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Pn(  6  S < p   p N N@bv'lSMǏ z/fck8^`Q NvN" _N1\/fl gS0RQYagN6R~v`Q NvMN"0.9 b /b(9 H  0޽h ? f3"V+Nfz *"`(    S h 0p   h ]NSuǏ z-N dN_̃SO _uir__/fYN6R~agN Nv N[hQ'lSr` ( incomplete oxidation under stress condition )0 ]_Sv]NSuǏ z YD-a|x0gjxTsT|vuN (WُNSuǏ z-N uÑyVagN~_g 'YϑRl]NSuvNir  _uir[hQ'lSv-NN"Nirv[_\0Nxb f fb `B0 0    F H  0޽h ? f3"V+Nfr "p(  z  S ` pp     N[hQ'lS/f_uirNNatQv'`( @b N[hQ'lS cv/f(WُǏ z-N؏l g[hQ'lSv g:gSTir\O:N]NSuv~zNircQ~ހ ُ*NǏ z[]NSu/fASR͑v02Yb$Qf$fY H  0޽h ? f3"V+Nfrd0;J_MkfX[Up0ke/[kWsP_uwwz5}ahc + #(.e147;O>;B  &Y+HMQUXy\b_ei'mptOwyO[cCe @EyKфy0I}ї?1 ;Oh+'0 px ( H T ` lxԪԭe ѧڶ˵2Ԫ ?D:\Microsoft Office\Templates\Presentation Designs\Ribbons.pot*\M249Microsoft PowerPointemp@`L~@@|^@00pju՜.+,0Y    Y 屏幕显示 )E JTimes New Roman宋体隶书幼圆RibbonsE第二节 微生物代谢网络 的向心板块 代谢网络中,胞外碳架物质跨膜并注入中心板块所流经的途径统称向心途径,向心途径群组成代谢网络的向心板块。 3.2.1 向心途径在代谢网络及其在细胞 机器工作模式中的位置 3.2.2 微生物的分解代谢途径和代谢网 络的向心途径 Z3.2.1 向心途径在代谢网络和细胞 机器工作模式中的位置 碳架物质从向心板块注入中心板块(前述中心代谢途径上的12个前体代谢物的大平台)时所流经的代谢途径统称向心途径。与整个代谢网络一样,向心途径的变化也取决于遗传与环境条件,本课程讨论的内容建立在微生物细胞相对稳定的代谢基础上。 在工业发酵的细胞机器工作模式中,向心途径首当其冲,是工业发酵载流路径的一部分。 PowerPoint 演示文稿R3.2.2 微生物的合成代谢途径和 代谢网络中的向心途径  化能异养型微生物可以广泛地利用各种各样的有机物进行生长和繁殖。在发酵工业上发酵生产的原料大多是农副产品。微生物必须能合成并分泌能分解这些原料的酶,把原料转化成可以进入细胞的有机营养物质。. 各种有机营养物质(包括经水解酶降解形成的有机营养物质)大多以主动输送的方式进入原核生物的细胞,以促进扩散和主动输送的方式进入真核微生物的细胞,这几种输送方式均需借助于细胞的蛋白质(载体蛋白或酶)。g 进入细胞的营养物质若是中心代谢途径的起始物或中间产物,即可纳入中心代谢途径进行代谢;进入细胞的营养物质若不是中心代谢途径的起始物或中间产物,则需经向心途径的胞内部分,转化成中心代谢途径的起始物或中间产物,才可纳入中心代谢途径进行代谢。j 化能异养型微生物细胞借助于微生物的向心途径将有机化合物降解或转化成中心途径上对应的的化合物,从而使这两种途径衔接起来。微生物的向心途径一般依次包括三个部分: (1)胞外降解途径(胞外酶催化) (2)跨膜系统(有载体蛋白参与) (3)胞内向心途径E 原料化合物经向心途径注入中心途径, 向心途径与中心途径的接合点(接口)原则上可以是中心途径上的任意化合物,实际上主要是12个代谢前体物。如果进入细胞的营养物质已经是中心代谢途径的起始物或中间产物,则胞内向心途径就缩成一个 “ 点 ” ,这个点也就是向心途径与中心途径的接口 。 代谢网络的向心途径往往是分解代谢途径,但它们是以中心大平台,或者更精确地说是以中心途径上对应的化合物为终点的。PowerPoint 演示文稿PowerPoint 演示文稿(3.2.2.1 胞外降解途径:胞外酶催化多 聚物的水解 3.2.2.2 有载体蛋白参与的跨膜系统 3.2.2.3 化能异养型微生物对各种有机 化合物的利用 3.2.2.4 细胞内源化合物的氧化降解 3.2.2.5 关于质粒编码的降解代谢活性 3.2.2.1 胞外降解途径:胞外酶对多聚物 的水解 微生物可以分泌胞外酶,大多数情况下是水解酶,把多聚物水解成可以进入细胞的分子。这些胞外酶游离于微生物细胞外,有些仍与细胞相联。细胞还会分泌一些附着物,使某些疏水化合物结合到细胞表面,以便于降解。多聚物水解不会产生可被生物利用的能量,但多聚物水解是向心途径的第一步。8 可作为营养的多聚物包括:淀粉、纤维素、果胶类物质(果胶酸、果胶、原果胶质 )、 几丁质( 甲壳质 )、半纤维素 (hemi cellulose)、木质素(1ignin)、蛋白质和多肽、核酸等等种类。 化能异养型微生物能分泌一系列胞外酶将外源性多聚有机化合物降解。还有些有机化合物虽然不是多聚物,也需要首先被微生物的胞外酶水解以后才能被微生物吸收。例如许多微生物能分泌脂肪酶,它能将甘油三脂水解成脂肪酸和甘油。S3.2.2.2 有载体蛋白参与的跨膜系统(第二章 2.2.2 已有叙述。) 3.2.2.3 化能异养型微生物对各种有机化 合物的利用 微生物通过一定的机制从培养基中吸收有机化合物,进入细胞的有机化合物再经过一段代谢途径的代谢作用,将碳架物质注入中心代谢途径。这些有机化合物可以是碳水化合物、烃类化合物、含氮有机化合物或其他有机化合物。 5 微生物通过一定的机制从培养基中吸收有机化合物,进入细胞的有机化合物再经过一段代谢途径的代谢作用,将碳架物质注入中心代谢途径。这些有机化合物可以是碳水化合物、烃类化合物、含氮有机化合物或其他有机化合物。⑴二糖的吸收和降解 关于二糖的吸收和降解,研究得最清楚的要数乳糖。大肠杆菌若要在以乳糖为碳源的培养基中生长,首先要诱导出 3 种酶:乳糖透性酶、β- 半乳糖苷酶和 β- 半乳糖苷乙酰基转移酶。 乳糖透性酶实际上是乳糖的载体蛋白,用于乳糖的主动输送,将乳糖送入细胞; β- 半乳糖苷酶将乳糖水解成葡萄糖和半乳糖; β- 半乳糖苷乙酰基转移酶的作用是催化AcCoA将乳糖及其它半乳糖苷乙酰化的反应,它可将未能代谢的乳糖及与乳糖结构类似的分子乙酰化,并将其排出细胞,因此,这个酶的生物学功能可能是起解毒作用。 乳糖水解生成的葡萄糖被磷酸化而进入酵解途径;水解生成的半乳糖又在细胞中诱导出参与半乳糖进一步代谢的 3 种酶,在这 3 种酶的协同作用下,使半乳糖转化成 G-6-P, 即可注入酵解途径。 