53 resultados para viral gene delivery system


Relevância:

100.00% 100.00%

Publicador:

Resumo:

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase1. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Chitosan has shown its potential as a non-viral gene carrier and an adsorption enhancer for subsequent drug delivery to cells. These results showed that chitosan acted as a membrane perturbant. However, there is currently a lack of direct experimental evidence of this membrane perturbance effect, especially for chitosans with low molecular weight (LMW). In this report, the interaction between a lipid (didodecyl dimethylammonium bromide; DDAB) bilayer and chitosan with molecular weight (MW) of 4200 Da was studied with cyclic voltammetry (CV), electrochemical impedance spectroscopy and surface plasmon resonance (SPR). A lipid bilayer was formed by-fusion of oppositely charged lipid vesicles on a mercaptopropionic acid (MPA)-modified gold surface to mimic a cell membrane. The results showed that the LMW chitosan could disrupt the lipid bilayer, and the effect seemed,to be in a concentration-dependent manner.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Injection and combustion of vaporized kerosene was experimentally investigated in a Mach 2.5 model combustor at various fuel temperatures and injection pressures. A unique kerosene heating and delivery system, which can prepare heated kerosene up to 820 K at a pressure of 5.5 MPa with negligible fuel coking, was developed. A three-species surrogate was employed to simulate the thermophysical properties of kerosene. The calculated thermophysical properties of surrogate provided insight into the fuel flow control in experiments. Kerosene jet structures at various preheat temperatures injecting into both quiescent environment and a Mach 2.5 crossflow were characterized. It was shown that the use ofvaporized kerosene injection holds the potential of enhancing fuel-air mixing and promoting overall burning. Supersonic combustion tests further confirmed the preceding conjecture by comparing the combustor performances of supercritical kerosene with those of liquid kerosene and effervescent atomization with hydrogen barbotage. Under the similar flow conditions and overall kerosene equivalence ratios, experimental results illustrated that the combustion efficiency of supercritical kerosene increased approximately 10-15% over that of liquid kerosene, which was comparable to that of effervescent atomization.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

A series of experiments were conducted to characterize the self-ignition and combustion of thermally cracked kerosene in both a Mach 2.5 model combustor with a combustor entrance height of 51 mm and a Mach 3.0 model combustor with an entrance height of 70 mm. A unique kerosene heating and delivery system was developed, which can prepare heated kerosene up to 950 K at a pressure of 5.5 MPa with negligible fuel coking. The extent of China no. 3 kerosene conversion under supercritical conditions was measured using a specially designed system. The compositions of gaseous products as a result of thermal cracking were analyzed using gas chromatography. The mass flow rates of cracked kerosene were also calibrated and measured using sonic nozzles. With the injection of thermally cracked kerosene, the ability to achieve enhanced combustion performance was demonstrated under a variety of airflow and fuel conditions. Furthermore, self-ignition tests of cracked kerosene in a Mach 2.5 model combustor over a range of fuel injection conditions and with the help of different amounts of pilot hydrogen were conducted and discussed.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Injection and combustion of vaporized kerosene was experimentally investigated in a Mach 2.5 model combustor at various fuel temperatures and injection pressures. A unique kerosene heating and delivery system, which can prepare heated kerosene up to 820 K at a pressure of 5.5 MPa with negligible fuel coking, was developed. A three-species surrogate was employed to simulate the thermophysical properties of kerosene. The calculated thermophysical properties of surrogate provided insight into the fuel flow control in experiments. Kerosene jet structures at various preheat temperatures injecting into both quiescent environment and a Mach 2.5 crossflow were characterized. It was shown that the use ofvaporized kerosene injection holds the potential of enhancing fuel-air mixing and promoting overall burning. Supersonic combustion tests further confirmed the preceding conjecture by comparing the combustor performances of supercritical kerosene with those of liquid kerosene and effervescent atomization with hydrogen barbotage. Under the similar flow conditions and overall kerosene equivalence ratios, experimental results illustrated that the combustion efficiency of supercritical kerosene increased approximately 10-15% over that of liquid kerosene, which was comparable to that of effervescent atomization.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

