酶生物燃料电池载体选择及其应用基础研究

酶生物燃料电池（EBFC）是利用酶作为催化剂将化学能转化为电能的装置。由于氧化还原蛋白质和酶通常具有复杂的空间结构，活性中心深埋在它们的肽链中，很难与基底电极进行直接电子传递，从而影响了电池的性能。但使用适当的载体对电极表面进行修饰，可以实现直接的、快速的电子传递。因此，开发稳定性好、成本低、能够有效促进氧化还原蛋白质或酶与基底电极进行直接电子传递的载体成为EBFC发展中的重要课题之一。 本论文主要集中于EBFC中蛋白质或酶载体的选择方面的研究。探讨不同性质的载体，包括半导体电物质、生物相容性物质和导电物质对氧化还原蛋白质或酶的直接电子传递的影响。同时以SiO2纳米粒子为例，探讨了载体促进氧化还原蛋白质直接电子传递的作用机理。通过对不同载体的考察，最终选择了一种合适的材料组装成葡萄糖/O2 EBFC，并考察了EBFC的性能。主要结果如下： 1．将SiO2纳米粒子固定在GC电极上，成功实现了细胞色素c（Cyt c）的准可逆的直接电化学反应，并在这基础上提出双功能机理模型，说明了半导体对氧化还原蛋白质和电极之间的直接电子迁移的影响。 2．发现Cyt c能够在SBA-15膜修饰的电极上实现准可逆的直接电化学反应，并能够对H2O2产生较好的电催化还原效果。 3．以生物相容性物质壳聚糖为载体，分别研究了Cyt c、微过氧化物酶（MP-11）和葡萄糖氧化酶（GOD）的准可逆的直接电化学反应。并发现固载在壳聚糖上的Cyt c和MP-11对H2O2和O2还原有很好的电催化活性，而固载在壳聚糖上的GOD对葡萄糖氧化有很好的电催化活性。 4．以碳纳米管（CNT）为载体，实现了GOD的准可逆的直接电化学反应。并在氧化还原媒介体的作用下实现了其对葡萄糖的电催化氧化。 5．将筛选出的最佳载体组装成葡萄糖/O2 EBFC，分别以葡萄糖氧化酶和漆酶作为阳极和阴极的催化剂，制得有隔膜和无隔膜的EBFC。

带侧羧基的脂肪族生物降解聚合物

生物降解脂肪族聚酷如聚乙交醋（PGA）、聚丙交醋（PLA）、聚。一己内酷（PCL）以及它们的共聚物由于具有良好的生物相容性和生物降解性而在外科手术缝合线、组织工程、药物控制释放、骨固定等领域得到很多应用。但是，它们自身缺乏功能基团，亲水性差，因而在应用上受到很大限制。因此，含有功能侧基生物降解聚合物的制备在过去十年中受到大家极大的关注。功能基团的引入对于调节聚合物的性能如：亲水性、生物降解性和药物的渗透性等非常重要。需要特别指出的是，功能基团的引入为把药物和其它生物活性物质与聚合物结合进而扩大聚合物的应用范围提供了机会。本文合成了几个功能化单体并且通过共聚的方法制备了几种新型的两亲性功能化聚合物。这些改性后的聚合物可以通过化学键接上药物或者其它生物活性物质，有望作为靶向控释药物载体和智能化的组织工程支架材料。具体的研究结果如下：1．以2，2-二羟甲基丙酸节酷为功能单体，通过两步法成功合成了一系列新的带功能基团的聚酷酞胺，并通过1H NMR和FTIR对聚合物的化学结构进行了表征，DSC结果表明所合成的聚酯酰胺的Tm和Tg分别在150℃和0℃左右；2．合成了功能化的环状单体（35）-3-［（苄氧羰基）乙基」吗啉-2，5-二酮（BEMD），并以PEG作为引发剂，Sn（Oct）2作为催化剂，通过L一LA和BEMD的开环共聚合得到共聚物PLGBG-PEG-PLGBG；随后用10％铭碳催化氢化得到带有侧梭基的两亲性嵌段共聚物PLGG-PEG-PLGG和其它两亲性嵌段共聚物一样，PLGG-PEG-PLGG在水溶液中能够自组装成胶束，用花作为荧光探针，通过荧光光谱法研究了其形成胶束的过程并测定了它们的临界胶束浓度，发现在总的分子量大致相当的情况下，PLGG-PEG4600-PLGG比PLGG-PEG2000-PLGG有较高的临界胶束浓度；场发射电子显微镜表征结果显示胶束具有均一的球形特征，动态光散射结果表明该胶束具有较窄的单峰粒径分布；蛋白酶K溶液中的降解研究表明带有侧梭基的PLGG-PEG-PLGG比PLA具有更快的降解速率；人胚关节软骨细胞培养结果表明所合成的聚合物PLGG-PEG-PLGG显示出较好的细胞相容性。3．在缩合剂DCC和催化剂DMAP存在下，带有侧梭基的两亲性嵌段共聚物PLGOPEG-PLGG和紫杉醇发生缩合反应得到两亲性嵌段共聚物一紫杉醇键合药P（LGG-paclitaxel）-PEG-P（LGG-paclitaxel）。它具有两亲性嵌段共聚物的性质，能够自组装成胶束，场发射电子显微镜表征结果表明胶束具有均一的球形特征，动态光散射结果表明该胶束具有较窄的粒径分布，平均粒径为119.4nm。该胶束的药物释放具有pH敏感性，酸性环境中比生理环境中（pH＝7.4）具有较快的释放速率。P（LGG-Paclitaxel）-PEG-P（LGG-paclitaxel）胶束的壳层由良好亲水性的PEG组成，避免了胶束纳米粒子在血液循环中被人体网状内皮系统吞噬，保证有充足的时间通过EPR效应在肿瘤部位聚集，进而通过细胞内吞进入细胞并在细胞内的酸性环境中释放药物，进一步的研究工作有待深入进行。4．合成了功能化的环状碳酸酷单体MBC，以MPEG作为引发剂，ZnEt2作为催化剂，LLA和MBC发生开环共聚合，以较高的转化率，得到高分子量共聚物MPEG-b-P（LA-co-MBC）。13C NMR表明LLA和确c发生了无规共聚合；DSC征结果表明MPEG-b-P（LA-co-MBC）为无定型态聚合物，Tg在20-50℃之间，随着MBc含量的增加而降低；MPEG-b-P（LA-co-MBC）脱保护后得到带侧梭基的MPEG-b-P（LA-co-MCC），它的Tg明显提高，可能是聚合物侧梭基之间强的氢键作用力以及梭基对水解反应的催化作用造成的；脱保护前后的共聚物在蛋白酶K溶液中的降解研究表明，MPEG-b-P（LA-co-MCC）的降解速率大于MPEG-b-P（LA-co-MBC）的降解速率；人胚关节软骨细胞培养结果表明，所合成的MPEG-b-P（LA-co-MCC）是一种具有良好生物相容性的新型生物降解材料。5．利用本实验室开发的一种新型有机氨锯引发剂Sr-PO在温和的条件下通过顺序加料聚合的方法合成了新的嵌段共聚物PCL-b-PMBC。WAXD结果表明 PCL-b-PMBc中PCL的衍射峰均可观察到，只是衍射峰的强度随着PMBC含量的增加而减弱。DSC结果表明PCL-b-PMBC中PCL的Tm在57到52℃之间，并且随着PMBC含量的增加而降低。PCL-b-PMBC的玻璃化转变在-41.6 到-23.3℃之间，随着PMBC含量的增加而增加，这表明PMBC和PCL之间 有着强的相互作用，尽管两段不是完全相容的。PCL-b-PMBC的侧节醋在10％把碳催化下氢化还原为带侧梭基的PCL-b-PMCC后，衍射峰和结晶焙大大降低，说明侧梭基的存在使得分子链间有着强的氢键相互作用而不利于结晶。由以上分析可知，PCL-b-PMCC侧梭基的存在将会使共聚物的降解速度大大提高，而且因为功能梭基的存在可以使共聚物通过化学键连接上药物、短肤、寡糖或者其它生物活性物质，从而扩大该聚合物在生物医学领域的应用范围。

