The human Rad9A checkpoint protein is required for genomic stability and proper execution of the DNA damage checkpoint. Previous work has shown Rad9A to be the key member of a heterotrimeric toroidal structure known as the 911 complex, along with Hus1A and Rad1, which is similar in structure to PCNA. Recent literature suggests Rad9A is a novel oncogene, found to be upregulated in a number of cancers and high mRNA levels are thought to have a protective effect on tumour growth. This thesis describes two novel functions for the Rad9A protein. The first is as a facilitator for the nuclear translocation of the Claspin adaptor protein, required for successful Chk1-mediated checkpoint activation. The second is as part of a novel nuclear structure containing important members of the homologous recombination DNA repair pathway. Work described herein also confirms the existence of a Rad9A paralogue, Rad9B, that partially rescues defects associated with Rad9A-deficiency and is expressed in both tumour and undifferentiated embryonic stem cell lines. This work discusses these findings in the context of current literature and provides future experiments to continue investigations into the function of this vital checkpoint protein. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-03-26 12:02:41.304

The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions: unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor which delays anaphase, thus buying cells time to fix attachment errors. Although the major checkpoint proteins were identified some 25 years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the crucial checkpoint signal. Much of this can be attributed to the difficulty associated with studying these proteins at the kinetochores, which are highly complex and thus often make clean dissection of function impossible. To circumvent this problem, a synthetic version of the spindle checkpoint was engineered on an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes how the co-targeting of only two checkpoint components, the outer kinetochore protein Spc7 and the checkpoint kinase Mph1, was found to be sufficient to successfully generate a checkpoint-dependent metaphase arrest and how this paves the way for a clearer, more joined-up understanding of how the checkpoint works.

Il a été montré récemment que l'instabilité génétique joue un rôle central dans les étapes précoces de la tumorigenèse. Celle-ci provoquerait une activation chronique des voies ATR/CHK1 et ATM/CHK2 dans les cellules précancéreuses, entrainant l'apoptose ou la sénescence des cellules concernées. Les mécanismes de checkpoint constituant une barrière contre la progression tumorale, toute mutation affectant ce checkpoint serait ainsi sélectionnée très tôt dans le processu s de tumorigenèse et faciliterait ensuite la progression tumorale. Ce modèle met en évidence le rôle central de l'instabilité génomique et du checkpoint dans la progression tumorale.Au cours de ma thèse, je me suis intéressé au complexe Claspine / Timeless / Tipin, initialement identifié comme médiateur de la voie ATR/CHK1 et qui est donc considéré comme ayant une fonction suppresseur de tumeur. Cependant, Claspine présente aussi des propriétés oncogéniques, puisque qu'elle est surexprimée dans de nombreuses lignées tumorales et cette surexpression est importante pour la prolifération cellulaire. Nous nous sommes donc demandé comment cette protéine pouvait être à la fois un oncogène et un suppresseur de tumeur. Chez la levure, l'homologue de Claspine est impliqué dans le maintien de la stabilité des fourches de réplication, indépendamment de sa fonction dans le checkpoint. Nous proposons que dans les cellules cancéreuses cette surexpression permette une meilleure stabilité des fourches, ce qui serait très important pour répliquer efficacement un génome soumis à un stress réplicatif constant. Au cours de ma thèse, nous avons construit et caractérisé un modèle de cellulescancéreuses HCT116 dans lesquelles nous avons diminué le niveau de Claspine ou deTimeless grâce à des shRNA, sans que cela n'affecte l'efficacité du checkpoint de réplication. Nous avons pu observer dans ces cellules un ralentissement de la progression des fourches de réplication et l'apparition d'une instabilité génétique. Il semblerait que spécifiquement dans le cas de Timeless, le ralentissement de la fourche de réplication et l'instabilité génomique se manifeste surtout dans les régions du génome répliquées tardivement. / The correct execution of the replication program is essential for the maintenance of genome integrity during S phase. Indeed, replication fork progression is frequently challenged by DNA lesions and by a variety of natural pause sites. Arrested forks are unstable structures, which represent a major threat for the genome integrity if they are not promptly stabilized and restarted. In response to replicative stress, cells activated the replication checkpoint to prevent collapse of stalled forks and promote fork recovery. Recent evidence indicates that spontaneous replication stress occurs in precancerous lesions and promotes the development of cancer. Identifying the origin of this replication stress would represent a better understanding of the early stages of tumorigenesis. We study the function of Claspin, a mediator of the replication checkpoint, which plays a key role in the response to replication stress. It is therefore acting as a tumor suppressor, preventing genomic instability during DNA replication. Intriguingly, recent evidence indicates that Claspin is overexpressed in different cancers and can also acts as an oncoprotein. Studies on Mrc1p, the yeast homologue of Claspin, have shown that Mrc1 is permanently associated to forks, where it forms a complex with two partners called Tof1p and Csm3p (homologues of human Tim and Tipin respectively). We have shown by DNA combing that the replicative function of Mrc1p, is critical for viability in the presence of a replication stress. In l ight of our results in yeast, we propose that the Claspin/Tim/Tipin complex may also play a direct role for the replication of human cells. If confirmed, this replication function may account for the role of this complex in the cancer process and tumor resistance to genotoxic agents commonly used in anticancer therapy.

INTRODUÇAO: O câncer de cólon é a terceira neoplasia mais prevalente no país, com aumento progressivo da incidência associada ao envelhecimento populacional. Os avanços nos tratamentos local e sistêmico do câncer de cólon metastático tem aumentado significativamente o tempo de sobrevida global. Entretanto, ainda não existem biomarcadores consolidados na literatura, capazes de predizer resposta a estes tratamentos ou o prognóstico. No processo da carcinogênese, uma das importantes vias que se encontra alterada é a via de reparo do DNA. A Chk2 é uma proteína quinase com atividade no reparo celular atuando de forma supressora no processo da carcinogênese, sendo que alterações em sua expressão e/ou função têm sido associadas à progressão tumoral em outras neoplasias como no câncer de mama, pulmão, vulva, bexiga, cólon, ovário, osteossarcoma e linfomas. OBJETIVO: Avaliar a expressão imunohistoquímica do Chk2 no câncer de cólon metastático e correlacionar sua expressão com características clínico-patológicas e sobrevida. PACIENTES E MÉTODOS: Foram incluídos 58 pacientes com diagnóstico confirmado de câncer de cólon metastático, tratados em primeira linha com quimioterapia baseada em fluorouracila e oxaliplatina. O tempo mínimo de seguimento foram de 2 anos pós-diagnóstico. Para análise da expressão do Chk2 foram utilizadas as técnicas de tissue microarray e imunohistoquímica. Estes resultados foram correlacionados com características clínicas, patológicas e de sobrevida. Para análise estatística, foi utilizado o programa SPSS17 e o valor de p<0,05 foi considerado estatisticamente significativo. RESULTADOS: A expressão de Chk2 foi positiva em 69% dos pacientes. Houve associação entre a expressão de Chk2 e o status linfonodal (p = 0,012) e entre a sobrevivência (p=0,034). A expressão negativa de Chk2 aumentaram as chances de envolvimento linfonodal (OR:10,2, p=0,03). O tempo de sobrevida global de pacientes Chk2 negativo foi maior (72 versus 59 meses, p=0,155), o mesmo foi observado com o tempo sobrevida livre de progressão (19 versus 13 meses, p=0,293). As curvas de sobrevida foram diferentes de acordo com a expressão do Chk2 em pacientes com ou sem envolvimento linfonodal, sendo menor nos pacientes com Chk2 positivo, p=0,028. Houveram mais óbitos em pacientes com Chk2 positivo. Análise multivariada identificou o performance status segundo a escala de ECOG (p=0,001 ); metástase sincrônica (p=0,037); diferenciação das células tumorais (p=0,029) e expressão de Chk2 (p=0,020) como fatores independentes para sobrevida global. CONCLUSÃO: A expressão positiva do Chk2 no adenocarcinoma de cólon metastático foi indicativa de maior agressividade e disseminação tumoral, impactando de forma negativa na sobrevida e desfecho dos pacientes. / INTRODUCTION: The DNA damage checkpoint pathway has been of interest to the field of cancer biology, since checkpoint defects result in the accumulation of altered genetic information, a central feature of carcinogenesis. Little is known about the role of Chk2 in colorectal cancer tumorigenesis. OBJECTIVE: The purpose of this study was to evaluate Chk2 expression in metastatic colon cancer and correlate this with clinicopathological features and patient survival. PATIENTS AND METHODS: Tissues were obtained from 58 patients with confirmed metastatic colon cancer diagnosis, treated with capecitabine and oxaliplatin chemotherapy as standard doses. Patients included had, at least, 2 years post diagnosis of clinical following. The tissue microarray immunohistochemistry was the technic to evaluate Chk2 expression. Statistics analysis used SPSS 17. A p-value <0,050 was considered to be statistically significant. Immunohistochemical expression of Chk2 and its relationship with clinical and pathological characteristics and survival data was reported. RESULTS: The expression of Chk2 was positive in 69%. There was association between expression of Chk2 and Iymph node status (p=0.012) and between survival (p=0.034). The negative expression of Chk2 enhanced the chances of linfonodal involvement (OR:10,2, p=0.03). The global survival time of Chk2 negative patients was higher (72 versus 59 months, p= 0.155); the same was observed with progression-free survival time (19 versus 13 months, p=0.293). The survival curves were different according to Chk2 expression in patients with or without Iymph node involvement, being lower in patients with Chk2 positive, p=0.028. There were more deaths in patients with Chk2 positive. Multivariate regression analysis identified performance status ECOG (p=0.001), synchronous metastasis (p=0.037), tumor cell differentiation (p=0.029) and expression of Chk2 (p=0.020) as independent factors to overall survival. CONCLUSION: This study demonstrated that the Chk2 positive expression in colon cancer indicates increased tumor spread and tumoral aggressiveness, impacting negatively on survival and outcome of patients.

