文昌鱼Dkk和Kremen基因家族表达图谱和功能的研究

文昌鱼长期作为脊索动物的祖先模型被研究。它与脊椎动物发育机制的比较为后者的发生和进化提供了大量证据。Wnt信号通路在动物胚胎发育中行使着多样而重要的功能：如胚胎轴系的建立，胚层分化，神经图式形成等。在腔肠动物胚胎发育的早期，Wnt/β-catenin主要参与动植物半球极性的形成和胚层分化——很可能是Wnt通路的祖先功能。而在高等脊椎动物胚胎发育的早期，Wnt/β-catenin通路对于背腹轴、前后轴和左右轴极性的建立发挥着至关重要的作用。我们的研究主要集中在文昌鱼Wnt/β-catenin信号通路的两种调节因子Dickkopf（Dkk）和Kremen，以探明这些Wnt信号调节因子的祖先功能，以及在脊椎动物中获得新功能的进化历程。分泌性蛋白Dkk是Wnt信号通路的抑制因子，协同它的高亲和性受体Kremen，在两栖类胚胎头部的发育中起着关键作用。基于脊椎动物Dkk和kremen的报道以及佛罗里达文昌鱼基因组序列信息，我们运用分子克隆的方法，得到白氏文昌鱼Dkk家族的两个基因：BbDkk124和BbDkk3，以及Kremen家族的5个基因：BbKremen-a，BbKremen-c，BbKremen-d，BbKremen-e，BbKremen-g，用整体胚胎原位杂交的方法研究了它们的表达图谱，并在293T细胞和非洲爪蟾胚胎这两个系统中检测了它们对Wnt信号活性的影响和胚胎发育表型的影响。结果表明文昌鱼Dkk和Kremen的表达区域与脊椎动物的同源基因并不相同，出现了较大分歧，但BbDkk124作为Wnt信号抑制因子的功能是保守的。Kremen家族的两个基因BbKremen-e和BbKremen-g在293T细胞内对Wnt通路的影响不显著，而在非洲爪蟾系统中，引起胚胎不同的畸形表型。我们的实验结果为脊椎动物Dkk和Kremen 基因家族的进化提供了一些资料。 此外我们还研究了文昌鱼GATA家族的基因，这个家族在脊椎动物和非脊椎动物的发育中行使重要的动能，在进化上也是非常保守的。脊椎动物的GATA基因分为两个亚群：GATA1/2/3和GATA4/5/6。通过生物信息分析，我们在文昌鱼的基因组中找到了三个GATA基因：一个GATA1/2/3亚家族基因，两个GATA4/5/6亚家族基因，另外还找到一个类GATA基因。我们克隆了白氏文昌鱼GATA123的一段序列并研究了它在早期胚胎发育中的特异性表达。结果表明GATA123在原肠胚的中内胚层表达，而在神经胚晚期和幼体早期，GATA123在脑泡和消化道中部区域表达。这种表达模式与头部发育的重要基因Otx相类似。我们的研究结果提示在文昌鱼脑泡的发育过程中GATA123和Otx很可能协同发挥着重要的作用。

Scophthalmus maximus cystatin B enhances head kidney macrophage-mediated bacterial killing

Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300 bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen. blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia colt exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

牙鲆肌肉发育调节基因的克隆、表达及功能分析

本文克隆了牙鲆的成肌因子MyoD和Myf5，以及牙鲆的Forkhead基因FoxD1、 FoxD3和FoxD5，并对其在牙鲆肌肉发育中的功能进行了分析。 牙鲆MyoD和Myf5基因都具有三个外显子，两个内含子。其编码的氨基酸序列都含有保守的bHLH；牙鲆FoxD1，FoxD3，FoxD5基因都只有一个外显子，编码的氨基酸序列都含有保守的翼状螺旋DNA结合结构域。 在胚胎发育早期，Myf5在近轴中胚层中表达，体节发生过程中，Myf5在体节中表达，MyoD基因最早在分节板的体节前细胞中表达，随后在近轴细胞、体节中表达；随着胚胎的发育，Myf5在成熟体节中表达量降低，在新生体节中表达较强；MyoD自30个体节时期后只在新生的尾部体节中表达，在成熟的体节中表达量降低；在孵化期，MyoD和 Myf5在头部及鳍的肌肉、尾部的体节中表达；生长期的牙鲆中，Myf5在骨骼肌和肠中表达，成体牙鲆中，Myf5只在肌肉中表达；生长期的牙鲆及成体牙鲆中，MyoD只在肌肉组织中表达。 牙鲆MyoD和Myf5的启动子可以驱动绿色荧光蛋白在斑马鱼肌肉纤维中表达，其包含了这两个基因正常表达所需的核心区域，并可以跨物种行使功能。 在胚胎发育早期，FoxD3在未迁移神经嵴前体细胞、体节、耳后的基板、头部和躯干的神经嵴细胞、松果体中表达。牙鲆FoxD1主要在脑，体节，肾脏及肠中表达。牙鲆FoxD5主要在体节、尾芽、前脑、耳泡中表达。 在斑马鱼中过量表达牙鲆FoxD3，并与斑马鱼的同源基因进行比较，结果表明注射牙鲆和斑马鱼FoxD3的斑马鱼胚胎表型一致，它们在中轴两侧的发育出现了不同步现象，MyoD和Myf5在近轴中胚层中的表达受到不同程度抑制。因此，FoxD3在不同物种之间保守，并且FoxD3在肌肉发育的调控通路中可能通过与MyoD和Myf5相互作用而行使功能。 在斑马鱼中过量表达FoxD1后，MyoD在一侧体节中的表达受到了严重抑制，而在近轴细胞中的表达未受影响，Myf5在体节前中胚层，近轴细胞，及体节中的表达都受到了抑制。FoxD1在胚胎发育的早期可能通过调控MyoD和Myf5的表达而参与肌肉发育的调控。 将牙鲆FoxD5在斑马鱼中过量表达，MyoD在一侧体节中的表达量有所升高，而在近轴细胞中的表达未受影响；Myf5在一侧体节和体节前中胚层中的表达也有所升高，表明牙鲆FoxD5可以调控肌肉调节因子MyoD和Myf5的表达而参与早期肌肉发育的调控。

皮层蛋白（CORTACTIN）在斑马鱼早期胚胎发育中的分布图式和功能分析

该文选择斑马鱼胚胎为实验材料,利用蛋白质电泳转印技术（WesternBlotting）、整装免疫组化染色方法及激光扫描共聚焦显微观察技术等,研究了皮层蛋白在斑马鱼早期胚胎中的分布图式,并通过显微注射抗皮层蛋白的抗体至斑马鱼受精卵中,初步研究了它在胚胎发育中的功能.

