No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23802号 / 医博第4848号 / 新制||医||1058(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 永井 洋士, 教授 羽賀 博典, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>This work used biotechnology to invent new caloxins - allosteric peptide inhibitors of plasma membrane Ca²⁺pumps (PMCA) needed to understand the Ca²⁺signalling in coronary artery.</p> <p>PMCA are encoded by genes PMCA1-4. Defects in PMCA expression have been associated with several pathologies. The major objectives of my thesis were to determine the expression of PMCA isoforms in the smooth muscle and the endothelium of coronary artery and to invent high affinity and specificity caloxins for the isoforms present in these tissues.</p> <p>In Aim 1 it was determined that the total PMCA protein and activity was much greater in smooth muscle than in endothelium. Both tissues expressed only PMCA1 and PMCA4, with PMCA4 > PMCA1 in smooth muscle and PMCA1 > PMCA4 in endothelium. Therefore, the search for PMCA1 and 4 selective caloxins using phage display technique was conducted.</p> <p>Aim 2 was to invent PMCA1 selective inhibitors. Caloxin 1b3 was invented as the first known PMCA1 selective inhibitor. It inhibited PMCA1 Ca²⁺-Mg²⁺-ATPase with higher affinity than PMCA2, 3 or 4. Aims 1 and 2 were consistent with the greater potency of caloxin 1b3 than a known PMCA4 selective caloxin 1b1 in increasing cytosolic Ca²⁺ concentration in endothelial cells.</p> <p>Aim 3 was to obtain ultrahigh selectivity and affinity PMCA4 bidentate inhibitor using the previously invented PMCA4 selective caloxins 1c2 and 1b2. In the first step the affinity of caloxin 1b2 was improved by limited mutagenesis to obtain caloxin 1c4. Caloxin 1c4 had 5-6 times higher affinity than caloxin 1b2 for inhibiting PMCA4 activity. Optimization of the bidentate caloxins from caloxin 1c2 and 1c4 was also attempted.</p> <p>The novel caloxins may aid in elucidating the role of PMCA1 and PMCA4 in the physiology and pathophysiology of coronary artery and other tissues.</p> / Doctor of Philosophy (PhD)

<p>Bronchial thermoplasty has recently been FDA approved as a novel therapy for use on adults suffering from severe asthma. The procedure uses radiofrequency energy to heat the airways to 65°C for 10 s. This has been shown in dogs to lead to a reduction of airway smooth muscle mass and in humans to improve quality of life and asthma control. Early cellular reactions to this treatment are unclear; as well, there is limited information regarding thermal sensitivity of airway smooth muscle when exposed to extreme temperatures (50-65°C). We examined the cellular impact of bronchial thermoplasty by investigating the response of airway smooth muscle to heat by immersing bovine tracheal strips and bronchial segments in heated Krebs. We confirmed dramatically decreased functionality over the temperature range 50-60°C at 1 h and 24 h in all tissues. TUNEL analysis noted significant cell death in all tissues heated to 65°C and limited cell death in bronchial tissues treated with <55°C. Immunohistochemical analysis showed an effect of temperature on caspase 3 activation in bronchi; tracheal strips demonstrated co-localization of caspase 3 and TUNEL at 55°C but not 65°C. These data suggests that cell death of airway smooth muscle contributes to the cellular effects observed following heating to 65°C; at lower temperatures, cell death may be limited. We conclude that bronchial thermoplasty (heat treatment to 65°C for ~30 seconds) leads to a number of structural and functional changes in the airway smooth muscle, which culminate in marked loss of function and cell death.</p> / Master of Health Sciences (MSc)

