionally, NOTCH3 promoter methylation status was significantly lower in CRC subjects than adenoma subjects (p ≤ 0.01). The results obtained allow conclude that MN frequency varies according CRC pathologic status and, together with other variables, is a valid biomarker for adenoma and CRC risk. Additionally, the plasma of patients affected with CRC not only serve as a biomarker for oxidative stress but also as biomarker of genetic damage correlated with the carcinogenic process that verifies in colon-rectum. SEPT9 and NOTCH3 promoter methylation status, at peripheral blood level, varies according hystopathological changes observed in colon-rectum, suggesting that promoter methylation profile of these genes could be a reliable biomarker for CRC risk.

On the cellular stress response to Staphylococcus aureus alpha-toxin

Staphylococcus aureus alpha-hemolysin was the first bacterial toxin recognized to form pores in the plasma membrane of eukaryotic cells. It is secreted as a water-soluble monomer that upon contact with target membranes forms an amphiphatic heptameric beta-barrel which perforates the bilayer. As a consequence, red cells undergo colloidosmotic lyses, while some nucleated cells may succumb to necrosis or programmed cell death. However, most cells are capable of repairing a limited number of membrane lesions, and then respond with productive transcriptional activation of NF-kB. In the present study, by using microarray and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), data from a previously performed serial analysis of gene expression (SAGE) were extended and verified, revealing that immediate early genes (IEGs) such as c-fos, c-jun and egr-1 are strongly induced at 2-8 h after transient toxin treatment. Activating protein 1 (AP-1: c-Fos, c-Jun) binding activity was increased accordingly. As IEGs are activated by growth factors, these findings led to the discovery that -toxin promotes cell cycle progression of perforated cells in an EGFR-dependent fashion. Although the amount of c-fos mRNA rose rapidly after toxin treatment, c-Fos protein expression was observed only after a lag of about 3 h. Since translation consumes much ATP, which transiently drops after transient membrane perforation, the suspicion arised that membrane-perforation caused global, but temporary downregulation of translation. In fact, eIF2α became heavily phosphorylated minutes after cells had been confronted with the toxin, resulting in shutdown of protein synthesis before cellular ATP levels reached the nadir. GCN2 emerged as a candidate eIF2α kinase, since its expression rapidly increased in toxin-treated cells. Two hours after toxin treatment, GADD34 transcripts, encoding a protein that targets the catalytic subunit of protein phosphatase 1 (PP1) to the endoplasmic reticulum, were overexpressed. This was followed by dephosphorylation of eIF2α and resumption of protein synthesis. Addition of tautomycetin, a specific inhibitor of PP1, led to marked hyperphosphorylation of eIF2α and significantly reduced the drop of ATP-levels in toxin-treated cells. A novel link between two major stress-induced signalling pathways emerged when it was found that both translational arrest and restart were under the control of stress-activated protein kinase (SAPK) p38. The data provide an explanation for the indispensible role of p38 for defence against the archetypal threat of membrane perforation by agents that produce small transmembrane-pores.

Cellular and molecular effects of cross reacting material 197 (CRM197) on HT-29 colon cancer cell line

Cross Reacting Material 197(CRM197) is a Diphteria toxin non toxic mutant that had shown anti-tumor activity in mice and humans. CRM197 is utilized as a specific inhibitor of heparin-binding epidermal growth factor (HB-EGF), that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. We evaluated the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible effects on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment, or changes in cell cycle distribution, in EGFR localization or activation, they show a change in gene expression profile analyzed by microarray. This is the first study where the CRM197 treatment on HT-29 show the alteration of a specific and selected number of genes.

From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.

Implicit computational complexity and probabilistic classes

The thesis applies the ICC tecniques to the probabilistic polinomial complexity classes in order to get an implicit characterization of them. The main contribution lays on the implicit characterization of PP (which stands for Probabilistic Polynomial Time) class, showing a syntactical characterisation of PP and a static complexity analyser able to recognise if an imperative program computes in Probabilistic Polynomial Time. The thesis is divided in two parts. The first part focuses on solving the problem by creating a prototype of functional language (a probabilistic variation of lambda calculus with bounded recursion) that is sound and complete respect to Probabilistic Prolynomial Time. The second part, instead, reverses the problem and develops a feasible way to verify if a program, written with a prototype of imperative programming language, is running in Probabilistic polynomial time or not. This thesis would characterise itself as one of the first step for Implicit Computational Complexity over probabilistic classes. There are still open hard problem to investigate and try to solve. There are a lot of theoretical aspects strongly connected with these topics and I expect that in the future there will be wide attention to ICC and probabilistic classes.

HIF1α regulates Mitochondrial biogenesis and Cellular senescence induced by Gamma Radiation

Cellular response to γ-rays is mediated by ATM-p53 axis. When p53 is phosphorylated, it can transactivate several genes to induce permanent cell cycle arrest (senescence) or apoptosis. Epithelial and mesenchymal cells are more resistant to radiation-induced apoptosis and respond mainly by activating senescence. Hence, tumor cells in a senescent state might remain as “dormant” malignant in fact through disruption of p53 function, cells may overcome growth arrest. Oncocytic features were acquired in the recurring neoplasia after radiation therapy in patient with colonrectal cancer. Oncocytic tumors are characterized by aberrant biogenesis and are mainly non-aggressive neoplasms. Their low proliferation degree can be explained by chronic destabilization of HIF1α, which presides to adaptation to hypoxia and also plays a pivotal role in hypoxia-related radio-resistance. The aim of the present thesis was to verify whether mitochondrial biogenesis can be induced following radiation treatment, in relation of HIF1α status and whether is predictive of a senescence response. In this study was demonstrate that mitochondrial biogenesis parameters like mitochondrial DNA copy number could be used for the prediction of hypoxic status of tissue after radiation treatment. γ-rays induce an increase of mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence. Mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a MDM2-mediated HIF1α degradation, leading to the release of PGC-1β inhibition by HIF1α. On the other hand, this protein blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally in vivo, post-radiotherapy mtDNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of senescence.