土壤氨基糖微生物转化过程与机理探讨

土壤氨基糖因其异源性和稳定性可用以指示微生物对土壤碳（C）氮循环的相对贡献。但由于氨基糖是微生物在土壤中长期残留的平衡结果，其数量变化无法准确反映在微生物作用下无机态氮（N）向氨基糖转化的动态过程和机制，从而使氨基糖对土壤氮内循环的指示作用受到限制。如果能够利用新的技术手段研究土壤氨基糖的微生物转化过程，将使土壤氮素内循环研究产生突破。同位素技术是研究土壤C，N转化过程的有效手段。但是研究特定化合物如氨基糖的微生物转化过程还需要新的技术支持，本研究首先建立了稳定同位素培养－气质联机技术测定土壤氨基糖同位素富集比例新方法。对于15N培养样品，由于氨基糖分子中只有一个N原子，15N富集比例可通过m/z（F+1）与F相对丰度的比值计算；对于13C培养样品，由于葡萄糖c整体掺入形成氨基糖c骨架，所以利用m／z（F＋n）与F相对丰度的比值计算13c在土壤氨基糖中的富集（n为质谱碎片中骨架C原子数）。同位素富集用原子百分超（APE）表示。EI和cI两种方式测得的APE有很好的同一性，且不受土壤基质的影响，表明方法的可靠性和广泛适用性。利用以上方法，进行了土壤样品的同位素培养与测定，以跟踪土壤氨基糖微生物合成动态，进行氨基糖的微生物转化与更新研究。主要结论如下：1．当以葡萄糖为碳源且每周施入底物时，NH4+和NO3-均可被微生物迅速同化并进行氨基糖的合成。但NO3-必须被还原成NH4＋才能被微生物利用，因而NO3．存在短暂的滞后期，之后被微生物快速利用。氨基葡萄糖（GluN）和胞壁酸（MurN）不同的同位素富集特征表明，N源形态对细菌增殖无显著影响，但真菌更倾向于利用NH4+。在NO3-培养中氨基糖的增量及微生物对C的截获均小于NH4＋。2．分别利用u一13c一glucose一NH4十和glLlcose一ISNH4＋进行样品培养时，同位素富集趋势相同，但APE（13C）大于APE（15N），这种差别反映了了土壤微生物利用C，N的时间特征及土壤有机质含量对C，N循环的影响。3．以gtucose-15NH4+为底物时，施入N素频率的改变也会影响微生物的活性。尤其是细菌的快速生长受到N素不足的限制，转而代之以细菌和真菌的持续的低速生长。微生物活性的降低减少了对有机c的截获。DCD的加入有效抑制了NH4+向NO3一的转化，但对氨基糖的合成无显著影响。4．土壤氨基糖反映的主要是土壤中已经死亡了的微生物的一种长期过程而产生的残留，同土壤微生物量无明显的相关性。但经外加底物培养后，氨基糖同位素富集比例的变化则来源于微生物的转化，因而与微生物量碳有直接的相关性。5．从原理上说，在氨基糖的微生物合成过程中，葡萄糖没有发生C骨架的断裂，而是直接转化成为氨基葡萄糖的骨架。但在复杂的土壤基质中，氨基葡萄糖的合成必然受到葡萄糖其他生物化学过程的影响。使少量葡萄糖经酵解后再次参与己糖胺的合成。以全取代葡萄糖为底物时，葡萄糖碳骨架的断裂与重排不影响13c同位素的富集。但对于单取代葡萄糖培养来说，必须要考虑因葡萄糖碳骨架断裂而产生的同位素的重新分配，Mass（F+1）和Mass（F+2）的丰度变化的总和真正代表了氨基葡萄糖的同位素富集。6．添加有机物料和N素进行土壤样品培养时，对外加氮素的同化远低于相应的葡萄糖培养。碳源尤其是能源不足限制了N的转化。微生物分解高C加的有机物料需吸收外加N源以满足自身生长需要。N素的加入频率影落响微生物对外加N素的利用。当加入的N不能满足微生物分解有机物料的需要，就会降低微生物对有机物料的分解速度，使无机N向氨基糖态N转化速度降低。应用稳定同位素技术，发现施到土壤中的无机氮素可被微生物快速转化成某种形态有机氮，这种有机态氮处于不断转化循环之中，构成土壤有效氮的暂存"过渡库"，其中氨基糖是重要成分之一。过渡库现象的发现为氮肥的有效利用提供了新的思路。根据土壤有效氮"过渡库"的模型，氮月巴高效利用调控实际上就是土壤氮素微生物转化过程的调控。提高土壤无机氮素向土壤有机氮的转化速率和转化强度可以有效减少无机氮在土壤中的积累，从而降低肥料和土壤氮素的硝化和反硝化损失，提高氮肥利用率。研究还发现，土壤有效氮"过渡库"容量与循环速率不仅取决于N源自身性质，碳源的可利用性显著影响施入土壤的N素微生物转化特征。只有适当提高可利用碳源即活性碳源的数量，才能提高氮素的微生物同化，土壤有效氮"过渡库"容量，从而减少氮素损失。

西南喀斯特典型坡地土壤有机碳库特征及碳同位素组成

喀斯特石漠化及其影响己经成为制约中国西南地区可持续发展最严重的生态环境问题，越来越受到人们的关注，已成为环境地球化学研究的一个重要领域。石漠化的本质就是土地退化，造成土壤肥力和生产力的下降。因此喀斯特地区现阶段主要面临如何管理好陆地生态系统，以保持原有碳储量，并尽可能沉积更多碳是当前面临的主要挑战。 峰丛洼地是喀斯特地区的典型景观，该景观的坡地是喀斯特地区自然环境最脆弱的区域，存在基岩大量裸露的情况，土地和植被退化最为严重。本文以西南喀斯特地区具有不同植被类型的典型峰丛坡地（草丛、灌草丛、稀疏灌丛和乔木林）作为研究对象，为了解喀斯特地区碳的循环机制，我们采集了4个典型坡地不同地形部位的表层和剖面土壤，主要分析了土壤可溶性有机碳（DOC）、整体土壤及不同粒径土壤组分中有机碳含量和稳定碳同位素（δ13C）值组成，同时测定了植物优势种叶片、枯枝落叶δ13C值组成。得到以下几点认识： 1、草丛和灌草丛坡地各地形部位的土壤有机碳（SOC）含量和碳氮比(C/N)值差异较大，其上坡位的土壤都具有较高SOC含量和C/N值。而稀疏灌丛和乔木林坡地SOC含量和C/N值比较稳定，其变幅较小。 2、各坡地剖面土壤中DOC含量分布和变化主要受到了有机质输入､坡地地形及土壤质量等影响｡草丛和灌草丛坡地各地形剖面中DOC含量变幅要大于乔木林和稀疏灌丛坡，对各个坡地几个剖面上层土壤（0～20cm）中DOC含量来看，其含量主要表现为：乔木林坡地＞稀疏灌丛坡地＞灌草丛坡地＞草丛坡地。但通过DOC与SOC进行比对发现，在剖面土壤中DOC/SOC值整体变化趋势主要表现为随剖面土层的加深而增加的趋势，但在土层较薄的剖面中主要呈现一直增加的趋势，而在土层较厚的剖面中则出现先增加后降低或保持稳定的现象。除了稀疏灌丛坡地最低值出现在下坡位剖面PG3外，在草丛、灌草丛和乔木林坡地最低值均出现在上坡位的剖面，其中HC1、HG1和HG2剖面的DOC/SOC值最低，范围都在0.2%以下，具有明显的高SOC和低DOC的含量特征；而乔木林坡地各剖面DOC/SOC值相对较高，范围都在0.3%以上，并且各剖面之间的差异较小。 3、草丛和灌草丛坡地上坡位HC1、HG1和HG2剖面土壤主要是基岩风化物和植物残体堆积而成的砂质新成土，砂粒含量较高，剖面土壤中有机碳含量虽然高于下坡位各个剖面土壤，但有机碳主要贮存在砂粒土壤中，其碳库处于不稳定状态。而下坡位剖面中土壤主要以粉粒和黏粒为主，土壤有机碳含量相对较低，但主要贮存在粉粒和黏粒土壤中，其碳库处于相对稳定的状态。稀疏灌丛和乔木林坡地各地形剖面中主要以粉粒和黏粒土壤为主，各个剖面之间变化幅度较小，有机碳也主要贮存在粉粒和黏粒土壤中，其碳库组成比较稳定。 4、对采样点的植物优势种叶片的碳同位素组成进行了测定。结果显示，其δ13C值主要受到遗传因素的（光合作用途经）控制，植物碳同位素组成表现出明显的差异性，C3植物的δ13C平均值为-28.2‰，C4植物的δ13C平均值为-12.7‰。另外，对不同坡地采集的同种植物叶片的δ13C值分析发现，δ13C值受环境影响因素较小。 5、对各个坡地枯枝落叶和相应表层土壤有机质δ13C值之间的相关性研究发现，灌草丛和乔木林坡地具有显著相关性，其相关系数分别为R2=0.93和R2=0.77，明显高于草丛和稀疏灌丛坡地的R2=0.28和R2=0.36。由于土壤剖面发育历史及地表植被的不同，草丛和灌丛坡地土壤有机质的δ13C值要明显高于稀疏灌丛和乔木林坡地，并且不同地形剖面的土壤有机质的δ13C值的差异较大。另外，大多数剖面的土壤有机质δ13C值在剖面深度上的变化主要表现为随土层深度的加深呈现先升高后降低或趋于稳定的变化趋势。δ13C值在土壤剖面中随深度变化，反映了作物残体输入和土壤累积特征，有助于鉴定剖面中土壤有机碳的迁移转化过程。 6、剖面中土壤δ13CDOC值与δ13CSOC值的变化规律具有相似性，表明了土壤中DOC与SOC的具有密切的关系；另外，从△(δ13CDOC－δ13CSOC)来看，大多数剖面中δ13CDOC值要高于δ13CSOC值，其中乔木林的变化幅度最低，而草丛坡地的最高。 7、对剖面中不同粒径土壤有机质δ13C值的研究发现，不同粒径土壤有机质的δ13C值在剖面深度上的差异，主要受到输入土壤中有机质变化和进入土壤后分解程度的影响。各个坡地砂粒土壤中有机质的δ13C值对地表植被的变化较敏感，同时也指示了输入土壤中较新或是分解速率较慢的有机质来源，而粉粒和粘粒土壤有机质的δ13C值则指示了土壤中分解速率较快或是分解程度较充分的有机质来源。不同粒径土壤有机质δ13C值组成特征能够较好地反映土壤有机质更新速率。

HARNESSING iNKT CELLS WITH RECOMBINANT CD1d FUSION PROTEINS TO REDIRECT INNATE AND ADAPTIVE IMMUNITY TO THE TUMOR

Les thérapies du cancer, comme la radiothérapie et la chimiothérapie, sont couramment utilisées mais ont de nombreux effets secondaires. Ces thérapies invasives pour le patient nécessitent d'être améliorées et de nombreuses avancées ont été faites afin d'adapter et de personnaliser le traitement du cancer. L'immunothérapie a pour but de renforcer le système immunitaire du patient et de le rediriger de manière spécifique contre la tumeur. Dans notre projet, nous activons les lymphocytes Invariant Natural Killer T (iNKT) afin de mettre en place une immunothérapie innovatrice contre le cancer. Les cellules iNKT sont une unique sous-population de lymphocytes T qui ont la particularité de réunir les propriétés de l'immunité innée ainsi qu'adaptative. En effet, les cellules iNKT expriment à leur surface des molécules présentes aussi sur les cellules tueuses NK, caractéristique de l'immunité innée, ainsi qu'un récepteur de cellules T (TCR) qui représente l'immunité adaptative. Les cellules iNKT reconnaissent avec leur TCR des antigènes présentés par la molécule CD1d. Les antigènes sont des protéines, des polysaccharides ou des lipides reconnus par les cellules du système immunitaire ou les anticorps pour engendrer une réponse immunitaire. Dans le cas des cellules iNKT, l'alpha-galactosylceramide (αGC) est un antigène lipidique fréquemment utilisé dans les études cliniques comme puissant activateur. Après l'activation des cellules iNKT avec l'αGC, celles-ci produisent abondamment et rapidement des cytokines. Ces cytokines sont des molécules agissant comme des signaux activateurs d'autres cellules du système immunitaire telles que les cellules NK et les lymphocytes T. Cependant, les cellules iNKT deviennent anergiques après un seul traitement avec l'αGC c'est à dire qu'elles ne peuvent plus être réactivées, ce qui limite leur utilisation dans l'immunothérapie du cancer. Dans notre groupe, Stirnemann et al ont publié une molécule recombinante innovante, composée de la molécule CD1d soluble et chargée avec le ligand αGC (αGC/sCD1d). Cette protéine est capable d'activer les cellules iNKT tout en évitant l'anergie. Dans le système immunitaire, les anticorps sont indispensables pour combattre une infection bactérienne ou virale. En effet, les anticorps ont la capacité de reconnaître et lier spécifiquement un antigène et permettent l'élimination de la cellule qui exprime cet antigène. Dans le domaine de l'immunothérapie, les anticorps sont utilisés afin de cibler des antigènes présentés seulement par la tumeur. Ce procédé permet de réduire efficacement les effets secondaires lors du traitement du cancer. Nous avons donc fusionné la protéine recombinante αGC/CD1d à un fragment d'anticorps qui reconnaît un antigène spécifique des cellules tumorales. Dans une étude préclinique, nous avons démontré que la protéine αGC/sCD1d avec un fragment d'anticorps dirigé contre la tumeur engendre une meilleure activation des cellules iNKT et entraîne un effet anti-tumeur prolongé. Cet effet anti-tumeur est augmenté comparé à une protéine αGC/CD1d qui ne cible pas la tumeur. Nous avons aussi montré que l'activation des cellules iNKT avec la protéine αGC/sCD1d-anti-tumeur améliore l'effet anti- tumoral d'un vaccin pour le cancer. Lors d'expériences in vitro, la protéine αGC/sCD1d-anti- tumeur permet aussi d'activer les cellules humaines iNKT et ainsi tuer spécifiquement les cellules tumorales humaines. La protéine αGC/sCD1d-anti-tumeur représente une alternative thérapeutique prometteuse dans l'immunothérapie du cancer. - Les cellules Invariant Natural Killer T (iNKT), dont les effets anti-tumoraux ont été démontrés, sont de puissants activateurs des cellules Natural Killer (NK), des cellules dendritiques (DC) et des lymphocytes T. Cependant, une seule injection du ligand de haute affinité alpha-galactosylceramide (αGC) n'induit une forte activation des cellules iNKT que durant une courte période. Celle-ci est alors suivie d'une longue phase d'anergie, limitant ainsi leur utilisation pour la thérapie. Comme alternative prometteuse, nous avons montré que des injections répétées d'αGC chargé sur une protéine recombinante de CD1d soluble (αGC/sCD1d) chez la souris entraînent une activation prolongée des cellules iNKT, associée à une production continue de cytokine. De plus, le maintien de la réactivité des cellules iNKT permet de prolonger l'activité anti-tumorale lorsque la protéine αGC/sCD1d est fusionnée à un fragment d'anticorps (scFv) dirigé contre la tumeur. L'inhibition de la croissance tumorale n'est optimale que lorsque les souris sont traitées avec la protéine αGC/sCD1d-scFv ciblant la tumeur, la protéine αGC/sCD1d-scFv non-appropriée étant moins efficace. Dans le système humain, les protéines recombinantes αGC/sCD1d-anti-HER2 et anti-CEA sont capables d'activer et de faire proliférer des cellules iNKT à partir de PBMCs issues de donneurs sains. De