<p>This thesis examines the possibility of designing a fully integrated and switched testing environment for a test laboratory which conducts automated testing. Execution of tests in this environment will make it possible to manage all test objects without requiring any manual intervention resulting in efficient utilization of machine hours and test objects. The thesis explores the concepts and requirements for designing such an environment. It also describes the methods to implement the environment. The result of the thesis work shows that it is possible to design and implement a fully integrated and switched testing environment which can reduce the lead time for delivery by a substantial amount along with a more efficient utilization of machine hours and resources. The exact information related to the instruments, devices under testing and tools are removed by the author according to NDA.</p>

ATM (Ataxia-Telangiectasia Mutated) i DNA-PKcs (DNA-dependent Protein Kinase, catalytic subunit) són dues proteïnes que pertanyen a la família de les PIKKs (PhosphatidylInositol 3-Kinase-related Kinase) i ambdues desenvolupen funcions importants en la via de resposta al dany en el DNA (DDR). ATM i DNA-PKcs són activades en resposta als trencaments de doble cadena del DNA (DSBs) que es produeixen a conseqüència de l'actuació de múltiples factors, entre ells les radiacions ionitzants. Un cop activades, les dues cinases realitzen funcions diferents -però significativament complementàries- en aquesta via: ATM és capaç de: (1) aturar el cicle cel·lular en qualsevol fase; (2) activar diverses proteïnes implicades en la via de reparació homòloga (HR) del DNA i (3) induir l'activació de la via de l'apoptosi cel·lular en cas que el dany sigui massiu i/o irreparable. Per la seva part DNA-PKcs és una proteïna mestra de la via de reparació no homòloga del DNA (NHEJ), on activa i regula a la resta de factors participants d'aquesta via. Finalment DNA-PKcs també pot induir la via de l'apoptosi en resposta a un dany massiu. L'absència d'alguna d'aquestes dues cinases comporta, en els individus afectes, l'aparició de síndromes d'inestabilitat cromosòmica caracteritzats per una major probabilitat de desenvolupar processos cancerígens. Les cèl·lules derivades dels individus afectes són extremadament radiosensibles i acumulen aberracions cromosòmiques. L'objectiu d'aquesta tesi doctoral és determinar quins factors contribueixen de manera específica a la radiosensibilitat i a la inestabilitat cromosòmica de les cèl·lules deficients en ATM i en DNA-PKcs. Per assolir aquest objectiu es va analitzar: (1) l'espectre d'aberracions radio-induïdes en ambdós tipus cel·lulars; (2) l'evolució i resolució d'aquestes aberracions al llarg del temps; (3) la possible implicació del metabolisme telomèric en aquestes aberracions i (4) la cinètica de reunió de DSBs de les cèl·lules deficients en ATM i la de les cèl·lules deficients en DNA-PKcs. Després de ser irradiats, ambdós tipus cel·lulars acumulen un nombre significativament elevat d'aberracions cromosòmiques. El metabolisme telomèric només contribueix de manera marginal a la inestabilitat cromosòmica en cèl·lules deficients en DNA-PKcs i no té repercussions en la línia cel·lular deficient en ATM emprada en aquest estudi. En analitzar la cinètica de reunió dels DSBs radio-induïts es va fer palès que la deficiència en DNA-PKcs indueix un alentiment de la mateixa. Una reparació més lenta de les lesions en el DNA afavoreix (1) l'acumulació de fragments cromosòmics i, alhora, (2) la reunió il·legítima dels mateixos. La cinètica alentida explica l'ampli espectre d'aberracions radio-induïdes obtingut en aquestes cèl·lules així com la seva persistència en el temps, esdevenint el principal factor responsable de la radiosensibilitat i inestabilitat cromosòmica en cèl·lules deficients en DNA-PKcs. En canvi les cèl·lules deficients en ATM reparen la majoria dels DSBs radio-induïts amb una cinètica comparable a la de les cèl·lules normals, però una fracció dels trencaments roman sense reparar inclús a llargs temps post-irradiació. Per tant, l'acumulació de trencaments pendents de ser reparats a llargs temps post-irradiació (48 i 72 h) apareix com el principal factor responsable de la radiosensibilitat i inestabilitat cromosòmica d'aquestes cèl·lules. Però com s'explica la persistència d'aquests trencaments durant vàries divisions post-irradiació? Un cop descartat un alentiment de la cinètica de reparació ens varem plantejar la