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Epidemiology and Prevention of Respiratory Syncytial Virus Infections
No full text人類呼吸道融合病毒的感染會引起細支氣管炎和肺炎等下呼吸道感染，而且是嬰兒與年幼兒童的首要住院原因。RSV相關住院發生率的高峰出現在1個月大到6個月大之間。在具有明確RSV流行季節的溫帶國家中，約有50%的RSV相關住院事件都是發生在未滿6個月大的嬰兒身上，幾乎所有兒童在2歲時都已經至少感染過RSV一次。發生嚴重RSV疾病的前置因子包括年齡較小、早產、慢性肺病（CLD）、先天性心臟病（CHD）、神經肌肉障礙、免疫缺乏，以及唐氏症等。RSV的流行模式會因地理位置而異：在溫帶氣候中，RSV的流行常出現於秋季並持續流行至春季，而在熱帶地區則通常與雨季一致。RSV呼吸道疾病的治療主要為支持性治療。目前一般建議在冬季期間對高風險兒童每個月施用一次palivizumab以預防RSV感染。對於在台灣針對高風險嬰兒施行的RSV免疫預防療程而言，所謂的「RSV流行季節」可能與RSV感染有季節性高峰的其他國家（如美國）有所不同。台灣的全國性流行病學數據有限，且RSV感染帶來的整體疾病負荷仍未有具體的數據。因此，本研究的目的包括：1. 進行人口群研究以測定台灣RSV相關住院事件的嚴重性、危險因子、季節性以及醫療費用。2. 針對過去連續10季在北台灣分離出之臨床病毒株的RSV F蛋白基因，探討其親緣關係、演化變異性及族群動力學特性。3. 測定連續6個月每月施用palivizumab一次的新型預防療程，在高風險早產兒中預防RSV感染時的臨床有效性及安全性。 首先，我們利用台灣的全民健康保險資料庫，針對台灣未滿5歲兒童自2004年至2007年間因RSV而住院的案例，分析其人口群（population-based）年發生率、共存疾病及各項特性。本研究總共探討11081名曾經歷RSV相關住院事件的兒童。人口群平均年住院率在未滿6個月大和未滿5歲的兒童中，分別為每100,000人年1077和232例。發生率尖峰值出現在1個月和2個月大之間。發生風險的男女比為1.4:1（P<0.001）。依季節區分時有顯著的分布模式：每年一致以春季和秋季為高峰（P<0.001）。共有373例（3.3%）曾再次感染RSV。患有共存疾病的943名兒童（8.5%）年齡較大（P =0.001）、必須在加護病房（ICU）內住較長的時間（P <0.001）、氣管插管的發生率較高（P <0.05），且醫療費用較高（P <0.001）。共有888名病患（8%）需要住進加護病房。年齡較小（P <0.001）和患有共存疾病（P <0.001）是需要住進加護病房的獨立預測因子。結論是在台灣RSV感染每年會有兩波流行（春季和秋季達到高峰）。患有共存疾病者，其住院天數、加護病房住院天數會比較長，醫療費用也比較高。年齡較小、早產、先天性心臟病和腦性麻痺都是入住加護病房的預測因子。 接著，我們研究2000-2010年間於北台灣分離出的臨床RSV病毒株，測定F蛋白基因的分子流行病學特性和種系演變特性。針對於2000年7月和2010年6月間因急性下呼吸道感染就診的兒童身上所分離出之RSV病毒株，首先根據F蛋白基因序列進行分型。接著利用鄰接法（neighbor-joining (NJ) method）和最大概似法（maximum likelihood (ML) method）進行親緣關係的重建和評估。RSV F蛋白基因的種系演變模式係以貝氏馬可夫鏈蒙地卡羅框架（Bayesian Markov Chain Monte Carlo framework）分析而得。F蛋白基因所承受的天擇壓力，乃利用Datamonkey網站的介面進行偵測。總數為325株的臨床RSV病毒株中，親緣分析顯示83種A亞群病毒株（RSV-A）可進一步分成3群，而58種B亞群病毒株（RSV-B）則無顯著的分群現象。在細胞毒性T淋巴細胞（CTL）的HLA-B*57和HLA-A*01限制性表位上，可觀察到RSV-A和RSV-B之間有三個胺基酸不同（111、113和114號位置）。在RSV-B中可觀察到一處發生正向選擇（positive selection）的位置，而RSV-A中則無。病毒的演化速率在2000年以前幾乎無變動，接著於2000年和2005年之間減速，並於2005年之後出現顯著較快的演化現象。在每次流行中最主要的RSV-A亞型都會在下一次的流行中被其他亞型取代。結論是在2004年以前，RSV-A感染參與了數次小規模流行，而且只有極少數的病毒株得以演化而在往後數年中再次興起。2005年以後，流通於人群中的RSV-A病毒株明顯有別於前幾年的病毒株，並持續演化至2010年。種系演變模式顯示RSV的演化歧異性在最近5年內顯著上升。這些觀察結果對於疫苗的設計和流行病學研究可能十分具有價值。 台灣自2010年12月開始，一項每月施用一劑palivizumab、連續施用6劑的新型RSV感染預防療程已獲准用於高風險早產兒。為了研究此項新型療程預防RSV感染時的臨床有效性及安全性。我們從2011年4月開始至2013年3月為止，於馬偕紀念醫院小兒科部進行一項前瞻性觀察研究。我們收錄接受palivizumab預防療法的高風險嬰兒作為研究組，並進行12個月的追蹤。我們以傾向分數配對的方式，找出於2000年7月和2008年6月間於同一所醫院內出生並接受追蹤的歷史對照組。主要評估指標為RSV相關住院事件，而次要評估指標包括住院天數和加護病房（ICU）住院天數。這段時間內，我們收錄了127名嬰兒 (108名於妊娠週數≤28週時出生的嬰兒，加上19名妊娠週數為29-35週時出生且患有慢性肺病〔CLD〕的嬰兒）。他們已按照時程完成6劑的palivizumab療程。在研究組內，RSV相關住院事件在出院後6個月內有2件（1.6%），在12個月內有5件（3.9%）。我們以傾向分數配對的方式，為研究組內127名嬰兒配對出對照組內的 127名嬰兒。RSV相關住院率的降幅在出院後6個月內為86%（10.2%比上1.6%，P =0.002），在出院後12個月內則為78%（15.7%比上3.9%，P =0.004）。相較於對照組，加護病房住院率在出院後6個月內由7.1%顯著降為0.8%（P =0.024），在出院後12個月內則由7.9%顯著降為0.8%（P =0.014）。在6.4%的注射次數中曾記錄到不良事件。結論是在RSV感染案例無單季高峰期的地區（如台灣），連續6個月、每月一次的palivizumab肌肉內注射療法能有效預防高危險群病人RSV相關住院。 為了能讓大多數沒有潛在疾病的健康嬰兒也能預防呼吸道融合病毒感染，建立呼吸道融合病毒DNA疫苗之研究平台。我們選用了C57/BL6的小鼠品系作為實驗對象：DNA疫苗由大腿肌肉注射，每次注射100μg (兩腿各50μg)，間隔10天後再注射一次，合計共免疫三針。每次注射前自小鼠尾端採血並保存血清，第三次免疫的10天後，犧牲部份小鼠取得脾臟細胞進行免疫學分析，剩餘的小鼠以106 PFUs RSV經呼吸道感染後記錄體重變化。我們以小鼠血清做為一級抗體 (primary antibody)進行同樣的分析時，三種DNA疫苗的組合都偵測不到F蛋白，實驗顯示小鼠經過三次F蛋白的DNA疫苗注射後，無法產生能辨認F蛋白的抗體。除了中和抗體的生成外，我們也藉分析細胞性免疫反應 (cellular immune responses)來評估疫苗的效果。實驗結果顯示，同時注射F蛋白及GM-CSF的重組DNA時，樹突細胞和巨噬細胞表面的MHC class II皆有上升的趨勢。顯示CD4 T細胞有一定程度的活化，也呼應了抗原呈獻細胞MHC class II表現量上升的結果。然而，進一步分析T細胞產生干擾素(interferon-γ)的能力時，卻看不到干擾素-γ表現量上升，經過抗CD3抗體刺激後，CD4和CD8 T細胞的增生能力也沒有顯著提高。經由以上的實驗結果，我們可以知道，同時注射F蛋白和GM-CSF的重組DNA時，脾臟中CD4 T細胞和抗原呈獻細胞會部分活化，但其程度可能沒有高到會促進細胞增生，而且由於干擾素-γ表現量沒有增加，表示CD4 T細胞免疫反應可能不是往Th1的方向。至於如何藉由改變與調整DNA的劑量、佐劑的使用、注射疫苗的次數，以及評估時使用的檢驗方法是否適宜，以期能得到效果更顯著的疫苗，則需要參考更多文獻和進行更進一步實驗。