从头起源的酵母新基因的功能和正反链编码的基因相互作用的新机制的研究

基因从头起源一直被传统观念认为是近乎不可能的事件。虽然近年来有一些 基因起源于非编码序列的实例的报道，但所有这些从头起源的基因都没有确凿的 编码蛋白的能力的证据。在本工作的前半部分，我们在酿酒酵母Saccharomyces cerevisiae 中发现了一个从头起源的蛋白编码基因MDF1。通过全面的遗传学、 细胞生物学和分子生物学等研究手段，我们细致地揭示了MDF1 在酿酒酵母中 获得的新功能。在营养充足的情况下，MDF1 编码的蛋白质Mdf1p 通过与一个 S. cerevisiae 交配型决定因子MATα2 结合抑制了酿酒酵母交配通路，从而极大程 度上抑制了S. cerevisiae 的交配行为，使S. cerevisiae 节省下更多的能量用于快速 的无性繁殖。我们的工作首次为从头起源的基因提供了确凿的蛋白编码能力的证 据，而且证明年轻的新基因也可以像保守的老基因一样在一些基本的生命过程发 挥关键的作用，为提高物种的适应性作出重要贡献。同时我们的工作在机制上阐 明一个新进化出的基因如何被一个已有的信号通路募集，这为更深刻理解信号通 路的进化提供了有益参考。 在本工作的后半部分，我们发现了一种新的正反链编码的基因对相互作用的 分子机制。最近全基因组转录谱的研究提示：在很多真核生物的基因组中，很大 一部分双链DNA 链都有编码能力，这些分别由正反两条链编码的基因之间可能 存在的相互作用已被公认为一种基因调控的重要方式。现在已知的正反链基因相 互作用的分子机制包括RNAi, 转录干扰，RNA-诱导的组蛋白去乙酰化，RNA 编辑等，所有这些反链基因对正链基因的调控机制都依赖于非编码的反链RNA 的存在，但编码蛋白的反链基因能否对正链基因行使调节功能还是未知。在本工 作中，我们发现编码新基因MDF1 的同一座位的反链基因可以编码一个保守的 基因ADF1, 而且MDF1 和ADF1 对酿酒酵母生长产生相反的影响（MDF1 可以 促进生长，但ADF1 抑制生长）提示MDF1 和ADF1 之间存在相互作用。对这种 相互作用的分子机制的深入研究揭示ADF1 编码的蛋白Adf1p 以转录抑制因子的 方式结合在MDF1 的启动子区，从而抑制MDF1 的转录。这种相互作用需要反 链编码的蛋白而不是RNA 参与，所以不同于任何一种已知的正反链相互作用机 制。我们还进一步发掘出这种抑制效应在S. cerevisiae 中起作用的生理条件。当 营养丰富时，Mdf1p 抑制性地结合非发酵碳源代谢的控制因子Snf1p, 从而促进可发酵碳源被快速利用，使S. cerevisiae 获得最快的生长速度。当营养减少时， Adf1p 抑制MDF1 表达，从而促进非发酵碳源的利用。我们前后两部分的工作还 为生殖代价提供了一种机制上的解释。有性生殖和无性繁殖是两种负相关的过 程，有性生殖总会以生长速度减慢为代价，但至今没有一种分子机制能把这两个 拮抗的过程联系起来。Mdf1p 同时处在有性生殖和无性繁殖两条信号通路中，抑 制交配行为而加速生长，所以Mdf1p 实现了两条信号通路之间的对话。

