No description available.

Primordial follicles are the first small follicles to appear in the mammalian ovary. Women are born with a fixed number of primordial follicles in the ovaries. Once formed, the pool of primordial follicles serves as a source of developing follicles and oocytes. The first aim of this thesis was to investigate the functional role of the intra-oocyte signaling pathways, especially the phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) pathways in the regulation of primordial follicle activation and survival. We found that a primordial follicle remains dormant when the PI3K and mTORC1 signaling in its oocyte is activated to an appropriate level, which is just sufficient to maintain its survival, but not sufficient for its growth initiation. Hyperactivation of either of these signaling pathways causes global activation of the entire pool of primordial follicles leading to the exhaustion of all the follicles in young adulthood in mice. Mammalian oocytes, while growing within the follicles, remain arrested at prophase I of meiosis. Oocytes within the fully-grown antral follicles resume meiosis upon a preovulatory surge of leutinizing hormone (LH), which indicates that LH mediates the resumption of meiosis. The prophase I arrest in the follicle-enclosed oocyte is the result of low maturation promoting factor (MPF) activity, and resumption of meiosis upon the arrival of hormonal signals is mediated by activation of MPF. MPF is a complex of cyclin dependent kinase 1 (Cdk1) and cyclin B1, which is essential and sufficient for entry into mitosis. Although much of the mitotic cell cycle machinery is shared during meiosis, lack of Cdk2  in mice leads to a postnatal loss of all oocytes, indicating that Cdk2 is important for oocyte survival, and probably oocyte meiosis also. There have been conflicting results earlier about the role of Cdk2 in metaphase II arrest of Xenopus  oocytes. Thus the second aim of the thesis was to identify the specific Cdk that is essential for mouse oocyte meiotic maturation. We generated mouse models with oocytespecific deletion of Cdk1  or Cdk2  and studied the specific requirements of Cdk1 and Cdk2 during resumption of oocyte meiosis. We found that only Cdk1 is essential and sufficient for the oocyte meiotic maturation. Cdk1 does not only phosphorylate the meiotic phosphoproteins during meiosis resumption but also phosphorylates and suppresses the downstream protein phosphatase 1, which is essential for protecting the Cdk1 substrates from dephosphorylation.

Herpes Simplex Virus Type 1 (HSV-1) establishes latent infections in neurons in vivo and lytic infections in epithelial cells and fibroblasts. During latent infections, HSV-1 transcription is restricted and the genomes are not replicated. Latent HSV-1 genomes are chromatinized, such that digestion with micrococcal nuclease (MCN) releases DNA fragments with sizes characteristic of nucleosomal DNA. During lytic infections, in contrast, all HSV-1 genes are expressed, the genomes are replicated, and their digestion produces primarily heterogeneously sized fragments. However, as evaluated by ChIP assays, HSV-1 DNA interacts with histones during lytic infections, although in most cases only a small percentage of HSV-1 DNA co-immunoprecipitates with histones (or is cleaved to nucleosome sizes following MCN digestion). Therefore, although current models propose that chromatin regulates HSV-1 transcription, it remains unclear how the association of histones with only a small percentage of HSV-1 DNA can globally regulate viral transcription. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. My objective was therefore to evaluate the biophysical properties of the HSV-1 DNA-containing complexes during lytic infection. Differing from pervious studies, however, I used classical chromatin purification techniques. I show that most HSV-1 DNA is in unstable nucleoprotein complexes and, consequently, more accessible to MCN than DNA in cellular chromatin. This HSV-1 DNA is protected from MCN redigestion only after crosslinking, similar to unstable cellular nucleosomes. HSV-1 DNA is in such complexes throughout lytic infection. Using unrelated small-molecule inhibitors, I further show that inhibition of HSV-1 transcription is associated with a decrease in MCN accessibility of HSV-1 DNA. Roscovitine, a cyclin-dependent kinase inhibitor, prevents activation but not elongation of IE, E, and L HSV-1 transcription. Consistent with a functional association between accessibility and transcription, roscovitine only decreases the accessibility of DNA templates of which it also inhibits transcription, independent of specific promoter sequences. In summary, I show that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infection and that accessibility to HSV-1 DNA likely plays a key role in regulating HSV-1 transcription.

No description available.

No description available.

