生物化學

去氧核糖核酸水解?，(deoxyribonuclease,簡稱DNase)為一種核酸內切?。目前研究得最透徹的去氧核糖核酸水解?為牛胰臟的DNase I，有四種以上的同功?存在。而牛和雞的DNA序列有極高的相似度，由之前的研究中在等電點焦集電泳法顯示雞胰臟DNase I 具A、B 兩種同功?，本實驗則希望能進一步了解兩種同功?的差異原因。在抽取RNA 後，以RT-PCR 方法合成cDNA，將其以限制?切割，再以大腸桿菌作為載體進行轉殖及繁殖培養，最後將其DNA 定序後進行分析比較。目前已完成第一次定序，但因引子接合過程中有部分位置錯亂造成殖體無法進行表現。另外在此次所得之序列中有發現一個胺基酸的轉變，而其是否為多型性之表現則有待進一步的實驗證實。The most advanced research on deoxyribonuclease in current states is on the bovine pancreatic DNase I, more than four of which isoforms have been found. It is shown that the DNA sequences of chicken and bovine have relatively high similarity. In addition, according to the result of isoelectric focusing from previous researches, chicken pancreatic DNaseI has two isoforms (A and B.) In this research, the author expects to establish more knowledge on the differences on the isoforms and the causes. After receiving the RNA, the RT-PCR is preformed to incorporate the cDNA, which is later cleaved by restriction enzyme and inserted into the plasmid DNA of the E. coli host cell to be cloned. So far a polynucleotide sequence has been deduced from clones of the cDNA, but it cannot be expressed successfully in E.coli due to few random mistaken splicing. There is an alternation of one amino acid, and whether it is the actual state of isoform or not still requires further proof.

細胞凋亡具有控制生物體細胞數目之功能，能讓特定的細胞走向死亡，因此若能掌握其作用機制，便可能藉由調控細胞凋亡的發生，進而應用於癌症治療。前人研究（2.）指出，經基因重組技術純化之口蹄疫病毒鞘蛋白rVP1，會導致BHK-21的Akt 蛋白質去活化，引起細胞凋亡的現象。然而在其後續的研究中，卻發現到在BHK-21中大量表現磷酸化的Akt 蛋白質，並無法反轉由rVP1 所誘發之細胞凋亡。因此本實驗利用二維蛋白質電泳，尋找Akt pathway 以外之細胞凋亡相關蛋白質。目前已證明Prohibitin 此一蛋白質，在由rVP1 所引起之細胞凋亡中，有自細胞核移動至細胞質的現象。此外，亦經由實驗排除Prohibitin 位於Akt pathway 的可能性。Western Blot 之結果更顯示，經rVP1 處理後，Prohibitin 在很短的時間內便出現遷移的現象，故推測其具有調控細胞凋亡上游反應的功能。Apoptosis can lead some specific cells to programmed death, thus, it is a major way for creatures to control their cells amounts. If we can command the mechanism of apoptosis, we may use it as a therapy for cancer by artificial regulation of apoptosis. VP1 is one of the capsid proteins of Foot and Mouth Disease Virus (FMDV). A research (2.) has indicated that the recombinant VP1 (rVP1) can result in dephosphorylation of Akt in BHK-21, and then lead the cells to apoptosis. However, in their follow-up experiments, they discovered that even if they expressed great amount of phospho-Akt in BHK-21, it still couldn’t reverse the apoptosis induced by rVP1. Therefore, this experiment takes the advantage of two-dimension protein electrophoresis (2D) in order to find apoptotic proteins excluded from the Akt pathway. I have found that Prohibitin exports from nucleus to cytosol after rVP1 treatment. Furthermore, I eliminate the possibility that Prohibitin’s may be located in Akt pathway. The results of Western Blot also shows that protein amount of Prohibitin in BHK-21 increase after rVP1 treatment, hence the purpose of nuclear export of Prohibitin might not be to degrade it. It might have some much more important function in the process of exportation. Besides, Prohibitin exports to cytosol in quite a short time after rVP1 treatment. According to this phenomenon, I suppose that Prohibitin has a role as a regulator of apoptotic up-stream reactions.

Background:\r Delivery of anticancer drugs in vivo can be achieved by using targetspecific\r drug carriers of various types – e.g. microspheres and liposomes –\r which can be applied intravenously, or subcutaneously via a hypodermal\r patch, etc. as appropriate to the type of drug carrier employed. Recently,\r interest has surged in the usage of amphiphilic polymeric micelles for this\r purpose, as they prove superior to other drug carriers in many ways. They\r have highly-hydrated hydrophilic shells, and hydrophobic cores wherein\r hydrophobic anticancer drugs such as adriamycin can be encapsulated\r during transport in the blood. Their main advantages include their\r smallness of size – enabling them to elude capture by the\r reticuloendothelial system (RES) – their stability in the bloodstream as\r shown in their low critical micelle concentration (CMC) values, their\r passive targeting of tumour sites via the enhanced permeability and\r retention (EPR) effect, and their ability to integrate an active targeting\r mechanism, e.g. antibody-antigen recognition, pH-sensitivity, etc.

IPNV 已是世界性分布的魚類病原體，其感染對象以鮭魚、?魚為主，但也在其他魚類分離出來，如鰻魚、吳郭魚、梭子魚、鱈魚、鱸魚、文蛤、泥鰍、虱目魚、香魚、金龍、石斑、等中皆曾發現IPNV之感染。足見此病毒廣泛地存在於魚貝類，常造成經濟魚貝類的重大損失。感染性胰臟壞死病毒之致病株，可造成魚類急性感染，而大量死亡。非致病株 ，不會造成大量死亡。國科會整合計劃之一其研究結果顯示，非致病株IPNV感染石斑魚死亡率只有10%，若病毒與重金屬同時存在時，會有55~92%死亡率; 在弧菌(Vibrio)與IPNV混合感染，亦有相似結果，此說明單一病毒感染魚貝類或單一環境因子，都不會造成大量死亡，但病毒和環境因子加在一起，引發大量死亡。所以對IPNV的防治是很重要的，所以針對病毒基因加以研究，IPNV是一種兩段雙股核糖核酸病毒，複製是在其細胞內的，但其團譯出的VP3中，已經證明與雙祋核糖核酸結合有關。近發現Michelle Donnelly及Gillain Elliott 的研究論文"Nuclear Localiziont and Shuttling of Herpes Simplex Virus Tegumnet Pretin"中，證明了HSV病毒所轉譯出的蛋白質VP13及VP14中，密集出現精胺酸(Arginine)的地方具有能將蛋白質帶入細胞核內的功能，而攜出細胞核的序列則有斷續的白胺酸(Leucine)出現。我們發現了傳染性胰臟壞死病毒(IPNV)病毒的轉譯出的VP3中也有能將蛋白質帶入或帶出細胞核的按基酸序列，故欲利用實驗證明是否有類似將蛋白質送進及送出的訊息，以便進一步探討VP3是否進入細胞核直接調控細胞核的某些功能。

Many biological processes in the human body take place in intracellular environments under crowded conditions. This means that such cellular activities occur in the presence of inert macromolecules. These macromolecules are thought to have large effects on reaction rates and equilibria. However, under conventional conditions, these same processes are studied in vitro under uncrowded conditions in dilute buffers and therefore not reflective of physiological conditions. Hence, this project aims to study the effect of macromolecular crowding on antigen-antibody reactions in an attempt to mimic intracellular environments. It also aims to highlight the importance of introducing crowders as a factor of study in conventional in vitro experiments involving cellular reactions. The model reaction investigated involves human collagen type 1 on skin samples and specific antibodies against these antigens. Inert macromolecules of defined hydrodynamic radius and charge were added into the reaction media while quantitative tests are conducted to determine the effect of crowding on the rate and extent of reaction. The macromolecular crowders used were Dextran Sulphate, Ficol170, Ficol1400 and Polyvinylpyrrolidone360. Results were than analysed with the Metamorph 7 imaging system to determine signal strength under crowded and non-crowded conditions. At high primary antibody dilutions, skin samples incubated with a primary antibody solution crowded with a combination of inert macromolecules showed improve signal strength. This could be because crowding tends to shift reaction equilibria such as to minimise the amount of excluded volume, leading to association of the antigen and its specific antibody. This is known as the excluded volume effect. In addition, crowding the primary antibody solution helped increase the rate of reaction. The results also suggest that the addition of crowders may reduce the amount of antibodies needed per sample and decrease the necessary incubation time. Therefore, antigen-antibody reactions can be done more cost-effectively, However, the presence of charged macromolecules such as Dextran Sulphate has an inhibitory effect. Further work is also needed to establish whether macromolecular crowding enhances specificity and thereby reduce autoflourescence of the skin sample.

One of the anthropogenic influences on the permafrost landscapes is deforestation and breach of the surface cover at the road constructions. On these areas a development of various cryogenic and postcryogenic processes and features (thermosubsidence, knobs (bilars, baydjarakhs), ravines, small lakes - djyodje) is being observed. Such features can be observed on the territory of Megino- Kangalassky Region, which is situated in the Central Yakutia, as well. During the period from 1998 to 2003 the author carried out monitoring studies of cryogenic features along three roads. Ten areas measuring twenty square metres were put in all. During 6 years of research about 1520 measurements of cryogenic formations parameters were made. Based on the results, it is concluded that elimination of the shadowing effect by trees and removal of the surface cover along the roads have caused thawing of shallow-lying ice wedges, as well as development of various cryogenic processes and features. In this paper, the author presents the basic technologies which are used in road construction in permafrost areas and, based on the research results, proposes a set of measures for rehabilitation of the roadside areas.

Successful calving is measured by the safe delivery of a healthy calf. Any factors that\r cause dystocia (difficult calving) are undesirable; as it will result in weak or dead calves, stress\r dams and a decrease in profit to the rancher. The most often identified reason for dystocia are\r calves that are too large at birth. Ranchers frequently use sires that will result in calves with\r small birth weights that will be born easy. This is especially true when breeding first calving\r heifers. The selection for small calves at birth results not only in less growth in the uterus but\r also less growth after birth, which means less beef to sell. This means ranchers try to balance\r reasonable growth and reasonable calf size when selecting their sires. Much data has been\r published on birth weight and its selection.

此研究是探討在目前全球因聖嬰現象後北極暖化，造成溫度持續在三十年內以每年上升攝氏零點五度，目前多數資料以顯示，對於地球內的生物生態產生了微妙的變化，在本文中將引述著名雜誌─科學人雜誌所刊登之關於全球暖化造成的生態環境影響；然而早在三億五千萬年前就已存在地球上的古老生物─蟑螂，順利的度過了多次的大滅絕，走過冥霜與煉獄。但是否會因為暖化作用而造成其生態影響呢？他又會不會成為少數存留並且大量繁殖的征服者呢？所以我們開始查詢白堊紀之資料，在研究、討論並製作改造完成實驗室氣溫控制冷熱溫差調節器，並從專業研究蟑螂生態的業界專業實驗室取得同一時期的實驗蟑螂物種，以期待本實驗更能具有更高的正確可信度；於特殊自行改造的觀察箱內進行整個實驗，已改變溫度並測量其進食狀況，瞭解蟑螂在溫度變化下的生態狀況。\r \r It’s easy to find the cockroach at any corner, such as school or house. They also hide in the refrigerator and stove.Thus,we are curious why they have durable vitality. This is the reason why we want to uncover the mysterious veil. According to data, we are curious about the environment of the cockroach and the temperature.Therefore,we want to imitate the situation of the ecosystem temperature at that time and inquire into its mystery. The purpose of Research is to make the violent changes, then discussing the meal which has attained its biggest existence rate. This uncontrolled experiment will influence the accuracy by factors.Therefore,we go to visit the laboratory personally and obtain some species of cockroach. Through the professional explanation and introductions, we make sure the direction of this experiment further. In this experiment we measure their appetite and the controls of the temperature everyday. They almost can crawl quickly along any material. We adopt the professional suggestion to measure with CO2 and O2.When the cockroach inhales CO2,we can observe the construction of the each part carefully. Through long-term observation, the food of the cockroach decreases, when the temperature rises to 20.6℃ or declines to 16.3℃,and it will stop moving when the temperature rises to 31.8℃ or declines to 8.7℃. When the temperature rises rapidly or reduce more than 15℃,the cockroach will look for shelter. Besides they easily get fainted when cockroach inhales CO2 without soil. The dinosaur were all buried underground, but why can the cockroach survive up to now? Probably, large land is their savior! Our conclusion is (1)The temperature that cockroach can exist from 49℃ to 3℃.(2)The suitable environment of cockroach growth is between 28.5℃ and 25℃.(3)The cockroach maintains their existence by eating under the low temperature 20℃ to 15 ℃.(4)Above 32℃ and under 7℃ the antenna is close to ground, its life is weaker.(5)The cockroach almost can live at any dilemma. But it can’t keep the prosperous life when it’s short of water.(6)From the above cockroach will be king of the world forever.

高中生物課本內對於轉譯機制所舉的例子，通常僅侷限於轉譯起始階段(initiation)所受的調控，例如色胺酸調控組。因此，我們想藉此研究更進一步探討：細胞在不同階段是否有調控轉譯的現象。我們利用冷光蛋白測定法(luciferase reporter assay)測出CPEB3的確對於轉譯有減緩的效果，並利用不同internal ribosome entry site(IRES)間接證明轉譯速率的變化主要是根據延長階段的不同而有所改變。另一方面，我們將多組CPEB3突變株進行交叉分析，找出cpeb3序列中兩百多個鹼基對的重要基因片段，並發現其為不連續的基因，且用Co-Immunoprecipitation(Co-IP)驗證我們的實驗結果。未來，我們將繼續探討在延長階段抑制的意義，以助於我們進一步了解細胞轉譯的過程。