Mammalian Egg Activation: The Roles of TRPV3 Channels and PLCZ1

Calcium channels at the plasma membrane have been suggested to mediate Ca2+ influx during egg activation. The transient receptor potential (TRP) Ca2+ channel, TRPV3, is differentially expressed in oocytes during maturation, being fully active at MII stage. Specific stimulation of TRPV3 channels in mouse eggs promotes Ca2+ influx sufficient to induce egg activation and parthenogenesis. In chapter 2, we explore the function and distribution dynamics of the TRPV3 channel protein during oocyte maturation. Using dsRNA, TrpV3 overexpression, and inhibitors of protein synthesis, we modified the native expression of the channel and showed that the TRPV3 protein is synthesized and translocated to the plasma membrane during maturation. We demonstrated that 2-APB at the concentration used to promote Ca2+ influx in eggs, specifically and reversibly targets TRPV3 channels, and does not block the IP3 receptor. Finally, we showed that TRPV3 channels functionally interact with the actin cytoskeleton indicating an actin-based regulation of its expression on the plasma membrane. Our results indicated that TRPV3 is a target of 2-APB in eggs, a condition that can be used to induce parthenogenesis. The association of endogenous TRPV3 channels with the actin cytoskeleton is a novel finding, and suggests that the rearrangements of actin that occur during maturation could regulate both plasma membrane presence and function of TRPV3 Ca2+ channels involved in oocyte maturation and fertilization. We show that TRPV3 is required for strontium influx because TRPV3-KO eggs failed to conduct Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation. In chapter 3, we conducted studies to determine if expression of the testis-specific PLCZ1 correlated with low success or fertilization failure after ICSI in patients with normal parameters after standard semen analysis (SA). The presence of PLCZ1 in sperm was ascertained using western blotting and immunofluorescence (IF) analysis. The ability of sperm to initiate changes in the intracellular concentrations of [Ca2+]i was also examined. Male partners of couples with failed or low success ICSI fertilization but with normal SA parameters showed low expression levels of PLCZ1 as determined by western blotting and reduced fluorescent signal during IF studies. In addition, fewer of these males’ sperm showed PLCZ1 expression and were able to initiate robust [Ca2+]i oscillations upon injection into eggs. In the second part of this chapter, we examined two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intra cytoplasmic sperm injection (ICSI). Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, converting Ile 489 into Phe (Ile489Phe). We showed that the mutation is deleterious, leading to absence of the protein in sperm. Injection of cRNA into GV and MII oocytes showed that the protein was mislocalized and produced highly abnormal Ca2+ transients and early embryonic arrest. Altogether these alterations are consistent with our patients’ sperm inability to induce oocyte activation and initiate embryo development. In contrast, no mutations were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans that the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes, opening the door for structure function studies to define the conserved amino acids on the b1 strand of the C2 domain that might regulate the selectivity of PLCZ1 for its lipid substrate(s)

ARHGAP18: A regulator of sprouting angiogenesis and junctional integrity

The formation of the vascular network requires a tightly controlled regulation of proangiogenic and stabilising signals, including VEGF and the endothelial junctions respectively. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies, including cancer. The Rho GTPases are major signaling regulators of the cytoskeleton and control functions such as cell migration and adhesion. We have identified that ARHGAP18 controls RhoC activity and functions as an endogenous negative regulator of angiogenesis by limiting pro-angiogeneic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumour vascularization and growth. Endogenous ARHGAP18 acts specifically via RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. The molecular mechanism governing the activation and translocation of ARHGAP18 to the cell periphery is at its infancy but phosphorylation and association with cortactin are involved. Aberrant Ras signaling is associated with cancer and vascular pathologies, such as angiosarcomas. Chronic HRas activation induces a transformed-like phenotype, with fibroblastic cell phenotype, increased cell migration and sprouting, dysregulated endothelial junctions and a partial alteration of endothelial-to-mesenchymal markers. This is associated with but not dependent on downregulation of ARHGAP18 expression. These findings highlight ARHGAP18 as a specific RhoGAP to fine-tune vascular morphogenesis, acting as a negative regulator to limit tip cell formation and promote junctional integrity to stabilize the angiogenic architecture

Perturbative QCD analysis of B(B→Xlνˉ)B(B\to Xl\bar{\nu}), charm yield <nc><n_c> in B decay, and τ(Λb)/τ(Bd)\tau (\Lambda_b)/\tau (B_d)

We apply perturbative QCD factorization theorems to inclusive heavy hadron decays, and obtain simultaneously a low semileptonic branching ratio $B(B\to Xl\bar{\nu})=10.16$, the average charm yield $=1.17$ per $B$ decay, a small lifetime ratio $\tau(\Lambda_b)/\tau(B_d)=0.78$, and the correct absolute decay widths of the $B$ meson and of the $\Lambda_b$ baryon.Comment: 13 pages in a latex file, numerical results are revised, We obtain $B(B\to Xl\bar{\nu})=10.16$, $=1.17$, $\tau(\Lambda_b)/\tau(B_d)=0.78$, and the correct absolute decay widths of the $B$ meson and of the $\Lambda_b$ baryo

