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  1. NTU Theses and Dissertations Repository
  2. 生命科學院
  3. 分子與細胞生物學研究所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95864
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor蔡皇龍zh_TW
dc.contributor.advisorHuang-Lung Tsaien
dc.contributor.author劉展廷zh_TW
dc.contributor.authorJhan-Ting Liuen
dc.date.accessioned2024-09-18T16:26:17Z-
dc.date.available2024-09-19-
dc.date.copyright2024-09-18-
dc.date.issued2024-
dc.date.submitted2024-08-09-
dc.identifier.citation1 Van Drunen, R. & Eckel-Mahan, K. Circadian rhythms of the hypothalamus: from function to physiology. Clocks & sleep 3, 189-226 (2021).
2 Fonken, L. K. & Nelson, R. J. The effects of light at night on circadian clocks and metabolism. Endocrine reviews 35, 648-670 (2014).
3 Golombek, D. A. & Rosenstein, R. E. Physiology of circadian entrainment. Physiological reviews 90, 1063-1102 (2010).
4 Walker, W. H., Walton, J. C., DeVries, A. C. & Nelson, R. J. Circadian rhythm disruption and mental health. Translational psychiatry 10, 1-13 (2020).
5 Fujii, S., Krishnan, P., Hardin, P. & Amrein, H. Nocturnal male sex drive in Drosophila. Current Biology 17, 244-251 (2007).
6 Kaniewska, M. M., Vaněčková, H., Doležel, D. & Kotwica-Rolinska, J. Light and temperature synchronizes locomotor activity in the linden bug, Pyrrhocoris apterus. Frontiers in Physiology 11, 242 (2020).
7 George, R. & Stanewsky, R. Peripheral sensory organs contribute to temperature synchronization of the circadian clock in Drosophila melanogaster. Frontiers in Physiology 12, 622545 (2021).
8 Chuman, Y., Matsushima, A., Shimohigashi, Y. & Shimohigashi, M. in Peptides: The Wave of the Future: Proceedings of the Second International and the Seventeenth American Peptide Symposium, June 9–14, 2001, San Diego, California, USA. 797-798 (Springer).
9 SAUNDERS, D. S. Circadian rhythms and the evolution of photoperiodic timing in insects. Physiological Entomology 34, 301-308 (2009).
10 Yoshii, T., Fujii, K. & Tomioka, K. Induction of Drosophila behavioral and molecular circadian rhythms by temperature steps in constant light. Journal of biological rhythms 22, 103-114 (2007).
11 Price, J., Dembinska, M., Young, M. & Rosbash, M. Suppression of PERIOD protein abundance and circadian cycling by the Drosophila clock mutation timeless. The EMBO journal 14, 4044-4049 (1995).
12 Sehgal, A. et al. Rhythmic expression of timeless: a basis for promoting circadian cycles in period gene autoregulation. Science 270, 808-810 (1995).
13 Ruoff, P., Christensen, M. K. & Sharma, V. K. PER/TIM-mediated amplification, gene dosage effects and temperature compensation in an interlocking-feedback loop model of the Drosophila circadian clock. Journal of theoretical biology 237, 41-57 (2005).
14 Leloup, J.-C. & Goldbeter, A. A model for circadian rhythms in Drosophila incorporating the formation of a complex between the PER and TIM proteins. Journal of biological rhythms 13, 70-87 (1998).
15 Abruzzi, K. C. et al. Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression. Genes & development 25, 2374-2386 (2011).
16 Rutila, J. E. et al. CYCLE is a second bHLH-PAS clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell 93, 805-814 (1998).
17 Kim, E. Y. & Edery, I. Balance between DBT/CKIε kinase and protein phosphatase activities regulate phosphorylation and stability of Drosophila CLOCK protein. Proceedings of the National Academy of Sciences 103, 6178-6183 (2006).
18 Teles-de-Freitas, R., Rivas, G. B., Peixoto, A. A. & Bruno, R. V. The summer is coming: nocte and timeless genes are influenced by temperature cycles and may affect Aedes aegypti locomotor activity. Frontiers in Physiology 11, 614722 (2020).
19 Costa, E. A. P. d. A., Santos, E. M. d. M., Correia, J. C. & Albuquerque, C. M. R. d. Impact of small variations in temperature and humidity on the reproductive activity and survival of Aedes aegypti (Diptera, Culicidae). Revista Brasileira de Entomologia 54, 488-493 (2010).
20 Yuan, Q., Metterville, D., Briscoe, A. D. & Reppert, S. M. Insect cryptochromes: gene duplication and loss define diverse ways to construct insect circadian clocks. Molecular biology and evolution 24, 948-955 (2007).
21 Lamia, K. A. et al. AMPK regulates the circadian clock by cryptochrome phosphorylation and degradation. Science 326, 437-440 (2009).
22 Ceriani, M. F. et al. Light-dependent sequestration of TIMELESS by CRYPTOCHROME. Science 285, 553-556 (1999).
23 Koh, K., Zheng, X. & Sehgal, A. JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS. Science 312, 1809-1812 (2006).
