Expression, purification of recombinant flounder MRF4 protein in Escherichia coli and analysis of its polyclonal antibodies

MRF4 is one of muscle regulatory factors and plays critical roles during skeletal muscle development. The muscle development is important for the fish growth which is an important economic factor for the fish culture. To analyze the function of MRF4 in fish, the founder MRF4 antibody was prepared. The flounder MRF4 was cloned, ligated into prokaryotic expression vector pET-30b and expressed in strain E. coli BL21 (130). The recombinant flounder MRF4 fusion protein was soluble and purified with cobalt IMAC resins. To prepare MRF4 polyclonal antibodies, rabbits were immunized with the soluble protein and the increasing level of antibodies was determined by Western blot. Also, the endogenous flounder MRF4 was recognized by the anti-serum. The result further proved the existence of the anti-MRF4 antibody in the anti-serum, which will be useful for studies on the function of flounder MRF4.

中华绒螯蟹（Eriocheir sinensis）Peroxiredoxin 6和Thioredoxin1基因的克隆及表达

中华绒螯蟹是我国重要的水产经济动物，近年来养殖规模不断扩大，产量持续增加。但是，伴随着养殖规模的扩大，养殖环境也日益恶化并导致了大量疾病的发生，严重制约了中华绒螯蟹养殖业的健康发展。因此，疾病预防和控制对中华绒螯蟹养殖业的可持续发展具有举足轻重的作用。与其他无脊椎动物一样，中华绒螯蟹的免疫系统没有免疫球蛋白和淋巴细胞，而是依靠由细胞免疫和体液免疫构成的固有免疫系统来对病原进行识别和清除。中华绒螯蟹的固有免疫机制的研究有助于推动中华绒螯蟹病害防治工作的开展。 本研究采用大规模EST测序方法，结合末端快速扩增（rapid amplification of cDNA ends，RACE）技术从中华绒螯蟹血淋巴中克隆到了过氧化物还原酶（peroxiredoxin，EsPrx6）和硫氧还蛋白（thioredoxin，EsTrx1）基因的cDNA 全长序列；采用实时荧光定量PCR 技术检测了这两个基因在健康个体中表达的组织分布情况以及鳗弧菌刺激后血淋巴细胞中的时序表达规律；同时，将这两个基因的编码区克隆到pET 系列载体，并在大肠杆菌中实现了重组表达，并进行了体外活性检测。 过氧化物还原酶是一个抗氧化蛋白超家族，在保护机体免受活性氧（reactive oxygen species，ROS）的伤害中发挥着重要作用。中华绒螯蟹Prx6（EsPrx6） 基因的cDNA 全长为1076 bp，5` UTR（untranslated region，UTR） 为69 bp，3` UTR 为347 bp，开放阅读框（open reading frame，ORF）为660 bp，编码219 个氨基酸的蛋白。mRNA 3`-端具有多聚腺苷酸加尾信号（polyadenylation signal）AATAAA 和polyA 尾巴。EsPrx6 的预测分子量为 24 kDa，理论等电点为6.21，具有一个保守的Prx 结构域、一个AhpC 结构域和过氧化物酶催化活性中心PVCTTE，表明EsPrx6 属于1-Cys 型Prx。在所检测的组织中均有EsPrx6 的表达，其中以肝胰腺表达量最高，为血淋巴细胞中表达量的17.4 倍。鳗弧菌刺激后，血淋巴细胞中EsPrx6 的表达下降，到12 h 时，实验组显著低于对照组（P<0.05）；随时间推移，表达水平逐渐回升，但在整个实验期间，都没有恢复到起始水平。将EsPrx6 进行体外重组并在大肠杆菌E. coli BL21（DE3）中实现表达，重组EsPrx6 具有预期的抗氧化活性和过氧化物酶活性，其中抗氧化活力为14.69 U/mg 蛋白，高于相同条件下GSH 的抗氧化力（P<0.05），过氧化物酶活力为23.46 U/mg 蛋白。结果表明，EsPrx6 作为一种重要的抗氧化剂，在中华绒螯蟹抵御ROS 可能引起的氧化损伤方面具有重要作用。 硫氧还蛋白是广泛存在于生物体内的一种具有硫醇依赖性的具有还原活性的蛋白。中华绒螯蟹Trx1（EsTrx1）基因的cDNA 全长为641 bp，5` UTR 为17 bp，3` UTR 为306 bp，开放阅读框为318 bp，编码105 个氨基酸。EsTrx1 的预测分子量为12.2 kDa，理论等电点为4.8。EsTrx1 不含信号肽，其氨基酸序列与其他动物的Trx1s 具有高度相似性，如与地中海黄蝎的Trx1 相似度达到73%；而与其他物种Trx2 的同源性很低，相似度仅为14.3-22.8%，表明EsTrx1 属于Trx1 亚族。实时荧光定量PCR 检测发现，EsTrx1 在鳃、性腺、肝胰腺、肌肉、心脏和血淋巴细胞中都有表达。血淋巴细胞中EsTrx1 mRNA 的表达量在菌刺激后上升，刺激后6 h，实验组表达量显著高于对照组和空白组（P<0.05），然后逐渐恢复到刺激前水平。为进一步探讨其生物学功能，将EsTrx1 进行体外重组并在大肠杆菌E. coli BL21（DE3）得到表达，重组EsTrx1 具有预期的氧化还原调节活性，抗氧化活力为3.06 U/mg，且抗氧化活力高于GSH（P<0.05）。rEsTrx1 的二硫键还原活力为5.03，低于凡纳滨对虾的二硫键还原活力（10.44），接近于大肠杆菌（4.93），小牛胸腺（6.50）和小牛肝脏（5.09），而高于鲍鱼Trx2（1.83）活力。结果表明，EsTrx1 在生理条件下能够作为一种重要的抗氧化剂，参与对细菌感染的免疫应答反应。

