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TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATION OF ADULT AND FETAL MOUSE NEURAL STEM CELL PROPERTIES: ROLE OF REPRESSOR ELEMENT 1-SILENCING TRANSCRIPTION FACTOR AND MICRORNAS IN NEURONAL AND GLIA DIFFERENTIATION
No full textNeural stem cells (NSCs) are a self-renewing population of cells that generate the neurons and glia of both the developing and adult central nervous system (CNS). The maintenance versus differentiation of NSCs relies on an intricate network of signalling regulators that finely control gene expression program in a spatial and temporal defined sequence.
One factor that has been implicated in neuronal differentiation is the repressor element-1 silencing transcription factor (REST). Recent evidences suggest that REST plays a much broader role that varies according to cell type and developmental stage. In this study, we address the role of REST in regulating the properties of NSCs derived from the mouse adult Subventricular Zone (SVZ). Rest silencing in a transiently or stable transfected adult NSC line (aNSC1) promoted cell differentiation into neurons and glia, despite the presence of growth factors in the culture medium. Interestingly REST silencing did not modify the neurogenic and gliogenic potential of aNSC1 induced to differentiate by growth factors withdrawal. These data suggest that REST is crucial to the maintenance of aNSC1 in an undifferentiated proliferative state but it does not affect their lineage-specific differentiation program. Consistently, knockdown of REST in primary neurospheres impaired their growth and clonogenic potential, as evidenced in proliferation- and colony-forming assays. Furthermore, genome- and transcriptome-wide analyses have shown that REST targets different genes implicated in fundamental biological regulatory networks. REST binds to several neuronal gene and microRNA regulatory regions and its inhibition affects the expression of various genes implicated in the control of cell growth and differentiation including Fgf1, Wnt5a and Notch3. The analysis of the regulatory network governed by REST may elucidate the molecular mechanisms involved in the regulation of the aNSC1 properties.
Posttranscriptional control by microRNAs (miRNAs), in addition to transcriptional regulation, has emerged as critical regulator of NSC biology. Although several miRNAs have been identified as specific regulators of neuronal and oligodendrocyte differentiation, the miRNAs involved in astrogliogenesis remain still unknown. Here we aimed to identify specific miRNAs involved in astrocyte fate specification of neural progenitor cells derived from mouse embryonic cortex (eNPCs). ENPCs in culture can fully differentiate into GFAP-positive astrocyte in the presence of FBS. We performed a large-scale analysis of more then 350 miRNAs on eNPCs under proliferating and differentiating condition. This global analysis has revealed that many miRNAs are differently expressed during differentiation in vitro. Among these, northern blot analysis showed that miR-23a/b, miR-27a/b, miR-24, miR-26a and miR-125b increased almost linearly during differentiation, instead the family members of miR-29 (miR-29a, miR-29b and miR-29c) are characterized by a late induction. These data suggest a potential role of different microRNAs at different times during the process of astrocyte differentiation. Furthermore, we demonstrated that miR-23a and miR-125b suppresses the expression of Musashi-1 (Msi1), a RNA-binding protein critical regulator of NSC proliferation and self-renewal. The miRNAs mediated suppression of Msi1 could play an important role in maintaining the NSCs in an undifferentiated state preventing astrocyte differentiation
Development of analytical methods for counterfeit drugs investigation
No full textDuring this PhD work new analytical methods for the analysis of counterfeit drugs were developed.
The Colorimeter is a simple instrument for preliminary and fast screening of suspicious counterfeit drug. It employs an “Eye one” spectrophotometer, that acquires the reflectance spectrum of solid surfaces in the visible; it is portable and very cheap. Since the color both of tablets and of peculiar regions of the packaging are specific to a drug, it is very unlikely that a counterfeit product can imitate them in an accurate way. Thus the reflectance spectrum of a suspicious drug can be compared to that of the original one, obtained through a statistical analysis of a representative amount of original samples. By this comparison it is evaluated if the analyzed drug is probably a counterfeit, hence it needs more in depth analyses, or if it is original.