possibilitat de que l'absència d'ATM impedís la correcta detecció d'aquests DSBs. Per tal d'avaluar aquesta hipòtesi es va realitzar un anàlisi de la presència de &#947;H2AX i Mre11 en els extrems cromosòmics trencats. La majoria de les delecions cromosòmiques presents en les cèl·lules deficients en ATM presenten marcatge amb les dues proteïnes en el punt de trencament, però una fracció considerable de les mateixes (25%) no presenta cap tipus de marcatge. El resultat obtingut suggereix que els trencaments no senyalitzats no estan essent correctament detectats i que la maquinària de reparació no és activa en ells. Proposem que l'acumulació de trencaments pendents de reparar són una característica de les cèl·lules deficients en ATM, i que aquesta acumulació contribueix de manera important a la seva radiosensibilitat i inestabilitat cromosòmica. Mentre que la fracció de trencaments correctament senyalitzats per &#947;H2AX i Mre11 podrà ser reparada al llarg del temps, la fracció de trencaments sense senyalitzar podria romandre sense reparar durant temps indefinit, contribuint especialment a la inestabilitat cromosòmica d'aquestes cèl·lules. / ATM (Ataxia-Telangiectasia Mutated) and DNA-PKcs (DNA-dependent Protein Kinase, catalytic subunit) belong to the PIKKs (PhosphatidylInositol 3-Kinase-related Kinase) family, and both proteins develop important functions in the DNA damage response pathway (DDR). ATM and DNA-PKcs are activated by the presence of DNA double strand breaks (DSBs), which are produced by multiple factors, ionizing radiations among these. Once activated, both kinases display different -but significantly complementary- functions in the DDR: ATM is able to: (1) halt the cell cycle; (2) activate several proteins implicated in the homologous repair pathway (HR) and (3) if the cell harbours massive and/or irreparable damage, ATM can initiate the apoptosis pathway. Meanwhile, DNA-PKcs is a master protein belonging to the non homologous end joining repair pathway (NHEJ), where it activates and regulates the remaining factors implied in this repair pathway. Finally, if the cell carries extensive damage DNA-PKcs can also induce the apoptosis pathway. Individuals affected by the absence of either kinase develop chromosomal instability syndromes, which are characterized by a special cancer predisposition. Cells obtained from the affected individuals are extremely radiosensitive and accumulate chromosomal aberrations.The main goal of this doctoral thesis is to determine which factors specifically contribute to the radiosensitivity and chromosomal instability of ATM and DNA-PKcs deficient cells. In order to attain this goal we analyzed: (1) the spectrum of radio-induced aberrations in both cell types; (2) the evolution and resolution of these aberrations over time; (3) the possible implication of telomeric metabolism in these aberrations and (4) the DSBs joining kinetics of ATM and DNA-PKcs deficient cells.After irradiation both cellular types accumulate a significant number of chromosomal aberrations. While telomeric metabolism contributes -although only marginally- to the chromosomal instability in DNA-PKcs deficient cells, it has no influence on the ATM deficient cell line employed in this study. Analysis of the DSBs joining kinetics demonstrates that DNA-PKcs deficiency induces a delay in the repair kinetics of radio-induced lesions. Slower DNA repair favours the accumulation of chromosomal fragments as well as their illegitimate joining displayed by DNA-PKcs deficient cells. Thus, the slower repair kinetics explain the broad aberration spectrum obtained in these cells, as well as their persistence in time, revealed to be the main factor responsible for radiosensitivity and chromosomal instability in DNA-PKcs deficient cells. On the other hand, ATM deficient cells are able to repair the majority of the radio-induced DSBs with normal joining kinetics except for a fraction of breaks, which remain unrepaired even at long post-irradiation times. Therefore, the accumulation of unrepaired breaks at long post-irradiation times (48 and 72 hr) is revealed to be the main factor responsible for the radiosensitivity and chromosomal instability of AT cells. But how can the persistence of these breaks in an unrepaired state during several cell divisions be explained? After discarding a delayed DSBs joining kinetic, we reflected upon the possibility of the absence of ATM preventing proper detection of unrepaired DSBs. In order to evaluate