Plus d'un siècle après la découverte des rayons X, les effets biologiques des radiations ionisantes restent encore méconnus. En particulier, une meilleure connaissance des phénomènes liés à la radiosensibilité individuelle permettrait une meilleure prédiction du risque radioinduit tant en ce qui concerne les réactions tissulaires que la formation de cancers.Dans le cadre des recherches menées par le Groupe de Radiobiologie de l'UMR 1052 Inserm (Centre de Recherche en Cancérologie de Lyon), l'accumulation de données radiobiologiques issues de patients radiosensibles a permis d'initier une théorie basée sur le transit cytonucléaire de la protéine ATM. Acteur majeur de la réponse aux radiations ionisantes ATM est muté dans l'Ataxie Telangiectasie, syndrome génétique rare associé à la plus forte radiosensibilité. Plus précisément, les chercheurs du Groupe ont proposé le modèle suivant : l'irradiation produit une monomérisation des formes cytoplasmiques de la protéine ATM. Les monomères d'ATM diffusent dans le noyau pour assurer la reconnaissance et la réparation des cassures double-brin de l'ADN (CDB), dommages-clés de la réponse aux radiations. Tout retard dans ce transit conduirait à une certaine radiosensibilité.Le but de cette thèse est d'identifier d'une part, les protéines (appelées X) qui freinerait ce transit en s'associant à ATM dans le cytoplasme ; d'autre part, les agents chimiques (métaux, pesticides) qui influeraient sur ce processus.Les protéines X identifiées dans le cadre de cette thèse sont notamment la huntingtine, la neurofibromine, la tubérine qui, lorsqu'elles sont mutées, causent respectivement la maladie de Huntington, la Neurofibromatose de type 1 et la Tubéreuse de Bourneville. Les métaux étudiés sont les chlorures d'aluminium, de cuivre, de zinc, de fer, de nickel, de palladium, de cadmium ainsi que le nitrate de plomb, le selenium et le chrome. Les pesticides sont l'atrazine, le glyphosate, la permetrine, le thiabendazole et le pentachlorophénol.Cette thèse introduit la notion de pro-, dys- ou anti-épiskévie, c'est-à-dire la capacité de certains agents, protéines ou drogues à accélérer, ralentir ou interdire le transit cytonucléaire de la protéine ATM / More than a century after the discovery of X rays, the effects of ionising radiation are still misunderstood. In particular, a better knowledge of individual radiosensitivity could lead to a better prediction of radio induced risk of cancer and acute reactions after radiotherapy. As part of the research conducted by the Radiobiology Group of UMR Inserm 1052 (Cancer Research Center of Lyon), the accumulation of radiobiological data from radiosensitive patients allowed to initiate a theory based on the ATM protein transit from cytoplasm to nucleus. ATM a the major actor in the response to ionising radiation and is mutated in Ataxia Telangiectasia, a rare genetic syndrome associated with the highest radiosensitivity. Specifically, the researchers of the Group proposed the following model: irradiation produces monomerization of cytoplasmic forms of ATM protein. ATM monomers diffuse into the nucleus to ensure the recognition and repair of DNA double-strand breaks (DSBs), the key damage response to radiation. Any delay in this transit would lead to radiosensitivity.The aim of this thesis is to identify in one hand, the proteins (called X proteins), which would slow the transit by interracting with ATM in the cytoplasm; on the other hand, chemical agents (metals, pesticides) that would affect this process.X proteins identified in this thesis include huntingtin, neurofibromin, tuberin which, when mutated, cause, respectively, Huntington's disease, Neurofibromatosis type 1 and Tuberous Sclerosis. Studied metals are aluminum, copper, zinc, iron, nickel, palladium and cadmium chlorides, lead nitrate, selenium and chromium. Pesticides are atrazine, glyphosate, permethrin, thiabendazole and pentachlorophenol.This thesis introduces the concept of pro-, dys or anti- episkévia, that is to say the ability of some agents, proteins or drugs to speed up, slow down or inhibit the the ATM nucleoshuttling

O complexo Mre11 (Mre11/Rad50/Nbs1) é uma componente chave da resposta celular ao dano ao DNA em humanos e recentes observações sugerem que estas proteínas são em parte responsáveis pela interface ente o ensoreamento do dano ao DNA, seu reparo e as funções das proteínas envolvidas nos pontos de checagem do ciclo celular. Em Aspergillus nidulans, a partir de um screening para o isolamento de letais sintéticos na ausência de dineína, o gene sldIRAD50 foi clonado como um desses letais sintéticos através da complementação do fenótipo de deficiência de conidiação do mutante. Foi identificada uma transversão G-C na posição 2509 (Ala-692-Pro) no mutante sldI1444 a qual está presente na região de dobradiça da proteína. Essa mutação causa sensibilidade a vários agentes mutagênicos. Uma linhagem mutante sldIRAD50::pyrG foi construída a qual apresentou também vários defeitos na reposta celular ao dano ao DNA incluindo sensibilidade a várias drogas mutagênicas, defeito no ponto de checagem de replicação do DNA e na viabilidade dos ascosporos. Além disso, o gene sldIRAD50 interage geneticamente com bimEAPC1 para o controle do spindle pole checkpoint durante a segregação cromossômica sugerindo um novo papel para o complexo Mre11. Em atuação paralela com o complexo Mre11, duas proteínas quinases ditas apicais, ATM e ATR coordenam a transdução do sinal do dano ao DNA para proteínas efetoras do reparo. A proteína ATM está mutada na síndrome de instabilidade cromossômica herdada Ataxia Telangiectasia. Para a caracterização do homologo de ATM em A. nidulans AtmA, uma linhagem mutante atmAATM foi isolada. Esse mutante apresentou falha na reposta ao dano ao DNA, como seus homólogos em vários outros organismos mostrando defeitos no ponto de checagem intrafase S e G2/M, além de sensibilidade a camptothecin e bleomicina. Ainda, o extrato protéico bruto desse mutante não foi capaz de fosforilar o homologo de NBS1 em A. nidulans, ScaA. Além das conhecidas funções de ATM na resposta ao dano ao DNA, foi verificado que o mutante atmAATM apresentou uma acelerada cinética de divisão nuclear e severos defeitos no estabelecimento e manutenção do eixo de crescimento polarizado, evidenciando uma função ainda não descrita para ATM no crescimento polar. Provavelmente, AtmA regula a função e/ou localização de proteínas chaves para a formação do eixo de polarização. Diante disso, para investigar as vias metabólicas que são controladas por esse gene, o perfil transcricional do mutante atmAATM, em comparação com a linhagem selvagem foi verificado em diferentes condições de crescimento. Os resultados indicaram um importante papel da via das pentoses fosfato na proliferação celular monitorada pela AtmA. Além disso, foram identificados vários genes com a expressão do mRNA diminuída envolvidos no crescimento polarizado, na síntese de ácido fosfatídico e de ergosterol e no tráfico intracelular, secreção e transporte vesicular. Buscando identificar genes que participam da resposta celular ao dano ao DNA causado pela droga anti topoisomerase I, camptothecin, foram utilizados filtros de macroarray de A. nidulans contendo 2787 genes deste organismo para monitorar a expressão gênica da linhagem selvagem e do mutante uvsBATR, num experimento de indução com CPT por 30, 60 e 120 minutos. Os resultados revelaram um total de 1512 e 1700 genes modulados na linhagem selvagem e uvsBATR respectivamente, em pelo menos um ponto experimental. Seis desses genes que apresentaram aumento da expressão de mRNA na linhagem selvagem e diminuição da linhagem uvsBATR foram caracterizados: fhdA (que codifica para uma proteína com domínio fork-head associated), tprA (uma proteína hipotética que apresenta o domínio tetratrico peptide repeat), mshA (um homólogo MutS6 envolvido em mismatch repair), phbA (um homólogo da prohibitina), uvsCRAD51 e cshA (homólogo da proteína CSB envolvida no reparo por excisão de nucleotídeos e ligada a Síndrome de Cockayne). A indução transcricional desses genes na presença de CPT requer a função de uvsBATR. Estes genes foram deletados e surpreendentemente apenas uvsCRAD51 apresentou sensibilidade a CPT, enquanto os outros mostraram sensibilidade a outros agentes que causam dano ao DNA e estresse oxidativo. Além disso, com exceção de uvsCRAD51, a deleção desses genes leva a supressão parcial da sensibilidade a menadiona e paraquat do mutante uvsBATR. Esses resultados indicaram um comportamento heterogêneo de sensibilidade durante o crescimento na presença de agentes que causam dano direto ou indireto ao DNA, evidenciando que o perfil transcricional não é determinante para predizer a função de um gene na proteção da célula a determinada droga que causa dano ao DNA. / The Mre11 protein complex (Mre11/Rad50/Nbs1) has emerged as a central