A solid state approach was developed by combining FT-IR, DSC, TGA. This approach is rapid as sample treatments are not necessary. By this approach it is possible to identify the ingredients of an unknown drug, in particular both active substances and excipients can be detected. First a data bank was built analyzing all the possible components of a suspicious drug: active substances, pharmaceutical excipients, low cost widespread materials (from food and construction industry) were analyzed and for each technique the peculiar signals of every ingredient were detected. Then suspicious samples were analyzed: matching the signals with the data bank it was possible to identify which ingredients were present. By this approach it was possible to detect the presence of inappropriate and dangerous ingredients like chalk.
A discriminating dissolution method for the analysis of sildenafil citrate tablets was developed and validated (sildenafil citrate is the active substance of Viagra®). By this dissolution method it was possible to highlight differences in the active substance release rate between originals and counterfeit drugs.
The dissolution medium was a phosphate buffer of pH 4,5; in this condition sildenafil citrate is weakly soluble, hence the dissolution is slow and informative. The Basket system (Apparatus I Ph.Eur.) was employed since the Paddle system caused a coning effect.
Counterfeit and suspicious samples were analyzed and compared to the originals. By this method it was possible to acquire valuable information about formulation and technology of production of these unknown drugs.
An NMR method for quantitative determination was validated according to the ICH guideline “Note For Guidance On Validation Of Analytical Procedures: Text And Methodology”.
Since a quantitative NMR method doesn’t require a reference standard of the investigated substance but only a generic internal standard, it can have important applications in the counterfeit drugs investigation. In particular it allows to quantitate substances whose reference standard is not available, like the omologues; these are new, not authorized, active substance with a structure that is similar to that of an authorized one; their presence in fake herbal product and food dietary supplements has recently been reported in literature.
Three different internal standards were tested during the validation: aciclovir, maleic acid, dinitrobenzoic acid. Deuterated DMSO was employed as solvent since most of the active substances are soluble in it.
This method resulted very accurate, precise, with an excellent linearity in the concentration range of interest
Transient Receptor Potential Vanilloid 1 (TRPV1) activation induces autophagy in thymocytes through ROS-regulated AMPK and Atg4C pathways.
No full textAutophagy is a highly conserved process involved in lymphocyte development and differentiation. Herein, we demonstrated for the first time that triggering of Transient Receptor Potential Vanilloid 1 (TRPV1) by the specific agonist capsaicin (CPS) induces autophagy in mouse thymocytes. TRPV1-dependent autophagy required calcium influx and ROS generation resulting in AMP-activated kinase (AMPK) activation. CPS specifically increased autophagy related 4C (Atg4C) mRNA expression and induced oxidation of Atg4C protein by ROS generation. TRPV1-triggered autophagy was Atg6/Beclin-1-dependent, as demonstrated by the use of Beclin-1+/- transgenic mice, and involved ROS- and AMPK-mediated up-regulation of Beclin-1 expression. Autophagy is activated as pro-survival process since its inhibition triggered apoptosis of thymocytes: this effect was accompanied by down-regulation of Atg4C, Bcl-XL and immunity-related GTPase family M (Irgm1) mRNA expression, decreased Bcl-XL and Beclin-1 protein levels and caspase-3 activation, suggesting the existence of a molecular interplay between autophagic and apoptotic programs.
TRPV1 activation by CPS altered the expression of CD4 and CD8α antigens, inducing the development of a double positive subpopulation expressing lower levels of both receptors (DPdull), representing an intermediate stage of thymocyte maturation.