Human respiratory syncytial virus (RSV) causes lower respiratory tract infection such as bronchiolitis and pneumonia and is the leading cause of hospitalization of infants and young children. The peak incidence of RSV-related hospitalization is between the second and the sixth month of age. In countries with temperate climates that have well defined RSV seasons, approximately 50% of RSV-related hospitalization occurs in infants younger than 6 months and almost all children are infected by RSV at least once by 2 years of age. Predisposing conditions for the development of serious RSV disease include young age, prematurity, chronic lung disease (CLD), congenital heart disease (CHD), neuromuscular impairment, immunodeficiency, and Down syndrome. RSV epidemics occur depending on geographic location; they cluster during the autumn and last until spring in temperate climate and generally coincide with the rainy season in tropical areas. Treatment of RSV respiratory illness is mainly supportive. Monthly palivizumab is currently recommended to high-risk children as a prophylaxis for RSV infection during winter season. The so-called “RSV season” for RSV immunoprophylaxis in high-risk infants in Taiwan might be different from other country with defined seasonal peaks of RSV infections such as United States. The nationwide epidemiologic data are limited and the total burden of RSV infection remains undefined in Taiwan Therefore, the aims of my study include: 1. Perform a population-based study to determine severity, risk factors, seasonality, and medical cost of RSV-associated hospitalization in Taiwan. 2. To investigate the phylogenetic relationship, evolutionary variability, CTL epitopes and population dynamics of the RSV F protein gene of clinical isolates in northern Taiwan over ten consecutive seasons. 3. To determine the clinical effectiveness and safety of the novel six consecutive monthly doses of palivizumab prophylaxis protocol for the prevention of RSV infection in high-risk preterm infants. 4. To estabilish the RSV DNA vaccine platform. Firstly, we analyzed the annual population-based incidence, underlying diseases and characteristics of hospitalizations due to RSV in Taiwanese children under 5 years of age from 2004 to 2007 by using Taiwan’s National Health Insurance database. A total of 11081 children with RSV-associated hospitalization were studied. Average annual population-based hospitalization incidences were 1077 and 232 per 100,000 children-year in children under 6 months and under 5 years of age, respectively. The peak incidence was between one and two month of age. The male-to-female incidence risk ratio was 1.4:1 (P<0.001). There was a significant seasonal distribution with consistent peaks in the spring and autumn every year (P <0.001). A total of 373 (3.3%) had repeated RSV infection. The 943 children (8.5%) with underlying diseases were older (P =0.001), required longer intensive care unit (ICU) stays (P <0.001), higher rate of endotracheal intubation (P<0.05), and higher medical cost (P <0.001). A total of 888 (8%) patients required ICU care. The younger age (P < 0.001) and underlying diseases (P < 0.001) were independent predictors of requiring ICU care. RSV infection occurred biennially with peaks in spring and fall in Taiwan. Patients with underlying diseases needed longer hospital stay, ICU stay and higher medical cost. Younger age, prematurity, congenital heart disease and cerebral palsy are predictors of ICU care. Secondly, we studied the molecular epidemiology and phylodynamics of the F protein gene in clinical RSV strains isolated in northern Taiwan from 2000–2010. RSV isolates from children presenting with acute respiratory symptoms between July 2000 and June 2010 were typed based on F protein gene sequences. Phylogeny construction and evaluation were performed using the neighbor-joining (NJ) and maximum likelihood (ML) methods. Phylodynamic patterns in RSV F protein genes were analyzed using the Bayesian Markov Chain Monte Carlo framework. Selection pressure on the F protein gene was detected using the Datamonkey website interface. From a total of 325 clinical RSV strains, phylogenetic analysis showed that 83 subgroup A strains (RSV-A) could be further divided into three clusters, whereas 58 subgroup B strains (RSV-B) had no significant clustering. Three amino acids were observed to differ between RSV-A and -B (positions 111, 113, and 114) in CTL HLA-B*57- and HLA-A*01-restricted epitopes. One positive selection site was observed in RSV-B, while none was observed in RSV-A. The evolution rate of the virus had very little changed before 2000, then slowed down between 2000 and 2005, and significantly evolved faster after 2005. The dominant subtypes of RSV-A in each epidemic were replaced by different subtypes in the subsequent epidemic. We found that before 2004, RSV-A infections were involved in several small epidemics and only very limited numbers of strains evolved and re-emerged in subsequent years. After 2005, the circulating RSV-A strains were clearly different from those of the previous years and continued evolving through 2010. Phylodynamic pattern showed the evolutionary divergence in RSV has significantly increased in recent 5 years. These observations may be valuable for vaccine design and epidemiological studies. Thirdly, a novel six consecutive monthly doses of palivizumab for RSV prevention protocol has been approved for high risk preterm infants since December 2010. We conducted an observational prospective study at Department of Pediatrics of Mackay Memorial Hospital from April 2011 to March 2013 to determine the clinical effectiveness and safety of this novel protocol for the prevention of RSV infection. We enrolled high-risk infants who received palivizumab prophylaxis as study group and followed up for 12 months. Historic control, those who were born and followed up in the same hospital between July 2000 and June 2008, were enrolled for propensity score matching. Primary endpoint was RSV-related hospitalization, and secondary endpoints included the length of hospital stay and intensive care unit (ICU) care. During the study period, we enrolled 127 infants (108 infants born at ≤ 28 weeks and 19 infants born at 29–35 weeks with CLD). They completed 6-dose palivizumab as scheduled. Among the study group, the RSV-related hospitalizations were 2 (1.6%) within 6 months and 5 (3.9%) within 12 months after discharge. We matched 127 infants in the control group with 127 infants in the study group by propensity score matching. The reductions of RSV-related hospitalization rates were 86% (10.2% vs 1.6%, P =0.002) within 6 months after discharge and 78% (15.7% vs 3.9%, P =0.004) within 12 months after discharge. Compared to the control group, the rate of ICU care significantly decreased from 7.1% to 0.8% (P =0.024) within 6 months after discharge and from 7.9 % to 0.8% (P =0.014) within 12 months after discharge. Adverse events were recorded in 6.4% injections. This six monthly intramuscular administration of palivizumab is effective for prevention of RSV hospitalization in regions with no single seasonal peak of RSV infection such as Taiwan. Fourthly, in order to protect most of the healthy baby with no underlying disease from respiratory syncytial virus infection, the establishment of respiratory syncytial virus DNA vaccines research platform is needed. We chose the mouse strain C57 / BL6 as experimental subjects: DNA vaccine by intramuscular vaccination to bilateral thigh with a total 100 μg (each leg 50 μg), for a total of three times of immunizations with an interval of 10 days. To measure HRSV-specific serum IgG, sera will be obtained from blood samples collected from the tail prior to each immunization. Ten days after the third immunization, mice were sacrificed for immunological analysis of spleen cells. Mice was challenged with 106 PFUs RSV and then recorded for weight change daily. We used the mouse serum to assay for antibody response. The experiments showed that mice can not generate F protein antibody after three F protein DNA vaccines. We also analyzed cellular immune response to evaluate the effectiveness of the vaccine. Experimental results showed that when injected with the F protein DNA and recombinant GM-CSF, the dendritic cells and macrophages cell surface MHC class II were upregulated. That CD4 T cells display a certain degree of activation also echoes the increases of MHC class II expression. However, further analysis of interferon-γ by T cells falied to demonstrate the increase of interferon-γ performance and CD4 and CD8 T cell proliferative ability were not significant increased through the anti-CD3 antibody stimulation. From above experimental results, when F protein DNA vaccine and simultaneous injection of recombinant GM-CSF DNA, the spleen CD4 T cells and antigen presenting cells partially activated, but the degree might not be so high as to promote cell proliferation. There were no increase in the amount of interferon-γexpressed by CD4 T cells; this implied there might not be good Th1 immune response. In order to get a more significant effect of the vaccine, we need to adjust the dose by DNA, the adjuvant, or the protocol of vaccination. We also need to search more literature and conducted further experiments
論程頤之理與事
No full text本論文主要從哲學議題出發，探討從先秦道器範疇至理事範疇的轉變與發展。以程頤理與事為討論的中心，觀看其思想上的邏輯發展與其賦予理事的新意。程頤認為，「理」是無形的，「事」具有具體的內容，並強調抽象之理與具體之事必須是合一的，二者之間體用不分，因此本論文最後一部分便針對理事概念的論說，找尋其思想上的內在實現。筆者以「禮」為例，說明程頤一方面賦予了「禮」之本質，一方面也實現其理事合一的哲學論說。目次
第一章 緒論……………………………………………………………1
第一節 研究動機、目的與方法 …………………………………1
第二節 前人研究成果 ……………………………………………3
第三節 研究內容 …………………………………………………6
第二章 由道器關係到理事關係….…………………………………13
第一節 道器關係 …………………………………………………14
第一小節 《老子》、《莊子》二者之道物關係 …………………14
第二小節 《易傳》、《淮南子》二者之道物關係 ………………18
第二節 王弼之理 …………………………………………………22
第一小節 至理為一 …………………………………………………22
第二小節 統宗會元之理 ……………………………………………25
第三小節 所以然之理 ………………………………………………29
第三節 郭象之理 …………………………………………………31
第一小節 物物自造之理 ……………………………………………31
第二小節 物物有理，事事有宜 ……………………………………33
本章小結 ……………………………………………………………37
第三章 程頤之理與事 ………………………………………………39
第一節 程頤之理 …………………………………………………40
第一小節 道即理 ……………………………………………………40
第二小節 性即理 ……………………………………………………47
第二節 程頤之事 …………………………………………………53
第一小節 物之理 ……………………………………………………54
第二小節 易之理 ……………………………………………………57
第三節 理一分殊 …………………………………………………… 67
本章小結 ………………………………………………………………72
第四章 程頤理與事的實現：以禮為例 ……………………………73
第一節 禮者，理也 ………………………………………………74
第一小節 禮 …………………………………………………………74
第二小節 禮與理 ……………………………………………………76
第二節 程頤之「禮」 ……………………………………………78
第一小節 程頤之「禮即是理」 ……………………………………78
第二小節 合內外之敬與義 …………………………………………87
第三節 程頤之「禮」：理事合一的具體實現 ……………………92
第一小節 「禮」：理事體用合一之表現 …………………92
第二小節 「禮」：理一分殊之表現 ………………………………93
本章小結 ………………………………………………………………95
第五章 結論 …………………………………………………………97
第一小節：論文成果 …………………………………………………97
第二小節：餘論 ………………………………………………………98
參考書目 ………………………………………………………………10
An interaction and an activation of ClpQ and ClpY protease in Escherichia coli
No full text大腸桿菌的ClpY/HslU為一種AAA 蛋白質（ATPase associated with various cellular activities），是具有ATPase活性的伴隨蛋白（chaperone）；ClpQ/HslV則是一種具胜肽酶（peptidase）活性的蛋白質。ClpY能與ClpQ形成具有蛋白酶活性的的多元體ClpYQ，其中ClpY扮演辨識基質並藉著消耗能量將基質解開送入ClpQ的角色；再經由ClpQ胜肽酶分解基質；前人發現ClpYQ與真核生物中的26S proteasome是具有同源性的。在結構上ClpY是以六元體環狀的形式，與兩個同為環狀的六元體ClpQ所構成的十二元體結合來作用。
在近年來的研究中指出，從已有的結晶結構來預測ClpYQ的作用區域可能在ClpY的羧基端以及ClpQ六元環之間形成的空間。而在2002年亦有實驗發現，若將ClpY最後十個胺基酸逐漸去除時，ClpYQ蛋白酶會失去其分解胜肽的能力；並且也會逐漸失去形成ClpYQ多元體的能力；更進一步甚至影響ClpY其本身形成六元環的能力；導致ClpY無法與胜肽酶 ClpQ結合而進行作用，因此失去ClpYQ蛋白酶的活性。
本實驗從已有的結晶結構預測可能之ClpQ與ClpY之交互作用點：ClpQ E61及K28及各自對應點ClpY R440及L443。並使用分子生物技術的方法，分別在胺基酸位置E61及K28建構全長為175個胺基酸之ClpQ點突變株，先使用RcsA及SulA相關系統來分析建構之突變株是否能與ClpY或是前人建構之ClpY突變株形成ClpYQ蛋白酶的活性，再分別使用酵母菌雙雜交系統 （Yeast two-hybrid system）觀察ClpY與ClpQ之間的作用力，以期望了解ClpY 羧基端結合與活化ClpQ功能的關係。發現ClpY之羧基端就如同一把鑰匙，插入ClpQ與相鄰ClpQ間產生的之孔隙，並開啟 ClpQ 的活性。藉著 ClpQ 結構改變的作用力可以幫助其緊密結合。其ClpY羧基端本身於長度、電荷以及其胺基酸構形上皆有適當的要求，且ClpQ孔隙由大量正電荷胺基酸所構成。ATP-dependent proteolysis plays an essential role in controlling the levels of key regulatory proteins and in the elimination of abnormal polypeptides. These tasks are carried out by architecturally related ATP-dependent proteases such as the 26S proteasome in eukaryotes and two component protease, the ClpAP, ClpXP, and ClpYQ (HslUV) in archea and eubacteria. The clpYQ/hslVU operon in Escherichia coli encodes two heat shock proteins, the HslV/ClpQ peptidase and HslU/ClpY ATPase. Both ClpY and ClpQ self-assemble into a hexameric rings. Until now, two substrates RcsA and SulA of another ATP-dependent protease Lon, and RpoH, can be recognized by ClpY.
In the clpYQ complex, the ClpY and ClpQ central pores are aligned, and the proteolytic active sites are sequestered in an internal chamber of ClpQ, with access to this chamber restricted to small axial pore. The roles of translocate peptides from ClpY to ClpQ and how interaction of ClpY and ClpQ to initiate ClpQ activity are not clear. In the research of Seong et al, an insertion of ClpY C-terminal tails into pockets at the ClpQ-ClpQ interface in the ATP-bound state might cause an opening of the central pore of ClpQ peptidase for an access of unfolded polypeptide substrates into the ClpQ proteolytic chamber. The ClpY C-terminal tail is essential for its interaction with ClpQ and for an activation of the peptidase. With ClpY molecule, the last 7 amino acids sequence of ClpY is highly conserved, and the lacking of the last four amino acids, ClpY leads to ClpYQ inactive.
In this study, from ClpYQ crystal structure, R440 and L443 in ClpY C-terminal are opposited to E61 and K28 in ClpQ. Moreover, ClpQ point mutants in the position E61, K28 were constructed. And these positions were substituted with other 19 Amino acids. Subsequently, two methods were used to funtionally assay the activation between ClpQ mutants and ClpY as well as ClpY C-terminal mutants in E.coli. The yeast two-hybrid system was used to analyze oligomerlization of mutants between ClpQ-ClpY and ClpQ-ClpQ. The aim of this study is to find the significance of E61 and K28 in ClpQ, and a relationship between an interaction and an activation of ClpY and ClpQ protease.中文摘要 7
Abstract 8
壹、前言 9
一、蛋白酶簡介 9
二、熱休克蛋白酶HslUV（ClpYQ）簡介 10
三、雙次元體ClpYQ 11
四、大腸桿菌ClpYQ蛋白酶之結構分析 12
五、大腸桿菌ClpYQ蛋白酶之基質 13
六、大腸桿菌ClpY羧基端與ClpQ之關係 15
七、研究動機及目的 17
貳、材料與方法 19
一、研究材料 19
1、實驗菌株及質體 19
（1）ClpYQ於細胞內之活性表現系統（Activation assay） 19
（2）酵母菌雙雜交系統（Interaction assay） 19
2、藥品及試劑 20
（1）相關酵素 20
（2）培養基 20
（3）實驗相關核酸引子 21
3、分析軟體 21
4、結構資料 21
二、實驗方法 21
1、質體製備與轉殖 21
（1）質體製備 22
Mini-preparation of plasmid DNA from bacteria（小量製備） 22
Midi-preparation of plasmid DNA from bacteria（大量製備） 22
（2）質體轉殖 22
限制酶(restriction enzyme )反應 22
DNA瓊脂膠體(agarose gel )電泳 22
DNA瓊脂膠體(agarose gel )電泳回收DNA 22
接合反應(DNA ligation ) 22
2、勝任細胞（competent cell）製備 23
MC1068（用於大量複製一般質體） 23
KC8（用於電穿孔法electroporation transformation） 23
3、轉形作用（transformation） 24
（1）E. coli transformation 24
Heat shock transformation 24
Electroporation transformation 24
TSS-Transformation（用於AC3112菌株之轉形） 25
4、ClpQ點突變基因之質體建構 26
（1）二次聚合酶連鎖反應（PCR, Polymerase chain reaction） 26
（2）選殖基因之質體製備（Cloning Technology） 28
pGilda（用於Yeast菌株之轉形） 28
pB42AD（用於Yeast菌株之轉形） 28
pBAD33（用於AC3112菌株之轉形） 28
5、ClpYQ於細胞內表現之活性測試（Activation assay） 29
（1）β-galactosidase活性測試 29
（2）MMS（Methyl methansulfonate）之抗性分析 30
6、酵母菌雙雜交系統報導基因之表現測試（Interaction assay） 31
（1）lacZ 表現：酵母菌β-galactosidase活性測試 31
（2）lacZ 表現：酵母菌X-gal測試 32
（3）leu2 表現：酵母菌生長測試 33
7、程式分析 33
3D結構之預測與分析 33
參、實驗結果 34
一、前人建構之ClpY羧基端之突變蛋白分析與整理 34
二、分析建構之ClpQ E61點突變蛋白 34
1、ClpQ E61突變蛋白之活性分析 35
（1）從β-galactosidase活性分析ClpQ E61突變蛋白與ClpY作用分
解RcsA的能力 35
（2）以MMS生長測試分析ClpQ E61突變蛋白與ClpY作用分解
SulA的能力 36
2、用Yeast two-hybrid分析建構的ClpQ E61突變蛋白與ClpY之間的交互作用力 36
3、用Yeast two-hybrid分析建構的ClpQ E61突變蛋白本身形成六元
體的能力 37
三、分析建構之ClpQ E61C點突變蛋白與前人建構之ClpY羧基端R440
之突變蛋白之間的活性與交互作用力 38
1、ClpQ E61C突變蛋白與ClpY羧基端R440突變蛋白之活性分析 38
2、用Yeast two-hybrid系統分析ClpQ E61C突變蛋白與ClpY羧基端R440突變蛋白之間的交互作用力 39
四、以建構之ClpQ E61C點突變蛋白與ClpY羧基端L443之突變蛋白進
一步分析活性與交互作用之關係 39
1、ClpQ E61C突變蛋白與ClpY羧基端L443突變蛋白之活性分析 39
2、用Yeast two-hybrid系統分析ClpQ E61C突變蛋白與ClpY羧基端
L443突變蛋白之間的交互作用力 40
五、分析建構之ClpQ K28點突變蛋白 41
1、ClpQ K28突變蛋白之活性分析 41
2、用Yeast two-hybrid分析建構的ClpQ K28突變蛋白與ClpY之間
的交互作用力 41
3、用Yeast two-hybrid分析建構的ClpQ K28突變株本身形成六元體
的能力 42
六、分析建構之ClpQ K28R點突變蛋白與前人建構之ClpY羧基端L443
之突變蛋白之間的活性表現 42
七、以結構軟體分析ClpQ蛋白之正電荷胺基酸分布位置 43
肆、討論 45
一、胺基酸E61在ClpQ蛋白質中扮演的意義 45
二、分析ClpQ E61與ClpY R440之間的關係 45
三、胺基酸K28在ClpQ蛋白質中扮演的意義 46
四、分析ClpQ K28與ClpY L443之間的關係 47
五、正電荷胺基酸在ClpYQ蛋白酶中的重要性 47
六、整理分析ClpQ與ClpY之間活性與交互作用的關係與重要性 48
七、結論 50
伍、參考文獻 52
陸、表 64
柒、圖 7
The Effect of Homophones on Mandarin-speaking Children's Lexical Organization
No full text中文有兩大特性，第一，物體名稱通常為雙音節複合詞 (disyllabic compounds) ，第二，同音詞比例很高。屬於同一個類別 (category) 的物體，通常其名稱的第二個音節相同，因此，中文的同音詞素 (homophone) 在這種情況下對兒童物體分類可能具有正面的影響，即同音會促進兒童抽取出物體相似的特性或是功能上的相似性。中文另一項特點為同音詞是一個常見現象，不同種類的物體可能在名稱上會包含相同或相似的聲音，這個特性使兒童在學習時可能會把不同種類的東西誤認為同一類。從這兩個特性來看，中文的同音詞在詞彙學習上有優點也有缺點。本研究以兩個實驗來探討同音詞素對於兒童的詞彙分類究竟會不會產生影響。九十六位學齡前的幼童參與本研究，平均年齡分別為四歲及六歲。受試者必須判斷詞組內的詞是否屬於同一類。在第一個實驗中，物體名稱及類別名稱皆為真詞，受試者可以根據之前的經驗或是實驗時提供的圖片作為判斷依據。而在第二個實驗中，類別名稱皆為新詞 (novel words)，受試者必須根據實驗者所提供的故事內容來判斷哪些物體屬於同一類。實驗材料可分為三種，第一種是類別不同語音也不同的詞組 (non-member with different form)，第二種是類別不同語音相同 (non-member with same form)，第三種是類別相同但語音不同的詞組 (member with different form)。結果發現，語音對於幼童詞彙分類有很大的影響。學齡前幼童（尤其是四歲組的幼童）確實傾向將名稱發音相似的物體當成同一類。另一方面，當兒童在學習新的類別，而此類別內部成員很少且名稱分歧時，孩童即減少使用語音作為分類的依據。此外，隨著年齡及經驗的增加，兒童會逐漸使用語義而非語音來做為詞彙分類的準則。In Mandarin, names of objects are usually disyllabic compounds. Items belonging to a category often share a final syllable that is the same as the name of the category, which often serves as the head of a compound. Since words ending with the same syllable are likely to belong to the same category, one strategy that can be used in lexical organization is judgment by the phonological forms of words. Nevertheless, another feature of Mandarin is the frequency of homophones. Words belonging to different categories often end with identical syllables. Therefore, children may mistake items of different categories as belonging to the same one. Given that there are both advantages and disadvantages in employing homophones in categorization, the present study is designed to examine whether homophonic heads of compounds influence children’s lexical organization when they are required to group items according to their meanings or perceptual features.
Ninety-six Mandarin-speaking four- and six-year-old preschoolers participated in this study. With pictures provided, the participants were asked to judge whether certain pairs of objects belonged to the same category. There are three types of stimuli in this study, the NMDF (non-member with different final syllable), NMSF (non-member with same syllable), and MBDF (member with different syllable) types. In all three types of materials, the first item in a pair is a member of a category and shares the final syllable with the name of that category. However, the items presented as the second of the pairs in each of the three types are different. In the NMDF type of stimuli, the second item is a non-member of the category and its final syllable is different from that of the first item. The second item of a pair in the NMSF type of material is a non-member sharing the same final syllable with the first item. As to the MBDF type, the second item belongs to the same category as the first one but contains a different final syllable.
The results showed that in addition to semantic knowledge, phonological form played an important role in children’s categorization. Homophonic morphemes were strong cues that lured children, especially the younger ones, to take items ending with the same syllable as belonging to the same category, even though visual cues (pictures) were provided. With increase of age and experience, children may find this phonology-driven strategy not always useful. They would gradually apply another more reliable strategy, the semantic one, in their judgment of category members.Acknowledgements ………………………………………………………………… i
English Abstract …………………………………………………………………… ii
Chinese Abstract …………………………………………………………………… iii
Table of Contents …………………………………………………………………… iv
List of Tables ………………………………………………………………………... vi
List of Figures …………………………………………………………...……….. vii
Chapter 1 Introduction ………………………………………………….... 1
1.1 Background ……………………………………………………………….. 1
1.1.1 Characteristics of Chinese Words …………………………………… 1
1.1.2 Homophones in Acquisition …………………………………………. 2
1.1.3 Compounds in Mandarin ……………………………………………. 3
1.1.4 Acquisition of Morphology ………………………………………… 8
1.1.5 Competition Model and Chinese Compounds ……………………… 9
1.2 Goal of the Study ………………………………………….………...… 10
1.3 Organization ……………………………………………………………11
Chapter 2 Literature Review ………………………………………...….. 12
2.1 Form and Function Mapping …………………………………………….. 12
2.2 Formation of Morphological Paradigms ……………………………….. 14
2.3 Mental Representation of Compounds ………………………………… 16
2.4 Compounds in Acquisition ………………………………………….….. 19
2.5 Phonological Effects on Categorization …………………………….….. 20
Chapter 3 Experiment 1 – Real Category …………………………….. 23
3.1 Design and Materials ……………………………………………………. 23
3.1.1 Design of the Study ………………………………………………… 23
3.1.2 Materials ……………………………………………………………. 26
3.2 Participants and Procedure ….………………………………………… 30
3.2.1 Participants …………………………………………………………. 30
3.2.2 Experimental Procedure ……………………………………………. 30
3.2.3 Scoring of Truth Value Judgment Task …………………………….. 32
3.3 Results …………………………………………………………………… 32
3.3.1 Results of Truth Value Judgment Task – By-Item Analyses ……….. 33
3.3.2 Results of Truth Value Judgment Task – By-Subject Analyses ….…. 38
3.3.3 Results of Picture Selection Task ……………………………….….. 41
Chapter 4 Experiment 2 – Novel Category ………………….…………. 44
4.1 Methodology …………………………………………………………….. 44
4.1.1 Participants …………………………………………………………. 44
4.1.2 Materials ………………………………………………………….. 44
4.1.3 Procedure ……………………………………………………………46
4.2 Results …………………………………………………………………….48
4.2.1 Results of Truth Value Judgment Task – By-Item Analyses ………...48
4.2.2 Results of Truth Value Judgment Task – By-Subject Analyses ……..53
4.2.3 Results of Picture Selection Task ……………………………………56
Chapter 5 Comparison between Experiment 1 and Experiment 2 …..59
5.1 Comparison of Truth Value Judgment Tasks ……………………………..59
5.1.1 Same/Different Category Judgment ………………………………...59
5.1.2 Category Membership Judgment …………………………………....60
5.1.3 By-Subject Analyses ………………………………………………...62
5.2 Comparison of Picture Selection Tasks …………………………………...65
5.2.1 Response Types in Picture Selection Tasks ………………………….65
5.2.2 Response Types Used Consistently ………………………………….66
5.3 Relation between Truth Value Judgment and Picture Selection Task …….68
5.3.1 Performance in Truth Value Judgment Task by Children Using Consistent Response Type ………………………………………….68
5.3.2 Phonological Type of Response and Truth Value Judgment – Experiment 1 ……………………………………………………….70
5.3.3 Phonological Type of Response and Truth Value Judgment – Experiment 2 ………………………………………………………..73
Chapter 6 Discussion and Conclusion ………………………………….76
6.1 Summary ………………………………………………………………….76
6.2 Phonological Effects …………………………………………………….79
6.3 Semantic/ Contextual Information …...…………………………………...81
6.4 Compound Learning and Category Formation ……………………………82
6.5 Conclusion ………………………………………………………………. .83
References …………………………………………………………………………..85
Appendices ………………………………………………………………………….90
Appendix 1 Sets of Items Selected ………………………………………....90
Appendix 2 Children’s answers to TVJ questions ….……………………….91
Appendix 3 Stories and Instruction in Experiment 2 ………………………..9
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
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Design and Implementation of an Oversampling Delta-Sigma Modulator
No full text在這篇論文中，我們設計並以交換電容電路實現了一個多位元的三角積分調變器。在多位元的調變器中存在一個提供回授信號的數位類比轉換器，由於該數位類比資料轉換器是由多個單位電容所構成，這些單位電容很容易因為積體電路製程或是電路的實體佈局時產生元件不匹配的效應，此一不匹配效應所帶來的非線性失真將直接由調變器輸出，而無法經由三角積分調變器本身的雜訊整形技術獲得任何的改善。為了改善這個問題，本調變器應用了動態元件匹配(Dynamic Element Matching)技術來降低不匹配誤差。其中，資料加權平均(DWA)演算法因與其他的動態元件匹配演算法相較，具有一階雜訊整形特性，能快速降低誤差與電路易於實現的優點，所以被我們採用於設計的調變器中。
設計的調變器信號頻寬為24kHz，可以應用於音頻的領域上。整個調變器是以混合訊號模式進行設計，並以Matlab進行調變器的建模與行為模擬。最後我們以TSMC 0.18um 1P6M 3.3V製程，利用Hspice與Spectre軟體進行電路的設計、模擬驗證與佈局。In this Thesis, we designed and implemented an oversampling multi-bit delta-sigma modulator. Multi-bit delta-sigma modulator uses an internal DAC to provide the feedback signal. However, elements mismatch in DAC due to process variation will results in non-linear distortion and cannot be noise shaped by the delta-sigma modulation loop, this will degrade the performance of a delta-sigma modulator very much.
In order to reduce the mismatch error of DAC, many dynamic element matching (DEM) algorithms have been proposed. Compared with other algorithms, the data weighting averaging (DWA) technique is used in our design due to it has the advantage of fast error cancellation and easy circuit implementation.
We use TSMC 0.18um 1P6M process and mixed-signal design methodology for our work. The designed modulator presents a 24 kHz signal bandwidth and can be used in audio application.ABSTRACT ................................................................................................................I
TABLE OF CONTENTS.................................................................................................. III
LIST OF FIGURES .........................................................................................................VII
LIST OF TABLES ............................................................................................................ XI
CHAPTER 1 INTRODUCTION ................................................................................ 1
CHAPTER 2 DELTA-SIGMA MODULATOR FUNDAMENTALS......................... 3
2.1 Quantization and Quantization Noise ........................................................... 4
2.2 Oversampling Techique ................................................................................ 8
2.3 Delta-Sigma Modulators............................................................................. 10
2.3.1 First-Order Delta-Sigma Modulator .................................................... 10
2.3.2 Second-Order Lowpass Delta-Sigma Modulators ............................... 17
2.3.3 Bandpass delta-sigma Modulators ....................................................... 19
2.4 The Third and High-Order Delta-Sigma Modulators ................................. 21
2.4.1 Single-Loop High-Order Delta-Sigma Modulators ............................. 21
2.4.2 Multi-Stage Noise Shaping Single-Loop Delta-Sigma Modulators .... 23
CHAPTER 3 DESIGN OF MULTIBIT DELTA-SIGMA MODULATORS ............ 25
3.1 Linearity Issues of Multibit of Multibit Delta-Sigma Modulators.............. 26
3.2 Multi-bit Noise-Shaping and Dynamic Element Matching ........................ 29
3.2.1 Dynamic Element Random Averaging................................................. 31
3.2.2 Conventional Clocked Averaging ........................................................ 33
3.2.3 Individual Level Averaging.................................................................. 35
3.2.4 Data Weighted Averaging ........................................................................ 36
CHAPTER 4 SYSTEM AND CIRCUIT SPECIFICATIONS.................................. 41
4.1 System Architectures and Specifications.................................................... 41
4.1.1 Noise Design........................................................................................ 43
4.1.2 Dynamic-Range Scaling ...................................................................... 46
4.2 Thermal Noise and Capacitor Sizing .......................................................... 48
4.3 OP Amplifier Specification......................................................................... 50
4.3.1 Slew Current ........................................................................................ 51
4.3.2 Linear Settling Time and Unit Gain Frequency................................... 51
4.3.3 Transconductance Specification .......................................................... 54
4.3.4 DC Gain of Op Amp ............................................................................ 55
4.4 MATLAB Behavior Modeling and Simulation........................................... 55
CHAPTER 5 CIRUIT DESIGN AND IMPLEMENTATION.................................. 59
5.1 Integrator..................................................................................................... 59
5.2 Operational Amplifier ................................................................................. 61
5.2.1 Folded-Cascode Amplifier................................................................... 62
5.2.2 Common-Mode Feedbacks .................................................................. 66
5.2.3 Bias Circuit for the Operational Amplifier .......................................... 68
5.3 Four-Bit Quantizer ...................................................................................... 71
5.3.1 Switched-Capacitor Comparator.......................................................... 73
5.3.2 Comparator .......................................................................................... 73
5.3.3 Fat Tree Thermometer-to-Binary Encoder........................................... 76
5.3.4 Simulation Result of Quantizer............................................................ 77
5.4 Clock Genterator......................................................................................... 78
5.5 DWA Control Unit ...................................................................................... 79
5.6 Whole System Simulation........................................................................... 81
CHAPTER 6 CONCLUSION................................................................................... 83
REFERENCE .............................................................................................................8
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