无课表2015(2) (1)
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2011—2012学年第一学期研究生课程表二○一一年九月目录法学院硕士研究生课程表............................................................................................................................................................1-2 马克思主义学院研究生课程表......................................................................................................................................................3-4 公共经管学院硕士研究生课程表 (5)经济与经管学院硕士研究生课程表................................................................................................................................................6-8 人文学院哲学系硕士研究生课程表.............................................................................................................................................9-10 人文学院历史学系硕士研究生课程表 (11)人文学院新闻与传播学系、影视艺术研究中心硕士研究生课程表 (12)人文学院中国语言文学系硕士研究生课程表.................................................................................................................................13-14 外国语学院硕士研究生课程表 (15)艺术与设计学院硕士研究生课程表 (16)教育学院硕士研究生课程表......................................................................................................................................................17-18 生命科学与食品工程学院硕士研究生课程表.................................................................................................................................19-22 理学院化学系硕士研究生课程表................................................................................................................................................23-24 理学院数学系硕士研究生课程表 (25)理学院数学系2010级硕士研究生课程表 (26)理学院物理系硕士研究生课程表................................................................................................................................................27-28 材料科学与工程学院、理学院经管科学与工程系硕士研究生课程表 (29)机电工程学院硕士研究生课程表.................................................................................................................................30-32 信息工程学院计算机系、信息经管系硕士研究生课程表 (33)信息工程学院计算中心、电子信息工程系硕士研究生课程表 (34)信息工程学院电气与自动化工程系硕士研究生课程表 (35)目录建筑工程学院硕士研究生课程表....................................................................................................................................36-38 环境与化学工程学院硕士研究生课程表...........................................................................................................................39-41 马克思主义学院、建筑工程学院、环境与化学工程学院博士研究生课程表 (42)机电工程学院博士研究生课程表 (43)理学院博士研究生课程表 (44)生命科学与食品工程学院博士研究生课程表 (45)材料科学与工程学院博士研究生课程表 (46)2 / 1041 / 1042 / 1043 / 1044 / 1045 / 1046 / 1047 / 1048 / 1049 / 10410 / 104人文学院哲学系2011—2012学年第一学期硕士研究生课程表人文学院哲学系2011—2012学年第一学期硕士研究生课程表11 / 104人文学院历史学系2011—2012学年第一学期硕士研究生课程表12 / 10413 / 104人文学院新闻与传播学系、影视艺术研究中心2011—2012学年第一学期硕士研究生课程表14 / 10415 / 10416 / 10417 / 104艺术与设计学院2011—2012学年第一学期硕士研究生课程表18 / 10419 / 10420 / 104生命科学与食品工程学院2011—2012学年第一学期硕士研究生课程表21 / 10422 / 104生命科学与食品工程学院2011—2012学年第一学期硕士研究生课程表23 / 10424 / 10425 / 10426 / 10427 / 10428 / 10429 / 104理学院物理系2011—2012学年第一学期硕士研究生课程表2 / 1043 / 1044 / 104机电工程学院2011—2012学年第一学期硕士研究生课程表5 / 1046 / 1047 / 1048 / 1049 / 10410 / 104建筑工程学院2010—2011学年第一学期硕士研究生课程11 / 104建筑工程学院2010—2011学年第一学期硕士研究生课程12 / 104环境与化学工程学院2011—2012学年第一学期硕士研究生课程表13 / 104环境与化学工程学院2011—2012学年第一学期硕士研究生课程表14 / 104环境与化学工程学院2011—2012学年第一学期硕士研究生课程表15 / 104马克思主义学院、建筑工程学院、环境与化学工程学院2011—2012学年第一学期博士研究生课程表16 / 10417 / 104机电工程学院2011—2012学年第一学期博士研究生课程表18 / 104。
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2010书名定价探索发现:世界未解之谜生命起源未解之谜19.8一目了然的世界战争史3古罗马的繁荣与分裂23.8一目了然的世界战争史4法兰克王国与拜占庭帝国23.8优秀青少年科普趣味读物丛书:远古生物19.8优秀学生必知的化学解读23.8优秀学生必知的科技发明23.8优秀学生必知的陆地奇观23.8优秀学生必知的气象奇观23.8优秀学生必知的珍禽异兽23.8激发孩子想象力的1000个奇思妙想不可思议的人体19.8激发孩子想象力的1000个奇思妙想动物王国大密探19.8激发孩子想象力的1000个奇思妙想令人惊奇的科学19.8激发孩子想象力的1000个奇思妙想千变万化大自然19.8激发孩子想象力的1000个奇思妙想日常生活大揭秘19.8激发孩子想象力的1000个奇思妙想异想天开好问题19.8激发孩子想象力的1000个奇思妙想有趣的植物世界19.8玩转历史—大腕传记书系莎士比亚和他的戏梦人生18.5大灾难未解之谜24.8中小学快乐沟通丛书—难忘的伙伴:如何与同学沟通?16.8健康日记—扔掉小眼镜16中学数理化知识拓展丛书-手机电池中的化学25《红色侵染-革命歌曲大家唱》-歌唱新生活(一)19.8《红色侵染-革命歌曲大家唱》-歌颂祖国19.8健康日记-心理更健康16.8健康日记-远离大虫牙16.8中小学快乐沟通丛书-守护的天使:如何与父母沟通?16.8健康日记-防止意外伤15健康日记-告别小胖墩16.8中小学快乐沟通丛书-化雨的春风:如何与老师沟通?16.8大自然未解之谜24.8激发孩子想象力的1000个奇思妙想-宇宙地球大探索19.8玩转历史—大腕传记书系猫王和他的摇滚麦克风19玩转历史—大腕传记书系丘吉尔和他的光荣战役19玩转历史—大腕传记书系亚历山大和他的磅礴远征18没事儿读点英文小说吧-杰克·伦敦短篇小说精选20探索发现:世界未解之谜地球起源未解之谜19.8健康的生活18.7神的平衡器24.8小学生笔顺规范字典10小学生组词造句(双色)20学生反义词词典(双色)22赠言小词典10土地上的诗庄稼-中国农民诗人诗选25最经典的科幻故事25最经典的情感故事25雷锋精神学习读本25雷锋日记13学生常用典故词典(双色)18小学生辨字辨音手册(双色)18【中华民族优秀传统文化教育丛书】义的系列故事(连环画)29【中华民族优秀传统文化教育丛书】智的系列故事(连环画)29科学巨人18.7神奇的细胞18.7生命的基础18.7多彩的生命18.7生生不息18.7探索发现:世界未解之谜宇宙起源未解之谜19.8优秀学生必知的奇花异木23.8没事儿读点英文小说吧-马克·吐温短篇小说精选20教师口才艺术25玩转历史-大腕传记书系维多利亚女王和她的宫廷娱乐18《红色侵染-革命歌曲大家唱》-歌唱新生活(二)19.8《红色侵染-革命歌曲大家唱》-军歌嘹亮19.8破译天下谜团-飞碟谜团24.8历史深处的忧虑(二版)-近距离看美国之一19总统是靠不住(二版)-近距离看美国之二23如彗星划过夜空21破译天下谜团-宇宙谜团24.8破译天下谜团-地球谜团24.8福尔摩斯全集 血字的研究24探索发现:世界未解之谜自然科学未解之谜19.8探索发现:世界未解之谜UFO与外星人之谜19.8探索发现:世界未解之谜百慕大未解之谜19.8鬼斧神工18.7生命的圣火18.7飞翔的中国18成语故事(彩绘版)13.8动物故事(彩绘版)13.8天鹅·光源24.8探索发现:世界未解之谜恐龙灭绝未解之谜19.8探索发现:世界未解之谜奇趣自然未解之谜19.8探索发现:世界未解之谜人类进化未解之谜19.8【中华民族优秀传统文化教育丛书】行的系列故事(连环画)29学生同义词近义词词典(双色版)18英文书法指南(修订)16最经典的经济学常识25最经典的军事常识25牵牛花25长大不容易28中国记忆-美文卷19.8换一种方式飞行25最经典的人与动物故事25最经典的世界历史常识25中国红:中国新诗90年红色经典20探秘中国汉字18成语小词典19与青少年谈观念22如何与他人交往22从哪里来到哪里去18.7世界未解之谜发明发现未解之谜19.8大自然给人类的礼物能源20分分合合的陆地和水20探索月球的奥秘+20外星人谜团18苦乐年华24还我河山19树枝分叉中的数学19足球中的物理学25细读弟子规25探索太阳系的奥秘18探索星座的奥秘18探索宇宙的奥秘18飞碟现象未解之谜24.8探索宝藏未解之谜24.8高速智能的计算机24次级生长的木材20五彩缤纷的海洋世界20优秀生必知的航天航空20优秀生必知的人体奥秘23优秀生必知的物理奥秘23优秀生必知的宇宙奥秘23优秀生必知的昆虫王国23环境与科学23人类奥秘20恕的系列故事20两个小八路13受益终生的处世精粹16在苏州国学讲习会的讲稿20实用五线谱乐理入门18摄影基础与入门18围棋战术布局技巧18梁遇春精品文集20与青少年谈情操22如何面对灾难22好习惯成就好人生22好心态才会更健康22恐龙帝国24心心相印的磁20新型的纳米技术20细胞20陨石20钢铁20电20可回收使用的废物20爬行动物20地球20探索UFO和外星人的奥秘18探索地球的奥秘18英文小说 欧·亨利短篇小说精选20近距离看美国之三25柯南·道儿短篇小说精选20载人宇宙飞船20神奇的人体结构20生活中离不开的化学20撬动地球的力20干旱缺水的荒漠-沙漠 20温暖千家的热20无处不在的纤维20五彩缤纷的光20生命的杀手 病毒20与人类最亲密的哺乳动物20运输大王火车20在学习中走向成熟22挫折是成长的必修课22拥有健康的心理22与青少年谈品质22习惯是生活的基石22国际象棋人门与提高22五子棋人门与提高18象棋入门与提高18游泳入门与提高18钟启泉教育思想访谈录32感伤的科学史29吉鸿昌的故事18江竹筠的故事18孔子拜师人物传说19董存瑞的故事18刘志丹的故事18左权的故事18黑狗哈拉诺亥23永远的民族精神22与青少年谈哲理22学会思考22受益终生的美术精粹16受益终生的文学精粹16品味热的世界21化学趣味探索实验22你身边的特种部队 真菌与人类22环保的过去现在22图形趣话21蓝色经济21激励小学生勤奋进取的励志故事25开拓小学生创意思维的创新故事25科学未解之谜24.8“事”说师生关系25班主任九项技能训练25优秀学生必知的水族万象23.8玩转科学-透视蓝天的秘密-飞机中的科学25玩转科学-在钢铁中注入灵魂-玩转机器人25破解科学-时间的第四维世界23.8破解科学-网络中的虚拟世界24.5科学就在你身边-探索微观世界的精灵-细菌与人类25科学就在你身边-在深海中与你同行-海洋生物点评22解码科学-微生物的世界23.8解码科学-非常探险20文化艺术大讲堂-美在自然25受益终生的诗词精粹16.9尽展青春风采-与青少年谈礼仪22酿出青春的琼浆-在成长中积累智慧22青春的哲思感悟-与青少年谈感悟22一场滋养精神的盛宴-读书与励志22黒鹤动物文学系列 狼谷的孩子20马本斋的故事18.5邱少云的故事18.5方志敏的故事18.5黄继光的故事18.5刘师培:中国中古文学史19.8孟森:在北大讲明史19.8好学生是这样培养出来的-北京八中初中部得教育思考之二30王若飞的故事18.5刘胡兰的故事18.5黒鹤动物文学系列 黑焰(插图版)19解码科学-图解南北极24.5科学就在你身边-让智慧点亮生活-影响你我的发明25玩转科学-做健康生活的领跑者:运动中的科学25玩转科学-再造另一个你自己-克隆与仿生25奥斯卡优秀影片阅读-大地惊雷25奥斯卡优秀影片阅读-圣安娜的奇迹24优秀青少年科普趣味读物丛书:宇宙探索19.8培养小学生真诚善良的品德故事25探究式科普丛书-最美丽的石头-宝石20探究式科普丛书-生物生存的重要能源-土壤20探究式科普丛书-天堑变通图:桥20探究式科普丛书-高超的猎手-猫科动物20探究式科普丛书-取之不尽的太阳能20探究式科普丛书-人的生物学信息-人类生物学20儿童围棋入门 启蒙篇(第三版)23.8地震与防震减灾知识200问答25国家自然科学基金科普项目《信息世界与人类》科普丛书14.3探究式科普丛书-人体内的电子机器-电子生化人20探究式科普丛书-高大巍峨的山20探究式科普丛书-巨大的天然冰体-冰川20探究式科普丛书-喜忧参半的细菌20探究式科普丛书-显微镜下的微世界-微生物20启迪小学生聪明才智的智慧故事25星火燎原全集普及本之二-强渡大渡河20科学就在你身边-何以构筑美好的家园-谈环境污染22魔幻科学-头脑的魔鬼训练与思维狂欢21解码科学-世博与能源21书写时代新风尚-学好八荣八耻22征服人心的魔力-培养出众的能力22砍断白魔的黑手-拒绝毒品22奏响青春的交响乐-成功与哲理22聆听青春的心跳-寻找激励的源泉22精神放松的艺术-健康的表达情绪22海参的爱情之歌(趣味自然史系列丛书)25孟森:在北大讲清史19.8章太炎:国学的精要19.8实用电子琴基础入门18围棋入门与提高18引领青少年了解世界-政治篇-美国可以说不25为科学献身的动物们19.8校园安全与危机处理(学校管理新探索丛书)32赵一曼的故事18.5鲁班传线人物传说19沉重的社会之痛-解读青少年犯罪22让青少年远离危险22奏响和谐的青春旋律-缓解成长的压力22科学就在你身边-40亿年的风雨历程动物进化22解码科学-墓室迷踪25破解科学-生活在数字时代19宇宙空间大探秘16.9青少年法律教育丛书-帮你活学活用:别让你受伤23.8青少年法律教育丛书-打造你的思维:养成法律思维小中版23.8青少年法律教育丛书-教你怎么行为:做个懂法的小中生22.8学生普法教育丛书-中华人民共和国义务教育法注释本9方法总比问题多-名师转变棘手学生的施教艺术25智慧诊所19.8星火燎原全集普及本之六-跟随毛主席长征18星火燎原全集普及本之一-“八一”的枪声19优秀学生必知的海洋奇观23.8星火燎原全集普及本之四-红十八师突围记19优秀女孩必读的101个故事19.5兄弟树19.8名师最有效的激励细节25让语文教学有趣简单高效25学学名师那些事25综合课的整合创新教学25。
华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:071英语系07级英语专业一班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:072英语系07级英语专业二班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:073英语系07级英语专业三班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:074英语系07级英语专业四班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:061英语系06级英语专业一班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:062英语系06级英语专业二班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:063英语系06级英语专业三班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:064英语系06级英语专业四班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:051英语系05级英语专业一班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:052英语系05级英语专业二班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:053英语系05级英语专业三班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:054英语系05级英语专业四班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:041英语系04级英语专业一班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:042英语系04级英语专业二班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:043英语系04级英语专业三班华东师范大学外语学院英语系课程表(2007-2008学年第二学期第一周开始)英语专业班号:044英语系04级英语专业四班。