plus, la protéine αGC/sCD1d-scFv a la capacité d'activer directement des clones iNKT humains en l'absence de cellules présentatrices d'antigènes (CPA), contrairement au ligand αGC libre. Mais surtout, la lyse des cellules tumorales par les iNKT humaines n'est obtenue que lorsqu'elles sont incubées avec la protéine αGC/sCD1d-scFv anti- tumeur. En outre, la redirection de la cytotoxicité des cellules iNKT vers la tumeur est supérieure à celle obtenue avec une stimulation par des CPA chargées avec l'αGC. Afin d'augmenter les effets anti-tumoraux, nous avons exploité la capacité des cellules iNKT à activer l'immunité adaptive. Pour ce faire, nous avons combiné l'immunothérapie NKT/CD1d avec un vaccin anti-tumoral composé d'un peptide OVA. Des effets synergiques ont été obtenus lorsque les traitements avec la protéine αGC/sCD1d-anti-HER2 étaient associés avec le CpG ODN comme adjuvant pour la vaccination avec le peptide OVA. Ces effets ont été observés à travers l'activation de nombreux lymphocytes T CD8+ spécifique de la tumeur, ainsi que par la forte expansion des cellules NK. Les réponses, innée et adaptive, élevées après le traitement avec la protéine αGC/sCD1d-anti-HER2 combinée au vaccin OVA/CpG ODN étaient associées à un fort ralentissement de la croissance des tumeurs B16- OVA-HER2. Cet effet anti-tumoral corrèle avec l'enrichissement des lymphocytes T CD8+ spécifiques observé à la tumeur. Afin d'étendre l'application des protéines αGC/sCD1d et d'améliorer leur efficacité, nous avons développé des fusions CD1d alternatives. Premièrement, une protéine αGC/sCD1d dimérique, qui permet d'augmenter l'avidité de la molécule CD1d pour les cellules iNKT. Dans un deuxième temps, nous avons fusionné la protéine αGC/sCD1d avec un scFv dirigé contre le récepteur 3 du facteur de croissance pour l'endothélium vasculaire (VEGFR-3), afin de cibler l'environnement de la tumeur. Dans l'ensemble, ces résultats démontrent que la thérapie médiée par la protéine recombinante αGC/sCD1d-scFv est une approche prometteuse pour rediriger l'immunité innée et adaptive vers le site tumoral. - Invariant Natural Killer T cells (iNKT) are potent activators of Natural Killer (NK), dendritic cells (DC) and T lymphocytes, and their anti-tumor activities have been well demonstrated. However, a single injection of the high affinity CD1d ligand alpha-galactosylceramide (αGC) leads to a strong but short-lived iNKT cell activation followed by a phase of long-term anergy, limiting the therapeutic use of this ligand. As a promising alternative, we have demonstrated that when αGC is loaded on recombinant soluble CD1d molecules (αGC/sCD1d), repeated injections in mice led to the sustained iNKT cell activation associated with continued cytokine secretion. Importantly, the retained reactivity of iNKT cell led to prolonged antitumor activity when the αGC/sCD1d was fused to an anti-tumor scFv fragments. Optimal inhibition of tumor growth was obtained only when mice were treated with the tumor-targeted αGC/CD1d-scFv fusion, whereas the irrelevant αGC/CD1d-scFv fusion was less efficient. When tested in a human system, the recombinant αGC/sCD1d-anti-HER2 and -anti-CEA fusion proteins were able to expand iNKT cells from PBMCs of healthy donors. Furthermore, the αGC/sCD1d-scFv fusion had the capacity to directly activate human iNKT cells clones without the presence of antigen-presenting cells (APCs), in contrast to the free αGC ligand. Most importantly, tumor cell killing by human iNKT cells was obtained only when co- incubated with the tumor targeted sCD1d-antitumor scFv, and their direct tumor cytotoxicity was superior to the bystander killing obtained with αGC-loaded APCs stimulation. To further enhance the anti-tumor effects, we exploited the ability of iNKT cells to transactivate the adaptive immunity, by combining the NKT/CD1d immunotherapy with a peptide cancer vaccine. Interestingly, synergistic effects were obtained when the αGC/sCD1d- anti-HER2 fusion treatment was combined with CpG ODN as adjuvant for the OVA peptide vaccine, as seen by higher numbers of activated antigen-specific CD8 T cells and NK cells, as compared to each regimen alone. The increased innate and adaptive immune responses upon combined tumor targeted sCD1d-scFv treatment and OVA/CpG vaccine were associated with a strong delay in B16-OVA-HER2 melanoma tumor growth, which correlated with an enrichment of antigen-specific CD8 cells at the tumor site. In order to extend the application of the CD1d fusion, we designed alternative CD1d fusion proteins. First, a dimeric αGC/sCD1d-Fc fusion, which permits to augment the avidity of the CD1d for iNKT cells and second, an αGC/sCD1d fused to an anti vascular endothelial growth factor receptor-3 (VEGFR-3) scFv, in order to target tumor stroma environment. Altogether, these results demonstrate that the iNKT-mediated immunotherapy via recombinant αGC/sCD1d-scFv fusion is a promising approach to redirect the innate and adaptive antitumor immune response to the tumor site.

