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关键词： Nutrition   Bioinformatics   Epidemiology  

摘要： Personalized dietary approaches to cardiovascular disease (CVD) prevention will require sensitive biomarkers of multiple types. First, direct CVD risk biomarkers will enable the prioritization of dietary interventions for specific individuals. Second, biomarkers of diet response will allow more effective dietary recommendations for maintaining optimal cardiovascular risk factor (CRF) levels. Both genomic variants and epigenomic factors such as DNA methylation have the potential to enable improvements in both of these biomarker types. Aim 1 of this work involved an investigation of mechanistic links between DNA methylation and incident CVD events. Differentially-methylated regions (DMRs) and the WGCNA module-based clustering approach were used to derive robust and interpretable epigenomic features before testing associations with CVD in the Women’s Health Initiative and Framingham Heart Study Offspring Cohort. Multiple novel signals were uncovered, including differential methylation near immune function-related genes (SLC1A5, SLC9A1, and TNRC6C) and of development- and immune-related epigenomic modules. Module methylation was also linked to cumulative CRF exposure, providing proof-of-concept for the use of DNA methylation as a molecular recorder of cumulative risk factors over the life course. Aim 2 expanded on these findings in seeking to directly optimize a predictor of CVD risk. Using time-to-CVD-event elastic net regression models, a methylation-based CVD risk score (MRS) was able to add predictive power after adjustment for traditional risk factors. A recently-introduced ensemble method for training predictive models in omic datasets showed comparable performance to a standard analysis pipeline. A preliminary analysis of interactions between the MRS and other CVD risk metrics (based on traditional risk factors or genomics) found that the MRS appears to perform better in lower-risk regions of these alternative metrics, suggesting its value in identifying individu

关键词： kappa opioid receptor   addiction   biased agonist  

摘要： Endogenous opioids mediate both analgesic and affective responses to stress. While the mu opioid receptor (MOR) produces the reinforcing euphoric effects responsible for the high abuse potential of traditional opioid analgesics, the kappa opioid receptor (KOR) is hypothesized to mediate the dysphoric component of stress. With increasing rates of opioid addiction, extensive research efforts are focused on better understanding each of these systems and how they interact, with the goal of developing less addictive pain management strategies and better approaches to addiction treatment. With this in mind, we conducted both clinical and pre-clinical studies aimed at developing better treatment strategies for opioid use and the management of patients with opioid use disorder (OUD). First, we assessed a local cohort of patients with (OUD) for a collection of single nucleotide polymorphisms (SNPs) related to reward processing, with the goal of furthering efforts to develop a gene-based screening mechanism for OUD risk assessment. Second, we conducted preclinical assessments of the G protein-biased KOR agonist nalfurafine as a potential adjuvant medication for increasing the therapeutic efficacy of opioid-based analgesia. Genetic analysis of OUD patients identified two SNPs within the gene encoding the mu opioid receptor, one SNP within the gene encoding the serotonin 2B (5-HT2B) receptor, and one SNP within the gene encoding Regulator of G protein Signaling 2 (RGS2), with the frequency of each SNP varying significantly from that observed in reference populations of European descent. Preclinical investigations with nalfurafine use in mice demonstrated a greater analgesic synergy when co-administered with morphine than morphine co-administered with the unbiased KOR agonist U50,488. As G protein-biased KOR agonists are hypothesized to produce less dysphoria (a therapeutically limiting side effect of KOR agonists), they may present a viable method for reducing the dose of MOR-t

关键词： Educational psychology   Science education  

摘要： As the public interacts more often and more consequentially with genetics in their lives, the goal of increasing students’ understanding of the central dogma of molecular biology becomes more of an imperative in our science classrooms. Many researchers are interested in how to do this, and this dissertation offers insights into the understandings of students taking a popular, high-level course in biology. Specifically, this dissertation explores the relations between the mechanisms of genetic inheritance, student abilities to apply these mechanisms across spatial and temporal scales, and the effects of student demographics on student outcome measures. The results indicate that the molecular mechanism of genetics, the central dogma of molecular biology, is the least accessible for students as measured by item difficulty. Students’ ability to reason across spatial and temporal scales, as measured by their scores on items coded to this science practice, is strongly related to students’ overall knowledge of biology and their understanding of the mechanisms of genetic inheritance at both the Molecule/Cell and Organism/Population scales. Students who are male, are from White or Asian(American)/ Pacific Islander backgrounds, or have guardians who have 4+ year degrees perform better on all items related to genetic inheritance on the AP Biology exam. While these results can support or challenge current theories about the instructional sequencing of genetic topics, they are limited in the types of questions that they can answer as well as their generalizability. Future research can expand this research to show if – and if so, how much – students’ ability to reason across scale impacts their understandings of the mechanisms of genetic inheritance as well as the speed at which they learn. The goal of increasing student understandings of science content and practices in genetics, as well as their interest in pursuing a STEM-based career, is an important one. Regardless of career

关键词： 神经系统   光遗传学   视紫红质通道蛋白2   突触可塑性   囊泡释放行为   钙离子通透性  

摘要： 视紫红质通道蛋白2（Channelrhodposin-2，ChR2）作为光控阳离子通道已被广泛地应用于神经科学研究。然而因其具有钙离子通透性，在光刺激下额外的钙离子会通过ChR2进入细胞，这些额外钙离子是否影响神经递质释放及短时程突触可塑性，至今尚未被系统地研究的科学问题。就此对光遗传学方法在突触传递研究中的适用性开展定量化的研究。以仅具单个输入的Calyx of Held突触为研究对象，通过对比传统的场电位电刺激，突触终末光刺激，轴突光刺激下的突触反应的影响，定量化地研究光遗传的刺激对于突触囊泡释放和突触可塑性的影响。发现，对突触终末直接给予光刺激时低光强度阈下照射（不产生动作电位）会提高突触囊泡自发释放频率;中强度的最小阈上光照射（可稳定诱发单个动作电位）显著改变突触传递及突触短时程可塑性;高强度照射（可持续诱发动作电位）引起更为显著的突触可塑性变化，主要表现为非生理性的囊泡释放的概率以及囊泡动员(priming)的增强。　　进一步的研究显示以上影响是由额外的钙离子内流引起的，额外钙离子内流主要源于光刺激诱发动作电位的形变所引起的电压门控钙离子通道电流的改变，以及ChR2的钙离子通透性所引入的钙离子。还发现，轴突光刺激诱发的突触传递以及短时程突触可塑性与电刺激无异。　　综上所述，在基于光遗传学的研究中，对突触终末的直接光刺激引起的钙离子内流，显著改变了囊泡动员和囊泡释放概率，直接改变突触传递可塑性，将影响以突触传递为基础的神经信息处理。而以轴突光刺激代替突触终末刺激，可消除这一非生理效应。本研究通过实验观测和理论分析对光遗传学方法应用的局限性和可靠性进行了定量研究，进而为光遗传学在突触传递和神经环路研究中的准确应用提供了可行的方法。

