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Optimisation of the bolometric performances of the CUORE-0/CUORE and LUCIFER detectors for the neutrinoless double beta decay search.
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CDKL5 e sindrome di Rett: un approccio molecolare per la definizione di una correlazione genotipo/fenotipo.
No full textLa sindrome di Rett è una grave malattia del neurosviluppo legata al cromosoma X che colpisce principalmente individui di sesso femminile. Le bambine affette da sindrome di Rett subiscono uno sviluppo postnatale apparentemente normale fino all'età di 6-18 mesi, per poi manifestare un marcato declino neurologico con decorso molto variabile. Circa il 25% delle pazienti sono affette da forme atipiche che si discostano dalla forma classica o per il fatto di essere più blande con un iter meno devastante o per un fenotipo più “aggressivo” con insorgenza precoce che può portare anche a crisi epilettiche non trattabili farmacologicamente.
La maggior parte dei casi di forma classica di Rett è causata da mutazioni nel gene MECP2, un repressore trascrizionale espresso maggiormente nel cervello che lega il DNA metilato e che causa la formazione di una struttura cromatinica compatta e trascrizionalmente inerte. Circa solo il 50% delle pazienti con varianti della sindrome di Rett però sono portatrici di mutazioni in questo gene. Per questa classe di fenotipi sono state descritte nel 2004 mutazioni a carico del gene CDKL5 codificante per una serin-treonina chinasi e nel 2008 altre a carico del gene FOXG1, un fattore trascrizionale. La ricerca scientifica ha quindi ampliato i propri obbiettivi al fine di comprendere se questi tre geni, apparentemente diversi fra loro, potessero essere accomunati da uno stesso pathway molecolare. A tutt'oggi però i risultati sono ancora scarsi e, a volte, contraddittori. Per quanto riguarda gli studi su CDKL5, un aspetto generalmente accettato risiede nel fatto che in tutti i casi l'attività catalitica di CDKL5 appare di estrema importanza. Con questa premessa, sarebbe quindi interessante osservare con quale modalità le mutazioni a carico di CDKL5 possano influire sulla sua funzione enzimatica.
L'obbiettivo della mia ricerca è stato quello di analizzare otto mutazioni puntiformi (A40V, N71D, V132G, R178W, W195R, E203D, L220P e T288I) giacenti sul dominio catalitico di CDKL5. Queste mutazioni sono state riscontrate in pazienti con la variante Hanefeld. Per ogni mutazione è stata valutata la localizzazione cellulare e l'attività chinasica sul motivo TEY. Inoltre, grazie all'ausilio di strumenti bioinformatici, è stato creato un modello tridimensionale del dominio chinasico di CDKL5 ed è stato possibile predire l'influenza che ogni mutazione ha sulla struttura della proteina.
L'analisi di localizzazione subcellulare ci ha permesso di concludere che in generale le mutazioni a carico del dominio catalitico non influenzano in maniera significativa la distribuzione di CDKL5 all'interno della cellula, a differenza delle mutazioni troncanti sulla coda carbossiterminale, precedentemente analizzate nel nostro laboratorio. Ciononostante per le mutazioni W195R e E203D si è osservato un accumulo della proteina nel compartimento citoplasmatico. E' stata inoltre osservata la perdita della tipica organizzazione in dots di CDKL5. Le analisi bioinformatiche collocano questi due residui nell' α-elica F del sottodominio IX del dominio chinasico della proteina. A quest'elica, comune alle MAP chinasi, era già stata precedentemente attribuita una cruciale importanza nell'orchestrazione dell'attività chinasica. E' quindi possibile che una sua precisa conformazione spaziale sia necessaria per una corretta interazione con le altre proteine e per una altrettanto corretta distribuzione della proteina all'interno della cellula.
L'analisi dell'attività catalitica invece ha permesso di evidenziare che le mutazioni in esame possono portare a perdita totale di fosforilazione (A40V), a una sua diminuzione (V132G, R178W, W195R, E203D, L220P e T288I) oppure a nessun effetto evidente (N71D). Per questa mutazione perciò è stato proposto un saggio di stabilità che ha evidenziato un'emivita minore rispetto alla forma canonica, dando un'ulteriore prova di come i livelli di CDKL5 probabilmente debbano essere finemente regolati per una sua corretta funzionalità neuronale.
Nell'ultimo periodo del mio dottorato ho avuto inoltre la possibilità di valutare un ipotetico ruolo di CDKL5 nella maturazione neuronale, in quanto è stata osservata la sua presenza a livello dei centrosomi in cellule proliferanti fissate in metafase.
E' ormai assodato il ruolo del centrosoma nelle tre fasi della maturazione neuronale e perciò ho approfondito questo aspetto verificando la colocalizzazione di CDKL5 nel centrosoma anche in colture di neuroni corticali in via di maturazione. Questa scoperta non chiarisce con certezza quale sia lo specifico ruolo di CDKL5 in questo fenomeno ma offre sicuramente una nuova chiave di lettura per stabilire quale meccanismo neuronale venga a mancare nelle pazienti portatrici di difetti in questo gene
The NK cells phenotype and function in resected non small cell lung cancer: differences between squamous and adenocarcinoma.
No full textThe tumor microenvironment has come to light as a key player in carcinogenesis and progression. Tumors affect many host cell types, in particular immune cells. The immune system appears to select particularly fit tumor cells in the process of immuno-editing, while the tumor cells influence the polarization of immune cells towards phenotypes that favor tumor growth and vascularization. Here we investigated the phenotype of tumor infiltrating natural killer (NK) cells, focusing on angiogenesis associated cytokines and activities in patient-derived material from non-small cell lung cancer (NSCLC). Samples from the tumor and adjacent normal issues, as well as peripheral blood and lung samples as well as from non-oncologic patients with bullous emphysema were collected and rapidly processed to obtain single cell suspensions. Flow cytometry (FC) analyses were performed to evaluate specific markers (CD3, CD56, CD16) to identify NK cell subsets. We observed that in the NSCLC samples, the CD56+CD16- NK phenotype, associated with cytokine production, predominated in the tumor samples while the CD56dimCD16+ cytotoxic phenotype dominated in the adjacent normal tissues and in lung tissue derived from non-oncologic patients. This was independent of tumor histotype and smoking status. We examined the angiogenic potential of tumor infiltrating NK cells by intracellular staining for production of VEGF, PlGF, IL-8 (CXCL8), IFNand other markers. The CD56+CD16- subset was clearly associated with production of angiogenic cytokines in all samples. However, patients with squamous carcinoma histotypes showed remarkably and significantly higher production of angiogenic factors in tumor infiltrating, adjacent tissue and especially in peripheral blood CD56+CD16- NK cells than patients with adenocarcinomas. Following surgical intervention, these levels were reduced in disease-free patients. Moreover, supernatants derived from the tumor infiltrating CD56+CD16- NK cells were able to induce endothelial cell chemotaxis and formation of capillary-like structures in vitro; this was particularly evident for NK cells isolated from squamous cell carcinomas. Our data suggest that squamous NSCLC tumors have a significant systemic effect on NK cells, enhancing angiogenic cytokine production in a manner dependent on the presence of disease. NK cells appear to participate in tumor neovascularization and could represent a peripheral marker for disease progression, angiogenesis and response to therapies in some tumor subsets
Host immune suppression by toxoneuron nigriceps polidnavirus: use of Drtosophila melanogaster as an experimental model system.
No full textHost regulation strategies adopted by parasitic Hymenoptera are increasingly attracting the scientific interest, since several data support the idea that these strategies include targeting of evolutionarily conserved pathways.
This is in particular emerging from molecular and functional analyses of several parasitoid-associated polydnaviruses (PDVs), whose gene products, expressed in the tissues of parasitized hosts, interact with key signaling molecules (Pennacchio and Strand. 2006; Espagne et al. 2004; Falabella et al. 2003; Falabella et al. 2007; Provost et al. 2004; Thoetkiattikul et al. 2005).
T. nigriceps bracovirus (TnBV) is the PDV associated with Toxoneuron nigriceps (Hymenoptera, Braconidae), the endophagous larval parasitoid of the tobacco budworm Heliotis virescens (Lepidoptera, Noctuide).
We recently identified a host gene, named 102, which is targeted by a small TnBV transcript. Several data indicate that this gene is involved in the immune response, but its mode of action is still unclear.
Since the 102 gene is highly conserved throughout evolution, to investigate the function of the protein encoded by the 102 gene, we decided to use Drosophila melanogaster as a model system, since it offers a wide range of molecular genetic tools not available in other systems: its genome is fully sequenced; detailed knowledge about the molecular pathways regulating cellular immune responses is available; several sophisticated genetic and molecular genetic techniques have been developed and can be used as powerful tools to dissect the functional mechanisms underlying a wide range of biological processes.
In Drosophila melanogaster two putative protein sequences, showing 38% and 34% identity, respectively, with the translation product of the 102 gene, were found by Blast analyses. One of these is highly expressed in larval hemocytes, like its Heliotis virescence counterpart. Moreover, as reported in the Flyatlas database (http://www.flyatlas.org/), the gene is strongly expressed also in the fat body.
I took advantage of the well-established RNAi strategies based on the GAL4/UAS binary system to interfere with the expression of this gene in immuno-competent tissues (i.e. hemocytes and fat body) of Drosophila melanogaster. First of all, I evaluated the potential impact of this tissue-specific RNA interference on viability and found a dramatic lethal effect approaching 100%. Lethal phase measurements indicated that mortality was prominent in larval and pupal stages. Morphological analyses of the larvae in which the 102 gene had been targeted by RNA interference showed the presence of huge melanotic masses freely floating in the hemocoel. This tumorous-like phenotype is usually associated with altered and/or excessive performance of the immune system. Accordingly, hemocyte counts indicated that hemocyte overproliferation occurred in larvae, and increased numbers of both plasmatocytes and lamellocytes were found in their hemolymph. Furthermore, in these larvae, the sessile hemocyte population, was also affected.
Finally, morphological analyses of melanotic masses clearly identified their association with hemocytes, forming a cellular capsule around them
Fine-roots dynamics, anatomy and carbon-nitrogen concentrations in relation to forest management and soil water content. Case studies in beech(Fagus sylvatica L.) and Turkey-oak (Quercus cerris L.) forests.
No full textUncertainties in estimates of fine root dynamics prevent a proper quantification of net primary productivity and belowground C allocation. Moreover, model studies for estimating carbon budgets are biased by the lack of fine roots datasets at forest stand level. This study shed some lights on fine root dynamics in two different Italian forests. In particular fine-root systems was investigated: 1) in three beech forest stands (Fagus sylvatica L.) located in Southern-Alps in relation to different forest management practices and age 2) in a mature Turkey-oak stand (Quercus cerris L.) located in the Southern Apennines in relation to soil moisture seasonal changes.
Data from beech forests showed that conversion from coppice to high forest practice induced considerable variations in fine-root traits. Reduction of stand tree density induced a reduction of total fine-root mass and an increase of both production and turnover rate. Both fine-root production and turnover rate increased in converted stands. When fine-roots Carbon and Nitrogen contents were analyzed, their ratio was significantly lower in converted stands, supporting the finding of a higher turnover rate. A histological study was carried to assess if also anatomical changes occurred due to conversion practices. Anatomy on fine roots showed a higher percentage of xylem cells in conversion stands explaining the lowest carbon concentration.
Turkey-oak fine-root biomass and length showed a bimodal pattern with a peak in summer and a peak in autumn. SRL had only one peak in summer. All fine root traits increased during the transition from the wet to dry season. These results indicate a pulse in root growth in order to increase the soil exploitation when soil water content is low. Moreover, during the summer period, Q. cerris change fine-root morphology leading an increase of fine root length per unit mass
On reputation evaluation in 2.0 communities.
No full textCurrent technology has disappointed many members of WEB2.0 communities by its slow and tough adaptability to specific usersʼ skills and competences. Most of the largest WEB2.0 communities rely on the work of their members to create the resources they are built on, a business model known under the acronym UGC (User Generated Contents), which is today very popular yet still very simple and immature. Presently the vast majority of the communities that relay on UGCs lack an accurate ranking system for their most proficient members and almost none of them provide an effective rewarding mechanism.
Sometimes they do provide means to cooperatively evaluate and classify the available resources, but very few of these mechanisms take in account the competence of the voters.
Although rough, the current technology seems sufficient to confer to some cooperative web communities a discrete success, which for the most part is due to their core-members passion and commitment. Unfortunately deficiencies like the ones described can involve that, over time, only a small amount of the members keep committed and willing to produce resources of a certain quality. This tendency leads to the situation in which, although the community grows in terms of numbers, in proportion its value decreases.
Considering the fast diffusion of web2.0 communities and the new emerging Enterprise2.0 communities phenomena, this problem needs no longer to be underestimated. 2.0 communities should start thinking about new means to raise their quality standards in order to stay effective and successful over time.
Providing communities with concrete rewarding systems like remunerative ones could fix the problem, nevertheless this approach rarely and loosely has been implemented so far.
The reason is that the task involves several and not trivial side problems, such as the necessity of an advanced ranking mechanisms to evaluate the members.
This method should be able to consider complex factors like competence and commitment, but at the same time it should be easily understandable and shared by the members of the community. In order to make sure that the members share and agree with the rating system it must involve their collaboration, which is a non trivial problem, especially in Entrprise2.0 communities.
For the aforementioned reasons I propose as a means to overcome the problem a method based on reputation rather than competence and commitment, which is named Reputation Community Evaluation (RCE). This new approach is not based solely on quantitative measurements but relies on an algorithm which exploits the community member reputations to evaluate the resources and the members themselves. In this method the reputation of the single member grows or decreases according to its activity in the community and the evaluations received from the other members over a certain period of time. All members opinions/votes are themselves weighted accordingly to the single member reputation. This approach makes possible to put in place new kinds of business models, which aim to use users reputations as a digital currency inside Web 2.0 or Enterprise 2.0 communities
Molecular analysis of persistent enterovirus infections.
No full textHuman enteroviruses (HEVs) are etiological agents of different clinical entities varying from asymptomatic to mild, severe, or fatal diseases. There is now growing evidence that HEVs can cause or contribute to common chronic diseases. Even though a specific therapies are not yet available, a correct diagnosis is necessary both for diagnostic and epidemiologic purposes. Gene amplification methods and sequencing greatly facilitates the laboratory diagnosis of these conditions and allows discovering previously unsuspected etiopathogenetic associations.
In this work, three clinical conditions possibly associated with persistent HEV infections have been investigated: type 1 diabetes in children, the post-polio syndrome in adult patients, as well as clinical cases of inflammatory cardiomyopathy. New methods for HEVs detection have been developed based on bioinformatic studies of HEV sequence databases. These methods have been tested with reference virus strains as well as a wide range of clinical specimens. Our most sensitive assay is based on the detection and sequencing of the viral polymerase (3Dpol) coding region.
Different cohorts of patients have been investigated using classical virology and molecular methods. The studies allowed to conclude that HEVs can persist for long periods in humans and that these agents can be associated with endocrine, neurological, and cardiovascular conditions
Comprendere il dissenso: aporie del realismo e pragmatica universale tra H. Putnam e J. Habermas.
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Experimental techniques for deflection and radiation studies with bent crystals
No full textWhat happens when a high energy charged particle crosses an amorphous material? It loses energy by ionization and its trajectory is affected by the multiple Coulomb scattering, being these phenomena originated by uncorrelated collisions with the atoms. If the atoms of the target were distributed according to an ordered scheme, the uncorrelated collisions would turn into a coherent interaction with the whole atomic structure. This is the case of an aligned crystal that, depending on the orientation, is seen as a set of atomic planes or strings by the impinging particles. Planes and strings produce potential wells able to confine the charged particles in a transversal region of the crystal, in the so called channeling condition, so that, bending the crystal, particles are forced to follow the curvature, being deflected.
This simple and powerful idea, dating 1979, is at the basis of many theoretical and experimental studies that have proven its effectiveness, described the possible applications and optimized the deflection performances.
The contribution of this thesis locates itself in this field as an attempt to provide a picture of the experimental techniques and the analysis procedures developed to investigate bent crystals in the last years.
In this period, bent crystal physics has witnessed a tremendous progress characterized by the increase of performances and the discovery of new phenomena. The driving forces of this process have been essentially three: bent crystals have been identified as a possible solution of the LHC collimation problem, bringing considerable resources to their research field; new bending techniques exploiting secondary deformations have been implemented and, finally, the single particle track reconstruction has become the core of the crystal testing procedures, making the measurements faster and more precise.
This thesis is a bridge across two complementary fields, that is the experimental techniques applied in the crystal study and the description of the observed phenomena from the phenomenological point of view as well as from the microscopic theoretical one, resulting in a complete overview on the bent crystal physics.
The bent crystals features are presented in the very beginning of the thesis, with the key elements of the research field described in the first chapter before going to an overview on the most important applications to show how bent crystals can be exploited in accelerator physics.
The last section of the chapter focuses on the microscopic bent crystal behaviour to describe the physics behind the effects induced by the crystal on the charged particles crossing it. The second chapter describes the state of the art of the bent crystals characterization and test. The first section presents the experimental setup, from the inspiring concepts to the description of the single components and procedures. The setup basic idea is that the combination of a silicon microstrip tracking system with a multi-stage goniometer allows to measure the relative alignment between the beam and the crystal as well as the crystal deflection angle.
The second section is dedicated to the analysis methods used to characterize the single crystal behaviour in terms of the channeling and the volume reflection phenomena. The described procedures are applied in a wide range of beam conditions from 13 GeV to 400 GeV of energy with positive and negative particles; all the data have been acquired at CERN on different beamlines during the past three years.
The third chapter explores a different bent crystal aspect, that is the radiation emission.
Coherent interaction with atoms, in fact, does not only mean deflection capabilities but also quasi-periodic trajectories that, in turn, produce an enhancement of the emitted electromagnetic radiation.
The description of the theoretical bases of this phenomenon both in channeling and volume reflection is presented in the first section of the chapter, while the second one is dedicated to the setup and the results obtained with a 120 GeV/c positron beam.
The last chapter goes back to the bent crystals deflection issue; going beyond channelling and volume reflection, it shows innovative bent crystals deflection schemes, developed in the last years in order to enhance the efficiency and the angular acceptance provided by channeling.
This result can be achieved creating a sequence of aligned crystals to increase the deflection angle induced by volume reflection as shown in the first section or exploiting the interaction with a crystalline axis that, as described in the second section, originates new phenomena such as the multi volume reflection in one crystal (MVROC) and the axial channeling able to deflect both positive and negative particles with high efficiency