斑马鱼肌肉发育相关基因克隆、表达与功能分析

本文主要研究在斑马鱼胚胎发育过程中参与肌肉发育的相关基因，克隆了四个在体节和肌肉中表达的基因全长，分析了基因的时空表达特征，并对其中两个基因进行了过表达，分析其在体节形成和肌肉发生过程中的功能。 从斑马鱼中克隆到甲状腺激素受体相关蛋白基因TRAP150。原位杂交分析TRAP150表达在近轴细胞的慢肌和体节的快肌，表达模式与MyoD的模式相近；并表现出肌肉特异性表达，TRAP150在体节形成和肌肉发生早期高水平说明TRAP150在肌肉分化过程发挥着重要的作用。此外，在胚胎的心脏中也检测到TRAP150的表达。在斑马鱼胚胎过量表达TRAP150造成MyoD在近轴中胚层的过量表达，而对MyoD在近轴细胞的表达影响不明显；由于MyoD在近轴中胚层的表达将诱导快肌的形成，因此过量表达TRAP150将可能导致快肌的增多；从过量表达的结果分析，TRAP150在MyoD的上游正向调控MyoD的表达，是诱导快肌的分化的重要基因。 从斑马鱼中克隆到双特性酪氨酸调控激酶新基因DYRK2。RT-PCR结果表明DYRK2具有母源性表达的特点，并且在36小时前各个时期都有表达。DYRK2在体节形成后表达在体节的近轴细胞中，大约在15个体节时检测到在快肌部位表达，18小时后在胚胎肌肉组织、脑部以及眼睛表达。DYRK2在斑马鱼胚胎发育过程中与体节和肌肉发生相关的基因之一，并可能参与脑和眼睛的发生。在斑马鱼胚胎中过量表达DYRK2导致肌肉标记基因MyoD的表达出现了很大的变化，注射侧MyoD在近轴细胞和近轴中胚层过量表达，尤其是在未形成体节的体节前体中胚层，注射侧的MyoD有大范围的高表达，而在正常一侧没有表达。MyoD的过量表达说明斑马鱼胚胎早期DYRK2通过调控MyoD的表达影响慢肌的分化。 克隆得到斑马鱼的血细胞生成的PBX1互作蛋白基因HPIP1。RT-PCR结果表明HPIP1在斑马鱼胚胎表达具有母源性，但是原位杂交的检测一直到10个体节时才检测到，说明HPIP1一直到肌肉分化后才大量表达，可能在肌肉的成熟阶段起作用。当胚胎发育到18小时，HPIP1表达在所有体节中，前端的较早形成的体节中表达量比晚形成的体节表达量高，也符合HPIP1参与肌肉成熟过程的判断。HPIP1还表达在胚胎的眼部周围，说明HPIP1可能参与到眼部肌肉的形成。 在斑马鱼中克隆的Chp-1相似蛋白基因CHORDC1。在3个体节时，CHORDC1表达在脊索两侧的近轴细胞中，而在5个体节时CHORDC1在表达在体节中，这个特点与MyoD的表达很相似，与MyoD在体节中表达不同的是，CHORDC1也在体节前体中胚层中表达，这与Myf5的表达特点相似，CHORDC1紧随着MyoD，Myf5的高表达说明CHORDC1在肌肉细胞的分化的早期即参与肌肉的发育，而其高表达量也说明CHORDC1在这个过程中可能起到非常重要的作用。CHORDC1在近轴中胚层的表达与MyoD，Myf5有不同的特点，这种不同表现在其在近轴中胚层的表达不仅仅限于快肌和慢肌，而且由后到前的逐渐扩展。而且，CHORDC1在心肌中也表达说明其不仅在骨骼肌中发挥作用。综合CHORDC1的表达特点可以认为其对肌肉的作用不限于特定肌肉类型，广泛参与到各种肌肉的发育过程

文蛤（Meretrix meretrix）幼虫生长发育相关基因的克隆和功能分析

贝类养殖是我国海水养殖业中最重要的组成部分之一。贝类人工大规模养殖中的关键环节是种苗的人工繁育，早期幼虫能否正常生长发育，对幼虫变态和变态后生长率及成活率有很大的影响，直接关系到生产产量和经济效益，所以，了解幼虫发育机制对于生产实践具有重要的指导意义。 文蛤（Meretrix meretrix）是一种在东亚各国沿海和滩涂地区广泛分布的双壳贝类，是我国一种重要的经济品种。本论文以文蛤幼虫为研究对象，分别对文蛤幼虫发育过程中贝壳形成相关的铁蛋白（MmeFer）、营养及变态相关的组织蛋白酶B（MmeCB）及变态过程中细胞凋亡相关的caspase三个基因进行了克隆，分析了基因及编码蛋白在担轮幼虫期（L1）、D形幼虫期（L2）、壳顶幼虫期（L3）和稚贝期（L4）的时空表达特征，解析了其可能的功能，并研究了相应酶类的特异性抑制剂作用对幼虫发育过程的影响，进行了目标蛋白的功能验证，详述如下： 研究结果显示，在文蛤胚胎发育到原肠胚时放入不含铁离子的人工海水中培养，发育成无壳的畸形，随着人工海水中铁离子添加浓度的升高，幼虫长出壳状组织接近正常状态；而发育到L1期幼虫放入不含铁离子的人工海水中培养却可以发育出正常的壳，推测铁和铁代谢相关蛋白在幼虫贝壳初始形成有重要的作用。根据构建的文蛤幼虫cDNA文库中提供的序列信息，从文蛤中克隆了与铁离子代谢密切相关的铁蛋白（MmeFer）的全长cDNA 序列；通过Real time PCR发现，MmeFer mRNA的表达量在贝壳形成前后有明显改变；整体原位杂交结果显示MmeFer mRNA在L1期的表达部位刚好是贝壳生成的起始部位，推断文蛤铁蛋白与文蛤幼虫贝壳初始形成密切相关。 利用文蛤幼虫cDNA文库中的EST信息在幼虫中克隆到文蛤组织蛋白酶B（MmeCB）全长cDNA序列；通过整体原位杂交分析发现MmeCB mRNA在L2至L4期幼虫的消化腺部位表达，而且在L2期的幼虫表皮也有表达，说明MmeCB可能和幼虫消化相关，而且可能参与幼虫从表皮摄取营养的过程。利用MmeCB特异性抑制剂（CA074Me）处理饥饿幼虫，发现其生长受到明显抑制，验证了MmeCB参与幼虫表皮营养代谢的推论。利用免疫组织化学技术，研究了MmeCB蛋白在文蛤幼虫中的时空分布，发现其在L2期幼虫表面和胃部有阳性信号，而在L3期，MmeCB在幼虫面盘基部有强烈的表达，提示MmeCB在文蛤幼虫中不仅起到营养的作用，而且可能和幼虫附着变态有关。利用CA074Me分别处理文蛤胚胎和变态期幼虫，发现抑制MmeCB的活性对胚胎发育和幼虫变态都有显著影响。研究结果提示MmeCB在幼虫发育各阶段均具有重要的生物学功能。 根据caspase在不同物种中的保守区域设计简并引物，在文蛤幼虫中扩增到cDNA序列片段；检测有活性的caspase在文蛤幼虫各发育时期的分布部位，发现其在L1至L3期幼虫中都有分布，说明整个幼虫形态变化过程中都有caspase的参与；细胞凋亡检测结果显示，幼虫主要发生细胞凋亡的部位在L3期幼虫的面盘，即变态过程中要退化的器官，说明细胞凋亡可能是文蛤幼虫变态过程中面盘退化的主要机制；用caspase特异性抑制剂处理变态前幼虫，发现幼虫变态率下降，初步验证了caspase在文蛤幼虫变态过程中的作用。 通过对上述三种基因的研究，分别探讨了文蛤幼虫发育阶段中的几个主要事件（L1期到L2期的贝壳形成、L2到L3期的幼虫营养摄食及L3到L4期的附着变态）相关的基因及其功能，为研究贝类生长发育调控的分子机理提供了新的线索。

