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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李士傑 | zh_TW |
| dc.contributor.advisor | Shyh-Jye Lee | en |
| dc.contributor.author | 熊俐伃 | zh_TW |
| dc.contributor.author | Li-Yu Hsiung | en |
| dc.date.accessioned | 2021-07-10T21:53:08Z | - |
| dc.date.available | 2024-08-15 | - |
| dc.date.copyright | 2019-08-23 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | Bordeaux, J., Welsh, A., Agarwal, S., Killiam, E., Baquero, M., Hanna, J., Anagnostou, V., Rimm, D., 2010. Antibody validation. Biotechniques 48, 197-209.
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Nakayama, T., Al-Maawali, A., El-Quessny, M., Rajab, A., Khalil, S., Stoler, J.M., Tan, W.H., Nasir, R., Schmitz-Abe, K., Hill, R.S., Partlow, J.N., Al-Saffar, M., Servattalab, S., LaCoursiere, C.M., Tambunan, D.E., Coulter, M.E., Elhosary, P.C., Gorski, G., Barkovich, A.J., Markianos, K., Poduri, A., Mochida, G.H., 2015. Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination. Am J Hum Genet 96, 709-719. Novorol, C., Burkhardt, J., Wood, K.J., Iqbal, A., Roque, C., Coutts, N., Almeida, A.D., He, J., Wilkinson, C.J., Harris, W.A., 2013. Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression. Open Biol 3, 130065. Park, D.W., Kim, S.S., Nam, M.K., Kim, G.Y., Kim, J., Rhim, H., 2011. Improved recovery of active GST-fusion proteins from insoluble aggregates: solubilization and purification conditions using PKM2 and HtrA2 as model proteins. BMB Rep 44, 279-284. Prykhozhij, S.V., Steele, S.L., Razaghi, B., Berman, J.N., 2017. A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish. Dis Model Mech 10, 811-822. Reversade, B., Escande-Beillard, N., Dimopoulou, A., Fischer, B., Chng, S.C., Li, Y., Shboul, M., Tham, P.Y., Kayserili, H., Al-Gazali, L., Shahwan, M., Brancati, F., Lee, H., O'Connor, B.D., Schmidt-von Kegler, M., Merriman, B., Nelson, S.F., Masri, A., Alkazaleh, F., Guerra, D., Ferrari, P., Nanda, A., Rajab, A., Markie, D., Gray, M., Nelson, J., Grix, A., Sommer, A., Savarirayan, R., Janecke, A.R., Steichen, E., Sillence, D., Hausser, I., Budde, B., Nurnberg, G., Nurnberg, P., Seemann, P., Kunkel, D., Zambruno, G., Dallapiccola, B., Schuelke, M., Robertson, S., Hamamy, H., Wollnik, B., Van Maldergem, L., Mundlos, S., Kornak, U., 2009. Mutations in PYCR1 cause cutis laxa with progeroid features. Nat Genet 41, 1016-1021. Shaughnessy, D.T., McAllister, K., Worth, L., Haugen, A.C., Meyer, J.N., Domann, F.E., Van Houten, B., Mostoslavsky, R., Bultman, S.J., Baccarelli, A.A., Begley, T.J., Sobol, R.W., Hirschey, M.D., Ideker, T., Santos, J.H., Copeland, W.C., Tice, R.R., Balshaw, D.M., Tyson, F.L., 2014. Mitochondria, energetics, epigenetics, and cellular responses to stress. Environ Health Perspect 122, 1271-1278. Shokolenko, I.N., Alexeyev, M.F., 2015. Mitochondrial DNA: A disposable genome? Biochim Biophys Acta 1852, 1805-1809. Szoka, L., Karna, E., Hlebowicz-Sarat, K., Karaszewski, J., Palka, J.A., 2017. Exogenous proline stimulates type I collagen and HIF-1alpha expression and the process is attenuated by glutamine in human skin fibroblasts. Mol Cell Biochem 435, 197-206. Tao, H., Liu, W., Simmons, B.N., Harris, H.K., Cox, T.C., Massiah, M.A., 2010. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. Biotechniques 48, 61-64. Terburgh, K., Lindeque, Z., Mason, S., van der Westhuizen, F., Louw, R., 2019. Metabolomics of Ndufs4(-/-) skeletal muscle: Adaptive mechanisms converge at the ubiquinone-cycle. Biochim Biophys Acta Mol Basis Dis 1865, 98-106. Zaki, M.S., Bhat, G., Sultan, T., Issa, M., Jung, H.J., Dikoglu, E., Selim, L., I, G.M., Abdel-Hamid, M.S., Abdel-Salam, G., Marin-Valencia, I., Gleeson, J.G., 2016. PYCR2 Mutations cause a lethal syndrome of microcephaly and failure to thrive. Ann Neurol 80, 59-70. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77260 | - |
| dc.description.abstract | 吡咯啉-5-羧酸鹽還原酶(Pyrroline-5-carboxylate reductase, PYCR)普遍被認為是管家酵素，可與NAD(P)H共同催化脯氨酸的生成。在斑馬魚有三種PYCR同源蛋白，分別是Pycr1a、Pycr1b與Pycr3。分別由Pycr1a/Pycr1b 和 Pycr3兩條路徑來催化脯氨酸的生成。Pycr1a與Pycr1b位於粒線體內而Pycr3位於細胞質中。研究顯示Pycr1a與Pycr1b可保護細胞抵禦氧化傷害，而Pycr1a與Pycr1b基因突變皆會影響粒線體的功能，進而造成早衰症及畸形小頭症等相關疾病，而本論文之目的乃探討Pycr1b在斑馬魚發育過程中之基因及蛋白質表現及分布。在此論文中，我以反轉錄聚合酶鏈式反應(RT-PCR)發現pycr1b基因從胚胎早期發育階段與成魚所有組織中皆有高度表現。而在原位雜合(in situ hybridization)實驗中我觀測到pycr1b基因在腦部及其特定區域有高度表達。我以Pycr1b C端約一半的序列製作了一個GST融合重組蛋白，並以之為抗原製作了一支抗體。利用西方墨點法，此Pycr1b抗體可以專一的辨識到一條約34 kDa的斑馬魚粒線體蛋白，而在一天大的斑馬魚胚胎粒線體蛋白中Pycr1b的條帶在Pycr1b (-/-)基因剔除魚比野生型的魚明顯減弱。此證明了此Pycr1b (-/-)突變魚是Pycr1b基因缺失突變魚，後續可更進一步用以檢測Pycr1b功能並進行其他實驗。 | zh_TW |