Simple and surprisingly effective one-step extraction-cleanup by Soxflo for DDT and its metabolites from environmental samples

A pilot study found that DDT breakdown at the GC inlet was extensive in extracts from some-but not all-samples with high organic carbon contents. However, DDT losses could be prevented with a one-step extraction-cleanup in the Soxflo instrument with dichloromethane and charcoal. This dry-column procedure took 1 h at room temperature. It was tested on spiked soil and peat samples and validated with certified soil and sediment reference materials. Spike recoveries from freshly spiked samples ranged from 79 to 111% at 20-4000 mug/kg concentrations. Recoveries from the real-world CRMs were 99.7-100.2% of DDT, 89.7-90.4% of DDD and 89.6-107.9% of DDE. It was concluded that charcoal cleanups should be used routinely during surveys for environmental DDX pollution in order to mitigate against unpredictable matrix-enhanced breakdown in the GC. (C) 2004 Elsevier B.V. All rights reserved.

Soil carbon and litter development along a reconstructed biodiverse forest chronosequence of South-Western Australia

Soil organic matter (SOM) increases with time as landscape is restored. Studying SOM development along restored forest chronosequences would be useful in clarifying some of the uncertainties in quantifying C turnover rates with respect to forest clearance and ensuing restoration. The development of soil organic matter in the mineral soils was studied at four depths in a 16-year-old restored jarrah forest chronosequence. The size-separated SOM fractionation along with δ13C isotopic shift was utilised to resolve the soil C temporal and spatial changes with developing vegetation. The restored forest chronosequence revealed several important insights into how soil C is developing with age. Litter accumulation outpaced the native forest levels in 12 years after restoration. The surface soils, in general, showed increase in total C with age, but this trend was not clearly observed at lower depths. C accumulation was observed with increasing restoration age in all three SOM size-fractions in the surface 0–2 cm depth. These biodiverse forests show a trend towards accumulating C in recalcitrant stable forms, but only in the surface 0–2 cm mineral soil. A significant reverse trend was observed for the moderately labile SOM fraction for lower depths with increasing restoration age. Correlating the soil δ13C with total C concentration revealed the re-establishment of the isotopically depleted labile to enriched refractory C continuum with soil depth for the older restored sites. This implied that from a pedogenic perspective, the restored soils are developing towards the original native soil carbon profile.

