丹尼爾寶寶的酗酒日記--酒精對斑馬魚胚胎頭骨與腦下垂體基因的影響
長期以來，臨床研究發現酒精會影響人類胚胎的正常發育，但是其分子機 制尚不清楚。在本研究中利用RNA 定位雜交的方式觀察酒精對於胚胎發育過程 中shh、sox9a、sox9b、col2a1、hand2 的影響，發現這些基因的表現均會受到酒 精的抑制。這項結果顯示在胚胎發育過程中，酒精透過對上述基因的影響，造成 神經脊細胞減少，細胞遷移異常，以及干擾軟骨細胞分化的現象，進而造成頭骨 發育的嚴重缺陷。此外，實驗中亦發現生長激素在腦下垂體的表現亦受到酒精抑 制。這項研究的結果成功地從基因的層次深入了解胎兒酒精中毒症候群造成頭骨 畸形及生長遲緩的病理機制。 It was known that prenatal alcohol exposure may cause serious birth defects and developmental disabilities. The molecular mechanism of this fetal alcohol syndrome still remains unclear. As revealed by whole mount RNA in situ hybridization, it was shown that expression of a number of craniofacial cartilage-related genes, including shh, sox9a, sox9b, col2a1 and hand2, were all inhibited in zebrafish embryo by alcohol exposure. It suggests that alcohol exposure may result in reducing neural crest cell production, interfering neural crest migration, preventing chondrogenesis and eventually cause craniofacial defects. In addition, the transcriptional profile of pituitary hormones were investigated by RNA in situ hybridization. It appears that only growth hormone, but not prolactin and thyroid stimulating hormone, was inhibited by alcohol exposure. The reduction of growth hormone transcription was also confirmed by real time PCR. It also appears that the expression of upstream transcription factor pit1 and downstream target gene igf1 remains unchanged. It suggests that the reduction of gh transcription is mediated by a PIT1-independent pathway. The transcriptional profile of alcohol-exposed embryo was investigated by gene microarray analysis. It appears that the expression profiles of a number of development, cellular signaling, cell growth and apoptosis related genes have be affected by 1.5% alcohol treatment. It was noted that a number of retinal-specific genes were all repressed significantly. It consists with histochemical observation that alcohol exposure results in loss lamination and disturbed differentiation. This study help us understanding the molecular mechanism of fetal alcohol syndrome.
斑馬魚 SULT2 ST2在早期胚胎發育的 RNA 表現
在哺乳動物裡，硫酸化是一種參與外來物解毒作用、內生組織的荷爾蒙調節、藥物代謝及膽汁解毒之重要路徑。其中， SULTZ ( hydroxysteroid sulfotransferase ）是能進行上述反應的酵素之一，為了對 SULTZ 的功能與早期發育所扮演的角色作進一步的研究，在本實驗中，我們以班馬魚為模式動物，希望藉由原位雜交法（ in situ hybridization ）找到 SULTZ 在班馬魚早期胚胎發育的 RNA 表現位置。由目前的實驗結果，發現從卵巢到1-cell、 dome 、 50 ％一 epiboly 、 12 小時，都可在胚胎細胞看到訊號表現 · 在 24 小時、 36 小時、 48 小時、 72 小時則可在脊椎兩側體節的肌肉、內胚層、嗅窩、頭部看到訊號表現，此外，在成腦的原位雜交染色結果中亦看到了訊號表現。由此可推論 SULTZ 在斑馬魚早期發育確實扮演了重要的角色。Sulphonation is an important pathway for detoxification of xenobioties, bile acids, drug metabolism, and the regulation of endogenous hormones. SULT2 (hydroxysteroid sulfotransferase) is one of the enzymes which catalyse sulphonation. Zebrafish has emerged as a popular animal model in recent years. Compared with other vertebrates, it provides advantages including ease to get embryos, rapid external development, virtually transparent embryos and ease of genetic manipulation. The above-mentioned strong points made zebrafish a good model animal for us to understand the function of SULT2 during early embryonic development. We performed in situ hybridization to find out the RNA expression of SULT2 during zerbrafish early development. According to our present results, we can detect expression signal on the edge of telencephalon and tectum opticum. the edge on the dorsal zone of corpus cerebelli and ventral zone of periventircular hypothalamus of the adult brain. Besides, we can observe signal evenly distributed in blastocytes of the embryo at 1-cell, dome. 50%-epiboly and 12 hours after fertilization. We also find signal on the muscle next to the spinal cord during the stages of 24, 36, 48 and 72hrs. There are also expressions on hypoblast of embryos at 24, 36 and 72hrs, the olfactory pit at 36 and 4hrs, and strong expression in head region at 48 and 72hrs. These results suggest that SULT2 may have some function at the early development of zebrafish.
分子篩與過氧化氫感測器
目前市面上缺乏簡單而精準的過氧化氫檢測法，我們參考Fenton Reaction 中，鐵離子對過氧化氫分解的催化模式，利用鐵、鈷、鎳、錳、鋅分子篩，測試他們催化過氧化氫分解反應的效率，從成本、毒性、活化能的多方考量下，選擇以分子篩來固定金屬催化離子，作為我們後續實驗的研究主軸。實驗的初步，我們選擇過氧化氫作為自由基，並著重於過氧化氫的分解反應。利用濃差電池的原理，設計出一套濃度檢測系統，由分子篩作為電極。鑒於粉末狀的分子篩容易流失，我們製備出陶土鐵分子篩，以陶土固定分子篩，並以此作為電極，搭配白金絲，透過能士特方程式，測出過氧化氫的濃度，且藉由電路調控放大倍率，可以直接控制檢測範圍。從實驗結果得知，鐵分子篩在處理過氧化氫的時候，不會有鐵離子溶出的現象，且其催化性在酸性液中可以維持，能不斷的使用，長時間來看，分子篩相當有經濟與環境保護上的價值。We attempted to provide a system for quickly determining the concentration of free radicals. The existing methods or techniques are inefficient or need expensive equipment, therefore, an inexpensive system is being sought for. As a preliminary study, we focused on the decomposition of hydrogen peroxide. Taking the Fenton reaction as reference, we designed a measuring system. This system includes a catalyst containing Fe or Pt ions for catalyzing hydrogen peroxide decomposition reaction. The Fe- and Pt-zeolite were prepared to hold Fe and Pt ions to avoid losing. Because the electrically induced potential would decrease with the decrease in the concentration of hydrogen peroxide, we could measure the concentration of hydrogen peroxide by monitoring the electrical potential. We determined the initial concentration of hydrogen peroxide in water from the initial electrical potential measured through the equation obtained from the calibration line. The practicability of this system has been assured after a series of experiments. We will further develop the technique for measuring other free radicals. We anticipate that this technique will be further developed for measuring other free radicals. Although there are several problems and limitations to be solved and conquered, one thing is for sure: this system is an environment-friendly and cost-effective facility for determining the concentration of free radicals in an aqueous solution.
大自然的飛行家--蝴蝶飛行之初部探討
本研究主要針對蝴蝶之飛行進行探討，研究中主要探討蝴蝶翅膀形狀、身體重量、翅膀面積、展弦比、拍翅頻率及環境溫度對飛行速率之影響，並利用自製之風洞裝置，觀察蝴蝶之翼翅運動，分析通過蝴蝶模型之氣流方向及相關氣動力。研究結果顯示：紋白蝶展翅約4.5~5 cm，平均展弦比(AR)為1.71 ± 0.12，身體重量約為0.06± 0.02 g，翅膀面積約0.0012 ± 0.0003 m2，當紋白蝶身體重量愈重，則翅膀面積愈大(R2=0.9586)。另外，紋白蝶身體重量愈重、展弦比愈小，則飛行速率亦愈快(R2=0.5559、R2=0.4726)。23℃時，紋白蝶飛行速率為1.01±0.24 m/s，當環境溫度愈高(5、16、23℃)，則飛行速率亦愈快(y=0.07x＋0.7733，R2=0.6967)。風洞實驗發現：蝴蝶會逆風而飛，當風洞的風愈強，蝴蝶翅膀拍動角度愈大，且快而持久，仰角也變大(45 度)；蝴蝶翼尖軌跡呈八字形，翼翅運動包含線性平移及旋轉；蝴蝶拍翅時，可在翅上方及前方產生低壓帶，在後方產生高壓帶，以利蝴蝶向前方飛行。另外，翅緣彎曲角度(上反角)愈大，蝴蝶模型之上升高度亦愈高，當上反角60°時，蝴蝶模型之上升高度最高(2.2±0.1cm)。This research approaches the flying ability of butterflies. Our research mainly discusses the weight, aspect –ratio of butterflies, frequency of flapping, and the shape, surface area of butterflies’ wings, and the connection between temperature and flying velocity. More over, we use the wind tunnel which was made by us to observe the movement of butterflies’ wings and analyzed the direction of airflow and aero-elastic which pass through the wind tunnel. Our research shows that Pieris canidia’s length of wings is about 4.5 to 5 cm. The average of aspect –ratio (AR) is 1.71±0.12 . Its weight is about 0.06±0.02 . And its surface area is about 0.0012±0.0003 m 2 . The heavier Pieris canidia is, the bigger its surface area will be (R2 =0.9586). In addition, the heavier it is, the smaller its aspect –ratio will be (R2 =0.5559, R2 =0.4726), and the swifter its flying velocity will be. When it is 23°C, the flying velocity of Pieris canidia is 1.01±0.24m/s. The hotter temperature is (5,16,23°C), the swifter it flies (y=0.07x+0.7733,R=0.6967). Accroding to the detect of the wind tunnel’s experiment , the butterflies will fly on luff. When the stronger the wind of the wind tunnel is, the larger the angles of wing’s flap are. And they are fast and lasting, the elevation also becomes larger (45°). The butterflies’ trochoids of wings mimic the word “eight”, and the movement of wingspan includes parallel movement of linearity and wheel. When butterflies flap, it will amount depression upon and in front of the wings, amounting the high pressure on the back so that butterflies can fly antrorsely. Furthermore, the larger the curvy angle of marginal wings (Dihedral) is, the higher the ascending height of model butterfly will be. When dihedral is 60°, the ascending height of model butterfly is the highest(2.2±0.1 ㎝).
全民攻笛
本實驗主要是研究閉管駐波的發聲原理。何謂「閉管駐波」？就是一個管子在相同長度下，用不同的力道吹，會有不同音高的聲音產生，這些音被稱為「諧音」。原管長所能發出的最低頻率稱作「第一諧音」，第二低的聲音稱作「第三諧音」，依此類推。在簫的演奏上，只要按住同樣的孔，用較大的力量吹，也同樣會發出較高的音；同樣地，在曲笛的演奏技巧上，有平吹、急吹等分別。為什麼吹越用力，音就越高呢？如果現在拿一個大吸管吹（要裝活塞），你會發現，只有在特定的位置（角度）下，才能吹出聲音。那麼，角度對於聲音也有影囉？這些現象的幕後黑手，就是在管口產生的「渦流」，渦流頻率也會隨著風速而增加；而且，渦流的頻率在特定風速下，會有特定的範圍。經由實驗可以大略歸納出，影響閉管駐波的三個主要變因，分別是「風速」、「風吹角度」、及「吹口至管口的距離」。吹得越急，風速就越快，渦流頻率越高，越易使諧音躍遷；吹的角度越小，越易產生渦流，亦易引發聲音；吹的距離越小，渦流越不?定，越易產生其他的擾動。以上就是本實驗的概略。This project is aimed to fine out how the closed tube can produce a sound. We know what harmonics are. When we hold a big straw and blow with increasing strength (the bottom should be in water), it will generate a higher sound. The high sound is called “harmonic”. The lowest sound it can make is “the first harmonic”, the second lowest sound is “the third harmonic”, and so forth. Likewise, when we press the same key on vertical bamboo flute with increasing strength, it’ll also produce a higher sound. But why do we use the strong air stream to blow the tube to cause the tone to transfer? Now let’s blow a straw flute. You will find that you need to blow in the particular position, and then the sound will be produced. So, is there any relationship between the blowing angle and the frequency? Actually, all these sound are produced by “vortex in the mouthpiece.” The frequency of vortex will increase with the wind speed. Moreover, the frequency of vortex has a range. In sum, the higher the wind speed is , the higher the frequency of the vortex is , and leads to the higher frequency of the sound. The smaller the blowing angle is, the easier the vortex will be produced; the easier the frequency will be made. The smaller the distance between the blowing angle and the frequency is, the more unstable the frequency will be. The above is the most important research in this project.
AtbZIPs 轉錄因子及其下游基因啟動子的特定序列之研究
Arabidopsis thaliana bZIPs（AtbZIPs）是一群影響層面相當廣泛的轉錄因子，一半以上的 AtbZIPs 基因表現受到光的調控，且近九成的分子機制尚未明瞭，因此探討 AtbZIPs 在植物光調控機制中所扮演的角色將是個有趣的課題。AtbZIP16 與 AtbZIP17 皆被推測會參與光的調控機制，然而迄今少有文獻針對這二個轉錄因子進行更多的研究。因此，我們想藉由細菌單雜合系統（Bacterial one hybrid system）的方法，找出能與 AtbZIP16 與 AtbZIP17轉錄因子結合的 DNA 序列，以瞭解此二轉錄因子調節下游基因表現的分子機制，並探討其在光訊息傳導途徑中所扮演的角色。針對 AtbZIP16 與 AtbZIP17，本實驗分別找到了 7 與10 種可能的結合序列。首先，經由資料庫比對分析，我們發現其序列上帶有的 motifs 功能，主要參與在光調控、環境逆境反應機制、組織發育、賀爾蒙調節、病原菌防禦、鈣離子訊息傳遞等方面，其中又以光調控佔最大的比例。再者，藉由將 motifs 的功能繪製成文氏圖，並與 HY5 （AtbZIP56）做比較，結果顯示，這三個轉錄因子雖同屬於 AtbZIP family，據推測皆受到光的調控，可能參與某些相似的生理調節過程，但都各自具備不同的功能，影響植物體的發育。如此的差異，表示他們有實質上的不同，值得我們更深入的研究。整體而言，本實驗結果除了說明 AtbZIPs 的功能確實廣泛之外，也顯示AtbZIP16 與AtbZIP17 是執行光訊號傳導很重要的調控因子。Arabidopsis thaliana bZIPs (AtbZIPs) is a group of transcription factors affecting a wide range of responses in Arabidopsis. The expression of more than half of the AtbZIPs is regulated by light, and the molecular mechanism for roughly 90% of these AtbZIPs remains unknown. Therefore, the roles AtbZIPs play in Arabidopsis light signal transduction is an interesting topic to pursue. AtbZIP16 and AtbZIP17 have been suggested to participate in the regulation mechanism mediated by light. However, only limited studies for these two transcription factors have been previously performed. For this reason, we intended to determine the DNA-binding sequences for AtbZIP16 and AtbZIP17 via the bacterial one hybrid system to reveal their target binding sites in the promoter region of their downstream genes and to speculate their possible biological function especially in light signal transduction pathway. We have identified 7 and 10 possible recognition sequences for AtbZIP16 and AtbZIP17, respectively. Using motif-finding programs analyses, we found the motifs identified are mainly involved in light and stress signaling, tissue development, hormone regulation, pathogen defense and Ca2+ signaling. Among these regulation pathways, sequences involved in light regulation owns the highest proportion. Furthermore, a Venn diagram was generated to compare functions of genes regulated by AtbZIP16, AtbZIP17 and HY5. Results revealed that, although these three transcription factors all belong to the AtbZIP family and are predicted to be regulated by some similar physiological regulation process (e.g. light), they still possess distict biological functions in plant development. Further studies are thus required to put these transcription factors into their shared and unique biological context. Taken together, the results of this experiment not only indicated light is a key regulation factor for AtbZIP16 and AtbZIP17, but also showed the function of AtbZIPs could be diverse.