Interestingly, we found that DPdull represent the thymocyte subpopulation undergoing autophagy upon CPS treatment and that, when autophagy is inhibited, it becomes apoptotic. Our findings suggest that DPdull cells are able to respond either to survival or death signals and that TRPV1 channel dependent autophagy/apoptosis could play a major role in thymocyte development
Tecniche integrate di modellazione della denudazione dei versanti e monitoraggio dell’erosione accelerata in siti chiave dell’Italia centrale
No full textThe growing interest in studying badland dynamics reflects the need to increase knowledge of geomorphologic processes and dynamics in subhumid badland areas, particularly because of their importance in generating extremes of water and sediment production. Field studies of soil erosion are expensive, time-consuming and data needs to be collected over many years. Though providing detailed understanding of the erosion processes, field studies have limitations because of the complexity of interactions and the difficulty of generalising from the results.
Cost-efficient methods of estimating erosion over whole catchments are required as ways of predicting erosion after disturbance or following various erosion management strategies. Thus, the indirect estimation and the prevision of erosion rates is still one of the main research topics of the scientific community in the field of geomorphology and is far from solved.
This Ph.D. research project is aimed at defining an integrated methodology of denudation intensity estimation and prevision, for areas greatly affected by badlands, and it is based on both quantitative geomorphic analysis and multivariate statistical investigations, in order to deepen the relationships between the main denudation effects and the potential causal factors favoring geomorphologic instability in badlands areas. The research have allowed to propose a statically based method for water erosion hazard assessment, conceived as a spatially distributed prevision of calanchi badlands, and associated erosion rate, occurrence. Direct measures of erosion intensity in badlands were used to validate the water erosion estimates and previsions.
As the research project is focused mainly on methodological objectives, well-known study areas of Tyrrhenian side of central Italy, included within the Tevere and Ombrone River Basins, have been selected, in order to compare the results gradually achieved with the earlier available data.
Erosion rate estimations were performed refining some empirical equations (“Tu Denudation index”), that estimate the suspended sediment yield (SSY) as a function of morphometric parameters related to drainage network and relief (Ciccacci et al., 1981, 1986). Tu denudation index was confirmed to be a good estimator of the suspended sediment yield (SSY) for catchments characterized by the prevalence of sedimentary and weakly coherent outcropping lithologies. The improvement of the regression relations contributed to better estimate sedimentary output for catchment widely affected by badland areas. In these basins, in fact, SSY is strongly correlated to the areal ratio affected by badlands to the total catchment area. Thus, using the not-projected drainage density parameter (D3d), instead of the traditional drainage density parameter (D), even if not improving the SSY estimation for large basins, was considered to better reflect the conditions predisposing erosions than D for smaller catchments, where large calanchi badlands and related high slope gradients are present. The dominant role of drainage density in estimating erosion rate for badland areas was confirmed by the attempt of zoning the estimated erosion rates using the Tu Grid Analysis. This attempt proved to be very efficient in estimating the erosion rate due to runoff within badland areas, as confirmed by the comparison between the estimated and the measured erosion rates. This result seems to increase the prospective of using Tu grid analysis when prolonged denudation monitoring is not possible. Moreover, even where punctual erosion rates are measured by pin monitoring, the estimated erosion rate map represents a validated continuous representation of water erosion rate for larger areas.
Geomorphological susceptibility evaluation was performed applying a multivariate statistical method based on conditional analysis (Bayesian interpretation of probability) integrated by a proposal of a new method for most influential causal factors selection. The procedure provided satisfactory results for the unbiased prediction of landslide and water erosion susceptibility for the Upper Orcia Valley. The method is conceptually simple but, at the same time, effective in evaluating the conditional probability of hazardous events given a certain combination of causal factors: the proposed factor selection procedure has proved to be a useful tool for the unbiased detection of the factors really discriminant for instability landforms in the study area, and can be very helpful when analyzing new areas. Moreover the use of vector datasets allow to create vector easy-to-read susceptibility maps, in which the fragmentation generally characterizing raster outputs is avoided. These characteristics make this susceptibility method easy to be understood and each resulting map easy to be read, thus suitable for policy makers in planning land management strategies.
The association of the estimated erosion rate for calanchi badland areas to the surveyed landforms allowed to use the susceptibility method to evaluate the water erosion hazard, since the temporal information about the erosion processes was related to the spatial data. This procedure is proposed when direct erosion rate measures are not available.
