Corticotropin releasing factor (CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter (NET) in vitro. SK-N-BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [3H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status.

Glucocorticoids affect cellular and molecular events in brains by modulating the expression of many genes during stress. In the present study, we examined the regulatory effect of corticosterone on the expression and function of the norepinephrine transporter (NET) in vitro. The results show that exposure of SK-N-BE(2)C cells to corticosterone for 14 days significantly increased mRNA (up to 43%) and protein (up to 71%) levels of NET in the concentration- dependent manner. Longer exposure (21 days) resulted in greater increases in the levels of mRNAs (up to about 160%) and proteins (up to about 250%) of the NET. The up-regulatory effect of corticosterone on NET expression lasted a persistent period after cessation of exposure. Associated with the corticosterone-induced enhancement in NET expression, there was a parallel increase in the uptake of [3H]norepinephrine by SK-N-BE(2)C cells. Increased NET expression and function were abolished after exposure of cells to corticosterone in combination with mifepristone or spironolactone, two specific antagonists of corticosteroid receptors. This is consistent with the hypothesis that corticosterone-induced NET up-regulation is mediated by corticosteroid receptors. Nevertheless, there was no synergistic effect for a combination of both corticosteroid receptor antagonists. A similar up-regulation of NET protein levels was also observed after exposing PC12 cells to corticosterone. The present findings demonstrate that corticosterone up-regulates the expression and function of NET in vitro, indicating the action of corticosterone on the noradrenergic phenotype may play an important role in the correlation between stress and the development of depression.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [ 3H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21223号 / 生博第392号 / 新制||生||52(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 根岸 学, 教授 松崎 文雄 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

To determine whether prolonged supersensitization of dopamine D-1 receptors could be produced during ontogeny, rats were treated daily, from birth, for 33 consecutive days with the D-1 receptor agonist, SKF 38393 HC1 (3.0 mg/kg per day i.p.). These rats were additionally treated at 3 days after birth with the neurotoxin, 6-hydroxydopamine HBr (6-OHDA; 200 μg, i.c.v., half in each lateral ventricle) or its vehicle. At 6 to 7 weeks from birth a challenge dose of SKF 38393 HCl (3.0 mg/kg i.p.) increased stereotypy scores for a number of behaviors in 6-OHDA-lesioned rats that were treated repeatedly during ontogeny with SKF 38393. These accentuated behaviors included licking, grooming, taffy pulling, jumping, paw treading and locomotion. Although the findings demonstrate an increased sensitivity of D-1 receptors to an agonist, there was no change in the Bmax or Kd for D-1 receptors in the striatum. In rats that were treated during postnatal development with SKF 38393, but not lesioned with 6-OHDA, SKF 38393-induced stereotyped behaviors were not substantially different from control. The neonatally primed rat model may be useful for probing mechanisms of receptor supersensitivity.

No description available.

A variety of soybean oils (SOs) were developed with improved functional properties. Some of the modified SOs contain altered fatty acid (FA) composition by selective breeding methods. Currently, low α- linolenic acid soybean oil (LLSO) and low α- linolenic acid and mid oleic acid soybean oil (LLMOSO) are available FA modified SOs in the market. The consumption of FA modified SOs has been increased because the United States Food and Drug Administration required listing trans fat content in food products sold in U.S. as an effort to reduce possible health risks caused by trans fat beginning 2006. However, the effects of these FA modified SOs on human chronic diseases including breast cancer (BC) have not been studied. BC has become the most frequently diagnosed cancer and is the second leading cause of cancer death among American women. The type of dietary fat, FA composition, and n-6/n-3 ratio are known to influence BC development. Therefore, it is possible that the changed FA composition and n-6/n-3 ratio in the FA modified SOs may affect BC progression, and its critical health concern needs to be investigated. Increased human epithelial growth factor receptor 2 (Her-2/neu) and fatty acid synthase (FAS) are associated with BC progression. In fact, FAS activity and expression are affected by dietary FA composition and FA metabolism. Hypothesis of this research is that LLSO and LLMOSO may affect Her-2/neu and FAS expressing human BC (SK-Br3) cell growth in vitro and in vivo. To test our hypothesis, we investigated the potential adverse or beneficial effects of LLSO and LLMOSO in comparison with conventional SO and lard on human BC cells and then examined the possible mechanisms of action by evaluating the expression level of genes markers involved in growth factor mediated signal transduction pathway, specifically Her-2/neu PI 3-kinase (phophoinositide 3- kinase)-FAS signal transduction pathway. In vitro study demonstrated that all the tested oils at 0-2 μl/ml level have cytotoxic effects. LLMOSO had less cytotoxic effects on the growth of SK-Br3 cells compared to SO. However, there was no difference in SK-Br3 cell growth between LLSO and SO. The apoptotic protein markers (mutant p53 and caspase-3) analysis revealed that the cell growth inhibition by oil treatments was cytotoxic by triggering apoptosis. Western blot analysis demonstrated that LLSO- and LLMOSO- induced changes on cell growth involve Her-2/neu and FAS signaling transduction pathway and sterol regulatory element binding protein-1 (SREBP-1), mitogen activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI 3-kinase) are possible down-stream effectors of Her-2/neu signaling pathway. We also evaluated the dietary effects of LLSO (20% fat of total calorie), SO (20%), and lard (20%) on the growth of SK-Br3 tumors implanted in athymic mice. Changes in tumor surface area, body weight, and food intake were monitored during the 6 months feeding study. After termination, tumor net weight, Her-2/neu and FAS mRNA expression in tumors, FAS protein expression in liver, lipid composition in diets, abdominal fat, and serum, as well as plasma total cholesterol and triglyceride levels were analyzed. In vivo study showed that there were no statistical differences in tumor size and tumor net weight among SO, LLSO, and lard groups. No differences in FAS mRNA and protein expression levels between the LLSO and SO groups were observed. Tumors from the lard group expressed higher Her-2/neu and FAS mRNA than those from the LLSO and SO group. The lipid analysis demonstrated that LLSO was not significantly distinct from SO in trans fat concentration after metabolism. Serum cholesterol and triglyceride levels were unchanged in LLSO fed compared to SO fed mice. In summary, LLSO which contained modification in αLA concentration showed similar effects on SK-Br3 as SO in both in vitro and in vivo. However, LLMOSO which contained more drastic modifications on FA composition exhibited less cytotoxicity compared to SO in vitro. / Master of Science

Diese Dissertation beschäftigt sich mit EDP-Konvergenz. Dabei handelt es sich um einen Konvergenzbegriff auf dem Gebiet der verallgemeinerten Gradientensysteme und metrischen Gradientensysteme, der geeignet ist für Gradientenflüsse, die von einem kleinen Parameter abhängen. EDP-Konvergenz liefert einen Algorithmus, der es erlaubt in der Energie und dem Dissipationspotenzial zum Grenzwert überzugehen. Es ist die fundamentale Frage evolutionärer Γ-Konvergenz, wie das Limes-Dissipationspotenzial berechnet werden kann. Das Ziel dieser Arbeit ist es aufzuzeigen, dass EDP-Konvergenz das mikro- und das makroskopische Dissipationspotenzial in einer sinnvollen und eindeutigen Art und Weise in Beziehung setzt. Anhand von drei Beispielen wird der Konvergenzbegriff untersucht: die Diffusionsgleichung auf einem dünnen, dreischichtigen Gebiet, die Poröse-Medien-Gleichung mit einer dünnen Membran und ein Modell mit oszillierender Energie. Es wird die Definition von relaxierter EDP-Konvergenz und EDP-Konvergenz mit Kippung motiviert. EDP-Konvergenz basiert auf dem Prinzip, dass es ein Gleichgewicht zwischen Energie und Dissipation gibt – das Energie-Dissipations-Prinzip (EDP). Mittels Γ-Konvergenz wird sowohl in der Energie, als auch dem totalen Dissipationsfunktional zum Grenzwert übergegangen. Durch die zusätzliche Entkopplung von Zustand und Triebkraft wird die Dissipationslandschaft erkundet und die kinetische Beziehung des Limessystems ermittelt. Das Modell mit oszillierender Energie zeigt die Bedeutung der kinetischen Beziehung – und damit der Kippung – für die Herleitung des Limes-Dissipationspotenzials auf. Die Modelle mit Wasserstein-Dissipation zeigen, dass das Limes-Dissipationspotenzial nicht der naive Grenzwert ist. Insbesondere können klassische Gradientensysteme mit quadratischer Dissipation zu verallgemeinerten Gradientensysteme konvergieren. / In the realm of generalized gradient systems and metric gradient systems we study a notion of convergence suited for gradient flows which depend on a small parameter. This notion is called EDP-convergence. In order to understand the convergence of gradient systems we need an algorithm to derive the limiting energy as well as the limiting dissipation potential. The fundamental question of evolutionary Γ-convergence is how to compute the limit dissipation potential. The aim of this thesis is to show that EDP-convergence connects the microscopic dissipation potential with the macroscopic, i.e. limiting, dissipation potential in a meaningful and unique way. As a proof of concept 3 different examples are presented: (i) the diffusion equation on a thin sandwich-like domain, (ii) the porous medium equation with a thin interface and (iii) a wiggly energy model. We show how the gradient flow concept that is used in this thesis can be used to obtain also gradient flows with respect to the Wasserstein metric. We motivate the definition of relaxed EDP-convergence and EDP- convergence with tilting. EDP-convergence is based upon the principle that there is an energy-dissipation-balance involving the total dissipation functional and the energy difference – the energy-dissipation-principle (EDP). The limit passage, in both the energy and the total dissipation functional, is performed in terms of Γ-convergence. By perturbing the flow as well as the driving force, the dissipation-landscape is explored and a kinetic relation for the limit system can be established. The wiggly energy model demonstrates the importance of the kinetic relation for the construction of the limiting dissipation potential and thus the introduction of tilts. The models with a Wasserstein dissipation show that the limiting dissipation potential is not the naive limit. In particular, classical gradient systems with a quadratic dissipation potential converge to a generalized gradient systems.

Ziel dieser Arbeit ist theoretische Eigenschaften verschiedener Bootstrap Methoden zu untersuchen. Als Ergebnis führen wir die Konvergenzraten des Bootstrap-Verfahrens ein, die sich auf die Differenz zwischen der tatsächlichen Verteilung einer Statistik und der Resampling-Näherung beziehen. In dieser Arbeit analysieren wir die Verteilung der l2-Norm der Summe unabhängiger Vektoren, des Summen Maximums in hoher Dimension, des Wasserstein-Abstands zwischen empirischen Messungen und Wassestein-Barycenters. Um die Bootstrap-Konvergenz zu beweisen, verwenden wir die Gaussche Approximations technik. Das bedeutet dass man in der betrachteten Statistik eine Summe unabhängiger Vektoren finden muss, so dass Bootstrap eine erneute Abtastung dieser Summe ergibt. Ferner kann diese Summe durch Gaussche Verteilung angenähert und mit der Neuabtastung Verteilung als Differenz zwischen Kovarianzmatrizen verglichen werden. Im Allgemeinen scheint es sehr schwierig zu sein, eine solche Summe unabhängiger Vektoren aufzudecken, da einige Statistiken (zum Beispiel MLE) keine explizite Gleichung haben und möglicherweise unendlich dimensional sind. Um mit dieser Schwierigkeit fertig zu werden, verwenden wir einige neuartige Ergebnisse aus der statistischen Lerntheorie. Darüber hinaus wenden wir Bootstrap bei Methoden zur Erkennung von Änderungspunkten an. Im parametrischen Fall analysieren wir den statischen Likelihood Ratio Test (LRT). Seine hohen Werte zeigen Änderungen der Parameter Verteilung in der Datensequenz an. Das Maximum von LRT hat eine unbekannte Verteilung und kann mit Bootstrap kalibriert werden. Wir zeigen die Konvergenzraten zur realen maximalen LRT-Verteilung. In nicht parametrischen Fällen verwenden wir anstelle von LRT den Wasserstein-Abstand zwischen empirischen Messungen. Wir testen die Genauigkeit von Methoden zur Erkennung von Änderungspunkten anhand von synthetischen Zeitreihen und Elektrokardiographiedaten. Letzteres zeigt einige Vorteile des nicht parametrischen Ansatzes gegenüber komplexen Modellen und LRT. / The objective of this thesis is to explore theoretical properties of various bootstrap methods. We introduce the convergence rates of the bootstrap procedure which corresponds to the difference between real distribution of some statistic and its resampling approximation. In this work we analyze the distribution of Euclidean norm of independent vectors sum, maximum of sum in high dimension, Wasserstein distance between empirical measures, Wassestein barycenters. In order to prove bootstrap convergence we involve Gaussian approximation technique which means that one has to find a sum of independent vectors in the considered statistic such that bootstrap yields a resampling of this sum. Further this sum may be approximated by Gaussian distribution and compared with the resampling distribution as a difference between variance matrices. In general it appears to be very difficult to reveal such a sum of independent vectors because some statistics (for example, MLE) don't have an explicit equation and may be infinite-dimensional. In order to handle this difficulty we involve some novel results from statistical learning theory, which provide a finite sample quadratic approximation of the Likelihood and suitable MLE representation. In the last chapter we consider the MLE of Wasserstein barycenters model. The regularised barycenters model has bounded derivatives and satisfies the necessary conditions of quadratic approximation. Furthermore, we apply bootstrap in change point detection methods. In the parametric case we analyse the Likelihood Ratio Test (LRT) statistic. Its high values indicate changes of parametric distribution in the data sequence. The maximum of LRT has a complex distribution but its quantiles may be calibrated by means of bootstrap. We show the convergence rates of the bootstrap quantiles to the real quantiles of LRT distribution. In non-parametric case instead of LRT we use Wasserstein distance between empirical measures. We test the accuracy of change point detection methods on synthetic time series and electrocardiography (ECG) data. Experiments with ECG illustrate advantages of the non-parametric approach versus complex parametric models and LRT.