Nonlinear Analysis of Submarine Pipelines During Single Point Lifting

基于管道微单元体平衡建立了海管单点提升的非线性力学模型的控制微分方程组,使用变弧长的无量纲代换将动边界问题化为固定边界的两点边值问题,利用maple环境下编制的两点边值问题的打靶法程序得到了该问题在各个提升阶段的数值解答和在单点提升过程中管道的极限弯矩约为0.71q~{1/3}(EI)~{2/3}。

Instability Analysis of Nonlinear Surface Waves in a Circular Cylindrical Container Subjected to a Vertical Excitation

Singular perturbation theory of two-time scale expansions was developed both in inviscid and weak viscous fluids to investigate the motion of single surface standing wave in a liquid-filled circular cylindrical vessel, which is subject to a vertical periodical oscillation. Firstly, it is assumed that the fluid in the circular cylindrical vessel is inviscid, incompressible and the motion is irrotational, a nonlinear evolution equation of slowly varying complex amplitude, which incorporates cubic nonlinear term, external excitation and the influence of surface tension, was derived from solvability condition of high-order approximation. It shows that when forced frequency is low, the effect of surface tension on mode selection of surface wave is not important. However, when forced frequency is high, the influence of surface tension is significant, and can not be neglected. This proved that the surface tension has the function, which causes free surface returning to equilibrium location. Theoretical results much close to experimental results when the surface tension is considered. In fact, the damping will appear in actual physical system due to dissipation of viscosity of fluid. Based upon weakly viscous fluids assumption, the fluid field was divided into an outer potential flow region and an inner boundary layer region. A linear amplitude equation of slowly varying complex amplitude, which incorporates damping term and external excitation, was derived from linearized Navier-Stokes equation. The analytical expression of damping coefficient was determined and the relation between damping and other related parameters (such as viscosity, forced amplitude and depth of fluid) was presented. The nonlinear amplitude equation and a dispersion, which had been derived from the inviscid fluid approximation, were modified by adding linear damping. It was found that the modified results much reasonably close to experimental results. Moreover, the influence both of the surface tension and the weak viscosity on the mode formation was described by comparing theoretical and experimental results. The results show that when the forcing frequency is low, the viscosity of the fluid is prominent for the mode selection. However, when the forcing frequency is high, the surface tension of the fluid is prominent. Finally, instability of the surface wave is analyzed and properties of the solutions of the modified amplitude equation are determined together with phase-plane trajectories. A necessary condition of forming stable surface wave is obtained and unstable regions are illustrated. (c) 2005 Elsevier SAS. All rights reserved.

Nonlinear Analysis of Oscillatory Indentation in Elastic and Viscoelastic Solids

Determining the mechanical properties at micro- and nanometer length scales using nanoindentation or atomic force microscopy is important to many areas of science and engineering. Here we establish equations for obtaining storage and loss modulus from oscillatory indentations by performing a nonlinear analysis of conical and spherical indentation in elastic and viscoelastic solids. We show that, when the conical indenter is driven by a sinusoidal force, the square of displacement is a sinusoidal function of time, not the displacement itself, which is commonly assumed. Similar conclusions hold for spherical indentations. Well-known difficulties associated with measuring contact area and correcting thermal drift may be circumvented using the newly derived equations. These results may help improve methods of using oscillatory indentation for determining elastic and viscoelastic properties of solids.

Time-Frequency Analysis of Nonlinear Systems: The Skeleton Linear Model and the Skeleton Curves

The joint time-frequency analysis method is adopted to study the nonlinear behavior varying with the instantaneous response for a class of S.D.O.F nonlinear system. A time-frequency masking operator, together with the conception of effective time-frequency region of the asymptotic signal are defined here. Based on these mathematical foundations, a so-called skeleton linear model (SLM) is constructed which has similar nonlinear characteristics with the nonlinear system. Two skeleton curves are deduced which can indicate the stiffness and damping in the nonlinear system. The relationship between the SLM and the nonlinear system, both parameters and solutions, is clarified. Based on this work a new identification technique of nonlinear systems using the nonstationary vibration data will be proposed through time-frequency filtering technique and wavelet transform in the following paper.

Nonlinear Analysis of m Method for Single Pile Under Lateral Loading

通过对大量模型试验结果的分析,提出了简便合理的水平荷载作用下单桩的计算方法。该方法将常用m法中单一m值与位移建立指数关系,并由b_m及K两参数来描述。将这一关系引入常用单桩m法计算中,则计算结果可考虑单桩非线性响应的影响。

A Nonlinear Threshold Model Applied to Spallation Analysis

Spallation in heterogeneous media is a complex, dynamic process. Generally speaking, the spallation process is relevant to multiple scales and the diversity and coupling of physics at different scales present two fundamental difficulties for spallation modeling and simulation. More importantly, these difficulties can be greatly enhanced by the disordered heterogeneity on multi-scales. In this paper, a driven nonlinear threshold model for damage evolution in heterogeneous materials is presented and a trans-scale formulation of damage evolution is obtained. The damage evolution in spallation is analyzed with the formulation. Scaling of the formulation reveals that some dimensionless numbers govern the whole process of deformation and damage evolution. The effects of heterogeneity in terms of Weibull modulus on damage evolution in spallation process are also investigated.

Derivation Of A Nonlinear Reynolds Stress Model Using Renormalization Group Analysis And Two-Scale Expansion Technique

Adopting Yoshizawa's two-scale expansion technique, the fluctuating field is expanded around the isotropic field. The renormalization group method is applied for calculating the covariance of the fluctuating field at the lower order expansion. A nonlinear Reynolds stress model is derived and the turbulent constants inside are evaluated analytically. Compared with the two-scale direct interaction approximation analysis for turbulent shear flows proposed by Yoshizawa, the calculation is much more simple. The analytical model presented here is close to the Speziale model, which is widely applied in the numerical simulations for the complex turbulent flows.

Nonlinear analysis of pile foundation jacket platform

In this paper, the nonlinear collapse of the BOHAI-8 pile foundation jacket platform has been analyzed. The ultimate load and collapse process of two computational models of the structure are given. One model is of fixed support whose length is eight times the pile leg diameter and the other considers the nonlinearity of the soil-pile interaction.

Dynamic analysis of arrest of buckle propagation on a beam on a nonlinear elastic foundation by FEM

Based on the dynamic governing equation of propagating buckle on a beam on a nonlinear elastic foundation, this paper deals with an important problem of buckle arrest by combining the FEM with a time integration technique. A new conclusion completely different from that by the quasi-static analysis about the buckle arrestor design is drawn. This shows that the inertia of the beam cannot be ignored in the analysis under consideration, especially when the buckle propagation is suddenly stopped by the arrestors.