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First insights on the signaling pathways related to CDKL5 regulation and on its possible involvement in synaptic plasticity
No full textRett syndrome (RTT) is an X-linked form of mental retardation that occurs sporadically once every 10,000-15,000 female births. After a period of normal development (6-18 months), the patients show a rapid regression of acquired speech and motor skills and the development of several symptoms including mental retardation, seizures, intermittent hyperventilation and stereotypic hand movements. This condition is mainly stable and signs of progressive neurodegeneration are absent. Almost 80% of Rett cases are associated with mutations in the
MECP2 (methyl CpG binding protein 2) gene. MeCP2 is a nuclear protein that binds methylated DNA and recruits histone deacetylases and co-repressor complexes to suppress transcription. It belongs to the MBD family of proteins involved in the epigenetic regulation of gene-expression.
Recently, mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients characterized by a subset of Rett clinical phenotypes and generally suffering of infantile spasms and severe mental retardation. The product of CDKL5 is a serine/threonine kinase that belongs to the CMGC family; the exact functions exerted by this kinase and its regulatory mechanisms remain mainly unknown.
CDKL5 is present in the nucleus and in the cytosol of neurons and its expression shows a continued increase during development; accordingly, CDKL5 is a critical regulator of neuronal morphogenesis, neurite growth and dendritic arborization. In the cytosol, CDKL5 phosphorylates NGL-1 (Netrin-G1 Ligand 1), a regulator of early synapse formation and maturation. In the nucleus, CDKL5 binds and phosphorylates in vitro MeCP2. Furthermore, in the nucleus CDKL5 colocalizes with nuclear speckles and is probably involved in the regulation of mRNA splicing.
Recently our group has demonstrated that the expression levels and the subcellular distribution of CDKL5 are modified by neuronal activation. In particular, a glutamate bath induces in cultured hippocampal neurons the rapid exit of the kinase from the nucleus and its proteasome-dependent degradation. The significance of this response remains to be elucidated. Furthermore, BDNF induces in rat cortical cultures, a rapid phosphorylation of CDKL5.
The main aim of this work was to study how neuronal depolarization or activation by BDNF affects Cdkl5 regulation, in terms of gene transcription, post translation modifications of its final protein product and the involved signaling pathway(s). We found that, both in primary murine neuronal cultures and cortical slices, depolarization affects the expression of the gene, both at the transcriptional and post-transcriptional levels, together with its phosphorylation state. The response is affected by the maturation stage of the treated neurons and the involved signaling pathways have been characterized. We speculate that the observed regulation of Cdkl5 during neuronal depolarization could be related to a role of the kinase in neuronal activation. Electrophysiological approaches will be required to confirm the involvement of CDKL5 in the regulation of neuronal activity; furthermore, the identification of novel interactors of Cdkl5 should help in understanding its physiological functions in the central nervous system and the pathological consequences of a malfunctioning CDKL5
Analysis and modeling of geological and environmental factors in site selection: the case of the Po Plain
No full textIl progetto di ricerca sviluppato nel corso del Dottorato ha l’obiettivo di illustrare come una rigorosa analisi geologica consenta di identificare, valutare e mitigare gli effetti di eventi naturali estremi, che possono costituire un serio fattore di rischio per gli impianti industriali e le aree metropolitane; a tale scopo, sono state selezionate due aree campione localizzate all’interno del bacino Padano, dove si sono condotte analisi geositologiche ed ambientali per caratterizzare le interazioni tra contesto naturale e impianto industriale: una discarica di rifiuti non pericolosi nel Comune di Cairo Montenotte (Sv) e l’area urbana di Como.
Nel caso di Cairo Montenotte, sono state condotte indagini geognostiche e geofisiche, prove in sito e di laboratorio e monitoraggio strumentale; altre indagini sperimentali sono state mirate alla caratterizzazione delle proprietà fisiche e meccaniche dei rifiuti, con particolare riferimento alla stabilità e all’evoluzione dei cedimenti del cumulo dei rifiuti.
La città di Como sorge su sedimenti di origine palustre molto recenti e soggetti a subsidenza; l’analisi di oltre 300 stratigrafie di sondaggio e delle prove geotecniche disponibili ha consentito di ricostruire l’assetto stratigrafico del sottosuolo mediante profili geologico-tecnici; il continuo monitoraggio della rete piezometrica comunale e dei parametri idrologici del bacino lacustre ha permesso di caratterizzare la circolazione idrica sotterranea. Sono stati poi approfonditi alcuni aspetti relativi alla presenza di lineamenti strutturali di importanza regionale, in particolare il Retroscorrimento di Monte Olimpino e infine sono state condotte alcune elaborazioni per ricostruire l’evoluzione paleo-ambientale dell’area a partire dalla deglaciazione.