Different techniques of direct monitoring of erosion rates and processes have been performed, with the aim of identifying the main geomorphic processes acting in the study areas and quantifying their intensity. In particular, direct measurements (erosion pin monitoring, geomorphologic survey, DGPS survey and digital photogrammetric analysis) were used to validate the results obtained after indirect erosion rate estimations and susceptibility and hazard assessment models application. Even though, interesting remarks have been concluded on the applicability of various methods of erosion monitoring.
The size of the study area, the time available, and the quality of the data required are perhaps the most critical issues to be considered when looking for the most appropriate technique. As well-known, the traditional erosion pin method generally allows to carry out very accurate punctual measures, whose error is measurable in few millimeters. So, it can be used to quantify very detailed temporal variations (monthly or after-event ground level changes). On the other hand, DGPS survey can be proper when a single hillslope of less than few hectares is being monitored, as the time and effort required would be acceptable. For larger areas or wider time interval, high resolution photogrammetric analysis could be more appropriate. However, all these methods are affected by many error sources, that limit their use to very specific time and spatial ranges.
Finally, some new contributions to the knowledge of the physical factors influencing the initiation and the development of different water erosion landforms in the studied badland areas have been achieved.
Comparison of pluviometric data and measured ground level variations for Bargiano site (Tevere River Basin) has highlighted that clay removal by water erosion is generally due to intense rainfall event preceded by quite long dry periods, while accumulation (due to gully banks collapsing) is favoured by intense rainfall after a certain number of rainy days (frequent in spring). Moreover, in inter-rill position, where almost the lonely water erosion acts, intense events are significantly more effective than long events.
Considering the distribution of calanchi and biancane landforms of Upper Orcia Valley (Ombrone River Basin) among the different classes of the main topographic and physiographic factors, it is a matter of fact that calanchi badlands develop on steeper slopes and where higher values of amplitude of relief occur, due to the morphoevolutionary processes. Moreover, observations on present embryonic biancane of Lucciolabella site confirm the leading role played by reticular systems of joints in the dissection of original, gently-dipping surfaces. Actually, a resolute difference on dispersivity level of the biancana parent material samples of La Piaggia subcatchment was not found with respect to calanchi badlands samples of the same subcatchment.
On the other hand, a significant influence of clay properties was observed on the different erosion rates measured during decadal monitoring investigations by means of erosion pins in the study areas. Calanchi badlands show lower erosion rates due to surface runoff. The major facility of biancane clays to be entrained at very low stream powers is reflected in their major dispersivity, while, in badlands, the morphoevolution and sediment removal is predominantly caused by widespread mudsliding from the rill and gully heads, as also confirmed by the mean positive variations of ground level recorded at some calanchi monitoring stations. This observation can be also related to the higher sand content in calanchi badlands, which may favour the infiltration processes to the detriment of runoff.
Finally, as already observed by several authors, the agricultural exploitation of these lands lead to a decrease of exchangeable cations concentration (and, thus, clay dispersivity), even if the permanent inhibition of chemical dispersion due to increase of soil stability hypothesized by Phillips (1998) cannot be completely agreed. Decadal monitoring and observation in the study areas and in other sites of central Italy have outlined that badlands initiation is even enhanced by agricultural manipulation: grazing and farming are among the most important triggers for accelerated water erosion, and tillage erosion has been recognized as an increasing factor of water erosion.
The performed investigations have allowed to carry out some new remarks about both the applied and proposed methodologies and the studied areas and related processes. In particular, results from this research have contributed to improve some methods useful to deepen the knowledge of processes and denudation intensity acting in badland areas of Mediterranean drainage basins
Romania, l'attuale situazione economica e le difficili prospettive di superamento della crisi
No full textAnalisi statistica delle abitudini alla suzione di tipo non nutritivo in un campione in età pediatrica
No full textObjective: Aim of this case-control study was to evaluate the prevalence of malocclusion related to non-nutritive sucking habits (pacifier or finger) in a sample of children at the Unit of Pediatric Dentistry, Department of Oral and Maxillo-facial Sciences of the “Sapienza”, University of Rome.