component in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA damage detection to DNA repair and cell cycle checkpoint functions. In Aspergillus nidulans, the sldI1444D mutant was isolated in a screen for dynein synthetic lethals. The sldIRAD50 gene was cloned by complementation of the sporulation deficiency phenotype of this mutant. A transversion G-C at the position 2509 (Ala-692-Proamino acid change) in the sldI1444D mutant causes sensitivity to several DNAdamaging agents. The mutation sldI1 occurs at the CXXC hinge domain of Rad50. An inactivation strain sldIRAD50::pyrG was constructed. Besides sensitivity to a number of DNA-damaging agents, this deletion strain was also impaired in the DNA replication checkpoint response and in ascospore viability. Also, sldIRAD50::pyrG geneticaly interacted with bimEAPC1, acting in the spindle pole checkpoint control during segregation, suggesting a new possible role of Mre11 complex. In parallel to the Mre11 complex, two apical quinases ATM and ATR respond to DNA damage and transduce the signal to effector proteins. In humans, mutations in ATM cause the devastating neurodegenerative disease Ataxia Telangiectasia. Here we characterized the homolog of ATM (AtmA) in the filamentous fungus A. nidulans. The deletion strain atmA presented defects in the DNA damage response as previously shown in other model organisms including intra S-phase and G2/M checkpoint defects, sensitivity to camptothecin and bleomycin. Also, the crude extract from the mutant strain did not phosphorylate the NBS1 homologue ScaA. In addition to its expected role in the DNA damage response, the atmA mutant showed increased nuclear division kinetics and severe defects in polarized hyphal growth, indicating a novel feature for the ATM gene. Probably, AtmA regulates the function and/or localization of landmark proteins required for the formation of a polarity axis. We extended these studies by investigating which pathways are controlled by AtmA during proliferation and polar growth by comparatively determining the transcriptional profile of A. nidulans wild type and atmA mutant strains in different growth conditions. Our results indicated an important role of the pentose phosphate pathway in the fungal proliferation during endogenous DNA damage and polar growth monitored by the AtmA kinase. Furthermore, we identified several genes that have decreased mRNA expression in the atmA mutant that are involved in the formation of polarized hyphae and control of polar growth; in the biosynthesis of phosphatidic acid and ergosterol; and intracellular trafficking, secretion, and vesicular transport. In order to identify genes that responded to the DNA damage mediated by the anti- toposomerase I drug, camptothecin, we used an A. nidulans macroarray carrying sequences of 2,787 genes from this fungus to monitor gene expression of both wild-type and uvsBATR in a time-point experiment where mycelium was exposed to 60, 90 and 120 minutes to the drug. The results revealed a total of 1,512 and 1,700 genes in the wild-type and uvsBATR deletion mutant strain that displayed statistically significant difference in at least one experimental time-point. We characterized six genes that have increased mRNA expression in the presence of CPT in the wild-type strain relative to the uvsBATR mutant strain: fhdA (encoding a fork head associated domain protein), tprA (encoding a hypothetical protein that contains a tetratrico peptide repeat), mshA (encoding a MutS homologue involved in mismatch repair), phbA (encoding a prohibitin homologue), uvsCRAD51 (the homologue of the RAD51 gene), and cshA (encoding a homologue of the excision repair protein ERCC-6 [Cockaynes syndrome protein]). The induced transcript levels of these genes in the presence of CPT required uvsBATR. These genes were deleted, and surprisingly, only the uvsCRAD51 mutant strain was sensitive to CPT; however, the others displayed sensitivity to a range of DNA-damaging and oxidative stress agents. Moreover, with the exception of UvsC, deletion of each of these genes partially suppressed the sensitivity of the uvsB strain to menadione and paraquat. These results indicated a very complex and heterogeneous sensitivity behavior during growth in the presence of agents that directly or indirectly cause DNA damage and the transcriptional response to DNAdamaging agents does not necessarily identify the genes that protect against these agents.