The main goal of the project is to illustrate how the rigorous geological analysis allows to identify, assess, and mitigate extreme geological hazards, which might be a serious factor of environmental risk for the local industrial plants and large metropolitan areas. We selected 2 study areas in the Po Plain, a non-hazardous waste landfill located in Cairo Montenotte (Savona province), and the Como urban area. At these sites we perform several analysis in order to characterize the interactions between natural environment and industrial plants or critical facilities.
In the Cairo Montenotte case, core drillings, geophysical analysis and laboratory tests were conducted; moreover, the physical and mechanical properties of the waste were analyzed, specifically concerning waste stability and landfill subsidence. The mutual impacts between the industrial waste treatment plant and the natural environment have been analyzed and assessed.
The Como urban area lies on very recent palustrine sediments, an environment showing remarkable Holocene subsidence, and high potential for liquefaction and coastal flooding; the stratigraphic setting was reconstructed analyzing more than 300 borehole logs and geotechnical tests, and building new geological sections; wells monitoring allowed to characterize groundwater circulation. Some aspects related to the structural setting (e.g., Monte Olimpino Backthrust) and the Late Quaternary environmental evolution were also closely examined. The results of this comprehensive geological analysis have been used in order to assess the environmental impact of the new integrated defense system against floods, presently under construction along the Como lakeshore. This critical facility consists of rows of mobile gates which are intended to hydrologically isolate the town during events of very high level of the Lake Como.
In both cases it is possible to show how a proper approach for the geological siting is a critical tool for A) ensuring the environmental safety of the local industrial plant or critical facility, and B) reducing the operational costs by preventing from repeated readjustment of the project due to poorly defined design parameters
Lo specchio de' governanti di Antonio Masucci, O.F.M.Conv. Per un'ermeneutica simbolica di un trattato politico del Seicento. The mirror of rulers from Antonio Masucci,O.F.M.Conv. A symbolic approach to hermeneutics of a political treatise of the Seventeenth century.
No full textOggetto della mia indagine è un trattato di morale politica del Seicento intitolato Specchio de’ governanti il cui autore è un frate francescano, vissuto a Napoli, Antonio Masucci. Il presupposto è stato svolgere, attraverso un approccio di ermeneutica simbolica, un parallelismo tra due periodi di crisi, quella “barocca” del XVII secolo e quella della società contemporanea.
Dopo una necessaria premessa sulla metodologia di ricerca, la simbolica e i concetti di simbolo, ho ritenuto opportuno ricostruire il percorso del contributo della cristianità alla formazione della coscienza, individuale e collettiva, europea e, per quanto riguarda il piano politico, nel cammino di evoluzione del moderno concetto di sovranità politica dal Medioevo sino alla modernità.
Ricostruito tale percorso storico-teorico e, approfondito lo scenario socio-culturale italiano del Seicento, con riferimento anche alle riflessioni sulla “ragion di Stato” e sulla sovranità dei principati italiani, ho ottenuto gli strumenti adatti per affrontare l’analisi del testo di riferimento.
L’opera era finalizzata ad ammonire i governanti dell’epoca sui loro errrori al fine di istruirli ad un miglioramento. Il testo è di stile barocco, quindi denso di ripetizioni e cenni dotti tratti da diverse fonti a cominiciare dalla Sacra Scrittura, ritenuta primaria fonte del diritto.
Il concetto cardine dell’opera è la giustizia, espressione della volontà divina ma anche della natura razionale quindi valevole per tutti gli uomini.
Il testo contiene, inoltre, diversi concetti simbolici, tra cui il centrale è quello dello specchio: espressione dell’Intelligenza creatrice della divinità, quindi strumento di edificazione del governante che, a sua volta, è definito come “specchio del suo popolo”, assumendo valore simbolico.
Object of my study is a treatise on political morality of the seventeenth century titled Mirror of the rulers whose author is a franciscan friar, who lived in Naples, Antonio Masucci. The assumption has been to carry out, through a symbolic approach to hermeneutics, a parallelism between two periods of crisis, the “baroque” of the seventeenth century and that of contemporary society.
After a necessary premise in research methodology, the symbolic and the concepts of symbol, I decided to trace the path of the contribution of christianity to the formation of european conscience, individual and collective, and, as regards the political level, in the process of evolution of the modern concept of political sovereignty from the Middle Ages until the modern times.
Reconstructed this historical-theoretical way and, after having described socio-cultural context of Italian seventeenth century, with reference to the reflections on “reason of State” and the sovereignty of the Italian principalities, I got the tools to deal with the analysis of the text reference.
The work was intended to warn the rulers of their mistakes in order to instruct them to an improvement. The text is baroque style, so full of repetitions and references taken from different sources, to start by Sacred Scripture, which is considered the primary source of law.
The key concept is the justice, expression of the divine will and of a rational nature then valid for all men.
