A short-term suspension culture system for bovine oviduct epithelial cells suitable for the study of embryo-maternal communication processes

A short-term (24 h) culture system for bovine oviduct epithelial cells (BOEC) suitable for co-culture experiments with embryos was established and evaluated. BOEC were obtained on Day 3.5 of the estrous cycle and processed by mechanical means only to obtain cell aggregates. Cell yields were 10-fold higher as described in former studies employing enzymatic treatment to achieve a single cell suspension for seeding to obtain a BOEC monolayer. Light microscopic examinations showed vigorously beating cilia on the apical side of the BOEC in aggregates and a rapid and constant motion of cell aggregates due to active ciliary beat. Scanning electron microscopy and transmission electron microscopy confirmed ultrastructural characteristics of BOEC at seeding and after 24 h in culture very similar to the situation in vivo. Both secretory cells with numerous secretory granules and ciliated cells with long, well-developed kinocilia were visible. The purity of the epithelial cell culture was > 95 %, as assessed by immunocytochemical methods. For further characterization of cultured BOEC, gene expression patterns were examined after different time spans in culture. Cultured BOEC isolated from ampullae ipsilateral to the ovulation site yielded significantly higher amounts of RNA than their contralateral counterparts (2.73 ± 0.98 versus 2.31 ± 0.14 µg per 10^6 cells). However, quantitative PCR did not detect significant differences in transcript levels between ipsi- and contralateral BOEC for the majority of marker genes (ESR1, ESR2, HMGCR, OVGP1, PGR, TRA1) throughout the 24 h culture period. The analysis of combined data obtained from different sampling time points during the culture period revealed an effect only for GPX4 (B. taurus non-selenium glutathione phospholipid hydroperoxide peroxidase), a gene known to be differentially expressed in vivo. Marker gene expression of five genes remained stable after 6h of cell culture, indicating only a short adaptation period of cultured cells. The use of two different sera (estrous cow serum versus cow serum obtained on Day 3.5 of the estrous cycle) in a concentration of 2 % did not affect gene expression patterns. Western blot analysis confirmed ESR1 (estrogen receptor α) and PGR (progesterone receptor) protein expression throughout the culture period. In agreement with cyclic differences in vivo, stimulation with 10 pg/ml estradiol-17β increased PGR transcript abundance in BOEC significantly. A response to the stimulation with 10 ng/ml progesterone was shown as INOS (inducible nitric oxide synthase) gene expression increased significantly after steroid treatment. Thus, the developed culture system provides functional BOEC with an unchanged morphology and maintained functionality as compared with cells in vivo. The system is rapidly available for use in co-culture experiments with bovine embryos and provides cultured cells in sufficient quantities for holistic transcriptome and proteome studies, thereby helping to decipher early embryo-maternal communication

Defining New Mechanisms for DNA Damage Tolerance in Cancer

Cancer cells rely on DNA damage tolerance pathways to cope with intrinsic oncogenic stresses and evade DNA-damaging environmental and therapeutic agents. However, the mechanisms by which neoplastic cells hijack tightly controlled DNA damage tolerance-signaling cascades to promote mutagenesis and chemoresistance are not understood. Thus, limitations in our knowledge of DNA damage tolerance and mutagenesis impede effective prevention and treatment of cancer. We have discovered two unique regulators of RAD18 and replication-associated DNA damage tolerance that are overexpressed in cancer: RNF168 (an apical mediator of double strand break signaling) and MAGEA4 (a cancer cell-specific protein with no known function). RNF168 is mutated in human RIDDLE syndrome, a disease characterized by severe immunodeficiency, developmental defects, radiosensitivity and a predisposition to cancer. We show here that RNF168 is a novel component of the RAD18 complex, facilitating its recruitment to stalled replication forks and promoting damage tolerance following replication stress. We have also identified the cancer/testis antigen (CTA) MAGEA4 as a stabilizing binding partner of RAD18 that promotes trans-lesion DNA synthesis. Thus, the findings in this thesis offer neomorphic cancer cell-specific roles for regulators of DNA damage tolerance. Identification of mechanisms of DNA damage tolerance that drive carcinogenesis and confer chemoresistance will allow for the development of more effective cancer treatment regimens. CTAs are absent from normal somatic cells but aberrantly overexpressed in many cancers. Interestingly, CTAs have been correlated with chemotherapeutic resistance and poor prognostic outcomes, though their contributions to carcinogenesis are not understood. We have found that depletion of several CTAs (MAGEA4, MAGEA10, or HORMAD1) sensitizes non-small cell lung cancer (NSCLC) cells to DNA-damaging therapies. These studies identify novel mechanisms by which NSCLC cells aberrantly overexpress germ cell proteins to alter genome maintenance, offering a cancer cell-specific mechanism by which neoplastic cells acquire chemoresistance and evade therapy. Accordingly, these CTAs are promising therapeutic targets whose inhibition should be innocuous to normal somatic cells while greatly sensitizing cancer cells to existing DNA damaging chemotherapeutic agents.Doctor of Philosoph

Data-driven aerodynamic shape design with distributionally robust optimization approaches

We formulate and solve data-driven aerodynamic shape design problems with distributionally robust optimization (DRO) approaches. Building on the findings of the work \cite{gotoh2018robust}, we study the connections between a class of DRO and the Taguchi method in the context of robust design optimization. Our preliminary computational experiments on aerodynamic shape optimization in transonic turbulent flow show promising design results

Mechanisms of Post-Replication DNA Repair

Accurate DNA replication is crucial for cell survival and the maintenance of genome stability. Cells have developed mechanisms to cope with the frequent genotoxic injuries that arise from both endogenous and environmental sources. Lesions encountered during DNA replication are often tolerated by post-replication repair mechanisms that prevent replication fork collapse and avert the formation of DNA double strand breaks. There are two predominant post-replication repair pathways, trans-lesion synthesis (TLS) and template switching (TS). TLS is a DNA damage-tolerant and low-fidelity mode of DNA synthesis that utilizes specialized ‘Y-family’ DNA polymerases to replicate damaged templates. TS, however, is an error-free ‘DNA damage avoidance’ mode of DNA synthesis that uses a newly synthesized sister chromatid as a template in lieu of the damaged parent strand. Both TLS and TS pathways are tightly controlled signaling cascades that integrate DNA synthesis with the overall DNA damage response and are thus crucial for genome stability. This review will cover the current knowledge of the primary mediators of post-replication repair and how they are regulated in the cell

An integral equation based numerical solution for nanoparticles illuminated with collimated and focused light

To address the large number of parameters involved in nanooptical problems, a more efficient computational method is necessary. An integral equation based numerical solution is developed when the particles are illuminated with collimated and focused incident beams. The solution procedure uses the method of weighted residuals, in which the integral equation is reduced to a matrix equation and then solved for the unknown electric field distribution. In the solution procedure, the effects of the surrounding medium and boundaries are taken into account using a Green’s function formulation. Therefore, there is no additional error due to artificial boundary conditions unlike differential equation based techniques, such as finite difference time domain and finite element method. In this formulation, only the scattering nano-particle is discretized. Such an approach results in a lesser number of unknowns in the resulting matrix equation. The results are compared to the analytical Mie series solution for spherical particles, as well as to the finite element method for rectangular metallic particles. The Richards-Wolf vector field equations are combined with the integral equation based formulation to model the interaction of nanoparticles with linearly and radially polarized incident focused beams

The effect of milling additives on powder properties and sintered body microstructure of NiO

The evolution of powder particle size, crystal structure, and surface chemistry was evaluated for micron scale NiO powders subjected to impact milling with commonly employed milling additives: methanol, Vertrel XF, and amorphous carbon. The effect of the different comminution protocols on sintered body microstructure was evaluated for high temperature sintering in inert atmosphere (N2). X-ray photoelectron spectroscopy showed that NiO powder surface chemistry is surprisingly sensitive to milling additive choice. In particular, the proportion of powder surface defect sites varied with additive, and methanol left an alcohol or alkoxy residue even after drying. Upon sintering to intermediate temperatures (1100 ℃), scanning electron microscopy (SEM) showed that slurry milled NiO powders exhibit hindered sintering behaviors. This effect was amplified for NiO milled with methanol, in which sub-500 nm grain sizes dominated even after sintering to 1100 ℃. Upon heating to high temperatures (1500 ℃), simultaneous differential scanning calorimetry/thermogravimetric analysis (DSC/TGA) showed that the powders containing carbon residues undergo carbothermal reduction, resulting in a melting transition between 1425 and 1454 ℃. Taken together, the results demonstrated that when processing metal oxide powders for advanced ceramics, the choice of milling additive is crucial as it exerts significant control over sintered body microstructure

DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

The mechanisms by which neoplastic cells tolerate oncogene-induced DNA replication stress are poorly understood. Cyclin-dependent kinase 2 (CDK2) is a major mediator of oncogenic DNA replication stress. In this study, we show that CDK2-inducing stimuli (including Cyclin E overexpression, oncogenic RAS, and WEE1 inhibition) activate the DNA repair protein RAD18. CDK2-induced RAD18 activation required initiation of DNA synthesis and was repressed by p53. RAD18 and its effector, DNA polymerase κ (Polκ), sustained ongoing DNA synthesis in cells harboring elevated CDK2 activity. RAD18-deficient cells aberrantly accumulated single-stranded DNA (ssDNA) after CDK2 activation. In RAD18-depleted cells, the G2/M checkpoint was necessary to prevent mitotic entry with persistent ssDNA. Rad18 −/− and Polκ −/− cells were highly sensitive to the WEE1 inhibitor MK-1775 (which simultaneously activates CDK2 and abrogates the G2/M checkpoint). Collectively, our results show that the RAD18–Polκ signaling axis allows tolerance of CDK2-mediated oncogenic stress and may allow neoplastic cells to breach tumorigenic barriers

A neomorphic cancer cell-specific role of MAGE-A4 in trans-lesion synthesis

Trans-lesion synthesis (TLS) is an important DNA-damage tolerance mechanism that permits ongoing DNA synthesis in cells harbouring damaged genomes. The E3 ubiquitin ligase RAD18 activates TLS by promoting recruitment of Y-family DNA polymerases to sites of DNA-damage-induced replication fork stalling. Here we identify the cancer/testes antigen melanoma antigen-A4 (MAGE-A4) as a tumour cell-specific RAD18-binding partner and an activator of TLS. MAGE-A4 depletion from MAGE-A4-expressing cancer cells destabilizes RAD18. Conversely, ectopic expression of MAGE-A4 (in cell lines lacking endogenous MAGE-A4) promotes RAD18 stability. DNA-damage-induced mono-ubiquitination of the RAD18 substrate PCNA is attenuated by MAGE-A4 silencing. MAGE-A4-depleted cells fail to resume DNA synthesis normally following ultraviolet irradiation and accumulate γH2AX, thereby recapitulating major hallmarks of TLS deficiency. Taken together, these results demonstrate a mechanism by which reprogramming of ubiquitin signalling in cancer cells can influence DNA damage tolerance and probably contribute to an altered genomic landscape