扇贝丝氨酸蛋白酶抑制剂基因CfKZSPI及Aikunitz的克隆与重组表达

扇贝养殖是我国重要的海水养殖产业，然而自1997 年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且严重影响了该产业的健康发展。丝氨酸蛋白酶抑制因子及丝氨酸蛋白酶在无脊椎动物的免疫应答中起着核心作用，它们的协同作用直接导致外界病源入侵的信号转导和级联放大，并进一步激活一系列防御体系，如黑化反应、血液凝结和抗菌肽的合成等。因此，克隆扇贝参与免疫防御的丝氨酸蛋白酶抑制剂基因并对其功能进行研究，将有助于进一步研究扇贝的免疫防御机制，丰富和发展无脊椎动物免疫学的内容。 运用大规模EST技术和RACE技术从栉孔扇贝中克隆出一个Kazal型丝氨酸蛋白酶抑制剂基因，定名为CfKZSPI。该基因cDNA序列全长1788bp，其中5' 非编码区（Untranslated Region, UTR）为97 bp，3' UTR161 bp，有一个典型的多聚腺苷酸信号序列（AATAAA）和一个ploy A 尾巴，开放阅读框（Open Reading Frame, ORF）含有1530 bp，编码509 个氨基酸残基。对其推测氨基酸序列进行分析，发现其中包括22个氨基酸残基组成的信号肽序列和12个Kazal型丝氨酸蛋白酶抑制剂结构域。采用QRT-PCR（quantitative real time PCR）对鳗弧菌浸泡刺激后栉孔扇贝血淋巴中CfKZSPI 的 mRNA表达量进行了检测，发现其mRNA 的表达量在鳗弧菌刺激后3h明显上升，达到空白组的43.6倍；然后在6h时有所下降，为空白组的15.0倍；随着菌刺激时间的增长，CfKZSPI基因的 mRNA 表达量急剧增加，在刺激后8h，12h，24h分别达到空白组的174.1，207.8，675.4倍。统计分析发现3h（P=0.019<0.05）和12h（P=0.020<0.05）时，CfKZSPI基因mRNA表达量与空白组差异均显著。为了研究栉孔扇贝CfKZSPI的蛋白活性，将其第十二个结构域克隆到pET-32a（+）载体中，转化大肠杆菌Rosetta-gami（DE3）表达菌株，获得可溶性表达的蛋白rCfKZSPI-12，对其进行抑制蛋白酶活性的分析，发现其对胰蛋白酶有很强的抑制活性，而对凝血酶没有抑制活性。当rCfKZSPI-12与胰蛋白酶分子比率为1：1时，约90%的蛋白酶活性被抑制。运用狄更斯作图法研究rCfKZSPI-12对胰蛋白酶的抑制能力，结果发现其对胰蛋白酶的抑制常数为173 nmol L-1。 采用同样方法从海湾扇贝cDNA文库中克隆出一个Kunitz型丝氨酸蛋白酶抑制剂基因，定名为Aikunitz。该基因全长632 bp，其中5' UTR 为105 bp，3' UTR 为 245 bp，有一个典型的多聚腺苷酸信号序列（AATAAA）和一个ploy A 尾巴，ORF 含有282 bp，编码93 个氨基酸残基。推测的氨基酸序列N末端有一个20个氨基酸残基组成的信号肽序列，成熟蛋白包括一个Kunitz型丝氨酸蛋白酶抑制剂结构域。采用QRT-PCR对鳗弧菌和藤黄微球菌感染后海湾扇贝血淋巴中Aikunitz 的mRNA的表达量进行了检测，结果发现其在鳗弧菌刺激后3h到9h持续上升，9h时表达量为PBS对照组的4.49倍（P=0.008<0.05），然后开始下降，在72h时表达量为对照组的0.24倍（P=0.021<0.05）；而在藤黄微球菌刺激后3h到12h其表达量上升，其中6h时为空白组的5.95倍（P=0.0004<0.01）；12h以后迅速下降，其中24h的表达量为对照组的0.38倍（P=0.028<0.05）。将Aikunitz基因编码的成熟蛋白按照重组CfKZSPI-12的方法进行重组表达，并对重组蛋白进行抑制蛋白酶和抑菌活性分析。结果发现其对胰蛋白酶和弹性蛋白酶两种丝氨酸蛋白酶都没有抑制作用。抑菌实验同样发现，重组Aikunitz 对供试的革兰氏阳性菌藤黄微球菌和革兰氏阴性菌鳗弧菌和大肠杆菌都不显示明显抑菌活性。