关键词： 骨髓增生异常综合征   进展   急性髓系白血病   表观遗传学   DNA甲基化  

摘要： 目的骨髓增生异常综合征(MDS)一种高度异质性的髓系肿瘤,约有30%的MDS患者可能进展为急性髓系白血病(AML,亦称sAML),一旦转变预后极差。截至目前为止,MDS疾病转变的机制尚未完全明确,遗传学和表观遗传学改变在其中扮演重要作用。本研究旨在探究MDS转AML中全基因组DNA甲基化改变,同时筛选和验证驱动疾病进展相关的表观分子,并分析其临床意义。方法应用全基因组简化甲基化测序(RRBS)分析4例对照和4例配对MDS/sAML患者不同阶段甲基化态势。同时应用生物信息等方法筛选具有预后意义和潜在生物学功能的相关基因。应用实时定量甲基化特异性PCR(RQ-MSP)、亚硫酸氢盐测序PCR(BSP)及MethylTarget靶向测序等方法进一步扩大标本验证和确定候选基因甲基化改变。实时定量PCR(RQ-PCR)及Western Blot(WB)等方法检测候选基因的表达水平。基于临床标本参数等进行临床意义和预后分析。同时通过细胞凋亡、细胞周期、细胞增殖等实验评价候选基因功能。结果在4例配对MDS/sAML患者中,随着疾病进展处于sAML阶段的全基因组甲基化水平显著高于MDS阶段。MDS阶段与对照相比,一共筛选出1090个差异性甲基化片段(DMFs),包括441个高甲基化片段及649个低甲基化片段。sAML阶段与MDS阶段相比,一共筛选出103个DMFs,其中包括96个高甲基化片段及7个低甲基化片段。针对4例配对MDS/sAML患者单独比较,我们分别筛选出5803个、3735个、4043个及2748个差异甲基化基因(DMGs)。结合初步筛选和生物信息分析发现目标分子ID4和DLX5不仅和AML预后密切相关,并且可能有参与肿瘤生成作用。此外,通过RQ-MSP方法进一步扩大标本确定MDS转AML中ID4和DLX5的高甲基化改变现象。MDS和AML患者中ID4甲基化水平显著高于对照组(P<0.001),此外,以IPSS危险程度分组比较,Int-2/High危险组患者ID4甲基化水平显著高于Low/Int-1危险组患者(P=0.004),sAML患者ID4甲基化水平也显著高于原发AML(pAML)患者(P=0.003)。ID4高甲基化组MDS患者的总体生存(OS)短于ID4低甲基化组MDS患者,差异具有统计学意义(P=0.038)。通过Cox回归多因素分析发现ID4高甲基化并不是MDS患者预后不良的独立影响因素(HR=1.643,P=0.433)。ID4高甲基化组AML患者的完全缓解率(CR)与ID4低甲基化组AML患者无差异(P=0.629)。在非M3患者中,ID4高甲基化组AML患者的CR率略低于ID4低甲基化组AML患者(P=0.202)。然而在正常核型AML(CN-AML)中,ID4高甲基化组AML患者的CR率显著低于ID4低甲基化组AML患者(P=0.030)。18例AML患者经过诱导化疗达到CR后ID4甲基化水平有不同程度下降(P<0.001)。通过Kaplan-Meier生存分析发现:ID4高甲基化组AML患者的OS和无白血病生存(LFS)短于ID4低甲基化组AML患者(P=0.002及P=0.001)。在CN-AML中,ID4高甲基化组AML患者的OS明显短于ID4低甲基化组AML患者(P=0.001);然而,两组之间的LFS差异并无统计学意义(P=0.326)。Cox回归多因素分析发现:ID4高甲基化是CN-AML患者预后不良的独立影响因素(HR=2.483,P=0.004),而在非M3患者中,趋近统计学意义(HR=1.507,P=0.081)。AML患者中ID4的表达水平较对照显著下调(P=0.003),且ID4甲基化和ID4表达呈负相关(R=-0.275,P=0.001)。其次,我们选取ID4高甲基化、低表达的白血病细胞株K562通过DAC进行去甲基化处理,随着DAC浓度提高,ID4甲基化水平逐步下降,同时ID4的mRNA和蛋白水平呈现上调。功能实验发现转染ID4后K562和HL60细胞增殖能力下降,细胞周期阻滞在G0/G1期,细胞凋亡增加。前期通过生物信息分析发现ID4不仅受到DNA甲基化调控,还可能受到miR-335调控,AML患者中miR-335水平较对照组显著上调(P=0.009),且和ID4表达呈负相关(R=-0.146,P=0.043)。与大多数报道不同,我们发现AML中miR-335甲基化与对照并无差异。MiR-335和ID4均异常表达组AML患者的CR率最低,miR-335或者ID4任何一个异常表达组次之,miR-335和ID4表达正常组最高,三组之间具有显著统计学差异(34%、40%和63%,P=0.024)。在非M3患者中,三组的CR率分别是25%、31%和55%,三组之间有显著统计学差异(P=0.0

