Cette thèse propose une modélisation aussi générique que possible de la combustion dans les moteurs automobiles dans un cadre de simulation aux grandes échelles. Une première étude aborde la fermeture du terme de transport non résolu pour la flamme de prémélange. Par la suite, un couplage entre les modèles ecfm-les et pcm-fpi est proposé et validé pour intégrer les effets de chimie complexe à la simulation de la flamme de prémélange. Ce travail est étendu par l'adjonction de modélisations spécifiques à l'allumage par bougie et de l'auto-inflammation, toujours en intégrant les effets de chimie détaillée. Des calculs d'application aux combustions anormales dans les moteurs à allumage commandé concluent ce travail.

Une transition épithélio-mésenchymateuse (TEM) et une expression élevée de la sphingosine kinase 1 (SPHK1) sont souvent observées dans les cancers. Notre étude du génome et du transcriptome d'adénocarcinomes pulmonaires (AP) montre que l'expression élevée de SPHK1 est en rapport, d'une part, avec des gains de la région incluant le locus SPHK1 et, d'autre part, avec une signature d'expression génique de TEM dans des tumeurs invasives. L'expression de SPHK1 est restreinte aux cellules tumorales. La surexpression de SPHK1 dans des cellules d'AP et l'exposition à son produit, la sphingosine-1-phosphate (S1P), entraînent une TEM, de manière réversible pour la S1P. La surexpression de SPHK1 active aussi NF-kB. La surexpression du facteur anti-apoptotique FLIP active NF-kB, induit une TEM et augmente l'expression de SPHK1, suggérant une boucle d'amplification entre NF-kB et SPHK1. Une TEM et la surexpression de FLIP ont été impliquées dans la résistance primaire aux inhibiteurs pharmacologiques de l'EGFR (EGFR TKI). Nous montrons que la surexpression de SPHK1 dans des cellules A549 diminue modestement la sensibilité au gefitinib, alors que l'inhibition de SPHK1 ou la déplétion du sérum en S1P l'augmentent modestement. L'invalidation de SPHK1 entraîne l'apoptose d'A549 y compris quand FLIP est surexprimé. L'activation et le maintien d'une TEM sont généralement attribués à des signaux contextuels du stroma. Cette thèse montre que les cellules tumorales elles-mêmes favorisent la surexpression de SPHK1 qui peut induire une TEM de façon autonome. De plus, la surexpression de FLIP impliquée dans la résistance aux EGFR TKI, n'empêche pas l'apoptose induite par l'invalidation de SPHK1. / Epithelial-mesenchymal transition (EMT) and sphingosine kinase 1 (SPHK1) high expression are often seen in cancers. Our study of genomic and gene expression data in pulmonary adenocarcinomas (AP) shows that SPHK1 high expression correlates with both gains in the region encompassing the SPHK1 locus, and an EMT gene expression signature in invasive tumors. SPHK1 expression is restricted to tumors cells. SPHK1 overexpression in AP cells, as well as exposure to its productsphingosine-1-phosphate (S1P),induce an EMT -in a reversible manner for S1P. SPHK1 overexpression also activates NF-kB. Overexpression of FLIP – an antiapoptotic factor - activates NF-kB, induces an EMT, and increases SPHK1 expression, suggesting an amplification loop between NF-kB and SPHK1. EMT and FLIP overexpression are known to favor primary resistance to EGFR pharmacological inhibitors (EGFR TKI). We show that SPHK1 overexpression in A549 cells slightly decreases cell sensitivity to gefitinib, while pharmacologic inhibition of SPHK1 or serum S1P depletionincrease it. Downregulation of SPHK1 expression induces apoptosis of A549 cells even when FLIP is overexpressed. Activation and maintenance of EMT are generally attributed to contextual signals from the stroma. Here, we show that tumor cells themselves favor SPHK1 overexpression, which can led to EMT in cell-autonomous manner. In addition, FLIP overexpression which is implicated in EGFR TKI resistance, cannot prevent apoptosis that is induced by SPHK1 invalidation.

