京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21029号 / 医科博第90号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 山下 潤, 教授 湊谷 謙司, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Every 37 seconds, someone in the United States dies from cardiovascular disease. Vascular calcification is one of the underlying causes of these fatal events. Medial calcification develops following arteriosclerosis, or hardening of the arteries. Medial calcification is characterized by the deposition of hydroxyapatite in the medial layer of the arteries after normal vascular smooth muscle cells undergo a phenotypic switch to resemble osteoblast-like cells. It is hypothesized that this switch is caused by the wingless related (WNT)-Signaling pathway. The WNT-Signaling pathway, upon activation, causes the upregulation of osteogenic markers for the development of osteoblast-like cells. Current treatments alleviate consequences of calcification but do not address the disease. Due to a lack of cures for calcification, a novel therapy for this disease is overdue. By studying human aortic smooth muscle cells and confirming the role of WNT-Signaling as it relates to calcification, a possible therapeutic target for calcification can be identified.

Modern large data sets inevitably contain outliers that deviate from the model assumptions. However, many widely used estimators, such as maximum likelihood estimators and least squared estimators, perform weakly with the existence of outliers. Alternatively, many statistical modeling approaches have matrices as the parameters. We consider penalized estimators for matrix-valued parameters with a focus on their robustness properties in the presence of outliers. We propose a general framework for robust modeling with matrix-valued parameters by minimizing robust loss functions with penalization. However, there are challenges to this approach in both computation and theoretical analysis. To tackle the computational challenges from the large size of the data, non-smoothness of robust loss functions, and the slow speed of matrix operations, we propose to apply the Frank-Wolfe algorithm, a first-order algorithm for optimization on a restricted region with low computation burden per iteration. Theoretically, we establish finite-sample error bounds under high-dimensional settings. We show that the estimation errors are bounded by small terms and converge in probability to zero under mild conditions in a neighborhood of the true model. Our method accommodates a broad classes of modeling problems using robust loss functions with penalization. Concretely, we study three cases: matrix completion, multivariate regression, and network estimation. For all cases, we illustrate the robustness of the proposed method both theoretically and numerically. / Statistics

Cardiovascular disease (CVD) is the leading cause of death in the United States, and hypertension has been recognized as a major contributor to its manifestation and progression. Vascular smooth muscle cells control the tone and elasticity of vessels and their dysfunction in hypertension contributes to arterial remodeling and subsequent end organ damage. Evidence has indicated that the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) may be compromised in hypertension, while the contribution of protein aggregate formation (a main driver of UPR activation) is undefined. Glucose regulated protein-78 (GRP78), a residential ER chaperone, acts to aid in the proper folding of nascent peptides during translation, while also acting as the primary signal transducer for UPR. We hypothesize overexpression of GRP78 can protect against Angiotensin II induced protein aggregation in vascular smooth muscle cells (VSMCs) to reduce pathological ER UPR signaling and hypertensive vascular remodeling. To test this hypothesis, we investigated protein aggregate induction by Ang II stimulation as well as ER UPR activation, and if overexpression of the ER-resident chaperone glucose regulated protein 78 (GRP78) could protect against these as well as VSMC remodeling markers: hypertrophy, collagen production and inflammation. Utilizing pre-amyloid oligomer (PAO) immunofluorescence staining to identify Ang II induction of amyloid in VSMCs, we found amyloid accumulation was maximal at 48h post stimulation, which could be prevented with adenoviral overexpression of GRP78. Ang II significantly induced ER stress markers p-IRE1α, p-PERK and cleaved ATF6 in VSMCs. Overexpression of GRP78 was able to attenuate these ER stress responders. Interestingly, shotgun proteomic analysis of triton X-100 insoluble aggregate fractions revealed proteostasis machinery enriched in Ang II treated VSMC aggregates (HSP70, VCP, CryAB), which were attenuated with GR78 overexpression. To investigate pathological VSMC remodeling markers, we found that Ang II induced VSMC collagen production, immune cell adhesion, VCAM-1 expression, and hypertrophy (via protein synthesis) which was attenuated by GRP78 overexpression. Utilizing a VSMC-promoter derived GRP78 overexpression mouse model (GRP78TG SM22α Cre-/- or GRP78TG SM22α Cre+/-), we investigated the effect of ER stress inhibition on Ang II induced vascular remodeling. Importantly, hypertrophy and fibrosis in the aorta and the cardiac vasculature were assessed by Masson’s Trichrome and Sirius red staining and found to be increased in Cre-/- mice, while Cre+/- were significantly protected. These effects were accompanied with a significant reduction in Ang II-induced aortic amyloid burden (PAO) and ER UPR signaling. Blood pressure was monitored via tail cuff which revealed GRP78 Cre+/- mice were not protected against Ang II induced hypertension. Overall, these findings indicate that VSMC protein aggregation activates the ER stress response and contribute to hypertensive vascular remodeling. Furthermore, therapeutically targeting this mechanism via overexpression of GRP78 may elude new pharmacological interventions for arterial stiffness, while addressing fundamental questions about the mechanisms involved. / Biomedical Sciences

