Discovery of Peptide and Aptamer Ligands for Targeted Drug Delivery to Hepatic Stellate Cells

Title from PDF of title page, viewed January 3, 2018Dissertation advisor: Kun ChengVitaIncludes bibliographical references (pages 99-109)Thesis (Ph.D.)--School of Pharmacy, Department of Chemistry and School of Medicine. University of Missouri--Kansas City, 2016The objective of this dissertation is to present two approaches to identify Hepatic Stellate Cells (HSCs) targeting ligands using phage-display library biopanning and systematic evolution of ligands by exponential enrichment (SELEX). These HSC-specific ligands can be used for diagnosis and therapy of liver fibrosis. The mechanism of fibrogenesis and potential treatments of liver fibrosis are summarized in Chapters 1 and 2. In Chapter 3, we identified HSC-specific peptides using a peptide phage display for diagnosis and treatment of liver fibrosis. Caused by chronic injuries, such as hepatitis, alcohol abuse, and nonalcoholic steatohepatitis, liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) in the liver. Activation of HSCs is the most critical step during liver fibrogenesis due to the production of excessive ECM and profibrogenic cytokines. Therefore, development of HSC-specific delivery systems is essential for the success of antifibrotic agents. The objective of this Chapter is to identify peptide ligands targeting the insulin like growth factor II receptor (IGFIIR), which is overexpressed in HSCs during liver fibrogenesis. A combinatorial phage display biopanning against IGFIIE protein- and HSC-T6 rat hepatic stellate cells was conducted to identify phage/peptide candidates. Phage ELISA, cellular uptake and cell viability assay were employed to evaluate the binding affinity and specificity of these peptide ligands to recombinant human IGFIIR and HSCs. IGFIIR siRNA was used to silence the IGFIIR expression in human HSCs (LX-2) to confirm the specificity of the identified peptide ligands. Among the identified peptide candidates, the peptide-431 shows the highest binding affinity and specificity to recombinant human IGFIIR protein and HSCs. The apparent equilibrium dissociation constant (Kd) of the peptide-431 is 6.19 µM for LX-2 cells and 12.35 μM for rat hepatic stellate cells HSC-T6. Cellular uptake of the peptide-431 in LX-2 cells is significantly reduced after silencing IGFIIR with siRNA. The peptide-431 also enhances the uptake of a proapoptotic peptide (KLA peptide) in LX-2 and HSC-T6 cells, indicating that the peptide-431 can be used as a targeting ligand to deliver antifibrotic agents into not only rat but also human HSCs. In order to improve the potential of the application of peptide-431 in liver fibrosis targeted delivery, we developed a dimerized peptide-431 which is also descripted in Chapter 3. The binding affinity of peptide-431 has improved 9 fold by forming dimer comparing with monomer peptide-431. We also screened aptamer ligands using SELEX for targeted delivery to HSCs. In Chapter 4, IGFIIR-specific aptamers were identified using SELEX. The binding affinity and specificity of the aptamers were studied by flow cytometry and Surface Plasma Resonance (SPR). The identified aptamer was annealed with the PCBP2 siRNA and delivered it into HSCs. The recovered aptamers result shows that the aptamers were enriched after seven rounds of SELEX. The binding affinity (Kd) of the aptamer-20 on IGFIIR protein is approximately 35.5 nM. Knocking down the expression of IGFIIR with siRNA decreased the binding affinity of aptamer-20, indicating the specificity of the aptamer to IGFIIR in HSCs. Cellular uptake of the aptamer-siRNA chimera is much higher than the siRNA. PCBP2 expression in HSCs is significantly downregulated after incubation with the aptamer-siRNA chimera in LX-2 cells. After systemic administration, the aptamer-siRNA chimera is primarily located in the liver of rats with CCl₄-induced liver fibrosis. Therefore, aptamer-20 is a very promising IGFIIR-specific ligand for drug delivery to HSCs.Introduction -- review of literature -- Discovery of peptide ligands for hepatic stellate cells using page display -- Discovery of aptamer ligands for hepatic stellate cells using SELEX -- Summary and conclusion -- Appendix letter of permissio