The Effect of Service Quality and Price on International Airline Competition

A game theoretic model, integrated with passenger’s international choice behaviour, for the competition between international airlines is developed and used to identify the role of competing service quality. The empirical evidence suggests that safety, convenience, and service quality have a major influence on the choice decision of air passengers. Passengers respond strongly to decreases in price, safety, service comprehensiveness and increases in convenience. In a Cournot model, airlines are predicted to increase service quality, with China Airlines, a dominant carrier, the winner on safety and service quality. Foreign companies are beneficiaries in providing convenient service quality. In a Stakelberg model, all the airlines will increase safety and diversify service quality to a reference point. Foreign airlines will additionally be winners in safety and convenience with China Airlines a winner in diversifying service quality. This research can be used by the airline companies as a reference for making tactical decisions and gaining a competitive advantage in the air transportation market. By this, we can raise the quality of service of the aviation market as a whole and deliver a win-win situation to all stakeholders

Airborne Measurements of High Pollutant Concentration Events in the Free Troposphere over the West Coast of South Korea between 1997 and 2011

Aircrafts enable the direct measurement of chemical components in the free troposphere (FT). This study employed airborne measurements to examine the occurrences of high concentrations of SO2 and NOx in the FT over the coastal region west of the Seoul metropolitan area, South Korea. The data from a long-term (1997-2011) airborne measurement campaign were used to determine the meteorological conditions favorable for carrying these pollutants into the Seoul area. The back trajectory analyses of 21 instances of high FT pollutant concentration events showed ascending patterns from the major pollutant sources, mainly the industrial complexes in eastern China, in 9 instances and passing patterns in 12 instances. In the ascending instances, developing low-pressure systems over the source regions provide favorable conditions to uplift air pollutants from the surface into the FT. In the passing instances, an anomalous low-pressure system near the surface prevented airflows from descending into the boundary layer and upper-level anticyclonic systems helped to keep the ascending airflows in the FT. This study proposes the basic mechanisms for predicting air quality in the Seoul area, considering that air pollutants in the FT often entrain into the boundary layer to increase local concentrationsopen0

Industrialisasi Berbasis Pertanian sebagai Grand Stratedy Pembangunan Ekonomi Nasional

EnglishAs a developing economy Indonesia, should have a comprehensive integrated long-term development plan which may be used as the guideline in implementing its national economic development as well as an instrument for evaluating government accountability and credibility. The New Order regime had prepared its first and second long-term development plan for 1969-1993 and 1993-2018 successively. The twin plans, however, has led Indonesia to the 1997-1999 multi dimensions crises and is considered in appropriate in the existing new era of total reformation. It must be totally reconstructed. For this, public discussions on the need for the government to formulated the new grand strategy of national development have emerged, but up and down, in the last two years. As an active contribution to the public debase, this paper reviews previous, Indonesia development plans, others\u27 countries experiences as well as grand theories of economic development. Then it is suggested that the agricultural based industrialization may be the most suitable one for Indonesia. The new grand strategy should be decided based on a national concensus in order to avoid the practice of just for political rhetoric\u27s as was during the New Order regime. IndonesianBagi negara berkembang seperti Indonesia, rencana pembangunan jangka panjang komprehensif-integratif sangat di perlukan sebagai acuan pelaksanaan pembangunan dan sebagai salah satu instrumen akuntabilitas dan kredibilitas pemerintah. Pemerintahaan Orde Baru telah menyusun rancangan pembangunan jangka panjang tahap I dan II masing-masing untuk peiode 1969-1993 dan 1993-2018. Rencana jangka panjang yang disusun rejim Orde Baru tersebut terbukti membawa Indonesia kedalam krisis tahun 1997-1999 dan sudah tidak sesuai dalam era Reformasi sehingga perlu dirancang ulang. Dalam dua tahun terakhir sesungguhnya telah muncul wacana publik yang menuntut agar pemerintah segera menyusun grand strategy ( strategi besar ) pembangunan nasional. Sebagai bagian dari wacana tersebut, tulisan ini mereview tentang konsepsi strategi pembangunan selama Orde Baru, pengalaman beberapa negara lain pemikiran teoritis tentang strategi pembangunan ekonomi. Berdasarkan hasil review tersebut, disarankan agar industrialisasi berbasis pertanian (agricultural based industrialization) dijadikan sebagai strategi besar (grand strategy) pembangunan nasional. Strategi tersebut haruslah dijadikan sebagai konsensus nasional, sehingga tidak sekedar retorika politik seperti pada masa Orde Baru