Les cellules cancéreuses sont sujettes à des défauts de reformation de l'enveloppe nucléaire (EN) après la mitose. BAF est l'une des premières protéines recrutées sur les chromosomes pour initier la reformation de l’EN. Chez l'humain, le recrutement de BAF nécessite sa déphosphorylation par la phosphatase PP2A et Ankle2, une protéine du réticulum endoplasmique (RE) interagissant avec PP2A. Cependant, les fonctions d’Ankle2 dans la reformation de l’EN ne sont pas complètement comprises. Pour les étudier, notre laboratoire utilise la drosophile comme organisme modèle. On ne sait pas si Ankle2 de drosophile fonctionne dans le NER. Nous avons constaté qu’Ankle2 est nécessaire au recrutement de BAF pour le réassemblage du noyau après la mitose chez la drosophile. Pour mieux comprendre son fonctionnement, nous avons identifié des protéines avec lesquelles BAF interagit : PP2A, Vap33 (une protéine du RE) et certaines Kinases Dépendantes des Cyclines (CDK). Nous avons cartographié les régions d’Ankle2 impliquées dans ces interactions protéiques grâce à une analyse mutationnelle, des co-purifications par affinité et des pulldowns GST. Nous avons ensuite généré des mutants d’Ankle2 spécifiquement déficients pour des interactions et testé leur capacité à sauver la prolifération et la reformation de l’EN dans des cellules où Ankle2 endogène est déplété. Nos résultats indiquent que l'interaction entre Ankle2 et PP2A est essentielle pour sa fonction dans la reformation de l’EN. Une analyse biochimique suggère qu’Ankle2 fonctionne comme une sous-unité régulatrice de PP2A. En utilisant une approche phosphoprotéomique, nous avons confirmé que la déphosphorylation de BAF dépend d’Ankle2 et nous avons aussi identifié de nouveaux substrats potentiels du complexe PP2A-Ankle2. Nous concluons que le complexe PP2A-Ankle2 est nécessaire à la déphosphorylation de BAF et à son recrutement pour le réassemblage du noyau. Les expériences en cours permettront de déterminer les exigences d'autres interactions d’Ankle2 pour ses fonctions dans la reformation de l’EN. La suite de ces travaux impliquera l’étude de la régulation de nouveaux substrats de PP2A-Ankle2 impliqués dans ce processus. Une reformation de l’EN défectueuse peut provoquer une 4 micronucléation, ce qui peut déclencher une réponse immunitaire innée. La perturbation de la reformation de l’EN dans les cellules cancéreuses pourrait donc être bénéfique dans le contexte de l’immunothérapie. / Cancer cells are prone to defects in Nuclear Envelope Reformation (NER) after mitosis. BAF is one of the first proteins recruited on chromosomes to initiate NER. In humans, BAF recruitment requires its dephosphorylation by PP2A and Ankle2, a PP2A-interacting protein of the endoplasmic reticulum (ER). However, the functions of Ankle2 in NER are incompletely understood. Our lab uses Drosophila as a model system. Whether Drosophila Ankle2 functions in NER is unknown. We found that Ankle2 is required for BAF recruitment to reassembling nuclei in Drosophila. To better understand how it functions, we identified its interactors, which include PP2A, Vap33 (an ER protein) and Cyclin-Dependent Kinases (CDKs). We mapped the regions of Ankle2 involved in these protein-protein interactions through a mutational analysis, affinity co-purifications and GST pulldowns. We then generated mutant forms of Ankle2 defective in individual interactions and tested their ability to rescue proliferation and NER in cells depleted from endogenous Ankle2. Our results indicate that the interaction of Ankle2 with PP2A is essential for its function in NER. A biochemical analysis suggests that Ankle2 functions as a regulatory subunit of PP2A. Using a phosphoproteomic approach, we confirmed that BAF dephosphorylation depends on Ankle2 and also identified novel candidate substrates of the PP2A-Ankle2 complex. We conclude that PP2A-Ankle2 complex is required for BAF dephosphorylation and recruitment to reassembling nuclei. Ongoing experiments will determine the requirements of other interactions of Ankle2 for its functions in NER. Future work will explore the regulation of novel PP2A-Ankle2 substrates in this process. Defective NER can cause micronucleation, which can elicit an innate immune response. Disrupting NER in cancer cells could be beneficial in the context of immunotherapy.

No description available.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25176号 / 医博第5062号 / 新制||医||1071(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 武藤 学, 教授 萩原 正敏, 教授 永井 純正 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Brain metastases (BMs) present a formidable obstacle across various primary cancer types, notably small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), melanomas, and breast cancers. In this investigation, we aim to evaluate the potential of genotype-guided targeted therapy while addressing the challenges of co-existing genomic alterations frequently encountered in BMs. This research explores the efficacy of adagrasib (MRTX849), a KRAS G12C inhibitor, and abemaciclib, a CDK 4/6 inhibitor, both individually and in combination against BMs originating from NSCLC cell lines harboring KRAS G12C and CDKN2A mutations. Utilizing a diverse array of methodologies encompassing cell viability assays, cell death assays, western blot analyses, and in vivo xenograft models, we elucidate both the therapeutic potential and underlying mechanisms. Distinct responses to adagrasib and abemaciclib monotherapies were observed across two different cell lines, underscoring the necessity for tailored treatment strategies. While adagrasib exhibited variable efficacy, abemaciclib consistently inhibited CDK 4/6 activity. Notably, the combination therapy demonstrated synergistic effects, suggesting a promising approach for enhanced therapeutic outcomes. Our findings from both in vitro assays and western blot analyses corroborate targeted pathway inhibition, although the observed pathway reactivation underscores the importance of optimizing dosing strategies. In vivo studies further support our in vitro findings, demonstrating efficacy but also raising concerns regarding toxicity with combination therapy. Pharmacokinetic / pharmacodynamic (PK/PD) analyses underscore potential advantages of combination therapy in terms of systemic exposure and brain penetration. Despite histological evidence of therapeutic effects, discrepancies between in vivo and in vitro caspase-dependent apoptosis results highlight the complexity of tumor biology and the challenges of translation. By Focusing on personalized treatment approaches and addressing therapeutic hurdles, this work establishes the foundation for clinical investigation in advancing the management of BMs and improving treatment outcomes in NSCLC patients.