Mantle cell lymphoma (MCL) is characterized by the presence of the t(11 ;14)(g13 ;g32) translocation which results in cyclin Dl over-expression. MCL is one of the most difficult lymphoproliferative disorders to manage with a median survival rate of 43 months from diagnosis. The poor prognosis associated with MCL is due in large part to its late classification as a separate clinical entity leading to a dearth in available pre-clinical models. The specific objectives of the research described in this thesis were (1) to establish MCL preclinical models of disease and (2) to evaluate deregulated cell signaling pathways in MCL that can impact treatment response. Pre-clinical models of MCL were established from pre-existing cell lines containing the t(11 ;14)(g13 ;g32). These cell lines were previously misclassified because they were developed prior to the classification of MCL as a distinct lymphoma subtype. With the establishment of MCL models, deregulated cell signaling pathways in MCL and response to different treatment strategies were investigated. These included an investigation of the cell signaling pathways activated in bcl-2 over-expressing MCL cells that were treated with oblimersen; a molecular gene silencing strategy that effectively suppresses bcl-2 in vitro and in vivo. Silencing bcl-2 provided insight into which pathways were influenced by bcl-2 over-expression in MCL. More specifically loss of cyclin D1, NF-KB, p53, bax and p27 were observed following bcl-2 silencing. Additional studies investigated how abnormal expression of CD40/CD40L and Fas/FasL along with bcl-2 family members contributes to B cell clonal expansion and influences Rituximab-mediated cell death in MCL models. Rituximab is a chimeric monoclonal antibody targeted against B cells and both Rituximab-sensitive and insensitive MCL models were defined. An abnormally high expression of bcl-2, bcl-x L, mcl-1, CD40/CD40L and Fas were observed in all MCL cells, as well as high levels of soluble FasL, capable of blocking Fas-mediated apoptosis. These deregulated pathways were associated with response to Rituximab treatment in a sensitive MCL model. These studies demonstrated some of the key pathways associated with treatment response in MCL, and the establishment of well characterized MCL models enables us to continue to explore new treatment strategies currently being studied in other lymphomas.

Mantle cell lymphoma (MCL) is characterized by the presence of the t(11 ;14)(g13 ;g32) translocation which results in cyclin Dl over-expression. MCL is one of the most difficult lymphoproliferative disorders to manage with a median survival rate of 43 months from diagnosis. The poor prognosis associated with MCL is due in large part to its late classification as a separate clinical entity leading to a dearth in available pre-clinical models. The specific objectives of the research described in this thesis were (1) to establish MCL preclinical models of disease and (2) to evaluate deregulated cell signaling pathways in MCL that can impact treatment response. Pre-clinical models of MCL were established from pre-existing cell lines containing the t(11 ;14)(g13 ;g32). These cell lines were previously misclassified because they were developed prior to the classification of MCL as a distinct lymphoma subtype. With the establishment of MCL models, deregulated cell signaling pathways in MCL and response to different treatment strategies were investigated. These included an investigation of the cell signaling pathways activated in bcl-2 over-expressing MCL cells that were treated with oblimersen; a molecular gene silencing strategy that effectively suppresses bcl-2 in vitro and in vivo. Silencing bcl-2 provided insight into which pathways were influenced by bcl-2 over-expression in MCL. More specifically loss of cyclin D1, NF-KB, p53, bax and p27 were observed following bcl-2 silencing. Additional studies investigated how abnormal expression of CD40/CD40L and Fas/FasL along with bcl-2 family members contributes to B cell clonal expansion and influences Rituximab-mediated cell death in MCL models. Rituximab is a chimeric monoclonal antibody targeted against B cells and both Rituximab-sensitive and insensitive MCL models were defined. An abnormally high expression of bcl-2, bcl-x L, mcl-1, CD40/CD40L and Fas were observed in all MCL cells, as well as high levels of soluble FasL, capable of blocking Fas-mediated apoptosis. These deregulated pathways were associated with response to Rituximab treatment in a sensitive MCL model. These studies demonstrated some of the key pathways associated with treatment response in MCL, and the establishment of well characterized MCL models enables us to continue to explore new treatment strategies currently being studied in other lymphomas.

