The complex interplay of RNA-binding proteins and RISC in neurons

An extensive network of RNA-binding proteins (RBPs) is at the center of posttranscriptional gene regulation. Importantly, different RBPs – including microRNA-loaded Argonaute (Ago) – can bind to a single mRNA resulting in antagonistic or cooperative mode of actions, thereby determining the fate and function of an mRNA. Here, I investigated the impact of two different RBPs, HuR and Staufen2 (Stau2), on microRNA/Ago-dependent gene expression homeostasis. The results of my thesis allow me to present a working model how these three RBPs might control neuronal function in a novel RNA-structure dependent manner. HuR protein binds to AU-rich elements within mRNAs. In the case of Regulator of G-protein signaling (Rgs4) mRNA, I find that HuR binding occurs close to a miR-26/RISC binding site, resulting in Rgs4 destabilization. As both binding sites are in close proximity within a predicted RNA hairpin structure, only synergistic action of HuR and miR-26 results in Rgs4 repression. I propose a novel mechanism involving the trifold combination of HuR, miR-26-loaded Ago and RNA secondary structure in governing functional regulation of Rgs4 mRNA in neurons. Certain RBPs such as Stau2 protein bind to double-stranded RNAs (dsRNAs), thereby shaping local and global secondary structures of mRNAs. Based on preliminary data linking Stau2 and the miRNA pathway, I investigated Stau2-dependent expression, localization and function of the miRNA-induced silencing complex (RISC) in neurons. Proteome and small RNA transcriptome analysis in Stau2 deficient primary neurons revealed significant upregulation of several RISC associated proteins, including Ago1/2, while global miRNA levels were unaffected. This upregulation was accompanied by decreased global translation and translocation of Ago2 from Processing-bodies, sites of mRNA storage, to translating polysomes. Phenotypically, depletion of Ago1/2 reduced dendritic branching. This effect could be rescued by simultaneous knockdown of Stau2, suggesting that Ago1/2 and Stau2 functionally counterbalance each other in neurons. I hypothesize that Stau2’s ability to bind to dsRNA stabilizes defined mRNA structures thereby governing association of RISC and mRNAs. Based on Stau2 hiCLIP experiments by our collaborator Jernej Ule, I was able to define a long-range RNA duplex in the 3’-untranslated region of Rgs4 mRNA bound by Stau2 in vivo. This RNA duplex is necessary and sufficient to drive Stau2-dependent ribonucleoprotein particle (RNP) assembly as well as dendritic RNA localization in neurons. Together, the data presented in my thesis support a model, in which balanced expression and interdependent action of RBPs, RISC and RNA structure shapes RNP assembly and gene expression homeostasis, important for neuronal function

Characteristics of the steam flow in the end seals of high pressure cylinders in the state of vacuum augmentation

Разработан алгоритм определения параметров пара в концевых уплотнениях на режиме набора вакуума, который позволяет учесть утечки пара из камер с дренажами и найти характеристики течения в каждой камере. Определен характер течения пара в каждой секции концевых уплотнений цилиндра высокого давления (ЦВД) турбины К-325-23,5 на этапе набора вакуума. Распределение давлений и расходов пара по секциям переднего и заднего концевых уплотнений свидетельствует о нерациональной схеме прогрева ротора и возможности возникновения высокого уровня термических напряжений. По рассчитанным параметрам пара вычислены коэффициенты теплоотдачи при одно- и двухфазном течении пара на поверхности ротора ЦВД в области концевых уплотнений для последующего термопрочностного анализа