Cell division requires accurate DNA replication and cells develop checkpoint mechanisms toensure the correct passage of the genetic material. Cells arrest by a checkpoint when DNAdamage is found. After the checkpoint is silenced, the cell cycle can be resumed. Polo-likekinase 1 (Plk1) and Aurora A kinase (AurA) are both important regulators for checkpointrecovery. The question how AurA is activated was studied by many researchers, but the exactmechanism stays unclear.We developed a new setup to study AurA activation during checkpoint recovery. Quantitativeimmunofluorescence of fixed cells as well as a FRET probe that monitors Plk1 activity intime-lapse filming were applied in this study as indirect readouts of Aurora A activation. Theresult suggests that a Plk1-AurA feedback loop exists during checkpoint recovery. It can alsobe concluded that the inhibition of Cdk1 reduces Plk1 and AurA activity during checkpointrecovery. We also investigated the effect of calcium interfering drugs on AurA activation butno conclusive result was obtained.

Dephosphorylation of the Cdc2 kinase by the Cdc25 tyrosine phosphatase is the universally conserved trigger for mitotic entry. Cdc25 is also the point of convergence for checkpoint signaling pathways which monitor the genome for damaged DNA and incomplete replication. In addition, Cdc25 is inhibited by a MAP kinase cascade in the event of osmotic, oxidative and/or heat stress. These pathways inhibit cell cycle progression by phosphorylating Cdc25 resulting in its association with 14-3-3 and nuclear export. Although Cdc25 can be observed leaving the nucleus following inhibitory signals it is controversial whether phosphorylation, 14-3-3 binding or export itself is required for checkpoint proficiency. In fission yeast, Cdc25 is phosphorylated in vitro on 12 serine and threonine residues by the effector kinase of the DNA replication checkpoint, Cds1. Nine of these residues reside in the N-terminal regulatory region, while three are found in the extreme C-terminus of the protein. We show here that phosphorylation the nine N-terminal residues, nor any of the 12 in vitro sites, are required for enforcement of the DNA replication checkpoint. In lieu of Cdc25 phosphorylation the phosphatase is rapidly degraded and mitotic entry prevented by the action of the Mik1 kinase, targeting Cdc2. Thus, multiple mechanisms exist for preventing mitotic entry when S-phase progression is inhibited. The three C-terminal in vitro phosphorylation sites have not previously been examined in fission yeast. However, homology exists between the S. pombe protein and the Cdc25 orthologues in humans, Xenopus and Drosophila in this region. We report here that in S. pombe these sites are required to prevent mitotic entry following replication arrest in the absence of Mik1, and in the maintenance, but not establishment, of arrest following DNA damage. Our previous work showed that Cdc25 nuclear import requires the Sal3 importin-β but at the time we were unable to show a direct interaction between these two proteins. The final chapter of this thesis proves physical interaction by co-immunoprecipitation. Cdc25 mutants lacking all twelve putative Cds1 sites show nuclear localization during mitosis in a sal3- background, effectively reversing the cell cycle regulated pattern of accumulation of the phosphatase. / Thesis (Ph.D, Biology) -- Queen's University, 2011-06-20 12:16:15.71

