1,893 research outputs found
Experimental studies of Hg(II)-Hg(0) transformations and their effects on Hg isotope fractionation
No full textUnderstanding the redox transformations of inorganic forms of mercury (Hg) is necessary for understanding the fate of mercury in environmental systems. In this study, the interactions of Hg(0) and Hg(II) with organic and inorganic substances were characterized using mercury stable isotopes. Interactions of a mixed Hg(0)-Hg(II) solution with thiol and humic substances were observed, with no net changes to redox speciation. In the presence of mercaptoacetic acid (MCA), an equilibrium isotope enrichment factor (ε202Hg = δ202HgHg(II) - δ202HgHg(0)) of 1.34‰ between the Hg(II) and Hg(0) fractions was observed, similar with previously published values for Hg(0) oxidation by thiols. The equilibrium ε202Hg values similarly determined for 2-mercaptopropionic acid (MPA) and Suwannee River humic acids were 2.03‰ and 1.50‰, respectively. Reduction of mercury by siderite (FeCO3) was also characterized with respect to isotope fractionation over the course of the reaction. This reaction resulted in an 87% reduction of Hg(II) over 30 minutes, with change in mercury isotope ratios of the reactant. Kinetic ε202Hg values for the reduction of Hg(II) by siderite (ε202Hg = δ202HgHg(0) - δ202HgHg(II)) were determined by closed-system model (-1.59‰) and Rayleigh distillation model (-1.07‰; product enrichment in light isotopes). The final equilibrium state exhibited an equilibrium ε202Hg of -0.67‰. The results from the experiments conducted suggest that there is equilibrium isotope exchange between thiol-bound Hg(II) and dissolved Hg(0), and that the reduction by siderite is rapid and may involve multiple processes through the reaction.M.S.Includes bibliographical reference
Molecular cloning for human granulocyte colony stimulating factor (hG-CSF) in Pichia pastoris
No full text碩士人類白血球顆粒細胞增生因子（Human granulocyte colony stimulating factor；hG-CSF）是人體嗜中性白血球（neutrophilic granulocyte）生長、分化所必須的細胞激素，是由174個氨基酸所構成。主要是應用在化療所引起之嗜中性白血球缺乏症（neutropenia）。本實驗以基因工程的方法，將hG-CSF基因轉殖到酵母菌，如酵母菌可使hG-CSF分泌到細胞外，即可簡化之後之純化程序。實驗中，我們以聚合酶連鎖反應(PCR) 從pET25b-hG-CSF中擴增hG-CSF，並轉殖此因子之基因片段。此DNA片段有522 bp，並且在5’端設計有6個組織氨酸（histidine tag）和可被腸激酶所辨識（enterokinase cleavage site）的序列，需要時可將此酵素切割成hG-CSF蛋白質。PCR之後挑選clone帶有hG-CSF基因，並且將此基因再接到表現質體（pPIC9K）上，完成質體pPIC9K-hG-CSF的建構。最後將質體轉殖到酵母菌SMD1168內，挑選出生長於含有4 mg/ml G-418的YPDS固態培養基上的菌落，這可能是含有high copy number的轉型菌。
將誘導後的細胞及培養基，分別以SDS-PAGE及西方點墨法（Western blotting）來鑑定結果。可發現不管是在細胞內或培養基都有hG-CSF，而分泌到細胞外的hG-CSF約為每公升10毫克，表現的hG-CSF確實其分子量在20 kDa左右。
將經由FPLC分子篩液相層析管柱（HiPerp Sephacryl S-200）初步純化後的Fraction I（12~19）和Fraction II（21~34），各分別添加到人類白血病細胞株（HL-60）中，發現Fraction II有助於HL-60細胞增生，而Fraction I卻不能使細胞增生，因此可判斷Fraction II具有生物活性。添加40 ng/ml的Fraction II，在第７天時，可以觀察到細胞數目比對照組增加了２倍；添加Fraction II由1 ng ~ 40 ng/ml有顯著增生，而在40 ng ~ 150 ng/ml達到最高的細胞數。Human granulocyte colony stimulating factor (hG-CSF) is a 18.7 kDa glycoprotein , consisting 174 amino acids. It can stimulate granulocyte colony formation and affects proliferation, differentiation and activation of mature neutrophilic granulocytes. It was widely for treatment of neutropenia in cancer therapy. We cloned the hG-CSF gene into P. pastoris and hope to see the recombinant hG-CSF can secrete into medium. It will make the purification procedure much easiler. We amplified the hG-CSF gene from pET25b-hG-CSF by polymerase chain reaction (PCR). The size of the hG-CSF DNA contains 522 bp, and histidine tag (His6) and enterokinase cleavage site (Asp4Lys) were added at the N-terminus of the protein. The PCR product was first cloned to pOPtima™ cloning vector (TA- Cloning). The “insert” was further cloned into pPIC9K vector. pPIC9K-hG-CSF plasmid was integrated into the alcohol oxidase region of the SMD1168 genome. We selected the transformants that were selected from medium contains 4 mg/ml of G-418. Based on it can be selected from the plate contains 0.2 mg/ml of G-418 having 1 copy of insertion. We can predict this is a multicopy clone.
