Functional Characterization of Microtubule Associated Proteins in ES Cell Division and Neuronal Differentiation

Microtubules are tubular polymers that are involved in a variety of cellular processes such as cell movement, mitosis and intracellular transport. The dynamic behavior of microtubules makes this possible because all of these processes require quick responses. Embryonic stem (ES) cells were first isolated from mouse embryos and they have two unique characteristics; they can be kept undifferentiated for many passages with a stable karyotype and they can be differentiated into any type of cells under appropriate conditions. The pluripotency of ES cells, their ease of manipulation in culture, and their ability to contribute to the mouse germ-line provides us a model of differentiation both in vitro and in vivo. In my thesis I focused on the cell division and neuronal differentiation of ES cells and developed two methods to understand the effects of microtubule dynamics in spindle assembly and chromosome segregation and to reveal the roles of different Microtubule Associated Proteins (MAPs) in the neuronal morphology formation. In the first part, we developed a live-cell imaging method for ES cells to visualize, track and analyze the single cell behavior in a cell population over a time period. So far many techniques have been adapted and combined for imaging of cell lines, mainly for the cancer or immortalized ones. However, because ES cells are very prone to apoptosis, tend to form spheres and hard to stably label, it is quite tricky to image them in culture conditions. In our system, we combined the BAC-based gene expression with wide-field deconvolution microscopy for ES cells that are plated onto the laminin-511 coated surface and kept in CO2 independent culture conditions. This combined technique does not interfere with the growth of cells and keeps them healthy up to 24 hours on the microscope stage. In the second part, we analyzed the effects of MAPs chTOG, EB1, Kif18A and MCAK in the overall spindle morphology and mitotic progression in mES cells. For this purpose, we utilized our stable TUBB-GFP and H2A-GFP cell lines along with our live-cell imaging set-up to reveal the effects of the above-mentioned proteins and the interplay among each other. By using RNAi method we either single or co-depleted the genes by siRNAs and measured the spindle length and width in RNAi conditions. We further analyzed the mitotic progression in H2A-GFP cell line in terms of the metaphase timing and the percentage of chromosome segregation errors. Our results showed that, EB1 depletion did not cause any significant changes in the overall spindle morphology or in the metaphase timing. However, the co-depletion of EB1 with chTOG partially rescued the sichTOG specific mini-spindle phenotype. siKif18A produced longer spindles without any change in the spindle width. Surprisingly, the co-depletion of antagonistic chTOG and Kif18A proteins had additive effects on the spindle dynamics and on mitotic progression in a way that spindle assembly was severely disrupted by the absence of these two proteins and as a result of this, both metaphase timing and chromosome missegregation levels increased significantly. These results overall indicate that MAPs have important roles in the regulation of dynamic instability and these proteins have an interplay among each other to be able to control the morphology of the spindle as well as the correct segregation of chromosomes into daughter cells. In the last part, I will introduce you a new ES cell based differentiation and morphology model, which brings the advantages of high resolution imaging capacity, control over development and easy genetic manipulation and culturing. We have generated Tet-induced shRNA cell lines against chTOG, Kif18A and MCAK, which are also stably expressing TUBB-GFP. These labeled cells were mixed with unlabeled wild-type mES cells before differentiation at 1:1000 ratio and then they were differentiated into mouse cortical cells and spinal motor neurons. Our results showed that, all of the three genes could be successfully knocked-down by shRNA after 48 hours of Tet induction. After mixing the labeled and unlabeled cells, single neurons could be imaged at high resolution and their skeletons could be generated afterwards. The RNAi studies in shchTOG cell line showed that, the knock-down of this gene in early differentiation interferes with the neuronal differentiation

A Comparative Study of the Structural Dynamics of Four Terminal Uridylyl Transferases.

African trypanosomiasis occurs in 36 countries in sub-Saharan Africa with 10,000 reported cases annually. No definitive remedy is currently available and if left untreated, the disease becomes fatal. Structural and biochemical studies of trypanosomal terminal uridylyl transferases (TUTases) demonstrated their functional role in extensive uridylate insertion/deletion of RNA. Trypanosoma brucei RNA Editing TUTase 1 (TbRET1) is involved in guide RNA 3' end uridylation and maturation, while TbRET2 is responsible for U-insertion at RNA editing sites. Two additional TUTases called TbMEAT1 and TbTUT4 have also been reported to share similar function. TbRET1 and TbRET2 are essential enzymes for the parasite viability making them potential drug targets. For this study, we clustered molecular dynamics (MD) trajectories of four TUTases based on active site shape measured by Pocket Volume Measurer (POVME) program. Among the four TUTases, TbRET1 exhibited the largest average pocket volume, while TbMEAT1's and TbTUT4's active sites displayed the most flexibility. A side pocket was also identified within the active site in all TUTases with TbRET1 having the most pronounced. Our results indicate that TbRET1's larger side pocket can be exploited to achieve selective inhibitor design as FTMap identifies it as a druggable pocket

Is Channel Estimation Necessary to Select Phase-Shifts for RIS-Assisted Massive MIMO?

Reconfigurable intelligent surfaces (RISs) have attracted great attention as a potential beyond 5G technology. These surfaces consist of many passive elements of metamaterials whose impedance can be controllable to change the characteristics of wireless signals impinging on them. Channel estimation is a critical task when it comes to the control of a large RIS when having a channel with a large number of multipath components. In this paper, we propose novel channel estimation schemes for different RIS-assisted massive multiple-input multiple-output (MIMO) configurations. The proposed methods exploit spatial correlation characteristics at both the base station and the planar RISs, and other statistical characteristics of multi-specular fading in a mobile environment. Moreover, a novel heuristic for phase-shift selection at the RISs is developed. For the RIS-assisted massive MIMO, a new receive combining method and a fixed-point algorithm, which solves the max-min fairness power control optimally, are proposed. Simulation results demonstrate that the proposed uplink RIS-aided framework improves the spectral efficiency of the cell-edge mobile user equipments substantially in comparison to a conventional single-cell massive MIMO system. The impact of several channel effects are studied to gain insight about which RIS configuration is preferable and when the channel estimation is necessary to boost the spectral efficiency.Comment: 30 pages, 9 figures, submitted to IEEE Journa

Reviewing assertiveness level of elite male and female Athletes in Different Sports Branches

The purpose of this research was to review the assertiveness levels of elite level athletes who study in İstanbul Aydın University and play different sports. For this purpose, the survey model was used in the study. A total of 121 students including 36 female (age average x=20,11±1,83) and 85 male (age average x=20,34±2,47) volunteered for this study. Rathus Assertiveness Schedule (RAE) that was developed by A.S Rathus (1973) was used as data collection tool to determine the assertiveness levels of participants (Voltan 1980). Adaption works for Turkish of this schedule was performed by Nilüfer Voltan Acar (1980). Independent T-test and One Way ANOVA Test in SPSS 24.0 packaged software were used in statistical analyses of the data obtained. It was found at the end of the research that the assertiveness level of 19 students is at timidity level, the assertiveness level of 102 students is at an aggressive level. While there were no statistically significant differences in variables of gender, branch, income level and educational background of the parent; there was found statistically significant difference by evaluation based on age ranges