Materials and methods: Two samples of children were selected from January 2009 to December 2010, all aged between 5 and 9 years in deciduous or early mixed dentition. The first sample (study group) consisted of 125 subjects that presented one or more malocclusions; the second sample (control group) consisted of 150 children that did not show any form of malocclusion. Increased overjet, anterior openbite, and posterior crossbite were recorded. Information about non-nutritive sucking habits in the two samples were obtained from a questionnaire directed to the parents.
Results: Collected data were analysed for correlations between the presence and type (pacifier or finger) of non-nutritive sucking habits and the development of malocclusions. Preliminary results from our analysis showed that children with non-nutritive sucking habits present a risk of developing malocclusions 2.5 times higher than children who did not suck the pacifier or the finger. Also, the use of pacifier was statistically correlated with all types of malocclusions, while finger sucking was only correlated with the development of overjet.
Conclusions: Our study confirms that non-nutritive sucking habits give a significant contribution to the development of malocclusions, as reported in previous studies
Speculative Protocols for Actively Replicated Transactional Systems
No full textNowadays, the role of transactions has become twofold:
- they are used in order to guarantee consistency and atomicity in applications manipulating data;
- they are used as a means to synchronize the activities of threads working concurrently within any software layer.
Overall, the concept of transaction, historically related to support data manipulation in the context of database systems, has been widened so to encapsulate synchronization aspects in the context of parallel and concurrent applications. The latter aspect found its expression via Software Transactional Memory (STM) technologies, which have been oriented to mask the complexity of synchronization to the application programmers, thus moving along the path of bringing the power of multi/many-core architectures into the hand of ordinary, non-specialized, software developers. Such a widened scope of transactions, together with significant technological innovations possibly impacting the execution profile/cost of traditional database transactions (e.g. the advent of SSD storage systems) and the level of transaction parallelism (e.g. the ad- vent of many-core architectures), raise the need for reconsidering the design of protocols supporting fault tolerance.
In this thesis, I focus on fault tolerant protocols based on the active repli- cation paradigm, which is done by systematically exploiting speculative computation approaches. More in detail, I worked on innovative speculative transactional replication protocols relying on Optimistic Atomic Broadcast group communication primitives, which have been used as a building block for replicas coordination. Some proposed results are mostly oriented to theory, while others have a more strict relation with pragmatic aspects associated with the design/implementation of replicated transactional systems
La rappresentazione dell'io nella corrispondenza privata di Baudelaire
No full textPartendo dall’ipotesi che la Correspondance di Baudelaire si situi alla frontiera tra la realtà autobiografica e la finzione letteraria, il mio lavoro si propone di analizzare le immagini che Baudelaire dà di sé nella sua corrispondenza privata con i nuovi strumenti dell’epistolario, che permettono di esaminare le lettere al di là della loro letterarietà e di individuare i modi di pensare, le convinzioni e le motivazioni profonde del soggetto epistolare. La prospettiva metodologica mi è stata suggerita dai lavori di analisi del discorso che sono stati già da tempo applicati allo studio delle lettere, e più precisamente dai lavori sull’ethos, sull’immagine di sé e dell’altro, condotti da Ruth Amossy nonché da altri specialisti dell’epistolario che si situano nell’orbita dell’analisi del discorso: Jürgen Siess, Georges-Elia Sarfati, Anna Jaubert, ecc. Dopo un primo capitolo a carattere teorico nel quale affronto tre problematiche diverse ma strettamente collegate tra di loro (la questione della verità nelle scritture intime, l’identità del soggetto epistolare e l’ethos epistolare) passo nel secondo capitolo all’analisi delle principali immagini che Baudelaire offre di