二糖的降解一般在细胞内发生,有如下两种降解方式:一种是被相应的二糖水解酶水解成单糖,如蔗糖酶将蔗糖水解成葡萄糖和果糖,乳糖酶( β- 半乳糖苷酶 )将乳糖水解成葡萄糖和半乳糖,麦芽糖酶将麦芽糖水解成两份葡萄糖。另一种分解方式是在相应的磷酸化酶的作用下,使二糖经磷酸化裂解(phosphorylytic cleavage),得到 1分子单糖和1分子磷酸葡萄糖。5 这种方式的裂解比起第一种方式更为经济,由这种方式产生磷酸葡萄糖即可以糖的磷酸酯的形式进入中心代谢途径,而且,此过程不消耗ATP,而游离的单糖大多需经激活(消耗高能键而被磷酸化)后才能进入葡萄糖的降解途径。⑵己糖和戊糖的降解 作为化能异养型微生物的碳源与能源,果糖像葡萄糖一样,受己糖激酶催化而被 ATP 激活,生成 F-6-P;或者借助由PEP参加的磷酸转移酶系统进入细胞,生成 F-1-P。 然后又在1-磷酸果糖激酶(该酶受果糖诱导)的催化下形成 F-1,6-2P。 F-6-P和 F-1,6-2P 均为 EMP 途径中间产物,可按EMP途径进一步降解。 甘露糖在甘露糖激酶的催化下,生成 6-磷酸甘露糖,再由 6- 磷酸甘露糖异构酶催化,生成F-6-P,进入EMP 途径。也有例外,如果没有甘露糖激酶, 也可以以另外的方式实现甘露糖的磷酸化。 半乳糖诱导 3 种酶的合成:半乳糖激酶、葡萄糖∶1- 磷酸半乳糖尿苷酰转移酶和 UDP-Glc差向异构酶。半乳糖在这些酶的顺序催化下,先生成 1-磷酸半乳糖,然后再生成 G-1-P。G-1-P 经G-6-P磷酸葡萄糖变位酶作用生成 G-6-P, 即可进入酵解途径。在嗜糖假单胞菌中,半乳糖可被直接氧化成半乳糖酸内酯,然后进入与 ED途径类似的途径,最终产物与 ED 途径相同。  戊糖,如核糖、木糖和阿拉伯糖,虽然可以被不少微生物利用,但常有适应现象。即它们或以自己独立的途径降解,或经转化后进入葡萄糖降解途径。 如核糖在激酶催化下生成R-5-P,即可进入HMP和PK途径。 阿拉伯糖在异构酶的催化下生成核酮糖,然后再磷酸化生成 Ru-5-P,即可进入HMP或PK途径。 木质纤维水解物中有40%是木糖,木糖可以经木糖醇而转化为木酮糖,也可以在木糖异构酶催化下直接生成木酮糖,木酮糖经激酶催化生成 Xu-5-P,即可进入 HMP或PK途径。⑶己糖醛酸的降解 果胶类物质在胞外酶作用下降解成半乳糖醛酸。半乳糖醛酸经进一步代谢即可转化成 ED 途径的中间产物 KDPG,即可进入 ED 途径。 这样的作用在假单胞菌、气单胞菌和土壤杆菌中比较明显。⑷甘露(糖)醇的降解 在不同的微生物中,可能经不同的途径将甘露醇转化为 EMP 途径的中间产物 F-6-P。 ⑸丙酸的降解 在需氧条件下, 大肠杆菌的丙酸降解至今未有明确的结论, 其丙酸代谢的第一步是生成丙酰 CoA,后者与乙醛酸缩合成α-羟基戊二酸,然后又裂解成乳酸和乙酸。乙酸经AcCoA进入TCA环或(和)DCA环。而乳酸经乳酰 CoA、丙酮酰 CoA,转化成羟基丙酮醛, 此化合物的进一步代谢情况尚待研究, 但已知当丙酸浓度较高时, 丙酸主要经乙酸代谢。U⑹葡萄糖酸的降解 某些微生物能利用葡萄糖酸作为唯一碳源,葡萄糖酸可经 6-磷酸葡萄糖酸脱羧,生成 5- 磷酸核酮糖而进入 HMP 途径;也可以经 2,5 -二酮葡萄糖酸,继续代谢生成α-KG而进入TCA环。在假单胞菌中,葡萄糖酸经 6-磷酸葡萄糖酸进入ED途径。⑺山梨(糖)醇的降解 山梨(糖)醇先被转化成果糖,然后生成F-6-P而进入EMP途径。 ⑻苹果酸的降解 在需氧情况下,苹果酸按TCA环氧化方向生成OAA,然后OAA脱羧生成PYR,然后按需氧途径降解。