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京都大学 / 新制・課程博士 / 博士(農学) / 甲第23247号 / 農博第2454号 / 新制||農||1084(附属図書館) / 学位論文||R3||N5337(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Indiana University-Purdue University Indianapolis (IUPUI) / The degradation of the resin-dentin interface after restoration placement is multifactorial and can be attributed in part to matrix metalloproteinases (MMPs) enzymes associated with recurrent and secondary caries progression. This dissertation aimed to synthesize and characterize the effects of Epigallocatechin-3-gallate (EGCG) from green tea extract as an MMP-inhibitor loaded into a dental adhesive using slow therapeutic compound release nanotubes as a reservoir to allow sustained and slow release. Loading efficiency and drug release were evaluated using a UV-vis spectrometer. The effects on the degree of conversion (DC), polymerization conversion (PC), and Vickers Micro-Hardness (VHN) tests were performed. MMP mediated β-casein (bCN) cleavage rate was used to determine the potency of the eluates contained EGCG to inhibit MMP-9 activity. The results indicated that HNTs could hold about 21.35% (±4.2%) of the EGCG used in the encapsulation process. The addition of 7.5% HNT or 7.5% EGCG-encapsulated HNT adhesive groups did not alter the curing efficiency indicated by the degree of conversion, polymerization conversion, and surface hardness results compared to the control group (p> 0.05). A statistically significant influence of adding HNTs was found to slow down the EGCG release measured up to 8 weeks (p< 0.05). There was a significant decrease in the degradation of β-casein mediated by pre-activated MMP-9 exposed to eluates from EGCG adhesives compared to non-EGCG adhesive groups (p< 0.05). The results suggested that using HNTs for EGCG encapsulating can remedy the negative impact of EGCG on the adhesive’s polymerization and still have the MMP-inhibitory effect and longer release period. Dentin adhesive containing EGCG-encapsulated HNT may contribute to the long-term preservation of restorations through slow and controlled release to maintain the dentin-resin interface's integrity by inhibiting MMP activity.

<P> Multi-site phosphorylation of protein targets by specific kinases is a common event used to propagate biological messages through signal transduction pathways in the context of the cellular environment and is a vital regulatory mechanism for many metabolic processes. Recent advances in the study of the protein glycogen synthase kinase-3 (GSK-3) have shed some light on the intricate role this enzyme plays within the framework ofmammalian cellular metabolism. Abnormal behaviour of GSK -3 profoundly impacts cellular function, and is implicated in Alzheimer's disease and the development of Type II Diabetes. A key issue in assaying the activity of GSK-3 is the ability to distinguish between singly and multiply phosphorylated substrates, as this enzyme has the ability to selectively phosphorylate a previously phosphorylated (primed) substrate. Given the serious nature of the disorders caused by the dysfunction of this kinase, high throughput screening of specific inhibitors from compound libraries is urgently needed. Unfortunately, many of the currently existing kinase screening technologies are geared towards monitoring single phosphorylation events and thus, are not be amenable to effective assaying of multiply phosphorylated substrates. In this thesis, a novel, solution-based assay method based on electrospray ionizationtandem mass spectrometry (ESI-MS/MS) is developed as a platform for inhibitor screening with full consideration being given to the specific nature of GSK-3 substrates and products. The semi-automated application of this assay is possible using an in-line autosampler, and is shown to be a potentially effective means for screening primed binding site inhibitors from compound mixtures, with subsequent deconvolution performed to isolate the effective molecule. Optimization of the MS-based assay required significant alterations in buffer conditions compared to those used in the standard GSK-3 radioassay based on y-32P ATP, owing to the inability of electrospray ionization to tolerate high buffer concentrations. Preliminary screening of mixtures was demonstrated, and expansion to screening of large compound libraries consisting of previously untested compounds and natural product extracts should be possible. </p> <p> To investigate the adaptation of the GSK-3 MS/MS assay to allow mixture deconvolution, a preliminary study was performed on the utilization of sol-gel technology for entrapment of GSK-3 to develop a solid-phase affinity assay for pull-down of bioactive ligands identified in enzyme activity assays. This method requires the preservation of enzyme function within the silica matrix, which has not been previously demonstrated for GSK-3. The sol-gel entrapment of GSK-3, however, proved to be problematic. Implementation of a flow-through assay using immobilized GSK-3 was hampered by issues such as non-specific adsorption of the cationic substrate and inhibitors, owing to electrostatic interactions with the anionic silica matrix used for enzyme entrapment. Future work aimed at further developing and optimizing the sol-gel materials and processing methods are proposed. </p> / Thesis / Master of Science (MSc)