Der Asynchronous Transfer Mode (ATM) stellt die zukünftige und einheitliche Basistechnologie für das Breitband-ISDN dar. Da nahezu alle wesentlichen Protokollfunktionen in Hardware realisierbar sind, soll nachfolgend ein Überblick über bereits angebotene VLSI-Schaltkreise gegeben werden. Eine Systematisierung und Einordnung vorhandener ATM-Chips hinsichtlich ihrer Leistungsfähigkeit und ihres Funktionsumfangs erfolgt in Hinblick auf das sogenannte B-ISDN-Referenzmodell. Dieses Schichtenmodell definiert die notwendigen Protokolle und Schnittstellen für den Asynchronous Transfer Mode. Zum grundlegenden Verständnis wird einleitend eine kurze Einführung in die Basisprinzipien von ATM gegeben.

The childhood cancer neuroblastoma mostly affects children under the age of 2 and comprises 6% of all childhood cancers. Neuroblastoma has very diverse phenotypes caused by both inter- and intra-tumour heterogeneities. The phenotypes are classified as being either low- or high-risk. This project focuses on high-risk NB cell lines with various chemotherapy sensitivity. Titration studies with chemotherapy agents cisplatin or doxorubicin showed a proneness of p53 mutated cell lines to arrest in either the S- and/or the G2/M-phase, depending on the drug and the drug dosage, indicating on a dose-dependent cell cycle response. To potentially inhibit the cells from arresting a treatment assay with 3 cell cycle key-components, pATM, Chk1 and Wee1 inhibitors was done. An initial immunocytochemistry staining of the expression levels of pATM and Wee1 showed that pATM was upregulated for 5 out 7 tested cell lines, namely SK-N-SH, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C, upon chemotherapy treatment with doxorubicin. Wee1 was however only upregulated for 3 out 7 cell lines; Kelly, SK-N-DZ and BE(2)-C. The upregulation of pATM and Wee1 showed a potential confirmation of their involvement in CT induced cell cycle arrest. Upon inhibition of pATM, Chk1 and Wee1 diverse effects were observed for each cell line (SK-N-SH, SK-N-AS, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C). Wee1 showed the most promising results were the cell viability decreased for all 5 p53 mutated cell lines and the confluency over time decreased for 4 out 5 p53 mutated cell lines. The p53 wild type cell line SK-N-SH was less sensitive towards Chk1 and Wee1 inhibition indicating that cell lines with functional p53 might not be as dependent on the Chk1 and Wee1 pathways compared to cell lines with non-functional p53. Thus, targeting the cell cycle arrest might be a promising therapeutic target for high-risk neuroblastoma. / Barndomscancern neuroblastom utgör 6% av all barncancer. Majoriteten av de drabbade är under 2 år. Neuroblastom har en stor mångfald av fenotypiska utryck som orsakas av dess inter- och intra-tumör heterogenitet. Fenotyperna klassificeras antigen som låg- eller högrisk. Här har 7 högrisks-neutoblastom cellinjer med varierande grad av känslighet mot kemoterapi analyserats. Titreringsstudier med kemoterapierna cisplatin och doxorubicin påvisade en benägenhet för de p53 muterade cellinjerna att arrestera i