Mantle cell lymphoma (MCL) is characterized by the presence of the t(11 ;14)(g13 ;g32) translocation which results in cyclin Dl over-expression. MCL is one of the most difficult lymphoproliferative disorders to manage with a median survival rate of 43 months from diagnosis. The poor prognosis associated with MCL is due in large part to its late classification as a separate clinical entity leading to a dearth in available pre-clinical models. The specific objectives of the research described in this thesis were (1) to establish MCL preclinical models of disease and (2) to evaluate deregulated cell signaling pathways in MCL that can impact treatment response. Pre-clinical models of MCL were established from pre-existing cell lines containing the t(11 ;14)(g13 ;g32). These cell lines were previously misclassified because they were developed prior to the classification of MCL as a distinct lymphoma subtype. With the establishment of MCL models, deregulated cell signaling pathways in MCL and response to different treatment strategies were investigated. These included an investigation of the cell signaling pathways activated in bcl-2 over-expressing MCL cells that were treated with oblimersen; a molecular gene silencing strategy that effectively suppresses bcl-2 in vitro and in vivo. Silencing bcl-2 provided insight into which pathways were influenced by bcl-2 over-expression in MCL. More specifically loss of cyclin D1, NF-KB, p53, bax and p27 were observed following bcl-2 silencing. Additional studies investigated how abnormal expression of CD40/CD40L and Fas/FasL along with bcl-2 family members contributes to B cell clonal expansion and influences Rituximab-mediated cell death in MCL models. Rituximab is a chimeric monoclonal antibody targeted against B cells and both Rituximab-sensitive and insensitive MCL models were defined. An abnormally high expression of bcl-2, bcl-x L, mcl-1, CD40/CD40L and Fas were observed in all MCL cells, as well as high levels of soluble FasL, capable of blocking Fas-mediated apoptosis. These deregulated pathways were associated with response to Rituximab treatment in a sensitive MCL model. These studies demonstrated some of the key pathways associated with treatment response in MCL, and the establishment of well characterized MCL models enables us to continue to explore new treatment strategies currently being studied in other lymphomas. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Das Mantelzelllymphom (MCL) gehört zu den aggressiven, mit bislang zur Verfügung stehenden Therapien nicht heilbaren, Non-Hodgkin-Lymphomen (NHL). Das MCL weist eine schlechte Prognose auf. Charakteristisch für das MCL ist die t(11,14)- Translokation, die das Cyclin D1- Gen betrifft. Darüber hinaus finden sich zahlreiche weitere genetische Alterationen mit Häufung bestimmter Zugewinne und Verluste von genetischem Material. Einer der am häufigsten chromosomal zugewonnene Abschnitte in MCL ist der kurze Arm von Chromosom 7 (7p). In Fällen mit dieser genetischen Veränderung fand sich das IMP3/IGF2BP3-Gen (Insulin-like growth factor 2 mRNAbinding protein 3) unter den am stärksten differentiell exprimierten Genen. In dieser Arbeit konnte in einer immunhistochemischen Analyse eine stark variable IMP3-Protein-Expression in einer Serie von insgesamt 172 primären MCL gezeigt werden. Darüber hinaus fand sich in diesem Kollektiv eine signifikante Korrelation der IMP3-Expression mit der Proliferationsfraktion (Ki67-Immunhistochemie) sowie auch eine Assoziation mit einer blastoiden Morphologie. Es konnte jedoch letztlich keine statistisch signifikante Assoziation der IMP-3-Protein-Expression mit einem chromosomalen Zugewinn von 7p, dem Genort von IMP3, nachgewiesen werden, so dass hier offenbar auch noch andere Mechanismen für die Regulation eine wichtige Rolle spielen. In einer darüber hinaus untersuchten Vergleichsgruppe von 20 Fällen von Lymphknoten mit Infiltraten durch ein small lymphocytic Lymphoma (SLL) zeigte sich insgesamt nur eine geringe IMP3-Expression. Der Befund einer vermehrten IMP3-Protein-Expression in einer Teilgruppe von MCL mit erhöhter Tumorzellproliferation unterstützt die Idee, dass eine Aktivierung des IGFSignalweges in MCL möglicherweise die Proliferation und biologische Aggressivität begünstigt. Daher könnte eine therapeutische Manipulation dieses Signalweges vermutlich eine zukünftige therapeutische Option für das MCL