PSU Knowledge Bank Community:http://kb.psu.ac.th:8080/psukb/handle/2010/56312024-02-28T04:48:04Z2024-02-28T04:48:04ZIn Vitro and In Silico Analyses of Biomolecular Regulation of Liposomal CRE-SD on Osteoclastogenesis via a Canonical NF-κB Signaling PathwaySompot Jantarawonghttp://kb.psu.ac.th:8080/psukb/handle/2016/192312023-12-20T04:01:27Z2023-01-01T00:00:00ZTitle: In Vitro and In Silico Analyses of Biomolecular Regulation of Liposomal CRE-SD on Osteoclastogenesis via a Canonical NF-κB Signaling Pathway
Authors: Sompot Jantarawong
Abstract: Curcuminoids, namely curcumin, demethoxycurcumin, and bisdemethoxycurcumin, are the major active compounds found in Curcuma longa L. (turmeric). Although their suppressive effects on bone resorption have been demonstrated, their pharmacokinetic disadvantages remain a concern. Herein, solid dispersion of a curcuminoid-rich extract (CRE), comprising such curcuminoids, was utilized to prepare CRE-SD; subsequently, liposome encapsulation of the CRE-SD was performed to yield liposomal CRE-SD. In vitro release assessment revealed that a lower cumulative mass percentage of CRE-SD was released from liposomal CRE-SD than from CRE-SD samples. After culture of murine RANKL-stimulated RAW 264.7 macrophages, in vitro examinations confirmed that liposomal CRE-SD may impede osteoclastogenesis by suppressing p65 and IκBα phosphorylation, together with nuclear translocation and transcriptional activity of phosphorylated p65. Blind docking simulations showed the high binding affinity between curcuminoids and the IκBα/p50/p65 protein complex, along with many intermolecular interactions, which corroborated the in vitro findings. Therefore, liposomal CRE-SD can inhibit osteoclastogenesis via the canonical NF-κB signaling pathway, suggesting its pharmacological potential for treating bone diseases with excessive osteoclastogenesis.
Description: Master of Science (Molecular Biotechnology and Bioinformatics), 20232023-01-01T00:00:00ZPreservation of Spermatogonia cell in ShrimpNatthida Rakbanjonghttp://kb.psu.ac.th:8080/psukb/handle/2016/191462023-12-06T07:22:48Z2022-01-01T00:00:00ZTitle: Preservation of Spermatogonia cell in Shrimp
Authors: Natthida Rakbanjong
Abstract: Prawns and shrimp are the most interesting group of resources, they are also one of the most widely traded fishery products on the international market and they are one of the few that can be considered a "commodity," generating significant economic benefits, especially for many developing countries. In Thailand, shrimp culture (such as giant freshwater prawn, banana shrimp and black tiger shrimp) is economically important of market. Nowadays, the number of shrimps in natural is decreased. Therefore, the conservation of shrimp species is important to prevention. Cryopreservation has been used to preserve germ cell of many endangered species. However, this method has not been established for cryopreservation of spermatogonia cell in shrimp. Therefore, in this study aimed to identify the spermatogonia cells developmental stage for cryopreservation and optimize the equilibration time (15, 30 and 60 min) for slow freezing method, type and concentration of cryoprotectants (dimethyl sulfoxide (DMSO), glycerol (GLY), and magnesium chloride (MgCl_2) at 5%, 10%, and 15%), and thawing temperature (10 and 27 °C) for preserve spermatogonia cell of giant freshwater prawn, banana shrimp and black tiger shrimp by compare between slow freezing and vitrification methods for a long-term preservation of spermatogonia cells. The viability and recovery percentage of fresh and frozen spermatogonia cell of shrimp were assessed by staining spermatogonia cell with trypan blue.
The result was obtained in equilibration time tested of giant freshwater prawn spermatogonia cells were incubated in cryomedium containing cryoprotectants (10% DMSO, 10% GLY and 10% MgCl_2) and extender for 15, 30 and 60 min, there was not significantly difference. In next experiment, among three cryoprotectants tested, the best result of viability and recovery percentage was obtained with 10% DMSO for spermatogonia cell of giant freshwater prawn and banana shrimp and 10% GLY for spermatogonia cell of black tiger shrimp. The best thawing was found at 10 ᵒC for giant freshwater prawn, banana shrimp and black tiger shrimp. For long-term cryopreservation, the recovery of spermatogonia cells preserved in liquid nitrogen at 6 months with vitrification method were significantly higher than of preserved with slow freezing method in all shrimps and the total cells were observed after preserved not apoptosis during cryopreservation for both methods. This study provide evidence that spermatogonia cell in giant freshwater prawn, banana shrimp and black tiger shrimp can be preserved long-term in liquid nitrogen. Importantly, our study demonstrate that cryopreservation can be successfully performed without requiring no special or expensive equipment (freezing container and -80 ᵒC freezer) by using vitrification method.
Description: Master of Science (Molecular Biology and Bioinformatics (International Program)), 20222022-01-01T00:00:00ZControlling of Reproductive Organs Maturation in ShrimpKunwadee Palasinhttp://kb.psu.ac.th:8080/psukb/handle/2016/190162023-11-07T02:37:21Z2018-01-01T00:00:00ZTitle: Controlling of Reproductive Organs Maturation in Shrimp
Authors: Kunwadee Palasin
Abstract: Ribosomal protein was known to play a function in protein synthesis. Recently, many studies reported the extraribosomal activity including DNA repair, apoptosis, regulation of translation, controlling of development and red blood cell development. Ribosomal protein L10a (Rpl10a) encoded by the rpl10a gene has been reported the secondary function during embryogenesis, organogenesis and ovarian development. In this study, the activity of Rpl10a protein on reproductive organs development was investigated. The recombinant His-Rpl10a protein (rRpl10a) was injected into shrimps to promote the ovarian development in shrimps. The concentration at 180 μg of His-rRpl10a per shrimp was the effective dose to stimulate ovaries to reach to stages I and II of ovarian maturation within 7 days post injection. In addition, the spermatogenesis in shrimp and mouse were stimulated by His-rRpl10a protein in vitro. The early stage marker gene expressions (Dmrt1 in shrimp or Rhau in mouse) were decreased, while Prm2, late-stage marker gene was upregulated. The cell proliferation was also confirmed in rRpl10a treated testis.
Furthermore, the other functions of rpl10a on anemia using rpl10a mutant zebrafish (Danio rerio) as a model were also studied. The rpl10a-deficient embryos displayed the abnormality phenotypes including thinner yolk extension, smaller eyes, shorter body length and bent tail at 25 hpf (hour-post fertilization). Besides, rpl10a gene deficiency showed the phenotypes as a bigger yolk sac, edema, smaller eyes, melanophore pigment reduction, and a curved tail at 50 hpf. These morphological abnormalities were recovered by rpl10a full-length mRNA. This result indicated that rpl10a gene is essential for early embryogenic development. Moreover, loss activity of rpl10a affected growth retardation and embryonic lethality within 3-7 dpf. Anemic phenotype and hemoglobin activity were observed both knockdown and knockout model. The hemoglobin marker genes including gata1, hbae3, and hbbe1 have declined expression, whereas tp53 transcript was increased. These findings supported that Rpl10a protein might play an extra-function in anemia. Knockdown of rpl10a gene also affected the low expression of primordial germ cell (PGC) marker genes (nanos1 and vasa) significantly. Interestingly, these findings suggested that Rpl10a protein is necessary on the early stage of reproductive organs development, primordial germ cells development, embryogenesis, and hemoglobin synthesis.
Description: Doctor of Philosophy (Molecular Biology and Bioinformatics), 20182018-01-01T00:00:00ZExtra-Function of Ribosomal Protein L10a via Insulin Signaling PathwayNetnapa Chaichanithttp://kb.psu.ac.th:8080/psukb/handle/2016/181612023-05-16T03:14:06Z2021-01-01T00:00:00ZTitle: Extra-Function of Ribosomal Protein L10a via Insulin Signaling Pathway
Authors: Netnapa Chaichanit
Abstract: Ribosomal protein L10a (RpL10a) is one of several ribosomal proteins which has functions besides protein synthesis in the ribosome. Many reports recently revealed the extra–ribosomal function of RpL10a, including a role in oogenesis, spermatogenesis, ovarian development, organogenesis, and embryogenesis. In Drosophila melanogaster, the overexpression of the shrimp RpL10a resulted in the loss of red pigment in the eye center. This finding suggests that RpL10a also plays a role in cell proliferation. In contrast, the lack of RpL10Ab (RpL10Ab-/-) in the germline of fruit flies showed the disappearance of the follicle cells surrounding the egg chamber, similar to the insulin receptor mutants (InR-/-), indicating that RpL10a plays a role in oogenesis. Normally, InR is the transmembrane receptor of the insulin signaling pathway that plays a key role in cell development, cell division, metabolism regulation especially the role in glucose homeostasis regulation. Therefore, RpL10a may be involved in the step of regulation of InR to regulate cell development and metabolism.
To determine whether RpL10a is involved in regulating glucose homeostasis, carbohydrate content was observed in the eyes of normal and overexpressed flies by using an anthrone-sulfuric acid colorimetric assay. In this study, the function of shrimp RpL10a involved in insulin receptor (InR) and carbohydrate metabolism were investigated. In the eyes of RpL10a–overexpressed flies, the expression of InR was extensively increased in mRNA level and protein level, determined by qPCR and immunohistochemistry, respectively. Moreover, In the RpL10a–overexpressed eyes of the mutant fly showed the highly phosphorylated insulin signaling mediators such as Akt and dFOXO, whereas the mutant flies showed the reduction of the glycogen. This finding revealed that shrimp RpL10a affects cell proliferation and glycogenolysis through the insulin signaling pathway. Besides, The interaction between RpL10a and InR was performed both in silico and in vitro binding. The shrimp RpL10a protein and the human InR protein were obtained as a silico binding model. The ClusPro software indicated that RpL10a could interact with InR at the difference site on fibronectin type III (FnIII) domain of insulin bound to InR. Then, to reduce the culture time and to control the culture condition easily, the study of shrimp RpL10a function was performed in CHO-S cell line. The in vitro study results, including pull-down assay and immunofluorescence assay, confirmed the binding of the proteins. The binding of the two proteins could stimulate the insulin signaling pathway by binding at the FnIII domain of InR and increased the carbohydrate utilization while the lipid metabolism was unchanged. Also, the effect of RpL10a on glucose metabolism was investigated in insulin resistance conditions. The CHO-S cells were induced to insulin resistance under high glucose conditions before stimulating with RpL10a. After the healthy cells and insulin-resistant cells (IRCs) were incubated with RpL10a, RpL10a could induce glucose uptake by the IRCs better than healthy cells.
Moreover, RpL10a could induce the expression of hexokinase in glycolysis and decreased the expression of glucose-6-phosphatase in gluconeogenesis. These findings suggested that RpL10a could alleviate the insulin resistance by inducing the glucose uptake into the cells, activating glycolysis, and suppressing glucose synthesis in IRCs induced under high glucose conditions. From this important function, RpL10a may have a function as an insulin-mimetic for insulin-resistant diabetes treatment.
Description: Doctor of Philosophy (Molecular Biology and Bioinformatics), 20212021-01-01T00:00:00Z