this hypothesis we analysed the presence of &#947;H2AX and Mre11 signalling in the broken chromosome ends scored in AT cells. The majority of the chromosome deletions displayed both &#947;H2AX and Mre11 labelling at the break point, but a significant fraction (25%) was devoid of any labelling. The results obtained suggest that unlabelled breaks are not being correctly detected and that the cell repair machinery is not active on them. We propose that the accumulation of breaks waiting for efficient repair is a hallmark of ATM deficient cells, and that this accumulation makes a major contribution to their radiosensitivity and chromosomal instability. While the fraction of correctly &#947;H2AX and Mre11-labelled breaks will eventually be repaired, the fraction of unlabelled breaks remains invisible to the DNA damage repair machinery, thus especially contributing to the chromosomal instability of these cells.

The Ataxia-telangiectasia mutated (ATM) protein is regarded as the major regulator of the cellular response to DNA double Strand Breaks (DSBs). In response to DSBs, ATM dimers dissociates into active monomers in a process promoted by Mre11-Rad50-Nbs1 (MRN) complex. ATM-deficient cells exhibit signs of chronic oxidative stress, suggesting that ATM plays an important role in the regulation of reactive oxygen species (ROS). I show for the first time that ATM can be activated by oxidative stress directly in the form of exposure to H₂O₂. In vitro kinase assays with purified ATM suggest that the activation by H₂O₂ is independent of DSBs and the MRN complex. In 293T cells, H₂O₂ induces ATM autophosphorylation on serine 1981. p53 and Chk2 are also phosphorylated by ATM after H₂O₂ treatment but not histone H2AX and heterochromatin protein Kap1, indicating that ATM activation by H₂O₂ in human cells is independent of DNA damage. I also show that the cysteine residue 2991 is critical for ATM activation by H₂O₂ in vitro. / text

Ce document propose un parcours de mes travaux de recherche en optimisation, en passant par l'optimisation mixte en variables entières, l'optimisation non-linéaire continue locale et le clustering dans les réseaux (graphes). Le premier chapitre traite de la programmation non linéaire mixte en variables entières et de l'optimisation globale déterministe. Il présente des contributions relatives à des investigations théoriques ainsi que des applications à des problèmes concrets. Nous discutons principalement de relaxations convexes et de reformulations automatiques de problèmes de programmation mathématique, dans le but d'améliorer l'efficacité des algorithmes de Branch-and-Bound. Dans le cadre de la programmation polynomiale, nous avons étudié des relaxations convexes pour les monômes multilinéaires et la génération de relaxations compactes de problèmes polynomiaux basés sur une technique spécifique de reformulation-linéarisation (RLT). Parmi les applications, une attention particulière est portée à des problèmes qui se posent dans la gestion du trafic aérien. Nous avons proposé de nouveaux modèles mathématiques et des approches de résolution basées d'une part sur l'optimisation mixte en variables entières et d'autre part sur le contrôle optimal. Deux thèmes de l'optimisation continue non-linéaire sont décrits au deuxième chapitre. Des méthodes de point intérieur pour la programmation quadratique et leurs noyaux d'algèbre linéaire (systèmes KKT) sont d'abord discutées. L'accent est mis sur les méthodes itératives pour les systèmes KKT et sur des questions connexes, telles que les techniques de préconditionnement et les propriétés de convergence. L'autre sujet discuté concerne, encore une fois, des problèmes de trafic aérien. Il porte sur les approches déjà mentionnées de contrôle optimal qui conduisent à des problèmes non-linéaires. Le troisième chapitre présente mes principaux résultats dans le domaine du clustering dans les réseaux. Le problème de l'identification de clusters dans les réseaux peut être formulé en utilisant la programmation mathématique et conduit généralement à un problème d'optimisation combinatoire. Mes contributions concernent les critères de classification et les méthodes de clustering correspondantes. Une attention particulière est portée aux méthodes exactes utilisées pour résoudre l'ensemble du problème d'optimisation ou, localement, les sous-problèmes survenant dans des heuristiques hiérarchiques, ou enfin dans le raffinement des solutions obtenues précédemment par d'autres méthodes.

