甘味受容体における呈味調節物質作用モデルの構築及びその検証

学位の種別: 課程博士審査委員会委員 : (主査)東京大学准教授 三坂 巧, 東京大学教授 伏信 進矢, 東京大学特任教授 朝倉 富子, 東京大学准教授 永田 宏次, 東京大学准教授 寺田 透University of Tokyo(東京大学

Change in teachers’ practice through the elaborationand testing of an informed teaching proposal

Educational research indicates that it is very difficult for experienced teachers, to modify their teaching approach, even after several disciplinary or educational courses. It is known that they, generally continue with the familiar methods used for years, remaining as the central figure that transmits knowledge, without fully considering the ideas, interests or knowledge of their students. We ask ourselves if a group of experienced teachers improve their edagogical content knowledge (PCK) after attending an interdisciplinary training program, in which the elaborated teaching proposal is tested in the classroom. In order to investigate this, a questionnaire was applied to master degree graduates with such characteristics of co-construction and application of a teaching proposal. Contrary to the results of short courses, we have found that all the surveyed teachers had significant advance in their PCK.La investigación educativa indica que es muy difícil para los profesores con experiencia para modificar su método de enseñanza, incluso después de varios cursos disciplinarios o educativos. Se sabe que por lo general continúan con los métodos habituales utilizados por años, manteniéndose como la figura central que transmite el conocimiento, sin examinar a fondo las ideas, los intereses o los conocimientos de sus alumnos. Nos preguntamos si un grupo de profesores con experiencia mejorarían su conocimiento del contenido pedagógico (PCK) después de asistir a un programa de entrenamiento interdisciplinario, en el que la propuesta didáctica elaborada se prueba en el aula. Para investigar esto, se aplicó un cuestionario a los graduados de maestría con características tales como co-construcción y aplicación de una propuesta de enseñanza. Contrariamente a los resultados de cursos de corta duración, se ha encontrado que todos los profesores encuestados tuvieron avance significativo en su PCK

Perilipin Isoforms and PGC-1α Are Regulated Differentially in Rat Heart during Pregnancy-Induced Physiological Cardiac Hypertrophy

Background and Objectives: Perilipins 1-5 (PLIN) are lipid droplet-associated proteins that participate in regulating lipid storage and metabolism, and the PLIN5 isoform is known to form a nuclear complex with peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) to regulate lipid metabolism gene expression. However, the changes in PLIN isoforms' expression in response to pregnancy-induced cardiac hypertrophy are not thoroughly studied. The aim of this study was to quantify the mRNA expression of PLIN isoforms and PGC-1α along with total triacylglycerol (TAG) and cholesterol levels during late pregnancy and the postpartum period in the rat left ventricle. Materials and Methods: Female Sprague-Dawley rats were divided into three groups: non-pregnant, late pregnancy, and postpartum. The mRNA and protein levels were evaluated using quantitative RT-PCR and Western blotting, respectively. TAG and total cholesterol content were evaluated using commercial colorimetric methods. Results: The expression of mRNAs for PLIN1, 2, and 5 increased during pregnancy and the postpartum period. PGC-1α mRNA and protein expression increased during pregnancy and the postpartum period. Moreover, TAG and total cholesterol increased during pregnancy and returned to basal levels after pregnancy. Conclusions: Our results demonstrate that pregnancy upregulates differentially the expression of PLIN isoforms along with PGC-1α, suggesting that together they might be involved in the regulation of the lipid metabolic shift induced by pregnancy

Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI)

Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization

Reuse of anion exchangers as supports for enzyme immobilization: Reinforcement of the enzyme-support multiinteraction after enzyme inactivation

β-Galactosidase from Aspergillus oryze has been immobilized on agarose beads coated with polyethyleneimine. The fresh enzyme was released from the support using 500. mM NaCl at pH 7. After thermal inactivation or inactivation in the presence of organic solvents, the active enzyme still could be easily released from the support using similar conditions. However, SDS-PAGE analysis of the enzyme contained in the support after enzyme desorption showed that enzyme molecules remained in the support (inactivated enzyme molecules). This effect was stronger on enzyme preparations inactivated in an organic medium. Now the conditions should be greatly strengthen to permit the full enzyme desorption: only after incubation in 2. M sodium phosphate at pH 2 and 50. °C full release of the enzyme molecules was achieved. This could be repeated several cycles with any difference neither in the immobilization performance nor on the SDS-PAGE analysis. Therefore, the reversibility of the immobilization is a real fact, but recovery of a support fully free of protein molecules is not an easy objective after enzyme inactivation, because the inactivated enzymes seemed to unfold increasing in a great way the interaction with the support, driving to a very strong enzyme-support multi-interaction that difficulty its desorption

Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites

Lipases from Candida antarctica (isoform B) and Rhizomucor miehei (CALB and RML) have been immobilized on octyl-agarose (OC) and further coated with polyethylenimine (PEI) and dextran sulfate (DS). The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed). The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme

Activation of Peripheral Cannabinoid Receptors Synergizes the Effect of Systemic Ibuprofen in a Pain Model in Rat

Pharmacological synergism is a current strategy for the treatment of pain. However, few studies have been explored to provide evidence of the possible synergism between a non-steroidal anti-inflammatory drug (NSAID) and a cannabinoid agonist, in order to establish which combinations might be effective to manage pain. The aim of this study was to explore the synergism between ibuprofen (IBU) and the synthetic cannabinoid WIN 55,212-2 (WIN) to improve pain relief by analyzing the degree of participation of the CB1 and CB2 cannabinoid receptors in the possible antinociceptive synergism using an experimental model of pain in Wistar rats. First, the effective dose thirty (ED30) of IBU (10, 40, 80, and 160 mg/kg, subcutaneous) and WIN (3, 10, and 30 µg/p, intraplantar) were evaluated in the formalin test. Then, the constant ratio method was used to calculate the doses of IBU and WIN to be administered in combination (COMB) to determine the possible synergism using the isobolographic method. The participation of the CB1 and CB2 receptors was explored in the presence of the antagonists AM281 and AM630, respectively. The combination of these drugs produced a supra-additive response with an interaction index of 0.13. In addition, AM281 and AM630 antagonists reversed the synergistic effect in 45% and 76%, respectively, suggesting that both cannabinoid receptors are involved in this synergism, with peripheral receptors playing a relevant role. In conclusion, the combination of IBU + WIN synergism is mainly mediated by the participation of the CB2 receptor, which can be a good option for the better management of pain relief