darstellen. / The mantle cell lymphoma (MCL) is one of the most aggressive non-Hodgkin´s lymphomas (NHL) with poor prognosis. As of today, no therapy is capable to cure this lymphoma. One of the key MCL characteristics is the t(11,14)-translocation, involving the cyclin D1 gene. In addition, there are numerous further genetic alterations, especially gains and losses of genetic material. One of the most commonly affected chromosomal sections is the short arm of chromosome 7 (7p). IMP3/IGF2BP3 gene (insulin-like growth factor 2mRNAbinding protein3) is among the strongest differentially expressed genes in cases with this genetic alteration. This work analyses a series of 172 primary MCL cases with the help of immunohistochemistry. These cases have highly variable IMP3 protein expression. This thesis showed significant correlation between the IMP3 expression and the proliferation levels (Ki67 immunohistochemistry), as well with the blastoid morphology of these cases. However, no statistically significant association was found between the IMP3 protein expression and the chromosomal gains of 7p (the locus of IMP3), suggesting that other mechanisms can be responsible for the regulation of IMP3 in MCL. The analysis of the control group (20 cases of lymphnodes infiltrated with a small lymphocytic lymphoma - SLL) showed overall only a small IMP3 expression. The study of seven secondary MCL cases showed no major differences in IMP3 protein expression between the primary diagnoses and later relapses. The findings of the correlation between the IMP3 protein expression and the increased proliferation support the idea that the activation of the IGF signaling pathway possibly favors proliferation and biological aggressiveness in MCL. Therefore, future MCL therapy research could include therapeutic manipulation of the IGF signaling pathway.

No / This paper presents a novel approach for probabilistic clustering, motivated by a real-world problem of modelling driving behaviour. The main aim is to establish clusters of drivers with similar journey behaviour, based on a large sample of historic journeys data. The proposed approach is to establish similarity between driving behaviours by using the Kullback-Leibler and Jensen-Shannon divergence metrics based on empirical multi-dimensional probability density functions. A graph-clustering algorithm is proposed based on modifications of the Markov Cluster algorithm. The paper provides a complete mathematical formulation, details of the algorithms and their implementation in Python, and case study validation based on real-world data.

The normal physiological response to bacterial infection or wounding with threat of infection, termed inflammation, has been shown to be dysregulated in certain human diseases including (but not limited to): idiopathic pulmonary fibrosis, acute lung injury, arthritis and glomerulonephritis. The earliest arriving and most abundant cell responding to an inflammatory stimulus is the neutrophil granulocyte. It has been shown that under inflammatory conditions neutrophil granulocytes have extended longevity, enhanced responsiveness and upregulated activation parameters. In the setting of non-infective, or prolonged, ineffectuallycleared infective disease where resolution of inflammation does not occur then neutrophil granulocytes may cause tissue damage which is mediated by excessive, misdirected exocytosis of toxic granule contents or by spillage of the same products from necrotic or netotic cell carcasses that have lost membrane integrity. A key process in the resolution of inflammation is the induction of apoptosis in recruited neutrophils following a successful response to an inflammatory stimulus. Cellular signalling from apoptotic cells and from professional phagocytes that have ingested apoptotic cells has been shown to favour resolution of inflammation and restoration of tissue homeostasis. Additionally, the removal of key inflammatory cells in a highly regulated, non-phlogistic fashion robustly assists the resolution process. Cyclin-dependent kinase (CDK) inhibitor drugs are being developed as anti-cancer agents as it is hypothesized that they should interfere with the enhanced cellcycling ability (increased proliferative capacity and extended longevity) which is such a key feature of cancer cell biology. The CDKs that drive the cell cycle are CDKs 1, 2, 4 and 6 and consequently agents were