At present reproductive biotechnology methods are on the rise, but their development and application in the broader management of reproduction is dependent on obtaining a sufficient number of quality oocytes cultured in vitro. The prerequisite for this requirement is the creation of the optimal conditions in the course of culturing oocytes. Understanding and knowledge of the processes that occur in oocyte during maturation is an important and necessary condition for optimizing the process of culturing oocytes in vitro and gaining a sufficient number of good quality oocytes in metaphase II of meiotic division. A large number of mechanisms that affect and control oocyte maturation are known, however it cannot be claimed that this process has been fully explained and studied. One factor which has a potential role in the regulation of meiotic maturation of oocytes is gasotransmitter hydrogen sulfide (H2S), a critical signaling molecule of endogenous origin. The study of H2S led to the hypothesis that H2S actively influences the course of meiotic maturation of pig oocytes by regulating key signaling cascades. The aim of this work was to determine the involvement of H2S in the regulation of the MEK1-MAPK signaling cascade, responsible for the initiation and progress of the meiotic maturation of oocytes and the MEK1-PARP-1 cascade as signaling that supports cell viability. For this purpose, pig oocytes cultured in modified media were used, supplemented with a specific combination of enzyme inhibitors (3Ki) or in a culture medium with donor H2S. The ocytes were then subjected to immunocytochemistry staining, fluorescence microscopy and image analysis. The results show that H2S is involved in the regulation of meiotic maturation. It confirmed the hypothesis of the endogenous production of H2S in the course of the meiotic maturation of pig oocytes and the influence of the MAPK signaling cascade. Based on the results, it is however likely that the MEK1-PARP-1 signaling cascade is not affected by H2S, unlike MAPK signaling, comprising the mentioned MEK1 as superior kinase. MAPK kinase activity is significantly lower in oocytes after treatment 3Ki. Further experiments are for a detailed understanding of these regulatory pathways and for the proper verification of the mechanism of the effects of H2S necessary, in particular for a full understanding of the target control factors by the post-translational modification of S-sulfhydration.

Extracellular signal-regulated kinases 1/2 (ERK1/2) are important kinases regulating cell proliferation and cell migration, and have been established as therapeutic targets for cancer treatment. Previously, we found that ERK1 phosphorylates histone deacetylase 6 (HDAC6) to regulate its enzymatic activity. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we have discovered that ERK1/2 are acetylated proteins and shown that HDAC6 manipulates ERK1’s kinase activity via deacetylation. We demonstrated that both ERK1 and ERK2 interact with HDAC6 physically. We showed that the acetylation level of GST-ERK1/2 increased in a dose- and time-dependent manner upon treatment with a pan-HDAC inhibitor, Trichostatin A. Furthermore, the treatment by HDAC6-specific inhibitor, ACY-1215, also increased the level of acetylated GST-ERK1/2. We also noted that ERK1/2 acetylation levels increased in HDAC6-knockout mouse embryonic fibroblasts and in HDAC6-knockdown A549 cell lines compared with controls. In addition, we determined that acetyltransferases CBP and p300 acetylate ERK1/2. We have identified novel acetylation sites located in ERK1 and ERK2 by mass-spectrometry analysis. Among these acetylation sites, ERK1 lysine 72 acetylation status is related to ERK1 phosphorylation. The acetylation-mimicking mutant exhibits a decreased kinase activity toward ELK1, while the deacetylation-mimicking mutant exhibits a similar level of kinase activity as the wild-type ERK1, suggesting that acetylation/deacetylation alters ERK1 enzymatic activities. Taken together, our results suggest that HDAC6 may regulate ERK1’s kinase activity via deacetylation of its lysine 72 residue.