INTRODUÇAO: O câncer de cólon é a terceira neoplasia mais prevalente no país, com aumento progressivo da incidência associada ao envelhecimento populacional. Os avanços nos tratamentos local e sistêmico do câncer de cólon metastático tem aumentado significativamente o tempo de sobrevida global. Entretanto, ainda não existem biomarcadores consolidados na literatura, capazes de predizer resposta a estes tratamentos ou o prognóstico. No processo da carcinogênese, uma das importantes vias que se encontra alterada é a via de reparo do DNA. A Chk2 é uma proteína quinase com atividade no reparo celular atuando de forma supressora no processo da carcinogênese, sendo que alterações em sua expressão e/ou função têm sido associadas à progressão tumoral em outras neoplasias como no câncer de mama, pulmão, vulva, bexiga, cólon, ovário, osteossarcoma e linfomas. OBJETIVO: Avaliar a expressão imunohistoquímica do Chk2 no câncer de cólon metastático e correlacionar sua expressão com características clínico-patológicas e sobrevida. PACIENTES E MÉTODOS: Foram incluídos 58 pacientes com diagnóstico confirmado de câncer de cólon metastático, tratados em primeira linha com quimioterapia baseada em fluorouracila e oxaliplatina. O tempo mínimo de seguimento foram de 2 anos pós-diagnóstico. Para análise da expressão do Chk2 foram utilizadas as técnicas de tissue microarray e imunohistoquímica. Estes resultados foram correlacionados com características clínicas, patológicas e de sobrevida. Para análise estatística, foi utilizado o programa SPSS17 e o valor de p<0,05 foi considerado estatisticamente significativo. RESULTADOS: A expressão de Chk2 foi positiva em 69% dos pacientes. Houve associação entre a expressão de Chk2 e o status linfonodal (p = 0,012) e entre a sobrevivência (p=0,034). A expressão negativa de Chk2 aumentaram as chances de envolvimento linfonodal (OR:10,2, p=0,03). O tempo de sobrevida global de pacientes Chk2 negativo foi maior (72 versus 59 meses, p=0,155), o mesmo foi observado com o tempo sobrevida livre de progressão (19 versus 13 meses, p=0,293). As curvas de sobrevida foram diferentes de acordo com a expressão do Chk2 em pacientes com ou sem envolvimento linfonodal, sendo menor nos pacientes com Chk2 positivo, p=0,028. Houveram mais óbitos em pacientes com Chk2 positivo. Análise multivariada identificou o performance status segundo a escala de ECOG (p=0,001 ); metástase sincrônica (p=0,037); diferenciação das células tumorais (p=0,029) e expressão de Chk2 (p=0,020) como fatores independentes para sobrevida global. CONCLUSÃO: A expressão positiva do Chk2 no adenocarcinoma de cólon metastático foi indicativa de maior agressividade e disseminação tumoral, impactando de forma negativa na sobrevida e desfecho dos pacientes. / INTRODUCTION: The DNA damage checkpoint pathway has been of interest to the field of cancer biology, since checkpoint defects result in the accumulation of altered genetic information, a central feature of carcinogenesis. Little is known about the role of Chk2 in colorectal cancer tumorigenesis. OBJECTIVE: The purpose of this study was to evaluate Chk2 expression in metastatic colon cancer and correlate this with clinicopathological features and patient survival. PATIENTS AND METHODS: Tissues were obtained from 58 patients with confirmed metastatic colon cancer diagnosis, treated with capecitabine and oxaliplatin chemotherapy as standard doses. Patients included had, at least, 2 years post diagnosis of clinical following. The tissue microarray immunohistochemistry was the technic to evaluate Chk2 expression. Statistics analysis used SPSS 17. A p-value <0,050 was considered to be statistically significant. Immunohistochemical expression of Chk2 and its relationship with clinical and pathological characteristics and survival data was reported. RESULTS: The expression of Chk2 was positive in 69%. There was association between expression of Chk2 and Iymph node status (p=0.012) and between survival (p=0.034). The negative expression of Chk2 enhanced the chances of linfonodal involvement (OR:10,2, p=0.03). The global survival time of Chk2 negative patients was higher (72 versus 59 months, p= 0.155); the same was observed with progression-free survival time (19 versus 13 months, p=0.293). The survival curves were different according to Chk2 expression in patients with or without Iymph node involvement, being lower in patients with Chk2 positive, p=0.028. There were more deaths in patients with Chk2 positive. Multivariate regression analysis identified performance status ECOG (p=0.001), synchronous metastasis (p=0.037), tumor cell differentiation (p=0.029) and expression of Chk2 (p=0.020) as independent factors to overall survival. CONCLUSION: This study demonstrated that the Chk2 positive expression in colon cancer indicates increased tumor spread and tumoral aggressiveness, impacting negatively on survival and outcome of patients.