Under on culturing condition, the recombinant hG-CSF was able to be secreted into the medium, 10 mg/L of culture medium. After the medium was concentrated, we obtained the protein from FPLC-gel filtration column (Hiperp Sephacryl S-200). There were two fractions . 「Fraction I and Fraction II」 From SDS-PAGE, both were located at 20 kDa. But from the biological activity study for stimulation of granulocyte. We only found Fraction II has the activity. We concluded that Fraction I was obtain in the early portion of the gel filtration column. So it may exist as the aggregated form.目 錄
封面內頁
口試委員審議通過簽名表
授權書
致謝
中文摘要 I
英文摘要 II
目錄 III
圖表目錄 V
第一章 緒論
第一節 前言 1
第二節 人類白血球顆粒細胞增生因子 3
第三節 嗜甲基酵母菌Pichia pastoris(P. pastoris) 5
第二章 實驗材料、設備
第一節 實驗材料 16
第二節 實驗設備 19
第三節 培養基配置 20
第四節 酵母菌表現質體及寄主選擇 23
第三章 實驗方法
第一節 hG-CSF基因選殖 26
第二節 pPIC9K表現質體和hG-CSF基因之重組 34
第三節 pPIC9K-hG-CSF質體轉殖到酵母菌 39
第四節 hG-CSF基因表現 42
第五節 hG-CSF重組蛋白質純化 49
第六節 hG-CSF對細胞增生的測定 52
第四章 結果與討論
第一節 製備人類白血球顆粒細胞增生因子基因 59
第二節 確認及純化P. pastoris所表現的hG-CSF蛋白質 61
第三節 檢測表現蛋白質hG-CSF的活性 64
第五章 結論與未來展望 81
第六章 參考文獻 83
圖表目錄
表1.1 生物技術工業常用的蛋白質表現系統 2
表1.2 利用P. pastoris表現的異源蛋白質 15
表3.1 PCR反應之各項成分 27
表3.2 PCR反應條件 27
表3.3 TA ligation之各項成分 31
表3.4 pOptima-hG-CSF DNA以限制酶(EcoRI、NotI)水解 34
之反應組成
表3.5 pPIC9K 質體DNA以限制酶(EcoRI、NotI)水解之反 35
應組成
表3.6 接合反應溶液 37
表3.7 pPIC9K-hG-CSF質體 DNA以限制酶(EcoRI、NotI)水 38
解之反應組成
表3.8 pPIC9K-hG-CSF質體 DNA以限制酶(SacI)水解之反應 40
組成
圖1.1 G-CSF蛋白質分子之三級結構 11
圖1.2 P. pastoris代謝甲醇路徑圖 12
圖1.3 異源蛋白質分泌至胞外示意圖 13
圖1.4 表現載體整合進入P. pastoris染色體DNA的方式 14
圖2.1 pPIC9K質體圖 23
圖2.2 pPIC9K multiple cloning site 24
圖2.3 pPIC9K-hG-CSF質體建構示意圖 25
圖3.1 TA vector：pOptima™ cloning vector質體圖 30
圖3.2 轉漬槽示意圖 48
圖3.3 血球計數盤 55
圖3.4 MTT還原成formazan示意圖 56
圖4.1 洋菜膠電泳分析PCR反應擴增的重組基因 66
圖4.2 TA cloning 藍白篩選 67
圖4.3 洋菜膠電泳分析重組基因限制酶片段 68
圖4.4 洋菜膠電泳分析pPIC9K-hG-CSF的限制酶片段 69
圖4.5 Insert基因（上）與資料庫中人類白血球顆粒細胞增生因子基因（下）定序結果 70
圖4.6 重組人類白血球顆粒細胞增生因子基因DNA全長氨基酸轉譯圖 71
圖4.7 以SDS-PAGE及西方點墨法來確認P. pastoris所表現之hG-CSF 72
圖4.8 以SDS-PAGE及西方點墨法分析，通過親和性管柱之重組hG-CSF（native的管柱結果） 73
圖4.9 以SDS-PAGE及西方點墨法分析，通過親和性管柱之重組hG-CSF（denatured的管柱結果） 74
圖4.10 FPLC分子篩液相層析（HiPrep Sephacryl S-200） 75
圖4.11 以SDS-PAGE及西方點墨法分析，經分子篩（HiPrep Sephacryl S-200）液相層析管柱純化之hG-CSF 76
圖4.12 測試於培養基中有無添加1.25％ DMSO對HL-60細胞增生的影響 77
圖4.13 HL-60細胞經hG-CSF（denatured，實驗3.5.2）處理後，以細胞計數觀察細胞增生的變化 78
圖4.14 HL-60細胞經hG-CSF（12~19）及hG-CSF（21~34）處理後，以細胞計數觀察細胞增生的變化 79
圖4.15 利用MTT assay方法分析不同濃度的hG-CSF（21~34）對HL-60細胞增生的影響 80學號: 693290495, 學年度: 9
Liquid structure of Rb-Hg alloys studied by neutron diffraction
Get PDFThe structures of liquid Rb–Hg alloys were studied as a function of composition by neutron diffraction. In the intermediate Rb concentration range, the obtained structure factors show a small prepeak, which may be an evidence of the formation of Hg polyanion units in liquids. The Reverse Monte Carlo (RMC) analysis was applied to separate the total radial distribution function into the corresponding partial radial distribution functions. Up to 10 at.% Rb, no obvious changes are found for the first peak position of the partial radial distribution functions of the Hg–Hg pair and that of the Hg–Rb pair. The first peak position between the Hg–Rb pairs increases above 20 at.% Rb. In addition to the first peak, a subpeak between Hg–Hg pairs can be seen in the large distance. At 60 at.% Rb, the nearest neighbor distance between Hg atoms shows the closest value in the concentration range studied. These results indicate that with the progress of charge transfer the solvation structure in the dilute Rb concentration range changes into the structure containing polyanions composed of Hg species