S- och/eller i G2/M-fasen, beroende på behandlingen samt behandlingsdosen, vilket indikerar på en dos-beroende cellcykel respons. En behandlingsanalys med de 3 nyckelkomponenterna fosforylerat ATM, Chk1 samt Wee1 gjordes för att potentiellt inhibera cellerna från att arrestera. Efter en initial immunocytokemi infärgning av pATM samt Wee1 visade 5 av 7 cellinjer (SK-N-SH, SK-N-FI, Kelly, SK-N-DZ samt BE(2)-C) en uppreglering av pATM-uttryck till följd av doxorubicin behandling. Däremot var Wee1 endast uppreglerat för 3 av 7 cell linjer (Kelly, SK-N-DZ samt BE(2)-C). Uppregleringen av pATM och Wee1 påvisar ett potentiellt samband mellan kemoterapi-inducerad cellcykelarrest och ökat utryck av pATM och Wee1. Vid inhibering av pATM, Chk1 samt Wee1 gav Wee1 de mest lovande resultaten där cellviabiliteten minskade för samtliga 5 p53-muterade cellinjer och där konfluensen över tid minskade för 4 av 5 p53-muterade cellinjer. SK-N-SH med funktionerande p53 var mindre känslig gentemot Chk1 och Wee1 inhibering, vilket indikerar att cellinjer med funktionerande p53 inte är lika beroende av reaktionsvägarna för Chk1 och Wee1 jämfört med cellinjer som har icke-funktionerande p53. Därmed kan riktad behandling mot cellcykelarrest vara en lovande behandling för högrisks-neuroblastom.

International Telemetering Conference Proceedings / October 25-28, 1999 / Riviera Hotel and Convention Center, Las Vegas, Nevada / Due to Cost and Safety considerations the Range Division of the 412th Test Wing is upgrading remote telemetry (TM) antenna sites to be operated and monitored remotely. This is possible, in part, due to the installation of fiber optic cable, and the use of ATM communications protocol. Both of these applications significantly reduce signal latency from the remote control station located at Ridley Mission Control Center (RMCC) and the Antenna site. This paper discusses the challenges associated with controlling these sophisticated systems remotely. We will also describe the decisions and how they were made, the concerns over system performance, and the impact to other systems. This paper also addresses the technologies chosen to support the requirements and overcome the challenges. The benefits of remote range sensors are also discussed. We will provide top-level block diagrams of the system architecture.

International Telemetering Conference Proceedings / October 22-25, 2001 / Riviera Hotel and Convention Center, Las Vegas, Nevada / Undersea warfare (USW) was perceived as a large-area, deep-water operation in the past therefore Fleet USW training ranges were designed to meet these requirements. Currently the bigger threat is the likelihood of regional conflict throughout the world by aggressive nations in littoral waters. The U.S. Navy must stand ready to respond to these regional conflicts when national interests are threatened. Consequently, naval forces must train to operate in the littoral environments where such regional conflicts are likely to occur. The West Cost Shallow Water Undersea Warfare Training Range (WC SWUWTR) is being developed to provide this training.

No description available.