长白山火山成因—物理机制和化学演化

This thesis is one of the contributions to NSFC project, “The Changbaishan Volcanism and its Links to the Northeast Asia Tectonic System”. The thesis presents our most recent works on Changbaishan Volcanism, on two aspects as (1) the chemical evolution of the Cenozoic volcanism and the physical links of magma genesis, (2) the Holocene activity of the Tianchi Volcano and risks of potential eruption. 1. Chemical evolution and physical links to the Changbaishan Volcanism Physical links to the Chanbaishan Volcanism, just like origins of most of the volcanisms in eastern China, has long been an enigma. A large scale of volcanic activity has dominated many places of eastern China in Meso-Cenozoic. Activity of these volcanisms in eastern China covers several quite different blocks, covers variety of tectonics, and covers a period of time over 200 million years. Such a large-scale and long-lived volcanism in a continental area challenges our knowledge on dynamics of the Earth’s interior. Some works on “Diwa” hypothesis and “lithospheric thinning” hypothesis present possible links between volcanisms and dynamic evolutions of the earth’s interior, but still cannot interpret where are the sources of the heat and fluid, which are essential to the volcanisms. Based on the study of this thesis, we suggest that dynamics of the deep subduction of western Pacific Plate is the critical factor to the Changbaishan Volcanism and volcanisms in NE China, and maybe even essential to most of the Meso-Cenozoic volcanisms in eastern China. In NE China, stagnant slabs flatted in the mantle transition zone (MTZ, ca. 660 km deep) transport and release significant hydrous fluid to the upper mantle. Metamorphism of the deep-subducted slabs and hence a series of mineral phases play an important role in the water transport, exchange, restore, and release. Dehydrated fluid of the wet slab ascending from the MTZ fertile the upper mantle, and also provide upward heat flow which is essential to the magma genesis. Then magma and volcanism occur with the deep subduction from Mesozoic to mordern time in eastern China. To discribe the exact chemical characteristics of the deep subduction releated volcanics is very difficult, because few researches has contributed to the chemical behaviors of fluid and trace elments in the very deep interior of the Earth, such 660 km deep, 410km or 350km where the fluid may ascend and react. However we can still find some chemical characteristics of oceanic subduction. Basalts of the Changbaishan Volcanism have siginficant characteristics of potassium rich, and even can be called a potassic igneous province. If there are only two possible ways, recycled continentical crust or oceanic crust, to fertile the mantle potossium element as we know now, it’s easy to attribute this to the deep-subducted of the west Pacific Plate. To the eastern China, fluid inclusions in mantle xenoliths from the Cenozic basalts also reveal potassium-rich characteristics. This reveals that the same potassium feritle agents may occur in the mantle sources of eastern China. 2. Holocene activiy of the Tianchi Volcano As one of the large volcanic center and complex volcanic cone, the Tianchi Volcano is a dangerous active volcano, with several Holocene eruptions. Among these eruptions, the Millennium Eruption is regardede as one of the biggist eruptions in the world in the last 2000 years. To estimate the potential danger of volcanic eruption, we discuss two essential factors, as (i) volcanic history of Holocene eruptions, including volcanic geolgy, chronnology and chemistry, (ii) state, evolution and relationship of the magma chambers in mantle and crust beneath the Tianchi Volcano.