<p>Allergic atopic asthma is a respiratory condition that involves immune responses to specific allergens resulting in coughing, wheezing, shortness of breath and tightness in the chest. During an atopic asthmatic attack, the immune system initiates cellular infiltration of lymphocytes and eosinophils, airway hyper-responsiveness and ECM remodeling, which manifests in lung dysfunction in chronic disease. ASMC have recently been shown to play a role in the inflammatory processes of asthma through the production of inflammatory mediators. Various cytokines and chemokines serve as stimulants for these pathways and therefore require further attention to examine inflammatory signaling. OSM, a member of the gp130 family of cytokines, is secreted by inflammatory cells and has been detected in the sputum of asthmatics. Previous findings have established the potential of OSM in induction of lung inflammation, its role in increasing ECM, and its potential role in asthma. Viral or bacterial infections cause asthma exacerbations which result in increased severity of symptoms. The innate immune system relies on pattern recognition receptors including the TLRs to recognize invading pathogens and activate cells such as macrophages and natural killer cells. Although there are a number of these TLRs, this project will focus on the role of TLR3 and TLR4 in ASMC. I generally hypothesized that OSM markedly increases lung cell airway smooth muscle cell responses to external stimulae, such as products of bacteria or viruses that activate toll-like receptors. This exacerbates inflammation and extracellular matrix remodeling which contributes to pathology in asthmatic patients. Findings in this thesis have demonstrated that OSM stimulation increases the production of various cytokines and chemokines and growth factors seen in asthma. Co-stimulations with OSM and TLR-ligands augmented the production of a variety of these inflammatory mediators in comparison to ligands alone. TLR responses were shown to be associated with TLR expression, at both the mRNA and protein level, as well through the activation of the JAK-STAT and NFκB pathways. These findings implicate ASMC in immunomodulatory roles in response to TLR-ligands and OSM, and could play a role in the increased severity of asthma seen during exacerbations.</p> / Master of Science (MSc)

The underlying cause of all vascular proliferative diseases is injury-induced activation of vascular endothelium and vascular smooth muscle cells (VSMC). Activated VSMC proliferate, than migrate from the arterial media to the intima, contributing to neointima formation. Activated immune cells, vascular cells, and their endogenous regulators mediate this complex process. One integral regulator of VSMC activation is allograft inflammatory factor-1 (AIF-1). AIF-1 is a cytoplasmic scaffold protein, expressed constitutively in lymphoid cells and induced in VSMC by injury. Stable over expression of AIF-1 increases VSMC proliferation and migration in vitro, causes increased injury-induced neointima formation, and increases Rac1 and p38 MAP Kinase activity. Recent studies show a correlation between VSMC expression of AIF-1 and atherosclerosis development. We hypothesize that VSMC over expression of AIF-1 contributes to atherosclerosis development by increasing activity of inflammatory signaling molecules, and that inhibiting VSMC AIF-1 expression will decrease injury-induced neointima formation. Rat carotid arteries transfected with AIF-1 si RNA adenovirus after balloon angioplasty developed significantly less neointima compared to controls. AIF-1 si RNA transfected VSMC proliferated significantly less than AIF-1 or GFP transfected VSMC, while AIF-1 si RNA transfection did not attenuate AIF-1-mediated migration. p38 inhibition showed that AIF-1-mediated proliferation is dependent on p38 activation while AIF-1-mediated migration is not. AIF-1 transgenic mice fed a high fat diet showed significantly more atherosclerotic lesions than WT littermates. Boyden Chamber assays showed OxLDL treatment increases VSMC migration but does not effect AIF-1-mediated migration. Expression of migration and inflammatory responsive genes in AIF-1 and XGal transfected VSMC after OxLDL treatment at various time points were examined. MMP-2 and -9 expression did not change. ICAM-1 and VCAM-1 expression increased in both groups. AIF-1 VSMC showed significantly higher ICAM-1 expression at baseline and early time points and elevated, but not significantly higher VCAM-1 expression at early time points. Western blots showed increased activation of NF-kB in AIF-1 transfected VSMC at baseline and 30 minutes after OxLDL stimulation compared to XGal transfected VSMC. Expression of the scavenger receptor receptors CD36 and SRA(I) expression increased after lipid treatment in AIF-1 and XGal transfected groups. AIF-1 VSMC showed sustained expression of both receptors after 16 hours of treatment compared to XGal VSMC, which showed decreased expression at that time point. CXCL16/PSOX expression increased with treatment, but differences in expression patterns were not seen between cell groups. Analysis showed significantly more OxLDL was taken up by AIF-1 VSMC compared to XGal VSMC. These data show that AIF-1 expression in VSMC is tightly linked to the vascular response to injury and development of vascular disease. Although AIF-1-mediated migration is not p38 dependent, AIF-1 may contribute to increased VSMC migration in part by upregulating NF- kB downstream effectors through increased NF-kB activity. AIF-1 may also speed the progression of atherosclerosis by increasing scavenger receptor expression and thereby increasing OxLDL uptake and foam cell formation. Although more study is required to fully elucidate the molecular mechanisms leading to AIF-1 mediated VSMC activation, these data have further established AIF-1 as an integral regulator of the VSMC response to injury. / Molecular and Cellular Physiology

<p> Calcium (Ca2+) is an important signaling molecule and hence its movement across cell membranes must be tightly regulated. The intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle and endothelium controls the coronary tone. After stimulation, decreasing the [Ca2+]i back to resting levels is achieved mainly by the sodium calcium exchanger (NCX), the plasma membrane calcium pump (PMCA) or the sarcoendoplasmic reticulum calcium pump (SERCA). The present study will focus on NCX and its interactions with SERCA in the smooth muscle and endothelium of pig coronary artery.</p> <p> Aim 1 of my thesis is determination of activity levels of NCX in smooth muscle cells (SMC) and endothelial cells (EC). The NCX activity in cultured cells was approximately 5 times greater in EC than in SMC. The NCX inhibitors KB-R7943 and SEA 0400 blocked the NCX mediated Ca2+ entry, as did collapsing the Na+ gradient with monensin. NCX1 is the isoform largely responsible for NCX activity in SMC and EC. NCX activity was also assayed as the Ca2+ efflux in cultured cells and as Ca2+ uptake in plasma membrane vesicles isolated from freshly isolated smooth muscle.</p> <p> Aim 2 is to assess the existence of a functional NCX mediated Ca2+ entry linked to SERCA in SMC. In the absence of thapsigargin, BAPTA loading SMC increased the NCX mediated uptake. Thapsigargin did not affect the Ca2+ uptake in BAPTA loaded cells but it inhibited the Ca2+ uptake in cells that were not loaded with BAPTA. These data are consistent with a model in which SER acts as a sink for the NCX mediated Ca2+ entry. However, with BAPTA chelation and the resulting lower intracellular Ca2+, the need for SER to act as a sink is eliminated, and NCX is driven in full force. EC did not demonstrate a NCX-SERCA linkage.</p> <p> Arterial SMC and EC differ in their structure and function. The function of SMC is the generation of tone which is achieved by the Ca2+ dependent contractile filaments. Since these filaments are distributed throughout the cell, Ca2+ must be transported to and removed from deep within the cell. As a result, the SER may play a large role in Ca2+ regulation in the SMC. Furthermore, SMC also contain higher levels of high affinity Ca2+ pumps (SERCA and PMCA) and thus Ca2+ is more tightly regulated. Endothelial cells release nitric oxide in response to an increase in [Ca2+]i, which relaxes the smooth muscle. The endothelial nitric oxide sythase produces nitric oxide and is located adjacent to the PM in EC. The SER that removes Ca2+ from deep within the cell cytosol may play a small role in Ca2+ dependent modulation of the endothelial nitric oxide synthase activity. Based on the Western blot data, EC contain a greater amount of the high capacity NCX, thus the larger quantities of Ca2+ can be removed from the cell and the vicinity of endothelial nitric oxide synthase.</p> / Thesis / Master of Science (MSc)

Integrated weed management involves the utilization of weed biology principles to develop effective and economical control strategies. This research involved investigations of herbicide-based weed management programs in potato (<i>Solanum tuberosum</i> L.) and winter wheat (<i>Triticum aestivum</i> L.) as well as investigations of the biological characteristics of smooth pigweed (<i>Amaranthus hybridus</i>), a troublesome species in many crops. Sulfentrazone is an herbicide registered for use in soybean [<i>Glycine max</i> (L.) Merr.] and tobacco (<i>Nicotiana tabacum</i> L.) that may also have potential for use in potato. In field experiments, potato tolerance to preemergence (PRE) applications of sulfentrazone at rates up to 0.21 kg/ha was similar to that from the registered herbicides metribuzin, metolachlor, or metribuzin plus metolachlor PRE. Potato generally did not tolerate sulfentrazone applications to foliage. Sulfentrazone effectively controlled common lambsquarters (<i>Chenopodium album</i> L.) at rates as low as 0.11 kg/ha and also controlled several annual grasses at higher application rates, but was slightly less effective on jimsonweed (<i>Datura stramonium</i> L.) and ineffective on common ragweed (<i>Ambrosia artemisiifolia</i> L.). Potato tuber yield and grade from sulfentrazone PRE applications was similar to yield of potato treated with registered herbicides. Laboratory research was also conducted to determine the mechanism of sulfentrazone selectivity between potato (a tolerant species), common lambsquarters (a sensitive species), and jimsonweed (an intermediate species). After 48 h root exposure to [14C] sulfentrazone, absorption by common lambsquarters was nearly two-fold that of jimsonweed and three-fold that of potato. Both weed species also exhibited nearly a two-fold increase in sulfentrazone translocation from roots to shoots compared to potato. Since the site of action of sulfentrazone, protoporphyrinogen oxidase, is located in shoot tissue, translocation to shoots is essential for sulfentrazone toxicity. Therefore, the proposed primary mechanisms of selectivity between these species are differential root absorption and differential translocation. Experiments were also conducted to investigate the potential of the experimental herbicide AE F130060 03 for Italian ryegrass (<i>Lolium multiflorum</i> Lam.) control in winter wheat. In laboratory research, foliar absorption of AE F130060 03 in Italian ryegrass was at least three times that in wheat. Additionally, herbicide metabolism was greater in wheat, particularly in wheat treated with the herbicide safener AE F107892. In field experiments, AE F130060 03 was as effective as diclofop-methyl for control of diclofop-sensitive Italian ryegrass and more effective than diclofop-methyl and all other herbicides tested for control of diclofop-resistant Italian ryegrass. Although wheat injury from AE F130060 03 was greater than from other herbicides, wheat recovered and yields were not affected. Postemergence AE F130060 03 applications controlled Italian ryegrass from emergence until the end of tillering, but applications made to four- to five-tiller Italian ryegrass resulted in the least amount of new Italian ryegrass emergence following application. To further define the utility of AE F130060 03 in winter wheat, ten wheat cultivars adapted to Virginia were evaluated for tolerance to AE F130060 03. Biomass production between cultivars was not influenced by AE F130060 03 application in the greenhouse, although slight yield decreases due to herbicide application were found in FFR 518, Coker 9663, AgriPro Patton, and VA98W593 under weed-free conditions in the field. Greenhouse, growth chamber, and field experiments were also conducted to investigate growth and seed production of one imidazolinone-susceptible (S) and five -resistant (R1, R2, R3, R4, and R5) smooth pigweed biotypes. Although the S biotype produced more total biomass than four of the five R biotypes, R4 displayed a more rapid growth rate at 3 to 5 wk after planting and a faster germination rate than S and all other R biotypes. Seed production in R4 was similar to S and greater than in all other R biotypes. Early rapid growth in R4 did not translate into increased biomass accumulation compared to S at the conclusion of the experiments. / Ph. D.

In this work we present a new tool for the convergence analysis of numerical optimization methods. It is based on the concepts of the Clarke derivative and set-valued mappings. Our goal is to apply this tool to minimization problems with non-smooth and noisy objective functions. After deriving a necessary condition for minimizers of such functions, we examine two unconstrained optimization routines. First, we prove new convergence theorems for Implicit Filtering and General Pattern Search. Then we show how these results can be used in practice, by executing some numerical computations. / Master of Science

No / T-type Ca2+ channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca2+ channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca2+ channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca2+ channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca2+ channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca2+ channel-mediated proliferation by H2S is independent of the channels’ sensitivity to H2S. / This work was supported by the British Heart Foundation (PG/11/84/29146).