Our cells are constantly dealing with DNA damage generated by endogenous cellular activity (e.g. DNA replication) and exogenous agents (e.g. ultraviolet and ionizing radiation (IR)). The cellular stress response to DNA damage requires strict co-ordination between cell cycle checkpoint control and DNA repair. In response to DNA double-strand breaks (DNA-dsbs), members of the phosphatidylinositol 3-kinase–related kinase family (e.g. ATM and DNA-PKcs kinases) have been shown to redundantly phosphorylate substrates including the DNA-dsb marker, gamma-H2AX, and the p53 tumour suppressor protein. The p53 protein is best known as the guardian of the genome through its transcriptional-dependent and -independent functions. Despite a clear link between ATM-dependent phosphorylation of p53 with cell cycle checkpoint control and various modes of DNA damage repair, the intracellular biology and sub-cellular localization of p53 and specifically its phosphoforms during DNA damage induction and repair remains poorly characterized. Using G0/G1 confluent primary human diploid fibroblast cultures, this thesis shows that endogenous p53, phosphorylated at serine 15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following the induction of DNA breaks. This biologically distinct sub-pool of p53Ser15 is ATM-dependent and resistant to 26S-proteasomal degradation. p53Ser15 co-localizes and co-immunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA-dsb rejoining. Sub-nuclear microbeam irradiation studies confirm that p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATMSer1981 and DNA-PKcsThr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX-association and altered DNA-dsb kinetics following DNA damage. We further hypothesized that the non-specific DNA binding activity of the p53 carboxy-terminus mediates chromatin anchoring at sites of DNA damage. YFP-p53 fusion constructs expressing carboxy-terminus deletion mutants of p53 were transfected into p53-null H1299 cells to determine the role of the carboxy-terminus in chromatin-binding pre- and post-IR, independent of transcriptional activity. Within this isogenic human cell system, we observed exogenous YFP-p53WT associated with ATMSer1981 and 53BP1 within cellular chromatin in a dynamic manner. We confirmed that these associations also occurred between endogenous WTp53 with ATMSer1981 and 53BP1 within the chromatin of primary human diploid fibroblasts. YFP-p53del1-299 fusion proteins, which lack transcriptional activity and the Ser15-residue, also associated within chromatin. Ser15-phosphorylation was found not to be essential for DNA damage-induced association of p53 with chromatin or with ATMSer1981 and 53BP1. These data suggest a unique biology for p53Ser15 phosphoforms in the initial steps of DNA damage signaling and implicates ATM-p53-53BP1 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair. And we propose a model whereby a pre-existing pool of p53 that constantly scans the genome, responds immediately to radiation-induced DNA damage by virtue of its association with chromatin through its carboxy-terminus. The consequences for these p53-ATMSer1981-53BP1 complexes following DNA damage remains to be investigated: could residual complexes be associated with decreased DNA-dsb rejoining or error-prone repair, or could these complexes signal for cell survival or cell death? Since altered p53 function and biology is an important factor in cellular carcinogenesis and response to cancer therapy, this study provides a step towards a greater understanding of WTp53 and MTp53 biology in tumour development and therapeutic resistance, in the hopes to contribute towards predicting therapeutic response and/or improving p53-targeted therapies.

<p>Approximately 50% of all patients with cancer receive radiation therapy at some point during the course of their illness. Despite advances in radiation delivery and treatment planning, normal tissue toxicity often limits the ability of radiation to eradicate tumors. The tumor microenvironment consists of tumor cells and stromal cells such as endothelial cells that contribute to tumor initiation, progression and response to therapy. Although endothelial cells can contribute to normal tissue injury following radiation, the contribution of stromal cells to tumor response to radiation therapy remains controversial. To investigate the contribution of endothelial cells to the radiation response of primary tumors, we have developed the technology to contemporaneously mutate different genes in the tumor cells and stromal cells of a genetically engineered mouse model of soft tissue sarcoma. Using this dual recombinase technology, we deleted the DNA damage response gene <italic>Atm</italic> in sarcoma and heart endothelial cells. Although deletion of <italic>Atm</italic> increased cell death of proliferating tumor endothelial cells, <italic>Atm</italic> deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. In addition, the ATM inhibitor NVP-BEZ235 selectively radiosensitized primary sarcomas, demonstrating a therapeutic window for inhibiting ATM during radiation therapy. Sensitizing tumor endothelial cells to radiation by deleting <italic>Atm</italic> prolonged tumor growth delay following a non-curative dose of radiation, but failed to increase local control. In contrast, deletion of <italic>Atm</italic> in tumor parenchymal cells increased the probability of tumor eradication. These results demonstrate that tumor parenchymal cells rather than endothelial cells are the critical targets that regulate tumor eradicaiton by radiation therapy.</p> / Dissertation

In our work, we have proposed a new framework for the classification and comparative study of ATM congestion control schemes. The different aspects on which we have classified the algorithms are control theoretic approach, action and congestion notification. These three aspects present of the classification present a coherent framework on which congestion control algorithms are to be classified. Such a classification will also help in developing new algorithms.