藏东及滇东南新生代钾质岩浆作用及其深部制约

The dissertation focuses on the petrology, geochemistry of the volcanic rocks in east Tibet and southeast Yunnan. It lucubrates the Magmatic process, forming mechanism and the possible tectonic settings of the volcanic rocks. The volcanic rocks of Nangqen basin in east Tibet, Qinghai province are mainly Cenozoic intermediate-acid shoshonites. The rocks are LREE enriched and the LREE/HREE = 3～34; (La/Yb)_N = 18.17-53.59, and ΣREE 222～1260μg/g. There are no Eu anomaly, and Nb, Ta, Zr, Hf, Ti are markedly depleted. The isotopic composition is ~(87)Sr/~(86)Sr = 0.70497～0.70614, ~(206)Pb/~(204)Pb = 18.622～18.974, ~(208)Pb/~(204)Pb = 38.431～38.996, ~(207)Pb/~(204)Pb = 15.511～15.613, respectively. K-Ar age of the whole rocks and the single mineral are between 32.0-36.5Ma. Based on the trace elements and isotopic elements, we get the conclusion that the partial melting is one of the dominated forming mechanisms for the volcanic rocks in Naneqen basin. The magma did not experience the crustal contamination en route to the surface; however, the complex mixture took place in the upper mantle before the melt was formed. There are at least two kinds of mixed sources that can be identified. The basalt in southeast Yunnan province is studied. They are distributed in Maguan, Tongguan, and Pingbian County, which is located on the both sides of the Red River belt, and the ultrabasic xenolith are cursory introduced. The volcanic rocks belongs to the alkali series, which can be subdivided into trachybasalt and basanite(Ol normal molecule >5). The volcanic rocks are characteristics by high Ti and low Mg#. According to the magma calculation model, the original rocks of the basalt in southeast Yunnan province are Spinel Lherzolite in Tongguan, Garnet Lherzolite in Pingbian and Maguan, while Togguan undergoes 2-5 percent and percent of partial melting, whereas volcanism in Maguan and Pingbian was so complex to calculate. The fractional crystallization took place during the magma evoltion in southeast Yunnan. The basalt is enriched in LREE with LREE/HREE=9.23-20.19. All of the trace elements display weak Nb, Ta peak, and the depletion of Zr, Hf and Ti in Maguan and pingbian represent the presence of Garnet in the source. The composition of the isotope ratio are ~(87)Sr/~(86)Sr = 0.70333-0.70427, ~(143)Nd/~(144)Nd = 0.512769-0.512940, ~(206)Pb/~(204)Pb = 18.104-18.424, ~(207)Pb/~(204)Pb = 15.483 -15.527; ~(208)Pb/~(204)Pb = 37.938-38.560, respectively, which shows the characteristics of the HIMU type OIB. The volcanic rocks of the southwest Yunnan are derived from the enriched, OIB type mantle sources by synthesizing all the data from trace and isotope elements. It is similar to that of the volcanic rocks in Hawaii, a typical kind of the mixtures of the recycled oceanic crust plume and depleted asthenosphere. To sum up, the volcanic rocks in southeast Yunnan are formed by the intraplate hotpot volcanism.

胶东地区金矿床成矿时代及其成矿地球动力学背景-兼论壳幔相互作用与成岩成矿

Jiaodong Peninsula is the largest repository of gold in China. Varieties of studies have been involved in the mechanism of metallogenesis. This thesis is a part of the project "Study of basic geology related to the prespecting of the supra-large deposits" which supported by National Climbing Program of China to Prof. Zhou. One of the key scientific problems is to study the age and metallogenic dynamics of ore deposit and to understand how interaction between mantle and crust constrains on metallogenesis and lithogenesis. As Jiaodong Peninsula to be study area, the Rb-Sr, Sm-Nd and Pb isotopic systematics of pyrite and altered rocks are measured to define the age and origin of gold. The elemental and Sr-Nd-Pb isotopic compositions of dikes and granites was studied to implicate the source and lithogenesis of the dike and granite and removal of lithosphere and the interaction between mantle and crust in the Jiaodong Peninsula. Considering the tectonic of Jiaodong Peninsula, basic on the time and space, this thesis gives a metallogenic dynamics of gold mineralization and discusses the constraints of the interaction between mantle and crust on the metallogenesis and lithogenesis. This thesis reports the first direct Rb-Sr dating of pyrites and ores using sub-sampling from lode gold deposit in Linglong, Jiaodong Peninsula and the results demonstrate this as a useful geochronological technique for gold mineralization with poor age constraint. The Rb-Sr data of pyrites yields an isochron age of (121.6-122.7) Ma, whereas, those of ore and ore-pyrite spread in two ranges from 120.0 to 121.8 Ma and 110.0-111.7 Ma. Studies of characteristic of gold deposit, microscopy of pyrite and quartz indicate that the apparent ages of ore and ore-pyrite are not isochron ages, it was only mixed by two end members, i.e., the primitive hydrothermal fluids and wall rocks. However, the isochron age of pyrite samples constrains the age of gold mineralization, i.e., early Cretaceous, which is in good consistence with the published U-Pb ages of zircon by using the SHRIMP technique. The whole rock Rb-Sr isochron age of altered rocks indicates that the age of gold mineralizing in the Xincheng gold deposit is 116.6 ± 5.3 Ma. The Sr, Nd and Pb isotopic compositions of pyrite and altered rocks indicate that the gold and relevant elements were derived from multi-sources, i.e. dikes derived from enriched lithospheric mantle and granites, granodiorites and metamorphic rocks outcropped on the crust. It also shows that the hydrothermal fluids derived from mantle magma degassing had play an important role in the gold mineralizing. The major and trace elements, Sr-Nd-Pb isotopic data of granites and granodiorites suggest that the Linglong Granite and Kunyushan Granite were derived from partial melting of basement rocks in the Jiaodong Peninsula at post-collision of North China Craton with South China Craton. Guojialing Granodiorite was considered to be derived from a mixture source, that is, mixed by magmas derived from an enriched lithospheric mantle and crust during the delamination of lithosphere induced by the subduction of Izanagi Plate and the movement of Tancheng-Lujiang Fault. There are kinds of dikes occurred in the Jiaodong Peninsula, which are accompanying with gold mineralization in time and space. The dikes include gabrro, diabase, pyroxene diorite, gabrrophyre, granite-porphyry, and aplite. The whole rock K-Ar ages give two age intervals: 120-124 Ma for the dikes that erupted at the gold mineralizing stage, and <120 Ma of the dikes that intruded after gold mineralizing. According to the age and the relationship between the dikes and gold mineralizing, the dikes could be divided into two groups: Group I (t = 120-124 Ma) and Group II (t < 120Ma). Group I dikes show the high Mg and K, low Ti contents, negative Nb anomalies and positive Eu anomalies, high ~(87)Sr/~(86)Sr and negative εNd(t) values and an enrichment in light rare earth elements, large ion lithosphile elements and a depletion in high field strength elements. Thus the elemental and isotopic characteristics of the Group I dikes indicate that they were derived from an enriched lithospheric mantle perhaps formed by metasomatism of the melt derived from the recycled crustal materials during the deep subduction of continent. In contrast, the Group II dikes have high Ti, Mg and K contents, no negative Nb anomalies, high ~(87)Sr/~(86)Sr and positive or little negative εNd(t) values, which indicate the derivation from a source like OIB-source. The geochemical features also give the tectonic constraints of dikes, which show that Group I dikes were formed at continental arc setting, whereas Group II dikes were formed within plate background. Considering the tectonic setting of Jiaodong Peninsula during the period of gold mineralizing, the metallogenic dynamics was related to the subduction of Izanagi Plate, movement of Tancheng-Lujiang Fault and removal of lithopheric mantle during Late Mesozoic Era.

Specialization of Perceptual Processes

In this report, I discuss the use of vision to support concrete, everyday activity. I will argue that a variety of interesting tasks can be solved using simple and inexpensive vision systems. I will provide a number of working examples in the form of a state-of-the-art mobile robot, Polly, which uses vision to give primitive tours of the seventh floor of the MIT AI Laboratory. By current standards, the robot has a broad behavioral repertoire and is both simple and inexpensive (the complete robot was built for less than $20,000 using commercial board-level components). The approach I will use will be to treat the structure of the agent's activity---its task and environment---as positive resources for the vision system designer. By performing a careful analysis of task and environment, the designer can determine a broad space of mechanisms which can perform the desired activity. My principal thesis is that for a broad range of activities, the space of applicable mechanisms will be broad enough to include a number mechanisms which are simple and economical. The simplest mechanisms that solve a given problem will typically be quite specialized to that problem. One thus worries that building simple vision systems will be require a great deal of {it ad-hoc} engineering that cannot be transferred to other problems. My second thesis is that specialized systems can be analyzed and understood in a principled manner, one that allows general lessons to be extracted from specialized systems. I will present a general approach to analyzing specialization through the use of transformations that provably improve performance. By demonstrating a sequence of transformations that derive a specialized system from a more general one, we can summarize the specialization of the former in a compact form that makes explicit the additional assumptions that it makes about its environment. The summary can be used to predict the performance of the system in novel environments. Individual transformations can be recycled in the design of future systems.

Synthesis of FeCoMnAPO-5 molecular sieve and catalytic activity in cyclohexane oxidation by oxygen

A new FeCoMnAPO-5 with AFI structure was synthesized under hydrothermal conditions and characterized by XRD, FT-IR, X-ray fluorescence, nitrogen adsorption and SEM. The oxidation of cyclohexane with molecular oxygen was studied over the catalyst at 403 K. It show d higher activity compared to FeAPO-5, CoAPO-5 and MnAPO-5. The FeCoMnAPO-5 catalyst was recycled twice without loss of activity or selectivity.

Selective oxidations on recoverable catalysts assembled in emulsions

Catalysts assembled in emulsions are found to be potentially recoverable and efficient for a number of catalytic reactions. The catalysts composed of polyoxometalate anions and quaternary ammonium cations have been designed and synthesized according to the catalytic reactions and by optimizing the structures of cations and anions. The catalysts act essentially as surfactants, which are uniformly distributed in the interface of the emulsion droplets, and accordingly behave like homogeneous catalysts. The catalysts show remarkable selectivity and activity in the oxidation of sulfur-containing molecules to sulfones in diesel and the selective oxidation of alcohols to ketones, using H2O2 as oxidant. For an example, the catalyst demonstrated over 96% efficiency of H2O2 and similar to 100% selectivity to sulfones for the selective oxidation of sulfur-containing molecules in real diesel. Moreover, the catalysts can be separated and recycled by a simple demulsification and re-emulsification.

Liquid phase oxidation of toluene to benzaldehyde with molecular oxygen over copper-based heterogeneous catalysts

We conducted the liquid phase oxidation of toluene with molecular oxygen over heterogeneous catalysts of copper-based binary metal oxides. Among the copper-based binary metal oxides, iron-copper binary oxide (Fe/Cu = 0.3 atomic ratio) was found to be the best catalyst. In the presence of pyridine, overoxidation of benzaldehyde to benzoic acid was partially prevented. As a result, highly selective formation of benzaldehyde (86% selectivity) was observed after 2 h of reaction (7% conversion of toluene) at 463 K and 1.0 MPa of oxygen atmosphere in the presence of pyridine. These catalytic performances were similar or better than those in the gas phase oxidation of toluene at reaction temperatures higher than 473 K and under 0.5-2.5 MPa. It was suggested from competitive adsorption measurements that pyridine could reduce the adsorption of benzaldehyde. At a long reaction time of 4 It, the conversion increased to 25% and benzoic acid became the predominant reaction product (72% selectivity) in the absence of pyridine. The yield of benzoic acid was higher than that in the Snia-Viscosa process, which requires corrosive halogen ions and acidic solvents in the homogeneous reaction media. The catalyst was easily recycled by simple filtration and reusable after washing and drying.

Cartilha de tecnologias: vitrine tecnológica de agroecologia "Vilson Nilson Redel".

Sumário: Os princípios da Agroecologia em 2.600 m²; Histórico da vitrine tecnológica de Agroecologia; Adubos verdes e consórcios; Agrofloresta; Como transformar uma propriedade convencional em agroecológica?; Como controlar pragas e doenças em sistemas agroecológicos?; Controle biológico de pragas; Homeopatia e agroecologia; Manejo de pragas e doenças ? as caldas e repelentes naturais; Armadilha para captura de percevejos em soja; Compostagem e vermicompostagem; Fruticultura ecológica; Fixação Biológica de Nitrogênio (FBN) - uso de inoculantes; Olericultura como alternativa de renda para agricultura familiar ecológica; Plantas alimentares não convencionais (PANC); Plantas medicinais; Bioconstruções; Cultivares de soja para sistemas de base ecológica; Meliponicultura-uma atividade essencialmente agroecológica; Milho QPM (alta qualidade proteica); Sistemas alternativos de irrigação; Irrigação com sistemas adaptados de baixo custo; Aspersor de garrafa PET com conexão de 3/4 de polegada; Irrigação alternativa por gotejamento e microaspersão; Carneiro hidráulico; Pastoreio Racional Voisin (PRV); Manejo nutricional em rebanhos de base agroecológica; Suplementação alimentar proteica de bovinos de leite em períodos de escassez (seca ou frio).

Micropropagation of Begonia and a study of genome stability in Begonia rex

The development of procedures and media for the micropropagation of B. rex are described. Media for the production of plantlets from a number of other Begonia hybrids are also provided. Growth analysis data is given for plants produced in vivo from leaf cuttings and in vitro from mature leaf petioles and immature leaves derived from singly and multiply recycled axenic plantlets. No significant difference was found in phenotype or quantitative vegetative characters for any of the populations assessed. The results presented from studies on the development of broad spectrum media for the propagation of a number of B. rex cultivars using axenic leaf explants on factorial combinations of hormones illustrate the major influence played by the genotype on explant response in vitro and suggest media on which a range of B. rex cultivars may be propagated. Procedures for in vitro irradiation and colchicine treatments to destabilize the B. rex genome have also been described. Variants produced from these treatments indicate the utility of in vitro procedures for the expression of induced somatic variation. Colour variants produced from irradiation treatment have been cultured and prove stable. Polyploids produced as variants from irradiation treatment have been subcultured but prove unstable. Media for the induction and proliferation of callus are outlined. The influence of callus subculture and aging on the stability of the B. rex genome is assessed by chromosomal analysis of cells, in vitro and in regenerants. The B. rex genome is destabilized in callus culture but attenuation of variation occurs on regeneration. Diploid cell lines are maintained in callus subcultures and supplementation of regenerative media with high cytokinin concentrations, casein hydrolysate or adenine failed to produce variants. Callus aging however resulted in the production of polyploids. The presence and expression of pre-existing somatic variation in B. rex pith and root tissue is assessed and polyploids have been produced from pith tissues cultured in vitro. The stability of the B. rex genome and the application of tissue culture to micropropagation and breeding of B. rex are discussed.

Reexamining the science of marine protected areas: Linking knowledge to action

Marine protected areas (MPAs) are often implemented to conserve or restore species, fisheries, habitats, ecosystems, and ecological functions and services; buffer against the ecological effects of climate change; and alleviate poverty in coastal communities. Scientific research provides valuable insights into the social and ecological impacts of MPAs, as well as the factors that shape these impacts, providing useful guidance or "rules of thumb" for science-based MPA policy. Both ecological and social factors foster effective MPAs, including substantial coverage of representative habitats and oceanographic conditions; diverse size and spacing; protection of habitat bottlenecks; participatory decisionmaking arrangements; bounded and contextually appropriate resource use rights; active and accountable monitoring and enforcement systems; and accessible conflict resolution mechanisms. For MPAs to realize their full potential as a tool for ocean governance, further advances in policy-relevant MPA science are required. These research frontiers include MPA impacts on nontarget and wide-ranging species and habitats; impacts beyond MPA boundaries, on ecosystem services, and on resource-dependent human populations, as well as potential scale mismatches of ecosystem service flows. Explicitly treating MPAs as "policy experiments" and employing the tools of impact evaluation holds particular promise as a way for policy-relevant science to inform and advance science-based MPA policy. © 2011 Wiley Periodicals, Inc.

Radium and barium removal through blending hydraulic fracturing fluids with acid mine drainage.

Wastewaters generated during hydraulic fracturing of the Marcellus Shale typically contain high concentrations of salts, naturally occurring radioactive material (NORM), and metals, such as barium, that pose environmental and public health risks upon inadequate treatment and disposal. In addition, fresh water scarcity in dry regions or during periods of drought could limit shale gas development. This paper explores the possibility of using alternative water sources and their impact on NORM levels through blending acid mine drainage (AMD) effluent with recycled hydraulic fracturing flowback fluids (HFFFs). We conducted a series of laboratory experiments in which the chemistry and NORM of different mix proportions of AMD and HFFF were examined after reacting for 48 h. The experimental data combined with geochemical modeling and X-ray diffraction analysis suggest that several ions, including sulfate, iron, barium, strontium, and a large portion of radium (60-100%), precipitated into newly formed solids composed mainly of Sr barite within the first ∼ 10 h of mixing. The results imply that blending AMD and HFFF could be an effective management practice for both remediation of the high NORM in the Marcellus HFFF wastewater and beneficial utilization of AMD that is currently contaminating waterways in northeastern U.S.A.

On-board Robotic Multi-pinhole SPECT System for Region-of-interest (ROI) Imaging

On-board image guidance, such as cone-beam CT (CBCT) and kV/MV 2D imaging, is essential in many radiation therapy procedures, such as intensity modulated radiotherapy (IMRT) and stereotactic body radiation therapy (SBRT). These imaging techniques provide predominantly anatomical information for treatment planning and target localization. Recently, studies have shown that treatment planning based on functional and molecular information about the tumor and surrounding tissue could potentially improve the effectiveness of radiation therapy. However, current on-board imaging systems are limited in their functional and molecular imaging capability. Single Photon Emission Computed Tomography (SPECT) is a candidate to achieve on-board functional and molecular imaging. Traditional SPECT systems typically take 20 minutes or more for a scan, which is too long for on-board imaging. A robotic multi-pinhole SPECT system was proposed in this dissertation to provide shorter imaging time by using a robotic arm to maneuver the multi-pinhole SPECT system around the patient in position for radiation therapy.

A 49-pinhole collimated SPECT detector and its shielding were designed and simulated in this work using the computer-aided design (CAD) software. The trajectories of robotic arm about the patient, treatment table and gantry in the radiation therapy room and several detector assemblies such as parallel holes, single pinhole and 49 pinholes collimated detector were investigated. The rail mounted system was designed to enable a full range of detector positions and orientations to various crucial treatment sites including head and torso, while avoiding collision with linear accelerator (LINAC), patient table and patient.

An alignment method was developed in this work to calibrate the on-board robotic SPECT to the LINAC coordinate frame and to the coordinate frames of other on-board imaging systems such as CBCT. This alignment method utilizes line sources and one pinhole projection of these line sources. The model consists of multiple alignment parameters which maps line sources in 3-dimensional (3D) space to their 2-dimensional (2D) projections on the SPECT detector. Computer-simulation studies and experimental evaluations were performed as a function of number of line sources, Radon transform accuracy, finite line-source width, intrinsic camera resolution, Poisson noise and acquisition geometry. In computer-simulation studies, when there was no error in determining angles (α) and offsets (ρ) of the measured projections, the six alignment parameters (3 translational and 3 rotational) were estimated perfectly using three line sources. When angles (α) and offsets (ρ) were provided by Radon transform, the estimation accuracy was reduced. The estimation error was associated with rounding errors of Radon transform, finite line-source width, Poisson noise, number of line sources, intrinsic camera resolution and detector acquisition geometry. The estimation accuracy was significantly improved by using 4 line sources rather than 3 and also by using thinner line-source projections (obtained by better intrinsic detector resolution). With 5 line sources, median errors were 0.2 mm for the detector translations, 0.7 mm for the detector radius of rotation, and less than 0.5° for detector rotation, tilt and twist. In experimental evaluations, average errors relative to a different, independent registration technique were about 1.8 mm for detector translations, 1.1 mm for the detector radius of rotation (ROR), 0.5° and 0.4° for detector rotation and tilt, respectively, and 1.2° for detector twist.

Simulation studies were performed to investigate the improvement of imaging sensitivity and accuracy of hot sphere localization for breast imaging of patients in prone position. A 3D XCAT phantom was simulated in the prone position with nine hot spheres of 10 mm diameter added in the left breast. A no-treatment-table case and two commercial prone breast boards, 7 and 24 cm thick, were simulated. Different pinhole focal lengths were assessed for root-mean-square-error (RMSE). The pinhole focal lengths resulting in the lowest RMSE values were 12 cm, 18 cm and 21 cm for no table, thin board, and thick board, respectively. In both no table and thin board cases, all 9 hot spheres were easily visualized above background with 4-minute scans utilizing the 49-pinhole SPECT system while seven of nine hot spheres were visible with the thick board. In comparison with parallel-hole system, our 49-pinhole system shows reduction in noise and bias under these simulation cases. These results correspond to smaller radii of rotation for no-table case and thinner prone board. Similarly, localization accuracy with the 49-pinhole system was significantly better than with the parallel-hole system for both the thin and thick prone boards. Median localization errors for the 49-pinhole system with the thin board were less than 3 mm for 5 of 9 hot spheres, and less than 6 mm for the other 4 hot spheres. Median localization errors of 49-pinhole system with the thick board were less than 4 mm for 5 of 9 hot spheres, and less than 8 mm for the other 4 hot spheres.

Besides prone breast imaging, respiratory-gated region-of-interest (ROI) imaging of lung tumor was also investigated. A simulation study was conducted on the potential of multi-pinhole, region-of-interest (ROI) SPECT to alleviate noise effects associated with respiratory-gated SPECT imaging of the thorax. Two 4D XCAT digital phantoms were constructed, with either a 10 mm or 20 mm diameter tumor added in the right lung. The maximum diaphragm motion was 2 cm (for 10 mm tumor) or 4 cm (for 20 mm tumor) in superior-inferior direction and 1.2 cm in anterior-posterior direction. Projections were simulated with a 4-minute acquisition time (40 seconds per each of 6 gates) using either the ROI SPECT system (49-pinhole) or reference single and dual conventional broad cross-section, parallel-hole collimated SPECT. The SPECT images were reconstructed using OSEM with up to 6 iterations. Images were evaluated as a function of gate by profiles, noise versus bias curves, and a numerical observer performing a forced-choice localization task. Even for the 20 mm tumor, the 49-pinhole imaging ROI was found sufficient to encompass fully usual clinical ranges of diaphragm motion. Averaged over the 6 gates, noise at iteration 6 of 49-pinhole ROI imaging (10.9 µCi/ml) was approximately comparable to noise at iteration 2 of the two dual and single parallel-hole, broad cross-section systems (12.4 µCi/ml and 13.8 µCi/ml, respectively). Corresponding biases were much lower for the 49-pinhole ROI system (3.8 µCi/ml), versus 6.2 µCi/ml and 6.5 µCi/ml for the dual and single parallel-hole systems, respectively. Median localization errors averaged over 6 gates, for the 10 mm and 20 mm tumors respectively, were 1.6 mm and 0.5 mm using the ROI imaging system and 6.6 mm and 2.3 mm using the dual parallel-hole, broad cross-section system. The results demonstrate substantially improved imaging via ROI methods. One important application may be gated imaging of patients in position for radiation therapy.

A robotic SPECT imaging system was constructed utilizing a gamma camera detector (Digirad 2020tc) and a robot (KUKA KR150-L110 robot). An imaging study was performed with a phantom (PET CT PhantomTM), which includes 5 spheres of 10, 13, 17, 22 and 28 mm in diameter. The phantom was placed on a flat-top couch. SPECT projections were acquired with a parallel-hole collimator and a single-pinhole collimator both without background in the phantom, and with background at 1/10th the sphere activity concentration. The imaging trajectories of parallel-hole and pinhole collimated detectors spanned 180 degrees and 228 degrees respectively. The pinhole detector viewed a 14.7 cm-diameter common volume which encompassed the 28 mm and 22 mm spheres. The common volume for parallel-hole was a 20.8-cm-diameter cylinder which encompassed all five spheres in the phantom. The maneuverability of the robotic system was tested by navigating the detector to trace the flat-top table while avoiding collision with the table and maintaining the closest possible proximity to the common volume. For image reconstruction, detector trajectories were described by radius-of-rotation and detector rotation angle θ. These reconstruction parameters were obtained from the robot base and tool coordinates. The robotic SPECT system was able to maneuver the parallel-hole and pinhole collimated SPECT detectors in close proximity to the phantom, minimizing impact of the flat-top couch on detector to center-of-rotation (COR) distance. In no background case, all five spheres were visible in the reconstructed parallel-hole and pinhole images. In with background case, three spheres of 17, 22 and 28 mm diameter were readily observed with the parallel-hole imaging, and the targeted spheres (22 and 28 mm diameter) were readily observed in the pinhole ROI imaging.

In conclusion, the proposed on-board robotic SPECT can be aligned to LINAC/CBCT with a single pinhole projection of the line-source phantom. Alignment parameters can be estimated using one pinhole projection of line sources. This alignment method may be important for multi-pinhole SPECT, where relative pinhole alignment may vary during rotation. For single pinhole and multi-pinhole SPECT imaging onboard radiation therapy machines, the method could provide alignment of SPECT coordinates with those of CBCT and the LINAC. In simulation studies of prone breast imaging and respiratory-gated lung imaging, the 49-pinhole detector showed better tumor contrast recovery and localization in a 4-minute scan compared to parallel-hole detector. On-board SPECT could be achieved by a robot maneuvering a SPECT detector about patients in position for radiation therapy on a flat-top couch. The robot inherent coordinate frames could be an effective means to estimate detector pose for use in SPECT image reconstruction.