Asynchronous Transfer Mode (ATM) Adaptation Layer Type 2 (AAL2) has been developed to carry low and variable bit rate traffic. It provides high bandwidth efficiency with low packing delay by allowing voice traffic from different AAL2 channels to be multiplexed onto a single ATM virtual channel connection. Examples of where AAL2 are used include the Code Division Multiple Access and the Third Generation mobile telephony networks. The main objective of this thesis is to study traditional and novel AAL2 multiplexing methods and to characterise their performance when carrying low and variable bit rate (VBR) voice traffic. This work develops a comprehensive QoS framework which is used as a basis to study the performance of the AAL2 multiplexer system. In this QoS framework the effects of packet delay, delay variation, subjective voice quality and bandwidth utilisation are all used to determine the overall performance of the end-to-end system for the support of real time voice communications. Extensions to existing AAL2 voice multiplexers are proposed and characterised. In the case where different types of voice applications are presented to the AAL2 multiplexer, it was observed that increased efficiency gains are possible when a priority queuing scheme is introduced into the traditional AAL2 multiplexer system. Studies of the voice traffic characteristics and their effects on the performance of the AAL2 multiplexer are also investigated. It is shown that particular source behaviours can have deleterious effect on the performance of the AAL2 multiplexer. Methods of isolating these voice sources are examined and the performance of the AAL2 multiplexer re-evaluated to show the beneficial effects of a particular source isolation technique. The extent to which statistical multiplexing is possible for real time variable VBR sources is theoretically examined. These calculations highlight the difficulties in multiplexing VBR real time traffic while maintaining guaranteed delay bounds for these sources. Based on these calculations, multiplexing schemes that incorporate data transfers within the real time traffic transfer are proposed as alternatives for utilising unused bandwidth caused by the VBR nature of the voice traffic.

Les cassures Double-brin de l'ADN (DSBs) sont une menace majeure pour la stabilité du génome. Afin de se protéger des effets délétères de ces dommages, les cellules activent une voie de réponse aux cassures double-brins (DDR) qui comprend des évènements qui conduisent à la reconnaissance et à la réparation de ces cassures ainsi qu'à un délai du cycle cellulaire. Cette DDR repose largement sur 2 membres de la famille des PI3K-like kinase, ataxia telangiectasia mutated (ATM) et DNA Protein Kinase (DNAPK) dont les fonctions respectives lors de la réparation restent controversées. Grâce à l'utilisation d'une lignée cellulaire contenant l'enzyme de restriction AsiSI combinée à de la cartographie par ChIP-chip, de l'analyse de la réparation de cassures séquence-spécifique ainsi qu'à de la microscopie haute résolution, j'ai pu, au cours de ma thèse mettre en évidence que aussi bien ATM que DNAPK sont recrutées sur une région confinée autour des DSBs. Cependant, une fois recrutées, elles présentent des fonctions non-redondantes que ce soit pour la ligation des cassures ou pour l'établissement des domaines yH2AX. Concernant la réparation, DNAPK est absolument requise pour la ligation des extrémités de la cassure alors que ATM est dispensable mais promeut la fidélité. En revanche, ATM est la principale kinase requise pour l'établissement des domaines yH2AX et ce quelque soit la cassure. J'ai aussi pu mettre en évidence le fait que plusieurs cassures induites par AsiSI sont capables de se regrouper au sein d'un "foyer de réparation" et ce de manière dépendante d'ATM et indépendante de DNAPK. Cette étude éclaircit les rôles respectifs des kinases ATM et DNAPK que ce soit pour la ligation des extrémités ou l'établissement des domaines yH2AX. Enfin elle a permis de mettre en évidence un nouveau rôle d'ATM dans l'organisation spatiale de la réparation et plus précisemment dans le regroupement de plusieurs DSBs au sein de "foyers de réparation" afin d'être réparées. / DNA Double Strand Breaks (DSBs) form a major threat to the genome stability. To circumvent the deleterious effects of DSBs, cells activate the DNA damage response (DDR), which comprises events that lead to detection and repair of these lesions, as well as a delay in cell cycle progression. This DDR largely rely on two members of the PI3K-like kinase family : ataxia telangiectasia mutated (ATM) and DNA Protein Kinase (DNAPK), whose respective functions during the DDR remains controversial. Using a cell line, expressing the AsiSI restriction enzyme, combined with high resolution ChIP-chip mapping, sequence-specific DSB repair kinetics analysis and advanced high resolution microscopy, we uncovered that both ATM and DNA-PK are recruited to a confined region surrounding DSBs. However, once present at the DSB site, they exhibit non-overlapping functions on end-joining and yH2AX domain establishment. At the repair level, DNAPK is absolutely required for end-joining while ATM is dispensable although promoting repair fidelity. By contrast, ATM is the main kinase required for the establishment of the histone mark yH2AX at all breaks. We also clearly demonstrated that multiple AsiSI-induced DSBs are able to associate within "repair foci", in a manner that strictly depends on ATM, but not DNAPK, activity. Our study shed light on the respective roles of ATM and DNAPK regarding end joining and yH2AX domain establishment. Lastly it allowed us to uncover a function of ATM in the spatial organisation of the repair, more precisely in the clustering of multiple breaks within "repair foci" in order to be repaired.