Background: Treatment options for poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinoma are unsatisfactory and prognosis is generally poor. Lenvatinib (LEN), a multi-tyrosine kinase inhibitor targeting fibroblast growth factor receptors (FGFR) 1-4 is approved for advanced radioiodine refractory thyroid carcinoma, but response to single agent is poor in ATC. Recent reports of combining LEN with PD-1 inhibitor pembrolizumab (PEM) are promising. Materials and Methods: Primary ATC (n=93) and PDTC (n=47) tissue samples diagnosed 1997-2019 at five German tertiary care centers were assessed for PD-L1 expression by immunohistochemistry using Tumor Proportion Score (TPS). FGFR 1-4 mRNA was quantified in 31 ATC and 14 PDTC with RNAscope in-situ hybridization. Normal thyroid tissue (NT) and papillary thyroid carcinoma (PTC) served as controls. Disease specific survival (DSS) was the primary outcome variable. Results: PD-L1 TPS≥50% was observed in 42% of ATC and 26% of PDTC specimens. Mean PD-L1 expression was significantly higher in ATC (TPS 30%) than in PDTC (5%; p<0.01) and NT (0%, p<0.001). 53% of PDTC samples had PD-L1 expression ≤5%. FGFR mRNA expression was generally low in all samples but combined FGFR1-4 expression was significantly higher in PDTC and ATC compared to NT (each p<0.001). No impact of PD-L1 and FGFR 1-4 expression was observed on DSS. Conclusion: High tumoral expression of PD-L1 in a large proportion of ATCs and a subgroup of PDTCs provides a rationale for immune checkpoint inhibition. FGFR expression is low thyroid tumor cells. The clinically observed synergism of PEM with LEN may be caused by immune modulation.

Chronic Myelogenous Leukemia (CML) is a blood cancer that arises when hematopoietic cells acquire an abnormal protein known as BCR-ABL. Current therapies for CML include drugs that inhibit BCR-ABL. However, these drugs only suppress the disease and do not cure it. One reason is that BCR-ABL drugs fail to kill the primitive population of CML cells, referred to as leukemia stem cells (LSCs), which are responsible for initiating and propagating CML. Since LSCs are not killed, the cancer is not cured and many affected patients eventually relapse. Recent studies suggest that LSCs are protected from current therapies by the bone marrow micro-environment where they reside. There, cytokine signaling molecules are present, which mediate processes that protect LSCs from BCR-ABL drugs. The stem cell factor (SCF) is one of these signaling molecules. It activates the receptor c-Kit located on the surface of LSCs, and this activation in turn allows proliferating LSCs to resist BCR-ABL drugs, even without prior exposure to these drugs, i.e., innate resistance is observed. In this thesis, the mechanism of this innate resistance is investigated, so that a suitable treatment strategy can be developed. To this end, a co-agent approach based on synthetic antibodies (sABs) is proposed to inhibit the receptor c-Kit, with the goal of disrupting its activation by the ligand SCF. This disruption should in turn block the SCF-mediated innate resistance, thus potentially restoring BCR-ABL drug apoptotic activity. The method for this disruption involves targeting the c-Kit structural susceptibility. Specifically, the sABs are designed via antibody phage display technology to target the D1–D2–D3 domains representing the SCF binding sites, hence preventing downstream pathway activation. The hypothesis is that, by blocking the SCF-mediated innate resistance, a suitable combination of such an sAB co-agent and a BCR-ABL drug should be conducive to suppressing LSCs, thereby providing a potential means to improve CML treatment. In addition, to assess the performance of the proposed treatment strategy, a set of in vitro tests is conducted, focusing on performance behaviors such as cell binding, cell death, and the progenitor inhibition. The experimental results support the hypothesis that the proposed combinatorial strategy is indeed a promising approach to mitigate the innate resistance, thus restoring BCR-ABL drug apoptotic activity.