The text also contains several symbolic concepts, including the central one is the mirror: Intelligence creative expression of divinity, and tool of improvement of the ruler who is defined as a “mirror of its people”, assuming symbolic value
Functional and structural MRI image analysis for brain glial tumors treatment
No full textThis Ph.D Thesis is the outcome of a close collaboration between the Center for Research in Image Analysis and Medical Informatics (CRAIIM) of the Insubria University and the Operative Unit of Neurosurgery, Neuroradiology and Health Physics of the University Hospital ”Circolo Fondazione Macchi”, Varese.
The project aim is to investigate new methodologies by means of whose, develop an integrated framework able to enhance the use of Magnetic Resonance Images, in order to support clinical experts in the treatment of patients with brain Glial tumor.
Both the most common uses of MRI technology for non-invasive brain inspection were analyzed. From the Functional point of view, the goal has been to provide tools for an objective reliable and non-presumptive assessment of the brain’s areas locations, to preserve them as much as possible at surgery.
From the Structural point of view, methodologies for fully automatic brain segmentation and recognition of the tumoral areas, for evaluating the tumor volume, the spatial distribution and to be able to infer correlation with other clinical data or trace growth trend, have been studied. Each of the proposed methods has been thoroughly assessed both qualitatively and quantitatively.
All the Medical Imaging and Pattern Recognition algorithmic solutions studied for this Ph.D. Thesis have been integrated in GliCInE: Glioma Computerized Inspection Environment, which is a MATLAB prototype of an integrated analysis environment that offers, in addition to all the functionality specifically described in this Thesis, a set of tools needed to manage Functional and Structural Magnetic Resonance Volumes and ancillary data related to the acquisition and the patient
Data quality evaluation through data quality rules and data provenance.
No full textThe application and exploitation of large amounts of data play an ever-increasing role in today’s research, government, and economy. Data understanding and decision making heavily rely on high quality data; therefore, in many different contexts, it is important to assess the quality of a dataset in order to determine if it is suitable to be used for a specific purpose. Moreover, as the access to and the exchange of datasets have become easier and more frequent, and as scientists increasingly use the World Wide Web to share scientific data, there is a growing need to know the provenance of a dataset (i.e., information about the processes and data sources that lead to its creation) in order to evaluate its trustworthiness. In this work, data quality rules and data provenance are used to evaluate the quality of datasets.
Concerning the first topic, the applied solution consists in the identification of types of data constraints that can be useful as data quality rules and in the development of a software tool to evaluate a dataset on the basis of a set of rules expressed in the XML markup language. We selected some of the data constraints and dependencies already considered in the data quality field, but we also used order dependencies and existence constraints as quality rules. In addition, we developed some algorithms to discover the types of dependencies used in the tool. To deal with the provenance of data, the Open Provenance Model (OPM) was adopted, an experimental query language for querying OPM graphs stored in a relational database was implemented, and an approach to design OPM graphs was proposed
Effetti del trattamento cronico con nandrolone decanoato in ratti maschi adolescenti ed adulti sul comportamento emotivo ed interazione con il sistema endocannabinoide.
No full textIn this work we investigated the effects of chronic treatment with nandrolone decanoate (ND), a widely abused anabolic androgenic steroid, on the emotional profile of both adult and adolescent male rats. ND induced a complex depressive-like phenotype when it was administered during adolescence but not in adulthood, suggesting a more vulnerability of the adolescent brain to steroid abuse. In adolescent male rats this phenotype is characterized by alterations in emotional and cognitive behaviour, as well as in biochemical parameters such as pCREB levels and monoaminergic activity.
Since the endocannabinoid system (ECS) seems to be involved in the etiopathogenesis of depression and we observed alterations in the endocannabinoid receptor CB1 in rats exposed to ND during adolescence, we investigated the ability of the indirect CB1 receptor agonist URB597 to recover the depressive-like phenotype induced by ND. We observed that a 2-week URB597 treatment fully recovered the behavioural alterations but only partially rescued the biochemical changes and the monoaminergic activity alterations.
Finally, since cannabis is a drug widely abused among adolescents we evaluated a possible coabuse of THC, the main psychoactive component of cannabis, and ND in adolescent male rats. The co-administration of THC/ND prevented the occurrence of alterations in emotional behaviour. Overall, these data suggest that adolescent exposure to ND triggers the development of a complex depressive-like phenotype and that manipulation of the ECS could modulate the adverse effects induced by steroids on emotional behaviour
Analysis of the interaction between the IGF system and HIF: rationale for cotargeting the two systems in breast cancer.
No full textThe observation that several RTKs can act as promoters of tumor development and growth has brought a revolution in the study of new targets, in the hope of developing more specific anticancer drugs: the Insulin-like Growth Factors system is among the most studied.