5 à 10% des cancers du sein sont héréditaires mais parmi ceux-ci seulement la moitié est expliquée par une altération constitutionnelle d’un gène de prédisposition connu tels que les gènes BRCA1 et BRCA2. L’importante hétérogénéité génétique qui caractérise les famillesBRCAx rend difficile la réalisation d’études familiales groupées et ne permet pas l’identification de nouveaux gènes de prédisposition au cancer du sein selon les méthodes classiques de liaison génétique ou d’association. Les techniques de séquençage de nouvelle génération (NGS) à l’échelle de l’exome ou du génome entier, autorisent en revanche l’étude de familles individuelles à la recherche de mutations constitutionnelles privées mais le nombre considérable de variants génétiques identifiés impose leur tri sur des critères de pathogénicité ou de récurrence. Un autre critère de tri peut être représenté par l’identification de régions candidates définies en fonction de réarrangements génomiques tumoraux communs à plusieurs tumeurs au sein d’une même famille. Le génotypage tumoral par puces SNP (pour single nucleotide polymorphism) permet en effet la détection d’haplotypes conservés dans des régions récurrentes de LOH (pour loss of heterozygosity) communes à plusieurs tumeurs familiales et donc l’identification de régions candidates suspectes d’abriter des mutations germinales dans des gènes de prédisposition au cancer. La combinaison de ces deux approches, génotypage tumoral puis NGS, a été appliquée à une série de 17 familles avec agrégation de cancers du sein pour lesquelles au moins deux échantillons tumoraux étaient disponibles. Aucun nouveau gène de prédisposition au cancer du sein n’a été identifié mais une mutation délétère constitutionnelle du gène ATM a ainsi été retrouvée, associée à une perte de l’allèle sauvage dans les 2 tumeurs d’une famille BRCAx. L’analyse de 17 tumeurs du sein supplémentaires provenant de 10 familles avec agrégation de cancers du sein et mutation constitutionnelle du gène ATM identifiée chez le cas index, a révélé que l’allèle sauvage d’ATM était fréquemment perdu dans ces tumeurs (>80% contre 20% attendu en situation sporadique ; p<0.001). Ce résultat plaide fortement en faveur de l’implication d’ATM dans la carcinogénèse de ces cancers du sein tel un gène suppresseur de tumeur et suggère que les mutations constitutionnelles d’ATM sont impliquées dans des formes familiales de cancer du sein. / Hereditary breast cancers (BCs) account for 5-10% of all diagnosed BCs, yet only 50% of such tumors arise in the context of a germline mutation in known tumor suppressor genes such as BRCA1 or BRCA2. The vast genetic heterogeneity which characterizes BRCAx families makes grouped studies impossible to perform. Next generation sequencing (NGS) techniques, however, allow individual families to be studied in order to identify private mutations. Single nucleotide polymorphism (SNP) arrays allow the detection of conserved haplotypes within recurrent regions of loss of heterozygosity, common to several familial tumors, therefore identifying genomic loci likely to harbor a germline mutation in cancer predisposition genes. The combination of both exome sequencing and SNP arrays for a series of 17 familial BC did not allow the identification of a novel BC predisposition gene, but revealed a germline ATM mutation associated with a loss of the wild-type allele in a BRCAx family. The analysis of 17 additional breast tumors from ten BC families in which a germline ATM mutation had been identified revealed a high frequency of wild-type allele loss in these tumors (>80% compared to the 20% expected in sporadic BC; p <0.001). This result argues strongly in favor of the involvement of ATM in the carcinogenesis of these tumors as a tumor suppressor gene and suggests that germline ATM mutations are involved in a subset of familial BC.

Depuis plus d'un siècle que les rayons X ont été découverts, les effets biologiques des radiations ionisantes ne sont pas encore entièrement expliqués. Pourtant, une description précise et la modélisation mathématique des événements physico-chimiques, moléculaires et cellulaires contribueraient significativement à l'effet des risques liés à une irradiation. Le groupe de Radiobiologie de l'UMR1052 Inserm (Lyon) a accumulé un nombre considérable de données sur la radiosensibilité individuelle et la réparation des dommages radioinduits de l'ADN qui nous permettent aujourd'hui de valider des modèles nouveaux qui sont souvent en contradiction avec les paradigmes actuels. En particulier, alors que les cassures double-brin de l'ADN semblent être les dommages clés de la létalité cellulaire, aucun protocole ni biomarqueur n'est considéré comme prédictif de la radiosensibilité. Le but de la thèse a donc été de déterminer les paramètres précis qui peuvent prédire la réponse aux radiations. Un grand nombre de protéines se relocalisent sous forme de foci nucléaire autour des sites de CDB. Dans une première étape, nous avons pu proposer une formule générale qui lie induction, reconnaissance et réparation des CDB, valable pour toutes les protéines relocalisantes après irradiation. Cette formule a été appelée « Formule de Bodgi ». La validité de cette formule a pu être vérifiée sur différents biomarqueurs et sur différents types de cellules de patients montrant des radiosensibilités différentes. Dans une deuxième étape, nous avons pu modéliser le processus de transit cytonucléaire de la protéine ATM. Nous avons pu alors apporter une interprétation nouvelle et cohérente des paramètres α et β du modèle linéaire-quadratique qui décrit la relation entre la dose de radiation et la survie cellulaire. Notre théorie s'est avérée également utile pour expliquer certaines autres énigmes de la radiobiologie, notamment le phénomène d'hypersensibilité aux faibles doses / More than a century after the discovery of X-rays, the biological effects of ionizing radiation are still not entirely explained. Nevertheless, a relevant description and a mathematical model of the physico-chemical, molecular and cellular events would significantly contribute to the evaluation of the related risks. The Radiobiology Group of the UMR1052 Inserm Unit (Lyon) has collected a considerable number of data concerning individual radiosensitivity and DNA damage repair, which allows us today to validate actual modeling approaches that are often contradicting actual paradigms. Particularly, while the DNA double-strand breaks (DSB) appear to be the key-damages of cell lethality, there is still no experimental protocol or biomarker that is considered to be predictive for radiosensitivity. The purpose of this thesis was to determine the precise parameters that can predict the response to radiation. An important number of proteins relocalize as nuclear foci in the DSB sites. As a first step, we proposed a general formula that links the DSB induction, recognition and repair, valid for all the relocalized proteins after irradiation. We called this formula the ‘’Bodgi’s formula’’. The validity of this model was verified with different biomarkers, but also on different cell types from patients showing different radiosensitivity. In a second step, we proposed a model for the whole process of the nucleo-shuttling of the ATM protein that occurs after irradiation. We provided a novel and coherent interpretation of the α and β parameters of the linear-quadratic model that describes the relation between radiation dose and cell survival. Our theory was also shown to be useful in explaining some other enigmas of radiobiology, including the hypersensitivity to lowdose phenomena

Development of a PCI card for PCI bus based host systems, which allows for analysis of ATM data streams on ATM transmission links with a data rate of 622Mbit/s. Implementation and previous deliberations and therefrom and from given specifications arisen partial tasks Fields of application of the ATM-Analyzer board Depiction of particular development steps Functional description of the ATM-Analyzer Explanations on how to put the PCB and the employed test software into operation Assessment of test results / Entwicklung einer Einsteckkarte für PCI-Bus-basierte Hostsysteme zur Analyse von Datenströmen auf ATM-Übertragungsstrecken mit einer Geschwindigkeit von 622 Mbit/s. Realisierung und damit verbundene Vorüberlegungen und daraus und aus gegebenen Spezifikationen erwachsene Teilaufgaben Anwendungsgebiete der Analyzer-Karte Darstellung der einzelnen Entwicklungsschritte Funktionsbeschreibung des ATM-Analyzers Erläuterungen zur Inbetriebnahme der Leiterkarte und der verwendeten Testsoftware Bewertung der Testergebnisse