Holocene peat Hg anomalies
No full textThis project examines the major explosive volcanic eruptions recorded in peat. Hg data from three peat profiles, the Hongyuan and Dangxiong peat from the Tibetan Plateau and the Pinet peat record from France, were synthesized here to support our conclusions. All the Hg data and the MATLAB script are prepared for the manuscript entitled "Tibetan peat saw Holocene global major explosive volcanic eruptions".Corresponding author: Haijun PENG, [email protected].</p
MATRIX ISOLATION ESR STUDIES OF Hg RADICALS: , , , and
No full textAuthor Institution: Furman University; University of Western AustraliaAtomic mercury vapor was passed through various rare gas plasma discharges and condensed in neon, argon, and krypton rare gas matrices at cryogenic temperatures for electron spin resonance (ESR) investigations. The first ESR spectrum of , observed in Ne, Ar, and Kr matrices is presented, with determination of its magnetic parameters, which exhibited excellent agreement with gas phase values. The ratio of the values for and is shown to vary slightly from to HgH/HgD. This variation can be attributed to electronic structure differences surrounding the mercury nucleus. Observation of in a Kr lattice also provides information on the required energy gap between the electron affinity of an isolated species and the ionization potential of the matrix host atom. HgH and HgD were observed in experiments where the plasma discharge was doped with a small percentage of gas. The HgH/HgD radicals were observed for the firs t time in a neon matrix, providing a comparison of the various magnetic parameters to earlier argon matrix results. The electronic structure and magnetic parameters of will be analyzed based upon these new matrix results for and HgH
Freedom of choice or force of circumstance? : Eastern European sex-workers in the Republic of Cyprus ; paper for the conference 'Alltag der Globalisierung. Perspektiven einer transnationalen Anthropologie', January 16-18, 2003, Institute of Cultural Anthropology and European Ethnology, Johann Wolfgang Goethe University, Frankfurt am Main
No full textThis paper focuses on Eastern European migrants who, since the beginning of the 1990s, are entering the Republic Cyprus as “artistes”. This is a visa permit status as well as an euphemism for short-term work permits in the local sex industry. In addition to exploring the migrational experiences of these women and their living and working conditions in the Republic of Cyprus, the paper reconstructs, empirically and analyt ically, the connection between immigration and the local sex industry. Here, several categories of social actors and institutions in Cyprus are actively involved. The rhetoric of government representatives, entrepreneurs and clients in the sex business on the one hand is contrasted with the discourse of local NGO representatives concerned with immigrants’ rights on the other hand. The paper comes to the conclusion that all of these discursive positions ultimately do not do justice to the complex process of decisionmaking that women undergo who migrate into the sex industry. Either, freedom of choice is emphasized – such as by entrepreneurs and the government – or the domination of women – as in the public statements of the NGO. In order to analyze the ambivalent tension between freedom of choice and submission to force by which the women’s decision is characterized, the author employs Michel Foucault’s concept of governmentality, which describes forms of political regulation that use the individual’s freedom of action as an instrument to exercise power
Enhanced neuronal excitability in the absence of neurodegeneration induces cerebellar ataxia
No full textComparison of system architecture and converter topology for a solar powered electric vehicle charging station
No full textAccepted Author ManuscriptOld - EWI-ESE-DC&S DC systems & StoragePhotovoltaic Materials and Device
The land surface mildly contaminated by Hg
No full textThe Hg content in the waters of Jiaozhou Bay was within 0.010-0.060μg/L in May, September and October 1993, which conforms to the seawater quality standards of Class I and II. It indicates that in the entire waters of Jiaozhou Bay, there were some water areas that did not get contaminated by Hg while some areas got mild contamination by Hg in May, September and October. In May, the variation range of Hg content in the waters of Jiaozhou Bay was 0.010-0.046μg/L. In September, it ranged within 0.007-0.011μg/L. And in October, the content of Hg detected in the waters of Jiaozhou Bay was 0.060μg/L. There were three sources to transport Hg content, open sea currents, Licun river and the surface runoff. The Hg content from the transportation of open sea currents was 0.046μg/L, from the Licun river was 0.011μg/L and from the surface runoff was 0.060μg/L. This paper builds a model block diagram to present the different paths and Hg contents that were input in the Jiaozhou Bay. In May, the open sea currents did not get any contamination of Hg; in August, the Licun river did not get any contamination of Hg as well; while in September, the surface runoff was mildly contaminated by Hg. The author concluded two points about the migration of Hg content: 1) Human activities discharge Hg to the land so that the Hg content could directly run to the ocean through the surface runoff. As the Hg content transported by surface runoff was relatively high, it resulted that the nearshore waters got mild contamination of Hg content. On the other hand, the surface runoff transported Hg to rivers, resulting relatively low content of Hg. Then rivers delivered the Hg content to nearshore waters, leading to a non-contaminated condition of rivers. 2) Human activities input Hg content to the ocean in a long period, resulting the increase of Hg content in the ocean. As the accumulation of Hg content in the ocean, the ocean got close to the mild contamination status
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