醫學與健康科學

Adducin蛋白在細胞骨架的調節上扮演著重要的角色。然而，近來有許多研究指出，骨架蛋白也會出現在細胞核並參與轉錄調控，因此本研究的目的即在探討adducin蛋白是否會進入細胞核中，並參與轉錄調控或具有其他功能。在本研究中，我們將綠色螢光蛋白(GFP)標示的adducin質體DNA，利用轉染技術送入老鼠纖維母細胞株NIH3T3中表現。NIH3T3細胞原本並無adducin蛋白的表現，在共軛焦顯微鏡下觀察，野生型的GFP-adducin蛋白會表現於細胞核與細胞質中。由於adducin蛋白尾端序列攜有可能往核內運輸的訊號，於是將位在此一訊號中的離胺酸718及離胺酸719進行突變，結果發現此一突變株只能在細胞質中表現。此外，蛋白磷酸脢C(protein kinase C)已知能磷酸化adducin蛋白在絲胺酸716及絲胺酸726的位置，於是假設其磷酸化是否與其在細胞內的分布有關。將adducin的絲胺酸726置換成丙胺酸，並不影響其在細胞內的分布。然而將絲胺酸716置換成丙胺酸後，則完全只在細胞核中表現。由於adducin可分布於細胞核，因此我們懷疑adducin蛋白可能與細胞分裂有關，於是本研究利用流式細胞儀分析adducin轉染後NIH3T3細胞的細胞週期。流式細胞儀的分析結果顯示，攜有GFP-adducin或其突變株的細胞與未經轉染的NIH3T3細胞的細胞週期並沒有顯著差異。其次，為了避免因轉染的效率不高而造成統計上的誤差，我們利用顯微鏡追蹤技術觀察攜有GFP-adducin的細胞株，結果顯示攜有adducin突變株的NIH3T3細胞株仍能正常分裂。再者，因為adducin能與細胞骨架中的肌動蛋白結合，所以adducin不同的分布位置可能影響細胞附著與細胞展延的效率。細胞展延試驗的結果顯示，adducin及其突變株對細胞附著與細胞展延的效率並無明顯的影響。本研究的結果證明，adducin的確帶有往核內運輸的訊號，其在細胞質中的分布可能也同時受到絲胺酸716磷酸化的影響。然而adducin的功用似乎與纖維母細胞的分裂與展延無明顯的關聯性。Adducin, an actin binding protein, is known to play an important role in the regulation of the membrane cortical cytoskeleton. More and more evidence indicates that proteins involved in the cytoskeletal regulation could also reside in the nucleus and participate in gene regulation. Thus, the goal of this study is to examine whether adducin is expressed in the nucleus and involved in certain nuclear events. In this study, adducin and its various mutants were fused with green fluorescent protein (GFP) and transfected into mouse NIH3T3 fibroblasts which do not have endogenous adducin for monitoring their subcellular distribution under a laser scanning confocal microscope. The wild-type GFP-adducin was found to be present both in the nucleus and in the cytoplasm. The COOH-tail of adducin contains a motif analogous to the nuclear localization signal (NLS). Mutation of two lysine residues (lysine 718 and lysine 719) located within this motif abolished the nuclear localization of adducin. Moreover, adducin is known to be phosphorylated by protein kinase C at serine 716 and 726. Substitution of adducin serine 726 with alanine had no effect on its subcellular localization. In contrast, substitution of adducin serine 716 with alanine led to only nuclear expression. Nuclear localization of adducin renders it possible that adducin may be involved in the regulation of cell division cycle. For cell cycle analysis, flow cytometry was applied. The results of flow cytometry indicated that expression of adducin and its mutants in NIH3T3 fibroblasts did not affect their cell cycle progression. To further examine the effect of adducin on cell division, NIH3T3 cells transiently transfected by adducin were monitored by time lapse video microscopy. The video clearly showed that the cells with GFP-adducin underwent cell division to generate two daughter cells. Since adducin is well known to bind to actin and thereby regulate microfilaments, we wondered that expression of adducin in NIH3T3 cells might affect their adhesion and spreading onto extracellular matrix proteins. The results of cell spreading assays showed that adducin appeared not to affect cell spreading. In conclusion, our results demonstrate that the subcellular distribution of adducin is likely regulated by two signals, one is the nuclear localization signal and the other is the phosphorylation status of the serine 716. However, enforced expression of exogenous adducin in fibroblasts such as NIH3T3 cells does not alter their cell cycle or cell spreading on fibronectin.

罹患神經母細胞瘤的兒科病人，尤其是罹患stage IVs 神經母細胞瘤者，他們有些伴隨著非常嚴重的貧血，但卻檢測不出神經母細胞瘤已經侵犯骨髓；有時病情來勢洶洶，尤其是腫瘤細胞中已可偵測到N-myc 基因增幅者，診斷時腫瘤細胞可能已在腹腔四處擴散並已侵犯大部分的肝臟。但是，某些這種病患，特別是腫瘤細胞中N-myc 基因沒增幅者，即使在沒有治療的狀況下卻可能有自然恢復的現象，也就是腫瘤細胞會自動消退，但原因仍待進一步的證實與探討。可是，這些病人在其病情最嚴重的時候，骨髓內紅血球母細胞形態上的改變顯示可能與病毒感染有關。但是關於病毒來源的研究，現有的資訊仍然十分有限，其中最重要的是，病毒感染與引發其後天之免疫作用是否有關，更需要深層的研究。因此，為更進一步了解罹患神經母細胞瘤之兒科病人的病毒感染及病毒蛋白表現的作用，我們這次研究的目的在檢驗罹患神經母細胞瘤及貧血之兒科病人與微小病毒B19 (PVB19)、Epstein-Barr Virus (EBV)、腸病毒71 型(EV 71)和巨細胞病毒(CMV)的關係，以及病毒蛋白表現對這些病人的作用與臨床意義。In pediatric patients with neuroblastoma, in particular, those with stage IVs neuroblastoma, sometimes the disease was combined with severe anemia. However, no tumor involvement was detected in the bone marrow. Although some of these patients may have N-myc gene amplification, and the disease could have invaded many abdominal organs, especially liver, interestingly, the disease might regress spontaneously in some of these patients. The medical reason of the spontaneous regression, nonetheless, remains to be determined. It is worth noting that morphological changes of erythroid progenitor cells in the bone marrow have suggested virus infection in these pediatric patients. However, the available information of viral origin is limited. Furthermore, it is possible that the virus infection in these patients could be associated with the revocation of immune responses related to the spontaneous regression of the tumor. In this study we will investigate the relationship of parvovirus B19 (PVB19), Epstein-Barr virus (EBV), enterovirus 71 (EV71) and cytomegalovirus (CMV) with neuroblastoma by PCR in Taiwanese pediatric patients. Moreover, we will study the effect and the clinical significance of viral gene expression as well as N-myc gene amplification in these patients.

茶由於其具有特殊的芳香氣味及口味，廣泛地被世人用為飲料，近年來的研究證據顯示茶具有促進身體健康和防癌功效，但茶中之成分，尤其兒茶素隨茶及茶沖泡方式而有所不同，因此對於茶中成分及沖泡方式的認知是一門重要的課題。 本實驗主要是探討茶沖泡方式及儲藏條件，對於茶液中成分變化的影響，探討的茶包含紅茶、烏龍茶、綠茶及其茶包。並使用質譜儀及高效能液相層析儀配備紫外光偵測器分析茶液中之成分，實驗結果顯示加蓋子的茶杯儲存茶時比沒蓋子的要好；成分變化速率又以未發酵的綠茶較發酵的紅茶和烏龍茶快；低溫儲存時，亦可延緩茶的成分變化，例如加蓋並存放於冰箱，茶甚至可置放至隔夜其成分均不會改變；尤其本實驗尚發現照光與否影響茶液成分變化甚鉅。;Tea is the most widely accepted and consumed beverage worldwide due to its characteristic aroma and taste. Recent studies have provided the strong scientific basis for understanding the health promoting effects and cancer preventive actions of tea. The components of tea especial the catechins are varied with the conditions of making tea. To understand and determine the chemical composition of tea is very important. Some investigations of the parameters on the storage and making of tea were carried on in this study. The kinds of tea studied were including black tea, oolong tea, green tea, and instant tea bags. A high performance liquid chromatograph combined with UV detector was used to analyze the components of tea. The results showed that the composition of tea solution is dependant of the exposure of light. The tea kept in cup with cover was better than that without cover. The ingredients in green tea were changed faster than those in fermentative black tea and oolong tea. The components of tea can be kept unchanged for a long time at low temperature. In refrigerator, the time can be extended to overnight.

DcR3是腫瘤壞死因子6B的成員（Tumor necrosis factor receptor superfamily），又稱為TR6或M68。DcR3會藉由與Fas Ligand, LIGHT或TL1A等細胞表面配位結合，阻擋T細胞的死亡訊息傳輸，保護該細胞免於細胞凋亡；另外，DcR3也會與抗原呈現細胞的硫化肝素糖蛋白結合，誘導未分化之T細胞發育成能減少自體免疫疾病發生的Th2細胞，顯示DcR3有充分作為自體免疫疾病預防之藥物的可能性。因此我藉本實驗探討DcR3對T細胞分化與發育造成的影響與造成此一現象的原因。 根據實驗結果，我們發現DcR3不僅有上述之功能，更能增加調節型T細胞（Treg）這種抑制免疫反應的特殊T細胞表現量，若能藉由Treg調控免疫系統活力，將可改善易般免疫抑制劑治療造成病人免疫力下降之缺點。因此我藉由NOD小鼠，實際檢測DcR3在活體中對抗自體免疫疾病發生的能力，並以DcR3與T 細胞之交互作用實驗，探討現象的成因。根據實驗結果，在Somatic gene therapy和Protein therapy的實驗中，皆可看出疾病延遲發生的現象。而在DcR3對T細胞存活率的影響方面，可看出DcR3並不會造成T細胞之異常死亡。故我們推測DcR3可藉由增加Treg表現量減緩或降低NOD小鼠糖尿病的發病。 因此我們認為DcR3非常有潛力作為未來自體免疫疾病預防或移植手術後用以抗排斥之藥物。

動脈硬化是個致病率和致死率相當高的慢性發炎疾病，為台灣十大死因之一。在病理過程中血中濃度過高的膽固醇為動脈硬化的一大危險因子，其會誘發一連串的發炎反應驅使血管壁內皮細胞功能喪失，血壓上升，平滑肌細胞增生等。Statin 是臨床上十分有效的降血脂藥物，雖然其作用機制已知在於抑制膽固醇合成酵素 HMG-CoA reductase 而有降血脂功效，但近年來探討 statin 在抗發炎方面的作用也漸受重視。nitric oxide synthase (NOS) 代謝產物如 nitric oxide (NO)，cyclooxygenase (COX) 代謝產物如 prostaglandin (PGE?、PGI?)，及 heme oxygenase-1 (HO-1) 代謝產物如 carbon monoxide (CO)，均有文獻指出可以改善血流，而可能在動脈硬化上扮演保護角色。相反的，matrix metalloproteinase (MMP) 的表現會誘使更多的免疫細胞浸潤到血管壁，並增加動脈硬化斑破裂，引起栓塞和中風的發生。在此實驗中，我們利用培養的大鼠主動脈血管平滑肌細胞作為研究材料，發現了 statin (lovastatin 、pravastatin、atorvastatin 、fluvastatin) 具有一些和降血脂無關的直接保護血管壁能力。包括會增加 interleukin-1β (IL-1β) 所誘導 iNOS 蛋白的表現及NO 的產生; statin 本身會增加 COX-2 和 HO-1 蛋白的表現及 PGE? 和PGI? 的產生，及抑制 MMP-2 和 MMP-9 蛋白活性的表現。此外分析調控 iNOS 基因轉錄最為關鍵的基因轉錄因子 NF-κB，發現適量的 statin 會增加 IL-1β 活化NF-κB 的作用。值得一提的是雖然適量 NO 有維持血管恆定的功能，過量時則會造成血壓過低休克的現象，這就是細菌感染後因內毒素 lipopolysaccharide (LPS) 作用引發敗血性休克的主要原因之一。為更進一步釐清 statin 是否會影響受細菌感染病人的生命危險，我們也探討 statin 對LPS 作用的影響。結果發現 statin 反而會抑制 LPS 誘導大量 iNOS 蛋白的表現，NO 的產生及 NF-κB 的活化。這些新的實驗結果提供更多證據支持 statin 可以藉由維持血管舒張，減緩血管壁的發炎反應，穩定動脈硬化斑的作用，以有效控制動脈硬化各個病程的進展。這發現能讓我們更透徹明白 statin 的作用，且對將來研發 statin 在心血管疾病方面新的臨床治療用途是有所助益的。 ;The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, statins, are potent inhibitors of cholesterol synthesis and have wide therapeutic use in cardiovascular diseases. Recent evidence, however, suggests that the beneficial effects of statins may extend beyond their action on serum cholesterol levels. In this study, we investigated the effects of lovastatin, pravastatin, atorvastatin, and fluvastatin on cultured rat vascular smooth muscle cells. We found statins can inhibit LPS-induced iNOS expression and NO production, while they can potentiate IL-1β-elicited responses. Moreover, statins themselves can stimulate COX-2 expression, PGE?, PGI? formation, and HO-1 induction. In contrast, statins can inhibit basal and IL-1β-induced enzyme activities of MMP-9 and MMP-2. In studying the activity of NF-κB, which plays an important role for iNOS gene induction, we found that statin can increase IL-1β-induced NF-κB activity, while inhibit that induced by LPS. All these results suggest that stimulation of iNOS expression in the presence of IL-1β, togeth1er with the increased COX-2 and HO-1 expression might contribute to the beneficial effects of statins in atherosclerotic process in terms of vasodilation and inhibition of smooth muscle cell proliferation. The inhibition of MMP activity might enhance plaque stability and reduce the development of atherosclerosis. All these results strengthen the pleiotropic actions of statins in anti-inflammation and anti-atherosclerosis.

a. Purpose of the Research Nowadays people are getting unhealthy, especially the heart. Since the outbreak of SARS, the Hong Kong citizens cared more about their health. In the past two decades, due to the technological advancement, many medical instruments that were used by doctors are now available to the public. One of the examples is the sphygmomanometer used for measuring blood pressure. On the other hand, very few heart monitoring devices are developed for public use. As a result, there is a need that such heart monitors should be available to the public. b. The device Our device is a modified stethoscope, which electronic components are added to this common medical instrument. The device mainly consists of 3 parts: 1. The sensor: modified from ordinary stethoscope, which a condenser microphone is added to change the heart sounds into electrical signals 2. The signal processor: integrated circuits and resistor-capacitor couples, which the cost is much lower than digital electronic components, are used to amplify and filter the noise in the electrical signals. The processor is divided into 3 stages: - Preamplifier: Amplifies the electrical signals converted from heart sounds - Low pass filter: Filters the noise in the signal. The cut-off frequency is 600Hz, which most of the heart sounds are below 600Hz. - Power amplifier: Amplifies the filtered signals before outputting into computers or earphones. 3. The output devices: it can be a computer or an earphone. In a computer, the heart sounds can be converted into graphs, enabling precise graphical analysis. Since many abnormal heart conditions will alter the heart sounds, with the aid of computers and graphs, people can know whether their hearts are normal or not, and can seek for medical support before developing any critical situation. Moreover, abnormal heart sounds are more significant in graphs, so any heart problems can be discovered more easily. c. Data During an exhibition in Hong Kong, about 1000 people tried the device. Among them approximately 150 people were confirmed to have heart problems with abnormal heart sounds. Using our device, we discovered 109 of them. As a result, the accuracy of the device is about 72.6% d. Conclusion With the low cost of our device (~€9.80/US$12.80), everyone would be able to afford it. As a result, people can check their conditions of their hearts more frequently, and would be able to discover any early heart problems.

My project is about investigation on traditional medicines in\r Namibia. This is all about plants and different herbs that different\r people in Namibia use for medicinal purposes. The objective of this\r project is to inform the world of how valuable nature is and how we can\r try to conserve nature’s treasure so that valuable knowledge cannot pass\r away with olden experienced herbalists. Understandably the enquiry\r into indigenous medicinal herbs arose from the need to expand the\r possibilities of ones own medical practice. It was very important for me\r to satisfy our farmer’s thirst for knowledge, which is what I want to do\r with the rest of the world.\r In this project I have included most but not all of the plants that we\r use in Namibia for medicinal purposes. Seeing that some of these plants\r do not grow in certain parts of the country, I have also tried to clone it in\r other parts of the country. I have also made a powder from one of the\r plant’s leaves and tested it on several people within the country.\r To conclude my findings and experiments I would like to say, let us\r try to take these precious knowledge left for us from our ancestors and\r maybe one day in the future we might need it in some ways.

To contribute to the feeding of the popular sector using a product of the basic basket of\r consumption and in simultaneous form to operate a natural resource, that when being combined\r will derive in a rich product in nutrients. With this project we try to offer to the population an\r innovating product, based on a food of daily but added consumption with all the nutrients of the\r nopal, of this form will be a better nutrition in the tortilla consumers. In Mexico, like in some\r countries of Central America, the maize products, like the tortilla, are the base of the popular\r feeding, its consumption is related closely to the obtaining of energy, calcium, fiber, iron and zinc,\r which usually display deficit levels in the population of the region. The contributions of the nopal\r are diverse, because it counts with some different nutritional and medicinal properties.

安非他命的濫用在台灣是非常嚴重的公眾健康及社會問題。安非他命會導致一連串的行為異常，包括在中腦紋狀體內釋放多巴胺及阻止多巴胺回收來增加使用者的活動力。由於安非他命會對腦細胞造成傷害，本研究的目的為探討低劑量、無立即毒性之安非他命(類似於人類使用習慣)長期施打下，是否會對C57BL6 小鼠大腦紋狀體內的蛋白質表現有影響。因此利用西方點墨法分析施打低劑量安非他命(2 到6 mg/kg) 約一星期之後，C57BL6 小鼠的大腦紋狀體中一些重要蛋白質(包括腺.酸受體A2A-R、第五亞型腺.酸環化.AC5、caspase-8 及PARP) 的表現是否有改變。實驗結果顯示，低劑量安非他命處理對這些蛋白質的表現並沒有明顯的差異。但利用二維電泳法可看到有少許蛋白質，在經過安非他命處理下有顯著的差別，如KIAA0193 homolog 、GOS-28、gammacrystallin A、malate dehydrogenase 和phosphoglycerate mutase isozyme B (PGAM-B)。這些蛋白質中，malate dehydrogenase 和PGAM-B 與代謝和產生ATP 有關，但前者是增加的，而後者減少，推測安非他命會影響神經細胞的能量代謝，因此長期施打安非他命對紋狀體造成的影響值得進一步探討。;The wide spreading use of amphetamine (AMPH) in Taiwan has become a serious public health and social problem. AMPH evokes a series of behavior abnormality including enhanced locomotor behavior by releasing dopamine and inhibiting dopamine-uptake in the striatum. Since AMPH is known to cause brain damage, the purpose of this study is to investigate the expression of several important proteins in the striatum of C57BL6 mice after chronic treatment with low and non-toxic dosages of AMPH (mimicking the common usage pattern of AMPH addict). C57BL6 mice were daily IP-injected with various dosages of AMPH (0 to 6 mg/kg) for one week. Expression levels of A2A adenosine receptor (A2A-R), adenylyl cyclase type V (AC5), caspase-8 and PARP in the striatum were analyzed by Western blotting analysis. Most proteins examined were not affected by this 1-week AMPH treatment. By the aid of two-dimensional gel electrophoresis, expressions of a few striatal proteins (such as KIAA0193 homolog, GOS-28, gammacrystallin A, malate dehydrogenase and phosphoglycerate mutase isozyme B (PGAM-B) in AMPH-treated mice were altered. Note that malate dehydrogenase and PGAM-B are two enzymes involved in energy metabolism and ATP generation. Interestingly, the former was increased and while the latter was decreased in AMPH-treated mice. Collectively AMPH may affect the energy metabolism in neuronal cells. These results suggest that the injury induced by long-term AMPH exposure warrants our further concerns and investigation.