sé nelle lettere private, ossia all’immagine del fainéant, dell’escluso e dell’eterno bambino. La figura dell’io epistolare si costruisce a partire da ciò che egli dice di se stesso, ma soprattutto attraverso le modalità della sua parola. È più sul piano dell’enunciazione che sul piano dell’enunciato che si realizza il suo ethos, il quale può essere analizzato secondo un duplice approccio metodologico: il primo tipo di metodo consiste nello studio delle tracce di soggettività nelle lettere, mentre il secondo, di impronta pragmatica, serve a indagare le interazioni tra il mittente e i suoi vari destinatari. Questi due aspetti risultano così strettamente correlati fra di loro da non poter essere oggetto di una trattazione separata. Infine, nell’ultima parte della tesi, a carattere conclusivo, accosto l’io della Correspondance all’io lirico delle Fleurs du mal, cercando di capire che fine fanno le immagini che Baudelaire dà di sé nelle lettere private nel momento in cui passa alla scrittura lirica destinata alla pubblicazione
THE ROLE OF STAT3 SIGNALING IN CARCINOGENESIS
No full textThe protein STAT3 (Signal Transducer and Activator of Transcription 3) plays a central role in a range of physiologic process, and when subverted in cancer, can be a central mediator of malignant cellular function. This protein resides at the critical junction between intracellular signaling events and the regulation of genes involved in apoptosis, differentiation, and cell proliferation. The present research has intended to investigate the role of STAT3 in the nuclear compartment 1) as transcription factor in the regulation of a new promoter sequence; 2) as protein hub in modulation of enhanceosomes STAT3-specific in response to different pathways; 3) as protein involved in onset and progression of human prostate cancer.
STAT3 acts as transcription factor in the regulatory region of TPX2 gene coding for a protein involved in the complex process of mitosis, and was identified as one of the microtubule-associated proteins. An inspection of the 5'-flanking region of the human BCL2L1 gene, coding for Bcl-xL, has many potentially binding sites for STAT proteins, but none of these correspond closely to the consensus sequence of Stat3. We identified a high-affinity binding sites for Stat3 located at -4305/-4297 base pairs from the transcription start of TPX2 and had a TTCCCGGAA sequence, which is identical to the sequence bound by activated Stat3 in the promoter of gene CDKN1A, coding for the protein p21WAF1/CIP1. By reporter gene assay, conducted in M14 cells treated with specific phosphopeptidic inhibitor of STAT3, we showed that this protein is recruited on the TPX2 gene promoter and regulated in vivo TPX2 expression.
Whereas STAT3 is tyrosine phosphorylated by three types of kinases, it uses a precise sequence of functional actions by multiple coactivator complexes and post-translational modifications (PTMs) for mediating gene activation. Considering that we found a different set of STAT3-associated proteins in tumoral cell lines in which the activation of STAT3 can be mediated by different pathways, we could speculate not only a tumor-specific, but also a signal-dependent composition of enhanceosome STAT3-specific. Following CoIP, ChIP and RT2-PCR assays, we hypotized a functional interplay between PARP-1 STAT3-associated proteins when the transcription factor was phosphorylated by Src-kinases or after EGF stimulation, instead the association with CBP/P300 is IL-6 inducted by JAK kinases.
In the study of STAT3 partners that affect its function, we demonstrated the importance of PTMs of STAT3 in prostate cancer. In this work we evaluated in parallel, by immunoblotting analysis, the variation of phosphorylation, acetylation and gluthationylation of STAT3 in cell lines and in human prostate tumor (FFPE). To investigate how differences in PTMs of STAT3 can influence gene expression and interactions with coactivators, immunoblottig analysis, Co-IP and ChIP experiments have been conducted in LNCaP and PC3 cells treated with IL-6 and H2O2, to simulate a physiological response to citokines or to oxidative stress. It has been shown that S-gluthationylation of STAT3 and the recruitment of coactivators such ERp57 and Ref-1, two protein involved in redox modification, is a response to oxidative stress, associated with a more advanced state of disease.This work was sponsored by "Istituto Pasteur-Fondazione Cenci Bolognetti