5-氟尿嘧啶(5-Fluorouracil, 5-FU)是一种抗代谢药物,广泛用于临床治疗结直肠癌、胃癌、乳腺癌等多种癌症,但其首过代谢显著、亲脂性较低,选择性差、毒副作用大。为克服这些缺点人们对5-FU进行了大量的修饰工作,包括小分子修饰以及与各种载体形成微球、微囊、纳米粒、共价前药等。 环糊精(Cyclodextrin,简称CD),可被结肠中的糖苷酶特异性地降解成小分子糖,而胃和小肠中由于缺乏相应的酶而使环糊精不被降解,这一特性在结肠药物的靶向输送及释放中有重要应用价值。环糊精中含有丰富的羟基,易进行化学修饰,将药物与环糊精通过共价键结合制成前药,使其在胃和小肠中不降解,而在盲结肠中被特异性的酶降解释出药物,达到结肠靶向释药的目的。研究表明,环糊精作为一种前药载体为结肠靶向释药和缓释、控释系统提供了一种有效的手段。 本工作选择5-氟尿嘧啶为模型药物、β-环糊精作为载体,通过中间体5-FU羧酸衍生物的制备及其与β-环糊精的偶联,合成了系列5-FU-β-CD前体药物,并利用紫外、红外、质谱、核磁、元素分析、热分析等手段对其进行结构表征。同时,还研究了前体药物的体外释药性质。具体内容包括: 1. 含有羧基的5-FU衍生物中间体的合成:(5-氟尿嘧啶-1-基)-乙酸(FUAC)、3-(5-氟尿嘧啶-1-基)-丙酸(FUPC)、5-(5-氟尿嘧啶-1-基)-戊酸(FUVC)的合成。 2. 中间体5-FU的羧酸衍生物与β-CD的偶联:分别通过以6-OTs-β-CD为中间体的取代法和活化酯法,合成了第一面取代和第二面取代的5-FU-β-CD大分子前体药物。在二面取代的前体药物制备中,通过改变原料的比例,合成了系列不同取代度(DS)的2-[(5-氟尿嘧啶-1-基)-乙酰基] -β-环糊精结合物。 3. 对上述前体药物进行体外释放研究:分别考察了前体药物在不同pH缓冲溶液中的水解行为及其在小鼠胃肠道人工体液中的酶解行为,并通过UV-Vis及HPLC对前体药物释放情况进行检测分析。 5-Fluorouracil(5-Fu), commonly known as a broad-spectrum antineoplastic drug, has been widely used in the treatment of various kinds of cancer including colon cancer for 40 years. However, this antitumor agent exhibits serious adverse effects, such as their marrow toxicity, gastrointestinal reaction and low selectivity in their clinical use. In order to improve its antitumor activity and reduce its toxicity, the compound was modified in various ways, including the formation of conjugated prodrugs with kinds of carrier, microsphere and nanoparticles etc. Cyclodextrins(CDs) are known to be barely capable of being hydrolyzed and only slightly absorbed in passing through the stomach and small intestine; however they are fermented into small saccharides by colonic microflora and thus absorbed as small saccharides in the large intestine. This biodegradation property of CDs may be useful as a colon-targeting carrier, and thus CD prodrugs may serve as a source of site-specific delivery of drugs to colon. It was demonstrated that prodrugs of CDs can provide a versatile means for construction of not only colon targeted delivery systems, but also delayed release systems. 5-Fluorouracil was taken as a model drug and β-CD as the carrier in this study. Series prodrugs of 5-FU was prepared through the preparation of reactive 5-FU derivatives containing carboxyl group and coupling to hydroxyl groups of CD. The structures of the conjugates were charactered by using IR, UV–vis, ESI-MS, 1H, 13C-NMR spectra, elemental analyses, and thermal analysis. In vitro hydrolysis behavior in aqueous solution and in rat gastrointestinal tract contents of the conjugates were also investigated. The main content of this dissertation includes following aspects: 1. The preparation of 5-FU derivatives containing carboxyl group: 5-Fluorouracil- acetic acid(FUAC)、3-(5-FU-1)-propionic acid (FUPC)、and 5-(5-FU-1)-valeric acid(FUVC). 2. The coupling of 5-FU derivatives to β-CD: 5-FU was selectively conjugated onto the primary or secondary hydroxyl groups of β-CD through an ester linkage, by the substitution of 6-OTs-β-CD and the activated ester method respectively. For the secondary face conjugation, the degree of substitution(DS) can be controlled by changing the mole ratio of the starting materials(FUAC and β-CD). 3. In vitro release behavior of the conjugates in aqueous solution and in rat gastro- intestinal tract contents of the conjugates were investigated, and the reaction was monitored and analyzed by using UV-Vis and HPLC methods.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

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.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Basic research related to heavy-ion cancer therapy has been done at the Institute of Modern Physics (IMP), Chinese Academy of Sciences since 1995. Now a plan of clinical trial with heavy ions has been launched at IMP. First, superficially placed tumor treatment with heavy ions is expected in the therapy terminal at the Heavy Ion Research Facility in Lanzhou (HIRFL), where carbon ion beams with energy up to 100 MeV/u can be supplied. The shallow-seated tumor therapy terminal at HIRFL is equipped with a passive beam delivery system including two orthogonal dipole magnets, which continuously scan pencil beams laterally and generate a broad and uniform irradiation field, a motor-driven energy degrader and a multi-leaf collimator. Two different types of range modulator, ripple filter and ridge filter with which Guassian-shaped physical dose and uniform biological effective dose Bragg peaks can be shaped for therapeutic ion beams respectively, have been designed and manufactured. Therefore, two-dimensional and three-dimensional conformal irradiations to tumors can be performed with the passive beam delivery system at the earlier therapy terminal. Both the conformal irradiation methods have been verified experimentally and carbon-ion conformal irradiations to patients with superficially placed tumors have been carried out at HIRFL since November 2006.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

A new gas delivery system is designed and installed for HIRFL-CSR cluster target. The original blocked nozzle is replaced by a new one with the throat diameter of 0.12mm. New test of hydrogen and argon gases are performed. The stable jets can be obtained for these two operation gases. The attenuation of the jet caused by the collision with residual gas is studied. The maximum achievable H-2 target density is 1.75x10(13) atoms/cm(3) with a target thickness of 6.3x10(12) atoms/cm(2) for HIRFL-CSR cluster target. The running stability of the cluster source is tested both for hydrogen and argon. The operation parameters for obtaining hydrogen jet are optimized. The results of long time running for H-2 and Ar cluster jets look promising. The jet intensity has no essential change during the test for H-2 and Ar.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

在辐射治疗应用方面,相比传统体外辐射疗法,高能量的重离子束流有着巨大的优势。近年来,世界上多数重离子治疗中心都对重离子的辐射特性已经进行了深入研究,从2006年起中国科学院近代物理所也开始了重离子辐射治疗肿瘤的临床实验。目前绝大多数重离子治癌中心都采用了包括一对独立的二极铁的束流配送系统,将从加速器引出的笔形束流在肿瘤的各层等深横截面上进行均匀照射。本文重点阐述了HIRFL-CSR重离子治癌装置中的束流配送系统的工作原理和分系统结构,包括深层治癌重离子束运线,终端扫描系统和根据治疗计划生成的扫描路径软件系统。第一部分简单介绍了世界上各大重离子医疗辐射工程,总结了医疗重离子加速器的设计经验,尤其对日本的HIMAC和德国GSI重离子治癌装置进行了详细介绍,同时对新型重离子治癌装置的特点和重离子治癌装置的发展方向进行了介绍。侧重分析研究了束流引出系统、控制系统和扫描系统的工作原理和相关在线设备,详细比较了两种扫描方式的优缺点。第二部分重点介绍了HIRFL-CSR加速器及其重离子辐射应用工程。CSR是中国第一台重离子冷却存储环,其主加速器CSRm是在兰州重离子治癌装置的核心,负责提供对应不同穿透深度不同能量的慢引出束流。兰州近代物理所的治癌临床实验分为三个阶段,其中第一阶段利用HIRFL辐照终端引出的重离子束流对浅层肿瘤进行适形照射。第二阶段利用CSRm引出的重离子束流开展对深层肿瘤的辐照实验,包括动物实验和临床实验。第三阶段在技术成熟后将小型医用重离子加速器向社会推广。第三部分中总结回顾了深层治癌重离子束运线的设计原理和和束运线的磁聚焦结构。对扫描系统(栅扫描和点扫描)进行了计算机模拟和束斑尺寸的控制方式进行了讨论。在重离子深层治癌进行第一次动物实验时,利用位于终端的分条电离室测试了治癌重离子束流的基本参量,得到了引出束流在垂直和水平方向以及束流微结构的品质信息,并用梯度法测量了束流的发射度。这些工作对于模拟不同引出束流情况对应的不同扫描方式时束流照射均匀度很有帮助,也给制定肿瘤的治疗计划提供了一些参考。最后论文还简单介绍了束流的共振引出系统,侧重说明引出束流的特性,提及重离子垂直治疗终端桶型旋转机架的设计

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Bioactive, luminescent and mesoporous europium-doped hydroxyapatite (Eu:HAp) was successfully prepared through a simple one-step route using cationic surfactant as template. The obtained multifunctional hydroxyapatite was performed as a drug delivery carrier to investigate the drug storage/release properties using ibuprofen (IBU) as a model drug

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Spherical MCM-41 particles with a diameter of about 150 nm have been successfully coated with CaWO4:Ln (Ln = Eu3+, Dy3+, Sm3+, Er3+) phosphor layers through a simple Pechini sol-gel process. The obtained CaWO4:Ln@MCM-41 composites, which kept the mesoporous structure of MCM-41 and the luminescent properties of phosphors, were investigated as a drug delivery system using aspirin (ASPL) as a model drug.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

In this work, we illustrate a simple chelation-based strategy to trigger DNA release from DNA-incorporated multilayer films, which were fabricated through the layer-by-layer (LbL) assembly of DNA and inorganic zirconium (IV) ion (Zr4+). After being incubated in several kinds of chelator solutions, the DNA multilayer films disassembled and released the incorporated DNA. This was most probably due to the cleavage of coordination/electrostatic interactions between Zr4+ and phosphate groups of DNA. Surface plasmon resonance (SPR), UV-vis spectrometry and atomic force microscopy (AFM) were used to characterize the assembly and the disassembly of the films.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

A layer-by-layer film composed of DNA and inorganic zirconium ion (Zr4+) was fabricated on the surface of gold thin film, and an electric field triggered disintegration of the multilayer film was studied by using electrochemical surface plasmon resonance (EC-SPR). EC-SPR results demonstrated that the film was disassembled upon the application of an electric field and the disassembly rate varied with the applied potential, leading to the controlled release of DNA. The electrodissolution could be switched off by removing the electric potential and reactivated by reapplying the potential.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Here, we demonstrated dimethyldioctadecylammonium bromide (DODAB), a cationic lipid, bilayer coated Au nanoparticles (AuNPs) could efficiently deliver two types of plasmid DNA into human embryonic kidney cells (HEK 293) in the presence of serum. The transfection efficiency of AuNPs was about five times higher than that of DODAB. The interaction of AuNPs with DNA was characterized with dye intercalation assay and agarose gel electrophoresis. The morphology of the complex of AuNPs with DNA was observed with scanning electron microscope (SEM). The intracellular trafficking of the complex was monitored with transmission electron microscope (TEM).