Desenvolvimento e validação de método analítico empregando cromatografia gasosa ultrarrápida para quantificação de teor total de ésteres em amostras de biodiesel

Pós-graduação em Química - IQ

Síntese e atividade anti-HCV, anti-candida e antibacteriana de nitrochalconas

Pós-graduação em Química - IBILCE

Land use change to perennial energy crops in Northern Italy: Effects on soil organic carbon sequestration and distribution, soil enzyme activities and microbial communities.

Studies on soil organic carbon (SOC) sequestration in perennial energy crops are available for North-Central Europe, while there is insufficient information for Southern Europe. This research was conducted in the Po Valley, a Mediterranean-temperate zone characterised by low SOC levels, due to intensive management. The aim was to assess the factors influencing SOC sequestration and its distribution through depth and within soil fractions, after a 9-year old conversion from two annual systems to Miscanthus (Miscanthus × giganteus) and giant reed (Arundo donax). The 13C natural abundance was used to evaluate the amount of SOC in annual and perennial species, and determine the percentage of carbon derived from perennial crops. SOC was significantly higher under perennial species, especially in the topsoil (0-0.15 m). After 9 years, the amount of C derived from Miscanthus was 18.7 Mg ha-1, mostly stored at 0-0.15 m, whereas the amount of C derived from giant reed was 34.7 Mg ha-1, evenly distributed through layers. Physical soil fractionation was combined with 13C abundance analysis. C derived from perennial crops was mainly found in macroaggregates. Under giant reed, more newly derived-carbon was stored in microaggregates and mineral fraction than under Miscanthus. A molecular approach based on denaturing gradient gel electrophoresis (DGGE) allowed to evaluate changes on microbial community, after the introduction of perennial crops. Functional aspects were investigated by determining relevant soil enzymes (β-glucosidase, urease, alkaline phosphatase). Perennial crops positively stimulated these enzymes, especially in the topsoil. DGGE profiles revealed that community richness was higher in perennial crops; Shannon index of diversity was influenced only by depth. In conclusion, Miscanthus and giant reed represent a sustainable choice for the recovery of soils exhausted by intensive management, also in Mediterranean conditions and this is relevant mainly because this geographical area is notoriously characterised by a rapid turnover of SOC.

Studies of the effects of molecular encounters on N.M.R. spin-lattice relaxation times

This thesis is concerned with investigations of the effects of molecular encounters on nuclear magnetic resonance spin-lattice relaxation times, with particular reference to mesitylene in mixtures with cyclohexane and TMS. The purpose of the work was to establish the best theoretical description of T1 and assess whether a recently identified mechanism (buffeting), that influences n.m.r. chemical shifts, governs Tl also. A set of experimental conditions are presented that allow reliable measurements of Tl and the N. O. E. for 1H and 13C using both C. W. and F.T. n.m.r. spectroscopy. Literature data for benzene, cyclohexane and chlorobenzene diluted by CC14 and CS2 are used to show that the Hill theory affords the best estimation of their correlation times but appears to be mass dependent. Evaluation of the T1 of the mesitylene protons indicates that a combined Hill-Bloembergen-Purcell-Pound model gives an accurate estimation of T1; subsequently this was shown to be due to cancellation of errors in the calculated intra and intemolecular components. Three experimental methods for the separation of the intra and intermolecular relaxation times are described. The relaxation times of the 13C proton satellite of neat bezene, 1,4 dioxane and mesitylene were measured. Theoretical analyses of the data allow the calculation of Tl intra. Studies of intermolecular NOE's were found to afford a general method of separating observed T1's into their intra and intermolecular components. The aryl 1H and corresponding 13C T1 values and the NOE for the ring carbon of mesitylene in CC14 and C6H12-TMS have been used in combination to determine T1intra and T1inter. The Hill and B.P.P. models are shown to predict similarly inaccurate values for T1linter. A buffeting contribution to T1inter is proposed which when applied to the BPP model and to the Gutowsky-Woessner expression for T1inter gives an inaccuracy of 12% and 6% respectively with respect to theexperimentally based T1inter.

Fatty-acids and their C-13 signatures in seep carbonates from hydrocarbon seeps on the upper (GC 185) and lower (AC 645) continental slope of the Gulf of Mexico

Here we reported the fatty-acids and their δ 13C values in seep carbonates collected from Green Canyon lease block 185 (GC 185; Sample GC-F) at upper continental slope (water depth: ∼540 m), and Alaminos Canyon lease block 645 (GC 645; Sample AC-E) at lower continental slope (water depth: ∼2200 m) of the Gulf of Mexico. More than thirty kinds of fatty acids were detected in both samples. These fatty acids are maximized at C16. There is a clear even-over-odd carbon number predominance in carbon number range. The fatty acids are mainly composed of n-fatty acids, iso-/anteiso-fatty acids and terminally branched odd-numbered fatty acids (iso/anteiso). The low δ 13C values (−39.99‰ to.32.36‰) of n-C12:0, n-C13:0, i-C14:0and n-C14:0 suggest that they may relate to the chemosynthetic communities at seep sites. The unsaturated fatty acids n-C18:2 and C18:1Δ9 have the same δ 13C values, they may originate from theBeggiatoa/Thioploca. Unlike other fatty acids, the terminally branched fatty acids (iso/anteiso) show lowerδ 13C values (as low as −63.95‰) suggesting a possible relationship to sulfate reducing bacteria, which is common during anaerobic oxidation of methane at seep sites.