HMG-CoA reductase inhibitors, known commonly as statins are one of the most widely prescribed medications worldwide. Statins reduce circulating cholesterol levels and are very effective at reducing one’s risk for all-cause and cardiovascular mortality. Though generally well tolerated, statin-associated muscle symptoms (SAMS) present in more than a quarter of statin users. The most common SAMS is myalgia or muscle pain. Statin myalgia often presents in the absence of myofibre damage, making its origin and treatment ambiguous. There are numerous rodent models for statin myopathy in the literature, but surprisingly there is no representation of statin myalgia that we are aware of. This is shocking given the high prevalence of statin myalgia compared to statin myopathy. Recently, our lab published an in vitro model of statin myalgia that focused on elevated xCT transporter activity and interstitial glutamate. This model explains that pain perceived in statin myalgia is the result of statins’ downstream ability to elevate skeletal muscle interstitial glutamate concentrations, thereby activating peripheral nociceptors. The studies herein aimed to create an in vivo rodent model of statin myalgia based on the aforementioned in vitro model. We hypothesized that glutamate, sampled by way of skeletal muscle microdialysis, would be elevated in the skeletal muscle interstitium of rats following statin treatment. Drawing conclusions on the role of glutamate in statin myalgia was not a straightforward process and required multiple model adjustments due to confounding variables. Additionally, many of the recognized effects of statins that were assumed from human and in vitro studies did not translate well to our rodent model. This was the first attempt at creating an in vivo model of statin myalgia and evidence suggests that a rodent model may not be an appropriate representation of what occurs in humans. While these studies also raised doubt on the efficacy of rodent models for SAMS investigations in general and highlighted the importance of having standardized models, certain limitations and assumptions of our model must be addressed before concrete conclusions can be drawn. / Thesis / Master of Science in Medical Sciences (MSMS) / Statins, a class of cholesterol-lowering medications, are one of the most widely prescribed medications worldwide. They have been demonstrated to be very effective at reducing one’s risk of cardiovascular-related death. Statins are generally very well tolerated, however, the most common negative side effects of their use are muscle related and include muscle pain, muscle inflammation and muscle damage. Muscle pain is the most common of these symptoms to present and interestingly, often presents without any clinical indication of muscle damage. The lack of a physical explanation for what is causing this pain makes treating statin-associated muscle pain quite difficult. A lot of effort has gone into determining the mechanism(s) for statin-associated muscle damage, however, there is a gap when it comes to investigating the mechanism(s) for statin-associated muscle pain. The studies herein, therefore, aimed to bridge this gap and investigated a potential mechanism for statin-associated muscle pain in a rodent model. The foundation for this model was built on a cell culture model that was previously developed in our lab. Our data suggest that a rodent model for statin-associated muscle pain may not be an appropriate representation of what occurs in humans. In particular, reduced blood cholesterol and substantial skeletal muscle oxidative stress were not demonstrated in our model as they have been in humans and in cell culture studies. This raised concern around the efficacy of rodent models for statin associated muscle symptoms in general and highlighted the importance of having standardized models. The differences between human/cell culture studies and rodent models also made it difficult to draw firm conclusions on whether the mechanism for statin myalgia investigated herein is supported.

Master of Science (MS)