A Note on Location Parameter Estimation using the Weighted Hodges-Lehmann Estimator

Robust design is one of the main tools employed by engineers for the facilitation of the design of high-quality processes. However, most real-world processes invariably contend with external uncontrollable factors, often denoted as outliers or contaminated data, which exert a substantial distorting effect upon the computed sample mean. In pursuit of mitigating the inherent bias entailed by outliers within the dataset, the concept of weight adjustment emerges as a prudent recourse, to make the sample more representative of the statistical population. In this sense, the intricate challenge lies in the judicious application of these diverse weights toward the estimation of an alternative to the robust location estimator. Different from the previous studies, this study proposes two categories of new weighted Hodges-Lehmann (WHL) estimators that incorporate weight factors in the location parameter estimation. To evaluate their robust performances in estimating the location parameter, this study constructs a set of comprehensive simulations to compare various location estimators including mean, weighted mean, weighted median, Hodges-Lehmann estimator, and the proposed WHL estimators. The findings unequivocally manifest that the proposed WHL estimators clearly outperform the traditional methods in terms of their breakdown points, biases, and relative efficiencies

Electronic Raman Scattering in copper oxide Superconductors: Understanding the Phase Diagram

Electronic Raman scattering measurements have been performed on hole doped copper oxide superconductors as a function of temperature and doping level. In the superconducting state coherent Bogoliubov quasiparticles develop preferentially over the nodal region in the underdoped regime. We can then define the fraction of coherent Fermi surface, $f_c$ around the nodes for which quasiparticles are well defined and superconductivity sets in. We find that $f_c$ is doping dependent and leads to the emergence of two energy scales. We then establish in a one single gap shem, that the critical temperature $T_{c} \propto f_{c}\Delta_{max}$ where $\Delta_{max}$ is the maximum amplitude of the d-wave superconducting gap. In the normal state, the loss of antinodal quasiparticles spectral weight detected in the superconducting state persists and the spectral weight is only restored above the pseudogap temperature $T*$. Such a dichotomy in the quasiparticles dynamics is then responsible for the emergence of the two energy scales in the superconducting state and the appearance of the pseudogap in the normal state. We propose a 3D phase diagram where both the temperature and the energy phase diagrams have been plotted together. We anticipate that the development of coherent excitations on a restricted part of the Fermi surface only is a general feature in high $T_c$ cuprate superconductors as the Mott insulating is approaching.Comment: 40 pages, 19 figures some new references and comments have been added with respect to the first version of march 30t

Vortex coupling in trailing vortex-wing interactions

The interaction of trailing vortices of an upstream wing with rigid and flexible downstream wings has been investigated experimentally in a wind tunnel, using particle image velocimetry, hot-wire, force and deformation measurements. Counter-rotating upstream vortices exhibit increased meandering when they are close to the tip of the downstream wing. The upstream vortex forms a pair with the vortex shed from the downstream wing, and then exhibits large displacements around the wing-tip. This coupled motion of the pair has been found to cause large lift fluctuations on the downstream wing. The meandering of the vortex pair occurs at the natural meandering frequency of the isolated vortex, with a low Strouhal number, and is not affected by the frequency of the large-amplitude wing oscillations if the downstream wing is flexible. The displacement of the leading vortex is larger than that of the trailing vortex, however it causes highly correlated variations of the core radius, core vorticity and circulation of the trailing vortex with the coupled meandering motion. In contrast, co-rotating vortices do not exhibit any increased meandering

Frequency hopping signal detection based on optimized generalized S transform and ResNet

The performance of traditional frequency hopping signal detection methods based on time frequency analysis is limited by the tradeoff of time-frequency resolution and spectrum leakage. Machine learning-based frequency hopping signal detection techniques have a high level of complexity. Therefore, this paper proposes a residual network and the optimized generalized S transform to detect frequency hopping signals. First, based on the time-frequency aggregation measure, the generalized S transform parameters $\lambda$ and $p$ are optimized using a multi-population genetic algorithm. Second, the optimized generalized S transform is used to determine a signal's time-frequency spectrum, which is then normalized to make this robust to noise power uncertainty. Finally, a residual network structure is designed which receives the time-frequency spectrum. To detect frequency hopping signals, the network automatically learns the time-frequency properties of signals and noise. Simulated findings indicate that the multi-population genetic algorithm not only increases optimization efficiency when compared to a regular genetic algorithm, but also has faster convergence and more stable optimization results. Compared with a hybrid convolutional network/recurrent neural network algorithm, the proposed technique is better at detection and has less computational and storage complexity

CircaKB: A Comprehensive Knowledgebase of Circadian Genes Across Multiple Species

Circadian rhythms, which are the natural cycles that dictate various physiological processes over a 24-h period, have been increasingly recognized as important in the management and treatment of various human diseases. However, the lack of sufficient data and reliable analysis methods have been a major obstacle to understanding the bidirectional interaction between circadian variation and human health. We have developed CircaKB, a comprehensive knowledgebase of circadian genes across multiple species. CircaKB is the first knowledgebase that provides systematic annotations of the oscillatory patterns of gene expression at a genome-wide level for 15 representative species. Currently, CircaKB contains 226 time-course transcriptome datasets, covering a wide variety of tissues, organs, and cell lines. In addition, CircaKB integrates 12 computational models to facilitate reliable data analysis and identify oscillatory patterns and their variations in gene expression. CircaKB also offers powerful functionalities to its users, including easy search, fast browsing, strong visualization, and custom upload. We believe that CircaKB will be a valuable tool and resource for the circadian research community, contributing to the identification of new targets for disease prevention and treatment. We have made CircaKB freely accessible at https://cdsic.njau.edu.cn/CircaKB

Focal shift of silicon microlens array in mid-infrared regime

采用严格数值算法对中红外硅微透镜阵列进行了模拟,该微透镜阵列特征尺寸小于波长工作波长.研究发现该微透镜阵列存在一个显著的离焦效应,其离焦量达到0; .4左右,超出了现有的传统理论模型预测范围.对微透镜阵列进行了制作和焦距测试,发现测试结果跟数值模拟基本吻合.微纳衍射光学集成系统中透镜离焦量是; 系统集成非常重要的一个参数,该研究结果为硅微透镜阵列和中红外探测器光学集成提供有效参考.In this study rigorous numerical model was utilized to characterize the focal shift of the diffractive mid infrared (MIR) silicon microlens arrays (MLAs) with the critical size smaller than the working wavelength. We found a more pronounced focal shift in mid-infrared wavelength which is out of the range predicted by existing models. Focal properties of the MLAs were also measured experimentally. The results agrees well with the simulation results. Our results provide a reference point in understanding the focal shift in MIR regime, which is important in terms of deciding the focal length of micro-nano lens systems, especially when dealing with the integration of diffractive devices in infrared optical system.Special Project on the Integration of Industry, Education and Research; of Aviation Industry Corporation of China [CXY2011XD24

Gold metasurfaces as saturable absorbers for all-normal-dispersion ytterbium-doped mode-locked fiber laser

Optical metasurfaces, i.e., thin planar structures with subwavelength metal or dielectric unit cells, have attracted great interest due to their intriguing capabilities of shaping light in both linear and nonlinear regimes. However, their saturable absorption properties and corresponding applications have so far been rarely exploited. Here we report on passively mode-locked ytterbium-doped fiber lasers utilizing saturable metasurfaces made of periodically arranged gold nanorods. Three 400-nm-period metasurfaces with gold nanorods of different lengths (from 210 to 240 nm) and plasmonic resonances (from 968 to 1044 nm) are fabricated and found to exhibit nonlinear absorption with decent modulation depths, facilitating the formation of mode-locking states at 1060 nm. The corresponding lasers generate typically mode-locked pulses with the duration of ∼ 62.3 ps, repetition rate of 10.33 MHz, and signal-to-noise ratio of ∼74 dB. Our experimental results demonstrate that the gold nanorod-based metasurfaces can be used as relatively broadband mode-lockers in the 1-μm-wavelength range. </p