《现代教育》2015年第3期目录序号作者论文题目年-期-页1 刘慧浙江省高中选修课问题分析与实施策略2015030012 谢露婷《写作教学模式论》研究创新探微2015030043 王丽媛PCK研究热点知识图谱——基于CNKI数据库的关键词共词分析2015030084 李晓萍论康德美学的“美”与“丑”2015030115 赵宇坤新课程背景下小学教师师德培养的探索2015030156 王欣怡潘玉进虚拟社交网络对初中生人际交往能力和价值观的影响2015030177 屠悦悦王睿阿地兰袁帆帆表情信号表达与接收的不一致性2015030218 陈连云陈雷敏“学为主”语文阅读课堂教学目标研究2015030269 薛建华基于学情视角的语文单元整组课堂作业设计——以人教版小学语文三年级上册第二组阅读教材为例20150303010 赖亦芬林怡萌碰撞乡土文学感悟园本课程20150303411 郑海山马赛花“3+2+x”班级管理机制的实践研究20150303712 陈开连基于内部变革的农村薄弱学校成长路径探索20150304113 潘进听高中语文当代文学课程资源的开发与利用20150304514 陈传敏小学语文课堂教学中想象力培养问题及对策研究20150304815 张亮小学语文科普文教学现状及对策20150305216 陈玉萍浅析现代教育技术在小学习作教学中的应用20150305717 李灵香浅谈小学低段语文课堂板书设计的切入点20150305918 李淑婵陈开连“小组合作自评表”在小学语文课堂教学中的实践研究20150306419 黄凤霞新人教版《鸡兔同笼》教学思考20150306720 王建程浅谈优化科学实验方法20150307121 陈世丰巧用数学日记促进有效教学20150307522 林永向祝光辉科学课堂教学整合综合实践活动的探索20150307723 吴秀培引领视障学生有效参与科学课堂的策略20150308024 叶光希高中物理趣味实验的实践探究20150308225 张乐如何在数学课堂中培养学生的创新思维20150308426 金全论幼儿园双手美术创作教学20150308727 姜伟幼儿园晨间“户外体能工坊”的建构与开展20150309328 李建中尊重幼儿自主活动促进幼儿有效学习20150309729 林红芸幼儿电子成长档案的制作与应用20150309930 戚强建基于小组合作的中职学前教育钢琴集体课教学探索20150310231 吴声孟浅谈图片在历史与社会教学中的运用20150310432 陈海华中学政治课堂探究性学习“失效”现象及其对策20150310733 黄发颖刘海霞从黄光耀小说《白河》看土家族文化20150310934 宋妍刘海霞回忆无禁区——从伤痕文学角度浅析刘心武新作《土茉莉》20150311235 陈钰荧刘海霞不分胜负的博弈——评荆永鸣新作《较量》20150311536 王秋莉刘海霞《当我看不到你的目光的时候》中镜像人生探析201503118 《现代物流学》复习资料第一章 现代物流概述论本章重点难点1、现代物流的概念;2、现代物流与流通的关系;3、现代物流的功能及其分类;4、物流活动;5、物流合理化的含义、作用及其模式;学习目的认识现代物流及物流活动,了解物流的发展和物流科学的产生,掌握物流的概念和物流的功能。
29 Jun 201529 June 2015P.U. (B) 263NOTIS PENENTUAN MUKTAMAD AFIRMATIFAKTA PELINDUNG 2006NOTIS PENENTUAN MUKTAMAD AFIRMATIF PENYIASATAN MENGENAI KELUARANPLAT GULUNGAN PANAS YANG DIIMPORT KE DALAM MALAYSIA(SM 01/14)PADA menjalankan kuasa yang diberikan oleh subseksyen 23(3) Akta Pelindung 2006 [Akta 657] dan peraturan 14 Peraturan-Peraturan Pelindung 2007[P.U. (A) 386/2007], Kerajaan telah membuat penentuan muktamad afirmatif berhubungdengan penyiasatan mengenai keluaran plat gulungan panas yang diimport ke dalamMalaysia sebagaimana yang diperihalkan dalam perenggan 1 (“keluaran”).Keluaran yang disiasat dan pengelasan tarif1.Keluaran yang disiasat dan pengelasan tarifnya adalah seperti yang berikut:(a)keluaran plat keluli gulungan panas besi atau keluli bukan aloi dankeluli aloi yang lain, yang mempunyai kelebaran 600 milimeter ataulebih, gulungan panas, tidak diliputi, disadur atau disalut, yangmempunyai ketebalan antara 6 milimeter hingga 75 milimeter; dan(b)keluaran yang dikelaskan di bawah Kod Sistem yang Diharmonikan(Kod H.S.) 7208.51.000, 7208.52.000, 7225.40.000, dan TatanamaTarif Berharmonis ASEAN (AHTN) 7208.51.0000, 7208.52.0000,7225.40.9000,yang aplikasi, standard antarabangsa, gred dan tajuknya adalah sebagaimana yang dinyatakan dalam Jadual Pertama.2. Bagi maksud subsubperenggan 1(b), Kod H.S. dan AHTN adalah hanya untuk makluman dan tidak mempunyai kesan mengikat terhadap pengelasan keluaran itu.Keluaran serupa atau keluaran yang bersaing secara langsung3. Keluaran serupa atau keluaran yang bersaing secara langsung ialah keluaran yang dikeluarkan oleh industri dalam negeri dan mempunyai perihalan yang sama dengan keluaran yang dinyatakan dalam perenggan 1.Sebab bagi penentuan muktamad afirmatif4. Berdasarkan penyiasatan, Pihak Berkuasa Penyiasat telah mendapati bahawa terdapat kenaikan secara mutlak import keluaran semasa tempoh penentuan kemudaratan iaitu mulai1 Januari 2013 hingga 31 Disember 2013. Pihak Berkuasa Penyiasat mendapati bahawa industri dalam negeri telah mengalami kemudaratan yang serius berkenaan dengan pengurangan syer pasaran, pengurangan dalam jualan dalam negeri, tahap produktiviti dan penggunaan kapasiti yang rendah, penurunan dalam aliran tunai, kemerosotan keuntungan dan inventori, dan pulangan pelaburan yang negatif. Perkara ini telah menyebabkan dan mengancam untuk menyebabkan kemudaratan yang serius kepada industri dalam negeri yang mengeluarkan keluaran serupa atau keluaran yang bersaing secara langsung.Langkah pelindung yang terpakai5. Langkah pelindung yang terpakai berhubung dengan keluaran hendaklah dalam bentuk duti pelindung muktamad dan dikenakan terhadap negara yang dinyatakan dalam Jadual Kedua.6. Duti pelindung muktamad hendaklah dikenakan bagi tempoh tiga tahun mulai 2 Julai 2015 hingga 1 Julai 2018 pada kadar yang berikut:Tempoh Duti Pelindung Muktamad (%)2 Julai 2015 – 1 Julai 2016 17.40 %2 Julai 2016 – 1 Julai 2017 13.90%2 Julai 2017 – 1 Julai 2018 10.40%JADUAL PERTAMA[Perenggan 1]KELUARAN PLAT GULUNGAN PANAS YANG DIKENAKAN DUTI PELINDUNG MUKTAMADJADUAL KEDUA[Perenggan 5]NEGARA YANG DIKENAKAN DUTI PELINDUNG MUKTAMAD1. Australia2. Kanada3. Republik Czech4. Republik Persekutuan Jerman5. Perancis6. Luxembourg7. Greece8. Hungary9. Ireland10. Itali11. Jepun12. Negara Belgium13. Negara Denmark14. Negara Norway15. Negara Sepanyol16. Negara Sweden17. Negara Belanda18. New Zealand19. Republik Rakyat China20. Republik Portugal21. Liechtenstein22. Republik Austria23. Republik Bulgaria24. Republik Cyprus25. Republik Estonia26. Republik Finland27. Republik Iceland28. Republik Indonesia29. Republik Korea30. Republik Latvia31. Republik Lithuania32. Republik Malta33. Republik Poland34. Republik Singapura35. Republik Slovenia36. Romania37. Slovakia38. Negara Israel39. SwitzerlandBertarikh 26 Jun 2015[MITI:ID/(S/AP/SG/045/3 Jld 2); PN(PU2)647/IV]40. Ukraine41. United Kingdom of Great Britain dan Ireland Utara 42.Amerika SyarikatDATO’ SRI MUSTAPA BIN MOHAMEDMenteri Perdagangan Antarabangsa dan IndustriSAFEGUARDS ACT 2006NOTICE OF AFFIRMATIVE FINAL DETERMINATION OF AN INVESTIGATION WITH REGARD TO HOT ROLLED PLATES PRODUCTS IMPORTED INTO MALAYSIA(SM 01/14)IN exercise of the powers conferred by subsection 23(3) of the Safeguards Act 2006 [Act 657] and regulation 14of the Safeguards Regulations 2007 [P.U. (A) 386/2007], the Government has made an affirmative final determination inrelation to the investigation with regard to the hot rolled plates products imported intoMalaysia as described in paragraph 1 (“products”).Products under investigation and tariff classification1. The products under investigation and its tariff classification are as follows:(a)the hot rolled steel plate products of iron or non-alloy steel andother alloy steel, of a width of 600 millimetres or more, hot rolled,not clad, plated or coated, of a thickness between 6 millimetresto 75 millimetres; and(b) the products classified under the Harmonised System Code(H.S. Code) 7208.51.000, 7208.52.000, 7225.40.000, and ASEANHarmonised Tariff Nomenclature (AHTN) 7208.51.0000,7208.52.0000, 7225.40.9000,which applications, international standards, grades and titles are as specified in theFirst Schedule.2.For the purposes of subsubparagraph 1(b), the H.S Code and AHTN are only for information and shall have no binding effect on the classification of the products.Like products or directly competitive products3. The like products or directly competitive products are products which are produced by the domestic industry and have the same description as the products specified in paragraph 1.Reasons for affirmative final determination4. Based on the investigation, the Investigating Authority has found that there wasan increase in imports of the products in absolute terms during the period of injury determination that is from 1 January 2013 until 31 December 2013. The Investigating Authority has found that the domestic industry has suffered serious injury in respect ofthe decline in market share, decline in domestic sales, low production and capacity utilisation, decline in cash flow, decline in profitability and inventory, and negative return on investment. These have caused and are threatening to cause serious injury tothe domestic industry that produces like or directly competitive products.Safeguards measures applicable5. The safeguards measures applicable in relation to the products shall take the formof definitive safeguards duties and shall be imposed on the countries specified in the Second Schedule.6. Definitive safeguards duties shall be imposed for a period of three years from 2 July 2015 until 1 July 2018 at the following rates:Period Definitive Safeguards Duties (%)2 July 2015 – 1 July 2016 17.40 %2 July 2016 – 1 July 2017 13.90%2 July 2017 – 1 July 2018 10.40%FIRST SCHEDULE[Paragraph 1]HOT ROLLED PLATES PRODUCTS WHICH ARE IMPOSED WITH THE DEFINITIVESAFEGUARDS DUTIESSECOND SCHEDULE[Paragraph 5]COUNTRIES IMPOSED WITH DEFINITIVE SAFEGUARDS DUTIES1. Australia2. Canada3. Czech Republic4. Federal Republic of Germany5. France6. Luxembourg7. Greece8. Hungary9. Ireland10. Italy11. Japan12. Kingdom of BelgiumDated 26 June 2015[MITI:ID/(S/AP/SG/045/3 Jld 2); PN(PU2)647/IV]13. Kingdom of Denmark 14. Kingdom of Norway 15. Kingdom of Spain 16. Kingdom of Sweden17. Kingdom of the Netherlands 18. New Zealand19. People's Republic of China 20. Portuguese Republic 21. Liechtenstein22. Republic of Austria 23. Republic of Bulgaria 24. Republic of Cyprus 25. Republic of Estonia 26. Republic of Finland 27. Republic of Iceland 28. Republic of Indonesia 29. Republic of Korea 30. Republic of Latvia 31. Republic of Lithuania 32. Republic of Malta 33. Republic of Poland 34. Republic of Singapore 35. Republic of Slovenia 36. Romania 37 Slovakia38. State of Israel 39. Switzerland 40. Ukraine41. United Kingdom of Great Britain and Northern Ireland 42.United States of AmericaDATO’ SRI MUSTAPA BIN MOHAMED Minister of International Trade and Industry。
ORIGINAL ARTICLEThe P2X7receptor is a key modulator of the PI3K/GSK3β/VEGF signaling network:evidence in experimental neuroblastomaF Amoroso 1,M Capece 1,A Rotondo 1,D Cangelosi 2,M Ferracin 1,A Franceschini 1,L Raffaghello 3,V Pistoia 3,L Varesio 2and E Adinol fi1INTRODUCTIONOne of the new frontiers in oncology is the understanding of microenvironment activity on tumor cells.Extracellular ATP,an abundant constituent of the oncogenic milieu,is emerging as a new and potent regulator of cancer growth progression and immune response modulation.1–4In the tumor microenvironment,ATP acts as a trophic factor,danger signal and main source of the immunosuppressant adenosine.5Extracellular ATP is the natural ligand of P2Y metabotropic and P2X inotropic receptors,among which P2X7is the best candidate responsible for cancer-associated ATP effects,as it has shown an intriguing ability to confer a growth advantage to cancer cells in in vivo models.1The growth-promoting activity of P2X7may be related to the proliferative advantage conferred to tumor cells under limiting growth conditions,such as serum and glucose deprivation,6–8to the stimulation of vascular endothelial growth factor (VEGF)secretion and to the facilitation of extracellular matrix invasion.1,2,9Although P2X7expression and activity has been reported in several cancers 10,11the tumor-promoting pathways activated by P2X7are largely unknown.Neuroblastoma (NB)is a common neuroendocrine childhood tumor,causing 15%of pediatric cancer deaths.This malignancypresents as a highly heterogeneous disease ranging from spontaneously regressing to refractory forms.The therapeutic intervention for advanced-stage patients is rarely successful.Advanced-stage patients show 42%survival rate at 5years,despite aggressive multimodality therapy.12Owing to the similarities of neuroblastoma cells to neurons,the activity of P2X7receptor in NB cell lines has been investigated by different studies.13P2X7has been attributed a role in NB cell exocytosis 14and in the regulation of neuronal differentiation.15,16We and others have previously shown that P2X7receptor is involved in NB cell proliferation both in vitro 16,17and in vivo .1However,an in-depth investigation of the signaling cascade,activated by P2X7and responsible for stimulation of NB growth,was still missing.The phosphatidylinositol-tris-phosphate kinase 3(PI3K)signal-ing network is a recognized oncogenic pathway activated in NB.18The PI3K pathway comprises PI3K itself,Akt and glycogen synthase kinase 3β(GSK3β).19PI3K/Akt signaling promotes increased GSK3βphosphorylation,which is associated with reduced GSK3βactivity.19In its unphosphorylated form,GSK3βmediates proteasome degradation of MYCN,the oncogene most frequently overexpressed in NB.20Moreover,aggressive NB shows high microvessel density,indicative of intense neo-angiogenesis,1Department of Morphology,Surgery and Experimental Medicine,Section of Experimental Pathology,Oncology and Biology,University of Ferrara,Ferrara,Italy;2Laboratory of Molecular Biology,Giannina Gaslini Institute,Genoa,Italy and 3Laboratory of Oncology,Giannina Gaslini Institute,Genoa,Italy.Correspondence:Dr E Adinol fi,Department of Morphology,Surgery and Experimental Medicine,Section of Experimental Pathology,Oncology and Biology,University of Ferrara,Via Luigi Borsari 46,Ferrara 44121,Italy.E-mail:elena.adinol fi@unife.itReceived 21July 2014;revised 21November 2014;accepted 1December 2014Oncogene (2015),1–12©2015Macmillan Publishers Limited All rights reserved 0950-9232//oncdue to PI3K-dependent VEGF activity.21However,the PI3K/Akt pathway is of pivotal importance also in non-cancerous cell survival,and the therapeutic activity of drugs interfering with these biochemical ways was lower than expected.22Therefore, novel tumor-specific upstream pharmacological targets able to modulate the PI3K/Akt axis are eagerly sought.In this study,we report the identification of P2X7as an upstream modulator of the PI3K/Akt pathway.We show that P2X7increases PI3K/Akt activation,HIF1αexpression,VEGF secretion and GSK3βinactivation,regulating MYCN oncogene and glycogen accumulation.Interestingly,two different P2X7 antagonists were found to be highly effective in reducing NB growth in vivo,in both xenogeneic and syngeneic murine models.Finally,high P2X7levels were associated with poor overall survival in a cohort of NB patients.Taken together,our data point to P2X7as a new oncogene and a promising therapeutic target in neuroblastoma.RESULTSP2X7increases PI3K/Akt activity in ACN human NB cellsPI3K,which plays a critical role in cancer cell growth,has been recently associated to NB progression.18Thus,we investigated whether P2X7could modulate PI3K activity.To this aim,we utilized the ACN human NB cell line,which proliferates both in vitro and in vivo in a P2X7-dependent manner.1,17Treatment of ACN cells with either P2X7synthetic agonist benzoyl-ATP or its natural ligand ATP caused a30%increase of PI3K activity,as assessed by measuring the phosphatidylinositol-tris-phosphate (PIP3)production(Figure1a).PI3K activation was followed by enhanced phosphorylation of Akt(PhAkt(Ser473))(Figure1b and c). PhAkt(Ser473)was dose dependently upmodulated upon benzoyl-ATP application,reaching a100%increase at the maximal dose applied(300μM).Akt activity was increased even more by millimolar concentrations of ATP(Figures1b and c).In a similar manner,P2X7Figure1.P2X7receptor stimulation increases PI3K/Akt,while decreasing GSK3βactivity in ACN cells.(a–d)ACN cells were treated with P2X7 agonists benzoyl-ATP(Bz-ATP)and ATP for48h.(a)PI3K activity was evaluated by measuring PIP3levels.Red:untreated,dark green:100μM Bz-ATP,light green:200μM Bz-ATP,blue:1m M ATP,cyan:2m M ATP.N=9,***P o0.001;**P o0.01.(b)Representative western blot showing the effect of P2X7stimulation on PhAkt(Ser473),total Akt,PhGSK3β(Ser9),total GSK3β,myosin II.(c)Densitometry of PhAkt(Ser473) normalized on myosin II.N=6,***P o0.001;**P o0.01,*P o0.05.(d)Densitometry of PhGSK3β(Ser9)normalized on myosin II.N=6;***P o0.001;*P o0.05.P2X7antagonism as neuroblastoma treatmentF Amoroso et al2Oncogene(2015)1–12©2015Macmillan Publishers Limitedactivation augmented GSK3βphosphorylation(PhGSK3β(Ser9)) (Figures1b and d),thus causing a decrease in its activity.P2X7silencing or blockade reduces extracellular ATP levelsIn an effort to determine whether P2X7receptor could affect the concentration of ATP in NB microenvironment,we evaluated the effect of P2X7loss or blockade on ATP secretion.P2X7silencing was obtained by stable transfection of two shRNA constructs (referred as shRNA1and shRNA2;see Supplementary Figures1a and b),while P2X7activity was antagonized by administration of AZ10606120and A740003at the lowest effective concentrations in blocking P2X7-dependent intracellular calciumflux(300n M AZ10696120;5μM A740003,Supplementary Figure1c).P2X7 down-modulation by either silencing or drug administration resulted effective in reducing ATP release from ACN cells (Figure2a),suggesting that the receptor would activate an autocrine/paracrine loop positively influencing NB growth.P2X7downmodulation decreases PI3K/Akt activity and cell growth while increasing GSK3βfunctionOnce ascertained that P2X7can influence the levels of its natural agonist ATP in NB,we evaluated if receptor downmodulation could also negatively affect the PI3K/Akt axis.In accordance with results obtained with P2X7agonists,production of PIP3was almost halved by P2X7silencing while antagonism with AZ10606120or A740003caused a reduction in PIP3amount of 33%and75%,respectively(Figure2b).P2X7blockade by shRNA2 or both antagonists tested reduced PhAkt(Ser473)slightly but significantly(Figures2c,d,f and g).On the contrary,GSK3βactivity was strikingly increased by P2X7downmodulation,as demon-strated by decrease in its phosphorylation at serine9(Figures2c, e,f and h).Increased activity of GSK3βranged from15%,obtained with AZ10606120,to60%,achieved by both shRNA1and shRNA2 silencing(Figures2e and h).In an effort to evaluate whether P2X7 antagonism was effective in substantially reducing PI3K/Akt-mediated NB cell proliferation,we measured ACN cell growth following administration of previously tested P2X7antagonists and PI3K inhibitors LY294002and NVP-BEZ235.All compounds caused a significant and comparable reduction of ACN cell growth (Figure2i).Interestingly,if compared to single-drug administra-tion,jointed treatment with P2X7and PI3K blocking drugs did not cause an additional reduction of cell growth,suggesting that,in our model,P2X7impacts on proliferation acting as an upstream regulator of the PI3K pathway.P2X7influences glycogen accumulation in NB cellsActive GSK3βblocks glycogen synthase activity,resulting in depletion of glycogen stores.23To confirm the link between P2X7 and GSK3βactivity,we evaluated whether P2X7could influence glycogen accumulation.ACN cells silenced for P2X7by shRNA1 and shRNA2showed an evident reduction of glycogen stores over control scramble shRNA,as assessed by Periodic Acid-Schiff(PAS) staining(Figures3a and c).P2X7antagonists determined a similar reduction in glycogen staining intensity in ACN cells (Figures3f and h).Notably,both P2X7silencing and antagonists induced a significant decrease in the number of PAS-positive cells(Figures3d and i)and in their total glycogen content, assessed by measuring glucose generated from glycogen hydro-lysis(Figures3e and j).P2X7silencing or blockade decreases HIF1α/VEGF levelsTo further extend our understanding of P2X7role in NB,we investigated whether this receptor could be involved in angiogen-esis,which is critical in NB progression and has been associated to PI3K/Akt axis.24Therefore,we tested whether P2X7could affect the most relevant pathway causing vessels sprouting,that is,HIF1α–VEGF.25Both P2X7partial loss and antagonism induced a striking reduction(≈50%)of HIF1αprotein content(Figure4a).As expected,secretion of VEGF was consequently reduced (Figure4b).P2X7antagonists cause NB regression in two different in vivomodelsTo investigate whether P2X7receptor could be a valuable pharmacological target for NB,we studied the effects of P2X7 antagonists on NB cell growth in vivo.As afirst model,we tested tumors derived by subcutaneous injection of human ACN cells in immune-compromised nude/nude mice.AZ10606120(300n M),A740003(5μM)or placebo(phosphate-buffered saline+0.005% dimethyl sulfoxide)was administered intraperitoneally every2days after the appearance of the tumor mass(day5from cells injection)for a total of28days.Treatment with either AZ10606120or A740003caused an evident(≈40%)reduction of excised tumorsize(Figures5a and b).Western blot analysis of tumor homogenates(Figure5c)confirmed the data obtained in vitro. Hence,P2X7antagonist administration decreased both Akt and GSK3βphosphorylation(Figures5c and e),suggesting a reductionand an increase,respectively,in the activity of these kinases. Considering the two different antagonists tested,A740003hadthe most relevant effect on the Akt/GSK3βaxis causing30% reduction in the phosphorylation of both kinases(Figures5c and e).AZ10606120in vivo administration caused a slight reduction(≈15%)of Akt/GSK3βphosphorylation.These results were consistent with the in vitro data,showing a stronger activity ofA740003versus AZ10606120(Figures2e and g).Treatment withboth P2X7antagonists obliterated MYCN content in ACN-derived tumors(Figures5c and f).Similarly to the in vitro data,P2X7 blockade by A740003caused a significant reduction of VEGF levels measured in tumor homogenates(Figure5g).We next tested the efficacy of P2X7antagonists against experimental NB obtained by injection of murine Neuro2A NB cells in syngeneic AlbinoJ mice.This fully immune-competent strain allowed us to assess whether P2X7antagonists interferedwith the mouse immune system,and how such interferenceinfluenced tumor growth,as P2X7activation has been associatedto antitumor immune response.26In Neuro2A cells,the lower concentration of A740003effective in blocking P2X7-evoked calcium rise was of10μM(Supplementary Figure1d);the drug was consequently in vivo administered at this concentration.Placeboor inhibitors were administered every2days from the appearanceof thefirst tumor mass(day8from the inoculum)until day15.The effect of P2X7antagonism in Neuro2A-bearing mice was even stronger than that seen in ACN-bearing animals.In fact,receptor blockade by both A740003and AZ10606120caused an almost 50%reduction of tumor growth rate in live animals(Figure6a), accompanied by a comparably strong reduction of excised tumor volume(Figures6b and c).The biochemical pathways affected byanti-P2X7drugs included PhGSK3β(Ser9),whose levels were halved by A740003(Figures6d and e),and HIF1α,which was strongly decremented by both drugs(Figures6d and f). Interestingly,P2X7antagonists strikingly reduced MYCN levels (Figures6d and g)similarly to that observed in the ACN model (Figures5c and f).Finally,HIF1αamount reduced by P2X7 antagonists(Figures6d and f)was paralleled by a decreased levelof VEGF in tumor homogenates,blood vessel numbers and staining with the endothelial marker von Willebrand factor (Figures6h–l).P2X7expression correlates with poor prognosis of stage IV NB patientsTo further investigate P2X7relevance as a therapeutic target,we correlated patient survival with P2X7levels in a NB patients’cohort.We performed the gene expression profile of131NBP2X7antagonism as neuroblastoma treatmentF Amoroso et al3©2015Macmillan Publishers Limited Oncogene(2015)1–12patients for which we collected information on International Neuroblastoma Staging System stage and overall survival.Patients were divided into two groups according to P2X7expression levels (high-expressing and low-expressing).Kaplan–Meier survival analysis was performed independently on56highly aggressive (stage4)and75not aggressive(stages1,2,3,4s)tumors using P2X7expression levels as a variable.We found that higher P2X7 levels significantly correlated with an unfavorable prognosisin P2X7antagonism as neuroblastoma treatmentF Amoroso et al4Oncogene(2015)1–12©2015Macmillan Publishers Limitedstage-4(Figure 7a,P 40.05),but not in other-stage (1,2,3,4s)NB patients (Figure 7b).In the examined cohort,we did not find any correlation between P2X7and PI3K,GSK3βor MYCN gene expression levels (data not shown),thus suggesting that P2X7acts post-transcriptionally in modulating both PI3K and GSK3βaxis.DISCUSSIONAn increasing body of literature strongly supports P2X7involve-ment in cancer cell proliferation,energy production and migration.1,2,7,8,27P2X7expression has been demonstrated in a wide spectrum of tumor types,11,28including colon carcinoma,melanoma and NB,all of which also showed P2X7-dependent growth in vivo .1With the present study,we further extended our findings,unveiling PI3K/GSK3β/MYCN /HIF1αaxis as a new path-way activated by P2X7in NB.The PI3K/Akt signaling cascade is a central player of tumor cell growth and bioenergetics that regulates aerobic glycolysis,cell cycle progression and autophagy.In the speci fic context of NB,PI3K/Akt has been also associated to progression and resistance to chemotherapy.18,29In this study,we demonstrated a P2X7-dependent positive regulation of the PI3K/Akt path in NB cell lines and derived tumors.Indeed,P2X7agonist benzoyl-ATP increased both PI3K and Akt activity,as P2X7antagonism caused a substantial inhibition of the PI3K/Akt pathway.Interestingly,P2X7antagonist reduced the levels of extracellular ATP released by NB cells,con firming a role for the receptor in the secretion of its own natural ligand in cancer.30We also provided the first evidence that P2X7not only modulated the PI3K/Akt pathway but also downstream effectors such as GSK3βand HIF1α.31,32In response to agonist binding,P2X7caused inactivation of GSK3β(Figure 1),whereas P2X7antagonists or receptor silencing had an opposite effect on kinase activity (Figures 2,3,5and 6).GSK3βis well known as a negative regulator of glycogen synthase,and drives consumption of cellular glycogen.Accordingly,wild-type ACN showed increased levels of glycogen in comparison to ACN cells either P2X7silenced or treated with P2X7antagonists (Figure 3).Clear glycogen-rich cells are present in many aggressive tumors,and glycogen is emerging as the energy supply for cancer cells,promoting their survival in metabolic stressing conditions and hypoxia.23,33,34Our data,showing a reduction of tumor cell glycogen following P2X7downmodulation,suggest that P2X7might favor cell survival acting at this energy source.By activating GSK3β,P2X7antagonists also destabilize another target of this kinase,35that is,MYCN (Figures 5b,e and 6d,g).MYCN is a well-characterized oncogene in NB and,owing to its frequent ampli fication in poor-prognosis patients,it represents a reliable prognostic marker.20Remarkably,in vivo administration of P2X7antagonists strongly reduced tumoral levels of MYCN in two different experimental NB models (Figures 5and 6).As far as we know,this is the first timethat an association between P2X7and MYCN was reported,further supporting an oncogenic role for P2X7.Moreover,in the patient cohort that we analyzed,the levels of P2X7mRNA did not correlate with those of MYCN (not shown),suggesting that destabilization of MYCN ,detected in experimental tumor samples (Figures 5c and f),occurred at a post-translational level,likely owing to GSK3βphosphorylation.35,36MYCN and its positive regulator PI3K have been postulated to increase NB vasculariza-tion,via upregulation of VEGF secretion.21Blood vessel formation is an absolute requisite for tumor progression and accounts for the oncogenic activity of angiogenic proteins such as HIF1αand VEGF.37In accordance with its effect on PI3K and MYCN ,P2X7also modulated HIF1αand VEGF expression in both in vitro and in vivo models.In ACN human NB cells,P2X7silencing or its pharmaco-logical blockade caused a remarkable reduction of both HIF1αand VEGF levels (Figure 4).A similar effect was observed in in vivo NB models,where P2X7antagonists downmodulated HIF1αand VEGF secretion (Figures 5and 6)and consequent blood vessel formation (Figures 6f –l).Despite its recognized oncogenic role in neuroblastoma,MYCN is a problematic pharmacological target.38Similarly,molecules developed to target the PI3K/Akt path in cancer patients,once tested in clinical trials,showed a limited ef ficacy,owing to toxicity on normal cells.22Our data point to P2X7antagonists as a valuable therapeutic alternative to MYCN and PI3K/Akt inhibitors in NB.In fact,in our experimental setting,treatment with either AZ10606120or A740003signi ficantly downsized tumor dimensions (Figures 5and 6).Reduction of tumor growth was also accompanied by MYCN ,HIF1αand VEGF downmodulation and upregulation of the antioncogenic kinase GSK3β.These data provide a strong preclinical evidence of the ef ficacy of P2X7inhibitors for NB therapy.Interestingly,when co-administered with P2X7antagonists,PI3K inhibitors did not show any signi ficant additional effect on cell proliferation (Figure 2i)suggesting that,in NB,P2X7blockers could effectively substitute PI3K inhibitors as therapeutic strategy.Different P2X7antagonists are under clinical trial for in flammatory pathologies and appear to be safely tolerated by humans.39–41Moreover,a clinical trial to cure cancer with a P2X7-blocking antibody was recently launched (Biosceptre International,North Ryde,NSW,Australia).Our results may pave the way to the investigation of the ef ficacy of such molecules in controlled studies enrolling patients affected by NB.One of the main arguments against the use of P2X7antagonists in tumor therapy is P2X7-dependent activation of antitumoral immune response,which could be lost upon receptor blockade,thus favoring tumor progression.4However,in our experimental setting,systemic administration of P2X7blockers caused tumor regression.Moreover,the antitumor effect of P2X7antagonists was higher in the fully immune-competent Neuro2A-bearing mice than in the immune-compromised ACN-bearing mice.This difference could be partially ascribed to blockade of P2X7expressed by suppressor immune cells such asmyeloid-derivedneuroblastoma cells,affecting their proliferation.(a )Extracellular ATP levels measured in supernatants of ACN cells.Red:ACN scrambled control,cyan:ACN transfected with anti-P2X7shRNA1,blue:ACN transfected with anti-P2X7shRNA2,ocher:untreated ACN control,light green:300n M AZ10606120,dark green:5μM A740003.N =10,***P o 0.001,**P o 0.01.(b )PI3K activity was evaluated measuring PIP 3levels.Red:ACN scrambled control,cyan:ACN transfected with anti-P2X7shRNA1,blue:ACN transfected with anti-P2X7shRNA2;ocher:untreated ACN control,light green:300n M AZ10606120,dark green:5μM A740003.N =9;**P o 0.01;*P o 0.05.(c –e )Western blot analysis of ACN cells silenced for P2X7receptor (shRNA1,shRNA2)or scramble control.(c )Representative immunoblot of PhAkt(Ser473),total Akt,PhGSK3β(Ser9),total GSK3β,myosin II.(d )Densitometry of PhAkt(Ser473)normalized on myosin II.N =6,*P o 0.05.(e )Densitometry of PhGSK3β(Ser9)normalized on myosin II.N =6,***P o 0.001.(f –h )Western blot analysis of ACN cells treated for 48h with either 300n M AZ10606120or 5μM A740003.(f )Representative immunoblot of PhAkt(Ser473),total Akt,PhGSK3β(Ser9),total GSK3β,myosin II.(g )Densitometry of PhAkt(Ser473)normalized on myosin II.N =6,*P o 0.05.(h )Densitometry of PhGSK3β(Ser9)normalized on myosin II.N =6,**P o 0.01;*P o 0.05.(i )Proliferation at 48h of ACN cells treated with P2X7antagonists AZ10606120(300n M ),A740003(5μM ),PI3K inhibitors NVP-BEZ235(1μM ),LY294002(1μM )administered singularly or in combination.Data shown represent fold increase in cell numbers on time 0after 48h in serum starvation.Reported signi ficance was calculated versus vehicle.N =15,***P o 0.001.P2X7antagonism as neuroblastoma treatment F Amoroso et al5©2015Macmillan Publishers LimitedOncogene (2015)1–12Figure 3.P2X7receptor downmodulation decreases ACN cells'glycogen stores.(a –d ,f –i )ACN cells were stained with PAS to reveal glycogen.(a )ACN transfected with scramble shRNA control.(b )ACN silenced for P2X7with shRNA1.(c )ACN silenced for P2X7with shRNA2.(d )Percentage of PAS-positive cells.Red:ACN scramble,cyan:ACN shRNA1,blue:ACN shRNA2.N =9,***P o 0.001.(e )Intracellular glycogen evaluated enzymatically following cell lysis (see Materials and Methods),red:ACN scramble,cyan:ACN shRNA1,blue:ACN shRNA2.N =15,*P o 0.05.(f )ACN untreated control.(g )ACN cells treated for 48h with 300n M AZ10606120.(h )ACN cells treated for 48h with 5μM A740003.(i )Percentage of PAS-positive cells.Ocher:untreated control,light green:AZ10606120,dark green:A740003.N =9,***P o 0.001.(j )Intracellular glycogen evaluated enzymatically following cell lysis (see Materials and Methods).Ocher:untreated control,light green:AZ10606120,dark green:A740003.N =15,***P o 0.001,*P o 0.05.P2X7antagonism as neuroblastoma treatmentF Amoroso et al6Oncogene (2015)1–12©2015Macmillan Publishers Limitedsuppressor cells.In fact,myeloid-derived suppressor cells have been shown to facilitate neuroblastoma growth negatively regulating tumor immune cell in filtration in a P2X7-dependent manner.42One of the open challenges in neuroblastoma therapy is the discovery of new and ef ficacious pharmacologic approaches for highly aggressive patients that show a really limited survival rate.The analysis of P2X7expression levels in a patients ’cohort allowed to identify an association between the receptor and clinical outcome.In fact,P2X7expression was associated with reduced overall survival of stage-4NB patients,whereas it did not stratify patients in stages 1,2,3,4s.Taken together,these data suggest that pharmacological treatment with anti-P2X7agents could be tailored to aggressive NB patients.Notably,previous studies related P2X7overexpression to poor outcome and metastatic dissemination in other malignancies such as chronic lymphocytic and acute leukemia,papillary thyroid and prostate carcinoma,43–46which,in light of our data,could be considered as eligible pathologies for P2X7-blocking therapy.Evidence reported in this study indicates P2X7as a clear oncogenic driver in neuroblastoma,affecting the best known pathways involved in tumor growth and progression (Supplementary Figure 2),and patient prognosis.We also gave preclinical demonstration of P2X7antagonist ef ficacy in neuro-blastoma treatment,prompting the use of these drugs in clinical trials.MATERIALS AND METHODS Reagents and antibodiesP2X7antagonists AZ10606120and A740003were purchased from Tocris Bioscience (Ellisville,MS,USA).The two compounds were dissolved in dimethyl sulfoxide,to reach a 100m M stock concentration,and subsequently diluted in phosphate-buffered saline.PI3K antagonists LY294002and NVP-BEZ235were from Selleckchem (distributed by Aurogene,Rome,Italy).Anti-P2X7receptor polyclonal antibody was from Sigma Aldrich (Milan,Italy).Human monoclonal anti-Akt,PhAkt(Ser473)and anti-myosin IIa antibodies (Abs)were acquired from CellSignalingFigure 4.Silencing or pharmacological inhibition of P2X7reduces HIF1αprotein content and VEGF release.(a )HIF1αamount revealed by ELISA assay in ACN scramble (red),ACN shRNA1(cyan),ACN shRNA2(blue),ACN untreated (ocher)or treated with 300n M AZ10606120(light green),5μM A740003(dark green).N =6;***P o 0.001;**P o 0.01.(b )VEGF levels measured by ELISA assay in ACN scramble (red),ACN shRNA1(cyan),ACN shRNA2(blue),ACN untreated (ocher)or treated with 300n M AZ10606120(light green)or 5μM A740003(dark green).Treatment with P2X7inhibitors was performed for 48h.N =6;***P o 0.001;**P o 0.01.P2X7antagonism as neuroblastoma treatment F Amoroso et al7©2015Macmillan Publishers LimitedOncogene (2015)1–12Figure 5.P2X7antagonist reduces growth of ACN cells injected in nude/nude mice.ACN-derived tumors were generated in nude/nude mice as described in Materials and Methods.300n M AZ10606120,5μM A740003or placebo (phosphate-buffered saline+dimethyl sulfoxide 0.005%)were intraperitoneally administered every 2days from first tumor mass appearance (day 5)for a total of 28days.(a –b )P2X7inhibitors reduce excised tumor dimensions.(a )Representative explants obtained from mouse treated with placebo,300n M AZ10606120or 5μM A740003.(b )Volume of excised tumor masses was calculated as described in Materials and Methods.Ocher:placebo;light green:AZ10606120;dark green:A740003.N =6;**P o 0.01;*P o 0.05.(c –f )Western blot analysis of tumor homogenates.(c )Representative immunoblot of PhAkt (Ser473),PhGSK3β(Ser9),total GSK3β,total Akt ,MYCN ,myosin II.(d )Densitometry of PhAkt(Ser473)normalized on myosin II.N =9,*P o 0.05.(e )Densitometry of PhGSK3β(Ser9)normalized on myosin II.N =9,*P o 0.05.(f )Densitometry of MYCN normalized on myosin II.N =9,***P o 0.001.(g )VEGF amount in tumor homogenates revealed by ELISA,ocher:placebo;light green:AZ10606120;dark green:A740003.N =9,***P o 0.001.P2X7antagonism as neuroblastoma treatmentF Amoroso et al8Oncogene (2015)1–12©2015Macmillan Publishers LimitedFigure 6.P2X7antagonists cause growth arrest of Neuro2A-derived experimental neuroblastoma.Neuro2A cell-derived tumors were generated in syngeneic AlbinoJ mice as described in Materials and Methods.AZ10606120300n M ,A74000310μM or placebo (phosphate-buffered saline+dimethyl sulfoxide 0.005%)was intraperitoneally administered every 2days from first tumor mass appearance (day 8)for four doses.(a )P2X7blockade slows down tumor growth.Tumors were measured in live animals with a manual caliper.Ocher:placebo;light green:AZ10606120;dark green:A740003.N =6,***P o 0.001.(b –c )P2X7antagonism reduces tumor dimensions.(b )Representative explants obtained from mouse treated with placebo,300n M AZ10606120or 10μM A740003.(c )Volume of excised tumors.Ocher:placebo;light green:AZ10606120;dark green:A740003.N =6,**P o 0.01,*P o 0.05.(d –g )Western blot analysis of tumor homogenates.(d )Representative immunoblot of PhGSK3β(Ser9),total GSK3β,MYCN ,HIF1α,myosin II.(e )Densitometry of PhGSK3β(Ser9)normalized on myosin II.N =6,*P o 0.05.(f )Densitometry of HIF1αnormalized on myosin II.N =6,*P o 0.05.(g )Densitometry of MYCN normalized on myosin II.N =6,*P o 0.05,***P o 0.001.(h )VEGF levels measured in tumor homogenates.Ocher:placebo;light green:AZ10606120;dark green:A740003.N =6,**P o 0.01,*P o 0.05.(i –k )Neuro2A-derived tumors stained with anti-von Willebrand Factor antibody to identify vascular endothelia.(i )Placebo.(j )AZ10606120.(k )A740003.(l )Number of blood vessels per microscopic field obtained with an ×10objective.Ocher:placebo;light green:AZ10606120;dark green:A740003.N =10,***P o 0.001.P2X7antagonism as neuroblastoma treatment F Amoroso et al9©2015Macmillan Publishers Limited Oncogene (2015)1–12。
Drift in phase space:a new variational mechanismwith optimal diffusion timeMassimiliano Berti,Luca Biasco and Philippe Bolle Abstract:We consider non-isochronous,nearly integrable,a-priori unstable Hamiltonian systems with a(trigonometric polynomial)O(µ)-perturbation which does not preserve the unperturbed tori.We prove the existence of Arnold diffusion with diffusion time T d=O((1/µ)log(1/µ))by a variational method which does not require the existence of“transition chains of tori”provided by KAM theory.We also prove that our estimate of the diffusion time T d is optimal as a consequence of a general stability result derived from classical perturbation theory.1Keywords:Arnold diffusion,variational methods,shadowing theorem,perturbation theory,nonlinear functional analysisAMS subject classification:37J40,37J45.1Introduction and main resultsTopological instability of action variables in multidimensional nearly integrable Hamiltonian systems is known as Arnold Diffusion.For autonomous Hamiltonian systems with two degrees of freedom KAM theory generically implies topological stability of the action variables,i.e.under theflow of the perturbed system the action variables stay close to their initial values for all times.On the contrary,for systems with more than two degrees of freedom,outside a large set of initial conditions provided by KAM theory, the action variables may undergo a drift of order one in a very long,butfinite time called the“diffusion time”.Arnoldfirst showed up this instability phenomenon for a peculiar Hamiltonian in the famous paper[2].As suggested by normal form theory near simple resonances,the Hamiltonian models which are usually studied have the form H(I,ϕ,p,q)=(I21/2)+ω·I2+(p2/2)+ε(cos q−1)+εµf(I,ϕ,p,q) whereεandµare small parameters,n:=n1+n2,(I1,I2,p)∈R n×R are the action variables and (ϕ,q)=(ϕ1,ϕ2,q)∈T n×T are the angle variables.In Arnold’s model I1,I2∈R,ω=1,f(I,ϕ,p,q)=√(cos q−1)(sinϕ1+cosϕ2)and diffusion is proved forµexponentially small w.r.t.1Supported by M.U.R.S.T.Variational Methods and Nonlinear Differential Equations.intersection between W uµ(TµI)and W sµ(TµI′):this is the so called“gap problem”.In[2]this difficulty is bypassed by the peculiar choice of the perturbation f(I,ϕ,p,q)=(cos q−1)f(ϕ),whose gradient vanishes on the unperturbed tori T I,leaving them all invariant also forµ=0.Thefinal step is to prove,by a “shadowing argument”,the existence of a true diffusion orbit,close to a given transition chain of tori, for which the action variables I undergo a drift of O(1)in a certain time T d called the diffusion time.Thefirst paper proving Arnold diffusion in presence of perturbations not preserving the unperturbed tori has been[12].Extending Arnold’s analysis,it is proved in[12]that,if the perturbation is a trigono-metric polynomial in the anglesϕ,then,in some regions of the phase space,the“density”of perturbed invariant tori is high enough to allow the construction of a transition chain.Regarding the shadowing problem,geometrical method,see e.g.[12],[15],[13],[14],and variational ones,see e.g.[9],have been applied,in the last years,in order to prove the existence of diffusion orbits shadowing a given transition chain of tori and to estimate the diffusion time.We also quote the important papers[7]-[8]which,even if dealing with Arnold’s model perturbation only,have obtained,by variational methods,very good diffusion time estimates and have introduced new ideas for studying the shadowing problem.For isochronous systems new variational results concerning the shadowing and the splitting problem have been obtained in[4],[5]and[6].In this paper we provide an alternative mechanism to produce diffusion orbits.This method is not based on the existence of a transition chain of tori:we avoid the KAM construction of the perturbed hyperbolic tori,proving directly the existence of a drifting orbit as a local minimum of an action functional. At the same time our variational approach achieves the optimal diffusion time.We also prove that our diffusion time estimate is the optimal one as a consequence of a general stability result,proved via classical perturbation theory.As in[12]we deal with a perturbation which is a trigonometric polynomial in the angles and our diffusion orbits will not connect any two arbitrary frequencies of the action space,even if we manage to connect more frequencies than in[12],proving the drift also in some regions of the phase space where transition chains might not exist.Clearly if the perturbation is chosen as in Arnold’s example we can drift in all the phase space with no restriction.The results proved here have been announced in [3].In this paper we will assume,as in Arnold’s paper,the parameterµto be small enough in order to validate the so called Poincar´e-Melnikov approximation,when thefirst order expansion term inµfor the splitting,the so called Poincar´e-Melnikov function,is the dominant one.For this reason,through this paper we willfix the“Lyapunov exponent”of the pendulumε:=1,considering the so called“a-priori unstable”case.Actually our variational shadowing technique is not restricted to the a-priori unstable case,but would allow,in the same spirit of[4],[5]and[6],once a“splitting condition”is someway proved, to get diffusion orbits with the best diffusion time(in terms of some measure of the splitting).We will consider nearly integrable non-isochronous Hamiltonian systems defined byHµ=I22+(cos q−1)+µf(I,ϕ,p,q,t),(1.1) where(ϕ,q,t)∈T d×T1×T1are the angle variables,(I,p)∈R d×R1are the action variables and µ≥0is a small real parameter.The Hamiltonian system associated with Hµwrites˙ϕ=I+µ∂I f,˙I=−µ∂ϕf,˙q=p+µ∂p f,˙p=sin q−µ∂q f.(Sµ) The perturbation f is assumed to be a real trigonometric polynomial of order N inϕand t,namely2f(I,ϕ,p,q,t)= |(n,l)|≤N f n,l(I,p,q)e i(n·ϕ+lt).(1.2)The unperturbed Hamiltonian system(S0)is completely integrable and in particular the energy I2i/2of each rotator is a constant of the motion.The problem of Arnold diffusion in this context is whether,for µ=0,there exist motions whose net effect is to transfer O(1)-energy among the rotators.A naturalf n,l(I,p,q)=f−n,−l(I,p,q)for all(n,l)∈Z d×Z with|(n,l)|≤N wherecomplementary question regards the time of stability(or instability)for the perturbed system:what is the minimal time to produce an O(1)-exchange of energy,if any takes place,among the rotators?For simplicity,even if it is not really necessary,we assume f to be a purely spatial perturbation, namely f(ϕ,q,t)= 0≤|(n,l)|≤N f n,l(q)exp(i(n·ϕ+lt)).The functions f n,l are assumed to be smooth.Let us define the“resonant web”D N,formed by the frequenciesω“resonant with the perturbation”D N:= ω∈R d ∃(n,l)∈Z d+1s.t.0<|(n,l)|≤N andω·n+l=0 =∪0<|(n,l)|≤NE n,l(1.3) where E n,l:={ω∈R d|ω·n+l=0}.Let us also consider the Poincar´e-Melnikov primitiveΓ(ω,θ0,ϕ0):=− R f(ωt+ϕ0,q0(t),t+θ0)−f(ωt+ϕ0,0,t+θ0) dt,where q0(t)=4arctan(exp t)is the separatrix of the unperturbed pendulum equation¨q=sin q satisfying q0(0)=π.The next Theorem states that,for any connected component C⊂D c N,ωI,ωF∈C,there exists a solution of(Sµ)connecting a O(µ)-neighborhood ofωI in the action space to a O(µ)-neighborhood of ωF,in the time-interval T d=O((1/µ)|logµ|).Theorem1.1Let C be a connected component of D c N,ωI,ωF∈C and letγ:[0,L]→C be a smooth embedding such thatγ(0)=ωI andγ(L)=ωF.Assume that,for allω:=γ(s)(s∈[0,L]),Γ(ω,·,·) possesses a non-degenerate local minimum(θω0,ϕω0).Then∀η>0there existsµ0=µ0(γ,η)>0and C=C(γ)>0such that∀0<µ≤µ0there exists a solution(Iµ(t),ϕµ(t),pµ(t),qµ(t))of(Sµ)and two instantsτ1<τ2such that Iµ(τ1)=ωI+O(µ),Iµ(τ2)=ωF+O(µ)and|τ2−τ1|≤CTheorem1.2Let f(I,ϕ,p,q,t)be as in(1.2),where the f n,l(|(n,l)|≤N)are analytic functions.Then ∀κ,r and|p(0)|≤ r,there results|I(t)−I(0)|≤κ∀t such that|t|≤κ0µ.(1.5)Actually the proof of Theorem1.2contains much more information:in particular the stability time (1.5)is sharp only for orbits lying close to the separatrices.On the other hand the orbits lying far away from the separatrices are much more stable,namely exponentially stable in time according to Nekhoroshev type time estimates,see(7.4)and(7.11).Indeed the diffusion orbit of Theorem1.1is found close to some pseudo-diffusion orbit whose(q,p)variables move along the separatrices of the pendulum.As a byproduct of the techniques developed in this paper we have the following result(proved in section6)concerning“Arnold’s example”[2]where Tω:={I=ω,ϕ∈T d,p=q=0}are,for allω∈R d, even forµ=0,invariant tori of(Sµ).Theorem1.3Let f(ϕ,q,t):=(1−cos q) f(ϕ,t).Assume that for some smooth embeddingγ:[0,L]→R d,withγ(0)=ωI andγ(L)=ωF,∀ω:=γ(s)(s∈[0,L]),Γ(ω,·,·)possesses a non-degenerate local minimum(θω0,ϕω0).Then∀η>0there existsµ0=µ0(γ,η)>0,and C=C(γ)>0such that∀0<µ≤µ0there exists a heteroclinic orbit(η-close toγ)connecting the invariant tori TωI and TωF.Moreover thediffusion time T d needed to go from aµ-neighbourhood of TωI to aµ-neighbourhood of TωFis bounded by(C/µ)|logµ|for some constant C.The method of proof of Theorem1.1(and Theorem1.3)relies on afinite dimensional reduction of Lyapunov-Schmidt type,variational in nature,introduced in[1]and later extended in[4],[5]and[6]tothe problem of Arnold diffusion.The diffusion orbit of Theorem1.1is found as a local minimum of theaction functional close to some pseudo-diffusion orbit whose(p,q)variables move along the separatrices of the pendulum.The pseudo-diffusion orbits,constructed by the Implicit Function Theorem,are truesolutions of(Sµ)except possibly at some instantsθi,for i=1,...,k,when they are glued continuouslyat the section{q=π,mod2πZ}but the speeds(˙ϕµ(θi),˙qµ(θi))=(Iµ(θi),pµ(θi))may have a jump.The time interval T s=θi+1−θi is heuristically the time required to perform a single transition during which the rotators can exchange O(µ)-energy,i.e.the action variables vary of O(µ).During each transition we can exchange only O(µ)-energy because the Melnikov contribution in the perturbed functional is O(µ).Hence in order to exchange O(1)energy the number of transitions required will be k=O(1/µ).We underline that the question offinding the optimal time and the mechanism for which we can avoidthe construction of transition chains of tori are deeply connected.Indeed the main reason for whichour drifting technique avoids the construction of KAM tori is the following one:if the time to perform a simple transition T s is,say,just T s=O(|lnµ|)then,on such“short”time intervals,it is valid to approximate the pseudo diffusion orbits with unperturbed solutions living on the stable and unstable manifolds of the unperturbed tori W s(Tω)=W u(Tω)={I=ω,ϕ∈T d,p2/2+(cos q−1)=0},when computing the value of the action functional.In this way we do not need to construct the true hyperbolic tori Tµω(actually for our approximation we only need the time for a single transition to be T s<<1/µ).The fact that it is possible to perform a single transition in a very short time interval like T s=O(|lnµ|)is not obvius at all.In[7]the time to perform a single transition,in the example of Arnold,is O(1/µ).This transition time arises in order to ensure that the variations of the kinetic part of the action functional associated with the rotators are small compared with the(positive definite)second derivative of the Poincar´e-Melnikov primitive at its minimum point.Unfortunately this time is too long to use a simple approximation of the functional.The key observation that enables us to perform a single transition in a very short time interval concerns the behaviour of the“gradientflow”of the unperturbed action functional of the rotators.This implies a sort of a-priori estimate satisfied by the minimal diffusion orbits, see remark6.1.We think that estimate(6.18)is interesting in itself.In this way we can show that the variations of the action of the rotators are small enough,even on time intervals T s<<1/µ,and do not “destroy”the minimum of the Poincar´e-Melnikov primitive.When trying to build a pseudo-diffusion orbit which performs single transitions in very short time intervals we encounter another difficulty linked with the ergodization time.The time to perform a single transition T s must be long enough to settle,at each instantθi,the projection(θi,ϕi)of the pseudo-orbit on the torus T d+1sufficiently close to the minimum of the Poincar´e-Melnikov function,i.e.the homoclinic point(in our method it is sufficient to arrive just O(1)-close,independently ofµ,to the homoclinic point).This necessary request creates some difficulty since our pseudo-diffusion orbit may arrive O(µ)-close in the action space to resonant hyperplanes of frequencies whose linearflow does not provide a dense enough net of the torus.The way in which this problem is overcome is discussed in section5:we observe a phenomenon of“stabilization close to resonances”which forces the time for some single transitions to increase.Anyway the total time required to cross these(finite number of)resonances is still T d=O((1/µ)log(1/µ)),see(5.13)and the proof of Theorem1.1.This discussion enables us to prove optimal fast-Arnold diffusion in large regions of the phase space and allows to improve the local diffusion results of[14].We need therefore some results on the ergodization time of the torus for linearflows possibly resonant but only at a“sufficiently high order”.We present these results in section4.We point out that the main result of this section,Theorem4.2,implies as corollaries Theorems B and D of[11],see remark4.1.It is of independent interest and could possibly improve the other results of[11].This work is a further step of a reaserch line,started in[4]-[5]and[6],forfinding new mechanisms to prove Arnold diffusion.We expect that the variational method developed in this paper could be suitably refined in order to prove the existence of drifting orbits in the whole action space and then to prove such results for generic analytic perturbations too.Another possible application of these methods could regard infinite dimensional Hamiltonian systems where the existence of“transition chains of infinite dimensional hyperbolic tori”is quite far for being proved.The paper is organized as follows:in section2we perform thefinite dimensional reduction and we define the variational setting.In section3we provide a suitable development of the reduced action functional.In section4we prove the new results on the ergodization time.In section5we define the unperturbed pseudo-orbit.In section6we prove the existence of the diffusion orbit.In section7we prove the stability result,that is to say the optimality of our diffusion time.Notations:Through this paper the notation a(z1,...,z k)=O(b(µ))will mean that,for a suitable positive constant C(γ,f)>0,|a(z1,...,z p)|≤C(γ,f)|b(µ)|.2The variational setting and thefinite dimensional reduction When the perturbation f(ϕ,q,t)= |(n,l)|≤N f n,l(q)exp(i(n·ϕ+lt))is purely spatial,3system(Sµ) reduces to the second order system¨ϕ=−µ∂ϕf(ϕ,q,t),−¨q+sin q=µ∂q f(ϕ,q,t)(2.1) with associated LagrangianLµ(ϕ,˙ϕ,q,˙q,t)=˙ϕ22+(1−cos q)−µf(ϕ,q,t).(2.2)Using the Contraction Mapping Theorem we will prove in lemma2.1that,near the unperturbed solutions (ω(t−θ)+ϕ0,q0(t−θ))living on the stable and unstable manifolds of the unperturbed tori Tω,there exist,forµsmall enough,solutions of the perturbed system(2.1)which connect the sections{ϕ=ϕ+,q=−π,t=θ+}and{ϕ=ϕ−,q=π,t=θ−}(under some assumptions).The diffusion orbit will be a chain of such connecting orbits.Wefirst introduce a few definitions and notations.Forλ:=(θ+,θ−,ϕ+,ϕ−)∈R2×R2d withθ+<θ−we define T λ:=θ−−θ+and the “mean frequency”ωλ∈R d as ωλ:=ϕ−−ϕ+c,c 1>0such that ∀0<µ≤µ2,∀λ=(θ+,θ−,ϕ+,ϕ−)such that C 0β2λ>µand C 1|ln µ|≤T λ≤C 0βλ/µthere exists a unique solution (ϕµ(t ),q µ(t )):=(ϕµ,λ(t ),q µ,λ(t ))of (2.1),defined for t ∈(θ+−1,θ−+1),satisfying ϕµ(θ±)=ϕ±,q µ(θ±)=∓πand (i )|ϕµ(t )−cµ(1+c 1µT 2λ)/β2λ,|˙ϕµ(t )−ω|≤cµ,|˙q µ(t )−˙Q T λ(t −θ+)|≤ϕ(t ):=ωλ(t −θ+)+ϕ+.Moreover ϕµ,λ(t ),˙ϕµ,λ(t ),q µ,λ(t )and ˙q µ,λ(t )are C 1functions of (t,λ).The proof of lemma 2.1is given in the Appendix.Remark 2.1Roughly,the meaning of the above estimates is the following.1)We have imposed C 1|ln µ|<T λ:=θ−−θ+so that by (2.4),on such intervals of time,the periodic solution Q T λis O (µ)close to “separatrices”q ∞of the unperturbed pendulum.2)Estimate (ii )implies that for t ≈(θ++θ−)/2the perturbed solution q µmay have O (µ)oscillations around the unstable equilibrium of the pendulum q =0,mod 2π,which is exactly what one expects perturbing with a general f .On the contrary for the class of perturbations considered in [2]as f (ϕ,q,t )=(1−cos q )f (ϕ,t )preserving all the invariant tori,estimate (ii )can be improved,getting max {|q µ(t )−Q T λ(t −θ+)|,|˙q µ(t )−˙Q T λ(t −θ+)|}=O (µmax {exp(−C |t −θ+|),exp(−C |t −θ−|)}).3)For βλ≈√µfor some 0<|(n,l )|≤N the perturbed transition orbits ϕµare no more well-approximated by the straight linesBy lemma2.1,for0<µ≤µ2,we can define on the setΛµ:= λ=(θ+,θ−,ϕ+,ϕ−) C0β2λ>µ,C1|lnµ|≤Tλ≤C0βλ12|˙ϕ(θ+)|2+12|˙ϕ(θ−)|2+12|˙ϕ|2(θ+)+Forβ>0fixed,denotingλi=(θi,θi+1,ϕi,ϕi+1),we define on the set:= λ=(θ1,...,θk,ϕ1,...,ϕk)∈R k×R kd ∀1≤i≤k−1,λi∈Λµ,βλi≥β ,Λµ,k:=Λβµ,kthe reduced action functional Fµ:Λµ,k→R asFµ(λ)=ωIϕ1−|ωI|2θk+µΓs(ωF,θk,ϕk)−µF(ωF,θk,ϕk)2whereΓu(ω,θ0,ϕ0):=− 0−∞ f(ωt+ϕ0,q0(t),t+θ0)−f(ωt+ϕ0,0,t+θ0)) dt,(2.7)Γs(ω,θ0,ϕ0):=− +∞0 f(ωt+ϕ0,q0(t),t+θ0)−f(ωt+ϕ0,0,t+θ0)) dt,(2.8)are called resp.the unstable and the stable Poincar´e-Melnikov primitive,andF(ω,θ0,ϕ0):=−f0,0θ0− 0<|(n,l)|≤N f n,l e i(n·ϕ0+lθ0)2θ1+µΓu(ωI,θ1,ϕ1)+µF(ωI,θ1,ϕ1)and−ωFϕk+|ωF|23The approximation of the reduced functionalIn order to prove the existence of critical points of the reduced action functional Fµthanks to the properties of the Poincar´e-Melnikov primitivesΓ(ω,·,·)we need an appropriate expression of Fµ,see lemma3.5.We shall express Fµas the sum of a function whose definition contains theΓ(ω,·,·)(for which we can prove the existence of critical points)and of a remainder whose derivatives are so small that it cannot destroy the critical points of thefirst function.Thefirst lemma gives an approximation of Gµ(defined in(2.6)).Lemma3.1For0<µ≤µ3,forλ∈Λµwe haveGµ(λ)=1(θ−−θ+)+µΓs(ωλ,θ+,ϕ+)+µΓu(ωλ,θ−,ϕ−)−µθ−θ+f(β2λTλ .