La fonction de cytolyse est un mécanisme majeur des effecteurs du système immunitaire pour éliminer les cellules infectées ou tumorales. Cette fonction associe l'activité de la perforine, qui forme des pores dans la membrane d'une cellule cible, à la sécrétion de protéases: les granzymes. Ces dernières sont des molécules pro-apoptotiques qui induisent la mort de la cellule cible. Les granzymes et en particulier granzyme B ciblent plusieurs voies intracellulaires complémentaires pour assurer l'efficacité de la cytolyse. Or il est difficile d'observer directement la fonction de cytolyse au cours de réponse immunitaire in vivo dans des conditions physiologiques. Dans les travaux présentés dans cette thèse, nous avons développé un nouveau modèle qui permet de suivre la fonction de cytolyse en temps réel par l'expression d'une protéine de fusion fluorescente GZMB-tdTomato. Les résultats obtenus par expression rétrovirale ont montré que la protéine de fusion est correctement exprimée dans les vésicules cytolytiques qui deviennent fluorescentes. Dans un second temps, nous avons réalisé un nouveau modèle murin qui exprime GZMB-tdTomato de manière substituée au GZMB natif par recombinaison homologue (Knock In). Nous avons mis en évidence que la protéine de fusion conserve l'activité catalytique de la protéine native et ses caractéristiques (conditions d'expression, de maturation, de sécrétion et demeure active après le passage dans la cellule cible lors de la cytolyse). En utilisant un modèle murin exprimant un TCR transgénique nous avons pu suivre le déroulement de la fonction de cytolyse de lymphocytes cytotoxiques en temps réel par video microscopies. / Cytolysis is a major function used by the immune system's effectors to kill infected or tumor cells. Cytolysis depends on the pore forming protein perforin and the secretion of proteases of the granzyme family. Granzymes, including granzyme B (GZMB) have pro-apoptotic features and induce target cell death. Several complementary pathways are triggered by granzymes to ensure efficient cytolysis. It remains difficult to directly observe cytolysis during in vivo immune responses under physiological conditions. In this PhD we developed a new model to visualize cytolytic function in real time by expression of a fusion protein: GZMB-tdTomato. Results obtained from retroviral transduction showed that the fusion protein is correctly expressed in cytolytic vesicles, which became fluorescent. We then constructed a new mouse model by homologous recombination (Knock In) that express GZMB-tdTomato substituted for the native GZMB. The fusion protein conserves the catalytic activity of GZMB and its features (expression, maturation, secretion conditions) and remains active after its passage into target cells. Using TCR transgenic OTI cells, we followed the sequence of events of cytolysis from lymphocytes in real time by videomicroscopy. We also observed the cytolytic vesicles relocalization towards the cell contact zone and the death of target cell by cytolysis. Finally, we studied in vivo differentiation of naïve lymphocyte to cytolytic effector cells (the acquisition of cytolysis) and target cell death after bacterial infection.

Les cancers du sein sont divisés en sous types définis par leur histologie, leur prolifération et l’expression du récepteur aux œstrogènes ER. Notre étude porte sur le gène FOXA1 dans le sous-type luminal caractérisé par des cellules bien différenciées, peu prolifératives et exprimant fortement les protéines FOXA1 et ER. Des études suggèrent que FOXA1 est impliqué dans le développement de la glande mammaire, dans la différenciation et la prolifération des cellules mammaires. Dans ce cadre, mon projet de thèse s’articulait autour de trois points ; développer un Knock-In au niveau du gène FOXA1, identifier le rôle de FOXA1 dans la différenciation mammaire et enfin rechercher le rôle des facteurs ER et FOXA1 dans la résistance à l’hormonothérapie. De nombreux tests d’intégration ciblée ont été réalisés à l’aide de différentes matrices de recombinaison et de nucléases spécifiques, les ZFNs. Aucune intégration ciblée n’a finalement été observée. Nous avons montré qu’en fonction du contexte cellulaire, FOXA1 jouait différents rôles dans la différenciation cellulaire et l’expression de la molécule d’adhérence E-Cadhérine. Ces résultats suggèrent que FOXA1 influence l’agressivité tumorale suivant le contexte cellulaire. Nous avons également identifié une amplification d’ER et de FOXA1 dans les cellules tumorales résistantes à l’hormonothérapie par une étude génomique. Les tests in vitro ont montré que la surexpression de FOXA1 augmenterait bien la résistance au fulvestrant mais la surexpression d’ER aurait l’effet inverse, suggérant l’implication d’autres facteurs. De futures recherches nous permettront d’identifier ces facteurs et de préciser les rôles de FOXA1 et d’ER dans la différenciation luminale, l’agressivité tumorale et dans la réponse cellulaire à l’hormonothérapie / Breast cancers are divided into subtypes defined by their histology, proliferation and expression of estrogen receptor ER. Our study focuses on the FOXA1 gene in the luminal subtype characterized by well-differentiated cells, low proliferative and strongly expressing FOXA1 protein and ER. Studies suggest that FOXA1 is involved in the mammary gland development and in the differentiation and proliferation of mammary cells. In this context, my thesis project was structured around three points, develop a knock-in at the FOXA1 gene, identify the role of FOXA1 in mammary differentiation and finally explore the role of ER and FOXA1 in resistance to hormone therapy. Many targeted integration tests were performed using different matrix of recombination and specific nucleases, the ZFNs. No direct integration was finally observed. We showed that depending on the cell context, FOXA1 played different roles in cell differentiation and expression of E-cadherin, an adhesion molecule. These results suggest that FOXA1 influence tumor aggressiveness depending on the cell context. We also identified amplification of ER and FOXA1 in tumor cell resistant to hormone therapy by a genomic study. Surprisingly, in vitro tests showed that overexpression of FOXA1 increased resistance to fulvestrant whereas overexpression of ER would have the opposite effect, suggesting the involvement of other factors. Future research will allow us to identify these factors and to clarify the roles of ER and FOXA1 in luminal differentiation, tumor aggressiveness and response to hormone therapy.

Engine knock has long been a well recognized phenomenon in the automotive industry. Detecting engine knock opens up the possibility for an indirect feedback of the engine's internal combustion without installing a pressure transducer inside the cylinder. Knock detection has mainly been used for spark advance control, making it possible to control the engine close to its knock limit in search for the optimal ignition timing. This application has to a lesser extent been applied to lightweight two-stroke engines, which is the focus of this study. The investigation features a modern chainsaw engine whose knock characteristics were first determined with a pressure transducer. The structural vibrations originating from the engine knock are filtered out of the signal from a remote located accelerometer. The knock intensity is compared with the signal from the pressure transducer which shows a correlation with an accepted extent between the two sensors. Parameters that affect the knock intensity have also been investigated. These include engine temperature, different types of fuel and ignition timings.

Ein durch „Ca2+ Release Activated Ca2+ (CRAC)“-Kanal vermittelter Ca2+-Einstrom ist unverzichtbar für die vollständige Aktivierung von T-Zellen und eine produktive Immunantwort. Im Jahr 2006 führte die Entdeckung des transmembranen Proteins Orai1, einer porenbildenden Untereinheit des CRAC-Kanals, zu einem besseren Verständnis dieses Signalweges. Eine Mutation in Orai1 hat durch die Aufhebung der CRAC-Kanal Funktion eine schwere kombinierte Immundefizienz (SCID) zur Folge (Feske, S. et al. 2006). Die im Rahmen dieser Arbeit präsentierten Experimente hatten die nähere Erforschung der Rolle von Orai1 in Bezug auf die Aktivierung und Entwicklung von Lymphozyten sowie auf die pathogenetische Bedeutung für humane Immundefektsyndrome zum Ziel. So konnte hier durch das Sequenzieren genomischer DNA mehrerer SCID-Patienten eine neue Mutation in Orai1 aufgedeckt werden. Mithilfe intrazellulärer Durchflusszytometrie und Real-Time-PCR gelang es, die Expression von Orai1 auf humanen und murinen Immunzellen, einschließlich T- und B-Lymphozyten, nachzuweisen. Darüber hinaus wurden Orai1 „knock-in“ Mäuse analysiert, welche transgen für eine bei zwei SCID-Patienten gefundene Mutation (R91W) (Feske, S. et al. 2006) sind. Dadurch war es möglich die Funktion von Orai1 und die des CRAC-Kanal vermittelten Ca2+-Einstroms für die Entwicklung und Aktivierung von Lymphozyten zu analysieren. Diese transgenen Mäuse stellen das zu diesem Zeitpunkt erste Tiermodell dar, mit dessen Hilfe die Rolle von CRAC-Kanälen in vivo studiert werden kann.

Pocket proteins and E2F transcription factors regulate the expression of cell cycle associated genes and play a central role in the coordination of cell division, differentiation, and apoptosis. Disorders of these pathways contribute to the development of various human tumor entities. Despite intensive research in the field of cell cycle regulation many details are not yet understood. The LIN complex (LINC / DREAM) is a recently discovered human multiprotein complex, which dynamically interacts with pocket proteins and E2F transcription factors. An essential component of the LIN complex is the LIN9 protein. In order to obtain a better insight into the function of this protein in cell cycle regulation and tumorigenesis, a conditional Lin9 knockout mouse model was established in our laboratory. The primary objective of this study was the phenotypic characterization of embryonic fibroblasts (MEFs) from these mice. Shortly after inactivation of Lin9 cell proliferation was massively impaired. Multiple types of mitotic defects such as structural abnormalities of the spindle apparatus, aberrant nuclei, failed nuclear segregation and cytokinesis failure have been observed in Lin9-depleted cells leading to a dramatic increase in polyploid and aneuploid cells. Ultimately these serious aberrations result in premature cellular senescence. If the senescence of Lin9-deficient cells is overcome by the Large T antigen the cells can adhere to the loss of Lin9, but show severe genomic instability and grow anchorage-independently in soft-agar as a sign of oncogenic transformation. In the second part of the thesis the gene expression of Lin9-deficient cells was assessed by quantitative real time PCR analyses to determine, whether the mitotic abnormalities are caused by transcriptional defects. Here a significant reduction of mitotic gene expression was observed in Lin9-depleted cells. Additionally chromatin immunoprecipitation experiments were performed to clarify the underlying molecular mechanisms. Compared to control cells epigenetic alterations at the promoters of mitotic target genes with regard to activating histone modifications were found in Lin9-deficient MEFs. In the last section of this study, the effects of Lin9 heterozygosity were analyzed. Lin9 heterozygous MEFs showed normal proliferation, although expression of different mitotic genes was slightly reduced. It appeared, however, that the mitotic spindle checkpoint of Lin9 heterozygous MEFs is weakened and thus over several cell generations an increase in polyploid cells was observed. Soft-agar assays showed that Lin9 heterozygosity contributes to oncogenic transformation. Taken together, these results document a crucial role of LIN9 in the regulation of cell cycle-associated gene expression. LIN9 is an essential factor for cell proliferation on one hand, while at the same time it functions as a tumor suppressor. / Pocket-Proteine und E2F-Transkriptionsfaktoren regulieren die Expression von Zellzyklus-assoziierten Genen und spielen eine zentrale Rolle bei der Koordination der Zellteilung, Differenzierung und Apoptose. Störungen dieser Signalwege tragen zur Entstehung zahlreicher Tumorentitäten beim Menschen bei. Trotz der intensiven Untersuchung der Zellzyklusregulation sind viele Details noch unverstanden. Der LIN-Komplex (LINC / DREAM) ist ein kürzlich entdeckter humaner Multiprotein-komplex, welcher dynamisch mit Pocket-Proteinen und E2F-Transkriptionsfaktoren interagiert. Eine essentielle Komponente des LIN-Komplexes ist das LIN9-Protein. Um die Funktion dieses Proteins bei der Zellzyklusregulation und Tumorentstehung genauer untersuchen zu können, wurde in unserer Arbeitsgruppe ein konditionelles Lin9-Knockout-Mausmodell etabliert. Primäres Ziel der Arbeit war es, den Phänotyp embryonaler Fibroblasten (MEFs) aus diesen Mäusen zu charakterisieren. Bereits kurz nach Inaktivierung von Lin9 konnte ein stark verlangsamtes Zellwachstums beobachtet werden. In Lin9-depletierten MEFs wurden multiple mitotische Defekte detektiert, die u. a. strukturelle Auffälligkeiten des Spindelapparates, aberrante Zellkerne, Störungen der Chromosomensegregation sowie zytokinetische Defekte umfassen und in einer dramatischen Zunahme polyploider und aneuploider Zellen resultieren. Im Langzeitverlauf führen diese erheblichen Aberrationen zu einer vorzeitigen zellulären Seneszenz. Wird diese durch das Large T-Protoonkogen durchbrochen, können sich MEFs an den Verlust von Lin9 adaptieren, zeigen dann jedoch eine hochgradige genomische Instabilität und Substrat-unabhängiges Wachstum im Weichagar als Zeichen onkogener Transformation. Im zweiten Abschnitt der vorliegenden Arbeit wurde die Genexpression in Lin9-defizienten MEFs mittels quantitativer Real Time-PCR untersucht um zu klären, ob die beschriebenen Defekte auf Veränderungen der transkriptionellen Aktivität zurück-zuführen sind. Dabei wurde eine erhebliche Reduktion der Expressionslevel mitotischer Gene nach Verlust von Lin9 beobachtet. Des Weiteren wurden zur Klärung der zu Grunde liegenden molekularen Mechanismen Chromatin-Immunpräzipitations-Experimente (ChIP) durchgeführt. Im Vergleich zu Kontrollzellen wurden dabei in Lin9-defizienten Zellen signifikante epigenetische Veränderungen bezüglich aktivierender Histon-Modifikationen an den Promotoren mitotischer Lin9-Zielgene festgestellt. Im letzten Abschnitt der Arbeit sollten die Auswirkungen des heterozygoten Verlustes von Lin9 analysiert werden. Dabei zeigte sich, dass Lin9-haploinsuffiziente Zellen normal proliferieren, obwohl die Expression verschiedener G2/M-Gene leicht vermindert war. Es wurde jedoch eine Schwächung des mitotischen Spindelkontrollpunktes und in der Folge über mehrere Zellgenerationen eine Zunahme polyploider Zellen beobachtet. Mit Weichagar-Assays konnte gezeigt werden, dass bereits der heterozygote Verlust des Lin9-Gens zur onkogenen Transformation beiträgt. Zusammengenommen dokumentieren diese Studien, dass LIN9 eine entscheidende Bedeutung bei der Regulation von Zellzyklus-assoziierten Genen spielt und sowohl einen essentiellen Faktor für die Zellproliferation darstellt als auch durch die Gewährleistung genomischer Stabilität tumorsuppressive Eigenschaften aufweist.

<p>In the report a model using a reduced reaction analysis has been used to see if it is possible to predict knock. The model is based on n-heptane combustion, but it is used for iso-octane. The model was supposed to be able to adapt to different fuels, but it is shown to be unable to do so. Further, the model has been compared to an existing method for predicting knock, known as knock index, to see if any improvements could be made. When comparing the model to the knock index, it has shown that no big advantages can be found using the new model. It is more time consuming and is not able to work with simulated input, instead of measured. It can however predict if knock occurs with a good reliability, but compared to the knock index it is not an improvement.</p>

In the report a model using a reduced reaction analysis has been used to see if it is possible to predict knock. The model is based on n-heptane combustion, but it is used for iso-octane. The model was supposed to be able to adapt to different fuels, but it is shown to be unable to do so. Further, the model has been compared to an existing method for predicting knock, known as knock index, to see if any improvements could be made. When comparing the model to the knock index, it has shown that no big advantages can be found using the new model. It is more time consuming and is not able to work with simulated input, instead of measured. It can however predict if knock occurs with a good reliability, but compared to the knock index it is not an improvement.

In response to the global concerns towards oil scarcity and climate change, the automotive industry is currently focusing on improving fuel economy and reducing exhaust emissions. Modern downsized gasoline engines that come with a package that includes a boosting system, variable valve train and direct fuel injection system is effective for fuel economy improvement and emission reduction. However, the knocking issue becomes severe at high load operations as a result of the high intake boosting pressure. In regard to the part load conditions, the gas exchange process requires extra work to draw in air into the cylinder due to a lower amount of pressure in the intake manifold caused by the restriction of the throttle plate. The Miller cycle is regarded as a potential strategy of knock control for downsized gasoline engines. Extensive works have sought to examine the performance improvement via the Miller cycle, yet only limited research has been conducted on the manner in which it can influence knock suppression. The focus of this thesis is to investigate early and late intake valve closing timings in terms of how they affect the compression process, the ability to suppress engine knock and meet the power output required at high loads for spark-ignited gasoline engines. Apart from that, this research also demonstrates the Miller cycle potential by utilising fully variable valve timing in controlling the load at the part load condition without using a throttle. The early intake valve closing with different valve lifts was tested in order to investigate the impact during the gas exchange process, particularly the pumping losses and the potential to improve fuel economy. This study includes both experimental and simulation studies. A Lotus single-cylinder research engine referred to as SCORE was mainly used for the experimental component of the study. The simulation work was conducted using a one-dimensional spark ignition engine model built in the Ricardo WAVE software for naturally-aspirated and downsized engines. The engine model values are validated against the experimental values from the Lotus SCORE and Lotus SABRE engines. The combustion model with a reduced kinetics mechanism was validated using a Rover K-series engine. A broad matrix of the engine operations has been investigated combining a variety of engine speeds and engine loads. The Miller cycle effects on knock suppression in a downsized engine environment have been investigated in three parts, namely the Miller cycle at different speed-load, knock suppression with extreme Miller cycle, and knock analysis with combustion kinetics. Through the works, the Miller cycle has demonstrated its capability to suppress engine knocking in a more efficient manner as opposed to the standard engine operation. This is contributed by the fact that early and late intake valve closings could affect the end gas condition at the end of the compression stroke, thus making it possible to suppress the engine knocking. The experimental study for controlling load without using throttle under the naturally-aspirated condition found that the Miller cycle with an early intake calve closing strategy able to improve fuel consumption by reducing pumping losses. The downsized engine condition, which has been evaluated via modelling work, also showed an improved performance trend using the unthrottled Miller cycle strategy. The open cycle and close cycle efficiencies have improved through the Miller cycle implementation. The contribution of this work is made in order to establish the comparison of the Miller cycle strategy in suppressing knocking between the early intake valve closing and late intake valve closing under a boosted environment. For the part load condition of the downsized engine, the research contributes to the existing body of knowledge by comparing the throttle-less Miller cycle and the standard throttled operation as a load control strategy.

This study investigates the impact of employing the Miller cycle on a high specific power downsized gasoline engine by means of Early Intake Valve Closing (EIVC) and Late Intake Valve Closing (LIVC). This investigation assesses the potential for the Miller cycle to improve fuel economy at part load points, as well as high load points with significantly elevated boost pressures (Deep Miller) of up to 4 bar abs. The impact of geometric Compression Ratio (CR) and Exhaust Back Pressure (EBP) has also been investigated. The knock mitigating qualities of Deep Miller have been assessed, and its ability to increase maximum engine load explored. Low Speed Pre-ignition (LSPI) and autoignition tendencies with reduced coolant flow rates and with aged and new fuels have also been studied. This study comprises both experimental and analytical studies. A Ricardo Hydra single cylinder thermodynamic engine was developed and used for the experimental component of the study. This engine features a high specific power output (120kW/l) cylinder head from the Mahle 1.2l 3 cylinder aggressively downsized engine. The analytical component was carried out using a 1-dimensional GT-Power model based on the Ricardo Hydra experimental engine. A Design of Experiments (DoE) based test plan was adopted for this analytical study. The experimental study found that EIVC was the optimal strategy for improving fuel economy at both part-load and high-load conditions. LIVC yielded a fuel economy penalty at part-load operations and a fuel economy improvement at high-loads. The unexpected part-load LIVC result was attributed to the engine breathing dynamics of the experimental engine. The analytical study found moderate LIVC to be the optimal strategy at lower speeds, unless compensation for the increased degree of scavenging experienced with EIVC was compensated for, in which case EIVC was optimum. At higher speeds EIVC was found to be optimum regardless of whether or not compensation for scavenging was employed. It was generally found that less sensitivity to EBP was exhibited the more extreme the EIVC and LIVC. It was also found that a higher geometric CR could be tolerated with extreme EIVC and LIVC, and a fuel economy benefit could be obtained through the elevation of Geometric CR.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20793号 / 医博第4293号 / 新制||医||1025(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 妻木 範行, 教授 鈴木 茂彦, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM