Trasduzione del segnale e poliamine nell'apoptosi di cellule cardiache

Introduction: Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, thus representing a potential therapeutic target. Apoptosis of cardiac cells can be induced experimentally by several stimuli including hypoxia, serum withdrawal or combination of both. Several lines of research suggest that neurohormonal mechanisms play a central role in the progression of heart failure. In particular, excessive activation of the sympathetic nervous system or the renin-angiotensin-aldosterone system is known to have deleterious effects on the heart. Recent studies report that norepinephrine (NE), the primary transmitter of sympathetic nervous system, and aldosterone (ALD), which is actively produced in failing human heart, are able to induce apoptosis of rat cardiomyocytes. Polyamines are biogenic amines involved in many cellular processes, including apoptosis. Actually it appears that these molecules can act as promoting, modulating or protective agents in apoptosis depending on apoptotic stimulus and cellular model. We have studied the involvement of polyamines in the apoptosis of cardiac cells induced in a model of simulated ischemia and following treatment with NE or ALD. Methods: H9c2 cardiomyoblasts were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation. Cardiomyocyte cultures were prepared from 1-3 day-old neonatal Wistar rat hearts. Polyamine depletion was obtained by culturing the cells in the presence of α-difluoromethylornithine (DFMO). Polyamines were separated and quantified in acidic cellular extracts by HPLC after derivatization with dansyl chloride. Caspase activity was measured by the cleavage of the fluorogenic peptide substrate. Ornithine decarboxylase (ODC) activity was measured by estimation of the release of 14C-CO2 from 14C-ornithine. DNA fragmentation was visualized by the method of terminal transferase-mediated dUTP nick end-labeling (TUNEL), and DNA laddering on agarose gel electophoresis. Cytochrome c was detected by immunoflorescent staining. Activation of signal transduction pathways was investigated by western blotting. Results: The results indicate that simulated ischemia, NE and ALD cause an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of caspase activity, a family of proteases that execute the death program and induce cell death. This effect was prevented in the presence of DFMO, an irreversible inhibitor of ODC, thus suggesting that polyamines are involved in the execution of the death program activated by these stimuli. In H9c2 cells DFMO inhibits several molecular events related to apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, down-regulation of Bcl-xL, and DNA fragmentation. The anti-apoptotic protein survivin is down-regulated after ALD or NE treatement and polyamine depletion obtained by DFMO partially opposes survivin decrease. Moreover, a study of key signal transduction pathways governing cell death and survival, revealed an involvement of AMP activated protein kinase (AMPK) and AKT kinase, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact polyamine depleted cells show an altered pattern of AMPK and AKT activation that may contrast apoptosis and appears to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. Conclusions: These results indicate that polyamines are involved in the execution of the death program activated in cardiac cells by heart failure-related stimuli, like ischemia, ALD and NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.

Development of bionanotechnological strategies for signal enhancement in nucleic acids biosensors

Nucleic acid biosensors represent a powerful tool for clinical and environmental pathogens detection. For applications such as point-of-care biosensing, it is fundamental to develop sensors that should be automatic, inexpensive, portable and require a professional skill of the user that should be as low as possible. With the goal of determining the presence of pathogens when present in very small amount, such as for the screening of pathogens in drinking water, an amplification step must be implemented. Often this type of determinations should be performed with simple, automatic and inexpensive hardware: the use of a chemical (or nanotechnological) isothermal solution would be desirable. My Ph.D. project focused on the study and on the testing of four isothermal reactions which can be used to amplify the nucleic acid analyte before the binding event on the surface sensor or to amplify the signal after that the hybridization event with the probe. Recombinase polymerase amplification (RPA) and ligation-mediated rolling circle amplification (L-RCA) were investigated as methods for DNA and RNA amplification. Hybridization chain reaction (HCR) and Terminal deoxynucleotidil transferase-mediated amplification were investigated as strategies to achieve the enhancement of the signal after the surface hybridization event between target and probe. In conclusion, it can be said that only a small subset of the biochemical strategies that are proved to work in solution towards the amplification of nucleic acids does truly work in the context of amplifying the signal of a detection system for pathogens. Amongst those tested during my Ph.D. activity, recombinase polymerase amplification seems the best candidate for a useful implementation in diagnostic or environmental applications.

Immunoconiugati contenenti tossine vegetali o siRNA per la deplezione selettiva di cellule leucemiche. Preparazione, citotossicità e studi di mutagenesi sito-specifica.

CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.

La ville comme labyrinthe: réécritures d'un mythe entre les années 1950 et 1980

Entre les années 1950 et 1980, émerge une nouvelle forme de labyrinthe chez des romanciers européens comme Michel Butor, Alain Robbe-Grillet, Italo Calvino, Patrick Modiano et Alasdair Gray : un labyrinthe insaisissable et non cartographiable. Pour en rendre compte nous avons recours au modèle du rhizome, issu de la philosophie de Gilles Deleuze et de Félix Guattari, aussi bien qu'au concept d'hétérotopie de Michel Foucault. La spatialité de nos romans nous pousse à prendre en compte également les réécritures ironiques du mythe de Thésée, Ariane, le Minotaure, Dédale. Les citations et les allusions au mythe nous font remarquer la distance d'avec le modèle traditionnel et les effets de ce qu'on peut considérer comme un « bricolage mythique », dans le cadre d'un regard ironique, parodique ou satirique. La représentation romanesque du labyrinthe accentue d'un côté l'absence d'un centre, et de l'autre côté l'ouverture extrême de cet espace qu'est la ville contemporaine. En même temps, la présence de nombreux « espaces autres », les hétérotopies de Foucault, définit l'égarement des protagonistes des romans. Au fur et à mesure que les écrivains acquièrent conscience des caractéristiques « labyrinthiques » de ces espaces, celles-ci commencent à informer l'œuvre romanesque, créant ainsi un espace métafictionnel. Entre les années Cinquante et le début des années Soixante-dix, les Nouveaux romanciers français accentuent ainsi l'idée de pouvoir jouer avec les instruments de la fiction, pour exaspérer l'absence d'un sens dans la ville comme dans la pratique de l'écriture. Calvino reformule cette conception du roman, remarquant l'importance d'un sens, même s'il est caché et difficile à saisir. Pour cette raison, à la fin de l'époque que nous analysons, des auteurs comme Modiano et Gray absorbent les techniques d'écriture de ces prédécesseurs, en les faisant jouer avec la responsabilité éthique de l'auteur.

La mediazione civile e commerciale come strumento di gestione del conflitto. Analisi a seguito dell'entrata in vigore del d.Lgs 4/3/2010 n. 28

Dopo aver analizzato il conflitto, le sue funzioni e le modalità di gestione, l'autore si sofferma dapprima sulle varie tipologie di mediazione per poi focalizzare l'attenzione sulla mediazione civile e commerciale evidenziando i dati disponibili dall'entrata in vigore del tentativo obbligatorio come condizione di procedibilità della domanda giudiziale per le materie civili, alla fine del 2013.

1967-1987: l'altra civiltà del testo. Impulsi storici e scrittura nel teatro d'innovazione francese

Lo studio analizza le svolte nel teatro di innovazione francese prodotte dalla rivolta sociale e dalla sua rivoluzione culturale del 1968. La ricerca si è concentrata su tre livelli di analisi: un'analisi – storica, politica, sociale, culturale – dell'anno-tournant; un'analisi delle reazioni nel mondo del teatro contemporanee alla rivolta; un'analisi delle prassi spettacolari di lunga durata originate dall'evento. Sono stati, quindi, esaminati criticamente i testi cardine degli intellettuali ispiratori, protagonisti e interpreti della breccia culturale, per poi rintracciare i valori emersi all'interno del mondo dello spettacolo. La contestazione sociale ha agito sul mondo del teatro producendo delle ripercussioni immediate – l'occupazione dell'Odéon, la contestazione al XXII Festival d'Avignon, la produzione della Déclaration de Villeurbanne – e delle prassi di lunga durata che hanno agito sui processi formativi e creativi delle compagnie francesi che, dalla fine degli anni Sessanta, hanno prodotto creazioni collettive (in particolare il Théâtre du Soleil e il Théâtre de l'Aquarium, ma anche la Nouvelle Compagnie d'Avignon, il Grand Magic Circus e il Théâtre Populaire di Lorraine) e sul corpus drammaturgico e teorico di Michel Vinaver. Sono state, quindi, analizzate le relazioni che si sono instaurate tra i protagonisti appena nominati e i differenziati contesti, sociali e teatrali, facendo emergere nuove istanze creative. Tali istanze hanno saputo rispondere alle spinte etiche ed estetiche del momento storico, le hanno declinate sul piano delle prassi teatrali e rilanciate verso modalità nuove che hanno favorito l'emersione di una nuova civiltà del testuale.