针式微萃取结合GC-IRMS测定水溶液中甲酸、乙酸的稳定碳同位素组成

甲酸和乙酸稳定碳同位素组成（δ^13C）的分析对环境、食品、制药和自然产品等的研究具有重要的应用价值。但目前尚缺乏有效的测定方法。本研究利用最近出现的针式固相微萃取技术（NeedlEX），以吹扫．捕集方式对水溶液中的有机酸进行了萃取，然后利用气相色谱．同位素比值质谱联用仪（GC—IRMS）对所萃取的有机酸分子进行了占δ^13C的测定。结果显示，质谱计的信号强度与水溶液中有机酸的浓度存在显著的线性相关关系（R^2〉0．99，P〈0．05），表明Needl EX对水溶液中有机酸具有稳定的萃取能力。在甲酸与乙酸含量分别不低于300μg／mL与200μg／mL的水溶液中，1000mL的吹扫体积可以使两者δ^13C多次分析结果的相对误差分别保持在3％和1％左右，且整个实验流程没有造成可检测的碳同位素分馏作用。低于这两个浓度界线，则分析误差随浓度的降低迅速增加。本研究虽然是针对水溶液中有机酸δ^13C的测定，其萃取方法对其他水溶性挥发和半挥发有机物δ^13C的分析也同样具有应用价值.

1H and 13C NMR spectral studies of C-2 substituted isomeric exo- and endo-5-methyl-bicyclo[3.2.1]octane-6,8-diones

A detailed analysis of the 1H and 13C NMR spectra of C-2 aryl and alkyl/desalkyl substituted isomeric exo- and endo-5-methylbicyclo[3.2.1]octane-6,8-diones is presented. The chemical shift of the C-5 angular methyl, the C-2 alkyl/olefinic (C-10)/C-2 methine protons, the aromatic proton shieldings and the characteristic AMX and ABX spectral pattern of the ketomethylene and bridgehead protons were found to be sensitive to the phenyl ring orientation (anisotropy). These distinctive features could be used for configurational distinction for this class of compounds. With increasing ortho-methoxy substitution on the phenyl ring, considerable deshilelding of the bridgehead proton was observed (ca. 0.6 ppm). Absence of the C-2 alkyl group in the desalkyl isomers resulted in substantial changes in the chemical shifts of different protons. A study of the NMR spectra of the corresponding bicyclic compounds with C-2 methoxy/hydroxy substitution instead of the aryl group revealed that the anisotropy of the phenyl ring and the electronegative oxygen substituents have opposite effects. The 13C NMR spectral assignment of each carbon resonance of C-2 aryl and alkyl/desalkyl substituted isomeric exo- and endo-5-methylbicyclo[3.2.1]octane-6,8-diones and the corresponding C-2 methoxy/hydroxy/chloro and methyl bicyclic compounds are reported. Additional ortho-methoxy substitution on the phenyl ring was found to produce considerable high field shifts of the C-10 and C-1 carbon resonances. A high-field shift was observed for the C-6 and C-8 carbonyl carbons, presumably due to 1,3-dicarbonyl interactions. The chemical shifts of C-1 aromatic, C-10 alkyl and C-2 carbons, which are sensitive to exo/endo isomerism, could be utilized in differentiating a pair of isomers.

Evidence for classification of c.1852_1853AA > GC in MLH1 as a neutral variant for Lynch syndrome

Background: Lynch syndrome (LS) is an autosomal dominant inherited cancer syndrome characterized by early onset cancers of the colorectum, endometrium and other tumours. A significant proportion of DNA variants in LS patients are unclassified. Reports on the pathogenicity of the c.1852_1853AA>GC (p.Lys618Ala) variant of the MLH1 gene are conflicting. In this study, we provide new evidence indicating that this variant has no significant implications for LS. Methods: The following approach was used to assess the clinical significance of the p.Lys618Ala variant: frequency in a control population, case-control comparison, co-occurrence of the p.Lys618Ala variant with a pathogenic mutation, co-segregation with the disease and microsatellite instability in tumours from carriers of the variant. We genotyped p.Lys618Ala in 1034 individuals (373 sporadic colorectal cancer [CRC] patients, 250 index subjects from families suspected of having LS [revised Bethesda guidelines] and 411 controls). Three well-characterized LS families that fulfilled the Amsterdam II Criteria and consisted of members with the p.Lys618Ala variant were included to assess co-occurrence and co-segregation. A subset of colorectal tumour DNA samples from 17 patients carrying the p.Lys618Ala variant was screened for microsatellite instability using five mononucleotide markers. Results: Twenty-seven individuals were heterozygous for the p.Lys618Ala variant; nine had sporadic CRC (2.41%), seven were suspected of having hereditary CRC (2.8%) and 11 were controls (2.68%). There were no significant associations in the case-control and case-case studies. The p.Lys618Ala variant was co-existent with pathogenic mutations in two unrelated LS families. In one family, the allele distribution of the pathogenic and unclassified variant was in trans, in the other family the pathogenic variant was detected in the MSH6 gene and only the deleterious variant co-segregated with the disease in both families. Only two positive cases of microsatellite instability (2/17, 11.8%) were detected in tumours from p.Lys618Ala carriers, indicating that this variant does not play a role in functional inactivation of MLH1 in CRC patients. Conclusions: The p.Lys618Ala variant should be considered a neutral variant for LS. These findings have implications for the clinical management of CRC probands and their relatives.