24 Yoshii, T. et al. Drosophila cryb mutation reveals two circadian clocks that drive locomotor rhythm and have different responsiveness to light. Journal of insect physiology 50, 479-488 (2004).
25 Wong, J. C. et al. Differential roles for cryptochromes in the mammalian retinal clock. The FASEB Journal 32, 4302 (2018).
26 Fribourgh, J. L. et al. Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK: BMAL1 to control circadian timing. Elife 9, e55275 (2020).
27 Au, D. D. et al. Nocturnal mosquito Cryptochrome 1 mediates greater electrophysiological and behavioral responses to blue light relative to diurnal mosquito Cryptochrome 1. Frontiers in Neuroscience 16, 1042508 (2022).
28 Bezerra, J. R. A., Bruno, R. V. & Araripe, L. O. Males of Aedes aegypti show different clock gene expression profiles in the presence of conspecific females. Parasites & Vectors 15, 374 (2022).
29 McAlear, S. D., Kraft, T. W. & Gross, A. K. 1 rhodopsin mutations in congenital night blindness. Retinal Degenerative Diseases: Laboratory and Therapeutic Investigations, 263-272 (2010).
30 Rao, V. R., Cohen, G. B. & Oprian, D. D. Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness. Nature 367, 639-642 (1994).
31 Humberg, T.-H. & Sprecher, S. G. Age-and wavelength-dependency of Drosophila larval phototaxis and behavioral responses to natural lighting conditions. Frontiers in Behavioral Neuroscience 11, 66 (2017).
32 Zhou, Y., Ji, X., Gong, H., Gong, Z. & Liu, L. Edge detection depends on achromatic channel in Drosophila melanogaster. Journal of Experimental Biology 215, 3478-3487 (2012).
33 Sakai, K. et al. Drosophila melanogaster rhodopsin Rh7 is a UV-to-visible light sensor with an extraordinarily broad absorption spectrum. Scientific reports 7, 7349 (2017).
34 Ni, J. D., Baik, L. S., Holmes, T. C. & Montell, C. A rhodopsin in the brain functions in circadian photoentrainment in Drosophila. Nature 545, 340-344 (2017).
35 Liu, X. et al. Opsin1 regulates light-evoked avoidance behavior in Aedes albopictus. BMC biology 20, 110 (2022).
36 Zhan, Y., San Alberto, D. A., Rusch, C., Riffell, J. A. & Montell, C. Elimination of vision-guided target attraction in Aedes aegypti using CRISPR. Current Biology 31, 4180-4187. e4186 (2021).
37 Hu, X., Whaley, M. A., Stein, M. M., Mitchell, B. E. & O'Tousa, J. E. Coexpression of spectrally distinct rhodopsins in Aedes aegypti R7 photoreceptors. PloS one 6, e23121 (2011).
38 Montelli, S. et al. period and timeless mRNA splicing profiles under natural conditions in Drosophila melanogaster. Journal of biological rhythms 30, 217-227 (2015).
39 Martin Anduaga, A. et al. Thermosensitive alternative splicing senses and mediates temperature adaptation in Drosophila. Elife 8, e44642 (2019).
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95864-
dc.description.abstract大多數生物體在其體內擁有特定的分子,能夠自主調節生理機能與行為。這種現象被稱為生物節律(Biological Rhythms),而這些內生性的生物鐘會受到來自外界的刺激而調控其震盪週期以適應周遭的環境,這些刺激包括溫度、進食、光線等等,其中又以光線造成的影響最為顯著。在先前的研究中已發現果蠅具有兩個影響其日週期節律(Circadian Rhythms)的光感受蛋白,分別是隱花色素(Cryptochrome, CRY)和視紫質7(Rhodopsin 7, Rh7),它們各自作為光感測器並對果蠅的生物節律具有重要影響。在埃及斑蚊中,已經找出兩個CRY基因,分別是CRY1和CRY2,且埃及斑蚊擁有一種和果蠅Rh7結構相似的視蛋白稱為GPROP10,儘管其在果蠅體內被報導和日週期節律有相關,但它在蚊子體內的功能仍然是未知的,本篇論文的主要目的在於研究Rh7是否在埃及斑蚊身上也具有調節日週期節律的功能。在本研究中我使用CRISPR/Cas9技術產生的CRY1(AAEL004146)和GPROP10(AAEL005322)突變蚊進行研究,在分子層次我使用西方墨點法發現Rh7突變體的兩個日週期中心蛋白TIM(timeless)和PER(period)會有上升的趨勢,並且產生了長度較短的PER蛋白異構體,且這種較短的PER專一的表現在Rh7突變體的複眼中,這些結果顯示Rh7可能在PER/TIM兩種蛋白的轉錄機制上有一定的功能。在行為分析上,我利用果蠅活動力測試儀器(locomotor activity monitor, LAM)實驗記錄埃及斑蚊一天的活動力並進行分析,發現Rh7突變體在早晨的活動力(Morning Peak)會顯著下降,另外我也在夜晚期間使用了不同波長的LED光源對埃及斑蚊進行光照刺激,並分析其活動軌跡,發現Rh7突變體在藍光的刺激下活動力顯著低下,這些結果指出Rh7在埃及斑蚊身上的確具有調控日週期節律的功能並且可能是一種接收藍光波段的蛋白。zh_TW
dc.description.abstractMost organisms possess specific molecules within their bodies that autonomously regulate physiological functions and behaviors. This phenomenon is referred to as Biological Rhythms, and these endogenous biological clocks are regulated by stimuli from the external environment to adapt their oscillation cycles, including factors such as temperature, feeding, and light, with light having the most significant impact. Previous studies have identified two light-sensitive proteins in fruit flies that influence their circadian rhythms, namely Cryptochrome (CRY) and Rhodopsin 7 (Rh7), each acting as photoreceptors and exerting significant effects on fruit fly biological rhythms. In Aedes aegypti, two CRY genes, CRY1 and CRY2, have been identified, along with a visual protein similar in structure to fruit fly Rh7, called GPROP10. While its relevance to circadian rhythms has been reported in fruit flies, its function within mosquitoes remains unknown. The primary aim of this study is to investigate whether Rh7 also plays a role in regulating circadian rhythms in Aedes aegypti mosquitoes. In this study, I use CRISPR/Cas9 generated mutant mosquitoes CRY1 (AAEL004146) and GPROP10 (AAEL005322) for research purposes. At the molecular level, using Western blot analysis, I found an upward trend in two circadian clock proteins, TIM (timeless) and PER (period), in Rh7 mutant variants, along with the production of shorter PER protein isoforms. This shorter PER isoform exhibited specific expression in the mutant's compound eyes, suggesting a potential role for Rh7 in the transcriptional mechanisms of PER/TIM proteins. In behavioral analysis, I recorded the daily activity of Aedes aegypti using a locomotor activity monitor (LAM) and found a significant decrease in morning peak activity in Rh7 mutants. Additionally, I stimulated mosquitoes with different wavelengths of LED light sources during the night and analyzed their activity patterns, and found significantly reduced activity in Rh7 mutants under blue light stimulation. These results indicate that Rh7 indeed regulates circadian rhythms in Aedes aegypti mosquitoes and may serve as a protein sensitive to blue light wavelengths.en
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dc.description.tableofcontents中文摘要 i
Abstract ii
Contents iv
Content of figures vi
Content of supplementary figures vii
Chapter 1. Introduction 1
1.1 Circadian 1
1.2 Cryptochrome 3
1.3 Rhodopsin 5
Chapter 2. Material and methods 7
2.1 Mosquito cultivation methods 7
2.2 Protein extraction 7
2.3 Western blot 7
2.4 RNA extraction 8
2.5 Reverse transcription and qPCR 8
2.6 Locomotor activity monitor assay 9
2.7 Light pulse assay 10
2.8 Mosquito brain immunostaining 10
Chapter 3. Results 12
3.1 TIM accumulates at night and degrades in the morning 12
3.2 CRY1 plays a primary role in degrading TIM in mosquitoes 12
3.3 PER is phosphorylated at night 13
3.4 Rh7 KO leads to the emergence of PER isoforms 13
3.5 The expression of PER isoforms is particularly pronounced in the compound eyes of Rh7 mutants 14
3.6 The impact of Rh7 occurs at the level of PER rather than at the level of CLK/CYC 14
3.7 PER isoform mRNA oscillation 15
3.8 Low temperatures do not induce the appearance of short-form PER 15
3.9 Dim light exposure in LL conditions still leads to PER degradation in Rh7 knockout mosquitoes 16
3.10 Rh7 mutants exhibit reduced activity under blue light conditions 16
3.11 The morning peak of Rh7 mutant female mosquitoes is weakened, while male mosquitoes remain unaffected 17
3.12 Single mutants of Cryptochrome or Rh7 do not affect the ability of Aedes aegypti mosquitoes to adapt to new light-to-dark transitions 17
Chapter 4. Discussion 19
4.1 The mechanism of PER short-form generation and its association with locomotion 19
4.2 Rh7 mutants exhibit differential locomotion effects in male and female mosquitoes 20
4.3 Mutations of a single photoreceptor do not affect the activity of Aedes aegypti mosquitoes 21
References 39
-
dc.language.isoen-
dc.title揭示在埃及斑蚊的生物節律和光敏感性中Rh7所扮演的功能zh_TW
dc.titleUncovering the Functions of Rh7 in Aedes aegypti Circadian Rhythms and Photosensitivityen
dc.typeThesis-
dc.date.schoolyear112-2-
dc.description.degree碩士-
dc.contributor.oralexamcommittee陳俊宏;涂世隆zh_TW
dc.contributor.oralexamcommitteeChun-Hong Chen;Shih-Long Tuen
dc.subject.keyword日週期節律,隱花色素,視紫質,活動力測試儀器,zh_TW
dc.subject.keywordCircadian rhythms,Cryptochrome 1 (Cryptochrome),Rhodopsin 7 (Rhodopsin),LAM assay,en
dc.relation.page43-
dc.identifier.doi10.6342/NTU202403665-
dc.rights.note未授權-
dc.date.accepted2024-08-12-
dc.contributor.author-college生命科學院-
dc.contributor.author-dept分子與細胞生物學研究所-
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