| dc.description.abstract | Pyrroline-5-carboxylate reductase (PYCR) is a housekeeping enzyme catalyzing the reduction of delta (1)-pyrroline-5-carboxylate (P5C) to proline with NAD(P)H as a cofactor. In zebrafish, there are three PYCR family genes, including pycr1a, pycr1b and pycr3, to catalyze the production of proline in two separate reactions by Pycr1a/Pycr1b or Pycr3. Pycr1a and Pycr1b are located in mitochondria and Pycr3 is a cytosolic protein. Pycr1a and Pycr1b can protect cells from overt oxidative stress. Mutations in the pycr1a or pycr1b gene can alter mitochondrial function and result in progeria and microcephaly-related diseases. The objective of this thesis is to understand the spatial and temporal expression of pycr1b gene during development in zebrafish. Using RT-PCR, I found that the Pycr1b is highly expressed from early developmental stages and different adult tissues. Embryos or larval zebrafish were fixed at designated stages and subjected to whole-mount in situ hybridization analysis against pycr1b. I found that pycr1b was highly expressed in the brain. To carry out the project, I also generated and characterized a Pycr1b antibody specifically recognizing a GST-fusion protein containing the C-terminal half of zebrafish Pycr1b. By Western blotting, the generated Pycr1b antibody can recognized a ~ 34 kDa protein from a mitochondria fraction of zebrafish embryos. I further observed that the Pycr1b band was weaker in pycr1b (-/-) than that of wildtype 1-day old zebrafish embryos. This suggests that the pycr1b (-/-) mutant is a null mutant which can be used for Pycr1b functional assay. The Pycr1b antibody may also be used to further analyze protein expression pattern and other functional test for Pycr1b. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:53:08Z (GMT). No. of bitstreams: 1 ntu-108-R06b21015-1.pdf: 5017828 bytes, checksum: f7ef378cb3cd61f86ea2680824ab1728 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | CHINESE ABSTRACT ................................................................................... 7
ABSTRACT …………………………………………………………………………………………8 INTRODUCTION …………………………………………………………………………………9 MATERIALS and METHODS .......................................................................13 RNA extraction and complementary DNA preparation ...............................13 Cloning of zebrafish pycr1b..........................................................................14 Induction and purification of GST-Pycr1b fusion protein............................15 Solubilization of bacterial protein................................................................16 Batch purification of GST fusion protein......................................................17 Zebrafish maintenance..................................................................................17 Microinjection...............................................................................................18 Preparation of mitochondrial fraction from zebrafish embryos...................18 Coomassie blue staining……………………………………………………………………..20 Negative staining……………………………………………………………………………….20 Protein identification by Mass spectrometry.................................................22 Western blotting.............................................................................................23 Production of polyclonal antibody.................................................................25 Whole-mount in situ hybridization.................................................................25 Screening of Pycr1b(-/-) mutant fish by PAGE analysis................................28 Immunohistochemistry (IHC) ........................................................................29 Immunoprecipitation…………………………………………………………………………..30 Statistical Analysis……………………………………………………………………………..31 RESULTS ..........................................................................................................32 Amino acid sequence alignment and phylogenetic tree of PYCR1, PYCR2 and PYCR3 orthologs.....................................................................................32 Spatial and temporal expression of PYCR1b....................................................32 Whole-mount in situ hybridization......................................................................34 Validation of zebrafish pycr1b mutants...............................................................35 Pycr1a(-/-) mutant females exhibited lower body weight and body length.........36 Reproduction efficiency of pycr1a(-/-) , pycr1b(-/- )and WT fish........................37 Pycr1b, Pycr1b N-terminal and Pycr1b C-terminal GST fusion protein induction, purification and antibody production.................................................38 Characterization of Pycr1b_C anti-serum…………………………………………………..40 Mass spectrometry……………………………………………………………………………………41 Pycr1b (-/-) mutant is a null mutant.....................................................................42 Efficacy testing