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https://ptsldigital.ukm.my/jspui/handle/123456789/500527| Title: | Study effects of the disruption of RAD50 on zebrafish embryo development and microglia cell lines |
| Authors: | Nahid Khalili (P86024) |
| Supervisor: | Mohd Shazrul Fazry Sa`ariwijaya, Dr. |
| Keywords: | DNA RAD50 Embryo Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia |
| Issue Date: | 8-Sep-2021 |
| Description: | DNA damage occurs daily in the cells of organisms. A variety of proteins and posttranslational events are involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs) to maintain genome integrity. MRE11-RAD50-NBS1 (MRN complex) play a key role in this process. The MRN complex rapidly recognizes and localizes DNA DSB where it recruits and assists in ATM activation. RAD50 mutations can lead to microcephaly, mental retardation and growth retardation in human. The ABC domain of RAD50 exhibits both ATPase and adenylate kinase activity, which are essential for all known functions of the MRN complex. In this study, the function of RAD50 during embryonic development was investigated using zebrafish, due to its fast development and transparency nature. In silico analysis revealed that zebrafish RAD50 protein sequence has 70% similarity with human RAD50 protein. Knock out (KO) RAD50 embryos with CRISPR-Cas9 show embryonic lethality. Two types of abnormalities were seen in the head in the injected CRISPR-Cas9 group. To further investigate the significance of RAD50 in DNA damage stress during embryogenesis, we treated zebrafish embryos with MIRIN (35μM) and irradiated them with Gamma ray (15 Gy). The result of the time point test shows that inhibition of RAD50 and ATM proteins in the early stage of embryonic development (1 hpf) has the greatest effect on growth and development. Inhibition of RAD50 at early embryonic stages has a stronger effect on zebrafish embryo growth and development. Embryos exposed to RAD50 inhibitor and Gamma ray, showed impaired brain development (microcephaly). The reduced mass of brain tissue in the midbrain and hindbrain, possibly caused by the increased number of apoptotic cells, resulted in microcephaly and insufficient development of zebrafish embryos. Additional study using BV2 microglial model cell lines showed that exposure of the cells to MIRIN caused a reduction in cell viability after 24, 48 and 72 hours. The morphology and phagocytic activity of BV2 cells under different culture conditions were highly variable, with the untreated cells being more amoeboid in shape. Flow cytometry showed that 24 h post treatment and irradiation, apoptosis was significantly increased. These data suggest that disruption of the RAD50 gene can affect the development of organisms and that the RAD50 protein in the zebrafish embryo is essential for proper brain development.,Ph.D. |
| Pages: | 165 |
| Publisher: | UKM, Bangi |
| Appears in Collections: | Faculty of Science and Technology / Fakulti Sains dan Teknologi |
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| File | Description | Size | Format | |
|---|---|---|---|---|
| ukmvital_123123+SOURCE1+SOURCE1.0.PDF Restricted Access | 3.72 MB | Adobe PDF |  View/Open |
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