designed to have enhanced specificity for these targets. CDK inhibitor drugs target the ATP-binding domain of CDKs and as a result usually have activity against more than one CDK. The CDK inhibitor drug, R-roscovitine which targets CDKs 2, 5, 7 and 9 was shown to promote neutrophil apoptosis and consequently resolution of inflammation. This thesis aims to investigate the mechanism by which apoptosis is induced in neutrophil granulocytes by CDK inhibitor drugs. The first experimental chapter of this thesis explores in detail the time-course and active concentration range of CDK inhibitor drugs in comparison to known promoters and inhibitors of neutrophil apoptosis. It then dissects the apoptotic machinery which is responsible for the effects of CDK inhibitor drugs before investigating their capacity to promote apoptosis even in the presence of survival mediators relevant to the context of inflammatory disease. Flow-cytometry, light and confocal microscopy as well as western blotting for caspases, mitochondrial dissipation assay, fluorometric caspase assay and the detection of DNA laddering demonstrate that CDK inhibitor drugs promote classical neutrophil apoptosis by the intrinsic pathway and show similar kinetics of apoptosis induction to drugs that inhibit transcription. The second experimental chapter investigates the key neutrophil survival protein and bcl-2 homologue Mcl-1. By flow cytometry, western blotting and RT-PCR it is demonstrated that Mcl-1 is down-regulated at the level of transcription and that this occurs even in the presence of inflammatory mediators that would normally promote neutrophil survival. Additionally, it is shown that pro-apoptotic bcl-2 homologues are affected to a lesser degree suggesting an imbalance of bcl-2 proteins is caused by effects at a transcriptional level mediated by CDK inhibitor drugs. The third experimental chapter identifies CDKs and their binding partner cyclins in neutrophil granulocytes and investigates the impact of CDK inhibitor drugs on CDK protein levels and cellular distribution by differential lysis and western blotting as well as by confocal microscopy. The key transcriptional enzyme RNA polymerase II is also identified and the effect of CDK inhibitor drugs on phosphorylation of this enzyme is documented. Western blotting and confocal microscopy demonstrate the presence of key CDKs 2, 5, 7, 9 and cyclin binding partners of CDKs 7 and 9. It is shown that the phosphorylation of RNA polymerase II mediated by CDKs 7 and 9 is inhibited by CDK inhibitor drugs. This suggests that a key mechanism by which neutrophil apoptosis is induced by CDK inhibitor drugs is the inhibition of transcription of key proteins and suggests that neutrophils require survival proteins for functional longevity. The fourth experimental chapter addresses the production and use of HIV-tat dominant negative CDK 7 and 9 proteins to knockdown CDKs 7 and 9 in neutrophil granulocytes in vitro to provide a molecular biology surrogate for the pharmacological data already presented. The cloning, production, purification and use of HIV-tat dominant negative CDK proteins are described. The final chapter describes the use of a more specific pharmacological inhibitor of CDKs 7 and 9, DRB, in the mouse bleomycin lung injury model. Resolution of inflammation by a compound specifically targeting CDKs 7 and 9 is described. This thesis identifies CDKs 7 and 9 as key targets of CDK inhibitor drugs in neutrophilic inflammation. It shows these drugs acting at the level of transcription to drive neutrophil apoptosis by exploiting the unique dependency of neutrophils on the short-lived survival protein Mcl-1. In so doing the presence of functional and essential transcriptional machinery is identified in neutrophils and the transcriptional profile of resting, stimulated and inhibited neutrophils is delineated. These findings suggest novel approaches to the pharmacological promotion of resolution of inflammation and indicate key new targets for rational drug design. In future, it will be important to further characterize the effects of CDK inhibitor drugs on other cell-types including epithelial cells, fibroblasts and mononuclear cells. This information should prove important to the continued investigation of CDK inhibitor drugs in resolution of inflammation and also to the ongoing experimental trial of these drugs in idiopathic pulmonary fibrosis.