Through-time 3D radial GRAPPA for whole heart cardiac imaging

Summary Through-Time 3D Radial GRAPPA can be used to reconstruct 3D CINE images covering the whole heart in a single breathhold. Background Through-Time Non-Cartesian GRAPPA has been previously demonstrated for real-time 2D cardiac imaging (Seiberlich, et al. Magn Reson Med. 2011 Feb;65(2):492-505.). This parallel imaging method works by acquiring several fully-sampled non-Cartesian datasets with a low temporal resolution, and using the coil sensitivity information from these datasets to reconstruct highly undersampled non-Cartesian data acquired in real-time. By modifying this through-time non-Cartesian GRAPPA method to reconstruct highly undersampled 3D data, whole heart 3D CINE images can be generated using data acquired in a single breathhold. Methods A total of 20 fully-sampled 3D stack-of-stars radial datasets were acquired during free-breathing with no EKG gating using a 1.5T Siemens Espree and the following parameters: bSSFP sequence, TE=1.52ms, TR=3.04ms, matrix size = 128x128x20, projections/partition=128, FOV=300x300x90mm3, Flip Angle=45°, 5/8 Partial Fourier, 18 receiver channels. Segmented undersampled data (using only 16 projections/partition, an acceleration factor of R=8) were acquired with EKG gating and the above parameters during a breathhold for 15 heartbeats, resulting in 15 CINE frames. In order to perform the calibration, each of the time frames and partitions were employed as separate sources of calibration information; thus, a total of 300 repetitions could be used to generate the through-time GRAPPA weight sets. After reconstruction, the undersampled data yielded fully-sampled 3D CINE images, each with a temporal footprint of 48ms, an in-plane resolution of 2.3mm2, and a through-plane resolution of 6mm. The total acquisition time was 116s for the calibration and approximately 15 s for the breathhold CINE acquisition. Results Example images from diastole and systole of one healthy volunteer are shown in Figures 1 and 2. It is important to note that these represent just two of the 15 CINE frames acquired in this dataset. Despite the high acceleration factor (R=8 in comparison to the fully-sampled calibration data), the images demonstrate only minor residual aliasing artifacts. Because a 3D dataset is acquired, the images from each partition can be shown in the same cardiac phase, which is challenging when using multiple breathholds to acquire several 2D CINE slices

Sensitivity analysis of magnetic field measurements for magnetic resonance electrical impedance tomography (MREIT)

Purpose Clinical use of magnetic resonance electrical impedance tomography (MREIT) still requires significant sensitivity improvements. Here, the measurement of the current-induced magnetic field (ΔBz,c) is improved using systematic efficiency analyses and optimization of multi-echo spin echo (MESE) and steady-state free precession free induction decay (SSFP-FID) sequences. Theory and Methods Considering T1, T2, and math formula relaxation in the signal-to-noise ratios (SNRs) of the MR magnitude images, the efficiency of MESE and SSFP-FID MREIT experiments, and its dependence on the sequence parameters, are analytically analyzed and simulated. The theoretical results are experimentally validated in a saline-filled homogenous spherical phantom with relaxation parameters similar to brain tissue. Measurement of ΔBz,c is also performed in a cylindrical phantom with saline and chicken meat. Results The efficiency simulations and experimental results are in good agreement. When using optimal parameters, ΔBz,c can be reliably measured in the phantom even at injected current strengths of 1 mA or lower for both sequence types. The importance of using proper crusher gradient selection on the phase evolution in a MESE experiment is also demonstrated. Conclusion The efficiencies observed with the optimized sequence parameters will likely render in-vivo human brain MREIT feasible

Human In-vivo MR Current Density Imaging (MRCDI) Based on Optimized Multi-echo Spin Echo (MESE)

MRCDI aims at imaging an externally injected current flow in the human body, and might be useful for many biomedical applications. However, the method requires very sensitive measurement of the current-induced magnetic field component ?Bz,c parallel to main field. We systematically optimized MESE to determine its most efficient parameters. In one of the first human in-vivo applications of MRCDI, the optimized sequence was successfully used to image the ?Bz,c distribution in the brain caused by a two-electrode montage, as confirmed by finite-element calculations of ?Bz,c. Further improvements will be performed to increase its robustness to field drifts