在弱氧化醋酸杆菌中,苹果酸经草酰苹果酸生成α-KG而进入TCA环,以便用于氨基酸合成。]⑼草酸的降解 草酸先被转化成乙醛酸,然后经甘油酸途径生成3-P-GA进入EMP途径。 ⑽乙醇酸(羟基乙酸)和乙醛酸的降解 乙醇酸先被氧化成乙醛酸,后者直接与AcCoA反应,生成苹果酸进入TCA环、DCA环;或者乙醛酸经甘油酸途径,生成3-P-GA而进入EMP途径。⑾乙醇和乙酸的降解 乙醇经乙醛氧化成乙酸,乙酸在乙酰硫激酶的催化下,与CoA、ATP作用,生成AcCoA而进入TCA环。 ⑿脂肪酸的降解 脂肪和脂肪酸也可以作为微生物的碳源和能源,但脂肪不能进入细胞,细胞内贮藏的脂肪也不可直接进入糖的降解途径,因此要在有关酶的作用下先行水解。 脂肪在微生物细胞合成的脂肪酶(胞外酶对胞外的脂肪,胞内酶对胞内脂肪)的作用下,水解成甘油和脂肪酸。 甘油经3-磷酸甘油或经二羟基丙酮转化成磷酸二羟丙酮(DHAP)进入EMP途径。 / 绝大多数细菌对脂肪酸的分解能力很弱,然而一经诱导,它们的脂肪酸氧化活性就会增强。如大肠杆菌就有一个可被诱导合成的,用来降解脂肪酸的酶系,并且,C6~C16脂肪酸靠酰基CoA合成酶参与的基团转移机制进入细胞。 脂肪酸的β-氧化是在原核细胞的细胞质和真核细胞的线粒体内进行的。 若脂肪酸分子的碳原子数为偶数,最终得AcCoA,若脂肪酸分子的碳原子数为奇数, 则同时也得到丙酰CoA。AcCoA直接进入TCA 环降解,丙酰 CoA 则可以经甲基丙二酸单酰CoA,然后甲基丙二酸单酰 CoA分子重排,形成ScCoA而进入TCA环。⒀脂肪族烃的降解 微生物对烃的降解,首先需要氧,因此,在烃上生长是专性需氧的过程。 在甲烷上生长的过程,需特殊的途径,因此甲烷氧化细菌不能在其它烃上生长。微生物对其它脂肪族烃的代谢情况大致是这样的:能够在C5~C8脂肪族烃中生长的细菌相对较少,如分支细菌、黄杆菌、诺卡氏菌等。。? 然而,能利用长链的脂肪族烃的微生物比较多,分布也比较广,其中特别是能利用 C10~C18 正烷烃的微生物,而且这些正烷烃被利用的速度特别快。能在长链脂肪族烃上生长的微生物包括谷氨酸棒状杆菌、荧光假单胞菌、铜绿假单胞菌、嗜石油诺卡氏菌、简单节杆菌、解脂假丝酵母、球拟酵母、粉红头孢等微生物。值得注意的是酵母和丝状真菌也能利用这类基质。大肠杆菌、产气肠杆菌和枯草芽孢杆菌不能在这类基质上生长。 脂肪族烃是水不溶性基质,因此,微生物对它们的吸收和催化作用就有一定的特殊性。微生物通常生活在水的环境中,不能生活在油中,因此,微生物对这类基质的作用只发生在油水界面上。对于这类基质的利用是从对它们的吸收开始的。在这类基质上生长的酵母细胞具有富含糖脂的相当厚的壁,在这类基质上生长的细菌则形成含海藻糖脂、鼠李糖脂或具有与这些糖脂类结构类似的化合物的细胞壁。  脂肪族烃溶在这样的细胞壁中并且被传送到质膜。换句话说,这些微生物能产生起乳化剂作用的物质,这些物质具有既亲油又亲水的分子结构,因而可以促进微生物对脂肪族烃的吸收。例如节杆菌、短杆菌、棒状杆菌和诺卡氏菌产生的这类物质是海藻糖脂(由1分子海藻糖与2分子。α-分支-β-羟基脂肪酸组成),嗜石油假丝酵母产生的这类物质是由多肽和脂肪酸组成的。 ! 当然也可以用人工的方法增加微生物细胞的这种吸收活性,比如,加入表面活性剂促进乳化作用,使烷烃呈胶状分散状态。对于固态烃(如固体石蜡),除了细胞直接黏附于其表面外,还可能有使其溶解的机制。  脂肪族烃被微生物吸收过程的最初的 3 个反应在膜中进行,催化这 3 个反应的酶是与膜结合的。