Drugs that alter microtubule dynamics are often used in chemotherapy regimes in combination with other agents in order to treat various cancers. Despite the success over many years, there remain problems in toxicity, resistance and predictability to the drugs. In order to overcome these problems, there is a need to gain an understanding of how these drugs kill cancer cells in cell culture. As microtubule function is particularly important for chromosome movement in mitosis, cells treated with these agents cause a mitotic arrest through activation of the spindle assembly checkpoint. Following induction of a mitotic arrest, cells can escape this arrest (mitotic slippage) or undergo mitotic death, determined in part by the response of the apoptotic network. Levels of an anti-apoptotic protein, Mcl-1, are often lost over time in mitosis. Using time-lapse analysis on a cell line unable to escape the mitotic arrest, this thesis shows that Mcl-1 protein contributes to cell death both in mitosis and the subsequent interphase in response to microtubule toxin, taxol. This suggests that inhibiting Mcl-1 may increase the efficacy of anti-mitotic agents. In addition, mitotic cell lines prone to mitotic slippage were found to have higher levels anti-apoptotic protein, Bcl-xL, in comparison to Mcl-1, indicating one way in which these cells can cope with loss of Mcl-1 during mitosis. Secondly, an evaluation of the contribution of the previously identified interaction between Mcl-1 and mitotic E3 ligase complex, the APC/C-Cdc20, to the rate of mitotic death and mitotic slippage was assessed. Inhibition of APC/C-Cdc20 activity or mutation of a Mcl-1 motif (RxxL) thought to engage with the APC/C-Cdc20 complex did not have a substantial effect on Mcl-1 degradation or mitotic death, thereby questioning the functional significance of this interaction. However, it appears that Mcl-1 protein levels can influence the rate of mitotic slippage and this influence was affected via Mcl-1’s RxxL motif within Mcl-1. This suggests that Mcl-1 protein may delay mitotic slippage via substrate competition for the APC/C-Cdc20 complex with Cyclin B1, whose degradation is required for mitotic exit. Further analysis of this effect showed that this interaction may not be a universal effect. This together with the specific functional effect on mitotic slippage rather than mitotic death, suggests that this is an indirect effect caused by network interference between the components of the death and slippage pathways.

Les glycosides cardiaques (GCs) utilisés en clinique contre les maladies cardiaques sont des inhibiteurs de la pompe sodium-potassium-ATPase. Récemment il a été montré que ces composés ont un effet anti-cancéreux. Nous avons étudié les effets de l’UNBS1450, un GC modifié partiellement extrait de la plante Calotropis procera. Nous avons analysé ses effets sur une protéine responsable de la résistance aux traitements anti-cancéreux : Mcl-1. L’UNBS1450 diminue son expression dans différents types de cancer à des concentrations très faibles. Cette modulation est capitale pour l’induction de la mort cellulaire. De plus, l’UNBS1450 diminue l’expression d’une protéine importante dans la prolifération cellulaire : c-Myc, cette modulation intervient dans les mêmes conditions, que celles qui permettent la diminution de Mcl-1. Nous avons observé que l’UNBS1450 est actif dans les cancers du sein, en bloquant le cycle cellulaire et la prolifération. Dans cette étude, nous avons montré que l’UNBS1450 et les CGs en général, d’une part induisent la mort cellulaire en diminuant l’expression de Mcl-1, et d’autre part affecte la prolifération cellulaire en modulant c-Myc / Cardiac glycosides (CGs) are used in clinics to treat heart diseases. They inhibit the sodium-potassium-ATPase. Recently, anti-cancers activities have been ascribed to these compounds. In this study, we investigated the anti-cancer effects of UNBS1450, a compound partially modified from a CG extracted from the plant Calotropis procera. We analyzed its activities against a protein responsible for the chemoresistance of cancer cells: Mcl-1. We found that UNBS1450 decreases the levels of Mcl-1 in many forms of cancer, at very low concentrations. This modulation is important to induce cancer cell death and is common to other CGs. Furthermore, UNBS1450 also decreases the intracellular levels of another protein important for the cancer cell proliferation: c-Myc. This modulation takes place in the same conditions determining Mcl-1 decrease. Finally, we found UNBS1450 active on breast cancer, by blocking the cell cycle and proliferation of the breast cancer cells. In this study, we have shown that UNBS1450 and CGs in general induce cell death by decreasing the expression of Mcl-1 protein and affect cell proliferation with the concomitant decrease of c-Myc protein