During mitosis, accurate chromosome segregation is essential: gain or loss of genetic information can be detrimental to cell viability, or promote tumourigenesis. The mitotic checkpoint (also known as the spindle assembly checkpoint or SAC) ensures accurate chromosome segregation by delaying cell cycle progression until accuracy can be guaranteed. Mps1 is a protein kinase that is crucial for mitotic checkpoint signalling and also for proper chromosome alignment at metaphase. However, the precise role of Mps1’s catalytic activity is still unclear. Here, I present AZ3146, a novel small molecule inhibitor of Mps1. AZ3146 inhibits recombinant Mps1 in vitro with an IC50 of ~35 nM, and has low activity against a panel of 50 kinases, suggesting a reasonable degree of selectivity. As predicted for an Mps1 inhibitor, AZ1346 treatment led to spindle checkpoint malfunction in cells, accelerated mitotic timing, and perturbed the kinetochore localisation of the checkpoint effector Mad2. AZ3146 has a negative effect on cell viability, suggesting it leads to detrimental missegregations. Thus, the cellular effects of AZ3146 are consistent with Mps1 inhibition, and I was able to use the compound confidently as a tool to further probe the role of Mps1 activity in cells.Strikingly, levels of Mps1 increased at unattached kinetochores following inhibition of its kinase activity, suggesting Mps1’s kinetochore localisation is regulated by its own activity. A kinase-dead GFP-Mps1 fusion protein only accumulated at kinetochores in the absence of endogenous, active Mps1, implicating intra-molecular interactions in regulation of Mps1’s kinetochore localisation. I confirm a role for Mps1 in the mechanism of chromosome alignment, but in contrast to previous reports I did not detect a decrease in Aurora B activity following Mps1 inhibition. On the contrary, both Mps1’s phosphorylation status and its kinetochore localisation were affected by treatment with the Aurora B inhibitor ZM447439, placing Mps1 downstream of Aurora B. As an alternative explanation for the alignment defect in cells with reduced Mps1 activity, I found that levels of the plus-end directed kinesin Cenp-E were markedly decreased at unaligned kinetochores. I propose a model in which catalytically active Mps1’s auto-release from kinetochores simultaneously promotes both mitotic checkpoint signalling and chromosome alignment by facilitating Mad2 dimerisation and Cenp-E binding at unattached kinetochores.

No description available.

In parallel computing, MPI is heavily used due to its support of popular cluster based parallel machines and the Single Program Multiple Data (SPMD) model. Normally cluster nodes are dedicated to a single parallel job/application but MPI could also be used with nodes that are concurrently shared by multiple users. In this case, nodes could become overloaded with work from other users. Even a few overloaded nodes can result in application slowdown. Thus, it is desirable to relocate affected processes in a running application to lightly loaded nodes by partial checkpointing and migrating of those processes. In some MPI applications, groups of processes communicate frequently with one another. Such groups must be near one another to ensure communication efficiency. Thus, if any member of a group is to be checkpointed and migrated, all should be. It must therefore be possible to identify such groups. I have built a prototype, using LAM/MPI, that supports partial checkpoint, migration and restart of MPI processes. To identify process groups for checkpoint and migration, I adapted TEIRESIAS (an algorithm for pattern discovery from bioinformatics) to identify frequent, recurring patterns of communication using data gathered by LAM/MPI. I then created predictors that use the discovered patterns to predict groups of communicating processes that should be checkpointed and migrated together. I have assessed the effectiveness of my technique using synthetic and real communication data (for a small set of representative applications) to show that my predictors can accurately predict process groups for those applications. Additionally, I have created a simple simulation system to allow me to explore scenarios related to network characteristics and overload conditions under which my system might provide useful speedup. Not all MPI applications will benefit from my approach (e.g. those with unpredictable communication patterns or large groups of frequently communicating processes). However, my experimental and simulation results suggest that my technique should be effective for a number of common application types, network characteristics and overload conditions. Using partial checkpoint and migration should therefore allow many long running applications to finish faster than if a subset of their processes was left running on overloaded nodes.