不同FGF-2表达量的饲养层对猕猴和兔胚胎干细胞的增殖和自我更新的影响

胚胎干细胞（ESC）培养是ESC研究的基础，饲养层的选择是ESC培养的一个重要方面。本实验曾对不同的猕猴饲养层进行研究，显示能够更好的支持猕猴ESC生长的饲养层FGF-2表达量也较高。FGF-2，又名bFGF（碱性成纤维生长因子），是ESC生长所需的重要因子，但其中的分子机制现在并未完全了解。本文一方面对ESC相关研究进展进行了综述，另一方面对以下内容进行了研究：用转基因和RNA干（RNAi）扰的方法建立不同FGF-2的表达量猕猴耳部皮肤细胞（MESF）系五组：过表达FGF-2（f1），过表达的阴性对照组（f2），FGF-2 RNA干扰组（f3），RNA干扰的阴性对照组（f4）以及未作任何处理的对照组（f5），这五组MESF的FGF-2表达量相对值为f1：f2：f3：f4：f5＝4：2：1：2：2；c-fos，TGF-β1，INHBA，Gremlin1在f1中表达量上升，在f3中表达量下降；BMP4，TGF-β2在f1中表达量下降，在f3中表达量上升；表明内源FGF-2能够作用于MESF的TGF-β信号通路，引起相关基因表达量的变化。用这些细胞作为饲养层分别培养两种ESC（猕猴ESC，R366. 4和兔ESC，RFESC） ，连续培养了10代，其中在f1上培养的两种ESC增殖速度都比对照组快，并且c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog，Sox2表达量均上升，BMP4表达下调；在f3上培养的猕猴ESC增殖较慢，BMP4表达上调，c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog，Sox2表达下调；f3上的兔ESC没有变化。表明ESC中的TGF-β信号通路也受到调节。五组猕猴和兔的ESC形成的EB均表达各胚层早期标记基因(marker)，但表达量有差异，f1上ESC形成的EB所有marker都低表达。实验结果表明饲养层中的FGF-2含量高低不仅影响自身相关基因的表达，还对ESC的增殖和维持自我更新有一定的作用。

小鼠Bicc1 基因的克隆、Bicc1 多克隆抗体制备及蛋白定位与表达的研究

双尾-C 基因 (Bicaudal-C)首先在果蝇（Drosophila melanogaster）中发现，其功能丧失导致果蝇胚胎滤泡细胞的错误迁移、头部的缺失和双尾结构的形成。后来发现多个物种都含有Bicaudal-C 的同源基因，其中小鼠中的同源基因Bicc1 的缺失导致小鼠产生肾脏等脏器的病变，其症状与人类多囊肾疾病高度相似，但其具体机制还不清楚。本研究以小鼠肾脏组织总RNA 为模板体外反转录为cDNA，通过分段巢式 PCR 及酶切连接的方法获得了全长约3Kb 的小鼠Bicc1 cDNA 序列。根据生物信息学分析全长的Bicc1 蛋白，选择两个免疫原性较好的区段作为抗原位点构建相应的原核表达载体；IPTG 诱导表达并纯化融合蛋白，制备两种兔抗Bicc1 蛋白多克隆抗体，并通过Western blot 证实这两种抗体具有高度特异性。用细胞免疫荧光方法及免疫组织化学方法对该蛋白的定位做了一些初步研究。发现Bicc1 蛋白定位于体外培养的小鼠肾细胞的细胞质内，并在胚胎发育于期表达仅在心脏，后来逐步地在各个组织器官内出现，并在出生后的小鼠体内表达稳定。Bicc1 mDNA 也表达于多个器官内，并且在肾脏中有明显较高的表达量。找到了的两个针对Bicc1 基因的RNAi 的序列，通过荧光强度变化和Western blot 均证明这两个序列能明显降低Bicc1 蛋白在体外培养细胞中的表达水平，为下一步建立稳定的细胞株奠定了良好的基础。