The immune system recognizes and eliminates an enormous array of pathogens due to the diverse antigen receptor repertoire of T and B lymphocytes. However, the development of lymphocytes bearing receptors with unique specificities requires the generation of programmed double strand breaks (DSB) coupled with bursts of proliferation, rendering lymphocytes susceptible to mutations and oncogenic transformation. Thus, mechanisms responsible for monitoring global genomic integrity, such as those coordinated by the ATM (ataxia-telangiectasia mutated) kinase, must be activated during lymphocyte development to limit the oncogenic potential of antigen receptor locus recombination. I show that ATM deficiency compromises TCRα recombination and the post-mitotic survival of T-cell receptor αβ (TCRαβ+) CD4+CD8+ (DP) thymocytes, providing a molecular and developmental basis for the immunodeficiency characteristic of ATM loss. Moreover, I show that in early thymocyte progenitors undergoing TCRβ recombination, ATM loss leads to cell cycle defects and developmental arrest, likely facilitating the acquisition of mutations that contribute to leukemogenesis. Using ATM deficiency as a murine model of T cell precursor acute lymphoblastic leukemia (T-ALL), I demonstrate that IL-7 signaling, a critical survival and proliferation signal during early stages of normal thymocyte development, is also required for leukemic maintenance. Moreover, we show for the first time that in normal and leukemic thymocyte precursors, interleukin 7 receptor (IL-7R) expression and function are controlled by Notch signaling, a key determinant of T cell fate. Collectively, these findings provide insight into the mechanisms by which ATM promotes normal lymphocyte development and protects from neoplastic transformation, while establishing the groundwork for assessing the molecular events that lead to the initiation and stepwise progression of T cell leukemogenesis.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Gliomas are the commonest primary tumours of the brain and glioblastoma multiforme (GBM) represents more than 50% of this group. GBM remains a neurosurgical conundrum since patients often succumb to the disease within one year. Surgery followed by radiation and medical regimens over the years have had minimal impact on the prognosis of patients with this cancer and hence, alternative and novel therapeutic modalities are required if the survival of patients with this disease is to be significantly improved. The ATM gene, which is mutated in the disease ataxia-telangiectasia (A-T), is implicated in response to radiation-induced DNA damage, leading to profound radiosensitivity. By reducing the levels of ATM in the radioresistant GBM cells through antisense or RNA interference (RNAi) technology delivered by lentiviruses, malignant GBM tumour cells were successfully sensitised to radiation treatment. In conjunction with surgery, this strategy will provide an enhanced therapeutic intervention especially in the case of GBM where the tumour is untreatable. In this thesis, analysis of the D-3-Phosphoglycerate dehydrogenase promoter in a GBM cell line as well as the development of a novel rat model for GBM using a bioluminescent F98 cell line will also be presented.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Techn. Universiẗat, Diss., 2000--München.