(3.2)Proof.By lemma2.1,we can writeϕµ,λ(t)=In the following,in order to avoid cumbersome notation,we shall use the abbreviations v,w,Q for v µ,λ,w µ,λ,Q T λ(·−θ+),the dependency w.r.t.λand µbeing implicit.We haveG µ(λ)=θ−θ+1ϕ(t )|2+˙2|˙v (t )|2+12˙w 2(t )+ θ−θ+[1−cos(Q (t )+w (t ))]−µf (ϕ(t )·˙v (t )dt =θ−θ+ωλ·˙v (t )dt =0and θ−θ+˙Q (t )˙w (t )dt =θ−θ+−¨Q(t )w (t )dt = θ−θ+−(sin Q (t ))w (t )dt.As a result,G µ(λ)=G 0µ(λ)+R 1(λ),where G 0µ(λ)= θ−θ+1ϕ|2+1ϕ,Q,t ),R 1(λ)= θ−θ+12˙w 2+(cos Q −cos(Q +w )−w sin Q )−µf (ϕ,Q,t ).We shall first prove that |∇R 1|=Oµ2(1+µT 2λ)dt(∂θ+v )−µ∂ϕf (dt (∂θ+w )+ sin(Q +w )−sin Q −µ∂q f (ϕ,Q,t )−∂ϕf (ϕ,r 5:=µ θ−θ+ ∂q f (ϕ+v,Q +w,t ) ∂θ+Q,r 6:=−12˙w (θ+)2.Now v and w satisfy−¨v (t )=µ∂ϕf (ϕ(t )+v (t ),Q (t )+w (t ),t )+sin Q (t ).Moreover,deriving w.r.t.θ+the equality v (θ+)=0we obtain that (∂θ+v )(θ+)=−˙v (θ+).Similarly (∂θ+w )(θ+)=−˙w (θ+),(∂θ+v )(θ−)=0and (∂θ+w )(θ−)=0.Therefore an integration by parts gives r 1=|˙v (θ+)|2,r 2=˙w (θ+)2hence |r 1|+|r 2|=O (µ2/β2).By the properties of Q T ,∂θ+Q is bounded in the interval [θ+,θ−]by a constant independent of λ.Moreover −sin Q (t )+sin(Q (t )+w (t ))−w (t )cos Q (t )=O (w (t )2).Therefore r 3=O (µ2T ).We have also,for some positive constant c ,|r 4|+|r 5|≤cµT sup t ∈[θ+,θ−]|∂θ+Q (t )|+|∂θ+ϕis bounded independently of λ,we have by lemma 2.1|r 4|+|r 5|=O µ2(1+µT 2λ)indent We now develop G0µ(λ)asG0µ(λ)=1(θ−−θ+)+µΓs(ωλ,θ+,ϕ+)+µΓu(ωλ,θ−,ϕ−)−µθ−θ+f(2˙Q2(t)+(1−cos Q(t))dt= Tλ01ϕ(t),Q(t),t)−f(ϕ(t),Q(t),t)dt−µΓs(ωλ,θ+,ϕ+)−µΓu(ωλ,θ−,ϕ−)=µ(a3(λ)+b3(λ))wherea3(λ):=− Tλ/20g(ωλt+ϕ+,Q Tλ(t),t+θ+)dt+ ∞0g(ωλt+ϕ+,q0(t),t+θ+)dt,b3(λ):=− 0−Tλ/2g(ωλt+ϕ−,Q Tλ(t+Tλ),t+θ−)dt+ 0−∞g(ωλt+ϕ−,q0(t),t+θ−)dt. We havea3(λ)=− Tλ/20 g(ωλt+ϕ+,Q Tλ(t),t+θ+)−g(ωλt+ϕ+,q0(t),t+θ+) + ∞Tλ/2g(ωλt+ϕ+,q0(t),t+θ+).Recalling that sup t∈(0,T/2)|∂T Q T(t)|=O(e−c2T),sup t∈(0,T/2)|Q T(t)−q0(t)|=O(e−c2T),it is easy to see that the derivatives of thefirst integral are O(Tλe−c2Tλ)=O(µ)(still provided C1is large enough). Moreover,using that(|g(ωλt+ϕ+,q0(t),t)|+|∂ϕg(ωλt+ϕ+,q0(t),t)|+|∂t g(ωλt+ϕ+,q0(t),t)|)=O(q0(t)−2π)=O(e−c2t)for t∈(Tλ/2,+∞),wefind that the derivatives of the second integral are O(µ)as well. Hence|∇a3(λ)|=O(µ).The same estimate holds for b3.We then conclude that∇R3(λ)=O(µ2),which completes the proof of lemma3.1.θi+b i,ϕi=ϕi,µ,∀i=1,...,k−1,(3.5)whereβi:=βλi :=β(ωi)andωi:=ωλi:=(ϕi+1−ϕi)/(θi+1−θi).Moreover we will assume(see(5.8))|ωi|≤ρµwhereϕi+1−θi(1≤i≤k−1),ω0:=ωI,ωk:=ωF(3.6)andρ>0is a small constant to be chosen later(see(6.3)).For the time being,assuming(3.5)and(3.6), we want to give a suitable expression of Fµin E.By lemma3.1,forλ∈E,we haveFµ(λ)=k−1i=11θi+1−θi+ωIϕ1−ωFϕk−|ωI|22θk+ki=1µ Γu(ωi−1,θi,ϕi)+Γs(ωi,θi,ϕi) +µF(ωI,θ1,ϕ1)−k−1i=1µ θi+1θi f(ωi(t−θi)+ϕi,0,t)dt−µF(ωF,θk,ϕk)+k−1 i=1R0(µ,λi),(3.7)where|∇λR0(µ,λ)|satisfies(3.2).We shall write Fµin an appropriate form thanks to the following lemmas.Thefirst one says how close the“mean frequencies”ωi are to the unperturbedωi|=O 1|lnµ| .(3.8) MoreoverΓu(ωi−1,θi,ϕi)+Γs(ωi,θi,ϕi)=Γ(ωi=−Lemma3.3For0<µ≤µ4µF(ωI,θ1,ϕ1)−ki=1µ θi+1θi f(ωi(t−θi)+ϕi,0,t)dt−µF(ωF,θk,ϕk)=k i=1R i5(µ,λi−1,λi),(3.10)where,for all i4∇R i5(µ,θi−1,ϕi−1,θi,ϕi,θi+1,ϕi+1)=O µβ2i(θi+1−θi)+µ|βi−βi−1|i 1(n·ωi+l)Now we prove (3.11).Let us consider for example ∂θi R i5.We have ∂θi R i5=µ∂θi F (ωi −1,θi ,ϕi )−F (ωi ,θi ,ϕi )=µ ∂ωF (ωi −1,θi ,ϕi ).−ωi −1(θi +1−θi )−µ0<|(n,l )|≤Nf n,l le i(n ·ϕi +lθi )(1(n ·ωi +l )) ,(3.12)where∂ωF (ω,θ0,ϕ0)=0<|(n,l )|≤Nf n,lne i(n ·ϕ0+lθ0)Finally,to get a suitable expression of F µ,we find convenient to introduce coordinates (b,c )∈R (1+d )k defined by (3.4)andc i =a i −ωi )+(0,c i )).Lemma 3.4We havek −1 i =11(θi +1−θi )+ωI ϕ1−ωF ϕk −|ωI |22θk =1∆θi :=θi and 5∇R i6(µ,θi −1,ϕi −1,θi ,ϕi ,θi +1,ϕi +1)=O (∆ωi (θi +1−θi )+γi .We can write ωI ϕ1−ωF ϕk asωI ϕ1−ωF ϕk=k −1 i =1 (ωi )ϕi −ωk −1−ωF )=k −1 i =1(ωi )ϕi −|ωi γi+ϕk (2θ1+|ωF |2ωi |2ωi −1|2ωi |22−|2θk ,(3.18)k −1 i =11(θi +1−θi )=k −1 i =1|2(θi +1−θi )+1(θi +1−θi )+2|ϕi +1−ϕi |22θ1+|ωF |22|γi |25Fori =k we have R k 6=R k 6(µ,θk ,ϕk ).k−1i=1 |2−|2 θi+(ωi)ϕi+ϕk(2−|2 θk.(3.20) Substitutingθi+b i forθi,we getγi=(a i+1−a i)−ωi−1−ωi|2ωi−1|2ωi).Finally,expressingγi in terms of(b i,c i)we getγi=(a i+1−a i)−ωi and then from(3.20),developing the square,we get(3.16).2k−1i=1|c i+1−c i|2θi+(b i+1−b i)+µki=1Γ(θi+b i,ωi b i+c i)+R7(b,c),(3.22)R7(b,c):=ki=1R i7(µ,b i−1,c i−1,b i,c i,b i+1,c i+1),(3.23)where6|∇R i7|≤C2ρµ.(3.24) Proof.It is easy to see that(3.6),(3.8)and(3.21)imply(providedµis small enough)thatβi−1θi−θi−1+1ωi∂ϕi +∂θi,estimate(3.24)follows from(3.2),(3.9),(3.11),(3.25)and(3.16).ϕi,6In the cases i=1,i=k we have R17=R17(µ,θ1,ϕ1,θ2,ϕ2)and R k7=R k7(µ,θk−1,ϕk−1,θk,ϕk).Theorem4.1∀l∈N there exists a positive constant a l such that,∀Ω∈R l,∀δ>0,T(Ω,δ)≤(α(Ω,a l/δ))−1.Moreover T(Ω,δ)≥(1/4)α(Ω,1/4δ)−1.In the above Theoremα−1is equal to0ifα=+∞and to+∞ifα=0.Remark4.1Assume thatΩis a C-τDiophantine vector,i.e.there exist C>0andτ≥l−1such that ∀k∈Z l|k·Ω|≥C/|k|τ.Thenα(Ω,R)≥C/Rτand so T(Ω,δ)≤aτl/Cδτ.This estimate was proved in Theorem D of[11].Also Theorem B of[11]is an easy consequence of Theorem4.1.Theorem4.1is a direct consequence of more general statements,see Theorem4.2and remark4.2. Let us introducefirst some notations.LetΛbe a lattice of R l,i.e.a discrete subgroup of R l such that R l/Λhasfinite volume.For allΩ∈R l we defineT(Λ,Ω,δ)=inf t∈R+ ∀x∈R l d(x,[0,t]Ω+Λ)≤δ(T(Λ,Ω,δ)is the time required to have aδ-net of the torus R l/Λendowed with the metric inherited from R l).For R>0,letΛ∗= p∈R l ∀λ∈Λ,p·λ∈Z andΛ∗R= p∈Λ∗ 0<|p|≤R(Λ∗is a lattice of R l which is conjugated toΛ).We defineα(Λ,Ω,R)=inf |p·Ω| p∈Λ∗R .The following result holds:Theorem4.2∀l∈N there exists a positive constant a l such that,for all latticeΛof R l,∀Ω∈R l,∀δ>0,T(Λ,Ω,δ)≤(α(Λ,Ω,a l/δ))−1.Remark4.2It is fairly obvious that T(Λ,Ω,δ)≥(1/4)α(Λ,Ω,1/4δ)−1.Indeed,assume thatΛ∗1/4δ=∅and let p∈Λ∗1/4δbe such that p·Ω=α:=α(Λ,Ω,1/4δ).Let x∈R l satisfy p·x=1/2.Then ∀t∈[0,1/4α),∀λ∈Λ,|x−(tΩ+λ)|≥|p·(x−tΩ−λ)|with M=8πa d+1/δ,then theflow of(ω,1)provides aδ/4-net of the torus T d+1.Moreover ifω/∈Q M then for all(n,l)∈Z d\{0}×Z,|n·ω+l|=|n|dist(ω,E n,l)≥dist(ω,E n,l)≥dist(ω,Q M)>0.(5.2) By Theorem4.1(or Theorem4.2),we deduce from(5.2)the estimateT((ω,1),δ/4)≤2πIfΓ(α(s),·,·)possesses,for each s,a non-degenerate local minimum(θα(s)0,ϕα(s)),then,by the Im-plicit Function Theorem,along any curveγsufficiently close toα,Γ(γ(s),·,·)possesses local minima(θγ(s)0,ϕγ(s))such thatD2(θ,ϕ)Γ(γ(s),θγ(s),ϕγ(s))>λId,∀s∈[0,L],(5.4)for some constantλ>0depending onα.Therefore,by the above lemma,it is enough to prove the existence of drifting orbits along admissible curvesγ.Property(5.4)will be used in lemma6.1.Given a Q M-admissible curveγ,let us call s∗1,...,s∗r the elements of I(γ),andω∗1=γ(s∗1),...,ω∗r=γ(s∗r)the corresponding frequencies.Since,∀m=1,...,r,(θω∗m0,ϕω∗m0)is a nondegenerate local minimum ofΓ(ω∗m,·,·),there is a neighborhood W m ofω∗m such that,∀ω∈W m,Γ(ω,·)admits a nondegeneratelocal minimum(θω0,ϕω0),the mapω→(θω0,ϕω0)being Lipschitz-continuous on W m.Therefore we shall assume without loss of generality that for all m=1,...,r,∀(ω,ω′)∈(W m∩γ([0,L]))2|(θω0,ϕω0)−(θω′0,ϕω′0)|≤K|ω−ω′|.(5.5) It is easy to prove that,ifγis an admissible curve,there exists d0>0such that(*){s∈[0,L]|dist(γ(s),Q M)≤d0}is the union of afinite number of disjoint intervals[S1,S′1],..., [S r,S′r];for all m=1,...,r each interval[S m,S′m]intersects I(γ)at a unique point s∗m and γ([S m,S′m])⊂W m.Moreover(s→dist(γ(s),Q M))is decreasing on[S m,s∗m),increasing on (s∗m,S′m],and dist(γ(s),Q M)≥(ν/2)|s−s∗m|for all s∈[S m,S′m].Now we are able to define the“unperturbed transition chain”:for some small constantρ>0which will be specified later we choose k∈N and k+1“intermediate frequencies”ωI=:ω1,...,ωk:=ωFwith2≤s i+1−s i≤ρµ,∀i=0,...,k−1.(5.6) By(5.6)there results thatLρµ,(5.7) moreover it follows from(a)that|ωi|≤ρµ,∀i=0,...,k−1.(5.8) This condition has been used before in lemma3.4.Given k time instantsω10<θi< ...<ϕi}i=1,...,k by the iteration formulaω1,ϕi+θi+1−θi}i=1,...,k is specified in the next lemma:the main request is that(ϕi) must arriveδ-close mod2πZ d+1,to the local minimum point(θωi0)of the Poincar´e-Melnikov primitive Γ(ωi is1/|lnµ|far from the set Q M of“non-ergodizing frequencies”we can reach this goal for“short”time intervalsθi≈|lnµ|.In order to cross the set Q M of“non-ergodizing frequencies”we need to use longer time intervalsθi≈1/ dist(Q M,µ/|lnµ|<dist(Q M,ωi are“close”(less than√θi+1−θk−θi+1−θi}i=1,...,k withω10 satisfying,•(i)if dist(µdist(θi+1−dist(ωi,Q M)≤√|lnµ|then C1|lnµ|<θi<2C1|lnµ|,。