贾第虫microRNA 及其靶基因的预测与验证

MicroRNAs (miRNAs)是一类长约21-25nt 的非编码小分子RNAs，通过与靶基因的互补结合在转录水平及转录后水平来负调控基因表达。人们已在众多高等多细胞生物中如人、果蝇、线虫、拟南芥等鉴定出众多microRNAs 分子。近来报道单细胞原生生物衣藻中也存在大量microRNAs。然而到目前为止，在被很多证据证实是最原始的单细胞真核生物贾第虫中却仍未有microRNAs 的报道。那么到底贾第虫这种具有特殊进化地位的单细胞原生动物是否存在有microRNAs 呢？如果存在的话，其microRNAs 的特点是什么？与高等多细胞生物及单细胞衣藻的 microRNA 相比又有何异同点呢？贾第虫的microRNAs 是否与其致病性相关呢？已有研究表明，贾第虫基因组中存在与RNAi 相关的Argonaute（AGO）家族蛋白和Dicer 酶。有意思的是，这些与siRNA 引起RNAi 作用关键的蛋白AGO 和Dicer 同样也是miRNA 系统的关键成份，这就提示我们在贾第虫中很有可能也存在有miRNA 并发挥功能。有研究发现在贾第虫基因组中存在大量的非编码转录物，这些大量的非编码转录物中，是否都是后来所认为的为双向启动子转录有用基因时的副产物，还是也存在一些起调控作用的RNA 分子（如miRNAs 等），需要进一步的研究。本文利用生物信息学的手段，依据miRNAs 的生物学特征，结合多种计算机预测的方法，在贾第虫基因组中筛选可能的microRNAs 分子，结果共鉴定出50 个miRNAs 候选分子，这50 个可能的贾第虫miRNAs 不具有保守性，在已知的其他物种的miRNAs 中找不到同源物。用这50 个microRNAs BLASTN 贾第虫的蛋白质编码序列及其相邻5’端和3’端各200bp 的序列，来寻找这些microRNAs 所调控的靶基因。结果表明，寻找到的贾第虫miRNA 的靶基因除很大一部分未知功能的蛋白外，还包括了很多涉及不同功能的蛋白，如VSP 蛋白（various surface proteins）这样一类表面抗原蛋白，提示我们贾第虫miRNA 可能与其致病性相关。接下来我们对其中14 个预测的贾第虫microRNAs 进行了RT-PCR 检测并克隆测序，结果表明gla-mir-6, gla-mir-35 在贾第虫滋养体细胞中稳定表达。我们的研究第一次用生物信息学结合实验的方法在贾第虫寻找到了microRNAs，为下一步深入研究这些microRNAs 在贾第虫中的功能提供了可能。

Microarray Analyses of Gene Expression during the Tetrahymena thermophila Life Cycle

Background: The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression. Methodology/Principal Findings: A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation. Conclusions/Significance: Of the,27,000 predicted open reading frames, transcripts homologous to only,5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