Non-small-cell lung cancer (NSCLC) accounts for 80–85% of all lung cancers and is associated with significant mortality. As epidermal-growth-factor receptor (EGFR) is over-expressed in 80-90% of NSCLC, its inhibition via EGFR-Tyrosine Kinase inhibitors (EGFR-TKIs) is a main therapeutic strategy. However, patients with mutations in KRAS are resistant to EGFR-TKIs. A study in mutant KRAS-driven lung cancer in transgenic mice showed that tumor growth was dependent on the activity of focal adhesion kinase (FAK). Therefore, we hypothesized that KRAS-mutant NSCLC will be sensitive to FAK-TKIs and, given known FAK-EGFR cross-talk, FAK inhibition will sensitize KRAS-mutant NSCLC to EGFR-TKIs. We performed cell viability assays of WT versus mutant KRAS NSCLC cell lines following treatment with FAK-TKI alone or in combination with a clinically relevant EGFR-TKI. We found that KRAS-mutant cells were more sensitive to FAK-TKI than KRAS-WT NSCLC. In addition, we found that the combination treatment including FAK and EGFR TKIs resulted in reduced tumor cell viability as compared to treatment with either drug alone. This enhanced anti-tumor response could be due to FAK-TKI’s ability to down-regulate EGFR downstream targets. Our preliminary data suggests that in KRAS-mutant cells the drug combination appears to more effectively inhibit Akt activity than single drug treatment alone. This suggests an enhanced ability to impair cell survival following treatment with the drug combination. We also found that treatment with FAK TKI in KRAS mutant NSCLC cells resulted in increased activation of EGFR which was due in part to modulation of EGFR recycling and production of endogenous EGFR ligands. Thus, the combination of FAK- and EGFR-TKIs may be more effective in KRAS mutant NSCLC as treatment with EGFR-TKI overcomes the unexpected ‘side effect’ of treatment with FAK-TKI, namely activation of the EGFR pathway by this drug. The findings of our study are novel and have uncovered previously unrecognized outcomes of FAK inhibition on EGFR activity. Moreover, our data support the notion that the combination of FAK- and EGFR-TKIs could be an effective treatment for KRAS mutant NSCLC patients.

Chronic myeloid leukaemia (CML) is characterised by the constitutively active tyrosine kinase BCR-ABL. Standard treatment with tyrosine kinase inhibitors (TKI) in the chronic phase (CP) of CML conveys excellent long-term prognosis but is associated with side effects and costs. Treatment free remission (TFR) is possible in a proportion of patients discontinuing treatment after obtaining deep treatment responses but it is not fully known how to select the right patients for stopping attempts. Treatment of accelerated phase (AP) and blast crisis (BC) is more complicated and the prognosis more dismal. In this thesis, we have studied factors of importance for outcome in CML patients with focus on immunological factors and clinical management. In a cohort of 32 newly diagnosed CP-CML patients, evidence of active immune escape mechanisms were found. These declined with the course of TKI treatment and at the same time, effector lymphocyte responses were elicited. These anti-leukaemia immune responses might help in the long-term control of CML. Multiple plasma protein markers were also measured with three multiplex platforms in a smaller cohort of patients (n=14). Inflammatory cytokines and other plasma proteins were affected by TKI treatment and multiplexing seems useful for finding potential biomarkers with biologic or prognostic significance in CML. Patients progressing to AP/BC were studied in a population-based material from the Swedish CML register. Approximately 4% of TKI-treated CP-CML patients transformed to AP/BC within 2 years of diagnosis. Monitoring of treatment responses was suboptimal in 1/3 of these patients and the median survival was 1.4 years after diagnosis of AP/BC. Thus, minimising the risk of disease progression through strict adherence to guidelines for monitoring and treatment is essential. In a cohort of patients (n=50) discontinuing TKI treatment within a large European trial, musculoskeletal pain was reported by 30% of patients, starting within 1- 6 weeks of TKI discontinuation and spontaneously resolving over time in most cases. Patients (n=56) were also evaluated with a multiplex platform with a total of 162 inflammation- and cancer-related plasma proteins. No predictive protein biomarkers for successful TKI discontinuation could be found. However, profound effects of TKI-treatment were seen and plasma proteomics could be useful for understanding effects of long-term TKI-treatment.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23775号 / 医博第4821号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 永井 純正, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM