http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/191 2026-10-03T18:27:05Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92183 標題: 黑腹果蠅長非編碼RNA特性研究; Characterizing Long Non-coding RNAs in Drosophila melanogaster 作者: 陳玫如; Mei-Ju Chen 摘要: 次世代定序技術(Next-generation sequencing; NGS)開啟RNA領域研究的新紀元。過往認為只是轉錄訊號擾動的長非編碼RNA (long non-coding RNA; lncRNA)，已由許多研究證實其在許多重要生理機制中扮演要角。然而，現今文獻對於重要模式生物—黑腹果蠅(Drosophila melanogaster)的lncRNA瞭解仍相當有限；究其原因，乃黑腹果蠅lncRNA的基礎資訊之稀缺所致。因此，本論文追根溯源，由四個面向對黑腹果蠅lncRNA進行系統性探究—(1) 收集與發現：本論文開發一生物資訊方法，自我們產生的組織特異性RNA-seq資料鑑定出為數不少的新lncRNAs，並與公開資訊可收集之已知lncRNAs整合，呈現迄今最新之黑腹果蠅lncRNA資料集；(2) 特性註解：本論文採用大量的RNA-seq與ChIP-seq資料集(總計93組)增進現有lncRNA的註解資訊如轉錄方向與染色質特徵之品質，並進而觀察摘要出黑腹果蠅lncRNA的一般特性；(3) 基因表現：本論文以RT-qPCR實驗驗證了挑選之lncRNA的基因表現，並彰顯RNA-seq技術平台用於發現lncRNA的結果具有相當的可信度；(4) 轉錄調控：本論文提出一結合序列特徵探勘之生物資訊方法，系統性分析轉錄因子結合位(Transcription factor binding site; TFBS)於lncRNA啟動子出現與否，以及其與lncRNA基因轉錄調控的關聯性。結果顯示，當使用核小體佔據與跨物種保留性資訊，於共表現之編碼基因集進行序列探勘，其所得的序列特徵(或稱順式因子；cis-element)，多數與已知的TFBS相似；此外，這些順式因子可在共表現之編碼基因與lncRNA基因的啟動子區域同時觀察得見(較常見於第三期幼蟲至雄蟲階段共表現群集)，顯示出共表現之編碼基因與lncRNA基因具有被共同調控的可能性。簡言之，本論文彰顯系統性整合研究的優點，透過基因體與轉錄體資料的整合，大幅加速鑑別lncRNA的特性；而所得之觀察結果可作為黑腹果蠅lncRNA功能研究的堅實基礎。; Recent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Thus, this thesis aims at chracterizing fruit fly lncRNAs from four aspects: (1) collection and discovery; (2) annotation; (3) expression; and (4) regulation. I developed a computational approach to discover novel lncRNAs from the newly generated tissue-specific RNA-seq data, and then I combined the discovered lncRNAs with previously published lncRNAs into a curated dataset. Next, numerous RNA-seq and ChIP-seq datasets (93 sets) were used to improve the lncRNA annotation such as transcriptional direction and presence of conventional chromatin signatures. With these efforts, I summerized general characteristics of fruit fly lncRNAs in the thesis. In addition, I used RT-qPCR experiments to validate the expression of some randomly selected lncRNAs and demonstrated that RNA-seq is a reliable platform to discover lncRNAs. Moreover, I proposed a method to incorporate de novo motif discoveries to systemically investigate the presence of TFBSs in lncRNA promoters and how it is related to the regulation of lncRNA expression. The result revealed that most of the motifs (cis-elements) discovered from the co-expressed coding gene promoters are similar to the annotated TFBSs, where the motif dicscovery procedure considerd the information of nucleosome occupancy and evolutionary conservation. I also found that common cis-elements were usually observed in the promoters of the co-expressed coding and lncRNA genes in the development stages from L3 to male adlut. In conclusion, this thesis demostrated that integration of genomic and transcriptomic data can largely facilitate lncRNA discovery and characterization, and provided a solid foundation for studying the functions of lncRNAs in D. melanogaster. 2016-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99524 標題: 馬口魚族基因體分化之機制：染色體裂變與環境適應; Mechanisms of Genomic Divergence in the Opsariichtyini: Chromosomal Fission and Environmental Adaptation 作者: 戴睿紘; Jui-Hung Tai 摘要: 　　馬口魚族（Tribe Opsariichthyini）為一群分布於東亞溪流的小型鯉科魚類，包含異鱲屬（Parazacco）、東瀛鯉屬（Nipponocypris）、鬚鱲屬（Candidia）、鱲屬（Zacco）及馬口魚屬（Opsariichthys）。其中，馬口魚屬物種不僅多樣性極高，亦廣泛分布於整個東亞地區。值得注意的是，相較於其他類群，馬口魚屬的染色體裂變次數顯著偏高，達 14–15 次之多。 　　本研究組裝了五種馬口魚族物種的染色體層級基因體，並分為兩部分探討不同演化議題。第一部分聚焦於染色體大規模裂變的潛在分子機制。儘管染色體裂變在演化歷程中屢見不鮮，其形成機制與演化意義迄今仍未明朗，且過去多被認為是具破壞性的事件。本研究提出一項可能廣泛存在的機制：大規模裂變與轉座子的大量擴增有關。當轉座子大量插入並累積於著絲點區域，可能導致該區域拓寬，產生額外的著絲點 DNA，進而誘發新的著絲點（kinetochore）形成，產生具雙著絲點的染色體（dicentric chromosome）。這類染色體在裂變後仍可維持減數分裂期間的正常分離。本研究亦觀察到，染色體裂變對物種本身並未造成明顯不良後果，反而可能與加速物種形成相關。 　　第二部分則探討臺灣特有魚類——臺灣鬚鱲（Candidia barbatus）之族群結構與南北分化的潛在驅動因素。我們分析了 59 個體的全基因體資料，結果顯示臺灣鬚鱲可劃分為北部、中部與南部三個主要族群。與高分化的粒線體基因不同，核基因體呈現相對較低的族群分化程度。根據全基因體資料推論，我們認為傳統的南北分界應往北修正，以涵蓋高屏溪流域。此外，過去被重新命名為屏東鬚鱲之族群，與其他南部族群之間幾無遺傳分化與生殖隔離，顯示其分類地位亟待重新評估。更重要的是，我們發現南北族群的分化並非僅源於地理隔離，亦反映環境適應的結果：北部族群顯示與缺氧與低溫環境相關的適應性基因訊號，而南部族群則呈現與高溫環境相關的遺傳變異。 　　綜合而言，本研究不僅釐清了轉座子促成染色體大規模裂變的潛在機制，也揭示環境選擇如何塑造臺灣特有淡水魚類的族群結構與適應性演化，對瞭解物種形成與制訂保育策略均具有重要意義。; 　　The tribe Opsariichthyini comprises a group of small cyprinid fishes distributed in East Asian stream habitats, including the genera Parazacco,Nipponocypris, Candidia, Zacco, and Opsariichthys. Among them, species in the genus Opsariichthys exhibit relatively high diversity and are widely distributed throughout East Asia. Notably, compared to other genera, Opsariichthys species have undergone as many as 14–15 chromosomal fission events. 　　In this study, we assembled and analysis chromosome-level genomes for five species of the Opsariichthyini and addressed two distinct questions. The first part focuses on the potential mechanism underlying large-scale chromosomal fission. Although chromosomal fission has repeatedly occurred in evolution, its molecular mechanism remains poorly understood, and it has long been regarded as a harmful event. Here, we identify a potentially widespread mechanism in which large-scale fission is associated with massive transposable element (TE) expansion. TE insertions lead to the expansion of centromeric regions, generating additional centric DNA and potentially inducing the formation of new kinetochores, resulting in dicentric chromosomes. These chromosomes, even after fission, can still maintain proper segregation during meiosis. Importantly, we found no evidence of deleterious effects caused by fission in these species; on the contrary, chromosomal fission may be associated with accelerated speciation. 　　The second part investigates the population structure and potential drivers of north-south divergence in Candidia barbatus, a freshwater fish endemic to Taiwan. We analyzed whole-genome data from 59 individuals and found that C. barbatus can be divided into three major populations: North, Central, and South. In contrast to the highly divergent mitochondrial genome, the nuclear genome shows relatively low levels of differentiation. Based on genome-wide data, we propose that the traditional north-south boundary should be adjusted northward to include the Kaoping River basin. Additionally, the population previously renamed as Candidia pingtungensis shows almost no genetic differentiation or reproductive isolation from other southern populations, suggesting that its taxonomic status requires re-evaluation. More importantly, we found that the north-south divergence is not solely caused by geographic isolation but is also associated with environmental adaptation: the northern population shows selection signals related to hypoxia and cold tolerance, while the southern population exhibits selection signals associated with adaptation to high-temperature environments. 　　This study not only clarifies the potential mechanism of large-scale chromosomal fission but also reveals how environmental selection shapes population structure and local adaptation in a freshwater species endemic to Taiwan. These findings have important implications for understanding evolutionary mechanisms and informing conservation strategies. 2025-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/100170 標題: 頻率調控視丘下核深層腦刺激對運動皮質動態的差異性調節; Differential Modulation of Motor Cortex Dynamics by Frequency-Tuned STN Deep Brain Stimulation 作者: 胡昱庭; Yu-Ting Hu 摘要: 腦深層電刺激（DBS）通過電極在大腦深部的視丘下核（STN）施加高頻電刺激緩解帕金森氏症引起的運動症狀。儘管 STN-DBS 的療效仰賴對運動皮質（MO）投射至 STN 的調節，但 MO 在 DBS 過程中的實際角色仍未完全釐清。 本研究探討不同 DBS 頻率對 MO 神經動態的影響，以深入了解其治療機制。我們利用光遺傳技術選擇性地活化皮質投射至 STN 的路徑，並在半側帕金森氏症小鼠中進行活體雙光子鈣成像。雖然在多種頻率下小鼠的移動表現皆有所改善，但 MO 神經群體的反應則呈現頻率依賴性。值得注意的是，僅有高頻刺激能較好的維持與動作相關的皮質活動模式。另外，我們也發現第五層（L5）神經元與第二/三層（L2/3）神經元對刺激頻率的反應不同，暗示不同皮質層在電刺激下有各自的功能貢獻。 本研究結果強調調整 DBS 參數以保留內源性運動訊號之重要性，並為發展更精準的帕金森氏症神經調控療法提供了新的見解。; Deep brain stimulation (DBS) of the subthalamic nucleus (STN) provides effective relief of motor symptoms in Parkinson’s disease (PD). Although the therapeutic benefits of STN-DBS depend in part on modulating the motor cortical (MO) inputs to the STN, the precise role of MO during STN-DBS is not fully understood. Here, we examined how MO neuronal dynamics are influenced by different DBS frequencies to gain insights into therapeutic mechanisms and optimize neuromodulatory strategies. We used optogenetic stimulation to selectively activate cortical projections to the STN while performing in vivo two-photon calcium imaging of motor cortex neurons in hemi-parkinsonian mice. Locomotor performance improved over a broad range of stimulation frequencies, yet the underlying MO population responses varied depending on the frequency. Layer-specific analyses revealed that layer 5 (L5) neurons, whose activity is attenuated in the Parkinsonian state, were robustly and consistently modulated across the tested frequencies. In contrast, layer 2/3 (L2/3) neurons were more sensitive to stimulation frequency, suggesting distinct circuit-level contributions. These findings highlight the importance of tailoring DBS parameters to preserve endogenous motor signals in the cortex while maximizing therapeutic effects. Our results provide insight into how frequency-dependent cortical dynamics under STN-DBS contribute to motor recovery, with implications for developing more precise neuromodulatory interventions for Parkinson’s disease. 2025-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66338 標題: 響應環境變化而發生的截然不同的剪接調控; Distinct splicing regulation in response to environmental changes 作者: Hsuan-Kai Wang; 王宣凱 摘要: 剪接內含子是普遍存在的非編碼核糖核酸序列，通常注定要從新合成的核糖核酸中去除並迅速降解。但是，最近的研究表明，當營養缺乏時，積累的內含子可以幫助酵母細胞存活。在這裡，我們描述了一個指數，簡稱為IA，可用以評估內含子積累水平和剪接效率。通過使用IA指數的篩選準則，可以在各種生長條件下鑑定出新的內含子和信使核糖核酸前驅物積累事件。令人驚訝的是，在所有檢查的生長條件下，其中包括在營養培養基中指數生長期間，都發現了許多信使核糖核酸前驅物積累事件。內含子水平的提高是由於這些轉錄物不能被有效剪接和/或這些內含子不能被降解導致的。此外，在不同生長條件下，轉錄物的剪接效率會隨之變動，這表示剪接是響應於環境而專門進行調節的。順式信息分析表明，剪接效率低下的轉錄本內含子短，啟動子弱。剪接效率會隨轉錄加強而增加。為了確定反式因子與剪接事件之間的相互作用，我們開發了細胞內調節子的高通量篩選技術（CHRES），並且套用於約4,300種不同單基因缺失的酵母菌珠中。有趣的是，剪接較差的轉錄本的調節子與標準剪接的轉錄本幾乎不同。我們發現Ras / PKA信號傳遞路徑參與了低效率剪接轉錄本（KIN28）的調控。當將飢餓的細胞轉移到含有葡萄糖的豐富培養基中時，它可使KIN28轉錄物迅速被剪接，而當葡萄糖受到限制時，KIN28轉錄物則不能被有效剪接，這有助於細胞在需要呼吸作用時生長。因此，本篇研究展示了我們鑑定新型內含子積累事件及其調控機制的有效方法。同時展示了具有轉錄物特定性的調控網絡，其中包括營養刺激物的感知和信號傳遞讓剪接開始進行，使細胞得以快速適應新環境。; Spliceosomal introns are ubiquitous non-coding RNA sequences that typically destined for removing from newly synthesized RNAs and rapidly degrading. However, recent studies suggest that accumulated introns help yeast cells survive when nutrients were scarc. Here, we describe an index, IA, to evaluate intron accumulation level and splicing efficiency. By using IA criteria, novel intron and pre-mRNA accumulation events were identified in various growth conditions. Surprisingly, lots of pre-mRNA accumulation events were found in all examined growth conditions including during exponential growth in rich medium. Increased levels of intron result from failures of these transcripts to be efficiently spliced and/or of these introns to be degraded. In addition, the splicing efficiency of transcripts varies with different growth conditions that suggest splicing is specifically regulated in response to environments. Cis-information analysis demonstrated the inefficiently spliced transcripts have short intron and weak promoters. Splicing efficiency increases with increased transcription. To identify interactions between trans-factors and splicing events, we developed a cell-based high-throughput regulator screening (CHRES) and implemented in the background of ~4,300 different gene deletions. Interestingly, regulators of the poorly spliced transcripts are distinct from the canonically spliced transcript. The Ras/PKA signaling transduction was found to involve in the regulation of inefficiently spliced transcript KIN28. It enables KIN28 transcript to be rapidly spliced when shifting starved cells to rich medium containing glucose but to be inefficiently spliced when glucose is limited, that help cell in respiratory growth. Thus, our work demonstrates the capacity of our approach to identifying novel intron accumulation events and their regulatory mechanisms. It exhibited transcript-specific regulatory networks that include sensing and signaling of the nutrient stimuli to activate splicing, allowing cells to quickly adapt to the new environments. 2020-01-01T00:00:00Z