Neuroblastom är en barncancer som uppstår ur det sympatiska nervsystemet och drabbar omkring 15 barn i Sverige varje år. Högriskvarianten är associerad med mycket hög dödlighet och risk för återfall, vilket tros ha att göra med att tumörernas ovanligt heterogena sammansättning tillåter resistenta subpopulationer att motstå konventionella behandlingsmetoder. Tidigare forskning har identifierat rubbade mekanismer för celldelning som ett tillvägagångssätt för tumörcellerna att överleva DNA-skador som kemoterapeutiska droger orsakar. I detta masterprojekt analyserades fem ultra-högrisk neuroblastomcellinjer i syfte att belysa deras progression genom celldelningen efter behandling med doxorubicin och/eller gemcitabin. Vidare identifierades ataxia telangesia mutated (ATM) serine/threonine kinase som ett essentiellt protein vid inhibering av celldelningen och i samband med reparation av DNA-skador, vilket bekräftades av förhöjt uttryck av fosforylerat ATM i alla fem cellinjer efter behandling med doxorubicin, gemcitabin, och/eller en kombination av båda. Återväxt av tumörcellerna efter inhibering av fosforylerat ATM i kombination med doxorubicin och gemcitabin analyserades sedan, och fördröjd återväxt noterades i en av cellinjerna efter kombinationsbehandling. Sammantaget har nya mekanismer för behandlingsresistens hos tumörceller identifierats och alternativa kombinationsbehandlingar har visat effekt på en av fem testade neuroblastomcellinjer. / Neuroblastoma is a pediatric cancer of the sympathetic nervous system that afflicts around 15 children annually in Sweden. Despite aggressive treatment, high-risk neuroblastoma is associated with a mortality of 50% and relapse rate of up to 60%, emphasizing the need for novel treatment options. In this study, fluoresce activated cell sorting was used to analyze cell cycle progression in five ultra-high-risk neuroblastoma cell lines: BE(2)-C, Kelly, SK-N-AS, SK-N-DZ, and SK-N-FI, post-treatment with doxorubicin and gemcitabine. In line with previous research, doxorubicin primarily induced cell cycle arrest in G2/M-phase and gemcitabine in the S-phase. Combined, the compounds induced varied effects, with accumulation primarily in the G1-and S-phase. Immunoblotting revealed elevated levels of phosphorylated ATM (pATM), a key regulator of cell cycle arrest and DNA damage signaling, across all five cell lines post-treatment to doxorubicin, gemcitabine, and/or the combination, indicating its vital role in their survival. Kelly stood out in both cell cycle progression and ATM phosphorylation, exhibiting minimal to no changes in cell cycle accumulation or pATM expression when exposed to the combined treatment, despite reacting to both monotherapies. These results may indicate that Kelly might implement an alternative mechanism of regulation compared to the remaining cell lines. To explore targeted inhibition of pATM, we employed the ATM kinase inhibitor KU-55933, which in BE(2)-C cells reduced expression levels of pATM when combined with doxorubicin, but not gemcitabine or the combination. Regrowth assays showed increased efficacy of doxorubicin and gemcitabine upon addition of the ATM kinase inhibitor KU-55933 in one of the tested cell lines in comparison to doxorubicin alone. However, longer incubation time is needed before the effect can be fully evaluated. These findings shed light on the differential cell cycle behavior in high-risk neuroblastoma cell lines exposed to combination therapy and suggest a vital role of ATM in the DNA damage response following doxorubicin and gemcitabine treatment. Further investigations are warranted to explore alternative strategies for enhancing the effectiveness of doxorubicin and gemcitabine in the treatment of this aggressive cancer subtype.

CD4 T-cell depletion is a hallmark of HIV/AIDS, but the underlying mechanism is still unclear. We have recently shown that ataxia-telangiectasia-mutated (ATM) deficiency in CD4 T cells accelerates DNA damage, telomere erosion, and cell apoptosis in HIV-infected individuals on antiretroviral therapy (ART). Whether these alterations in ART-treated HIV subjects occur in vitro in HIV-infected CD4 T cells remains unknown. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the telomeric DNA damage response (DDR) and cellular apoptosis in highly permissive SupT1 cells, followed by the validation of our observations in primary CD4 T cells with active or drug-suppressed HIV infection. Specifically, we established an in vitro HIV T-cell culture system with viral replication and raltegravir (RAL; an integrase inhibitor) suppression, mimicking active and ART-controlled HIV infection in vivo. We demonstrated that HIV-induced, telomeric DDR plays a pivotal role in triggering telomere erosion, premature T-cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This in vitro model provides a new tool to investigate HIV pathogenesis, and our results shed new light on the molecular mechanisms of telomeric DDR and CD4 T-cell homeostasis during HIV infection. IMPORTANCE The hallmark of HIV infection is a gradual depletion of CD4 T cells, with a progressive decline of host immunity. How CD4 T cells are depleted in individuals with active and virus-suppressed HIV infection remains unclear. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the chromosome end (telomere) DNA damage response (DDR) and cellular apoptosis in a T-cell line (highly permissive SupT1 cells), as well as in primary CD4 T cells with active or drug-suppressed HIV infection. We demonstrated that HIV-induced telomeric DDR plays a critical role in inducing telomere loss, premature cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This study sheds new light on the molecular mechanisms of telomeric DDR and its role in CD4 T-cell homeostasis during HIV infection.

Apresenta-se um framework que permite a caracterização, conformação e escalonamento de todas as categorias de serviços A TM, dentro do ambiente de usuário (CEP). Propõem-se um modelo de tráfego otimizado para este ambiente que oferece condições de garantias de QoS individuais para fluxos VBR, tempo real ou não, CBR, ABR e UBR. O modelo proposto, denominado de PCSTS (priority class services traffic shaping), inclui um módulo que permite a obtenção do descritor de tráfego de fluxos desconhecidos. Os fluxos são conformados de forma individual, e em tempo real, segundo um conformador baseado num algoritmo de escalonamento virtual duplo baseado no GCRA do ITU/ ATM Forum. Para a multiplexação dinâmica dos diferentes fluxos do ambiente de usuário, no enlace de acesso, é proposto um algoritmo de escalonamento EDD, modificado através de um sinal de realimentação entre conformador e escalonador, e desta forma tem-se condições de oferecer garantias de limite de atraso e jitter para fluxos individuais de serviços rt-VBR ou nrt-VBR. São apresentadas simulações, tanto da caracterização dos fluxos desconhecidos como da arquitetura do escalonador, que comprovam o comportamento esperado do modelo de tráfego PCSTS. / We present a framework that enhances the characterization, shaping and scheduling of ATM traffic flows for ali ATM service categories in the customer prernise equipment (CPE) environment. We propose an optirnized traffic model, capable to guarantee QoS parameters at per connection levei for VBR services, real time or not, CBR, ABR and UBR services. The model proposed, narned PCSTS (priority classes services traffic shaping), includes a module that performs the characterization of services with unknown traffic descriptor. The flows are shaped individually, in real time, through a shaper based on the double discrete time scheduling algorithm of the GCRA from ITU/ ATM Forum. For the dynarnic multiplexing of the different service flows , at the link levei, a modified EDD scheduling algorithm is proposed with a feedback signal between the scheduling and shaping modules. As a result, it is demonstrated that it is possible to grant jitter and delay bounds for the rt-VBR and nrt-VBR services. We present simulations of the traffic characterization module and of the scheduling architecture, for different services, which confirms the expected behavior of the PCSTS model.

Apresenta-se um framework que permite a caracterização, conformação e escalonamento de todas as categorias de serviços A TM, dentro do ambiente de usuário (CEP). Propõem-se um modelo de tráfego otimizado para este ambiente que oferece condições de garantias de QoS individuais para fluxos VBR, tempo real ou não, CBR, ABR e UBR. O modelo proposto, denominado de PCSTS (priority class services traffic shaping), inclui um módulo que permite a obtenção do descritor de tráfego de fluxos desconhecidos. Os fluxos são conformados de forma individual, e em tempo real, segundo um conformador baseado num algoritmo de escalonamento virtual duplo baseado no GCRA do ITU/ ATM Forum. Para a multiplexação dinâmica dos diferentes fluxos do ambiente de usuário, no enlace de acesso, é proposto um algoritmo de escalonamento EDD, modificado através de um sinal de realimentação entre conformador e escalonador, e desta forma tem-se condições de oferecer garantias de limite de atraso e jitter para fluxos individuais de serviços rt-VBR ou nrt-VBR. São apresentadas simulações, tanto da caracterização dos fluxos desconhecidos como da arquitetura do escalonador, que comprovam o comportamento esperado do modelo de tráfego PCSTS. / We present a framework that enhances the characterization, shaping and scheduling of ATM traffic flows for ali ATM service categories in the customer prernise equipment (CPE) environment. We propose an optirnized traffic model, capable to guarantee QoS parameters at per connection levei for VBR services, real time or not, CBR, ABR and UBR services. The model proposed, narned PCSTS (priority classes services traffic shaping), includes a module that performs the characterization of services with unknown traffic descriptor. The flows are shaped individually, in real time, through a shaper based on the double discrete time scheduling algorithm of the GCRA from ITU/ ATM Forum. For the dynarnic multiplexing of the different service flows , at the link levei, a modified EDD scheduling algorithm is proposed with a feedback signal between the scheduling and shaping modules. As a result, it is demonstrated that it is possible to grant jitter and delay bounds for the rt-VBR and nrt-VBR services. We present simulations of the traffic characterization module and of the scheduling architecture, for different services, which confirms the expected behavior of the PCSTS model.

Apresenta-se um framework que permite a caracterização, conformação e escalonamento de todas as categorias de serviços A TM, dentro do ambiente de usuário (CEP). Propõem-se um modelo de tráfego otimizado para este ambiente que oferece condições de garantias de QoS individuais para fluxos VBR, tempo real ou não, CBR, ABR e UBR. O modelo proposto, denominado de PCSTS (priority class services traffic shaping), inclui um módulo que permite a obtenção do descritor de tráfego de fluxos desconhecidos. Os fluxos são conformados de forma individual, e em tempo real, segundo um conformador baseado num algoritmo de escalonamento virtual duplo baseado no GCRA do ITU/ ATM Forum. Para a multiplexação dinâmica dos diferentes fluxos do ambiente de usuário, no enlace de acesso, é proposto um algoritmo de escalonamento EDD, modificado através de um sinal de realimentação entre conformador e escalonador, e desta forma tem-se condições de oferecer garantias de limite de atraso e jitter para fluxos individuais de serviços rt-VBR ou nrt-VBR. São apresentadas simulações, tanto da caracterização dos fluxos desconhecidos como da arquitetura do escalonador, que comprovam o comportamento esperado do modelo de tráfego PCSTS. / We present a framework that enhances the characterization, shaping and scheduling of ATM traffic flows for ali ATM service categories in the customer prernise equipment (CPE) environment. We propose an optirnized traffic model, capable to guarantee QoS parameters at per connection levei for VBR services, real time or not, CBR, ABR and UBR services. The model proposed, narned PCSTS (priority classes services traffic shaping), includes a module that performs the characterization of services with unknown traffic descriptor. The flows are shaped individually, in real time, through a shaper based on the double discrete time scheduling algorithm of the GCRA from ITU/ ATM Forum. For the dynarnic multiplexing of the different service flows , at the link levei, a modified EDD scheduling algorithm is proposed with a feedback signal between the scheduling and shaping modules. As a result, it is demonstrated that it is possible to grant jitter and delay bounds for the rt-VBR and nrt-VBR services. We present simulations of the traffic characterization module and of the scheduling architecture, for different services, which confirms the expected behavior of the PCSTS model.