http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76 2026-03-12T05:22:52Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73874 標題: 鼠腦前額葉皮質對具功能性連結之中間神經元和藍斑核上而下之伴隨調控; Concomitant Top-down Innervations from the Prefrontal Cortex onto Synaptically Connecting Interneurons and Norepinephrine Neuronsin Locus Coeruleus in Mice 作者: Yu-Shan Kuo; 郭育姍 摘要: 在這份研究中我們試圖探討中間神經元 (interneurons, IN) 是否會整合來自大腦皮質送往位在藍斑核(locus coeruleus, LC)去甲基正腎上腺素(norepinephrinergic, NE)神經細胞的資訊。在形態研究上，LC-NE神經細胞會對大腦發送全面性的投射，並作為中樞神經系統主要的NE提供者。在行為研究上，LC-NE系統能夠促進整體細胞活性(反應度、警覺度)，參與睡眠-清醒週期(sleep-wake cycle)的轉換，和與任務相關表現的提升有關。近期研究指出LC-NE系統能夠作為暫時性的過濾器，允許跟任務相關的訊息能通過感官門控(sensory gating)，進而促進行為本身，並與擁有高度認知大腦皮質所判定結果協調一致。LC神經細胞的'phasic activity'與神經網絡的增益值(gain)有高度關聯性，我們猜想phasic activity可能受到LC附近的INs調控，或是受到來自大腦皮質，如腦前額葉(prefrontal cortex, PFC)上而下的神經調節。位在PFC的腦區在評估獎賞的機制上扮演重要功能，並且這些功能與LC-NE系統的功能有高度的重疊。先前在猴子與大鼠的研究已經指出，PFC對LC的投射主要發生在核邊樹突區域(peri-cerulear dendritic zone, peri-LC)，該區域密布著LC樹突的投射。由於大部分的INs也位在peri-LC，我們猜想是否這些INs會接收PFC的投射，作為PFC和LC之間整合器。為了回答這問題，我們製作了cre酵素依賴的病毒 (AAV2-DIO-WGA)，該病毒同時擁有橫跨神經細胞突觸(trans-neuronal)的能力，藉此來揭露與LC有突觸連結的INs。我們也決定先將焦點放在研究抑制性中間神經元(inhibitory interneurons, I-IN)上面，因為在INs的群體中同時也存在著興奮性神經元(excitatory interneurons, E-IN)。AAV2-DIO-WGA被打入兩種基因型老鼠的後代，第一種老鼠為TH-cre會表現cre酵素在兒茶酚胺神經細胞(catecholaminergic neurons)中，第二種老鼠為GAD-GFP會表現綠螢光蛋白於γ-氨基丁酸神經細胞(GABAergic neurons)中，這份組合使後代能夠進行位在橋腦背側LC細胞專一性的感染，並且使目標I-INs能夠發出自體螢光。透過免疫染色，我們如預期在LC細胞上觀察到了TH和WGA的免疫活性，並在非LC細胞(沒有TH免疫活性)上觀察到了WGA的免疫活性。由於此cre酵素在兒茶酚胺神經細胞以外的細胞表現量極低，擁有很高的專一性感染可信度，我們認為這些沒有TH免疫活性、卻有WGA活性的神經細胞正是橫跨神經突觸標定到的INs，這些INs與LC細胞具有功能性連結。這些INs被發現大量聚集於LC樹突周邊的內側區域，約10.33 ± 0.12% (n=2)的INs有GFP被認定為I-INs，約22.89 ± 2.26% (n=1)的INs擁有FoxP2 轉錄因子的活性。為了證實是否這些INs接收來自大腦皮質的投射，我們重複了以上實驗並搭配了新的病毒 (AAV9-Syn-ChrimsonR-tdT)，注射於PFC以進行順行的追蹤。我們發現PFC的投射與LC和INs都有接觸。總之，這些結果支持了我們的論點，位在LC周邊樹突區域內側的E-INs和I-INs位與LC神經細胞有具功能性的連結，並且這群INs會接收、整合來自PFC送給LC神經細胞的訊息。; In this study, we aim to test whether interneurons (IN) could integrate cortical signals to norepinephrinergic (NE) neurons in locus coeruleus (LC). In morphology, LC-NE neurons sends out global axonal projections and provide major NE supply to the central nervous system. In behavior, the LC-NE system promotes general cellular activity (responsiveness, vigilance) in the brain, participating in shift of sleep-wake cycle, and relates to improvement of task-related performance. Recent studies have suggested that LC-NE system serves as a temporary filter, allowing task-related signals passing through sensory gating to facilitate behavior, in alignment of decision made by cortical areas of high cognitive function. Phasic activity of LC is highly correlated to increasing gain of neuronal network, which we suggest that it might be regulated by INs in local circuit and top-down innervating signals from cortical areas like the prefrontal cortex (PFC). Brain regions in PFC play significant roles in evaluating rewards and their functions are overlapping with those attributed to the LC-NE system. Previous studies in monkeys and rats have shown that PFC projection to LC terminates at peri-cerulear dendritic zone (peri-LC), where dendrites of LC-NE neurons are located. Since most local INs are also located in this peri-LC region, we wondered if INs received concomitant top-dwon innervations from the PFC, serving as integrators of cortical signals before passing them to LC. To answer this question, we produced a cre-dependent virus, AAV2-DIO-WGA, with trans-neuronal capacity to reveal INs synaptically connecting with LC neurons. And we decided firstly to focus on inhibitory interneurons (I-IN), besides from excitatory interneurons (E-INs). AAV2-DIO-WGA was injected into offspring of TH-cre mouse (expressing cre enzyme in catecholaminergic neurons) crossed with GAD-GFP mouse (expressing green fluorescence protein in GABAergic neurons), this allows cell-specific infection in LC neurons in dorsal pons and provides intrinsic GFP label on I-INs. Using immunohistochemistry (IHC) staining, we observed TH- and WGA- immuno-reactive (ir) within LC as expected, but also abundant neurons, which are WGA-ir but not TH-ir, in peri-LC. With high confidence of low leakage rate of cre expression in cells other than catecholaminergic population, we reason these WGA-ir and TH-negative neurons are trans-synaptically labelled and connected with LC neurons. These INs were located predominantly in the medial aspect of peri-LC region, about 10.33 ± 0.12% (n=2) of INs are found labelled with GFP being I-INs, and about 22.89 ± 2.26% (n=1) of INs with FoxP2 transcriptional factor. To confirm whether these INs receive top-down cortical inputs, we repeated experiments described above with an additional injection of AAV carrying reading frame of Crimson, AAV9-Syn-ChrimsonR-tdT, into the PFC for anterograde tracing. We found that the PFC fibers made contact with both LC neurons and INs. Together, these results support our arguments that there are local excitatory and inhibitory INs in medial peri-LC forming functional connections with LC-NA neurons and integrating the PFC inputs onto LC neurons. 2019-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56034 標題: 點帶石斑魚非特異性毒殺細胞受器蛋白分子的選殖與定性; Molecular Cloning and Characterization of Nonspecific Cytotoxic Cell Receptor Protein-1 (NCCRP-1) from Orange-Spotted Grouper (Epinephelus coioides) 作者: Chih-Hsuan Teng; 滕致萱 摘要: 從點帶石斑魚的脾臟選殖出非特異毒殺細胞受器蛋白分子(NCCRP-1) cDNA，包括132 bp 5’端非轉譯區( 5’ untranslated region, 5’ UTR)、699 bp 編碼區 (open reading frame, ORF) 及108 bp 3’UTR，共可轉譯出232個胺基酸。根據Expert Protein Analysis System (ExPASy)網站上的Motif Scan、美國國家生物技術資訊中心 (The National Center for Biotechnology Information, NCBI)的Position-Specific Iterative BLAST進行domain 預測結果顯示，NCCRP-1在第30~228個胺基酸區域為F-box associated (FBA) domain，內含三個功能區塊，分別是N端的proline-rich 區塊、中間的antigen binding 區塊及C端的signal transduction 區塊;但並沒有預測到穿膜區。 NCCRP-1蛋白質分子量推測為26 kDa、等電點是5.06。胺基酸序列多重比對顯示，石斑魚和其他5種海水魚的NCCRP-1一致性在56~84%，和紅笛鯛 (Lutjanus sanguineus)最相近。Q-PCR結果顯示石斑魚受到海水白點蟲感染後第一天NCCRP-1轉錄表現顯著上升，第三天達到最高，第七天降至背景值。利用昆蟲細胞Sf21和桿狀病毒表現系統合成石斑魚NCCRP-1重組蛋白，經SDS-PAGE和西方墨點轉漬法檢驗，分子量約為29 kDa，顯示NCCRP-1 分子經過轉譯後修飾，在Asn171有N-linkage，可能進行醣基化反應;在Gly93及Gly158各有一段N-myristoylation的片段 (GIPAGV及GADKST) ，在Cys10 推測為palmitoylation位置，可能進行脂化反應(Lipidation)。LC Mass/Mass比對重組蛋白確定為石斑魚NCCRP-1 。將NCCRP-1重組蛋白免疫紐西蘭大白兔八週後取得抗血清，硫銨沉澱(部份純化)的免疫球蛋白(Ig)耦合上FITC，進行流式細胞分析周邊及組織內白血球;顯示寄生蟲感染後第四天，細胞膜上RP-1平均表現量(MFI)顯著增加。顯示NCCRP-1是一種可被誘發 (inducible) 的基因，且表現量由低至高。 從流式細胞儀分析及原位雜交皆顯示，石斑魚NCCRP-1+細胞為直徑3-6 μm的白血球。石斑魚受到寄生蟲感染前後，以流式細胞儀測量血液、脾臟、胸腺、鰓、皮膚及頭腎NCCRP-1+細胞佔全部白血球的比例，顯示感染前約佔1%，而感染後則增加到3~19.3%。取石斑魚受海水白點蟲感染後第三天的鰓組織切片，用NCCRP-1 antisense riboprobe進行原位雜交(in situ hybridization)，發現海水白點蟲營養體(trophont)周圍及內部有NCCRP-1+細胞。NCCRP-1+細胞具有毒殺海水白點蟲纖毛幼蟲和被神經壞死病毒(nervous necrosis virus, NNV)感染的細胞的能力。 2014-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71266 標題: 黑腹果蠅管家基因之研究-以生物特性及分子演化之觀點; Study on Housekeeping Genes in Drosophila melanogaster-Based on Biological Characteristics and Molecular Evolution 作者: Yi-Wen Lu; 盧怡文 摘要: 管家基因是維持細胞基礎功能及細胞存活的重要基因，因為管家基因在生物體的重要性，故本論文採用昆蟲的模式生物-黑腹果蠅來進行管家基因之相關研究。本論文的研究目標為: 1)研究黑腹果蠅管家基因的生物特性， 2) 研究影響黑腹果蠅管家基因演化速率的可能因子，以及 3) 研究影響黑腹果蠅管家基因密碼子使用偏性的可能因子。本論文使用生物資訊的方法，對黑腹果蠅1,107個管家基因及1,084 個高組織特異性基因來進行相關分析,在比較分析這兩群基因的各種生物特性時，發現這兩群基因存在很大的差別，管家基因的演化速率比高組織特異性基因要慢，表示管家基因在演化較為保守。進一步分析可能影響管家基因的演化速率的因子，發現可能的相關因子為：密碼子使用偏性、基因表現量、世系年齡、蛋白質交互作用的情形、蛋白質作用部位的占比、以及蛋白質的多功性，利用淨相關分析發現密碼子使用偏性是影響管家基因的演化速率最主要的可能因子。本論文再進階分析影響管家基因密碼子使用偏性的因子，分析結果發現偏性突變及轉譯篩選可能是影響管家基因密碼子使用偏性的重要因子，而蛋白質交互作用的情形以及蛋白質作用部位的占比同樣為影響管家基因密碼子使用偏性的可能因子。本論文探討昆蟲模式生物-黑腹果蠅其管家基因的生物特性及分子演化的研究結果，期能作為未來生醫及農業相關應用研究之基礎。; Housekeeping genes (HK genes) are required for cell survival and the maintenance of basic cellular functions. This thesis focused on the research of HK genes in Drosophila melanogaster. The aims of this thesis were: 1) investigation of biological characteristics in HK genes, 2) investigation of factors affecting evolution rates in HK genes, 3) investigation of factors affecting codon usage patterns in HK genes. In this thesis, bioinformatics approaches were employed to analyze 1,107 HK genes and 1,084 high tissue specificity genes (HTS genes) of D. melanogaster. The comparisons of multiple biological characteristics between these two gene groups suggested that these characteristics were significantly different between these two groups. The average evolutionary rate of HK genes is slower than that of HTS genes. To identify the possible factors that constrain the evolutionary rates of HK genes, several factors were analyzed herein. Codon usage bias (CUB), gene expression level, phyletic age, protein connectivity, proportion of protein interacting length and protein multifunctionality were found to be related to the influence on evolutionary rates of HK genes. Partial correlation analysis revealed that CUB has the strongest influence than other factors on influencing the evolutionary rate variation between these two gene groups. Therefore, further analyses were made to identify the factors that potentially influence the CUB of HK genes. The results suggested that mutation pressure and natural selection highly correlate with CUB in the HK genes and two topological properties of HK proteins (proportion of protein interacting length and protein connectivity) also correlate with CUB in the HK genes. The results provide more insight into biological characteristics and the molecular evolution in the HK genes of D. melanogaster, and the results may support future investigations of potential applications in agricultural and biomedical field. 2018-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95571 標題: 黑水虻替換成分飼料配合地衣芽孢桿菌對美國螯蝦生長表現、免疫反應及飼料效益的影響; Effects of black soldier fly replacement feed with Bacillus licheniformis supplement on growth, immunity and feed efficiency of Procambarus clarkii 作者: 李晏菱; Yen-Ling Lee 摘要: 美國螯蝦(Procambarus clarkii)是近年來水產養殖中一種重要的飼養物種，於世界各地作為食用或觀賞用。因大量的飼養使養殖業對魚粉的需求增加，導致漁業資源的減少及魚粉價格的升高，因此找到適合的飼料替代成分是目前水產養殖業的挑戰之一；黑水虻(Hermitia illucens)是近年最常被使用的替代材料之一，具有飼養容易、蛋白質含量高等優點，但因其有幾丁質組成的外骨骼，過量的添加會使宿主的腸道受到損害並降低攝食率。 本實驗使用脫脂黑水虻粉做為魚粉的替代材料，並且為提高飼養的效益及解決替代成分的缺陷，挑選具有對幾丁質、蛋白質優秀分解能力的地衣芽孢桿菌(Bacillus licheniformis)作為益生菌進行添加。以0%（控制組）、21%(B21)及30%(B30)的黑水虻比例取代魚粉用量製成飼料，並將地衣芽孢桿菌培養至3x109(CFU/ml)後添加至上述條件的各組飼料中進行投餵，分別為BB0、BB21與BB30，共六組。56天飼養後測量成長指標，採集各組螯蝦的肝胰腺、腸道及尾部肌肉進行基因表現、菌相分析與組織切片，以及肌肉蛋白質含量檢測。結果顯示進食21%黑水虻替換飼料的螯蝦(B21)在各種成長表現的指標皆有提升的趨勢，與添加益生菌的BB21組相比，BB21組的螯蝦成長表現提升更加顯著，腸道健康表現優於其他組別，並且對外在感染源的抗性增加。這證明以一定比例的黑水虻替換魚粉並添加益生菌能有效改善螯蝦的成長及健康表現。; The American crayfish (Procambarus clarkii) is extensively cultivated for both food and ornamental purposes. The rising demand for fish meal in aquaculture has led to decreased wild fish resources and increased fish meal prices, creating a need for alternative feed ingredients. The black soldier fly (Hermetia illucens) known for it’s high protein content and easy for cultivation, has become one of the most commonly used materials in recent years. However, its exoskeleton composed of chitin may potentially damage the crayfish's intestines and reduce feeding efficiency. This study explored the use of defatted black soldier fly powder as a fish meal replacement in crayfish feed and evaluated the effects of supplementing Bacillus licheniformis, a probiotic that can degrade chitin and protein. The crayfish were fed diets containing 0% (B0), 21% (B21), 30% (B30) black soldier fly powder, with same formula and additional receiving Bacillus licheniformis (BB0, BB21, BB30). After 56 days, we assessed growth indicators and collected hepatopancreas, intestines, and tail muscles for gene expression analysis, metagenomics, tissue sections, and tail muscle protein content measurement. Results showed that the BB21 group, which received 21% black soldier fly powder and Bacillus licheniformis, demonstrated significantly better growth performance and intestinal health compared to the B0 and B30 groups. This suggests that replacing fish meal with proportion of black soldier fly, combine with probiotics supplement, can enhance the growth and health of crayfish. 2024-01-01T00:00:00Z