反应由单加氧酶催化开始: 基质-H + O2 + AH2 → 基质-OH + A + H2Or 上式中 AH2 是指一种可被氧化的辅基质( cosubstrate ),这里是还原性的红氧( 化 )还( 原 )蛋白 ( reduced rubredoxin ), 这种含铁的蛋白质小分子 ( Fe2+ 或Fe3+与多肽链上的半胱氨酸残基相联),先被可溶性的依赖 NADH 的酶系统还原, 然后才能作为单加氧酶的辅基质。 然后,经单加氧酶催化形成的伯醇被依赖于 NAD+ 的伯醇脱氢酶进一步氧化成对应的醛,最后醛又被依赖于 NAD+ 的醛脱氢酶氧化成对应的脂肪酸。此外,还发现有脂肪族烃分子两端均被氧化的,结果生成二羧酸。 在棒状杆菌的一些种和酵母中,辅基质则是还原性细胞色素 P450,它经黄素蛋白和铁硫蛋白,从NADH获得还原力。( 在某些微生物中(如诺卡氏菌中的某些种)还发现了另外一种氧化机制。从脂肪族烃的亚末(即第二个碳)开始氧化,生成仲醇,继续氧化成酮;在第二个单加氧酶的催化下转化成乙酸酯;乙酸酯水解成乙酸和长伯链醇;这个长链伯醇将进一步降解。 脂肪族烯也能被降解。或是在双键处加水;或是先形成环氧化物,然后水解成二羟基烷。此外,环烷烃、二环烷烃、萜烯烃类等均可被不同的微生物降解。 总之,不同种类的长链脂肪族烃通过不同途径均可被转变成长链脂肪酸,长链脂肪酸经 β- 氧化,生成AcCoA而进入TCA环。F⒁氨基酸的降解 由蛋白酶或肽酶水解蛋白质或多肽生成的低分子质量的肽和氨基酸,能被许多种微生物主动吸收,并用于生长。一些氨基酸的分子结构与中心代谢途径上某些中间化合物(central intermediates)相关,所以它们的降解非常容易。  大多数情况下,氨基酸首先转变成相关的酮酸。如Glu→α-KG、Asp→OAA、Ala →PYR,以及Val→2-氧代异戊酸、Leu →2-氧代异己酸、I1e → 2-氧代-3-甲基戊酸。 这就是氧化脱氨。α- KG、OAA 和 PYR 属于中心代谢途径的中间化合物。2- 氧代异戊酸和2-氧代-3-甲基戊酸还需经特异性的降解途径转变成中心代谢途径的中间化合物,已发现细菌中有与动物中类似的反应将这两者转化为AcCoA和丙酰CoA。  脱氨也可以引发氨基酸的降解。发生脱氨反应的条件是: α- 氨基羧酸分子中β-碳原子上的取代基 ( 如羟基 )促进α- 氨基的离去,生成酮酸。 因而诸如 Ser、Thr 和 His 都可以进行脱氨降解。因为脱氨降解是从脱水开始的,所以把反应所用的酶叫做 脱水酶 ( 即通常所说的脱氨酶 )。P 在大肠杆菌中降解代谢的Thr 脱水酶与合成代谢的 Thr脱水酶在性质上是不同的(特别是在调节性能上),后者催化从 Thr合成I1e的第一步反应。如果要在 Asp 上需氧生长,许多微生物会合成天冬氨酸酶,催化 Asp 脱氨。His 的降解也由类似的反应引发。P 以上所述脱氨机制是微生物降解氨基酸的主要机制,此外还有其他降解机制,如 Arg 可通过多种途径降解成 SCA,Phe可以降解成 FMA 和乙酰乙酸。因此,许多微生物能在营养液和其他含有多种氨基酸和肽的培养基中很好地生长的事实,是可以理解的。 但应注意到,氨基酸也可以在脱羧酶的催化下脱羧生成相应的胺。含 1 个氨基的氨基酸脱羧后生成单胺;含 2 个氨基的氨基酸脱羧后生成二胺,二胺有毒。u⒂核苷酸、核苷和嘌呤、嘧啶的降解 化能异养型微生物可以分泌胞外酶,如核酸酶(磷酸二酯酶)、核苷酸酶(磷酸单酯酶)和核苷酶,将胞外的核酸降解成核苷酸、核苷、嘌呤和嘧啶。