Cardiovascular disease leads in morbidity and mortality in Western societies with no known cure. NADPH oxidase (Nox) contributes to atherosclerosis through the indirect activation of macrophages leading to the internalization of oxidized low density lipoproteins (oxLDL). Chronic inflammation in activated macrophages contributes to atherosclerosis. Because macrophages are positioned at the cross-roads of lipid metabolism in vessel walls, they are important in the cellular pathology of atherosclerosis. Components of the endocannabinoid (eCB) system are vital to atherosclerotic development, since the eCB system has been found to play an important role in the amelioration of atherosclerosis. The eCB system has several components, including the G-protein-coupled cannabinoid receptors (CB1 and CB2); their endogenous ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA); and biosynthetic enzymes that produce and degrading these compounds. CB2 signaling has been shown to upregulate immunoprotective and anti-oxidative pathways, whereas CB1 signaling has opposite effects. We hypothesized a mechanistic link between scavenger receptor activation and Nox activity, which leads to enhanced 2-AG biosynthesis via a signaling pathway that activates diacylglycerol lipase beta (DAGLB). Activation of CB2-mediated signaling by enhanced “eCB tone” can potentially reduce oxidative stress in macrophages. The released 2-AG is subsequently catabolized hydrolytic enzymes, leading to enhanced 2-AGbiosynthesis via activated DAGLB. We first proved that macrophage treated with oxLDL can activate Nox and increase reactive oxygen species production. We used human and mouse macrophages to demonstrate cause and effect. Secondly, we demonstrated that increased levels of superoxide causes enhanced 2-AG biosynthesis within the macrophage, and that upregulation in eCB production is an adaptive response to oxidative stress. Finally, we identified and quantified the serine hydrolases found in smooth muscle cells (SMCs) using an activity-based protein profiling (ABPP)-MudPIT approach that our laboratory has previously done using human macrophages. Additionally, the catabolism of 2-AG by primary SMCs was explored to demonstrate SMCs can hydrolyze 2-AG to its metabolites arachidonic acid and glycerol by the known hydrolytic enzymes. We demonstrated that enhancing endocannabinoid tone within the vessel wall is a valuable strategy to reduce the occurrence of inflammation that leads to atherosclerosis.

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Kalman-type filtering methods are mostly designed based on exact knowledge of the system’s model with known parameters. In real applications, there may be considerable amount of uncertainties about the model structure, physical parameters, level of noise, and initial conditions. In order to overcome such difficulties, robust state estimation techniques are recommended. This PhD thesis presents a novel robust state estimation method that is referred to as the 2nd-order smooth variable structure filter (2nd-order SVSF) and satisfies the first and second order sliding conditions. It is an extension to the 1st-order SVSF introduced in 2007. In the 1st-order SVSF chattering is reduced by using a smoothing boundary layer; however, the 2nd-order SVSF alleviates chattering by preserving the second order sliding condition. It reduces the estimation error and its first difference until the existence boundary layer is reached. Then after, it guarantees that the estimation error and its difference remain bounded given bounded noise and modeling uncertainties. As such, the 2nd-order SVSF produces more accurate and smoother state estimates under highly uncertain conditions than the 1st-order version. The main issue with the 2nd-order SVSF is that it is not optimal in the mean square error sense. In order to overcome this issue, the dynamic 2nd-order SVSF is initially presented based on a dynamic sliding mode manifold. This manifold introduces a variable cut-off frequency coefficient that adjusts the filter bandwidth. An optimal derivation of the 2nd-order SVSF is then obtained by minimizing the state error covariance matrix with respect to the cut-off frequency matrix. An experimental setup of an electro-hydrostatic actuator is used to compare the performance of the 2nd-order SVSF and its optimal version with other estimation methods such as the Kalman filter and the 1st-order SVSF. Experiments confirm the superior performance of the 2nd-order SVSF given modeling uncertainties. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24782号 / 医博第4974号 / 新制||医||1066(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 濵﨑 洋子, 教授 湊谷 謙司, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / The contractile activation of airway smooth muscle tissues stimulates actin polymerization and the inhibition of actin polymerization inhibits tension development. Actin depolymerizing factor (ADF) and cofilin are members of a family of actin–binding proteins that mediate the severing of F–actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of airway smooth was evaluated in intact canine tracheal smooth muscle tissues. Two–dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated (inactivated). Phospho–ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho–cofilin mimetic (cofilin S3E), but not WT cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh–induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh–induced dephosphorylation of ADF/cofilin required the Ca2+–dependent activation of calcineurin (PP2B). Expression of Slingshot (SSH) inactive phosphatase (C393S) decreased force development and cofilin dephosphorylation. Activation of ADF/cofilin was also required for the relaxation of tracheal muscle tissues induced by forskolin and isoproterenol. Cofilin activation in response to forskolin was not Ca2+–dependent and was not inhibited by calcineurin inhibitors, suggesting it was regulated by a different mechanism. Cofilin activation is required for actin dynamics and tension development in response to the contractile stimulation of tracheal smooth muscle and is regulated by both contractile and relaxing stimuli. These concepts are critical to understanding the mechanisms of smooth muscle contraction and relaxation, which may provide novel targets for therapeutic intervention in the treatment of abnormal airway responsiveness.