Dramatic extension of tumor latency and correction of neurobehavioral phenotype in Atm-mutant mice with a nitroxide antioxidant

Mutations in the ATM gene (mutated in ataxia telangiectasia) in both humans and mice predispose to lymphoid tumors. A defect in this gene also causes neurodegeneration in humans and a less severe neurological phenotype in mice. There is some evidence that oxidative stress contributes to these defects, suggesting that antioxidants could alleviate the phenotype. We demonstrate here that the antioxidant 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO) dramatically delays the onset of thymic lymphomas in Atm(-/-) mice which is not due to an enhancement of apoptosis by CTMIO. We also show that this compound corrects neurobehavioral deficits in these mice and reduces oxidative damage to Purkinje cells. The likely mechanism of action of CTMIO is due to a reduction in oxidative stress, which is protective against both the tumor progression and the development of neurological abnormalities. These data suggest that antioxidant therapy has considerable potential in the management of ataxia telangiectasia and possibly other neurodegenerative disorders where oxidative stress is implicated. (c) 2006 Elsevier Inc. All rights reserved.

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

Optimization of ATM Telecommunication Networks

ATM network optimization problems defined as combinatorial optimization problems are considered. Several approximate algorithms for solving such problems are developed. Results of their comparison by experiments on a set of problems with random input data are presented.

Resolução em tomografia de tempo de trânsito poço-a-poço: a dependência da iluminação

The key aspect limiting resolution in crosswell traveltime tomography is illumination, a well known result but not as well exemplified. Resolution in the 2D case is revisited using a simple geometric approach based on the angular aperture distribution and the Radon Transform properties. Analitically it is shown that if an interface has dips contained in the angular aperture limits in all points, it is correctly imaged in the tomogram. By inversion of synthetic data this result is confirmed and it is also evidenced that isolated artifacts might be present when the dip is near the illumination limit. In the inverse sense, however, if an interface is interpretable from a tomogram, even an aproximately horizontal interface, there is no guarantee that it corresponds to a true interface. Similarly, if a body is present in the interwell region it is diffusely imaged in the tomogram, but its interfaces - particularly vertical edges - can not be resolved and additional artifacts might be present. Again, in the inverse sense, there is no guarantee that an isolated anomaly corresponds to a true anomalous body because this anomaly can also be an artifact. Jointly, these results state the dilemma of ill-posed inverse problems: absence of guarantee of correspondence to the true distribution. The limitations due to illumination may not be solved by the use of mathematical constraints. It is shown that crosswell tomograms derived by the use of sparsity constraints, using both Discrete Cosine Transform and Daubechies bases, basically reproduces the same features seen in tomograms obtained with the classic smoothness constraint. Interpretation must be done always taking in consideration the a priori information and the particular limitations due to illumination. An example of interpreting a real data survey in this context is also presented.

Resolução em tomografia de tempo de trânsito poço-a-poço: a dependência da iluminação

The key aspect limiting resolution in crosswell traveltime tomography is illumination, a well known result but not as well exemplified. Resolution in the 2D case is revisited using a simple geometric approach based on the angular aperture distribution and the Radon Transform properties. Analitically it is shown that if an interface has dips contained in the angular aperture limits in all points, it is correctly imaged in the tomogram. By inversion of synthetic data this result is confirmed and it is also evidenced that isolated artifacts might be present when the dip is near the illumination limit. In the inverse sense, however, if an interface is interpretable from a tomogram, even an aproximately horizontal interface, there is no guarantee that it corresponds to a true interface. Similarly, if a body is present in the interwell region it is diffusely imaged in the tomogram, but its interfaces - particularly vertical edges - can not be resolved and additional artifacts might be present. Again, in the inverse sense, there is no guarantee that an isolated anomaly corresponds to a true anomalous body because this anomaly can also be an artifact. Jointly, these results state the dilemma of ill-posed inverse problems: absence of guarantee of correspondence to the true distribution. The limitations due to illumination may not be solved by the use of mathematical constraints. It is shown that crosswell tomograms derived by the use of sparsity constraints, using both Discrete Cosine Transform and Daubechies bases, basically reproduces the same features seen in tomograms obtained with the classic smoothness constraint. Interpretation must be done always taking in consideration the a priori information and the particular limitations due to illumination. An example of interpreting a real data survey in this context is also presented.

Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial.

PURPOSE: The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. PATIENTS AND METHODS: We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. RESULTS: We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). CONCLUSION: The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.