The role of this system in the development and progression of a large number of malignancies is well documented. Hence the development of different classes of compounds that can affect this system, in particular directly targeting the type 1 receptor (IGF-1R). Some of these compounds, both monoclonal antibodies (mAbs) against the receptor or small molecules that inhibit the TK activity of IGF-1R, are already undergoing clinical trials, because of their effectiveness in inhibiting the signal.
Unfortunately, cancer cells show a high degree of plasticity and redundancy in the proliferative and anti-apoptotic signals, which may contribute to the creation of resistance to specific inhibitors: the identification of these mechanisms, and their overcome, are two key goals in the development of these drugs.
Within this study, we encountered a similar problem: our experiments show that the compound in use, NVP-AEW541 (a specific, low molecular weight inhibitor of IGF-1R catalytic activity), is perfectly capable of inhibiting the signal transduced by the receptor following stimulation with IGF-1, but it appears to lose effectiveness following stimulation with IGF-2. Starting from the observation that IGF- 2 seems to be the key factor in this resistance, literature search showed us a growing number of experimental evidences in support of the role of variant A of the insulin receptor (IR-A) in the maintenance of a proliferative signal similar to that classically attributed to IGF-1R: our experiments confirmed the activation of this receptor following exposure to IGF-2, justifying the loss of effectiveness of NVP-AEW541 in regard of the migratory potential of MDA-231 cell line. While the dual inhibition of IGF-1R and IR could overcome this redundancy in the signal, it remains to consider the problems that may occur due to the inhibition of a receptor as essential for cellular metabolism as IR.
A different and probably more practicable strategy would be the inhibition of the key player in the dual activation, IGF-2. The one proposed is a strategy that reflects the physiological mechanism of controlling the levels of IGF-2: in fact normal cells are able to control the levels of circulating IGF-2 by binding the ligand to IGF-2R, followed by its subsequent transport to lysosomes for degradation.
During this study we then concentrated also on IGF-2R, observing no significative differences between the cell lines in study.
Following the ligand-based targeting of the IGF system, which effectiveness seems to be proven by an ongoing phase I clinical trial using MEDI-573, actually the only mAb in clinical testing that exerts its effects by neutralizing not only IGF-2 but also IGF-1, we used a neutralizing antibody against IGF- 2 (MAB292), making it impossible for it to bind both IGF-1R and IR-A. The use of MAB292 proved to be effective both in combination with NVP-AEW541 and alone: MDA-231 cells seem in fact unable to respond to stimulation with IGF-2 and almost completely lose the ability to migrate.
This strategy could be particularly effective especially considering that, in hypoxic conditions (pO2 <2.5%), stabilization of HIF-1α leads to an increase in the levels of IGF-2. This increase in fact results in the resistance of MDA-231 to NVP-AEW541, while MAB292 still retain its effectiveness. We also observed the lack of a direct and defined relationship between HIF-1α and IGF-2R levels: cells seemed not able to compensate to IGF-2 increase by increasing also the receptor levels.
From this observation it’s therefore evident that another strategy to overcome the resistance to IGF-1R inhibitors may be the combination with HIF-1α inhibitors. At the present days, the fundamental problem is the lack of specific inhibitors: many chemotherapic drugs, in fact, have a complete or partial inhibitory activity of HIF-1α, that is however accompanied by a number of off-target effects.
One of these compounds is Topotecan, a Top-1 poison in use in the treatment of ovarian and small cell lung cancer that inhibits HIF-1α translation by a Top1-dependent but DNA damage-independent mechanism, suggesting that its effect as HIF-1α inhibitor could be mechanistically distinguished from those characterizing its cytotoxic activity. In our case the dual inhibition was obtained with success through the use of Topotecan in combination with NVP-AEW541.
We also introduced the combination between NVP-AEW541 and Luteolin, a compound of natural origin belonging to the flavonoid class that possess anti-proliferative and anti-migratory activity.
Despite the more complex comprehension of its effect on HIF-1, which resulted only slightly decreased, the most interesting feature of this compound resulted to be its anti-migratory potential: the use of Luteolin in fact completely inhibited migration in MDA-231.
As a last point in our study, we focused our attention on the metabolic alteration determined by the modulation of insulin/IGF system. Since deregulation of cellular energy metabolism is considered an increasingly important hallmark of cancer, IR and its related metabolic syndromes have become another major focus in the breast cancer research and treatment field. Preliminary analysis of the oxidative and glycolitic profiles of three cell lines showed in first stance an interesting difference at the basal level as well as a modulation of the two processes after treatment with NVP-AEW541 together with MAB292 or with Luteolin. Further experiments would be needed to assess the effective impact of these compounds on cellular metabolism, as well as the metabolic processes and responses in hypoxia.
In conclusion we can say that the dual targeting of IGF-1R and HIF-1 appears to be an interesting therapeutic strategy in breast cancer, especially given the close relationship between these two systems