关键词： 细胞生物学   遗传学  

ISBN: (纸本)9787561095539

摘要： 本书内容分细胞生物学和遗传学两篇。细胞生物学部分包括细胞生物学概述, 细胞的基本概念和分子基础, 细胞的增殖, 细胞的分化、衰老与死亡等 ; 遗传学部分包括遗传学概述、人类染色体与染色体病、遗传的分子基础、遗传变异与生物进化等。

摘要： Introduction: Osteoarthritis (OA) is the most prevalent musculoskeletal disease and a leading cause of disability worldwide. It affects 40% of individuals over the age of 70. The health economic burden of OA is rising, commensurate with longevity and obesity rates, and there is currently no curative therapy. The genetic component of OA is ~50% and previous genetic studies have identified associated polymorphisms, traversing hip, knee and hand OA with limited overlap. The heritability explained by these loci is relatively low. Aims: The aim of the work conducted in this thesis was to unravel the genetic component of OA by conducting a series of GWAS followed by additional analyses and by examining established OA loci in so far non-studied populations. Methods: Initially a knee OA GWAS in a Greek population is described. Further, GWAS were performed using genotype data across 16.5 million variants from UK Biobank, followed by gene-based and gene-set analyses. OA was defined based on both self-reported status and through linkage to Hospital Episode Statistics data, and on joint-specificity of disease (knee and/or hip OA). In silico replication and meta-analysis of promising signals was carried out in further cases and controls. Finally, a hip and/or knee OA GWAS was conducted in an independent Greek ethnic group. Results: Confirmatory evidence for previously reported OA risk loci was found in the Greek population datasets, while three loci showed suggestive evidence for association. The UK Biobank analyses identified nine novel OA loci, located within TGFA, ANXA3, PLEC, MAP2K6, JPH3, ZNF345, near SLC30A10, between MPB3B and EQTN and near LTN1 respectively. Most of these signals are common at frequency and reside in or near genes reported to be associated with skeletal and OA relevant phenotypes by functional and animal model studies. Gene and gene based analyses identified genes and biological processes associated with OA. Conclusions: My findings contribute to a bette

关键词： 骨髓增殖性肿瘤   基因突变   T淋巴细胞亚群   细胞因子   纤维化   芦可替尼  

摘要： 背景和目的:基因突变、细胞因子、成纤维细胞均参与了MPNs的演进。骨髓纤维化(MF)是MPNs各亚型的共同疾病特征,与骨髓间充质细胞(BMMSC)、肿瘤成纤维细胞(CAF)和细胞因子有关。但目前有关影响骨髓纤维化的机制尚未阐明。本研究通过结合基因突变、T淋巴细胞亚群和细胞因子探讨MPNs的异质性,并采用芦可替尼(RUX)处理MPNs细胞,分析CAF、LOXL-2与MF演进的可能关系,为MPNs患者的治疗提供有力的理论依据。方法:1.采用高通量测序检测125例MPNs患者中127个相关基因的突变情况。2.采用流式细胞仪检测123例MPNs患者中T淋巴细胞亚群的表达水平,采用高通量蛋白芯片检测与MPNs相关的33个细胞因子的表达情况,并采用ELISA进一步验证与MF发生相关的细胞因子的含量。3.采用qPCR检测正常人和MPNs在RUX治疗前后血清中LOXL-2 mRNA的表达水平,采用Western-blot检测正常人和MPNs在RUX治疗前后骨髓中有核细胞CAF标志物(α-SMA、FAP)和血清中LOXL-2蛋白的表达水平,并分析BMMSC中a-SMA和FAP的表达水平。4.分别观察正常人和MPNs患者在RUX治疗前后BMMSC培养后细胞的形态,并评价细胞免疫表型及成骨、成脂能力的变化,检测各组细胞中a-SMA和FAP mRNA和蛋白的表达水平。结果:***2、CALR、TET2、ASXL1 和DNMT3A是MPNs患者最常见的5个基因突变;按基因功能分类,MPNs易检测到信号通路和表观调节基因突变;附加突变的异质性明显。***患者的CD3+和CD4+T细胞在ET、PV和PMF中均减少(P<0.05),PMF较ET和PV更低(P<0.05);JAK2突变者的CD3+和CD4+T细胞、JAK2和CALR突变者的CD8+CD28-细胞均低于对照组(P<0.05);有和无附加突变MPNs的CD3+、CD4+和CD8+CD28-细胞均较对照组减少,但后者更低(P<0.05)。***患者的IL-8、VEGF等12种细胞因子水平均较正常组高(P<0.05),IL-15较正常组低(P<0.05);MF明显组的IL-8和VEGF较不明显组更高(P<0.05),ELISA复核IL-8和VEGF与蛋白芯片结果一致。***患者骨髓中a-SMA、FAP及LOXL-2的mRNA和蛋白的表达水平均显著增加(P<0.05),RUX治疗后a-SMA、FAP及LOXL-2的mRNA和蛋白的表达水平均显著降低(P<0.05)。***治疗前后MPNs的BMMSC免疫表型和a-SMA、FAP mRNA水平较正常人无明显差异(P>0.05);与正常人相比,MPNs的BMMSC中a-SMA和FAP的蛋白水平均显著增加(P<0.05),经RUX治疗后,a-SMA和FAP的蛋白水平均下降(P<0.05)。结论:***驱动基因突变类型有限,而附加突变按功能分类的异质性较明显,在判断预后和疾病演进中具有重要价值,表观调节基因突变是MPNs各亚型中最常见的附加突变类型。2.本研究发现的少见基因突变与疾病的可能关系包括:SH2B3和GNAS与发病;CUX1和IKZF1与进展为白血病;CEBPA、STAG2、NF1和PTEN与疾病发展:KMT2C、PTPN11、ZRSR2和IL7R的作用未明,其中ZRSR2和IL7R在MPNs患者中鲜有报道,可为进一步研究提供线索。3.动态监测MPNs患者的淋巴细胞亚群、IL-8和VEGF的变化,在了解疾病进展、MF的程度和判定预后方面具有临床应用价值。4.在乏氧环境下RUX可能通过降低LOXL-2的表达,减少刺激MSC转化为CAF,改善MPNs患者的MF进程。

关键词： 叶绿体基因组   单核苷酸多态性   网状进化   群体遗传多样性   生物地理学  

摘要： 独蒜兰属Pleione ***主要分布于东亚地区,是亚热带与温带的分界指示植物;全世界约31种(含7个天然杂交种),具有极高的观赏和药用价值,该属分布区为全球植物多样性热点区域的东亚植物区系,备受研究者的关注;然而该属类群间的界定和系统关系一直存在争论,给该属的深入研究、保护及利用造成了困扰。本研究利用高通量测序技术,对独蒜兰属22个种进行低盖度测序,通过生物信息手段提取叶绿体基因组和单核苷酸多态性(SNP)数据,构建系统发育和网状进化关系,并结合分子钟和地理分布信息,探讨该属的起源及演化历史。此外,采用形态学分析、核基因与叶绿体基因以及简化基因组RAD-seq的SNP位点,对网状进化关系明显的台湾独蒜兰及其近缘类群开展花形态演化、群体遗传学研究,探讨其物种多样化形成机制。主要研究结果如下: (1)独蒜兰属叶绿体基因组特征与系统发育关系 22个种的叶绿体全基因组无基因丢失、重排和倒位现象,其中6个编码区片段和12个非编码区片段,可开发作为该属系统发育和物种鉴定的有效分子标记。通过转录组和二代测序数据共筛选得到223,820个单拷贝基因SNP位点,利用该位点和叶绿体全基因组分别构建的系统发育树均支持全属分为5个主要进化支;属内存在网状进化现象,clade V为全属网状进化最明显类群,二叶独蒜兰***和岩生独蒜兰***为早期杂交起源种,小叶独蒜兰***为台湾独蒜兰***与陈氏独蒜兰***杂交渐渗种,***为台湾独蒜兰与美丽独蒜兰***杂交渐渗种。 (2)台湾独蒜兰及其近缘类群的鉴定与花形态演化 基于核基因片段nr ITS和叶绿体基因片段cp DNA(mat K+trn T-trn L+trn L+trn L-trn F)构建台湾独蒜兰及其近缘类群的系统发育关系,结果显示台湾、福建、浙江、安徽南部和江西东部为台湾独蒜兰的主要分布区,南岭东南段至江西井冈山为台湾独蒜兰与陈氏独蒜兰杂交渐渗区,南岭西北段至贵州雷山为美丽独蒜兰与陈氏独蒜兰杂交渐渗区,江西安福为台湾独蒜兰与美丽独蒜兰杂交渐渗区。基于系统发育分析结果对7个台湾独蒜兰居群、2个台湾独蒜兰与陈氏独蒜兰的杂交种居群、1个***居群,共计170个植株的花部形态进行分析,结果支持杂交促进了该类群的花部形态变异,台湾独蒜兰与陈氏独蒜兰的杂交种与台湾独蒜兰形态呈分离现象,***的花显著小于台湾独蒜兰,且花色多态性高于其它所有群体。 (3)台湾独蒜兰及其近缘类群的种群分化与遗传多样性 利用RAD-seq获得的693,684个SNP位点对台湾独蒜兰及其近缘类群开展群体遗传分析,结果显示该类群分为台湾独蒜兰复合体和独蒜兰复合体两大类群,前者包括台湾独蒜兰、台湾独蒜兰与陈氏独蒜兰的杂交渐渗群、小叶独蒜兰和***;后者包括金华独蒜兰***、美丽独蒜兰、美丽独蒜兰与陈氏独蒜兰的杂交渐渗群、独蒜兰***。结构分析显示部分群体存在明显的杂交渐渗,尤其以杂交类群最为明显。遗传多样性分析显示台湾独蒜兰复合体的***遗传多样性水平较高,而台湾独蒜兰及其杂交渐渗群在快速扩张前存在明显瓶颈效应,遗传多样性水平较低;独蒜兰复合体各类群未发现明显瓶颈效应,各类群的遗传多样性水平较高。 (4)独蒜兰属生物地理与台湾独蒜兰及其近缘种谱系地理历史 分子钟估算显示独蒜兰属出现在约18.45 Ma,第一次快速分化期在晚中新世的8.87至7.83 Ma,而后在约4.05 Ma开始第二次快速分化,并持续至整个更新世。两次快速分化期均与青藏高原的抬升和亚洲季风气候的增加有关,进入更新世后种间分化主要与第四纪气候波动有关。祖先分布区重建显示该属最大可能起源于横断山脉—中国西南地区,并往中南半岛、喜马拉雅和中国中东部山区扩散。台湾独蒜兰复合体和独蒜兰复合体在约2.39 Ma的中国东部地区发生分化,该分化可能与青藏高原的最后一次大范围隆起有关,第四纪的气候波动影响了该类群现代分布区的形成,第二、三阶梯之间的平原和南岭山脉是目前两大复合体的主要分界线。此外,冰期时部分群体原地避难导致独蒜兰复合体呈现谱系间断分布的现象。