一般认为核苷酸难以被吸收,腺嘌呤核苷酸借助特异性的移位酶的跨膜交换比率为1∶1。  核苷和嘌呤、嘧啶碱基可以通过基团转移等机制进入胞内。胞内的核酸、核苷酸、核苷可被胞内相应的酶降解成嘌呤和嘧啶。这个分解过程可以概括为: 核酸←→核苷酸←→(核苷 + 磷酸 )→ [ 嘌呤或嘧啶 +(1-磷酸戊糖)] 嘌吟、嘧啶若要作为能源,则需要降解。嘌呤与嘧啶降解的最后产物是有机酸(氨基酸)、NH3和CO2。⒃芳香族化合物的降解 芳香族化合物在环境和食品中的累积会危及人类的健康(芳香族氨基酸无毒),人类希望能利用微生物来降解芳香族化合物,使它们重新进入大自然的碳素循环。 芳香族化合物(萘、蒽、菲、苯乙醇酸、色氨酸、奎尼酸等)大多先被转变成儿茶酚( 邻苯二酚 )和原儿茶酸(3,4-二羟苯甲酸)。然后儿茶酚和原儿茶酸再经邻位分解 ( 3- 氧代己酸途径 ) 降解成SCA和AcCoA;或儿茶酚和原儿茶酸经间位分解(间位分解途径)降成PYR和乙醛。因此,儿茶酚和原儿茶酸堪称芳香族化合物分解的“中心代谢物”。 微生物也可以不经过以上途径降解芳香族氨基酸。Phe和Tyr经尿黑酸( 2,5-二羟苯乙酸 )降解为延胡索酸和乙酰 CoA,即可进入TCA环。 B3.2.2.4 细胞内源化合物的氧化降解 当没有外源能源化合物供应时,微生物可以氧化其细胞内组成物质(即内源营养物质),获得维持生命活动的能量。 内源营养物质包括细胞内的各种多糖、类脂和多聚 β- 羟基丁酸、细胞蛋白质和RNA等。U 当前面 3 种能被较快降解的内源营养物质用完后,细胞常常能利用细胞蛋白质和 RNA 。 以上内源营养物质的降解途径除 β- 羟基丁酸外,前面均已讨论过了。 多聚β-羟基丁酸的降解始于解聚,得到β-羟基丁酸,然后β- 羟基丁酸被最终降解成AcCoA。s3.2.2.5 关于质粒编码(Plasmid-encoded) 的降解代谢活性 前面所述的各种降解途径均是由染色体 DNA 编码的。 近年来发现在恶臭假单胞菌和一些相关的种的微生物细胞中含有降解性质粒(degradative plasmids),它们包含某些特殊的降解代谢的酶(酶系)合成的遗传信息。S 首先发现的是编码樟脑降解酶系的质粒,后来还发现恶臭假单胞菌的烷烃氧化作用,受正辛烷质粒( octane plasmid ) 的控制,这个质粒为可诱导的烷烃羟化酶和伯醇脱氢酶编码,而染色体DNA则为用于伯醇、脂肪醛和脂肪酸降解的组成型的氧化酶编码。 以上所述的氧化降解过程都是正常情况下的代谢,也就是说没有受到内外条件制约的情况下的降解代谢。 工业发酵过程中,除了要获得菌体,微生物往往是处于制约条件下的不完全氧化状态 ( incomplete oxidation under stress condition )。 已开发的工业发酵过程,诸如D-葡萄糖酸、柠檬酸和味精的生产,在这些发酵过程中,生产菌种因条件约束,被迫大量分泌工业发酵的产物——被微生物完全氧化的中间代谢产物相对很少。 不完全氧化是微生物令人感兴趣的性质,所谓不完全氧化,指的是在这过程中还没有被完全氧化的有机化合物作为工业发酵的终端产物排出细胞,这个过程对工业发酵是十分重要的。 已用的字体演示文稿设计模板幻灯片标题E_**  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~Root EntrydO)PicturesbCurrent UserSummaryInformation(PowerPoint Document(DocumentSummaryInformation8Y