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic, inflammatory condition, characterised by airflow limitation. The use of glucocorticoids (GC) as an anti-inflammatory treatment in COPD has limited clinical benefits, and as such, new treatments are needed. Identifying key pathways involved in the inflammatory response in COPD may enable the development of novel treatments. The aims of this thesis were to examine the steroid sensitivity of an in vitro mixed sputum culture cell model, comparing COPD cells to smoking and non-smoking controls, examine expression of the intracellular signalling molecule p38 Mitogen Activated Protein Kinase (MAPK) in COPD lungs compared with controls, examine the GC and p38 MAPK inhibitor and dual therapy sensitivity of a bronchial epithelial cell line and finally, to understand the mechanisms by which a p38 MAPK inhibitor in combination with a GC synergistically inhibit pro-inflammatory mediator production in a bronchial epithelial cell line. Dexamethasone inhibits mixed sputum cell pro-inflammatory mediator release, with no differences in sensitivity observed between COPD and control cells. Isolated sputum neutrophils demonstrate modest sensitivity to dexamethasone, which is in contrast to blood neutrophils. There are increased numbers of cells positive for activated p38 MAPK in COPD lungs compared with controls, specifically localised to follicular B and CD8+ T cells, bronchial epithelial cells and alveolar and sputum macrophages. Lung and sputum neutrophils are devoid of activated p38 MAPK, and a pharmacological p38 MAPK inhibitor has no effect on pro-inflammatory mediator production from these cells. This is in contrast to blood neutrophils, whereby p38 MAPK activation can be induced following LPS stimulation and in vitro cell culture, and pro-inflammatory mediator release is inhibited by a p38 MAPK inhibitor. Dexamethasone and birb 796 inhibit stimulated pro-inflammatory mediator release from a bronchial epithelial cell line in a dose-dependent manner. Sensitivity to either drug is dependent on stimuli and the pro-inflammatory mediator analysed. There is additive and synergistic inhibition of pro-inflammatory mediator production when combination therapy comprising dexamethasone and birb 796 is used compared with either drug alone. This may be due to Birb 796 enhancing dexamethasone-mediated nuclear translocation of the glucocorticoid receptor, which may enhance the GC-mediated anti-inflammatory effects. Combination therapy may therefore be a useful therapeutic in the treatment of COPD.

Plants are sessile organisms and constantly encounter a myriad of pathogens; therefore, they rely on highly effective defense system for their survival. Our understanding of how plant immunity is triggered and regulated has seen tremendous progress over the last two decades, with many important players identified in the model systems, Arabidopsis thaliana. Mitogen activated protein kinases play a central role in signal transduction in biotic and abiotic stresses. MAPK pathways are regulated by three-interlinked protein kinases (MAPKKK, MAPKK, MAPK), which are sequentially activated by phosphorylation. The activation of the three MAPKs MPK3, MPK4 and MPK6 is one of the earliest cellular responses following pathogen attack leading to the phosphorylation of appropriate cytosolic or nuclear targets to regulate cellular processes. However, only few targets of MPK3, MPK4 and MPK6 have been identified and validated so far and many MAPK substrates remain to be discovered. We performed largescale phosphoproteomics on mock treated and flg22 treated WT and the three loss-of-function mutants mpk3, mpk4 and mpk6 to identify novel MAPKs substrates and their cellular functions in response to pathogen attack. We identify and validated some of the differentially phosphorylated cytosolic and chromatin targets of MPK3, MPK4 and MPK6. DEK2, a nuclear protein involved in multiple chromatin-related processes, was identified in the phosphoproteomics screen as an in vivo target of MPK6 and it interacts in planta and is phosphorylated in vitro by the three immune MAPKs. dek2 loss-of-function mutants were susceptible to bacterial as well as fungal pathogens. Additionally, transcriptome data of the dek2-1 mutant show that DEK2 is a transcriptional repressor inclusive of defense related genes and hormone synthesis and signaling genes. We determined that DEK2 is a reader of the histone mark, H3K9me1, by Microscale thermophoresis. From ChIP-Seq analysis, DEK2 was found to be enriched at class I TCP binding motif regions. We further need to determine whether DEK2 binds to TCP transcription factors directly or indirectly. Finally, based on our data we postulate a hypothetical working model for the function of DEK2 as a transcriptional repressor and a reader of H3K9me1 mark.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24914号 / 農博第2577号 / 新制||農||1103(附属図書館) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 佐々木 努, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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