for Pycr1b_C anti-serum for immunoprecipitation....................43 Expression pattern analysis by immunohistochemistry against Pycr1b in developing zebrafish embryos and larvae............................................................43 DISCUSSION ........................................................................................................45 REFERENCES.......................................................................................................48 TABLES ………………………………………………………………………………………………….51 Table 1. Primers used in PYCR1b C’ fusion protein construct production………51 Table 2. Primers used in PYCR1a and PYCR1b zebrafish sequencing…………….52 Table 3. Primers used in Real-Time PCR…………………………………………………….53 Table 4. Mass spectrometry (MS) results to zebrafish whole lysate protein……..54 FIGURES …………………………………………………………………………………………………56 Figure 1. Amino acid sequence and phylogenetic tree analyses of pycr family genes.....................................................................................................................56 Figure 2. RT-PCR expression analysis of pycr1b during embryogenesis............58 Figure 3. RT-PCR expression analysis of pycr1b in adult tissues…………………..60 Figure 4. Pycr1b protein expression during embryonic development and larvae stages…………………………………………………………………………………………..61 Figure 5. Expression pattern analysis by whole-mount in situ hybridization (WISH) against pycr1b in developing zebrafish embryos and larvae..................63 Figure 6. Validation of pycr1b (-/-) mutation.......................................................65 Figure 7. Reduced body weights and lengths in pycr1a (-/- ) fish........................67 Figure 8. Comparison of embryo clutch size, fertilization and abnormality rate among pycr1a-/- , pycr1b-/- and wildtype zebrafish………………………………………70 Figure 9. Examination of survival rate and head development in pycr1a and pycr1b mutants......................................................................................................72 Figure 10. Multiple sequence alignments among Pycr1a, Pycr1b and Pycr3......73 Figure 11. Schematic pycr construct designs........................................................74 Figure 12. PCR cloning, subcloning and induction of GST-pycr1b......................76 Figure 13. PCR cloning and sequence validation of 5’- and 3’-terminal halves of Pycr1b GST-fusion proteins...............................................................................78 Figure 14. Induction and analysis of N- and C-terminal halves of Pycr1b GST-fusion proteins…………………………………………………………………………………..80 Figure 15. Induction and solubilization of GST-Pycr1b_C fusion protein………..81 Figure 16. Failure in purification GST-Pycr1b_C fusion protein.........................83 Figure 17. Preparation of SDS-PAGE gel containing GST-Pycr1b_C fusion protein as immunogen for antibody production.....................................................84 Figure 18. Preparation of gel for protein identification by Mass spectrometry....85 Figure 19. Characterization of Pycr1b-C anti-serum............................................86 Figure 20. Quantitation of Pycr1b/Pycr1a by immunoblotting.............................88 Figure 21. Testing of immunoprecipitation (IP) by anti-Pycr1b_C…………………..90 Figure 22. Epi-fluorescent microscope examination of Pycr1b expression in zebrafish embryos and larvae by immunohistochemistry…………………………………92 Figure 23. Confocal microscope examination of Pycr1b expression in zebrafish embryos and larvae by immunohistochemistry.......................................................94 | - |
| dc.language.iso | en | - |
| dc.subject | 斑馬魚 | zh_TW |
| dc.subject | pycr1a | zh_TW |
| dc.subject | pycr1b | zh_TW |
| dc.subject | 脯氨酸 | zh_TW |
| dc.subject | 衰老 | zh_TW |
| dc.subject | 早衰症 | zh_TW |
| dc.subject | 畸型小頭症 | zh_TW |
| dc.subject | microcephaly | en |
| dc.subject | aging | en |
| dc.subject | pycr1a | en |
| dc.subject | pycr1b | en |
| dc.subject | progeria | en |
| dc.subject | proline | en |
| dc.subject | zebrafish | en |
| dc.title | 二號吡咯啉-5-羧酸鹽還原酶之表達與功能性分析 | zh_TW |
| dc.title | Expression and functional analysis of Pyrroline-5-carboxylate reductase 1b | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 郭典翰;蔡素宜 | zh_TW |
| dc.contributor.oralexamcommittee | Dian-Han Kuo;Su-Yi Tsai | en |
| dc.subject.keyword | 斑馬魚,pycr1a,pycr1b,脯氨酸,衰老,早衰症,畸型小頭症, | zh_TW |
| dc.subject.keyword | zebrafish,pycr1b,pycr1a,proline,aging,progeria,microcephaly, | en |
| dc.relation.page | 95 | - |
| dc.identifier.doi | 10.6342/NTU201902510 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-13 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生命科學系 | - |
| 顯示於系所單位： | 生命科學系 | |
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