転写因子SIRT1およびHAND2の着床機序への関与についての分子的解明

学位の種別:課程博士University of Tokyo(東京大学

Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells: an implicative role of SIRT1 in the ovary

<p>Abstract</p> <p>Background</p> <p>Resveratrol is a natural polyphenolic compound known for its beneficial effects on energy homeostasis, and it also has multiple properties, including anti-oxidant, anti-inflammatory, and anti-tumor activities. Recently, silent information regulator genes (Sirtuins) have been identified as targets of resveratrol. Sirtuin 1 (SIRT1), originally found as an NAD⁺-dependent histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. To date, the presence and physiological role of SIRT1 in the ovary are not known. Here we found that SIRT1 was localized in granulosa cells of the human ovary.</p> <p>Methods</p> <p>The physiological roles of resveratrol and SIRT1 in the ovary were analyzed. Immunohistochemistry was performed to localize the SIRT1 expression. SIRT1 protein expression of cultured cells and luteinized human granulosa cells was investigated by Western blot. Rat granulosa cells were obtained from diethylstilbestrol treated rats. The cells were treated with increasing doses of resveratrol, and subsequently harvested to determine mRNA levels and protein levels. Cell viability was tested by MTS assay. Cellular apoptosis was analyzed by caspase 3/7 activity test and Hoechst 33342 staining.</p> <p>Results</p> <p>SIRT1 protein was expressed in the human ovarian tissues and human luteinized granulosa cells. We demonstrated that resveratrol exhibited a potent concentration-dependent inhibition of rat granulosa cells viability. However, resveratrol-induced inhibition of rat granulosa cells viability is independent of apoptosis signal. Resveratrol increased mRNA levels of SIRT1, LH receptor, StAR, and P450 aromatase, while mRNA levels of FSH receptor remained unchanged. Western blot analysis was consistent with the results of quantitative real-time RT-PCR assay. In addition, progesterone secretion was induced by the treatment of resveratrol.</p> <p>Conclusions</p> <p>These results suggest a novel mechanism that resveratrol could enhance progesterone secretion and expression of luteinization-related genes in the ovary, and thus provide important implications to understand the mechanism of luteal phase deficiency.</p

Renormalized Bosonic Interaction of Excitons

An effective bosonic Hamiltonian of $1s$ excitons with ``spin'' degrees of freedom in two dimension is obtained through a projection procedure, starting from a conventional electron-hole Hamiltonian ${\cal H}_{eh}$. We first demonstrate that a straightforward transformation of ${\cal H}_{eh}$ into a Hamiltonian of bosonic excitons does not give the two-body interaction between an ``up-spin'' exciton and a ``down-spin'' exciton, which are created by the left- and right-circularly polarized light beams, respectively. We then show that this interaction is generated through a projection procedure onto the subspace spanned by $1s$ excitons, as a renormalization effect coming from higher exciton states. The projection also renormalizes the interaction between $1s$ excitons with the same spins by a large amount. These renormalization effects are crucial for the polarization dependence of the optical responses from semiconductors. The present theory gives the microscopic foundation of the phenomenology that was successfully applied to the analysis of four-wave mixing experiments in GaAs quantum wells strongly coupled to the radiation field in a high-Q micro cavity.Comment: 2 figure

Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach

The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA) library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR) dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A) under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS) resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA) library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses