In Saccharomyces cerevisiae DNA postreplication repair (PRR) functions to bypass replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis (TLS) has been well characterized, little is known about the molecular events involved in error-free bypass although it has been assumed that homologous recombination (HR) is required for such a mode of lesion bypass. We undertook a genome-wide, synthetic genetic array (SGA) screen for novel genes involved in PRR and observed evidence of genetic interactions between error-free PRR and HR. We were screening for synthetic lethality which occurs when the combination of two mutations leads to an inviable organism, however, either single mutation allows for cell viability. In addition, we screened for conditionally synthetic lethal interaction which occurs when the combination of two mutations is inviable only in the presence of a DNA-damaging agent. This screen identified and assigned four genes, CSM2, PSY3, SHU1 and SHU2, whose products form a stable Shu complex, to the error-free PRR pathway. Previous studies have indicated that the Shu complex is required for efficient HR and that inactivation of any one of these genes is able to suppress the severe phenotypes of top3 and sgs1. We confirmed and further extended some of the reported observations and demonstrated that error-free PRR mutations are also epistatic to sgs1. Based on the above analyses, we propose a model in which error-free PRR utilizes the Shu complex to recruit HR to facilitate template switching, followed by double-Holliday junction resolution by Sgs1-Top3. Null mutations of HR genes including rad51, 52, 54, 55 and 57 are known to confer characteristic synergistic interactions with TLS mutations. To our surprise, null mutations of genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which is also required for HR, are epistatic to TLS mutations. The MRX complex confers an endo/exonuclease activity required for the detection and processing of DNA double-strand breaks (DSBs). Our results suggest that the MRX complex functions in both TLS and error-free PRR and that this function requires the nuclease activity of Mre11. This is in sharp contrast to other known HR genes that only function downstream of error-free PRR. Furthermore, we found that inactivation of SGS1 significantly inhibits proliferating cell nuclear antigen (PCNA) monoubiquitination and is epistatic to mutations in TLS, suggesting that Sgs1 also functions at earlier steps in DNA lesion bypass. We also examined the roles of Sae2 and Exo1, two accessory nucleases involved in DSB resection, in PRR. We found that while Sae2 is primarily required for TLS, Exo1 is exclusively involved in error-free PRR. In light of the distinct and overlapping activities of the above nucleases in the resection of DSBs, we propose that the distinct single-strand nuclease activities of MRX, Sae2 and Exo1 dictate the preference between TLS and error-free PRR for lesion bypass. While both PRR pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes non-canonical Lys63-linked polyubiquitinated PCNA to signal lesion bypass. This mechanism is dependent on the Mms2-Ubc13 complex being in close proximity to PCNA, a process thought to be dependent on Rad5. Rad5 is a member of the SWI/SNF family of ATPases that contains a RING finger motif characteristic of an E3 Ub ligase. Previous in vitro experiments demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase/ATPase activity. We therefore created site-specific mutants defective in either Rad5 RING finger or helicase/ATPase activity, or both, in order to examine their genetic interactions with known TLS and error-free PRR genes. Our results indicate that both the Rad5 RING finger motif and the helicase/ATPase activity are exclusively involved in error-free PRR. To our surprise, like the Rad5 RING finger, lack of the helicase/ATPase activity also abolishes the Lys63-linked PCNA polyubiquitin chain formation, suggesting that either the Rad5 helicase/ATPase-promoted replication fork regression signals PCNA polyubiquitination or this domain has a yet unidentified activity. In summary, results obtained from this thesis dissertation have revealed novel mechanisms of yeast PRR in S. cerevisiae, a mechanism that appears to be evolutionarily conserved throughout eukaryotes, from yeast to humans.

O Objetivo deste trabalho foi desenvolver uma estratégia vacinal de utilizando linhagens de salmonellas capazes de expressar uma forma atoxica da principal toxina associada a linhagens de EHEC que causa a síndrome hemolítica urémica. Estas linhagens de salmonellas diferem na expressão de flagelina, a principal unidade estrutural do flagelo. As linhagens foram capazes de expressar uma forma atóxica da proteína \"Shiga-like\" no interior da bactéria e no espaço extracelular, apresentaram alta estabilidade plasmidial in vitro e in vivo e foram capazes de aumentar o grau de colonização intestinal. Além disso, avaliamos o potencial imunogênico de estas linhagens e encontramos que a imunização com três doses de salmonelas foram capazes de gerar anticorpos sistêmicos (IgG) e locais (IgA) com propriedades neutralizantes in vitro e proteção parcial em condições in vivo frente a ensaios de desafios com a toxina nativa. Não fomos capazes de encontrar diferencias significativas em lãs propriedades imunogênicas entre as linhagens que diferem na expressão de flagelina. Nossos resultados indicam que formas atóxicas da proteína Stx2 expressadas em vetores biológicos podem ser uma alternativa de estratégia vacinal para controlar a SHU. / The goal of this study was to develop a vaccine strategy of using Salmonella strains capable of expressing a nontoxic form of the main toxin associated with EHEC strains that cause hemolytic uremic syndrome. These strains of Salmonella differ in flagellin expression, the main structural unit of the flagellum. The strains were capable of expressing a nontoxic form of the protein \"Shiga-like\" within the bacteria and the extracellular space, showed high plasmid stability in vitro and in vivo and were able to increase the degree of intestinal colonization. Furthermore, we evaluated the immunogenic potential of these strains and found that immunization with three doses of salmonella were able to generate (IgA) humoral and (IgG) systemic antibodies with neutralizing properties in vitro and partial protection in vivo conditions facing trials challenges with the native toxin. We were unable to find significant differences in the immunogenic properties between these strains that differ in flagellin expression. Our results indicate that nontoxic forms of Stx2 expressed in biological vectors can be an alternative vaccine strategy to control the SHU.

No description available.

本文研究之目的，在於釐析四庫提要經部之義例，藉由目錄學之探討，從四庫總目豐 富的材料中，詳為分析，即可供撰寫目錄學之參究，亦可從中探得清代樸學之精蘊。 蓋提要之作，成於通儒碩學之士，雖絀於時日，草率成編，然其校勘得失，考證詳確 ，識詣即真，鑒裁亦卓，以之為讀群書之門徑，其霑溉後學，功用良多。 近人姚名達利用清人輯本別錄、七略之有限資料，分析其義例，為目錄學之研習，闢 一途徑，而四庫提要經部則有四十四卷之多，內容可謂豐富，故本文之撰寫方式，首 章為緒論，敘述四庫提要與學術研究之關係，並說明撰述之體例。次章至七章則分就 書名，卷數，撰者，板刻，辨偽，批評與價值等項，加以分析，並歸納成若干義例， 融合目錄學與經學於一爐，翼收因書究學之效。八章論館臣對目錄學之見解。九章探 討經義考與四庫提要之關係。十章比較各種提要之差異，藉以了解館臣前刪潤筆削之 權衡，亦可為學術研究之資料。末章結論，說明本文之特點。創意，並如何著手進行 後續之工作。

一、顏李學派倡「實踐主義」，於清初經世致用之學風中，尤獨樹一格；而其主張， 於政治、社會，誠有救弊之效。然後人研究顏學者多，研究李學者寡，致鮮有人知李 學之修訂補足顏學者，故為文闡述之。 二、斯編之作，以李恕谷遺著為主要資料，次及顏習齋遺著、宋明清有關之學術著作 及史料，並參以近人相關之著作。 三、斯編之作，首先蒐集有關文獻、擬訂大綱，即據大綱將原典資料及閱讀心得分門 別類以卡片記錄之；至著手為文，以論述恕谷學術思想之本質為中心，間及於其與顏 學之異同。 四、期編之作，共分九章，約十萬言。首章述恕谷之生平，重其為學之歷程及思想之 發展；二章述恕谷於著作之觀點，並簡介其著作之內容，以見其思想之梗概；三章述 恕谷於交遊之態度，並擇其交遊中影響其思想較深者略述之；四章述明末清初之學風 ，明恕谷於當時之學術背景下如何自處，自倡實學至入於考據，皆有論述。五章述恕 谷之心性論；六章述恕谷之修養論；七章述恕谷之實學論；八章述恕谷之政教論；此 四章為恕谷學術思想之重點論述。九章結論，綜合學者之評論及筆者之心得，得失俱 見，予恕谷學術思想之價值以定位。 五、觀茲世之現象，誠有如顏、李所指斥之病態，故其說亦可用於當前，唯顏學較為 保守固陋，復古之說實有時代之限制；而恕谷能通達時變，其學應可提醒世人，並指 引世人努力之方向。

Résumé confidentiel / Confidential abstract

Les toxines Shiga (Stx) sont produites par Shigella dysenteriae et certaines espèces d’E. coli transmisent aux humains par la consommation d'aliments contaminés et causant des maladies graves. La toxine Stx est libérée par les bactéries dans l'intestin et par la suite, traverse les vaisseaux sanguins en aval pour atteindre leurs principaux organes cibles, notamment les reins. Les dommages causés aux reins peuvent entraîner des complications graves notamment Le syndrome hémolytique urémique (SHU). A ce jour, il n’existe aucun traitement disponible contre le SHU. Les toxines Stx usent du transport rétrograde intracellulaire pour infester les cellules endothéliales rénales et atteindre leur cible cytosolique, l'ARN ribosomal 28S. Via un screening à haut débit, il a été démontré que le composé Rétro-2 bloque le trafic rétrograde de Stx à l'interface Endosome-TGN, sans affecter la morphologie des organites cellulaires et le trafic des protéines endogènes. Au cours de cette thèse, une analyse des relations structure fonction du composé Retro-2 nous a permis d’identifier les régions de l'inhibiteur qui sont critiques pour l'activité de protection. Nous avons identifié un dérivé dihydroquinazolinone nommé Rétro-2.1 qui est à ce jour l'inhibiteur le plus puissant contre les toxines Stx. Afin d’identifier la cible moléculaire de Retro-2.1, nous avons développé des sondes photo-activables bio-actives. En outre, les données de diffraction des rayons X ont révélé que de l'activité antitoxine réside principalement dans l’énantiomère S. (S) -Retro-2.1 est 500 fois plus puissant contre Stx (50 nM) que la molécule initiale. Cette étude peut donner lieu à un nouveau concept thérapeutique ciblant la voie de transport rétrograde de la toxine à l'intérieur de la cellule hôte. Une telle stratégie thérapeutique pourrait donc être étendue à d'autres agents pathogènes qui usent également du trafic rétrograde pour une intoxication des cellules hôtes. Ce nouveau concept thérapeutique qui permet de cibler les cellules hôtes et non l'agent pathogène représente une véritable percée dans la découverte de médicaments à large spectre et réduit le risque de développement d’une résistance chez l’agent pathogène. / Shiga toxins (Stx) are produced by Shigella dysenteriae and certain species of E. coli that can be transmitted to humans primarily through consumption of contaminated foods and may cause severe disease. Stx is released by the bacteria in the intestine and subsequently, could cross the downstream blood vessels to reach their main target organs such as kidney. Damage to the kidney can result in serious life-threatening complication hemolytic uremic syndrome, for which there is no proven safe treatment available other than supportive care. Stx invades renal endothelial cells in a retrograde manner from cell surface to the endoplasmic reticulum in order to gain access to its cytosolic target, 28S rRNA. By using HTS, it was previously demonstrated that the compound Retro-2 blocks retrograde trafficking of Stx at the early endosome-TGN interface, without affecting the morphology of cellular organelles and trafficking of other endogenous proteins. In this work, different regions of the lead inhibitor Retro-2 that are critical for the protective activity have been determined by systematic structure-activity relationship studies. It allowed us to identify a dihydroquinazolinone derivative, named Retro-2.1 that is the most potent inhibitor of Stx to date and also to develop bio-active photo-activatable probes with the aim of identifying the molecular target of Retro-2 derivatives. Further, crystal X-ray diffraction data revealed that the antitoxin activity resides mainly in the S-enantiomer. (S)-Retro-2.1 has displayed 500 fold more potency (50 nM) than parent molecule against Stx cytotoxicity. This study may result in a new therapeutic concept - targeting the retrograde transport route of toxin inside host cell - for the treatment of Stx-producing E. coli infections and could therefore be extended to other pathogens that also traffic via the retrograde transport. Such a new therapeutic concept that target the host cells and not the pathogen itself would represent a real breakthrough in drug discovery leading to broad spectrum drugs.

Infecções associadas a cepas da Escherichia coli enterohemorrágica (EHEC), podem causar manifestações clínicas sendo a Síndrome Hemolítica Urêmica (SHU), a complicação mais severa. SHU está relacionada a presença da toxina de Shiga do tipo 2 (Stx2) e até o momento não se dispõe de uma vacina ou tratamentos efetivos para uso em humanos. Assim, este trabalho teve por objetivo desenvolver uma vacina baseada no derivado atóxico contendo a subunidade B e a porção A2 da subunidade A denominado Stx2ΔAB. Após expressão em linhagens de E.coli e tentativas iniciais de purificação, resultaram na formação de agregados proteicos. Ajustes nas condições de cultivo e purificação permitiram obter a proteína na forma de monômero da subunidade B, mas sem a presença da porção A2. O antígeno foi incorporado a lipossomas multilamelares (MLVs), combinados ao lipídio A e administrados por via subcutânea a camundongos. Animais imunizados desenvolveram anticorpos sistêmicos específicos contra Stx2 capazes de neutralizar a toxina in vitro e conferir proteção parcial a animais desafiados com dose letal da toxina. Em conclusão, o trabalho confirmou o potencial vacinal do antígeno e validou a estratégia baseada na incorporação do antígeno às MLVs como estratégia de imunização. / Infections associated with strains of enterohaemorrhagic Escherichia coli (EHEC), can cause clinical manifestations are the hemolytic uremic syndrome (HUS), the most severe complication. HUS is related to the presence of Shiga toxin type 2 (Stx2) and yet do not have a vaccine or effective treatments for use in humans. This work aimed to develop a vaccine based on non-toxic derivative containing the B subunit and the A2 portion of the subunit called Stx2ΔAB. After expression in E. coli strains and initial purification attempts resulted in the formation of protein aggregates. Adjustments in the cultivation and purification conditions have enabled the protein as the monomer subunit B but without the presence of the A2 portion. The antigen was incorporated into multilamellar liposomes (MLVs), the combined lipid A and administered subcutaneously to mice. immunized animals develop systemic antibodies specific against Stx2 able to neutralize toxin in vitro and to confer partial protection when challenged with a lethal dose of toxin. In conclusion, the study confirmed the potential vaccine antigen and validated strategy based on antigen incorporation into MLVs as immunization strategy.

書儀是研究唐、五代社會應用文書的重要資料也是敦煌文學中重要的一環。本文在大陸學者已有相關資料匯集之後進一步分析書儀中應用文書的特性。文中先探討「書儀」及「應用文書」的定義並探討二者的共同特性及相異之處，以此作為分析敦煌寫本書儀應用文書的準則並簡單介紹敦煌寫本書儀的作者與年代；其次為敦煌寫本書儀進行分類，依使用狀況的不同，分為一般應用文書、吉儀、凶儀、書、啟、狀再依其使用者的不同細分成類闡明各類書儀的形式與內容勾勒出敦煌所存唐、五代寫本書儀的一個模型。最後則探討敦煌寫本書儀在應用文上的特性，並與唐、五代的應用文書相比較檢視其異同之處。就應用文書的特性來看敦煌寫本書儀保存了當時書信形式及語言也反映當時人對於書信範本的需求及使用應用文書時的普遍性規則。其中表、啟、狀等書牘的基本架構與全唐所載的相關文獻是相符的。雖寫本年代前後不一其用語、形式上卻有相當的一致性。此外在書儀可以見到當時人的人際關係、風俗禮儀、語言文學等。

本篇論文以將國民政府視為革命新政權之假設為論述基礎，試圖對北伐階段的歷史進行更清楚的分析。首先列入討論的是革命政權，或謂新政府的行為模式探討。其中包括：革命的意義及發生背景、革命政權決策的產出及新政府之外交取向，末了再以上述理論基礎為根據，闡明國民政府之革命政權本質。以後論述內容均以國民政府此一特質為分析要點。在第三章中將影響國民政府外交決策的外部因素做一分析，首先提到的是自辛亥革命以來的國內局勢及國民黨發展空間之背景，接著對談到國民黨與英、美、日三國的關係，進一步則對影響北伐時期國府甚鉅的聯俄容共政策有所說明，蘇聯對國民政府而言，有著根本內在的影響，而英、美、日三國則左右著廢除不平等條約談判。第四章則述及國府內部的決策考量，例如：排外運動之風起雲湧、南北對峙的時代背景及國民黨內分裂狀況對決策產出之影響。除此之外，執行政策者如陳友仁、伍朝樞及王正廷三人本身的考慮亦為決策重點。分別提及影響決策制定的外在環境與內在因素後，將提出幾件較為人注意的對外交涉個案，以佐證上述之分析，租界之收回、關稅自主及治外法權之撤廢是論述重點，不過基因於五卅慘案和南京事件分別亦帶有群眾外交之始末意義，因此也附帶一提。而在結論中將對所謂「革命外交」之意義重新審定，進而對整個外交成果做一回顧。