瓦山安息香中苯并呋喃促雌激素合成的机理研究

雌激素是人体内重要的激素之一，具有广泛的生理功能。雌激素缺乏与许多疾病相关，如卵巢功能低下，更年期综合征以及骨质疏松等；雌激素过剩也将导致某些疾病，如乳腺癌、卵巢癌、子宫内膜癌等。目前，如何降低肿瘤组织中的雌激素水平而达到治疗肿瘤的目的，已经得到广泛的研究，但促雌激素生成或调节卵巢功能药物或其相关研究则很少。 本实验室前期的研究发现，瓦山安息香属植物果实中的乙醇提取物具有促雌激素生成作用，通过活性追踪和结构鉴定，确认促E2 生成的主要成分为苯并呋喃类化合物。苯并呋喃类化合物的作用与芳香酶有关，但其确切的作用机理有待证实和深入研究。 为了探讨安息香苯并呋喃类化合物的促雌激素合成的作用机理，拟采用如下的实验方案： 1、细胞学方面，对小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-5、OVCAR-8、IGROV1 等细胞株，采用RT-PCR 和ELISA 方法研究芳香酶Aro基因的表达和雌二醇E2 的生成，芳香酶抑制剂Formestane 作为阳性对照，研究时效曲线和量效曲线，确定安息香苯并呋喃类化合物SP25 的有效浓度和作用时间。 2、RNAi 方面，设计合成了针对人芳香酶Aro基因的3 对RNAi 序列，转染入细胞，芳香酶促进剂Forskolin 和地塞米松、芳香酶抑制剂Formestane 作为阳性对照，采用实时定量PCR 技术，研究RNA 干扰后，安息香苯并呋喃类化合物SP25 对人芳香酶Aro基因表达水平瓦山安息香苯并呋喃促雌激素合成的机理研究的影响。 3、雌激素受体方面，设计一段ERE 的雌激素调控元件，构建重组荧光素酶报告基因载体，瞬时转染人乳腺癌细胞株MDA-MB-231，建立针对雌激素受体的报告基因筛选模型，观察安息香苯并呋喃类化合物SP25 对雌激素受体的选择性和亲和力，从受体水平考察安息香苯并呋喃类化合物SP25 促进雌激素生成的药理学机理。 实验结果显示： 1、分化后的小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7 、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-8 等细胞株具有芳香酶基因的表达。睾酮向雌二醇的转化能够被芳香酶抑制剂Formestane 所阻断，其中OVCAR-3 最适合进行下一步的RNAi研究。 2、RNAi 实验结果显示，设计的3 对RNAi 序列中R2 的干扰效果最强，相应的阴性对照C2 与R2 的表达量相差118 倍（24 小时）和19 倍（48 小时），显示R2/C2 这组序列可用于进一步的RNAi 试验。以R2 干扰OVCAR-3 细胞株，药物作用24、48 小时后，芳香酶抑制剂Formestane 与R2 相对表达量相比分别为0.83 倍和0.04 倍；芳香酶促进剂Forskolin 与R2 相对表达量相比分别为3.61 和1.84 倍；芳香酶促进剂地塞米松与R2 相对表达量相比分别为5.76 倍和3.49倍；苯并呋喃类化合物SP25 与R2 相对表达量相比分别为8.13 倍和4.59 倍。实验证实安息香苯并呋喃类化合物SP25 能够促进因RNAi 而发生基因沉默的人芳香酶Aro表达水平的上调。 3、雌激素受体实验结果显示，构建成功重组pERE-pGL3-promoter 荧光素酶报告基因载体和基于报告基因系统的雌激素受体激动剂或拮抗剂的细胞筛选模型。实验结果表明安息香苯并呋喃类化合物SP25 与雌激素受体ERα和ERβ亲和力选择性之比约为3：1 ，SP25通过与雌激素受体ERα结合作用其受体，刺激芳香酶的表达。 本课题通过RNA 干扰、ELISA、荧光实时定量PCR、报告基因筛选模型等技术手段，从细胞水平、蛋白酶水平和基因表达水平、雌激素受体水平等方面系统地研究了从瓦山安息香属植物果实中提取的苯并呋喃SP25 促进促雌激素生成的机理研究。试验结果显示苯并呋喃类化合物SP25 促雌激素生成的主要作用机制是直接促进芳香酶基因表达水平，以及与雌激素受体a 结合，刺激芳香酶活性。 Estrogen is an important hormone that has versatile physiologicalfunctions. Lack of estrogen will lead to many diseases such as lower ovarianfunction, climacteric syndrome and osteoporosis. Excessive estrogen alsoinduces breast carcinoma, oophoroma and endometrial carcinoma and otherdiseases. To depress the estrogen level in tumor tissue to cure carcinomawas widely studied, but there is only few studies reported on the induction ofestrogen and on the regulation of ovary function. We found that the extracts from seeds of Styrax perkinsiae couldpromote the synthesis of estrogen. The active compounds benzofurans wereidentified. Effect of benzofurans may be related to aromatase, but the mechanism was not clear. To reveal the mechanism of these benzofurans to promote estrogensynthesis, the following protocols were adopted: 1 Cytology: 3T3-L1 preadipocytes,human ovary carcinoma celllines OVCAR-3,OVCAR-4,OVCAR-5,OVCAR-8,IGROV1 andbreast carcinoma cell lines MCF-7 and MDA-MB-231 were usedto determine Aro gene expression and estrogen production withRT-PCR AND ELISA methods. Formestane, an aromataseinhibitor, was used as positive control. And dose-curve,time-curve and the effective concentration of SP25 were also studied. 2 Designed 3 pairs of RNAi for human aromatase gene, andtransfected into cell. Aromatase inducer Forskolin andDexamethasone, and aromatase inhibitor Formestane were usedas positive controls. We studied the change of Aro expressionlevel with SP25 by using real-time PCR after RNA interfering. 3 Estrogen Receptor: We constructed the recombined Luciferasereport vector and establish a screening system for estrogenagonist and antagon. With this system, we studied the affinity ofSP25 and estrogen receptor. Results: 1 Differentiated 3T3-L1 preadipocytes¡¢human ovary carcinomacell lines:OVCAR-3, OVCAR-4, OVCAR-8 and breast carcinomacell lines MCF-7, MDA-MB-231 had detected aromatase geneexpression.And OVCAR-3 is more suitable for further aromatasegene function research. 2 In RNAi assay, R2 has a strong interfering effcet in OVCAR-3 cellline, and ratio of C2 (the negative control) to R2 were 118 times(24 hours) and 19 times (48 hours). This means sucessful inRNA interfering. After R2 acted on OVCAR-3 cell line, the ratiosof formestane to R2 were 0.83 and 0.04 times, 5.76 and 3.49times (Dex), 3.61 and 1.84 times (forskolin) and 8.13 and 4.59times (sp25) after drug treated 24 or 48 hours respectively.These results indicated that SP25 can directly induce aromatasegene up-regulation. 3 We had constructed pERE-pGL3-promoter recombined vectorand the Luciferase report gene screening system. Luciferasereport gene assay showed that sp25 had a higher affinity with strogen receptor alpha than estrogen receptor beta, this indicated that SP25 can act on estrogen receptor and induce aromatase. Our results revealed that the mechanisms of benzofuran to promoteestrogen were the upregulation aromatase gene expression and promotion ofaromatase activity and have partially elective affinity with estrogen receptoralpha.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Regulation of the Edwardsiella tarda Hemolysin Gene and luxS by EthR

Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur (Fur(Et)) was overproduced, and enhanced when Fur(Et) was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: Identification and functional characterisation of Dicer-1

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629 bp in length, including a 51 untranslated region (UTR) of 130 bp, a 3' UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P 0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P > 0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular characterization of an ecdysone inducible gene E75 of Chinese shrimp Fenneropenaeus chinensis and elucidation of its role in molting by RNA interference

Ecdysone inducible gene. E75 is a primary target of ecdysone receptor (EcR). and is found to play a critical role in the molting process of arthropods In this study, a cDNA encoding the E75 of Chinese shrimp Fenneropenaeus chinensis (FcE75) was cloned using RT-PCR and RACE techniques FcE75 cDNA was 3611 bp in length with an ORF of 2394 bp. The deduced amino acid sequence of FcE75 had the highest sequence identity to E75 from a land crab Gecarcinus lateral's and E75 of the shrimp Metapenaeus crisis Quantitative real-time PCR revealed a prominently high expression of FcE75 mRNA in the whole body RNA extract of late premolt period (D3) juvenile shrimp. The role of E75 in the process of shrimp molting was investigated using the RNA interference technique Long double-stranded RNA corresponding to the FcE75 (dsE75) efficiently silenced the FcE75 transcript levels in juvenile F. chinensis. Further, injection with dsE75 completely arrested the molting process in experimental shrimp which eventually caused death Setogenic analysis of the uropods from molt-arrested shrimp, showed defective epidermal retraction, poor development of setae and new cuticle. These results indicate that E75 might be related to the molting process and is essential for proper molting and survival of shrimp This is the first report demonstrating the use of double stranded RNA to elucidate the possible role of E75 in the molting of decapod crustaceans (C) 2010 Elsevier Inc All rights reserved

中华绒螯蟹（Eriocheir sinensis）cDNA文库的构建、EST分析及其酚氧化酶系统关键基因的研究

中华绒螯蟹（Eriocheir sinensis）是我国的特色物种，具有重要的经济和科研价值。酚氧化酶系统作为节肢动物特有的免疫机制，在中华绒螯蟹的免疫反应中发挥重要作用。本研究构建了一个中华绒螯蟹的cDNA文库，利用表达序列标签 (Expressed Sequence Tag，EST) 技术，对中华绒螯蟹表达序列进行了大规模测序分析，并利用cDNA末端快速扩增（rapid amplification of cDNA ends，RACE）、实时定量PCR、原核重组和RNAi等技术研究了其酚氧化酶免疫系统的分子基础及其相应功能。 用鳗弧菌和金黄色葡萄球菌同时感染中华绒螯蟹，提取血细胞的RNA构建了一个库容为3.3×106 克隆cDNA文库。随机测序后获得7535条高质量的EST序列，其中在GenBank数据库中未发现同源序列的为4593 条，而具有较高同源性2942条可以分为20个功能类别，参与了23个生物学反应。 进一步分析发现，969 条（32.9% ）EST与免疫相关，可拼接成221个免疫基因。这个比例高于其它任何一个已公布的甲壳动物cDNA文库。在免疫相关EST中，抗菌肽比例最高，约占总数的20.1%（195条EST）。免疫基因的高比例和抗菌肽的高表达，证明细菌刺激是提高cDNA 文库中免疫基因丰度的有效方法。 EST序列的获得和免疫基因的富集，丰富了中华绒螯蟹的基因组信息，初步了解了中华绒螯蟹固有免疫系统的概况, 为进一步克隆和研究中华绒螯蟹免疫防御功能基因提供了序列基础。 本研究在EST分析的基础上，克隆获得了中华绒螯蟹酚氧化酶系统10个基因的cDNA全长序列, 它们分别是前酚氧化酶（EsproPO），丝氨酸蛋白酶同源物（EsSPH), 丝氨酸蛋白酶抑制剂pacifastin, serpin, PAPII (EsPLC, Es serpin, EsPAPII), 模式识别丝氨酸蛋白酶（EsPRSP），peroxinectin (Esperoxinectin)和3个前酚氧化酶激活酶 (EsPAP1, 2, 3)。它们与相近物种的酚氧化酶系统相应基因均具有较高同源性，并含有胰酶催化结构域，CLIP结构域，PLD结构域，KAZAL结构域，Serpin结构域以及酚氧化酶结构域等酚氧化酶系统相应基因典型的特征结构域。分析发现，PAPs的CLIP结构域和PRSP，Pacifastin，Proxinectin，proPO基因是节肢动物特有的，是酚氧化酶系统作为节肢动物特有免疫机制的分子基础。本研究从多个基因的3′UTR区发现了调控元件，如15-LOX-DICE，K-box和 Brd-Box。在所推断的蛋白中，EsPAP3和EsPAPII的等电点呈碱性，Esperoxinetin的为中性，而EsPRSP，EsSPH，EsproPO, EsPAPII, Esserpin，EsPAP1的等电点在酸性区间。健康中华绒螯蟹 EsPAP1，EsPAP2，EsPAPII基因在肌肉中的表达量最高，而在血细胞中的表达量相对较低；EsPAP3，EsproPO，EsPLC基因在血细胞中表达量较高，在肌肉中的表达量最低。其中，EsPAP3在血细胞中的表达量是其在肌肉组织中表达量的526.35倍。调控元件和多种激活酶与抑制剂的存在、组织分布和等电点的差异，说明中华绒螯蟹酚氧化酶系统在转录、翻译、激活等多个层次上受到了调控。在中华绒螯蟹受到鳗弧菌刺激后，EsPAP1，EsPAP2，EsPAP3，EsPLC和EsPAPII基因的表达量呈上升或下降的趋势，但表达量的极限值均出现在2小时和12小时，这一规律与EsproPO应激后的mRNA表达和酶比活力的变化特点相吻合，说明中华绒螯蟹酚氧化酶系统各因子相互协调共同参与中华绒螯蟹对入侵细菌的防御反应。同时EsPAP2，EsPAP3，EsproPO，EsPAPII，EsPLC在中华绒螯蟹受到鳗弧菌刺激后的表达呈现反复多次上升，表明酚氧化酶系统可能参与了多种免疫反应。研究还发现EsPAP1参与中华绒螯蟹血液凝集过程，而EsPAP3是蟹血细胞中的有效的前酚氧化酶激活因子。研究结果初步揭示了中华绒螯蟹酚氧化酶系统的分子基础、对微生物的响应机制及其调控机制和演化趋势，为节肢动物固有免疫系统研究奠定了良好基础。

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。

Optimisation of immune effector function within the tumour microenvironment

Cancer represents a leading of cause of death in the developed world, inflicting tremendous suffering and plundering billions from health budgets. The traditional treatment approaches of surgery, radiotherapy and chemotherapy have achieved little in terms of cure for this deadly disease. Instead, life is prolonged for many, with dubious quality of life, only for disease to reappear with the inevitable fatal outcome. “Blue sky” thinking is required to tackle this disease and improve outcomes. The realisation and acceptance of the intrinsic role of the immune system in cancer pathogenesis, pathophysiology and treatment represented such a “blue sky” thought. Moreover, the embracement of immunotherapy, the concept of targeting immune cells rather than the tumour cells themselves, represents a paradigm shift in the approach to cancer therapy. The harnessing of immunotherapy demands radical and innovative therapeutic endeavours – endeavours such as gene and cell therapies and RNA interference, which two decades ago existed as mere concepts. This thesis straddles the frontiers of fundamental tumour immunobiology and novel therapeutic discovery, design and delivery. The work undertaken focused on two distinct immune cell populations known to undermine the immune response to cancer – suppressive T cells and macrophages. Novel RNAi mediators were designed, validated and incorporated into clinically relevant gene therapy vectors – involving a traditional lentiviral vector approach, and a novel bacterial vector strategy. Chapter 2 deals with the design of novel RNAi mediators against FOXP3 – a crucial regulator of the immunosuppressive regulatory T cell population. Two mediators were tested and validated. The superior mediator was taken forward as part of work in chapter 3. Chapter 3 deals with transposing the RNA sequence from chapter 2 into a DNA-based construct and subsequent incorporation into a lentiviral-based vector system. The lentiviral vector was shown to mediate gene delivery in vitro and functional RNAi was achieved against FOXP3. Proof of gene delivery was further confirmed in vivo in tumour-bearing animals. Chapter 4 focuses on a different immune cell population – tumour-associated macrophages. Non-invasive bacteria were explored as a specific means of delivering gene therapy to this phagocytic cell type. Proof of delivery was shown in vitro and in vivo. Moreover, in vivo delivery of a gene by this method achieved the desired immune response in terms of cytokine profile. Overall, the data presented here advance exploration within the field of cancer immunotherapy, introduce novel delivery and therapeutic strategies, and demonstrate pre-clinically the potential for such novel anti-cancer therapies.

Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

Molecular mechanisms underpinning IFN-gamma and TNF-alpha synergy in intestinal epithelial cells

Cytokine-driven signalling shapes immune homeostasis and guides inflammatory responses mainly through induction of specific gene expression programmes both within and outside the immune cell compartment. These transcriptional outputs are often amplified via cytokine synergy, which sets a stimulatory threshold that safeguards from exacerbated inflammation and immunopathology. In this study, we investigated the molecular mechanisms underpinning synergy between two pivotal Th1 cytokines, IFN-γ and TNF-α, in human intestinal epithelial cells. These two proinflammatory mediators induce a unique state of signalling and transcriptional synergy implicated in processes such as antiviral and antitumour immunity, intestinal barrier and pancreatic β-cell dysfunction. Since its discovery more than 30 years ago, this biological phenomenon remains, however, only partially defined. Here, using a functional genomics approach including RNAi perturbation screens and small-molecule inhibitors, we identified two new regulators of IFN-γ/TNF-α-induced chemokine and antiviral gene and protein expression, a Bcl-2 protein BCL-G and a histone demethylase UTX. We also discovered that IFN-γ/TNF-α synergise to trigger a coordinated shutdown of major receptor tyrosine kinases expression in colon cancer cells. Together, these findings extend our current understanding of how IFN-γ/TNF-α synergy elicits qualitatively and quantitatively distinct outputs in the intestinal epithelium. Given the well-documented role of this synergistic state in immunopathology of various disorders, our results may help to inform the identification of high quality and biologically relevant druggable targets for diseases characterised by an IFN-γ/TNF-α high immune signature