La thématique du laboratoire de l’équipe d’Adam Telerman porte sur la réversion tumorale, un processus rare au cours duquel les cellules cancéreuses perdent leur phénotype malin, et deviennent des cellules dites révertantes. Plusieurs protéines clefs impliquées dans cette transformation ont été mises en évidence, dont TCTP (Translationally Controlled Tumor Protein). La protéine TCTP est également impliquée dans la régulation de l’apoptose en interagissant et en renforçant l’activité anti-apoptotique de Mcl-1 et de Bcl-xl, deux protéines appartenant à la famille des Bcl-2. Ce projet s’attache à comprendre en termes moléculaires le mode d’action de TCTP au cours de l’apoptose. / Adam Telerman’s team research focuses on tumor reversion, a rare process in which cancer cells lose their malignant phenotype, and therefore become revertant. Many key proteins were showed to be involved in this transformation, including TCTP (translationally Controlled Tumor Protein). TCTP protein is also involved in apoptosis regulation by interacting and strengthening the anti-apoptotic activity of Mcl-1 and Bcl-xl, two proteins from Bcl-2 family.

Point estimates of the Standard Chain Ladder method (CLM) and of the more complex Munich Chain Ladder method (MCL) are compared to real data on 38 different datasets in order to evaluate if MCL produces better predictions on average with a dataset from an arbitrary insurance portfolio. MCL is also examined to determine if the future paid and incurred claims converge as time progresses. A bootstrap model based on MCL (BMCL) is examined in order to evaluate its possibility to estimate the probability density function (PDF) of future claims and observable claim development results (OCDR). The results show that the paid and incurred predictions by MCL converge. The results also show that when considering all datasets MCL produce on average better estimations than CLM with paid data but no improvement can be seen with incurred data. Further the results show that by considering a subset of datasets which fulfil certain criteria, or by only considering accident years after 1999 the percentage of datasets in which MCL produce superior estimations increases. When examining BMCL one finds that it can produce estimated PDFs of ultimate reserves and OCDRs, however the mean of estimate of ultimate reserves does not converge to the MCL estimates nor do the mean of the OCDRs converge to zero. In order to get the right convergence the estimated OCDR PDFs are centered and the mean of the BMCL estimated ultimate reserve is set to the MCL estimate by multiplication. / Punktskattningar gjorda med Standard Chain Ladder (CLM) och den mer komplexa Munich Chain Ladder-metoden (MCL) jämförs med verklig data för 38 olika dataset för att evaluera om MCL ger bättre prediktioner i genomsnitt än CLM för en godtycklig försäkringsportfölj. MCLs prediktioner undersöks också för att se om de betalda och de kända skadekostnaderna konvergerar. En bootstrapmodell baserad på MCL (BMCL) undersöks för att utvärdera om möjligheterna att estimera täthetsfunktionen (probability density function, PDF) av framtida skadekostnader och av ”observable claim development results (OCDR)”. Resultaten visar att MCLs estimerade betalda och kända skadekostnader konvergerar. Resultaten visar även att när man evaluerar alla dataseten så ger MCL i genomsnitt bättre prediktioner än CLM med betald data, men ingen förbättring kan ses med CLM med känd skadekostnadsdata. Vidare visar resultaten även att genom att bara titta på dataset som uppfyller vissa krav, eller genom att bara använda olycksår efter 1999, så ökar andelen dataset där MCL ger bättre prediktioner än CLM.Vid evaluering av BMCL ser man att den kan producera estimerade PDF:er för ultimo-reserver och OCDR:er, men att medelvärdet av ultimo-reserv prediktionerna från BMCL inte konvergerar mot MCL-prediktionerna och att medelvärdet av OCDR:erna inte konvergerar mot noll. För att få rätt konvergens så centreras OCDR PDF:erna och ultimo-reservernas medelvärden sätts till motsvarande MCL-prediktionens värde genom multiplikation.