Functional analysis of the ATM-p53-p21 pathway in the LRF CLL4 trial: blockade at the level of p21 is associated with short response duration.

PURPOSE: This study sought to establish whether functional analysis of the ATM-p53-p21 pathway adds to the information provided by currently available prognostic factors in patients with chronic lymphocytic leukemia (CLL) requiring frontline chemotherapy. EXPERIMENTAL DESIGN: Cryopreserved blood mononuclear cells from 278 patients entering the LRF CLL4 trial comparing chlorambucil, fludarabine, and fludarabine plus cyclophosphamide were analyzed for ATM-p53-p21 pathway defects using an ex vivo functional assay that uses ionizing radiation to activate ATM and flow cytometry to measure upregulation of p53 and p21 proteins. Clinical endpoints were compared between groups of patients defined by their pathway status. RESULTS: ATM-p53-p21 pathway defects of four different types (A, B, C, and D) were identified in 194 of 278 (70%) samples. The type A defect (high constitutive p53 expression combined with impaired p21 upregulation) and the type C defect (impaired p21 upregulation despite an intact p53 response) were each associated with short progression-free survival. The type A defect was associated with chemoresistance, whereas the type C defect was associated with early relapse. As expected, the type A defect was strongly associated with TP53 deletion/mutation. In contrast, the type C defect was not associated with any of the other prognostic factors examined, including TP53/ATM deletion, TP53 mutation, and IGHV mutational status. Detection of the type C defect added to the prognostic information provided by TP53/ATM deletion, TP53 mutation, and IGHV status. CONCLUSION: Our findings implicate blockade of the ATM-p53-p21 pathway at the level of p21 as a hitherto unrecognized determinant of early disease recurrence following successful cytoreduction.

IGF-1R inhibition sensitizes breast cancer cells to ATM-Related Kinase (ATR) inhibitor and cisplatin

The complexity of the IGF-1 signalling axis is clearly a roadblock in targeting this receptor in cancer therapy. Here, we sought to identify mediators of resistance, and potential co-targets for IGF-1R inhibition. By using an siRNA functional screen with the IGF-1R tyrosine kinase inhibitor (TKI) BMS-754807 in MCF-7 cells we identified several genes encoding components of the DNA damage response (DDR) pathways as mediators of resistance to IGF-1R kinase inhibition. These included ATM and Ataxia Telangiectasia and RAD3-related kinase (ATR). We also observed a clear induction of DDR in cells that were exposed to IGF-1R TKIs (BMS-754807 and OSI-906) as indicated by accumulation of γ-H2AX, and phosphorylated Chk1. Combination of the IGF-1R/IR TKIs with an ATR kinase inhibitor VE-821 resulted in additive to synergistic cytotoxicity compared to either drug alone. In MCF-7 cells with stably acquired resistance to the IGF-1R TKI (MCF-7-R), DNA damage was also observed, and again, dual inhibition of the ATR kinase and IGF-1R/IR kinase resulted in synergistic cytotoxicity. Interestingly, dual inhibition of ATR and IGF-1R was more effective in MCF-7-R cells than parental cells. IGF-1R TKIs also potentiated the effects of cisplatin in a panel of breast cancer cell lines. Overall, our findings identify induction of DDR by IGF-1R kinase inhibition as a rationale for co-targeting the IGF-1R with ATR kinase inhibitors or cisplatin, particularly in cells with acquired resistance to TKIs.

Applicazione della Tomografia con raggi X per la lettura virtuale di antichi manoscritti veneziani

Questo lavoro di tesi si inserisce in un progetto di ricerca internazionale denominato “Venice Time Machine” dove collaborano fianco a fianco l’Ecole Polytechnique Fédérale de Lausanne e l’Università Cà Foscari di Venezia. Grazie al coinvolgimento dell’Archivio di Stato di Venezia, decine di chilometri di documenti verranno digitalizzati e indicizzati, al fine di creare un database open access da utilizzare per la ricerca e l’istruzione. Molti di questi documenti tuttavia sono particolarmente fragili oppure, come nel caso di diversi testamenti, non sono mai stati aperti, per cui le tecniche tradizionali di digitalizzazione non sono applicabili. Di qui deriva l’interesse per sperimentare nuove tecniche non invasive al fine di digitalizzare e quindi rendere fruibili al pubblico anche questi documenti altrimenti inaccessibili. Lo scopo dell’analisi tomografica è quello di creare un modello 3D del documento, su cui effettuare successive elaborazioni al fine di ottenere una separazione virtuale delle pagine e quindi permetterne la lettura anche se il manoscritto è chiuso e non può essere aperto. In particolare in questo lavoro di tesi sono stati analizzati due testamenti: un testamento del 1679, usato come campione di prova per verificare la migliore sorgente di raggi X ai fini della ricostruzione tomografica e anche per valutare l’efficacia della tecnica, e il testamento Alchier-Spiera (dai nomi dei testatori), datato 1634, di maggiore interesse poiché ancora chiuso. I risultati ottenuti sono molto soddisfacenti, poiché elaborando le ricostruzioni tomografiche è possibile la lettura virtuale sia di parole che di intere frasi. Questo risultato porta nuova linfa al progetto che, di base, si pone l’obiettivo di convertire in formato digitale decine di km di testi conservati in Archivio, ma che ha trovato, in questo tipo di testamenti chiusi, un ostacolo molto difficile da superare.

Tomografia a coerenza ottica: principi ed applicazioni

La tomografia a coerenza ottica (Optical Coherence Tomography, OCT) rappresenta fondamentalmente una nuova modalità di diagnostica per immagini non invasiva e ad alta risoluzione. L’OCT fornisce immagini, sotto forma di sezioni trasversali o tomografiche, delle microstrutture dei tessuti biologici tramite la misura del ritardo dell’eco e dell’intensità della luce retrodiffusa o riflessa dal campione in analisi. L’OCT è una potente tecnica poiché fornisce in tempo reale immagini in situ delle strutture tissutali normali o patologiche, con una risoluzione da 1 a 15 micron, che è da uno a due ordini di grandezza maggiore rispetto alle tecniche per immagini convenzionali come ultrasuoni, risonanza magnetica o tomografia computerizzata. Tutto questo senza la necessità di ottenere e analizzare un campione tramite biopsia e studio istopatologico. Sin dall’inizio della sua utilizzazione nel 1991, l’OCT è stata impiegata in un vasto spettro di applicazioni cliniche, principalmente l'oftalmologia. In strutture non trasparenti, diverse da quelle oculari, la profondità massima esplorabile è limitata a 2-3 mm a causa dei fenomeni di attenuazione secondari e alla dispersione della luce. Inoltre, i numerosi sviluppi fatti dalla tecnologia OCT hanno portato ad alte velocità di acquisizione e, grazie all’utilizzo di sorgenti laser di ultima generazione, risoluzioni assiali dell’ordine di 1 micrometro. Dunque, la tomografia a coerenza ottica può essere sfruttata: - Quando la biopsia escissionale è pericolosa o impossibile. - Quando si rischiano errori di campionamento, come guida alla biopsia in modo da ridurre errori nel prelievo del campione. - Come guida alla procedura d’intervento, in associazione alle procedure di cateterismo, endoscopia e laparoscopia.

Estudo teórico da tomografia por impedância elétrica

O presente trabalho descreve um estudo sobre a metodologia matemática para a solução do problema direto e inverso na Tomografia por Impedância Elétrica. Este estudo foi motivado pela necessidade de compreender o problema inverso e sua utilidade na formação de imagens por Tomografia por Impedância Elétrica. O entendimento deste estudo possibilitou constatar, através de equações e programas, a identificação das estruturas internas que constituem um corpo. Para isto, primeiramente, é preciso conhecer os potencias elétricos adquiridos nas fronteiras do corpo. Estes potenciais são adquiridos pela aplicação de uma corrente elétrica e resolvidos matematicamente pelo problema direto através da equação de Laplace. O Método dos Elementos Finitos em conjunção com as equações oriundas do eletromagnetismo é utilizado para resolver o problema direto. O software EIDORS, contudo, através dos conceitos de problema direto e inverso, reconstrói imagens de Tomografia por Impedância Elétrica que possibilitam visualizar e comparar diferentes métodos de resolução do problema inverso para reconstrução de estruturas internas. Os métodos de Tikhonov, Noser, Laplace, Hiperparamétrico e Variação Total foram utilizados para obter uma solução aproximada (regularizada) para o problema de identificação. Na Tomografia por Impedância Elétrica, com as condições de contorno preestabelecidas de corrente elétricas e regiões definidas, o método hiperparamétrico apresentou uma solução aproximada mais adequada para reconstrução da imagem.