CORE B- HEART BIOLOGY CORE

核心 B- 心脏生物学核心

基本信息

批准号：
7526857
负责人：
ENRICO STEFANI
金额：
$ 36.73万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7526857
关键词：
Accounting Acetone Achievement Acids Acromion Acute Adenovirus Vector Adenoviruses Adult Aftercare Aging Agitation Air Alamethicin Alexa594 Algorithms Alleles Ambulatory Monitoring Amplifiers Anesthesia procedures Animal Model Animals Anoxia Anterior Antibiotics Antibodies Antibody Specificity Antigen-Antibody Complex Antigens Antithymoglobulin Aorta Apex of the Heart Area Arteries Aspartic Acid Attention Back Be++ element Beryllium Binding Biological Biological Assay Biological Models Biology Blood Flow Velocity Blood Pressure Blood flow Blur Body Temperature Body Weight Bovine Serum Albumin Breeding Buffers Calcium Calcium Signaling Calcium-Activated Potassium Channel Caliber Calibration Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone Cardiac Cardiac Myocytes Cardiac Output Cardiomyopathies Cardiovascular system Carotid Arteries Caspase Catheterization Catheters Cations Caveolins Cell Line Cell Nucleus Cell Separation Cell membrane Cells Centrifugation Cerebrum Chest Chimera organism Chimeric Proteins Chronic Circadian Rhythms Cloning Collaborations Collection Color Commercial Sources Complex Computer Systems Computer software Computers Condition Confocal Microscopy Constriction procedure Consumption Contracts Contracture Coronary artery Count Coupled Coupling Creatine Cultured Cells Cyclosporine Cyclosporins Cytochromes Cytolysis Cytoplasm DNA DOCA Data Data Analyses Data Quality Detection Development Devices Diastole Diastolic blood pressure Diazoxide Diet Diffuse Digestion Digitonin Dimensions Dimethylarsinate Discrimination Dissection Distal Doppler Echocardiography Dose Durcupan Durcupan ACM Dyes EFRAC Echocardiography Egtazic Acid Electrocardiogram Electrodes Electron Microscopy Electron Transport Elements Embryo Ensure Equipment Ethanol Evaluation Exercise Exercise Tolerance Exercise stress test Experimental Models Fee-for-Service Plans Fiber Fiber Optics Figs - dietary Flax Floor Fluorescein-5-isothiocyanate Fluorescence Force of Gravity Functional disorder Gases Gel Gene Activation Gene Silencing Gene Transfer Generations Generic Drugs Genes Genetic Genetic Recombination Genetic Screening Genomics Genotype Genus Capra Glass Glucose Glucosephosphate Dehydrogenase Glutamates Glutaral Goals Goat Graph Greater sac of peritoneum Green Fluorescent Proteins HEPES Halothane Hand Heart Heart Atrium Heart Mitochondria Heart Rate Height Horseradish Peroxidase Hour House mice Housing Hypertrophy Ice Image Image Analysis Imagery Immunoblotting Immunoglobulin G Immunoglobulins Implant In Situ Incubated Individual Infarction Inferior vena cava structure Infusion procedures Injection of therapeutic agent Injury Intercept International Intervention Intraventricular Invasive Ischemia Ischemic Preconditioning Isoflurane Italy Knock-in Mouse Knock-out Label Laboratories Lateral Left Left Ventricular Ejection Fraction Left Ventricular Hypertrophy Left Ventricular Mass Letters Light Literature Localized MAP3K7IP1 gene MAPK14 gene Macromolecular Complexes Maintenance Malates Manuscripts Masks Measurement Measures Mechanics Mediating Membrane Membrane Potentials Membrane Proteins Metabolic Methodology Methods Microinjections Microscope Microscopy Minor Mitochondria Mitochondrial Proteins Mitomycin Mitral Valve Modeling Molds Molecular Molecular Analysis Molecular Biology Molecular Genetics Molecular Medicine Molecular Probes Monitor Morphology Motion Mus Muscle Cells Myocardial Myocardial Infarction Myocardium Myosin Heavy Chains N-Type Calcium Channels NADP NRG1 gene Needles Neonatal Nephrectomy Nerve Neuregulins New York Nitrogen Noise None or Not Applicable Nuclear Numbers Oceans Operative Surgical Procedures Optics Organ Organelles Oryctolagus cuniculus Osmium Tetroxide Oxidative Phosphorylation Oxygen Oxygen Consumption Pathology Patient currently pregnant Patients Pattern Peptides Perfusion Permeability Pharmaceutical Preparations Phosphorylation Physical activity Physical condensation Physiologic intraventricular pressure Physiologic pulse Physiological Physiology Plant Roots Poaceae Polymerase Chain Reaction Positioning Attribute Pregnancy Preparation Pressoreceptors Principal Investigator Probability Procedures Process Production Property Protein Isoforms Protein Kinase C Proteins Protocols documentation Publications Publishing Pulse taking Pump Purpose Pyruvate Pyruvates Radio Range Rate Rattus Reagent Recombinants Recovery Reflex action Regulation Relative (related person)Reperfusion Injury Reporting Research Research Infrastructure Research Personnel Resistance Resolution Resources Respiration Rest Rhodamine Rhodamines Robin bird Rodent Role Running Sample Size Sampling Sarcolemma Sarcoplasmic Reticulum Science Score Screening procedure Sectioning technique Secure Series Serum Services Shapes Shortening Fraction Signal Transduction Silk Simulate Site Slide Sodium Sodium Chloride Solutions Source Specific qualifier value Speed Staining method Stains Standardization Standards of Weights and Measures Statistical Methods Statistically Significant Stem cells Sterility Stream Stress Tests Structure Sucrose Sum Supervision Surface Surgical incisions Surgical sutures Suspension substance Suspensions Swelling Switch Genes System Systole Systolic Pressure Tail Tamoxifen Taurine Techniques Telemetry Temperature Testing Tetracycline Tetracyclines Thick Thoracotomy Three-Dimensional Image Tidal Volume Time Tissues Transducers Transfection Transgenic Animals Transgenic Organisms Triton X100 Tube Tyrode&apos s solution Ultrasonography Value Meaning Venous Pressure level Ventilator Ventricular Ventricular Cardiac alpha-Myosin Ventricular Function Ventricular septum Vial device Walking Water Week Weight Western Blotting Work Workload animal breeding animal care artery occlusion awake base blind blood gas analyzer calcein AM calcium green caveolin 1 caveolin-3 cell fixation cell fixing cellular imaging collagenase conditioning cost effective cryostat cytochrome c day design detector digital egg electrical property embryonic stem cell expectancy wave experience expression vector fluorexon fluorophore foot formycin triphosphate gene therapy gene transfer vector genetic manipulation handbook heart cell hemodynamics hexanoic acid hexokinase homologous recombination immunocytochemistry implantation improved in vivo indexing insight instrument interest intraperitoneal knockout animal laboratory facility laminin-10 large-conductance calcium-activated potassium channels light microscopy light scattering malate mathematical model medical schools member mitochondrial membrane mitochondrial permeability transition pore mouse model myometrium osmotic minipump papillary muscle paraform pericardial sac picric acid plasmid DNA potassium ferrocyanide pressure presynaptic processing speed programs promoter protein distribution research study response restoration retinal rods sample fixation seal sensor size stoichiometry subcutaneous success syntaxin 1 tetramethylrhodamine methyl ester tool transgene expression tribromoethanol uptake uranyl acetate vector voltage wound zygote

项目摘要

In our experimental conditions, we are determining "colocalization" in the resolution limit of light microscopy. In this PPG, the "colocalization" term will imply that two molecules may share the same microenvironment and/or can be elements of the same set of macromolecular complexes which are resolved by the optical system. Forexample, two proteins forming part of two different molecular complexes may show positive "colocalization" tests due to the lack of the optical resolution to detect each molecular complex. Still, the positive test will indicate that the two proteins are located proximally within the optical resolution. Thus, in "colocalization" determinations it is critical to achieve the maximal x,y,z resolution. In this PPG, we will use developed methodologies in the laboratory to achieve the highest resolution attainable with optical microscopy. We have maximized the optical linearity and the quantity of light recorded by the detectors in conjunction with image restoration analysis to recover the light lost by the optical imperfections of the system. This methodology has allowed us to reach a resolution in the x,y plane of about 100-200 nm (see Figs. 1-3). Definition of "colocalization" and random "colocalization". In a cell labeled with two fluorophores, one red (R) and one green (G), the number of voxels above threshold that are labeled with the red fluorophore is nRand the number labeled with the green fluorophore is nG. The number of colocalized voxels, i.e. those voxels containing intensity signals above threshold from both fluorophores is ncoioc. The percentage of "colocalization" of G with R is given by 100*^^- (eq. 1); and the percentage of "colocalization" of R with G is given by 100*^^ (eq. 2). In n* nc these measurements we will estimate the probability that the measured "colocalization" could occur by chance by calculating % random "colocalization" from ((area_1x area_2)/total areaA2) x100, where area_1 and area_2 are the areas above threshold (eq. 3). The "colocalization" method is generally based on the user ability to set the intensity threshold, and has the major draw back of the lack of a mathematical model to adjust the threshold of both images that will define the degree of paired pixel overlap. Most importantly the pixel overlap method has the limitation of being a binary test that determines whether the two paired pixels in two images have or not intensities above the intensity threshold and does not consider whether the two stained proteins have a landscape of intensity staining with a high degree of correlation, as it would be expected if they are elements of a common complex. The application of a correlation measurement was recently described by Li et al. (2004)7. In the following sections, I will discuss and apply developed algorithms to quantify the degree of protein-protein association by two methods, the INTENSITY CORRELATION ANALYSIS and the INTENSITY THRESHOLD "COLOCALIZATION"ANALYSIS. Intensity correlation analysis. In the present analysis, we acquire high resolution images to compare the correlation of pixel intensities in equivalent x,y coordinates of paired images from cells double stained for two different proteins. The prediction is that if two proteins are elements of the same macromolecular complex, the intensity staining landscape of the two images should have a x,y pixel to pixel positive correlation. On the contrary, if the two proteins are localized in distinct compartments the result will be a negative correlation. Finally, if the proteins in the two images are labeled in a diffuse non structured pattern (random), the correlation will tend to 0. This method is based on the principle that for any set of values the sum of the differences from the mean equal zero, i.e., ?N(A-a)=0, where a is the mean of the distribution with N values of Al. In the experiment N is the number of pixels, and A is the intensity for each pixel. If we have two set of values in two arrays 1 and 2 with N pixels per array having a random distribution of intensities A-,and 6, for arrays 1 and 2 , the sum of the product of their differences will also tend to zero, thus IN(A-a)(S,-Jb)~0. On the other hand, if the two intensities are positively correlated, the product will tend to be a positive value (^(Ara)(Brb}>G) and if they are negatively correlated the product will tend to a negative value (LN(Ara)(Brb)<0).

在我们的实验条件下，我们是在光学显微镜的分辨率极限中确定“共定位”。在此PPG中，“共定位”术语将暗示两个分子可能共享相同的微环境和/或可以是同一集的元素由光学系统解决的大分子复合物的。例如，两个蛋白质形成部分由于缺乏光学分辨率，两种不同的分子复合物可能显示出阳性的“共定位”测试检测每个分子复合物。尽管如此，阳性测试仍表明这两种蛋白质位于近端在光学分辨率中。因此，在“共定位”确定中，达到最大x，y，z至关重要解决。在此PPG中，我们将使用实验室中开发的方法来实现最高分辨率可通过光学显微镜完成。我们最大程度地提高了光学线性和由检测器与图像恢复分析结合使用，以恢复由光学缺陷丢失的光系统。这种方法使我们能够在约100-200 nm的x，y平面中达到分辨率（见图1-3）。 “共定位”和随机“共定位”的定义。在带有两个荧光团标记的细胞中，一个红色（r）和一个绿色（g），用红色荧光团标记的阈值高于阈值的体素数为nrand 用绿色荧光团标记的数字为Ng。共定位体素的数量，即那些含有的体素来自两个荧光团阈值高于阈值的强度信号为NCOIOC。 G与R的“共定位”的百分比为由100*^^ - （等式1）; R与G的“共定位”的百分比由100*^^（等式2）给出。在 n* nc 这些测量结果我们将估计测量的“共定位”可能会偶然发生的概率计算％随机的“共定位”（（区域_1x afore_2）/rate Areaa2）x100，其中abret_1和abreat_2是高于阈值的区域（等式3）。 “共定位”方法通常基于用户设置强度阈值的能力，并且具有主要借助缺乏数学模型来调整这两个图像的阈值，以定义的程度配对的像素重叠。最重要的是，像素重叠方法的局限性是二进制测试确定两个图像中的两个成对像素是否具有强度高于强度阈值的强度，并且不要考虑两种染色蛋白是否具有高度相关性的强度染色景观，正如预期的那样，如果它们是共同复合物的元素。相关测量的应用最近由Li等人描述。（2004）7。在以下各节中，我将讨论并应用开发的算法来量化蛋白质蛋白质的程度通过两种方法的关联，强度相关分析和强度阈值 “共定位”分析。强度相关分析。在目前的分析中，我们获得了高分辨率图像以比较等效X中的像素强度的相关性，来自两个细胞的成对图像的y坐标，两种不同的染色蛋白质。预测是，如果两种蛋白质是同一大分子复合物的元素，则强度这两个图像的染色景观应具有x，y像素到像素正相关。相反，如果这两个蛋白质位于不同的隔间中，结果将是负相关。最后，如果蛋白质中的蛋白质两个图像以弥漫性非结构化模式（随机）标记，相关性将趋于0。该方法基于以下原理：对于任何一组值，差异的总和来自平均值相等的零，即，？n（a-a）= 0，a是n值n值的分布的平均值。在实验中n是像素，A是每个像素的强度。如果我们在两个数组1和2中有两组值，每个数组的n像素对于阵列1和2，具有强度A-和6的随机分布，其差异的产物总和将也倾向于零，因此在（a-a）（s，-jb）〜0中。另一方面，如果两个强度正相关，则该产品将往往是一个正值（^（ara）（brb}> g），如果它们是负相关的，则产品将趋向于负值（LN（ARA）（BRB）<0）。